Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management, 2nd Edition

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DISORDERS OF HEMOGLOBIN Genetics, Pathophysiology, and Clinical Management SECOND EDITION This book is a completely revised new edition of the definitive reference on disorders of hemoglobin. Authored by world-renowned experts, the book focuses on basic science aspects and clinical features of hemoglobinopathies, covering diagnosis, treatment, and future applications of current research. While the second edition continues to address the important molecular, cellular, and genetic components, coverage of clinical issues has been significantly expanded, and there is more practical emphasis on diagnosis and management throughout. The book opens with a review of the scientific underpinnings. Pathophysiology of common hemoglobin disorders is discussed next in an entirely new section devoted to vascular biology, the erythrocyte membrane, nitric oxide biology, and hemolysis. Four sections deal with ␣ and ␤ thalassemia, sickle cell disease, and related conditions, followed by special topics. The second edition concludes with current and developing approaches to treatment, incorporating new agents for iron chelation, methods to induce fetal hemoglobin production, novel treatment approaches, stem cell transplantation, and progress in gene therapy. Martin H. Steinberg is Professor of Medicine, Pediatrics, Pathology and Laboratory Medicine at Boston University School of Medicine and Director of the Center of Excellence in Sickle Cell Disease at Boston Medical Center. He received his BA from Cornell University and an MD from Tufts University School of Medicine. Dr. Steinberg is a diplomat of the American Board of Internal Medicine in the subspecialty of Hematology, a Fellow of the American Association for the Advancement of Science and a member of the American Society for Clinical Investigation and Association of American Physicians. Dr. Steinberg’s research and clinical interests are focused on disorders of the red blood cell with a special emphasis on sickle cell disease and inherited disorders of hemoglobin. His current work focuses on genotypephenotype relationships in sickle cell disease and thalassemia, and how multiple genes influence the phenotype of disease. Dr. Steinberg has published nearly 300 articles in his areas of interest and has edited three textbooks that focus on the basic science and clinical aspects of sickle cell disease and other disorders of the hemoglobin molecule. He has served as a scientific consultant for the American Heart Association, FDA, NIH, NSF, Doris Duke Charitable Foundation, US-Israel Binational Science Foundation, Wellcome Trust, Telethon2002, ISERM, Accreditation Council for Graduate Medical Education, and the Department of Veterans Affairs, and served on the editorial boards of the American Journal of Hematology, American

Journal of the Medical Sciences, BMC Medical Genetics, Haematologica, Journal of Laboratory and Clinical Medicine and Hemoglobin. Bernard G. Forget is a distinguished physician scientist in Hematology, nationally and internationally recognized for research accomplishments in the field of Molecular Hematology pertaining to the molecular biology of gene expression in blood cells and the molecular basis of hereditary disorders of the red blood cell, including hemoglobinopathies. He is the co-author with Dr. H. F. Bunn of a highly respected textbook entitled Hemoglobin: Molecular, Genetic and Clinical Aspects, (WB Saunders Co., Philadelphia, 1986). He is the senior author of a large number of scientific publications in the field of Molecular Hematology and red blood cell disorders, published in leading journals He has also co-authored a number of chapters on thalassemia and other red blood cell disorders in various leading hematology textbooks. Douglas R. Higgs qualified in medicine at King’s College Hospital Medical School in 1974 and trained as a haematologist. He joined the MRC Molecular Haematology Unit (Oxford) in 1977 and is currently Professor of Molecular Haematology at the University of Oxford and Director of the MRC Molecular Haematology Unit. The current interests of the Unit are (i) to understand the processes of lineage commitment in haemopoiesis with particular emphasis on erythropoiesis (ii) to understand how the globin genes are activated and regulated during normal erythropoiesis (iii) to study the human genetic diseases affecting these processes. The main interest of his own laboratory has been to understand how the human alpha globin genes are regulated from their natural chromosomal environment in the telomeric region of 16p13.3. Recently the group has characterised the terminal 2 Mb of chromosome 16 and concentrated on understanding how gene expression is influenced by epigenetic modifications of this region (e.g. chromatin structure, histone acetylation, methylation, timing of replication, nuclear positioning) and the proteins that mediate these processes. David J. Weatherall is currently Regius Professor of Medicine Emeritus, University of Oxford and Chancellor, Keele University, Keele, UK. His major research contributions have been in the elucidation of the clinical, biochemical and molecular characteristics of the thalassaemias and their related disorders, the population genetics of these conditions, and the application of this information to the development of programmes for the prevention and management of these diseases in the developing countries.

DISORDERS OF HEMOGLOBIN Genetics, Pathophysiology, and Clinical Management SECOND EDITION Edited by Martin H. Steinberg Boston University School of Medicine

Bernard G. Forget Yale University School of Medicine

Douglas R. Higgs University of Oxford

David J. Weatherall University of Oxford

CAMBRIDGE UNIVERSITY PRESS

Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo, Delhi, Dubai, Tokyo Cambridge University Press The Edinburgh Building, Cambridge CB2 8RU, UK Published in the United States of America by Cambridge University Press, New York www.cambridge.org Information on this title: www.cambridge.org/9780521875196 © Cambridge University Press 2009 This publication is in copyright. Subject to statutory exception and to the provision of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published in print format 2009

ISBN-13

978-0-511-59618-6

eBook (NetLibrary)

ISBN-13

978-0-521-87519-6

Hardback

Cambridge University Press has no responsibility for the persistence or accuracy of urls for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate. Information regarding prices, travel timetables, and other factual information given in this work are correct at the time of first printing, but Cambridge University Press does not guarantee the accuracy of such information thereafter. Every effort has been made in preparing this book to provide accurate and up-todate information that is in accord with accepted standards and practice at the time of publication. Although case histories are drawn from actual cases, every effort has been made to disguise the identities of the individuals involved. Nevertheless, the authors, editors, and publisher can make no warranties that the information contained herein is totally free from error, not least because clinical standards are constantly hanging through research and regulation. The authors, editors, and publisher therefore disclaim all liability for direct or consequential damages resulting from the use of material contained in this book. Readers are strongly

Contents

List of Contributors Foreword, by H. Franklin Bunn

page ix xv

Preface

xvii

Introduction, by David J. Weatherall

xix

SECTION ONE. THE MOLECULAR, CELLULAR, AND GENETIC BASIS OF HEMOGLOBIN DISORDERS

Douglas R. Higgs and Bernard G. Forget

1 A Developmental Approach to Hematopoiesis Elaine Dzierzak

3

2 Erythropoiesis Sjaak Philipsen and William G. Wood

24

3 The Normal Structure and Regulation of Human Globin Gene Clusters Bernard G. Forget and Ross C. Hardison

46

4 Nuclear Factors That Regulate Erythropoiesis Gerd A. Blobel and Mitchell J. Weiss

62

5 Molecular and Cellular Basis of Hemoglobin Switching George Stamatoyannopoulos, Patrick A. Navas, and Qiliang Li

86

6 Structure and Function of Hemoglobin and Its Dysfunction in Sickle Cell Disease Daniel B. Kim-Shapiro 7 Hemoglobins of the Embryo, Fetus, and Adult Martin H. Steinberg and Ronald L. Nagel

101 119

SECTION TWO. PATHOPHYSIOLOGY OF HEMOGLOBIN AND ITS DISORDERS

Martin H. Steinberg

8 Rheology and Vascular Pathobiology in Sickle Cell Disease and Thalassemia Dhananjay K. Kaul

139

9 The Erythrocyte Membrane Patrick G. Gallagher and Clinton H. Joiner

158

v

vi

Contents

10 The Biology of Vascular Nitric Oxide Jane A. Leopold and Joseph Loscalzo 11 Mechanisms and Clinical Complications of Hemolysis in Sickle Cell Disease and Thalassemia Gregory J. Kato and Mark T. Gladwin 12 Animal Models of Hemoglobinopathies and Thalassemia Mary Fabry

185

201 225

SECTION THREE. ␣ THALASSEMIA

Douglas R. Higgs

13 The Molecular Basis of ␣ Thalassemia Douglas R. Higgs

241

14 The Pathophysiology and Clinical Features of ␣ Thalassaemia Douglas R. Higgs

266

15 Unusual Types of ␣ Thalassemia Douglas R. Higgs, Veronica J. Buckle, Richard Gibbons, and David Steensma

296

SECTION FOUR. THE ␤ THALASSEMIAS

Bernard G. Forget

16 The Molecular Basis of ␤ Thalassemia, ␦␤ Thalassemia, and Hereditary Persistence of Fetal Hemoglobin Swee Lay Thein and William G. Wood

323

17 Clinical Aspects of ␤ Thalassemia and Related Disorders Nancy F. Olivieri and David J. Weatherall

357

18 Hemoglobin E Disorders Suthat Fucharoen and David J. Weatherall

417

SECTION FIVE. SICKLE CELL DISEASE

Martin H. Steinberg

19 Clinical and Pathophysiological Aspects of Sickle Cell Anemia Martin H. Steinberg, Kwaku Ohene-Frempong, and Matthew M. Heeney

437

20 Sickle Cell Pain: Biology, Etiology, and Treatment Samir K. Ballas and James R. Eckman

497

21 Hemoglobin SC Disease and Hemoglobin C Disorders Martin H. Steinberg and Ronald L. Nagel

525

22 Sickle Cell Trait Martin H. Steinberg

549

23 Other Sickle Hemoglobinopathies Martin H. Steinberg

564

SECTION SIX. OTHER CLINICALLY IMPORTANT DISORDERS OF HEMOGLOBIN

Martin H. Steinberg

24 Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants of Clinical and Biological Interest Martin H. Steinberg and Ronald L. Nagel

589

Contents

25 Dyshemoglobinemias Neeraj Agarwal, Ronald L. Nagel, and Josef T. Prchal

vii 607

SECTION SEVEN. SPECIAL TOPICS IN HEMOGLOBINOPATHIES

Martin H. Steinberg

26 Population Genetics and Global Health Burden David J. Weatherall and Thomas N. Williams

625

27 Genetic Modulation of Sickle Cell Disease and Thalassemia Martin H. Steinberg and Ronald L. Nagel

638

28 Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders Mary Fabry and John M. Old

658

SECTION EIGHT. NEW APPROACHES TO THE TREATMENT OF HEMOGLOBINOPATHIES AND THALASSEMIA

Martin H. Steinberg

29 Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease Janet L. Kwiatkowski and John B. Porter 30 Induction of Fetal Hemoglobin in the Treatment of Sickle Cell Disease and ␤ Thalassemia Yogen Saunthararajah and George F. Atweh

689

745

31 Novel Approaches to Treatment Kirkwood A. Pritchard Jr., Alicia Rivera, Cheryl Hillery, and Carlo Brugnara

755

32 Stem Cell Transplantation Emanuele Angelucci and Mark Walters

774

33 Prospects for Gene Therapy of Sickle Cell Disease and Thalassemia Derek A. Persons, Brian P. Sorrentino, and Arthur W. Nienhuis

791

Index

815

Bernard G. Forget, MD Professor of Medicine and Genetics Director, Hematology Training Program Section of Hematology Department of Medicine Yale University School of Medicine New Haven, CT

List of Contributors

Chapter 1: A Developmental Approach to Hematopoiesis Elaine Dzierzak, PhD Professor of Developmental Biology Erasmus Stem Cell Institute Erasmus Medical Center Rotterdam, The Netherlands

Chapter 2: Erythropoiesis Foreword H. Franklin Bunn, MD Professor of Medicine Division of Hematology Brigham and Women’s Hospital Harvard Medical School Boston, MA

Preface Martin H. Steinberg, MD

Sjaak Philipsen, PhD Professor of Genomics of Cell Differentiation Department of Cell Biology Erasmus University Medical Center Rotterdam, The Netherlands William G. Wood, PhD Professor of Haematology MRC Molecular Haematology Unit Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington, Oxford, UK

Bernard G. Forget, MD Douglas R. Higgs, MD, FRS Sir David J. Weatherall, MD, FRS

Introduction Sir David J. Weatherall, MD, FRS Emeritus Professor of Medicine (University of Oxford) Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington, Oxford, UK

SECTION ONE. The Molecular, Cellular, and Genetic Basis of Hemoglobin Disorders Douglas R. Higgs, MD, FRS Professor of Molecular Haematology and Director of the MRC Molecular Haematology Unit (University of Oxford) MRC Molecular Haematology Unit Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington, Oxford, UK

Chapter 3: The Normal Structure and Regulation of Human Globin Gene Clusters Bernard G. Forget, MD. Ross C. Hardison, PhD T. Ming Chu Professor of Biochemistry and Molecular Biology The Pennsylvania State University University Park, PA

Chapter 4: Nuclear Factors That Regulate Erythropoiesis Gerd A. Blobel, MD, PhD Professor of Pediatrics Division of Hematology The Children’s Hospital of Philadelphia University of Pennsylvania School of Medicine Philadelphia, PA Mitchell J. Weiss, MD, PhD Associate Professor of Pediatrics Division of Hematology The Children’s Hospital of Philadelphia University of Pennsylvania School of Medicine Philadelphia, PA ix

x Chapter 5: Molecular and Cellular Basis of Hemoglobin Switching George Stamatoyannopoulos, MD, Dr Sci Professor of Medicine and Genome Sciences Director, Markey Molecular Medicine Center University of Washington School of Medicine Seattle, WA Patrick A. Navas, PhD Research Assistant Professor Division of Medical Genetics Department of Medicine University of Washington School of Medicine Seattle, WA Qiliang Li, PhD Research Professor of Medicine Division of Medical Genetics Department of Medicine University of Washington School of Medicine Seattle, WA

Chapter 6: Structure and Function of Hemoglobin and Its Dysfunction in Sickle Cell Disease Daniel B. Kim-Shapiro, PhD Professor of Physics Department of Physics Wake Forest University Olin Physical Laboratory Winston Salem, NC

Chapter 7: Hemoglobins of the Embryo, Fetus, and Adult Martin H. Steinberg, MD Professor of Medicine Pediatrics, Pathology and Laboratory Medicine Boston University School of Medicine Boston, MA

Contributors Chapter 9: The Erythrocyte Membrane Patrick G. Gallagher, MD Professor of Pediatrics Section of Perinatal Medicine Yale University School of Medicine New Haven, CT Clinton H. Joiner, MD, PhD Professor of Pediatrics Children’s Hospital Medical Center Cincinnati, OH

Chapter 10: The Biology of Vascular Nitric Oxide Jane A. Leopold, MD Associate Professor of Medicine Cardiovascular Medicine Division Department of Medicine Brigham and Women’s Hospital Boston, MA Joseph Loscalzo, MD, PhD Hersey Professor of the Theory and Practice of Medicine Chairman, Department of Medicine Brigham and Women’s Hospital Boston, MA

Chapter 11: Mechanisms and Clinical Complications of Hemolysis in Sickle Cell Disease and Thalassemia Gregory J. Kato, MD Director, Sickle Cell Vascular Disease Unit Vascular Therapeutic Section Vascular Medicine Branch National Institutes of Health Bethesda, MD

Martin H. Steinberg, MD

Mark T. Gladwin, MD Professor of Medicine Division Chief, Pulmonary, Allergy, and Critical Care Medicine University of Pittsburgh Medical Center Director, Hemostasis and Vascular Biology Research Institute University of Pittsburgh Pittsburgh, PA

Chapter 8: Rheology and Vascular Pathobiology in Sickle Cell Disease and Thalassemia

Chapter 12: Animal Models of Hemoglobinopathies and Thalassemia

Dhananjay K. Kaul, PhD Professor of Medicine Division of Hematology Albert Einstein College of Medicine Bronx, NY

Mary Fabry, PhD Professor of Medicine Division of Hematology Albert Einstein College of Medicine Bronx, NY

Ronald L. Nagel, MD New York, NY

SECTION TWO. Pathophysiology of Hemoglobin and Its Disorders

Contributors SECTION THREE. ␣ Thalassemia Douglas R. Higgs, MD, FRS

Chapter 13: The Molecular Basis of ␣ Thalassemia

xi Division of Clinical Investigation and Human Physiology University Health Network Toronto General Hospital Toronto, ON, Canada

Douglas R. Higgs, MD, FRS

Sir David J. Weatherall, MD, FRS

Chapter 14: The Pathophysiology and Clinical Features of ␣ Thalassemia

Chapter 18: Hemoglobin E Disorders

Douglas R. Higgs, MD, FRS

Chapter 15: Unusual Types of ␣ Thalassemia Douglas R. Higgs, MD, FRS. Veronica J. Buckle, MD MRC Senior Scientist MRC Molecular Haematology Unit Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington, Oxford, UK Richard Gibbons, MD University Lecturer and Honorary Consultant Clinical Geneticist Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington, Oxford, UK David Steensma, MD Associate Professor of Medicine and Oncology Consultant, Division of Hematology Mayo Clinic Rochester, MN

SECTION FOUR. The ␤ Thalassemias Bernard G. Forget, MD

Chapter 16: The Molecular Basis of ␤ Thalassemia, ␦␤ Thalassemia, and Hereditary Persistence of Fetal Hemoglobin Swee Lay Thein, MD Professor of Molecular Haematology Head, Division of Gene and Cell Based Therapy King’s College London School of Medicine and King’s College Hospital London, UK

Suthat Fucharoen, MD Director, Thalassemia Research Center Institute of Science and Technology for Research and Development Mahidol University Salaya Campus Puttamonthon, Nakornpathom, Thailand Sir David J. Weatherall, MD, FRS

SECTION FIVE. Sickle Cell Disease Martin H. Steinberg, MD

Chapter 19: Clinical and Pathophysiological Aspects of Sickle Cell Anemia Martin H. Steinberg, MD. Kwaku Ohene-Frempong, MD Professor of Pediatrics Hematology The Children’s Hospital of Philadelphia Philadelphia, PA Matthew M. Heeney, MD Instructor in Pediatrics Harvard Medical School Boston, MA

Chapter 20: Sickle Cell Pain: Biology, Etiology, and Treatment Samir K. Ballas, MD Professor of Medicine and Pediatrics Thomas Jefferson University Philadelphia, PA James R. Eckman, MD Professor of Medicine Comprehensive Sickle Cell Center Emory University School of Medicine Atlanta, GA

William G. Wood, PhD

Chapter 17: Clinical Aspects of ␤ Thalassemia and Related Disorders Nancy F. Olivieri, MD Senior Scientist

Chapter 21: Hemoglobin SC Disease and Hemoglobin C Disorders Martin H. Steinberg, MD Ronald L. Nagel, MD

xii Chapter 22: Sickle Cell Trait Martin H. Steinberg, MD

Contributors Chapter 28: Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders Mary Fabry, PhD

Chapter 23: Other Sickle Hemoglobinopathies Martin H. Steinberg, MD

SECTION SIX. Other Clinically Important Disorders of Hemoglobin

John M. Old, MD Consultant Clinical Scientist National Haemoglobinopathy Reference Laboratory Oxford Haemophilia Centre Churchill Hospital Oxford, UK

Martin H. Steinberg, MD

Chapter 24: Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants of Clinical and Biological Interest Martin H. Steinberg, MD Ronald L. Nagel, MD

Chapter 25: Dyshemoglobinemias Neeraj Agarwal, MD Assistant Professor, Oncology Division University of Utah, School of Medicine Salt Lake City, UT Ronald L. Nagel, MD Josef T. Prchal, MD Professor of Medicine Internal Medicine Hematology Division University of Utah Salt Lake City, UT

SECTION SEVEN. Special Topics in Hemoglobinopathies Martin H. Steinberg, MD

Chapter 26: Population Genetics and Global Health Burden Sir David J. Weatherall, MD, FRS Thomas N. Williams, PhD Wellcome Trust Senior Clinical Fellow Kenya Medical Research Institute/Wellcome Trust Programme Centre for Geographic Medical Research Kilifi District Hospital Kilifi, Kenya

Chapter 27: Genetic Modulation of Sickle Cell Disease and Thalassemia Martin H. Steinberg, MD Ronald L. Nagel, MD

SECTION EIGHT. New Approaches to the Treatment of Hemoglobinopathies and Thalassemia Martin H. Steinberg, MD

Chapter 29: Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease Janet L. Kwiatkowski, MD Assistant Professor of Pediatrics Division of Hematology The Children’s Hospital of Philadelphia Philadelphia, PA John B. Porter, MA, MD, FRCP, FRCPath Professor of Haematology Department of Haematology University College London London, UK

Chapter 30: Induction of Fetal Hemoglobin in the Treatment of Sickle Cell Disease and ␤ Thalassemia Yogen Saunthararajah, MD Associate Professor Cleveland Clinic/University of Illinois at Chicago Twissing Cancer Institute Cleveland, OH George F. Atweh, MD Koch Professor of Medicine Director, Division of Hematology/Oncology Director, Barrett Cancer Center University of Cincinnati College of Medicine Cincinnati, OH

Chapter 31: Novel Approaches to Treatment Kirkwood A. Pritchard Jr., PhD Professor of Pediatric Surgery Medical College of Wisconsin Milwaukee, WI Alicia Rivera, PhD Instructor of Pediatrics Harvard Medical School Boston, MA

Contributors Cheryl Hillery, MD Blood Center of Wisconsin Associate Professor Pediatrics and Medicine Medical College of Wisconsin Milwaukee, WI Carlo Brugnara, MD Professor of Pathology Harvard Medical School Children’s Hospital Boston, MA

Chapter 32: Stem Cell Transplantation Emanuele Angelucci, MD Associate Professor Head, Hematology Department and BMT Centre Armando Businco Cancer Centre Cagliari, Italy Mark Walters, MD Director, Blood and Marrow Transplantation Program Children’s Hospital Oakland Research Institute Oakland, CA

xiii Chapter 33: Prospects for Gene Therapy of Sickle Cell Disease and Thalassemia Derek A. Persons, MD Assistant Member Department of Hematology Division of Experimental Hematology St. Jude Children’s Research Hospital Memphis, TN Brian P. Sorrentino, MD Member Department of Hematology Director, Division of Experimental Hematology St. Jude Children’s Research Hospital Memphis, TN Arthur W. Nienhuis, MD Member Department of Hematology Division of Experimental Hematology St. Jude Children’s Research Hospital Memphis, TN

Foreword H. Franklin Bunn

The study of hemoglobin continues to be a rewarding endeavor. Cumulative progress since the turn of the last century has laid cornerstones in protein chemistry and molecular genetics and has provided a wealth of insight into the pathogenesis of some of the world’s most prevalent and devastating disorders. The first edition of Disorders of Hemoglobin, published 8 years ago, was a comprehensive compilation and analysis of the basic science of hemoglobin and its application to the thalassemias, sickle cell disease, and other globin mutants that spawn a wide range of clinical phenotypes. This second edition now presents an updated overview of all aspects of the hemoglobin story as well as a detailed account of the impressive advances that have been made in biochemistry, genetics, and clinical investigation. Hemoglobin boasts a proud history. By the end of the nineteenth century, it was well established that hemoglobin was a composite of protein and heme that could reversibly bind oxygen and that this substance was found in almost all living creatures. Entry into the twentieth century marked the dawn of quantitative physiology, biochemistry, and the application of the scientific method to medicine. All three of these developing disciplines owe their early impetus to hemoglobin and the lessons learned from this remarkable molecule. Physiologists from Scandinavia (Bohr and Krogh) and England (Barcroft, the Haldanes, and Roughton) made accurate equilibrium and kinetic measurements of oxygen– hemoglobin binding as a function of pH and thereby provided a mechanistic understanding of the reciprocal transport of oxygen from lung to tissues and of acid waste from tissues to lung. These early contributions set the stage for an appreciation of how the homeostasis of the organism depends on the orderly integration of its organ systems. The fledgling science of biochemistry was given a jump start by the studies of Adair and Svedberg, which established that hemoglobin is a uniform protein with a large but narrowly defined molecular weight and was therefore,

like sodium chloride and glucose, a bona fide molecule. Hemoglobin and its cousin myoglobin were the first proteins whose structures were solved at high resolution by X-ray crystallography by Perutz and Kendrew, respectively, thereby, providing an opportunity for detailed exploration of structure–function relationships. Hemoglobin was the first multisubunit protein to be understood at the molecular level and therefore was the model system used by Monod, Changeux, and Wyman for establishing the principles of allostery, which dictate the regulation of a broad range of enzymes, receptors, transcription factors, and so on. The linkage of specific diseases to abnormalities of specific molecules began with Pauling’s demonstration in 1949 that patients with sickle cells have hemoglobin with an altered surface charge. Within 8 years, Ingram demonstrated that sickle hemoglobin differs from normal hemoglobin only by a substitution of valine for glutamic acid in the sixth residue of the ␤-globin subunit. This was the first example of how an abnormal gene can change the structure of a protein and, therefore, verified in a most satisfying way the Beadle–Tatum one gene–one enzyme hypothesis. During the last quarter of the twentieth century, with the development of recombinant DNA technology and genomics, hemoglobin again became primus inter pares among biological molecules. Indeed, the human globin genes were among the first to be molecularly cloned and sequenced. This soon led to the identification of a wide range of globin gene mutants responsible for the ␣ and ␤ thalassemias. Understanding the mechanisms by which these genotypes impair globin biosynthesis provided insight into the diverse clinical manifestations encountered in patients with different types of thalassemia. In addition, the evolving knowledge of human globin genes enabled the development of molecular techniques for antenatal diagnosis and polymorphism-based population studies, both of which were then applied to many other disorders. To date, more than 1,000 hemoglobin variants have been discovered and characterized. Study of these variants, so amply documented in this book, established the principle of how a mutant genotype alters the function of the protein it encodes, which in turn can lead to a distinct clinical phenotype. This linkage is at the heart of how molecular genetics impacts our understanding of pathophysiological mechanisms. Thus, hemoglobin held center stage in the biomedical discoveries of the twentieth century, and, in the new millenium, there is no indication that the pace has slackened. This book begins with authoritative and up-to-date coverage of all aspects of hemoglobin, beginning with overviews of erythropoiesis, globin gene regulation, and structure– function relationships. Subsequent sections of the book are devoted to in-depth coverage of the thalassemias, sickle cell disease, and other hemoglobinopathies. A recurrent theme is how understanding pathophysiology at the molecular xv

xvi level has informed the design and development of novel, rationally based therapy. This second edition incorporates a number of advances that have been made in the past 8 years. Chapter 4 describes the important insights that have accrued from the discovery of ␣-hemoglobin stabilizing protein (AHSP), the chaparone that protects the ␣-hemoglobin subunit during assembly of the tetramer. Chapters 6, 10, and 11 include new information on nitric oxide and its controversial roles in allosteric modulation of hemoglobin function and in the pathophysiology of sickle cell disease and other types of hemolytic anemia. Chapter 27 presents recent information on the contribution of genetic polymorphisms to the clinical phenotypes of sickle cell disease and thalassemia. The last 4 chapters cover the development of oral iron chelators

Foreword as well as bolder therapeutic strategies, including impressive progress in globin gene therapy. The creative energy that continues to bear down on all aspects of hemoglobin research is well represented by the impressive list of basic and clinical investigators who have contributed to this book. As in any field at the cutting edge of science, controversies enrich the scientific dialogue among hemoglobinologists. In carefully reading chapters on closely related topics, the thoughtful reader will adopt a policy of caveat emptor, appreciating that strongly held opinions need to be vetted by both experimentation and alternative hypotheses. This proviso notwithstanding, Disorders of Hemoglobin offers authoritative and comprehensive coverage of one of the most exciting and fruitful areas at the interface of bioscience and clinical medicine.

Preface

Eight years have passed since this monograph first appeared, and the advances in basic, translational, and clinical research during this interval justify a new edition. To conserve space and avoid duplicating our first edition, we review very briefly historical aspects, summarize established older information, and focus on the progress of the past 8 years. Although some older references are retained, we have tried to focus on the literature since 2001. In expanding our coverage of clinical issues, we also have decreased the length of the book by considering together pathophysiological features common to many hemoglobin disorders such as vasculopathy, erythrocyte membrane damage, and mechanisms of hemolysis. More than half of the contributors to this volume are either new authors or previous authors addressing different topics; David Weatherall has joined the editorial team. Hemoglobin has been an interest of basic and translational scientists, clinicians, and clinical diagnostic laboratories. So, we continue to address the molecular, cellular, genetic, diagnostic, and clinical aspects of hemoglobin disorders. When applicable, we provide practical recommendations for diagnosis and treatment. The first section of the book again focuses on molecular, cellular, and genetic aspects of hemoglobin and includes discussions of developmental hematopoiesis, erythropoiesis, globin genes and their regulation, minor normal hemoglobins, and an update on new structural and functional features of normal and variant hemoglobins. Pathophysiology of hemoglobin disorders follows, with new chapters on vascular biology, the erythrocyte membrane, the biology of nitric oxide, mechanisms of hemolysis, and how animal models

of disease provide new pathophysiological insights. Four sections deal with diagnosis, complications, and treatment of ␣ thalassemia, ␤ thalassemia, and related conditions, including hemoglobin E diseases, sickle cell disease, and less common genetic and acquired hemoglobin disorders. This is followed by special topics such as population genetics and the health burden of hemoglobin disorders, the genetic modulation of sickle cell disease and thalassemia, and developments in laboratory detection, including antenatal diagnosis. Finally, current and developing approaches to treatment, incorporating new agents for iron chelation, methods to induce fetal hemoglobin production, novel treatment approaches such as antioxidants, antiinflammatory agents, enhancement of nitric oxide effects, and agents that modulate membrane cation and water transport are discussed, concluding with the use of stem cell transplantation and progress in gene therapy. Ronald L. Nagel (pictured), a coeditor of the first edition, has retired as Irving D. Karpas Professor of Medicine, Physiology and Biophysics and Head of the Division of Hematology at Albert Einstein College of Medicine. Although no longer a coeditor of this monograph, his influence in the field is felt in most chapters. His contributions to the structure, function, pathophysiology, and genetics of hemoglobin disorders are vast and time tested. The editors, and the field of hematology, will miss his scientific insight and originality. The Editors

xvii

Introduction David J. Weatherall

A few years ago, an eminent British professor of medicine, while reviewing a new edition of a well-known textbook of medicine, suggested that works of this type were becoming valueless because they were already out of date by the time they were published. His derogatory comments went further: Having taken the trouble to weigh the book, he suggested that volumes of this type would suffer the same fate as dinosaurs and become extinct by collapsing under their excessive weight. Even allowing for this bizarre and completely erroneous view of the biological fate of the dinosaurs, does this argument carry any weight beyond its metaphorical context? Undoubtedly, there is feeling rife among medical publishers that the day of the major monograph in the biological sciences may be coming to an end. They argue that there is so much information online that the need for works of this type is becoming increasingly limited. Is this really the case? Although it is impossible to deny that the long gestation of monographs of this type may lead to the omission of the occasional “breakthrough” in a field, it seems very important that in any rapidly moving area of the biomedical sciences there is a regular and broad critical review of where it has got to and how it has been modified by recent advances. Not uncommonly in medical research and practice, today’s breakthrough is tomorrow’s breakdown. Is the hemoglobin field moving rapidly? This was another question that had to be considered by the editors of this new edition. As judged by the amount of space given to disorders of the red cell in current journals, the volume of work in this field seems to have declined considerably over recent years. A visitor from outer space, browsing through the journals, might be excused for wondering how Homo sapiens transfers oxygen to their tissues. Hence, it might have been perceived that there is insufficient material to warrant this new edition. A broader review of the field over recent years suggests, however, that this is not the case. There undoubtedly have

been major advances in our understanding of the regulation of hematopoiesis, some of which have important implications for a better understanding of the pathophysiology of the hemoglobin disorders that may, in the longer term, lead to more definitive approaches to their management. Furthermore, there have also been dramatic developments in many areas of genome technology that have direct application to the many unanswered questions of the hemoglobin field, not in the least the reasons for the remarkable phenotypic variation of its diseases. Of even greater importance, there has been a genuine increase in the appreciation of the major public health burden that these diseases are likely to cause in the future. This is particularly relevant to the poorer countries of the world in which the epidemiological transition following improvements in nutrition and basic public health is resulting in a reduction in neonatal and childhood mortality; many babies with severe hemoglobin disorders who would previously have died in early life are now surviving to present for diagnosis and management. It is only in the last few years that these public health issues have been recognized by the major international health agencies. In 2002, the World Health Organization (WHO) published a report, Genomics and World Health, in which the hemoglobin disorders were described as a prime example of how the new technology of molecular genetics can be applied for the benefit of poorer countries. At the 118th session of the WHO Executive Board, held in 2006, the sickle cell disorders and thalassemias were formally recognized as major health burdens that required immediate action. In 2007, it was decided to include the hemoglobin disorders in the Global Burden of Disease Program, an international study conducted under the auspices of several universities, the WHO, the Bill and Melinda Gates Foundation, the World Bank, and others that attempts to define the relative global burden posed by each of the major diseases. Previous versions of this work have undoubtedly had a major influence on developing healthcare policies by governments and international healthcare agencies. Clearly, this new edition is appearing at the same time as a major drive to define the most appropriate ways of controlling and managing the hemoglobin disorders, particularly in the developing countries, and to determine the most cost-effective and efficient ways of approaching this problem. We hope, therefore, that this updated distillation of knowledge about the scientific, clinical, and epidemiological aspects of this field will be of value to scientists and clinicians, not only to those in wealthier countries but particularly to those who are attempting to cope with these diseases with limited resources in the developing countries of the world. There is also an important message for our younger readers. There are still some extraordinarily exciting areas of this field to be pursued, not in the least a better understanding of the reasons for the remarkable clinical

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xx diversity of all the hemoglobin disorders; a better appreciation of their pathophysiology at the molecular level with respect to novel approaches for their more definitive management; and an understanding of the long-neglected role of the environment in their clinical diversity, the cellular mechanisms whereby protection against malaria has resulted in their extremely high frequency, how current knowledge of their diagnosis and control may be applied in the poorer countries of the world, and many other stimulating questions. Currently, the hemoglobin field offers challenges ranging from basic cell and molecular biology

Introduction through clinical research at the bedside to epidemiology, public health, and the social sciences. Finally, we thank Cambridge University Press and particularly Beth Barry and more recently Larry Fox for continued support of this project. We are also extremely grateful to the authors from many parts of the world who have willingly given their time to writing parts of this new edition, and for the personal help that we have received from Liz Rose, during its preparation. It is particularly gratifying to be able to report that the marriages of the four editors have survived another edition.

SECTION ONE

THE MOLECULAR, CELLULAR, AND GENETIC BASIS OF HEMOGLOBIN DISORDERS Douglas R. Higgs and Bernard G. Forget

Over the past 30 years we have become familiar with the way in which different types of hemoglobin are expressed at different stages of development. In the human embryo the main hemoglobins include Hb Portland (␨ 2 ␥ 2 ), Hb Gower I (␨ 2 ε 2 ), and Gower II (␣2 ε 2 ). In the fetus, HbF (␣2 ␥ 2 ) predominates and in the adult, HbA (␣2 ␤2 ) makes up the majority of hemoglobin in red cells. These simple facts belie the complexity of the cellular and molecular processes that bring about these beautifully coordinated changes in the patterns of globin gene expression throughout development. To understand these phenomena we have to consider the individual components including 1) the origins of erythroid cells in development, 2) the processes by which erythroid cells differentiate to mature red cells at each developmental stage, and 3) the molecular events that produce the patterns of gene expression we observe. Two different types of erythroid cells are observed during development. The first erythroid cells to be seen in the developing embryo are located in the blood islands of the yolk sac. These primitive erythroid cells are morphologically different from the definitive erythroid cells made in the fetal liver and bone marrow and contain predominantly embryonic hemoglobins. Somewhat later during embryonic development, definitive erythroid and other hematopoietic cells originate from multipotent cells identified in a part of the embryo that lies near the dorsal aorta, in the region close to where the kidneys first develop: the so-called aorta-gonads-mesonephros (AGM) region. It is thought that the cells that are destined to provide fetal(liver) and adult- (bone marrow) derived red cells originate from AGM cells, although the ultimate origin of hematopoietic stem cells is still a matter of controversy. In the first trimester of pregnancy, fetal erythroid cells derived predominantly from hematopoiesis in the liver contain mainly HbF with small amounts of embryonic hemoglobin. There are no circumstances in which expression of embryonic globins persists at high levels or becomes substantially

reactivated in fetal or adult life, although low levels of ␨ -globin chains are present in the most severe form of ␣ thalassemia. Until approximately the time of birth, fetal cells continue to make predominantly HbF but switch to making HbA between 30 and 40 weeks postconception. In contrast to the situation in embryonic cells, there are many conditions in which HbF synthesis persists or becomes reactivated in adult red cells. The simplest explanation for all of these observations is that the switch from embryonic to fetal–adult patterns of hemoglobin synthesis involves the replacement of embryonic cells (with one program of expression) by definitive cells (with a different program of expression). In contrast, the switch from fetal to adult hemoglobin expression takes place in definitive cells so this represents a true change in the molecular program within a single lineage of erythroid progenitor cells. At present we do not know when during development the embryonic and fetal programs are established in the differentiating hematopoietic cells. Furthermore we do not fully understand by what mechanisms the programs of globin gene expression are initiated or maintained. Perhaps the greatest progress toward such an understanding has been to identify key regulatory molecules, including transcription factors, cofactors, and chromatin-associated proteins that play important roles in specifying the formation of erythroid cells from multipotent hematopoietic stem cells. Of greatest importance in this area has been the characterization of the tissue-restricted zinc finger proteins (GATA-1 and GATA-2), their cofactors (FOG-1 and FOG2), the b-Zip family of proteins (NF-E2, Nrf1, Nrf2, Nrf3, ¨ Bach1 and Bach2), and the erythroid Kruppel-like factors (EKLF and FKLF). Experiments in which GATA-1, GATA-2, and FOG-1 have been inactivated in the mouse genome show that these proteins play a major role in establishing the erythroid lineage and allowing differentiation to mature red cells. A major focus of interest over the past 20 years has been to understand how these developmental programs are played out on the ␣- and ␤-globin gene clusters. We now know that in most mammals in each cluster the globin genes are arranged along the chromosome in the order in which the genes are expressed in development: the ␣like globin gene cluster on chromosome 16 (␨ -␣2 -␣1 -) and the ␤-like globin gene cluster on chromosome 11 (ε-G ␥ A ␥ -␦-␤-), suggesting that gene order may be important in unfolding this program. Expression of each cluster is dependent on remote regulatory elements, originally identified as DNase I hypersensitive sites in the chromatin of nucleated erythroid cells. In the ␣-globin gene cluster there is a single regulatory element (RE or HS -40) that lies 40 kb upstream of the gene complex, and in the ␤-globin gene cluster there are five major hypersensitive sites, collectively referred to as the ␤-globin locus control region (␤-LCR) lying 5–20 kb upstream of the locus. Again, many details remain unknown but it appears that the ␨ and ε genes are switched on in embryonic cells and are largely 1

2 off in definitive cells in which they cannot be substantially reactivated. With regard to the switch from ␥ - to ␤-globin gene expression during fetal development and neonatal life, the situation is complex. There is strong evidence for autonomous silencing of the ␥ genes, in a manner analogous to that of the ε gene, but it also appears that there may exist some degree of competition between the ␥ and ␤-globin genes that is modified by the transcriptional milieu, which can change dramatically during this time of development, with the balance tipped toward ␥ -globin gene expression in fetal life and ␤-globin gene expression in adult life. The balance between ␥ - and ␤-globin gene expression may be altered in vivo (in hereditary persistence of fetal hemoglobin and other hemoglobinopathies) as well as in various experimental systems. Changes in the repertoire or amounts of transcription factors may influence the switch from ␥ - to ␤-globin gene expression. For example, without EKLF the ␤-globin genes cannot be fully activated during development. Alternatively, alterations in the arrangement of the ␤-LCR and the ␥ - and ␤-like genes with respect to each other may alter the pattern of switching. The precise molecular mechanisms underlying these changes are still poorly understood but it seems unlikely that changes in the patterns of globin gene expression are only brought about through changes in the repertoire of trans-acting factors present in embryonic, fetal, and adult red cells, as originally proposed; however, they may be influenced by other epigenetic changes in the chromosome (e.g., chromatin structure and modification, replication timing, and methylation). Despite our continuing interest and frustrated attempts to fathom how the entire globin clusters are regulated, we do know a lot about the structure and function of individual genes. The globin genes have provided the paradigm for understanding the general arrangement of mammalian genes including their promoters, exons, introns, and processing signals. Furthermore, the mechanisms by which these genes are transcribed into pre-RNA, processed into mature RNA, and translated into protein are now understood in detail. This brings us back in a full circle to where modern molecular biology started by establishing the structure and function of the proteins that are expressed by globin genes. Hemoglobin was one of the first proteins

Douglas R. Higgs and Bernard G. Forget whose amino acid sequence and crystal structure were solved, which in turn led to a complete understanding of how it captures, transports, and releases oxygen. Given the very large number of natural mutants of hemoglobin that have now been identified it also provides an unsurpassed example of how mutations can give rise to “molecular diseases,” the best example still being sickle cell disease. Even with this apparent depth of knowledge, there are still surprises. We know from theory and experiment that erythrocytes containing embryonic hemoglobins and fetal hemoglobins have a higher affinity for oxygen than those containing adult hemoglobin. Traditionally we have surmised that this enables the developing fetus to acquire oxygen more efficiently from the maternal circulation, a seemingly important consideration. We have known for many years, however, that the babies of mothers whose blood contains mainly fetal (high-affinity) hemoglobin are entirely normal. Similarly, thanks to experimental work in model systems, we know that mice, which by design only make embryonic hemoglobin throughout fetal and adult life, survive normally and thrive as adults. Presumably the complex system of hemoglobin switching that keeps investigators so busy has been molded in very subtle ways by natural selection. So why do we pursue this subject with such enthusiasm? There are two main reasons. The first is that the globin system still provides the most thoroughly studied and comprehensively understood example of mammalian gene expression we have. If there are undiscovered general principles governing the regulation of mammalian genes, then analysis of globin gene expression is likely to elucidate them. The second is that understanding how these genes are controlled offers the best hope of developing strategies to ameliorate or cure the many thousands of severely affected patients who inherit defects in the structure or production of the ␣- and ␤-like globin chains that make up embryonic, fetal, and adult hemoglobins. The following seven chapters trace the genesis of hemoglobin, from the earliest appearance of erythroid cells during development, through the nuclear factors that govern its synthesis, the evolution of globin genes, their organization and switching, to the production of hemoglobin and its functions in the erythrocyte.

1 A Developmental Approach to Hematopoiesis Elaine Dzierzak

INTRODUCTION AND GENERAL CONSIDERATIONS During mammalian development, the first morphologically recognizable blood cells in the conceptus are those of the erythroid lineage. The early production of erythroid lineage cells in the yolk sac is required for the development of the vertebrate embryo. These blood cells are shortlived, however. In contrast, long-term adult hematopoiesis results from a complex cell lineage differentiation hierarchy that produces at least eight functionally distinct lineages of differentiated blood cells. The founder cells for this hierarchy are the hematopoietic stem cells (HSCs), which undergo progressive differentiation, proliferation, and restriction in lineage potential. The adult blood system is constantly replenished throughout adult life from rare HSCs harbored in the bone marrow. The field of “developmental hematopoiesis” investigates how this complex adult system is generated in the conceptus. Current research interests in this field include 1) the embryonic origins, cell lineage relationships, and functions of the cells within the multiple embryonic hematopoietic compartments; 2) the changing developmental microenvironments that support hematopoietic (stem) cell growth; and 3) the molecular programming of the hematopoietic system during ontogeny. This chapter will focus on our current knowledge concerning the embryonic beginnings of the adult hematopoietic system. Insights emerging from such a developmental approach should lead to novel molecular and cellular manipulations that could aid in the ex vivo generation and/or expansion of HSCs and progenitors for clinical use in transplantations for leukemias or blood-related genetic disease.

ONTOGENY OF THE HEMATOPOIETIC SYSTEM Developmental studies provide insight into the initiation, growth, and function of cells in the wide variety of adult tissues. The cellular interactions and molecular programs

governing tissue development are conserved throughout evolution, as revealed in a variety of animal models ranging from invertebrates to mammalian vertebrates. Similarly, conserved developmental principles also govern the generation of the hematopoietic system. Our current knowledge of the embryonic origins of the adult hematopoietic system has been gained from the study of nonmammalian vertebrate embryos such as frogs and birds1,2 and the widely used mammalian vertebrate model, the mouse.3 These cumulative results have provided wide support for multiple de novo hematopoietic specification events, at least three independent embryonic origins of hematopoiesis, and for the colonization theory of hematopoiesis. The variety of in vivo and in vitro hematopoietic assays and the ease of genetic manipulation of mice have significantly expanded our molecular knowledge of mammalian blood development. Studies of human embryonic hematopoiesis are further facilitated through xenotransplantation studies of human cells into mice4 and induced hematopoietic differentiation of embryonic stem cells (ESCs) (mouse and human).5,6 Thus, a more dynamic view of human embryonic hematopoiesis has been realized.

Initiation and Appearance of Hematopoietic Cells Mesoderm The hematopoietic system is one of the earliest tissues to develop during ontogeny. It is derived from the mesodermal germ layer of the conceptus, and in the human this embryonic stage is referred to as the “mesoblastic” period.7 The mesoderm forms through an inductive interaction between the ectodermal and endodermal germ layers during the midblastula stage (Fig. 1.1A). Much of our knowledge of mesoderm induction comes from studies of amphibian embryos in which the manipulation, grafting, and culture of embryos are facilitated by their large size and development outside the mother. Nieuwkoop8 was the first to demonstrate that culture of the amphibian midblastula stage animal cap (ectoderm) alone leads to the production of epidermis, whereas coculture of the animal cap with the vegetal pole (endoderm) leads to the generation of mesodermal structures such as muscle, notochord, heart, pronephros, and blood (Fig. 1.1B). Cell lineage mapping studies show that mesodermal cells are formed from the presumptive ectoderm that receives signals from the underlying vegetal component and presumptive endoderm.9,10 Recent studies suggest that the earliest hematopoietic mesoderm is derived from a specialized mesendodermal layer of cells.11,12 Animal cap assays have identified mesoderm-inducing factors including transforming growth factor–␤1 (TGF␤1 ) family members BMP-4, activin, and Vg1, and members of the fibroblast growth factor (FGF) family.11–13 The production (by endodermal cells) of these factors and their graded distribution suggest that they act as morphogens. Together with the 3

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Figure 1.1. Schematic diagram of germ layer development in vertebrate embryos. (A) Mesoderm arises from an inductive interaction between ectoderm and endoderm. (B) Experimental scheme in Xenopus embryos that shows that mesodermal cells arise from the ectoderm (animal cap cells) under the inductive influence of the endodermal vegetal fragment.10 (See color plate 1.1.)

extensive rearrangements of cell movement during gastrulation, different lineages of mesoderm are formed: dorsal, paraxial, lateral, and ventral. Numerous secreted factors (as well as transcription factors and adhesion molecules) play roles in this patterning of mesoderm.12,14 Similarly, mesoderm induction is the first step leading to the specification of hematopoietic cells in the mammalian conceptus. Mesoderm induction occurs in the primitive streak of the mouse conceptus beginning at embryonic day (E) 6.5/7.0. Single-cell marking of the presumptive mesoderm in the mouse epiblast showed that the first mesoderm emerging from the posterior primitive streak contributes to extraembryonic hematopoietic tissue, that is, yolk sac and allantois15 (Fig. 1.2A). Mesodermal derivatives within the rostral embryo body arise from epiblast cells that ingress through the anterior primitive streak. Thereafter, cells that give rise to lateral blood-forming mesoderm of the anterior trunk (Fig. 1.2B) transit through the primitive streak. Mesoderm emigrating from more caudal regions of the streak forms the mesoderm of the remaining trunk regions.16 Interestingly, the entire epiblast of the early- and midstreak stage mouse embryo contains hemogenic potential, but that potential is later restricted to the trunk and posterior region of the embryo.17 Thus, induction of prospective hematopoietic mesoderm is conserved between vertebrate species.16

Elaine Dzierzak produced by the endoderm in mouse embryo cultures22,23 play inductive roles in patterning hematopoietic mesoderm. The close temporal and spatial appearance of hematopoietic and endothelial cells in the yolk sac has led to speculation of a common mesoderm precursor cell for these two lineages, the hemangioblast.24,25 Indeed, the shared expression of markers such as Flk-1 (KDR), SCL, and CD34 by hematopoietic cells and endothelial cells and the complete lack of endothelial and hematopoietic cells in Flk-1deficient embryos support the existence of hemangioblasts in the mammalian conceptus.26–28 ESC hematopoietic differentiation cultures have facilitated the isolation and characterization of hemangioblasts. Stepwise differentiation of ESCs toward the mesodermal lineage and thereafter to hematopoietic and endothelial lineages closely parallels such development in the yolk sac.29 Under controlled culture conditions, ESCs differentiate to form cells expressing Brachyury, a well-known mesodermal marker. Brachyury expression in mouse ESCs is upregulated following exposure to mesodermal inducing factors such as FGF, TGF-␤1 , and BMP-4. Shortly thereafter these cells express Flk-1 and have potential to differentiate to angioblasts and SCL+ CD34+ blast colony–forming cells (BLCFC)30 The ESC-derived BLCFCs are considered to be hemangioblasts. This cell type has also been identified in the early mouse embryo. At E7.5 Brachyury+ cells become Flk1+ . When put in culture, these cells (and a small fraction of the Brachyury+ Flk1− cells) exhibit the functional properties of BLCFC.31 Additional studies have

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Extraembryonic Hematopoiesis: Yolk Sac Yolk sac blood islands containing primitive erythrocytes are detectable in the mouse conceptus at E7.518 and in the human conceptus at approximately 16–20 days of gestation.19 Mesodermal cells migrate to this extraembryonic site and come in close contact with the endoderm. As shown in avian embryos, interaction with the endoderm is required for the initiation of hematopoiesis.20 Several endodermally produced developmental factors and morphogens in the chick21 and the Indian hedgehog factor

Figure 1.2. Mesodermal migration during mouse embryogenesis. (A) Schematic diagram of a mouse conceptus at the early primitive streak stage. Emerging from the posterior primitive streak are waves of yolk sac mesoderm migrating to form this extraembryonic tissues. Slightly later, this mesoderm also forms the allantois. Hemangioblasts are found in the posterior primitive streak. (B) Schematic diagram of a mouse conceptus at the mid–late primitive streak stage. Mesoderm emerging from the anterior primitive streak forms the paraxial and lateral mesoderm of the trunk region of the embryo (mesoderm for the prospective PAS/AGM region). At this stage the allantois is visible, as are the first primitive erythroid cells in the yolk sac blood islands. (Drawings adapted from ref. 3.) (See color plate 1.2.)

A Developmental Approach to Hematopoiesis established that subsequent SCL expression can be used to isolate the hemangioblast from angioblasts.26 Surprisingly, hemangioblasts in vivo are localized not in yolk sac but in the posterior primitive streak31 (Fig. 1.2A). As they migrate to the yolk sac they become committed endothelial and hematopoietic progenitors and several of these cells contribute to the formation of each blood island.32 Studies with human ESCs and other animal models further demonstrate the existence of hemangioblasts in the earliest stages of mesoderm and blood development, and there are some suggestions that hemangioblasts may persist in postnatal stages.26

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Extraembryonic Hematopoiesis: Chorion, Allantois, and Placenta The placenta has long been recognized as a site where hematopoietic cells are harbored and circulate; however, it is only recently that this tissue was shown to possess hemogenic properties.33,34 Placenta organogenesis is initiated through the fusion of the chorionic membrane with the allantois, both derived from the extraembryonic mesoderm. The growth of this highly vascularized extraembryonic tissue is a cooperative effort between fetus and mother, allowing nutrients to be delivered to the fetus and wastes exported to the mother. The hemogenic properties of the allantois were initially studied in avian embryos. The avian allantois, before it becomes vascularized, contains clusters of hematopoietic cells resembling blood islands35 and, upon grafting, forms adult blood and endothelium.36 In contrast, initial grafting studies of the mouse allantois did not reveal erythroid lineage contribution in vivo, although a small population of erythroid cells was found in cultured tissues.37 Recently, both the mouse allantois and chorion have been shown to possess intrinsic hematopoietic potential that is not dependent on chorioallantoic fusion.33,34 Mouse allantois and chorion tissues contain multilineage hematopoietic potential as shown by colony–forming unit-culture (CFUC) assays. They express the Runx1 transcription factor, a molecule required for hematopoietic induction. The rudiments of the prospective placenta are hemogenic before the vascular continuity between the allantois and yolk sac is established, and thus are thought to generate de novo hematopoietic cells. In addition, soon after the formation of the placenta, potent hematopoietic progenitor and stem cell activity can be found at high frequency in this tissue.38–40 It remains to be determined what percentages of these hematopoietic cells are de novo generated in the placenta and whether placental cells contribute long term to the adult hematopoietic system.

Intraembryonic Hematopoiesis: Paraaortic Splanchnopleura/Aorta-Gonad-Mesonephros In the mid-1970s, amphibian and avian embryo culture and grafting approaches were used extensively to study

Figure 1.3. Nonmammalian vertebrate embryo–grafting experiments used for determining the origin of the adult hematopoietic system. (A) A schematic diagram of the avian embryo grafting strategy in which quail embryo bodies were grafted onto chick yolk sacs at the precirculation stage of development. (B) A schematic diagram of the amphibian embryo grafting strategy in which genetically marked dorsal lateral plate (DLP) or ventral blood island (VBI) regions were transplanted onto unmarked Xenopus or Rana embryos. (C) Genetic marking experiment in 32 blastomere Xenopus embryo (left). Marking of the C3 blastomere, D4 blastomere, and C1 and D1 blastomeres allowed the tracing of progeny cells to the DLP, pVBi, and aVBI, respectively, at the larval stage (right). (Drawings adapted from ref. 3.) (See color plate 1.3.)

cell fate, morphogenesis, and organogenesis. In the avian species, grafts between quail and chick embryos or between different strains of chicks were used to create chimeras in which the embryonic origins of adult blood cells were determined.1,41 Donor-specific nucleolar or immunohistochemical markers determined whether the differentiated adult blood cells were derived from the graft or the recipient. For example, yolk sac chimeras were constructed by grafting a quail embryo body onto the extraembryonic area of a chick blastodisk (Fig. 1.3A). The combined results of many such experiments41–44 led to the following conclusions: 1) the first emergence of hematopoietic cells is extraembryonic, in the yolk sac; 2) slightly later, hematopoietic cells emerge both extraembryonically and intraembryonically; and 3) intraembryonically derived hematopoietic cells are permanent contributors to the adult hematopoietic system. Most extraembryonically derived hematopoietic cells become extinct. Furthermore, multipotential hematopoietic progenitors as assayed in in vitro clonal cultures are associated with the dorsal aorta of avian embryos.45 The close association of hematopoietic cell clusters and endothelial cells on the ventral aspect of the dorsal aorta41 led to the hypothesis that hematopoietic cells are derived from endothelial cells. Indeed, when chick aortic endothelial cells are labeled in situ with lipophilic dye during prehematopoietic stages,46,47 labeled intraaortic hematopoietic clusters are found 1 day later, thus

6 demonstrating a precursor–progeny relationship between endothelial cells and hematopoietic clusters.2 Similarly, chimeric embryo studies in amphibians have demonstrated independent intraembryonic and extraembryonic mesodermally derived sites of hematopoiesis.48–50 Using DNA content as a marker, chimeric frog embryos were generated by reciprocal grafting of the ventral blood island (VBI) region (a region analogous to avian and mammalian yolk sac) and the dorsal lateral plate (DLP) (a region analogous to the avian intraembryonic region containing the dorsal aorta) from diploid and triploid embryos (Fig. 1.3B). Again, the ventral mesodermal yolk sac analog produces the first hematopoietic cells, and slightly later, the dorsal mesodermal intrabody compartment generates adult hematopoietic cells. Unlike birds, some ventrally derived hematopoietic cells persist to adult stages and appear to contribute to red and white blood cell populations.48,49 The specific localization of intrabody hematopoiesis has been found to be associated with the dorsal aorta and pronephros, with the most abundant hematopoiesis in the pronephros.51 Indeed, lineage-tracing experiments in which individual blastomeres in the 32-cell stage embryo are marked show that the blastomeres contributing to the formation of the VBIs (anterior and posterior) are distinct from each other and from the blastomere that contributes to the formation of the DLP52 (Fig. 1.3C). Moreover, in early embryos the prospective hematopoietic cells in the VBI (primitive) and DLP (adult) can be reprogrammed to an adult or primitive hematopoietic fate. The programs become fixed at a later time point and are thought to become restricted through regulatory interactions from the local environment.53 Thus, there are three distinct origins of prospective hematopoietic cells in Xenopus that are influenced by the local microenvironment. Similarly, in the early-stage mammalian embryo, there are at least three distinct mesodermal tissue origins of hematopoietic cells, the yolk sac, intraembryonic aorta-gonad-mesonephros (AGM) region, and the chorioallantoic placenta (and possibly the vitelline and umbilical vessels) (Fig. 1.4A). The AGM region de novo produces the first adult type HSCs54–55 (reviewed in Dzierzak56 ). This intraembryonic region contains a single central aorta surrounded by the differentiated urogenital tissue (Fig. 1.4B). At early developmental stages, the AGM is identified as the paraaortic splanchnopleura (PAS)57 ) and consists of the paired dorsal aortae and the surrounding mesenchyme adjacent to the gut endoderm. The establishment of the vascular connection between the mouse embryo body and the extraembryonic sites at E8.2537 precludes the identification of extra-versus intraembryonically derived hematopoietic cells. Potent hematopoietic progenitors CFU-spleen (S)58 B lymphoid,59 and multipotent (erythroid-myeloid-lymphoid) hematopoietic progenitors60 have been found in the E9 PAS/AGM region. At slightly later stages of mouse embryogenesis (E10), adult-type HSCs are autonomously generated in the AGM region54,55 and more specifically the dorsal aorta.61

Elaine Dzierzak

Figure 1.4. Sites of hematopoietic activity in the midgestation mouse conceptus. (A) A whole E10.5 mouse conceptus is shown. The placenta, AGM, yolk sac, and the vitelline (V) and umbilical (U) vessels harbor and/or generate hematopoietic cells at this time. (B) Transverse section through the AGM region of an E10.5 mouse embryo is shown. The dorsal aorta is located in the midline, with the neural tube on the dorsal and gut on the ventral side. The urogenital ridges laterally flank the aorta. Hematopoietic cell clusters are found in the lumen of the dorsal aorta as they emerge from the ventral hemogenic endothelium. (See color plate 1.4.)

As reported in a wide range of species,41,52,62,63 hematopoietic foci appear as clusters adhering tightly along the ventral wall of the dorsal aorta (Fig. 1.4B). Cell surface markers, such as CD34 and CD31,63,64 are shared between the hematopoietic cell clusters and endothelial cells. Both cell types also express the Runx1 (AML1, CBF␣2) transcription factor,65 which is required for definitive hematopoiesis66,67 and the Sca-1 marker used for sorting adult HSCs.68 Thus, the PAS/AGM region plays an important role as an early and potent intraembryonic site of hematopoiesis. Hemogenic potential is localized to a subset of endothelial cells lining the wall of the dorsal aorta. Interestingly, the other major vasculature (umbilical and vitelline vessels) of the mouse embryo also contain hematopoietic clusters and it is thought that potent hematopoietic cells emerge from hemogenic endothelium in the midgestation vasculature.

Secondary Hematopoietic Territories: Liver and Bone Marrow In mammalian species, the liver serves as a temporary hematopoietic territory during fetal stages of development. The colonization theory of hematopoiesis first suggested that the hematopoietic cells generated within the extraembryonic yolk sac migrate and colonize the fetal liver and then later move to the bone marrow where they contribute to adult hematopoiesis.69 Now included in the colonization theory of the fetal liver are the potent hematopoietic cells generated in the PAS/AGM and allantois/chorion/placenta (Fig. 1.5). Abundant evidence from coculture experiments and quantitative temporal and spatial analyses of hematopoietic progenitors/stem cells supports the currently accepted dogma that fetal liver does not de novo generate hematopoietic cells but instead is seeded with cells from these generating tissues.3 Moreover, the demonstration that mouse embryos with a deficiency of ␤1 -integrin contain normal yolk sac hematopoiesis but lack fetal liver hematopoiesis provides the first genetic

A Developmental Approach to Hematopoiesis

7 the hematopoietic cells again migrate and colonize the newly established trabecular spaces in the long bones, the so-called bone marrow. In the human fetal liver, CD34+ hematopoietic progenitors appear at 30 days of gestation and hematopoiesis continues in this tissue only until 20 weeks of gestation. At week 10, the bone trabeculae are being established and marrow hematopoiesis commences 1 week later.19

The Embryonic Hematopoietic Hierarchy

Figure 1.5. Sites of hematopoiesis and possible migration and colonization events during mouse embryonic development. It is generally accepted that migration to the fetal liver and adult bone marrow occurs, as indicated by the solid arrows. Cell migration between the embryonic tissues (yolk sac, AGM, and placenta) generating different types of hematopoietic cells is as yet undetermined (dotted arrows).

evidence that adhesion/homing molecules play a role in the colonization process.70,71 In addition to providing a niche for harboring hematopoietic cells, the fetal liver expands and differentiates the newly emigrated cells, particularly directing differentiation toward the erythroid lineage.72 Colonization with hematopoietic progenitors begins at late E973,74 and HSCs appear at E11 in the mouse fetal liver.56,75 The liver remains a hematopoietic niche until birth when

The complex lineage relationships of the cells within the adult mammalian hematopoietic hierarchy are well known and are based on results of in vivo and in vitro differentiation assays of bone marrow cells76 (see Table 1.1 for assay descriptions). These assays measure the maturational progression of cells at the base and branch points of the hematopoietic system all the way through to the terminally differentiated cells of all the distinct blood lineages. The stem cells and progenitors measured by in vitro hematopoietic assays such as CFU-C, fetal thymic organ culture, stromal cocultures, CAFC and LT-CIC, and in vivo transplantation approaches for CFU-S and short-term and long-term repopulating HSCs have led to a placement of these cells within the “textbook” depiction of the hierarchy for adult hematopoiesis. Molecules expressed by distinct hematopoietic lineages and undifferentiated hematopoietic progenitor and stem cells have been instrumental

Table 1.1. Assays to detect hematopoietic cells in the mouse cconceptus

Cell type

Hematopoietic assay

Erythroid–myeloid progenitor Erythroid–myeloid progenitor

CFU-C

T-lymphoid progenitor

Fetal thymic organ culture/OP9-delta coculture Stromal coculture

B-lymphoid progenitor Multipotent progenitor

CFU-S

Single-cell multipotential assay

Neonatal repopulating HSC

Neonatal liver transplantation

Adult repopulating HSC

Adult transplantation

Method

Lineage

Reference

In vitro culture for 5–14 d in semisolid medium with growth factors In vivo transplantation into lethally irradiated adult recipients leading to macroscopic spleen colony formation at 8–16 d In vitro culture with T-depleted thymus for 9–21 d or coculture with delta producing stromal line In vitro 14-d coculture with IL-7 and stromal cells A two-step in vitro culture. Tissue explants/cells cultured on S17 or OP9 cells followed by CFU-C and B/T lymphoid assay or in vivo transplantation to immunodeficient adults In vivo transplantation directly in the liver of 1-day-old hematopoietic ablated recipients. Yields long-term, multilineage repopulation In vivo transplantation into lethally irradiated adult recipients. Yields long-term, high-level, multilineage repopulation

Erythrocytes, macrophages, granulocytes, mast Erythrocytes, macrophages, granulocytes

77

T lymphoid

60, 94

B lymphoid

60, 95

Erythroid, myeloid, B and T lymphoid

60

All hematopoietic lineages

111

All hematopoietic lineages

55, 75, 123

58

8 in assigning direct precursor–progeny relationships and prospectively isolating the cells within the adult hierarchy. The adult hierarchy begins with the HSC and proceeds unidirectionally, with restrictive events occurring throughout hematopoietic differentiation to produce all the differentiated cells in the hematopoietic system. Although these events are represented by discrete cells in the hierarchy, it is most likely that there is a continuum of cells between these landmarks. Indeed, use of the Flt3 receptor tyrosine kinase surface marker along with many other well-studied markers has redefined the early branch points of the adult hierarchy and the subsets of cells committing to myeloid and lymphoid lineages.77 With the description of further markers to identify additional intermediate cell subsets, it may be possible to determine all the molecular events needed for the differentiation of entire adult hematopoietic system and the transit time necessary for differentiation to the next subset. Until recently, little was known about the embryonic hematopoietic hierarchy.3 Although the adult hematopoietic system is usually in a state of equilibrium, the hematopoietic system of the embryo is vastly different: It must de novo generate the entire hematopoietic system, generate these cells within a short span of time in several mesodermally derived microenvironments (yolk sac, amnion/chorion/ placenta, and PAS/AGM), and promote the sequential migration, colonization, and maintenance of hematopoietic cells in yet other microenvironments (liver, circulation, other) before they are finally localized in the bone marrow of the adult (Fig. 1.5). Additionally, different subsets of hematopoietic cells exist in the embryo, possess unique functions, and are not long-lived. Thus, to model the embryonic hematopoietic hierarchy cell origins, precursor– progeny relationships and lifespans of the hematopoietic cells throughout ontogeny must be established. A description of the types of terminally differentiated cells, committed progenitors, immature progenitors, and HSCs existing within the mouse conceptus, and in some cases the human conceptus, is provided here.

Erythropoiesis Histological sectioning reveals that cells of the erythroid lineage are the earliest differentiated hematopoietic cells in the human and mouse conceptus. Primitive erythroblasts are observed in the yolk sac blood islands of the human at E16–20,78 and mouse at E7.0/7.5.18,79,80 In human embryos, up to 100% of all nucleated blood cells at 4–8 weeks of gestation are erythropoietic. These cells are found in the chorial and umbilical vessels, liver sinusoids, and other intraembryonic blood vessels. A switch to enucleated definitive erythropoietic cells occurs at 7–10 weeks of gestation in the blood, and slightly earlier in the fetal liver78 (Fig. 1.6). Similarly, in the mouse, nucleated primitive erythropoietic cells predominate in the yolk sac and fetal liver until a switch

Elaine Dzierzak

Figure 1.6. Developmental expression of the human globin genes. Sites of primitive and definitive hematopoiesis throughout development are shown. Sequential waves of ε (epsilon), ␥ (gamma), and ␤ (beta) globin synthesis begin with ε-globin expression in the first month of human development, followed by ␥ -globin expression in the fetal stage, to just after birth when ␤-globin becomes the predominant hemoglobin type in definitive erythroid cells. The chromosomal organization of the genes of the human ␤-globin locus is in a linear arrangement that correlates with developmental expression. The arrows indicate the DNase1 hypersensitive sites of the LCR (locus control region), which is a region important for globin gene regulation.

from primitive to definitive cell types occurs between E10 and E12.81,82 In both species, the switch from primitive to definitive erythropoiesis is characterized by changes in the expression of the developmentally regulated fetal and adult globin genes (reviewed in refs. 83, 84 [Fig. 1.6]). Individual erythroid progenitors from ESC differentiation cultures can give rise to both fetal and adult erythroid cells85 and single fetal liver cells can switch from a fetal to adult globin gene expression program.86 The general populations of mature erythroid cells, however, are derived from developmentally separate stem cell populations in the embryo.80,87 Moreover, the receptor tyrosine c-kit appears to be required for fetal liver hematopoiesis but not yolk sac erythropoiesis,88 suggesting the origins of primitive and definitive erythroid cells from distinct and differentially regulated hematopoietic progenitor/stem cell populations. Additional molecular differences in primitive and definitive erythropoietic programs, particularly in the requirements for erythropoietic growth factors such as erythropoietin and transcription factors (GATA-1 and EKLF) are well documented.89

Myelopoiesis The first cells of the monocyte–macrophage lineage appear in human conceptuses at 4–5 weeks in gestation.78 Monocytes are routinely represented in human embryos at approximately a 1%–4% frequency in nucleated blood

A Developmental Approach to Hematopoiesis populations after 11 weeks of gestation. Interestingly, macrophages can be found in early human blood smears only until approximately 14 weeks of gestation. This is consistent with findings in the mouse that two separate lineages of macrophages are thought to develop in ontogeny: primitive macrophages and the monocytic lineage of macrophages.90 In the mouse, primitive macrophages (which begin to appear at E9 in the yolk sac) are thought to arise from a local precursor and not a monocytic progenitor. These primitive macrophages proliferate and colonize other embryonic tissues. In contrast, adult macrophages do not circulate through the blood. These cells of the monocytic lineage begin to appear in the fetal liver and yolk sac at E10. Thus, the ontogeny of the monocyte–macrophage is different in the early embryo compared with its later developmental stages and it has been suggested that adult macrophages are the progeny of monocytic precursors from the AGM.91

Lymphopoiesis The production of lymphoid cells begins in the human at 7–10 weeks of gestation.78 Small lymphocytes are found in the blood: 0.2% of nucleated cells at weeks 9–10 and 14% after 14 weeks. Large lymphocytes represent 3%–5% of nucleated blood cells after 11 weeks of gestation. No lymphoid cells are found in the yolk sac, although the presence of lymphoid progenitors has not been examined. Lymphopoiesis begins in the human fetal liver, thymus, gutassociated lymphoid tissue, and lymph plexuses at approximately 7 weeks of gestation, whereas the bone lymphocytes are found only at week 12. Extensive analyses on the development of lymphoid progenitors have been performed in the mouse. Although no functional lymphocytes are found in the mouse conceptus at early gestational stages, cells with lymphoid potential are present. E8.5 yolk sac contains T lymphoid potential when cultured in depleted fetal thymic explants.92,93 B lymphoid potential is found in the embryo body (E9.5) and subsequently the yolk sac (E10) of the mouse conceptus by coculturing such cells in the presence of stromal cells.94 Dissection of the PAS/AGM region has revealed the presence of an AA4.1-positive progenitor for the B1a lineage of B cells as early as E8.5.59,60 A two-step culture system with E7.5 mouse embryo tissues has demonstrated multipotential lymphoid progenitors in the intraembryonic PAS but not in the yolk sac. Only beginning at E8.5 does the yolk sac acquire such multipotential lymphoid activity,57 suggesting that PAS-generated multipotential lymphoid progenitors may migrate to the yolk sac after E8.5 when the intraand extraembryonic circulation is connected. Alternatively, the yolk sac may be capable of producing such progenitors de novo but 1 day later than the PAS. At E10, multipotent B-lymphoid progenitors are found in the circulation, reach a maximum number at E12, and are undetectable in the

9 blood at E14.95 B-cell precursors are detected in the fetal liver at E14 and in the embryonic marrow at E15. Interestingly, adult mouse bone marrow and fetal liver HSC–enriched populations exhibit different T- and Blymphoid lineage potentials. In the T-lymphoid lineage, fetal liver but not bone marrow HSCs produce V␥ 3 and V␥ 4 T-cell receptor–positive subsets.96 Such T cells can also be cultured from yolk sac after E8.5.93,96 Similarly in the Blymphoid lineage, the B1a subset of cells is produced by fetal liver,97 yolk sac,98 and PAS,59 but not by adult bone marrow. It is interesting to propose that the distinct B1a– B cell subset, as well as V␥ 3–4 T-cell subsets, may be the product of a special subset of developmentally regulated progenitors or HSCs in the PAS of the early embryo. It is not known whether such lymphoid subsets and progenitors exist in human embryos.

Erythroid–Myeloid Progenitors: CFU-C The early presence of hematopoietic progenitors within the developing mouse yolk sac was established using in vitro culture approaches developed initially for measuring the hematopoietic potential of adult mammalian bone marrow. The culture of yolk sac cells in semisolid medium in the presence of colony-stimulating factors revealed the presence of erythroid and granulocyte–macrophage progenitors beginning at E7.69,99 Burst-forming unit-E (BFUE) and CFU-Mix are also found in the yolk sac at E8,99 and mast cell precursors are found at E9.5.100 At E8.25, following the first wave of primitive erythropoiesis and before the circulation is established, myeloid progenitors are detected in the yolk sac.101 After the circulation is established myeloid progenitors are also found in the trunk region.89 Tissue explant culture prior to CFU-C assay reveals that both the E8 yolk sac and E8 PAS contains cells with potential to become myeloid progenitors.57 Similar cultures of precirculation allantoides34 also revealed cells with myeloid potential. By E9 the placenta contains an abundance of myeloid progenitors.38 Analyses of two mutant mice, Cdh5−/− and Ncx1−/− , have provided strong in vivo evidence for the de novo production of definitive myeloid progenitors in the yolk sac. In Cdh5−/− conceptuses there is no vascular connection, whereas in Ncx1−/− conceptuses the vitelline vessels are intact but there is no heartbeat to promote the circulation between the yolk sac and embryo body. Similar numbers of myeloid progenitors were found in the E9.5 Cdh5−/− yolk sac compared with wild-type conceptuses, although macrophage and mixed colony–forming progenitors were decreased in number.102,103 In Ncx1−/− conceptuses, the numbers of myeloid progenitors of all types in the yolk sac were found to be equivalent to the cumulative number of progenitors in the Ncx1+/+ conceptuses in all anatomical sites.103 No progenitors were found in the Ncx1−/− PAS, suggesting that the yolk sac normally generates all of these progenitors and

10 distributes them to the PAS and liver. Alternatively, Ncx1deficient conceptuses, which lack hemodynamic stress, do not produce the proper signals to induce myeloid progenitor formation in the PAS.104 Thus, several types of definitive myeloid progenitors are generated de novo in the yolk sac and also in the chorioallantoic placenta and PAS/AGM. In the human, yolk sac hematopoiesis covers the period from midweek 3 in gestation to week 8. BFU-Es have been found at early stages in the yolk sac but begin to decrease in frequency at week 5, when the fetal liver BFU-E frequency increases,105 thus suggesting a colonization of the fetal liver by yolk sac progenitors. Along with erythroid progenitors, the yolk sac and embryo body have been found to contain clonogenic myeloid progenitors and erythroid– myeloid multipotent progenitors at 25–50 days into human gestation.106,107 At the 4- to 5-week stage of gestation, a discrete population of several hundred cells bearing the cell surface phenotype of immature hematopoietic cells (CD45+ , CD34+ , CD31+ , and CD38− ) are found adhering to the ventral endothelium of the dorsal aorta.63,107 These clusters are similar to those described in the chick and mouse. Interestingly, when these cell clusters are cocultured with bone marrow stromal cells and assayed in methylcellulose for CFU-Cs, they yield many progenitors and large multilineage hematopoietic colonies.63

Erythroid–Myeloid Progenitors: CFU-S To determine whether the more immature hematopoietic progenitor compartment of the adult hierarchy is present early during embryonic development, in vivo transplantation analyses for CFU-S have been performed in irradiated mice. CFU-S are immature erythroid–myeloid progenitors that yield macroscopic colonies on the spleens of lethally irradiated mice 9–14 days following transplantation.108,109 Beginning at E9, statistically significant numbers of CFU-S are found both in the yolk sac and PAS/AGM.58,69 It is difficult to determine from which tissue these in vivo progenitors originate because the vascular connection between the yolk sac and embryo body is made at E8.5. The absolute numbers of CFU-S from the developing mouse embryo up to late E10 reveal that the AGM region contains more CFUS than the yolk sac.54,58 When an organ culture step is used before in vivo transplantation of yolk sac or AGM, the numbers of AGM CFU-S increase substantially, whereas only a slight increase in yolk sac CFU-S numbers is observed.54 Thus, the AGM region is the more potent generator of CFUS. CFU-S are localized to both the aorta subregion and urogenital subregion of the AGM and are also found in the vitelline and umbilical arteries.109

Erythroid-Myeloid-Lymphoid Multipotential Progenitors Within the mouse embryo, these in vitro progenitors are found at E7.5 within the intrabody PAS/AGM region by

Elaine Dzierzak a two-step culture system, 1 full day earlier than in the yolk sac.57 Results of temporal studies suggest that preliver intrabody hematopoiesis is more complex and potent than extraembryonic yolk sac blood formation, and such PASgenerated multipotent progenitors may seed the yolk sac after the circulation is established at E8.5. The multipotential progenitors in the E8–9 PAS have also been tested in vivo for CFU-S and adult repopulating HSC activity. In vivo, these cells do not repopulate lethally irradiated adult recipient mice short term or long term after transplantation; however, they do contribute to long-term, low-level hematopoiesis following transplantation into immunocompromised adult recipients. These results suggest that they are not fully competent adult HSCs but could be candidates pre–stem cell population. Similarly, the human AGM but not the yolk sac contains multipotent progenitors beginning at day 24 in gestation. They express CD34, and CD34+ hematopoietic cell clusters begin to appear on the ventral wall of the dorsal aorta at day 27. These multipotent cells could be HSCs or precursors of such cells.19,28,110

Neonatal Repopulating Hematopoietic Stem Cells A more potent in vivo repopulating multilineage hematopoietic cell has been described through the use of another transplantation assay. Neonatal mice from pregnant dams treated with busulfan (for myeloablation to enhance engraftment) were injected (directly into the liver at the time of birth) with yolk sac or PAS/AGM cells. When E9 CD34+ c-kit+ cells from E9 yolk sac and E9 PAS/AGM were transplanted in this manner, both were capable of multilineage engraftment and secondary engraftment into adult lethally irradiated recipient mice.111 Neither of these sorted populations could repopulate primary adult lethally irradiated recipients nor engraft the hematopoietic system of the primary neonatal recipient to 100%. Because the yolk sac contains more neonatal repopulating cells than the PAS/AGM, these investigators suggest that the yolk sac may be the generating source. Previous studies have suggested that the early-stage yolk sac cells can indeed lead to long-term hematopoiesis when transferred into embryonic recipients, either transplacentally or into the yolk sac cavity.112,113 These studies showed donor yolk sac–derived cells in the erythroid and lymphoid lineages, respectively, of fully developed adults. Thus, neonatal/fetal repopulating cells are long-lived multilineage progenitors that have the potential to become competent adult-type HSCs when exposed to the appropriate microenvironment.

Hematopoietic Stem Cells At the base of the adult hematopoietic hierarchy are HSCs. They are defined by their ability to high-level, multilineage, long-term repopulate irradiated adult mouse recipients. The presence of differentiated hematopoietic cells

A Developmental Approach to Hematopoiesis and many restricted, multipotent and in vivo immature hematopoietic progenitors in the PAS/AGM region, yolk sac, and chorioallantoic placenta of the mouse conceptus leads to the prediction (within the context of the adult hematopoietic hierarchy) that HSCs should be present from the onset of embryonic hematopoiesis at E7.0/7.5. In mouse embryos, however, the first adult repopulating HSCs are found only beginning at E10 in the AGM region55 and at E11 in the yolk sac55,69,70 and placenta.39,40 Organ explant culture before in vivo transplantation has revealed that the AGM region is the first tissue to generate autonomously HSCs.54 The yolk sac and placenta may subsequently be seeded by AGM-generated HSCs, or alternatively, these tissues may be capable of de novo generating their own HSCs.

Direct Precursors to the Hematopoietic Lineages Primitive erythroid cells arise from hemangioblasts, whereas the “definitive” classes of hematopoietic progenitor stem cells are thought to arise through different precursors, the so-called “hemogenic endothelium.” Discrete subsets of vascular endothelial cells in the conceptus exhibit hemogenic potential.25 Cross-species immunohistochemical studies have shown hematopoietic clusters tightly adherent to the ventral endothelium of the dorsal aorta and that of the umbilical and umbilical arteries2 (Fig. 1.4B). The first appearance of hematopoietic clusters is in parallel to the appearance of the first definitive HSCs that can be detected. In the chick embryo, metabolic lineage tracing (AcLDL-DiI) or retroviral labeling of endothelial cells prior to hematopoietic cell appearance has confirmed the endothelial–hematopoietic lineage relationship of aortic hematopoietic clusters.46,47 Similar marking attempts in ex utero cultured E10 mouse embryos show AcLDL-DiI+ definitive erythroid cells in the circulation 12 hours after intracardiac injection and marking of aortic endothelium.114 The phenotypic profile and spatial localization of HSCs in the AGM are also supportive of hemogenic endothelium as the direct precursor to definitive hematopoietic cells. All AGM HSCs are CD45+ , Ly-6A (Sca-1) GFP+ , c-kit+ CD34+ , Runx1+ , SCL+ , and Gata2+ .68,115–119 These markers (with the exception of CD45) are also expressed by some or all endothelial cells in the ventral aspect of the dorsal aorta at E10/11. Most or all AGM HSCs express cell surface vascular endothelial cadherin,117,120 which is typically thought of as an endothelial marker. Interestingly, not all the cells in the hematopoietic clusters express the same hematopoietic markers: Only some cells express CD41+121 or the Ly-6A GFP transgene,122 indicating that some cells in the clusters take on the HSC fate whereas others are fated to be progenitors. Studies in the mouse conceptus have identified hematopoietic clusters on both the ventral and dorsal aspects of the dorsal aorta.123 Functional studies indicate that definitive hematopoietic progenitors reside on both

11 aspects of the aorta, but only the ventral aspect contains fully potent HSCs.123 Thus, there appear to be subsets of hemogenic endothelium. In contrast, some studies suggest that HSCs are derived from mesenchyme located directly underneath endothelial cells in the ventral aspect of the dorsal aorta, or in discrete patches ventral–lateral to the dorsal aorta (subaortic patches). In Runx1-haploinsufficient AGMs, HSCs are present within the Runx1-expressing mesenchymal cells underlying the ventral aspect of the dorsal aorta (as defined by the phenotype CD45− , CD31− , and vascular endothelial cadherin− ). AGM cells similarly sorted from wild-type embryos did not contain HSCs,117 suggesting that HSCs are normally localized in the aortic endothelium. When cells from the subaortic patches (CD45− ckit+ AA4.1+ ) are transplanted into immunodeficient adult recipients, some long-term repopulating activity was found but the level of engraftment was low, ranging from 0.4% to 1.9%.124 These cells are not as potent as the Runx1+ or Ly-6A (Sca-1) GFP+ aortic endothelial/cluster HSCs that provide up to 100% engraftment of irradiated adult recipients.68,117 The hematopoietic cells localized in the subaortic patches may be precursors to the fully potent HSCs found in the aortic endothelial hematopoietic clusters or may represent differentiated progeny of hemogenic endothelium that have ingressed (as in the chick embryo) into this site. Together, these mouse data strongly indicate that the direct precursors of HSCs are predominantly hemogenic endothelial cells. In addition, the vascular endothelium of the human embryo has blood-forming potential.125

A Model of the Embryonic Hematopoietic Hierarchy The appearance of terminally differentiated primitive erythrocytes in the mouse conceptus 3 days before the appearance of adult-type HSCs is the antithesis of the adult hematopoietic hierarchy. In the conceptus, the stepwise progressive appearance of distinct cells with increasingly complex hematopoietic potential supports a model in which the embryonic hematopoietic system is not a single-lineage differentiation hierarchy but is instead many hierarchies. It is a continuum of hematopoietic fate determining events occurring within distinct subsets of presumptive hemogenic mesoderm that specify a variety of temporally and spatially separate precursor cells – hemangioblasts and hemogenic endothelium (Fig. 1.7). Dependent on developmental time and position within the extra- and intraembryonic tissues (yolk sac, placenta, and AGM), cells emerge with different hematopoietic potentials. Thus, the embryonic hematopoietic hierarchy is modeled on the appearance of functionally different cells without indications for lineage relationships. Although it is clear that “hemangioblasts” and hemogenic endothelium play roles, further results are necessary to determine whether the wide range of hematopoietic activities in the conceptus are achieved directly through hematopoietic fate

12

Elaine Dzierzak Hematopoietic Colonization and Migration During Development

Figure 1.7. The early embryonic/developmental hematopoietic hierarchy is unlike that of the adult. The temporal appearance of hematopoietic cells in the mouse conceptus suggests that many of these cells do not arise from an HSC but instead they arise directly from mesodermal populations that go through a hemangioblast and/or hemogenic endothelial intermediate. Thereafter, hematopoietic fate is acquired and hematopoietic cells are generated. The sequential appearance of primitive erythroid–myeloid cells, followed by increasingly more complex definitive hematopoietic cells and finally the appearance of definitive HSCs is contrary to adult hematopoietic differentiation hierarchy with the expected precursor–progeny relationships. Instead, the hematopoietic system in the embryo is generated at least five independent times in different mesodermal populations.

determination events in a variety of nonhematopoietic precursors (hemangioblasts and different subsets of hemogenic endothelium) or through the acquisition of more complex hematopoietic activities imposed by the microenvironment after hematopoietic fate determination of a small cohort of similarly active cells. In the human conceptus, the sequential appearance of differentiated and more complex hematopoietic progenitors is consistent with what has been observed in the mouse conceptus.106,107 Moreover, the distribution of these hematopoietic cells occurs similarly in the yolk sac, AGM, liver, spleen, and bone marrow (the hematopoietic activity of the placenta is predicted but is as yet uncertain (Fig. 1.8).

Figure 1.8. The sites of hematopoietic activity and the temporal appearance of the distinct hematopoietic lineages in the human embryo. There is a general correspondence of the hematopoietic sites and the temporal appearance of hematopoietic cells between the human and the mouse embryo. (Figure adapted from ref. 196.)

Until 1965 it was thought that the hematopoietic populations found in adult vertebrates were intrinsically generated in tissues such as the liver, spleen, bone marrow, thymus, and bursa of Fabricius (only found in avian species). A paradigm shift occurred when it was shown that hematopoietic cells generated in earlier embryonic tissues colonized these secondary hematopoietic tissues. The generating sources of the hematopoietic cells are likely to be one or more of these tissues: the yolk sac, PAS/AGM, and chorioallantoic placenta. In this section a summary of the findings demonstrating the migration of hematopoietic cells during development is provided.

Avian The results of experiments by Moore and Owen,126 in which parabiosed chick embryos were examined, suggested that the thymus, spleen, bursa of Fabricius, and bone marrow were colonized by blood-borne cells. Definitive proof that the hematopoietic cells in adult tissues are extrinsically derived comes from the quail–chick and chick–chick embryo grafting experiments. Initial experiments focused on the colonization of the grafted thymus and spleen rudiments with embryonic hematopoietic precursors.41,44 Each tissue rudiment provided the stroma or microenvironment for the seeding and differentiation of extrinsic precursors. Interestingly, it was found that several short periodic waves of lymphoid precursors enter the thymus, whereas a single long wave of precursors enter and colonize the bursa.127 These studies showed that the tissue rudiments exhibited limited times of receptivity for emigrating hematopoietic cells and suggested the emergence of progenitors at several discrete developmental times. Similarly, the ontogeny of the multilineage hematopoietic system was examined in embryo grafting experiments in which yolk sac chimeras were made (reviewed in ref. 1 and references therein) (Fig. 1.3A). The sites of de novo hematopoietic cell emergence were determined to be the yolk sac and the intraembryonic region containing the dorsal aorta. Only very briefly in early stages of embryogenesis do yolk sac–born erythrocytes predominate in the blood. Subsequently, intrabody-born erythrocytes rapidly predominate and red cells from the yolk sac disappear completely by the hatching stage. At least two generations of hemoglobin-producing cells were observed: the first from yolk sac–derived cells and the second from yolk sac– and intrabody-derived cells. During embryonic stages a small number of intrabody-derived cells can be found in the yolk sac and likewise, a small number of yolk sac–derived macrophage-like (microglial) cells can be found intraembryonically in the eye and in the brain. These cellular exchanges are thought to occur through the circulation of small populations or subsets of hematopoietic cells that

A Developmental Approach to Hematopoiesis may serve a specialized, short-lived function. In the adult, the originating source of adult blood was confirmed to be the intrabody region. When the prevascularized quail allantoic bud was grafted in the coelom of a chick host, cells of both the hematopoietic and endothelial lineages were found in the bone marrow of the host.36 Thus, the bone marrow is seeded by hematopoietic and endothelial precursors that arise in situ in the allantois. Hence, the allantoisas well as the paraaortic-derived cells of the avian embryo migrate and seed the adult blood system.

Amphibians Waves of colonization are also observed in the amphibian model system. Embryo grafting experiments show that the larval liver is colonized by intrabody-derived hematopoietic cells.128 The liver is thought to be seeded by intrabody cells that migrate through the interstitium because intrabody cells are not found in the circulation. Interstitial migration of cells is an efficient means of cell distribution within the amphibian embryo body and has been found to occur even before the completion of the vascular network.50 Support for interstitial migration has been provided recently in the zebrafish. CD41+ hematopoietic cells in the interstitium enter the circulation by intravasation via the posterior cardinal veins.129 In later stages of amphibian development, near the time of metamorphosis, intrabody-derived hematopoietic clones fluctuate in their contribution to the liver and some ventral blood island– derived clones are detected128,130 but do not become the predominant cell type.

Mammals In contrast to the ease of in vitro culture and manipulation of amphibian and avian embryos for the analysis of hematopoietic cell migration and colonization, the in utero inaccessibility of the mouse conceptus necessitates the use of other approaches for these studies. Some of the first experiments probing hematopoietic migration and colonization involved culturing whole E7 mouse embryo bodies in the presence or absence of the yolk sac.69 After 2 days, tissues were dissected and analyzed for granulocyte– macrophage colony formation. Only embryo bodies that retained their yolk sac were able to give rise to hematopoietic cells, suggesting that the yolk sac is the only embryonic site producing hematopoietic cells that colonize the liver rudiment. This experiment, as well as those examining the kinetics of CFC production in the yolk sac and fetal liver, suggests a dependence of early fetal hepatic hematopoiesis on an influx of exogenous yolk sac–derived cells.69,80,99 Other researchers have demonstrated that at late E9 fetal liver is populated by yolk sac–derived erythroid cells when these tissues are cultured adjacent to each other.131 Recently, studies in mouse conceptuses deficient for Cdh5 and Ncx1 genes suggest migration of yolk sac–

13 derived myeloid progenitors to the embryo body.102,103 In the absence of a vascular connection or heartbeat to promote the circulation, myeloid progenitors of all types were found in the yolk sac but not the embryo body. This suggests that the yolk sac normally generates all of these myeloid progenitors and distributes them to the PAS/AGM and liver. It is possible, however, that the PAS/AGM requires the normal stimulus of hemodynamic stress present in the wild-type conditions to generate these progenitors. Moreover, these experiments are limited to analysis of only very early tissues, and thus cannot take into account the multiple waves of hematopoietic cell generation and migration seen in the nonvertebrate species, particularly those that give rise to the adult hematopoietic system. Spatial and temporal quantitative analyses for CFU-C, CFU-S, and HSCs in the mouse conceptus provide strong support for migration of AGM, yolk sac, and placentalderived cells to the fetal liver.39,54,58,75 Data on B lymphopoiesis in the mouse conceptus suggest migration of these cells to the fetal liver through the circulation.95 The spleen and thymus are seeded either directly from the generating tissues or from the fetal liver.132,133 As found in avian embryos, the early classes of mouse hematopoietic cells (those defined by hematopoietic activity less potent than an HSC’s and with limited life span) may provide maturation signals to the rudiments of the secondary hematopoietic territories in the mouse to promote their growth and receptivity for the later generated HSCs.134,135 There is convincing evidence that integrins play an important role in the embryo in the colonization of secondary hematopoietic territories. Mouse embryos lacking ␤1 -integrin die during preimplantation stages of gestation.70 Chimeric embryos with ␤1 -integrin−/− ESCs were generated to examine its role during later stages of hematopoietic ontogeny.71 Although the yolk sac was found to contain normal numbers of hematopoietic cells derived from the ␤1 -integrin−/− ESCs, the fetal liver did not contain any ␤1 -integrin–deficient hematopoietic cells. The clonogenic potential of the yolk sac hematopoietic cells was normal, and such cells were found in the circulation of embryos until E15. These results strongly suggest that ␤1 -integrin is required for the successful migration of hematopoietic cells to the fetal liver. Additionally, and in accordance with a role for ␤1 -integrin in adult hematopoietic cell migration, no ␤1 -integrin–deficient hematopoietic cells were found in the thymus, bone marrow, or blood of adult chimeric mice. To trace the lineage of cells in the mouse conceptus that give rise to the permanent hematopoietic system in the adult, molecular marking using the Cre-lox recombination system136 has been attempted. This in vivo marking technology is based on the expression of a marker transgene (Rosa 26 locus inserted fluorescent or enzymatic gene) that is activated through the excision of a stop sequence positioned between two lox recombination sites. Cre recombinase performs the recombination event in specific cells depending on transcriptional regulatory elements

14 driving its expression and the activity of Cre recombinase (Cre-ERT), which can be controlled in a temporal manner by administration of tamoxifen. Thus, hematopoietic cells can be marked within a specific window of developmental time and the progeny of these marked cells can be followed through later fetal and adult stages. When the SCL (expressed in endothelial cells and definitive HSCs118 ) and Runx1 (expressed in all definitive hematopoietic cells, hemogenic endothelium, and some mesenchymal cells117 ) regulatory elements were used to direct Cre-ERT expression and early and midgestation mouse conceptuses were exposed to tamoxifen, approximately 10% of the bone marrow cells in the adult expressed the marker.137,138 These results indicate that the progeny of SCL- and Runx1expressing cells in the mouse conceptus migrate to the adult bone marrow and contribute to adult hematopoiesis. Thus, the progeny of hematopoietic cells generated in the embryo (tissue origin as yet unknown) migrate to the bone marrow where they reside and contribute to hematopoiesis through adult life.

Molecular Aspects of Embryonic (Primitive) and Adult (Definitive) Hematopoiesis Molecular interactions regulate the generation of the hematopoietic system. Some of these interactions include developmental signaling pathways, transcription factors, and chromatin remodeling factors. Induction events are orchestrated by the signaling pathways that “turn on or off ” transcription factors that regulate the expression of specific panels of genes (genetic programs) associated with hematopoietic fate and function. Moreover, the genetic programs are controlled by a limited number of epigenetic regulators (chromatin modifiers) that confer a “cell-specific molecular memory” and thus maintain the hematopoietic fate of the cell. The microenvironments in the mouse conceptus where hematopoietic cells are generated (yolk sac, PAS/AGM, and chorioallantoic placenta) differ from each other and from the secondary hematopoietic territories (fetal liver and adult bone marrow) that promote maintenance, self-renewal, and/or differentiation of hematopoietic progenitor and stem cells. Thus, beginning with the cell extrinsic influences of morphogens and factors emanating from the surrounding cellular environment, developmental signaling pathways are triggered and activate distinct but overlapping genetic (and epigenetic) programs to direct hematopoietic development in the mouse conceptus. Our understanding of the molecular programming of the hematopoietic system throughout ontogeny has been profoundly influenced by the use of gene-targeting technologies in mouse ESCs.139 The ability to generate mice with mutations in any chosen gene has resulted in the identification of numerous signaling pathways, transcription factors and epigenetic regulators that are critical for the development of the hematopoietic system. The most striking hematopoietic defects found are genetic mutations

Elaine Dzierzak that affect both primitive and definitive hematopoiesis and mutations that profoundly affect definitive but not primitive hematopoiesis. Because the deletion of some hematopoietic genes results in anemia and early embryonic lethality, study of the affects of such genes at the later developmental stages is facilitated by chimeric mouse generation with homozygous mutant ESCs and also conditional gene targeting strategies. Functional differences in the cells that make up the primitive (embryonic) and definitive (adult) hematopoietic systems predicted differences in molecular programming through development. Prime examples include the developmental regulation of the globin genes (␣ and ␤)83 in primitive and definitive erythroid cells and the T- and Bcell receptor genes (V␥ 3-V␥ 4 and B1a, respectively) in fetal lymphoid cells.96,97 These programs are regulated at the level of the HSC and thus suggest distinct developmental subsets of HSCs (dependent on the stem cell source and/or local microenvironment). Genes involved in hematopoietic specification also may be developmentally regulated in mesodermal cells as they emerge from the primitive streak and move to the extraembryonic yolk sac (ventral mesoderm) and intraembryonic PAS/AGM (lateral mesoderm). Thus, the genetic programs leading to hematopoietic specification overlap to a large degree but also possess unique features related to hematopoietic potential, function, site, and life span. This section and Table 1.2 summarize some of the signaling pathways, transcription factors, and epigenetic factors that most affect the development of the embryonic and adult hematopoietic systems.

Signaling Pathways Hematopoietic specification occurs shortly following the onset of mesoderm formation. The effects of various factors of the TGF␤1 superfamily and FGF family of genes in mesoderm and blood formation13,140 have been revealed in the Xenopus embryo model. The TGF␤1 superfamily member, BMP-4, acts as a ventralizing molecule within the mesoderm (the region known to form hematopoietic cells). BMP4 also induces the expression of Mix.1, a gene that has been shown to induce hematopoiesis in the Xenopus animal cap assay.53,141 Interestingly, the three blood compartments (aVBI, pVBI, and DLP) are specified from mesoderm that encounters different concentrations of BMP-4142 and the timing of expression of pivotal hematopoietic transcription factors (SCL, LMO2, and Runx1) is controlled by FGF.143 Similar to the interactions between endoderm and prospective hematopoietic mesoderm in Xenopus, such interactions are also necessary for hemogenic induction in the chick embryo. Blood island generation occurs only when the mesothelial and endoderm germ layers are cultured together – when cultured separately no primitive erythroblasts form.20,21,144 Somitic mesoderm, which normally only contributes to endothelium in the dorsal aspect of

A Developmental Approach to Hematopoiesis

15

Table 1.2. Molecules involved in early mouse hematopoietic development Class

Gene

Phenotype

Reference

Developmental growth factor/signaling pathway

TGF␤1

Lethal at E9.5–11.5, defects in hematopoiesis and vascular network formation Lethal at gastrula stage, no mesodermal differentiation. Later embryonic lethal, decreased yolk sac (YS) mesoderm formation and decreased erythropoiesis. Expressed in cells underlying aortic hematopoietic clusters Lethal at E8.5–9.5, defective in YS blood island and vessel formation, severe decrease in YS progenitor cell number and no definitive hematopoiesis Conceptuses die at E10, almost normal numbers of YS primitive erythroid and erythroid–myeloid progenitors, but no AGM hematopoiesis or HSCs. Notch1 and Delta-like 4, and Jagged 1 and 2 expressed in aortic endothelium Severe mutants lethal at E16, deficiencies in hematopoietic cells, CFU-S, primordial germ cells and melanocytes Lethal at E9.5, deficient in primitive and definitive hematopoiesis and defective in angiogenesis Lethal at E10.5, severe FL anemia. Relatively normal YS hematopoiesis but decreased CFU-C. No AGM HSCs or aortic clusters Lethal at E12, complete absence of definitive progenitors and HSCs and aortic clusters. Primitive hematopoiesis relatively normal Enhanced HSC self-renewal Embryonic lethal due to insufficient hematopoiesis Self-renewal defect in FL HSCs Complete lack of definitive hematopoiesis in the conceptus and early embryonic lethality

149

BMP-4

Flk-1/VEGF

Notch1

c-kit/SF Transcription factor

SCL GATA-2

Runx1

Epigenetic factor

Mel-18, Mph1/Rae28 Bmi-1 Mll

the dorsal aorta and not to the ventral endothelium or hematopoietic clusters, can be reprogrammed to assume the latter fates following transient exposure to endoderm prior to grafting.21 Several signaling molecules, including vascular endothelial growth factor (VEGF), basic (b)FGF, and TGF␤1 could substitute for this endodermal signal21 and the overexpression of BMP-4 has been found to influence mesodermal subtype formation.145 Thus, graded expression patterns of factors specify unique subsets of mesoderm including the presumptive hematopoietic mesoderm.14 Studies in the mouse conceptus also show that contact with visceral endoderm is necessary for primitive hematopoiesis in yolk sac explants. Exposure of prospective neurectoderm to endoderm or heparin–acrylic beads soaked in Indian Hedgehog (Ihh) could respecify this normally nonhematopoietic tissue to hematopoietic fate.22,23 Ihh is normally produced by the visceral endoderm, and this expression pattern, together with the explant data, suggests that Ihh signaling is essential for primitive erythropoiesis. Ihh signaling is essential for hematopoiesis in the zebra fish equivalent of the AGM and is at the beginning of

122, 148, 152

27, 154, 155

157, 158

88, 161, 162 165, 169 115, 116, 178

65, 66, 67, 117, 181

190 191 192 195

a signaling cascade for blood cell formation in the dorsal aorta that includes the downstream effectors VEGF, Notch, GATA-2, and Runx1.146 Although deletion of Ihh or its receptor Smoothened (Smo) in mice does not eliminate primitive erythropoiesis in the yolk sac, it does profoundly affect yolk sac vascularization147 and may also affect the AGM region. In the mouse VEGF/Flk-1, FGF, and TGF␤1 (and family members) are generally thought of as ventralizing factors. ESC differentiation cultures and gene-targeting studies reveal a role for the VEGF/Flk-1 and TGF␤1 signaling axes in vasculogenesis and hematopoiesis.27,148 In the TGF␤1 homozygous null condition, perinatal lethality occurs in 50% of the embryos between E9.5 and E11.5.149 The initial differentiation of endothelial cells from mesoderm occurs, but there is no organization of these cells into a vascular network. Defects in yolk sac vasculogenesis and hematopoiesis appear to be responsible for embryonic death, although the severity of the endothelial and hematopoietic cell defects do not always correlate. TGF␤1 signaling in the hematopoietic system suggests complex effects (indirect and/or redundant), because it is a member

16 of the large TGF␤1 superfamily that interacts through an array of receptors and intracellular Smad proteins.150 Another TGF␤1 family member, BMP-4, plays a role in early stages of mouse hematopoiesis. It induces the in vitro hematopoietic differentiation of ESCs.151 Gene targeting supports a role for BMP-4 in specification of hematopoietic mesoderm. Mouse embryos deficient for BMP-4 usually die at the time of gastrulation with little or no mesodermal differentiation.148 The few BMP-4–deficient embryos that do survive to slightly later ontogenic stages show profound decreases in mesoderm formation and erythropoiesis in the yolk sac, indicating a strict requirement for BMP-4 in the formation of the ventral-most mesoderm. BMP-4 can influence hematopoietic cell formation from the presumptive anterior head fold, normally a nonhematopoietic portion of the mouse epiblast.17 When added to AGM explant cultures, BMP-4 increases the number of HSCs.122 Interestingly, BMP-4 is localized in the mesenchyme underlying aortic clusters in the mouse122 and human152 embryo and thus appears to be an important effector in hematopoietic specification and growth. It controls the expression of some pivotal hematopoietic transcription factors such as SCL and GATA-1 (reviewed in ref. 153). Mouse embryos deficient in the VEGF/Flk-1 signaling axis exhibit more severe and consistent defects than TGF␤1 -deficient embryos. All Flk-1–deficient embryos die between E8.5 and E9.5.27 They are defective in the production of yolk sac blood islands and vessel formation, and the numbers of hematopoietic progenitors are dramatically reduced. A LacZ marker gene inserted in the Flk-1 gene allowed tracking of endothelial and hematopoietic cell formation in embryos. Those embryos lacking functional Flk-1 expressed the LacZ marker appropriately in the developing mesoderm. However, these expressing cells accumulated in the amnion instead of the areas of blood island formation, suggesting the requirement for Flk-1 as early as the formation and/or migration of the yolk sac mesodermal cells. The gene for VEGF, the ligand of Flk-1, has also been mutated. The generation of chimeric embryos with VEGF +/− ESCs results in embryonic lethality at E11, defective vasculogenesis, and a substantially reduced number of yolk sac red blood cells.154,155 VEGF can direct the in vitro differentiation of ESCs to both endothelial and hematopoietic lineages.156 Flk-1 is expressed by presumptive hemangioblasts, as shown by ESC studies and analyses of earlystage mouse conceptuses in the posterior region of the primitive streak.30,31,156 Although gene targeting of all these signaling molecules results in defects in both primitive and definitive hematopoiesis, Notch1 signaling in the mouse conceptus has been found to be selectively important for AGM (adult definitive) but not yolk sac (primitive) hematopoiesis. Notch1deficient mouse conceptuses die at E10 and contain almost normal numbers of yolk sac primitive erythroid and erythroid–myeloid progenitors, but have no AGM hematopoiesis or HSCs.157 Notch1, Notch4, and their ligands Delta-

Elaine Dzierzak like 4, Jagged 1, and Jagged 2 are expressed in endothelial cells lining the dorsal aorta.158 Mutations that affect Notch signaling in zebra fish eliminate Runx1 expression and hematopoietic cluster formation in the AGM.146,159 Overexpression of Runx1 in Notch signaling mutants in both zebrafish and mice restores AGM hematopoiesis.159,160 Thus, Notch1 appears to be a unique and pivotal factor in the onset of AGM definitive hematopoiesis. c-kit is a receptor tyrosine kinase closely related to Flk1. Many natural mutations for c-kit, and its ligand, steel factor (SF), have been found in mice. W and Sl strains of mice, respectively, mutant for these genes,161,162 exhibit deficiencies in hematopoiesis, primordial germ cells, and melanocytes. The most severe mutations result in embryonic lethality beginning at E16. Yolk sac primitive erythropoiesis is not affected, but definitive CFU-S progenitors and mast cells are absent. SF has been shown to act as a proliferative88,163 or antiapoptotic164 agent in hematopoietic progenitors and CFU-S. Thus, c-kit/SF signaling is required for normal definitive hematopoiesis and may play a role in clonogenicity of early hematopoietic progenitors or, as in primordial germ cells and melanocytes, play a role in definitive hematopoietic progenitor migration.

Transcription Factors The SCL transcription factor (basic helix-loop-helix family) is known to play a pivotal role in the production of all hematopoietic cells in the embryo as shown by gene targeting in the mouse.165–169 SCL−/− mouse conceptuses die at E9.0 of a complete absence of blood formation. Unlike TGF␤1 and Flk-1, SCL is not required for all endothelial cell and vascular formation; yolk sac capillaries are initiated. Vitelline vessel formation, however, is blocked and subsequent angiogenesis in the yolk sac is defective. A transgenic rescue of the hematopoietic defects in SCL−/− embryos confirms that SCL is necessary for embryonic angiogenesis.169 Interestingly, ectopic injection of RNA encoding the SCL hematopoietic transcription factor specifies normally nonhematopoietic pronephric mesoderm to become hematopoietic.170 The graded expression of this hematopoietic transcription factor may initiate the normal spatial borders of hematopoiesis in the different mesodermally derived regions of the embryo. Differentiation studies using SCL−/− ESCs indicate that this factor is essential for hematopoietic differentiation and vascular remodeling, playing a role in the hematopoietic commitment of the hemangioblast.171 Similarly gene-targeted deletion of the LMO2 gene results in a phenotype identical to that of SCL−/− embryos.172,173 It has been found that the LMO2 protein heterodimerizes with the SCL protein forming a transcriptional regulatory complex.174,175 GATA-2 is a member of GATA (DNA-binding motif ) transcription factor family of genes. The GATA factors are highly conserved among all vertebrate species. Along with GATA-1 and GATA-3, studies in mammalian cell lines have

A Developmental Approach to Hematopoiesis shown that GATA-2 plays a role in transcriptional regulation within the hematopoietic system.176,177 Specifically, GATA-2 is thought to act in HSCs and progenitors, due to its specific expression pattern. Mice lacking the transcription factor GATA-2 suffer from slightly reduced primitive erythropoiesis and a complete lack of other committed progenitors and HSCs and die at E10.5.178 GATA-2 is expressed in the aortic endothelium116 and is thought to affect the expansion of the hemogenic population emerging from these cells.115 Interestingly, GATA-2 haploinsufficiency profoundly decreases the number of AGM HSCs, but yolk sac HSCs are only slightly affected. The tissue differences suggest a developmental timing component in the requirement of HSCs for GATA-2, different tissue-specific interacting partners for GATA-2, and/or different downstream targets. Nonetheless, GATA-2 is strictly required for adult (definitive) hematopoiesis and is expendable for embryonic (primitive) erythropoiesis. GATA-2 is thought to work together with SCL and the Ets transcription factor Fli-1 in recursive gene regulatory circuit in early mouse hematopoietic development.179,180 The CBF transcription factor genes are the most frequent targets of chromosomal rearrangements in human leukemias and were thus suggested to function in the hematopoietic system. Runx1 (also called CBF␣2 and AML1) and CBF␤1 form a heterodimeric factor that interacts through Runx1 DNA-binding domain to bind the core enhancer motif present in a number of hematopoieticspecific genes. Targeted mutagenesis revealed that Runx1 and CBFβ 1 genes66,67,181 are required for definitive but not primitive hematopoiesis – embryos present with a complete lack of definitive hematopoietic progenitors and HSCs, fetal liver anemia, and embryonic lethality occurring after E11.5.66,67,182 Yolk sac vessels and primitive erythropoiesis appear normal in these embryos. Runx1 appears to act at the level of proliferation, generation, or maintenance of definitive hematopoietic progenitor and/or stem cells. Insertion of a LacZ maker gene into the Runx1 locus65 shows Runx1 expression ventrally in the mesenchyme, endothelium, and hematopoietic clusters of the dorsal aorta,65,117 confirming a role in the establishment of the first adult-type HSCs. Haploinsufficiency of Runx1 leads to an early increase in AGM HSCs when these are directly isolated from the embryo and transplanted into irradiated adult mice.65,117,182,183 When hematopoietic tissues of Runx1+/− conceptuses are first cultured as explants and then transplanted, they display interesting differential responses to Runx1 haploinsufficiency. HSCs were profoundly decreased in AGM explants but were increased in both yolk sac and placenta, suggesting that different regulatory networks, downstream targets, interacting molecules, or altered developmental timing are operative in these tissues.183 The Ets family transcription factor, PU.1, which is required for definitive hematopoiesis, is a critical downstream target of Runx1.184 Also, studies have shown that the hematopoi-

17 etic cytokine gene IL-3 is a target of Runx1 and that IL-3 affects AGM HSC numbers.183 As a pivotal factor in HSC ontogeny, transcriptional regulation of Runx1 requires the recruitment of a SCL/LMO2/Ldb-1 complex to its intronic enhancer sequence. This enhancer targets all definitive HSCs in the mouse embryo, suggesting that it integrates the other major hematopoietic transcriptional networks to initiate HSC generation.179 Thus, an understanding of how the master regulators are controlled and fine-tuned with respect to their levels in different hematopoietic subpopulations and sites will provide insight into the genetic network that governs hematopoietic emergence in the conceptus. By analogy to the ESC program,185 it may be possible to establish hematopoietic identity in nonhematopoietic cells with just a small set of factors (Runx1, GATA, Ets, and SCL).

Epigenetic Factors Lineage-specific gene expression programs are not only controlled at the level of transcription factor recruitment, but are coordinated and maintained in an active or repressed state of expression through the involvement of chromatin modifiers.186 Cellular memory enables cells to maintain a specific lineage fate over many cell divisions and involves epigenetic modifications that include DNA methylation and histone acetylation. Groups of proteins called the polycomb group (PcG) and trithorax group (trxG) proteins, recruit histone deacetylases and methyltransferases and are well conserved in evolution in many different species. PcG proteins are transcriptional repressors and trxG proteins are transcriptional activators during development. These proteins associate with chromatin at specific loci but their core proteins do not bind DNA. The importance of PcG protein in development was recognized through their role in maintaining a silent state of Hox gene expression.187 Hox genes are known to be important in the hematopoietic system. When Hox genes are overexpressed in HSCs, they proliferate extensively, increasing their pool size.188 Although PcG proteins are involved in many loci in a variety of stem cells, most studies investigating the role of PcG and trxG proteins in HSCs focus on fetal liver and adult bone marrow–derived HSCs. The PcG protein Ezh2 is found in a complex with histone deacetylases. Ezh2, together with Eed protein, also binds DNA methyltransferases.187 These complexes are thought to be involved in the initiation of gene repression and act functionally to preserve HSC quality and prevent HSC exhaustion after trauma.189 Several other PcG proteins affecting HSC self-renewal are thought to maintain gene repression. These include Mel-18, Mph1/Rae28, and Bmi-1.190–192 Homozygous deficiency of Mel-18 leads to enhanced HSC self-renewal; Mph1/Rae28 deficiency is embryonic lethal due to insufficient hematopoiesis during development; and Bmi1-deficient fetal liver HSCs are impaired in self-renewal.

18 An example of a trxG protein involved in hematopoiesis is the Mll gene. MLL1 is a histone methyltransferase.193,194 It is misexpressed following chromosomal translocation in acute leukemias. Moreover, gene targeting of Mll1 in the mouse results in a complete lack of definitive hematopoiesis in the conceptus and early embryonic lethality.195 Thus, the control of HSC self-renewal by PcG and trxG proteins supports a role for epigenetic modifications in the homeostasis of the hematopoietic system as it is initiated in the mouse conceptus, and such mechanisms most likely play a role in the initiation and maintenance of some leukemias.

IMPLICATIONS OF EMBRYONIC HEMATOPOIESIS FOR POTENTIAL CLINICAL APPLICATION IN HUMAN BLOOD-RELATED THERAPIES Significant progress continues to be made in the field of developmental hematopoiesis. The previous dogma concerning the origins of the adult mammalian hematopoietic system in the yolk sac has given way to a new understanding of multiple and independent sites of hematopoietic generation in the early- and midgestation conceptus. The initiation of the first multipotential hematopoietic progenitors and adult-type HSCs is now known to occur in the intraembryonic PAS/AGM. The placenta has been shown to be a potent generator of hematopoietic cells, and perhaps other yet untested embryonic tissues may also possess hematopoietic potential. The molecular programming within the variety of hematopoietic cells and embryonic compartments begins to reveal differences in developmental levels and timing of expression of pivotal hematopoietic transcription factors and the heritable genetic program that defines specific hematopoietic fate. The in vitro production of hematopoietic cells from factor-directed ESC differentiation cultures is improving due to knowledge obtained from results of molecular and cellular studies on the normal in vivo embryonic development of hematopoietic cells. Together with the long-anticipated direct precursor to hematopoietic cells, the hemangioblast, the rapid acceptance of hemogenic endothelium as the predominant precursor to definitive adult hematopoietic cells suggests a new strategy for hematopoietic cell production – one that would involve the isolation, expansion, and induction of hemogenic endothelium, perhaps from adult vasculature, to establish HSC fate. Through further knowledge of the cells and molecules that lead to the normal generation of the adult hematopoietic system, we can continue to improve medical strategies for the treatment of bloodrelated genetic diseases and leukemia.

ACKNOWLEDGMENTS The author thanks laboratory members (past and present) and researchers in the field for insightful discussions leading to this chapter. The research in my laboratory is sup-

Elaine Dzierzak ported by the Netherlands Medical Sciences Research Organization (VICI 916-36-601), the Netherlands Innovative Research Program (BSIK SCDD 03038), and the National Institutes of Health (R37 DK54077).

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2 Erythropoiesis Sjaak Philipsen and William G. Wood

mammals. The establishment of blood circulation is important to provide oxygen and nutrients to the developing embryo. Primitive erythrocytes are relatively large cells characterized by the expression of embryonic globins. In mammals, these are the only erythroid cells that retain their nucleus when they enter the circulation. Definitive hematopoiesis gives rise to all hematopoietic lineages and replenishes the hematopoietic compartment throughout the lifespan of the organism. In fish, amphibians, and birds the definitive erythrocytes remain nucleated. Mammalian definitive erythrocytes expel their nucleus before they enter the circulation. Definitive erythrocytes are smaller than primitive erythrocytes and express fetal/adult globins. We will now describe the main features of erythropoiesis in fish (zebrafish, Danio rerio), amphibians (African clawed frog, Xenopus laevis), birds (chicken, Gallus gallus) and mammals (mouse, Mus musculus, and human, Homo sapiens).

INTRODUCTION

ZEBRAFISH (Danio rerio )

Erythropoiesis involves the production of mature enucleated erythrocytes from committed erythroid progenitor cells, which in turn are derived from multilineage progenitors and ultimately from the hematopoietic stem cell (HSC). In human the mature erythrocytes turn over at a rate of approximately 1% per day and it can be estimated that maintaining the red blood cell count in an adult requires approximately 2.4 × 106 new erythrocytes to be produced each second. It is not surprising, therefore, that the regulation of erythropoiesis is a complex, multifaceted process that has to cope with not only maintaining the steady state but also with providing reserves to cope rapidly with increased demand as a result of physiological or pathological demands. In this chapter we will consider the developmental origins of red cell production, their differentiation from HSCs as well as production of the hormone erythropoietin. We will examine how erythropoietin responds to tissue hypoxia and exerts its effect through cell surface receptors on erythroid cells to trigger a number of cell signaling cascades to maintain, through critical transcription factors, the survival, proliferation, and maturation of the erythron.

The zebrafish has become a popular model organism to study early vertebrate development in particular. Large numbers of fertilized eggs can be obtained easily. The eggs are transparent and development of the embryos can therefore be monitored without any interference. Development proceeds rapidly: from the fertilized egg to hatched fry takes only 72 hours. Large collections of mutants are available that have been generated through forward genetic screens, using insertional, radiation-induced and N-ethylN-nitrosourea–mediated mutagenesis.1 Effective “knockdown” of specific proteins can be achieved by injecting antisense-modified oligonucleotides, known as morpholinos, into fertilized eggs.2 The morpholinos are designed to bind to the translation initiation site or a splice junction of a particular RNA molecule, thereby preventing the synthesis of protein. With the zebrafish genome sequence at hand, one can thus perform a very quick functional analysis of any protein of interest. To distinguish the morpholino-injected fish from genetic mutants, they are called “morphants”.3 Complementary proteins can be overexpressed through the injection of RNA synthesized in vitro. Finally, fish transmitting transgenes through the germline can be obtained by injection of linearized plasmids, albeit with low efficiency.4 Nevertheless, useful reporter strains have been generated in this way, for instance lines expressing green fluorescent protein in the endothelial cells of the vasculature5 and in erythroid cells.6,7 Such transgenic reporter fish provide easy visualization of mutants and morphants in which erythroid development is disturbed. From the forward genetic screens, approximately 25 complementation groups affecting blood formation have been identified.10 These groups fall into categories ranging from defective HSC generation (e.g., the cloche mutant affecting an as yet unidentified gene11 ), arrested erythroid development (e.g., the vlas tepes mutant affecting the GATA-1 gene12 ) to structural defects in erythrocytes (e.g., the sauternes mutant affecting the alas2

ERYTHROPOIESIS DURING DEVELOPMENT The first erythrocytes appearing during vertebrate development are known as primitive erythrocytes. These cells are produced by a transient first wave of hematopoiesis, which is almost entirely dedicated to the production of primitive red cells. Primitive erythropoiesis has been studied in evolutionary distant vertebrates, in particular in fish, amphibians, birds, and mammals. Despite the considerable anatomical differences between the developing embryos of these phyla, primitive erythropoiesis appears to be a remarkably conserved process allowing observations made in lower vertebrates to be extrapolated – with care – to 24

Erythropoiesis gene13 ) and the retsina mutant affecting the band3 gene.14 Often, mutations in the orthologous genes are associated with human hematological disorders, which has led to the notion that the zebrafish provides useful models for human diseases.15

SITES OF ERYTHROPOIESIS IN THE ZEBRAFISH The first erythroid cells arise in an area known as the intermediate cell mass, first evident at 16 hours postfertilization. This structure is a derivative of the lateral plate mesoderm that first appears approximately 10 hours postfertilization, at the end of gastrulation and the start of somatogenesis. The first erythroid cells become visible at 22 hours postfertilization and enter the circulation at 24 hours postfertilization. The primitive erythroid cells express embryonic α-like and β-like globin genes.16 Although the anatomical location of the intermediate cell mass is not obviously related to the extraembryonic location in the yolk sac of the primitive erythroid progenitors in mammals, the intermediate cell mass is derived from two paraxial stripes of mesoderm arising during gastrulation, a location analogous to the mammalian site. Similar to mammals, the first definitive hematopoietic cells appear in the ventral wall of the dorsal aorta, approximately 48 hours postfertilization.17 These cells can be identified by the expression of transcription factors such as runx1 and myb.18–20 setting them apart from the primitive erythroid cells, which can be identified by the expression of embryonic globins.16 In the adult zebrafish, the kidney is the site of erythropoiesis. This is clearly different from the situation in mammals, in which the bone marrow is the main site of adult erythropoiesis. Possibly, the production of erythropoietin (Epo), the main hormone regulating erythropoiesis, in the mammalian kidney is a remnant of the erythropoietic function of this organ in their ancestors.21

ERYTHROPOIESIS IN Xenopus There is a long tradition of using the African clawed frog Xenopus laevis as a model system to study vertebrate development.22 Xenopus eggs are polarized, and unlike mammals, the cells in the early embryo are highly organized as a result of oriented cleavage planes.23 Thus, lineage-tracing experiments can be performed in 32-cell stage embryos by injecting single blastomeres with a reporter, such as a fluorescent dye or in vitro synthesized RNA encoding ␤-galactosidase. This has been applied to demonstrate that primitive hematopoiesis and definitive hematopoiesis are derived from independent cell lineages.24,25 Xenopus laevis has a tetraploid genome, which limits its use in genetic experiments due to the presence of a duplicate copy of each gene, which may or may not have identical functions. Its close relative Xenopus tropicalis has a diploid genome and is therefore increasingly used by developmental biologists.26 The full scala of molecular

25 tools can be applied to Xenopus, similar to zebrafish. An advantage of Xenopus is that morpholino-mediated knockdown and RNA-mediated protein overexpression can be targeted to single blastomeres at the 32-cell stage. In this way, gene function can be studied more specifically in the lineage giving rise to the tissue of interest, without interfering directly with the rest of the embryo. Primitive erythropoiesis in Xenopus occurs in structures known as ventral blood islands (VBIs), which can be further subdivided into anterior and posterior VBIs. VBIs are analogous to the mammalian yolk sac blood islands, although they are an integral part of the embryo. Anterior VBIs are derived from mesodermal cells originating from the C1 and D1 blastomeres, whereas posterior VBIs are derived from the D4 blastomere. Definitive hematopoietic cells are derived from a single blastomere, C3, which gives rise to a mesodermal structure known as the dorsal lateral plate.24 The dorsal lateral plate serves as an intermediate structure; after extensive cell migration and tissue remodeling, the first definitive hematopoietic cells are observed as hematopoietic clusters closely associated with the ventral wall of the dorsal aorta.25 Anatomically, the dorsal lateral plate is the equivalent of the paraaortic splanchnopleura in mammals.27 Later in development and during adult life, the liver and spleen are the main sites of erythropoiesis; there is no evidence for hematopoietic activity in the bone marrow.28 At all stages, erythroid cells of Xenopus remain nucleated.

ERYTHROID DEVELOPMENT IN THE CHICKEN (Gallus gallus ) Developing avian embryos are easily accessible and can be subjected to experimental manipulation in ovo. A particularly powerful procedure is the grafting of quail tissue in orthopic or ectopic locations in the chick embryo. Quailderived cells can be traced later in the developing chimeric embryos with species-specific monoclonal antibodies.29,30 The most extreme version of this grafting procedure is the replacement of the entire chick embryo by the quail embryo. Such experiments performed with embryos isolated before the onset of circulation revealed that definitive hematopoiesis arises intraembryonically, independent of the first wave of extraembryonic primitive hematopoiesis.31 Thus, primitive erythrocytes are formed in the yolk sac blood islands from stem cells generated in situ. Definitive HSCs are born in the ventral side of the dorsal aorta. Furthermore, it has been demonstrated that the allantois, an endodermal and mesodermal embryonic appendage, is also a source of definitive HSCs.32 The bone marrow is seeded with HSCs as soon as it is formed and is the location of erythropoiesis in the adult bird.33,34 Like in the other model organisms, primitive chicken erythrocytes express embryonic globins, whereas definitive cells express adult-type globins.35,36 Remarkably, definitive erythrocytes of birds remain nucleated, despite the high demand for

26 oxygen during flight. Possibly, the highly efficient respiratory system of birds alleviates the need for enucleated erythrocytes to support their high metabolic rate. Although the chicken is a great experimental system to investigate early developmental processes, genetic approaches can only be applied to a very limited extent in this organism. The chicken has therefore not become a widely used model system to study erythropoiesis in vivo. Nevertheless, lineage-tracing studies combined with detailed morphological analyses are still expected to contribute significantly to the understanding of the ontogeny of vertebrate hematopoiesis.37

MAMMALIAN ERYTHROPOIESIS The first erythroid cells appearing during mammalian development emerge in the extraembryonic location of the yolk sac (Fig. 2.1a,b). These cells are formed in close association with the endothelial lining of the emerging blood vessels, before the vasculature is connected to the embryo and the onset of blood circulation.38 Once released in the bloodstream, the macrocytic primitive erythrocytes retain proliferative capacity and mitotic figures are observed in the circulating blood of early mammalian embryos (Fig. 2.1c).39 Intravascular erythropoiesis is not normally observed at any other developmental stage; both fetal and adult erythrocytes are enucleated before they enter the circulation. The view has long been held that primitive erythrocytes remain nucleated and that they disappear from the circulation very quickly during the embryonic to fetal transition period. Until recently, the fate of these cells was a mystery, but more recent work has shown that the primitive cells in fact enucleate very efficiently between days 12.5 and 14.5 of mouse development, resulting in macrocytic, enucleated, erythrocytes.40 At this stage, the first fetal liverderived definitive erythrocytes appear in the circulation and their numbers increase rapidly (Fig. 2.1e–h). This has made it particularly difficult to trace the remaining primitive erythrocytes. The use of transgenes that specifically label the primitive cells with a green fluorescent reporter protein has demonstrated that the primitive cells are a stable population that persist through the end of gestation.40 The primitive cells are characterized by the expression of embryonic globins (εy, ␤h1, and ␨ in the mouse, ε, ␥ , and ␨ in human) resulting in a variety of hemoglobin tetramers in man (␨ 2 ε 2 (Gower1); ␣2 ε 2 (Gower2), ␨ 2 ␥ 2 (Portland1) ␨ 2 ␤2 (Portland2)). Mice immediately switch to adult globins when definitive erythropoiesis starts in the fetal liver. Expression of a specific fetal ␤-like-globin (␥ -globin) is a feature of anthropoid primates. Hemoglobin tetramers consisting of ␣- and ␥ -globin chains (␣2 ␥ 2 ) are known as fetal hemoglobin (HbF) in humans. These specialized hemoglobins allow the developing fetus to extract oxygen more efficiently from the maternal blood. Near the time of birth, the site of erythropoiesis switches to the bone marrow and the spleen. Humans rely mainly on the bone

Sjaak Philipsen and William G. Wood marrow for steady-state adult erythropoiesis, but in mice the spleen remains an important erythropoietic organ during adult life (Fig. 2.1k,l). Under stress conditions, for instance caused by low oxygen pressure or anemia, the spleen is used to expand the erythropoietic capacity in both species.41 Fetal globin expression is silenced in adult erythropoiesis. Hemoglobin tetramers composed of ␣- and ␤globin (␣2 ␤2 , HbA) account for approximately 97% of all hemoglobin in adult erythrocytes. HbA2 (␣2 ␦2 ) and HbF account, respectively, for approximately 2% and T impairs the interaction of the proximal CACCC box with both erythroid and nonerythroid factors. Blood. 1996;88(8):3248–3249. Feng WC, Southwood CM, Bieker JJ. Analyses of betathalassemia mutant DNA interactions with erythroid Kruppel-like factor (EKLF), an erythroid cell-specific transcription factor. J Biol Chem. 1994;269(2):1493–1500. Orkin SH, Antonarakis SE, Kazazian HH, Jr. Base substitution at position -88 in a beta-thalassemic globin gene. Further evidence for the role of distal promoter element ACACCC. J Biol Chem. 1984;259(14):8679–8681. Donze D, Townes TM, Bieker JJ. Role of erythroid Kruppellike factor in human gamma- to beta-globin gene switching. J Biol Chem. 1995;270(4):1955–1959. Perkins AC, Sharpe AH, Orkin SH. Lethal beta-thalassaemia in mice lacking the erythroid CACCC transcription factor EKLF. Nature. 1995;375(6529):318–322. Nuez B, Michalovich D, Bygrave A, Ploemacher R, Grosveld F. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature. 1995;375(6529):316– 318. Zhou D, Pawlik KM, Ren J, Sun CW, Townes TM. Differential binding of erythroid Krupple-like factor to embryonic/fetal

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globin gene promoters during development. J Biol Chem. 2006;281(23):16052–16057. Yang B, Kirby S, Lewis J, Detloff PJ, Maeda N, Smithies O. A mouse model for beta-thalassemia. Proc Natl Acad Sci USA.1995;92(25):11608–11612. Perkins AC, Peterson KR, Stamatoyannopoulos G, Witkowska HE, Orkin SH. Fetal expression of a human Agamma globin transgene rescues globin chain imbalance but not hemolysis in EKLF null mouse embryos. Blood. 2000;95(5):1827– 1833. Chen X, Bieker JJ. Stage-specific repression by the EKLF transcriptional activator. Mol Cell Biol. 2004;24(23):10416– 10424. Siatecka M, Xue L, Bieker JJ. Sumoylation of EKLF promotes transcriptional repression and is involved in inhibition of megakaryopoiesis. Mol Cell Biol. 2007;27(24):8547– 8560. Drissen R, von Lindern M, Kolbus A, et al. The erythroid phenotype of EKLF-null mice: defects in hemoglobin metabolism and membrane stability. Mol Cell Biol. 2005; 25(12):5205–5214. Hodge D, Coghill E, Keys J, et al. A global role for EKLF in definitive and primitive erythropoiesis. Blood. 2006;107(8): 3359–3370. Nilson DG, Sabatino DE, Bodine DM, Gallagher PG. Major erythrocyte membrane protein genes in EKLF-deficient mice. Exp Hematol. 2006;34(6):705–712. Tallack MR, Keys JR, Perkins AC. Erythroid Kruppel-like factor regulates the G1 cyclin dependent kinase inhibitor p18INK4c. J Mol Biol. 2007;369(2):313–321. Armstrong JA, Bieker JJ, Emerson BM. A SWI/SNF-related chromatin remodeling complex, E-RC1, is required for tissuespecific transcriptional regulation by EKLF in vitro. Cell. 1998;95(1):93–104. Kadam S, McAlpine GS, Phelan ML, Kingston RE, Jones KA, Emerson BM. Functional selectivity of recombinant mammalian SWI/SNF subunits. Genes Dev. 2000;14(19):2441– 2451. Bultman SJ, Gebuhr TC, Magnuson T. A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in beta-globin expression and erythroid development. Genes Dev. 2005;19(23):2849–2861. Zhang W, Kadam S, Emerson BM, Bieker JJ. Site-specific acetylation by p300 or CREB binding protein regulates erythroid Kruppel-like factor transcriptional activity via its interaction with the SWI-SNF complex. Mol Cell Biol. 2001;21(7):2413–2422. Gregory RC, Taxman DJ, Seshasayee D, Kensinger MH, Bieker JJ, Wojchowski DM. Functional interaction of GATA1 with erythroid Kruppel-like factor and Sp1 at defined erythroid promoters. Blood. 1996;87(5):1793–1801. Drissen R, Palstra RJ, Gillemans N, et al. The active spatial organization of the beta-globin locus requires the transcription factor EKLF. Genes Dev. 2004;18(20):2485–2490. Vakoc CR, Letting DL, Gheldof N, et al. Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1. Mol Cell. 2005;17(3):453–462. Goh SH, Josleyn M, Lee YT, et al. The human reticulocyte transcriptome. Physiol Genomics. 2007;30(2):172–178.

Erythropoiesis 203. Keller MA, Addya S, Vadigepalli R, et al. Transcriptional regulatory network analysis of developing human erythroid progenitors reveals patterns of coregulation and potential transcriptional regulators. Physiol Genomics. 2006;28(1): 114–128. 204. Goodman SR, Kurdia A, Ammann L, Kakhniashvili D, Daescu O. The human red blood cell proteome and interactome. Exp Biol Med (Maywood). 2007;232(11):1391–1408. 205. Kakhniashvili DG, Bulla LA, Jr., Goodman SR. The human erythrocyte proteome: analysis by ion trap mass spectrometry. Mol Cell Proteomics. 2004;3(5):501–509. 206. Pasini EM, Kirkegaard M, Mortensen P, Lutz HU, Thomas AW, Mann M. In-depth analysis of the membrane and cytosolic proteome of red blood cells. Blood. 2006;108(3):791– 801.

45 207. Beug H, Palmieri S, Freudenstein C, Zentgraf H, Graf T. Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus. Cell. 1982;28(4):907–919. 208. Richmond TD, Chohan M, Barber DL. Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol. 2005;15(3):146–155.

3 The Normal Structure and Regulation of Human Globin Gene Clusters Bernard G. Forget and Ross C. Hardison

The genes encoding the different globin chains of hemoglobin are members of an ancient gene family. In this chapter we will review the structural features of the globin genes, with particular attention to the sequences needed for proper regulation of gene expression. Some of these have been well conserved during mammalian evolution and therefore are likely to provide a common function in many mammals. Others are only found in higher primates and may play roles in lineage-specific regulation. We will first describe the structural characteristics of the human globin genes and then provide a comparative analysis of the genomic contexts, regulatory regions, and evolutionary conservation of features present in the globin gene clusters.

NUMBER AND CHROMOSOMAL LOCALIZATION OF HUMAN GLOBIN GENES Hemoglobin is a heterotetramer that contains two polypeptide subunits related to the ␣-globin gene subfamily (referred to here as ␣-like globins) and two polypeptide subunits related to the ␤-globin gene subfamily (␤-like globins). Globin polypeptides bind heme, which in turn allows the hemoglobin in erythrocytes to bind oxygen reversibly and transport it from the lungs to respiring tissues. In humans, as in all vertebrate species studied, different ␣-like and ␤-like globin chains are synthesized at progressive stages of development to produce hemoglobins characteristic of primitive (embryonic) and definitive (fetal and adult) erythroid cells (Fig. 3.1). Before precise knowledge of globin gene organization was gained by gene mapping and molecular cloning, a general picture of the number and arrangement of the human globin genes emerged from the genetic analysis of normal and abnormal hemoglobins and their pattern of inheritance. The number and subunit composition of the different normal human hemoglobins (Fig. 3.1) suggested that there must exist at least one globin gene for each of the different globin chains: ␣, ␤, ␥ , ␦, ε, and ␨ . Evidence from 46

the study of hemoglobin variants and the biochemical heterogeneity of the chains in fetal hemoglobin (HbF) showed that the ␣- and ␥ -globin genes were duplicated. Persons were identified whose red cells contained more than two structurally different ␣-globin chains that could be best explained by duplication of the ␣-globin gene locus, and the characterization of the structurally different G ␥ - and A ␥ globin chains of HbF imposed a requirement for duplication of the ␥ -globin gene locus. Studies of the pattern of inheritance of hemoglobin variants from persons carrying both an ␣ chain and a ␤ chain variant revealed that the ␣- and ␤-globin genes are on different chromosomes (or very widely separated if on the same chromosome). Variants of ␣-globin and ␤-globin chains were always observed to segregate independently in offspring of doubly affected parents (reviewed in ref. 1). Linkage of the various ␤-like globin genes to one another was established from the study of interesting hemoglobin variants that contained fused globin chains, presumably resulting from nonhomologous crossover between different ␤-like globin genes. Characterization of Hb Lepore,2 with its ␦␤ fusion chain, established that the ␦-globin gene was linked to and located on the 5
(or N-terminal) side of the ␤-globin gene. Analysis of Hb Kenya,3 with its A ␥ ␤ fusion chain, provided evidence for linkage of the A ␥ gene, and presumably the G ␥ gene as well, to the 5
side of the ␦- and ␤-globin genes. Thus, the general arrangement of the globin genes that emerged from these various genetic analyses can be represented as illustrated in Figure 3.1. It was also assumed, but unsupported by genetic evidence, that the embryonic ␣-like (␨ ) and ␤-like (ε) globin genes were likely to be linked to the loci encoding their adult counterparts. By using rodent–human somatic hybrid cells containing only one or a few human chromosomes, Deisseroth and colleagues4,5 clearly established that the human ␣- and ␤-globin genes resided on different chromosomes. The ␣like globin genes are located on chromosome 16, whereas the ␤-like globin genes are on chromosome 11. The latter results were obtained by hybridizing a solution of total cellular DNA from the various somatic hybrid cells to radioactive cDNAs, synthesized from ␣- and ␤-globin mRNAs by reverse transcriptase. These results were later confirmed and extended by various groups using the gene mapping procedure of Southern blot analysis with DNA from various hybrid cell lines containing different translocations or deletions of the involved chromosomes. These studies also localized the globin gene loci to specific regions on their respective chromosomes: the ␤-globin gene cluster to the short arm of chromosome 11, and the ␣-globin gene cluster to the short arm of chromosome 16 (Fig. 3.1). These chromosomal assignments were further confirmed and refined by in situ hybridization of radioactive cloned globin gene probes to metaphase chromosomes and by fluorescence-based in situ hybridization. Thus, the ␤-globin gene cluster was assigned to 11p15.5 and the

The Normal Structure and Regulation of Human Globin Gene Clusters

47

150 bp, those of the ␨ and ␺ ␨ genes are larger.8 Furthermore, the first introns of the ␨ and ␺ ␨ genes are much larger than their second introns; in fact they are 8–10 times A G larger than the first introns of any other CEN CEN TEL TEL globin gene. The presence of intervening sequences Embryonic Fetal Adult that interrupt the coding sequences of Hb Gower 1: 2 2 Hb F: 2 2 Hb A: 2 2 structural genes imposes a requirement Hb A 2: Hb Gower 2: 2 2 2 2 for some cellular process to remove these Hb Portland: 2 2 sequences in the mature mRNA. As illustrated in Figure 3.2.B, intervening sequences are Figure 3.1. Basic organization of human globin gene complexes. The locations of the ␣-globin gene complex very close to the telomere of the short arm of chromosome 16 and the ␤-globin gene transcribed into globin (and other) precursor complex on the short arm of chromosome 11 are shown at the top. The genes are shown as boxes mRNA molecules,9 but they are subsequently on the second line, named according to the globin polypeptide that is encoded. In both diagrams, excised and the proper ends of the codthe 5
–3
transcriptional orientation is from left to right. Note that the orientations with respect to the ing sequences joined to yield the mature centromere (CEN) and telomere (TEL) are opposite; the ␣-like globin genes are transcribed toward mRNA.10 This posttranscriptional processing CEN, whereas the ␤-like globin genes are transcribed toward TEL. The composition of hemoglobins produced at progressive developmental stages is given at the bottom. of mRNA precursors to remove introns has been termed splicing. A crucial prerequisite for the proper splicing of globin (and other) precursor ␣-globin gene cluster to 16p13.3. Subsequent DNA mRNA molecules is the presence of specific nucleotide sequencing of entire human chromosomes and alignment sequences at the junctions between coding sequences with maps of chromosome bands places the ␤-globin (exons) and intervening sequences (introns). Comparison gene cluster in 11p15.4. The ␣-globin gene cluster is only of these sequences in many different genes has permitted approximately 150 kb from the telomere of the short arm of the derivation of two different consensus sequences, which chromosome 16. are almost universally found at the 5
(donor) and 3
(acceptor) splice sites of introns.11,12 The consensus sequences thus derived are shown in Figure 3.2A, along with the GLOBIN GENE STRUCTURE: INTRONS consensus surrounding the branch point A involved in the AND THEIR REMOVAL initiation of splicing. The dinucleotides GT and AG shown The coding region of each globin gene in humans and other in boldface, at the 5
and 3
ends, respectively, of the intron, vertebrates is interrupted at two positions by stretches are essentially invariant and are thought to be absolutely of noncoding DNA called intervening sequences (IVSs) or required for proper splicing. This is the so-called GT-AG introns.6 In the ␤-like globin genes, the introns interrupt the rule. Rare examples have been described in which GC sequence between codons 30 and 31 and between codons instead of GT is found at the donor splice site junction. 104 and 105; in the ␣-globin gene family, the intervening The importance of these consensus sequences is undersequences interrupt the coding sequence between codons scored by the fact that mutations that either alter them or 31 and 32 and between codons 99 and 100 (Fig. 3.2.A). create similar consensus sequences at new sites in a globin Although the precise codon position numbers at which gene can lead to abnormal processing of globin mRNA the interruption occurs differ between the ␣- and ␤-like precursors; these constitute the molecular basis for many globin genes, the introns occur at precisely the same positypes of thalassemia (Chapters 13 and 16). Throughout this tion in the aligned primary sequence of the ␣- and ␤chapter we will refer to human mutations that affect some globin chains. Thus, given the likely possibility that the ␣aspect of the pathway for gene expression. Readers desiring and ␤-globin gene families originally evolved from a single more information may want to use databases such as HbVar ancestral globin gene,7 these gene sequences are homolo(http://www.bx.psu.edu)13 or the Phencode project (http: gous, and we infer that the presence of the introns at these //phencode.bx.psu.edu)14 to find positions, genotypes, and positions predates the separation of ␣-globin and ␤-globin phenotypes for the greater than 1,000 known globin gene genes approximately 500 million years ago (in an ancesvariants. tral jawed vertebrate). The first intervening sequence (IVS1) is shorter than the second intervening sequence (IVS-2) DETAILED CHROMOSOMAL ORGANIZATION in both ␣- and ␤-globin genes, but IVS-2 of the human ␤OF THE HUMAN GLOBIN GENES globin gene is much larger than that of the ␣-globin gene (Fig. 3.2.A). A precise picture of the chromosomal organization of the The pattern of intron sizes of the ␨ -like globin genes dif␣- and ␤-like human globin gene clusters, with respect to fers from that of the other ␣-like globin genes. Whereas the the number of structural loci and intergenic distances, was introns in the ␣ and ␺ ␣ genes are small, that is, fewer than obtained by a number of different techniques: 1) restriction p13.3

p15.5/15.4

Chromosome 16

Chromosome 11

48

Bernard G. Forget and Ross C. Hardison

A.

5′

3′

1

31

32

99

1

30

31

104

100

141 codons

105

146 codons

CAG GTRAGT...YNYYRAG...YYYYYNYAG G 5′ splice site

branch

3′ splice site

B. pre-mRNA

post-transcriptional modifications cap

AAAAAA splicing

mRNA

cap

AAAAAA translation

globin polypeptide with heme

Figure 3.2. Structure and expression pathway of globin genes. (A) General structure of globin genes. The coding sequences of all globin genes in humans and other animals are separated by two introns (white boxes) into three exons. The first exon has a short 5
untranslated region (gray box) followed by a coding region (black box). All of the central exon codes for protein, whereas the third exon begins with coding sequences and ends with a 3
untranslated region. The relative sizes of the portions of the genes are indicated by the sizes of the boxes, and codon numbers are given above the boxes. The consensus sequence for critical sequences used in splicing are shown under the second intron of the ␤-globin gene, and similar sequences are present in all introns. The vertical arrows show the splice site junctions within the consensus sequences where cleavage occurs during the process of joining the exons. (B) The pathway for expression of globin genes. The RNA transcript is shown with short boxes corresponding to the untranslated regions (gray), coding regions (black), and introns (white) as in (A), with processing and splicing steps occurring in the nucleus to form the mature mRNA. The mRNA is translated in the cytoplasm to generate a globin polypeptide to which the heme (gray disk) will bind. The diagram of the folded globin structure was provided by Dr. John Blamire at the Brooklyn College of the City University of New York.

endonuclease mapping of genomic DNA (e.g., refs. 15, 16) using the gel blotting procedure of Southern,17 and, 2) gene isolation and sequencing using recombinant DNA technology (e.g., ref. 18). Sets of overlapping genomic DNA fragments spanning the entire ␣- and ␤-globin gene clusters were obtained by gene cloning, initially in bacteriophage λ and larger fragments in cosmid vectors. Detailed analysis of these recombinant DNA clones and complete DNA sequencing led to the determination of the gene organization illustrated in Figure 3.3. Some results were expected, such as the finding of single ␦- and ␤-globin gene loci and duplication of the ␣- and ␥ -globin gene loci. In addition, single loci for the embryonic ␨ - and ε-globin chains were found linked to the ␣- and ␤-globin gene clusters, respectively. It is noteworthy that the genes in each cluster are in the same transcriptional orientation and are arranged, in a

5
to 3
direction, in the same order as their expression during development. An unexpected finding was the presence in the globin gene clusters of additional gene-like structures with sequence homology and an exon–intron structure similar to the actively expressed globin genes. These DNA segments have been called pseudogenes.19 One, called ␺ ␤1, is in the ␤-like globin gene cluster between the ␥ - and ␦globin genes. At least two (and possibly four) are in the ␣like globin gene cluster. The two clear examples are ␺ ␨ 1 and ␺ ␣1, located between the active ␨ -globin and ␣-globin genes (Fig. 3.3). All three (␺ ␤1, ␺ ␨ 1, and ␺ ␣1) are characterized by the presence of one or more mutations that render them incapable of encoding a functional globin chain. This inability to encode a functional globin polypeptide does not necessarily render the pseudogenes inactive for transcription. The pseudogene ␺ ␤1 is transcribed and spliced, as shown by several spliced expressed sequence tags, whereas no evidence has been provided that ␺ ␣1 is transcribed. These pseudogenes appear to have arisen by gene duplication events within the globin gene clusters followed by mutation and inactivation of the duplicated gene and subsequent accumulation of additional mutations through loss of selective pressure. Two other ␣-like globin genes have been identified and characterized in the ␣-globin gene cluster, but their roles, if any, in encoding globin polypeptides are still uncertain. The ␪-globin gene is located to the 3
or Cterminal side of the duplicated ␣-globin genes.20 It is more closely related to the ␣-globin genes than to the ␨ -globin genes and is expressed at low levels in erythroid cells.21,22 Clear homologs to the ␪-globin gene are found in the homologous position in other mammalian ␣-like globin gene clusters. The ␮-globin gene is located just 3
of the ␺ ␨ 1-globin pseudogene;23,24 it was initially called ␺ ␣225 but with more accurate sequencing it is clear that this gene does not contain mutations that would render it inactive. It is a distant relative, being equally divergent from both ␣-globin and ␨ -globin genes. Its closest relatives are the ␣D -globin genes, which are actively expressed in red cells of reptiles and birds.24,26 DNA sequences similar to that of the human ␮-globin gene are found in other mammals, but in some species, such as mouse, the sequence has diverged so much that no obvious gene structure is found. Thus the presence of the ␪-globin gene is conserved in all mammals examined but the ␮-globin gene has been lost in some but not all lineages. Transcripts from both the ␪-globin gene and the ␮-globin gene are produced and spliced in erythroid cells, albeit at much lower levels than the ␣-globin gene. Curiously, no hemoglobin containing the ␪-globin chain or the ␮-globin chain has been identified, even by sensitive mass spectrometry.23 Furthermore, the predicted structure (translated amino acid sequence) of the ␪-globin chain suggests that it would be unlikely to function normally as a hemoglobin subunit.27 Thus these genes remain a puzzle. They tend to be retained over mammalian

The Normal Structure and Regulation of Human Globin Gene Clusters

A.

49

Human Mar. 2006 chr11:5,100,001-5,325,000 (225,000 bp) 5300000

chr11:

5250000

5200000

5150000

5100000

G A

CEN... OR51B5 OR51B2

OR51B4

...TEL

HBE1 HBG2 HBG1 HBD HBB

OR51V1

OR52A1 OR52A5

Genes OR

OR

OR

OR OR

Pseudogenes LCR HSs

54 32 1

3’HS1

Distal elements Promoters Enhancers Regulatory Potential

Conservation

0.3 _ 0_ 1.0 _ 0_ HPFH-1; Black HPFH-2; Ghanaian HPFH-6 HPFH-3; Indian

Deletions causing thalassemia and HPFH

B.

Human Mar. 2006 chr16:40,001-220,000 (180,000 bp) chr16:

50000

100000

150000

200000

TEL...

Genes

...CEN

POLR3K C16orf33

RHBDF1

HBZ HBM HBA2 HBQ1 HBA1 MPG

LUC7L

C16orf35

Pseudogenes Promoters Distal erythroid HSs 0.3 _ Regulatory Potential 0_ 1.0 _

-48 -40 -33

-10,-8

Conservation 0_ Ti~ - - (MC) - - (CAL) - - (THAI) - - (MED-II) - - (FIL) - - (RT) - - (CL) 0

- - (BRIT) - - (MA) - - (SA) - - (MED-I) - - (SEA) - -(CANT) - - (SPAN) - - (GEO) +

Figure 3.3. Detailed maps of the human globin gene complexes, including genomic features and representative deletions. (A) Detailed map of the ␤-like globin gene complex and surrounding olfactory receptor genes. The globin genes are named both by the encoded globin polypeptide and the official gene name. Pseudogenes are shown on a line below the genes. The known cis-regulatory modules are separated into distal elements such as the locus control region (shown as five DNase hypersensitive sites or HSs), promoters and enhancers close to the 3
ends of HBG1 and HBB. The next two tracks show two features derived from multiple alignments of the human genomic sequence with sequences from six other placental mammals (chimpanzee, rhesus macaque, mouse, rat, dog, and cow). The regulatory potential measures the similarity of patterns in the alignments to those that are distinctive for known regulatory regions versus neutral DNA.57 The conservation score estimates the likelihood that an alignment is in the most constrained portion of the genome, likely reflecting purifying selection (phastCons).56 Positions of deletions that cause ␦␤ thalassemia or hereditary persistence of fetal hemoglobin (HPFH) are shown in the lower portion. (B) Detailed map of the ␣-like globin gene complex and surrounding genes. The conventions and tracks are similar to those in (A) Positions of the distal erythroid HSs are from Hughes et al.26 The deletions are grouped by those with deletion of a single ␣-globin gene (␣+ thalassemia), deletion of both ␣-globin genes (␣0 thalassemia), and a representative deletion (Ti∼) that removes the distal enhancer (HS-40) but no structural genes. Coordinates of the deletions were provided by Dr. Jim Hughes. These figures were generated starting with output from the UCSC Genome Browser,121 using the following tracks in addition to ones already mentioned: UCSC Known Genes,122 ORegAnno for cis-regulatory modules,123 and Locus Variants for the deletions.14 For panel A, the Genome Browser output was rotated 180◦ so that the 5
–3
transcriptional orientation is left to right (note that the genome coordinates are decreasing from left to right). Both figures were edited for clarity.

50 evolution, suggesting that their sequences are constrained to preserve some function. They are expressed at the RNA level but do not appear to be translated into a polypeptide. Perhaps they or their RNA transcripts play some role that has yet to be discovered.

GENOMIC CONTEXT OF THE ␣-GLOBIN AND ␤-GLOBIN GENE CLUSTERS The separation of ␣- and ␤-globin gene clusters to different chromosomes has allowed them to diverge into strikingly different genomic contexts, with paradoxical consequences for our understanding of their regulation. Given that all contemporary vertebrates have developmentally regulated hemoglobin genes encoding proteins used for oxygen transport in erythrocytes, it would have been reasonable to expect that the molecular mechanisms of globin gene regulation would be conserved in vertebrates. Certainly, the coordinated and balanced expression of ␣- and ␤-globin genes to produce the heterotypic tetramer ␣2 ␤2 in erythrocytes should be a particularly easy aspect of regulation to explain. Because the two genes would have been identical after the initial duplication in the ancestral vertebrate, with identical regulatory elements, it is parsimonious to expect selection to keep the regulatory elements very similar. Much has changed between the ␣- and ␤-like globin gene clusters since their duplication. Not only are they now on separate chromosomes in birds and mammals, but in mammals they are in radically different genomic contexts.28 A major determinant of the genomic environment is the G+C content. A G+C-rich DNA segment has a high mole fraction of the nucleotides guanidylic acid (G) and cytidylic acid (C), whereas an A+T-rich DNA segment has a high mole fraction of the nucleotides adenylic acid (A) and thymidylic acid (T). The G+C content for the human genome on average is low (∼41%) but some segments can be much lower or higher, ranging from 30% to 65% in 20-kb windows.29 Regions that are G+C rich tend to be enriched in genes, and those genes tend to be expressed in a broad range of tissues. They also tend to have islands with an abundance of the dinucleotide CpG.30 This is in stark contrast to the bulk of the genome, which has very few CpGs because these are the sites for DNA methylation, and substitution of CpG to TpG or CpA is very rapid on an evolutionary time scale (as much as 10 times faster than the rates of other substitutions). The CpG islands are thus short regions (a few hundred base pairs) in which the CpG dinucleotides are not methylated; these have been associated with important functions such as promoters for transcription. The ␤-globin gene clusters in humans and other mammals are A+T rich, with no CpG islands,31 whereas the ␣like globin gene clusters are highly G+C rich, with multiple CpG islands.32 This correlates with several important differences in the structure and regulation of the two gene

Bernard G. Forget and Ross C. Hardison clusters. Tissue-specific gene expression of the ␤-like globin genes is correlated with an increased accessibility of the chromatin only in expressing cells,33 and hence “opening” of a chromatin domain is a key step in activation of these genes. In contrast, there are the ␣-like globin genes, which are in constitutively open chromatin.28 The ␤-globin gene cluster is subject to tissue-specific DNA methylation,34 but, in keeping with the presence of CpG islands, the ␣-globin gene cluster is not methylated in any cell type.35 The ␤-globin gene clusters are replicated early in S phase only in cells expressing them, whereas the human ␣-globin genes are replicated early in all cells.36–38 Thus, the mammalian ␣-globin genes have several characteristics associated with constitutively expressed “housekeeping” genes. The strikingly different genomic contexts of the two gene clusters affect several aspects of DNA and chromatin metabolism, including timing of replication, extent of methylation, and the type of chromatin into which the loci are packaged. Rather than selecting for similarities to ensure coordinate and balanced expression, the processes of evolution at these two loci have made them quite different. The full implications of these differences may not yet be known. For instance, the two “healthy” genes with no known function in the ␣-like globin gene cluster, ␪ and ␮, are themselves CpG islands. Could this be a clue to a role for these genes outside the conventional one of coding for proteins? The types of genes that surround the ␣-like and ␤-like globin gene clusters are quite different (Fig. 3.3). The ␤like globin gene cluster is surrounded by olfactory receptor (OR) genes, which encode G protein–coupled receptors expressed in olfactory epithelium.39 Several OR gene clusters containing approximately 1,000 genes and pseudogenes are found in the human genome. The OR gene cluster surrounding the ␤-like globin genes is a particularly large one, with approximately 100 genes extending almost 1 million bp (Mb) past HBB (the ␤-globin gene) and over 3 Mb toward the centromere from HBE1 (the ε-globin gene). This arrangement is found in homologous regions in mammals and in chickens. Thus the erythroid-specific regulation of the ␤-like globin gene cluster is exerted in a chromosomal environment that is largely devoted to olfactory-specific expression. Perhaps this has had an impact on selection for a particularly powerful enhancer, to override the olfactoryspecific regulation. As shown in Figure 3.3A, some deletions causing ␦␤ thalassemia or hereditary persistence of fetal hemoglobin not only remove ␤-like globin genes, but they also fuse the remaining genes with sequences close to an OR gene. The phenotype of patients carrying such deletions may be explained in part by bringing positive or negative regulatory elements normally associated with OR genes into proximity of the ␤-like globin genes40–41 (see Chapter 16). In contrast, the ␣-like globin genes are surrounded by a variety of genes (Fig. 3.3.B), many of which are widely expressed and carry out fundamental roles in cellular

The Normal Structure and Regulation of Human Globin Gene Clusters metabolism and physiology, such as MPG (encoding the DNA repair enzyme methyl purine glycosylase) and POLR3K (encoding a subunit of RNA polymerase III).42 Although the ␣-like globin gene cluster and surrounding DNA is in constitutively open chromatin, histones are hyperacetylated (another mark of active loci) in erythroid cells in a more restricted region encompassing the globin genes and their regulatory sequences.43 The regions homologous to that surrounding the ␣-like globin gene cluster have undergone inter- and intrachromosomal rearrangements in various vertebrate lineages, but the genes from POLR3K through HBQ1 have remained together in all species examined from fish to mammals.44 This suggests that this region encompasses all the sequences needed in cis for appropriate regulation of the ␣-like globin genes. Despite these many differences between ␣-like and ␤like globin gene clusters in mammals, the appropriate genes are still expressed coordinately between the two loci, resulting in balanced production of ␣-like and ␤-like globins needed for the synthesis of normal hemoglobins. The mechanisms that accomplish this task still elude our understanding. One important aspect that is common to the genomic contexts of both gene clusters is the presence of distal strong enhancers. The discovery of these enhancers was aided by mapping of deletions that result in ␤ thalassemia or ␣ thalassemia, which are inherited deficiencies in the amount of ␤-globin or ␣-globin, respectively (see Chapters 13 and 16). A number of these deletions removed distal sequences but retained all the globin genes, such as the deletions associated with Hispanic (ε␥ ␦␤)0 thalassemia and the Ti∼ ␣0 thalassemia (Fig. 3.3), as well as other deletions (Figs. 13.7 and 16.5). Within the deleted intervals are critical long-range enhancers needed for high-level expression of any gene in the linked globin gene clusters. These are the locus control region (LCR) for the ␤-globin gene cluster and HS-40 or major regulatory element for the ␣-globin gene cluster. Thus regulation of expression of globin genes involves DNA sequences both close to the genes (proximal) and as much as 70 kb away from the genes (distal). These will be examined in more detail in the next section.

EVOLUTIONARY INSIGHTS INTO REGULATION OF GLOBIN GENE CLUSTERS Motivation One avenue for improving the conditions of patients with hemoglobinopathies could involve regulation of expression of the globin genes. This hope is based on the normal human variation in phenotypes presented for a given mutant genotype. For example, patients with naturally higher concentrations of HbF (␣2 ␥ 2 ) in their erythrocytes tend to have milder symptoms of either sickle cell disease or ␤ thalassemia (Chapters 17 and 19). The ␣-globin gene

51

status can affect the severity of ␤ thalassemia, with more balanced production of ␣-globin and ␤-globin associated with milder disease. Thus considerable effort has gone into studying the stage-specific expression of the globin genes, with a long-term goal of enhancing or restoring production of embryonic or fetal hemoglobins in adult life or reducing expression of deleterious alleles. Although no current treatment by gene therapy is in practice as of this writing, much effort continues in this area. The use of hydroxyurea in the treatment of sickle cell disease is an outgrowth of studies on mechanisms of regulation of globin genes. Current studies aim to discover more sophisticated and directed pharmacological methods for enhancing production of embryonic and fetal hemoglobins. Studies over the past three decades have revealed much about the regulation of the human globin genes. In this section, we will summarize some of the information about DNA sequences needed in cis (i.e., on the same chromosome) for regulation of the globin genes. Chapter 4 will cover the proteins interacting with these regulatory DNA sequences.

Common versus Lineage-specific Regulation Comparison of noncoding genomic DNA sequences among related species is a powerful approach to identifying and better understanding cis-regulatory modules (CRMs). It is important to distinguish, however, what is similar and what is distinctive about the patterns of regulated expression of the genes in the species being compared. If one is searching for CRMs that perform a function common to most or all mammals, then conservation across all mammals and evidence of strong constraint in noncoding DNA will provide good candidates for further experimental tests (e.g., refs. 45–47). Such constrained noncoding sequences can have within them short, almost invariant regions that frequently correspond to transcription factor binding sites. These have been called phylogenetic footprints.48 If one is studying a type of regulation that only occurs in higher primates, then searching for sequences conserved in other mammalian orders will be futile. Instead, the search should focus on sequences conserved in the species with a common mode of regulation but which differ from the homologous regions in species with a different regulation. These have been called differential phylogenetic footprints.49 Regulatory features of globin genes common to many vertebrate species include tissue specificity and some aspects of developmental specificity. Expression of the ␣like and ␤-like globin genes in all vertebrate species examined is restricted to the erythroid lineage. Thus some determinants of tissue specificity should be common to all these genes. One example is binding by the transcription factor GATA-1. As will be detailed in the following sections, the promoter, enhancers, or both for all globin genes have binding sites for GATA-1. Another feature common to all mammals is the expression of the ε-globin and ␨ -globin

52 genes exclusively in primitive erythroid cells, which are produced during embryonic life. Thus one might expect determinants of embryonic expression to be conserved in many species. Indeed, conservation of the upstream promoter regions of these genes in eutherian mammals is more extensive than is seen for other promoters in their globin gene clusters.50 An example of lineage-specific regulation is the recruitment of the ␥ -globin genes for expression in fetal erythroid cells. In most eutherian mammals, the ␥ -globin genes are expressed in primitive erythroid cells, similar to the εglobin gene, and the ␤-globin gene is expressed in definitive erythroid cells both during fetal and adult life. Simian primates, including humans, express the ␥ -globin genes during fetal erythropoiesis, and the expression of the ␤-globin gene is delayed. The extent of delay varies in different primate clades, but in humans it is largely delayed until just before birth. Thus when examining interspecies alignments of the regulatory regions of the ␤-globin gene (HBB) and the ␥ -globin genes (HBG1 and HBG2), one will be seeing a combination of CRMs used in common (e.g., for adult erythroid expression of HBB) and in a lineage-specific manner (e.g., fetal expression of HBG1).

Quantitative Analysis of Sequence Alignments Alignments of genomic DNA sequences reveal the segments that are similar between species, and often these reflect homology (descent from a common ancestor). These sequence matches tend to have the highest similarity in the protein-coding exons, but significant stretches of noncoding sequences also align between mammalian species (for globin gene complexes, see refs. 51–53). Further analysis is required to discern which sequence matches simply reflect common ancestry (aligned neutral DNA) versus those in sequences that are under constraint (sequences with a common function).54,55 Several bioinformatic tools have been developed to help interpret the alignments of multiple sequences. Results from two of these, each analyzing alignments of several mammals (human, chimpanzee, rhesus macaque, mouse, rat, dog, cow, and sometimes additional ones), are shown in Figure 3.3. The Conservation track plots the phastCons score at each position of the human sequence. This score is an estimate of the posterior probability that a given nucleotide is in the most strongly constrained (i.e., most slowly changing) portion of the genome.56 Higher scores are associated with a greater likelihood that a position or region is under strong purifying selection. Sequences that are needed for a feature that is common to these several placental mammals would be expected to have a high Conservation score. A discriminatory analysis of the multiple alignments was used to generate a Regulatory Potential score.57 This machine-learning approach estimates the likelihood that a given aligning segment is a CRM, given the frequency of

Bernard G. Forget and Ross C. Hardison patterns in the alignments that are distinctive for CRMs as opposed to neutral DNA. The patterns are strings of alignment columns, and their discriminatory power is determined by the frequency of the patterns in training sets of alignments in CRMs compared with alignments in neutral DNA. Although the Regulatory Potential score is influenced by features in addition to constraint, it is designed for finding CRMs that are common among species.

Basal Promoters Promoters are DNA sequences needed for accurate initiation of transcription. For some promoters including the globin gene promoters, one DNA segment interacts with RNA polymerase II and its accessory factors (such as TFIID and TFIIB) to determine the start site of transcription; this is the basal promoter.58 Five motifs have been associated with basal promoters, and these are found in the promoters of human globin genes (Fig. 3.4.A). They include the familiar TATA box to which TBP binds, along with the BRE to which TFIIB binds and the Inr and DPE motifs to which components of TFIID bind.58 Early studies revealed the presence of the ATAAA motif approximately 25–30 bp 5
to the start site of transcription of the globin genes,59 and this is by far the most restricted in its consensus, that is, this motif appears to be under evolutionary constraint in globin genes. Recent studies on other promoters are revealing the roles of additional motifs close to the start site of transcription, but on both sides. Matches to these motifs can be found readily at the appropriate positions in the human globin genes (Fig. 3.4.A). The motifs other than TATA do not have well-defined consensus sequences, either for genes in general or for the human globin genes, and thus their presence alone may not signify function. Also, only the TATA box, Inr, and DPE show evidence of constraint in homologs in other mammalian species (Fig. 3.5.A, conservation track). Each of the motifs except BRE has been implicated in function by finding a mutation in at least one case of ␤ thalassemia. Every base in the TATA box has been altered in one or another ␤ thalassemia, and mutations in Inr, MTE, and DPE also are associated with ␤ thalassemia (Fig. 3.5.A, Compilation of Human Disease Variants and Other Mutations). The BRE overlaps with the ␤-direct repeat element (␤DRE), which is a cis-regulatory element bound by ␤DRF and demonstrated to function in regulation of the ␤-globin gene by mutagenesis and expression in transfected cells.60 Thus, the mutagenesis data (natural and directed) indicate that all five motifs are important for appropriate expression of the ␤-globin gene. The presence of similar motifs in the basal promoters for other human globin genes suggests that they are active in these genes as well. Although it is common to describe promoters recognized by RNA polymerase II by the motifs shown in Figure 3.4.A, it is important to realize that this is true for only a minority of human genes. Globin gene promoters

The Normal Structure and Regulation of Human Globin Gene Clusters

53

′

′

Figure 3.4. Motifs and binding sites in cis-regulatory modules of globin genes. (A) Motifs in the basal promoter, based on those defined in the review by Maston et al.58 Numbers along the top are relative to the transcription start site as +1, and ATG denotes the translation start site. The top consensus sequence is from Maston et al. Corresponding positions in the globin genes are given for each motif, followed by the consensus derived for the globin genes. Symbols for ambiguous nucleotides are S = C or G, W = A or T, R = A or G, Y = C or T, D = A or G or T, H = A or C or T, V = A or C or G, and N = A or C or G or T. (B) Motifs in the regulatory regions immediately upstream of the basal promoters. Motifs are indicated by sequence (CCAAT, CACC, and GATA), the name of the element (␤DRE, ␣IRE, ␥ PE, and OCT) or the protein name followed by bs for “binding site” (BP2bs, NF1bs, and BB1bs). Boxes for motifs found in several upstream regions are shaded. The boxes were placed in the correct order but spacing is not indicated. The thick line for the HBA upstream regions (both HBA1 and HBA2 ) denotes that it is a CpG island. (C) Motifs in the proximal enhancers. (D) Motifs in distal positive regulators, including three hypersensitive sites of the ␤-globin LCR and HS-40 for the ␣-globin gene cluster.

fall into the category of promoters with well-defined TATA boxes at a restricted location and one major start site for transcription. Recent studies show that these comprise a small minority of promoters, perhaps only 10%–20%. Most promoters are CpG islands with no obvious TATA box, and in some cases they have a broad distribution of start sites.61

Upstream Regulatory Sequences Adjacent to the basal promoter is the upstream regulatory region,58 which in globin genes runs from approximately positions -40 to -250 (Fig. 3.4.B). Only one motif in this region is found in all the highly expressed globin genes: the CCAAT box. Proteins such as NF-Y and CP1 bind to this

54

Bernard G. Forget and Ross C. Hardison

Figure 3.5. Conservation and mutations in globin gene promoters. (A) Basal promoter and (B) upstream promoter for HBB. In each panel, the sequence of an 80-bp segment is shown, along with positions of mutations associated with ␤ thalassemia, conservation scores, and alignments with many mammals, chicken, and frog (X. tropicalis). The display is from the UCSC Genome Browser in genome coordinates (top line), and the direction of transcription is from right to left (opposite that used in previous figures). The start site of transcription is denoted by the vertical line leading to a leftward arrow. Boxes are drawn around motifs, which are labeled by name and proteins that bind to them (bottom line in each panel).

motif,62,63 and it has been implicated in promoter function because of its presence in many promoters and the results of mutagenesis and binding studies.59 It is missing from the ␦-globin gene (HBD) promoter, but this gene is expressed at a low level (∼1%–2% of HBB).

Two motifs are found in many but not all promoters. One is the CACC box, which is bound by transcription factors in ¨ the Kruppel-like zinc finger class (KLF). The first erythroid ¨ KLF discovered was erythroid Kruppel-like factor, which binds to the CACC box in the HBB promoter and is needed

The Normal Structure and Regulation of Human Globin Gene Clusters for erythropoiesis.64,65 The CACC boxes in globin promoters tend to be highly conserved in other mammals, albeit not as constrained as the CCAAT box (Fig. 3.5.B). Mutations in almost every position in the proximal CACC box have been associated with ␤ thalassemia (Fig. 3.5.B). Thus many lines of evidence point to the importance of this motif. Other KLFs may bind to the CACC boxes in other globin gene promoters, such as FKLF or KLF1366 for the HBG1 and HBG2 promoters. The other motif occurring frequently in upstream regulatory regions is WGATAR, the binding site for GATA-1 and related proteins (Fig. 3.4.B). GATA-1 plays a critical role in erythroid-specific gene activation and repression,67–69 and the binding sites in these upstream regions have been implicated in positive regulation of the respective genes.70,71 The GATA-1 binding sites upstream of HBE1, HBG1, HBG2, and HBZ2 are conserved in most mammals, but the ones upstream of HBB are not. GATA-1 binds to the promoter regions of ␤-globin genes in both human63 and mouse,72 but the binding site motif occurs in different places in the two promoters.73 This is an example of alterations in the binding site being associated with changes in the pattern of regulation, such as the delay in onset of expression in humans. A different set of binding sites is distinctive to each type of gene. For instance, ␤DRF60 and BB1-binding protein72,74 have been implicated in the regulation of the ␤-globin gene but not other globin genes (Fig. 3.4.B). Both binding sites are conserved in many placental mammals (Fig. 3.5.B).73 Likewise, binding of OCT1 and ␥ PE has been shown for the upstream regions of ␥ -globin genes but not others.75 The cis-elements close to the ␥ -globin genes are key determinants of fetal compared with embryonic expression. One of the clearest demonstrations of this is from transgenic mouse experiments in which a construct containing an LCR is used to enhance expression of globin genes. The ␥ -globin gene of prosimians, that is, the bushbaby galago, is expressed embryonically, and when it is included in the test construct in transgenic mice, the transgene is also expressed embryonically. In contrast, a human ␥ -globin gene, normally expressed during fetal life in humans, is expressed fetally when transferred into transgenic mice in an otherwise identical construct.76 Thus one would expect to find alterations in the regulatory regions of anthropoid (monkey, ape, and human) ␥ -globin genes that are associated with this change in stage specificity (i.e., sequences that are conserved in anthropoid primates but are different in prosimians and nonprimate mammals). Examination of aligned sequences for differential phylogenetic footprints49 led to the identification of a stage selector element in the human ␥ -globin gene promoter (Fig. 3.4.B). The stage selector element is a binding site for a factor called the stage selector protein, which has been implicated in the differential expression of ␥ - and ␤-globin genes.77 Additional DNA sequences that bind several proteins have been implicated in fetal silencing of the ␥ -globin gene.49

55

Parallel protein-binding and mutagenesis studies led to the discovery of a novel protein that binds to an element called the ␥ PE, in the upstream regulatory region of the ␥ -globin genes, which has also been implicated in regulation of this gene.75 The most distinctive globin gene promoters are those of the ␣-globin genes (HBA1 and HBA2). These promoters are CpG islands, and among the hemoglobin genes, only those encoding ␣-globin have this feature. (The ␪-globin and ␮globin genes also have promoters in CpG islands, but as discussed previously, it is not clear that they encode components of hemoglobin.) Although the majority of mammalian promoters are CpG islands,61 most of the associated genes are expressed in multiple tissues and few if any are expressed at such a high level as the ␣-globin gene. Thus the presence of a CpG island in the promoter for a globin gene is curious, and it leads to several unanswered questions about the ␣-globin gene promoters. What prevents their expression in nonerythroid tissues? What sequences in addition to the CpG island lead to very high-level expression in erythroid cells? No GATA-1-binding site is found in the ␣-globin gene promoters of most placental mammals (the mouse ␣-globin genes is a notable exception), so sequence-directed binding of this protein to the proximal sequences is not the answer. Several studies have shown that the CpG island is a key component of the cis-regulatory elements for the ␣-globin gene of humans and rabbits, possibly through its effects on chromatin structure.78,79 The differences in the arrays of proteins functioning at ε-, ␥ -, ␤-, and ␣-globin genes indicate that a distinct battery of proteins functions in the promoter for each type of gene. Indeed, this is consistent with the observation that cis-acting sequences needed for stage-specific regulation of expression map close to the genes.80

Proximal Enhancers Enhancers are DNA sequences that increase the activity of promoters; they can be located on either side of a gene or internal to it, and they can act at considerable distances from genes.81 Two enhancers have been found close to genes in the ␤-globin gene cluster, one that is 3
to HBB and one that is 3
to HBG1 (Fig. 3.3.A). In both cases the enhancers are less than 1 kb downstream of the polyA additional signal for the respective genes. The HBB enhancer was discovered by its effect on developmental timing of expression of globin transgenes when introduced into mice. High-level expression of human ␥ - or ␤-globin transgene constructs in fetal erythroid cells (the normal onset of expression of mouse ␤-globin genes) is dependent on the presence of the enhancer.74,82–84 The HBG1 enhancer was discovered as the only DNA segment in a 22-kb region surrounding the ␥ -globin genes that boosted expression of a reporter gene driven by a ␥ -globin gene promoter in transfected erythroid cells.85 Deletion of this enhancer from a large construct containing the human LCR and ␤-like

56

Bernard G. Forget and Ross C. Hardison A. 3′ enhancer for HBG1 Human Mar. 2006 chr11:5,225,146-5,225,240 (95 bp) 5225150

5225160

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5225240

CTTCTGATA AGGAAAATA ATTTTATGATGGGGATCTGCTCTTATGAGCTCATCTAAACCTAAT TACTTTTCAAAAGCCTCCCCCACAGATA AGG Vertebrate Multiz Alignment & Conservation (17 Species) Conservation human g c t t c t g a t aag g aaaa t a a t t t t a t ga t gg g ga t c t g c t c t t a t gag c t c a t c t a a a c c t aa t t a c t t t t c aaa a g c c t c c c c c a c a g a t aag g chimp g c t t c t g a t aag g aaaa t a a t t t t a t ga t gg g ga t c t g c t c t t a t gag c t c a t c t a a a c c t aa t t a c t t t t c aaa a g c c t c c c c c a c a g a t aag g rhesus g c t t c t a a t aag g aaa t a a a t t t t a t ga t gg g ga t a t g c t c t t a t gag c t c a t c t a a a c c t ag t t a c t t t t c aaa a g t c t c c c c t a c a a a t aag g mouse rat dog cow armadillo elephant opossum Repeating Elements by RepeatMasker SINE LINE LTR DNA Simple Low Complexity Satellite RNA Other Unknown

GATA GATA-1

GATA GATA-1

B. Distal enhancer for HBA Human Mar. 2006 chr16:103,591-103,670 (80 bp) 103590

103600

103610

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ACTTGAGGGAGCAGATA ACTGGGCCAACCATGACTCAGTGCTTCTGGAGGCCAACAGGACTGCTGAGTCAT CCTGTGGGG Compilation of Human Disease Variants and Other Mutations Ti~ Vertebrate Multiz Alignment & Conservation (17 Species) Conservation human chimp rhesus mouse rat dog cow armadillo opossum

ACTTGAGGGAGCAGATA ACTGGGCCAACCATGACTCAGTGCTTCTGGAGGCCAACAGGACTGCTGAGTCAT CCTGTGGGG ACCTGAGGGAGCAGATA ACTGGGCCAACCATGACTCAGTGCTTCTGGAGGCCAACAGGACTGCTGAGTCAT CCTGTGGGG ATCTGAGGGAGCAGATA ACTGGGCCAACCATGACTCAGTGCTTCTGGAGGCCAACAGGACTGCTGAATCAT CCTGTGGGG GCTTGAACGAGCAGATA ACTAAGCCAAGCATGACTCAGAGTTTCTAGAGGCCACTAGGACTGCTGAGTAAT ACT - TGGGG GCTTGAATGAGCAGATA ACTGAGCCGAACATGACTCAGAGTTTCTAGA -GCCACCAGGACTGCTGAGTAACACT - TGGGG ACTCCACACAGCAGATA ACTG-GCCAACCATGACTCAGCATTGCAGGAGGCCAACAGGGCTGCTGAGTCACCCA - - - -GG GCCCC - CAGAGCTGATA ACC - - ACCTGCCGTGACTCAGCACCCCAGGA -GCCGACAGGGAGGCTGAGTCAT CCC - - - -GG - CTCCAGGGAGCAGATA AGGGGGCCAG- CGTGACTCAGCGTGGCCGGGGGCCAGCAGG- CTGCTGAGTCAGTCC - TGGGG GCTTAAAGGACCAGATA AACCAGGAAACCATGACTTAGGCTTTCTGAAGGCCAAGAGTACTGCTGAGTCATGGC -GGGGA

GATA GATA-1

MARE NF-E2

MARE NF-E2

Figure 3.6. Wide range of conservation in globin gene enhancers. (A) Proximal enhancer for HBG1, showing the sequence of part of the 3
enhancer, alignments with sequences of other anthropoid primates, the encompassing repetitive element, and binding motifs. (B) Distal enhancer for the ␣-globin gene cluster, HS-40. The panel shows an 80-bp segment of the enhancer, along with the Ti∼␣ thalassemia deletion that removes this DNA and more, the conservation track and alignments with several eutherian mammals and the marsupial opossum. Binding sites are boxed and labeled by name and proteins binding to them.

globin genes had no effect on expression levels in transgenic mice,86 which could mean that it actually has no function, or that other sequences compensate for its loss, or that its function is not apparent in mice. Indeed, comparative sequence analysis of these proximal enhancers strongly supports the conclusion that both play roles in higher primates but not in other species. As illustrated in Figure 3.4.C, both enhancers contain binding sites for GATA-1,87,88 and the HBG1 enhancer also binds to the ␥ PE protein.75 The DNA homologous to the HBB enhancer in other mammals is not strongly conserved, even in the GATA motifs. Furthermore, two of the GATA1–binding sites in the HBG1 enhancer were introduced via an LTR-type transposable element that is present only in higher primates (Fig. 3.6.A). Thus the presence of the HBG1 proximal enhancer correlates with the fetal recruitment of ␥ -globin gene expression in anthropoids, and its function

may not be observed in transgenic mice. Likewise, the presence of GATA-1–binding sites only in higher primates suggests that the function of the HBB proximal enhancer may also be lineage-specific, perhaps related to the delay in expression of HBB in higher primates. In this case, an effect on developmental timing is readily demonstrable in transgenic mice, but because of the differences in timing of HBB expression in humans (the source of the transgene) and mouse (the host species), it is difficult to understand fully this function.

Distal Enhancers In addition to the proximal promoters and enhancers, both the ␣-like and ␤-like globin gene clusters are regulated by distal control regions. The ␤-like globin cluster is regulated by the distal LCR (reviewed in refs. 89, 90), and the ␣-like

The Normal Structure and Regulation of Human Globin Gene Clusters globin gene cluster is regulated by HS-40.91 In both cases, deletion of the distal control region is associated with thalassemia (Fig. 3.3). Addition of the distal control regions has profound effects on expression of linked genes in transgenic mice. Without the LCR, erythroid expression of a ␤-globin transgene is not seen in all mouse lines,92 presumably because of integration in a repressive region of a chromosome (a position effect). With the LCR, the ␤-globin transgene is expressed at a high level in erythroid cells in almost all mouse lines, indicating strong enhancement and a reduction in position effects.93 HS-40 of the ␣-globin gene complex is a strong enhancer of globin gene expression, both in transgenic mice91,94 and in transfected cells.95 The ␤-globin LCR is a very large regulatory region, containing at least five DNase hypersensitive sites in humans spread over approximately 17 kb96–98 between HBE1 and an OR gene (Fig. 3.3.A). This region is highly conserved in mammals, with highly similar sequences indicative of constraint found both in the hypersensitive sites and between them.50,90 This can be seen in Figure 3.3.A as the string of peaks of conservation and RP in this region. The distal enhancer for the ␣-globin gene, HS-40, is much smaller than the LCR. It is approximately 250 bp in length,99 located in a widely expressed gene called C16orf35 (Fig. 3.3.B). Additional erythroid DNase hypersensitive sites are present in this large gene, but none has been shown to play a role in regulation of globin genes.26 HS-40 is sufficient for strong enhancement and high activity in erythroid cells of transgenic mice, especially during embryonic and fetal development.91 It is very strongly conserved in mammals, with obvious matches to species as distant as opossum (Figs. 3.3.B and 3.6.B). Functional tests have shown that the homologous regions of chicken and fish also have enhancer activity, despite considerable divergence outside the protein-binding sites.44 Regulatory activities in addition to tissue-specific enhancement have been attributed to the ␤-globin LCR, but they are not seen consistently in multiple lines of investigation.100 Examination of chromatin structure after deletion of the LCR led to the inference that the LCR is needed for tissue-specific chromosomal domain opening.101 Chromosome 11 from a patient with the Hispanic (ε␥ ␦␤)0 thalassemia (missing most of the LCR and some adjacent sequences, but leaving all of the ␤-like globin genes intact) (Fig. 3.3A) was transferred through multiple somatic cells to generate a hybrid murine erythroleukemia cell line containing the mutant human chromosome. The ␤-globin gene cluster in this hybrid cell line is inactive and is insensitive to DNase, indicating that the LCR is needed for opening a chromosomal domain.101 An engineered mouse line carrying a deletion of the mouse ␤-globin LCR and the sequences homologous to those lost in the Hispanic deletion retains an open chromatin conformation (accessible to DNase) in the mouse ␤-globin gene.102 Although expression of the mouse ␤-globin genes is reduced substantially, the locus is not silenced. Thus the repressive heterochromatin seen in the hybrid murine erythroleukemia cells

57

carrying human chromosome 11 with the Hispanic deletion may have been produced during the chromosome transfers between cell lines. Currently, the DNA sequence determinants of chromatin opening have still not been discovered. The ␤-globin LCR has also been implicated in overcoming position effects in transgenic mice,103 in keeping with the inferred effect on opening a chromatin domain. Transgene constructs containing the ␤-globin can still show position effect variegation.104 Both the ␤-globin LCR and the ␣-globin HS-40 are very strong, erythroidspecific enhancers needed for the expression of any of the linked globin genes. They also can overcome some but not all repressive effects after integration at a variety of chromosomal locations. This could be a consequence of the strong enhancement. Three transcription factor–binding motifs are present in almost all DNase hypersensitive sites that have a strong function in the distal enhancers (Fig. 3.4.D). All contain Maf-response elements (MAREs) to which transcriptional activator proteins of the basic leucine zipper class can bind.105 A subfamily of proteins related to AP1, such as NFE2, LCRF1/Nrf1, and Bach1, bind to this element (reviewed in refs. 106, 107). All are heterodimers containing a Maf protein as one subunit, which is the basis for the name of the response element. All the hypersensitive sites have GATA motifs, to which GATA-1 and its relatives bind.108 The third common motif is CACC, to which a family of ¨ Zinc-finger proteins including erythroid Kruppel-like factor can bind.64 At HS3 in the ␤-globin LCR, there is evidence that motifs related to CACC are bound by additional KLFs, such as Sp1.109 HS2 of the ␤-globin LCR also has three Eboxes, which are the binding sites for TAL-1 and its heterodimeric partners.47 This protein has been implicated in regulation of hematopoiesis, and it appears to also play a role in enhancement by HS2. Initial studies of protein binding at these and other CRMs used various in vitro methods and in vivo footprinting.99,110–112 Recent experiments using chromatin immunoprecipitation have demonstrated occupancy of the CRMs by several of these proteins in erythroid cells.113–116 Many of the sites have been implicated directly in activity by mutagenesis and gene transfer.47,117–119 The protein binding sites in the distal positive regulators show some common patterns (Fig. 3.4.D). A MARE plus two GATA motifs is present in most of the CRMs, and this arrangement has been shown to be needed for formation of a hypersensitive site at HS4.120 The strongest enhancers (as assayed by gene transfer in somatic cells) are HS2 and HS40. Both of these have two MAREs, and mutation of those MAREs removes much of the enhancing activity.117,119 Thus the MAREs and proteins binding to them are critical for high-level enhancement, but the other binding sites contribute to function as well. The CRMs marked by these hypersensitive sites in the distal positive regulators are conserved across almost all mammals.26,90 The portion of the alignments for HS-40 shown in Figure 3.6.B indicates the very strong constraint

58 seen in the known binding sites and additional short segments both for this enhancer and for HS2. Most of the binding sites in HS3 are also highly conserved, but some are not, likely reflecting both common and lineage-specific functions. HS4, with the MARE and two GATA motifs, is conserved across a wide span of placental mammals, but this DNA sequence is part of an LTR-type repeat, a member of the ERV1 repeat family. This appears to be an old transposable element (predating most of the mammalian radiation), but one that continues to provide a regulatory function.

CONCLUDING REMARKS Molecular clones containing mammalian globin gene clusters were isolated approximately 30 years ago. Intense study since then has revealed much about their structure, evolution, and regulation; however, understanding sufficient to lead to clinical applications continues to elude us. The myriad levels of regulation and function that operate within these gene clusters certainly confound attempts to find simplifying conclusions. Despite these challenges, studies of the globin gene clusters have consistently provided new insights into function, regulation, and evolution. The lessons being learned as we try to integrate information from classic molecular biology and genetics, new highthrough-put biochemical assays, and extensive interspecies sequence comparisons are paving the way for applying these approaches genome wide. The globin gene clusters illustrate the need to distinguish common from lineagespecific regulation. Although simple generalizations are rare, the extensive information that one needs for interpreting data in the context of comparative genomics is readily accessible. Throughout this chapter, we have illustrated points using output from the UCSC Genome Browser (http: //genome.ucsc.edu), with special emphasis on the tracks showing Conservation, Regulatory Potential, and Locus Variants. Deeper information on the variants associated with disorders of the hemoglobins can be obtained from HbVar (http://www.bx.psu.edu). We hope that the examples presented here will be helpful in guiding interpretation of the multitude of data available to the readers now and in the future.

ACKNOWLEDGMENTS RH was supported by NIH grant R01 DK065806 and BGF was supported by NIH grants R01 DK19482 and P01 HL63357.

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to the breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer activity and can modify the developmental expression of the human fetal A gamma-globin gene in transgenic mice. J Biol Chem. 1995;270:10256–10263. Flint J, Thomas K, Micklem G, et al. The relationship between chromosome structure and function at a human telomeric region. Nat Genet. 1997;15:252–257. Anguita E, Johnson CA, Wood WG, Turner BM, Higgs DR. Identification of a conserved erythroid specific domain of histone acetylation across the alpha-globin gene cluster. Proc Natl Acad Sci USA. 2001;98:12114–12119. Flint J, Tufarelli C, Peden J, et al. Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster. Hum Mol Genet. 2001;10:371–382. Gumucio DL, Heilstedt-Williamson H, Gray TA, et al. Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human ␥ and ε globin genes. Mol Cell Biol. 1992;12:4919–4929. Gumucio D, Shelton D, Zhu W, et al. Evolutionary strategies for the elucidation of cis and trans factors that regulate the developmental switching programs of the beta-like globin genes. Mol Phylog Evol. 1996;5:18–32. Elnitski L. Conserved E boxes in the locus control region contribute to enhanced expression of beta-globin genes via TAL1 and other basic helix-loop-helix proteins. The Pennsylvania State University; 1998. Tagle DA, Koop BF, Goodman M, Slightom J, Hess DL, Jones RT. Embryonic ε and ␥ globin genes of a prosimian primate (Galago crassicaudatus): Nucleotide and amino acid sequences, developmental regulation and phylogenetic footprints. J Mol Biol. 1988;203:7469–7480. Gumucio DL, Shelton DA, Blanchard-McQuate K, et al. Differential phylogenetic footprinting as a means to identify base changes responsible for recruitment of the anthropoid ␥ gene to a fetal expression pattern. J Biol Chem. 1994;269:15371–15380. Hardison R, Miller W. Use of long sequence alignments to study the evolution and regulation of mammalian globin gene clusters. Mol Biol Evol. 1993;10:73–102. Margot JB, Demers GW, Hardison RC. Complete nucleotide sequence of the rabbit beta-like globin gene cluster: Analysis of intergenic sequences and comparison with the human beta-like globin gene cluster. J Mol Biol. 1989;205:15–40. Shehee R, Loeb DD, Adey NB, et al. Nucleotide sequence of the BALB/c mouse ␤-globin complex. J Mol Biol. 1989; 205:41–62. Hardison R, Krane D, Vandenbergh D, et al. Sequence and comparative analysis of the rabbit alpha-like globin gene cluster reveals a rapid mode of evolution in a G+C-rich region of mammalian genomes. J Mol Biol. 1991;222:233–249. Hardison RC. The nucleotide sequence of the rabbit embryonic globin gene ␤4. J Biol Chem. 1983;258:8739–8744.

60 55. Cooper GM, Brudno M, Stone EA, Dubchak I, Batzoglou S, Sidow A. Characterization of evolutionary rates and constraints in three Mammalian genomes. Genome Res. 2004;14:539–548. 56. Siepel A, Bejerano G, Pedersen JS, et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 2005;15:1034–1050. 57. Taylor J, Tyekucheva S, King DC, Hardison RC, Miller W, Chiaromonte F. ESPERR: Learning strong and weak signals in genomic sequence alignments to identify functional elements. Genome Res. 2006;16:1596–1604. 58. Maston GA, Evans SK, Green MR. Transcriptional regulatory elements in the human genome. Annu Rev Genomics Hum Genet. 2006;7:29–59. 59. Efstratiadis A, Posakony JW, Maniatis T, et al. The structure and evolution of the human ␤-globin gene family. Cell. 1980;21:653–668. 60. Stuve LL, Myers RM. A directly repeated sequence in the ␤-globin promoter regulates transcription in murine erythroleukemia cells. Mol Cell Biol. 1990;10:972–981. 61. Carninci P, Sandelin A, Lenhard B, et al. Genome-wide analysis of mammalian promoter architecture and evolution. Nat Genet. 2006;38:626–635. 62. Cohen RB, Sheffery M, Kim CG. Partial purification of a nuclear protein that binds to the CCAAT box of the mouse ␣1-globin gene. Mol Cell Biol. 1986;6:821–832. 63. deBoer E, Antoniou M, Mignotte V, Wall L, Grosveld F. The human ␤-globin promoter; nuclear protein factors and erythroid specific induction of transcription. EMBO J. 1988;7:4203–4212. 64. Miller IJ, Bieker JJ. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear factors. Mol Cell Biol. 1993;13:2776–2786. 65. Perkins AC, Sharpe AH, Orkin SH. Lethal ␤-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature. 1995;375:318–322. 66. Asano H, Li XS, Stamatoyannopoulos G. FKLF, a novel Kruppel-like factor that activates human embryonic and fetal beta-like globin genes. Mol Cell Biol. 1999;19:3571–3579. 67. Pevny L, Simon MC, Robertson E, et al. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991;349:257– 60. 68. Simon MC, Pevny L, Wiles MV, Keller G, Costantini F, Orkin SH. Rescue of erythroid development in gene targeted GATA1-mouse embryonic stem cells. Nat Genet. 1992;1:92–98. 69. Welch JJ, Watts JA, Vakoc CR, et al. Global regulation of erythroid gene expression by transcription factor GATA-1. Blood. 2004;104:3136–3147. 70. Martin D, Orkin S. Transcriptional activation and DNA binding by the erythroid factor GF-1/NF-E1/Eryf 1. Genes Dev. 1990;4:1886–1898. 71. Gong Q-H, Dean A. Enhancer-dependent transcripion of the ε-globin promoter requires promoter-bound GATA-1 and enhancer-bound AP-1/NF-E2. Mol Cell Biol. 1993;13:911– 917. 72. Macleod K, Plumb M. Derepression of mouse ␤-major-globin gene transcription during erythroid differentiation. Mol Cell Biol. 1991;11:4324–4332. 73. Hardison R, Chao K-M, Schwartz S, Stojanovic N, Ganetsky M, Miller W. Globin gene server: A prototype E-mail data-

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base server featuring extensive multiple alignments and data compilation. Genomics. 1994;21:344–353. Antoniou M, deBoer E, Habets G, Grosveld F. The human ␤– globin gene contains multiple regulatory regions: Identification of one promoter and two downstream enhancers. EMBO J. 1988;7:377–384. Lloyd JA, Case SS, Ponce E, Lingrel JB. Positive transcriptional regulation of the human ␥ -globin gene: ␥ PE is a novel nuclear factor with multiple binding sites near the gene. J Biol Chem. 1994;269:26–34. TomHon C, Zhu W, Millinoff D, et al. Evolution of a fetal expression pattern via cis-changes near the ␥ -globin gene. J Biol Chem. 1997;272:14062–14066. Jane SM, Ney PA, Vanin EF, Gumucio DL, Nienhuis AW. Identification of a stage selector element in the human ␥ -globin gene promoter that fosters preferential interaction with the 5
HS2 enhancer when in competition with the ␤-promoter. EMBO J. 1992;11:2961–2969. Pondel M, Murphy S, Pearson L, Craddock C, Proudfoot N. Sp1 functions in a chromatin-dependent manner to augment human alpha-globin promoter activity. Proc Natl Acad Sci USA. 1995;92:7237–7241. Shewchuk BM, Hardison RC. CpG islands from the ␣-globin gene cluster increase gene expression in an integrationdependent manner. Mol Cell Biol. 1997;17:5856–5866. Trudel M, Magram J, Bruckner L, Costantini F. Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice. Mol Cell Biol. 1987;7:4024–4029. Tjian R, Maniatis T. Transcriptional activation: A complex puzzle with few easy pieces. Cell. 1994;77:5–8. Trudel M, Costantini F. A 3
enhancer contributes to the stage-specific expression of the human ␤-globin gene. Genes Dev. 1987;1:954–961. Behringer RR, Hammer RE, Brinster RL, Palmiter RD, Townes TM. Two 3
sequences direct adult erythroid-specific expression of human beta-globin genes in transgenic mice. Proc Natl Acad Sci USA. 1987;84:7056–7060. Liu Q, Bungert J, Engel JD. Mutation of gene-proximal regulatory elements disrupts human epsilon-, gamma-, and betaglobin expression in yeast artificial chromosome transgenic mice. Proc Natl Acad Sci USA. 1997;94:169–174. Bodine D, Ley T. An enhancer element lies 3
to the human A gamma globin gene. EMBO J. 1987;6:2997–3004. Liu Q, Tanimoto K, Bungert J, Engel JD. The A gammaglobin 3
element provides no unique function(s) for human beta-globin locus gene regulation. Proc Natl Acad Sci USA. 1998;95:9944–9949. Wall L, deBoer E, Grosveld F. The human ␤-globin gene 3
enhancer contains multiple binding sites for an erythroidspecific protein. Genes Dev. 1988;2:1089–1100. Puruker M, Bodine D, Lin H, McDonagh K, Nienhuis AW. Structure and function of the enhancer 3
to the human A␥ globin gene. Nucl Acids Res. 1990;18:7407–7415. Grosveld F, Antoniou M, Berry M, et al. The regulation of human globin gene switching. Philos Trans R Soc Lond. 1993;339:183–191. Hardison R, Slightom JL, Gumucio DL, Goodman M, Stojanovic N, Miller W. Locus control regions of mammalian ␤-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights. Gene. 1997;205:73–94.

The Normal Structure and Regulation of Human Globin Gene Clusters 91. Higgs D, Wood W, Jarman A, et al. A major positive regulatory region located far upstream of the human ␣-globin gene locus. Genes Dev. 1990;4:1588–1601. 92. Chada K, Magram J, Costantini F. Tissue- and stage-specific expression of a cloned adult beta globin gene in transgenic mice. Prog Clin Biol Res. 1985;191:305–319. 93. Grosveld F, van Assendelft GB, Greaves D, Kollias G. Positionindependent, high-level expression of the human ␤-globin gene in transgenic mice. Cell. 1987;51:975–985. 94. Sharpe JA, Chan-Thomas PS, Lida J, Ayyub H, Wood WG, Higgs DR. Analysis of the human ␣-globin upstream regulatory element (HS-40) in transgenic mice. EMBO J. 1992;11:4565–4572. 95. Ren S, Luo X-n, Atweh G. The major regulatory element upstream of the ␣-globin gene has classical and inducible enhancer activity. Blood. 1993;81:1058–1066. 96. Tuan D, Abelovich A, Lee-Oldham M, Lee D. Identification of regulatory elements of human b-like globin genes. In: Stamatoyannopoulos G, Nienhuis AW, eds. Developmental Control of Globin Gene Expression. New York: A.R. Liss; 1987:211– 220. 97. Forrester W, Takegawa S, Papayannopoulou T, Stamatoyannopoulos G, Groudine M. Evidence for a locus activating region: The formation of developmentally stable hypersensitive sites in globin-expressing hybrids. Nucl Acids Res. 1987;15:10159–10177. 98. Dhar V, Nandi A, Schildkraut CL, Skoultchi AI. Erythroidspecific nuclease-hypersensitive sites flanking the human bglobin gene cluster. Mol Cell Biol. 1990;10:4324–4333. 99. Jarman A, Wood W, Sharpe J, Gourdon G, Ayyub H, Higgs D. Characterization of the major regulatory element upstream of the human ␣-globin gene cluster. Mol Cell Biol. 1991;11:4679–4689. 100. Higgs DR. Do LCRs open chromatin domains? Cell. 1998;95: 299–302. 101. Forrester WC, Epner E, Driscoll MC, et al. A deletion of the human b-globin locus activation region causes a major alteration in chromatin structure and replication across the entire b-globin locus. Genes Dev. 1990;4:1637–1649. 102. Bender MA, Byron R, Ragoczy T, Telling A, Bulger M, Groudine M. Flanking HS-62.5 and 3
HS1, and regions upstream of the LCR, are not required for beta-globin transcription. Blood. 2006;108:1395–1401. 103. Fraser P, Hurst J, Collis P, Grosveld F. DNase I hypersensitive sites 1, 2 and 3 of the human b-globin dominant control region direct position-independent expression. Nucl Acids Res. 1990;18:3503–3508. 104. Alami R, Greally JM, Tanimoto K, et al. beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice. Hum Mol Genet. 2000;9:631–636. 105. Motohashi H, Shavit JA, Igarashi K, Yamamoto M, Engel JD. The world according to Maf. Nucl. Acids Res. 1997;25:2953– 2959. 106. Orkin S. Regulation of globin gene expression in erythroid cells. Eur J Biochem. 1995;231:271–281. 107. Baron MH. Transcriptional control of globin gene switching during vertebrate development. Biochim Biophys Acta. 1997;1351:51–72.

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108. Evans T, Felsenfeld G, Reitman M. Control of globin gene transcription. Annu Rev Cell Biol. 1990;6:95–124. 109. Shelton DA, Stegman L, Hardison R, et al. Phylogenetic footprinting of hypersensitive site 3 of the ␤-globin locus control region. Blood. 1997;89:3457–3469. 110. Talbot D, Philipsen S, Fraser P, Grosveld F. Detailed analysis of the site 3 region of the human ␤-globin dominant control region. EMBO J. 1990;9:2169–2178. 111. Strauss EC, Andrews NC, Higgs DR, Orkin SH. In vivo footprinting of the human ␣-globin locus upstream regulatory element by guanine and adenine ligation-mediated polymerase chain reaction. Mol Cell Biol. 1992;12:2135–2142. 112. Reddy PMS, Stamatoyannopoulos G, Papayannopoulou T, Shen C-KJ. Genomic footprinting and sequencing of human ␤-globin locus: Tissue specificity and cell line artifact. J Biol Chem. 1994;269:8287–8295. 113. Forsberg EC, Downs KM, Bresnick EH. Direct interaction of NF-E2 with hypersensitive site 2 of the beta-globin locus control region in living cells. Blood. 2000;96:334–339. 114. Sawado T, Igarashi K, Groudine M. Activation of beta-major globin gene transcription is associated with recruitment of NF-E2 to the beta-globin LCR and gene promoter. Proc Natl Acad Sci USA. 2001;98:10226–10231. 115. Letting DL, Rakowski C, Weiss MJ, Blobel GA. Formation of a tissue-specific histone acetylation pattern by the hematopoietic transcription factor GATA-1. Mol Cell Biol. 2003;23:1334– 1340. 116. Anguita E, Hughes J, Heyworth C, Blobel GA, Wood WG, Higgs DR. Globin gene activation during haemopoiesis is driven by protein complexes nucleated by GATA-1 and GATA-2. EMBO J. 2004;23:2841–2852. 117. Ney P, Sorrentino B, McDonagh K, Nienhuis A. Tandem AP1-binding sites within the human ␤-globin dominant control region function as an inducible enhancer in erythroid cells. Genes Dev. 1990;4:993–1006. 118. Caterina JJ, Ciavatta DJ, Donze D, Behringer RR, Townes TM. Multiple elements in human ␤-globin locus control region 5
HS2 are involved in enhancer activity and position-independent transgene expression. Nucl Acids Res. 1994;22:1006–1011. 119. Gong Q, McDowell JC, Dean A. Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the ε-globin gene in vivo by 5
hypersensitive site 2 of the ␤-globin locus control region. Mol Cell Biol. 1996; 16:6055–6064. 120. Stamatoyannopoulos JA, Goodwin A, Joyce T, Lowrey CH. NFE2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human ␤-globin locus control region. EMBO J. 1995;14:106–116. 121. Kent WJ, Sugnet CW, Furey TS, et al. The human genome browser at UCSC. Genome Res. 2002;12:996–1006. 122. Hsu F, Kent WJ, Clawson H, Kuhn RM, Diekhans M, Haussler D. The UCSC known genes. Bioinformatics. 2006;22:1036– 1046. 123. Montgomery SB, Griffith OL, Sleumer MC, et al. ORegAnno: an open access database and curation system for literaturederived promoters, transcription factor binding sites and regulatory variation. Bioinformatics. 2006;22:637–640.

4 Nuclear Factors That Regulate Erythropoiesis Gerd A. Blobel and Mitchell J. Weiss

more distant regulatory elements (Chapters 3 and 5). For example, the ␤-globin locus control region (␤–LCR) encompasses approximately 20 kb of DNA situated upstream of the ␤-globin gene cluster. Originally identified as a set of erythroid-specific DNase hypersensitive sites (HS), the ␤-LCR is now known to be essential for high-level erythroid expression of ␤-globin genes.11–14 Detailed analysis of globin gene promoters and the ␤-LCR has revealed a number of conserved DNA motifs important for globin expression. Among these motifs, the best studied are the “GATA,” “CACCC,” and “TGA(C/G)TCA” (NF-E2/AP-1-like) elements (Fig. 4.1). Not surprisingly, identical motifs also function in the promoters and enhancers of many other erythroid genes such as heme biosynthetic enzymes, red cell membrane proteins, and ␣-globin. One or more transcription factors has been discovered to bind each of these cis elements in erythroid cells.

INTRODUCTION

GENERAL PRINCIPLES

Studies of erythroid transcription factors originate from efforts to identify and characterize the numerous tissuespecific and ubiquitous proteins that bind cis-regulatory motifs within the globin gene loci (Chapters 3 and 5). In addition to elucidating mechanisms of globin gene regulation and erythroid development, this approach has led to the discovery of nuclear proteins that function in a wide range of developmental processes. Experimental approaches and insights gained through studies of the globin loci have broad implications for understanding how transcription factors regulate the expression of individual genes and work together to coordinate cellular differentiation. Erythrocyte formation in the vertebrate embryo occurs in several distinct waves1,2 (see also Chapter 1). The first erythrocytes, termed primitive (EryP), arise in the extraembryonic yolk sac at mouse embryonic day 7.5 (E7.5) and weeks 3–4 in the human embryo. Later, erythropoiesis shifts to the fetal liver where adult-type (EryD, definitive) erythrocytes are produced. Finally, at birth, blood formation shifts to the bone marrow, and also the spleen in mice. EryPs and EryDs are distinguished by their unique cellular morphology, cytokine responsiveness, transcription factor requirements, and patterns of gene expression.3–10 Most notably, the expression of individual globin genes is developmentally regulated (Chapter 3). Understanding how transcription factors regulate the temporal control of ␤-like globin genes during mammalian development is of general interest to the study of gene regulation in higher eukaryotes and could eventually lead to new approaches to reactivate the human fetal ␥ -globin genes in patients with ␤ chain hemoglobinopathies, such as sickle cell anemia and ␤ thalassemia. The primary cis-acting determinants of individual globin gene expression reside in the promoter regions immediately upstream of each gene and act in concert with

General studies of transcription factors have conveyed several important concepts and experimental approaches applicable to studies of erythroid nuclear proteins.

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1) Transcription factors are modular proteins with distinct domains mediating DNA binding, transcriptional activation, repression, and protein interactions.15 However, a single domain may have more ¨ than one function. For example, GATA and Kruppel zinc fingers mediate both DNA binding and protein interactions. Typically, domains are analyzed by determining the effects of various mutations and “domain swaps” on the ability to activate or repress synthetic promoter–reporter constructs in transient transfection assays using heterologous cells, such as 3T3 or COS. Such studies are useful but fail to provide the native chromosomal and cellular contexts in which a lineage-specific factor normally operates. In this regard, the availability of more biologically relevant cellular and in vivo models complements the use of conventional promoter–reporter studies. 2) Transcription factors function within multiprotein complexes.16,17 Defining these complexes in erythroid cells is critical to understanding the mechanisms that underlie globin gene expression and erythroid differentiation. Several approaches, including yeast two-hybrid screening, classic biochemical purification, and affinity purification with molecular tags are commonly used to identify interacting proteins and delineate higher order transcription factor networks in erythroid cells. 3) Posttranslational alterations, such as phosphorylation, acetylation, ubiquitination, and sumoylation, can modulate transcription factor function. These chemical modifications establish additional levels of

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Figure 4.1. Cis-acting elements and corresponding transcription factor families important for erythroid gene expression.

control through which gene expression may be regulated rapidly in response to changes in the nuclear environment and/or extracellular signals. Examples relevant to erythroid biology are discussed later in this chapter. 4) Most transcription factors can both activate and repress gene expression. Among erythroid transcription factors, GATA-1, EKLF, and SCL/TAL-1 all have the capacity to activate and repress gene expression. These dual functions enhance the utility of transcription factors in several key stages of tissue development. For example, during terminal maturation, a single nuclear protein can simultaneously activate genes associated with the differentiated phenotype and silence those associated with the immature state. In addition, tissue-restricted transcription factors may participate in cell fate decisions of multipotent progenitors by activating genes for one lineage and silencing those of alternative lineages. 5) Cellular environment and target promoter context influence transcription factor activities by regulating the assembly of specific multiprotein complexes at individual genes. For example, the megakaryocytespecific ␣-IIb gene contains a promoter that is activated by GATA-1 alone, but inhibited by GATA1 bound to its cofactor FOG-1.18,19 Binding of megakaryocyte-expressed Ets transcription factor, such as Fli1, to an adjacent DNA element converts FOG-1 into a coactivator. This identifies a mechanism by which GATA-1 and FOG-1 regulate the same gene differently in separate lineages.

6) As discussed in more detail later, transcription factors exert their functions in part by modifying chromatin, either directly or by assembling multiprotein complexes to establish and maintain active or repressive chromatin states. Many erythroid transcription factors associate with histone acetyltransferases. Hyperacetylation is typically found at sites of open chromatin that surround active genes. More recent work shows that erythroid nuclear factors facilitate the formation of long-range chromatin loops that bring critical regulatory elements into physical proximity.20,21 How erythroid transcription factors regulate chromatin and DNA accessibility is an active area of research.

EXPERIMENTAL APPROACHES FOR STUDYING TRANSCRIPTION FACTORS Several recently developed technologies have revolutionized the study of transcription factor function. Some of the technologies that have accelerated our understanding of erythropoiesis and globin gene expression are reviewed here.

Biochemical Purification Transcription factors invariably interact chemically with cofactor complexes to modify histone proteins, to remodel nucleosomes, and to recruit basal transcription factors. Thus, one fruitful approach to define the functions of individual nuclear factors is to identify interacting

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proteins. In particular, yeast two-hybrid screens and in vitro purification of transcription factor complexes by using conventional biochemistry have elucidated gene regulation by defining higher order regulatory networks. Such studies are greatly facilitated by recent advances in mass spectrometry that permit identification of small amounts of proteins in complex mixtures.

Tissue Culture Models for Erythroid Differentiation Numerous tissue culture models recapitulate selected aspects of erythroid maturation of multiple species, including chicken, mouse, and human. These include murine erythroleukemia (MEL) cells, which represent definitive (adult-type) erythroid precursors and K562, a human erythroleukemia line that expresses embryonic and fetal globins. A variety of chemical agents can be used to induce erythroid maturation of K562 and MEL cells.23,24 Mouse G1E cells are arrested at the proerythroblast stage due to a lack of transcription factor GATA-1.25 Conditional expression of GATA-1 in these cells induces synchronous erythroid maturation. Numerous avian cell lines have also provided useful models for erythropoiesis.26,27 Established erythroid cell lines provide unlimited quantities of material for biochemical purification studies and frequently allow for synchronous differentiation on exposure to chemical compounds. Moreover, in established cell lines it is relatively simple to manipulate the expression of key transcription factors via overexpression or siRNA silencing and determine the effects on erythroid maturation and/or gene expression. Biological studies in cell lines are confounded by the effects of immortalization or outright transformation necessitating the use of primary cells for some studies. In mice and humans, primary erythroid progenitors can be purified from yolk sac, fetal liver, spleen, or bone marrow and expanded or differentiated in short-term cultures by using appropriate growth and differentiation factors.28–32 It is also possible to generate primary erythroid cultures from in vitro differentiation of embryonic stem cells (ESCs).33,34

Chromatin Immunoprecipitation Historically, interactions between DNA-binding proteins and their target sequences were demonstrated by electrophoretic mobility shift assay, in which binding of a transcription factor retards the migration of an oligonucleotide during polyacrylamide gel electrophoresis (see Fig. 4.6 for an example). This assay does not, however, assess transcription factor binding to chromatinized DNA in live cells. Chromatin immunoprecipitation (ChIP) is used to determine whether a nuclear factor is physically associated with a specific region of DNA sequence in vivo. The topic is described by Orlando and Paro35 and experimental protocols are outlined by Boyd et al.36 To perform ChIP, cells are treated with a crosslinking reagent that covalently attaches DNA to its associated proteins. Then, the cells are lysed and chromatin is purified and fragmented into defined

Figure 4.2. ChIP analysis. ChIP can identify histone and nonhistone proteins and protein modifications associated with genomic regions of interest. The first step of ChIP is to cross link proteins to DNA or other proteins. Following lysis of cells, extracts are sonicated to shear the DNA. Micrococcal nuclease can also be used to fragment the DNA. Proteins are immunoprecipitated with specific or control antibodies. Cross links are reversed; DNA is purified and amplified by PCR. For quantification, the amounts of PCR product are compared with those of unprecipitated PCR-amplified DNA (input). PCR reactions with primers against regions not bound by protein are used as additional controls.

sizes (typically 0.5–1 kb) by sonication or partial nuclease digestion. The resulting material is immunoprecipitated with antibodies against the nuclear protein of interest or control antibodies. Protein–DNA crosslinks are reversed and the protein-associated DNA is purified and analyzed by quantitative polymerase chain reaction (PCR) using specific primer pairs that flank putative transcription factor binding sites or control regions where binding is not expected to occur (Fig. 4.2). Global analysis of ChIP products can be performed by hybridizing them to DNA-based microarrays (see later) or by using high throughput DNA sequencing technologies.37,38 In addition to determining transcription factor binding to specific genes, ChIP can be used to examine whether histone proteins or transcription factors are acetylated, methylated, or phosphorylated at specific chromosomal positions. Currently, ChIP analysis is the gold standard to determine transcription factor binding and posttranslational protein modifications at gene loci in vivo and has been used extensively at the globin loci to examine erythroid development.

MICROARRAY ANALYSIS TO IDENTIFY TRANSCRIPTION FACTOR TARGET GENES In microarray or “gene chip” analysis, genomic DNA or cDNA derived from cells of interest is labeled with fluorescent probes and incubated with nucleic acids specifying

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A

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B

Complementary DNAs or representative oilgonucleotides immobilized on a microarray “chip”

hybridize wash quantify retained signals

promoter region

Coding region

Oligonucleotides representing contiguous segments of genomic DNA

fluorescently labeled cDNA pool from tissue source of interest

fluorescently labeled genomic DNA from Chip experiment hybridize wash quantify retained signals

Figure 4.3. Microarray analysis. (A) Messenger RNA profiling. Complementary DNAs from a tissue source of interest are prepared, labeled with a fluorescent tag, and incubated with a slide or “chip” containing immobilized oligonucleotides or DNA segments that hybridize to specific cDNAs. The retained fluorescent signal at a fixed position or “address” on the chip reflects the relative expression level of a specific mRNA transcript. (B) ChIP–chip analysis. DNA from ChIP (Fig. 4.2) is amplified, labeled with a fluorescent tag, and incubated with a “tiled” microarray chip containing oligonucleotides that hybridize to contiguous segments of genomic DNA. Microarray chips representing all known promoter regions or even entire genomes are available commercially. In addition, it is possible to prepare microarray chips that specifically interrogate smaller genomic regions of interest.

unique chromosomal loci or mRNA transcripts that are immobilized on a solid surface (Fig. 4.3). Hybridization of the labeled cellular nucleic acids to sequences at specific locations on the chip is quantified and studied by optical scanning and computational analysis. This technology has advanced to a point where many thousands of different sequences can be examined in a single experiment. Microarrays are used in two general approaches to identify transcription factor targets. First, transcriptome analysis can identify mRNAs that are up- or down-regulated in response to altered transcription factor activities (Fig. 4.3A). For example, it is possible to manipulate specific transcription factors in biologically relevant cells through gene targeting (discussed later in this chapter), viral transduction, dominant negative mutants, and by creating conditional alleles that are activated or silenced by drugs. Then, microarray studies can be used to compare mRNA expression patterns between identical cells in which the transcription factor function is specifically altered. Remarkably, commercially available microarray platforms can interrogate most or all expressed genes in many species including mice and humans. Using this approach, it is possible to define the actions of any transcription factor on global gene expression in biologically relevant contexts. Followup studies using ChIP can investigate whether effects on the expression of specific genes are direct or indirect consequences of transcription factor activities. Examples of mRNA profiling using microarrays or other methods to identify erythroid transcription factor targets are described.39–42

Another approach to identify transcription factor targets combines ChIP with microarrays (ChIP–Chip) (Fig. 4.3B). DNA derived from ChIP using a transcription factor specific antibody is labeled to generate probes for microarrays containing genomic DNA. For this purpose, microarrays containing promoter regions of most expressed genes are available. In addition, it is possible to represent large chromosomal regions of interest, or even the entire genome, in “tiling arrays” that contain contiguous segments of genomic DNA. In this fashion, it is possible to screen for regions of genomic DNA in which the transcription factor binds in vivo. Currently, these experiments, particularly those that survey the entire genome, are expensive and technologically challenging, but the field is advancing rapidly. One limitation of this assay is that occupancy of a genomic sequence by a transcription factor does not necessarily reflect function – a transcription factor bound to DNA in vivo can activate, repress, or have no effect. An example of ChIP–chip analysis using a tiled microarray representing the ␣-globin locus is described by De Gobbi et al.43

DEFINING PHYSICAL INTERACTIONS BETWEEN DISTANT DNA ELEMENTS Several models are invoked to explain how transcription factors enhance or inhibit gene expression over substantial genomic distances. Tracking models propose that transcription factors bound at distant regulatory sites recruit RNA polymerase and/or basal transcription factors, which then move along the chromatin fiber until a promoter

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homologous recombination in ESCs and mice has been instrumental in assessing transcription factor function by providing a means to inactivate (knockout) genes of interest or to modify them (knockin) and examine the biological consequences. Murine embryonic stem ESCs derived from the inner cell mass of blastocyst stage mouse embryos provide the basis for gene targeting.52–54 ESCs can be maintained in a pluripotent state in culture and contribute to somatic and germ line tissues when introduced into blastocysts by microinjection. The first step toward studying a gene of interest is to disrupt a single allele by homologous recombination in ESCs B to create a heterozygous, or “single knockout” state.55,56 Several complementary experimental approaches are then available for further study (Fig. 4.4). First, genetically altered ESCs may be injected into host blastocysts to produce chimeric mice, which may transmit the mutant allele to progeny. Through interbreeding of heterozygous offspring, homozygous null animals can be created for analysis. One limitation of this approach is that mutations Figure 4.4. (A) Experimental strategies for studying gene knockouts. (B) In vitro differentiation of causing early embryonic death can obscure ESCs to obtain pure hematopoietic colonies. Reprinted from Weiss and Orkin100 with permission the analysis of later developmental events. from Elsevier Science, Copyright 1995. For example, direct examination of definitive hematopoiesis is difficult to assess in embryos that die prior to development of the fetal liver. is reached. Looping models posit that distal elements Another potential problem in interpreting the phenoare brought in physical proximity with their dedicated types of knockout animals is failure to distinguish whether promoters through the formation of chromatin loops. observed defects are cell autonomous or an indirect conseAlthough the latter model clearly applies to the ␣ and ␤quence of lesions in other cell types (noncell-autonomous). globin gene loci,44–46 tracking intermediates that precede Both of these problems may be circumvented through loop formation remain a distinct possibility. Evidence to chimera analysis or in vitro ESC differentiation assays (see support this is found at the human ε-globin gene.47 The later). In addition, more recent technology now permits most commonly used method to detect physical interdevelopmental stage and tissue-specific gene targeting by actions among chromosomal fragments is called chroexpressing specific recombinases to excise or modify the mosome conformation capture ([3C] or nuclear ligation target gene in a controlled spatiotemporal fashion.57–60 assay).48,49 If performed with the appropriate controls, it can be used to demonstrate interactions among chromoSecond, heterozygous mutant ESCs can be converted to somal fragments located in cis and on different chromoa homozygous-null state.61–63 These mutant ESCs may be somes. 3C analysis has demonstrated that transcription facinjected into wild-type host blastocysts to create chimeric tors GATA-1 and EKLF both promote folding of the ␤-globin animals in which the ability of the mutant donor ESCs to locus to ensure physical proximity between the LCR and contribute to various tissues is assessed using polymorphic the active globin gene promoters.20,21,50 3C has also been markers. For loci that are X-linked, such as Gata1, a single targeting event renders male ESCs null for the gene of used in the context of transgenic mice carrying versions of interest. Failure of homozygous or hemizygous null ESCs to the human ␤-globin locus to delineate cis-acting sequences contribute to a specific cell type or tissue indicates a cellthat organize the ␤-globin locus.51 autonomous requirement for the disrupted gene in the formation of that tissue. ELUCIDATING GENE FUNCTION BY TARGETED Finally, the hematopoietic potential of genetically modiMUTAGENESIS fied ESCs may be examined by in vitro techniques (Fig. 4.4). Under appropriate conditions, ESCs form embryoid bodies, Transcription factor functions identified in vitro must spherical aggregates containing numerous differentiated be examined in the context of primary cells and whole cell types, including mature hematopoietic cells that can be organisms. The advent of targeted gene disruption using

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Nuclear Factors That Regulate Erythropoiesis

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studied directly.64 Embryoid bodies may be disaggregated into a single cell suspension and analyzed for hematopoietic progenitors by using standard methylcellulosebased colony assays.33 Wild-type and genetically manipulated ESCs can also be induced to form hematopoietic lineages by cocultivation on the stromal line OP9.65,66 More recently, in vitro differentiation techniques have been used to generate hematopoietic cells from human ESCs.67–72 Of note, human ESC-derived definitive erythroid cells produced by current methods express mainly embryonic and fetal globins, but not adult globins. In the future, this experimental system may provide a useful tool to study the mechanisms of globin gene switching.

SPECIFIC ERYTHROID TRANSCRIPTION FACTORS GATA-1 and Related Proteins The abundant erythroid nuclear protein GATA-1 was identified through its ability to bind the (T/A)GATA(A/G) consensus motif found in regulatory regions of virtually all erythroid-specific genes including ␣- and ␤-globins, heme biosynthetic enzymes, red cell membrane proteins, and transcription factors.73,74 GATA-1 recognizes DNA through two related, tandemly arranged zinc fingers of the configuration Cys-X2-Cys-X17-Cys-X2-Cys. The carboxyl (C) finger is necessary and sufficient for DNA binding, whereas the amino (N) finger stabilizes protein–DNA interactions at a subset of sites, in particular those that contain two GATA motifs arranged as direct or inverted repeats.75–78 In addition, both zinc fingers serve as docking sites for various protein interaction partners.79,80

GATA-1 is Required for Terminal Erythroid Maturation and Platelet Formation Gene targeting studies demonstrate that GATA-1 is essential for the production of mature erythrocytes. In chimeric mice, Gata1-donor ESCs contribute to all tissues examined except red blood cells; reintroduction of GATA-1 cDNA into the mutant ESCs restores their ability to contribute circulating red blood cells.81,82 Gata1 embryos die of anemia between E10.5 and E11.5 (Fig. 4.5A).83 Examination of these embryos, combined with in vitro differentiation of Gata1-ESCs revealed a block to erythroid maturation and apoptosis at the proerythroblast stage (Fig. 4.4B).84,85 Together, these experiments demonstrated an essential, cell-autonomous role for GATA-1 in the production of mature erythrocytes. Subsequently, additional studies showed that GATA-1 is also important for the formation and/or function of platelets,86–88 eosinophils,89 mast cells,90 and dendritic cells.91

The GATA Protein Family The discovery of GATA-1 led to the identification of several related proteins with highly conserved zinc finger

Figure 4.5. Loss of GATA-1 blocks erythroid maturation. (A) Impaired primitive erythropoiesis in Gata1-embryos. (B) Developmental arrest and apoptosis of cells within definitive erythroid (EryD) colonies generated by in vitro differentiation of Gata1-ESCs. Modified from Weiss et al.84 and Fujiwara et al.83 Copyright 1995 and 1996, National Academy of Sciences, U.S.A. Photographs in panel A provided by Yuko Fujiwara and Stuart Orkin. (See color plate 4.5.)

domains but little similarity outside of this region.92–98 (for reviewed see refs. 99–102). Six vertebrate GATA proteins, named in the order of their discovery, function in the development of various tissues. GATA-1 and GATA2 are most relevant for erythroid maturation and appear to act sequentially and coordinately during this process. Both are expressed in hematopoietic stem cells and multipotential progenitors, although GATA-2 function predominates at these early stages.103–105 Concurrent with erythroid differentiation, GATA-2 expression declines as that of GATA-1 increases. Most likely, GATA-2 initiates the erythroid program in early progenitors and subsequently becomes replaced by GATA-1 during terminal maturation.100,106 Presumably, these two transcription factors have both unique and overlapping functions at different stages of erythropoiesis. In this regard, GATA-2 probably activates its own gene by binding to an upstream enhancer.107 GATA-1 displaces GATA-2 at this position to repress GATA-2 transcription.107 These studies highlight molecular crosstalk between the GATA factors during erythropoiesis and illustrate one target gene (Gata2) where GATA-1 and GATA-2 have opposing functions.

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GATA-1 Represses Transcription

Posttranslational Modifications of GATA-1

GATA-1 was originally viewed as a positive regulator of erythroid gene expression. As noted earlier, GATA-1 also functions as a transcriptional repressor. For example, GATA-1 negatively regulates human ε-globin expression by interacting with one or more silencer elements within the 5
flanking region of the ε gene.108,109 Interestingly, GATA1 binding to a region upstream of the G␥ -globin gene is required for developmental silencing of fetal globin synthesis. Although there is no obvious GATA consensus site in this region, patients with a mutation that abrogates GATA-1 binding display hereditary persistence of fetal hemoglobin.110 Transcriptome analysis in erythroid cells indicates that the repertoire of GATA-1 repressed target genes may be quite extensive.39 Among these targets are Gata2, Kit, Myc, and Myb, which mark early progenitors.107,111–113 The latter three are all protooncogenes that stimulate proliferation and their repression likely reflects a mechanism through which GATA-1 coordinates division arrest with terminal maturation.

Murine GATA-1 is phosphorylated constitutively on six serine residues within the amino terminus. An additional serine, at position 310, which lies in a conserved region near the carboxyl boundary of the DNA binding domain, is phosphorylated upon chemically induced differentiation of MEL cells. Extensive mutagenesis experiments have shown that phosphorylation at these sites does not significantly influence DNA binding, DNA bending, or transcriptional activation by GATA-1.120 Phosphorylation of GATA-1 has been reported to influence DNA binding in human K562 cells.121 GATA-1 is also phosphorylated through erythropoietin-mediated activation of AKT in erythroid cells,122–124 although mutation of the target serine residues in mice has minimal effects on erythroid development.125 GATA-1 is acetylated in vivo at two highly conserved, lysine-rich motifs at the C-terminal tails of both fingers, adjacent to regions that contact DNA. Acetylation within these regions is mediated by interaction with the ubiquitous transcriptional cofactors CREB-binding protein (CBP) and its relative, P300.126,127 These modifications appear to be functionally important as mutation of the acetylated lysine motifs reduces the ability of GATA-1 to rescue erythroid maturation in a tissue culture model.127 It has been proposed that acetylation augments the affinity of GATA1 for DNA,126 although this was not confirmed by another study.127 Rather, acetylation of GATA-1 might be required for its stable association with chromatin.128 Finally, ubiquitination and sumoylation of GATA-1 are reported, although it remains uncertain to what extent these modifications affect GATA-1 function in vivo.129,130

Structure–Function Analysis of GATA-1 GATA-1 acts as a potent transcriptional activator when cotransfected into heterologous cells (such as COS or 3T3) with a reporter gene containing a promoter with one or more GATA motifs.75,114 In this assay, several domains of murine GATA-1 are required for activity.75 In particular, the amino terminus contains an acidic domain that is required for transactivation of reporter constructs, and functions as an independent activator when fused to a heterologous GAL4 DNA binding domain. This domain is a target for somatic mutations associated with megakaryoblastic leukemias in patients with Down syndrome (discussed later in this chapter). The GATA-1 C-finger, which is required for DNA binding, is also essential for reporter gene activation. A strikingly different view emerges from structurefunction analyses that exploit GATA-1’s ability to influence hematopoietic lineage selection or maturation. Remarkably, the GATA-1 zinc finger region alone is sufficient to induce megakaryocytic differentiation of 416B myeloid cells115 and restore erythropoiesis in GATA-1–embryoid bodies.116 Hence, the amino terminal activation domain that is critical for activity in promotor–reporter assays is dispensable for at least some functions in hematopoietic cells. These findings demonstrate that structure–function relationships within the GATA-1 protein are contextdependent, and reveal potent biological activity within the zinc finger region. Further dissection of the GATA-1 DNA binding domain revealed that the N-finger is essential for activity in erythroid cells.25,117 One critical role of the Nfinger is to mediate the interaction between GATA-1 and FOG-1.118 The N-finger also functions through its ability to stabilize in vivo DNA interactions at a subset of bipartite GATA-1 motifs.75–78,119

GATA-1–Interacting Proteins GATA-1 participates in erythroid gene activation and repression through interactions with numerous erythroid specific and ubiquitous nuclear factors (for review see ref. 80). For instance, GATA-1 physically interacts with zinc finger proteins such as GATA-1 itself, other GATA factors, EKLF, and SP1.131–134 In each case, protein interactions occur through the zinc finger regions of the respective proteins and potentiate GATA-1 transcriptional activity at defined promoters. Unique combinations of interacting transcription factors might establish target gene specificity by synergistically enhancing transcription at erythroid enhancers. It is also possible that they mediate physical interactions between erythroid gene promoters and more distant regulatory regions (Chapter 3). For example, promoters of genes within the ␤-globin locus and the core elements of the LCR each contain CACCC and GATA motifs. Interactions between proteins bound to these elements appear to facilitate direct communication via looping between the LCR and specific globin genes.20,21

Nuclear Factors That Regulate Erythropoiesis Interaction between the N-terminal finger of GATA-1 and FOG-1 (Zfpm1) particularly important for erythroid and megakaryocytic development.118,135 Disruption of the Fog1/2fpm1 gene produces an erythroid defect similar to that of GATA-1 loss, albeit not as severe, suggesting the possibility of both FOG-1-dependent and-independent functions of GATA-1 in red blood cells. In contrast, Fog1/2fpm1−/− mice and ESCs exhibit a complete block to megakaryocytopoiesis, indicating that GATA-1-independent functions for FOG-1 exist in megakaryocytes.135 The mechanisms of FOG-1 actions are likely to be complex; for example, FOG-1 can either activate or repress transcription, depending on promoter and cell context.118,136 FOG-1 itself participates in several important protein interactions that help to assemble higher order complexes at GATA-1 target sites. For example, different regions of FOG-1 interact with the corepressor CtBP2 and the NuRD corepressor complex.136–138 NuRD is present at GATA1-repressed genes and is required for efficient repression of the GATA-1 target gene Kit.138,139 Point mutations that abrogate the FOG–CtBP2 interaction have no obvious erythroid effects in mice, indicating the possibility of functional redundancy with other repressors, including NuRD.140 Preliminary gene targeting studies indicate that the FOG-1–NuRD interaction is essential for normal erythroid and megakaryocytic development (Blobel GA, unpublished data). Tissue-restricted nuclear factors must communicate with the general transcriptional machinery. For GATA1, a direct and functionally important interaction with TRAP220, a component of the basal transcription factor complex called “mediator,” has been described.141 Moreover, GATA-1 interacts with the highly related general coactivators CBP and p300, which both interact with numerous basal transcription factors.126,142–144 In addition, CBP and p300 possess intrinsic and associated histone acetyltransferase activities. Histone acetylation is associated with an “open” chromatin configuration characteristic of the ␤globin locus in erythroid cells. Indeed, GATA-1 may stimulate histone acetylation at this locus and other active genes by recruiting CBP.144,145 As noted earlier, CBP-mediated acetylation of GATA-1 itself is also of functional importance in erythroid cells. Moreover, CBP interacts with additional erythroid transcription factors (see later) and may therefore participate in the formation of large multiprotein complexes. GATA-1 also physically interacts with the Ets family transcription factor PU.1, which is normally expressed in multipotential progenitors, myeloid cells, and B lymphocytes and is required for normal myelopoiesis and lymphopoiesis (for review see ref. 146). Inappropriate expression of PU.1 by retroviral insertion and transgenesis causes erythroleukemia147–149 and forced expression of PU.1 blocks differentiation in erythroid cell lines and primary progenitors.150–153 Hence, it has been postulated that downregulation of PU.1 is essential for normal ery-

69 thropoiesis. One underlying mechanism stems from recent observations that PU.1 and GATA-1 cross antagonize each other through direct physical interaction.154,155 In support, overexpression of GATA-1 relieves the PU.1-induced block to chemical-induced maturation of erythroleukemia cells. It is proposed that inhibition of PU.1 inhibits GATA-1 via formation of a RB-containing corepressor complex.156,157 PU.1 has also been shown to inhibit CBP-mediated GATA1 acetylation.158 Conversely, GATA-1 (and GATA-2) inhibit PU.1 transactivation activity, in part by displacing the PU.1 coactivator, c-Jun.159 Together, these data suggest that GATA-1 and PU.1 oppose each other’s actions and that their relative stoichiometry may influence differentiation decisions in multipotent myelo–erythroid progenitors. The SCL protein complex binds DNA directly, but can also associate with genes indirectly through interaction with GATA-1.160 This complex, which contains numerous proteins including LMO2, Ldb, and E2A is recruited to GATA-1-regulated genes via the LMO2 subunit. These proteins were also shown to cooccupy some erythroid regulatory elements in vivo.106,161 It is likely that SCL and associated proteins function as activators of GATA-1-dependent transcription, likely by recruiting additional coregulators. The role of SCL in erythroid development is discussed in greater detail later in this chapter.

GATA-1 and Human Disease Two major classes of human disease are caused by mutations in the X-linked GATA1 gene. A comprehensive review can be found at the following URL: http://www.ncbi. nlm.nih.gov/books/bv.fcgi?rid=gene.chapter.gata1. First, germline GATA1 missense mutations cause various cytopenias.119,162–167 Most commonly, these mutations occur in the N-finger, either at the FOG-1-interaction surface or at the region involved in DNA binding. Affected patients usually exhibit anemia and/or thrombocytopenia of variable severity. Interestingly, the nature of the phenotype varies considerably depending on the exact mutation, presumably reflecting varying structural requirements for GATA-1 at different target genes. For example, some mutations spare the erythroid lineage and affect platelet function and production more prominently. One interesting mutation, R216W, causes congenital erythropoietic porphyria due to reduced production of the GATA-1 target gene uroporphyrinogen III synthase.168 The same patient was also noted to have very high levels of fetal hemoglobin, consistent with the possibility that GATA-1 may be involved in the ␥ - to ␤-globin gene switch. Second, somatic mutations in the GATA1 are associated with transient myeloproliferative disorder and acute megakaryoblastic leukemia in patients with trisomy 21 (Down syndrome).169–175 All of these mutations occur in the first coding exon (exon 2) and cause splicing abnormalities or premature termination of translation that interferes with the production of full-length protein. In these cases,

70 translation initiation at an internal methionine results in the production of a GATA-1 variant (termed “GATA-1 short” or “GATA-1s”) that is truncated at the amino terminus. Because the GATA1 gene is X-linked, a single mutation leads to exclusive production of GATA-1s in male cells and in female cells with unfavorable Lyonization. Of note, one extended pedigree with a similar exon 2 mutation in the germline has been described.176 Affected males, who do not have trisomy 21, exhibit anemia and neutropenia, but do not develop leukemias. This suggests that altered GATA1 somehow synergizes with trisomy 21 to cause leukemia through unknown mechanisms. One interesting problem relates to the cellular functions of the amino terminus of GATA-1, which is absent in the short form. In gene-targeted mice this domain is dispensable for erythropoiesis, but is required to restrain the proliferation of embryonic megakaryocytic precursors.

Stem Cell Leukemia (SCL, TAL1, TCL5) The SCL/TAL1 gene was originally identified via chromosomal rearrangements involving 11p13 in acute T-cell leukemias (for review see ref. 177) SCL, a member of the basic helix-loop-helix class of transcription factors, functions as a heterodimer in association with a variety of widely expressed partner proteins including E2A, E2-2, or HEB (for review see ref. 178). The SCL complex recognizes a cognate DNA element termed E box (consensus CANNTG). Gene-targeting studies have demonstrated that SCL is critical for the establishment of all primitive and definitive blood lineages and for organization of the yolk sac vasculature in early embryos.179–183 These studies indicate that SCL functions at the onset of hematopoiesis, possibly within the hemangioblast, a bipotential hematopoietic endothelial cell precursor (Chapter 2). Genetic studies indicate that SCL is dispensable for formation of hemangioblasts, but required for their subsequent maturation.184–186 Interestingly, although SCL is required for the onset of hematopoiesis in the embryo, conditional gene-targeting studies indicate that SCL is dispensable for the maintenance of adult hematopoietic stem cells.187–189 The role of SCL in erythroid development is supported by numerous lines of investigation. First, SCL is expressed at a relatively high level in erythroid precursors and erythroid cell lines.190–192 Second, overexpression of SCL stimulates erythroid differentiation of murine erythroleukemia cells and the multipotential myeloid cell line, TF-1.193,194 Moreover, forced expression of SCL in human CD34+ cells stimulates formation of erythroid and megakaryocyte progenitors and an increase in the size of erythroid colonies.195,196 Third, ablation of the SCL gene in adult hematopoietic stem cells causes erythroid and megakaryocytic defects.183,188,189 Notably, the erythroid defects resemble that of GATA-1 or FOG-1 loss, consistent with physical and functional interactions between GATA-1, SCL, and associated proteins. Fourth, conserved, functionally

Gerd A. Blobel and Mitchell J. Weiss important E box motifs are present at numerous erythroid genes including EKLF/KLF1, the HS2 core of the ␤globin LCR, Band 3/SLC4A1, Band 4.2/EPB42, Glycophorin A/GYPA and the GATA1 gene itself.106,197–202 In some cases these E boxes are juxtaposed to GATA sites, and binding of both factors is thought to facilitate assembly of a larger complex containing GATA-1 (or GATA-2), SCL, E2A, and two non-DNA-binding nuclear proteins, Lmo-2 and Ldb-1.160,203 SCL complexes can activate and repress transcription in part through recruitment of coactivators such as p300/CBP or corepressors such as Sin3A or Eto-2.204–209 How these activities are controlled is unclear, although acetylation of SCL by PCAF, which reduces binding to Sin3A, might play a role in this process.206 Moreover, Eto-2 binding to SCL diminishes during erythroid differentiation, thus changing the composition of SCL-associated complexes, perhaps tipping the balance in favor of transcriptional activation.207,208 Transcriptional activation by SCL–GATA complexes also appears to be enhanced by single-stranded DNA-binding proteins, which bind and protect Lmo2 and Lbd1 from proteosomal degradation.209 Given the combinatorial complexity of SCL- and GATA-1-associated proteins, understanding the exact functional interplay among all of these subunits in vivo remains a long-term challenge.

EKLF and Other CACCC Box-binding Proteins Functionally important GC-rich elements, also referred to as CACCC boxes, are found in many erythroid gene regulatory elements including several globin gene promoters and the LCR (Chapter 3). The importance of an intact CACCC box in the ␤-globin gene promoter is underscored by the observation that certain thalassemias are associated with mutations in these elements.210–212 CACCC boxes, which vary somewhat in their sequence, are recognized by a diverse set of transcription factors that share a related DNA-binding domain composed of three zinc fingers with ¨ homology to the Drosophila melanogaster Kruppel protein (for reviews see refs. 213–215). These factors include the Sp1 family and proteins related to EKLF (Fig. 4.6). EKLF is of particular interest to studies of globin gene regulation because its expression is restricted mainly to erythroid cells, with low-level expression in mast cells.216,217 EKLF binds to the ␤-globin CACCC box with high affinity and mutations found in CACCC boxes of ␤ thalassemia patients abrogate EKLF binding.218

EKLF is Required for ␤-globin Gene Expression The presence of numerous erythroid factors that bind the same CACCC elements suggested considerable functional redundancy at a given promoter or enhancer in vivo. Therefore, it was surprising to discover that targeted disruption of the Eklf (Klf1) gene leads to significant loss of adult-type ␤-globin expression with resultant anemia

Nuclear Factors That Regulate Erythropoiesis

Figure 4.6. Gel mobility shift experiment showing multiple CACCC box–binding proteins in erythroid cells. In this assay, CACCC binding proteins present in nuclear extracts of murine erythroleukemia cells bind a radiolabeled oligonucleotide containing a single CACCC box, retarding its electrophoretic mobility. Migration of individual protein–DNA complexes is altered by incubation with specific antisera, as shown (Pre, preimmune serum). Note the presence of four major complexes, the most prominent one being Sp1. Despite the low abundance of EKLF in this assay, loss of EKLF function leads to a pronounced defect in ␤-globin gene transcription, which cannot be compensated for by other CACCC box–binding factors (see text). Although gel mobility shift experiments such as this identify factors that can bind to a CACCC box in vitro, they do not permit conclusions as to which factor(s) binds to a given CACCC box–containing promoter in vivo. Photograph provided by Merlin Crossley.

and embryonic lethality of homozygous null animals at E14–E16.219,220 Klf1−/− definitive erythrocytes exhibit molecular and morphological features typical of severe ␤ thalassemia including hypochromia, poikilocytosis and markedly elevated ␣/␤ globin ratio with Heinz body formation and ineffective erythropoiesis. Although EKLF can be detected at the embryonic ␤-like globin genes ␤H1 (Hbb-hh1) and Epsilon (Hbb-y) by ChIP,221 their expression is unaffected by the loss of EKLF, and primitive erythropoiesis appears to be normal in the mutant mice. Notably, there is a loss of the low, but detectable, levels of adult-type ␤-globin (beta adult major, Hbb-b1) in E11 yolk sac.220 Hence, EKLF appears to be selectively required for high-level expression of adult ␤-globin. One mechanism may be to facilitate the formation of a DNA loop that brings the ␤-globin gene into contact with LCR.20 Recently, EKLF has been found to occupy the ␣-globin locus and participate in its expression, although to a lesser extent than for ␤-globin.222

A Role for EKLF in ␤-Globin Switching Selective loss of adult ␤-globin gene expression in mutant embryos suggested that EKLF might participate in the switch from ␥ - to ␤-globin in humans. Indeed, when

71 transgenic mice bearing an extended human ␤-globin gene cluster were crossed with Klf1−/− mice, the resulting EKLFdeficient fetal liver cells displayed dramatically reduced human ␤-globin levels with a concomitant increase in the levels of ␥ -globin.223,224 In addition, human ␥ - to ␤-globin switching was delayed in Klf1−/+ heterozygous mice224 and accelerated by transgenic overexpression of EKLF.225 These studies are consistent with a model in which the ␥ - and ␤globin gene promoters compete for the action of the LCR. Hence, EKLF might contribute to a more stable interaction between the LCR and the ␤-globin promoter to accelerate shutoff of ␥ -globin. ␥ -Globin gene silencing can also occur independent of a competing ␤ promoter.226 Loss of EKLF also leads to reduced DNase1 hypersensitivity at HS3 and the ␤-globin promoter of both transgenic human and endogenous globin loci.224 This suggests that EKLF might contribute to changes in the chromatin configuration at selected sites at the ␤-globin gene locus. These alterations might facilitate binding of transcription factors to DNA or increase the interaction between the ␤-globin promoter and the LCR. Alternatively, they might merely be a secondary consequence of promoter–LCR interactions and transcriptional activity. The former possibility is supported by observations that EKLF associates with factors that have chromatin remodeling activity (see later).

Broader Roles for EKLF in Erythroid Development Initially, it was believed that EKLF might only regulate adult-type ␤-globin gene expression. Subsequent studies, however, demonstrated that EKLF controls numerous other erythroid genes. This possibility was raised initially by experiments showing that enforced expression of ␥ globin fails to rescue the defects in survival and maturation of Klf1−/− erythroid precursors.227,228 Subsequently, microarray-based studies examining mRNA expression in Klf1−/− erythroblasts identified numerous potential EKLF targets with important roles in erythropoiesis. These include genes encoding ␣-hemoglobin stabilizing protein AHSP (Eraf ), the erythroid membrane skeletal protein band 4.9, ankyrin, and heme biosynthetic enzymes.40,228 In follow-up studies, ChIP experiments demonstrated that EKLF directly occupies its regulatory regions of Ahsp/Eraf and Band 4.9 genes in erythroid cells.41,42,229,230 Of note, microarray studies suggested that EKLF functions predominantly as an activator of gene expression. However, EKLF interacts with corepressor proteins and has been shown to repress transcription in several experimental contexts. Recent overexpression and loss of function studies raised the possibility that EKLF not only promotes erythropoiesis, but also suppresses megakaryocyte formation.231 This indicates that EKLF might play a role in the developmental bifurcation between these two lineages from common bipotential megakaryocyte–erythroid progenitors. One mechanism may be through EKLF-mediated repression of the gene encoding the megakaryocyte Ets

72 nuclear factor Fli1, the first candidate target for biologically relevant EKLF-mediated transcriptional repression.231 An interesting question remains as to why some erythroid genes containing CACC boxes are more sensitive to the loss of EKLF than others. In particular, adult-type globin genes require EKLF but the embryonic ones do not, despite findings that EKLF binds to these genes in primitive erythroid cells in vivo.221 This cannot simply be explained by general loss of EKLF function in a primitive environment because adult ␤-globin expression is selectively impaired in Klf1−/− primitive erythroid cells.220 Early biochemical studies suggest that this selectivity might be explained by the higher affinity of EKLF for the ␤-globin CACCC box when compared with the ␥ -globin CACCC box.232 In GAL4 fusion constructs, however, the activation domain of EKLF, but not that of Sp1, can activate a ␤-globin–containing reporter construct in erythroid cells, suggesting that DNA binding affinity is not the sole determinant of EKLF specificity.233 In agreement with this interpretation, when the ␤- and ␥ globin CACCC boxes are switched, EKLF still only activates the ␤- but not ␥ -globin gene promoter. Hence, the specificity of EKLF depends, at least in part, on the surrounding DNA and protein context of its binding site. It is also noteworthy that the activation domains of EKLF and other ¨ Kruppel proteins such as Sp1 share no obvious homology, the former being proline rich and the latter being glutamine rich, suggesting that they interact with different coactivator/adaptor molecules.

Posttranslational Modifications of ELKF Terminal differentiation of MEL cells is accompanied by dramatic increases in ␣- and ␤-globin gene expression, whereas EKLF protein levels remain largely unchanged.233 This raises the possibility that EKLF activity might be subject to regulation by posttranslational modifications. Indeed, EKLF is phosphorylated at its N-terminal activation domain, and mutation of the phosphorylation site leads to reduced activity.234 Furthermore, EKLF is also acetylated by CBP and p300. CBP and p300 bind to EKLF and stimulate its activity in transient transfection assays.235 Although acetylation does not alter EKLF DNA binding it does regulate its interaction with SWI/SNF, an adenosine triphosphate– dependent chromatin-remodeling complex.236 EKLF is also sumoylated, and this modification appears to be important for its function as transcriptional repressor, which may relate to inhibition of megakaryopoiesis.237

EKLF Remodels Chromatin Structure ELKF interacts with the mammalian SWI/SNF chromatinremodeling complex (also referred to as EKLF coactivatorremodeling complex 1, E-RC1).238 E-RC1 is required for EKLF-dependent formation of a DNase1 hypersensitive, transcriptionally active, chromatinized ␤-globin promoter

Gerd A. Blobel and Mitchell J. Weiss template in vitro. Another mechanism by which EKLF could modify chromatin structure is by recruiting the acetyltransferases CBP and p300, similar to what has been described for GATA-1 (as described previously) and NF-E2 (see later).

EKLF-related Transcription Factors If EKLF acts at the ␤-globin gene promoter to participate in its stage-specific activation, what are the factors that control expression of the embryonic and fetal globin genes at their respective CACCC boxes? Candidates include ¨ fetal Kruppel-like factors (FKLFs), which share homology to EKLF.239,240 FKLFs activate the ε-, ␥ -, and ␤-globin gene promoters in transient transfection assays with the embryonic and fetal gene promoters showing the strongest response. This suggests that FKLFs might be important for embryonic/fetal globin gene expression in vivo. In contrast to the globin genes, regulatory regions of several other erythroid-expressed genes that contain functional CACCC boxes are not activated by FKLF.239 Another major CACCC box–binding activity found in embryonic yolk sac and fetal liver erythroid cells is basic ¨ Kruppel-like factor (BKLF).240 BKLF is a widely expressed protein that activates or represses transcription depending on cell and promoter context. Repression by BKLF is mediated through the association with a corepressor, CtBP2.137 Of note, EKLF-deficient erythroid cells display dramatically reduced BKLF levels,220,240 and EKLF directly activates BKLF expression.241 In light of the complexity of proteins bound to the ␤-globin CACCC box, this finding underscores the difficulty in directly linking a transcription factor to a specific target gene in vivo and in interpreting the phenotype of a gene knockout experiment on a molecular level. Targeted mutation of BKLF does not dramatically alter globin gene expression, suggesting that the ELKF null phenotype is not solely attributable to secondary loss of BLKF.242 The role of BKLF, if any, in regulating ␤-globin gene expression remains to be determined. Sp1, which was the first CACCC binding factor to be cloned, is expressed in a wide variety of cell types. Mice lacking Sp1 die approximately day 10 of embryogenesis with a multitude of defects.243 Embryonic ␣- and ␤-like globin genes are expressed normally in the mutant mice, which was somewhat surprising considering the relative abundance of Sp1 in erythroid cells. Mouse embryos that are heterozygous for null mutations in Sp1 and Sp3, a ¨ related Kruppel protein, exhibit multiple defects in organogenesis, including anemia.244 This underscores the complex functional interactions and redundancy of CACC binding factors for global tissue development, including erythropoiesis. It is clear from these previously noted studies that a formidable effort is required to establish which transcription factor operates at any given CACCC box. Combined gene knockouts are one approach to address this issue.

Nuclear Factors That Regulate Erythropoiesis Of equal importance will be to investigate further the mechanisms by which CACCC factors regulate transcription. The identification of novel interacting proteins and their analysis in vivo and in vitro will contribute to the understanding of the function of CACCC box binding proteins.

NF-E2 and Related Proteins AP-1-like motifs [(T/C)GCTGA(G/C)TCA(T/C)], now called maf recognition elements (MAREs), are functionally important cis elements within HS2 and HS3 of the β-globin LCR (Chapters XX). Although GATA elements are mostly associated with the position independence conferred by the LCR, MAREs contribute to LCR-mediated enhancer activity.245 Factors binding to MAREs contribute to the formation of DNase1 hypersensitivity, suggesting that these proteins modify chromatin.246–249 MAREs are also found in some nonglobin genes such as those encoding porphobilinogen deaminase and ferrochelatase.250,251 Careful analysis of these elements led to the realization that they are bound by an erythroid-specific transcription factor, called NF-E2.250,252 Affinity purification of NF-E2 from erythroleukemia cells identified a simple heterodimer with subunits named according to their molecular weights: p45 and p18 (now referred to as MafK).253–255 Both subunits contain a basic-zipper (b-Zip) domain, which mediates dimerization and DNA binding. p45 is expressed predominantly in erythroid cells and megakaryocytes, whereas p18 is found in a variety of cell types. It is now appreciated that both p45 and p18 belong to multiprotein families that are expressed in distinct but overlapping patterns, generating a large number of possible combinations of NF-E2–related protein–DNA complexes in different cell types.

The p45 Family of Proteins p45 is the founding member of a family of proteins that contain a region of similarity to the Drosophila Cap’n’collar (CNC) protein. This family includes Nrf-1 (LCRF1, TCF11), Nrf-2 (ECH), Nrf-3, Bach1 and Bach2. These proteins bind DNA as obligate heterodimers with Maf proteins (see later) (for reviews see refs. 256, 257). Expression of p45 is restricted to the hematopoietic system.253,255 Erythroid cells and megakaryocytes express high levels of p45 mRNA whereas little or no p45 mRNA is found in macrophages and B and T cells. This expression pattern suggests that p45 is a critical regulator of globin gene expression. Consistent with this idea, the murine erythroleukemia cell line CB3, which lacks both functional alleles of p45, expresses very low levels of ␣- and ␤-globin. Upon introduction of an intact p45 gene, globin gene expression is restored.258,259 Surprisingly, however, targeted inactivation of the p45 gene in mice has little effect on erythropoiesis or globin gene expression. In contrast p45 null mice exhibit a profound defect in megakaryocyte matu-

73 ration resulting in severe thrombocytopenia and frequent fatal hemorrhage.260,261 Given the large body of evidence implicating MARE elements in globin gene transcription, the minimal effect of p45 gene disruption on erythropoiesis suggested the potential for compensation by other CNC family members; however, homozygous disruption of the Nfe2/2/Nrf 2 gene does not reduce globin gene expression in mice,262 and the combined loss of Nrf-2 and p45 is no more severe than the p45 knock out alone.263,264 Disruption of the Nrf1 gene causes anemia and embryonic lethality, but the defect in erythropoiesis is not cell autonomous.265,266 Thus, the exact contribution of each CNC-b-Zip protein to globin gene expression in vivo remains to be determined. Bach1 and Bach2 are additional p45-related molecules that bind MAREs as heterodimers with Maf family members.267 Bach1 is expressed in hematopoietic cells starting at the earliest progenitor stages.267,268 Bach-Maf complexes are transcriptional repressors that bind MARE elements in a heme-regulated fashion.269 Heme binding to Bach–Maf complexes stimulates their release from DNA, export from the nucleus, ubiquitination, and subsequent proteolysis.270,271 In this fashion, Bach transcription factors provide an elegant mechanism to coordinate heme availability with gene expression in numerous tissues. For example, in erythroid cells depleted of heme, Bach1–Maf complexes bind MARE elements in the ␣- and ␤-globin genes to repress their transcription.272,273

The Maf Family The small subunit of NF-E2 (p18, MafK) belongs to the Maf family of proteins, which share homology with the c-Maf protooncoprotein. The small Maf proteins (MafF, MafG, and MafK) heterodimerize with CNC-b-Zip family and exhibit distinct temporal and spatial expression patterns. MafG and MafK are highly expressed in megakaryocytes and erythroid cells with a predominance of MafG in megakaryocytes and MafK in erythroid cells.256,257 Small Maf proteins lack an activation domain and are thought to stimulate transcription as heterodimers with CNC-b-Zip (p45-like) molecules. Small Maf proteins can also form homodimers on DNA and repress transcription, presumably by competing with activating transcription factor complexes.274 Surprisingly, MafK−/− mice develop normally and display no obvious defects in erythroid maturation, globin gene expression, or platelet formation,275 and NF-E2like DNA binding activity is still detected in fetal liver erythroid cells, consistent with the presence of other compensating Maf family members. Moreover, MafK−/− p45/Nfe2−/− compound mutant mice exhibit minimal defects in erythropoiesis.275 Presumably, other members of the p45/Nfe2 and MafK gene families can provide sufficient NF-E2-like activity to support globin production in vivo. Targeting of the MafG gene produces no obvious defects in

74 erythropoiesis, but impairs megakaryocytic differentiation, although to a lesser extent than in p45 knockout mice.276 The exchange of partners for Maf proteins is critical for the control of MARE activity (see later). It has also been observed that MafG is sumoylated. Mutation of the critical N-terminal sumoylation sites impairs the ability of MafG to repress transcription in megakaryocytes but leaves intact its ability to interact with p45 NF-E2 and activate gene expression.277

Mechanisms of NF-E2 Action Structure–function analysis of p45 in transient transfection experiments and in gene complementation assays using p45 null CB3 cells revealed that full NF-E2 activity requires an intact N-terminal activation domain and CNC domain.259,278 The N terminus of p45 interacts with numerous proteins including CBP/p300,279 ubiquitin ligase,280,281 and the TBP-associated factor TAFii130.282 As is the case for GATA-1 and EKLF (noted previously), the implications for NF-E2 interactions with CBP/p300 are twofold: First, CBP and p300 might link NF-E2 with basal transcription factors (for review see ref. 283). p45 may also communicate with basal transcription machinery via interactions with TAFii130.282 Second, recruitment of CBP/p300 and associated histone acetyltransferase activity to the LCR and other erythroid gene regulatory elements could promote the formation of “open” chromatin structure through histone acetylation. An additional role for NF-E2 in chromatin modification is indicated by the finding that NFE2 can disrupt chromatin structure on in vitro assembled chromatinized templates containing the ␤-globin LCR HS2 site.284 This adenosine triphosphate–dependent chromatin opening activity facilitates binding of GATA-1 to its nearby cis elements. It is not known whether this activity also contains histone acetyltransferases. The N terminus of p45 harbors two PPXY motifs that mediate interactions with several ubiquitin ligases.280,281 Mutations in these motifs reduce transcriptional activity of NF-E2,281 raising the possibility that ubiquitin ligases might modify nearby histones to regulate chromatin structure. In addition, NF-E2 interacts with the MLL2 methyltransferase complex.285 This complex is related to the MLL1 complex and methylates lysine 4 of histone H3, a chromatin mark that is found at most active genes. Thus, NF-E2 is linked with several histone-modifying enzymes, similar to other transcriptional regulators. In an erythroid cell line, MafK associates with Bach1 in the immature state and with p45 NF-E2 after chemicalinduced erythroid maturation. Notably, the exchange of MafK partner proteins was accompanied by redistribution of coregulator complexes.286 Thus, components of the NuRD and SIN3A repressor complexes copurified with Bach1 in immature cells, whereas the p45 NF-E2– containing complex associated with a transcriptional activators including CBP/p300. This suggests that MARE

Gerd A. Blobel and Mitchell J. Weiss binding proteins not only function during transcriptional activation but might also be involved in actively suppressing the expression of globin genes and perhaps other erythroid-specific genes in immature cells, consistent with findings that Bach1 binds and represses globin synthesis in low heme states.272,273 The onset of high level globin gene expression is coordinated with cellular differentiation and proliferation arrest, suggesting that these pathways may be mechanistically linked. Consistent with this idea is that NF-E2 is regulated by the MAP kinase pathway. In MEL cells, activation of MAP kinase potentiates NF-E2 DNA binding and transcriptional activation.287 In addition, NF-E2 binding sites are required for MAP kinase inducibility of the HS2 region within the LCR.288 The p45 subunit of NF-E2 is also phosphorylated by protein kinase A, but the physiological significance of this modification is unclear.289 It is interesting to consider that signals which trigger erythroid maturation might act in part by facilitating the exchange of partners for the small Maf proteins and their coregulator complexes. In addition, posttranslational modifications can modulate the activity of MARE binding proteins. For example, similar to certain Maf proteins, p45 NF-E2 is also subject to sumoylation, which reduces transcriptional activation by impairing the association of NF-E2 with its target sites in vivo.290 Whether sumoylation of Maf and p45 NF-E2 can occur simultaneously within the same complex or whether it is targeted to distinct complexes is unknown. It is possible that cellular signaling events influence the targeting of the SUMO modification to the appropriate subunit. In summary, MARE-associating factors are a heterogeneous group of proteins with distinct but overlapping functions and expression patterns. As an additional complexity, MARE factors activate transcription as heterodimers, leading to increased diversity through combinatorial associations. The major future challenge is to determine which combinations of MARE binding proteins act at a given gene regulatory element in vivo. In particular for erythroid biology, it will be important to learn the full complement of NF-E2-like proteins that activate the ␤-globin locus during normal erythropoiesis and in the background of various targeted mutations of MARE binding protein subunits.

Candidate Nuclear Factors Involved in Globin Switching One model to account for developmental regulation of gene expression within the ␤-globin locus is based on the principle that individual globin genes compete for ␤-LCR enhancer activity, which is available only to a single gene at any given time.291–293 These competitive interactions are believed to be influenced by variations in the relative concentrations and/or posttranslational modifications of transcription factors that are expressed at all developmental stages.294,295

Nuclear Factors That Regulate Erythropoiesis In addition to EKLF and GATA-1, other protein complexes have been invoked to play direct roles in hemoglobin switching. One example is human stage selector protein (SSP), which recognizes a DNA motif, termed stage selector element (SSE), found in the proximal ␥ -globin gene promoter. SSE was identified through its ability to allow the ␥ -promoter to function in preference to a linked ␤globin gene in plasmid constructs containing the HS-2 portion of the ␤-LCR.296 SSP DNA binding activity appears to be relatively restricted to fetal erythroid cells. Thus, it is believed that ␥ -globin synthesis is stimulated in part by expression of SSP, which binds SSE to impart a competitive advantage for recruitment of the LCR to the ␥ -promoter. This is supported by recent studies showing that transgenic overexpression of the p22 NF-E4 SSP subunit can increase the ratio of ␥ -globin to ␤-globin gene expression in mice carrying the human ␤-globin locus.297 The SSE, however, is neither necessary nor sufficient for competitive inhibition of ␤-globin gene expression in immortalized erythroid cell lines.296,298,299 Therefore, it is particularly important to determine the extent to which this cis-acting element influences ␥ gene expression in vivo. Another protein complex with potential roles in ␤-globin gene switching is direct repeat erythroid definitive (DRED). DRED was isolated through its affinity for direct repeat (DR) elements that cluster near the ε-globin and ␥ -globin promoters and contains the orphan nuclear receptors TR2 and TR4.300 Notably, another nuclear receptor, COUP-TFII also binds the DR elements.301 DR sequences are of interest because mutations in this region are associated with several cases of hereditary persistence of fetal hemoglobin.302 Gain- and loss-of-function studies support a model in which DRED subunits TR2 and TR4 cooperate to silence directly endogenous embryonic mouse ␤-globin genes and transgenic human embryonic and ␥ -globin genes. As is the case for studies on SSP, the effects of altered transcription factor levels on globin gene expression are gradual but not absolute, suggesting that multiple protein complexes operate in concert to modulate hemoglobin switching.

Summary and Perspective Studies of globin gene regulation are paradigms for investigating tissue-specific and developmental control of eukaryotic gene expression. Therefore, it is no surprise that pursuit of nuclear factors that coordinate globin gene transcription has produced a complexity of information with important implications for a variety of developmental processes. For example, GATA-1 regulates many aspects of terminal erythropoiesis and megakaryocyte maturation, presumably by controlling a number of as yet unidentified target genes. Moreover, discovery of GATA-1 led to the identification of several related proteins important for the formation of hematopoietic stem cells, T lymphocytes, heart, nervous system, and endodermally derived tissues. NF-E2, originally believed to be red blood cell specific,

75 was shown by knockout studies to be largely dispensable for globin synthesis and erythroid development, yet critical for platelet formation. In addition, studies of NF-E2 have focused attention on the large family of MARE binding proteins that participate in numerous processes including cognitive development and formation of early embryonic mesoderm. Likewise, the discovery and characterization of ¨ several Kruppel-related proteins with diverse functions was initiated largely by studies of globin regulation. Discovery of numerous tissue-restricted and widely expressed transcription factors that function in red blood cells provides a solid foundation for understanding globin gene expression and erythroid differentiation. The current challenge is to better understand the mechanistic basis for transcription factor function in intact organisms. Presumably, insights will be gained through continued investigation of the dynamic developmental stage and tissue-specific regulatory networks that exist among erythroid nuclear factors, basal transcription machinery, and chromatin. In addition, it is important to examine the hierarchical order by which transcription factors regulate each other’s expression. For example, GATA-1 activates its own expression but represses GATA-2, which may be prerequisite for terminal erythroid maturation.100 Prior to repression by GATA-1, GATA-2 appears to autoregulate.107 Moreover, GATA-1 positively regulates expression of the EKLF gene,303 and EKLF is required for full expression of BKLF.220,240 Such cross-regulation imposes a tissue-restricted and developmental order on the erythroid gene expression program. Analysis of cis-acting regulatory regions of erythroid transcription factor genes is beginning to explore how their expression is regulated. One ultimate goal is to exploit basic knowledge of transcription factor function for manipulating gene expression in the treatment of human diseases. In this regard, pharmacological alteration of transcription factor–DNA interactions may be difficult because these usually occur over extended surfaces and the affinities are usually high. Transcription cofactor complexes, however, typically contain one or more enzymatically active subunits (adenosine triphosphatases, deacetylases, acetyltransferases, and methyltransferases etc.) that might lend themselves to pharmacological intervention. For example the drug butyrate, which is used to activate fetal globin gene expression in patients with sickle cell anemia or ␤ thalassemia (Chapter 3) inhibits histone deacetylases.304 More potent agents with similar activity are now under study.305,306 Histone deacetylase inhibitors have also been shown to reactivate silenced globin transgenes delivered by retroviral vectors designed for gene therapy.307 Together, these results define an interface through which basic studies of gene regulation might ultimately impinge on clinical management of hematological disorders. Recent genetic association studies indicate that polymorphisms in the BCL11A gene influence fetal hemoglobin

76 levels significantly. BCL11A encodes a zinc finger nuclear protein that binds to DNA but can also associate with other erythroid nuclear factors such GATA-1 and FOG-1. Using a knock-down approach in human erythroid cells, it was shown that deplection of BCL11A leads to a significant upregulation of gamma-globin expression. These levels could be therapetatic if achieved in hemoglobinopathy patients. How BCL11A regulates gamma globin expression in still nuclear. Thus, BCL11A represents a newly discovered target protein for better understanding and manipulating fetal hemoglobin expression.308–312

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5 Molecular and Cellular Basis of Hemoglobin Switching George Stamatoyannopoulos, Patrick A. Navas, and Qiliang Li

INTRODUCTION Hemoglobin switching is characteristic of all animal species that use hemoglobin for oxygen transport. Most species have only one switch, from embryonic to adult globin formation. Humans and a few other mammals have two globin gene switches, from embryonic to fetal globin coinciding with the transition from embryonic (yolk sac) to definitive (fetal liver) hematopoiesis and from fetal to adult globin formation, occurring around the perinatal period (Fig. 5.1; see Chapters 1 and 2). The switch from ε- to ␥ -globin production begins very early in gestation, as fetal hemoglobin (HbF) is readily detected in 5-week-old human embryos,1,2 and it is completed well before the 10th week of gestation.1,3 ␤-globin expression starts early in human development, and small amounts of adult hemoglobin (HbA) have been detected by biosynthetic or immunochemical methods even in the smallest human fetuses studied. In these fetuses ␥ - and ␤-globins are present in the same fetal red cells.4 ␤-chain synthesis increases to approximately 10% of total hemoglobin by 30–35 weeks of gestation. At birth, HbF comprises 60%–80% of the total hemoglobin. It takes approximately 2 years to reach the level of 0.5%–1% HbF that is characteristic of adult red cells. HbF in the adult is restricted to a few erythrocytes called “F cells” (see chapter 7).5,6 Approximately 3%–7% of erythrocytes are F cells6 and each contains approximately 4–8 pg of HbF.5 Hemoglobin switching has been the target of intensive investigation for two reasons. First, it provides an excellent model for studying the control of gene activity during development. Indeed, until the late 1970s, hemoglobin switching was the only developmental system that could be investigated in detail at the protein level. Second, understanding of the control of switching is expected to lead to the development of treatments of hemoglobinopathies. The ␤-chain hemoglobinopathies, sickle cell disease, and thalassemia are unique among genetic disorders in that nature has shown an effective means of treatment: the 86

production of HbF that can compensate for the loss of ␤-chain activity or can decrease the propensity for sickling. Research on the cell and molecular control of switching is expected to lead to discoveries that will cure these disorders through abundant production of HbF in the patient’s red cells.

CELLULAR CONTROL OF SWITCHING Before the era of molecular biology, insights on the cellular mechanisms of hemoglobin switching were obtained through phenomenological observations in human and animal models and from cell biological studies. The observation that human fetuses have different hemoglobin than adults was made more than 100 years ago when it was discovered that the hemoglobin of neonates is alkali resistant. The observation that amphibia have different hemoglobins in the embryonic and the adult stages was made in the 1930s when the oxygen affinity of frog and tadpole blood was examined. The two types of hemoglobin were actually separated by Svedberg while he was developing the ultracentrifuge. Hemoglobin switching was more intensely investigated when the introduction of electrophoretic techniques allowed detailed studies of hemoglobin during the development of many species. Several questions on the cellular control of switching were asked during that time and, amazingly, clonal models of switching (see later) were proposed even before it became possible to analyze hemoglobin switching at the protein level. Systematic investigation of the cellular control of switching, however, started only when modern methods of cell biology became available in the 1970s.

Models of Cellular Control The first models of hemoglobin switching assumed that it represents an epiphenomenon due to replacement of hematopoietic stem cell lineages. The model was eloquently formulated by the late Vernon Ingram.7 To explain hemoglobin switching in the mouse or in the chicken, it was postulated that there is an embryonic stem cell lineage that is committed to embryonic globin gene formation and this is replaced by an adult stem cell lineage committed to expression of the adult globin genes. In the case of the human hemoglobin switching, three lineages were thought to exist: an embryonic, a fetal, and an adult stem cell lineage. The fetal (␥ -) to adult (␤-) switch was attributed to the replacement of the fetal stem cell lineage by the adult stem cell lineage.8–12 The transitions in major erythropoietic sites during ontogeny (see Chapter 1) seemed to support the clonal hypothesis of switching. The clonal hypothesis was also appealing because of the restriction of HbF in few red cells, the F cells, in the adult blood. When, in adult individuals, HbF was elevated, the number of F cells was elevated. Hence it was thought that F cells and A cells (i.e., cells that did not contain HbF) were derived from two distinct stem cell lineages.9

Molecular and Cellular Basis of Hemoglobin Switching

Figure 5.1. Hemoglobin switching in humans and mice. The human ε gene is homologous to murine εy. The ␥ -globin gene is homologous to ␤h1 whereas the ␤-globin gene is homologous to murine ␤minor and ␤major.

An alternative model was elaborated in the mid-1970s.13 It proposed that fetal to adult globin gene switching is not due to changes in stem cell populations but to changes in programs of gene expression that occur in the progeny of a single stem cell population. A fetal program is activated in the progenitor cells of the fetus and an adult program in the progenitor cells of the adult. Finding out which of the two models (i.e., changes in stem cell populations or changes in programs) is correct was important from the theoretical and the therapeutic point of view. In the 1970s it was thought that it was difficult to manipulate stem cell populations; on the other hand, it was possible that manipulation of gene expression programs could be achieved with pharmacological means. Therefore, a systematic investigation of the two models was conducted. The lineage models assume an absolute restriction of embryonic globins to primitive cells and of adult globins to definitive cells. During switching in chickens,14 in

87 the mouse,15 and in quail–chick chimeras,16,17 there are cell populations coexpressing both embryonic and adult hemoglobins. The hematopoietic cells of human embryos can be used to produce erythroid colonies, each of which originates from a single progenitor cell; typically, these colonies coexpress ε- and ␥ -globins.18,19 Thus, a single progenitor cell can form progeny producing adult and embryonic globins, contrary to the expectations of the lineage models. Three types of experiments provided evidence against the model of replacement of stem cell lineages as an explanation of the ␥ - to ␤-switch. First, studies of individuals with clonal hemopoietic stem cell disorders (polycythemia vera, chronic myelogenous leukemia, or paroxysmal nocturnal hemoglobinuria) clearly showed that both F cells and A cells are produced by a single stem cell clone (summarized in Stamatoyannopoulos and Grosveld20 ). Second, studies in culture showed that erythroid colonies derived from single progenitor cells of fetuses, neonates, or adults, contain both fetal and adult globins.20 Third, direct evidence came from analyses of somatic cell hybrids produced by fusion of mouse erythroleukemia (MEL) cells with human cells. These hybrids initially synthesize only (or predominantly) fetal human globin, and after 20–40 weeks in culture they switch to ␤-globin chain formation. Since each hybrid originated from a single cell, these results provided direct evidence that ␥ - to ␤-switching can occur in cells of a single lineage.21 It is thus clear that the fetal to adult hemoglobin switching takes place in the progeny of a single stem cell lineage. It represents changes in transcriptional environments at the level of committed cells rather than changes in stem cell populations “frozen” in a single gene expression program. It is of interest that despite the extensive evidence, even today the cellular phenotypes of HbF elevations in the adult are attributed by some authors to the presence of a fetal stem cell population in the adult marrow!

The Question of Developmental Clock of Switching If switching takes place in the cells of a single lineage, how do these cells know when to switch their globin gene expression program? Many changes occur during development and there is ample evidence that the cell’s microenvironment can determine the fate of a cell. Initially, inductive mechanisms were thought to trigger hemoglobin switching, and several experiments have been done to test whether changes in the environment of the developing fetus, especially hormonal changes, are responsible for the ␥ - to ␤-switch. The summary of this work indicates that there is no evidence that there exists a specific environmental signal that is responsible for the switch. On the other hand, there is evidence that the environment can influence the rate of the ␥ - to ␤-switching. Thus, in sheep, removal of the adrenals abolishes the increase in plasma cortisol that precedes birth.22 The ␥ - to ␤-switch in such

88 adrenalectomized animals is delayed, although the animals are normal with respect to developmental progression. Administration of cortisol allows the switch to progress with normal kinetics. Also, external factors can influence the rate of the ␥ - to ␤-switch in MEL/fetal erythroid hybrids: serum deprivation or addition of dexamethasone in the culture media strikingly accelerates, while addition of butyrate inhibits, the ␥ - to ␤-switch.23,24 Considerable evidence suggests that the rate and the timing of switching is inherently controlled, perhaps through the action of a developmental clock type of mechanism. Three arguments in favor of a clock-type of mechanism will be mentioned here. First, in vivo observations in humans indicate that the level of HbF in newborns is related to their developmental age from conception rather than to the time of birth itself.25,26 Thus, the switch is independent of the intrauterine or extrauterine status of the individual; rather, the degree of developmental maturity of the fetus determines the rate as well as the timing of the ␥ - to ␤-switch. Second, the rate of ␥ - to ␤-switching of the MEL/human fetal erythroid hybrids correlates with the age of the fetus from which the human erythroblasts were derived.21 Thus, hybrids produced using cells of younger fetuses switch more slowly than do hybrids produced using cells of older fetuses, as if the human fetal erythroid cells “know” whether they belong to an early or to a late developmental stage, and transmit this information to the hybrid cells. Third, transplantation experiments of hematopoietic stem cells have been done in sheep to determine whether the hematopoietic environment can influence the rate of the switch in transplanted cells. Adult stem cells were transplanted into fetuses and fetal stem cells into adult animals, and hemoglobin production in the engrafted donor cells was monitored. The adult cells transplanted into fetuses continued to produce adult globin, suggesting that the fetal environment cannot change the program of the adult cells.27 Transplantation of fetal sheep stem cells into lethally irradiated adult recipients showed that the donor cells switch.28 The rate of switching of the transplanted fetal cells, however, depended on the gestational age of the donor fetus, suggesting that switching reflects the action of a mechanism that in some fashion can count developmental time. Presumably, a clock determining the rate of switching is set sometime during embryogenesis and proceeds to execute a preset program as development advances. It has been difficult to test experimentally the molecular basis of this phenomenon. There are several examples of developmental clocks in drosophila, but these are usually associated with circadian rhythms. It is difficult to conceive how a “clock” that can operate for several months (as in the case of human ␥ - to ␤-globin gene switching) is controlled; although hypotheses on how cells can count developmental time have been proposed.29 The available evidence suggests that the clock of human ␥ - to ␤-switching is located on chromosome 11.30 It acts in cis and certain findings,31

George Stamatoyannopoulos, Patrick A. Navas, and Qiliang Li although interpreted differently by these authors, suggest that the clock may be controlled through sequences located in the ␤-globin gene cluster.

MOLECULAR CONTROL OF SWITCHING The last 20 years have witnessed considerable progress in the understanding of the molecular control of globin gene switching. Several tools have been used. Transgenic mice have provided information on the sequences of the locus that are responsible for developmental control and on the mechanisms that control switching in vivo. Traditional biochemistry and gene cloning techniques have led to the discovery of trans factors that interact with motifs of globin gene promoters and the locus control region (LCR). Essentially, we know today, in very broad terms, the mechanisms that regulate globin gene activity during development. There is, however, a vast amount of specific information that still needs to be learned until the phenomenon is completely understood at the molecular level.

Regulatory Elements of the ␧-Globin Gene In vitro experiments indicate that the CACCC and CCAAT boxes in conjunction with the GATA sites of the ε-globin gene promoter are required for expression expression.32–35 However, it is not known which factors interact with these sequences in vivo. The CACCC box binds the ubiquitous factor Sp1,36 but inactivation of Sp1 in vivo37 does not result in defective ε-gene expression. Two factors belong¨ ing to the erythroid Kruppel-like factor (EKLF)/Sp1 fam¨ ily, designated fetal Kruppel-like factor (FKLF)38 and FKLF2,39 have been shown to interact with the ε-gene CACCC box and activate gene transcription in transient expression assays and in stably transfected red cells. The CAAT box of the ε gene binds CP1; binding of CP1 activates in vitro gene expression. In the region of the CCAAT box of the embryonic and fetal, but not of adult, globin-gene promoters there exist direct repeats of a short motif that is analogous to DR-1 binding sites for nonsteroid nuclear hormone receptors.35 In vitro experiments and studies in transgenic mice have demonstrated that COUP-TF, an orphan nuclear receptor, binds to the DR-1 element of the gene promoter and acts as a developmental repressor.35 The role of the DR-1 element in ε-gene silencing was confirmed in a study performed in ␤YAC transgenic mice.40 Furthermore, this study demonstrated that the DR-1 element binds a 540-kD complex named DRED (direct repeat erythroid-definitive), which contains nuclear orphan receptors TR2 and TR4.40,41 TR2 and TR4 form a heterodimer and are able to bind to the ε- and ␥ -globin gene promoters. In TR2 and TR4 null mutant mice, silencing of both the ε- and ␥ -globin genes is delayed in definitive erythroid cells. In transgenic mice expressing a dominant-negative TR4, the ε gene is activated in primitive and definitive erythroid cells.42 Forced expression of wild-type TR2 and TR4 leads to precocious

Molecular and Cellular Basis of Hemoglobin Switching repression of the ε-globin gene; however, ␥ -globin expression is increased in definitive erythroid cells.42 The ε-globin gene promoter also contains a number of GATA sites. Studies in transgenic mice suggest that when GATA-1 binds at the −163 or −269 site it acts as a gene activator, but when it binds to the −208 site it acts as a repressor.43 Several binding sites for factors that can act either as repressors or activators in vitro have been identified in the upstream ε-gene promoter.44,45 Sox6, a member of the Sox transcription factor family, is able to bind at the proximal promoter of the mouse εy-globin gene and to silence directly expression of the gene in definitive erythroid cells.46,47 It remains to be seen whether Sox6 is involved in the autonomous silencing of the human ε-globin gene in adult erythropoiesis.

Regulatory Elements of the ␥-Globin Genes Evidence that the ␥ -globin gene promoter contains elements important for developmental control is provided by the point mutations that produce phenotypes of hereditary persistence of fetal hemoglobin (HPFH) (see Chapter 16). Most of these HPFH mutations occur in transcription factor binding motifs. Between the CAAT box and the TATA box of the ␥ -gene promoter there exists a G-rich sequence designated as stage selector element. This sequence is conserved in species that express the ␥ gene in the fetal stage, but diverges in species in which the ␥ -gene homolog is expressed in embryonic cells.48 A binding activity, called stage selector protein,49,50 binds to this sequence. Stage selector protein is composed of the ubiquitously expressed factor CP2 and a recently cloned protein, NF-E4, which is erythroid specific and activates ␥ -gene expression in transfection experiments in vitro.51,52 Several proteins bind to the CAAT box region of the promoter.53–58 CP1, a ubiquitously expressed protein, acts as a positive transcriptional activator in vitro. CAAT displacement protein (CDP) binds to both CAAT boxes, competitively displacing CP1 and, in vitro, acts as a transcriptional repressor.59 NF-E3 and GATA-1 bind in the CAAT box region53,54,58,60 and are considered to act as gene suppressors, but this hypothesis is not supported by experiments in transgenic mice.61 Studies in transgenic mice indicate that the CACCC box plays an important role in gene expression at the fetal stage of definitive hematopoiesis when the major synthesis of fetal hemoglobin takes place in humans.62 FKLF38 and FKLF-239 bind to the ␥ -globin gene CACCC box in vitro but their in vivo role has not yet been determined. As mentioned previously, a DR-1 element is also identified in the ␥ -globin gene promoter.35,40 The DR-1 binding site is disrupted by the HPFH-117 mutation in support of the hypothesis that the DR-1 element is implicated in ␥ -gene silencing. Other developmentally important sites have been revealed in the upstream promoter by HPFH mutants. GATA and octamer 1 sites are located near position −175. The

89 −175 HPFH mutation alters the interaction with GATA-1 and removes the binding site for octamer 1,63,64 but the relevance of these in vitro effects to the HPFH phenotype remains unknown. Several HPFH mutations are located in the −200 region. This region of the promoter is capable of forming a triple-stranded structure, which is thought to be the binding site for a repressor complex that is displaced by the transcription factors that bind to the novel sequences created by the HPFH mutations mutations.65,66 Other potential binding sites are located further upstream in the promoter promoter.48,67 Transgenic mouse experiments have localized a potential silencer element in the – 382 to –730 region.68 Also, this region contains a butyrate response element element.69 That the −382 to −730 region may contain a silencer has also been shown by the finding of an HPFH mutation at position −567. This mutation alters a GATA site and in vitro experiments showed a complete loss of GATA-1 binding,70 a phenotype recapitulated in transgenic mice.71 Chromatin immunoprecipitation experiments using fetal liver tissue from ␤YAC transgenic mice showed a recruitment of GATA-1, FOG-1, and Mi2 to the –567 GATA site late in fetal development when ␥ gene expression is silenced.71 Mi2 is a member of the NuRD complex whose functions include nucleosome remodeling and histone deacetylase activities resulting in transcription repression.72–74 An “enhancer” has been located downstream from the A ␥ gene on the basis of transient transfection experiments.75 This element contains binding sites for various transcription factors,76,77 but it appears to have no effect on ␥ -globin gene expression in vivo.78 In transgenic mice, presence of this 3
element protects the ␥ gene from position effects,79,80 suggesting that its likely role is stabilization of the interaction between the ␥ -globin gene and the LCR. The effects of the three basic cis elements of the ␥ -globin gene promoter, CACCC, CCAAT, and TATA, on the transcriptional potentials of the promoter at different developmental stages have been studied in transgenic mice. Mutations in each box disrupt ␥ -gene expression in adult erythropoiesis, but have no effect on ␥ -gene expression in embryonic erythropoiesis.62,81–83 These results imply that the transcriptional machinery in embryonic and adult erythroid cells may differ; thus, an intact promoter is required for highly effective transcription in adult erythroid cells whereas a partially defective ␥ -globin promoter can initiate high levels of transcription in embryonic erythroid cells.

Regulatory Elements of the ␤-Globin Gene Several factors have been shown to bind in the CAAT box region of the ␤-globin gene;84–86 CP1 behaves as a positive regulator of the CAAT box in vitro. The CACCC box binds several factors in vitro87 but the protein that appears to be the most important in vivo is EKLF.88,89 The ␤-globin CACC box has a higher binding affinity for EKLF than the ε- or ␥ -globin CACC boxes.90

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Studies using transgenic mice have identified two regions that could enhance ␤-globin gene expression.84,91–93 An enhancer is located downstream from the poly A site of the ␤-globin gene.84,91–93 Its deletion markedly decreases ␤-gene expression in transgenic mice,94 indicating that this element plays an important role in ␤-globin gene expression. Another enhancer is located in intron 2 of the ␤ gene.95,96

normal, suggesting that a hypersensitive site other than HS3 interacts with the ␥ promoter in embryonic cells. However, ␥ -globin gene expression, is totally absent in fetal liver cells, indicating that the core of HS3 is necessary for ␥ -gene transcription in the fetal stage of definitive erythropoiesis. These results are also compatible with the possibility that the LCR changes conformation during the course of development.114

The ␤-Globin Locus Control Region

Molecular Control of Switching

This region is described in Chapter 3 of this book. It is located 6–25 kb upstream from the ε-globin gene and contains a series of developmentally stable DNase I hypersensitive sites.97,98 A large body of data indicates that the activities of the LCR are mostly localized to the core elements of the hypersensitive sites, which are approximately 300 bp long. The regions flanking the hypersensitive site core elements of the LCR are also important for function. The current concept is that the LCR functions as a complex formed by interaction of the transcriptional factors that bind to the individual hypersensitive site elements. The unique property of the LCR is its activating function, which “opens” the chromatin domain and provides the possibility for gene transcription. In transgenic mice, the LCR is recognized by its capacity to confer integration site- or position-independent expression of a linked gene.99,100 Position effects are always overcome by the LCR in a dominant manner.101,100 Experiments in knockout mice have been recently interpreted to indicate that the LCR is not required for opening the chromatin domain.102–104 In ε-␥ -␦-␤ thalassemia mutants due to LCR deletions,105–109 there is total inactivation of the ␤-locus chromatin and total absence of transcription of the ␤-cluster genes in cis. However, when the LCR is deleted from the endogenous murine locus by homologous recombination, the globin genes continue to show some low levels of expression, and the chromatin of the ␤ locus remains in the open configuration.102,104,110 Why the phenotypes of the LCR deletions in humans and the LCR knockouts in mice differ is still unknown.111,112 Among the possible reasons are differences in the composition and organization of the murine and the human LCRs. Alternatively, the total silencing of the ␤ locus in the human LCR deletions might not be due to the deletion of the LCR per se, but the juxtaposition to the locus of heterochromatic regions, located upstream, that silence the genes of the locus. The DNase I hypersensitive sites of the LCR have developmental specificity.113 This was unequivocally shown in the studies of transgenic mice carrying ␤ locus yeast artificial chromosomes (YAC mice). Deletion of the core element of HS3 in the context of a ␤ locus YAC results in total absence of ε-globin gene expression in day-9 embryonic cells,114 suggesting that sequences of the core element of HS3 are necessary for activation of ε-globin gene transcription. ␥ -Gene expression in embryonic cells is

Major insights on the molecular control of switching have been obtained through studies of transgenic mice. As mentioned earlier, in the mouse there is only one switch during development – the switch from embryonic to definitive globin gene expression, which coincides with the transition from yolk sac to definitive, fetal liver, erythropoiesis. The murine εy and ␤h1 genes are expressed exclusively in the yolk sac and they are silenced in the fetal liver where ␤major- and ␤minor-globin gene expression occurs. The εy gene is homologous to human ε whereas the ␤h1 is homologous to human ␥ . Studies of transgenic mice carrying human ␥ - or ␤-globin transgenes, performed before the discovery of the LCR, have shown that the human ␥ and ␤ transgenes are regulated similarly to their murine homologous genes (references in Stamatoyannopoulos and Grosveld20 ). Thus, the ␥ genes, like the murine ␤h1, are expressed only in the yolk sac cells whereas the ␤ genes are expressed only in the definitive cells, indicating that all the elements required for correct developmental regulation are included in the sequences of the genes or their flanking sequences. With the discovery of the LCR, questions arose about how the globin genes are developmentally regulated in the presence of this powerful regulatory element. Studies in transgenic mice revealed that two mechanisms, gene silencing and gene competition, control hemoglobin switching.

Globin Gene Silencing The studies of cis elements and trans factors involved in turning off the embryonic globin gene provide a good example of the complexity of the control of gene silencing during development. ε-Globin gene expression is totally restricted in the embryonic yolk sac cells and its developmental control is autonomous, that is, all the sequences required for silencing of the ε gene in definitive erythropoietic cells are contained in the sequences flanking the gene.115,116 Regulatory sequences mediating this autonomous silencing have been mapped to the distal and proximal ε-gene promoter.40,43,44,117,118 Controversy has been generated with the studies of a putative negative regulatory element initially identified in the upstream gene promoter by using transient transfection assays.119 This element is located between −182 and

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adult erythroid cells. The function of this sequence of the ε-gene promoter is thus still unclear. As mentioned earlier, COUP-TF and/or DRED binding in the DR repeats near the CAAT box has suppressive effects and there is evidence that sequences having silencing properties are located further upstream in the ε-gene promoter. The mechanism that turns off the ␥ -globin gene has been more difficult to determine. Initially, the silencing of the ␥ gene was attributed solely to gene competition.124,125 Other experiments in transgenic mice suggested that the gene turns off solely through an autonomous silencing mechanism.126 It seems that autonomous silencing is the main mechanism whereby the ␥ genes are turned off during development. Evidence Figure 5.2. Globin gene silencing. The middle diagram shows the sequence of the upstream gene was provided by two types of experiments. promoter, which when deleted results in continuation of ␥ -gene expression in the adult. The lower diagram shows the binding sites for transcriptional factors contained in this silencer. First, in transgenic mice carrying ␤YAC constructs from which the ␤ gene has been deleted,127 the ␥ genes turn off after birth, even though the −467 bp from the initiation site and contains three binding motifs: a GATA site at −208, a YY1 site at −269, and ␤ genes are absent, thus arguing against the hypothesis a CACC motif at −37943,120 (Fig. 5.2). Deletion of the elethat ␥ -gene silencing is solely the result of competition for trans factors and/or the LCR by the ␤ gene. Second, when ment resulted in ε gene expression in the red cells of adult the ␤-globin gene is placed close to the LCR, it is expressed transgenic mice carrying an ε gene with an upstream micro throughout development.128,129 When the ␥ gene is placed LCR.121 Disruption of either the −208 GATA-1 or the −269 YY1 binding site also resulted in ε-gene expression in adult in the same position, it is expressed in the embryonic and transgenic mice.43 Presumably, several transcriptional facthe early fetal liver cells, but it is turned off postnatally, as expected if ␥ -gene silencing is autonomous.129 However, tors interact to form the silencing complex and disruption of any of these factors results in inhibition of silencing. the story is not that simple: other transgenic studies130 as ε-Gene silencing, therefore, is probably combinatorial well as the increase in ␥ -globin gene expression in patients (Fig. 5.3). The fact that GATA-1 binding at −208 results in εwith ␤ thalassemia due to ␤-gene promoter deletions,20 gene suppression was subsequently shown using a binary suggest that competition by the ␤-gene promoter, in additransgenic mouse system: Overexpression of GATA-1 in tion to autonomous silencing, contributes to the turning transgenic mice carrying a human ␤ locus off of the ␥ -globin gene. YAC resulted in a specific decrease of human ε-globin expression.122 The function of this ε-gene silencer was, however, questioned by studies in transgenic mice containing an intact human ␤-globin locus. Thus, deletion of a portion (125 bp) of the sequence of the ε silencer in a ␤YAC construct did not lead to expression of the ε gene in definitive erythropoietic cells.94 In contrast, the deletion resulted in a significant decrease of ε-gene expression in the yolk sac, suggesting that the deleted sequence could harbor a cryptic activity that is required for stimulation of εglobin RNA synthesis. Transgenic mice carrying a ␤YAC construct harboring a slightly larger (224 bp vs. 125 bp) deletion of the silencer123 had no abnormalities in ε-gene Figure 5.3. Evidence that the silencing of the ε gene is combinatorial. Mutations that affect binding expression in either embryonic or in definiof GATA-1 at −208 or YY1 at −269 or a CACCC binding protein at −379 result in continuation of tive erythropoietic cells, and there was no ε-globin gene expression in the adult. Other transcriptional factors involved in silencing include continuation of ε-gene expression in fetal or COUP-TF that binds to the DR-1 element near the gene’s CAAT box.

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George Stamatoyannopoulos, Patrick A. Navas, and Qiliang Li demonstrate that actively transcribed globin genes are located in proximity to the LCR, suggesting that a chromatin loop is formed when a globin gene is enhanced by the LCR.135–137 The formation of the loop between the LCR and the ␤-globin gene requires erythroid-specific trans-acting factors EKLF, GATA-1, and its cofactor FOG-1, but not NFE2.138–140 Binding of these factors to their cognate cis elements is not sufficient for loop formation,140 and binding of trans-acting factors represents an independent event that occurs prior to loop formation.139 Thus, although EKLF, GATA-1, and FOG are indispensable for loop formation, they use a complex pathway to regulate the process.

Control of HbF in the Adult Figure 5.4. Model of the competitive control of hemoglobin switching. “S” indicates the activity of a silencer element.

Gene Competition The initial observation that led to the formulation of the competition model was made in transgenic mice carrying either the ␥ - or the ␤-globin gene or both genes linked to the LCR. When the genes were alone, developmental control was lost. When the genes were linked together, developmental control was restored. Such findings led to the proposal that the ␥ -globin gene is regulated through competition with the ␤-globin gene and vice versa.124,125 The hypothesis is that in the embryonic stage, the LCR interacts with the ε-globin gene; the downstream genes are being turned off competitively. In the fetus, the ε gene is silenced, and the LCR interacts with the G ␥ and A ␥ genes. In the adult, the ␥ genes are silenced, and the LCR now interacts with the ␤-globin gene, the last gene of the locus (Fig. 5.4). Two conditions influence the probability of interaction of a gene with the LCR: the prevailing transcriptional environment and the distance from the LCR. Among the transacting factors that are likely to facilitate the interaction of the LCR with the ␥ - or ␤-gene promoters is EKLF and perhaps other factors of the KLF/SP1 family. In addition to the trans-acting factors, gene order and proximity to the LCR are important in determining a gene’s competitive advantage for interaction with the LCR.131,132 The closer the gene, the higher is the advantage. Its placement at the 3
end of the locus might explain why the ␤ gene is totally shut off in embryonic cells when it is located in its normal chromosomal position, whereas it is always expressed in the embryo if it is placed next to the LCR. In situ hybridization methods have allowed the visualization of the interaction of globin genes with the LCR.100,133 This element interacts with only one promoter of the locus at a given time, and switching essentially represents a change in frequency of interaction of the LCR with either the ␥ - or the ␤-gene promoter. Results from newly developed technologies, such as 3C134 and RNA trap assays,

One of the most interesting characteristics of human ␥ - to ␤-switching is its leakiness and the continuation of synthesis of small amounts of HbF in the adult. This has been known since the time the alkali denaturation method was used for HbF quantitation, but its significance was only realized when immunofluorescent methods were used to stain peripheral blood smears. These methods were first applied in the mid-1960s and they were rediscovered in the 1970s. It was then realized that this residual ␥ -globin expression is restricted to a minority of cells, the F cells. The question was then raised about how these F cells are formed. Initially, clonal hypotheses (reviewed earlier in this chapter) were proposed to explain the origin of F cells: They could be the progeny of fetal stem cell clones. Major insights into the understanding of the control of HbF in the adult were obtained through analyses of HbF expression in erythroid cultures and through observations in patients with activated erythropoiesis. The first clue on mechanisms came from studies in erythroid cultures, which showed that high levels of HbF are characteristic of colonies produced by erythroid burstforming units (BFU-E) of adult origin.13,141 In erythroid cells of these colonies, HbF was not uniformly distributed but the colonies were usually composed of erythroblasts that contained both HbF and HbA, and erythroblasts that contained only HbA. These observations were interpreted to indicate that the production of F cells was related to the phenomenon of erythroid cell differentiation.13 The second clue on mechanisms came from studies showing that rapid regeneration of the erythroid marrow induces F-cell production (reviewed in Stamatoyannopoulos et al.142 ). For example, increased F-cell production is characteristic of bone marrow regeneration following bone marrow transplantation,10 or following recovery from the aplastic phase of erythroblastopenia of childhood,143 or following chemotherapeutic ablation of the bone marrow,144 and following acute hemolysis.143 Experimental acute bleeding in baboons activated ␥ -globin production.145,146 Acute phlebotomy and decrease of hematocrit in humans stimulated F-cell production.143 Proof that acute erythropoietic stress can induce HbF production was obtained

Molecular and Cellular Basis of Hemoglobin Switching when baboons were treated with high doses of recombinant erythropoietin: These animals responded with striking elevation of F-cell production.147,148 It should be mentioned that in contrast to the consistent activation of HbF in acute erythropoietic expansion, with the exception of hemoglobinopathies and congenital hypoplastic anemias, there is no elevation of HbF in most patients with chronic anemias.149 Administration of low doses of erythropoietin to baboons increases the hematocrit but fails to induce HbF.147 Following acute bleeding, there is a surge of F-reticulocyte production, but when chronic anemia is instituted, the number of F-reticulocytes falls.142,146 The difference in the rates of F-cell formation between acute and chronic erythropoietic stress provided strong evidence that the kinetics of erythroid regeneration determine whether a cell will become an F cell or an A cell. The mechanism proposed to explain the induction of HbF in response to erythropoietic stress assumes that early progenitors encode a program allowing expression of fetal globin genes, but this program is changed to one allowing only adult globin expression during the downstream differentiation of erythroid progenitor cells (Fig. 5.5).13,150 Presumably, the earlier progenitor cells contain a combination of trans-acting factors that favors ␥ -globin gene expression, whereas the late progenitors have a combination of trans-acting factors that favors ␤-globin gene expression. F cells are produced when earlier progenitors become committed to terminal differentiation prematurely.150 In acute erythropoietic stress, the accelerated erythropoiesis increases the chance of premature commitment of early progenitors, resulting in increased production of F cells. Experimental evidence in support of this hypothesis was obtained by daily measurements of erythroid progenitor pools in baboons treated with high doses of recombinant erythropoietin.148 The major effect of erythropoietin in vivo is an acute expansion of colony-forming unit (CFU-E) and a mobilization of BFU-E. Umemura et al.148 showed that following the administration of high doses of erythropoietin, an increase in F-programmed CFU-E accounts for almost all of the expansion of CFU-E. The increase in these Fprogrammed CFU-E is followed by a striking increase in F-positive early erythroblasts, which precedes the appearance of F reticulocytes in the circulation.148

THE CONCEPTUAL BASIS OF PHARMACOLOGICAL INDUCTION OF FETAL HEMOGLOBIN SYNTHESIS The pharmacological induction of HbF synthesis was a direct consequence of the studies on the cellular control of HbF production in the adult. Cytotoxic drugs were initially used to test, in primates, whether acute regeneration will induce HbF synthesis in the adult. The use of cytotoxic drugs in patients with sickle cell disease or with ␤ thalassemia followed. The origin of the use of cytotoxic drugs for HbF induction can be traced to the debate about the mechanism

93

Figure 5.5. Model of regulation of fetal hemoglobin and F-cell production in the adult following acute erythroid regeneration or treatment with cytotoxic drugs such as hydroxyurea.

whereby 5-azacytidine stimulates HbF production. To test the hypothesis that DNA demethylation can activate ␥ globin gene expression, DeSimone et al.151 treated anemic juvenile baboons with escalating doses of 5-azacytidine; a striking augmentation of HbF production was observed. Induction of HbF synthesis was subsequently demonstrated in ␤ thalassemia patients treated with 5azacytidine.152 At this stage, a debate about the mechanism of this phenomenon started. 5-azacytidine, a cytotoxic compound, is expected to kill the most actively cycling erythroid cells. The resulting decrease in late erythroid progenitor cells could trigger rapid erythroid regeneration and induce F-cell formation. Therefore, it was argued that

94 the induction of HbF was not simply due to the demethylating effect of 5-azacytidine but to its cytotoxicity that triggers secondary erythroid regeneration. Measurements of erythroid progenitor cell pools in baboons treated with 5-azacytidine supported this hypothesis.153 To test whether cytoreduction and the ensuing secondary erythroid regeneration were the cause of HbF induction by 5-azacytidine, Papayannopoulou et al.154 asked whether other cytotoxic compounds producing erythroid regeneration but not DNA demethylation would also induce F-cell formation. Baboons were treated with cytotoxic doses of ara-C and responded with striking elevations of F reticulocytes, with kinetics indistinguishable from those elicited by 5-azacytidine.154 Induction of ␥ -globin gene expression was also observed in monkeys or baboons treated with hydroxyurea.154,155 Vinblastine, a cell cycle– specific agent that arrests cells in mitosis, also produces secondary erythroid regeneration and stimulates HbF synthesis in baboons.156 Following these studies, hydroxyurea was used for induction of HbF production in humans (see Chapter 30). Although other hypotheses for the mechanisms of action of hydroxyurea have been proposed, its activation of HbF synthesis through stimulation of erythroid regeneration is broadly accepted, although the initial rational for using cytotoxic drugs for stimulation of HbF production has been forgotten.157–159

George Stamatoyannopoulos, Patrick A. Navas, and Qiliang Li discovering HbF inducers that can be administered orally and are more potent than butyrate. The prevailing hypothesis is that short-chain fatty acids activate ␥ -globin gene expression through inhibition of histone deacetylases. Histone acetyltransferases catalyze histone acetylation through the transfer of acetyl groups to lysine residues of the core histones.172–174 It is believed that histone acetylation leads to gene activation by weakening the binding of histones to nucleosomal DNA, which makes the DNA subsequently accessible to transcription factors.175 Conversely, histone deacetylases are believed to largely mediate gene repression, as deacetylation of histones would allow the histone to bind more tightly to the nucleosomal DNA and displace transcription factors. Thus, histone deacetylase inhibitors may induce ␥ -globin gene activity by increasing the accessibility of chromatin around the ␥ -globin gene promoter to activating transcription factors. The exact mechanism whereby the short-chain fatty acids affect gene transcription remains unknown. Studies in transgenic mice are compatible with the assumption that the stimulation of HbF synthesis reflects inhibition of silencing rather than activation of transcription,176 but the evidence is indirect. It is obvious that the delineation of the mechanisms of stimulation of HbF synthesis by short-chain fatty acids will provide new insights into the control of silencing or activation of ␥ -globin gene expression.

Short-Chain Fatty Acids The seminal observation that eventually led to the discovery that short-chain fatty acids induce the synthesis of HbF was the finding by Perrine et al.160 that the ␥ - to ␤-switch is delayed in infants of diabetic mothers. Perrine and coworkers hypothesized that a metabolite in the blood of diabetic mothers was responsible for this finding and, using experiments in clonal erythroid cell cultures, they showed that ␥ -aminobutyric acid, which is elevated in the blood of diabetic mothers, is an inducer of HbF production.161 Subsequent studies showed that butyrate stimulated ␥ -globin chain production in adult baboons,162 and it induced ␥ globin gene expression in erythroid progenitors of adult animals or of patients with sickle cell anemia.162,163 Several other short-chain fatty acids were found to increase HbF in adult BFU-E cultures and in baboons.164,165 Derivatives of short-chain fatty acids such as phenylbutyrate166 and valproic acid165,167 induce HbF production in vivo. Increased levels of HbF were also recorded in patients with metabolic disorders resulting in accumulation of shortchain fatty acids.168,169 Butyrate and various short chain fatty acid derivatives have been used in a number of clinical trials (see Chapter 30). The induction of HbF production by short-chain fatty acids is very interesting from the practical and biological points of view. The practical significance lies in the fact that there are very large numbers of short-chain fatty acid derivatives that are potential inducers of HbF synthesis.170,171 Therefore, there are ample opportunities for

Role of the BCL11A Locus Recent studies have identified the BCL11A locus as a major locus regulating the levels of fetal hemoglobin in ␤thalassemia or sickle cell disease. A SNP located in the second intron of the BCL11A gene was found to be correlated with HbF levels in patients with ␤-thalassemia suggesting that this genetic polymorphism is an important indicator of disease severity.177,178 The BCL11A gene encodes three isoforms of a multi-zinc finger transcription factor and is developmentally regulated such that only the two largest isoforms (X and XL) are exclusively expressed during adult erythropoiesis.179 BCL11A binds to GG-rich motifs and has been shown to function as a transcription repressor.180,181 BCL11A knockdown experiments in adult erythroid progenitor cells resulted in a dramatic increase in F-cells numbers and HbF levels suggesting that BCL11A is involved in ␥ -globin gene silencing.179 Chromatin immunoprecipitation experiments showed that BCL11A directly binds to several locations of the ␤-globin locus in adult erythroid progenitor cells.179 Electromobility shift assays using extracts from BCL11A over expressing K562 cells showed BCL11A binding to a GGCCGG motif at position −56 to −51 of the G ␥ gene promoter.182 Collectively the studies of patients and the biochemical investigations strongly suggest that BCL11A acts as a stage specific repressor of ␥ -globin expression. Thus, BCL11A has emerged as an attractive target for reactivation of HbF in patients with ␤-thalassemia or sickle cell disease.

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HS1, and regions upstream of the LCR, are not required for ␤-globin transcription. Blood. 2006;108:1395–1401. 111. Higgs DR. Do LCRs open chromatin domains? Cell. 1998; 95:299–302. 112. Grosveld F. Activation by locus control regions? Curr Opin Genet Dev. 1999;9:152–157. 113. Fraser P, Pruzina S, Antoniou M, Grosveld F. Each hypersensitive site of the human ␤-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev. 1993;7:106–113. 114. Navas PA, Peterson KR, Li Q, et al. Developmental specificity of the interaction between the locus control region and embryonic or fetal globin genes in transgenic mice with an HS3 core deletion. Mol Cell Biol. 1998;18:4188–4196. 115. Raich N, Enver T, Nakamoto B, Josephson B, Papayannopoulou T, Stamatoyannopoulos G. Autonomous developmental control of human embryonic globin gene switching in transgenic mice. Science. 1990;250:1147–1149. 116. Shih DM, Wall RJ, Shapiro SG. Developmentally regulated and erythroid-specific expression of the human embryonic ␤-globin gene in transgenic mice. Nucl Acids Res. 1990;18:5465–5472. 117. Wada-Kiyama Y, Peters B, Noguchi CT. The ε-globin gene silencer. Characterization by in vitro transcription. J Biol Chem. 1992;267:11532–11538.

George Stamatoyannopoulos, Patrick A. Navas, and Qiliang Li 118. Li Q, Blau CA, Clegg CH, Rohde A, Stamatoyannopoulos G Multiple ε-promoter elements participate in the developmental control of ε-globin genes in transgenic mice. J Biol Chem. 1998;273:17361–17367. 119. Cao SX, Gutman PD, Dave HP, Schechter AN. Negative control of the human ε-globin gene. Prog Clin Biol Res. 1989;316A:279–289. 120. Peters B, Merezhinskaya N, Diffley JF, Noguchi CT. ProteinDNA interactions in the ε-globin gene silencer. J Biol Chem. 1993;268:3430–3437. 121. Raich N, Papayannopoulou T, Stamatoyannopoulos G, Enver T. Demonstration of a human ε-globin gene silencer with studies in transgenic mice. Blood. 1992;79:861–864. 122. Li Q, Clegg C, Peterson K, Shaw S, Raich N, Stamatoyannopoulos G. Binary transgenic mouse model for studying the trans control of globin gene switching: evidence that GATA-1 is an in vivo repressor of human ε gene expression. Proc Natl Acad Sci USA. 1997;94:2444–2448. 123. Navas PA, Li Q, Peterson KR, Stamatoyannopoulos G. Investigations of a human embryonic globin gene silencing element using YAC transgenic mice. Exp Biol Med (Maywood). 2006;231:328–334. 124. Behringer RR, Ryan TM, Palmiter RD, Brinster RL, Townes TM. Human ␥ - to ␤-globin gene switching in transgenic mice. Genes Dev. 1990;4:380–389. 125. Enver T, Raich N, Ebens AJ, Papayannopoulou T, Costantini F, Stamatoyannopoulos G. Developmental regulation of human fetal–to-adult globin gene switching in transgenic mice. Nature. 1990;344:309–313. 126. Dillon N, Grosveld F. Human ␥ -globin genes silenced independently of other genes in the ␤-globin locus. Nature. 1991;350:252–254. 127. Peterson KR, Li QL, Clegg CH, et al. Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of ␤-globin locus YAC mice carrying human globin developmental mutants. Proc Natl Acad Sci USA. 1995;92:5655–5659. 128. Dillon N, Trimborn T, Strouboulis J, Fraser P, Grosveld F. The effect of distance on long-range chromatin interactions. Mol Cell. 1997;1:131–139. 129. Harju S, Navas PA, Stamatoyannopoulos G, Peterson KR. Genome architecture of the human ␤-globin locus affects developmental regulation of gene expression. Mol Cell Biol. 2005;25:8765–8778. 130. Yu M, Han H, Xiang P, Li Q, Stamatoyannopoulos G. Autonomous silencing as well as competition controls ␥ globin gene expression during development. Mol Cell Biol. 2006;26:4775–4781. 131. Hanscombe O, Whyatt D, Fraser P, et al. Importance of globin gene order for correct developmental expression. Genes Dev. 1991;5:1387–1394. 132. Peterson KR, Stamatoyannopoulos G. Role of gene order in developmental control of human ␥ - and ␤-globin gene expression. Mol Cell Biol. 1993;13:4836–4843. 133. Wijgerde M, Grosveld F, Fraser P. Transcription complex stability and chromatin dynamics in vivo. Nature. 1995;377:209– 213. 134. Dekker J, Rippe K, Dekker M, Kleckner N. Capturing chromosome conformation. Science. 2002;295:1306–1311. 135. Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W. Looping and interaction between hypersensitive sites in the active ␤-globin locus. Mol Cell. 2002;10:1453–1465.

Molecular and Cellular Basis of Hemoglobin Switching 136. Palstra RJ, Tolhuis B, Splinter E, Nijmeijer R, Grosveld F, de Laat W. The ␤-globin nuclear compartment in development and erythroid differentiation. Nat Genet. 2003;35:190–194. 137. Carter D, Chakalova L, Osborne CS, Dai YF, Fraser P. Longrange chromatin regulatory interactions in vivo. Nat Genet. 2002;32:623–626. 138. Drissen R, Palstra RJ, Gillemans N, et al. The active spatial organization of the ␤-globin locus requires the transcription factor EKLF. Genes Dev. 2004;18:2485–2490. 139. Vakoc CR, Letting DL, Gheldof N, et al. Proximity among distant regulatory elements at the ␤-globin locus requires GATA1 and FOG-1. Mol Cell. 2005;17:453–462. 140. Kooren J, Palstra RJ, Klous P, et al. B-globin active chromatin Hub formation in differentiating erythroid cells and in p45 NF-E2 knock-out mice. J Biol Chem. 2007;282:16544–16552. 141. Papayannopoulou TH, Brice M, Stamatoyannopoulos G. Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals. Proc Natl Acad Sci USA. 1976;73:2033–2037. 142. Stamatoyannopoulos G, Veith R, Galanello R, Papayannopoulou T. Hb F production in stressed erythropoiesis: observations and kinetic models. Ann NY Acad Sci. 1985; 445:188–197. 143. Papayannopoulou T, Vichinsky E, Stamatoyannopoulos G. Fetal Hb production during acute erythroid expansion. I. Observations in patients with transient erythroblastopenia and post-phlebotomy. Br J Haematol. 1980;44:535–546. 144. Sheridan BL, Weatherall DJ, Clegg JB, et al. The patterns of fetal haemoglobin production in leukaemia. Br J Haematol. 1976;32:487–506. 145. DeSimone J, Biel SI, Heller P. Stimulation of fetal hemoglobin synthesis in baboons by hemolysis and hypoxia. Proc Natl Acad Sci USA. 1978;75:2937–2940. 146. Nute PE, Papayannopoulou T, Chen P, Stamatoyannopoulos G. Acceleration of F-cell production in response to experimentally induced anemia in adult baboons (Papio cynocephalus). Am J Hematol. 1980;8:157–168. 147. Al-Khatti A, Veith RW, Papayannopoulou T, Fritsch EF, Goldwasser E, Stamatoyannopoulos G. Stimulation of fetal hemoglobin synthesis by erythropoietin in baboons. N Engl J Med. 1987;317:415–420. 148. Umemura T, Al-Khatti A, Papayannopoulou T, Stamatoyannopoulos G. Fetal hemoglobin synthesis in vivo: direct evidence for control at the level of erythroid progenitors. Proc Natl Acad Sci USA. 1988;85:9278–9282. 149. Beaven GH, Ellis MJ, White JC. Studies on human foetal haemoglobin. II. Foetal haemoglobin levels in healthy children and adults and in certain haematological disorders. Br J Haematol. 1960;6:201–222. 150. Stamatoyannopoulos G, Papayannopoulou T. Fetal hemoglobin and the erythroid stem cell differentiation process. In: Stamatoyannopoulos G, Nienhuis AW, eds. Cellular and Molecular Regulation of Hemoglobin Switching. New York: Grune & Stratton; 1979:323–349. 151. DeSimone J, Heller P, Hall L, Zwiers D. 5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons. Proc Natl Acad Sci USA. 1982;79:4428–4431. 152. Ley TJ, DeSimone J, Anagnou NP, et al. 5-azacytidine selectively increases ␥ -globin synthesis in a patient with b+thalassemia. N Engl J Med. 1982;307:1469–1475. 153. Torrealba de Ron AT, Papayannopoulou T, Knapp MS, Fu MF, Knitter G, Stamatoyannopoulos G. Perturbations in the

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erythroid marrow progenitor cell pools may play a role in the augmentation of HbF by 5-azacytidine. Blood. 1984;63:201– 210. Papayannopoulou T, Torrealba de Ron A, Veith R, Knitter G, Stamatoyannopoulos G. Arabinosylcytosine induces fetal hemoglobin in baboons by perturbing erythroid cell differentiation kinetics. Science. 1984;224:617–619. Letvin NL, Linch DC, Beardsley GP, McIntyre KW, Nathan DG. Augmentation of fetal-hemoglobin production in anemic monkeys by hydroxyurea. N Engl J Med. 1984;310:869– 873. Veith R, Papayannopoulou T, Kurachi S, Stamatoyannopoulos G. Treatment of baboon with vinblastine: insights into the mechanisms of pharmacologic stimulation of Hb F in the adult. Blood. 1985;66:456–459. Fibach E, Burke LP, Schechter AN, Noguchi CT, Rodgers GP. Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or ␤-thalassemia. Blood. 1993;81: 1630–1635. Platt OS, Falcone JF. Membrane protein interactions in sickle red blood cells: evidence of abnormal protein 3 function. Blood. 1995;86:1992–1998. Steinberg MH, Lu ZH, Barton FB, Terrin ML, Charache S, Dover GJ. Fetal hemoglobin in sickle cell anemia: determinants of response to hydroxyurea. Multicenter study of hydroxyurea. Blood. 1997;89:1078–1088. Perrine SP, Greene MF, Faller DV. Delay in the fetal globin switch in infants of diabetic mothers. N Engl J Med. 1985; 312:334–338. Perrine SP, Miller BA, Greene MF, et al. Butryic acid analogues augment ␥ globin gene expression in neonatal erythroid progenitors. Biochem Biophys Res Commun. 1987;148: 694–700. Constantoulakis P, Papayannopoulou T, Stamatoyannopoulos G. ␣-Amino-N-butyric acid stimulates fetal hemoglobin in the adult. Blood. 1988;72:1961–1967. Perrine SP, Miller BA, Faller DV, et al. Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and b thalassemia. Blood. 1989;74:454–459. Stamatoyannopoulos G, Nienhuis AW. Hemoglobin switching. In: Stamatoyannopoulos G, Nienhuis AW, Majerus P, Varmus H, eds. Molecular Basis of Blood Diseases. 2nd ed. Philadelphia: W.B. Saunders Co.; 1994:107–154. Liakopoulou E, Blau CA, Li Q, et al. Stimulation of fetal hemoglobin production by short chain fatty acids. Blood. 1995;86:3227–3235. Dover GJ, Brusilow S, Charache S. Induction of fetal hemoglobin production in subjects with sickle cell anemia by oral sodium phenylbutyrate. Blood. 1994;84:339–343. Collins AF, Dover GJ, Luban NL. Increased fetal hemoglobin production in patients receiving valproic acid for epilepsy. Blood. 1994;84:1690–1691. Little JA, Dempsey NJ, Tuchman M, Ginder GD. Metabolic persistence of fetal hemoglobin. Blood. 1995;85:1712–1718. Peters A, Rohloff D, Kohlmann T, et al. Fetal hemoglobin in starvation ketosis of young women. Blood. 1998;91:691– 694. Cao H, Stamatoyannopoulos G, Jung M. Induction of human ␥ globin gene expression by histone deacetylase inhibitors. Blood. 2004;103:701–709.

100 171. Pace BS, White GL, Dover GJ, Boosalis MS, Faller DV, Perrine SP. Short-chain fatty acid derivatives induce fetal globin expression and erythropoiesis in vivo. Blood. 2002;100:4640– 4648. 172. Kuo MH, Brownell JE, Sobel RE, et al. Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. Nature. 1996;383:269–272. 173. Mizzen CA, Yang XJ, Kokubo T, et al. The TAF(II)250 subunit of TFIID has histone acetyltransferase activity. Cell. 1996;87:1261–1270. 174. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell. 1996;87:953–959. 175. Vettese-Dadey M, Grant PA, Hebbes TR, Crane- Robinson C, Allis CD, Workman JL. Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro. EMBO J. 1996;15:2508–2518. 176. Pace B, Li Q, Peterson K, Stamatoyannopoulos G. ␣-Amino butyric acid cannot reactivate the silenced ␥ gene of the ␤ locus YAC transgenic mouse. Blood. 1994;84:4344–4353. 177. Uda M, Galanello R, Sanna S, et al. Genome-wide association study shows BCL11A associated with persistent

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fetal hemoglobin and amelioration of the phenotype of ␤-thalassemia. Proc Natl Acad Sci USA. 2008;105:1620– 1625. Lettre G, Sankaran VG, Bezerra MA, et al. DNA polymorphisms at the BCL11A, HBS1L-MYB, and ␤-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease. Proc Natl Acad Sci USA. 2008;105:11869– 11874. Sankaran VG, Menne TF, Xu J, et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science. 2008;322:1839–1842. Senawong T, Peterson VJ, Leid M. BCL11A-dependent recruitment of SIRT1 to a promoter template in mammalian cells results in histone deacetylation and transcriptional repression. Arch Biochem Biophys. 2005;434:316–325. Liu H, Ippolito GC, Wall JK, et al. Functional studies of BCL11A: characterization of the conserved BCL11A-XL splice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells. Mol cancer. 2006;5:18–34. Chen, Z, Luo, HY, Steinberg, MH, Chui DH. BCL11A represses HBG transcription in K562 cells. Blood Cells Mol Dis. 2009;42:144–149.

6 Structure and Function of Hemoglobin and Its Dysfunction in Sickle Cell Disease Daniel B. Kim-Shapiro

INTRODUCTION Hemoglobin has evolved to be an efficient oxygen (O2 ) transporter. Its function, understood in terms of a two-state model of allostery, serves as a paradigm for many other proteins. A single ␤-globin gene (HBB glu6val) point mutation resulting in sickle hemoglobin (HbS) is the proximate cause of sickle cell disease (Chapter 19). The primary cause of the disease is HbS polymerization that injures and deforms the sickle erythrocyte, causing many pathological consequences discussed elsewhere in this book.

STRUCTURAL ASPECTS OF HEMOGLOBIN Hemoglobin is a 64-kD, nearly spherical protein with a diameter of approximately 5.5 nm. Its three-dimensional structure was solved by Max F. Perutz who discussed the molecular anatomy and physiology of hemoglobin in the first edition of this book.1 It is a dimer of dimers, with two ␣ subunits and two ␤ subunits (Fig. 6.1). The ␣ chains have 141 amino acid residues and the ␤ chains have 146 residues. Each of the ␣ and ␤ chains resemble each other closely in both secondary (␣ helical) and tertiary structure. Moreover, even though the primary amino acid sequence is different, each subunit also resembles myoglobin, a heme-containing globin having only one subunit in both secondary and tertiary structure. Generally, nonpolar groups are found in the interior of the subunits and polar residues are found on the surface. The SH group of the cysteine at position 93 of the ␤ chain is exposed to solvent in the oxygenated form of hemoglobin, but it is partially hidden when hemoglobin is deoxygenated. This is due to the change in quaternary structure of the protein when hemoglobin binds O2 . One ␣␤ dimer rotates approximately 120 with respect to the other and moves approximately 0.1 nm along the rotation axis.

Each of the subunits of the tetramer contains a heme prosthetic group (Figs. 6.1 and 6.2). Hemes are attached to the globin protein via a histidine side chain (Fig. 6.2). Heme is an iron-containing protoporphyrin IX, a tetrapyrrole with an iron atom at its center. The iron is usually ferrous, having a valency of +2. It can be oxidized to the ferric form (+3) and is then commonly referred to as methemoglobin. In the ferrous form, the heme group can bind to gaseous ligands including O2 , CO, and NO and can also bind alkylisocyanides.2 In the ferric form, hemoglobin does not bind to O2 or CO. It binds to NO, but with a much lower affinity than ferrous heme. Ferric hemoglobin also reversibly binds nitrite, nitrate, azide, and binds to cyanide very tightly, forming cyanomethemoglobin. In addition to the heme group, there are several other sites within hemoglobin through which it interacts with small molecules. Bisphosphoglycerate (BPG) and inositol hexaphosphate (IHP) bind in the central cavity of hemoglobin, crosslinking the four subunits. The ␤-93 cysteine binds N-ethylmaleimide, iodoacetamide, and nitrosonium ion (NO+ ), the latter forming S-nitrosated hemoglobin or SNO–hemoglobin. Carbon dioxide binds to the terminal amino groups.

NORMAL HEMOGLOBIN FUNCTION Oxygen Transport The primary function of hemoglobin is to transport O2 from the lungs to the tissues. The pressure and solubility of O2 in liquids make it such that only 200 ␮mol/L, at most, could be carried by blood in the absence of an O2 -carrying protein such as hemoglobin. Whole blood contains approximately 10 mmol/L hemoglobin (in heme), thus greatly increasing the O2 -carrying capacity of blood. The ability of hemoglobin to transport O2 effectively is illustrated by plotting its fractional O2 saturation (hemoglobin bound to O2 /total hemoglobin) against O2 pressure (Fig. 6.3). Hemoglobin binds O2 cooperatively, a phenomenon discovered by Christian Bohr, the father of the famous physicist Niels Bohr.3 Cooperative binding means that the affinity of a hemoglobin tetramer for O2 increases as more O2 is bound. Myoglobin binds O2 noncooperatively. In Figure 6.3, we see that at pressure of 20 mm Hg (close to that of metabolically active tissue), myoglobin is almost completely saturated with O2 , whereas hemoglobin is less than 40% saturated. Thus, hemoglobin has a lower affinity for O2 at this pressure. As the O2 pressure is raised to 90 mm Hg, which is close to that in the lungs, both hemoglobin and myoglobin are fully saturated with O2 so that the hemoglobin–O2 affinity has caught up to that of myoglobin. If myoglobin were contained in red blood cells instead of hemoglobin, then the red blood cells would be fully O2

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Daniel B. Kim-Shapiro 1.0 Hb Oxygen Saturation

Mb 0.75

Hb (pH 7.4) Hb (pH 7.2)

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Figure 6.3. Hemoglobin and myoglobin oxygen binding curves. The myoglobin oxygen binding curve was drawn according to Equation 6.1 with P50 taken as 2 mm Hg. The hemoglobin oxygen binding curves were drawn using Equation 6.2 with n = 2.8 and P50 taken as 26 mm Hg at pH 7.4 and as 35 mm Hg for pH 7.2. (See color plate 6.3.)

The myoglobin O2 saturation, Y, as a function of O2 pressure can be described by the simple relation Figure 6.1. Ribbon diagram of a sickle cell hemoglobin tetramer. Each of the four subunits is shown in a different color. Four heme groups (yellow-orange) are shown with an iron (red) atom in the middle. The valine residues resulting from the single point mutation causing sickle cell disease are shown at the ␤6 position on each ␤ subunit (purple). The molecule is shown looking down the axis where 2,3 bisphosphoglycerate binds. Except for the substitution of valine for glutamate, normal HbA would appear the same as the molecule shown. (The illustration was derived from the Protein Explorer (http://www.umass.edu/microbio/rasmol/) and data from the Protein Data Bank.) (See color plate 6.1.)

loaded in the lungs, but they would not release sufficient O2 in the tissues. By combining four myoglobin-like chains into a single tetramer, hemoglobin is able to function as an efficient O2 transporter.

Figure 6.2. Close up of oxygen bound to the heme. Looking down the heme, the iron atom (yellow-orange) is shown bound to an oxygen molecule (red). The proximal histidine side chain is also shown bound to the iron and the distal histidine is also clearly visible on the other side of the proximal one. (The illustration derived from the Protein Explorer (http://www.umass.edu/microbio/rasmol/) and data from the Protein Data Bank.) (See color plate 6.2.)

Y=

MbO2 pO2 , = MbO2 + Mb pO2 + P50

(6.1)

where pO2 is the O2 pressure and P50 is the O2 pressure where Y = 0.5 (the myoglobin is half saturated with O2 ). The hemoglobin O2 saturation dependence on O2 pressure is more complicated and can be described by Y=

HbO2 (pO2 )n , = HbO2 + Hb (pO2 )n + (P50 )n

(6.2)

where the exponent n is called the Hill coefficient. The Hill coefficient describes the degree of cooperativity in O2 binding. For myoglobin, where there is no cooperativity, n = 1. For hemoglobin, several factors could affect the value of n, but it is usually found to be approximately 2.8 under normal conditions. The ability of hemoglobin to bind O2 cooperatively is well-described in terms of a two-state model developed by Monod, Wyman, and Changeux (MWC).4 According to the model, there are two states of hemoglobin defined by the quaternary structure: the relaxed, high-O2 affinity R-state and the tense, low-O2 affinity T-state. When hemoglobin is completely deoxygenated, it is essentially all in the T-state and thereby has a low affinity. As O2 binds, a hemoglobin tetramer that has 2–3 O2 molecules bound will be likely to undergo the allosteric transition to the R-state, gaining a higher affinity for O2 . Thus, the allostery, whereby binding at one heme site affects binding at another site, explains the cooperative O2 binding of hemoglobin. One of the beautiful aspects of the MWC model is its simplicity. It is assumed that the affinity of a particular subunit heme group is only a function of the quaternary state (R or T) of the tetramer. Only three parameters are needed to apply the model. These are KR , the R-state association constant; KT , the T-state association constant; and L, the quaternary equilibrium constant between unligated tetramers (how much T-state there is vs. R-state in the absence of O2 or

Structure and Function of Hemoglobin and Its Dysfunction in Sickle Cell Disease 1.0 T0 R4

Fraction Species

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Hb Oxygen Saturation Figure 6.4. Fraction of hemoglobin states. The fraction of each state is plotted vs. hemoglobin oxygen saturation. Only the species T0 , T1 , R3 , and R4 are present at large enough fractions to be visible. At zero oxygen saturation the hemoglobin is virtually all in the T0 state and at 100% oxygen saturation it is all in the R4 state. The parameters used were L = 2 × 106 , c = 0.001, and KT = 1/(75 mm Hg). (See color plate 6.4.)

other ligands). The equilibrium constants between Rx and Tx are determined by Lcx , where x represents the number of ligands bound (so R3 is a hemoglobin molecule with three ligands bound in the R quaternary state) and c = KT /KR . As hemes in R-state hemoglobin have a much higher affinity than T-state hemes, the only species that are effectively present at any O2 tension are T0 , R4 , T1 , and R3 (Fig. 6.4). The phenomenon of allostery, action at a distance, whereby binding at one heme site affects the affinity at another is explained by motions of the heme iron coupled to the globin and communicated to other subunits via salt bridges and other interactions.5,6 When O2 binds to the heme iron, the iron moves approximately 0.05 nm into the plane of the heme, pulling along the proximal histidine. This movement is transmitted to the subunit interfaces and leads to disruption of the salt links. Binding of the first O2 molecule to hemoglobin is the most difficult because the many salt links must be broken. As these salt links break, the (tense) tetramer relaxes so that there are fewer salt links. At this point, binding of O2 to the R-state molecule is easier. This relative “relaxed” nature of R-state hemoglobin is evidenced by the fact that the dissociation constant for ligated hemoglobin tetramers into dimers is approximately 1 mM but deoxygenated (T-state) hemoglobin has an extremely low tendency for dimer formation. The MWC–Perutz model is supported by a large amount of theoretical and experimental evidence.7 One of the key elements comes from kinetics studies showing that the rate of ligand binding by a heme depends on the quaternary state of the hemoglobin and not on the number of ligands bound.8 The equilibrium constant describing the ligand affinity, such as that plotted for O2 in Figure 6.3, depends on the rate of association and dissociation, K = kon /koff . The

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cooperativity in equilibrium binding of O2 to hemoglobin is mainly due to the differences in the rate of O2 dissociation, that is approximately 100 times slower for R-state than T-state, rather than differences in the rate of O2 association, that is approximately 10 times faster for R-state than T-state.9,10 Several compounds greatly affect the ligand-binding properties of hemoglobin. These are classified as homotropic effectors (those that effect like ligands) and heterotrophic effectors, such as BPG, protons, chloride, and phosphate. Without BPG, the P50 of hemoglobin (partial pressure of O2 at which the hemoglobin molecule is half saturated) for O2 binding would be approximately 2 mm Hg, rather than approximately 25 mm Hg. According to the MWC– Perutz model, effectors alter the ligand binding by affecting L. BPG binding in the central cavity stabilizes the T-state. N-ethylmaleimide or NO+ binding at the ␤93 cysteine stabilizes the R-state. Thus, SNO–hemoglobin has a higher O2 affinity than hemoglobin that is not nitrosated.11 Lowering the pH also stabilizes the T-state, so that more O2 can be given off under acidic conditions (Fig. 6.3). The two-state MWC–Perutz model is capable of explaining many of the phenomena associated with ligand binding. When applied with more rigor to a variety of phenomenon, however, the need for modification is clear. This should not be a surprise as hemoglobin is not a homotetramer. Thus, a clearly necessary modification of the MWC–Perutz model is to account for chain differences.12,13 The ␣ subunits have a higher equilibrium affinity for O2 than the ␤ subunits, mainly due to faster dissociation rates from ␤ subunits.10,14 These differences in chain affinities are not consistent with a strict interpretation of a two-state model in which the ligand affinity is only a function of quaternary state (T or R). A further, commonly accepted modification involves a slight cooperativity within ␣␤ dimers in the T quaternary state.15,16 This modified two-state model is sufficient to explain a large variety of quantitative equilibrium and kinetic data. Exceptions to these have lead to further extended or alternative models.16–18 The effect of hemoglobin binding of gaseous ligands on O2 affinity is particularly interesting. CO2 reduces the ligand affinity of hemoglobin, similarly to protons. This combination leads to effective O2 delivery to metabolically active tissue. When NO is bound to the ␣ subunits forming ␣ nitrosyl hemoglobin, it acts as a negative allosteric effector, lowering the O2 affinity of the ␤ subunits.19 This is an example of how, in some cases, hemoglobin function at vacant hemes is dependent on the subunits to which ligand is bound and the type of ligand. Thus, ␣ nitrosyl hemoglobin function is not consistent with the MWC–Perutz model. The two-state model is formulated in terms of two structures obtained from x-ray crystallography. In 1992, a new crystal structure of liganded hemoglobin was discovered called R2.20 More recently, other liganded crystal structures have been determined.21 One might wonder which of these is the one present in solution and how

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this information relates to the two-state model.22,23 Using multidimensional and multinuclear nuclear magnetic resonance, it has been found that the solution structure of liganded hemoglobin is actually a dynamic ensemble of states that include those determined by x-ray crystallography.23–25 Similarly, the structure of deoxyhemoglobin in solution is likely to comprise several quaternary states that include the many ones found by x-ray crystallography.26 Thus, the actual picture of how hemoglobin functions is significantly more complicated than that described by a two-state model. For many applications, however, a two-state MWC– Perutz model is sufficient to explain biological phenomena. Nevertheless, it should be kept in mind that, like all models, especially simple ones that are applied to many complex behaviors, it has limitations.

Transport of Other Gases Hemoglobin also transports CO2 , which binds more tightly to deoxyhemoglobin than to oxyhemoglobin, so it is taken up in the tissues and given off in the lungs. In addition, deoxyhemoglobin uptake of protons helps transport CO2 as bicarbonate, HCO− 3 , which is more soluble than CO2 . + CO2 + H2 O ↔ HCO− 3 +H

(6.3)

Without uptake of protons by deoxyhemoglobin, the equilibrium in Equation 6.3, would shift to the left, limiting bicarbonate formation. Thus, cooperative binding of O2 links to that of CO2 so that hemoglobin is an effective transporter of both molecules. CO is produced by heme oxygenase during heme metabolism. The equilibrium affinity of hemoglobin for CO is approximately 200 times higher than that for O2 . This is due to the slow dissociation rate of CO from hemoglobin; O2 actually binds to hemoglobin faster than does CO. Due to its high O2 affinity and slow dissociation rate, CO has been recognized as a poison that disturbs O2 delivery (Chapter 24); however, potential beneficial effects of CO have recently been recognized.27–30 CO has been shown to have antiinflammatory effects and diminish apoptosis.28–30 Recently, infusion of red cells saturated with CO at 25% of blood volume was shown to be effective in hemorrhagic shock resuscitation.27 These beneficial effects of CO and hemoglobin’s role in transporting such activity demand more study. The ability of hemoglobin to destroy NO activity was an important element in the identification of NO as the endothelial-derived relaxing factor.31,32 This is due to the rapid dioxygenation of NO with oxyhemoglobin to form nitrate (Chapter 10). NO can also bind to the heme, and the degree to which this reaction preserves biological activity has been debated. One certainty is that the equilibrium binding affinity of hemoglobin for NO is extremely high, approximately 1,500 times stronger than CO and 500,000 times stronger than O2 .2

Because of its high affinity, little knowledge about NO binding to deoxyhemoglobin can be obtained from equilibrium studies. Virtually any NO added to molar excess hemoglobin will bind the heme – there will be essentially none left in solution. Thus, binding studies have focused on kinetics. Early studies showed that the rate of dissociation of NO from T-state hemoglobin is 100-fold faster than from R-state hemoglobin, with the T-state rate being approximately 10−3 /s.9,33,34 A difference in the dissociation rates from different subunits was also recognized.33,34 A unique feature of NO binding to the ␣ subunits is that when the ␣ nitrosyl hemoglobin is in the T-state, a proportion of iron– proximal histidine bonds break, resulting in a characteristic triplet hyperfine structure in electron paramagnetic resonance spectra.19,34–38 Recently, the dissociation rate of NO from this pentacoordinate ␣ nitrosyl hemoglobin was measured to be 4 × 10−4 /s.39 Thus, the rate of dissociation of NO from hemoglobin is faster for T-state hemoglobin and faster for ␤ subunits than ␣ subunits. Early stopped-flow absorption experiments mixing NO and deoxyhemoglobin found that the association rate of NO with hemoglobin is noncooperative and occurs at a rate of 3 × 107 M/s.40 Experiments examining the rate of release of a fluorescent BPG analog and the rate that partially NO-ligated hemoglobin binds CO indicated that a T- to R-state transition does take place after two–three NO molecules bind a tetramer.40 The rate of NO binding to ␣ and ␤ chains was also found to be identical.41 One study has suggested that although NO binds to Rstate hemoglobin at the same rate as T-state hemoglobin, when the R-state transition has been caused by binding of two–three NO molecules, the rate of R-state association of NO is 100 times faster when the R-state transition has been invoked by O2 binding.42 In other words, NO would bind R3 at the same rate as T0 when the three ligands on R-state are NO but it would bind 100 times faster if they were O2 . Such a phenomenon would violate the tenet of the two-state model whereby binding properties at one heme only depends on the quaternary state of the protein. Subsequent studies have challenged the idea that the binding rate of NO to R-state oxyhemoglobin is faster than to R-state NO hemoglobin.43,44 In addition, photolysis studies using a commonly accepted model of CO bound hemoglobin for oxyhemoglobin have also found that R-state hemoglobin binds NO at the same rate as T-state hemoglobin.45,46 Thus, the preponderance of evidence indicates that the association rate of NO to hemoglobin is independent of the quaternary state. This is likely to be because once in the heme pocket, NO binds the heme extremely quickly in both cases so that the rate-limiting step in NO binding is diffusion of the ligand through the protein to the heme pocket. Examination of both association and dissociation rates of NO shows that hemoglobin binds NO cooperatively, with all of the cooperativity being manifest in the dissociation rates. This is similar to O2 where most of the cooperativity is in the dissociation rates. Due to the faster dissociation

Structure and Function of Hemoglobin and Its Dysfunction in Sickle Cell Disease rate from ␤ subunits, ␣ nitrosyl hemoglobin is the primary form found in equilibrium. The association rate of NO to hemoglobin is only approximately 1.5-fold slower than the rate of the dioxygenation reaction.47 Thus, even at high O2 tensions, some NO will escape destruction via the dioxygention reaction to form NO bound hemoglobin. Another mechanism of potential preservation of NO activity via the formation of SNO–hemoglobin is discussed in Chapter 10, as it is a mechanism whereby NO activity is created via hemoglobin reduction of nitrite. Simple binding of NO to the heme is unlikely to constitute a mechanism of transport due to the very slow dissociation rate. However, a recent study suggests that NO-bound hemoglobin might be dislodged more quickly because of oxidation of the heme due to concurrent reactions of nitrite with oxyhemoglobin.48 The potential of hemoglobin to transport NO, discussed in more depth in Chapter 10, is an area of current intense study.

Methemoglobin In normal physiology, approximately 0.25% of hemes contain ferric iron (methemoglobin). Free heme oxidizes rapidly and aggregates. Incorporation of the heme into hemoglobin prevents aggregation and greatly slows autooxidation, facilitating O2 transport, as methemoglobin does not bind O2 . Low levels of methemoglobin are also maintained by reducing systems within the red blood cell.49–51 Although excessive formation of methemoglobin has been viewed strictly in terms of pathology (Chapter 24), some potential positive roles for methemoglobin have been discussed. Two studies have suggested a role of NO bound to methemoglobin (methemoglobin–NO) in potential transport or delivery of NO activity.52,53 It is likely that methemoglobin–NO forms transiently in the reaction of nitrite with deoxyhemoglobin, in analogy to some bacterial nitrite reductases.54 The dissociation rate of NO from methemoglobin is, however, relatively fast (∼1 s−1 )55 and methemoglobin also undergoes reductive nitrosylation to form ferrous iron nitrosyl hemoglobin.56 The overall affinity of ferrous hemoglobin for NO is approximately 1 million times higher than that of ferric hemoglobin for NO.55 Given that more than 99% of hemoglobin in the red blood cell is ferrous hemoglobin and that NO in the red cell is quickly destroyed via the dioxygenation reaction, it is extremely unlikely that there is any stable methemoglobin–NO in a red cell. The contention that NO is transported in the red cell bound to methemoglobin is untenable due to the relative stability of this species, as demonstrated recently.57 Some ligands bind methemoglobin more tightly than ferrous hemoglobin. Given recent evidence for a role of nitrite in physiology, disease, and therapeutics58 and the potential involvement of hemoglobin (see Chapter 10), the role of nitrite-bound methemoglobin might be worth exploring. At the very least, this could be one way that nitrite is stored in a red blood cell because, even though

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oxyhemoglobin and deoxyhemoglobin are in great excess to methemoglobin, nitrite will preferentially bind to methemoglobin given the relative affinities.

SICKLE CELL HEMOGLOBIN HbS differs from normal adult hemoglobin (HbA) by a single amino acid residue (Fig. 6.1). A variety of physical methods including x-ray diffraction, nuclear magnetic resonance, and circular dichroism all indicate that the protein conformation of a HbS tetramer in solution is the same or at least very similar to that to that of HbA.59 Some evidence exists for subtle changes in the structure of central cavity BPG binding site.60 Similar structure in solution phase HbS and HbA is supported by similar function. The equilibrium binding of solution phase HbS is the same as HbA.61 The bimolecular ligand rebinding rates of solution phase HbS are also the same as those of HbA.62,63 Finally, tertiary and quaternary changes that are induced on CO photolysis of solution phase HbS–CO exhibit the same kinetics as HbA–CO.64 The pathological consequences typical of sickle cell disease must derive, in part, due to a difference in function of HbS compared with HbA. Only one notable exception to the notion that, in the solution phase, HbS tetramers have very similar function as HbA exists: the propensity for HbS autooxidation.65–68 Although increased propensity to form methemoglobin and associated oxidative damage could contribute to several aspects of the disease, this propensity is not likely to be of primary importance in sickle cell disease and HbS polymerization seems paramount.

HbS Polymer The HbS polymer is made of seven twisted double stands (Fig. 6.5).59,69–71 It has a diameter of 21 nm and a mean helical pitch length of 270 nm.59 Each of the double strands is believed to be similar to ones formed by deoxyHbS when it crystallizes (Fig. 6.6). Having the structure of the double strand at 0.2 nm resolution greatly aids in understanding the structure of the 21 nm fiber because information on these larger structures cannot be obtained directly at the atomic level. A variety of techniques including mutational analysis, linear dichroism spectroscopy, resonance Raman spectroscopy, and x-ray diffraction support the idea that the basic building block of the polymer is the double strand with each of these twisted around one another.59,72 Contact sites between tetramers within double strands are known in the most detail.70 Valine at the ␤6 position makes a lateral contact with a hydrophobic pocket formed by Leu ␤88, Phe ␤85, and the heme of tetramer on the other strand within the double strand. Only one of the two Val ␤6 residues per tetramer is involved in the double strand formation. In addition to these hydrophobic interactions, there are some neighboring hydrophilic ones and bridging water contacts that have been recently observed.70 Lateral

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A

B

Figure 6.5. (A) Electron micrograph of a fiber. The pitch of the fiber is not fixed, but varies as indicated by the different distances between the minimum diameter points. (B) Fiber model with double strands. The model is built according to the description of Watowich et al.75 This 14-strand model (whose end is shown in the inset) was first proposed by Dykes et al. (1978) and is now universally accepted as the basic fiber description.17 Note the double strands that are a basic structural element of the fiber and are based on a structure determined by crystallography (see Fig. 6.6). The wrapping of the 14 strands leads to a structure that gently varies from narrow to wide along the fiber.

and axial contacts are mostly between ␤ subunits. Nonpolar interactions involving Pro ␣114 and Ala ␣115 of one tetramer form an axial contact with His ␤116, His ␤117, and Phe ␤118 of a second tetramer. In deoxy Hbs crystals, the double strands are linear. How these double strands twist around each other to make up the 21-nm fiber is incompletely understood. Interdouble strand contacts are believed to mainly involve ␣ subunits,59 but the second Val ␤6 has also recently been proposed to play a role.73 Electron microscopy has provided useful information that has lead to two detailed models that agree on overall architecture.74,75 but differ in some details, including overall density and water content.76 Recent theoretical calculations show that the twist in the HbS polymer plays an important role in its stabilization.77 Confirmed by experimental observations, the torsional rigidity of HbS fibers is found to be approximately 100-fold less than the bending rigidity.77,78 The resistance to twisting compared with bending is usually approximately the same for isotropic materials. Linear double strands are the lowest equilibrium form and the relative ease for these to twist is proposed as an explanation for the metastability of the 21-nm fibers.78

Higher Order Aggregation of HbS Further aggregation of the 21-nm fibers can take several forms. Understanding the nature of these aggregates has been aided recently by novel applications of differ-

ential interference (DIC) microscopy.79–81 Analysis of DIC microscopy data collected on two 21-nm fibers zippering up has allowed estimations of the interaction energies between two fibers. Two such fibers can be strongly bonded to each other.81 As described in detail later, HbS polymerization involves both de novo fiber formation through homogeneous nucleation and fiber formation on the surface of a second one through heterogeneous nucleation.82,83 It has been proposed that the same intermolecular mutation contact sites that are involved within a fiber are available in 4 of 10 HbS surface tetramers in each layer of the 21-nm fiber.84 This proposal has recently been confirmed by studying cross-linked hybrid molecules.85 HbS polymer formation deforms the red cell, decreases its deformability, and increases its fragility. Aggregation of fibers into fascicles or bundles is of great interest because these are likely to exacerbate these phenomena. The fascicles are composed of twisted 21-nm fibers as shown in Figure 6.7.86 These fascicles form crystals in vitro, probably through release of twist in the double strands with concomitant loss of polarity.86 The fascicles always form first. The system of aligned fibers and HbS tetramers is referred to as a gel and this is thought to be what is formed inside of red blood cells. Crystals are not formed even though they are the lowest energy state. The gel is highly viscous and semisolid and, due to alignment of the polymers, it is birefringent. Because the hemes of hemoglobin are largely parallel to each other, and the hemoglobin tetramers are arranged so that the hemes are nearly perpendicular to the

Structure and Function of Hemoglobin and Its Dysfunction in Sickle Cell Disease

Figure 6.6. (a) The double strand of deoxyHbS, based on the crystal structure of Harrington et al.70 The tetramers of HbS have been drawn with the central region excluded for clarity; one tetramer illustrates the exclusion region as a solid green sphere in the center of the molecule. Another tetramer is shown with the four subunits colored differently to differentiate them. Red and purple are ␤-globin chains; blue and orange are ␣-globin chains. Contacts along the axis of the double strand (vertical here) are denoted as axial, whereas those that connect diagonally are denoted as lateral. The ␤6 mutation site is in a lateral contact. Note that both the axial and the lateral contacts are dominated by interactions between the beta chains. (b) An end view of the double strand. The two molecules, with all amino acids now showing, are colored differently to aid the eye. The ␤6 contact is shown on the bottom (expanded view in (E)), and the salt bridge between ␣His50 and ␤Asp79 is above (expanded view in (d)). (c). The axial contact region in A has been enlarged to allow a better view. Unlike the lateral contact, no single amino acid dominates the geometry. Carbon atoms that are filled to van der Waals radii are yellow, oxygen atoms are red, and nitrogen atoms are blue. (d) The salt bridge between Asp ␤79 and His ␣50 in the lateral contact area viewed from the ␣-globin chain. The His is shown as a green licorice stick drawing in the foreground. (e) The lateral contact region showing the ␤6 Val (green stick figure, foreground) in the receptor pocket on its complementary chain. (Note that in the crystal there are two such regions.) ␤88Leu is just forward and above Val; ␤85Phe is then just below ␤88 Leu and behind the Val. (See color plate 6.6.)

A

B Figure 6.7. Sickle hemoglobin assembly creates structures larger than the fibers shown in Fig. 6.5. (A) Fibers can associate in bundles or fascicles.86 Fascicles ultimately form into crystals. The fascicle shown here has a twist, which also appears in crystals. (B) A macrofiber with six fibers extending from the end. Macrofibers are composed of double strands in antiparallel rows, and such structures appear at low pH (below 6.7 in 0.05 mol/L phosphate buffer). (This macrofiber is from the unpublished work of Wellems and Josephs.)

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Daniel B. Kim-Shapiro of radially symmetrical polymer domains has been confirmed using DIC microscopy (Fig. 6.9).

HbS Polymer Rheology

Figure 6.8. Polymer domains. As seen in the DIC images in the left sequence of panels, polymerization produces fibers in attached arrays called domains. In these pictures laser photolysis (as evidenced by the light-colored circles, of approximately 15 ␮m diameter) creates deoxyHb, which generates fibers.80 The attached fibers form twofold symmetrical patterns that spread to form larger structures with nearly radial symmetry. These patterns are also visible in birefringence seen as transmission of light when the sample is placed between crossed polarizers and is shown in panels a through c on the right. Each cross or bow-tie defines a polymer domain. Each domain is formed from a single homogeneous nucleation event. The size of the polymer domains is inversely related to the speed of their formation, and speed of formation in turn is related to concentration. The concentrations were 23.4 g/dL, 25.7 g/dL and 27.4 g/dL, respectively. Samples were gelled by temperature jump from 3◦ C to 23◦ C.

fiber, the index of refraction is greater perpendicular to the fiber axis than parallel, resulting in birefringence and linear dichroism (where light is more strongly absorbed when polarized perpendicular to the fiber axis than parallel). The result is that, as observed both in solution and in red cells, gels are visible when viewed through crossed polarizers. Because of the way that clusters of polymers or domains form, they can have a large degree of radial symmetry, which produces a Maltese cross pattern when viewed through crossed polarizers (Fig. 6.8). The formation

As rigidification of the red blood cell is probably the most important immediate affect of polymerization, it is useful to understand the rheology of the gel. A full understanding of the mechanical properties of the gel begins with understanding those of single fibers. Recently, DIC microscopy was used to determine the intrinsic Young modulus as well the persistence lengths of individual fibers and bundles.87 The Young modulus (a measure of stiffness) was found to be approximately 0.1 GPa, much less than structural proteins like actin fibers and microtubules but greater than fibers that are meant to bend, like elastin. The persistence length was found to vary from 0.24 to 13 mm, increasing as the radius of bundles increased. These values are much larger than the length of a red blood cell so that one can conclude that the fibers are stiff on the scale of a red blood cell. Macroscopic measurements of gel rheology are difficult due to the fact that shear applied in the measurement can disturb the mechanical properties of the gel itself. The rheology of the gel will depend on the number of cross links, which will be different for a few long fibers compared with many short ones. Breaking fibers, followed by additional growth, changes rigidity.88–90 In the absence of shear, the gel behaves like a solid and at low shear it behaves like an elastic solid in which all deformations are reversible.59 At higher shear, the gel can become irreversibly deformed. The rheological properties of sickle cell gels and their understanding in terms of single-fiber rheology, gel architecture, and quantitative contribution to vasoocclusion events remains an area in need of investigation.

HbS Thermodynamics It has been widely accepted that the gel is made of two phases: a polymer phase and a solution phase where the

Figure 6.9. The double nucleation mechanism.83 Polymers may form by homogeneous nucleation or heterogeneous nucleation onto other polymers. In either case, the initial steps are unfavorable, as indicated by the arrows, until a critical nucleus is formed. The critical nucleus is the first aggregrate that is equally likely to add monomers or to lose them. No special structure is assumed for the nuclei.

Structure and Function of Hemoglobin and Its Dysfunction in Sickle Cell Disease solution phase contains HbS tetramers (which can be referred to as monomers in the context of single building blocks of the polymer). In equilibrium, no intermediate aggregates are generally observed.59,91 The thermodynamics of polymerization can be understood within this twophase model in terms of the solubility of HbS known as cs or csat . When the total concentration of HbS, c0 , is below the solubility, there will be no polymers. In equilibrium, when c0 > cs , the concentration of HbS tetramers in the solution phase is cs , and the concentration of HbS tetramers in the polymer phase is equal to the total concentration of HbS minus the solubility (c0 − cs ). Although individual tetramers might exchange between the two phases, once equilibrium is reached, the concentration in each phase will not change unless environmental conditions that affect the solubility (discussed later) are altered. The solubility is easily measured by sedimenting the polymers in an ultracentrifuge, for example, at 150,000 g for 2 hours, and taking the concentration of HbS in the supernatant as the solubility. Recently, evidence from light scattering and DIC microscopy has been presented that suggests the existence of a third phase prior to and during HbS gelation.92–96 This phase consists of metastable clusters of liquid phase molecules or dense liquid droplets that have been implicated in the initial formation of homogeneous nuclei discussed further later.94 The clusters form within a few seconds of solution formation and are several hundred nm in diameter.94,95 These clusters also form in solutions of HbA and oxygenated HbS but do not lead to polymer nucleation as in HbS.94,95 A major factor that must be taken into account when evaluating polymerization is crowding.97 In most biochemical experiments, protein solutions are dilute enough so that they can be considered to be ideal, that is, when interactions between the molecules can be ignored, like in an ideal gas. The concentration of hemoglobin in a red cell is so high, however, (∼ 20 mmol/L in heme which is 32 g/dL or 0.32 g/cm3 ) that the solution is nonideal and interactions need to be accounted for. Theoretical treatments of nonideality in sickle cell hemoglobin polymerization have been worked out and agree very well with experiments.59,97 Generally, one needs to include the activity coefficient, ␥ , when evaluating the potential for HbS to polymerize so that c0 → ␥ c0 and cs → ␥ s cs , where ␥ s is the activity coefficient at the solubility concentration. These activity coefficients are close to one in dilute solutions. For hemoglobin concentrations found in red blood cells, ␥ is quite large, equal to 70 for 0.35 g/cm3 .59 For HbS concentrations found in very dense cells with 0.45 g/cm3 , ␥ is 900! The relevance of these crowding effects to sickle cell hemoglobin polymerization thermodynamics and kinetics cannot be overstated (for a fuller discussion see references 59, 97, 98). Increased crowding leads to increased polymerization, so that dehydration of red cells can have a dramatic effect where by ␥ c0 increases much faster than c0 . Any other solutes that

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take up significant volume also increase ␥ . Thus, replacing HbS with hemoglobin molecules that do not polymerize can reduce cs but ␥ remains unchanged, diminishing the effect of the substitution.

Effectors of Polymerization Generally, only T-state HbS molecules will polymerize.99,100 Because only T-state hemoglobin polymerizes, any effectors that stabilize the T-state tend to decrease HbS solubility or increase polymerization.59 Thus, BPG and IHP increase polymerization. In the physiological pH range, increasing proton concentration increases polymerization, but as the pH is lowered below 6.5, the solubility increases. The solubility is lowest around body temperature. This fact has been used extensively to prepare HbS gel samples where the solutions are prepared at 0◦ C and then temperaturejumped to 37◦ C. The effect of phosphate on solubility is quite interesting and useful. The solubility of HbS decreases dramatically in concentrated phosphate buffers.101–105 In 1.8 M phosphate the solubility is 0.04 g/dL (4 × 10−4 g/cm3 or 0.025 mmol/L) at 30◦ C.103 The effect of phosphate is likely to be due largely to increasing the activity by volume exclusion but there are also likely to be electrostatic interactions.106 The ability to study polymerization at such low concentrations is beneficial as the volume of HbS is required for studies under physiological conditions is large and this requirement is very restrictive when studying new modified hemoglobins and those from mouse models. Another method to study polymerization with lower total hemoglobin concentrations is to use dextran to exclude volume and decrease the solubility.106 With 12 g/dL of dextran, the solubility can be decreased approximately 5-fold.106 This is a much smaller effect than using 1.8 M phosphate but some differences in polymerization in high phosphate and physiological phosphate have been reported.106–111 It has recently been noted that a small amount of protein aggregates form in high phosphate that are not due to polymerization so that care is warranted in making sure that these are not misconstrued as HbS polymers.111 In general, use of high phosphate can be recommended as an excellent initial screening method for effects on polymerization with subsequent experiments with dextran being more likely to provide physiologically relevant data. Finally, all such effects should be confirmed using physiological conditions. The most important physiologically relevant variable involved in HbS polymerization is the O2 pressure. As O2 binding promotes R-state hemoglobin, it decreases polymerization. The effect of CO on polymerization is very similar to that of O2 – the solubility as a function of solution phase hemoglobin ligand saturation is the same for O2 as it is for CO.99 This is consistent both with the idea that only T-state HbS polymerizes and the MWC–Perutz model of hemoglobin cooperativity. To understand fully the effect of O2 saturation on solubility, the affinity of polymer

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phase HbS for O2 must be determined. Using linear dichroism spectroscopy, because the linear dichroism of solution phase HbS averages out, it was found that polymer phase HbS has approximately one third the O2 affinity as solution phase T-state hemoglobin.99 Using data for the solubility of HbS at a variety of temperatures and ligand saturations, Eaton and Hofrichter59 derived an empirical equation for the solubility as a function of these variables, cs (g/cm3 ) = 0.321 − 0.00883 T + 0.000125 T2 + 0.0924 Ys + 0.098 Ys3 + 0.235 Y15 s ,

(6.4)

where T is the temperature in degrees Celsius and Ys is the fractional hemoglobin O2 saturation of the solution phase HbS. In the absence of O2 (Ys = 0), the solubility is calculated to be 0.17 g/cm3 (10 mmol/L in heme) at 37◦ C, and 0.32 g/cm3 (20 mmol/L in heme) at 0◦ C. When the solution phase O2 saturation is 0.7, the solubility is 0.26 g/cm3 (17 mmol/L in heme) at 37◦ C. The effects of NO on polymerization have been controversial. NO inhalation therapy is being studied as treatment for sickle cell disease. One of the main ways of benefiting patients is likely to be by reducing the NO scavenging ability of cell-free hemoglobin that results from intravascular hemolysis (Chapter 11)112 and possibly due to induction of fetal hemoglobin (HbF) production.113,114 It has also been proposed that NO binding to the heme would reduce HbS polymerization, like O2 does.115 In early work, Briehl and Salhany showed that tetranitrosyl hemoglobin (where all four hemes have NO bound) polymerizes in the presence of IHP but does not polymerize when the hemoglobin is stripped of organic phosphates. More recently, it was shown that when HbS polymerization is studied in conditions mimicking those in vivo (with BPG present), the solubilizing or sparing effect of NO binding to the heme is much less than that of O2 .38 The minimal effects of iron nitrosylation can be understood in terms of the ability of NO compared with that of O2 , to convert T-state hemoglobin to R-state hemoglobin, because R-state hemoglobin has a much higher affinity for O2 than T-state hemoglobin, and 25% oxygenated hemoglobin will be nearly 25% R-state (Fig. 6.3). On the other hand, NO tends to favor binding to the ␣ subunits and ␣ nitrosyl hemoglobin has properties like T-state hemoglobin. Thus, a given amount of NO will tend to reduce polymerization less than the same amount of O2 . Another important factor to consider is that iron nitrosylation of hemoglobin through NO inhalation or other means is not likely to ever yield a significant fraction of the total hemoglobin bound to NO ( dense discocytes and ISCs. Dense sickle red cells contributed maximally to microvascular obstruction, as shown by their selective trapping in postcapillary venules in which deformable light-density sickle red cells preferentially adhered. Sickle cell adhesion has been confirmed using a variety of assay systems.58,60,63,64 Studies using the cremaster muscle microcirculation of transgenic sickle mice expressing HbS and HbS-Antilles (S+S-Antilles) confirmed venules as the exclusive sites of red cell adhesion in vivo.65 In contrast to S+S-Antilles mice, red cell adhesion is less obvious in BERK mice. In studies of C57BL mice transplanted with BERK marrow, adhesion was not observed, although transient interactions between leukocytes and red cells were noted.45 In contrast to S+S-Antilles mice, BERK mice have fewer dense red cells and have erythrocyte features of ␤ thalassemia, with microcytic red cells having reduced mean cell hemoglobin.66 BERK mice, however, have erythrocytes that sickle, hemolyse and undergo oxidative stress. These mice are more suitable for studying the role of hemolysis and inflammation than sickle cell adhesion and illustrate the imperfection of sickle mice and that different sickle mouse strains must be used for understanding different features of this disease (Chapter 12).

Mechanisms of Sickle Red Cell Adhesion Sickle erythrocytes adhere to endothelium by multiple mechanisms via adhesion molecules expressed on both sickle red cells and endothelium. Although repeated sickling–unsickling cycles result in red cell membrane damage and exposure of red cell membrane components like sulfated glycolipids and phosphatidylserine (PS) that might mediate adhesion, increased red cell destruction results

Rheology and Vascular Pathobiology in Sickle Cell Disease and Thalassemia

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Perfusion Fluid Gas Port Vein

Ppa 0.8

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24 32 16 40 Venular Diameter (µm)

48

D

C

Figure 8.2. Adhesion of sickle red cells in venules of the ex vivo mesocecum vasculature infused with a bolus of human sickle red cells during perfusion with Ringers albumin. (A) Artificially perfused mesocecum preparation of the rat (Asc. Colon, ascending colon; I.A. inj. port, arterial injection port; Ppa, arterial perfusion pressure; and Pv, venous outflow pressure). (B) Adherent sickle red cells of discocyte morphology are seen deformed in the direction of the flow (arrow). (C) Increased adhesion of sickle red cells at venular bending and at junctions of small-diameter immediate postcapillary venules. In this instance, the immediate postcapillary venules are completely blocked (arrows). (D) The inverse relationship between vessel diameter and sickle red cell adhesion in venules of the ex vivo mesocecum vasculature. The regression fits the equation y = aX−b , r = −0.81, P < 0.001). (Modified from ref. 60.) (See color plate 8.2.)

in excessive production of stress reticulocytes that display adhesion molecules. The adhesion molecules implicated in sickle red cell adhesion are broadly categorized as 1) red cell receptors; 2) adhesive bridging proteins; 3) endothelial adhesion molecules; 4) extracellular matrix adhesion molecules (Fig. 8.3). Red Cell Receptors: Two subcategories of receptors have been recognized in sickle erythrocytes. The first consists of receptors that are expressed on stress sickle reticulocytes. Two well-characterized receptors on reticulocytes are verylate-activation-antigen-4 (VLA-4/␣4 ␤1 ) and CD36.63,67,68 The integrin ␣4 ␤1 binds its endothelial ligand VCAM-1.

Figure 8.3. Schematic representation of adhesion molecules involved in sickle red cell–endothelium interactions. B-CAM/Lu = basal cell adhesion molecule/Lutheran; EC matrix = extracellular (subendothelial) matrix; IAP = integrin-associated protein; ICAM-4 = intercellular adhesion molecule-4; and Sulf. Glycolipids = sulfated glycolipids.

VCAM-1 is not constitutively expressed on endothelium but can be induced by cytokines or hypoxia.67,69 Importantly, VCAM-1 interactions with ␣4 ␤1 are enhanced under hypoxic conditions, and this interaction might play an important role in sickle acute chest syndrome, which is characterized by infiltration and retention of sickle red cells into the lung microcirculation.70,71 CD36 interacts with soluble thrombospondin (TSP), an adhesive bridging protein.68 Plausibly, CD36-expressing reticulocytes could also interact with endothelial ␣V␤3 integrin receptor via soluble TSP. Exposed membrane sulfated glycolipids could facilitate sickle red cell interaction with von Willebrand factor (vWF), an endothelial ultra-largemolecular weight protein and TSP, contributing to sickle cell–endothelium adhesion.72,73 The second subcategory of receptors includes more recently described molecules that are activated by signal transduction. Activation of integrin-associated protein (IAP/CD47) on sickle erythrocytes can induce signal transduction to activate still unidentified red cell receptors for TSP.74 A receptor, intercellular adhesion molecule4 (ICAM-4 (Landsteiner–Weiner protein or CD 242) has been identified on sickle erythrocytes, which can mediate adhesion by binding endothelial ␣v␤3 integrin.75 Activation of ICAM-4 by the physiological stress mediator epinephrine enhances sickle red cell adhesion. The epinephrine-induced activation of ICAM-4 involves a cyclic adenosine monophosphate–dependent signaling pathway, probably via stimulation of the red cell ␤-adrenergic receptor.75 Previous studies have ascribed a role of cyclic adenosine monophosphate–dependent pathways in sickle

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Adherent SS RBC/ 100 µm2

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Adherent SS RBC/ 100 µm2

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1.0 r = -0.81 p = 0.001

0.8

r = -0.18 p = 0.172

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Figure 8.4. Regression plots for the number of adherent sickle red cells (SS RBC)/100 ␮m2 relative to venular diameters in ex vivo preparations treated as follows: (A) PAF alone, (B) PAF and peptide FWV, (C) PAF and peptide ATSR, and (D) PAF and control peptide AWSS. The regression lines represent the multiplicative equation of the form Y = aX−b for the best fit. In preparations treated with PAF alone, adhesion of sickle erythrocytes showed a strong correlation with the venular diameter. Preparations treated with peptide FWV or ATSR showed a marked inhibition of sickle erythrocyte adhesion in venules of all diameters, with ATSR having the maximal inhibitory effect, especially in small-diameter venules, the sites of frequent blockage. In contrast, in the presence of the control peptide AWSS, the resulting adhesion was essentially similar to that observed in PAF-treated preparations. (Modified from ref. 61.)

red cell adhesion wherein epinephrine-induced activation of basal cell adhesion molecule/Lutheran (B-CAM/Lu) increased adhesion to immobilized laminin, an extracellular matrix molecule.76 Although epinephrine can activate both B-CAM/Lu and ICAM-4 in sickle red cells, the latter is specifically involved in adhesion to endothelial cells, as antibodies to B-CAM/Lu and laminin are ineffective in the ICAM-4–mediated interactions.75 Epinephrine enhances sickle cell adhesion to the extracellular matrix via B-CAM/LU and laminin and to endothelium via ICAM-4 and ␣V␤3, suggesting a role for physiological stress in vasoocclusion. Among the ICAM-4 family of adhesive proteins, ICAM-4 is unique in its expression on erythroid cells. ICAM-4 can bind diverse arrays of integrins including several ␣V integrins, ␤2 integrins expressed on leukocytes and ␣4␤1 integrin,77,78 suggesting multiple functions of this adhesion molecule. Although exposed PS might mediate adhesion,79 the tenacity of this interaction has not been evaluated. Adhesive Bridging Proteins: Adhesive proteins released by platelet and vascular endothelium are implicated in sickle red cell adhesion. Unusually large molecular weight forms of vWf and soluble TSP are known to enhance adhesion of sickle red cells to endothelium.80,81 Soluble TSP is likely to contribute to adhesion of sickle red cells via its Cterminal cell-binding domain.82 TSP and vWf, both present

in platelets and endothelial cells, can be released into the local environment under appropriate stimulation.83 Elevated levels of both these adhesive bridging proteins have been identified in the plasma of patients with sickle cell disease.68,84,85 vWf and TSP can act as bridging proteins because they can bind specific red cell receptors and endothelial ␣V␤3 integrin. Inhibition of sickle erythrocyte adhesion to TSP by vWf involved the use of plasma vWf that lacked the extra-large molecular weight forms of vWf released by endothelial cells and were implicated in sickle red cell adhesion.86 Endothelial Adhesion Molecules: Among the most prominent endothelial adhesion molecules, in addition to TSP and vWf, are P-selectin, CD36, and integrin ␣V␤3. P-selectin is expressed in activated endothelial cells in transgenic mouse models of sickle cell disease. P-selectin might facilitate a weak adhesion via interaction with red cell sialyl Lewis moieties.87 P-selectin–mediated transient interaction could affect local wall shear rates followed by a more tenacious adhesion via high affinity adhesion mechanisms. Antibodies to P-selectin can partially inhibit sickle red cell adhesion to human endothelial cells in a flow system.88 Moreover, P-selectin–mediated sickle red cell adhesion was inhibited by unfractionated heparin. Heparin and other anionic polysaccharides can inhibit TSP-mediated adhesion of sickle cells in the ex vivo mesocecum preparation,

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and in human endothelial cells under flow conditions,72 suggesting that an inhibitory effect of anionic polysaccharides is not limited to P-selectin–mediated adhesion. An agonist peptide for murine protease–activated receptor-1 (PARS-1), which selectively activates mouse endothelial cells, but not platelets, resulted in flow stoppage of infused BERK sickle red cells in the microcirculation, an effect not observed in P-selectin knockout mice.89 The contribution of leukocytes, however, was not confirmed. Moreover, the endothelial activation by murine protease– activated receptor-1 could concomitantly release vWF as both P-selectin and vWf are stored in endothelial Weibel–Palade bodies. Additional studies are needed to clarify the relative roles of P-selectin and extra-large forms of endothelial vWF in sickle red cell adhesion. CD36, an 88-kD protein, is expressed by microvascular endothelial cells90 in addition to platelets and sickle reticulocytes. CD36 has Figure 8.5. Top panel: Colocalization of fluoresceinated ATSR peptide with vascular endothelium been implicated as a ligand to the exposed of the ex vivo mesocecum preparation pretreated with PAF (A–C). (A) The presence of the PS on sickle red cells.79 Moreover, endothelial fluorescent peptide is shown in green. (B) Blood vessels were identified by a polyclonal primary antibody to vWF and a secondary TRITC-conjugated antibody (red). (C) Merged image signals CD36 can interact with soluble TSP to proshowed colocalization (yellow) of ATSR with the endothelial lining. Middle panel: The effect of a mote TSP-mediated adhesion. control antibody OC125 (D–F) on the colocalization of fluoresceinated ATSR peptide with vascular The vitronectin receptor, ␣V␤3 integrin, endothelium of PAF-treated ex vivo preparation. (D) The presence of the fluorescent peptide is expressed on activated endothelium, is likely shown in green. (E) Blood vessels were identified by a polyclonal antibody to vWF as in B (red). to play an important role in stable sickle red (F) Merged image signals showed colocalization (yellow) of ATSR with the vessel wall. Bottom panel: The effect of 7E3 antibody to ␣V␤3 (G–I) on the colocalization of fluoresceinated ATSR cell adhesion. ␣V␤3 integrin can interact peptide with vascular endothelium in PAF-treated ex vivo preparation. (G) In the presence of 7E3 with several adhesive bridging proteins (TSP, antibody, there was a marked decrease in ATSR localization with the vessel wall. ATSR infusion vWf, and possibly soluble laminin),85,91,92 and resulted in weak green staining likely attributable to autofluorescence or a low level of binding also with ICAM-4 (a receptor for ␣V␤3) on of ATSR peptide. (H) Vessel was identified by immunofluorescent staining for vWF (red). (I) No sickle red cells.75 Antibodies directed against colocalization of ATSR with vessel wall in the presence of 7E3. (Modified from ref. 61.) (See color plate 8.5.) this integrin can dramatically inhibit sickle red cell adhesion in the ex vivo mesocecum preparation83 and to human endothelial cells.75 sive therapies.95 In addition to direct interaction between Peptides (ATSR and FWV) based on ␣V-binding domains red cell ICAM-4 and endothelial ␣V␤3 integrin, possible of ICAM-4 markedly decreased sickle cell adhesion and formation of tripartite complexes involving the red cell vasoocclusion in the PAF-treated ex vivo mesocecum 61 receptor, adhesive protein, and endothelial receptor with vasculature under shear flow conditions (Fig. 8.4A-D). ␣V␤3 might contribute to sickle red cell adhesion (Fig. 8.6), PAF is a potent endothelial activating and inflammatory 93 agent that is elevated in sickle cell disease. The infused fluoresceinated ATSR peptide is colocalized with vascular endothelium and pretreatment with function-blocking antibody (7E3) to ␣V␤3 markedly inhibited this interaction (Fig. 8.5). These studies show that ICAM-4 on sickle red cells binds endothelium via ␣V␤3 and that this interaction contributes to vasoocclusion, suggesting that peptides or small molecules based on ␣V-binding domains of ICAM-4 might have a therapeutic potential. Moreover, smallmolecule cyclic ␣V␤3 antagonists containing the integrin recognition motif RGD are potent inhibitors of sickle cell adhesion.94 Because of its ability to bind several adhesive Figure 8.6. Endothelial ␣V␤3 integrin in sickle red cell adhesion. proteins, ␣V␤3 is a potential target in designing antiadhe-

146 for example, CD36-TSP-␣V␤3, IAP-TSP-␣V␤3, sulfated glycolipids-TSP-␣V␤3, and sulfated glycolipids-vWF-␣V␤3. Endothelial oxidant generation induced by elevated inflammatory stimuli and intermittent vasoocclusion events will cause endothelial activation and up-regulation of adhesion molecules, particularly ␣V␤3. PAF-induced endothelial oxidant generation resulted in markedly enhanced sickle red cell adhesion to vascular endothelium, which was abrogated by SOD and catalase.55 The Effect of Hydroxyurea and NO on Adhesion: One therapeutic approach to treat sickle cell disease is to increase fetal hemoglobin (HbF) concentration (Chapter 30).96,97 An inverse relationship exists between CD36positive reticulocytes and F cells98 and patients with higher levels of F cells have fewer adherent cells. Hydroxyurea decreases red cell adhesion and down-regulates endothelial adhesion molecules such as sVCAM-1 and sICAM-1.99,100 Hydroxyurea is an NO donor and by the activation of soluble guanylate cyclase (cGMP) might induce HbF expression.101 NO generation by hydroxyurea could be a critical factor for its reported antiinflammatory action and down-regulation of endothelial adhesion molecules such as sVCAM-1 and sICAM-1.99 Sickle cell adhesion to TNF␣treated endothelial monolayers is markedly inhibited by the NO donor DETA-NO.102 The relative roles of HbF and NO in the therapeutic efficacy of hydroxyurea have yet to be clarified. Extracellular Matrix Adhesion Molecules: Indirect evidence indicates the potential exposure of subendothelial matrix of the vascular intima where endothelial cell damage and detachment occurs, probably due to rheological insult and adhesion of sickle cells. Endothelial detachment is probably exemplified by the presence of circulating endothelial cells.52,53 Matrix proteins, including TSP, vWf, and laminin have been implicated in sickle cell adhesion (Fig. 8.7). Such interactions could be relevant to the pathophysiology of this disease but might not represent a generalized phenomenon.

Pathophysiological Implications of Sickle Erythrocyte Adhesive Interactions Sickle red cell adhesion in postcapillary venules increases microvascular transit times, induces hypoxia, and promotes HbS polymerization in the adherent and trapped cells, as shown by direct intravital microscopy and by increased peripheral resistance caused by sickle red cell adhesion.55,61 It was proposed that sickle red cell adhesion– induced vasoocclusion is a two-step process in which preferential adhesion of deformable, light-density cells in postcapillary venules is followed by reduced effective lumen diameter and selective trapping of dense cells causing vessel blockage.60,103 Dense cell trapping permits rapid HbS polymerization due to the high MCHC of these cells. Postcapillary obstruction might proceed to involve whole

Dhananjay K. Kaul

Figure 8.7. Schematic representation of matrix adhesive proteins involved in potential adhesion of sickle red cells to extracellular (subendothelial) matrix.

feeding capillary networks in a retrograde fashion.104–107 The obstructive behavior of dense sickle red cells has also been shown in a perfused rat leg model108 corroborating findings in the ex vivo mesocecum. That obstruction results in a disproportionate trapping of dense sickle red cells was suggested by the analysis of cells eluted in venous effluents under high perfusion pressure.109 Selective trapping of dense cells might occur during the evolution of the acute painful episode (Chapter 20). Following an initial increase in the circulating dense cells at the onset of the acute painful event,111 an observation explained by the sequestration of deformable light-density sickle red cells as they interact with endothelium, the highest density fraction of sickle erythrocytes disappears from the peripheral circulation during the course of the event. Together, these observations support the proposed two-step model of sickle vasoocclusion.110

Sickle Red Cell Adhesion and the Endothelial Response Sickle cell adhesion and intravascular sickling are likely to contribute not only to endothelial damage and upregulation of vasoactive molecules such as prostaglandins and endothelin-1 (ET-1)112 but also to up-regulation of adhesion molecules involved in blood cell–endothelium interactions.27 In vitro studies have shown that sickle cell contact and adhesion to cultured endothelial cells can inhibit endothelial DNA synthesis, increase ET-1 mRNA,113 impair NO synthesis,114 and stimulate arachidonic acid metabolism and prostacyclin release115,116 and up-regulate expression of endothelial adhesion molecules.46 Adhesion might promote release of hemoglobin and adenosine diphosphate. Damaged endothelium also releases adenosine diphosphate,117 a potent platelet activator that causes aggregation. Although of potential importance, the significance of these observations remains to be validated in the context of pathophysiology of sickle cell disease.46,118,119

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Figure 8.8. A model for vasoocclusion in sickle cell disease. (A) Adhesion of deformable sickle red cells and leukocytes (light colored) in postcapillary venules. (B) Adhesion of these cells is followed by reduction in local wall shear rates and selective trapping of dense sickle red cells, which could result in HbS polymerization in the trapped (dark red) and adhered sickle cells and obstruction of the affected vessels. (See color plate 8.8.)

Leukocytes and Vasoocclusion Neutrophils are increased in the peripheral circulation of most patients with sickle cell anemia and could be an important factor modulating microvascular flow.120,121 Because neutrophils are relatively large and less deformable than erythrocytes, an increase in their numbers, activation and recruitment to sites of injury will increase intravascular resistance and adversely affect microvascular flow.122,123 Increases numbers of neutrophils might increase red cell transit time and impair O2 delivery. In vivo studies have shown that microcirculatory flow is significantly influenced not only by sickle erythrocytes but also by increased leukocyte recruitment due to reperfusion injury and oxidant generation. In transgenic sickle mice, hypoxia/reoxygenation generated by transient occlusion might induce an inflammatory endothelial phenotype resulting in increased leukocyte–endothelium interactions.26 When bone marrow from BERK mice was transplanted into C57BL mice, adherent leukocytes facilitated mechanical trapping of elongated sickled red cells in venules.45 This log jamming of sickled red cells among adherent leukocytes was similar to the pattern of dense cell and ISC trapping among adherent sickle erythrocytes.60 These observations suggest that both erythrocyte and leukocyte adhesion to endothelium can help entrap sickled or dense red cells, potentially initiating vasoocclusion (Fig. 8.8). The results from C57BL mice that underwent BERK mouse bone marrow transplantation were used to further address the role of leukocytes in sickle vasoocclusion.124 Pretreatment with intravenous human gamma globulin (IVIG) followed by an inflammatory stimulus by using TNF␣ caused a significant reduction in leukocyte recruitment and associated red cell trapping in recipient mice. Pretreating the BERK marrow recipient C57BL mice with TNF␣ followed by IVIG administration reversed TNF␣-induced vasoocclusion.125 These studies suggested a role for IVIG for

treatment of acute painful episode, nevertheless, the underlying mechanism(s) of IVIG action are unresolved.

Vascular Function Vascular tone adaptations in sickle cell disease are a response to hemolytic anemia, intravascular sickling, and vasoocclusive events, all of which can contribute to tissue hypoxia. Subclinical transient vasoocclusive events or “microcrises” triggered by intravascular sickling and cell adhesion not only contribute to an inflammatory state and local tissue hypoxia, but are likely to cause endothelial dysfunction as reported for other inflammatory diseases.35 Chronic anemia is compensated for by hyperperfusion to maintain O2 delivery.126,127 Hyperperfusion is not limited to large conduit arteries but is also observed in the microcirculation of transgenic knockout sickle mice as a consequence of arteriolar dilation.35 In sickle cell disease and transgenic knockout sickle mice, relatively reduced systemic blood pressure35,128 is a likely consequence of the dilation of resistance arterioles. In patients, vasodilation and reduced blood pressure are associated with a 50%–60% decrease in the peripheral resistance.126 Vascular Tone Response to Red Cell Rheology: Abnormal rheology of sickle red cells might require appropriate adjustments in the vascular tone to facilitate their capillary passage. Increased intravascular pressure caused by less deformable sickle red cells could potentially trigger an oscillatory vasomotion pattern, or intermittent periodic flow, to facilitate microvascular passage of rheologically abnormal sickle cell blood.129,130 Vasomotor response is depressed following postocclusive hyperemia.130 The effect of red cell rheology on vascular tone was studied in transgenic sickle mice exposed to different levels of oxygenation.65 Experiments performed to determine the effect of local, transient hyperoxia revealed striking differences in the microvascular responses in control and

148 transgenic sickle mice. In S+S-Antilles transgenic sickle mice, Vrbc was significantly reduced under hypoxic conditions with pO2 of 15–20 mm Hg, consistent with increased viscosity due to in vivo HbS polymer formation. During transient hyperoxia, an altered microvascular tone and response was observed. In control animals, O2 caused approximately 70% arteriolar constriction, accompanied by 75% reduction in Vrbc. In contrast, in transgenic sickle mice, hyperoxia resulted in only an 8% decrease in the arteriolar diameter and a 70% increase in Vrbc. The altered response in transgenic mice to hyperoxia was probably due to an improved flow behavior of red cells as a consequence of HbS depolymerization and cell unsickling, although possibly intrinsic or acquired differences in the endothelial/vascular smooth muscle might also contribute to this response. This attenuated response to O2 was later validated in patients who showed a considerably smaller decrease in the brachial artery diameter when exposed to 100% O2 .127 NO Bioavailability, Non-NO Vasodilators and Vascular Reactivity: Vascular function in sickle cell disease has been facilitated by recent studies of NO bioavailability.23,26,28,38,131 Reduced NO bioavailability in patients and in transgenic sickle mice,35,132,133 results in vascular tone adaptations and attenuated vascular responses to NOmediated stimuli. The biological functions of NO are discussed in detail in Chapters 10 and 11. NO has diverse biological functions and its altered metabolism is a feature of many diseases.31,131,134–136 NO consumption by plasma hemoglobin and O2 radicals potentially results in excess consumption of arginine substrate by eNOS to compensate for reduced NO availability. In both patients and sickle mouse models, the evidence shows depleted levels of Larginine and NO metabolites (NOx).37,137–142 NO depletion attenuates vascular reactivity to NOmediated vasodilators and NOS antagonists in transgenic sickle mice35,143,144 and in patients with high plasma hemoglobin levels.145,146 Impaired NO bioavailability is suggested by attenuated vascular reactivity in sickle mouse models to endothelium-dependent vasodilators like acetylcholine (ACh), and in particular to sodium nitroprusside (SNP), a NO donor.28,35,143,144 Reduced flow-mediated vasodilation in sickle cell disease patients also suggests reduced bioavailability of NO.133,146 Nevertheless, plethysmographic measurements of the forearm flow in patients show an increased blood flow greater or an enhanced vasodilatory response to ACh compared with normal individuals.127,146 This response to ACh might imply upregulation of non-NO vasodilators because ACh augments the release of NO, prostaglandins and endothelium hyperpolarizing factors.146 Interestingly, responses to both ACh and SNP are reduced in men but not women with sickle cell disease,146 suggesting sex differences in NO bioavailability.

Dhananjay K. Kaul

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Figure 8.9. Arteriolar diameter (percentage of increase) responses to topical application of acetylcholine (ACh, 10−6 M) and sodium nitroprusside (SNP, 10−6 M) in C57BL, BERK-trait, BERK, and BERK+␥ mice. Note the blunted response of arterioles in BERK mice to ACh (A) and SNP (B). ACh and SNP caused significant increases in arteriolar diameters of BERK+␥ mice as compared with that in BERK mice (∼33% and ∼50% increases, respectively). * P < 0.005–0.000001 compared with C57BL and BERK-trait mice. + P < 0.00– 0.002 compared with the diameter increase in BERK mice. (Reproduced with permission from ref. 35.)

Because vascular resistance to blood flow is determined mainly by the vascular tone of arterioles, BERK mice were used to determine the role of NO and non-NO-related mechanisms in affecting the arteriolar tone.35 Arteriolar responses to NO-mediated vasoactive stimuli such as ACh and SNP were attenuated in BERK mice compared with C57BL and hemizygous BERK (BERK-Hemi) controls (Fig. 8.9). NG -nitro-L-arginine methyl ester (L-NAME) had no appreciable effect on blood pressure in these mice. Almost complete attenuation of arteriolar dilation to SNP, reflected inactivation and/or destruction of NO. As shown in Figure 8.10B, the arteriolar diameter response to SNP is strongly correlated with hemolytic rate. This observation is consistent with the ability of plasma heme to consume NO (Fig. 8.10A).38 The greater plasma heme level in BERK mice caused blunted vessel diameter response. In contrast, the low plasma heme levels in control mice were associated with maximal arteriolar dilation. BERKHemi and BERK mice expressing 20% HbF that had intermediate levels of plasma heme showed improved vessel diameter response to SNP. Sickle cell disease patients have a diminished response to SNP.38 The BERK model differs markedly from ischemic coronary artery disease, which is

Rheology and Vascular Pathobiology in Sickle Cell Disease and Thalassemia

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Figure 8.10. Plasma heme and its effect on NO consumption and microvascular response to sodium nitroprusside (SNP). (A) Heme concentration within plasma of sickle cell disease patients shows a significant correlation with NO consumption (r = 0.9, P < 0.0001). (B) Relationship between plasma heme levels and the arteriolar diameter response to SNP in the cremaster microcirculation of C57BL (control), BERK, BERK-trait, and BERK+␥ mice. A strong correlation is observed between the percent arteriolar diameter increase in response to SNP and the extent of hemolysis (plasma heme). With a greater hemolysis in sickle (BERK) mice the diameter response was blunted. Low plasma heme levels in controls were associated with maximal arteriolar dilation, whereas BERK mice expressing 20% HbF showed a lower plasma heme and an improved diameter response compared with BERK mice. (A is reproduced from ref. 38 by permission, and B is based on the published data in ref. 61.)

characterized by blunted responses to L-NG -monomethyl arginine but normal responses to SNP.54 BERK mice have more than a 2.5-fold increase in the plasma hemoglobin compared with control mice.35 They have an increase in the endothelial-bound XO,28 which might catalyze the increased generation of O2 .− and H2 O2 , altering the vascular response to NO-mediated vasodilators. Increased O2 .− generation will consume NO forming ONOO− , resulting in the increased formation of nitrotyrosine.35 Additional studies are needed to differentiate the relative contributions of oxidative stress and cellfree plasma hemoglobin in NO consumption and sickle vasculopathy. With chronic hypoxia and hemolysis, induction of non-NO vasodilators such as prostaglandins and heme oxygenase-1 (HO-1) could compensate for reduced NO bioavailability.131,146 HO-1, also a marker of hemolysis, catalyzes degradation of excess heme to produce carbon monoxide, a vasodilator. Also, elevated levels of cyclic guanosine monophosphate caused by HO-1 induction144 in response to excess plasma heme could contribute to the blunted effect of SNP in BERK mice. The second non-NO vasodilator enzyme, cyclooxygenase-2 (COX-2), is induced under the conditions of chronic hypoxia and oxidative stress. ONOO− has been implicated in the induction of COX-2.147 In BERK mice, reduced NO bioavailability and increased nitrotyrosine formation are associated with COX2 induction in microvascular endothelium,35 suggesting that the reported increase in prostaglandin E2 levels in sickle cell disease112 might be due to COX-2 activity. More-

over, COX-2 induction in BERK mice is associated with dilation of arteriolar resistance vessels, hyperperfusion, and hypotension as reported in sickle patients.35,26,127 Activation of non-NO vasodilator mechanisms in the BERK model might compensate for NO deficiency and help maintain optimal O2 delivery in the face of chronic anemia. Depletion of the substrate arginine and cofactor BH4 results in uncoupling of electron flow from L-arginine oxidation and NO production.148 This uncoupling of eNOS leads to its inactivation and O2 radical generation.149,150 Increased inflammatory effects were seen when bone marrow from transgenic sickle mice was transplanted into eNOS overexpressing mice.151 This was likely an effect caused by excessive ONOO− formation and BH4 depletion. Enhanced nitrotyrosine formation in transgenic sickle mice (Fig. 8.11A)35 has been supported by recent studies23 that showed increased ONOO− formation was associated with impaired eNOS activity with a loss of eNOS dimerization (Fig. 8.11B and C). The role of BH4 supplementation in microvascular tone and reactivity has not been examined, except for its role in leukocyte adhesion.151 NO depletion also up-regulates endothelin-1, a potent vasoconstrictor whose levels are increased in sickle cell disease. Altered Vascular Responses to Vasoconstrictors: Enhanced oxidative stress, particularly the formation of nitrotyrosine, might cause chronic vascular injury and impaired vascular reactivity. Nitrotyrosine infusion in rats attenuated the hemodynamic responses to epinephrine, norepinephrine, and angiotensin II,152,153 which is comparable to the blunted blood pressure response to norepinephrine

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Hypercoagulability Under normal conditions, vascular endothelium exerts a potent anticoagulant effect. Inhibition of coagulation by endothelium involves the expression of thrombomodulin that activates protein C; heparan sulfate that activates antithrombin III; annexin V that prevents binding of procoagulation factors such as PS; NO that has inhibitory effect on platelet activation and leukocyte adhesion; and prostacyclin, a vasodilator and an inhibitor of platelet aggregation. In sickle cell disease, inflammatory cytokines, hypoxia, sickle red cell–endothelium interaction, and perhaps apoptosis of injured endothelial cells can shift endothelium to a prothrombotic state. During acute painful episodes, there is excessive thrombin generation, platelet activation, and plasma fibrinolytic activity.27 The expression of tissue factor, a trigger of coagulation, is abnormally increased in sickle monocytes and circulating endothelial cells.27 Furthermore, procoagulant properties of sickle red cells are linked to externalization of PS, which is a consequence of repeated cycles of sickling and unsickling. PS exposure and thrombin generation are correlated in sickle patients.155 Thrombotic events in large vessels of the brain are implicated in sickle cell strokes156 and thrombi in the pulmonary vessels might be associated with acute chest syndrome and are often found in postmortem.

Figure 8.11. Elevated nitrotyrosine levels and endothelial nitric oxide synthase (eNOS) monomerization in BERK mice. (A) Western blot analysis of cremaster muscle lysates for the expression of nitrotyrosine. Two prominent bands of nitrated proteins (66 and 26 kD) were detected by the antibody to nitrotyrosine. BERK mice showed increased tyrosine nitration of both 66- and 26-kD proteins (i.e., average increase: fivefold and ∼twofold, respectively), whereas the BERK+␥ mouse showed a smaller increase compared with C57BL controls. The nitrotyrosine levels in BERK-trait and ␤ thalassemia mice showed no increase as compared with C57BL controls. Control lane depicts positive nitrotyrosine controls provided by the antibody manufacturer. (B) Western blots of lung homogenates under nondenaturing conditions demonstrate 280-kD dimer (active form) and 140-kD eNOS monomer. Wild-type (WT) and hemizygous (Hemi) sickle mice had more eNOS dimer than monomer, but sickle mice show almost a complete lack of dimerized eNOS. Positive monomer controls show eNOS dissociated completely to monomeric form by boiling. (C) Lung nitrotyrosine, evidence of NO scavenging by superoxide, was elevated in sickle mice. (Figure A is reproduced with permission from ref. 61. Figures B and C are reproduced from ref. 23 by permission.)

THALASSEMIA Red Cell Rheology An imbalance in globin chain synthesis is the major cause of red cell abnormalities in thalassemia and affects cell survival and deformability.157,158 In ␣ and ␤ thalassemia, the relative excess of unpaired ␤- and ␣-globin chains, respectively, result in accumulation of excessive amounts of unpaired globin chains that precipitate, and by different mechanisms, damage the cell membrane and cause their premature destruction (Chapters 14 and 17). Globin chain inclusion bodies are “pitted” from erythrocytes by reticuloendothelial cells. Erythrocytes in ␤-thalassemia major are less deformable than normal cells.159 Analysis of cellular and membrane deformability characteristics has shown that red cells of both ␣ and ␤ thalassemia have excess surface area in relation to cell volume, increased membrane dynamic rigidity, and a decreased ability to undergo cellular deformation under hypertonic osmotic stress.160 Nevertheless, when blood from thalassemia is analyzed on density gradients, erythrocytes of HbH disease, characterized by the presence of ␤4 tetramers, appeared less dense than normal red cells. In contrast, erythrocytes from ␤ thalassemia intermedia and major showed a much broader range of density distribution with cell populations showing both lower and higher densities than that observed for normal red cells.160 Therefore, the presence of dense cells in ␤ thalassemia indicates cellular dehydration owing to abnormal membrane transport. The membrane

Rheology and Vascular Pathobiology in Sickle Cell Disease and Thalassemia rigidity of ␤ thalassemia cells from nonsplenectomized and splenectomized patients showed a progressive increase with increasing cell density. The greater membrane rigidity in splenectomized patients is associated with increased pathological interactions of hemichromes with the membrane protein band 3,161 and with ankyrin, spectrin, and protein 4.1 of the membrane skeleton.162 Overall, the mechanical stability of ␣-thalassemia red cell membranes is normal or slightly decreased, and that of ␤ thalassemia membranes is markedly decreased.160 The membrane instability in ␤ thalassemia was attributed to decreased binding of spectrin to protein 4.1. The coexistence of ␣ thalassemia with sickle cell disease has a salutary effect on MCHC, dense cell numbers and the erythrocyte deformability (Chapter 23).163,164 The deformability of sickle cell anemia ␣ thalassemia erythrocytes is inversely correlated with the number of ␣-globin genes.164 Introduction of a ␤S -globin gene into ␤ thalassemic mice was associated with a significant improvement in red cell deformabilities.165 This improvement, however, is probably due to a slight excess synthesis of ␣-globin chains in these mice, suggesting that a mild decrement in ␤-globin synthesis might have a beneficial effect on hemoglobin concentration in sickle cell anemia by normalizing red cell density distribution profiles; however, reduced anemia might promote the viscosity/vasoocclusion features of sickle cell anemia (Chapters 11 and 19).

151

Hemostatic changes have been reported in patients with ␤ thalassemia major and ␤ thalassemia intermedia and HbH disease. An increased incidence of thromboembolic events, mainly in ␤ thalassemia intermedia, and the occurrence of prothrombotic hemostatic anomalies in the majority of the patients suggest the existence of a hypercoagulable state.166 Thalassemia is associated enhanced platelet and endothelial and monocyte activation.158 Thalassemia red cells show increased expression of PS as in sickle cell disease167 and patients have decreased levels of proteins C, S, and antithrombin III.166

cause elevation of pulmonary artery pressure. Pulmonary hypertension in hemolytic anemia is discussed in detail in Chapter 11. Cardiac and Arterial Abnormalities: Chronic hemolysis, the release of iron from lysed red cells, blood transfusions, and the resulting iron overload in thalassemia leads to the formation of O2 free radicals. Increased oxidative stress and lipid peroxidation can have detrimental effects on cell membranes. Moreover, in thalassemia, the irontransport protein transferrin becomes saturated, causing a marked increase in nonheme- and non-transferrin-bound iron in the plasma (Chapter 29).158 In ␤ thalassemia major, iron overload constitutes the major cause of heart disease because unbound iron is readily taken up by cardiac monocytes, causing heart failure, structural alterations of arteries, and deleterious effects on endothelial function.173,174 Iron overload might result in left ventricular systolic and diastolic dysfunction.174 Alterations of arterial components with disruption of elastic tissue and calcification also occur in ␤ thalassemia major. These arterial changes could translate functionally into altered arterial stiffness in vivo.175 Arterial stiffness is related to vascular impedance and, in turn, to the afterload that is presented to left ventricle, resulting in decreased mechanical efficiency of the heart. Endothelial dysfunction and vasoconstriction might be promoted by reduced NO bioavailability, leading to diffuse elastic tissue injury.174,176 Although right ventricular dysfunction is a prominent feature in ␤ thalassemia intermedia, left ventricular impairment also develops consequent to an increased state of volume and pressure overload needed to maintain high cardiac output through a dilated and yet rigid vascular bed.174 A uniform feature of ␤ thalassemia intermedia patients is high cardiac output. Echocardiographic measurements in these patients show an almost two-fold increase in the cardiac output levels when compared with normal individuals.176 ␤ Thalassemia mice have significantly increased peripheral vascular resistance, suggesting that altered arteriolar diameters, endothelial dysfunction and abnormal rheology of red cells contribute significantly to the increased resistance.177

Vascular Pathobiology

CONCLUSIONS

Pulmonary Hypertension: Pulmonary hypertension is a feature of ␤ thalassemia and the same risk factors as in sickle cell disease are involved. These include platelet activation, hypercoagulability, and chronic hemolysis and reduced NO bioavailability.168–170 Chronic hypoxia and lung injury caused by infections and iron deposition could also contribute to this complication.171,172 In ␤ thalassemia, the incidence of pulmonary hypertension is increased following splenectomy,170 probably due to increased hypercoagulability consequent to erythrocyte PS exposure.167 Plausibly, reduced NO bioavailability, chronic hypoxia, and hypercoagulability might act in concert or independently to

The presence of HbS in the sickle cell makes this disorder unique. Nevertheless, sickle cell disease and thalassemia share certain vascular abnormalities, although the majority of experimental work has focused on sickle cell disease. Some common features are a result of hemolysis and diminished NO bioavailability, reduced or absent splenic function, and damage to the erythrocyte membrane. The commonality of certain vascular abnormalities suggests that common approaches to treatment, for example, restoration of NO bioavailability or antiinflammatory agents, might be useful. These approaches are discussed in disease-specific chapters and in Chapter 31.

Hypercoagulation

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120. Boggs DR, Hyde F, Srodes C. An unusual pattern of neutrophil kinetics in sickle cell anemia. Blood. 1973;41(1):59– 65. 121. Platt OS. Sickle cell anemia as an inflammatory disease. J Clin Invest. 2000;106(3):337–338. 122. Helmke BP, Bremner SN, Zweifach BW, Skalak R, SchmidSchonbein GW. Mechanisms for increased blood flow resistance due to leukocytes. Am J Physiol. 1997;273(6 Pt 2): H2884–H2890. 123. Lipowsky HH, Scott DA, Cartmell JS. Leukocyte rolling velocity and its relation to leukocyte-endothelium adhesion and cell deformability. Am J Physiol. 1996;270(4 Pt 2):H1371– H1380. 124. Turhan A, Jenab P, Bruhns P, Ravetch JV, Coller BS, Frenette PS. Intravenous immune globulin prevents venular vasoocclusion in sickle cell mice by inhibiting leukocyte adhesion and the interactions between sickle erythrocytes and adherent leukocytes. Blood. 2004;103(6):2397–2400. 125. Chang J, Shi PA, Chiang EY, Frenette PS. Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion. Blood. 2008;111(2):915–923. 126. Lonsdorfer J, Bogui P, Otayeck A, Bursaux E, Poyart C, Cabannes R. Cardiorespiratory adjustments in chronic sickle cell anemia. Bull Eur Physiopathol Respir. 1983;19(4):339– 344. 127. Belhassen L, Pelle G, Sediame S, et al. Endothelial dysfunction in patients with sickle cell disease is related to selective impairment of shear stress-mediated vasodilation. Blood. 2001;97(6):1584–1589. 128. Johnson CS, Giorgio AJ. Arterial blood pressure in adults with sickle cell disease. Arch Intern Med. 1981;141(7):891– 893. 129. Rodgers GP, Schechter AN, Noguchi CT et al. Periodic microcirculatory flow in patients with sickle-cell disease. N Engl J Med. 1984;311(24):1534–1538. 130. Lipowsky HH, Sheikh NU, Katz DM. Intravital microscopy of capillary hemodynamics in sickle cell disease. J Clin Invest. 1987;80(1):117–127. 131. Nath KA, Katusic ZS, Gladwin MT. The perfusion paradox and vascular instability in sickle cell disease. Microcirculation. 2004;11(2):179–193. 132. Gladwin MT, Kato GJ. Cardiopulmonary complications of sickle cell disease: role of nitric oxide and hemolytic anemia. Hematol Am Soc Hematol Educ Program. 2005;51– 57. 133. Eberhardt RT, McMahon L, Duffy SJ, et al. Sickle cell anemia is associated with reduced nitric oxide bioactivity in peripheral conduit and resistance vessels. Am J Hematol. 2003;74(2):104–111. 134. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA. 1991;88(11):4651–4655. 135. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43(2):109–142. 136. Ogawa T, Nussler AK, Tuzuner E, et al. Contribution of nitric oxide to the protective effects of ischemic preconditioning in ischemia-reperfused rat kidneys. J Lab Clin Med. 2001;138(1):50–58.

156 137. Morris CR, Morris SM Jr, Hagar W, et al. Arginine therapy: a new treatment for pulmonary hypertension in sickle cell disease? Am J Respir Crit Care Med. 2003;168(1):63–69. 138. Romero JR, Suzuka SM, Nagel RL, Fabry ME. Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity. Blood. 2002;99(4):1103– 1108. 139. Enwonwu CO. Increased metabolic demand for arginine in sickle cell anemia. Med Sci Res. 1989;17:997–998. 140. Morris CR, Kato GJ, Poljakovic M, et al. Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease. JAMA. 2005;294(1):81–90. 141. Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest. 1997;100(9):2153– 2157. 142. Gladwin MT, Lancaster JR Jr, Freeman BA, Schechter AN. Nitric oxide’s reactions with hemoglobin: a view through the SNO-storm. Nat Med. 2003;9(5):496–500. 143. Kaul DK, Liu XD, Fabry ME, Nagel RL. Impaired nitric oxidemediated vasodilation in transgenic sickle mouse. Am J Physiol Heart Circ Physiol. 2000;278(6):H1799–H1806. 144. Nath KA, Shah V, Haggard JJ, et al. Mechanisms of vascular instability in a transgenic mouse model of sickle cell disease. Am J Physiol Regul Integr Comp Physiol. 2000;279(6):R1949– R1955. 145. Gladwin MT, Kato GJ. Cardiopulmonary complications of sickle cell disease: role of nitric oxide and hemolytic anemia. Hematol. Am Soc Hematol Educ Program. 2005;51–57. 146. Gladwin MT, Schechter AN, Ognibene FP, et al. Divergent nitric oxide bioavailability in men and women with sickle cell disease. Circulation. 2003;107(2):271–278. 147. Landino LM, Crews BC, Timmons MD, Morrow JD, Marnett LJ. Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. Proc Natl Acad Sci USA. 1996;93(26):15069–15074. 148. Alp NJ, Channon KM. Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease. Arterioscler Thromb Vasc Biol. 2004;24(3):413–420. 149. Landmesser U, Dikalov S, Price SR, et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003;111(8):1201–1209. 150. Katusic ZS, d’Uscio LV. Tetrahydrobiopterin: mediator of endothelial protection. Arterioscler Thromb Vasc Biol. 2004;24(3):397–398. 151. Wood KC, Hebbel RP, Lefer DJ, Granger DN. Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction. Free Rad Biol Med. 2006;40(8):1443–1453. 152. Kooy NW, Lewis SJ. Nitrotyrosine attenuates the hemodynamic effects of adrenoceptor agonists in vivo: relevance to the pathophysiology of peroxynitrite. Eur J Pharmacol. 1996;310(2–3):155–161. 153. Kooy NW, Lewis SJ. The peroxynitrite product 3-nitroL-tyrosine attenuates the hemodynamic responses to angiotensin II in vivo. Eur J Pharmacol. 1996;315(2):165–170. 154. Hatch FE, Crowe LR, Miles DE, Young JP, Portner ME. Altered vascular reactivity in sickle hemoglobinopathy. A possible protective factor from hypertension. Am J Hyperten. 1989;2(1):2–8.

Dhananjay K. Kaul 155. Setty BNY, Rao AK, Stuart MJ. Thrombophilia in sickle cell disease: the red cell connection. Blood. 2001;98(12):3228– 3233. 156. Ataga KI, Orringer EP. Hypercoagulability in sickle cell disease: a curious paradox. Am J Med. 2003;115(9):721–728. 157. Weatherall DJ. The thalassemias. In: Stamatoyannopoulos G, ed. Molecular Basis of Blood Diseases. Philadelphia: WB Saunders; 1994:157–205. 158. Urbinati F, Madigan C, Malik P. Pathophysiology and therapy for haemoglobinopathies. Part II: thalassaemias. Expert Rev Mol Med. 2006;8(10):1–26. 159. Lacelle PL. Behavior of abnormal erythrocytes in capillaries. In: Cokelet GR, Meiselman HJ, Brooks DF, eds. Erythrocyte Mechanics and Blood Flow. New York: Alan R. Liss; 1980:195– 211. 160. Schrier SL, Rachmilewitz E, Mohandas N. Cellular and membrane properties of alpha and beta thalassemic erythrocytes are different: implication for differences in clinical manifestations. Blood. 1989;74(6):2194–2202. 161. Waugh SM, Low PS. Hemichrome binding to band 3: nucleation of Heinz bodies on the erythrocyte membrane. Biochemistry. 1985;24(1):34–39. 162. Shinar E, Rachmilewitz EA, Lux SE. Differing erythrocyte membrane skeletal protein defects in alpha and beta thalassemia. J Clin Invest. 1989;83(2):404–410. 163. Noguchi CT, Dover GJ, Rodgers GP, et al. Alpha thalassemia changes erythrocyte heterogeneity in sickle cell disease. J Clin Invest. 1985;75(5):1632–1637. 164. Embury SH, Clark MR, Monroy G, Mohandas N. Concurrent sickle cell anemia and alpha-thalassemia. Effect on pathological properties of sickle erythrocytes. J Clin Invest. 1984;73(1):116–123. 165. Rubin EM, Kan YW, Mohandas N. Effect of human beta (s)-globin chains on cellular properties of red cells from beta-thalassemic mice. J Clin Invest. 1988;82(3):1129– 1133. 166. Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood. 2002;99(1):36–43. 167. Borenstain-Ben Y, V, Barenholz Y, Hy-Am E, Rachmilewitz EA, Eldor A. Phosphatidylserine in the outer leaflet of red blood cells from beta-thalassemia patients may explain the chronic hypercoagulable state and thrombotic episodes. Am J Hematol. 1993;44(1):63–65. 168. Singer ST, Kuypers FA, Styles L, Vichinsky EP, Foote D, Rosenfeld H. Pulmonary hypertension in thalassemia: association with platelet activation and hypercoagulable state. Am J Hematol. 2006;81(9):670–675. 169. Morris CR, Kuypers FA, Kato GJ, et al. Hemolysis-associated pulmonary hypertension in thalassemia. Ann NY Acad Sci. 2005;1054:481–485. 170. Aessopos A, Farmakis D. Pulmonary hypertension in betathalassemia. Ann NY Acad Sci. 2005;1054:342–349. 171. Factor JM, Pottipati SR, Rappoport I, Rosner IK, Lesser ML, Giardina PJ. Pulmonary function abnormalities in thalassemia major and the role of iron overload. Am J Respir Crit Care Med. 1994;149(6):1570–1574. 172. Zakynthinos E, Vassilakopoulos T, Kaltsas P, et al. Pulmonary hypertension, interstitial lung fibrosis, and lung iron deposition in thalassaemia major. Thorax. 2001;56(9):737– 739.

Rheology and Vascular Pathobiology in Sickle Cell Disease and Thalassemia 173. Link G, Pinson A, Hershko C. Heart cells in culture: a model of myocardial iron overload and chelation. J Lab Clin Med. 1985;106(2):147–153. 174. Aessopos A, Kati M, Farmakis D. Heart disease in thalassemia intermedia: a review of the underlying pathophysiology. Haematologica. 2007;92(5):658–665. 175. Cheung YF, Chan GC, Ha SY. Arterial stiffness and endothelial function in patients with beta-thalassemia major. Circulation. 2002;106(20):2561–2566.

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176. Aessopos A, Farmakis D, Karagiorga M, et al. Cardiac involvement in thalassemia intermedia: a multicenter study. Blood. 2001;97(11):3411–3416. 177. Stoyanova E, Trudel M, Felfly H, Garcia D, Cloutier G. Characterization of circulatory disorders in beta-thalassemic mice by noninvasive ultrasound biomicroscopy. Physiol Genom. 2007;29(1):84–90.

9 The Erythrocyte Membrane Patrick G. Gallagher and Clinton H. Joiner

INTRODUCTION Hemoglobinopathies, including the thalassemia syndromes and sickle cell disease, are complex disorders with protean manifestations. Their pathophysiology is influenced by environmental and genetic factors in addition to the pleiotropic effects of the globin gene mutations themselves. The erythrocyte membrane plays a critical role in these disorders because of the effects of its structural and functional perturbations and alterations in ion and water homeostasis regulated by membrane proteins.1 The first portion of this chapter reviews the structural and functional characteristics of the erythrocyte membrane; this is followed by a review of the alterations in ion and water homeostasis observed in the erythrocytes of sickle cell disease and thalassemia.

MEMBRANE STRUCTURE AND FUNCTION The erythrocyte membrane is a complex, multifunctional structure. Although providing a protective layer between hemoglobin and other intracellular components and the external environment, it provides the erythrocyte with the deformability and stability required to withstand its travels through the circulation. The erythrocyte is subjected to high sheer stress in the arterial system, dramatic changes in size in the microcirculation, and wide variations in tonicity, pH, and pO2 as it travels throughout the body. It facilitates the transport of cations, anions, urea, water and other small molecules in and out of the cell, but denies entry to larger molecules, particularly if charged. A unique anucleate cell, the erythrocyte has a limited capacity for self-repair.

Membrane Structure The erythrocyte membrane is composed of a lipid bilayer linked to an underlying cortical membrane skeleton.2 Membrane proteins are classified as integral, penetrating or crossing the lipid bilayer and interacting with the hydro158

phobic lipid core, or peripheral, interacting with integral proteins or lipids at the membrane surface, but not penetrating the bilayer core. Integral membrane proteins of the erythrocyte include the glycophorins, the Rh proteins, transport proteins such as band 3, the sodium pump, Ca2+ – adenosine triphosphatase (ATPase) and Mg2+ -ATPase. Peripheral membrane proteins include the structural proteins of the spectrin-actin–based membrane skeleton.

Membrane Pathobiology The membrane is in intimate contact with excess unpaired globin chains found in the thalassemia syndromes and sickle hemoglobin (HbS) in sickle cell disease, leading to membrane distortion by physical effects. Membrane proteins are also subjected to the toxic byproducts of the oxidative stress induced by the presence of excess unpaired globin, and hemoglobin, particularly that induced by the decompartmentalization of erythrocyte iron.3 Oxidant Stress. The normal erythrocyte is under continuous oxidative stress, with cyclic hemoglobin oxygenation and deoxygenation constantly generating oxidants in the form of reactive oxygen species. Because the mature, anucleate erythrocyte lacks the ability to synthesize proteins or lipids damaged by reactive oxygen species, it has many antioxidants, including superoxide dismutase, catalase, thiol species such as glutathione and peroxyredoxin, and vitamin E, to combat this ongoing oxidant stress. Thalassemic and sickle erythrocyte are especially challenged by oxidants. The instability of reactive, hemecontaining ␣- or ␤-globin chains and HbS, which is relatively unstable compared with normal HbA, leads to autooxidation,4–7 generating additional peroxide and oxygen radicals. As a result, the erythrocyte’s normal defenses against reactive oxidant species begins to be overwhelmed, and irreversible oxidative damage of both membrane and cytoplasmic proteins and lipids occurs.8,9 Excess globin chains and HbS precipitate on the inner membrane surface. This most likely is mediated by the formation of insoluble hemichromes that are oxidation products of methemoglobin. They prompt formation of reactive oxygen species and lead to the release of iron from heme in the form of highly reactive free, or molecular iron.10 The interaction between HbS and phosphatidylserine (PS) present on the inner leaflet of the lipid bilayer also promotes the formation of methemoglobin, hemichromes, and the release of heme into the lipid phase.11,12 In the presence of peroxidation byproducts, this process liberates free iron and causes additional lipid peroxidation. The rate of HbS oxidation becomes 3.4-fold faster in the presence of lipids, which corresponds to a doubling rate for the oxidation rate for HbS in solution (1.7-fold greater for HbS than HbA).11 Thalassemia and sickle erythrocytes constantly generate excessive amounts of superoxide, peroxide, and hydroxyl radicals. Furthermore, levels or activities of scavengers and enzymes involved in antioxidant defenses are perturbed,

The Erythrocyte Membrane with decreased levels of glutathione, vitamin C, glutathione reductase, possibly glutathione peroxidase and catalase,13 decreased levels of plasma and erythrocyte vitamin E,14 variable levels of superoxide dismutase,13,15 and increased levels of glucose-6-phosphate dehydrogenase. Sickle erythrocytes also demonstrate decreased NADH redox potential, decreased hexose monophosphate shunt activity, and increased ATP catabolism, attributed to the ongoing need to regenerate intracellular glutathione. Membrane-associated Iron. In living cells, iron is typically separated from lipids. Thus, the association of iron with the lipid bilayer would be expected to lead to significant pathological effects, particularly by iron-catalyzed lipid oxidation. In the hemoglobinopathies, membraneassociated iron plays a major role in erythrocyte pathobiology by its damaging oxidative effects.12,16–18 On the cytoplasmic face of thalassemic and sickle erythrocyte membranes, abnormal membrane association of iron is compartmentalized into heme-derived iron, as hemeprotein or as free heme, or non-heme-derived iron, as molecular free iron or as ferritin iron. Quantitatively, these erythrocyte membranes contain approximately three times more total heme (heme proteins plus free heme) than normal erythrocytes.19 The deposition of heme proteins is primarily in the form of hemichromes.20,21 These heme proteins are associated with integral or membrane skeletal proteins, partially through disulfide bonding, with a fraction nonrandomly associated with membrane aggregates of the protein, band 3.22–24 Normal erythrocytes exposed to oxidant stress also develop clusters of denatured hemoglobin and band 3.25 Sickle membranes also contain free heme, at approximately 5% of total membrane heme.26 Nonheme-derived iron associates with the erythrocyte membrane as ferritin iron or as molecular iron (nonheme, nonferritin iron). Ferritin iron is present in small amounts and its significance is unknown. Molecular iron, which reacts rapidly with ferrozone and is removed from ghost membranes by high-affinity free iron chelators, is present in larger amounts than heme iron.21,27 Membrane affinity for molecular iron is extremely high, predicting that binding sites on the membrane are able to maintain cytosolic free iron at a concentration below 10−20 .16,28 Molecular iron is bioactive and accounts for most of the ability of these variant erythrocyte to generate highly reactive hydroxyl radicals.29 It is likely that molecular iron plays an important role in promoting oxidation of hemoglobin to methemoglobin and deposition of denatured hemoglobin onto the red cell membrane. Thus, catalytic iron associated with the membrane can valence cycle between ferric and ferrous states, allowing it to participate in redox reactions. Molecular iron is associated with the membrane either as a chelate with bilayer phospholipids, particularly PS, or nonrandomly associated with clusters of hemichrome and band 3 aggregates. The specific amounts of molecular iron associated with phospholipids or hemichromes are

159 unknown. Interestingly, denatured hemoglobin on sickle erythrocyte membranes can be coated with phospholipids, suggesting that some membrane-associated molecular iron is associated with hemichromes because of the phospholipids that cover them.30 The amount of membrane-associated molecular iron is unrelated to systemic iron status; however, the removal of iron from the sickle membrane has several potential benefits for the erythrocyte and might have potential therapeutic benefit. The iron chelator deferiprone (L1) is effective in removing free iron from sickle and thalassemic erythrocytes (Chapter 29).31,32 This has been attributed to the neutral charge of L1, allowing it to penetrate erythrocyte membranes. In contrast, the chelator deferoxime, which is charged, does not penetrate the membrane.33 L1 therapy has also been associated with a significant reduction in the activity of the potassium-chloride (KCl) cotransporter (KCC), whose activity is abnormally increased as the result of oxidative damage present in homozygous ␤ thalassemia erythrocytes.34 Development of a safe, effective, and highaffinity iron chelator would be useful to reduce the membrane damage associated with the deposition of free iron in sickle erythrocytes.

The Membrane Skeleton The membrane skeleton (Fig. 9.1) is composed of an intricately interwoven meshwork of proteins that interact with the lipid bilayer and integral membrane proteins. The major proteins of the membrane skeleton include spectrin, actin, ankyrin, protein 4.1R, and protein 4.2. Spectrin is the primary structural component of the membrane skeleton and its most abundant protein. Spectrin functions include provision of structural support for the lipid bilayer, maintenance of cellular shape, and regulation of lateral movement of integral membrane proteins. ␣␤-Spectrin heterodimers self-associate with other ␣␤-spectrin heterodimers to form tetramers, the functional spectrin subunit in the erythrocyte. Tetramers provide significant flexibility and structural support for the lipid bilayer, helping maintain cellular shape. Spectrin heterodimer–tetramer interconversion is a moderate affinity, temperature-dependent association that favors tetramer formation. The membrane skeleton is linked to the plasma membrane by several interactions. These include binding of spectrin tetramers to ankyrin, which in turn binds to the integral protein band 3 and binding of spectrin to the “junctional complex,” a multiprotein complex that includes actin, adducin, protein 4.1R, tropomyosin, tropomodulin, and dematin. Another membrane skeleton linkage to the plasma membrane is mediated via binding of a multiprotein complex containing the Rh proteins, the RH-associated glycoproteins, CD47, LW, glycophorin B, and protein 4.2 to ankyrin. Finally, direct interactions of several skeletal proteins with the anionic phospholipids provides another linkage of the membrane skeleton to the lipid bilayer.

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Glycophorin C/D Band 3 Band 3

Band 3 CD-47 Rh

Glycophorin A

Rh AG

4.2 Ankyrin-1

Band 3

LW p55

Glycophorin A Glycophorin B

Dematin 4.1R Actin

β Spectrin α Spectrin

Adducin 4.1R Tropomyosin

Tropomodulla

Figure 9.1. Schematic model of the red cell membrane. (Reprinted with permission from ref. 2.)

Membrane Skeleton in Thalassemia and in Sickle Erythrocytes. Numerous alterations in the membrane skeleton have been described in thalassemic and sickle erythrocytes. In thalassemic erythrocytes, alterations in the membrane skeleton vary depending on the type of thalassemia.35–39 ␣ Thalassemia erythrocyte membranes, isolated from patients with HbH disease (Chapter 14), have approximately a 50% decrease in high-affinity spectrinbinding sites, thought to be due to perturbed or aggregated ankyrin. ␤ Thalassemia membranes, isolated from patients with ␤ thalassemia intermedia, have abnormal protein 4.1R.35,38 These findings are highly selective; protein 4.1R function is normal in ␣- thalassemia erythrocytes and spectrin binding is normal in ␤ thalassemia erythrocytes. In both types of erythrocytes, spectrin function is normal.40 In normal erythrocytes, hemoglobin binds to the cytoplasmic surface of band 3, which is linked to the membrane skeleton via ankyrin,41–44 HbS, especially its deoxy form, binds more avidly than HbA.45–48 The precipitation of byproducts derived from excessive ␣- or ␤-globin chains or HbS on the inner surface of the membrane, followed by the associated oxidative damage leads to the formation of Heinz bodies. Denatured hemoglobin, the major constituent of Heinz bodies, binds to the cytoplasmic surface of band 3 with extremely high avidity, leading to aggregation of band 3 into clusters followed by binding of autologous immunoglobulin G and erythrocyte removal.22–24,30,49,50 Sickle erythrocytes lack the typical large Heinz bodies associated with HbH molecules in ␣ thalassemia or oxidant-challenged glucose-6-phosphate dehydrogenase– deficient erythrocytes. Instead there are smaller inclusions

composed of hemichrome,20 called by some “micro-Heinz bodies.” In the regions of sickle erythrocyte membrane associated with micro-Heinz bodies, protein topography and distribution are drastically altered, with aggregation and clustering of band 3, glycophorin and ankyrin.22 As sickle erythrocytes dehydrate, there is progressive membrane protein redistribution, particularly near micro-Heinz bodies, leading to irregular, negatively charged clumps on the membrane surface. A similar process of band 3 aggregation and altered topography occurs in thalassemic red cell membranes. Continuous polymerization of HbS with formation of long spicules can physically dissociate fragments of the lipid bilayer from the membrane skeleton.51 This uncoupling of membrane from skeleton is most likely due to the growth of the sickle polymer though gaps in the membrane skeletal network. The membrane that comprises the spicule contains band 3, but not spectrin, which is limited to the body of the cell and the base of the spicule. The detachment of the lipid bilayer containing band 3 and possibly other integral membrane proteins from the skeleton likely facilitates membrane release from the erythrocyte as spectrin-poor vesicles (see later). Other changes have been observed in membrane proteins in sickle cell disease. The lateral mobility of band 3 and glycophorin is abnormal, becoming increasingly and progressively more aggregated in erythrocytes of increasing density.52 Ankyrin exhibits abnormal binding properties in sickle erythrocytes, both in binding band 3 and binding spectrin,53,54 similar to defects observed in erythrocytes containing Heinz bodies due to unstable

The Erythrocyte Membrane

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Band 3 spectrin

-S-S-

ankyrin

Fe denatured hemoglobin

free heme

Fe molecular iron

Figure 9.2. Pathological iron compartments on sickle red cell membranes. These compartments include (left to right): denatured hemoglobin associated by disulfide bonding or noncovalently, free heme associated with protein or lipid, and molecular iron associated with membrane aminophospholipid and with denatured hemoglobin. (Reprinted with permission from ref. 16.)

hemoglobinopathies (Chapter 24).55 These changes could be induced by changes brought about by oxidation, for example the thiol redox status of spectrin, ankyrin, and protein 4.1R is abnormal in sickle erythrocytes,56 or by direct deposition of denatured hemoglobin on the inner membrane. Functional abnormalities of protein 4.1R have been reported in sickle erythrocytes on one study57 but are unconfirmed.53 Oxidative changes of skeletal proteins might lead to loss of membrane flexibility.58

cell anemia (Chapter 19; Fig. 19.1). These dense, HbFpoor cells contain virtually no polymerized hemoglobin when they are fully oxygenated; they survive only a few days. ISCs maintain a deformed shape when exposed to oxygen or other factors that result in reversal of HbS polymerization;71,72 they were one of the first indicators of membrane perturbation in sickle erythrocytes. The number of ISCs is greatest in the dense, dehydrated cell population and correlates well with hemolysis and spleen size, but not with the prevalence of vasoocclusive complications.73,74 ISCs can be produced by ATP depletion, calcium accumulation, and oxy-deoxy cycling.75–78 The abnormal morphology of the ISC is maintained by irreversible deformation of the spectrin–actin membrane skeleton.79 Spectrin dimer–tetramer self-association could play a role in this permanent deformation, by the dissociation of spectrin tetramers into dimers, which then reassociate into tetramers in a permanently deformed configuration.80 It has also been proposed that the rigidity of the ISC is due to a defect in ␤-actin, which is brought about by oxidative changes that lead to formation of a disulfide bridge between two cysteine residues critical for actin function.81,82 Diminished content of reduced glutathione and other crucial antioxidants in ISCs might play a role in facilitating oxidant damage and posttranslational modification of ␤-actin in sickle erythrocytes.13,83,84

Membrane Lipids Membrane Loss and Vesiculation Sickle erythrocytes shed part of their membrane as spectrin-deficient vesicles during cyclic oxygenation and deoxygenation.59,60 These vesicles are likely derived from spicules that are apparently completely uncoupled from the underlying membrane skeleton.51 This vesicular shedding is viewed by some as the ultimate stage in membrane deformation induced by erythrocyte sickling. Some vesicles contain phosphoinositol-anchored membrane proteins,60–62 leading to depletion in the residual erythrocytes of the complement regulatory proteins acetylcholinesterase and decay accelerating factor (CD55). It has been hypothesized that this leaves the erythrocyte susceptible to complement-mediated intravascular hemolysis,63,64 facilitating erythrocyte recognition and removal by macrophages.65 Vesicles are also thought to play a role in the hypercoagulability of sickle cell disease.66 Vesicles produced by erythrocyte shedding shorten in vitro clotting times67,68 dramatically enhance generation of thrombin from the prothrombinase complex in vitro,69 and bind protein S in vitro, possibly accounting for decreased protein S levels in vivo.70 Irreversibly Sickled Cells. Irreversibly sickled cells (ISCs) are elongated, pointed erythrocytes that are found in the well-oxygenated peripheral blood smears of most patients with sickle cell disease, especially individuals with sickle

The human erythrocyte contains approximately 455 million lipid molecules within the lipid bilayer, where they comprise approximately half the weight of the plasma membrane. This bilayer is composed predominantly of phospholipids intercalated with unesterified cholesterol in nearly equimolar concentrations, and small amounts of glycolipids. The major membrane phospholipids are phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), sphingomyelin (SM), and PS. Small quantities of phosphatidic acid, phosphatidyl inositol (PI) and lysophosphatidyl choline (lysoPC) are also found. Cholesterol is distributed unequally between the inner and outer monolayers,85 and

HbS polymers Lipid bilayer Membrane skeleton Site of skeletonlipid bilayer uncoupling Figure 9.3. Model of the long spicule in the deoxygenated sickled cell. Hemoglobin S polymers penetrate the membrane skeleton, and uncouple the lipid bilayer from the skeleton. (Reprinted with permission from ref. 51.)

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The precise identity of all the proteins maintaining phospholipid asymmetry in the erythbudding thrombin vesicle rocyte is still unclear.87 Moreover, despite Fc rec exhaustive searching, the identity of the PS igG receptor has not yet been discovered in any xa b2-Gp va biological system.89 A recent study demonPS rec strated the existence of a novel functional adhesion receptor for PS on the microendothelium upregulated by hypoxia, cytokines, and heme.90 Membrane Lipid Alterations. Mature erythscramblase translocase floppase rocytes are unable to synthesize fatty acids, cytoskeleton 2+ phospholipids or cholesterol; thus exchange Ca calpain ATP pathways account for lipid modifications. ADP Cholesterol is rapidly exchanged with unesFigure 9.4. The regulation and physiology of membrane phospholipids asymmetry. This model terified cholesterol from plasma lipoproteins describes how membrane phospholipids asymmetry is generated, maintained, and perturbed as a in the circulation. PC and SM are slowly prerequisite to various PS-related pathophysiologies. Membrane lipid asymmetry is regulated by the cooperative activities of three transporters: 1) the ATP-dependent aminophospholipid-specific exchanged with plasma lipids, whereas PS translocase, which rapidly transports PS and PE from the cell’s outer-to-inner leaflet; 2) the and PE do not participate in lipid exchange. ATP-dependent nonspecific lipid floppase, which slowly transports lipids from the cell’s innerAnother potential lipid renewal pathway, fatty 2+ to-outer leaflet; and 3) the Ca -dependent nonspecific lipid scramblase, which allows lipids to acid acylation, is an ATP-dependent process move randomly between both leaflets. The model predicts that the translocases are targets for in which fatty acids combine with lysophosCa2+ that directly regulates the transporter’s activities. Elevated intracellular Ca2+ induces PS randomization across the cell’s plasma membrane by providing a stimulus that positively and phatides to remake the native phospholipids, negatively regulates scramblase and translocase activities, respectively. At physiological Ca2+ renewing damaged or lost fatty acid side concentrations, PS asymmetry is promoted because of an active translocase and floppase by chains. The composition of red cell phospho2+ inactive scramblase. Depending on the type of cell, elevated intracellular Ca levels can be lipids is quite distinct from that of plasma 2+ achieved by cellular stores. Increased cytosolic Ca can also result in calpain activation, which phospholipids, suggesting that specific pathfacilitates membrane blebbing and the release of PS-expressing procoagulant microvesicles. The appearance of PS at the cell’s outer leaflet promotes coagulation and thrombosis by providing ways exist in red cells to remodel phosa catalytic surface for the assembly of the prothrombinase and tenase (not shown) complexes pholipids to optimize their function. Dietary and marks the cell as a pathological target for elimination by phagocytes. Recognition of the PSchanges have only a minimal effect on the expressing targets can occur by both antibody-dependent and direct receptor-mediated pathways. composition of erythrocyte membrane phos(Amino phospholipids are shown with dark polar head groups and choline phospholipids with pholipids. The inability of the erythrocyte lights polar head groups b2-Gp, B2-glycoprotein-1; rec, receptor). (Reprinted with permission from ref. 106.) from individuals with sickle cell disease and thalassemia to maintain normal lipid composition and repair or renew oxidized lipids, particularly fatty the phospholipids are also asymmetrically organized, with acids, during periods of oxidative stress, a process essenPC and SM, the choline phospholipids, primarily in the tial for erythrocyte survival, leads to a variety of changes in outer monolayer, with most of PE, all of PS, the amino phosmembrane structure and function.91 pholipids, and the phosphoinositides, in the inner monolayer. Loss of Phospholipid Asymmetry. Maintenance of phosMaintenance of Phospholipid Asymmetry. The asympholipid asymmetry, particularly localization of PS and metrical distribution of membrane bilayer phospholipids, phosphoinositides to the inner monolayer, has important first recognized in erythrocytes,86 is universal in eukaryfunctional consequences.92 Typically, PS is exposed when a otic cells. This asymmetrical distribution of phospholipids signal for activation of a specific biological process, such as is a dynamic system involving a constant exchange between blood clotting or cell recognition and removal, is required. phospholipids of the two bilayer leaflets. Generated priIn thalassemia and sickle cell disease,93–97 outward expomarily by selective synthesis of lipids on one side of the sure of PS in subpopulations of erythrocytes leads to membrane, a number of proteins participate in the mainteactivation of blood clotting, increased cellular destrucnance or dissipation of this lipid gradient.87,88 “Flippases,” tion, increased adhesion to endothelial and mononuclear phagocytic cells1,98,99 and other effects. In sickle or aminophospholipid translocases, move phospholipids, particularly PS, from the outer to the inner monolayer using cell disease, morphological sickling upon deoxygenation Mg++ -ATP, keeping them sequestered from the cell surresults in exposure of external PS, especially in membrane spicules.94 In some cells, PS exposure persists after reoxyface, and “floppases” do the opposite against a concentration gradient in an energy-dependent manner. “Scramgenation. These PS-exposing cells are in the densest and blases” are bidirectional, ATP-independent transporters very lightest or reticulocyte-rich erythrocyte fractions100–102 that move phospholipids bidirectionally down their conwith the number of PS-exposing cells varying among centration gradients in an energy-independent manner. patients, changing during time in individual patients, and prothrombin

MACROPHAGE

The Erythrocyte Membrane decreasing after transfusion.95 Normal and sickle reticulocytes both exhibit a moderate degree of externalized PS, and the reticulocytosis of sickle cell disease complicates so some degree the interpretation of studies on PS exposure. Unlike mature erythrocytes, however, some “mature” sickle erythrocytes have moderate PS exposure, and a subset of the dense, ISC-rich population shows much higher degrees of PS exposure than that seen in reticulocytes.101 PS-exposing surfaces propagate the proteolytic reactions that result in thrombin formation and activation of fibrinolysis by conversion of prothrombin to thrombin by providing docking sites for assembly of coagulation factors on their surfaces.103,104 Exposure of PS also participates in feedback inhibition of thrombin formation via activation of the protein C pathway. These PS-exposing surfaces also can promote anticoagulation by providing a catalytic surface for factor Va inactivation by activated protein C.105,106 In sickle cell disease, the number of circulating erythrocytes with exposed PS has been correlated with the risk of stroke.107 It has been suggested that other coagulation abnormalities observed in thalassemia and sickle cell disease, including decreased protein C and S activity and increased anti-PS antibodies, might be caused by circulation of PS-exposing erythrocytes.108–110 In support of this hypothesis, the number of PS-exposing sickle erythrocytes correlated with plasma 1.2 (F1.2), d-dimer, and plasmin– antiplasmin complexes, but not the number of PS-positive platelets in pediatric sickle cell disease patients, suggesting that sickle erythrocytes and not platelets are responsible for the hemostatic activation.111 High levels of HbF are associated with decreased erythrocyte PS exposure and decreased levels of thrombin generation and microvesicle formation.112 Splenectomized HbE–␤ thalassemia patients exhibit significant levels of circulating plasma thrombin– antithrombin III complex associated with increased numbers of PS-exposing erythrocytes (Chapter 18).113 PS-exposure has been thought to be a signal for recognition by and attachment of these cells by macrophages of the reticuloendothelial system, marking them for destruction.114,115 This mechanism of erythrocyte removal, shown in a murine model of sickle cell disease, is thought by some to contribute to the reduced red blood cell survival observed in sickle cell disease and the thalassemias.91,108 In vivo studies using autologous, biotin-labeled sickle cells are not consistent with rapid removal of PS-exposing erythrocytes.101 PS exposure has other effects on sickle erythrocytes. Increased PS and PE exposure has been associated with activation of the alternative complement pathway.116 Highly PS-positive sickle erythrocytes, including the densest sickle cells, cause an increase in endothelial cell tissue factor expression in vitro, not due to erythrocyte– endothelial interactions, but rather to increased levels of cell-free hemoglobin due to hemolysis (Chapter 11). Finally, PS-exposing erythrocytes can become targets for phospholipases. For example, secretory phospholipase A2 (sPLA2 )

163 will hydrolyze lipids of PS-exposing but not normal erythrocytes,117 generating lysophospholipids and free fatty acids. In the presence of sPLA2, PS-exposing erythrocytes generate lysophosphatidic acid, which effects vascular integrity.117 sPLA2 levels appear to predict impeding acute chest syndrome in sickle cell disease,118 which would potentially allow intervention to prevent or ameliorate this devastating condition.119 Strategies to bind PS or inhibit sPLA2 activity could prove to be therapeutic targets in sickle cell and other diseases.91 Although initially attributed to oxidative damage to the membrane, oxidative damage per se is not the cause of increased PS-exposure in sickle erythrocytes.120 Repeated cycles of sickling and unsickling associated with HbS polymerization and depolymerization with resulting changes to the erythrocyte membrane likely contributes to the production of terminal spicules and vesicles with increased PS exposure.60 In normal erythrocytes, neither “flippase” nor “floppase” activity is influenced by cellular oxygenation. Deoxygenation of sickle erythrocytes, however, is associated with increased PS and PE exposure and decrease in “flippase” activity,121 particularly in PS-exposing erythrocytes.100 Decreased “flippase” activity has been attributed to oxidative stress and sulfhydryl modifications. Deoxygenation of sickle cells results in both exposure of PS and the disruption of the membrane skeleton in membrane spicules, suggesting a role of skeletal proteins in the maintenance of phospholipid asymmetry. “Flippase” inactivation alone, however, will not precipitate PS exposure. Activation of the “scramblase” is also required via increased levels of cytosolic calcium and/ or enhanced calcium influx.120 Not surprisingly, sulfhydryl modifications of the “scramblase” leads to increased PS exposure and a lower calcium requirement for scrambling,122 leading to the suggestion that oxidative modifications of sulfhydryl groups in both the “flippase” and “scramblase” contribute to increased PS exposure by sickle erythrocytes. Phosphoinositides. Phospholipids with a phosphoinositol-containing polar head group, which may be mono(PIP or PI-4-monophosphate) or biphosphorylated (PIP2 or PI-4,5-biphosphate), make up the phosphoinositides. Comprising only approximately 2.5% of membrane phospholipids, they have significant biological activity, including a role in maintenance of erythrocyte red cell shape and deformability. Some membrane proteins, including proteins involved in complement regulation, are anchored to the red cell membrane through a phosphoinositol lipid domain.123 This allows these proteins to move laterally in the membrane, preventing complement-mediated membrane damage. Phosphoinositol-anchored proteins are lost through the release of lipid-enriched vesicles from the cell during the membrane remodeling that accompanies reticulocyte maturation or cell aging. This process of vesiculation and loss of complement regulatory proteins is accelerated in sickle cell anemia by repeated cycles of

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and resulting in the persistence of the polymer in the oxygenated state.124–126 Dehydrated sickle cells also exhibit increased adhesion to endothelial cells, leukocytes, and other sickle erythrocytes, promoting endothelial damage and facilitating vasoocclusion.98,127–129 Experimental evidence for enhanced adherence of dense cells to endothelium is dependent on assay conditions, particularly shear stress (Chapter 8) In addition, dehydration directly impairs the rheological integrity of sickle cells, reducing deformability and increasing fragility.130–133 A distinguishing feature of sickle cell disease is the heterogeneity in the volume and water content of erythrocytes (Fig. 9.5).72,134 In addition to large numbers of reticulocytes and young red cells with increased volume Figure 9.5. Dense erythrocytes in sickle cell blood. Whole blood samples on continuous density and normal to low CHC, the blood of patients gradients reveal a range of densities for normal blood (AA). Sickle cell anemia (SS) blood contains with sickle cell disease contains dense, dehya broader distribution with more low-density cells, mostly reticulocytes, and variable numbers of drated erythrocytes and reticulocytes. The dehydrated cells with extremely high hemoglobin. (From ref. 148.) number of dehydrated cells with high CHC can be estimated by flow cytometry as the sickling, making these cells sensitive to complementnumber or percentage of cells with CHC more than mediated lysis.64 41 g/dL or by centrifugation on density gradients (density > 1.1100).134–137 This dense cell population is rich in ISCs. Early work established that the cation content of sickle erythrocytes was abnormal and was perturbed ALTERATIONS IN CATION CONTENT AND by deoxygenation.138–140 Later studies established that the CELLULAR HYDRATION dense, ISC-rich fraction of sickle cells was due to severe The critical cellular function of maintaining cell volume potassium depletion, with a lesser variable degree of is accomplished by regulation of water content. Because sodium loading.141,142 More recently, the presence of overwater is at osmotic equilibrium in most cells, cellular hydrahydrated, sodium-loaded cells has been found in sickle tion state is a function of the content of cations (Na+ , cell disease.143–145 These low-density cells with low CHC + 2+ 2+ − − K , Ca , and Mg ) and anions (Cl , HCO3 , 2,3-BPG, are older than most other sickle erythrocytes, appear to hemoglobin). In red cells, hemoglobin content, which is be derived from dehydrated cells, exhibit greater oxidative similar in sickle and normal cells, is determined by synthedamage than other sickle cells or normal cells, and have sis during erythroid differentiation, and monovalent anion very short in vivo survival.143,145–147 High cation permeabilcontent is fixed by Donnan effects. Thus, cation content ity of these cells144 supports the idea that they represent is the major variable determinant of cell volume and is a population of end-stage cells with damaged memsubject to regulation by several specialized transport sysbranes undergoing swelling that culminates in intravastems. Substantial volume reduction occurs after release cular hemolysis. Such osmotic lysis may be a source of of normal reticulocytes from the marrow: within approxfree plasma hemoglobin contributing to the perturbaimately 2 days, cell volume drops from 115 to 85 f L and tions of nitric oxide metabolism that foster endothelial cell cell hemoglobin concentration (CHC) increases from 26–28 dysfunction and inflammation (Chapter 11). to 32–34 g/dL. Thereafter, cell volume and hemoglobin The fraction of dense, dehydrated sickle cell ranges from concentration remain remarkably stable over the cell’s 0% to 40% and varies among patients and over time in 100–120 day lifespan. each patient. The number of dense cells decreases durIn sickle cells, dysregulation of cell volume is evident ing pain episodes,148–151 suggesting their selective removal in the presence of dehydrated cells with high CHC. This during vasoocclusion. Dense cells are more susceptible to abnormal hydration state is an important factor in the hemolysis,130 and dense cell numbers correlate with the pathogenesis of sickle cell disease, because the polymerizadegree of hemolysis.74 Coincident ␣ thalassemia is assotion of HbS is exquisitely dependent on its cellular concenciated with reduced numbers of dense sickle cells and tration (Chapter 6).124 Increased CHC resulting from erythmilder hemolytic disease.152–154 Individuals with the greatrocyte dehydration markedly enhances the tendency of est numbers of dense cells appear to have the fewest pain HbS to polymerize, reducing delay time for polymerization episodes,134,152–157 a paradox that might be explained by

The Erythrocyte Membrane the selective destruction of dense cells during vasoocclusive episodes. Because of the importance of cellular dehydration in sickle cell disease pathophysiology, understanding the mechanisms of dehydration has potential for stimulating new therapeutic approaches to the disease. Proof of principle of this approach has been provided by several studies in humans and mouse models using specific inhibitors for cation transport pathways involved in sickle cell dehydration (Chapter 31).158–163 Four major transport mechanisms have been implicated in the dehydration of sickle erythrocytes.

Deoxygenation-induced Cation Leak, Sodium–Potassium ATPase, Cell Sodium, and Sodium/Hydrogen Exchange in Sickle Erythrocytes Deoxygenation-induced Cation Permeability. Seminal studies established that deoxygenation of sickle cells was associated with potassium loss and sodium gain and that increased permeability took place through a diffusional pathway and was accompanied by stimulation of the sodium–potassium pump.138–140 The deoxygenationinduced increase in cation permeability of sickle cells has been amply confirmed75,164–166 and extensively characterized.167–172 Deoxy sodium influx and potassium efflux are activated when oxygen tension drops below 40–50 mm Hg (Fig. 9.6), correlating with deoxygenation of HbS and cell sickling.171,173 Cation flux via the activated deoxygenation-induced pathway is dependent on external and internal pH, reaching maximal values at pH 6.9– 7.0.171 The deoxygenation-induced pathway is not selective among the alkali metal cations lithium, sodium, potassium, rubidium or cesium, and also permits passage of calcium and magnesium.174–176 Deoxygenation does not, however, increase membrane permeability to organic cations such as tetramethyl- or tetraethyl-ammonium,171,177 or sugars such as erythritol, arabinose, or mannitol,177 reflecting a selectivity for metal cations. The basal permeability of the erythrocyte membrane to anions is roughly three orders of magnitude higher than that for cations, so that the permeability of the deoxygenation-induced pathway to anions cannot be assessed with accuracy.170 These properties are suggestive of ion movement via a diffusion pathway, with restriction to monovalent or divalent cations. Under conditions of very low osmotic strength, deoxygenation induces an increase in sucrose permeability,178 but its relationship to the sickling-induced cation pathway is not clear. Inward transport of sodium and outward transport of potassium are balanced in deoxygenated sickle cells and do not lead directly to cell dehydration (Fig. 9.6);166–168,179 however, the presence of external calcium and other divalent cations inhibits sodium influx more that potassium efflux, resulting in an imbalance between the two fluxes and a net potassium loss.172,179 No evidence of Gardos

165 channel activation was found in these experiments, indicating that the net potassium loss was indeed mediated by the deoxygenation-induced pathway. Interestingly, this effect was enhanced by the presence of heparin, suggesting modulation by a receptor–ligand interaction.179 The sodium–potassium pump might also play a role in sickle cell dehydration. Early work in normal human red cells indicated that activation of the pump in conditions of high sodium content lead to cell dehydration.180 In vitro evidence for erythrocyte dehydration mediated by activation of the sodium–potassium pump has been provided for deoxygenated sickle cells and for red cells in hereditary xerocytosis that exhibit a balanced cation leak.167 In deoxygenated sickle cells, increased cell sodium, even though initially balanced by potassium loss, stimulates the sodium–potassium pump to effect net cation loss due to its 3 Naout /2 Kin stoichiometry. The integrated red cell model, discussed later, predicts that this mechanism cannot fully account for the extreme potassium depletion of sickle cells181 because its contribution diminishes as the potassium gradient dissipates. The activation of deoxygenation-induced permeability pathway is associated with morphological sickling171,173 and can be impeded by agents that interfere with HbS polymerization.182,183 High cation fluxes are associated with deoxygenation under conditions producing marked morphological changes and extensive spicule formation, such as gradual deoxygenation, alkaline pH, low hemoglobin concentrations, and reticulocyte deoxygenation.171,173,179 This suggests that activation of this pathway is triggered by spicule formation, which is associated with disruption of spectrin–band 3 associations and perturbation of phospholipid and cholesterol domains.51,69,184 A relationship between deoxygenation-induced cation pathway and the nonselective cation leak induced by shear stress and membrane deformation is suggested by their similar physiological and pharmacological characteristics.185 The nature of these associations and the identification of the deoxygenation-induced pathway deserve further study. Deoxygenation-induced fluxes of sodium, potassium, and calcium are reduced by the anion exchange inhibitor diisothiocyanostilbene disulfonate (DIDS) without affected morphological sickling.169,175,177 Dipyridamole, an anion transport inhibitor, also blocks deoxygenation-induced cations fluxes in vitro, although other anion transport blockers are ineffective.176 DIDS-sensitive cation fluxes, presumably mediated by the anion exchanger, can be elicited in normal red cells by incubation in low chloride media.186 Single amino acid substitution in the anion exchange protein associated with stomatocytosis syndromes has been shown to be associated with increased erythrocyte cation permeability, sensitive to DIDS and dipyridamole; expression of these mutant anion exchangers in xenopus oocytes conferred increased cation permeability with similar inhibitor sensitivity.187 These findings

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Figure 9.6. Deoxygenation-induced permeability increase to mono- and divalent cations in sickle red cells. (A) Sodium and potassium influx as a function of pO2 . Increased permeability below 40 mm Hg corresponds to morphological sickling. (from ref. 168, used with permission) (B) Ca permeability, assessed as 45 Ca content of cells containing the chelator quin2 to minimize efflux. Increased cellular calcium uptake in deoxygenated sickle cells (
) compared with oxygenated cells ( ); DIDS partially inhibits calcium uptake in deoxygenated (), but not oxygenated cells ( ). (from ref. 175, used with permission) (C). Changes in cellular magnesium content upon deoxygenation in sickle cell incubated in high external Mg (5 mmol
), normal Mg (0.5 mmol
) or no Mg (5 mmol EDTA ). Oxygenated cells ( ) had stable Mg contents under all conditions. (Used with permission from ref. 174.). Normal red cells exhibit minimal changes in cation permeability upon deoxygenation.

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support the notion that the anion exchange protein, at least in some altered states, could mediate deoxygenationinduced fluxes in sickle erythrocytes. Regardless of the mechanism, pharmacological inhibition of deoxygenationinduced cation movements provides a potential avenue for therapeutic intervention to improve sickle cell hydration (see Chapter 31). Alterations in Cation Permeability by Shear Stress and Oxidation in Sickle Erythrocytes. Marked mechanical deformation of normal cells leads to a reversible increase in cation permeability.188–191 In the absence of external

calcium, the pathway mediates equivalent sodium and potassium movement,189,190 but in the presence of calcium, potassium loss is accelerated by activation of the calciumdependent Gardos pathway,192 suggesting that the pathway also mediates calcium influx. Cation fluxes induced by mechanical stress are chloride-independent. The pathway is activated in sickle erythrocytes at lower shear stress than normal cells,193 perhaps as a consequence of the oxidant damage to the membrane. When normal cells are mildly oxidized with t-butyl hydroperoxide, the leak is increased and activated at lower shear stress.188,189 Under

The Erythrocyte Membrane hypotonic conditions that induce cell swelling, the deformation-induced leak is increased, with potassium loss in excess of sodium gain, and is partially inhibited by bromide.185 Interestingly, deoxygenation-induced cation leaks in sickle cells was also reduced by bromide.185 Mechanically induced cation fluxes are partially blocked by DIDS, independently from the drug’s effect on anion permeability.185,194 Thus, the mechanically induced cation leak and the deoxygenation-induced pathway share a number of physiological and pharmacological characteristics, including an apparent origin in membrane deformation. It is conceivable that they represent the same mechanism and that the abnormal oxidation state of the sickle membrane increases the deoxygenation-induced cation leak in sickle erythrocytes. Sodium Permeability and Cell Sodium Content in Sickle Erythrocytes. The potassium depletion responsible for sickle erythrocyte dehydration is associated with variable degrees of sodium loading, especially marked in high-density cell populations rich in ISCs,195 which could result from increased sodium permeability or decreased activity of the sodium–potassium pump. Pump activity is abnormally decreased in dehydrated sickle cells, although ATPase activity in membranes derived from these cells is not,142 suggesting that there is abnormal downregulation of sodium–potassium pump activity in dense cells. One possible inhibitory factor might be the increased magnesium to phosphate ratio in these cells; when this ratio was normalized in vitro, the activity of the sodium–potassium pump was restored to normal.174 Increase sodium permeability could also contribute to high cell sodium content of dense cells. Several sodium influx pathways have been identified, including Na/H exchange,196 NaKCl cotransport,197 Na/Mg exchange,198 but assessment of their activities and contribution to net sodium influx has been variable. Earlier estimates of very high Na/H exchange rates in sickle erythrocytes196 have not been reproduced,199 and the sodium permeability in the dense cell population does not appear abnormally elevated. Sodium/hydrogen exchange is not stimulated by deoxygenation, indicating that the increase in intracellular calcium associated with deoxygenation is not sufficient to activate phosphokinase C and stimulate the Na/H exchanger. Likewise, there are no data to suggest that reduction in a sodium influx pathway contributes to dehydration in any sickle cell population. The presence of a subpopulation of extremely sodium loaded cells in the least dense fraction of sickle blood was revealed by the failure of these cells to undergo dehydration upon exposure to the potassium ionophore valinomycin.143 These cells are older sickle cells that arise from the dense cell population145,146 and might represent a terminal stage on their way to osmotic lysis (see below). Some of the sodium loading apparent in both the high- and low-density population of sickle cells could be due to this subpopulation of sodium loaded cells.

167 Cell Calcium and Calcium-activated Potassium Channel (Gardos Pathway) in Sickle Erythrocytes Cell Calcium and Calcium Pump Activity. Early studies indicated very high cell calcium in sickle cells, ranging from 110 to 300 ␮mol/L cells,200,201 two orders of magnitude higher than in normal cells (0.9–2.8 ␮mol/L cells).202 Free ionized cytoplasmic calcium measured by a variety of techniques, such as ionophore-induced equilibration of intracellular chelator and 45 Ca, calcium-sensitive fluorescent dyes, fura-2 or benz-2, and nuclear magnetic resonance, ranges from 11 to 30 nmol/L and is similar in sickle and normal erythrocytes.175,203–205 This apparent discrepancy was explained by the demonstration of compartmentation of calcium in sickle erythrocytes into cytoplasmic vesicles, first demonstrated by electron probe x-ray analysis of cryosections.206–208 These vesicles are derived from the plasma membrane and contain integral membrane proteins, including the Ca-ATPase, in an inside-out configuration. The Ca-ATPase pumps calcium from the cytoplasm into these vesicles, creating a very high intravesicular calcium concentration.206,207 Most of the calcium contained in sickle erythrocytes is sequestered into vesicles. Deoxygenation of sickle, but not normal, red cells increases the permeability of the membrane to calcium, resulting in enhanced calcium influx (Fig. 9.6).175,176,209,210 The effects on cellular ionized calcium are complex and heterogeneous within a population of cells, depending on the balance between the influx rate in an individual cell and its capacity to extrude calcium via the Ca–ATPase pump. Deoxygenation enhanced calcium influx rate in sickle cells is increased five fold, and reduced calcium pump activity by as much as 28%.210 The net effect produced a threefold increase in cellular ionized calcium level from 10 to 30 nmol in the discocyte fraction. Although these levels do not rise to the 40-nmol concentration estimated as the threshold for activation of the Gardos channel,211 it was suggested that cellular heterogeneity could account for higher calcium levels in certain cell populations. Indeed, sickling-induced permeability changes were greatest in reticulocytes,179 which are known to exhibit the most dramatic morphological changes upon deoxygenation and to deform at higher oxygen tensions, despite their relatively low CHC.212 It was later shown that the deoxygenationinduced permeability change occurred in a subset of cells (see later).213 The deoxygenation-induced fluxes of calcium, sodium and potassium have similar pharmacological sensitivity to DIDS and dipyridamole, suggesting mediation by a common pathway.169,175,176 Calcium-activated Potassium Channel (Gardos Pathway). In 1958, the Hungarian physiologist, Gyorgy G´ardos, described calcium-dependent potassium efflux from ATPdepleted red cells.214 These fluxes are now known to be mediated by a specific type of potassium channel activated by increased cytoplasmic calcium and known by several designations. Small (or intermediate) conductance

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Figure 9.7. Dehydration of sickle cells via the Gardos pathway. Frequency distributions of cellular hemoglobin concentrations (CHC) were measured by Bayer Advia automated cell counter; normal range of 28–41 g/dL is shown vertical markers. In vitro activation of the Gardos pathway by incubation of sickle cells in the presence of external calcium plus ionophore A23187 induces shift to higher CHC (upper left), which is blocked by chelation of calcium or by the Gardos channel inhibitor, clotrimazole (CLT). Cyclic deoxygenation (3 hours, 1 min O2 , 4 min N2 ) also produces a calcium-dependent (upper right) shift to higher CHC, absent in EGTA and inhibited by CLT. (Modified from ref. 239.)

calcium-activated potassium channel, IK1, SK4, all describe the product of the KCCN4 gene,215–217 which codes for a protein of 428 amino acids with six transmembrane domains and a pore region with the canonical GYGD sequence that determines K+ selectivity in numerous potassium channels.218 mRNA for KCNN4, but not KCNN3, increases during erythroid differentiation and is present in reticulocytes; protein is detected by KCNN4 protein antibodies in mature erythrocyte membranes.215 The peptide toxin charbydotoxin (ChTx) is a specific inhibitor of the human red cell Gardos channel.219,220 Binding studies with 125 I-ChTx demonstrated that normal human erythrocytes possess approximately 150 of these channels per cell.221 Upon uniform, maximal activation of Gardos channels via controlled ionophore-induced permeabilization of red cells to calcium, rapid, but remarkably uniform dehydration of both sickle and normal erythrocytes occurs.222 These results suggest a uniform distribution of channels among erythrocytes and are consistent with estimates of several hundred channels per cell. Generation of dense ISCs under conditions of ATP depletion was dependent on external calcium and an outwardly directed potassium gradient.75 When sickle cells are deoxygenated under conditions in which ATP levels are maintained, calcium-dependent formation of dense, dehydrated cells was observed (Fig. 9.7).76,158,223–226 Sickle erythrocyte dehydration produced by rapid in vitro deoxygenation–oxygenation cycles that mimicked in vivo circulatory times was predominantly calcium dependent, suggesting it was predominantly mediated by the

Patrick G. Gallagher and Clinton H. Joiner Gardos channel.227 The integrated red cell model181,228 has examined the different modalities of dehydration for reticulocytes and provided theoretical and indirect experimental evidence for a calcium dependent process based on transient activation of the Gardos pathway upon deoxygenation.179 Nevertheless, although Gardos channel potassium fluxes can be readily elicited in red cells in vitro, calcium-dependent potassium fluxes blocked by the specific Gardos channel inhibitors have been difficult to demonstrate directly upon deoxygenation of sickle cells. An elegant set of experiments helps to explain this apparent paradox. When sickle discocytes were deoxygenated, only 10%–40% became dense, and thus had evidence for calcium permeabilization and Gardos channel activation upon deoxygenation. The process was rapid and transient, and the resultant dense cell fraction did not increase with prolonged deoxygenation. If those dehydrated cells were removed, however, and the procedure was repeated, a similar fraction of cells became dense. These studies suggest that the activation of the sicklinginduced permeability pathway that permits calcium influx in deoxygenated sickle cell is a stochastic process affecting a small fraction of cells during each deoxygenation event. Thus Gardos-mediated potassium efflux is rapid and transient in only a few cells upon a given deoxygenation. This explains why oxy–deoxy cycling has generally been more effective than continuous deoxygenation in eliciting calcium-dependent density shifts in sickle cells and why Gardos potassium fluxes have been difficult to measure directly in vitro. Modulation of Gardos Channel Activity in Sickle Cell Disease. Gardos channel activity is subject to regulation in vitro by cytokines and lipid mediators of inflammation known to be elevated in persons with sickle cell disease as a result of the inflammatory vasculopathy associated with oxidative stress, endothelial cell damage and leukocyte activation (Chapters 8, 10, and 11). In murine erythrocytes, endothelin-1 (ET-1), a cytokine released from endothelial cells under oxidative or other stresses, increases both the internal calcium affinity and the Vmax of the Gardos channel. Pharmacological studies indicated that this effect was mediated by the ET-1 receptor B and involved activation of protein kinase C.229 Treatment of SAD sickle mice (Chapter 12) with a specific inhibitor of the ET-1 receptor B but not A, reduced sickle cell dehydration in vivo and Gardos channel fluxes measured ex vivo.161 The ability of ET-1 to augment Gardos channel activity was also demonstrated in human sickle cells in vitro. The increase in sickle cell density produced by oxy/deoxy cycling in vitro was enhanced by ET-1,230 indicating increased activation of the Gardos channel via sickling-induced calcium influx. In addition, two inflammatory cytokines – interleukin-10 and RANTES (Regulated upon Activation, Normal T lymphocyte Expressed and Secreted) – and the inflammatory

The Erythrocyte Membrane phospholipid mediator, platelet activating factor, had similar effects on Gardos channel activity in sickle cells. Other lipid mediators could augment Gardos channel activity by enhancing calcium influx. Treatment of normal erythrocytes with subnanomolar concentrations of prostaglandin E2 (PGE2 ) activated the Gardos channel in vitro in approximately 15% of cells, producing reduced cell volume and osmotic resistance,231 apparently as a result of an increase in calcium uptake by PGE2 , which has been demonstrated independently.232 Lysophosphatidic acid, a lipid mediator released from activated platelets, stimulates calcium uptake, detected by fluorescent dyes, in approximately 25% of red cells, as does activation of protein kinase C by phorbol esters and diacylglycerol. Calcium influx stimulated by lysophosphatidic and protein kinase C are both inhibited by ␻-agatoxin-TK, suggesting mediation by a P-type calcium channel and are modulated by inhibitors of tyrosine kinases (TK), but in subtly different ways, indicating that multiple signaling pathways might be involved.233,234 The modulation of Gardos channel activity by inflammatory cytokines and other mediators could be particularly relevant in sickle cell disease. Endothelial cells are stimulated to produce endothelin by interactions with sickle cells and activated leukocytes. Plasma levels of ET-1 and PGE2 are abnormally elevated in patients with sickle cell disease in the “steady state”235–237 and increase further with acute chest syndrome or other vasoocclusive events.236,238 It is possible that local levels of ET-1 and/or PGE2 in the microcirculation are even higher and potentiate Gardos channel activity of sickle cells during vasoocclusive or adhesive interactions. Such receptor ligand interactions could be exploited pharmacologically, as several specific blockers, such as ET-1 receptor antagonists, have been found to have clinical benefit in other disorders. Direct blockade of the Gardos channel is possible using the imidazole antimycotic clotrimazole and its derivatives, which acts by binding to the external pore of the channel (Chapter 31).239 Early studies indicated the ability of clotrimazole to reduce the number of dense cells in sickle cell patients.158 More recently, compounds lacking the imidazole ring have been shown to be effective Gardos channel blockers.240 One of these, senicapoc, has been tested in phase II and phase III clinical trials in sickle cell disease. Patients treated with a daily oral dose of senicapoc exhibited fewer dense cells, increased hemoglobin levels, reduced reticulocyte counts, lower bilirubin and lactate dehydrogenase levels, which was consistent with reduced hemolysis,163 and a predictable outcome of the mitigation of cellular dehydration. A phase III (ClinTrials. gov/, NCT00102791) study of senicapoc was recently terminated, as it was unlikely that its chosen endpoint, a reduction in pain episodes, could be reached. Nevertheless, given that vasoocclusion and hemolysis represent different aspects of sickle cell pathology, that dense cell numbers are

169 most closely associated with hemolysis, and that hemolysis appears to be associated with long-term complications, such as pulmonary hypertension, a drug such as senicapoc that reduces hemolysis might ultimately have important long-term benefit in sickle cell disease. In summary, the ionized calcium level in red cells in vivo is a dynamic balance between calcium influx and the compensatory capacity of the calcium pump and is normally maintained well below the threshold for activation of the Gardos pathway. Calcium influx can increase, especially in sickle cells, by a variety of mechanisms – triggering of the deoxygenation-induced pathway by sickling, modulation of calcium channels by cytokines or inflammatory mediators, or activation of stretch-induced cation channels by circulatory shear stress. Such influx events appear to produce calcium transients in red cells sufficient to activate the Gardos pathway. Compromise of the capacity of the calcium pump by physiological regulation or pathological damage would make such calcium transients greater in magnitude and/or more prolonged, increasing the probability of potassium channel activation and enhancing its adverse effects on cell volume. Modulation of the kinetic properties of the Gardos channel by inflammatory mediators might also enhance potassium loss in some cells. A substantial body of data now exists to support the occurrence of such events in vivo, at least in some populations of sickle erythrocytes. The quantitative integration of these events and their pathophysiological modulation in vivo remains a fruitful area of study.

Potassium Chloride Cotransport in Sickle Erythrocytes KCC was first described in red cells as a chloride-dependent potassium efflux stimulated by the sulfhydryl alkylating agent N-ethylmaleimide.241,242 Other activators of KCC in vitro include cell swelling, acid pH, urea, sulfhydryl oxidation, reduced cellular magnesium, and hyperbaric conditions.243–246 The activity of KCC is maximal in normal reticulocytes and young cells and is progressively reduced to negligible values in mature and dense normal red cells.197,247–250 Early reports established that sickle cell disease blood samples had high KCC activity (Fig. 9.8),197,251,252 although the quantitative comparison to normal cells is complicated by the presence of large number of reticulocytes and young cells in patient blood. KCC activity is highest in the least dense sickle cells, which contain most of the reticulocytes, and is least active in the dense cell fractions.197,252,253 The relative importance of activating stimuli for KCC and volume regulation in vivo is not known. Even reticulocyte rich fractions of sickle erythrocytes have minimal KCC activity in the absence of stimulation, and reticulocyte volume and CHC are stable upon in vitro incubation under these conditions.254 KCC fluxes are inversely proportional to whole blood MCHC over the range from 24 to 34 g/dL, but the relative activation of KCC in sickle and normal cells is indistinguishable

170

Figure 9.8. High levels KCl cotransport activity in sickle cells. High rate of acidstimulated potassium efflux in various density fractions reflects high activity of KCC in sickle cell disease blood. The highest activity is in the “top” density fraction containing the most reticulocytes, but other density fractions contain more reticulocytes than normal blood. Given that KCC activity declines with reticulocyte maturation, higher reticulocyte counts in sickle cell blood and the absence of older sickle cells means that cell age must be taken into account in any comparison of flux rates between sickle and normal erythrocytes, even in density fractionated cells. (From ref. 252.)

(Fig. 9.9). KCC activation by acid pH is exaggerated in sickle cells compared with normal cells (Fig. 9.9), so that if acidic conditions occur in the circulation, sickle cells would be more vulnerable to KCC-mediation dehydration than normal cells. Likewise, sickle cells are more sensitive to KCC activation by urea than normal cells (Fig. 9.9), showing activation at lower concentrations, well within those found in the medulla of the kidney. The heightened sensitivity of KCC in sickle cells to activation by acid pH and urea is due at least in part to reversible sulfhydryl oxidation, as suggested by its normalization on treatment by the sulfhydryl agent dithiothreitol. This raises the possibility of the therapeutic potential of reducing agents, such as N-acetyl cysteine, which has been shown to block in vitro dehydration of sickle cells,255 and, in a limited trial, to improve sickle cell hydration.256 Activation of KCC results in potassium, chloride and water loss, with reduction in cell volume and increase in CHC.252–254,257–260 This can be demonstrated in the phthalate density profile of sickle cell disease blood,257 but changes in normal erythrocytes with low reticulocyte counts are minimal. Measurement of the rapid reduction in reticulocyte CHC upon activation of KCC permits direct

Patrick G. Gallagher and Clinton H. Joiner comparison of sickle and normal cells, as shown in Figure 9.9. Whether activated by swelling, acid pH, or urea, sickle cells exhibit more extensive volume reduction than normal cells, achieving in each case a higher CHC. Swellinginduced volume regulation is not altered by sulfhydrylreducing agents, but abnormal sickle cell volume reduction triggered by acid pH and urea is partially normalized by dithiothreitol.254,261 Thus the CHC ‘set point’ for KCCmediated volume regulation appears to be abnormal in sickle cells and could result in part from their abnormal oxidation state.3,262,263 Deoxygenation of sickle erythrocytes produces complex changes in KCC activity. In normal human cells, and in fish and horse cells, KCC activity stimulated by urea, acid pH, or cell swelling is inhibited as pO2 falls.264,265 In sickle cells, activity initially declines with deoxygenation, but begins to increase again at approximately 40 mm Hg, with the onset of sickling; blockade of sickling with dimethyl adipimidate abolishes this effect.182 Nevertheless, activated KCC fluxes in fully deoxygenated cells are lower than in oxygenated cells, so that the overall effect of deoxygenation on activated KCC in sickle cells appears inhibitory. Part of this inhibition, although probably not all, results from the increase in cellular free magnesium concentration associated with binding of 2,3-BPG to deoxyhemoglobin.249 In sickle cells suspended in isotonic media at normal pH, in which KCC activity is minimal at high pO2 , deoxygenation activates KCC, especially if deoxygenation-induced increase in ionized magnesium is prevented by use of divalent cation ionophores.266 This behavior might explain other observations that cycles of oxygenation and deoxygenation produced chloride-dependent potassium loss and shifts toward higher density in sickle cells, especially reticulocytes. It has been suggested that on deoxygenation, changes in phosphorylation activate KCC, but the activity is masked by the increase in cellular magnesium, which is known to inhibit KCC. On reoxygenation, magnesium levels are restored to normal more rapidly than the changes in phosphorylation are reversed, providing a brief pulse of KCC activity that produces cumulative dehydration upon repeated cycling. If this mechanism occurs in vivo, cyclic deoxygenation in the circulation might be responsible for activation of KCC and dehydration of sickle cells, especially in reticulocytes which can linger in the venous circulation due to abnormal adhesive interactions.127 Cellular Magnesium and KCC in Sickle Cells. Although total erythrocyte magnesium content is reduced, especially in dense sickle cells, during deoxygenation the binding of 2,3-BPG, a major chelator of magnesium, to deoxy hemoglobin results in a large increase in free magnesium concentration. Cell sickling increases membrane permeability to magnesium and the transient outwardly directed magnesium gradient during deoxygenation produces magnesium efflux, resulting in reduced total magnesium content in sickle erythrocytes.174 Human erythrocytes also

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Figure 9.9. Abnormal regulation of KCC in sickle reticulocytes. Upper panels show activation of KCC-mediated fluxes as a function of initial MCHC (cell swelling), external pH, and urea concentration in normal (AA; open symbols) and sickle (SS; filled symbols) red cells. Fluxes are expressed as a percentage of the maximal volume stimulated flux to normalize for differences in the age distribution of the cells. Although SS proportionate activation by cell swelling is “normal,” response to acid pH is exaggerated, and activation by urea occurs at lower concentrations than in AA cells. (From refs. 254, 260.) Lower panels depict regulatory volume decrease, reflected as an increase in reticulocyte CHC with time upon activation of KCC. Reticulocyte CHC was measured by Advia cell counter. Regardless of how KCC is activated, the final MCHC achieved is higher in SS than in AA reticulocytes (From CH Joiner, unpublished data.)

possess a specific sodium–magnesium exchanges system, whose activity produces slow loss of magnesium from the erythrocyte.267 Sickle erythrocytes exhibit markedly increased activity of the sodium–magnesium exchanger, which could theoretically contribute to their reduced total magnesium content. Magnesium depletion of sickle cells is pathophysiologically relevant in view of the sensitivity of KCC to cellular magnesium. KCC is stimulated by the reduced levels of cellular magnesium found in sickle erythrocytes.159,174,268,269 The inhibition of KCC by increasing cellular magnesium content provides a new potential opportunity for preventing dehydration in thalassemic270,271 and sickle erythrocytes.160,272 Oral magnesium supplementation corrects the deficit in cellular magnesium in sickle cells, inhibits KCC cotransport, and reduces cell dehydration.160,272 Small pilot studies of magnesium supplementation demonstrate that oral magnesium supplementation can improve sickle cell hydration and decrease dense cell numbers, and phase I and II trials are in progress in sickle cell anemia and in HbSC disease (NCT00143572, NCT00532883). Large-scale trials have not been reported. Interestingly, magnesium supplements also reduced the

activity of the sodium–magnesium exchanger in sickle cells (Chapter 31).160 Molecular Basis for KCC. KCC is mediated by members of the cation-chloride cotransporter (SLC12) family,273,274 which includes the thiazide-sensitive NaCl cotransporters, the bumetanide-sensitive Na-K-Cl cotransporter and the volume-sensitive KCCs. These electroneutral transporters play three important physiological roles: transepithelial movement of solute, maintenance of intracellular ion concentrations (especially chloride) in electrically excitable cells, and regulation of cell volume. In erythrocytes, the KCC mediates the volume reduction and resultant increase in CHC that accompanies reticulocyte maturation.244,245,275,276 The prototype KCC (KCC1, SLC12A4)277 is expressed in most tissues. Three other KCC genes code for additional isoforms with more limited tissue distributions. The neuronal-specific KCC2 (SLC12A5 ) appears to function primarily as a regulator of chloride concentrations in neurons.278,279 KCC3 (SLC12A6) is expressed predominantly in kidney, lung, skeletal muscle, and brain, with a unique splicing isoform present in kidney.280–283 KCC4 (SLC12A7 ) is highly expressed in heart, kidney, and pancreas.282,284

172 The functional characteristics of the four isoforms are generally similar, although anion selective is subtly different and KCC1, KCC3, and KCC4 respond to hypotonic stimuli, whereas KCC2 does not. Human, sheep, and mouse red cell membranes contain both KCC1 and KCC3.285–288 In mouse cells, deletion of the KCC1 gene has little effect on KCC activity or cell volume; deletion of KCC3 results in a reduction of KCC activity, associated with an increase in KCC1 expression. Dual deletion abolishes KCC activity and results in overhydration of red cells with normal hemoglobin and mitigation of the dehydrated red cell phenotype found in the SAD mouse (Chapter 12).287 These data suggest that KCC3 is the predominant KCC transporter in mouse red cells. Human erythroid cells also express KCC4288,289 in addition to KCC1 and KCC3. The relative contribution of each of these transporters to KCC activity in human cells is unknown. It is possible that they interact with each other to modulate activity, as has been shown with artificially truncated KCC constructs,290 naturally occurring splicing isoforms of the sodium-potassium-chloride cotransporter (NKCC),291 and interactions between KCC and NKCC.292 Recent reports reveal that KCC isoforms interact differently with various regulatory kinases.293,294 Differences in the relative expression of KCC isoforms between sickle and normal red cells could conceivably produce increased KCC activity and/or abnormal regulation of KCC activity in sickle cells (Fig. 9.9). Interindividual differences in KCC isoform expression could also be a source of genetic variation that affects the phenotype of sickle cell disease (Chapter 27). Activation of KCC is associated with a serine/threonine dephosphorylation event, as protein phosphatase inhibitors such as okadaic acid and calyculin A block activation. Membrane stretch or shape change is not a signal transduction mechanism for KCC activation.295 Rather, studies of activation/inactivation kinetics in response to volume changes have suggested that cell swelling inhibits the putative inactivating protein kinase, shifting the kinase/ phosphatase equilibrium toward dephosphorylation and activating the transporter.296–299 Perhaps changes in cellular hemoglobin concentration associated with swelling produce dramatic alteration in the activity of cellular enzymes through macromolecular crowding effects in the nonideal thermodynamic conditions of concentrated protein solutions as present in erythrocytes (Chapter 6).300 The ubiquitous and promiscuous protein phosphatases 1 and 2A (PP1, PP2A) activate KCC and might function redundantly.269,301–304 Neither the phosphorylation sites on KCC nor the inhibitory kinase in red cells have been identified. In KCC1, threonine phosphorylation sites on the N terminus have been shown to be altered in response to changes in cell volume and to modulate the activity of the transporter.305 Several ST kinases have been shown to interact with NKCC1, including the stress-related kinase SPAK (STE20-related-proline-alanine-rich kinase, also known as PASK), OSR1 (oxidative stress response kinase), and the

Patrick G. Gallagher and Clinton H. Joiner WNK (with-no-lysine) kinases. Using a yeast two-hybrid system, SPAK and OSR1 have been shown to interact with KCC3, but not KCC1 or KCC4,293 although SPAK was unable to modulate KCC3 activity294 when coexpressed in xenopus oocytes. Coexpression of WNK 4 with KCC1, KCC3, or KCC4 resulted in inhibition of hypotonic activation of these transporters, whereas coexpression of a kinase-inactive mutant WNK4 activated KCC3 although not KCC1 and KCC4, under isotonic conditions. Interestingly, although inactivated SPAK was not able to activate KCC3 alone, it enhanced the activation of KCC3 by inactivated WNK4, suggesting interaction of these kinases, as has been shown in the regulation of NKCC1.306 The behavior of the WNK4 kinase in these in vitro systems is consistent with that expected from the putative swelling-inhibited kinase responsible for modulating KCC activity in red cells, but this identity remains to be demonstrated. Identification of the regulatory sites of KCC and the associated kinases would be an important step in delineating its dysregulation in sickle cell disease. The activities of PP1 and PP2A are themselves regulated by TKs. TK inhibitors such as staurosporine and celerythine activate KCC, probably by maintaining PP1/PP2A in the dephosphorylated, active state. Mice with genetic knockout of two src tyrosine kinases, fgr and hck, have constitutively activated KCC and exhibit dehydrated erythrocytes,307 elimination of either kinase alone was not sufficient to produce this phenotype, suggesting redundant function of the src kinases. Interestingly these animals do not show the normal decline in KCC activity with red cell aging, which could be explained by age-associated reduction in the paired TK. Deoxygenation alters protein phosphorylation state of the erythrocyte membrane, decreasing phosphorylation of several high abundance membrane proteins.308 Deoxygenation increased the activity of the syk kinase, and inhibitors of syk blocked the stimulation of KCC that accompanied deoxygenation. Activity of the src kinase expressed in human cells (lyn) was not changed by deoxygenation and lyn inhibitors did not alter the deoxystimulation of KCC.309 Thus, src-family TKs are negative regulators of KCC activity, probably via effects on the activating phosphatases, and syk-family TKs appear to be positive regulators. This complex pattern of regulation probably explains why some TK inhibitors stimulate and others inhibit KCC.310,311

MULTITRACK MODEL OF SICKLE CELL VOLUME REGULATION PATHOBIOLOGY Cell heterogeneity is a hallmark of sickle cell disease,134 with high numbers of dense cells and many low-density reticulocytes. In general, low-density sickle cells have high KCC activity and dehydrated cells exhibit low activity.249,252 Even if fractionated by age or density, sickle cells exhibit considerable heterogeneity. Some reticulocytes are found in the dense cell fraction, suggest very rapid dehydration in the circulation, or, fast-track dehydration. Within the

The Erythrocyte Membrane low-density fraction containing most of the sickle reticulocytes is a pool of cells exhibiting enhanced capacity to dehydrate via KCC.179 A similar fraction of reticulocytes/young cells was found with decreased F cell content.258 Transferrin receptor–positive sickle reticulocytes present in the dense cell fraction had greater KCC activity than sickle reticulocytes which had normal hydration in vivo;253 both studies demonstrated that KCC activity did not correlate with HbF content.259 Sickle erythrocyte maturation and density changes have been followed in vivo by using biotin-labeled erythrocytes.146,312,313 After ex vivo labeling and reinfusion, biotin-labeled cells exhibit increased density and dehydration within the first week, with loss of the least dense fractions and relative increases in high-density populations. This suggests that density changes in vivo occur soon after release of young cells into the circulation, supporting the notion of rapid dehydration. Sickle cells surviving longer than 1 week in the circulation, which account for 50%–66% of the population, are all strikingly dehydrated, with densities exceeding the densest normal cells.312 The low HbF content of dense reticulocytes suggests a sickling-induced mechanism for fast-track dehydration.179,181 This model envisions initial dehydration of a population of reticulocytes via activation of the Gardos channel by calcium influx through the sickling-induced pathway. Incremental dehydration would result in a slight intracellular acidification, which in turn would activate KCC in susceptible cells. Mathematical modeling of this mechanism predicts rapid volume collapse after cycles of deoxygenation.181 Reticulocyte heterogeneity in susceptibility to KCC activation by intracellular acidification could account for a subset of rapidly dehydrating cells. This notion is supported by the increased susceptibility of KCC activation to acid pH in sickle cells compared with normal cells.179,254 An alternative model for fast-track dehydration is that reticulocytes reach a state of intermediate dehydration via KCC activity, with heterogeneity derived from cellular differences in KCC capacity, function, or regulation. Reticulocytes with CHC thus increased would be “set up” for sickling, calcium permeabilization via the sicklinginduced pathway, Gardos channel activation and subsequent severe dehydration.226 This model is supported by the finding that formation of transferrin receptor–positive cells of intermediate density, which are dehydrated compared with normal reticulocytes, is not dependent on HbF concentration.259 Presently, the data do distinguish between these two models of initial dehydration through sickling-induced Gardos activation followed by KCC activation compared with dehydration by abnormal KCC activation potentiating sickling-induced Gardos activation. The lifespan of sickle erythrocytes that contain little or no HbF is approximately 2 weeks, compared with 6–8 weeks for F cells,312 confirming the selective survival of F cells inferred from the number of F cells and F reticulocytes in the circulation (Chapter 7).314 Unexpectedly high levels of

173 HbF, either naturally occurring or induced by hydroxyurea, were found to be associated with shortened survival times of non-F cells.313 The survival of dense sickle cells in vivo is extremely short, with 50% survival times ranging from 40 to 60 hours for dehydrated non-F cells and 120–330 hours for dense F cells.146 Their fragility and selective involvement in vascular occlusion and hemolysis, plus their rapid clearance during vasoocclusive episodes, supported the notion that dense sickle cells represented an end-stage in the life of sickle cells. That model has recently expanded to accommodate the existence of significant numbers of low-density, potassium-depleted, and sodium-loaded sickle erythrocytes resistant to dehydration by in vitro treatment with valinomycin, as discussed previously. The majority of lowdensity sickle cells that were older as assessed by biotin labeling were in fact, valinomycin-resistant, sodium-loaded cells. Such low-density cells arose spontaneously in vitro upon incubation of dense sickle erythrocytes under oxygenated conditions, and this process was accelerated by cyclic deoxygenation.145 The steady-state in vivo levels of valinomycin-resistant cells in sickle blood is approximately 3%–10%.143 Together with their short survival, this suggests that a significant proportion of sickle cells pass through this phase of sodium loading and over hydration prior to their destruction. A new model of the sickle cell “hydration” cycle thus includes pathological rehydration following pathological dehydration. The potassium loss that produces initial dense cell production deprives the cell of the ability to offset cation uptake driven by Donnan forces. Progressive sodium loading would then ensue, by virtue either of the deoxygenation-induced permeability increase or in response to dehydration, as has been shown experimentally under other conditions. Provided that the combination of sodium influx and potassium efflux exceeded the capacity for compensation by the sodium pump, the cell would be destined to swell to the point of osmotic lysis. This process of osmotic volume regulatory failure could contribute to intravascular hemolysis in sickle cell disease, now appreciated as an important aspect of the pathophysiology in light of the perturbations in nitric oxide metabolism brought about by free hemoglobin in the plasma (Chapter 11).315

Pharmacological Inhibition of Sickle Cell Dehydration The pathological dehydration of sickle cells and its contribution to hemolysis and vasoocclusion raised the possibility of a therapeutic benefit from improving sickle cell hydration.316 Attempts to rehydrate cells by infusions of hypotonic fluids or treatment with antidiuretic hormone proved impractical.317 More recently, drugs targeting specific pathways contributing to dehydration have undergone preliminary testing and some are discussed in Chapter 31. A trial of dipyridamole, an inhibitor of the deoxygenation-induced cation leak,176 is currently underway (NCT00276146).

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Cation Transport and Volume Regulation in Other Hemoglobinopathies and Thalassemia Specific and nonspecific interactions of hemoglobin with components of the red cell membrane can have important functional effects, which can be pathological when abnormal hemoglobins are involved. HbC is capable of powerful stimulation of KCC, resulting in excessive volume reduction of erythrocytes to produce elevated MCHC.252,318–321 The pathological consequences of elevated MCHC could contribute to crystal formation in the case of HbC disease (Chapter 21). Dehydration is particularly significant in HbSC disease, in which cellular dehydration produces conditions that permit sickling, even thought the participation of HbC in polymer formation is no greater than HbA.319 An argument has been made that KCC stimulation is specifically related to mutations around the sixth amino acid position of HBB, as HbS and HbC. Hb Siriraj (HBB glu7lys) and Hb San Jose (HBB glu7gly) produce slight elevations in KCC activity in heterozygotes, which were not observed in heterozygotes with HbO Arab or HbD (Chapter 23).320 Other studies, however, showed marked KCC stimulation and dehydration of both mature red cells and reticulocytes in homozygotes with HbO Arab and compound heterozygote with HbS and HbO Arab.322 In thalassemia, both hemoglobin content and abnormal cation transport affect cell volume. Although the responsible mechanisms are poorly understood, total hemoglobin content is an important determinant of cell volume so any condition that reduces hemoglobin synthesis produces microcytic, hypochromic erythrocytes. Indeed, the red cell “phenotype” in ␣ thalassemia, where one or two ␣-globin genes are deleted (Chapters 13 and 14) is virtually indistinguishable from that in iron deficiency. MCHC in these conditions and in HbH disease is slightly less than in normal erythrocytes. In contrast, despite their reduced hemoglobin content, ␤-thalassemia erythrocytes, especially those of ␤-thalassemia intermedia (Chapter 17), exhibit a substantial population of dense erythrocytes (Fig. 9.10).36 This elevated hemoglobin concentration results in increased cellular viscosity, which contributes to increased dynamic rigidity. KCC is increased in both ␤ and ␣ thalassemia erythrocytes, in proportion to the severity of disease. Swelling-induced KCC activity is substantially increased in both types of thalassemia.323 Although high reticulocyte counts might contribute to some of the elevation of KCC activity in these samples, several lines of evidence suggest that pathological activation might also occur. In vitro treatment of thalassemia red cells with dithiothreitol markedly reduced KCC activity, suggesting KCC activation by oxidative stress, consistent with the presence of hemichromes and other oxidant products in membrane. This connection is strengthened by the finding that treatment of patients with the iron chelator L1, which reduced iron content of red cell membranes, also lowered KCC activity and improved cellular potassium content.324 Like sickle cells,

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Figure 9.10. Density gradient analysis of thalassemic erythrocytes on discontinuous Stractan gradients. Density range 1.065–1.130 g/mL in 0.0045 g/mL increments. (A) Normal red blood cells. (B) ␤ thalassemia intermedia, unsplenectomized. (C) ␤ thalassemia intermedia, splenectomized. (D) HbH disease. (From ref. 36.)

␤ thalassemia red cells are depleted of cellular magnesium, and oral supplementation with magnesium pidolate has been shown to restore cellular magnesium, reduce KCC activity, and improve cellular potassium content.271 Although these findings support a link between abnormal KCC regulation and cellular dehydration in thalassemia, erythrocytes, especially in HbH disease, show quantitatively similar increases in KCC activity, with no increase in cellular hemoglobin concentration (Fig. 9.10). Whether mechanisms other than KCC contribute to dehydration of ␤ thalassemia red cells is unknown. Calcium content of ␤ thalassemia red cells is elevated, but most is sequestered in intracellular vesicles or retained organelles, and cytoplasmic calcium levels appear to be normal, as do active and passive calcium fluxes.325,326 In contrast, in a mouse model of ␤ thalassemia, treatment of animals with the Gardos channel blocker, clotrimazole, resulted in fewer dense cells, higher MCHC, and higher potassium content, but no change in hemoglobin or reticulocyte count.327 This suggested that the Gardos pathway plays a role in cellular dehydration in ␤ thalassemia red cells, but that dehydration does not shorten red cell survival. As yet there are no experimental data to support the hypothetical possibility of K channel activation via increase calcium influx due to shear stress on the mechanically unstable thalassemic membrane.

SUMMARY The erythrocyte membrane is a complex dynamic structure with multiple regulated functions. Cytoskeletal proteins maintain the structural integrity of a membrane that must simultaneously be flexible enough to deform in the microcirculation and durable enough to resist high shear stresses in large vessels. Multiple ligands and receptors interact with the external surface of the membrane, and some of these interactions trigger signaling cascades that regulate cell function. Specific proteins maintain a highly ordered lipid structure, which in turn, might modulate other membrane

The Erythrocyte Membrane protein functions or cellular interactions. Multiple regulated transport systems control solute and water fluxes across the membrane to facilitate the transport of respiratory gases, provide metabolic substrates, and regulate cell volume. Substantial energy is required to detoxify oxidant molecules that arise as an occupational hazard of the erythrocyte’s major tasks, the transport of oxygen. Given the physiological interactions of hemoglobin with the erythrocyte membrane, it is perhaps not surprising that abnormal hemoglobins can elicit significant pathological effects on membrane structure and function. A significant source of membrane damage in hemoglobinopathies is the increased level of oxidant species produced by reactive sulfhydryl on unstable or denatured hemoglobin molecules, membrane bound heme, or free iron. Oxidation affects cytoskeletal function, leads to externalization of PS, increases membrane rigidity and fragility, and perturbs cation transport and volume regulation. HbS polymerization elicits major perturbations of membrane structure and function. Polymer formation, in addition to increasing cellular rigidity and blood viscosity, results in formation of membrane spicules in which the cytoskeleton is dissociated from its membrane connections. Associated with this membrane disruption is externalization of PS and an increase in cation permeability via a nonselective cation pathway. Calcium influx via this deoxygenation-induced pathway results in activation of the Gardos pathway, which mediates selective potassium loss that results in cation depletion. KCC, normally involved in establishing reticulocyte hemoglobin concentration, is excessively active in the red cells of certain hemoglobinopathies and thalassemias, and leads to dehydrated, dense cells with high hemoglobin concentrations. In cells containing HbS, elevated hemoglobin concentrations greatly potentiates the rate and extent of polymer formation. The pleiotropic effects of abnormal hemoglobins on erythrocyte membrane structure and function contribute to the pathophysiology of sickle cell disease and thalassemia. Strategies targeting specific membrane pathology offer novel avenues to develop new therapy for these diseases. REFERENCES 1. Hebbel RP. Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology. Blood. 1991;77(2):214–237. 2. Perrotta S, Gallagher PG, Mohandas N. Hereditary spherocytosis. Lancet. 2008;372:1411–1426. 3. Hebbel RP. The sickle erythrocyte in double jeopardy: autooxidation and iron decompartmentalization. Semin Hematol. 1990;27(1):51–69. 4. Asakura T, Agarwal PL, Relman DA, et al. Mechanical instability of the oxy-form of sickle haemoglobin. Nature. 1973;244(5416):437–438. 5. MacDonald VW, Charache S. Drug-induced oxidation and precipitation of hemoglobins A, S and C. Biochim Biophys Acta. 1982;701(1):39–44.

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Patrick G. Gallagher and Clinton H. Joiner human erythrocyte Ca(++)-activated K+ channel: the imidazole ring is not required for inhibitory activity. J Pharmacol Exp Ther. 1995;273(1):266–272. Lauf PK, Theg BE. A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes. Biochem Biophys Res Commun. 1980;92(4):1422– 1428. Dunham PB, Stewart GW, Ellory JC. Chloride-activated passive potassium transport in human erythrocytes. Proc Natl Acad Sci USA. 1980;77(3):1711–1715. Brugnara C. Sickle cell disease: from membrane pathophysiology to novel therapies for prevention of erythrocyte dehydration. J Pediatr Hematol Oncol. 2003;25(12):927–933. Adragna NC, Fulvio MD, Lauf PK. Regulation of K-Cl cotransport: from function to genes. [erratum appears in J Membr Biol. 2006 Apr;210(3):213]. J Membr Biol. 2004;201(3):109– 137. Lauf PK, Adragna NC. K-Cl cotransport: properties and molecular mechanism. Cell Physiol Biochem. 2000;10(5– 6):341–354. Joiner CH. Cation transport and volume regulation in sickle red blood cells. Am J Physiol. 1993;264(2 Pt 1):C251–270. Brugnara C, Tosteson DC. Cell volume, K transport, and cell density in human erythrocytes. Am J Physiol. 1987;252(3 Pt 1): C269–276. Hall AC, Ellory JC. Evidence for the presence of volumesensitive KCl transport in ‘young’ human red cells. Biochim Biophys Acta. 1986;858(2):317–320. Canessa M, Fabry ME, Nagel RL. Deoxygenation inhibits the volume-stimulated, Cl(-)-dependent K+ efflux in SS and young AA cells: a cytosolic Mg2+ modulation. Blood. 1987;70(6):1861–1866. Ellory JC, Hall AC, Ody SA. Factors affecting the activation and inactivation of KCl cotransport in ‘young’ human red cells. Biomed Biochim Acta. 1990;49(2–3):S64–69. Canessa M, Spalvins A, Nagel RL. Volume-dependent and NEM-stimulated K+,Cl- transport is elevated in oxygenated SS, SC and CC human red cells. FEBS Letters. 1986;200(1):197–202. Brugnara C, Bunn HF, Tosteson DC. Regulation of erythrocyte cation and water content in sickle cell anemia. Science. 1986;232(4748):388–390. Franco RS, Palascak M, Thompson H, Joiner CH. KCl cotransport activity in light versus dense transferrin receptorpositive sickle reticulocytes. J Clin Invest. 1995;95(6):2573– 2580. Joiner CH, Rettig RK, Jiang M, Franco RS. KCl cotransport mediates abnormal sulfhydryl-dependent volume regulation in sickle reticulocytes. Blood. 2004;104(9):2954–2960. Gibson XA, Shartava A, McIntyre J, et al. The efficacy of reducing agents or antioxidants in blocking the formation of dense cells and irreversibly sickled cells in vitro. Blood. 1998;91(11):4373–4378. Pace BS, Shartava A, Pack-Mabien A, Mulekar M, Ardia A, Goodman SR. Effects of N-acetylcysteine on dense cell formation in sickle cell disease. Am J Hematol. 2003;73(1):26–32. Brugnara C, Van Ha T, Tosteson DC. Acid pH induces formation of dense cells in sickle erythrocytes. Blood. 1989;74(1):487–495. Fabry ME, Romero JR, Buchanan ID, et al. Rapid increase in red blood cell density driven by K:Cl cotransport in a subset

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of sickle cell anemia reticulocytes and discocytes. Blood. 1991;78(1):217–225. Franco RS, Thompson H, Palascak M, Joiner CH. The formation of transferrin receptor-positive sickle reticulocytes with intermediate density is not determined by fetal hemoglobin content. Blood. 1997;90(8):3195–3203. Joiner CH, Rettig RK, Jiang M, Risinger M, Franco RS. Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes. [erratum appears in Blood. 2007;109(7):2735]. Blood. 2007;109(4):1728–1735. Joiner CH, Rettig RK, Jiang M, Risinger M, Franco RS. Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes. Blood. 2007;109(4):1728– 1735. Hebbel RP, Ney PA, Foker W. Autoxidation, dehydration, and adhesivity may be related abnormalities of sickle erythrocytes. Am J Physiol. 1989;256(3 Pt 1):C579–583. De Franceschi L, Beuzard Y, Brugnara C. Sulfhydryl oxidation and activation of red cell K(+)-Cl- cotransport in the transgenic SAD mouse. Am J Physiol. 1995;269(4 Pt 1):C899–906. Gibson JS, Speake PF, Ellory JC. Differential oxygen sensitivity of the K+-Cl- cotransporter in normal and sickle human red blood cells. [see comment]. J Physiol. 1998;511(Pt 1):225–234. Gibson JS, Khan A, Speake PF, Ellory JC. O2 dependence of K+ transport in sickle cells: the effect of different cell populations and the substituted benzaldehyde 12C79. FASEB J. 2001;15(3):823–832. Joiner CH, Franco RS. The activation of KCL cotransport by deoxygenation and its role in sickle cell dehydration. Blood Cell Mol Dis. 2001;27(1):158–164. Feray JC, Garay R. An Na+-stimulated Mg2+-transport system in human red blood cells. Biochim Biophys Acta. 1986;856(1):76–84. Olukoga AO, Adewoye HO, Erasmus RT, Adedoyin MA. Erythrocyte and plasma magnesium in sickle-cell anaemia. East African Med J. 1990;67(5):348–354. De Franceschi L, Villa-Moruzzi E, Fumagalli L, et al. K-Cl cotransport modulation by intracellular Mg in erythrocytes from mice bred for low and high Mg levels. Am J Physiol Cell Physiol. 2001;281(4):C1385–1395. De Franceschi L, Brugnara C, Beuzard Y. Dietary magnesium supplementation ameliorates anemia in a mouse model of beta-thalassemia. Blood. 1997;90(3):1283–1290. De Franceschi L, Cappellini MD, Graziadei G, et al. The effect of dietary magnesium supplementation on the cellular abnormalities of erythrocytes in patients with beta thalassemia intermedia. Haematologica. 1998;83(2):118–125. De Franceschi L, Bachir D, Galacteros F, et al. Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease. J Clin Invest. 1997; 100(7):1847–1852. Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev. 2005;85(2):423–493. Hebert SC, Mount DB, Gamba G. Molecular physiology of cation-coupled Cl- cotransport: the SLC12 family. Eur J Physiol. 2004;447(5):580–593. Haas M, Forbush B 3rd. The Na-K-Cl cotransporter of secretory epithelia. Annu Rev Physiol. 2000;62:515–534. Haas M, Forbush B 3rd. The Na-K-Cl cotransporters. J Bioenerg Biomembr. 1998;30(2):161–172.

The Erythrocyte Membrane 277. Gillen CM, Brill S, Payne JA, Forbush B 3rd. Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cationchloride cotransporter family. J Biol Chem. 1996;271(27): 16237–16244. 278. Payne JA, Stevenson TJ, Donaldson LF. Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-specific isoform. J Biol Chem. 1996;271(27):16245– 16252. 279. Song L, Mercado A, Vazquez N, et al. Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter. Brain Res Mol Brain Res. 2002;103(1–2): 91–105. 280. Race JE, Makhlouf FN, Logue PJ, Wilson FH, Dunham PB, Holtzman EJ. Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter. Am J Physiol. 1999;277(6 Pt 1):C1210–1219. 281. Hiki K, D’Andrea RJ, Furze J, et al. Cloning, characterization, and chromosomal location of a novel human K+-Cl- cotransporter. J Biol Chem. 1999;274(15):10661–10667. 282. Mount DB, Mercado A, Song L, et al. Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family. J Biol Chem. 1999;274(23):16355–16362. 283. Mercado A, Vazquez N, Song L, et al. NH2-terminal heterogeneity in the KCC3 K+-Cl- cotransporter. Am J Physiol Renal Physiol. 2005;289(6):F1246–1261. 284. Boettger T, Hubner CA, Maier H, Rust MB, Beck FX, Jentsch TJ. Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4. Nature. 2002;416(6883):874– 878. 285. Pellegrino CM, Rybicki AC, Musto S, Nagel RL, Schwartz RS. Molecular identification and expression of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells. Blood Cell Mol Dis. 1998;24(1):31–40. 286. Lauf PK, Zhang J, Delpire E, Fyffe RE, Mount DB, Adragna NC. K-Cl co-transport: immunocytochemical and functional evidence for more than one KCC isoform in high K and low K sheep erythrocytes. Comp Biochem Physiol. 2001;130(3):499– 509. 287. Rust MB, Alper SL, Rudhard Y, et al. Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice. J Clin Invest. 2007;117(6):1708–1717. 288. Crable SC, Hammond SM, Papes R, et al. Multiple isoforms of the KC1 cotransporter are expressed in sickle and normal erythroid cells. Exp Hematol. 2005;33(6):624–631. 289. Joiner C, Papes R, Crable S, Pan D, Mount DB. Functional Comparison of Red Cell KCl Cotransporter isoforms, KCC1, KCC3, and KCC4. Blood. 2006;108:a. 290. Casula S, Shmukler BE, Wilhelm S, et al. A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and Cterminal cytoplasmic domains are required for K-Cl cotransport activity. J Biol Chem. 2001;276(45):41870–41878. 291. Plata C, Mount DB, Rubio V, Hebert SC, Gamba G. Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP. Am J Physiol. 1999;276(3 Pt 2):F359–366. 292. Gillen CM, Forbush B, 3rd. Functional interaction of the KCl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells. Am J Physiol. 1999;276(2 Pt 1):C328–336.

183 293. Piechotta K, Lu J, Delpire E. Cation chloride cotransporters interact with the stress-related kinases Ste20-related prolinealanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). J Biol Chem. 2002;277(52):50812–50819. 294. Garzon-Muvdi T, Pacheco-Alvarez D, Gagnon KB, et al. WNK4 kinase is a negative regulator of K+-Cl- cotransporters. Am J Physiol Renal Physiol. 2007;292(4):F1197–1207. 295. Jennings ML, Schulz RK. Swelling-activated KCl cotransport in rabbit red cells: flux is determined mainly by cell volume rather than shape. Am J Physiol. 1990;259(6 Pt 1):C960– 967. 296. Jennings ML, al-Rohil N. Kinetics of activation and inactivation of swelling-stimulated K+/Cl- transport. The volumesensitive parameter is the rate constant for inactivation. J Gen Physiol. 1990;95(6):1021–1040. 297. Jennings ML, Schulz RK. Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or Nethylmaleimide. J Gen Physiol. 1991;97(4):799–817. 298. Kaji DM, Tsukitani Y. Role of protein phosphatase in activation of KCl cotransport in human erythrocytes. Am J Physiol. 1991;260(1 Pt 1):C176–180. 299. Colclasure GC, Parker JC. Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells. J Gen Physiol. 1992;100(1):1–10. 300. Parker JC, Colclasure GC. Macromolecular crowding and volume perception in dog red cells. Mol Cell Biochem. 1992;114(1–2):9–11. 301. Mallozzi C, De Franceschi L, Brugnara C, Di Stasi AM. Protein phosphatase 1alpha is tyrosine-phosphorylated and inactivated by peroxynitrite in erythrocytes through the src family kinase fgr. Free Radic Biol Med. 2005;38(12):1625–1636. 302. Bize I, Taher S, Brugnara C. Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes. Am J Physiol Cell Physiol. 2003;285(1):C31–38. 303. Bize I, Guvenc B, Buchbinder G, Brugnara C. Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg++. J Membr Biol. 2000;177(2):159–168. 304. Bize I, Guvenc B, Robb A, Buchbinder G, Brugnara C. Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes. Am J Physiol. 1999; 277(5 Pt 1):C926–936. 305. Lytle C, Forbush B 3rd. The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation. J Biol Chem. 1992;267(35):25438–25443. 306. Dowd BF, Forbush B. PASK (proline-alanine-rich STE20related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1). J Biol Chem. 2003;278(30):27347–27353. 307. De Franceschi L, Fumagalli L, Olivieri O, Corrocher R, Lowell CA, Berton G. Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport. J Clin Invest. 1997;99(2):220–227. 308. Fathallah H, Coezy E, de Neef RS, Hardy-Dessources MD, Giraud F. Inhibition of deoxygenation-induced membrane protein dephosphorylation and cell dehydration by phorbol esters and okadaic acid in sickle cells. Blood. 1995;86(5):1999– 2007. 309. Merciris P, Claussen WJ, Joiner CH, Giraud F. Regulation of K-Cl cotransport by Syk and Src protein tyrosine kinases

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Patrick G. Gallagher and Clinton H. Joiner in deoxygenated sickle cells. Pflugers Archiv – Eur J Physiol. 2003;446(2):232–238. Flatman PW, Adragna NC, Lauf PK. Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport. Am J Physiol. 1996;271(1 Pt 1):C255–263. Bize I, Dunham PB. Staurosporine, a protein kinase inhibitor, activates K-Cl cotransport in LK sheep erythrocytes. Am J Physiol. 1994;266(3 Pt 1):C759–770. Franco RS, Lohmann J, Silberstein EB, et al. Time-dependent changes in the density and hemoglobin F content of biotinlabeled sickle cells. J Clin Invest. 1998;101(12):2730–2740. Franco RS, Yasin Z, Palascak MB, Ciraolo P, Joiner CH, Rucknagel DL. The effect of fetal hemoglobin on the survival characteristics of sickle cells. Blood. 2006;108(3):1073–1076. Dover GJ, Boyer SH, Charache S, Heintzelman K. Individual variation in the production and survival of F cells in sickle– cell disease. N Engl J Med. 1978;299(26):1428–1435. Reiter CD, Gladwin MT. An emerging role for nitric oxide in sickle cell disease vascular homeostasis and therapy. Curr Opin Hematol. 2003;10(2):99–107. Bookchin R, Tieffert JT, Daives SC, Vichinsky E, Lew, VL. Magnesium therapy for sickle cell anemia: a new rationale. In: Beuzard Y, Lubin, B, Rosa, J, eds. Sickle Cell Disease and Thalasssaemias: New Trends in Therapy. Paris, London: John Libby; 1995. Rosa RM, Bierer BE, Thomas R, et al. A study of induced hyponatremia in the prevention and treatment of sickle-cell crisis. N Engl J Med. 1980;303(20):1138–1143. Lawrence C, Fabry ME, Nagel RL. The unique red cell heterogeneity of SC disease: crystal formation, dense reticulocytes, and unusual morphology. Blood. 1991;78(8):2104–2112.

319. Nagel RL, Fabry ME, Steinberg MH. The paradox of hemoglobin SC disease. Blood Rev. 2003;17(3):167–178. 320. Olivieri O, Vitoux D, Galacteros F, et al. Hemoglobin variants and activity of the (K+Cl-) cotransport system in human erythrocytes. Blood. 1992;79(3):793–797. 321. Brugnara C, Kopin AS, Bunn HF, Tosteson DC. Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease. J Clin Invest. 1985;75(5):1608–1617. 322. Nagel RL, Krishnamoorthy R, Fattoum S, et al. The erythrocyte effects of haemoglobin O(ARAB). Br J Haematol. 1999;107(3):516–521. 323. Olivieri O, De Franceschi L, Capellini MD, Girelli D, Corrocher R, Brugnara C. Oxidative damage and erythrocyte membrane transport abnormalities in thalassemias. Blood. 1994;84(1):315–320. 324. de Franceschi L, Shalev O, Piga A, et al. Deferiprone therapy in homozygous human beta-thalassemia removes erythrocyte membrane free iron and reduces KCl cotransport activity. J Lab Clin Med. 1999;133(1):64–69. 325. Bookchin RM, Ortiz OE, Shalev O, et al. Calcium transport and ultrastructure of red cells in beta-thalassemia intermedia. Blood. 1988;72(5):1602–1607. 326. Rhoda MD, Galacteros F, Beuzard Y, Giraud F. Ca2+ permeability and cytosolic Ca2+ concentration are not impaired in beta-thalassemic and hemoglobin C erythrocytes. Blood. 1987;70(3):804–808. 327. de Franceschi L, Rouyer-Fessard P, Alper SL, Jouault H, Brugnara C, Beuzard Y. Combination therapy of erythropoietin, hydroxyurea, and clotrimazole in a beta thalassemic mouse: a model for human therapy. Blood. 1996;87(3):1188–1195.

THE BIOLOCHEMISTRY, GENETICS, AND VASCULAR CELL BIOLOGY OF NITRIC OXIDE

10 The Biology of Vascular Nitric Oxide Jane A. Leopold and Joseph Loscalzo

The role of nitric oxide (NO) as the key mediator of endothelial function and vascular tone was initially recognized by Furchgott and Zawadski1 over two decades ago when they discovered that an intact endothelium was required for acetylcholine-stimulated vasodilation. From these studies, they determined that the endothelium released a potent vasodilator substance that they termed endothelium-derived relaxing factor;1 several years later, this factor was identified as NO.2,3 Since that time, NO has been shown to modulate a host of functions that maintain the integrity of the endothelium as well as regulate interactions between circulating blood components and the vessel wall. Through its chemical reactions with a variety of species, including heme iron, NO is uniquely positioned to regulate these vascular homeostatic processes. Endothelium-derived NO serves as a paracrine regulator of vascular function. NO is released to the vascular smooth muscle cells where it activates soluble guanylyl cyclase to generate cyclic guanosine monophosphate (cGMP) and modulate cation flux which, in turn, induce vasodilation and adjust vascular tone accordingly.4,5 NO is also released to the bloodstream where it encounters erythrocytes, platelets, and plasma components. Here, the metabolic fate of NO is determined by a complex series of reactions that both consume and preserve stores of bioavailable NO. Owing to the relative abundance of erythrocytes compared with other circulating cell types, interactions between NO and redox-active hemoglobin achieve biological significance. In this chapter, we will discuss NO synthesis and biological chemistry, with particular focus on NO reactions with hemoglobin as well as the physiological relevance of NO-hemoglobin derivatives. In addition, we will detail the importance of NO for endothelial, vascular smooth muscle, and platelet homeostatic functions.

Nitric Oxide Synthesis NO is synthesized in numerous cell types and tissues by NO synthases (NOS). NOSs exists as two main isoform classes: the constitutive enzyme identified in the endothelium (eNOS or NOS3) and neuronal cells (nNOS or NOS1) and the inducible enzyme (iNOS or NOS2) found in smooth muscle cells, neutrophils, and macrophages (as well as many other cell types) following exposure to inflammatory cytokines or bacterial endotoxin.6 NO is generated via the five-electron oxidation of L-arginine to form L-citrulline and stoichiometric amounts of NO.3 This reaction requires molecular O2 and nicotinamide adenine dinucleotide phosphate as cosubstrates and flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin as cofactors to facilitate electron transfer to the NOS heme moiety.7 Each of the NOS isoforms possesses specific structural and functional characteristics that determine the degree to which cofactors regulate enzyme activity and NO production. Comparison of the amino acid sequences reveals that there is 50%–55% homology among NOS isoforms with the greatest conservation between sequences for the two main catalytic domains.8 All NOS isoforms are under the regulatory control of Ca2+ and calmodulin for effective electron transfer between the reductase and oxygenase domains of the enzyme; however, the affinity of NOS for the Ca2+ –calmodulin complex differs among isoforms and accounts, in part, for the difference in regulation of NO production. In endothelial cells, eNOS is membrane-bound within caveoli and binds calmodulin in a strongly Ca2+ -dependent manner that is reversible.9 Following stimulation with agonists such as acetylcholine or bradykinin, inositol 1,4,5-trisphosphate production is increased to promote the release of intracellular Ca2+ stores.10 This transient increase in intracellular Ca2+ enhances the formation of a Ca2+ –calmodulin complex, which, in turn, activates eNOS to facilitate dissociation from caveolin-1.11 Once activated, eNOS generates continuously low levels of NO until Ca2+ stores are depleted.9,12 Although eNOS is constitutively expressed, it is now recognized that expression may be differentially regulated under physiological and pathophysiological conditions and is subject to both posttranscriptional and posttranslational modification.6 In contrast, iNOS binds calmodulin in states of low intracellular Ca2+ , is regulated at the transcriptional level, and, therefore, requires several hours to effect a physiological response.13 Concentrations of NO (per mole of enzyme per minute) generated by iNOS are substantially greater than those achieved by eNOS,14 are potentially cytotoxic, and indicate that iNOS may play an integral role in both the immune response and apoptosis.15,16

185

186 In the vascular endothelium, a number of signaling molecules as well as hemodynamic forces modulate eNOS expression to influence NO production. For example, transforming growth factor-␤1, lysophosphatidylcholine, hydrogen peroxide, tumor necrosis factor–␣ (TNF␣), oxidized low-density lipoprotein, laminar shear stress, and hypoxia all mediate eNOS expression by regulating gene transcription. In addition, TNF␣, lipopolysaccharide, oxidized lowdensity lipoprotein, hydroxymethylglutaryl coenzyme A reductase inhibitors (statins), thrombin, and hydrogen peroxide have been shown to regulate eNOS expression by influencing mRNA degradation.17 NO production may be influenced further by a number of polymorphisms of the eNOS gene that have been identified and evaluated to determine the consequences for eNOS activity and association with vascular disease. Among these polymorphisms, a single nucleotide polymorphism in the promoter region (−786T/C) and in exon 7 resulting in the conversion of glutamate to aspartate at position 298 (Glu298Asp) and a variable number of tandem repeats in intron 4 (b/a) have been the most extensively studied. In a meta-analysis of 26 studies that examined these three polymorphisms, homozygosity for the Asp298 (odds ratio = 1.31; 95% confidence interval = 1.13−1.51) or the intron-4a allele (odds ratio = 1.34; 95% confidence interval = 1.03−1.75) was associated with an increased risk of ischemic heart disease, whereas no association was demonstrated for the −786C allele.18 Despite these findings, individual studies examining the functional significance of the Glu298Asp polymorphism have yielded conflicting results. Select studies of subjects homozygous for Asp298 demonstrate impaired endothelium-dependent brachial artery flow–mediated dilation, suggesting decreased bioavailable NO,19 whereas other studies fail to make this association.20–22 Although direct measures of eNOS activity and NO production were not determined in these studies, in vitro studies performed in primary human endothelial cells with the Glu298Asp polymorphism revealed enhanced eNOS protein cleavage, implying that this polymorphism should be associated with decreased NO production.23 A second polymorphism that has been studied is located in the promoter region and results from a T-to-C substitution, which may influence eNOS transcriptional activity, although this effect has not yet been confirmed in vivo. In contrast to what was found in the meta-analysis,18 individuals with the −786C promoter polymorphism had impaired endothelium-dependent vascular reactivity24 that was associated with a significant increase in death from cardiovascular causes at the end of a 2,000-day follow-up period.25 Interestingly, among the hemoglobinopathies, eNOS polymorphisms have been associated with disease status only in individuals with sickle cell disease.26 In AfricanAmerican women with sickle cell disease, the presence

Jane A. Leopold and Joseph Loscalzo of the −786C promoter polymorphism was significantly associated with acute chest syndrome (relative risk = 8.7; 95% confidence interval = 1.76–42.92).27 A mechanism to explain this association has not yet been elucidated; however, the −786T/C polymorphism adversely influences erythrocyte deformability.28

Bioreactivity of Nitric Oxide NO, as a free radical, is only modestly reactive compared with other biological free radicals (i.e., it can diffuse over ˚ micron rather than Angstrom distances before encountering another coreactant). NO may exist in one of three closely related redox forms, each with discrete properties and reactivities: NO• , NO+ (nitrosonium, formed by singleelectron oxidation of NO• ), and NO− (nitroxyl, formed by single-electron reduction of NO• ).29 These NO species react further with O2 -derived free radicals, redox metals, and thiols to generate NO compounds that have unique biological effects (Fig. 10.1).30 For example, the reaction of NO with the heme iron of guanylyl cyclase results in enzyme activation, whereas the reaction of NO derivatives (NO+ , N2 O3 , or ONOO− ) with –SH (or –S− derivatives)containing low-molecular-weight molecules and proteins generates S-nitrosothiols, a stable reservoir of bioavailable NO.31 In plasma, S-nitroso-albumin serves as an NO adduct and limits the inactivation of NO by reactive oxygen species.29 NO also forms N-nitroso adducts with amine moieties and nitrosyl adducts with heme groups to yield N-nitrosamines and nitrosylheme, respectively.32,33 Human plasma contains an approximately fivefold higher concentration of N-nitrosamine species (32.3 +/− 5.0 nmol/L) than S-nitrosothiols (7.2 +/− 1.1 nmol/L);34 however, functional studies in animal models studies suggest that Snitrosothiols are biologically more active by an order of magnitude compared to N-nitrosamine species.35 Dinitrosyl iron complexes also possess biological activity, inhibiting platelet aggregation and decreasing vascular tone in experimental models.36,37 In an O2 -rich environment, NO can be oxidized to nitrite (NO2 − ) and nitrate (NO3 − ), stable end products of its metabolism.30 Although nitrite has been reported to have no intrinsic vasodilator activity,38 nitrite does serve as a physiologically important source of NO, which is released in the circulation through the nitrite reductase activity of hemoglobin (vide infra).39 Under ischemic conditions, where the pH is in the acidic range, nitrite may be reduced directly to NO through a nonenzymatic mechanism.40 NO may also interact with reactive oxygen species, including superoxide, hydrogen peroxide, and lipid peroxyl radicals, formed during normal cellular metabolism or states of increased oxidant stress. In this manner, bioavailable NO is inactivated through the formation of peroxynitrite (ONOO− ), nitrous acid (HNO2 ), and lipid peroxynitrites (LOONO), respectively.30

The Biology of Vascular Nitric Oxide

187 RSNO S-nitrosothiols

-Tyr

3-NO2 -Tyr 3-nitrotyrosine

ONOO-

HNO2

.O-

Peroxynitrite

RSH

Nitrous acid

2

H2O2

NO2-/ NO3-

O2

.

.

Nitrite / Nitrate

LOONO Lipid peroxynitrites

LOO

NO

NO-Hb

Hb

Nitrosyl heme

.

L-Arginine + O2 eeNOS

NADP

.

L-Citrulline + NO

FADH-

.

BH4

z n Fe2+

NADPH

Fe3+

Ca2+/CaM

BH4 Ca2+/CaM

FMNH2

FMNH Endothelial cell

FADH2

Oxygenase

Reductase

Figure 10.1. Biological source and reactions of nitric oxide. Nitric oxide (NO• ) is synthesized in endothelial cells by the endothelial isoform of nitric oxide synthase (eNOS) via the five-electron oxidation reaction of L-arginine to L-citrulline. eNOS is activated by Ca2+ /calmodulin (Ca2+ /CaM) and the reaction requires molecular O2 and NADPH as cosubstrates and flavin adenine dinucleotide (FADH+ ), flavin mononucleotide (FMNH+ ), and tetrahydrobiopterin (BH4 ) as cofactors to facilitate electron transfer to the NOS heme moiety. Once generated, NO• diffuses into the interstitium and the bloodstream where it reacts with molecular O2 , superoxide anion (• O2 − ), R-SH groups, hydrogen peroxide (H2 O2 ), lipid oxides (LOO• ), and hemoglobin (Hb) to generate NO species – some of which have biological activity. (See color plate 10.1.)

Bioreactivity of Nitric Oxide with Hemoglobin NO can also bind to transition metals within heme groups, including myoglobin and hemoglobin itself. Endothelialderived NO diffuses readily from the endothelial cell into the blood pool, where it first encounters platelets, which are enriched in the blood lamina nearest to the endothelial monolayer, and then erythrocytes, in which it can react with hemoglobin (Fig. 10.2). The reaction of NO with hemoglobin was first carefully characterized by Drabkin and Austin,41 who recorded the absorption spectrum of nitrosyl-hemoglobin under anaerobic (i.e., deoxygenated) conditions: Hb(II) + NO → Hb(II)NO

(10.1)

This reaction occurs with a second-order rate constant of 2–6 × 107 M/s,42,43 and an extremely slow off-rate of approximately 10−3 –10−5 s.44,45 This slow off-rate renders the bound NO effectively irreversibly complexed to deoxygenated hemoglobin. Under normal conditions in circulating whole blood, there is a large pool of oxyhemoglobin with which NO also readily reacts to form methemoglobin [Hb(III)] and nitrate: Hb(II)O2 + NO → Hb(III) + NO3 −

(10.2)

The second-order rate constant for this reaction is 6–8 × 107 M/s,46,47 leading to an estimated half-life in the erythrocyte of approximately 0.5 ␮s. The kinetics of the reaction of NO with deoxyhemoglobin and with oxyhemoglobin suggests that the erythrocyte should serve as an highly efficient sink for NO; however, if this were the case, erythrocytic hemoglobin would significantly limit NO’s bioavailability for vascular smooth muscle cell and platelet homeostatic functions. This is best explained by the finding that erythrocytic hemoglobin is far less efficient than cell-free hemoglobin at scavenging NO, owing to the time required for NO to diffuse from the endothelial cell to the erythrocyte (accounted for by the erythrocyte-free zone nearest the endothelial monolayer and by the unstirred fluid layer surrounding the erythrocyte).48 Furthermore, the erythrocyte membrane provides a physical diffusion barrier that effectively compartmentalizes hemoglobin and limits its access to the endothelial monolayer or vascular interstitium.49,50 As shown in ex vivo studies performed in the absence of flow, 1,000 times more erythrocyteencapsulated hemoglobin is required to inactivate NO compared with cell-free hemoglobin.51 Nitrite has recently been recognized as a reservoir of bioactive NO in mammals. Under ischemic or acidic conditions, nitrite can be reduced to NO directly or enzymatically via xanthine oxidase.52,53 Recent data suggest that nitrite is

188

Jane A. Leopold and Joseph Loscalzo

.

NO

NO2-

.

NO

NO3-

NO3X-NO?

.

NO

MetHb

B3 AQPRN CA

NO2-

Rh

MetHb

Hb(II)O2

Hb(II)O2

Hb(II)

Hb(II)

?

NOHb

NOHb

MetHb

Figure 10.2. Reactions between NO and hemoglobin in erythrocytes. Nitric oxide (NO• ), synthesized by the endothelium, or nitrite (NO2 − ) circulating in plasma, diffuses into the erythrocyte where it reacts with oxyhemoglobin [Hb(II)O2 ] to yield methemoglobin (MetHb) and nitrate (NO3 − ), or with deoxyhemoglobin [Hb(II)] to generate nitrosylhemoglobin (NOHb) and NO adducts (X-NO). Nitrite also diffuses into the erythrocyte and reacts with deoxyhemoglobin and, via a possible intermediate species (?), produces nitrosylhemoglobin and methemoglobin. Once formed, NO• and NO• adducts are thought to exit the cell through a functional metabolon that is composed of oxy- and deoxyhemoglobin, Rh channels (Rh), aquaporin (AQPRN), band-3 complex (B3), and carbonic anhydrase (CA). These channels theoretically may transport NO• through the erythrocyte membrane. (See color plate 10.2.)

reduced by deoxygenated hemoglobin and that this reaction yields bioactive NO and methemoglobin.39,54 In this construct, hemoglobin can be viewed as a nitrite reductase that mediates hypoxic vasodilation and in this way contributes to homeostatic regulation of tissue perfusion and O2 delivery.50 The reactions involved in this process are: HONO + 2Hb(II) → Hb(III) + Hb(II)NO + OH− Hb(II)NO → Hb(II) + NO

(10.3) (10.4)

Hb(II)NO can also transfer NO to glutathione to yield S-nitrosoglutathione. These reactions are further complicated by hemoglobin allostery, as indicated by their sigmoidal (rather than pseudo-first-order) kinetics when nitrite is in excess over hemoglobin,55 and derives from the fact that the relaxed- (R-) state hemoglobin reduces nitrite faster than does tense- (T-) state hemoglobin. This allosteric dependence of the reaction coupled with its stoichiometry leads to accelerated kinetics as hemoglobin is converted from the R- to T-state during the reaction.56 Consistent with an allosteric mechanism, hemoglobinmediated nitrite reduction proceeds most rapidly at the hemoglobin P50 (where only half the hemoglobin is O2 bound). At the P50 , the reduced rate of the reaction caused by fewer deoxygenated hemes being present than at zero O2 is compensated for by the presence of more deoxygenated hemes in R-state hemoglobin tetramers.56 Recent data suggest that NO dissociation from hemoglobin (Eq. 10.4) alone cannot account for an exportable pool of

NO within the erythrocyte that can manifest bioactivity.57 Either compartmentalization or a reaction intermediate (an S-nitrosothiol?) may provide a physiological explanation for the phenomenon, but neither mechanism has been proven to date. Another hypothesis that has been advanced to explain the role of erythrocytic hemoglobin in NO bioactivity is that of S-nitrosation of its ␤-93 cysteinyl residue to form Snitrosohemoglobin (SNO-Hb).58 According to this view, NO binds allosterically to a deoxyheme moiety on a partially oxygenated hemoglobin tetramer, and is then transferred to the cysteinyl residue upon transition from the T- to the R-state with reoxygenation. Upon deoxygenation, the process is reversed with release of NO (possibly transferred to a protein or thiol-carrier), according to the following reaction mechanism: cys93

Hb(II)NO + O2 → Hb(II)O2 cys93 Hb(II)O2 SNO

SNO

→ Hb(II) + O2 + NO

(10.5) (10.6)

or cys93

Hb(II)O2

SNO + GSH → Hb(II) + O2 + GSNO

(10.7)

Based on this hypothesis, NO is then exported from the cell as an “X-NO.” The identity of the X-NO species and the mechanism of export have not been elucidated. Although this hypothesis received much attention when first proposed, it remains controversial, owing largely to published experimental data that reveal the preferential (if not exclusive) binding of NO to deoxygenated heme groups on

The Biology of Vascular Nitric Oxide R-state hemoglobin,59,60 an inability to detect cyclic transfer of NO from the heme group to the ␤-93 cysteinyl residue and vice versa,61 and failure to determine the O2 dependency of SNO-Hb instability in the presence of erythrocyte concentrations of glutathione (i.e., the S-NO linkage decays independent of O2 tension in the presence of millimolar concentrations of glutathione). Most important, the high intracellular concentration of glutathione would limit the stability of the ␤-93 S-NO bond.62 Thus, it is our opinion that the preponderance of data support the nitrite reductase hypothesis as the principal mechanism for the generation of bioactive NO by erythrocytes.

Nitrite Reductase Activity of Hemoglobin Nitrite, a stable end-product formed by the oxidation of NO, is generated and/or accumulates in the blood, measurements of which in human plasma isolated from healthy volunteers demonstrate levels of approximately 0.20 ± 0.02 ␮mol/L.34 Although nitrite itself does not possess intrinsic vasodilator properties at physiological concentrations, it may be considered an important vasodilator substance via its reduction to NO by the nitrite reductase activity of hemoglobin. Evidence to support the role of hemoglobin-mediated nitrite reduction as a physiological source of NO was first demonstrated when Gladwin and colleagues59 observed a plasma gradient of nitrite across vascular beds of human subjects under basal conditions and following the inhalation of NO gas. They next infused nitrite into the forearm brachial artery at near physiological levels (0.9–2.5 ␮mol/L) and noted an increase in blood flow as measured by strain gauge plethysmography.39 When subjects were pretreated with L-NG -monomethyl arginine citrate to inhibit eNOS and endogenous NO synthesis, infusion of near physiological levels of nitrite resulted in an increase in forearm blood flow during exercise. Nitrite infusion was associated with a detectable increase in erythrocyte Hb(II)NO that formed rapidly indicating that nitrite was being reduced to NO during one artery-to-vein transit in a manner that was inversely proportional to oxyhemoglobin saturation.39 When NO gas production was measured by chemiluminescence, the rate of NO production following the addition of nitrite to erythrocytes was significantly greater when hemoglobin was in a deoxygenated than oxygenated state. The ability to measure NO gas production in this system demonstrated further that some fraction of the released NO escaped autocapture by free heme groups and that NO generation is augmented under anaerobic conditions, consistent with a nitrite–deoxyhemoglobin reaction.39 The bioactivity of NO generated by the reaction between nitrite and hemoglobin was confirmed in a rat aortic ring bioassay where it was demonstrated that this reaction was, indeed, responsible for the observed vasodilation. In these studies, the aortic rings were found to relax spontaneously only at very low O2 tensions (10–15 mm Hg); however, in

189 the presence of nitrite (0.5–2 ␮mol/L) and erythrocytes, the vessel tension–O2 threshold curve was left-shifted to higher O2 tensions (∼40 mm Hg). This effect was mediated by the nitrite reductase activity of deoxygenated hemoglobin as oxygenated hemoglobin had no effect on vasodilation, even in the presence of excess nitrite. Together, these studies demonstrate that hemoglobin serves as a nitrite reductase under hypoxic conditions to release NO from nitrite and effect vasodilation.50

Nitric Oxide Export from Erythrocytes Although the aforementioned studies convincingly demonstrate a role for hemoglobin as an effective nitrite reductase to generate bioavailable NO from nitrite, the mechanism by which NO is exported from the erythrocyte remains to be determined. As noted, the half-life of NO in the erythrocyte is estimated to be approximately 5 ␮s, suggesting that it is unlikely that NO itself freely diffuses across the erythrocyte membrane.63 One proposed mechanism for NO export suggests that erythrocyte membrane proteins comprise a potential nitrite reductase metabolon containing deoxyhemoglobin and methemoglobin, anion exchange protein, carbonic anhydrase, aquaporin, and Rh channels that reside within the erythrocyte lipid raft, a caveola homolog. This metabolon would facilitate NO export by localizing nitrite, proton, and deoxyheme with highly hydrophobic channels at the membrane complex. Another proposed mechanism suggests that the reaction of nitrite with deoxyhemoglobin yields an intermediate species that could be stabilized by and transported through the red blood cell membrane (via lipid raft, Rh channels, or aquaporin); candidate species include S-nitrosothiols, nitrogen dioxide, peroxynitrite, and nitrated lipids.63,64 Measurements obtained in simulation studies support the existence of a NO intermediate species. Here, when the rate of NO formation was as high as 100 nM/s, the maximal NO concentration in blood was less than 0.012 nM in the setting of erythrocyte membrane permeability to NO of 4.5 cm/s at a hematocrit of 45%. Thus, it is unlikely that NO is exported directly from the erythrocyte because the resident plasma concentration would be too low to have physiological effects.65 At present, it remains to be determined which systems of export and candidate intermediate signaling molecule(s) are operative.

NITRIC OXIDE–DEPENDENT REGULATION OF TISSUE OXYGEN LEVELS Nitric Oxide and Hemoglobin–Oxygen Affinity By virtue of its reaction(s) with hemoglobin, it has been suggested that NO directly influences hemoglobin–O2 affinity; however, it is difficult to predict a priori the effect of NO binding on net hemoglobin–O2 affinity owing to the different hemoglobin species present at any given time that react

190 with NO. For example, NO oxidizes oxyhemoglobin to form methemoglobin, which increases O2 affinity,66 although the reaction of NO with deoxyhemoglobin yields nitrosylhemoglobin, which has a markedly decreased affinity for O2 .67 It has also been reported that when NO reacts with the ␤-Cys93 residue to form SNO-Hb, its O2 affinity increases compared to underivatized hemoglobin.68 As such, the overall net effect of NO on hemoglobin–O2 affinity may result, in part, from the relative abundance of each of these NO-hemoglobin derivatives. Despite the recognized differences in hemoglobin–O2 affinity of these NO-hemoglobin products, evidence indicates that the reaction of NO and hemoglobin may not, in fact, significantly influence hemoglobin–O2 affinity by a mechanism other than by increasing methemoglobin formation. In a study that examined the effect of NO gas (80 ppm) inhalation for 2 hours on hemoglobin–O2 affinity, inhaled NO did not alter hemoglobin–O2 affinity in normal subjects, and methemoglobin levels rose to only 1%, suggesting that the level of NO bound to hemoglobin was too low to influence overall O2 affinity.69 In vitro studies that exposed erythrocytes to NO gas (80 ppm) or the NO donors diethylamine NONOate and S-nitrosocysteine reported similar findings. Here, exposure to NO did not affect hemoglobin–O2 affinity per se, but a significant rise in methemoglobin formation was detected that was associated with a leftward shift in the P50 .70 These observations, therefore, imply that low concentrations of NO do not alter the hemoglobin–O2 affinity of erythrocytes directly, and perceived changes likely result from an increase in methemoglobin formation and loss of cooperativity (Hill coefficient).70,71

Jane A. Leopold and Joseph Loscalzo One explanation for these findings is that O2 consumption by the arteriole wall is, in part, dependent upon vascular tone; vasoconstriction increases vessel wall O2 consumption whereas vasodilation has the opposite effect.74,75 Further study of this model during NO-dependent and NOindependent vasodilation revealed that the NO-mediated reduction in O2 consumption resulted principally from a decrease in the mechanical work of vascular smooth muscle cells.76 Although the aforementioned studies demonstrate that NO regulates tissue and vessel wall O2 consumption, it has also been demonstrated that O2 determines vascular NO catabolism. Under normoxic conditions, rat aortas incubated in a solution injected with NO at constant O2 tension demonstrated a 50% increase in NO consumption as compared to what was observed in the absence of the vessel. Furthermore, aorta NO consumption declined as the O2 concentration was decreased to mimic hypoxic conditions, suggesting that mitochondria may be involved in the vessel wall interaction between O2 and NO. This may result from the direct reaction of NO with O2 in the mitochondrial membrane or with cytochrome c oxidase. In support of this theory, increased aorta NO consumption under anaerobic conditions was inhibited by the cytochrome c oxidase inhibitor sodium cyanide. These studies imply that decreased aorta NO catabolism that occurs under low O2 concentrations may preserve bioavailable NO levels to promote vasodilation. In contrast, increased NO consumption observed under anaerobic conditions suggests that this phenomenon may limit formation or accumulation of toxic NO metabolites in the vessel wall.77

HYPOXIC VASODIL ATION Tissue Oxygen Consumption The interaction between NO and O2 is of physiological importance: NO has been shown to regulate tissue O2 consumption and, conversely, O2 levels may determine the rate of NO catabolism. Endothelium-derived NO limits tissue O2 consumption, an effect that is independent of the rate of O2 delivery and associated with a decrease in both intravascular and tissue pO2 as well as a rightward shift in the hemoglobin–O2 dissociation curve.72 Other studies have confirmed that endothelium-derived NO modulates tissue oxygenation by decreasing vessel wall O2 consumption. Although it has been shown that the pO2 drops in arterioles, the observed decreases were too great to be explained by diffusion alone suggesting active consumption of O2 by the vessel wall. In a series of studies performed in vivo to examine the O2 consumption rate of arteriolar walls in rat cremaster muscle, inhibition of NO synthesis with NG -nitro-L-arginine methyl ester (L-NAME) resulted in a 42% increase in vessel wall O2 consumption. In contrast, enhancement of flow-mediated NO release resulted in a 34% decrease in O2 consumption by the vessel wall.73

NO plays an integral role in hypoxic vasodilation, a physiological response that regulates blood flow to deliver O2 and meet tissue metabolic demands. It has been hypothesized that this response results from a feedback mechanism that signals alterations in O2 or pH levels resulting from a discordance between the basal delivery rate of O2 and tissue O2 consumption.78 To ensure tissue O2 demands in the setting of perceived hypoxia, vasodilator substances such as NO are released to increase blood flow and, thereby, maintain tissue oxygenation. In mammalian systems, hypoxic vasodilation occurs concomitant with the desaturation of hemoglobin from 60% to 40% corresponding to partial pressures of O2 from 40 to 20 mm Hg.64 Interestingly, in skeletal muscle tissue O2 extraction occurs mainly in resistance arterioles implying that hypoxic sensing takes place in the vasculature at a location proximal to the arterioles and arteriolar capillaries.79 In this vascular bed, erythrocytes, which traverse the artery-toarteriole-to-capillary in approximately 10 s, form a column of moving blood such that the hemoglobin and O2 concentrations remain relatively constant at any given

The Biology of Vascular Nitric Oxide location within an arteriole.64 As such, it is likely that there is one anatomical site within the microcirculation that contains the greatest number of R3 tetramers with maximal nitrite reductase activity. It is at this anatomical location that hemoglobin-mediated nitrite reduction to release NO will be maximal in response to tissue O2 consumption and metabolic demands, and hypoxic vasodilation will ensue.64 In contrast, in the heart and brain, tissue O2 is delivered via the capillary bed through a mechanism that involves retrograde signaling. Here, it is believed that the capillaries or venous circulation provide the vasodilator signal.80,81 One proposed mechanism to explain vasodilation in these vascular beds involves the diffusional shunting of NO from veins to an adjacent arteriole. In fact, when cotransport of NO and O2 in a paired arteriole-venule surrounded by capillary-perfused tissue was modeled, it was found that the capillary bed connecting the arteriole and venule facilitates the release of O2 from the vessel pair to the surrounding tissue. In this model, decreasing the distance between the arteriole and venule resulted in a higher local NO concentration in the venule than in the arteriole wall, suggesting that transvalvular diffusional shunting of NO is plausible.82 Although the identity of the O2 sensor and released vasodilator has not yet been confirmed definitively, evidence suggests that hemoglobin serves as the O2 sensor and, owing to its nitrite reductase properties, reduces nitrite to release NO, the vasodilator. Hemoglobin is well poised to serve as the O2 sensor; the O2 -linked allosteric structural transition from the R-state to the Tstate may function to signal for release of NO from the erythrocyte.83 NO as the candidate vasodilator agent to regulate hypoxic vasodilation is intuitively sound. NO is a paracrine-signaling molecule synthesized by the endothelium and released into the bloodstream where it reacts at a nearly diffusion-limited rate (107 M/s) with both oxy- and deoxyhemoglobin to yield methemoglobin/nitrite and iron-nitrosyl-hemoglobin, respectively. Although these reactions limit the half-life of NO in blood (∼5 ␮s half-life in blood) as well as the diffusion distance (0.1 ␮mol/L) stimulated endothelial cell apoptosis. These findings were attributed to the influence of statins on eNOS expression; low doses of statins did not influence eNOS expression whereas higher doses markedly increased eNOS expression and NO generation.117 NO has also been shown to modulate endothelial cell senescence, recognized as the limited ability of cells to proliferate in vitro accompanied by phenotypic changes in morphology, gene expression, and function. As such, cellular senescence is believed to contribute to the pathogenesis of vascular disease. Endothelial cell senescence, measured as senescence-associated ␤-galactosidase and telomerase activity, is inhibited in cells treated with the NO donor diethylamine NONOate or gene transfer of eNOS. In contrast, in cells treated with L-NAME, transfection with eNOS had no effect, indicating that endothelium-derived NO was essential to maintain the proliferating phenotype.118

was elucidated using siRNA to decrease PDE1A expression; increased cGMP levels were associated with p27kip1 upregulation, cyclin D1 downregulation, and p53 activation to decrease proliferation and promote apoptosis.122 Similarly, NO inhibits vascular smooth muscle cell proliferation via a number of mechanisms that are cGMPindependent. For example, NO mediates two distinct cell cycle arrests; an immediate cGMP-independent block in Sphase followed by a shift back in the cell cycle from G1 –S to a quiescent G0 -like state.123 NO also decreases the activity of arginase and ornithine decarboxylases, resulting in reduced formation of polyamines that are required for DNA synthesis and increased expression of p21waf1/Cip1 .124,125 NO further increases p21waf1/Cip1 expression by inhibition of the small GTPase RhoA by S-nitrosation.126 Together, the divergent signaling pathways regulated by NO that converge to inhibit vascular smooth muscle cell proliferation highlight the importance of NO in maintaining the vascular smooth muscle cell contractile phenotype.

Vascular Smooth Muscle Cell Proliferation

Platelet Adhesion and Aggregation

In contrast to its stimulatory effects on endothelial cell proliferation, endothelium-derived NO inhibits vascular smooth muscle cell proliferation to maintain the normal architecture of the vascular wall and preserve the vessel lumen. When the endothelium is injured, as occurs in vascular disease states such as atherosclerosis or mechanical disruption by angioplasty, the resultant decrease in bioavailable NO is permissive for vascular smooth muscle cell phenotype transition from a contractile state to a dedifferentiated synthetic phenotype. Once this phenotype transition occurs, vascular smooth muscle cells proliferate, migrate, and elaborate extracellular matrix proteins to fashion the neointima and thereby narrow the vessel lumen. NO maintains a constant inhibition of vascular smooth muscle cell proliferation via cGMP-dependent and -independent signaling pathways. NO increases cAMP levels in a cGMP-dependent manner to activate protein kinase A and decrease intracellular Ca2+ stores; increased levels of intracellular Ca2+ have been shown to promote vascular smooth muscle proliferation.119 Protein kinase A activation limits cell proliferation further by inhibiting Raf-1 activation of mitogen-activated protein kinase signaling cascades to decrease DNA synthesis, and studies performed in eNOS-transfected vascular smooth muscle cells demonstrate increased expression of the cyclindependent kinase inhibitor p21waf1/Cip1 , which inhibits proliferation.120,121 Recently, it has been shown that cyclic nucleotide phosphodiesterases (PDE), which catalyze the hydrolysis of cGMP to 5
GMP, participate in the regulation of vascular smooth muscle cell proliferation. In quiescent cells in the contractile state, PDE1A is primarily localized in the cytoplasm of the cell, whereas in synthetic vascular smooth muscle cells PDE1A is translocated to the nucleus. The functional significance of PDE1A nuclear translocation

NO synthesized by the endothelium as well as by platelets importantly limits platelet adhesion, aggregation, and recruitment (Fig. 10.4). Although the formation of a hemostatic plug by platelets is a physiological response to vessel wall damage, platelet hyperreactivity, as occurs in NO-deficient states, results in pathophysiological arterial thrombosis. Under basal conditions, platelets remain in an inactive state that is regulated by NO, prostacyclin, and ecto-AD(T)Pase (CD39).127 In the bloodstream, platelets circulate in the erythrocyte-free low-shear boundary near the endothelial surface and are in optimal position to be affected by endothelium-derived NO.128 Platelets are also exposed to NO via a circulating pool of S-nitrosothiols; however, here, NO uptake is in part dependent on protein disulfide isomerase activity for S-nitrosothiol metabolism and transmembrane NO transfer.129 Once NO reaches the platelet cytosol, it activates soluble guanylyl cyclase to increase cGMP levels resulting in a decrease in intracellular Ca2+ flux and inhibition of platelet activation.130 In resting platelets, intracellular Ca2+ levels are maintained between 50 and 100 nM by the collaborative actions of the cGMP-responsive sarcoplasmic reticulum ATPase and plasma membrane Ca2+ ATPase pumps.131,132 NO also inhibits inositol-1,4,5-trisphosphate– induced intracellular Ca2+ release by stimulating cyclic GMP-dependent kinase to phosphorylate the inositol1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG). IRAG is expressed in platelets and phosphorylated at Ser664 and Ser677 to negatively regulate inositol-1,4,5-trisphosphate-induced intracellular Ca2+ flux.133 NO-dependent regulation of the intracellular Ca2+ flux limits the conformational change to the active state of the heterodimeric fibrinogen-binding integrin glycoprotein IIb/IIIa as well as decreases the number (by 50%) and

The Biology of Vascular Nitric Oxide

195

Unactivated platelets

Activated platelets Recruitable platelets

Activation ADP Serotonin

NO

.

NO.

.

GP IIb/IIIa

GP la/IIa

NO GP lb/IX/V VWF

Figure 10.4. NO and platelet activation. Under basal conditions, the vascular endothelium elaborates NO• to maintain a nonthrombogenic surface. In contrast, when the endothelium is disrupted and NO• levels are decreased, platelets become activated and adhere to collagen via the cell surface receptor glycoprotein Ib/IIa (GP Ia/IIa) and glycoprotein Ib/IX/V (GP Ib/IX/V) that binds to the interstitium by von Willebrand factor (VWF). These activated platelets undergo shape change and release adenosine diphosphate (ADP) and serotonin to recruit and activate additional circulating platelets to the growing thrombus, and increase expression of the conformationally active fibrinogen receptor, glycoprotein IIb/IIIa (GP IIb/IIIa) to increase platelet aggregation and thrombus formation; the concomitant release of NO limits platelet aggregation. Platelet-derived NO limits recruitment of platelets to the growing platelet-rich hemostatic plug (or thrombus). (See color plate 10.4.)

the affinity (2.7-fold increase in Kd ) of fibrinogen binding sites on the platelet surface.134,135 Another key component of platelet activation that is inhibited by NO is the exocytosis of platelet granules, which release mediators that modulate platelet interactions with the endothelium. Exocytosis is regulated, in part, by Nethylmaleimide-sensitive factor (NSF), an ATPase that promotes disassembly of soluble NSF attachment protein– receptor complexes. In human platelets, it has been shown that NO inhibits exocytosis of dense, lysosomal, and ␣granules by S-nitrosation of NSF. Furthermore, platelets isolated from an eNOS−/− mouse model demonstrate increased exocytosis in vivo.136 In fact, it is this mechanism that may explain the observation that eNOS-deficient mice do not demonstrate enhanced thrombosis in vivo. In these mice, it was shown that fibrinolysis is enhanced owing to the lack of NO-dependent inhibition of the release of endothelial Weibel–Palade body storage granule contents, which include von Willebrand factor, P-selectin, and tissue plasminogen activator.137,138 NO donors have also been shown to inhibit platelet adhesion by interfering with the interaction between von Willebrand factor and glycoprotein IIb/IIIa.139 In fact, an S-nitrosated derivative of the recombinant von Willebrand factor fragment, AR545C, has been shown to decrease platelet adhesion in both in vitro and in vivo studies. Furthermore, poly-S-nitrosated bovine serum albumin, an NOreleasing protein that increases platelet cGMP levels, has also been shown to limit platelet adhesion to collagen.140 In addition to the effects of endothelium-derived NO on platelet function, human platelets and megakaryoblastic cells express eNOS and synthesize NO.141,142 NO release has been measured in resting platelets and reported to

be approximately 11.2 pmol NO min/108 cells, suggesting that the amount of NO generated and released by platelets approaches that of endothelial cells.143 Based on these observations, it is therefore important to note the relative contribution of endothelium-derived as compared to platelet-derived NO to platelet activation and aggregation. To examine this phenomenon, platelets were isolated from eNOS knockout mice or wild-type mice, and transfused into a thrombocytopenic eNOS knockout mouse model. In mice transfused with eNOS knockout platelets, bleeding time was decreased significantly compared to what was observed in mice transfused with wild-type platelets ( bleeding time = 24.6 ± 9 s vs. 3.4 ± 5 s; P < 0.04), indicating that platelet-derived NO contributes significantly to limit platelet recruitment and thrombus formation.144 The importance of platelet-generated NO has also demonstrated in studies of patients with clinical atherothrombotic vascular disease. In 87 patients with symptomatic coronary artery disease referred for coronary angiography, platelets isolated from patients with acute coronary syndromes generated significantly less NO than those isolated from patients with stable angina (0.26 ± 0.05 pmol/108 platelets vs. 1.78 ± 0.36 pmol/108 platelets, P = 0.0001).144 This finding suggests that platelet-derived NO, in addition to endothelium-derived NO, importantly modulates platelet function and thereby affects vascular disease risk.

Leukocyte Adhesion Endothelium-derived NO also prevents leukocyte adhesion to the endothelium and transmigration into the vessel wall. NO donors, or endogenous NO, limit(s) leukocyte

196 adherence to the endothelium, whereas inhibition of eNOS results in increased adhesion and emigration from the bloodstream.145 Following exposure to cytokines such as interleukin (IL)-1␤ or TNF␣, the endothelium is activated and expresses endothelial-leukocyte adhesion molecules including vascular cell adhesion molecule-1, E-selectin, P-selectin, and intercellular adhesion molecule-1.146 In addition, activated endothelial cells produce leukocyte chemoattractants (chemokines), such as IL-8 and monocyte chemotactic protein-1. In this manner, activated endothelium both recruits and traps circulating leukocytes. NO regulates leukocyte chemotaxis by inhibiting expression of adhesion molecules and synthesis of chemoattractant proteins.146,147 NO also inhibits exocytosis of Weibel–Palade bodies and release of P-selectin through Snitrosylation of NSF.148 NO limits leukocyte recruitment and adhesion further by decreasing the synthesis of IL1␤, TNF␣, IL-6, and interferon-␥ in lymphocytes and monocytes. These effects are mediated, in part, by Snitrosation of transcription factors, including NF-KB/IkB and JAK/STAT, to inhibit upregulation of adhesion molecule expression.149

CONCLUSION NO, a paracrine mediator of vascular homeostasis, is synthesized by the endothelium and released continuously to the vascular interstitium to regulate basal vascular tone. NO also diffuses into the bloodstream where it undergoes a complex series of reactions and may be oxidized to nitrite and nitrate, or react with redox metals and thiols to yield NO compounds, including dinitrosyl iron, N-nitrosamines, and S-nitrosothiols that have unique biological effects. Notably, NO also reacts with erythrocyte deoxy- and oxyhemoglobin to generate nitrosyl-hemoglobin or methemoglobin and nitrite, respectively. Moreover, owing to the nitrite reductase activity of hemoglobin, NO is released in the vasculature response to tissue ischemia and, thereby, modulates tissue O2 consumption and hypoxic vasodilation. Endothelium-derived NO is also a key determinant of vascular permeability, endothelial and vascular smooth muscle cell proliferation and apoptosis, platelet adhesion and aggregation, as well as leukocyte recruitment and adhesion. As such, NO serves as an integral mediator of vessel wall integrity and vascular homoeostasis.

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11 Mechanisms and Clinical Complications of Hemolysis in Sickle Cell Disease and Thalassemia Gregory J. Kato and Mark T. Gladwin

OVERVIEW OF HEMOLYSIS IN SICKLE CELL DISEASE AND THALASSEMIA Anemia is the most basic clinical characteristic of sickle cell disease and thalassemia. In sickle cell disease, the polymerization of sickle hemoglobin (HbS) causes profound changes in the integrity and viability of the erythrocyte, leading to both extravascular and intravascular hemolysis. The lifespan of the erythrocyte in sickle cell disease is often shortened to less than one-tenth of normal. In ␤-thalassemia intermedia and major, but not in sickle cell disease, a substantial portion of the hemolysis occurs in the intramedullary space before the developing erythrocytes can even exit the bone marrow, referred to as ineffective erythropoiesis. In either case, erythropoiesis is markedly increased, but insufficient to compensate completely for the accelerated hemolysis, resulting in chronic anemia. This chapter examines the mechanisms that give rise to the accelerated hemolysis characteristic of these hemoglobinopathies and considers emerging data suggesting that chronic intravascular hemolysis produces endothelial dysfunction and a progressive vasculopathy. The latter mechanism of disease contributes to a clinical subphenotype of complications shared by many of the hemolytic anemias, including pulmonary arterial hypertension, cutaneous leg ulceration, priapism, and perhaps stroke. The mechanisms and consequences of hemolysis differ by two main anatomical compartments: extravascular hemolysis, which primarily involves phagocytosis by macrophages in the reticuloendothelial system, and intravascular hemolysis, which occurs within the blood vessel lumen. Approximately two-thirds of hemolysis in sickle cell disease is extravascular and one-third intravascular.1 Extravascular hemolysis occurs primarily through mechanisms involving cell surface phosphatidylserine exposure, adherent immunoglobulin G (IgG) and splenic entrapment of

rigid red cells. Intravascular hemolysis decompartmentalizes the red cell contents into blood plasma, releasing hemoglobin, arginase-1, erythroid isoforms of lactate dehydrogenase (LDH), and other intraerythrocytic enzymes. In sickle cell disease, the development of irreversibly sickled cells (ISCs), and oxidative injury to red cell membrane proteins and lipids are believed to contribute to intravascular hemolysis (Chapter 9).

Irreversibly Sickled Cells Aging sickle erythrocytes become increasingly dense and noncompliant.2 Dehydration of the red cell (Chapter 9) involves leakage of intracellular potassium and sodium, and dysfunction of K:Cl cotransport and calcium-dependent potassium transport. This loss of solute and water increases the mean corpuscular hemoglobin concentration (MCHC), which promotes HbS polymerization (Chapter 6) (Fig. 11.1).3 This effect can be inhibited by the coinheritance of ␣ thalassemia which decreases the MCHC, or increased fetal hemoglobin (HbF) levels, which inhibits HbS polymerization.4 Cycles of polymerization and depolymerization give rise to ISCs.5 ISCs cells are rigid and prone to being removed from the circulation by physical entrapment in the microvasculature, including peripheral blood vessels and the spleen, where presumably the lysis occurs.6 The ISC has a short survival and its numbers reflect the hemolytic rate.7,8 In fact, the severity of hemolysis correlates with the extent of HbS polymerization, as estimated from the MCHC and the relative proportion of hemoglobin fractions.9,10 Following red cell dehydration and polymerization of HbS, the formation of ISCs involves oxidation of erythrocyte components, and loss of aminophospholipid asymmetry, with subsequent binding of IgG or complement.

Oxidation of Erythrocyte Proteins and Lipids In sickle erythrocytes, oxygen radicals are formed at rates twice that of control erythrocytes, and membrane-bound hemichrome (oxidized hemoglobin precipitates) greatly enhances superoxide and peroxide-driven hydroxyl radical generation (Fig. 11.1).11 Data from many laboratories suggest that in sickle cell disease the red cell is exposed to high levels of superoxide produced by intravascular enzymes such as xanthine oxidase,12 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase13 and uncoupled nitric oxide (NO) synthase.14 Signs of oxidative injury are seen in the cytoplasm and membrane of the red cell, in association with membrane bound iron.15,16 The sickle erythrocyte membrane has reduced abundance of reduced sulfhydryl groups and increased lipid peroxidation.17–21 Cytoskeletal proteins, particularly protein 4.1, are also oxidized.22,23 These oxidative changes in the red cell cytoskeleton appear to contribute to the development of ISCs

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Figure 11.1. Factors leading to sickling and oxidant stress in erythrocytes in sickle cell disease. Solute loss and increased intracellular calcium promote red cell dehydration, which raises the intracellular concentration of sickle hemoglobin. This promotes polymerization of sickle hemoglobin, which is associated with generation of increased levels of oxidant species and depletion of cellular antioxidants, leading to a heightened state of oxidative stress. Each of these steps is described in the text, although the exact sequence of these events is somewhat speculative. (See color plate 11.1.)

(Fig. 11.2),24 as the red cell membrane ghosts and the cytoskeletons of ISCs remain deformed after removal of hemoglobin.24,25 Oxidation-induced defects in ␤-actin and spectrin appear to slow the dissociation of their ternary complex with protein 4.1. In ISCs, formation of a disulfide bridge is favored in ␤-actin involving cysteines at the 284 and 373 positions.24 The intense oxidative stress places high demands on metabolic pathways that provide compensatory reducing capacity in the red cell, such as the hexose monophosphate shunt, which produces NADH and reduced glutathione (Fig. 11.1).26,27 The enzymes in these pathways are inhibited by the excess free heme present in sickle11 and thalassemic red cells.28 As glutathione becomes oxidized, identified by a high ratio of oxidized to reduced glutathione or as a low level of total glutathione,11,29–31 cysteine residues in ␣-spectrin become glutathiolated.24 This blocks the normal ubiquitin-conjugating and ligating activity of ␣-spectrin, impairing its autoubiquitination in sickle erythrocytes.32,33 The lack of ubiquitination prevents normal dissociation of ␣-spectrin and protein 4.1, diminishing erythrocyte cytoskeletal flexibility and contributing to the rigid shape of the ISC.34

Consistent with a role for oxidative stress in ISC formation, the reducing agent N-acetylcysteine in vitro can convert ISCs into biconcave discs.35 A pilot trial of Nacetylcysteine in 16 patients with sickle cell disease did not, however, produce a significant change in the hemoglobin level or reticulocyte count, despite achieving higher erythrocyte glutathione levels and a lower percentage of ISCs at the highest dose tested.36 This suggests that the mechanisms of hemolysis are not solely related to erythrocyte shape. Some investigators have found deficiency of the naturally occurring antioxidant vitamin E in patients with sickle cell disease or thalassemia,37–39 although this has been inconsistent.40 Pilot studies have suggested that vitamin E supplementation may reduce hemolysis in sickle cell disease.41,42 Future approaches will likely attempt to inhibit extracellular sources of reactive oxygen species, including xanthine oxidase, NADPH oxidase, and uncoupled endothelial NO synthase.

Membrane Phospholipid Asymmetry In erythrocytes and other cell types, active mechanisms maintain asymmetry of membrane phospholipids. Over

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Figure 11.2. Factors promoting hemolysis in sickle cell disease. Decompensated oxidative stress in the sickle erythrocyte is associated with multiple lesions in its cytoskeleton and membrane. These lesions are responsible for binding of immunoglobulin to the membrane, promoting Fc receptor–mediated endocytosis of the damaged red cell by reticuloendothelial macrophages, considered extravascular hemolysis. Oxidative damage to the flippase enzyme is proposed to cause externalization of phosphatidylserine and phosphatidylethanolamine that also stimulate uptake of the red cell by macrophages, in addition to adhesion to endothelium. Oxidative damage and glutathiolation of the cytoskeleton can trigger endovesiculation of the membrane and release of membrane microparticles, and mechanical fragility of the damaged red cell. These latter events tend to produce intravascular hemolysis, resulting in the decompartmentalization of erythrocyte contents into plasma. (See color plate 11.2.)

75% of cellular phosphatidylcholine and sphingomyelin normally are maintained in the outer leaflet of the red cell membrane, whereas more than 80% of total phosphatidylserine and phosphatidylethanolamine are maintained in the inner leaflet (Chapter 9). This is accomplished by adenosinetriphosphate (ATP)–dependent aminophospholipid translocase, or flipase, which actively transports phosphatidylserine and phosphatidylethanolamine from the outer to the inner monolayer. Flippase can become dysfunctional with hypoxia-induced sickling (Fig. 11.2).43,44 The mechanism of flippase inactivation in murine sickle cells involves its oxidation,45 although this has not been confirmed in human sickle cells.46 Inactivation of flipase permits phosphatidylserine exposure on the outer leaflet of the red cell membrane.47 Phosphatidylserine and phosphatidylethanolamine also are transported actively to the outer leaflet by activation of a Ca2+ -dependent scramblase. With increased Ca2+ flux, this scramblase can become activated, resulting in abundant phosphatidylserine exposure on the red cell surface in sickle cell disease and ␤ thalassemia.48 Thus Ca2+ flux and accumulated oxidant stress may both play a role in red cell aging and destruction, mediated though phosphatidylserine exposure.49

Cytoskeletal abnormalities in sickled cells, particularly spiculation, also appear to promote localized membrane phospholipid asymmetry.50–52 Similar localized phospholipid asymmetry occurs at sites of Heinz bodies.53 Reticuloendothelial system macrophages bind to and engulf these phosphatidylserine-externalized red cells, contributing to extravascular hemolysis (Fig. 11.2).45,54 Phosphatidylserine exposure also induces binding of red cells to endothelial cells,55–57 likely leading to sequestration of phosphatidylserine-exposing cells in peripheral blood vessels. Other mechanisms also induce adhesion of young sickled cells to endothelial cells. Immobilization of ISCs in flowing blood onto vascular endothelium may lead to increased shear stress and intravascular hemolysis (Chapter 8).

Fragility of ISCs to Mechanical and Shear Stress The numbers of circulating ISCs decreases during the latter stages of vasoocclusive crisis, suggesting that the rigid, adhesive red cells might become sequestered in the microvasculature.58,59 ISCs are also implicated in intravascular hemolysis during mechanical membrane fragmentation. Repeated cycles of sickling in vitro generate

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dehydrated red cells with membrane spiculation apparent on electron microscopy, which appear to give rise to spectrin-free, hemoglobin-containing microparticles.60,61 Increased mechanical fragility of sickle erythrocytes has been documented in vitro by increased lysis with application of shear stress, and confirmed in vivo with the increased shear stress due to exercise-induced increase in blood flow.62 Cell fractionation experiments indicate that the dense, dehydrated sickle erythrocytes are the most sensitive to mechanical or shear-induced hemolysis62–66 and that rehydration restores resistance to shear stress, except in the densest red cells.62 The membrane structure of ISCs is weakened and may contribute to intravascular fragmentation (Fig. 11.2).67 Mechanical stress also induces hemoglobin denaturation and loss of hemin, which contributes to oxidative stress.68 ISCs may be the most sensitive circulating red cells to this mechanical lysis induced by shear stress.

Chronic extravascular hemolytic anemias in general are associated with the gradual development of splenomegaly that occurs frequently in patients with ␤-thalassemia intermedia and major. Although most adults and older children with sickle cell anemia have functional asplenia and splenic atrophy due to chronic subclinical splenic infarction, there are cases of sickle cell disease in which splenomegaly is present. These include young children homozygous for the HbS gene and patients with clinically milder sickling syndromes such as HbSC disease and HbS-␤+ thalassemia.85,86 Patients with splenomegaly and sickle cell disease or ␤ thalassemia often are more anemic than similar patients without splenomegaly, presumably due to chronic hypersplenism.87,88 In some of these cases, chronic hypersplenism has been documented by improvement in anemia following splenectomy.89,90

Adherent Immunoglobulin G

CLEARANCE OF HEMOGLOBIN

A subpopulation of erythrocytes in sickle cell disease binds IgG.20,69–71 Cyclic oxygenation-deoxygenation, or oxidant stress in sickle cell disease, indicated by malondialdehyde production stimulates binding of IgG to the red cell surface.20,72 Part or all of this effect may be related to Heinz bodies, oxidatively denatured hemoglobin, which generates a hemichrome that cross-links the major red cell membrane spanning protein, band-3.73 A cryptic antigen exposed on the clustered band-3 complexes on the erythrocyte surface is recognized and bound by specific IgG antibodies, which in turn bind avidly to the Fc receptors on reticuloendothelial system macrophages.74,75 This culminates in phagocytosis and lysis of the antibody-coated sickle erythrocytes by macrophages, a normal consequence of aging of red cells that is accelerated in sickle cells (Fig. 11.2).72 This Fc receptor–mediated mechanism is a second significant contributor to oxidant stress-related, extravascular hemolysis in sickle cell disease.

Extracellular hemoglobin is toxic to vascular health, and humans have developed multiple redundant pathways to facilitate its rapid clearance.91 During intravascular hemolysis, large amounts of hemoglobin are released from the lysed red cells into plasma. During extravascular hemolysis, the red cell is engulfed by a reticuloendothelial macrophage, and hemoglobin is degraded directly in the macrophage, releasing small amounts of hemoglobin into plasma. Upon its dilution into plasma, the hemoglobin tetramers decompose into ␣␤ dimers. Dimeric hemoglobin rapidly forms complexes with the soluble plasma protein haptoglobin, the principal hemoglobin scavenging protein, which prevents hemoglobin from crossing the glomerular membrane. Upon formation of the complex, haptoglobin displays a previously hidden binding site for its cognate receptor on macrophages, CD163, the hemoglobin scavenger receptor (Fig. 11.3).92 This high-affinity binding promotes endocytosis of the CD163-haptoglobin-hemoglobin complex, where it is degraded, disposing of the hemoglobin and along with it, haptoglobin, which is not recycled. Even with the lower-grade hemolysis seen in HbSC disease, serum haptoglobin levels are low. During the robust hemolysis of sickle cell anemia or ␤-thalassemia intermedia, haptoglobin is completely depleted from plasma. Thus, an undetectable plasma haptoglobin clinically signifies high-level hemolysis and pathophysiologically indicates that the primary, rapid mechanism for hemoglobin clearance has become saturated. The binding capacity of haptoglobin to hemoglobin is reported to be 0.07– 0.15 g/dL, depending on different genetic variants of haptoglobin. Hemopexin plays a complementary role, clearing plasma of free heme. Plasma hemoglobin that becomes oxidized to methemoglobin is prone to lose the oxidized heme

Complement-mediated Hemolysis Sickle cells are particularly sensitive to attack by complement-inducing intravascular hemolysis. Similar to erythrocytes in paroxysmal nocturnal hemoglobinuria, sickle cells show a defect in activity of the membrane attack complex, C5b-9.76 This occurs due to increased binding of complement C5b-7 and C9 to sickle cells, particularly to the densest cells. This binding leads to C5b-9-mediated lysis initiated by C5b-6, especially on ISCs. Because it has been found that anionic lipids on the surface of the red cell can induce binding of C5b-6,77 it is possible this binding occurs via the increased phosphatidylserine exposure that has been described on the outer membrane of the sickle and thalassemic erythrocyte.45,78–84

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Figure 11.3. Pathophysiological consequences of intravascular hemolysis. Decompartmentalization of red cell contents results in ectopic localization of hemoglobin and arginase into plasma. Hemoglobin is rapidly bound to haptoglobin, and this complex binds to CD163 on macrophages, resulting in endocytosis and clearance of the whole complex. If the utilization of haptoglobin exceeds its replacement by hepatic synthesis, plasma cell–free hemoglobin accumulates, which stoichiometrically inactivates NO. Plasma cell–free arginase converts plasma arginine to ornithine, reducing availability of arginine, the obligate substrate for NOS. Depletion of NO results in pulmonary vasoconstriction, platelet activation, smooth muscle dystonias, and reduced antioxidant capacity. (Reproduced with permission of the publisher from ref. 110.)

porphyrin ring, known as hemin. Free heme is capable of inserting into cell membranes and producing hydroxyl and nitrogen dioxide radicals via Fenton and peroxidase chemistry, respectively, and a specific clearance pathway exists to avoid this oxidative insult. Hemin is bound by another ␤-globulin plasma glycoprotein, hemopexin, and the complex is slowly cleared by hepatic parenchymal cells. After saturation of hemopexin, free hemin is adsorbed to albumin as the brown pigment methemalbumin.

Depletion of plasma hemopexin is another indicator of hemolysis. Disposal of plasma hemoglobin via the haptoglobin– CD163 pathway activates a program that helps to counteract the adverse effects of plasma hemoglobin on vascular homeostasis. Binding of hemoglobin-haptoglobin to CD163 activates the antiinflammatory cytokine interleukin-10 and the heme oxygenase-1 heme catabolic enzyme, which also has antioxidant activities (Fig. 11.3).93

206 Heme oxygenase-1 breaks down heme into biliverdin, free iron, and carbon monoxide. Carbon monoxide induces vasodilatory, antioxidant, antiinflammatory, and antiproliferative responses.94–96 Biliverdin is converted by biliverdin reductase to bilirubin, which also has antioxidant properties.97 Carbon monoxide, produced in the body solely through the heme oxygenase reaction and eliminated via the lungs, may be monitored in exhaled breath as an indicator of the rate of heme turnover.98 The hemoglobin scavenging activity of haptoglobin is supplemented by the haptoglobin-related protein (Hpr). This plasma protein also binds to plasma hemoglobin with high affinity, but this complex does not bind to CD163. Instead, the hemoglobin–Hpr complex is directed to specialized high-density lipoprotein particles called HDL3 OR TLF-1, which contain apolipoprotein A-I, L-I, and others.99 Whether this sequestration protects the vascular system against the toxic effects of plasma hemoglobin is unknown. Unlike haptoglobin, Hpr is not depleted from plasma of patients with sickle cell disease or other forms of intravascular hemolysis.100 It is not known whether hemoglobin– Hpr complexes in HDL particles are capable of scavenging NO. High-grade intravascular hemolysis may saturate the capacity of the haptoglobin-hemopexin-Hpr system, forcing hemoglobin clearance through alternative mechanisms. The relatively small size of ␣␤ globin dimers allows their penetration of the glomerulus into filtrate.91 Hemoglobinuria results when the maximal tubular reabsorption rate of 1.4 mg/min is exceeded.101 Hemoglobin reabsorbed into renal tubular cells is degraded, generating bilirubin and iron. Iron stored in ferritin in the renal tubular cells can accumulate to high levels, generating insoluble hemosiderin.102,103 The hemosiderin-laden renal epithelial cells are eventually sloughed into urine, where they may be identified in urinary sediment by Prussian blue staining and are indicative of high-grade chronic intravascular hemolysis. Such hemolytic rates are common in paroxysmal nocturnal hemoglobinuria, and may occur in sickle cell disease. In states of haptoglobin depletion, plasma hemoglobin also undergoes endocytosis by hepatic macrophages via direct binding to CD163.92 There is also indirect evidence of similar activity in macrophages of neovascularized atherosclerotic lesions. This might suggest a protective role for this pathway in the development of proliferative vasculopathy related to cases of chronic intravascular hemolysis or to intraplaque hemorrhage.

CLINICAL CONSEQUENCES OF INTRAVASCULAR HEMOLYSIS When the rate of intravascular hemolysis exceeds the capacity of the hemoglobin scavenging mechanisms, hemoglobin and other red cell constituents accumulate in plasma compartment. Several of these constituents are toxic to vascular health, and a clinical vasculopathy

Gregory J. Kato and Mark T. Gladwin syndrome has been identified in sickle cell disease, thalassemia, and other hemolytic anemias.

Hemolysis-associated Vascular Dysfunction: A Unique State of NO Resistance in Hemolytic Diseases NO is a free radical molecule produced in endothelium by the endothelial NO synthase enzyme, via the oxygen-dependent five-electron oxidation of L-arginine to citrulline.104–106 Once produced, NO diffuses as a paracrinesignaling molecule to adjacent smooth muscle where it binds avidly to the heme moiety of soluble guanylate cyclase. This activates the enzyme which in turn converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), activating cGMP-dependent protein kinases, which ultimately leads to Ca2+ sequestration and relaxation of the perivascular smooth muscle to produce vasodilation (Chapter 10). NO-dependent vasodilation can be stimulated by shear stress and direct activation of muscarinic receptors by agonists such as acetylcholine. Vascular NO production is also tonic and controls approximately 25% of our resting blood flow.107,108 This steady-state vascular NO flux promotes general vascular homeostasis and health. NO tonically down-regulates transcription of endothelial adhesion molecules such as VCAM-1, ICAM-1, P-selectin and E-selectin109 and inhibits platelet activation, tissue factor expression and thrombin generation.110 NO modulates the expression of endothelin receptors (promoting a vasodilator effect by increasing endothelial endothelin receptor B expression) and decreases expression of endothelin-1, a potent mitogen and vasoconstrictor.111,112 Finally, NO reacts in a nearly diffusion limited reaction with the superoxide radical, critically modulating vascular redox balance. All these pathways, normally inhibited by NO, are pathologically activated in sickle cell disease and perhaps other hemolytic anemias. The concentration of NO available for the activation of soluble guanylate cyclase depends on the rate of production from endothelial NO synthase and the extent of scavenging reactions with superoxide and hemoglobin (or other high affinity heme-globins such as myoglobin).113 NO reacts with oxy- and deoxy-hemoglobin at the near diffusion limit (107 Ms) to produce methemoglobin and nitrate or iron-nitrosyl-hemoglobin, respectively (Equations 11.1 and 11.2).114,115 NO will react even faster with superoxide (at the diffusion limit, 109–10 Ms), formed by the enzymes xanthine oxidase, NADPH oxidase, uncoupled NO synthase, as well as by hemoglobin autooxidation (HbFe+2 -O2 → HbFe+3 + O2 .− ), to form peroxynitrite (Equation 11.3).106 Although the reaction of NO with superoxide is approximately 100 times faster than that with hemoglobin, the concentration of hemoglobin in the plasma of patients with sickle cell disease (approximate mean concentrations of 4 ␮M116 ) is approximately more than 100 times that of superoxide, suggesting that

Mechanisms and Clinical Complications of Hemolysis in Sickle Cell Disease and Thalassemia both pathways have the potential to limit NO bioavailability in vivo. NO + HbFe+2 -O2 [oxyhemoglobin] → HbFe+3 [methemoglobin] + NO3 − [nitrate]

(11.1)

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(11.3)

Because mammals do not possess nitrate reductase enzymes and the off-rate of NO from iron-nitrosylhemoglobin is so slow, these three reactions represent irreversible scavenging reactions. Indeed, the half-life of NO in a free (i.e., not encapsulated by a red cell membrane) solution of 10 mM oxyhemoglobin (the concentration of hemoglobin in whole blood) is estimated to be 1 ␮s and this NO could only diffuse 1 ␮m.117 Based on the kinetics of these reactions (nearly diffusion limited) and the concentration of intravascular hemoglobin in red blood cells (10 mM concentration in heme in whole blood), theoretical calculations suggest that the diffusion radius of NO from endothelium would be severely limited.117 This effect is only slightly diminished by the fact that the smooth muscle cells are on one side of the endothelium and the blood is on the other.105 Because the net flux of NO is always defined by the three dimensional spatial gradient in its concentration, the presence of hemoglobin on one side of the endothelium decreases the concentration on the other side. Conceptually, one can appreciate that NO diffusion is random from its source of production, so that a particular NO molecule can diffuse luminally and then back abluminally; if a trap is present on one side, any molecule that diffuses in that direction is eliminated and the concentration of NO on the other side will thus diminish. Indeed, recent modeling studies reveal that as little as one micromolar cellfree intraluminal hemoglobin in plasma will dramatically reduce NO concentrations that reach smooth muscle.118 This effect is magnified in hemolytic anemia, where the total level of NO scavenging by the red cells is reduced as the red cell mass decreases, so that the relative contribution of NO scavenging by the plasma hemoglobin compartment increases.118 These chemical reactions create a paradox in vascular biology: How can NO be the endothelium derived relaxing factor if the massive concentrations of intravascular hemoglobin should scavenge it and limit its ability to diffuse from endothelium to smooth muscle? This paradox has been largely solved by the understanding that there are major diffusional barriers for NO between the source of production, endothelial NO synthases, and hemoglobin in the red blood cell (Fig. 11.4).113 There exist several major diffusional barriers for NO in the unstirred layer around the erythrocyte,119,120 in the cell free zone that forms along the endothelium in laminar flowing blood,121–123 and possibly

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in the red cell submembrane, formed from the protein lattice of actin, spectrin, methemoglobin, hemichromes, band-3, and other components of the inner membrane scaffolding.124–126 These three major diffusional barriers reduce the reaction of NO with intracellular hemoglobin by approximately 300–1,000 fold and allow sufficient NO diffusion for paracrine signaling from endothelium to smooth muscle. For example, with a cell-free zone of 5 micrometers, the lifetime of NO would increase from 1 microsecond to approximately 7.5 ms before it reached the red cell rich zone and would be scavenged; thus the lifetime of NO increases by a factor of almost 10,000 in this situation.117 Understanding this balance between NO production, diffusion to smooth muscle, and scavenging reactions with intracellular hemoglobin helps explain the clear toxicity observed in the clinical development of the stroma-free hemoglobin-based blood substitutes (recently reviewed110 and illustrated in Figure 11.4). The infusion of cell free hemoglobin solutions into normal volunteers and patients immediately disrupts the NO diffusion barriers and produces dose-dependent vasoconstriction (systemic and pulmonary hypertension),127–136 smooth muscle dystonias (gastroparesis, esophageal spasm and abdominal and chest pain),127,128,130,133,137 platelet activation,138–141 and death.142,143 The toxicity of cell free hemoglobin solutions has resulted in serious morbidity (myocardial infarction) and excess mortality in most clinical trials of blood substitutes in at-risk patients.110 In sickle cell disease, thalassemia, malaria and other acquired, iatrogenic, infectious and hereditary hemolytic conditions, intravascular hemolysis similarly disrupts the NO diffusional barriers created by the red cell membrane that limit NO reactions with hemoglobin, and the cell-free plasma hemoglobin destroys NO at a rate 1,000-fold faster than intraerythrocytic hemoglobin.110,113,116,144 As a result of hemolysis, hemoglobin is released into plasma where it reacts with and destroys NO, resulting in abnormally high rates of NO consumption and produces a state of resistance to NO bioactivity. Consequently, smooth muscle guanylyl cyclase is not activated and vasodilation is impaired. In support of this mechanism, plasma from patients with sickle cell disease contains cell-free ferrous oxyhemoglobin, which stoichiometrically consumes micromolar quantities of NO and abrogates forearm blood flow responses to NO donor infusions.116 This NO resistance syndrome is a unique form of vascular dysfunction. In coronary artery disease and its risk factors, diabetes, obesity, hypertension, smoking, increasing age, and hyperlipidemia, NO production is reduced. This is clinically characterized by the demonstration that infusions of the competitive NO synthase inhibitor, L-NMMA, into the brachial artery or coronary arteries exhibits a blunted vasoconstrictor response, suggesting that tonic NO synthase activity is impaired.107,145 However, in these patients with the metabolic syndrome, the infusion of sodium

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Smooth muscle cells Endothelial cells

Blood vessel

Figure 11.4. Role of diffusion barriers in intact red cells and plasma cell–free hemoglobin. Intact red cells carry a reservoir of NO in the form of nitrite. Nitrite can be reduced by deoxyhemoglobin to NO, although it remains to be determined how NO escapes from the erythrocyte. Xanthine oxidoreductase may also have the potential to perform this reaction. S-nitrosohemoglobin has also been proposed as another erythrocyte storage form of NO. NO is produced principally by NOS from arginine and binds to soluble guanylyl cyclase in vascular smooth muscle cells, producing tonic vasodilation. Diffusion barriers to consumption of NO by intact erythrocyte hemoglobin are provided by the cell-free zone along the endothelium associated with laminar flow of blood, and by the unstirred layer surrounding the red cell. (Reproduced with permission from ref. 113.)

nitroprusside or nitroglycerin, NO donor medications, produces normal vasodilation effects and are used as controls to show that the vessels are capable of vasodilation. In striking contrast, in patients with sickle cell disease with high hemolytic rates and high plasma hemoglobin levels, the vasodilatory effect to L-NMMA and the NO donor sodium nitroprusside are both blunted. This unique state of resistance to NO-dependent vasodilation146 has been recapitulated in numerous mouse and human studies (Fig. 11.5).

r In patients with sickle cell disease and high plasma hemoglobin concentrations the blood flow responses to infusions of the NO synthase inhibitor L-NMMA are blunted and blood flow responses to the NO donor sodium nitroprusside are nearly abolished.116,147 This effect appears to be more pronounced in males, consistent with estrogenic effects on NO synthase expression and activity.

r Endothelium-dependent, NO-dependent blood flow is impaired in patients with sickle cell disease, when measured by flow-mediated vasodilation. The responses to the exogenous NO donor, nitroglycerin are impaired, compared with control subjects with non-hemolytic anemia.148 r A similar state of resistance to exogenous NO (the NO donor NONOate or sodium nitroprusside) in different transgenic mouse models of sickle cell disease has also been described.149,150 NO resistance was highly correlated with plasma hemoglobin levels suggesting that NO resistance in this model was linked to hemolytic rate and oxidant stress.151 r NO is inhibited in the vasculature of transgenic sickle cell mice with sickle cell disease by a diffusion-limited reaction with superoxide produced from xanthine oxidase on endothelium.12,152 Increased xanthine oxidase expression in the lung of the transgenic mouse has also been reported to scavenge NO in this vascular system.153

Mechanisms and Clinical Complications of Hemolysis in Sickle Cell Disease and Thalassemia

209

metHb Intravascular Hemolysis

Nonhemorrhagic Stroke

Hb

A

NO3-

NO

ONOO-

LDH

Pulmonary Hypertension

Nitric Oxide Synthase L-Arginine

LDH Marker

B

Decreased NO Bioactivity

C

L-Citrulline

Arginase

Priapism

O2-

Ornithine

Xanthine Oxidase

XO

Leg ulceration NADPH OX

NADPH Oxidase Figure 11.5. Intravascular hemolysis and decreased NO bioactivity. Arginine is converted by NOS to NO plus citrulline. Intravascular hemolysis releases cell-free hemoglobin into blood plasma, where it stoichiometrically can react with NO (reaction A), producing methemoglobin and inert nitrate. In addition, erythrocyte arginase released into plasma catabolizes plasma arginine (reaction B), reducing its availability to NOS. In reactions not directly related to hemolysis, increased xanthine oxidase and NADPH oxidase activities found in sickle cell disease produce oxygen radicals, which can react with and further deplete NO, producing highly oxidative peroxynitrites (reaction C). Consequent decreased NO bioactivity contributes to the clinical risk of pulmonary hypertension, nonhemorrhagic stroke, priapism, and leg ulceration. (Reproduced with permission of the publisher from ref. 183.)

Recent studies have suggested a role for vascular NADPH oxidase in enhanced superoxide mediated NO scavenging in the sickle cell cerebral vasculature.154 r Sickle cell transgenic mice develop spontaneous pulmonary hypertension associated with a global impairment in both the production of NO (from uncoupled eNOS) and from NO inactivation by plasma hemoglobin and superoxide.155 Both pulmonary and systemic impairment in the vasodilatory responses to inhaled NO, sodium nitroprusside, NONOates and even phosphodiesterase-5 inhibitors were observed. A similar state of NO resistance developed in an alloimmunized intravascular hemolysis mouse model.156 r In a canine model of acute intravascular hypotonic water hemolysis,157 pulmonary and systemic vasoconstriction was associated with the degree of hemoglobinemia and the development of resistance to the NO donor sodium nitroprusside. Similar effects of hemolysis on NO bioavailability and endothelial function have been considered in paroxysmal nocturnal hemoglobinuria,110 in primate models of thrombotic thrombocytopenic purpura,158 and have recently been described in animal models of malaria.159,160 In the latter malaria studies, the degree of hemoglobinemia, which reduced systemic NO bioavailability, was even more closely linked to risk of death than the severity of parasitemia.

Arginasemia Limitations on NOS Substrate Bioavailability In addition to release of hemoglobin from the red cell into plasma, hemolysis releases erythrocyte arginase, which converts L-arginine, the substrate for NO synthesis, to ornithine (Figs. 11.4 and 11.5).161–163 Arginase activities in the plasma of patients correlated significantly with cell-free plasma hemoglobin and was increased in the plasma and red cells of patients with sickle cell disease (Fig. 11.6). Consistent with this observation, the arginine:ornithine ratio decreased significantly as plasma arginase activity rose. Low arginine:ornithine ratios were found in patients with sickle cell disease and comorbid pulmonary hypertension and were associated with increasing mortality.163,164

Hemolysis, Coagulation and the Spleen Intravascular hemolysis has the potential to drive a procoagulant state. Platelet activation is profoundly inhibited by NO and such NO-dependent inhibition may in turn be blocked by plasma hemoglobin-mediated NO scavenging.139,141,165,166 Activation of platelets in sickle cell disease correlated with pulmonary artery pressures and indices of hemolysis. In vitro experiments suggested that cell-free hemoglobin could directly activate platelets and inhibit the modulatory effects of NO on platelet activation.141 High hemolytic rate, reflected by reticulocytosis, was also associated with hemoglobin desaturation (ventilation/perfusion inhomogeneity) and adhesion molecule

210

Gregory J. Kato and Mark T. Gladwin Sources of Plasma L-Arginine Endogenous Synthesis in Kidney From Citrulline Protein Turnover Diet

CELLULAR COMPARTMENT

VASCULAR COMPARTMENT Competes With L-Arginine for Cellular Uptake Increased Ornithine Synthesis Increased L-Ornithine

Decreased L-Arginine Available for Cellular Uptake Decreased Plasma L-Arginine

Plasma L-Arginine

L-Arginine

Urea

Release of RBC Arginase Plasma

Arginine

O2

Hemolysis Decreased NO Synthesis

Cell-free Hemoglobin L-Ornithine

Polyamines

LUNGS

NOS

L-Citrulline

NO Scavenging

Proline

Uncoupled Reaction

NO

Decreased NO

Superoxide Peroxynitrite

Smooth Muscle Proliferation

Collagen Production and Deposition

Airway Remodeling

Pulmonary Hypertension Figure 11.6. Alterations in arginine metabolism in sickle cell disease and thalassemia. Plasma L-arginine is derived from synthesis in the kidney, from protein turnover, and from dietary sources. Intravascular hemolysis results in ectopic localization of erythrocyte arginase into blood plasma, where it can convert L-arginine into L-ornithine, reducing the availability of L-arginine as a substrate for NO synthesis. Under such conditions of limiting substrate, the subunits of NO synthase can become uncoupled, producing superoxides that can react with NO to form peroxynitrites. Excess production of ornithine has been proposed to provide substrate for production of polyamines and proline. Polyamines can fuel DNA production and cell proliferation, and proline can stimulate collagen production. These processes are proposed along with decreased NO bioavailability to contribute to pulmonary hypertension in patients with chronic intravascular hemolysis. (Reproduced with permission of the publisher from ref. 163.)

expression;167,168 it is possible that such a hypoxic state can induce hypoxia-inducing factor-1 (HIF-1) dependent factors such as erythropoietin, vascular endothelial growth factor (VEGF), and endothelin-1. Splenectomy has been reported to be a risk factor for the development of pulmonary hypertension, particularly in patients with hemolytic disorders.169–173 Perhaps the loss of splenic function increases the circulation of platelet derived mediators and that senescent and abnormal erythrocytes in the circulation trigger platelet activation, promoting pulmonary microthrombosis and red cell adhesion to the endothelium.169 A role for intensification of intravascular

hemolysis by splenectomy has also been suggested by the demonstration of significantly higher plasma hemoglobin and erythrocyte-derived microvesicles levels patients with ␤-thalassemia intermedia who have undergone splenectomy, compared with those who have not.174 It is likely that splenic reticuloendothelial cells subserve a critical function in the removal of senescent and damaged erythrocytes and that following surgical or autosplenectomy, the rate of intravascular hemolysis increases, resulting in increased plasma hemoglobin and NO scavenging, and increased circulating red cells with phosphatidylserine exposed on their membranes. Consistent with such a mechanism, the

Mechanisms and Clinical Complications of Hemolysis in Sickle Cell Disease and Thalassemia

211

Spectrum of Sickle Cell Complications Figure 11.7. Spectrum of sickle cell subphenotypes affected by hemolytic rate. The viscosity-vasoocclusion subphenotype is associated with a lower hemolytic rate, marked by a higher hemoglobin level, and low plasma hemoglobin, lactate dehydrogenase, bilirubin, and arginase levels. Patients with these features have a higher incidence of vasoocclusive pain crisis, the acute chest syndrome, and osteonecrosis. In contrast, patients with the hemolysis-endothelial dysfunction subphenotype exhibit markers of high hemolytic rate, including low hemoglobin level, high plasma hemoglobin, LDH, bilirubin, and arginase, culminating in low NO bioavailability and high prevalence of pulmonary hypertension, leg ulceration, priapism, and stroke. Coinheritance of ␣ thalassemia trait with sickle cell disease reduces the hemolytic rate, reducing the risk of hemolysis-associated complications and increasing the risk of viscosity-related complications. (Adapted with permission from ref. 183.)

HemolysisEndothelial Dysfunction

Viscosity-Vasoocclusion

Higher Hemolytic Rate

Lower Hemolytic Rate e

Higher plasma hemoglobin & arginase Higher reticulocyte count Higher serum LDH High bilirubin

Pulmonary Hypertension Leg ulceration Priapism Stroke?

Higher hemoglobin Higher plasma arginine Higher nitric oxide bioactivity

Osteonecrosis Acute Chest Syndrome Vasoocclusive Pain Crisis

α-thalassemia trait shifts risk

experimental intravenous injection of hemolysate promotes the formation of platelet-rich thrombi in the pulmonary vascular bed of rabbits after ligation of the splenic artery, without any thrombus formation in the animals without splenic artery ligation.175,176

HEMOLYTIC ANEMIA-ASSOCIATED CLINICAL SUBPHENOTYPES Recent epidemiological reexamination of the clinical complications of sickle cell disease suggests that the clinical manifestations of sickle cell disease may fall into two partially overlapping subphenotypes (Fig. 11.7). The first subphenotype encompasses the more classic manifestations of the disease: vasoocclusive crisis, acute chest syndrome and osteonecrosis. These morbidities are epidemiologically associated with high steady-state white blood cell counts, high steady-state hemoglobin levels and low HbF concentrations.177 These complications are largely mediated by microvascular obstruction by sickle erythrocytes and the pathogenesis characterized by ischemia-reperfusion injury, adhesion, infarction and inflammation.178,179 The second subphenotype encompasses clinical complications shared by other hemolytic anemias and includes pulmonary arterial hypertension, systemic systolic arterial hypertension, cutaneous leg ulceration, priapism, sudden death and possibly stroke.116,164,180–182 Consistent with this formulation, coinheritance of ␣ thalassemia, which reduces hemolytic rate in sickle cell disease, reduces the risk of leg ulceration, priapism and stroke, and increases the risk of vasoocclusive pain crisis, acute chest syndrome, and osteonecrosis as hemolysis is reduced and blood viscosity increases.183 Pulmonary hypertension is an increasingly recognized complication of chronic hereditary and acquired

hemolytic anemias, including sickle cell disease,164,184–192 ␤ thalassemia (in particular ␤-thalassemia intermedia and inadequately transfused and chelated patients with ␤-thalassemia major),169,193–201 paroxysmal nocturnal hemoglobinuria,202–204 hereditary spherocytosis and stomatocytosis,205–211 microangiopathic hemolytic anemias,212,212–218 pyruvate kinase deficiency,219 red cell alloimmune–mediated hemolytic anemia,220 unstable hemoglobin variants,221 and possibly malaria.222–224 Additionally, certain conditions are associated with both intravascular hemolysis and risk of pulmonary hypertension, such as schistosomiasis,225,226 iatrogenic hemolysis from mechanical heart valves,227,228 left ventricular assist devices, and cardiopulmonary bypass procedures.93,229–232 These studies are consistent with growing appreciation for a distinct syndrome of hemolysis-associated pulmonary hypertension.

Priapism and Hemolytic Anemia Priapism has been reported in patients with sickle cell disease, ␤-thalassemia intermedia, red cell enzymopathy, unstable hemoglobin disorders, and other hemolytic anemias.233–241 In one study, patients with a history of priapism had evidence of increased hemolytic rate and were five-fold more likely to have pulmonary hypertension, supporting a mechanistic and epidemiological link between these complications.164 Further analysis of this cohort based on stratum of hemolysis defined by relative levels of LDH confirmed an association between the intensity of hemolysis and the prevalence of priapism, cutaneous leg ulceration, and pulmonary hypertension.181 In a case-control analysis of data from the Comprehensive Study of Sickle Cell Disease (CSSCD), priapism was associated with laboratory markers of high hemolytic rate and

212

Gregory J. Kato and Mark T. Gladwin Table 11.1. Laboratory characteristics of cases with priapism and controls in a population of patients with sickle cell disease

Age at last follow-up, y ± SD Hemoglobin, g/dL HbF, g/dL Bilirubin, mg/dL Urea nitrogen (BUN), mg/dL Mean corpuscular volume, ␮m3 LDH, U/L Reticulocytes AST, U/L ALT, U/L WBC count, × 109 /L

Case subjects n = 273

Control subjects n = 979

26.2 ± 12.28 8.64 ± 0.13 0.44 ± 0.04 3.52 ± 0.13 9.49 ± 0.39 89.82 ± 0.48 526.19 ± 13.08 11.67 ± 0.35 50.34 ± 1.44 36.70 ± 2.96 11.62 ± 0.20

22.8 ± 12.72 9.51 ± 0.07 0.50 ± 0.02 2.92 ± 0.07 10.09 ± 0.20 87.18 ± 0.25 459.23 ± 6.92 9.37 ± 0.18 45.78 ± 0.76 35.18 ± 1.55 10.18 ± 0.10

P .001 1%–2%) in the peripheral blood on routine hemoglobin electrophoresis (Fig. 14.7) are said to have HbH disease. HbH inclusions are always detectable in the peripheral blood of such individuals (Fig. 14.5). Not surprisingly, patients defined in this ad hoc way span a wide range of clinical and hematological phenotypes. The majority are clinically well and in these the epithet HbH “disease” may be inappropriate. Some have thalassemia intermedia. The most severe forms of HbH disease may be lethal late in gestation or in the perinatal period, causing a condition referred to as HbH hydrops fetalis (see later). Extensive surveys have demonstrated that most cases of HbH disease occur in patients from southeast Asia, the Mediterranean basin, and the middle East (see Chapter 26).

Douglas R. Higgs This geographical distribution is easily explained now that we understand the molecular basis of this disorder (Table 14.1). HbH disease most commonly results from the interaction of ␣0 and ␣+ thalassemia. Although ␣ thalassemia is common throughout all tropical and subtropical regions, ␣0 determinants (and hence HbH disease) are predominantly found in the Mediterranean and southeast Asia. In southeast Asia the most common genotype associated with HbH disease is --SEA /-␣ whereas in the Mediterranean --MED /-␣ and -(␣)20.5 /-␣ are the most frequent (reviewed in Chui et al.1 ). Less often HbH disease results from the interaction of ␣0 thalassemia with nondeletional forms of ␣+ thalassemia (genotype --/␣T ␣) or in homozygotes for some nondeletional forms of ␣+ thalassemia (genotype ␣T ␣/␣T ␣).51,60–67 Again these molecular interactions are most frequently seen in southeast Asia and the Mediterranean but also occur at high frequencies in some areas of the middle East (see Chapter 26). Despite these useful geographical “rules of thumb,” one should be aware that patients with ␣ thalassemia trait and HbH disease have been described in almost every racial group. On detailed examination, patients originating from regions where ␣ thalassemia is otherwise rare are often found to have unusual and biologically interesting molecular defects.

The Pathophysiology of HbH Disease In HbH disease there is a moderately severe reduction in ␣ globin RNA and ␣ globin chain synthesis (see later). During fetal life excess ␥ chains form ␥ 4 tetramers (Hb Bart’s). Similarly, in adults (after the ␥ to ␤ switch, Chapter 5) excess ␤-globin chains form ␤4 tetramers (HbH). Both of these homotetramers have high oxygen affinity, lack heme–heme interaction, and do not exhibit any Bohr shift.68 Therefore, neither of these hemoglobins contributes to oxygen transport and their presence compounds the effects of anemia in patients with HbH disease. The reduced synthesis of HbA together with the production of nonfunctional Hb thus cause anemia and provoke an appropriate response of an increased level of erythropoietin.62,69 A second component of the pathophysiology arises from the fact that HbH is unstable and when oxidized forms intracellular precipitates, which cause cell death in a proportion of erythroblasts leading to ineffective erythropoiesis. This is reflected in an increase in the level of serum transferrin receptors (sTfR) reflecting the increased number of erythroid precursors in the bone marrow.62,69 Thirdly, and most importantly, when HbH precipitates it attaches to the cell membrane in circulating red cells as they age. This in turn causes local oxidative damage and membrane dysfunction. Thus the erythrocytes in HbH disease are rigid and their membrane is more stable than normal.70,71 Loss of normal membrane phospholipid asymmetry, exposure of phosphatidylserine, and the presence of increased amounts of Immunoglobulin G on the cell surface may

The Pathophysiology and Clinical Features of ␣ Thalassemia also enhance the engulfment of abnormal, ageing red cells by macrophages. Together these properties are thought to slow the passage of red cells through the microvasculature and promote erythrophagocytosis causing extravascular hemolysis, which is reflected in an increase in the reticulocyte count.62,69,72,73 The red cell survival, as judged by 51 Cr studies is reduced in patients with HbH disease; reported figures range from 8–17 days.74–77 External scanning indicates that most of the red cell destruction occurs in the spleen.76,77 Srichaikul et al.78 performed full erythrokinetic studies on nine, nonsplenectomized patients with HbH disease. They demonstrated a reduced red cell volume, increased plasma volume, and a reduced red cell survival of 6–19.5 days (normal range 25–32 days) with sequestration of 51 Cr-labeled red cells in the liver and spleen. In addition they showed that patients with HbH disease have a rapid clearance of 59 Fe with relatively good 59 Fe incorporation into red cells compared with patients with ␤ thalassemia. They also found that the patient’s hematocrit was correlated to the red cell survival. Together these findings suggest that both hemolysis and ineffective erythropoiesis contribute to anemia in HbH disease but most studies have concluded that the predominant mechanism is extravascular hemolysis.

␣/␤ mRNA and Globin Synthesis Ratios in Patients with HbH Disease As one would expect from the studies of ␣/␤ mRNA ratios in carriers of ␣ thalassemia (see previously), the red cell precursors of patients with HbH disease contain approximately one half to one quarter of the amount of ␣globin mRNA present in normal red cell precursors11,16,19,21 as demonstrated in Figure 14.1. Again this is generally reflected in the ␣/␤-globin chain synthesis ratios of patients with the deletional forms of HbH disease (average of 0.44, standard deviation 0.2, see Table 14.2 and Fig. 14.2. Also see Kanavakis et al.62 ). Excess ␤-globin chains synthesized during erythroid maturation mainly form ␤4 tetramers but in addition supply a small intracellular pool of ␤-chains that combine with newly synthesized ␣ chains as they become available. Nevertheless, it is clear that HbH is not present in the peripheral blood in amounts reflecting the rate at which it is synthesized indicating that it must be lost from the red cells while they are in the circulation consistent with the pathophysiology set out previously.

Red Cell Indices and Hematological Findings in HbH Disease As before, we will first consider the effect of ␣-globin deletions on red cell indices, comparing individuals with one functional ␣ gene (--/-␣) with normal individuals (␣␣/␣␣). Those who inherit only a single ␣ gene have lower levels of total hemoglobin, MCH and MCV but higher RBC counts than nonthalassemic (␣␣/␣␣) individuals (Table 14.2 and

277 Fig. 14.3). Similar trends have been shown by others.1,62,73 These differences in hematological indices are seen at all stages of development (see Table 14.2) although at present there are only anecdotal data on infants with HbH disease in the perinatal period or during the early months of life.73,79 Perhaps the most important hematological finding is that, using data accumulated from a variety of surveys (see Table 14.2) patients with HbH disease are anemic with, on average, approximately 2–g/dL less hemoglobin than age and sex matched normal individuals. It has been noted in some surveys that there may be striking fluctuations in the level of hemoglobin measured sequentially in the same individual over the course of 1–2 years,34,80 although in our experience, and that of others73 this is not common. The peripheral blood film shows hypochromia and polychromasia with variable anisopoikilocytosis and target cells (Fig. 14.5). The reticulocyte count is usually raised to approximately 3%–6%, although higher counts may be observed.72,73,81 Nucleated red cells and basophilic stippling may be present34 but in our more limited experience this is quite rare. Although bone marrow examination is rarely necessary in the investigation of patients with HbH disease, when analyzed it shows erythroid hyperplasia with only slight or absent deposition of hemosiderin.34 The erythroid hyperplasia is reflected increased levels of sTfR in the peripheral blood.62,69 Over the past 10–15 years the precise genotype of many patients with HbH disease has been established. In addition to the common deletional forms (--/-␣) discussed previously, HbH disease may also result from interactions involving nondeletional determinants (--/␣T ␣ and ␣T ␣/␣T ␣). Using data from several studies (Table 14.2 and Fig. 14.3), patients with nondeletional HbH disease and the ␣T ␣/␣T ␣ genotype have hematological indices that are similar to those with the deletional type of HbH disease (--/-␣), whereas those with the --/␣T ␣ are slightly more anemic with lower RBC counts and higher MCVs (Table 14.2 and refs. 1, 72, 73, 82). Limited data (Table 14.2) indicate that these differences are present throughout development. Several studies have compared the hematological findings in patients with deletional forms of HbH disease (--/-␣) and those with specific nondeletional defects including [--/␣cs ␣] (HbH-Constant Spring),72,79,83 [--/␣Nco ␣] and [--/␣Hph ␣]61,73,84 and anecdotally many other rarer mutations (reviewed in refs. 1, 73). In all of these nondeletional genotypes one finds lower levels of hemoglobin and RBC counts but higher MCVs than in patients with the pure deletional types of HbH disease.

Hemoglobin Analysis in HbH Disease Infants who go on to develop HbH disease later in life produce large amounts (19%–27%) of Hb Bart’s (␥ 4) at birth.34,85,86 During the first few months of development Hb Bart’s falls and is replaced by variable amounts of HbH

278 in adult life. The level of Hb Bart’s at birth often exceeds that of HbH in adult life. This is consistent with other observations87 showing that HbH (␤4 ) is less stable than Hb Bart’s (␥ 4). Adults with HbH disease have 0.8–40% HbH in the peripheral blood. It has been consistently noted that patients with the nondeletional type of HbH disease (--/␣T ␣) produce larger amounts of HbH.1,61,62,72,73,88,89 Hb Bart’s may still be detected in some adults with HbH disease but HbH usually predominates; occasionally the fetal pattern, with an excess of Hb Bart’s, persists.90 The reasons why some patients with HbH disease produce significant amounts of Hb Bart’s are not clear. It is possible that some have co-inherited ␤-globin clusters with point mutations that are associated with increased ␥ globin synthesis (see ref. 91 and Chapters 5 and 16). HbH and Hb Bart’s are easily detected as fast migrating bands on hemoglobin electrophoresis (Fig. 14.7). In addition HbH can be precipitated from peripheral blood red cells after incubation for 3 hours at room temperature (see ref. 43 for details). These characteristic inclusions are artefacts produced by the redox action of the dye (Fig. 14.5). The proportion of cells containing HbH inclusions is directly related to the level of HbH detected in the peripheral blood. Again patients with nondeletional ␣ thalassemia have a higher proportion of HbH cells than those with the deletional types of HbH disease.92 Even after prolonged incubation it is unusual to find inclusions in every cell; the reason for this heterogeneity is not clear. Splenectomized patients have large numbers of preformed inclusions in the red cells that can be detected by methyl violet staining. Other minor changes in the hemoglobin composition found in patients with HbH disease include a tendency to low levels of HbA2 (1%–2%) probably due to the lower affinity of ␣-chains for ␦- than ␤-chains; when the supply of ␣ chains is limited less HbA2 is formed (Chapter 7). In addition, variant chains creating abnormal hemoglobins may be detected in patients with chain termination mutants (for example see ref. 93) and some unstable mutants associated with ␣ thalassemia (e.g., Hb QuongSze and Hb Agrinio, see Chapter 13). Finally some ␣ chain variants, such as Hb J Tongariki and Hb G Philadelphia may be linked in cis to ␣ thalassemia variants (see Table 13.2 in Chapter 13).

Clinical Features of HbH Disease Although HbH disease is quite a common genetic disorder in the Mediterranean, Middle East, and southeast Asia, there have been relatively few systematic studies addressing the natural history of this condition or the relationship between genotype and phenotype. Most physicians caring for such patients agree that there is a remarkably wide clinical spectrum but often comment on the mild nature of this condition. Even from the biased perspective of hospitalbased studies, the majority of patients with HbH disease appear to have little disability.1,9,61,62,72,73,94–97 However, it has emerged over the past 10 years or so that a minority of

Douglas R. Higgs patients with HbH disease may be severely affected, requiring regular blood transfusion and rare cases may present as hydropic, newborn infants (see later). The largest clinical experience of HbH disease, including data from 500 adults and 502 children, was summarized over 30 years ago by Wasi et al.34 More recent studies have reviewed relatively large numbers patients whose molecular defects have been accurately defined,1,62,72,73,79,82,98 allowing us to make some predictions about the severity of HbH disease based on genotype. The following discussion is largely based on these reviews.

Presentation At birth, infants destined to develop HbH disease may have near-normal levels of hemoglobin with no hepatosplenomegaly,34 whereas other newborn infants may already show evidence of hemolytic anemia.73,79 In many infants, the clinical features of HbH disease (see later) develop in the first year of life. The age at which patients with HbH disease first present varies from birth to older than 70 years and in more than half of the patients the finding of HbH disease is incidental (e.g., associated with health checks or prenatal screening) or found during investigation for an unrelated illness. Some patients may first present at the time of an acute fall in the level of Hb,62,72 as will be discussed. Anecdotally, survival of patients with HbH disease into adult life appears to be the rule but there are no actuarial data to quantify this assertion.

Episodes of Severe Anemia The level of Hb in most patients appears to be relatively stable and above approximately 8 g/dL; however, the Hb level may fall (2–3 g/dL) quite dramatically,34 causing episodes of profound weakness and pallor requiring hospital admission and blood transfusion. The cause of such events is not always understood; they may recur and may vary from one environment to another. They are often thought to arise from increased hemolysis associated with pregnancy intercurrent infection/pyrexia or administration of oxidant drugs such as sulphonamides76 or transient aplasia due to B19 parvovirus infection.73 Such events may also occur in patients with hypersplenism.

Blood Transfusion Blood transfusion is often used in the management of patients who have an acute fall in the level of Hb (see previous discussion). In many studies up to 50% of patients with HbH disease have had a few transfusions during such episodes. In general, those with the lowest steady state levels of Hb (with nondeletional HbH disease) more frequently require such transfusions. It is unusual for patients with HbH disease to require regular blood transfusion and even in cases in which this has been thought necessary, it is not

The Pathophysiology and Clinical Features of ␣ Thalassemia always clear what criteria have been used to make such a decision. Nevertheless, nearly all such examples occur in patients with nondeletional types of HbH disease.

Hepatosplenomegaly and Jaundice In addition to pallor and jaundice most patients with HbH disease have enlarged livers and spleens, although clinically significant hepatomegaly is unusual in patients with uncomplicated HbH disease unless they have iron loading. Liver enlargement and spleen enlargement are both more common in patients with nondeletional types of HbH disease. Hypersplenism, which can significantly aggravate the anemia and lead to reduced platelet and white blood cell counts, occurs in approximately 10% of patients;34 splenectomy may be of benefit to such patients with persistent anemia. Severe liver disease has occasionally been reported in patients with HbH disease but it is not clear that this was directly attributable to thalassemia. Gallstones are quite frequent (up to ∼40%) in patients with HbH disease their frequency increasing with age and possibly modified by the co-inheritance of predisposing alleles of the uridine diphosphate glucuronosyl transferase locus.73,99 Complications of gallstones appear relatively infrequent; for example, in 95 patients followed by Piankijagum et al.80 for 2 years, there were four episodes of cholecystitis.

Growth and Bone Changes Approximately one third of patients with HbH disease were said to have bone changes associated with thalassemia.86 In general these are mild but may affect the facial features. In one study from Thailand 17% of children with deletional HbH disease and almost half of patients with HbH/CS disease had thalassemic facies.79 In the latter group half of the patients had moderately severe changes with maxillary overgrowth. Approximately 13% of children with HbH disease in Hong Kong and Sardinia had growth rates below the third percentile.72,73 A study from Thailand found more than half of children with HbH disease had growth impairment.79 Clinically significant extramedullary hemopoiesis rarely occurs in HbH disease.9,34,100

Hemoglobin H Disease and Pregnancy The normal physiological changes associated with pregnancy are even more challenging in patients with HbH disease than those with ␣ thalassemia trait (see previous discussion). In patients with HbH disease, there is usually an increasing severity of anemia and the level of Hb may fall to approximately 6.0 g/dL1,73,101 or even less.34,102 Some patients with the most severe anemias may also be iron deficient.34 In patients with severe anemia (60% –

Hb Bart’s %

Hb Portland %

Reference [119] [119] [240]

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The Pathophysiology and Clinical Features of ␣ Thalassemia 214. Eddleman K. In utero transfusion and transplantation in ␣thalassaemia. In: Migliaccio, AR ed. Stem Cell Therapy of Inherited Disorders. Rome; 1996. 215. Paszty C, Mohandas N, Stevens ME, Loring JF, Liebhaber SA, Brion CM, et al. Lethal alpha-thalassaemia created by gene targeting in mice and its genetic rescue. Nat Genet. 1995;11:33–39. 216. Paszty C. Transgenic and gene knock-out mouse models of sickle cell anemia and the thalassemias. Curr Opin Hematol. 1997;4:88–93. 217. Huisman THJ, Carver MFH, Efremov GD. A Syllabus of Human Hemoglobin Variants. Augusta, GA: The Sickle Cell Anemia Foundation; 1996. 218. Bruzdzinski CJ, Sisco KL, Ferrucci SJ, Rucknagel DL. The occurrence of the alpha G-Philadelphia-globin allele on a double-locus chromosome. Am J Hum Genet. 1984;36:101– 109. 219. Molchanova TP, Pobedimskaya DD, Ye Z, Huisman TH. Two different mutations in codon 68 are observed in Hb GPhiladelphia heterozygotes. Am J Hematol. 1994;45:345–346. 220. Milner PF, Huisman TH. Studies of the proporation and synthesis of haemoblogin C Philadelphia in red cells of heterozygotes, a homozygote, and a heterozygote for both haemoglobin G and alpha thalassaemia. Br J Haematol. 1976;34:207–220. 221. Pardoll DM, Charache S, Hjelle BL, et al. Homozygous alpha thalassemia/Hb G Philadelphia. Hemoglobin. 1982;6:503– 515. 222. Sancar GB, Tatsis B, Cedeno MM, Rieder RF. Proportion of hemoglobin G Philadelphia (alpha 268 Asn leads to Lys beta 2) in heterozygotes is determined by alpha-globin gene deletions. Proc Natl Acad Sci USA. 1980;77:6874–6878. 223. Rieder RF, Woodbury DH, Rucknagel DL. The interaction of alpha-thalassaemia and haemoglobin G Philadelphia. Br J Haematol. 1976;32:159–165. 224. Schwartz E, Atwater J. alpha-thalassemia in the American negro. J Clin Invest. 1972;51:412–418. 225. Liebhaber SA, Rappaport EF, Cash FE, Ballas SK, Schwartz E, Surrey S. Hemoglobin I mutation encoded at both alphaglobin loci on the same chromosome: concerted evolution in the human genome. Science. 1984;226:1449–1451. 226. Bunn HF, McDonald MJ. Electrostatic interactions in the assembly of haemoglobin. Nature. 1983;306:498–500. 227. Bunn HF. Subunit assembly of hemoglobin: an important determinant of hematologic phenotype. Blood. 1987;69:1–6. 228. Whitten WJ, Rucknagel DL. The proportion of Hb A2 is higher in sickle cell trait than in normal homozygotes. Hemoglobin. 1981;5:371–378.

295 229. Stallings M, Abraham A, Abraham EC. a-thalassemia influences the levels of fetal hemoglobin components in new born infants. Blood. 1983;62:75a. 230. Rombos J, Voskaridou E, Vayenas C, Boussiou M, Papadakis M, Loukopoulos D. Hemoglobin H in association with the Greek type of HPFH. Paper presented at International Congress on Thalassemia. Sardinia, 1989. 231. Giordano PC, Harteveld CL, Michiels JJ, et al. Atypical HbH disease in a Surinamese patient resulting from a combination of the -SEA and -alpha 3.7 deletions with HbC heterozygosity. Br J Haematol. 1997;96:801–805. 232. Thonglairuam V, Winichagoon P, Fucharoen S, Wasi P. The molecular basis of AE-Bart’s disease. Hemoglobin. 1989;13: 117–124. 233. Matthay KK, Mentzer WC Jr, Dozy AM, Kan YW, Bainton DF. Modification of hemoglobin H disease by sickle trait. J Clin Invest. 1979;64:1024–1032. 234. Svasti S, Yodsowon B, Sriphanich R, et al. Association of Hb Hope [beta136(H14)Gly–>Asp] and Hb H disease. Hemoglobin. 2001;25:429–435. 235. Vichinsky E. Hemoglobin e syndromes. Hematology Am Soc Hematol Educ Program. 2007:79–83. 236. Su CW, Liang S, Liang R, Wen XJ, Tang CN. Hb H disease in association with the silent beta chain variant Hb Hamilton or alpha 2 beta 2(11)(A8)Val -- lie. Hemoglobin. 1992;16:403– 08 237. Rahbar S, Bunn HF. Association of hemoglobin H disease with Hb J-Iran (beta 77 His -- Asp): impact on subunit assembly. Blood. 1987;70:1790–1791. 238. Chan V, Chan TK, Tso SC, Todd D. Combination of three alpha-globin gene loci deletions and hemoglobin New York results in a severe hemoglobin H syndrome. Am J Hematol. 1987;24:301–306. 239. Wilkie AOM. The a thalassaemia/mental retardation syndromes: model systems for studying the genetic contribution to mental handicap. Doctor of Medicine, 1991, University of Oxford. 240. Dallman PR. The red cell. In: Dallman, PR ed. Blood and Blood-forming Tissues. New York: Appleton-Century-Crofts; 1977:1109–1113. 241. Dallman PR, Siimes MA. Percentile curves for hemoglobin and red cell volume in infancy and childhood. J Pediatr. 1979;94:26–31. 242. Lubin BH. Reference values in infancy and childhood. In: Nathan DG, OskiFA, eds. Hematology of Infancy and Childhood. Philadelphia: W.B. Saunders; 1987:1677–1697. 243. Llewellyn-Jones D. Obstetrics. London: Faber and Faber; 1969.

15 Unusual Types of ␣ Thalassemia Douglas R. Higgs, Veronica J. Buckle, Richard Gibbons, and David Steensma

the first condition (ATR-16, OMIM: 141750) there are large (1–2 Mb) chromosomal rearrangements that delete many genes, including the ␣-globin genes from the tip of the short arm of chromosome 16 and this is an example of a contiguous gene syndrome.4 In the second syndrome (ATR-X, OMIM 301040), a complex phenotype, including ␣ thalassemia, results from mutations in an X-encoded factor (now called the ATRX protein), which is a putative regulator of gene expression. Mutations in this gene down regulate ␣ globin gene expression and also perturb the expression of other as yet unidentified genes.

THE ATR-16 SYNDROME

INTRODUCTION In this chapter we describe three relatively rare, clinically complex syndromes in which the occurrence of ␣ thalassemia provided the clue to understanding the molecular basis of each condition. These conditions exemplify the important interplay between clinical observation and human molecular genetics. Two of these syndromes (ATR16 [OMIM: 141750] and ATR-X [OMIM: 301040]) in which ␣ thalassemia is associated with multiple developmental abnormalities (including mental retardation, MR) are inherited. The third condition (ATMDS [OMIM: 300448]) is an acquired disorder in which ␣ thalassemia appears for the first time in the context of myelodysplasia.

To date we know of 40 individuals (from 32 families) who have well-characterized ATR-16 syndrome (Table 15.1a and b). Often one is alerted to this condition by observing the unusual association of ␣ thalassemia and MR in individuals originating from outside of the areas where thalassemia commonly occurs (see Chapters 13 and 14). There are two common patterns of inheritance. In many cases neither parent has ␣ thalassemia (␣␣/␣␣ × ␣␣/␣␣) and the affected offspring has the phenotype of severe ␣ thalassemia trait (genotype --/␣␣). Less commonly, one parent has the phenotype of mild ␣ thalassemia trait, the other parent is nonthalassemic (-␣/␣␣ × ␣␣/␣␣) and the child has HbH disease (genotype --/−␣). In addition to ␣ thalassemia, these patients have variable degrees of facial dysmorphism (Fig. 15.1) and a wide spectrum of associated developmental abnormalities (Table 15.2a and b). In all such cases, initial molecular genetic analyses have shown that affected individuals fail to inherit the entire ␨ –␣ globin cluster from one or other of the parents.

␣ THALASSEMIA ASSOCIATED WITH MENTAL RETARDATION AND DEVELOPMENTAL ABNORMALITIES The rare association of ␣ thalassemia and mental retardation (MR) was recognized more than 25 years ago by Weatherall and colleagues.1 It was known that ␣ thalassemia arises when there is a defect in the synthesis of the ␣-globin chains of adult hemoglobin (HbA, ␣2 ␤2 ). When these authors encountered three mentally retarded children with ␣ thalassemia and a variety of developmental abnormalities, their interest was stimulated by the unusual nature of the ␣ thalassemia. The children were of northern European origin, where ␣ thalassemia is uncommon, and although one would have expected to find clear signs of this inherited anemia in their parents, it appeared to have arisen de novo in the affected offspring. It was thought that the combination of ␣ thalassemia with MR (ATR), and the associated developmental abnormalities represented a new syndrome and that a common genetic defect might be responsible for the diverse clinical manifestations. What emerged was the identification of two quite distinct syndromes in which ␣ thalassemia is associated with MR.2,3 In

296

Figure 15.1. The facial appearance of patients with the ATR-16 syndrome. Common features include relative hypertelorism, a small chin and mouth, a “beaked” nose, downslanting palpebral fissures, and crowded teeth.

297

--/␣␣ --/␣␣ --/-␣␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/-␣ --/␣␣ --/␣␣ --/␣␣ --/-␣ na --/␣␣ --/␣␣

--/␣␣ --/-␣ na --/␣␣ --/␣␣ --/␣␣ --/-␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/-␣ --/␣␣ --/␣␣ --/␣␣ --/␣␣ --/-␣ --/␣␣ --/␣␣ --/-␣␣

Genotype

Abnormal Abnormal Normal Normal Normal Abnormal Abnormal Normal Normal Abnormal Normal Normal Normal Normal Normal Abnormal Abnormal Abnormal

Normal Normal na Normal Normal Normal Normal na Normal Normal Normal Normal Normal Normal∗ Normal Normal Normal Normal Normal Normal Deleted Normal

Conventional cytogenetics

46,XY, -16,+der(16)t(9;16)(p13;p13.3) 46,XX, -16,+der(16)t(9;16)(p21.2;p13.3) 46.XX, -16,+der(16)(qter->q24::p13.3->qter)mat 46,XX, -16,+der(16)(qter->q24::p13.3->qter) 46,XY, -16,+der(16)(qter->q24::p13.3->qter) 46,XX, der(16)(qter->p13.3::p13.3->p13.13:) 46,XY, -16+der(16)t(9;16)(p21.2;p13.3) 46,XX, -16,+der(16)t(16;20)(p13.3;q13.3)mat 46,XX, -16,+der(16)t(16;20)(p13.3;q13.3)mat 46,XX, -16,+der(16)t(10;16)(q26.13;p13.3)mat 46,XY, -16,+der(16)(qter->q24::p13.3->qter) 46,XY, -16,+der(16)t(16;21)(p13.3;p13) mat 46,XX, -16,+der(16)t(16;21)(p13.3;p13)mat 46,XY, -16, +der(16)t(16;20)(p13.3;p13) 46,XY, -16, +der(16)t(1;16)(p36;p13.3) 46,XY, -16+der(16)t(X;16)(p11.4;p13.3)mat 45,XY, -15, --16 +der (16)t(15;16)(q13.1;p13.3) 46XY, -16, +der (16)(qter->q22::p13.3->qter)

46.XX, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XY, del(16)(p13.3) 46.XX, del(16)(p13.3) 46.XX, del(16)(p13.3)

Chromosomal abnormality

na Paternal Maternal Unknown Unknown Paternal Maternal Maternal Maternal Maternal Paternal Maternal Maternal Paternal Maternal Maternal Paternal Maternal

Maternal Paternal na Paternal Unknown Unknown Paternal Maternal Maternal Maternal Maternal Maternal Unknown Maternal Maternal Maternal Unknown Unknown Paternal Unknown Paternal Unknown

Parental origin

na = data not available ∗ at low resolution ∗∗ Although LIN had 30% Hb Bart’s at birth, neither parent appears to have an inherited form of ␣ thalassemia that would account for the HbH chains reported in LIN. Additional cases of ATR-16 whose deletions have not been fully characterized at the molecular level have been described in refs. 114–118.

Trait Trait HbH Trait Trait Trait Trait Trait Trait Trait HbH Trait Trait Trait HbH na Trait Trait

Phenotype

(b) Translocation patients JPS M na MR F Mild BE(C) F Mild BE (Ch) F Mild BE(W) M Mild SS F na CU M Mild WA(C) F Borderline WA (Cj) F Mild Aa F Borderline HA M Borderline GR(M) M Mild GR (J) F Mild Wl M Borderline OD M Moderate LF M na DA M Mild BAR M Mild

MR

Trait HbH na Trait Trait Trait HbH Trait Trait Trait Trait Trait Trait HbH Trait Trait Trait Trait HbH Trait Trait∗∗ HbH

Sex

(a) Pure monosomy patients JT F Normal OY F Normal AB M Normal TY(MI) F Normal TY(Mi) M Normal YA F na BA F Normal GZ M Normal TN (Pa) F Borderline TN (Pe) M Mild TN (AI) M Mild SH (Pa) M Moderate SH (Ju) F Normal DO F Mild CJ M Mild MY F Mild PV M Mild FT F Mild BO M Mild HN M Mild LIN F Mild IM F Mild

Case

Table 15.1. Cytogenetic and hematological data and origin of ATR-16 mutations

na De novo Inversion/deletion Inversion/deletion Inversion/deletion De novo duplication with deletion De novo Derived from parental balanced translocation Derived from parental balanced translocation Derived from parental balanced translocation De novo inversion/deletion Derived from parental balanced translocation Derived from parental balanced translocation De novo Derived from parental balanced translocation Derived from parental balanced translocation De novo De novo inversion/deletion

De novo interstitial deletion 268 kb De novo truncation na Inherited truncation Unknown truncation Unknown De novo truncation Inherited deletion De novo truncation Inherited truncation Inherited truncation Inherited Unknown Unknown De novo interstitial deletion 1258 kb De novo truncation De novo deletion De novo deletion De novo truncation De novo deletion De novo Unknown

Mechanism

A Villegas (personal communication) [112] [113] and unpublished [113] and unpublished [113] and unpublished Unpublished [2,112] Unpublished Unpublished [2,8] [2] [18] [18] [2] [2,9] K May (personal communication) [2] [114]

[19] Unpublished [16] Unpublished Unpublished Unpublished [18] [15] [18] Unpublished Unpublished Unpublished Unpublished [2] Unpublished Unpublished [15] [15] [2,17,18] [15] [18,109] [18,110,111]

Reference

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Douglas R. Higgs, Veronica J. Buckle, Richard Gibbons, and David Steensma

Table 15.2. Clinical findings in patients with ATR-16 syndrome

Case

MR

Speech delay

(a) Pure monosomy patients − JT Normal∗ − OY Normal∗ − AB Normal∗ − TY(MI) Normal∗ − TY(Mi) Normal∗ YA na +

Developmental delay

Short stature

Facial dysmorphism

Genital abnormalities

Skeletal abnormalities

− − − − − +

− − − − − +

− − − − − +

− − − − − −

− − − − − −

Miscellaneous abnormalities

Macroglossia Supernumery nipples, umbilical hernia and developmental delay. Fx of umbilical hernia, pyloric stenosis and omphalocele. Poor motor skills Pyloric stenosis Less affected than sons

BA GZ TN (Pa) TN (Pe) TN (Al) SH (Pa)

Normal∗ Normal∗ Borderline Mild Mild Moderate

− − + + + na

− − + + + +

− − − − − −

− − + + + +

− − − − − +

− − − − − +

SH (Ju)

Normal∗

−

−

−

−

−

−

DO CJ

Mild Mild

+ +

+ +

+ −

+ +

− −

− −

MY

Mild

+

+

na

+

−

−

PV

Mild

+

+

−

+

−

+

FT BO

Mild Mild

+ na

+ +

− +

+ +

− +

+ +

HN

Mild

+

+

−

+

−

+

LIN IM

Mild Mild

+ na

+ +

− na

+ +

− −

na +

Bilat clubfoot

+

+ + + +

+ − na na

− na na na

+ na na na

SPC s na na

(b) Translocation patients JPS na MR Mild + BE (C) Mild BE (Ch) Mild BE (W) Mild

Left iris coloboma Fine motor problems, asthma, bronchitis, myopic Severe anxiety/depression IC, seizures, Heart murmur, macrocephalic, no speech at age 4, slow cognitive, social, and motor development Developmental delay. Plagiocephaly. Patient and normal sister had ASD. Recurrent chest infections and asthma, epilepsy, pectus excavatum Pectus excavatum IC, P, microcephaly, clubfoot, ductus arteriosus, pneumonia Recurrent chest infections and asthma, L clubfoot, arachnoid cyst in R temporal lobe

Unusual Types of ␣ Thalassemia

299

Table 15.2 (continued )

Case

MR

Speech delay

Developmental delay

Short stature

Facial dysmorphism

Genital abnormalities

Skeletal abnormalities

Miscellaneous abnormalities

SS

na

na

+

+

+

na

na

CU WA (C)

Mild Borderline

+ +

+ +

− +

+ +

− −

+ +

WA (Cj)

Mild

na

na

+

+

na

na

Aa HA GR (M)

Borderline Borderline Mild

+ + +

+ + +

− − −

+ + +

− − −

− + +

GR (J) WI OD LF DA BAR

Mild Borderline Moderate na Mild Mild

+ + + na

+ + + na

+

+

− − − na − −

+ + + + + +

− − + + + −

+ + − na + +

Rash, recurrent chest and ear infections, multiple developmental abnormalities SPC S, PVC, LFW, SD, NW, asthma, special school Broadly spaced, wide-open eyes, not sloping, rash UG, HPN E, clubfoot Bronchitis, pneumonia, reactive airway disease, heart murmur CHD AN, IC CAL T, CS, CHD, H died at 49 d SPC, HT Bilat equinovarus

∗ normal phenotype, included to define critical regions. na = data not available; U = unable to assess at time of death; CAL = cafe-au-lait patches; SPC = single palmar crease; HT = hypoplastic enamel of teeth; UG = unsteady gait; HPN = high placed nipples; IC = impaired coordination; P = ptosis; E = epilepsy; AN = accessary nipple; T = tracheobronchomalacia; CS = choanal stenosis; CHD = congenital heart disease; H = hydrocephalus; S = strabismus; PVC = paralyzed vocal cord (unilateral); LFW = left facial weakness; SD = sacral dimple; NW = neck webbing; M = myopia; TS = tuberous sclerosis; RC = renal cysts; PL = pigmented lesions (hypo & hyper). ASD = atrial septal defect; Fx = family history.

Defining the Genetic Abnormalities in Patients with ATR-16 Syndrome In some cases, conventional cytogenetic analysis demonstrated the underlying genetic abnormality. Because the ␣–globin complex lies very close to the 16p telomere (16p13.3, Fig. 15.2) any chromosomal abnormality affecting this region may give rise to ␣ thalassemia.2 In some patients with putative ATR-16 syndrome, gross chromosomal abnormalities resulting in deletions,2 formation of ring chromosomes,5–7 and translocations8 have been observed. Although such abnormalities may arise as de novo genetic events, often one parent carries a preexisting balanced translocation, which the child inherits in an unbalanced fashion (Fig. 15.3, summarizing the findings in ref. 9), resulting in monosomy for 16p and loss of the ␣ cluster. In many cases of ATR-16, initial high-resolution cytogenetic analysis appeared entirely normal. In some of these cases the pattern of inheritance of polymorphisms (such as variable number tandem repeats) within the ␣-cluster revealed the nature of the underlying molecular defect. In the example given in Fig. 15.3 the parental 16p alleles could be distinguished from each other. The mother in this family was shown to carry a balanced 1:16 translocation, which both of her children inherited in an unbalanced fashion. Her son OD (Tables 15.1 and 15.2) was monosomic for 16p,

and therefore was shown to have ␣ thalassemia (in this case HbH disease), whereas her daughter was trisomic for 16p. Both children had MR, dysmorphic facies, and a variety of associated developmental abnormalities. Fluorescence in situ hybridization (FISH) studies have also been used to analyze ATR-16 families. In this type of study, large segments (∼40 kb) of chromosome 16 in cosmid vectors are used as probes to demonstrate the presence or absence of the corresponding sequences in the 16p telomeric region by using fluorescence microscopy.10 By analyzing the chromosomes of both parents and the affected child it has been possible to define the extent of 16p monosomy and the mechanism by which it has arisen. In the example shown in Figure 15.4, FISH analysis demonstrated that the mother of an affected child with the ATR16 syndrome carries a balanced 16:20 translocation, which was inherited in an unbalanced fashion (as in Fig. 15.3) by her offspring. Some chromosomal abnormalities can only be detected by FISH or molecular analyses and they are referred to as “cryptic.” Over the past 10 years other methods have also been applied systematically to the characterization of the chromosome abnormalities in these patients. M-TEL FISH11 involves the hybridization of a panel of subtelomeric probes from each human chromosome to detect both loss of material from and additional material on the short arm

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16 0k

100k

CYXorf1 c16orf8 gs3 PolR3k IL9R3ps MPG c16orf33 C16orf35

200k HBZ HBA2 HBZps HBD Luc7L HBA1ps HBA1 HBQ

300k

400k

500k

600k

700k

RGS11 DPIA2 AXIN1

MRPL28 RAB11FIP3 TMEM8 NME4 DECR2

SOLH PIGQ WFIKKN1

METRN

900k

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CHTF18

MSLN

LOC388199

WDR24 RAB40C C16orf24 RPUSD1 LMF1 C16orf13 FBXL16 MPFL C16orf14 GNG13 LOC339123 WDR90 LOC388199 CCDC78 RHOT2

JT

800k

RHBDL1 plus108 ARHGDIG

SOX8

SSTR5 C1QTNF8

HAGHL

CACNA1H TPSB2 TPSG1 TPSD1

BAIAP3 GNPTG

TPSAB1

LOC283951 NP2IFT140

C16orf42 UNKL

TELO2 CLCN7

TMEM204

CRAMP1L

MAPK8IP3 HN1L

1900k

EME2 HAGH

NME3

FAHD1

MRPS34 C16orf73 SPSB3 NUBP2 IGFALS

HS3ST6

2000k

2100k

NDUFB10 NTHL1 PKD1 NPW

SEPX1 ZNF598 TSC2 TBL3 RPL3L NOXO1

SLC9A3R2 RPS2 SYNGR3 RNF151 GFER

STUB1 NARFL

[19]

AB

[16]

GZ

[15]

OY

[unpublished]

TY

[unpublished]

BA

[18]

TN

[18]

MR

[112]

BE

[113, unpublished]

SS

[unpublished]

SH

[unpublished]

CU

[2,12]

WA

[unpublished]

DO~

[2]

Aa

[2,8]

HA

[2]

CJ

[unpublished]

MY

[unpublished]

GR

[18]

WI

[2]

PV

[15]

FT

[15]

BO

[2,17,18]

HN

[15]

OD~

[2,9]

LF~

[K May pers.comm]

LIN

[18,109]

IM~

[18,110,111]

Figure 15.2. Summary of known ATR-16 deletions. The positions of the ␣-globin cluster and other genes within this region are indicated. Below, the extent of each deletion is shown with the patient code alongside (see Table 15.1). Deletions known to result from pure monosomy for this region are shown in either green (no abnormalities other than thalassemia) or red (ATR-16 phenotype). Chromosomal translocations (all with ATR-16 phenotypes) are shown in blue. Solid bars indicate regions known to be deleted and fine lines indicate the region of uncertainty of the breakpoints. The ␣ genes and the genes that when mutated are associated with tuberous sclerosis and adult polycystic kidney disease are shown (shaded boxes). (See color plate 15.2.)

of chromosome 16. For example, abnormal chromosome 16 in HA (Table 15.1) was shown by this method to be not only monosomic for part of 16p13.3 but also trisomic for part of 16q24 (derived from an inversion/deletion event). Recently there have been several technical developments that have narrowed the gap between cytogenetics and molecular analyses, enabling high-resolution analysis of the entire human genome. In the first of these types of analyses a large proportion of the genome is interrogated using microarrays consisting of DNA from bacterial artificial chromosomes spanning the genome. Using a competitive genome hybridization (CGH) approach, comparing DNA from one genome with another, it has been possible to detect large regions of monosomy in patients with ATR-16 syndrome.12 More recently using oligo-based microarrays or bead technologies (originally made to identify single nucleotide polymorphisms) with a modified analysis it has

been possible to identify deletions and duplications of greater than approximately 1 kb.13 The resolution of this type of analysis is continuously improving. One of these approaches (using microbead technology14 ) has recently been applied to previously characterized (and new) cases of ATR-16, nicely demonstrating the large deletions from 16p13.3 and identifying any associated aneuploidy (V. Buckle et al., in preparation). Figure 15.5 shows an example of this type of analysis in a patient (BO, Tables 15.1 and 15.2) with ATR-16 syndrome due to a deletion of 1900 kb from 16p13.3. In addition to these approaches Harteveld et al.15 have developed a rapid and simple high-resolution approach to identify and characterize deletions of the terminal 2 Mb of 16p13.3 using the multiplex ligation–dependent probe amplification technique. An established panel of specific, synthetic oligonucleotides can now be used to detect

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301

a)

mild α thal

mild α thal

OD MR HbH disease

MR

b)

c)

α

α α

α

α

α

α

α

16 1 Genotype -α/αα

-α/αα

−/−α

− α/−α/αα

Figure 15.3. Familial subcytogenetic translocation (from ref. 9). (a) Pedigree indicating parents with mild ␣ thalassemia only; son (OD) with MR and severe ␣-thalassemia (HbH disease) and daughter with MR. (b) Schematic representation of restriction fragment length polymorphism analysis using a fully informative marker closely linked to the ␣-globin cluster. Each track corresponds to the individual shown above. (c) Segregation of 1:16 translocation and ␣-globin complex (␣) in each family member. The resulting genotype is shown. Note that both children have inherited the paternal chromosome carrying the (-␣) allele; it has not been determined whether the mother’s normal or translocated chromosome 16 bears her (-␣) allele.

deletions in 16p13.3.15,16 Clearly the resolution of this technique is only limited by the spacing of the appropriate oligonucleotides. An example of this type of analysis applied to a patient with ATR-16 syndrome is shown in Figure 15.5. Using a combination of conventional cytogenetics, FISH, and molecular analysis at least three types of chromosomal rearrangements (translocation, inversion/deletion, and truncation) have now been found in ATR-16 patients (Fig. 15.2 and Table 15.1). To date, few breakpoints have been fully characterized. The telomeric truncations seen in BO, BA, and MY (Table 15.1 and Fig. 15.2) have been fully analyzed17 (Green et al., unpublished) and it appears that these chromosomes have been broken, truncated, and “healed” by the direct addition of telomeric repeats (TTAGGG)n as described for some less extensive 16p

deletions in patients with ␣ thalassemia but no MR (e.g., TY in Fig. 13.2 and Chapter 13). In addition, another 11 ATR16 cases, shown to have pure monosomy for 16p13.3 (Fig. 15.2), are likely to fall into this same class. Although many cases of ATR-16 have doubtless been overlooked, it is nevertheless likely that this syndrome is rare, because each case found to date has been the result of a unique and independent chromosome mutation.

How Do Chromosomal Abnormalities Give Rise to the ATR-16 Syndrome? Because individuals with ATR-16 syndrome may have quite different degrees of chromosomal imbalance, there is considerable variation in the associated phenotypes

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Figure 15.4. High-resolution chromosome analysis using FISH. (a and b) Examples of FISH on metaphase chromosomes of deletion patient JT. In each case chromosome 16 is identified by a chromosome 16–specific centromere probe. In (a) using a cosmid (cGG1) located close to the telomere, fluorescent signal was seen on the normal chromosome (arrow) but not the abnormal copy of chromosome 16 (arrowhead). (b) Using a second cosmid located closer to the centromere (not deleted in JT) a signal is seen on both normal and abnormal chromosomes (arrows). (c and d) FISH on metaphase chromosomes of the mother of WA showing a balanced translocation. In (c) and (d) both homologs of chromosome 16 are indicated by arrows and chromosome 20 by arrowheads. In (c) the subtelomeric probes for 16p and 16q were hybridized to metaphase chromosomes. Green fluorescent signal was seen on the q arm of both homologs of chromosome 16. Red fluorescent signal was seen on the p arm of the normal chromosome 16 and the q arm of the derivative chromosome 20 (der 20) but was absent from the deleted chromosome 16 (der 16). In (d) the subtelomeric probe for 20p (red) was seen on both homologs of chromosome 20. Fluorescent signal for 20q (green) was seen on the normal chromosome 20 and the p arm of the derivative 16 (der 16). (See color plate 15.4.)

(Table 15.2). The degree of monosomy in 16p13.3 varies from 0.3 to 2 Mb (Fig. 15.2) but many patients have additional chromosomal aneuploidy and in some cases imbalance of the non-16 material may dominate the clinical picture. For example in DA (Table 15.1) loss of material from chromosome 15 while forming the abnormal derivative t(15:16) chromosome produced the striking phenotype associated with the Prader–Willi syndrome. Because many patients with ATR-16 have complex genomic rearrangements, to determine the role played by any gene(s), within 16p13.3, in the developmental abnor-

malities associated with ATR-16 syndrome, future analysis should focus on patients who have pure monosomy for 16p13.3. Clearly, patients with the common forms of ␣ thalassemia have small deletions (∼5–40 kb) from the ␣-globin cluster (Chapter 13) with no abnormalities other than ␣ thalassemia. Nevertheless, rare individuals with much more extensive deletions have been identified. Surprisingly, these studies have shown that patients with deletions of up to 900 kb of 16p13.3 (including 52 genes) have ␣ thalassemia but may be minimally affected18 (unpublished data) or even normal2,15–19 (unpublished data). At the other

Unusual Types of ␣ Thalassemia a)

303

BO

Log Ratio

2 0 -2 -4 -6 -8

GZ b)

PV

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40

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0

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Figure 15.5. Detection of deletions. (a) Analysis of chromosome 16 in patient with ATR-16 using the QuantiSNP protocol.14 The Y-axis indicates the log ratio between normal and the test and the X-axis represents the distance along chromosome 16. (b) Analysis of two patients (GZ and PV15 and Fig. 15.2) with large deletions of 16p13.3 using the multiplex ligation–dependent probe amplification protocol15 ). The Y-axis represents the ratio peak height of the test divided by the normal control and the X-axis represents the distance along chromosome 16. (See color plate 15.5.)

extreme, patients with 16p monosomy for the entire terminal region of 16p (e.g., BO, LIN, and IM) have a relatively severe phenotype with ␣ thalassemia, MR, and dysmorphic features and skeletal abnormalities. All patients with deletions from 900 to 1700 kb have some degree of MR and shared but variable dysmorphic features (Table 15.2). In patients whose deletions extend beyond 2000 kb the clinical picture is dominated by more severe MR, tuberous sclerosis, and polycystic kidney disease.20 How might monosomy for 16p13.3 cause such developmental abnormalities? One possibility is that deletion of a large number of genes from one copy of chromosome 16 may unmask mutations in its homolog; the more genes that are deleted the greater the probability of this occurring. This is unlikely to be the explanation for most ATR-16 cases because it is estimated that normal individuals only carry a few harmful mutations of this type in the entire genome.21 A further possibility is that some genes in 16p are imprinted22 so that deletions could remove the only active copy of the gene. At present there is no evidence for imprinting of the 16p region (reviewed in ref. 23) and in the relatively few ATR-16 cases analyzed there appears to be no major clinical differences between patients with deletions of the maternally or paternally derived chromosomes (Tables 15.1 and 15.2). It therefore seems more likely that there are some genes in the 16p region that encode proteins whose effect is critically determined by the amount produced; so-called dosage-sensitive genes.24 Examples of such genes include those encoding proteins that form heterodimers, those

required at a critical level for a rate-determining step of a regulatory pathway, and tumor suppressor genes (e.g., TSC2). Removal of genes from one copy of 16p13.3 consistently reduces their levels of expression to approximately 50% of normal (Buckle et al., unpublished). If the deletion includes one or more dosage-sensitive genes this could account for the clinical effects seen in ATR-16 patients. The region lying between 900 and 1700 kb from the 16p telomere, deleted in all patients with the characteristic features of ATR-16 syndrome, contains 16 genes and gene families of known function,15,18 which have been implicated in a wide range of disorders with few or no features in common with ATR-16. One of these (SOX8) was considered as strong candidate because it is involved in the regulation of embryonic development and is strongly expressed in the brain.25 A recently described Brazilian patient with a deletion that removes both the ␣-globin locus and SOX8 was not associated with MR or any dysmorphism however (Harteveld, personal communication, 2007). Clearly, further examples of ATR-16 due to monosomy for 16p13.3 must be characterized to identify the gene(s) responsible for the MR and other developmental abnormalities associated with this condition.

Summary of the ATR-16 Syndrome The ATR-16 syndrome has served as an important model for improving our general understanding of the molecular basis for MR. It provided the first examples of MR due to

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a cryptic chromosomal translocation and truncation. Further work has shown that such telomeric rearrangements may underlie a significant proportion of unexplained MR.26 The current challenge is to understand in detail the mechanisms by which monosomy causes developmental abnormalities; the ATR-16 syndrome provides an excellent model for addressing this issue.

THE ATR-X SYNDROME As additional patients with ␣ thalassemia and MR were identified throughout the 1980s, it became clear that a second group of affected individuals existed in whom no structural abnormalities in the ␣-globin cluster or 16p could be found. In contrast to those with ATR-16 syndrome, this group of patients was all male, presented with a much more uniform phenotype, and had a remarkably similar facial appearance.3 That this group had a distinct and recognizable dysmorphism was underscored when additional cases were identified on the basis of their facial features alone.27,28 Ultimately, it was shown that this unusual syndrome of ␣ thalassemia with severe MR results from an X-linked abnormality (see later) and the condition is now referred to as the ATR-X syndrome (OMIM: 301040).

The Clinical Findings of the ATR-X Syndrome Cases of ATR-X syndrome from over 150 families have now been characterized, and in contrast to ATR-16 a definite phenotype is emerging. The cardinal features of this condition are severe MR and developmental abnormalities associated with a characteristic facial appearance and ␣ thalassemia. The frequency and nature of the most commonly encountered clinical features are summarized in Tables 15.3 and 15.4. Neonates with ATR-X syndrome usually have marked hypotonia and associated feeding difficulties. Seizures have

Table 15.3. Summary of the major clinical manifestations of the ATR-X syndrome Clinical feature

Total∗

%

Profound MR Characteristic face Skeletal abnormalities HbH inclusions Neonatal hypotonia Genital abnormalities Microcephaly Gut dysmotility Short stature Seizures Cardiac defects Renal/urinary abnormalities

160/168 138/147 128/142 130/147 88/105 119/150 103/134 89/117 73/112 53/154 32/149 23/151

96 94 90 88 84 79 77 76 65 34 21 15

∗

Total represents the number of patients for whom appropriate information is available and includes patients who do not have ␣-thalassemia but in whom ATRX mutations have been identified.

also been noted in some patients. In early childhood, all milestones are delayed. Many patients do not walk until late in childhood, and some never do so. Most have no speech, although some patients have a handful of words or signs. With some notable exceptions29–32 most patients with ATRX syndrome have only situational understanding and are dependent for almost all activities of daily living. Although head circumference is usually normal at birth, postnatal microcephaly usually develops. As affected individuals age, there is often a tendency toward spasticity and seizures occur in approximately one-third of cases. Vision usually appears normal but sensorineural deafness may occur in some patients. Computed tomography or magnetic resonance brain imaging is generally unremarkable, although mild cerebral atrophy may be seen. In two cases, partial or complete agenesis of the corpus callosum was

Table 15.4. Clinical manifestations of the ATR-X syndrome Genital abnormalities

Skeletal abnormalities

Renal/urinary abnormalities Cardiac defects Gut dysmotility

Miscellaneous

Small/soft testes, cryptorchidism, gonadal dysgenesis, inguinal hernia, micropenis, hypospadias, deficient prepuce, shawl scrotum, hypoplastic scrotum, ambiguous genitalia, female external genitalia (male pseudohermaphroditism) Delayed bone age, tapering fingers, drumstick distal phalanges, brachydactyly, clinodactyly, bifid thumb, fixed flexion deformities of joints, overriding toes, varus or valgus deformities of feet, scoliosis, kyphosis, hemivertebra, spina bifida, coxa valga, chest wall deformity Renal agenesis, hydronephrosis, small kidneys, vesicoureteric reflux, pelvoureteric junction obstruction, exstrophy of bladder, urethral diverticulum, urethral stricture Atrial septal defect, ventricular septal defect, patent ductus arteriosus, tetralogy of Fallot, transposition of the great arteries, dextrocardia with situs solitus, aortic stenosis, pulmonary stenosis Discoordinated swallowing, eructation, gastroesophageal reflux, vomiting, gastric pseudovolvulus, hiatus hernia, hematemesis, recurrent ileus/small bowel obstruction, volvulus, intermittent diarrhea, severe constipation Apneic episodes, cold/blue extremities, blepharitis, conjunctivitis, entropion, cleft palate, pneumonia, umbilical hernia, encephalitis, iris coloboma, optic atrophy, blindness, sensorineural deafness, prolonged periods of screaming/laughing, self injury

Unusual Types of ␣ Thalassemia

Figure 15.6. The facial appearance of patients with ATR-X syndrome as described in the text.

reported.33 There have been autopsy reports in only three cases. The brain was small in each; in two the morphology was normal, and in one the temporal gyri on the right were indistinct, with hypoplasia of the cerebral white matter. No systematic study of behavior has been performed in ATR-X syndrome but some recurrent themes are emerging.34,35 Patients are usually described by their parents as content and of a happy disposition but may exhibit unprovoked emotional outbursts with sustained laughing or crying. Whereas many individuals are affectionate to their caregivers, others exhibit autistic-like behavior. They may be restless, exhibiting choreoathetoid movements and some have repetitive, stereotypic movements (pill-rolling or hand-flapping). Frequently, they put their hands into their mouths and may induce vomiting and sometimes they may engage in self-injurious behavior. The characteristic facial appearance in ATR-X syndrome is most readily recognized in early childhood (Fig. 15.6). The frontal hair is often upswept and there is telecanthus, epicanthic folds, flat nasal bridge, midface hypoplasia, and a small triangular upturned nose with the alae nasi extending below the columella and septum. The upper lip is tented and the lower lip full and everted, giving the mouth a “carplike” appearance. The frontal incisors are frequently widely spaced, the tongue protrudes, and there is prodigious dribbling. The ears may be simple, slightly low-set, and posteriorly rotated. Urogenital abnormalities are seen in 80% of children. These may be very mild, for example, undescended testes or deficient prepuce; the spectrum of abnormality extends through hypospadias to micropenis to ambiguous or external female genitalia. The most severely affected children, who are clinically defined as male pseudohermaphrodites, are usually raised as females. It is of interest that these

305 abnormalities breed true within families.36 Renal abnormalities (hydronephrosis, renal hypoplasia or agenesis, polycystic kidney, and vesicoureteric reflux) may present with recurrent urinary tract infections. Various relatively mild skeletal abnormalities have been noted; some of which are probably secondary to hypotonia and immobility.37 Fixed flexion deformities, and other abnormalities, particularly of the fingers, are common. Short stature is seen in two-thirds of cases. A wide range of gastrointestinal abnormalities have been reported in patients with this syndrome. Vomiting, regurgitation, and gastroesophageal reflux are particularly common in early childhood. An apparent reluctance to swallow has been reported by several parents and probably reflects the discoordinated swallowing that has been observed radiologically (unpublished). The tendency for aspiration is commonly implicated as a cause of death in early childhood. Excessive drooling is very common, as is frequent eructation. Constipation occurs often, and in some individuals is a major management problem. Martucciello et al.38 demonstrated ultra-short Hirschsprung disease and colonic hypoganglionosis in two affected children. This may be a consequence of a widespread abnormality in the enteric nervous system leading to abnormal gut motility. Affected individuals may be susceptible to peptic ulceration. Esophagitis, esophageal stricture, and peptic ulcer have been observed endoscopically in single cases. Finally, a wide range of cardiac abnormalities have also been noted including septal defects (10 cases), patent ductus arteriosus (6 cases), pulmonary stenosis (3 cases), aortic stenosis (2 cases), tetralogy of Fallot (2 cases), and single cases of transposition of the great arteries, dextrocardia with situs solitus, and aortic regurgitation. Of the 168 patients described to date, there have been 25 deaths. The cause was established in just over half, of which there were 6 cases of pneumonia and four of aspiration of vomitus. Cases appear to be particularly vulnerable in early childhood, with 19 of the deaths occurring before the age of 5 years; this may be associated with the fact that gastroesophageal reflux and vomiting are often more severe in the early years. There are no long-term longitudinal data in this relatively newly described syndrome, but a number of affected individuals are fit and well in their 30s and 40s.

The Hematological Findings of the ATR-X Syndrome The presence of ␣-thalassemia (in the form of thalassemia trait or mild HbH disease) with HbH inclusions (see Chapter 14) was one of the original diagnostic criteria for ATR-X syndrome; however, with the identification of further cases, it became clear that the hematological findings (e.g., levels of Hb, mean corpuscular volume [MCV], and mean corpuscular hemoglobin [MCH]) were different from those seen in the common types of ␣ thalassemia. Now

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a)

b) 40

18

Affected Individuals Mean

14

2 SD below mean 12

10

8 0

10

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40

50

Age (yrs)

Mean cell hemoglobin (pg)

Hemoglobin (g/dL)

Affected Individuals

35

16

Mean 2 SD below mean

30

-α/−

25

20

15 0

10

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30

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50

Age (yrs)

Figure 15.7. (a) Hemoglobin levels in subjects with ATR-X syndrome at various ages. Solid line indicates the mean and dashed line is 2 standard deviations below the mean.119 For any given subject, only one result within each consecutive 5-year period is given. (b) MCH levels in subjects with ATR-X syndrome at various ages. Mean values for individuals with ␣ thalassemia resulting from an -␣/-- genotype are shown.

that the disease can be identified via the clinical phenotype or the ATR-X genotype, it is clear that there is considerable variation in the hematological manifestations associated with mutations of the ATRX gene. In fact, a number of families have been identified in which some or all of the affected members with mutations of ATRX, and the characteristic phenotype described previously, have no signs of ␣ thalassemia.39,40 Nevertheless, when the family history and phenotype are strongly suspect, a careful search for HbH inclusions should be made in all affected individuals and repeated if necessary as they may be very infrequent. The hematology is often surprisingly normal considering the presence of ␣ thalassemia (Fig. 15.7). Neither the level of hemoglobin nor the mean cell hemoglobin is as severely affected as in the classic forms of ␣ thalassemia associated with cis-acting mutations in the ␣-globin complex (see Chapters 13 and 14), and this probably reflects the different pathophysiology of the conditions. In most cases of ATR-X, there is insufficient HbH to be detected by electrophoresis and the number of HbH inclusions is quite variable among different patients, although relatively constant over time in any affected individual.

The ATR-X Syndrome is an X-linked Condition The five original “nondeletion” cases described by Wilkie et al.3 were sporadic in nature, and apart from all being male, there were no immediate clues to the genetic etiology. Somatic cell hybrids composed of a mouse erythroleukemia cell line containing chromosome 16 (wherein the ␣-globin genes lie) derived from an affected boy produced human ␣-globin in a manner indistinguishable from a similar hybrid containing chromosome 16 from a normal individual. It therefore seemed likely that the defect in globin synthesis lay in trans to the globin cluster. This

was confirmed in a family with four affected siblings in whom the condition segregated independently of the ␣globin cluster.41 Because affected individuals were always related via the female line, this suggested that the syndrome mapped to the X chromosome and hence the condition was named the ATR-X syndrome. Subsequently linkage analysis in 16 families mapped the disease interval to Xq13.1-q21.1, confirming that the associated ␣ thalassemia results from a transacting mutation.42 Early genetic studies showed that the ATR-X syndrome behaves as an X-linked recessive disorder; boys are affected almost exclusively. Furthermore, almost all female carriers have a normal appearance and intellect, although approximately one in four carriers has subtle signs of ␣ thalassemia with very rare cells containing HbH inclusions.42 The majority of carriers have a highly skewed pattern of X inactivation in leukocytes (derived from mesoderm), hair roots (ectoderm), and buccal cells (endoderm). In each case the disease-bearing X chromosome is preferentially inactivated. There is evidence from a recently reported mouse model of ATR-X syndrome43 that skewed X inactivation results from selection, at key steps during development, against cells that are deficient for ATRX.44 Together, these findings showed that ATR-X is an Xlinked disease and when the gene is mutated, among many other effects, this leads to downregulation of expression of the ␣-globin genes on chromosome 16.

Identification of the ATR-X Disease Gene The isolation of cDNA fragments mapping to Xq13.1-q21.1 provided an opportunity to study candidate genes for ATRX.45 A number of these cDNA fragments were hybridized to DNA from a group of affected individuals. An absent

Unusual Types of ␣ Thalassemia

307

35 2 2 2 2

9 2 2 2

ATR-X syndrome mutations 3 2

2

4 3

2

2

2

2

3

3

2

3‘ untranslated region

5‘ utr

ATMDS mutations

Scale:

ATRXt

200 amino acids

ATRX ADD

Helicase domains

PQ

Figure 15.8. Schematic diagram of the ATRX gene: boxes represent the 35 exons (excluding the alternatively spliced exon 7); thin horizontal lines represent introns (not to scale). The 3
and 5
untranslated regions (utr) are shown flanking the open reading frame. The two protein products ATRX and ATRXt are shown as rectangles. The principal domains, the zinc finger motif (ADD), and the highly conserved helicase motif are indicated, as are the P box (P) and a glutamine-rich region (Q). In the lower part of the figure is a graphic representation of the amino acid similarity between human and mouse ATRX proteins. In the upper part of the figure is illustrated the spectrum of ATRX mutations described in boys with ATR-X syndrome (above the gene) and in ATMDS (below the gene). The positions of the mutations are shown by circles: filled circles represent mutations (nonsense or leading to a frameshift) that would cause protein truncation; open circles represent missense mutations and small deletions that maintain the reading frame; deletions are indicated by horizontal lines. Recurrent mutations are illustrated by larger circles, and the number of independent families is indicated.

hybridization signal was noted in one patient when an 84-bp cDNA fragment (E4) was used. E4 was shown to be part of a gene known as XH2/XNP.46 Subsequently, a 2-kb genomic deletion was demonstrated in this individual with ATR-X syndrome. Subsequently, analysis of a segment of cDNA corresponding to XH2/XNP identified diseasecausing mutations in several individuals with the clinical and hematological features of ATR-X syndrome. The Xlinked gene was thus renamed as the ATRX gene.

Characterization of the ATRX Gene and Its Protein Product We now know that the ATRX gene spans approximately 300 kb of genomic DNA and contains 36 exons,47 although exon 7 may be nonfunctional. It encodes at least two alternatively spliced, 10.5-kb mRNA transcripts that differ at their 5
ends and are predicted to give rise to slightly different proteins of 265 and 280 kD (Fig. 15.8). A further transcript of approximately 7 kb represents an isoform that retains intron 11 and truncates at this point. This gives rise to a truncated protein isoform (ATRXt) that is conserved between mouse and human.48 Within the N-terminal region lies a complex cysteinerich segment (called the ADD, domain, Fig. 15.8). This comprises a PHD-like zinc finger and an additional C2 C2 motif just upstream, which is structurally similar to the GATA-1

zinc finger.49,50 The PHD finger is a zinc-binding domain (Cys4 -His-Cys3 ), 50–80 amino acids in length and is a common feature of chromatin-related proteins and thought to mediate protein–protein interactions.51 The structure of the ADD domain shows that it is highly related to the zinc finger domains of DNA methyltransferases.50 Several lines of evidence suggest that this domain may bind the N-terminal tail of histone H3 and it is also possible that the upstream GATA-1-like motif in this domain may bind DNA.52 Both of these issues are under investigation. The functional importance of the ADD segment in ATRX is clear. It is highly conserved throughout evolution and it contains over 50% of all mutations found in patients with ATR-X syndrome (Fig. 15.8 and see later). The central and C-terminal regions of ATRX show the greatest conservation between murine and human sequences (94%).53 The central portion of the molecule contains motifs that identify ATRX as a member of the SNF2 group of proteins. These proteins are characterized by seven highly conserved, colinear helicase motifs. Other members of the SNF2 subfamily are involved in a wide variety of cellular functions, including regulation of transcription (SNF2, MOT1, and brahma), control of the cell cycle (NPS1), DNA repair (RAD16, RAD54, and ERCC6), and mitotic chromosomal segregation (lodestar). An interaction with chromatin has been shown for SNF2 and brahma and this may be a common theme for all members of this group

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(reviewed by Carlson and Laurent54 ). The ATRX protein, although showing higher sequence homology to RAD54 than other members of this group, does not obviously fall into a particular functional category by virtue of homology in these flanking segments. RAD54 is a DNA repair enzyme, but there is no clinical evidence of ultraviolet sensitivity or the premature development of malignancy in the ATR-X syndrome, which might point to this being a defect in DNA repair. Furthermore, cytogenetic analysis has not demonstrated any evidence of abnormal chromosome breakage or segregation. Rather, the consistent association of ATRX with ␣ thalassemia suggests that the protein normally exerts its effect at one or more of the many stages involved in gene expression.

Mutations of the ATRX Gene and Their Associated Phenotype In addition to ATR-X syndrome, mutations in the ATRX gene have now been found in many other forms of syndromal X-linked MR (reviewed in ref. 55) and it is also the disease gene associated with the occurrence of ␣ thalassemia in myelodysplasia (discussed later). To date, 113 different constitutional mutations have been documented in 182 independent families with ATRX syndrome (reviewed in ref. 55). Missense mutations are clustered in two regions: the ADD domain and the helicase domain. Analysis of the mutations and their resulting phenotypes allows important conclusions to be drawn. It seems likely that complete absence of ATRX, a true null, would be lethal because in a mouse ATRX knockout (null) model no affected embryos develop beyond approximately 8.5 dpc.43 Therefore it was surprising to find a number of mutations, predicted to cause protein truncation, scattered throughout the gene in patients with ATR-X syndrome (Fig. 15.8). Such mutations would be expected to result in a major loss of function and yet they are clearly not lethal. In fact, their phenotypes are similar to (and in some cases milder than) those seen with other mutations. For example, one premature stop mutation (R37X) predicted to make a very small, truncated protein produced almost fulllength protein.32,56 This could result from translational initiation at a downstream codon56 or by skipping the mutation via alternate splicing;32 whatever the mechanism, this mutation was associated with a remarkably mild phenotype. In fact, similar phenotypic rescue has been seen for all the stop codons upstream of the catalytic domain studied to date.55 ATR-X syndrome is frequently caused by missense mutations (Fig. 15.8) and the levels of ATRX protein were shown to be substantially reduced in patients with such mutations involving the ADD domain.52,57 The structure of the ADD domain casts light on how mutations might affect protein folding and stability.50 Most mutations affect zincbinding cysteine residues or residues in the tightly packed hydrophobic core thus reducing its stability. Of greater

interest, there is a small cluster of surface mutations (e.g., R246C, G249D) that are associated with higher levels of stable protein. These mutations may interfere with protein function, possibly by disrupting an important protein– protein interaction. Missense mutations affecting the helicase domain are located adjacent to, rather than within, the seven highly conserved motifs that characterize the SNF2 helicase/adenosine triphosphatase (ATPase) proteins. In other SNF2 proteins, mutations that fall in the motifs completely abolish activity. It is possible that the ATRX mutations alter, rather than abolish, the protein activity. Together, these findings are consistent with a view that mutations seen in patients who survive with ATR-X syndrome decrease rather than abolish ATRX activity.

Is There Any Relationship Between the Types of ATRX Mutation and the Phenotype? Since the discovery of the ATRX gene, most new cases have been defined on the basis of severe MR, with the typical facial appearance associated with a mutation in the ATRX gene. This allows a less biased evaluation of the effect of ATRX mutations on the commonly associated clinical manifestations. The severity of three aspects of the phenotype (MR, genital abnormality, and ␣ thalassemia) is quantifiable, to some degree. The greatest variation in intellectual handicap is associated with a truncating mutation (R37X) at the N terminus of the protein.30 As discussed previously, protein analysis by Western blotting has shown small amounts of full-length protein for each patient affected by this mutation. This may be associated with the use of an alternative, downstream translational initiation codon or alternative splicing. Nevertheless, there is no correlation between the degree of retardation and the amount of full-length protein seen in lymphoblastoid cell lines derived from these patients. A number of other cases have been described with mild to moderate learning difficulties (reviewed in ref. 55). In a recent small study of 22 patients it was noted that cases with mutations in the ADD domain were less likely to walk than those with mutations affecting the helicase domain.58 We have now confirmed this finding in a cohort of 83 affected individuals. Whereas 75% of cases with helicase mutations were able to walk only 25% of those with ADD mutations were able to do so (Fig. 15.9). There are now eight different mutations associated with the most severe urogenital abnormalities.55 In five, the protein is truncated, resulting in loss of the C-terminal domain, including a conserved element (P in Fig. 15.8) and polyglutamine tract (Q in Fig. 15.8). From the available data, it appears that, in the absence of the C-terminal domain, severe urogenital abnormalities are likely (although not inevitable as one mutation in this region was associated with cryptorchidism), suggesting that this region may play a specific role in urogenital development. Consistent with

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this, in families with such mutations, severe urogenital abnormalities breed true,36,59 and a nonsense mutation (R2386X) gives rise to a similar phenotype in three unrelated families. In other regions of the protein, however, there is no obvious link between phenotype and genotype, and there is considerable variation in the degree of abnormality seen even in individuals with identical mutations.

The Relationship Between the Types of Mutation in ATRX and Thalassemia The relationship between ATRX mutations and ␣ thalassemia is unclear. Because the presence of excess ␤ chains

(HbH inclusions) was originally used to define the ATR-X syndrome, current observations are inevitably biased. Nevertheless, there is considerable variability in the degree to which ␣-globin synthesis is affected by these mutations, as judged by the frequency of cells with HbH inclusions. Up to 15% of patients do not have HbH inclusions39,40,60 (Fig. 15.10), although this does not rule out downregulation of ␣-globin expression because inclusions may not appear until there is 30%–40% reduction in ␣-chain synthesis (Chapter 14). It is interesting that patients with identical mutations may have very different, albeit stable, degrees of ␣ thalassemia, suggesting that the effect of the ATRX protein

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on ␣-globin expression may be modified by other genetic factors. This is most clearly illustrated by comparing the hematology of cases with identical mutations. Comparison of the 41 cases from 34 pedigrees with the common 736C>T; R246C mutation shows a wide range in the frequency of cells with HbH inclusions (0%–14%). Preliminary studies indicate that the haplotype of the ␣-globin locus influences the severity of the thalassemia and presumably there is a feature within this region that is variable and confers different degrees of ATRX dependency (unpublished observation).

What is the Normal Functional Role of ATRX? In the adult, ATRX mRNA is widely expressed early in development, and continues to be widely expressed throughout development with particularly high expression in the brain, heart, and skeletal muscle.61 Both isoforms, ATRX (280 kD) and ATRXt (200 kD), are readily detected on Western blots.48,57 Immunocytochemical analysis and indirect immunolocalization demonstrate that both isoforms are nuclear proteins that predominantly associate with heterochromatin (in interphase and metaphase).48,57 A signifi-

cant proportion of ATRX (but not ATRXt) is also found in nuclear speckles (called promyelocytic leukemia bodies) in human cells.62,63 One additional, striking finding in human metaphase preparations is that ATRX antibodies localize to the short arms of acrocentric chromosomes associated with the rDNA arrays.64 Undoubtedly, a significant proportion of ATRX can be seen to be associated with heterochromatin and it is possible that the remainder of ATRX, which cannot yet be visualized, may be associated with euchromatic regions of the genome. The next questions are how is ATRX recruited to chromatin and what does it do when it gets there? Like other members of the SNF2 family, ATRX appears to be part of a multiprotein complex. Preliminary experiments show that ATRX fractionates as a very large complex (approximately 1 Md) by Superose 6 gel filtration. These protein interactions could mediate both recruitment and contribute to ATRX function. One of the proteins with which ATRX has been shown to interact is the heterochromatin protein HP1, a structural adapter, implicated in both gene silencing and supranucleosomal chromatin structure. This interaction has been observed by the yeast two-hybrid experiments,65 immunoprecipitation,62

Unusual Types of ␣ Thalassemia and indirect immunofluorescence,57 and the observations are supported by genetic manipulation of the system (SuVar39H1) by which HP1 (and possibly ATRX) is recruited to heterochromatin.66 ATRX may also be recruited to heterochromatin via other, similar interactions. For example it has been shown that in postmitotic neurons ATRX is recruited to heterochromatin via MeCP2 .67 The ADD domain of ATRX is another potential site for protein interactions that may recruit ATRX to chromatin. One component of this domain is a PHD zinc finger that, in other proteins, appears to mediate an interaction with chromatin.51 Recent studies have shown that the PHD finger domains of at least two chromatin-associated proteins (BPTF and ING1) specifically recognize H3 histone tails (H3K4me3).68,69 In addition, the ADD domain of Dnmt3a binds to the sequence-specific DNA-binding protein RP58.70 A similar interaction between ATRX and other sequence-specific DNA-binding proteins may be responsible for targeting ATRX to specific loci or alternatively recruiting specific loci to repressive heterochromatin. What role might ATRX play at chromatin? Like other members of the SNF2 family, both recombinant ATRX71 and endogenous ATRX (isolated from cells by immunoprecipitation)63 have been shown to have ATPase activity and weak nucleosome remodeling activity.63 The ATRX protein (complex) exhibits an impressive ability to move (translocate) along double-stranded DNA63 (and Mitson et al. in preparation), suggesting that ATRX may be able to move nucleosomes to facilitate access of transcription factors or DNA modifying enzymes, for example. Identifying the preferred substrate that is associated with maximal ATPase activity and the assay best suited to reveal the manner by which ATRX interacts with chromatin requires further work. Finally a variety of other potential protein interactions with ATRX may be relevant. The strongest candidate is the protein Daxx,63 which has been implicated in both apoptotic pathways and in the regulation of transcription. It has also been shown that the ADD domain of Dnmt3a interacts with the histone deacetylase HDAC1.70 Interestingly, the PHD motif of Mi2b also binds HDAC1, and it is possible that this component of the ATRX-associated ADD domain may also bind HDAC. In a directed use of the yeast twohybrid system, Cardoso et al.72 investigated the interaction of ATRX with a variety of heterochromatin-associated proteins. An interaction was demonstrated between ATRX (475–734) and the SET domain of polycomb group protein EZH2, an enzyme that modifies chromatin. More work is required to substantiate the importance of these observations in vivo.

Functional Consequences of Mutations Some clues to the normal role of ATRX may come from observing what happens when the gene is mutated. Per-

311 haps the most striking observation is that mutations in ATRX are associated with changes in gene expression (hence ␣ thalassemia) but the mechanism underlying this is currently unknown. The effects of ATRX mutations on the chromatin structure of the rDNA arrays located at the tips of acrocentric chromosomes have been studied. Although no gross changes in DNase l, micrococcal nuclease, or endonuclease accessibility were detected, striking differences were noted in the pattern of rDNA methylation between normal controls and patients with ATR-X syndrome.64 In normal individuals, approximately 20% of the transcribed units were methylated, whereas in ATR-X patients, rDNA genes were substantially unmethylated. The hypomethylated regions in ATR-X individuals localized within the CpGrich region of the rDNA repeat, which contains the transcribed 28S, 18S, and 5.8S genes and resembles a large CpG island. Because ATRX is also associated with heterochromatin, which contains a substantial proportion of the highly repetitive DNA in mammalian genomes, these methylation studies were extended to other repetitive sequences containing CpG dinucleotides known to be epigenetically modified by methylation. In this way, two additional sequences were identified that were abnormally methylated in ATR-X patients. Y-specific repeats (DYZ2) were almost all methylated in ATR-X patients, whereas approximately 6% were unmethylated in peripheral blood of normal individuals. Subtle changes in the pattern of methylation were also observed in the TelBam3.4 family of repeats, which are mainly found in the subtelomeric regions. Perturbations in CpG methylation have been observed with mutations in another mammalian SWI-SNF protein PASG (also known as Lsh and HELLS)73 and its plant homolog, DDM1.74 This raises the possibility that ATPasedependent chromatin-remodeling activities are involved in the establishment or maintenance of DNA methylation and that different SWI/SNF proteins (e.g. ATRX and PASG) may be required for different chromatin environments. To date, no change in the pattern of methylation has been detected in the ␣-globin gene cluster to explain the presence of ␣ thalassemia.

Summary of the ATR-X Syndrome Despite considerable progress in defining the phenotype in ATR-X syndrome, describing the properties of the protein and the consequence of mutations, little is known about the function of this protein. In particular, its role in ␣–globin expression remains elusive. A key puzzle to be solved is how a protein that associates with heterochromatin, and is required for a normal pattern of DNA methylation at repetitive DNA, influences the expression of euchromatic genes. Further progress needs to be made to identify ATRX

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¨ Figure 15.11. (a) May-Grunwald-Giemsa–stained peripheral blood smear from an elderly woman with ATMDS (original magnification, 400X). Many red cells are hypochromic, microcytic, or both, whereas others look relatively normal. As expected, the red blood cell distribution width (RDW) was elevated (19%; normal range, 11%–15%) in this patient. Iron studies were unremarkable. (b) Brilliant cresyl blue–stained peripheral blood smear from a 56-year-old man with ATMDS who had characteristic microcytic, hypochromic red cell indices: in this case, an MCH of 19 pg and MCV of 66 fL (original magnification, 600X). Abnormal inclusion-containing “golf ball cells” are easily distinguished from normal reticulocytes and red cells and confirm the presence of HbH (␤-globin tetramers.) In this case, supravital staining was performed by incubating fresh blood for 24 hours with 1% brilliant cresyl blue in 0.9% normal saline. (c) and (d) Bone marrow samples from patients with ATMDS showing dyserythropoietic erythroblasts including binucleate cells. (See color plate 15.11.)

target genes and understand the role it plays at these loci.

AN ACQUIRED FORM OF ␣ THALASSEMIA ASSOCIATED WITH MYELOID MALIGNANCY (ATMDS SYNDROME) Occasionally, patients who previously exhibited normal erythropoiesis will develop an acquired form of ␣ thalassemia, which most commonly arises within the context of hematological malignancy.75 The first cases of this “acquired hemoglobin H (HbH) disease” were described in 1960.76,77 Over the past half century, it has become clear that all cases of acquired ␣ thalassemia arise in the context of an underlying clonal disorder of hematopoiesis, most commonly a form of the myelodysplastic syndromes (MDS) as defined by the latest World Health Organization hematopoietic neoplasia classification scheme.78 Therefore, this condition is now commonly referred to

as ␣ thalassemia–myelodysplastic syndrome (ATMDS; Mendelian Inheritance in Man (OMIM) #300448).79 ]. Although other acquired hemoglobin synthetic defects such as acquired ␤ thalassemia or perturbations in the level of HbF and HbA2 can complicate MDS or other chronic myeloid disorders, these appear to be less common than ␣ thalassemia, and they have not received a specific syndromic designation.80–83 The most dramatic and easily recognized ATMDS phenotype is that of a severe form of HbH disease, characterized by striking hypochromic microcytic anemia, numerous HbH inclusions detectable by supravital staining of peripheral blood (Fig. 15.11), and measurable amounts of HbH in the hemolysate.84 There are also milder forms of ATMDS, in which rare HbH-containing erythrocytes can be detected on the peripheral smear. In these cases, inclusion-containing cells make up less than 1% of anucleate erythrocytes, and HbH represents such a small fraction

Unusual Types of ␣ Thalassemia of the total hemoglobin that it is not easily demonstrable by routine chromatographic or gel electrophoretic techniques.85 Some of these mildly affected patients – along with others with more severe forms of ATMDS who have been recently transfused with red blood cells from healthy donors – exhibit normocytic or even macrocytic red cell indices, and thus may not be recognized by clinicians. In the past, most mildly affected ATMDS cases were detected incidentally when a supravital stain of the peripheral blood was performed for another reason, usually to evaluate the reticulocyte count. Many clinical hematology laboratories now quantify reticulocytes by dye-based flow cytometric methods rather than by supravital staining, and thus it is likely that many ATMDS cases, especially those cases with small numbers of HbH cells, now go undetected.86

General Clinical Features More than 80 well-documented patients with ATMDS have been described to date; a global ATMDS Case Registry is maintained in Oxford, and can be viewed at: http://www.imm.ox.ac.uk/groups/mrc molhaem/ home &break;pages/Higgs/ATMDS.xls. Case clustering among clinical groups who have developed an interest in the disorder suggests that the condition is likely to be underreported. Additionally, most described patients with ATMDS have been of European origin (88% of cases in the Registry), yet current understanding of the molecular pathology of the disease (see later) does not offer a reason for such an imbalanced geographical distribution. Instead, the global distribution of reported cases may reflect detection and reporting bias. Patients diagnosed with MDS who have microcytic red cell indices, and who originate from regions of the world where inherited forms of thalassemia are common, might reasonably be assumed to have a previously undetected inherited disorder of hemoglobin synthesis. Regardless, in all ATMDS cases for which archival data are available, there has been no evidence of a preexisting inherited form of ␣ thalassemia.87 At present, other than the disease-defining red cell changes, there are no clinical features that clearly distinguish ATMDS from MDS more generally, although there is a greater male predominance than is observed in chronic myeloid disorders overall (85% of ATMDS cases have been men, whereas the male/female ratio in MDS in general is ∼1.5:1).75 The reason for this dramatic sex imbalance in ATMDS is unclear. Patients with ATMDS are diagnosed at similar ages to patients with chronic myeloid disorders who lack thalassemia (median age, 68 years), have similar marrow findings and karyotypic results, have a median survival typical for MDS overall (2–3 years), and die of the same complications – primarily death from infection and, in approximately 25% of cases, of complications of progression to acute myeloid leukemia.75 When ATMDS progresses to acute myeloid leukemia, and the ability of hematopoietic

313 cells to differentiate is further impaired, some patients still continue to have detectable HbH, whereas in other cases the HbH inclusions disappear entirely. Most patients with acquired ␣ thalassemia have MDS (>80%), but a few have chronic idiopathic myelofibrosis or another form of myeloproliferative disease, and rarely patients with acquired HbH disease present with acute myeloid leukemia without an apparent antecedent chronic myeloid disorder.75 There was also a single case report of acquired ␣ thalassemia in association with TdTpositive acute lymphoblastic leukemia; this case predated the modern era of molecular diagnostics and leukemia immunophenotyping.88 No other cases of acquired thalassemia have been described in association with a lymphoproliferative disorder or plasma cell dyscrasia.

␣/␤ Globin mRNA and Globin Chain Synthesis Ratios in Patients with ATMDS The ␣- to ␤-globin mRNA ratio has only been studied in a few patients with ATMDS, but it was severely reduced in all these cases (range, 0.06–0.50).89 Likewise, the reticulocyte ␣/␤-globin chain synthesis ratio was similarly reduced in all 25 patients in whom this ratio has been analyzed (mean 0.28, range 0.05–0.67; normal 0.9–1.2). In the most severely affected individuals, ␣-chain synthesis is almost completely abolished (Fig. 15.12). The ␣/␤-chain synthesis ratio may vary quite considerably during the course of the disease, and this is reflected in varying proportions of HbHcontaining cells on serial supravital staining.89 In contrast to the typical findings in ATMDS, the ␣/␤-chain synthesis ratio was reported as elevated in a small series of MDS patients without thalassemic red cell indices.90

Red Cell Indices and Hematological Findings in ATMDS Patients with ATMDS are always anemic at presentation (mean hemoglobin 8.5 g/dL), with a reduced red blood cell count (mean 4.3 × 1012 /L) and markedly hypochromic and microcytic red cell indices (average MCH 22 pg and MCV 75 fL).75 These abnormalities are even more striking when compared with a control population of patients with MDS without thalassemia, who at presentation tend to have slightly higher than normal MCH and MCV values (average MCH 31 pg and MCV 97 fL) (Fig. 15.13). The red cell distribution width has been increased in all ATMDS patients studied. These hematological findings are reflected in the typical peripheral blood red cell morphology (Fig. 15.11). All patients examined have had at least some hypochromic microcytic red cells, and in many cases there is marked anisopoikilocytosis. In some cases, it appears that there are at least two populations of red cells: some are quite well hemoglobinized and of relatively normal appearance, whereas others contain scant hemoglobin and are often

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little more than “ghost cells.” This picture is likely to be a consequence of mixed clonality of the bone marrow in MDS and ATMDS, with residual normal hematopoietic cells coexisting with dysplastic clones, including (in ATMDS) progenitor cells with thalassemic erythrocyte progeny that have HbH inclusions and synthesize little or no normal hemoglobin.

Hemoglobin Analysis in ATMDS All patients with ATMDS by definition have detectable amounts of HbH in their peripheral red blood cells at some Mean Corpuscular Volume (MCV) in MDS and ATMDS

stage of the disease. In general, levels of HbH measured by column chromatography or agarose gel electrophoresis are lower than the proportion of anucleate erythrocytes that contain HbH inclusions on a supravital stained-peripheral blood smear. At the time of first detection of ATMDS, the proportion of red cells containing HbH inclusions after incubation with brilliant cresyl blue varies between less than 0.1% and 95% (median, 30%). HbH as a proportion of total hemoglobin ranges between 1% and 57% (median, 15%). Often it is not possible to obtain accurate estimations of HbH in a given ATMDS case because of prior blood transfusions. In some patients, trace amounts of Hb Bart’s

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Figure 15.13. Distribution of red cell indices in patients with ATMDS compared with a more general population of patients with MDS. (A) MCV at the time of initial presentation in ATMDS (n = 55) vs. MDS (n = 282). Boxes enclosed 25th through 75th percentiles; middle bar denotes the median; whiskers outline the 10th through 90th percentiles. (B) MCH in ATMDS vs. MDS. (C) Scattergram of MCH vs. MCV distribution in patients with ATMDS (open circles) vs. MDS (closed circles). Dashed lines highlight limits of normal range reported in most clinical laboratories. (Reprinted from ref. 75 with permission.)

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Unusual Types of ␣ Thalassemia (␥ globin tetramers) can be found. In the presence of these high oxygen affinity hemoglobins, the whole blood oxygen dissociation curve is shifted to the left, exacerbating the physiological consequences of anemia.1 Levels of HbA2 and HbF in ATMDS are variable and inconsistently abnormal.

The Molecular and Cellular Basis for ATMDS For many years, the molecular etiology of ATMDS was mysterious, but recently, considerable progress toward a mechanistic explanation has been made.91 It is now clear that most patients with ATMDS have an acquired, somatic point mutation or an mRNA intron–exon splicing abnormality involving ATRX, the X-linked gene that encodes the chromatin remodeling factor ATRX (Fig. 15.8).92,93 This exciting finding provides a partial explanation for the ATMDS phenotype. Although almost all of the common inherited forms of ␣ thalassemia worldwide are due to deletions or, less commonly, point mutations involving the ␣-globin gene cluster and its major upstream regulatory element (HS-40), this class of mutation is not typically found in ATMDS.94 ATMDS cases selected for initial molecular evaluation were those with less than 10% of the normal levels of ␣-globin mRNA and ␣-globin synthesis. This extreme degree of impairment of ␣-globin synthesis is even greater than one typically sees in patients with only a single functional ␣globin gene, suggesting that all four ␣ genes are downregulated in ATMDS. Detailed structural analysis of the ␣-globin genes and approximately 130 kb of their flanking regions on chromosome 16p in these cases consistently revealed no abnormalities, even in the most severely affected patients.79 In addition, direct sequencing of the globin genes was unremarkable. In a single reported ATMDS case, the telomeric end of chromosome 16p, including the ␣-globin cluster, was deleted in a clonally restricted fashion as part of a complex MDS-associated karyotype with numerous large chromosomal rearrangements.95 This case – in which the extent of the deletion was mapped by FISH – proved exceptional, and no other ATMDS cases have been found to harbor rearrangements of chromosome 16p. Because the presence of multiple cryptic cis-acting mutations seemed an unlikely basis for ATMDS, investigators’ attention focused next on the possibility of a trans-acting mutation, either involving a factor that normally controls ␣-globin gene expression or a novel gene that exerts a dominant negative effect. Further support for the possibility of a trans-acting defect came from the generation of an interspecific hybrid between bone marrow from an ATMDS patient and adenine phosphoribosyl transferase (APRT)–deficient mouse erythroleukemia (MEL) cells.96 (APRT is encoded on chromosome 16q24 in humans.) After selection for restored APRT activity, such hybrids retained human chromosome 16 derived from the ATMDS patient in a murine erythroid background, and the human chromosome directed normal

315 levels of human ␣-globin synthesis. Although these findings would appear to exonerate chromosome 16p in ATMDS, it is also possible that the human cell that originally fused with the MEL cell could have been from an unaffected hematopoietic clone in view of the mixed clonality of MDS. Thus, even this result was not considered definitive evidence of a trans-acting defect. The major clue that led directly to identification ATRX mutations as the specific trans-acting molecular defect underlying most ATMDS cases came from a cDNA microarray experiment, in which granulocyte RNA from a patient with severe, newly diagnosed ATMDS was compared with pooled granulocyte RNA from seven healthy donors.92 Granulocyte RNA was chosen because it is likely to be enriched for mutant clones in chronic myeloid neoplasia, compared with other easily isolated blood cell populations that might also have been suitable for genetic analysis. Strikingly, in this pilot cDNA microarray study, ATRX was one of the genes with the very lowest expression in the patient with ATMDS compared with the healthy controls. Sequencing of granulocyte-derived genomic DNA from the ATMDS patient demonstrated a G>A point mutation in the canonical splice donor motif of ATRX intron 1, probably resulting in nonsense-mediated decay of the ATRX transcript.92 In contrast to granulocytes, the patient’s buccal cells were ATRX wild type, supporting the clonally restricted nature of the mutation. In view of the important role that ATRX mutations play in congenital ATR-X syndrome (including an association with variable degrees of ␣ thalassemia), ATRX mutations seemed biologically plausible as the cause of ␣-globin downregulation in ATMDS.97 Precisely how mutations in ATRX result in down-regulation of ␣-globin expression is the subject of active investigation, and our current understanding is discussed in detail elsewhere in this text. Subsequent molecular analysis of archival material from 18 other ATMDS patients revealed ATRX mutations or premRNA splicing abnormalities in almost all cases (14 of 19 total).93 There is considerable allelic heterogeneity (Fig. 15.8). Reassuringly, when new ATMDS cases with substantial amounts of HbH have come to light, they have also consistently had point mutations involving evolutionarily conserved regions of ATRX.98–100 This is true not only for men, who have hemizygous ATRX mutations, but also for the rare women with ATMDS, who can be demonstrated to have heterozygous mutations in ATRX.100 Presumably, the retained wild-type ATRX allele in these women is located on the copy of the X chromosome that was inactivated during embryogenesis, and it is not expressed at a high enough level to “rescue” ␣-globin expression in the progeny of the ATRX-mutant clone.

Implications of ATRX Mutations in ATMDS In general, patients with ATMDS have a more severe form of ␣ thalassemia than boys with inherited ATR-X syndrome.

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More than 30% of described ATMDS patients have had more than 50% of circulating erythrocytes containing HbH inclusions, whereas 90% of boys with ATR-X syndrome have less than 10% HbH-containing cells.93,101 Even when an identical or similar ATRX mutation is detected in the germline in ATR-X syndrome and as a somatic change in the hematopoietic cells in ATMDS, the thalassemic phenotype is consistently more severe in ATMDS than in ATR-X. For instance, a boy with germline c.576G>S, p.L192F ATRX mutation had 0.1% HbH, whereas a man with ATMDS who had the identical mutation as an acquired phenomenon had 50% HbH. The reason for this striking difference is unclear and is suspected to be dependent on the cellular context, perhaps pointing to an interaction between ATRX mutations and a disturbed genetic or epigenetic background in MDS cells that is due to other mutations not present in boys with inherited ATR-X syndrome. In a few patients with ATMDS, an ATRX point mutation has not been detected, despite careful investigation. Most of these patients have had less than 10% HbH, and issues related to sensitivity of the tools used for mutation detection may account for the negative results, although it remains possible that mutations in another as yet undefined trans-acting factor can cause an ATMDS phenocopy. Direct fluorescent dye chemistry-based DNA sequencing can only detect mutations at a level of approximately 20%–30% mutant DNA in a wild-type background, and may miss acquired mutations that are present at lower levels of clonality.93 Other mutation screening techniques, such as denaturing high-performance liquid chromatography and denaturing-gradient gel electrophoresis, offer higher analytical sensitivity (down to ∼1% mutant DNA), but these techniques also do not detect ATRX mutations in all ATMDS cases. In some ATMDS cases, only genomic DNA has been available for screening, and it is possible that some of these cases actually have an alteration in ATRX premRNA splicing, as has been described in several ATMDS cases in which both DNA and RNA were accessible for analysis.92,93,98,102 Supravital staining of unselected MDS cases (i.e., without regard for red cell indices) demonstrates small numbers of HbH inclusion-containing cells in 8% of cases; these cells are not present in other forms of anemia not associated with a clonal myeloid disorder, and therefore are not simply a byproduct of disordered erythropoiesis.85 It seems likely that ␣ thalassemic clones arise fairly commonly in the marrow milieu of MDS, for unclear reasons, and that these changes alone do not give the cells that bear them any sort of competitive advantage. Additional mutations are probably required to provide a clone that happens to be thalassemic with a survival or proliferative advantage, leading ultimately to clonal dominance, and these secondary changes may account for the more dramatic ATMDS phenotypes.

Remaining Questions About ATMDS One of the most exciting prospects for the discovery of ATRX mutations in ATMDS is that this finding may yield some new general insights into the pathobiology of MDS. Such advances would be welcome, as MDS has proven to be a rather intractable disorder, both from the standpoint of analysis of disease mechanism and from a therapeutic perspective.103 Since the discovery of ATRX mutations in ATMDS, candidate gene analysis in MDS has focused on genes encoding known or suspected ATRX binding partners (HP1, DAXX, EZH2) or homologous SWI/SNF2 family members implicated in leukemogenesis (HELLS/LSH/SMARCA6), but these studies have thus far been unrevealing. The reason for the sex imbalance in ATMDS remains unclear. The fact that ATRX is an X-linked factor can provide only a partial answer, as other conditions that are caused by acquired mutations in genes on the X chromosome (e.g., paroxysmal nocturnal hemoglobinuria) do not display such a striking sex distinction. The fact that germline ATRX mutations are known to be associated with diverse alterations in DNA methylation across the genome has particular relevance in MDS, in which methylation changes are common and which some investigators have labeled an “epigenetic disease.”64,104,105 Two of the first three drug therapies approved by the United States Food and Drug Administration for the treatment of MDS – azacitidine (approved in 2004) and decitabine (approved in May 2006) – are aza-substituted cytosine nucleoside analogs that irreversibly bind to DNA methyltransferase 1 (DNMT1) and inhibit its enzymatic activity.106–108 DNMT1 inhibition that persists through the cell cycle results in alteration in the 5
methylation status of cytosine in CpG dinucleotides in gene promoters, with subsequent changes in gene expression. Anecdotally, one ATMDS patient treated with hypomethylating therapy experienced unusually profound and prolonged marrow aplasia, but it is uncertain whether this is merely a coincidence or is mechanistically related.99

Summary of ATMDS Syndrome Over the past few years, considerable progress has been made in understanding the molecular basis for the rare syndrome of ATMDS, when ␣ thalassemia is acquired as a “passenger” mutation (rather than a causative mutation) in MDS. We now know that the associated ␣ thalassemia results from mutation in the X-encoded ATRX gene, which is also the disease gene in the inherited condition ATRX syndrome. A remaining puzzle is why the same mutation in ATRX causes a mild form of thalassemia, whereas in ATMDS it causes severe HbH disease. It seems likely that the ATRX mutation is somehow made more severe when it interacts with one or more of the common genetic or

Unusual Types of ␣ Thalassemia epigenetic mutations in MDS. Understanding the mechanism underlying the severe ␣ thalassemia in this fascinating syndrome may shed some light on the molecular basis of the common forms of MDS.

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SECTION FOUR

THE ␤ THALASSEMIAS Bernard G. Forget

Over the years, study of the thalassemia syndromes has served as a paradigm for gaining insights into the factors that can regulate or disrupt normal gene expression. The thalassemias constitute a heterogeneous group of naturally occurring, inherited mutations characterized by abnormal globin gene expression resulting in total absence or quantitative reduction of ␣- or ␤-globin chain synthesis in human erythroid cells. ␣ Thalassemia is associated with absent or decreased production of ␣-chains, whereas in the ␤ thalassemias, there is absent or decreased production of ␤-chains. In those cases in which some of the affected globin chain is synthesized, early studies demonstrated no evidence of an amino acid substitution. In all cases in which genetic evidence was available, the thalassemia gene appeared to be allelic with the structural gene encoding ␣- or ␤-globin. The elucidation of the nature of the various molecular lesions in thalassemia has been a fascinating process, and full of surprises. Increase in our knowledge of the molecular basis of ␤ thalassemia has closely followed and depended on progress and technical breakthroughs in the fields of biochemistry and molecular biology. In particular, recombinant DNA and polymerase chain reaction– based technologies have contributed to a virtual explosion of new information on the precise molecular basis of most forms of thalassemia. The accrual of this knowledge has, to a great degree, paralleled the acquisition of new, detailed information on the structure, organization, and function of the normal human globin genes, as described in the preceding chapters. Historically, as new techniques have been developed for the study of protein synthesis and gene expression, they have been rapidly applied to the study of normal human hemoglobin synthesis and the abnormalities associated with abnormal globin gene expression in thalassemia. As a result, there has gradually emerged a progressively clearer and increasingly complex picture of the molecular pathology of this group of genetic disorders. One major conclusion that was drawn as this mystery unfolded was that a relatively limited number of pheno-

types can result from a surprisingly large number of varied genotypes. The chapters that follow will describe in detail the knowledge and progress that have accrued over the last 3– 4 decades on the understanding of the pathophysiology, the molecular genetics, and the clinical manifestations and management of the various ␤ thalassemia syndromes and related disorders. There are many primary and secondary causes for the anemia observed in ␤ thalassemia. It is easy to understand how reduced synthesis of ␤-globin chains of Hb A (␣2 ␤2 ) will result in an overall deficit of hemoglobin accumulation in red cells and cause a hypochromic, microcytic anemia with a low mean corpuscular hemoglobin concentration in affected erythrocytes. This is true in both the heterozygous and homozygous states. In the homozygous state, however, another pathophysiological process worsens the anemia and is responsible for the major clinical manifestations in the syndrome referred to as ␤ thalassemia major or Cooley’s anemia. The continued synthesis in normal amounts of normal ␣-globin chains results in the accumulation, within the erythroid cells, of excessive amounts of these chains. Not finding complementary globin chains with which to bind, these chains form insoluble aggregates and precipitate within the cell, causing membrane damage and premature destruction of the red cells. The ␣-chain aggregates are called inclusion bodies or, perhaps improperly, Heinz bodies. In contrast to true Heinz bodies, which are made up of total precipitated hemoglobin tetramers, these inclusion bodies have been shown to consist only of ␣-globin chains, which do have some attached heme in the form of hemichromes. The process of inclusion body formation occurs not only in mature erythrocytes, but in particular in the erythroid precursor cells of the bone marrow. As a result, there is extensive intramedullary destruction of erythroid precursor cells, a process that is called ineffective erythropoiesis. The severity of the clinical manifestations in ␤ thalassemia generally correlates well with the size of the free ␣-chain pool and the degree of ␣- to non-␣-globin chain imbalance. Therefore, the fortuitous coinheritance of ␣ thalassemia together with homozygous ␤ thalassemia reduces the degree of ␣- to non-␣-globin chain imbalance and leads to a milder clinical course. Similarly, coinheritance of ␤ thalassemia with conditions that are associated with increased levels of synthesis of ␥ -chains of HbF (␣2 ␥ 2 ) leads to less imbalance between ␣- and non-␣-globin chain synthesis, resulting in decreased formation of ␣-chain inclusion bodies, increased effective production of red cells, and their prolonged survival in the circulation. The clinical course in most cases of homozygous ␤ thalassemia is severe. Although anemia is not evident at birth, severe hypochromic, microcytic, hemolytic anemia develops during the first year of life and a regular transfusion program must be undertaken to maintain an adequate circulating hemoglobin level. The clinical manifestations 321

322 of homozygous ␤ thalassemia in childhood have changed considerably over the last 3–4 decades, owing to changes in the philosophy and practice of transfusion therapy. With modern transfusion therapy, most children will develop normally, with few or no skeletal abnormalities, and will have a reasonably good quality of life. To avoid iron overload from transfusional hemosiderosis, transfusion therapy is usually coupled with a vigorous program of iron chelation, typically using parenterally administered desferrioxamine and more recently, orally effective iron-chelating agents. Although it is possible to maintain iron balance with such a management program, compliance is often difficult to achieve. Iron overload eventually develops in most patients and is the major cause of morbidity and mortality in young adults. The one therapy that is curative is bone marrow or stem cell transplantation, which is being increasingly practiced when feasible. The hope for the future is the development of even more effective oral iron-chelating agents and improved approaches to gene therapy. Studies of the molecular basis of ␤ thalassemia have demonstrated that the gene defects responsible for the disorder are quite heterogeneous. In contrast to ␣ thalassemia, in which deletions in the ␣-globin gene cluster account for most of the mutations, the molecular defects associated with ␤ thalassemia are usually point mutations involving only one (or a limited number of ) nucleotide(s), but resulting in a major defect of ␤-globin gene expression either at the transcriptional or posttranscriptional level, including translation. Practically every conceivable type of defect in gene expression has been identified in one form or another of ␤ thalassemia. Over 175 point mutations have been identified. Some deletion types of ␤ thalassemia have also been described. In cases of ␤ thalassemia in which ␤-globin gene expression is not totally absent (so-called ␤+ thalassemia), the ␤-chain that is synthesized is usually structurally nor-

Bernard G. Forget mal. There is a syndrome called dominant ␤ thalassemia in which a highly unstable, structurally abnormal ␤-globin chain is synthesized, resulting in inclusion body formation in the heterozygous state. The coinheritance of HbE (␣2 ␤2 glu26lys) with ␤ thalassemia is very prevalent in southeast Asia and results in markedly variable and heterogeneous clinical manifestations, the basis for which is poorly understood. Finally, there are a number of ␤ thalassemia-like disorders, called ␦␤ thalassemia and hereditary persistence of fetal hemoglobin, that are distinguished from the more typical forms of ␤ thalassemia by the presence of a substantial elevation of HbF in heterozygotes, as well as in homozygotes and compound heterozygotes. These disorders are usually due to deletions of different sizes involving the ␤globin gene cluster, although nondeletion types of these disorders have also been identified. The great heterogeneity of molecular lesions causing ␤ thalassemia may appear at first glance to create insurmountable problems in putting this knowledge to practical use in the form of prenatal diagnosis and genetic counseling. The availability of rapid and accurate polymerase chain reaction–based assays for the detection of specific mutations in small samples of DNA has resulted in a number of surveys for the detection of the prevalence of different ␤ thalassemic mutations in various population groups. The results of these surveys indicate that a given mutation is usually found only within one racial group and not another. Furthermore, a small number of different mutations, usually five or six, frequently accounts for 90% or more of the cases of ␤ thalassemia in a given population group. Thus, it is possible to devise efficient and precise prenatal diagnosis programs using DNA-based approaches. Such programs have led to a striking decrease in the number of births of infants with homozygous ␤ thalassemia in many countries where the disease is prevalent.

16 The Molecular Basis of ␤ Thalassemia, ␦␤ Thalassemia, and Hereditary Persistence of Fetal Hemoglobin Swee Lay Thein and William G. Wood

INTRODUCTION The ␤ thalassemias and related disorders are characterized by a quantitative reduction in the production of ␤-globin chains of HbA. More than 200 ␤ thalassemia alleles have now been characterized (http://globin.cse.psu.edu) involving mutations affecting any of the steps in the transcription of the ␤-globin gene, posttranscriptional processing of its pre-mRNA, or the translation of its mRNA into protein. The vast majority of simple ␤ thalassemias are caused by point mutations within the gene or its immediate flanking sequences, although small deletions involving the ␤ gene may also occur. If ␤-chain production is totally abolished by the mutation it is referred to as ␤0 thalassemia, whereas reduced output of ␤-chains (of normal structure) produces ␤+ thalassemia, with the mildest forms sometimes referred to as ␤++ or “silent” ␤ thalassemia. These common forms of ␤ thalassemias are inherited as haploinsufficient mendelian recessives. Some structurally abnormal ␤-chain variants are also associated with quantitative deficiencies of ␤-globin chain production and have a phenotype of ␤ thalassemia, in which case they are referred to as “thalassemic hemoglobinopathies,” for example, HbE (␤26 Glu→Lys). In others, the ␤-globin variants are so unstable that they undergo very rapid postsynthetic degradation giving rise to a functional deficiency. These hyperunstable ␤-chain variants act in a dominant negative fashion, causing a disease phenotype even when present in the heterozygous state, and hence have been referred to as “dominantly inherited ␤ thalassemia.”1 ␤ Thalassemia mutations that segregate independently of the ␤-globin cluster have been described in occasional families. In such cases, trans-acting regulatory factors are implicated.2–4 The ␦␤ thalassemias and hereditary persistence of fetal hemoglobin (HPFH) are disorders related to the ␤ thalassemias that also involve down-regulation of ␤-globin

gene expression. In ␦␤ thalassemia the ␦ gene is also affected and there is variable compensation from increased HbF production. These disorders result from more extensive deletions within the ␤-globin gene cluster, as do the deletion forms of HPFH in which HbF compensation is sufficient to minimize hematological abnormalities. Other forms of HPFH result from mutations in the promoters of the ␥ -globin genes that not only increase HbF production in adult life but are accompanied by reduced ␤-chain production. This chapter outlines the molecular mechanisms underlying the different types of ␤ thalassemia. The common denominator is absent or decreased synthesis of ␤-globin chains, resulting in the accumulation of excess ␣-globin chains that are responsible for the pathophysiology of the disorder. The severity of the phenotype is usually related to the degree of imbalance between ␣- and non-␣-globin chain synthesis, and the size of the free ␣-chain pool. Hence severity is related to the type of ␤ allele (␤0 , ␤+ , ␤++ ), ameliorated by an interacting ␣ thalassemia (by reducing the ␣-chain excess) and any increased production of ␥ -chains (that decrease the excess of free a ␣-chains by binding to them to form HbF). The molecular bases of ␤ thalassemia and related conditions have provided a paradigm for our understanding of much of human genetics. Detailed references to earlier work are not provided here but readers are referred to the chapters by Forget,2,5 Thein,6 and Wood7 in the first edition of this book, as well as to the comprehensive monograph by Weatherall and Clegg.4

The ␤ THALASSEMIAS Nondeletion Forms of ␤ Thalassemia These defects account for the majority of the ␤ thalassemia alleles. They involve single base substitutions, small insertions or deletions within the gene or its immediate flanking sequences, and affect almost every known stage of gene expression. Allele frequency varies widely from one population to another but within any population there are usually a small number of common alleles together with many alleles that are rare for that population (see Chapter 15). They are listed in Table 16.1 according to the mechanism by which they affect gene function: transcription, RNA processing, or RNA translation. The mutations have been cataloged by Huisman et al.8 and the updated listing is accessible electronically through the Globin Gene Server Website:9 http://globin.cse.psu.edu. Heterozygotes have minimal anemia but hypochromic (mean corpuscular hemoglobin [MCH] 18–24 pg), microcytic (mean corpuscular volume [MCV] 65–80 fL) red blood cells. They are characterized by an increased proportion of HbA2 (normal 30

134 52 63 12 7

35.45 38.81 47.01 8.96 5.22

Blood transfusion (U) 20 Unknown

231 115 47 22 11 12 24

61.11 49.78 20.35 9.52 4.76 5.19 10.39

First menstruation (y) 10–14 15–19 20–24 None No record

modest splenomegaly, despite having hemoglobin levels that are only 2–3 g/dL higher than those with more severe disease. Yet some of these patients pass through a normal puberty with good growth and sexual development, whereas others who have been asymptomatic through early childhood have delayed puberty with or without defective growth. Detailed studies in a group of children with HbE–␤ thalassemia in Sri Lanka, performed over 10 years,

Hemoglobin E Disorders have emphasized the instability of the phenotype during early development.31,40,42

Complications Expanded Erythropoiesis. Erythrpoiesis is massively increased to 10–15 times normal because anemia stimulates erythropoietin production. Extensive erythropoiesis can be found in the liver, spleen, and bone and in extramedullary sites. Erythropoietic masses in the spinal canal can cause spinal cord compression and paraplegia, and when they occur intracranially convulsions may result.43–45 Massive erythropoiesis leads to fragility and distortion of the bones and decreases bone density because of osteoporosis and osteomalacia, as observed in irregularly transfused thalassemia major patients.46 Bone marrow expansion also increases blood volume, leading to high-output cardiac failure. Iron Overload. Iron overload occurs without exception.47 Excessive iron accumulates because of blood transfusions and enhanced gastrointestinal absorption.48 The skin is darkened and iron deposition occurs in the bone marrow, liver, spleen, heart, pancreas, and elsewhere.49–51 Arrhythmias are not as frequently encountered as in thalassemia major and although liver fibrosis from iron overload is common, ascites and other signs of cirrhosis are very rare. Diabetes mellitus secondary to iron deposition in the pancreas frequently develops in untreated adult patients if they live long enough.52 We have observed a terminal wasting stage in some patients who lived into their third and fourth decades. These patients developed more skin pigmentation, poor appetite, weight loss, and increasing anemia, and eventually died. This is believed to result from organ failure caused by uncontrolled tissue oxidation from chronic, severe iron overload. As iron overload is a constant complication of thalassemia and iron is a strong oxidant, reduced levels of antioxidants such as vitamins C and E are common in these patients.53 Heart Disease. Half of the patients with HbE–␤ thalassemia in Thailand die of heart failure. This is associated with failure of other organs, delayed growth and sexual maturation, hepatomegaly, and endocrinopathies. Organ failure results from iron deposition in the heart and other tissues.49–52 Myocardial iron deposition is mostly slight, occurring primarily as small granules in perinuclear areas, with later accumulation throughout the fibers, predominantly subepicardial, occasionally subendocardial.54 The small amount of iron deposited in the heart is in marked contrast to enormous iron deposition in the liver and pancreas. Other causes of death are anemia, infection, constrictive pericarditis, and pulmonary artery occlusion. Cardiomegaly is proportional to the severity of anemia and systolic murmurs are frequently present.55–57 Chronic pericarditis following upper respiratory tract infection is frequently encountered, more so in splenec-

425 tomized patients. A pericardial rub may be detected, often transiently. Intractable pericardial effusion may follow, causing cardiac tamponade and failure, and requires aspiration. In a very few cases chronic constrictive pericarditis develops, requiring surgical intervention. Histological examination of the pericardium shows nonspecific pericarditis.54 Viral infection has been suspected as the cause of this pericarditis but has not been proven. Infections. Prospective studies showed increased susceptibility to viral, bacterial, and fungal infection that may be causes of death in severe HbE–␤ thalassemia.58–61 In splenectomized patients, septicemia can be very acute and overwhelming, leading to death in a short period. Gramnegative and Gram-positive bacteria are frequent causes of septicemia. Fungal infection with Pythium can lead to arterial occlusion and gangrene of the legs.60,61 Investigators have not yet pinpointed the mechanisms that cause increased susceptibility to infections but iron overload and severe anemia may be involved. Recent studies have suggested that patients with HbE–␤ thalassemia may be more prone to infection by both Plasmodium falciparum and Plasmodium vivax malaria and that those who have undergone splenectomy may be even more susceptible. The clinical significance of these findings remain to be determined.62 Jaundice and Gallstones. Stones are found in approximately 50% of patients.63 As discussed earlier, they occur most frequently in a genetic subset of individuals with very high bilirubin levels in some populations.38,39 For the detection of biliary calculi, ultrasonography is more sensitive than oral cholecystography and plain abdominal films. Cholecystitis and ascending cholangitis may occur with abdominal pain, fever, and increasing jaundice.64 Antibiotics alone are usually not effective and cholecystectomy is necessary. Hypertension, Convulsions, and Cerebral Hemorrhage. After multiple blood transfusions some patients in Thailand developed hypertension, convulsions, and cerebral hemorrhage after transfusion of 2 U or more of blood and many of them died.65 This complication may develop as late as 2 weeks after multiple transfusions, suggesting that blood volume overload is not the cause of hypertension. Monitoring blood pressure during and after blood transfusions with prompt antihypertensive intervention has reduced deaths from this complication. This complication has not been reported in other populations. Hypoxemia. A great majority of splenectomized HbE–␤0 thalassemia patients in Thailand develop hypoxemia with low arterial pO2 .66 Platelet counts in splenectomized thalassemia patients are double that of nonsplenectomized patients; young and larger platelets are also observed in the absence of the spleen. Platelet microaggregates have been detected in the circulation of these splenectomized patients.67 One hypothesis for the pathogenesis of hypoxemia in HbE–␤0 thalassemia is that platelets increase in

426 number, are younger and more active after splenectomy, and aggregate in the circulation and in the pulmonary vasculature. Substances released during platelet aggregation may cause constriction of the terminal bronchioles leading to decreased oxygenation and hypoxemia. A canine model showed that induction of platelet aggregation in the circulation reproduced the hypoxemia observed in splenectomized thalassemia patients. Administration of aspirin to inhibit platelet aggregation reduces the degree of hypoxemia in the majority of cases,68 suggesting that these agents should be routinely given to splenectomized patients with HbE–␤ thalassemia. Interestingly, the combination of pulmonary hypertension and consequent hypoxemia has not been observed so frequently in other populations, suggesting that other factors may be involved in the Thai population. Thromboembolism. Autopsy findings in a large number of patients with HbE–␤ thalassemia revealed striking pulmonary artery occlusion. Serial sections of the lungs revealed in some patients as many as 24 lesions/cm2 , the distribution of which indicated an embolism.69,70 Thromboembolism in HbE–␤ thalassemia seems to involve platelets, a reactive thalassemic red cell surface, coagulation factors, and abnormal endothelium, but this problem is still under study. Autoimmune Hemolytic Anemia. Some patients develop autoimmune hemolytic anemia with worsening anemia and a positive Coombs’ test.71 The condition is responsive to corticosteroids. Studies of HbE thalassemia patients with this condition showed that their red cell surface is an active site of complex immune reactions that are likely to be associated with many pathophysiological phenomena.72

Treatment Because HbE–␤ thalassemia has such a variable phenotype and patients with this disorder, probably because they have relatively lower levels of HbF and reflecting the oxygen affinity of HbE, are able to adapt to anemia better than patients with other forms of ␤ thalassemia intermedia, it is vital to observe young children with this condition after presentation for a reasonable period before deciding on the best approach to management. It is important to remember that they may present with a particularly low hemoglobin level consequent to a recent infection and it is particularly important therefore not to establish them on a regular transfusion until their steadystate hemoglobin level and level of growth and degree of splenomegaly has been assessed. Particularly in areas where malaria is endemic it is important to exclude chronic P. vivax infection as a possible cause of rapidly progressive splenomegaly. The hemoglobin level alone should not be the major factor in initiating transfusion. Rather, the broader picture should be taken into account with particular attention to growth failure, lack of activity, and the earlier

Suthat Fucharoen and David J. Weatherall appearance of skeletal change. If it is clear that the patient will require regular transfusion the regimen to be followed, including chelation, is similar to that for the management of ␤ thalassemia major (see Chapter 17). Those who do not require transfusion should be maintained on folic acid supplements and advised about the early treatment of infective episodes. Although some patients with increasing splenomegaly and evidence of hypersplenism may benefit from splenectomy, this should be avoided when possible because of the particularly high risk of infection. Patients who do not require regular transfusion have serum ferritin estimations at least twice per year. Increased iron levels should be controlled by intermittent courses of chelating agents to maintain safe ferritin levels (see Chapter 17). Hydroxyurea therapy may increase HbF levels,73 although recent studies in other populations have shown that this effect is not great, even when combined with erythropoietin.74 For those who present early with severe disease bone marrow transplantation remains an important option75,76 (see Chapters 31 and 32). Rapidly expanding extramedullary hemopoietic masses, particularly involving the brain or spinal cord, require urgent treatment by blood transfusion, hydroxyurea, or possibly, radiotherapy. Limited experience in those with profound jaundice due to genetic inability to conjugate bilirubin suggest that at least in some cases very low doses of phenobarbitone may be helpful.

Conclusions HbE-␤ thalassemia is a major public health problem in Southeast Asia and in other Asian countries. Although some progress has been made toward a better understanding of its pathophysiology and clinical management a great deal remains to be learned. Recent work has made it absolutely clear that there must be other genetic modifiers to be discovered that are responsible for the variable phenotype. A better approach to predicting the phenotype is urgently required, particularly if prenatal diagnosis is to be widely used for the control of this condition and, even more so, if experimental forms of gene therapy become available in the future. Because it may be some time before there are more definitive forms of treatment it is important to utilize the information that we already have more effectively. For example, in malarious areas it will be very important to conduct trials of malaria prophylaxis with particular respect to the phenotype of patients with this condition early in life. Because recent evidence suggests that the erythropoietin response to anemia tends to decline with age, the possibility of transient periods of transfusion during maximum erythroid expansion should be seriously considered.29 Because genetic evidence indicates that the phenotype in this condition may be improved quite dramatically with only a modest increase in steady-state hemoglobin level,

Hemoglobin E Disorders 30%

427 30%

F

FA

20%

30%

F

20%

EFA

EF

20%

10%

10%

F

10%

A A

E E

0%

0% 0

1

2

3

4

5

6

0

1

2

3

A

4

5

0%

6

0

2

B

30%

30%

5

6

F Bt’s FA Bart’s

20%

EFA Bart’s

20%

A

A

10%

4

30%

Bt’s Hb Bart’s hydrops

3

C

F

Bart’s

20%

1

10%

10%

E 0% 0

1

2

3

4

5

6

0% 0

0% 1

2

D

3

4

5

6

0

1

2

E

3

4

5

6

F

Figure 18.5. Chromatograms of newborns with HbE syndromes detected by automated HPLC. (A) Healthy newborn; (B) newborn with HbE trait; (C) newborn with HbE–␤ thalassemia; (D) newborn with Hb Bart’s hydrops fetalis; (E) newborn with HbH disease; (F) newborn with HbAE Bart’s disease.

more efforts should be directed at trying to raise the HbF level in these patients.

NEONATAL AND PRENATAL DIAGNOSIS The amount of HbA2 in normal newborns is lower than in adults and usually is not visualized by hemoglobin electrophoresis. Pootrakul et al.77 observed a “slow” hemoglobin component at the position of HbA2 that proved to be HbE. The mean HbE level, quantitated by cellulose-acetate electrophoresis, was 3.7%. An automatic high-performance liquid chromatography (HPLC) system (VariantTM , BioRad) was used to study various HbE disorders in cord blood (Fig. 18.5). HbE concentrations in homozygous HbE ranged between 3.9% and 14.9%. In HbE heterozygotes, HbE was between 2.1% and 10.3% with a mean of 4.5% (Table 18.6).78 These data suggest that newborns with homozygous HbE have a tendency to have higher concentration of HbE than those with heterozygous HbE, but some overlap occurs. Furthermore, both homozygous HbE and HbE–␤0 thalassemia patients had similar chromatograms composed of HbE and HbF with similar amounts of HbE. DNA analysis is necessary to differentiate between these two syndromes.

Diagnosis of HbE syndromes can also be performed prenatally by cordocentesis at the gestational ages of 16–24 weeks.79 The chromatograms obtained from cordocentesis using the automatic HPLC system were similar to those of the cord blood specimens. In the HbE heterozygote, 0.8%–1.5% of HbE was detected in addition to HbA. HbA was not present in fetuses homozygous for HbE or with HbE–␤0 thalassemia (Table 18.7). These two conditions were distinguished only by DNA analysis. Prenatal diagnosis of Hb–␤ thalassemia is now performed, if requested, by chorion-villus sampling and DNA analysis.

LABORATORY DIAGNOSIS IN ADULTS The diagnosis of HbE is based on the electrophoretic or chromatographic separation of hemoglobins from peripheral blood. In alkaline buffer (pH 8.6), HbE migrates like HbA2 on cellulose-acetate membranes but can be distinguished from HbA2 by its higher concentration, more than 10% of the total hemoglobin. HbE also migrates like HbA2 on HPLC columns.78 Variable concentrations of HbE may reflect the genotypes of HbE disorders. In general, as

428

No.

326 1 1 1 9 114 1 1 5 1 3

Condition

Normal EA Bart’s disease EF Bart’s disease ␤ thal/HbE disease HbE homozygote HbE trait EE-␣+ thal trait EE-␣o thal trait HbE trait ␣o thal trait HbE trait ␣+ thal/Hb CS HbE trait Homozygous ␣+ thal

MCV fL 105 ± 6.2 72 70 99 103 ± 6.7 104 ± 7.5 96 82 89 ± 4.8 106 78 91 98

Hb g/dL

15.4 ± 1.7 13.0 11.2 13.1 14.5 ± 2.1 15.4 ± 1.6 13.8 13.5 14.0 ± 1.3 15.0 13.2 14.3 14.9

35 ± 2.2 22 21 33 34 ± 2.5 35 ± 6.6 31 27 28 ± 1.5 32 25 28 31

MCH pg 33 ± 1.0 30 30 34 33 ± 1.2 33 ± 1.0 33 33 32 ± 0.5 30 32 31 31

MCHC g/dL

Table 18.6. Hematologic data and hemoglobin analysis in cord blood samples

FA EFA Bart’s EF Bart’s EF EF EFA EF Bart’s EF Bart’s EFA Bart’s EFA Bart’s EFA Bart’s EFA Bart’s EFA Bart’s

Hb type 0.6 ± 0.4 3.6 30.1 2.0 8.0 ± 3.6 4.5 ± 1.5 11.6 7.6 5.1 ± 1.1 3.0 5.6 4.2 2.2

A2 (E) 74.1 ± 6.4 80.6 64.4 93.0 81.4 ± 4.5 76.7 ± 4.8 76.1 81.3 72.8 ± 4.4 66.0 72.0 79.3 88.2

F

17.8 ± 6.3 12.2 0.9 0.1 0.6 ± 0.6 10.1 ± 3.5 0.4 0.3 15.6 ± 3.6 22.1 15.9 12.3 4.9

A

% Hemoglobin (HPLC)

0.7 ± 0.4 27.9 17.5 1.0 0 0.7 ± 0.4 1.9 8.8 8.6 ± 1.7 9.3 3.7 5.3 2.8

Barts

␣␣/␣␣-␤A /␤A --/-␣-␤A /␤E --/-␣-␤E /␤E ␣␣/␣␣-␤thal /␤E ␣␣/␣␣-␤E /␤E ␣␣/␣␣-␤A /␤E -␣/␣␣-␤E /␤E --/␣␣-␤E /␤E --/␣␣-␤A /␤E -␣/␣CS ␣-␤A /␤E -␣/-␣-␤A /␤E -␣/-␣-␤A /␤E -␣/-␣-␤A /␤E

Genotype

Hemoglobin E Disorders

429

coelectrophoreses with HbE (␣2 ␤6Glu→Lys 2 ) in the standard alkaline buffer. These two variants can be distinguished by agar gel elecHb Analysis (%) trophoresis at acid pH, or by reverse phase HPLC, and by the presence of approximately Phenotype No. Hb Type A2 /E F A Bart’s 45% HbC in the heterozygous state.80 Normal 1 FA 0 94.0 5.8 – The blue dye dichlorophenolindophenol ␤ Thal trait 6 FA 0 94.8–96.3 4.0–5.2 – can be used as a screening test for HbE, which ␤ Thal/HbE 7 EF 1.0–1.7 89.4–98.6 0 – has a weakened ␣1 ␤1 contact and precipitates Homozygous HbE 2 EF 3.0, 2.1 96.8, 97.3 0 – on incubation with the dye at 37◦ C.27 HomozyHbE trait 4 EFA 0.8–1.4 94.0–97.2 2.0–2.8 – Hb Bart’s hydrops 10 Bart’s 0 0 0 @100% gous HbE produces heavy sediments at the bottom of the tube whereas heterozygous HbE, HbH disease, and HbE–␤ thalassemia produce mentioned previously, HbE is 25%–30% of the hemolysate a cloudy or evenly distributed particulate appearance. from heterozygotes and lower amounts of HbE are found with coinheritance of ␣ thalassemia (Table 18.2) or with coexistence of iron-deficiency anemia. Lower proportions INTERACTIONS OF HEMOGLOBIN E WITH of HbE in heterozygotes indicate a concomitant inheritance OTHER ␤-GLOBIN CHAIN VARIANTS of the more pronounced defects of ␣-globin chain syntheAs well as the common interactions of hemoglobin E sis as in HbAE Bart’s disease.10–12 In contrast, the amount of with the ␣ and ␤ thalassemias described in this chapter HbE is higher in HbE–␤ thalassemia and HbE levels of 85%– there have been occasional reports of interactions between 95% of total hemoglobin are found with homozygous HbE. HbE and other ␤-chain structural hemoglobin variants Compound heterozygotes with HbE and HbC also appear (Table 18.8). Although most of these interactions result in a to have very high HbE levels because HbC (␣2 ␤6Glu→Lys 2 )

Table 18.7. The amounts of Hbs A2 (E), F, and a from a normal fetus and fetuses with thalassemias and HbE

Table 18.8. Hemoglobin analysis of HbE/other ␤ variants Hb variants

␣-Genotype

Hb (g/dL)

MCV (fL)

HbE (%)

Hb variant (%)

HbF (%)

References

Hb Pyrgos

ND -␣3.7 /␣␣ ␣CS ␣/␣␣ --/␣␣ -␣3.7 /␣␣ ␣␣/␣␣ ND ND ␣␣/␣␣ ND ND ND ND ND ND ND ND ND ␣␣/␣␣ ND ND ND -␣3.7 /␣␣ ND ␣␣/␣␣ -␣/␣␣ ND ND ND

9.5 12.4 14.1 11.1

76.5 86 72.6 68.4

26.5 25.5 22.8 19.4

71.2 63.6 77.2 73.9

ND 5.1 ND 2.2

[82–84]

14.1 12 10.5 10.5 14.6 12.3 12.8, 15.5 12.1 14.3 17.8 14 9.5, 10.1 11.8 11.3 (11.0–12.0) 12.7 14.6 12.9 9.5, 12.2 10.1 10.2, 8.9 10.1 8.3, 6 10.1 11.4

77.2 84 63 78.3 ND 79.7 95, 84 77 77.4 80 ND 61, 59.5 59 81.7(74.1–88.0) 71 ND 74 77.5, 73.2 64 62.5, 61.3 58.5 60.3, 54.1 64.7 70

29.7 33.5 40 32.3 33.2 32.3 31.9, 30.4 38.9 24.1 35.2 37 35.2, 36.9 40.6 35.6(32.0–39.7) 34.6 32 36 21.8, 22.4 57.3 51.7, 50.6 59.4 64, 75 67.6 53

73.3 59 60 60.9 60 67.7 68.9, 69.6 55 58.8 62.2 50 55.9, 53.6 56 56.4(53.7–57.5) 64.2 60 60 71.1, 69.3 39.3 9.0, 11.0 20.3 10, 8 ND 12.7

Lys] in a Thai male. Hemoglobin. 1993;17:419–425.

Suthat Fucharoen and David J. Weatherall 81. Viprakasit V, Chinchang W. Two independent origins of Hb Dhonburi (Neapolis) [beta 126 (H4) Val-->Gly]: an electrophoretically silent hemoglobin variant. Clin Chim Acta. 2007;376:179–183. 82. Fucharoen S, Singsanan S, Sanchaisuriya K, Fucharoen G. Molecular and haematological characterization of compound Hb E/Hb Pyrgos and Hb E/Hb J-Bangkok in Thai patients. Clin Lab Haematol. 2005;27:184–189. 83. Jetsrisuparb A, Sanchaisuriya K, Fucharoen G, Fucharoen S, Wiangnon S, Komwilaisak P. Triple heterozygosity of a hemoglobin variant: hemoglobin Pyrgos with other hemoglobinopathies. Int J Hematol. 2002;75:35–39. 84. Sawangareetrakul P, Svasti S, Yodsowon B, et al. Double heterozygosity for Hb Pyrgos [beta83(EF7)Gly-->Asp] and Hb E [beta26(B8)Glu-->Lys] found in association with alphathalassemia. Hemoglobin. 2002;26:191–196. 85. Fucharoen S, Changtrakun Y, Surapot S, Fucharoen G, Sanchaisuriya K. Molecular characterization of Hb D-Punjab [beta121(GH4)Glu-->Gln] in Thailand. Hemoglobin. 2002; 26:261–269. 86. Svasti S, Winichagoon P, Svasti J, Fucharoen S. 2008: in press. 87. Chunpanich S, Fucharoen S, Sanchaisuriya K, Fucharoen G, Kam-itsara K. Molecular and hematological characterization of hemoglobin Hope/hemoglobin E and hemoglobin Hope/alpha-thalassemia 2 in Thai patients. Lab Hematol. 2004;10:215–220. 88. Pillers DA, Jones M, Head C, Jones RT. Hb Hope [beta 136(H14) Gly---Asp] and HbE [beta 26(B8)Glu----Lys]: compound heterozygosity in a Thai Mien family. Hemoglobin. 1992;16: 81–84. 89. Pootrakul S, Wasi S, NaNakorn S, Dixon GH. Double heterozygosity for hemoglobin E and hemoglobin New York in a Thai family. J Med Assoc Thai. 1971;54:688–697. 90. Hoyer JD, Wick MJ, Thibodeau SN, Viker KA, Conner R, Fairbanks VF. Hb Tak confirmed by DNA analysis: not expressed as thalassemia in a Hb Tak/HbE compound heterozygote. Hemoglobin. 1998;22:45–52. 91. Fucharoen S, Fucharoen G, Sanchaisuriya K, Surapot S. Compound heterozygote states for Hb C/Hb Malay and Hb C/Hb E in pregnancy: a molecular and hematological analysis. Blood Cells Mol Dis. 2005;35:196–200. 92. Sanchaisuriya K, Fucharoen G, Sae-ung N, Siriratmanawong N, Surapot S, Fucharoen S. Molecular characterization of hemoglobin C in Thailand. Am J Hematol. 2001;67:189– 193. 93. Schroeder WA, Powars D, Reynolds RD, Fisher JI. Hb-E in combination with Hb-S and Hb-C in a black family. Hemoglobin. 1977;1:287–289. 94. Altay C, Niazi GA, Huisman TH. The combination of Hb S and Hb E in a black female. Hemoglobin. 1976;1:100–102. 95. Gupta R, Jarvis M, Yardumian A. Compound heterozygosity for haemoglobin S and haemoglobin E. Br J Haematol. 2000;108:463. 96. Changtrakun Y, Fucharoen S, Ayukarn K, Siriratmanawong N, Fucharoen G, Sanchaisuriya K. Compound heterozygosity for Hb Korle-Bu (beta(73); Asp-Asn) and HbE (beta(26); Glu-Lys) with a 3.7-kb deletional alpha-thalassemia in Thai patients. Ann Hematol. 2002;81:389–393. 97. Hutt PJ, Fairbanks VF, Thibodeau SN, et al. Hb T-Cambodia, a beta chain variant with the mutations of Hb E and

Hemoglobin E Disorders Hb D-Punjab, confirmed by DNA analysis. Hemoglobin. 1997;21:205–218. 98. Waye JS, Eng B, Patterson M, et al. Hb E/Hb Lepore-Hollandia in a family from Bangladesh. Am J Hematol. 1994;47:262– 265. 99. Edison ES, Shaji RV, Srivastava A, Chandy M. Compound heterozygosity for HbE and Hb Lepore-Hollandia in India; first report and potential diagnostic pitfalls. Hemoglobin. 2005;29:221–224. 100. Viprakasit V, Pung-Amritt P, Suwanthon L, Clark K, Tanphaichtr VS. Complex interactions of deltabeta hybrid

433 haemoglobin (Hb Lepore-Hollandia) Hb E (beta(26G-->A)) and alpha+ thalassaemia in a Thai family. Eur J Haematol. 2002;68:107–111. 101. Boontrakoonpoontawee P, Svasti J, Fucharoen S, Winichagoon P. Identification of Hb Lepore-Washington-Boston in association with Hb E [beta 26(B8)Glu----Lys] in a Thai female. Hemoglobin. 1987;11:309–316. 102. Fucharoen S, Sanchaisuriya K, Fucharoen G, Surapot S. Molecular characterization of thalassemia intermedia with homozygous Hb Malay and Hb Malay/HbE in Thai patients. Haematologica. 2001;86:657–658.

SECTION FIVE

SICKLE CELL DISEASE Martin H. Steinberg

PATHOPHYSIOLOGY A ␤-hemoglobin gene mutation results in the synthesis of the sickle ␤-globin chain. Sickle hemoglobin (HbS) polymerizes when deoxygenated, and polymer-associated injury to the sickle erythrocyte is the proximate cause of sickle cell disease. The principal pathophysiological features of this disease are shown in the figure and can be grouped as vasoocclusive/blood viscosity related and hemolysis/vasculopathy related. This complex pathophysiology involves diverse molecular and cellular defects that include abnormal erythrocyte volume regulation, impaired nitric oxide bioavailability, reperfusion injury and inflammation, altered hemostasis, defects of intercellular interactions, endothelial cell damage, leukocyte and platelet activation, and in all probability, other perturbations of normal physiology.

ic, disabling, and cause premature death. For example, pulmonary hypertension is usually silent but is associated with a grave prognosis. The clinical features of sickle cell disease are heterogeneous. Fetal hemoglobin, ␣ thalassemia, and compound heterozygosity for other variant hemoglobins such as HbC or ␤ thalassemia are well-known determinants of the phenotype. Acute painful episodes are the most common clinical events. Most often they begin with little warning. In some patients it is difficult to distinguish among new episodes of acute pain, chronic pain with acute exacerbations, therapyinduced pain, and pain made worse by major psychosocial issues. Insufficient doses of opioid analgesics, given at infrequent intervals, is the most common deficiency in pain treatment. The acute chest syndrome, with fever, chest pain, cough, hypoxia, and lung infiltrates affects more than half of all sickle cell anemia patients. It is most common but least severe in young children, in whom it is often secondary to infection. In adults, pain often precedes this event, and mortality is higher than in children. Fat embolism from necrotic bone marrow is a common cause of the most severe acute chest events. Sometimes, fat embolization is accompanied by dramatic falls in the hemoglobin and platelet levels, marked leukocytosis, and multiorgan failure. Recognizing acute chest syndrome is critical because aggressive treatment with oxygen, blood transfusions, bronchodilators, and antimicrobials can be lifesaving. Strokes occur in approximately 10% of children with sickle cell anemia and are more rare in other genotypes.

DIAGNOSIS Sickle cell disease is a constellation of similar but not identical disorders, all of which have at least 50% HbS in the blood. The phenotype of sickle cell disease is caused by several common and some less common genotypes; homozygotes for the HbS gene are said to have sickle cell anemia; common compound heterozygous forms of disease include HbSC disease and HbS-␤ thalassemia. The cornerstone of the clinical laboratory diagnosis is the detection and quantification of HbS. Depending on the context of the diagnostic situation, direct detection of the HbS and other globin gene mutations can be warranted. Sickle cell trait is clinically benign and not considered a form of sickle cell disease.

CLINICAL FEATURES The complications of sickle cell disease can occur acutely, producing dramatic clinical findings, or they can be chron-

Pathophysiology of sickle cell disease. The nucleotide and amino acid substitution of HbS leads to the replacement of a glutamic acid residue by a valine residue. On deoxygenation, HbS polymer forms, causing cell sickling and damage to the erythrocyte membrane. Some cells adhere to the endothelium and cause vasoocclusion. Other cells are destroyed within the circulation, releasing hemoglobin and arginase and depleting bioavailable nitric oxide (Chapters 10 and 11).

435

436 Prophylactic blood transfusion can prevent a stroke in highrisk individuals. A current focus is how better to predict who will have clinically significant cerebrovascular disease and how this might best be managed. Survival decreases and the risk of stroke increases as blood pressure increases, even though it is within the “normal” range. Osteonecrosis, seen in approximately half of all patients, most often affects the heads of the femur and humerus. In some individuals, it culminates in a severely painful or a useless joint that requires surgery. Pulmonary hypertension affects approximately a third of adults with sickle cell anemia. Although usually mild, when defined in terms of pulmonary artery systolic pressure, its presence portends a poor prognosis as at least a quarter of patients die within 2 years of its detection. Other severe complications are leg ulcers, priapism, nephropathy with renal failure and severe anemia with advancing age, and sickle retinopathy, especially in HbSC

Martin H. Steinberg disease and liver disease. Infections are common and major causes of death in children and adults. Treatment. Treatment for sickle cell disease is evolving and many clinical trials of new agents are in progress. With the exceptions of transfusions to prevent stroke, hydroxyurea for prevention of painful episodes, and prophylactic penicillin for prevention of pneumococcal disease in children, controlled clinical trials have not established the superiority of any treatment modality. Nevertheless, much can be done to promote better health of the individual, including good nutrition and avoidance of extremes of temperature and dehydration, genetic counseling for couples at risk for having affected children, and neonatal screening to identify infants with sickle cell disease and directing their parents toward comprehensive care programs that provide recommended immunizations and prophylactic penicillin. The following five chapters discuss in detail the topics outlined previously.

any age. As in other chapters, because of space constraints, many early references are omitted to allow inclusion of new material. Most of these citations can be found in the first edition of this book.10

19 PREVALENCE OF DISEASE AND LABORATORY DIAGNOSIS

Clinical and Pathophysiological Aspects of Sickle Cell Anemia Martin H. Steinberg, Kwaku Ohene-Frempong, and Matthew M. Heeney

INTRODUCTION Many authors have recounted the history of sickle cell disease in Africa and its first recognition in the United States1–3 Sickle-shaped red cells were first described in 1910 in the blood of a sick, anemic student from Grenada.4,5 Sickle hemoglobin (HbS) was identified in 1949 and the mechanism of inheritance of sickle cell anemia was established afterward.6–8 A single amino acid difference was found to distinguish the sickle ␤-globin chain from the normal one.9 The breadth of clinical and laboratory manifestations of sickle cell disease and its multitudinous complications still challenge the pediatrician, internist, general surgeon, obstetrician, orthopedist, ophthalmologist, psychiatrist, and subspecialists in each of these disciplines. The features of sickle cell anemia change as life advances. Life’s first decade, with declining fetal hemoglobin (HbF) levels, is typified by a risk of severe lifethreatening infection, dactylitis, acute chest syndrome, splenic sequestration, and stroke; pain is often the torment of adolescence. If the worst of childhood and adolescent problems are survived or escaped, young adulthood can be a time of relative clinical quiescence, but sickle vasculopathy is likely to progress despite producing few symptoms. Chronic organ damage leading to pulmonary hypertension, deteriorating pulmonary function, renal failure, and late affects of previous cerebrovascular disease, including neurocognitive impairment, become paramount as years advance. Sickle cell anemia is noted for its clinical heterogeneity (Chapter 27). Any patient can have nearly all known disease complications; some have almost none, but die with a sudden acute problem. Some skip one or more phases of the disease but suffer intensely from others. For convenience, when discussing the most common clinical events of sickle cell anemia, we have grouped these by the age group in which a complication is most likely to occur, although nearly every complication can occur at

Prevalence Outside Africa, the prevalence of HbS depends primarily on gene flow from Africa to the Middle East, Asia, Europe, and the New World, modulated by genetic admixture with indigenous populations. Variations in the prevalence of HbS from within a country or geographic region are due to the presence of malaria, population isolates, altitude, and miscegenation. Although HbS is widely distributed in Caucasian populations, this is usually not the result of selection, but of ancient wars and migration, the slave trade, and generations of genetic admixture. In India, Greece, Turkey, Italy, and elsewhere in the Old World, HbS, excluding recent migrants from Africa or the Caribbean, is found mainly in Caucasians. In the United States, the prevalence of HbS in Caucasians varies according to the population examined, but it is usually less than 0.1% and is invariably found with African or Arab–Indian ␤-globin gene cluster haplotypes (Chapter 27). Table 22.1 in Chapter 22 shows the prevalence of the HbS gene in some African locations and throughout the world. In African Americans, the incidence of sickle cell anemia at birth was estimated to be approximately 1 in 600 and the incidence of all genotypes of sickle cell disease approached 1 in 300.11 These estimates and the distribution of disease in the United States have surely changed because of new immigrants from the Caribbean region, Central and South America, the Middle East, Africa, and India. In Paris, 1 in 900 newborns has sickle cell disease and in Salvador, Bahia, Brazil, approximately 1 in 500 newborns is affected. Although the treatment of complications of sickle cell disease differs little among its genotypes, knowing the genotype is critical for genetic counseling.

Diagnosis Imprecision defining sickle hemoglobinopathies has permeated the medical literature. Sickle cell disease is a phenotype, expressed in patients with different genotypes. We use the following definitions:

r Sickle cell disease – at least half the hemoglobin is HbS and patients have distinguishing clinical and hematological features r Sickle cell trait (HbAS; Chapter 22) is clinically benign and blood counts are normal, so it is not a form of sickle cell disease r Individuals homozygous for the HbS gene (HBB, glu6val) are said to have sickle cell anemia 437

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Figure 19.1. Blood films in patients with sickle cell anemia and average or low HbF levels. HbF in these patients are A-0.5%, B-1%, C-4%, D-5%, E-7%, and F-10%. Patients C and F were taking hydroxyurea. Although some of these patients have more ISCs than patients depicted in Figure 19.2 who have high HbF concentrations, note that patient A with the lowest HbF level has very few sickled cells. See color plates.

r Compound heterozygous forms of sickle cell disease include HbSC disease (HBB glu6val, glu6lys), HbS-␤ thalassemia (HBB glu6val and a ␤ thalassemia mutation), and less common types (Chapters 21 and 23). Diagnosing sickle cell disease is not difficult but determining the correct genotype can be problematic if family studies and access to molecular diagnostic methods are not available. Examining the blood of parents or siblings of affected patients is the least costly way of establishing the genotype and can be done with simple combinations of blood counts and quantitative studies of hemoglobin fractions.

Blood Counts and Erythrocyte Indices In sickle cell anemia, the erythrocytes are normocytic or macrocytic, depending on the reticulocyte count and the presence or absence of confounding conditions such as iron or folic acid deficiency and coincident ␣ thalassemia (Chapter 23). Microcytosis in a suspected case of sickle cell anemia can be seen very early in life before erythropoiesis has fully matured, when iron deficiency has developed or when ␣ thalassemia is present. HbS–␤0 thalassemia, a phenocopy of sickle cell anemia but with microcytosis, and sickle cell anemia–␣ thalassemia are addressed in Chapter 23. These two conditions are very alike hematologically and clinically and their distinction from each other is difficult lacking genetic testing or family studies. In an individual, they cannot be separated using hemoglobin analysis or blood counts alone. Typical blood counts and erythrocyte

indices in these genotypes are shown in Table 23.2 of Chapter 23.

Blood Films Sickled cells are nearly always seen in sickle cell anemia and HbS–␤0 thalassemia but are less common in other genotypes. Typical blood films from patients with sickle cell anemia with high, average, and low HbF concentrations are shown in Figures 19.1 and 19.2. Some patients have many irreversibly sickled cells (ISCs), nucleated red cells, Howell– Jolly bodies, and polychromatophilic cells, whereas others have far fewer. In adults, ISCs remain relatively constant over time, although their percentage increases early in a painful episode (Chapter 20).12

Detecting HbS and Measuring Hemoglobin Fractions From neonatal life through early adult life, there is a slow but continual fall in HbF, whereas HbA2 levels increase until ages 1–2 years. With few exceptions, the hemoglobin fractions present at age 1 year are sufficiently stable to be relied on for diagnosis. In untreated sickle cell anemia, HbS nearly always forms more than 80% of the hemolysate, except in infancy when the ␥ - to ␤-globin gene switch is incomplete. HbS can be detected by isoelectric focusing, hemoglobin electrophoresis, or high-performance liquid chromatography (HPLC). Hemoglobin fractions are best measured by HPLC or capillary electrophoresis. Sickling hemoglobins can be detected chemically because they are insoluble and precipitate in high-molarity phosphate buffer when

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Figure 19.2. Blood films in patients with sickle cell anemia and high HbF levels. HbF in these six patients are A-19%, B-18%, C-19%, D-21%, E-20%, and F-23%. All patients were receiving hydroxyurea. All still have sickled cells in the blood and these are particularly prominent in patient A. Also note nucleated red cells in A and E. (See color plate 19.2.)

reduced with sodium dithionite, but these sickle solubility tests should not be used as the sole means of detecting HbS or other sickling variants because their results are not quantifiable and they cannot reliably distinguish among genotypes of sickle cell disease and HbAS. DNA-based methods of detecting the HbS mutation are now widely available and are definitive (Chapter 28).

PATHOPHYSIOLOGY Major features of the pathophysiology of sickle cell disease such as HbS polymer, erythrocyte membrane abnormalities, sickle vasculopathy, and hemolytic anemia are discussed in Chapters 6, 8, 9, 10, and 11. Sickle vasoocclusion and hemolytic anemia are the two major features of disease pathophysiology. Reappraisals of the contributions of hemolysis and sickle vasoocclusion to the phenotypes of the disease and novel insights into the role of nitric oxide (NO) in sickle vasculopathy have led to new pathophysiological insights (Chapters 10 and 11).

A New View of Sickle Cell Pathophysiology: Hemolysis and Viscosity–Vasoocclusive Phenotypes (for a more detailed discussion, see Chapter 11) Hemolysis, long discounted as a critical measure of sickle cell disease severity when compared with sickle vasoocclusion, might be the proximate cause of some severe disease complications and a major predictor of mortality. Hemolytic anemia is most severe in patients with sickle cell anemia, less severe in individuals with sickle cell anemia and concurrent ␣ thalassemia, and least severe in patients with HbSC disease and HbS–␤+ thalassemia. Even within a

single genotype, the hemoglobin concentration is variable because of different rates of hemolysis.13–17 Intravascular heme reduces NO bioavailability. NO binds soluble guanylate cyclase, which converts guanosine triphosphate to cyclic guanosine monophosphate (cGMP), relaxing vascular smooth muscle and causing vasodilation. A state of reduced endothelial NO bioavailability in sickle cell disease impairs downstream homeostatic vascular functions of NO, such as inhibition of platelet activation and aggregation and transcriptional repression of the cell adhesion molecules, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, Pselectin, and E-selectin. Hemoglobin, heme, and heme iron catalyze the production of oxygen radicals, further limiting NO bioavailability and activating endothelium. Lysed erythrocytes also liberate arginase that destroys L-arginine, the substrate for the NO synthases. Reactive oxygen species, generated at high rates in patients with sickle cell anemia, also consume NO. The normal balance of vasoconstriction to vasodilation is therefore skewed toward vasoconstriction, endothelial activation, and proliferation. Both hemolysis and splenectomy are associated with red cell membrane damage, phosphatidylserine exposure at the red cell membrane surface, activation of tissue factor, and thrombosis. Chronic anemia and tissue ischemia might also contribute to a proliferative vasculopathy via activation of hypoxia inducible factor (HIF)-1␣–dependent factors such as inducible nitric oxide synthase (iNOS), erythropoietin, and vascular endothelial growth factor (VEGF).18 Clinical studies suggest a close association of hemolysis with the subphenotypes of pulmonary hypertension, priapism, leg ulceration, and perhaps ischemic stroke.19–26 Individuals with the highest rates of hemolysis also had a

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greater risk of death.27 The frequency of painful episodes has also been associated with mortality.28 Perhaps this association is mediated by increased hemolysis during painful episodes with a rise in pulmonary artery systolic pressure.29 Hemolysis-related complications occur less often in patients with HbSC disease and sickle cell anemia– ␣ thalassemia. HbF appears to have a lesser effect in modulating these phenotypes compared with the viscosity– vasoocclusive phenotypes of painful episodes, acute chest syndrome, and osteonecrosis. Hemolysis might also be linked to vasoocclusive disease via the adhesive properties of the sickle reticulocyte. Sickle reticulocytes, whose numbers reflect in part the bone marrow response to hemolysis, adhere to endothelium and to leukocytes and are the most adherent of the heterogeneous population of sickle erythrocytes. Sickle erythrocyte adherence varies according to the hemoglobin genotype being most manifest in sickle cell anemia. Even among patients with this genotype, adherence varies approximately 20-fold. Because most of the endothelial cell adhesion molecules that bind sickle reticulocytes are normally suppressed by NO, decreased NO bioavailability might also contribute to sickle erythrocyte adherence. Distinct from the hemolysis-related phenotypes are ones associated with increased blood viscosity and vasoocclusion, such as osteonecrosis, acute chest syndrome and painful episodes. Adversely affected by ␣ thalassemia, their prevalence is directly associated with hemoglobin concentration; high HbF levels have a protective effect. Hemolytic anemia–induced phenotypes are likely to be improved by transfusion, by agents that increase NO bioavailability and drugs that reduce intravascular hemolysis, but helped to a lesser extent by drugs that induce HbF expression.30,31 Drugs that induce the production of HbF appear to reduce the incidence of painful episodes and acute chest syndrome.32,33 Hemolytic and viscosity–vasoocclusive phenotypes must have substantial areas of overlap and HbF is likely to play a role in all disease complications. Nevertheless, this dichotomization of pathophysiology helps place subphenotypes of sickle cell disease into a new context with implications for therapeutics, for example, by devising drug combinations that target multiple limbs of the pathophysiological tree.18 One can envision combining a drug or drugs that induce HbF expression with agents that reduce sickle erythrocyte density, adhesive interactions of sickle cells, and the inflammatory response.34 Chronic activation of the hemostatic system has also been considered part of the pathobiology of sickle cell disease and might be the consequence of the exposure of aminophospholipids in the sickle erythrocyte, endothelial dysfunction with thrombin generation, and platelet activation, among other factors.35,36 Platelet activation was correlated with the severity of pulmonary hypertension and also with reticulocyte count but not with lactate dehydrogenase (LDH). When exposed to cell-free hemoglobin, basaland agonist-stimulated platelet activation was increased. In

patients, sildenafil reduced platelet activation. These findings suggested a possible interaction among hemolysis, decreased NO bioavailability, and pathological platelet activation that might contribute to thrombosis and pulmonary hypertension.37

CLINICAL FEATURES OF SICKLE CELL ANEMIA In the following discussion of the clinical features of sickle cell anemia complications are grouped by the age at which each is paramount. Painful episodes and their management are discussed separately in Chapter 20. Two large observational studies, one in Jamaica and the other in the United States, have provided much of our most reliable information on the clinical course and complications of sickle cell disease in developed countries. The Cooperative Study of Sickle Cell Disease (CSSCD) followed nearly 4,000 patients, including a newborn cohort of 694 patients, over a span of 20 years.38–40 In Jamaica, a newborn cohort of more than 300 individuals and their matched controls, have been followed even longer.41 Both studies have substantial patient numbers, longitudinal data collection, and biological sample repositories that can be used for genetic studies and measurement of newly developed disease markers. The inclusion of newborn cohorts in these studies eliminated the bias of studying only patients seeking medical care. Nevertheless, these studies are now decades old and their databases do not include complications of disease that were more recently recognized and do not report current methods of clinical and laboratory testing. Newly ascertained patient databases with their associated biological samples are needed. Few controlled clinical trials of the treatment of the complications of sickle cell anemia are published and progress in this area has been slow. There were a number of questions posed in the first edition of this book: How should acute chest syndrome be managed? Is exchange transfusion superior to simple transfusion? Can preoperative transfusion sometimes be avoided? Do angiotensin-converting enzyme inhibitors delay sickle nephropathy? How is priapism best managed? How aggressively should blood pressure be lowered to decrease the chance of stroke? These questions have yet to be answered. We will summarize recent information about the complications of sickle cell disease and provide explicit recommendations for treatment based on controlled clinical trials, in the few instances where they are available. In most cases, however, lacking the results of such studies, we will recommend management strategies based on experience with similar problems in other diseases, data from observational studies, expert opinion, and our understanding of the pathophysiology of sickle cell disease.

Assessing the Severity of Sickle Cell Disease Knowing the elements of disease that put patients at risk for selected complications, overall disease severity and death

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Figure 19.3. A Bayesian network model of the risk of death in sickle cell disease.44 (See color plate 19.3.)

would have great value for patient counseling, planning therapy, and the development of novel treatments. A study was done of 392 infants with sickle cell anemia or HbS– ␤0 thalassemia from the CSSCD. Nine years of clinical and laboratory data were available for analysis and an additional 115 infants were used for validation. Severe outcomes were defined as death, stroke, two or more painful episodes per year for 3 consecutive years, and one or more acute chest events per year for 3 years. Seventeen percent of the newborn cohort was identified as severe. A severe endpoint was achieved at a mean age of 5.6 ± 3.8 years. Hemoglobin levels of less than 8 g/dL, leukocyte counts more than 20,000/mm2 , an episode of dactylitis in patients aged younger than 1 year, and an elevated percentage of “pocked” red blood cells by age 1 year were significant predictors of adverse outcomes and combined identified 10.5% of the cohort, with a 0.35–0.71 probability of a severe outcome by age 10 years. Application of the severity model to the validation cohort yielded 100% positive predictive value and an 88% negative predictive value.42 Unfortunately, in another study of children, this result could not be replicated.43 In 168 children followed for up to 7.1 years, no relationship existed between these early clinical predictors and later adverse outcomes, with the possible exception of leukocyte count. At this moment, clinical and laboratory features seem unreliable means of making prognoses in young children. Using Bayesian network modeling and data from 3,380 sickle cell disease patients of all ages and multiple genotypes, a severity score was constructed that estimated the risk of death within 5 years.44 The predictive value of this score was validated in two independent patient groups: 140 adults and children in whom severity was

assessed by expert clinicians and 210 adults in whom severity was also assessed by the echocardiographic diagnosis of pulmonary hypertension and death. A network of 24 variables described complex associations among complications of sickle cell disease and its laboratory variables (Fig. 19.3). In addition to known risk factors, the severity of hemolytic anemia and clinical events associated with hemolytic anemia were major contributors to risk for death. By capturing multiple effects that could be synergistic or antagonistic, according to the overall clinical presentation, the model could be used to compute a personalized measure of disease severity. A model calculator can be used to assess the clinical severity of patients with sickle cell disease, given any clinical profile (www.bu.edu/sicklecell/downloads/Projects). This analysis suggested that the intensity of hemolytic anemia, estimated by LDH, reticulocyte count, and aspartate aminotransferase (AST), is an important contributor to death. Systolic hypertension, associated with pulmonary hypertension in patients with sickle cell disease, is an important predictor of mortality and might reflect the decrease in NO bioavailability that characterizes hemolytic anemia. Polymorphisms in key genes that modulate the pathobiology of disease that might underlie severity and the propensity to develop subphenotypes of disease are discussed in Chapter 27.

THE NEONATE At birth and for the first few months of life, children with sickle cell disease usually do not have typical vasoocclusive complications. This is likely to be due to the persistence of very high HbF levels. The CSSCD reviewed birth outcome

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Martin H. Steinberg, Kwaku Ohene-Frempong, and Matthew M. Heeney

Table 19.1. Incidence of major clinical events in children with sickle cell anemia in the first decade

the frequency of the most common complications and their prevalence or incidence at different ages in children with sickle cell Acute anemia anemia up to age 10 years. With the excepHand–foot Painful Acute tion of acute chest syndrome, which occurAge (y) syndrome event chest CVA Spleen Other Bacteremia red more frequently in boys than in girls < 0.5 14.6 2.9 6.8 0.0 1.0 2.9 1.9 (29.1 vs. 20.1 per 100 person-years), and 0.5–1 31.3 9.5 16.4 0.0 5.5 5.0 9.9 painful events, which had a higher inci1 20.0 24.0 26.8 0.3 6.2 1.7 6.5 dence in girls (35.7 vs. 28.9), there were no 2 11.0 38.3 26.3 1.3 5.3 3.3 8.7 sex differences in the occurrence of most of 3 3.5 42.4 34.2 0.4 2.0 5.9 4.7 these complications. Constant or episodic 4 2.0 49.6 25.5 1.5 1.5 3.9 2.0 scleral icterus and the pattern of recur5 0.0 40.8 22.5 0.7 1.4 2.0 0.0 rent pain in limbs and abdomen often be6 0.0 39.2 28.9 2.1 1.0 8.3 4.1 7 0.0 41.6 20.8 1.5 0.0 3.0 1.5 come established in the first year of life. 8–10 0.0 37.9 15.2 0.0 0.0 1.9 0.0 Splenomegaly, another manifestation of sickle cell disease in children, is present in Incidence rates per 100 person-years from 427 children with sickle cell anemia observed in the a few infants with sickle cell anemia aged 38 Cooperative Study of Sickle Cell Disease, from October 1978 to October 1988. younger than 6 months. The spectrum of complications was the same in children and neonatal clinical course of 480 infants with sickle cell with HbSC disease but complications occurred at lower disease. Rates of preterm birth (aged G polymorphism in the human mu-opioid receptor gene may increase morphine requirements in patients with pain caused by malignant disease. Acta Anaesthesiol Scand. 2004;48:1232– 1239. 123. Mehta S, Kutlar F, Bailey L, et al. Mu opioid receptor 1 (MOR 1) polymorphisms among patients with sickle cell disease. Oral presentation. In: 27th Annual Meeting of the National Sickle Cell Disease Program. Los Angeles, CA; April 18–21, 2004. 124. Darbari DS, Minniti CP, Rana S, Van Den Anker J. Pharmacogenetics of morphine: potential implications in sickle cell disease. Am J Hematol. 2008;83:233–236. 125. Darbari DS, van Schaik RH, Capparelli EV, Rana S, McCarter R, Van Den Anker J. UGT2B7 promoter variant -840G>A contributes to the variability in hepatic clearance of morphine in patients with sickle cell disease. Am J Hematol. 2008;83:200– 202.

521 126. Kopecky EA, Jacobson S, Joshi P, Koren G. Systemic exposure to morphine and the risk of acute chest syndrome in sickle cell disease. Clin Pharmacol Ther. 2004;75:140–146. 127. Buchanan ID, Woodward M, Reed GW. Opioid selection during sickle cell pain crisis and its impact on the development of acute chest syndrome. Pediatr Blood Cancer. 2005;45:716– 724. 128. Weber ML, Hebbel RP, Gupta K. Morphine induces kidney injury in transgenic sickle cell mice. Blood. 2005;106: 884a–5a. 129. King T, Vardanyan A, Majuta L, et al. Morphine treatment accelerates sarcoma-induced bone pain, bone loss, and spontaneous fracture in a murine model of bone cancer. Pain. 2007;132:154–168. 130. Poonawala T, Levay-Young BK, Hebbel RP, Gupta K. Opioids heal ischemic wounds in the rat. Wound Repair Regen. 2005;13:165–174. 131. Ferrante MF. Nonsteroidal anti-inflammatory drugs. In: Ferrante MF, Vade Bancoeur TR, eds. Post Operative Pain Management. New York: Churchill Livingstone; 1993a:133–143. 132. Perlin E, Finke H, Castro O, et al. Enhancement of pain control with ketorolac tromethamine in patients with sickle cell vaso-occlusive crisis. Am J Hematol. 1994;46:43–47. 133. Crain SM, Shen KF. Ultra-low concentrations of naloxone selectively antagonize excitatory effects of morphine on sensory neurons, thereby increasing its antinociceptive potency and attenuating tolerance/dependence during chronic cotreatment. Proc Natl Acad Sci USA. 1995;92:10540– 10544. 134. Winkelmuller M, Winkelmuller W. Long-term effects of continuous intrathecal opioid treatment in chronic pain of nonmalignant etiology. J Neurosurg. 1996;85:458–467. 135. Definitions Related to the Use of Opioids for the Treatment of Pain. American Academy of Pain Medicine American Pain Society American Academy of Addiction Medicine 2001. Available at http://www.ama-assn.org/ama1/pub/ upload/mm/455/opioiddefinitions.pdf. Accessed March 31, 2008. 136. Benjamin LJ, Payne R. Pain in sickle cell disease: a multidimensional construct. In: Pace B, ed. Renaissance of Sickle Cell Disease Research in the Genomic Era. London: Imperial College Press; 2007:99–118. 137. Mercadante S, Ferrera P, Villari P, Arcuri E. Hyperalgesia: an emerging iatrogenic syndrome. J Pain Symptom Manage. 2003;26:769–775. 138. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science. 2000;288:1765–1769. 139. Gottschalk A, Smith DS. New concepts in acute pain therapy: preemptive analgesia. Am Fam Physician. 2001;63:1979– 1984. 140. Baliki MN, Geha PY, Apkarian AV, Chialvo DR. Beyond feeling: chronic pain hurts the brain, disrupting the defaultmode network dynamics. J Neurosci. 2008;28:1398–1403. 141. Song I, Huganir RL. Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci. 2002;25:578–588. 142. Carroll RC, Beattie EC, von Zastrow M, Malenka RC. Role of AMPA receptor endocytosis in synaptic plasticity. Nat Rev Neurosci. 2001;2:315–324. 143. Perez-Otano I, Ehlers MD. Homeostatic plasticity and NMDA receptor trafficking. Trends Neurosci. 2005;28:229–238.

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Samir K. Ballas and James R. Eckman 164. Shapiro B, Schechter NL, Ohene-Frempong K. The Genetic Resource, Sickle Cell Related Pain: Assessment and Management. Mt. Desert, ME: New England Regional Genetics Group; 1994. 165. Frei-Jones MJ, Baxter AL, Rogers ZR, Buchanan GR. Vasoocclusive episodes in older children with sickle cell disease: emergency department management and pain assessment. J Pediatr. 2008;152:281–285. 166. Ballas SK. Current issues in sickle cell pain and its management. Hematology Am Soc Hematol Educ Program. 2007; 110: 97–105. 167. Tanabe P, Myers R, Zosel A, et al. Emergency department management of acute pain episodes in sickle cell disease. Acad Emerg Med. 2007;14:419–425. 168. Pain Episodes. Georgia Comprehensive Sickle Cell Center, 2006. Accessed at http://www.scinfo.org/painepi.htm. 169. The Georgia Comprehensive Sickle Cell Center at Grady Health System in Atlanta, Georgia 2006. (Accessed 2008) 170. Payne R. Pain management is sickle cell disease: rationale and techniques. Ann NY Acad Sci. 1989;565:189–206. 171. Platt A, Eckman JR, Beasley J, Miller G. Treating sickle cell pain: an update from the Georgia comprehensive sickle cell center. J Emerg Nurs. 2002;28:297–303. 172. Gonzalez ER, Bahal N, Hansen LA, et al. Intermittent injection vs patient-controlled analgesia for sickle cell crisis pain. Comparison in patients in the emergency department. Arch Intern Med. 1991;151:1373–1378. 173. Holbrook CT. Patient-controlled analgesia pain management for children with sickle cell disease. J Assoc Acad Minor Phys. 1990;1:93–96. 174. Melzer-Lange MD, Walsh-Kelly CM, Lea G, Hillery CA, Scott JP. Patient-controlled analgesia for sickle cell pain crisis in a pediatric emergency department. Pediatr Emerg Care. 2004;20:2–4. 175. Trentadue NO, Kachoyeanos MK, Lea G. A comparison of two regimens of patient-controlled analgesia for children with sickle cell disease. J Pediatr Nurs. 1998;13:15–9. 176. van Beers EJ, van Tuijn CF, Nieuwkerk PT, Friederich PW, Vranken JH, Biemond BJ. Patient-controlled analgesia versus continuous infusion of morphine during vaso-occlusive crisis in sickle cell disease, a randomized controlled trial. Am J Hematol. 2007;82:955–60. 177. Schechter NL, Berrien FB, Katz SM. PCA for adolescents in sickle-cell crisis. Am J Nurs. 1988;88:719, 21–2. 178. Schechter NL, Berrien FB, Katz SM. The use of patientcontrolled analgesia in adolescents with sickle cell pain crisis: a preliminary report. J Pain Symptom Manage. 1988;3:109– 113. 179. Ballas SK. Ethical issues in the management of sickle cell pain. Am J Hematol. 2001;68:127–132. 180. Elander J, Lusher J, Bevan D, Telfer P, Burton B. Understanding the causes of problematic pain management in sickle cell disease: evidence that pseudoaddiction plays a more important role than genuine analgesic dependence. J Pain Symptom Manage. 2004;27:156–169. 181. Elander J, Marczewska M, Amos R, Thomas A, Tangayi S. Factors affecting hospital staff judgments about sickle cell disease pain. J Behav Med. 2006;29:203–214. 182. Elander J, Midence K. A review of evidence about factors affecting quality of pain management in sickle cell disease. Clin J Pain. 1996;12:180–193.

Sickle Cell Pain: Biology, Etiology, and Treatment 183. Armstrong FD, Pegelow CH, Gonzalez JC, Martinez A. Impact of children’s sickle cell history on nurse and physician ratings of pain and medication decisions. J Pediatr Psychol. 1992;17:651–164. 184. Todd KH, Green C, Bonham VL, Jr., Haywood C, Jr., Ivy E. Sickle cell disease related pain: crisis and conflict. J Pain 2006;7:453–458. 185. Ballas SK. Current issues in sickle cell pain and its management. Hematology Am Soc Hematol Educ Program. 2007;2007:97–105. 186. Quill TE. Partnerships in patient care: a contractual approach. Ann Intern Med. 1983;98:228–234. 187. Donovan MI, Evers K, Jacobs P, Mandleblatt S. When there is no benchmark: designing a primary care-based chronic pain management program from the scientific basis up. J Pain Symptom Manage. 1999;18:38–48. 188. Gil KM, Carson JW, Porter LS, et al. Daily stress and mood and their association with pain, health-care use, and school activity in adolescents with sickle cell disease. J Pediatr Psychol. 2003;28:363–373. 189. Smith WR, Bovbjerg VE, Penberthy LT, et al. Understanding pain and improving management of sickle cell disease: the PiSCES study. J Natl Med Assoc. 2005;97:183–93. 190. Ennis JT, Serjeant GR, Middlemiss H. Homozygous sickle cell disease in Jamaica. Br J Radiol. 1973;46:943–950. 191. Milner PF, Kraus AP, Sebes JI, et al. Sickle cell disease as a cause of osteonecrosis of the femoral head. N Engl J Med. 1991;325:1476–1481. 192. Milner PF, Kraus AP, Sebes JI, et al. Osteonecrosis of the humeral head in sickle cell disease. Clin Orthop Rel Res. 1993:136–143. 193. Riggs W Jr, Rockett JF. Roentgen chest findings in childhood sickle cell anemia. A new vertebral body finding. Am J Roentgenol Radium Ther Nucl Med. 1968;104:838–845. 194. Diggs LW. Bone and joint lesions in sickle-cell disease. Clin Orthop Rel Res. 1967;52:119–143. 195. Diggs LW, ed. Anatomic Lesions in Sickle Cell Anemia. St. Louis: C.V. Mosby Co; 1973. 196. Neumayr LD, Aguilar C, Earles AN, et al. Physical therapy alone compared with core decompression and physical therapy for femoral head osteonecrosis in sickle cell disease. Results of a multicenter study at a mean of three years after treatment. J Bone Joint Surg Am. 2006;88:2573–2582. 197. Hernigou P, Habibi A, Bachir D, Galacteros F. The natural history of asymptomatic osteonecrosis of the femoral head in adults with sickle cell disease. J Bone Joint Surg Am. 2006;88:2565–2572. 198. Bishop AR, Roberson JR, Eckman JR, Fleming LL. Total hip arthroplasty in patients who have sickle-cell hemoglobinopathy. J Bone Joint Surg Am. 1988;70:853–855. 199. Hernigou P, Zilber S, Filippini P, Mathieu G, Poignard A, Galacteros F. Total THA in adult osteonecrosis related to sickle cell disease. Clin Orthop Rel Res. 2008;466:300–308. 200. Koshy M, Entsuah R, Koranda A, et al. Leg ulcers in patients with sickle cell disease. Blood. 1989;74:1403–1408. 201. Serjeant GR. Leg ulceration in sickle cell anemia. Arch Intern Med. 1974;133:690–694. 202. Eckman JR. Leg ulcers in sickle cell disease. Hematol Oncol Clin North Am. 1996;10:1333–1344. 203. Ballas SK. Pain management of sickle cell disease. Hematol Oncol Clin North Am. 2005;19:785–802.

523 204. Adams RJ. Neurologic complications. In: Embury SH, Hebbel RP, Mohondas N, Steinberg MH, eds. Sickle Cell Disease: Basic Principles and Clinical Practice. 1st ed. New York: Raven Press; 1994:599–621. 205. Konotey-Ahulu FI. Mental-nerve neuropathy: a complication of sickle-cell crisis. Lancet. 1972;2:388. 206. Asher SW. Multiple cranial neuropathies, trigeminal neuralgia, and vascular headaches in sickle cell disease, a possible common mechanism. Neurology. 1980;30:210– 211. 207. Sommer C. Painful neuropathies. Curr Opin Neurol. 2003;16: 623–628. 208. Woolf CJ, Mannion RJ. Neuropathic pain: aetiology, symptoms, mechanisms, and management. Lancet. 1999;353: 1959–1964. 209. Harden RN. Chronic neuropathic pain. Mechanisms, diagnosis, and treatment. Neurologist. 2005;11:111–122. 210. Irving GA. Contemporary assessment and management of neuropathic pain. Neurology. 2005;64:S21–27. 211. Siddall PJ, Cousins MJ. Persistent pain as a disease entity: implications for clinical management. Anesth Analg. 2004;99: 510–520. 212. Edwards RR. Individual differences in endogenous pain modulation as a risk factor for chronic pain. Neurology. 2005;65: 437–443. 213. Krantz MJ, Mehler PS. Treating opioid dependence. Growing implications for primary care. Arch Intern Med. 2004;164: 277–288. 214. Portenoy RK, Foley KM. Chronic use of opioid analgesics in non-malignant pain: report of 38 cases. Pain. 1986;25:171– 186. 215. Schofferman J. Long-term use of opioid analgesics for the treatment of chronic pain of nonmalignant origin. J Pain Symptom Manage. 1993;8:279–288. 216. Ballantyne JC, Mao J. Opioid therapy for chronic pain. N Engl J Med. 2003;349:1943–1953. 217. Ballantyne JC. Opioids for chronic pain: taking stock. Pain. 2006;125:3–4. 218. Kalso E, Edwards JE, Moore RA, McQuay HJ. Opioids in chronic non-cancer pain: systematic review of efficacy and safety. Pain. 2004;112:372–380. 219. Eriksen J, Sjogren P, Bruera E, Ekholm O, Rasmussen NK. Critical issues on opioids in chronic non-cancer pain: an epidemiological study. Pain. 2006;125:172–179. 220. Medicine AAoP, Society AP. The use of opioids for the treatment of chronic pain: a consensus statement. Clin J Pain. 1997;13:6–8. 221. Fishman SM, Wilsey B, Yang J, Reisfield GM, Bandman TB, Borsook D. Adherence monitoring and drug surveillance in chronic opioid therapy. J Pain Symptom Manage. 2000;20:293–307. 222. States Federation of Medical Boards, Model policy for the use of controlled substances for the treatment of pain. Available at: http://www.fsmb.org/pdf/2004 grpol Controlled Substances.pdf. 2004. 223. Fishman SM, Bandman TB, Edwards A, Borsook D. The opioid contract in the management of chronic pain. J Pain Symptom Manage. 1999;18:27–37. 224. Consent for chronic opioid therapy. American Academy of Pain Medicine, 2001. Available at: http://www.painmed. org/pdf/opioid consent form.pdf.

524 225. Long-term Controlled Substances Therapy for Chronic Pain. Sample Agreement. 1997 Available at: http://www.painmed. org/pdf/controlled substances sample agrmt.pdf. 226. Arnold RM, Han PK, Seltzer D. Opioid contracts in chronic nonmalignant pain management: objectives and uncertainties. Am J Med. 2006;119:292–296. 227. Hariharan J, Lamb GC, Neuner JM. Long-term opioid contract use for chronic pain management in primary care practice. A five year experience. J Gen Intern Med. 2007;22:485– 490. 228. Zylicz Z, Twycross R. Opioid-induced hyperalgesia may be more frequent than previously thought. J Clin Oncol. 2008;26:1564. 229. Doverty M, White JM, Somogyi AA, Bochner F, Ali R, Ling W. Hyperalgesic responses in methadone maintenance patients. Pain. 2001;90:91–96. 230. White JM. Pleasure into pain: the consequences of long-term opioid use. Addict Behav. 2004;29:1311–1324. 231. Angst MS, Clark JD. Opioid-induced hyperalgesia: a qualitative systematic review. Anesthesiology. 2006;104:570–587. 232. Chang G, Chen L, Mao J. Opioid tolerance and hyperalgesia. Med Clin North Am. 2007;91:199–211. 233. Mao J. Opioid-induced abnormal pain sensitivity: implications in clinical opioid therapy. Pain. 2002;100:213–217. 234. Mao J. Opioid-induced abnormal pain sensitivity. Curr Pain Headache Rep. 2006;10:67–70. 235. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, Revised. 4th ed. Washington, DC: American Psychiatric Association; 1994. 236. Kalivas PW, O’Brien C. Drug addiction as a pathology of staged neuroplasticity. Neuropsychopharmacology. 2008;33: 166–180. 237. O’Brien CP. Research advances in the understanding and treatment of addiction. Am J Addict. 2003;12 Suppl 2:S36–47. 238. Jackson JL, Kroenke K. Difficult patient encounters in the ambulatory clinic: clinical predictors and outcomes. Arch Intern Med. 1999;159:1069–1075.

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21 Hemoglobin SC Disease and Hemoglobin C Disorders Martin H. Steinberg and Ronald L. Nagel

INTRODUCTION HbC (HBB glu6lys), along with HbS (HBB glu6val) and HbE (HBB glu26lys), is one of the three most common hemoglobin variants in humankind. Its positive charge that allows it to bind the erythrocyte membrane, and perhaps other unique features of this variant, lead to loss of cell K+ and water, thereby increasing erythrocyte density. HbC disease, defined as homozygosity for the HbC gene, causes mild hemolytic anemia; simple heterozygosity for HbC (HbC trait, HbAC) is innocuous. In HbSC disease, in which the erythrocyte concentration of HbS and HbC is nearly equal, the dehydrated, dense erythrocyte accentuates the deleterious properties of HbS by producing a milieu favoring HbS polymerization. HbSC disease causes vasoocclusive disease and hemolytic anemia, albeit on average both less severe than found in sickle cell anemia (homozygosity for HbS). Like sickle cell anemia, the hematological and clinical features of HbSC disease are heterogeneous, but all of the complications that make sickle cell anemia notorious can be present; some even appear more often in HbSC disease.

HbC and HbC Disease

their haplotypes, HbC in sub-Saharan Africa and North Africa, appears to have had the same origin as in Burkina Faso. Strong gene flow, influenced by the selective pressure of Plasmodium falciparum malaria, has distributed HbC throughout central West Africa and toward North Africa, particularly Morocco. Absence of a Hpa I restriction site 3
to the HbC gene made it doubtful that HbC arose from mutations in a HbS gene.1 HbC, which is found in Africa in a restricted distribution, reaches its highest frequency in central West Africa and its gene frequency decreases concentrically outward from there.2,3 Interestingly, HbC is found in Africa almost exclusively in areas where HbS exists. Although the possibility of a second origin of the HbC gene in Africa has been raised by the presence of a 3
␤-globin gene Hpa I site in several patients, this site is in the middle of an extensive L1 repeat where a high probability of rearrangement exists. Recombination is expected to reduce the effect of selection on the extent of linkage disequilibrium. It was estimated that the HbC mutation originated less than 5,000 years ago, but despite strong selection and this recent origin, crossing-over or gene conversion present in the “hotspot” 5
to the ␤-globin gene locus in more than a third of the HbC chromosomes sampled led to rapid decay in linkage disequilibrium upstream of the HbC allele mitigating the effects of positive selection. Two instances of independent origin of the ␤C have been found in Thailand and in Oman.4 In both, a strong case for independent origin is made by the presence of the mutation in a different chromosomal framework. Haplotypes of the HbC Gene. Haplotypes of ␤C -globin gene–bearing chromosomes are shown in Figure 21.1A. Three major haplotypes of the ␤C -globin gene, termed CI, CII, and CIII are found. Several minor unusual haplotypes have also been described (Fig. 21.1B). Of 90 ␤C -globin gene chromosomes, 70% were CI, 20% CII, and 10% had other haplotypes that were either CIII or compatible with recombination events. Heterogeneity of haplotypes associated with the ␤C gene is likely to result from crossovers in the 5
portion of the ␤-globin gene cluster, an event that is common and results in atypical haplotypes of the ␤S gene. In Africa, most ␤C chromosomes from Burkina Faso and Benin had the C I haplotype.

Origins, Selection, and Distribution of HbC HbC, the second hemoglobin variant discovered, was described in 1950, and the first homozygous case was reported in 1953. The ␤C -globin gene contains a GAG→AAG transition and codes for lysine instead of glutamic acid. Shortly after its description in African Americans, HbC was found to be common in Africa. Origins. The HbC mutation originated on a ␤A -globin gene in West Africa and by means similar to the dispersion of the HbS gene (Chapter 27) spread throughout the world. The ␤C -globin gene migrated northward through ancient trans-Saharan trade routes. Predicated on the identity of

HbC and Malaria HbC provides some protection from malaria.5 In a casecontrol study in Burkina Faso, HbC was associated with a 29% reduction in risk of clinical malaria in carriers and a 93% reduction in HbC disease.6 It was also associated with reduced parasitemia and protection from malarial attacks.7 Among children with severe malaria, asymptomatic parasitemia and no malaria studied in Ghana, HbC trait did not prevent infection with P. falciparum but reduced the odds of developing severe malaria and anemia, albeit to a lesser extent than sickle cell trait (HbAS). The multiplication rate 525

526

Martin H. Steinberg and Ronald L. Nagel

A

B

Figure 21.1. ␤-Globin gene cluster haplotypes associated with the ␤C -globin gene. Above each section are depicted the ␤-like globin genes with approximate size markers in kilobases. (A) Haplotypes of ␤C -globin gene–bearing chromosomes were determined using eight restriction fragment length polymorphisms (RFLPs). Shown are the “typical” haplotypes CI, CII, and CIII. Arrows indicate, from left to right, the polymorphic restriction sites, Hind III (2 sites), Hinc II (2 sites), Hinf I, Ava II, Hpa I, and Bam HI. (A) (+) indicates cleavage and a (−), lack of cleavage by the designated enzyme. (B) Two atypical haplotypes associated with the ␤C -globin gene (A1 ␤C and A2 ␤C ). These haplotypes were determined using additional RFLPs as shown.107,108

of P. falciparum in HbC disease erythrocytes is lower than in normal cells with a high proportion of ring forms and with trophozoites disintegrating. Also, knobs present on the surface of infected cells are fewer and morphologically aberrant when compared with those on normal cells.8 These data support natural selection acting on the HbC gene because of the relative resistance it confers against severe malaria but not resistance to acquiring an infection.9,10

HbC had even higher solubility than HbA. Oxygen affinity of HbC “stripped” of 2,3-BPG was normal. HbC Crystallization. When concentrated solutions of purified hemoglobins are incubated in high-molarity

A

B

PATHOPHYSIOLOGY OF HbC DISORDERS Biochemical Features of HbC HbC was first detected because of its slow migration compared with HbA and HbS during cellulose acetate electrophoresis at pH 8.6, a difference caused by the net increase of two units of positive charge per molecule. Newer techniques of hemoglobin separation now allow high-performance liquid chromatography (HPLC) separation of variants with similar positive charges such as HbE and HbO-Arab (HBB glu121lys) and also the normal HbA2 , from HbC (Chapters 7, 23, and 28). Carboxy HbC has decreased solubility in phosphate buffers, whereas deoxy HbC has nearly the same solubility as deoxy HbA. In concentrated phosphate buffers, deoxy

Figure 21.2. Crystal forms observed in hemolysates of patients with HbC. (A) Cubic crystals, generated in hemolysates of compound heterozygotes for HbC and Hb Korle-Bu. (B) Tetragonal crystals observed intracellularly and in hemolysates of patients homozygous for HbC, with HbSC disease and compound heterozygotes for HbC and HbA and other mutants. (See color plate 21.2.)

Hemoglobin SC Disease and Hemoglobin C Disorders phosphate buffer, hemoglobin crystals usually form (Fig. 21.2). In these crystals, hemoglobin can be liganded either as cyanmet, oxy, or CO hemoglobin. HbC crystals form within 25 minutes in a 100% HbC solution and HbF appears to inhibit crystallization.11 In HbC disease erythrocytes, cells with crystals isolated from density gradients had very low HbF concentrations.12 In the dense cell fraction, F cells did not contain HbC crystals. HbA2 inhibited HbC crystallization to a greater extent than HbF. The critical residue involved is at position 87 in the ␦- and ␥ -globin chains where a gln replaces thr. This is the same residue that might account for the inhibition of HbS polymerization by HbF and HbA2 .11 An examination of the interactions of HbC with Hb Lepore-Boston, which inhibits HbC crystallization to a lesser extent than HbF and HbA2 , suggests that a reduction in inhibition could arise from the slight difference in hemoglobin conformation due to the shift of oxygen equilibrium observed in Hb Lepore.11,13 Other amino acids located between positions 88 and 146 could add to the inhibitory effect of both the ␥ -and the ␦-globin chains on HbC crystallization. Other mutant hemoglobins that are present with HbC can affect its in vitro crystallization. Compound heterozygotes having HbC-HbN-Baltimore (HBB lys95glu) and HbC-Hb Riyadh (HBB lys120asn) suggest that ␤120 and ␤95 are additional contact sites in the crystal. Hb Riyadh inhibited the in vitro crystallization of HbC explaining the lack of overt pathology, with the exception of microcytosis, in a compound heterozygous infant. In contrast, HbN-Baltimore accelerated the crystallization of HbC and contributed to abnormal red cell morphology, suggesting that the crystal is sustained by hydrophobic interactions.14 Hb Korle-Bu (HBB asp73asn) accelerates HbC crystallization.15 Compound heterozygotes for Hb Korle-Bu and HbC have mild microcytic hemolytic anemia and in vitro acceleration of crystal formation, where precrystal hemoglobin structures convert rapidly into cubic-like crystals as opposed to the typical tetragonal crystal structure. In vitro crystallization studies led to the conclusion that ␤87 and ␤73 are contact sites of the oxyhemoglobin crystal.13 Hemoglobin ␣2 G-Philadelphia ␤2 C has an increased rate of crystal nucleation compared with HbC, implying that position ␣68, the mutation site of HbG-Philadelphia, is a contact site in the crystal of HbC.16 Mixtures of HbA and HbC crystallize with a different morphology than the classic HbC crystal habit, raising the question of the hemoglobin composition of crystals generated in mixtures of HbC with HbF or HbA2 . As the HbA concentration of the mixture increases, more tetragonal crystals are formed as opposed to typical orthorhombic crystals. HbA alone does not form crystals under these conditions, making it likely that hybrid tetramers (␣2 ␤C ␤A ) are incorporated into these crystals. It also suggests strongly that these two hemoglobins cocrystallize. HbS also cocrystalizes with HbC.

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Figure 21.3. View of the F, E, and A helix in the ␤ chains of hemoglobin. Also depicted are the heme, the ␤15 trp - ␤72 ser bond and the ␤6 Lys (the mutation of HbC). The arrows depict the likely movement of the A helix away from the E helix, which is compatible with conformational changes in the central cavity of the tetramer.13

A weakening of trp␤15–ser␤72 bond most likely leads to a displacement of the A helix away from the E helix.13 The central cavity (between the two ␤-globin chains of HbC, is altered compared with HbA17 (Fig. 21.3). HbC crystals do not grow by the alignment of the preformed strand but by the attachment of single molecules to suitable sites on the surface. These sites are located along the edges of new layers generated by two-dimensional nucleation. During growth, the steps propagate with random velocities, with the mean being an increasing function of the crystallization driving force.18 Static and dynamic light scattering characterization of the interactions between the R-state (CO) of HbC, HbA, and HbS molecules in low-ionic-strength solutions showed that the interactions are dominated by the specific binding of solutions’ ions to the proteins. Crystals of HbC nucleate and grow by the attachment of native molecules from the solution and concurrent amorphous phases. Spherulites and microfibers are not building blocks for the crystal. HbC crystallization is possible because of the huge entropy gain stemming from the release of up to 10 water molecules per protein intermolecular contact–hydrophobic interaction, suggesting that the higher crystallization propensity of R-state HbC is attributable to increased hydrophobicity.18 Deoxy HbC formed aggregates and twisted macroribbon forms similar to those seen in the oxy liganded state. In contrast to oxy HbC, deoxy HbC favored the formation of a greater morphological variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. These results suggest that the glu6lys substitution evokes a crystallization process dependent on ligand state conformation. Oxy HbC is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. In contrast, deoxy HbC energetically equally favors multiple pathways

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Figure 21.4. Scanning microscopy of HbC disease fractions. (A and B) Whole blood, in B a folded cell. (C and D) Middle density red cell fraction, notice the abundance of folded cells. (E and F) The highest density red cell fraction, arrows point to crystal containing red cells.

of aggregation, not all of which might culminate in crystal formation.19 Intracellular Crystals in HbC Disease. HbC crystalizes in its oxy configuration and crystals probably exist in vivo. Nevertheless, they are unlikely to play an important role in the pathophysiology of this disorder because deoxygenation will induce melting before or as they enter the microcirculation. Observations of crystals in vivo were first made under circumstances in which crystal formation could have been induced by cell processing. When blood from splenectomized HbC disease patients was examined by continuous density gradients, a new, very dense band of cells containing crystals 1 ␮m or more in size were observed. This cell fraction was absent from most HbC disease individuals with intact spleens but present in smaller amounts in some HbC disease patients older than 55 years. Scanning electron microscopy of HbC cells isolated from density gradients and freeze-fracture preparations show intracellular crystals (Figs. 21.4 and 21.5). Circulating crystals can be detected in unperturbed wet preparations from individuals with HbC disease but are rare in unsplenectomized cases.20 HbC crystals in the red cells of a splenectomized HbC disease patient were found to be in the oxy state and melted after deoxygenation (Fig. 21.6).12 When cells from venous and arterial blood were fixed and counted, a small but significant difference in the mean percentage of crystalcontaining cells in the arterial circulation versus the venous

Figure 21.5. Freeze fracture of an HbC disease cell from a splenectomized patient containing a highly ordered crystal (XC) and tail portion (T) that correspond to the membrane of the cell devoid of hemoglobin. This is a negative print (light areas have accumulation of platinum) of a predominantly cross-fractured cell.

Hemoglobin SC Disease and Hemoglobin C Disorders

Figure 21.6. Melting of oxy HbC crystal intracellularly by deoxygenation. Time sequence (in seconds) recording of oxy HbC intracellular melting after the addition of isotonic dithionite solution for deoxygenation. Demonstration that the crystal habit of the intracellular crystal is indeed oxy, which is incompatible with the deoxy conformer of HbC.

circulation was present. HbC crystals were not observed either in vitro or in vivo in HbC trait.

PROPERTIES OF THE HbC ERYTHROCYTE Morphology HbC disease erythrocytes are noted for their bizarre morphology. In wet preparations, microcytic and hyperchromic cells are present, whereas in dried stained blood films, target cells, microspherocytes, and cells with crystalline inclusions are found. Target cells, a diagnostically useful artifact, are presumably the consequence of the greater surface/ volume ratio of HbC disease cells, which is in turn the consequence of their reduced water content.

Ion Transport and Cation Content Erythrocytes that contain HbC have a characteristic volume-stimulated K+ leak. Consequently, intracellular cation and water content of HbC disease cells are strikingly reduced, whereas the cation content of HbC trait and HbSC disease cells is less prominently depleted (Chapter 9).21,22 When the cation and water content of HbC disease cells were increased by making the membrane permeable to cations and returning the cells to an isotonic medium, they returned to their original volume by gaining K+ , a behavior that was not observed in control cells. Volumestimulated K+ efflux was neither inhibited by ouabain and butamide nor blocked by inhibitors of Ca2+ -activated K+ permeability. A volume-regulated decrease in cell water in HbC disease cells stands in sharp contrast to the usual description of the human red cell as a passive, slightly imperfect osmometer and probably explained the reduced response of HbC disease cells to changes in the extracellular osmotic pressure. From these observations, it was concluded that HbC disease cells have a potassium transport mechanism that is either absent from, or inactive in, normal cells, and raised the possibility that the presence of the abnormal hemoglobin was responsible for the

529 activity of this transporter. Volume-stimulated K+ efflux occurred when osmolarity and pH were both reduced, an effect that was chloride–dependent, N-ethylmaleimde stimulated, and similar to previously reported K+ Cl− transporters.23–25 Transporters of this sort are present in normal and sickle reticulocytes under oxygenated conditions and decrease in activity with cell aging.26 Although red cell lifespan is shortened to approximately 40 days in HbC disease, this is more than three times the lifespan of sickle cell anemia erythrocytes, suggesting that factors beyond a high proportion of young cells contribute to the high activity of the K+ Cl− transporter in HbC disease. Total cellular calcium was elevated in HbC disease cells as it was in sickle cell anemia and was sequestered in endocytic vesicles. K:Cl cotransport is a powerful volume regulator in young erythrocytes. The delay time for activation of K+ efflux in sickle cell anemia cells was sixfold shorter than for HbC disease cells and 5.1-fold shorter than for normal cells. HbC cells were first swollen to activate the K:Cl cotransport and then shrunk by reestablishing normal osmolarity. The delay time for K+ efflux was eightfold longer than sickle cell anemia cells. When K:Cl cotransport was activated by acidification, similar differences in the delay times for activation and deactivation were observed. Moreover, the delay time for activation increased markedly with cell density, but the delay time for deactivation was approximately equal in all density fractions. These studies suggest that K:Cl cotransport is very active in young HbC disease cells but has a longer delay time for deactivation by cell shrinkage than in sickle cell anemia or normal red cells. This suggests that transient activation of K:Cl cotransport in HbC disease reticulocytes, followed by a slow deactivation, can gradually decrease hydration and increase mean corpuscular hemoglobin concentration (MCHC). It implies that K:Cl cotransport regulation by phosphorylation/dephosphorylation might be altered either by a direct effect of HbC and/or by HbC-dependent alteration of enzymes controlling the phosphorylated state. Many of the same abnormalities in K:Cl cotransport can be found in HbC transgenic mice.27–30

Oxygen Affinity P50 of HbC disease cells was increased to 29.5 mm Hg (normal, 26.5 mm Hg). The 2,3-BPG content of HbC disease cells was equal to that of normal cells, whereas the difference between the intracellular and extracellular pH was increased for HbC disease cells. Decreased oxygen affinity of HbC disease cells was attributed primarily to reduced intracellular pH, but the “normal” 2,3-BPG content failed to take into account the diminished intracellular water content. When this is considered, the effective 2,3-BPG content is considerably higher than normal and should further decrease oxygen affinity. Decreased oxygen affinity could modulate the anemia of HbC disease by increasing oxygen delivery to tissues.

530 Osmotic Response and Cell Density High MCHC and low intracellular water content are characteristics of HbC cells, and the cells in HbC trait and HbC disease are denser than normal. Average MCHC in HbC disease, HbSC disease, HbC trait, and HbA cells were 38, 37, 34, and 33 g/dL, respectively. The youngest and lightest cells differ from the oldest and most dense cells by only 3–4 g/dL. In contrast, cells of sickle cell anemia have a very wide density distribution. HbC disease, HbSC disease, and HbC trait reticulocytes are denser than normal reticulocytes. Either these cells are denser than normal when they first enter the circulation or their density changes within 24 hours while they are still recognizable as reticulocytes. Reduced osmotic fragility in HbC disease is consistent with an increased surface to volume ratio.

Binding of HbC to Red Cell Membrane HbC, like HbS, interacts more strongly with the erythrocyte membrane than HbA. This interaction has been studied using changes in the fluorescence intensity of the membrane-imbedded fluorescent probe, whose fluorescence is quenched when it is approached by hemoglobin. At pH 6.8, the affinity of HbC for the erythrocyte membrane was approximately five times greater than that of HbA. Deoxy HbA and HbC were less strongly bound than the oxy form. When the NaCl concentration was increased, HbC was approximately 90% dissociated from the membrane. At higher hemoglobin concentrations, HbC binding was stronger, and higher salt concentrations were required to displace the hemoglobin from the membrane. The cytoplasmic portion of band 3 was implicated as the binding site for both HbA and HbC. HbO-Arab is more strongly bound than HbS and is even more tightly membrane bound than HbC, suggesting that both electrostatic charge and the protein conformation in the vicinity of the charged groups play roles in membrane binding. Membrane binding of abnormal hemoglobins could affect membrane proteins involved in anion or cation movement and play a role in altering red cell density.

Rheological Properties of HbC Disease Erythrocyte HbC disease cells were less filterable than normal cells. Although the optimum filterability of normal cells occurred at 300 mOsm, HbC disease cells were maximally filterable at 200 mOsm, an observation consistent with increased density. By measuring cellular elongation in response to a fluid shear stress, cellular deformability was examined as a function of buffer osmolarity. HbC disease cells were poorly deformable in isotonic media but deformability improved when osmolarity was reduced to 100–200 mOsm. HbC disease cells caused a 10%–20% increase in peripheral resistance when studied in a rat mesoappendix model. This minimal change is compatible with the benign clinical

Martin H. Steinberg and Ronald L. Nagel course of HbC disease and also suggests that the adverse affects density could be offset by their microcytosis.

HbC Disease, HbC Trait, HbC–␤ Thalassemia, and HbC–␣ Thalassemia HbC trait is found in 2% of African Americans.31 In parts of West Africa, the prevalence of the HbC gene can reach 0.125.3,32 Among African Americans, the prevalence of HbC disease is 1 in 6,000. Vasoocclusive episodes are not a feature of HbC trait or HbC disease. Although poorly deformable, HbC cells are rheologically competent. Any oxy HbC crystals formed will tend to melt when cells become lodged in the microcirculation.

Laboratory Features and Diagnosis Both HbC trait and HbC disease are characterized by target cells and other abnormal erythrocytes. Individuals with HbC trait are not anemic, have normal mean corpuscular volume (MCV) and normal red cell lifespan. MCHCs were approximately 2 g/dL higher than normal cells and 1 g/dL more than HbAS cells.33 HbC trait erythrocytes contain approximately 40% HbC and 2%–3% HbA2 . In HbC disease, HbC is the predominant hemoglobin with normal amounts of HbA2 and HbF. Many of the older commonly used methods of hemoglobin separation did not resolve HbC from HbA2 and positively charged variants like HbE and HbO-Arab. ␣ Thalassemia can be present in individuals with HbC trait or in homozygotes with HbC disease. Trimodality in the percentage of HbC in carriers of HbC trait first suggested the presence of ␣ thalassemia as a modulator of HbC concentration. HbC plus HbA2 concentration was 44%, 38%, and 32% in HbC trait carriers presumed to have four, three, and two ␣-globin genes, respectively. In other cases in which the carrier was presumed to have heterozygous ␣+ thalassemia, MCV was reduced and HbC and HbA2 together were approximately 33%; however, the ascertainment of ␣ thalassemia did not rely on DNA analysis. A case of HbC trait–HbH disease, caused by compound heterozygosity for the -␣3.7 and SEA deletions (Chapter 13), had splenomegaly, mild anemia, MCV of 59 fl, and 24% HbC.34 HbC disease–␣ thalassemia has been described with a phenotype similar to HbC disease, but again, ␣-globin gene mapping was not done. In HbC trait–␣ thalassemia, the concentration of HbC is reduced similarly to HbS levels in HbAS–␣ thalassemia and this reduction depends on the number of deleted ␣-globin genes (Chapter 23). mRNA isolated from individuals with simple HbC trait and HbC trait with either homozygous ␣ thalassemia-2 or HbH disease, supported balanced expression of ␤C and ␤S globin favoring a post translational mechanism for the modulation of HbC concentration.35 Compared with the wealth of information on the effects of the interaction of ␣ thalassemia and HbAS,

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there are few available data on the hemato- Table 21.1. Hematological findings in HbC disease and HbC–␤0 thalassemia∗ logical and clinical aspects HbC trait or HbC Hemoglobin MCV HbF Reticulocytes disease with ␣ thalassemia. Because both HbC (g/dL)/PCV (fL) (%) (%) Spleen trait and the -␣3.7 type of ␣ thalassemia are clin- Genotype ically innocuous there is no reason to believe HbC Disease 10–15/30–45 60–90 2–4 2–7 ↑↑ that their concordance should be any different. 8–12/25–35 55–70 3–10 5–20 ↑↑↑ Hb C–␤0 HbC crystals, very prominent target cells, Thalassemia and anemia are present in HbC disease. Mild hemolytic anemia is customary: the packed ∗ These data are a composite of many cases reported in the literature. cell volume (PCV) is usually 32%–34% and hemoglobin concentration between 9 and 12 g/dL, with a resembling HbC–␤+ thalassemia because of the presence of moderate reticulocytosis of between 5% and 10%. CharHbF and Hb Lepore. acteristically, the MCV is between 55 and 65 fL. Using DF 32 P(di-isopropylfluoropropyl phosphate)-labeled red cells, Clinical Features and Treatment of HbC Trait a mean red cell lifespan of 38.5 and 35.1 days was found in and HbC Disease two individuals with a 51 Cr red cell survival of 19 days. Hemolysis is unlikely to be the sole mechanism generHbC trait is not associated with clinical disease. Hematuria ating anemia in HbC disease. A reduced oxygen affinity of and isosthenuria, the best-characterized clinical abnormalHbC cells, could account for a portion of the reduction in ities of HbAS, are not associated with HbC trait. PCV as these red cells would more readily unload oxygen Individuals with HbC disease have hematological and in the tissues. In turn, erythropoietin production would be clinical evidence of their hemoglobinopathy but appear to stimulated at a lower hemoglobin level than normal. Anehave a normal lifespan and few signs or symptoms besides mia might therefor appear to be uncompensated because splenomegaly that are attributable to their disease. They do of the decreased oxygen affinity of HbC disease red cells. not have vasoocclusive events typical of sickle cell disease. Dyserythropoietic features, including nuclear disorganizaSplenomegaly has been reported in most adult patients tion and altered chromatin staining, have been found in with HbC disease and is usually asymptomatic. In the past, erythroblasts of HbC disease but not sickle cell anemia. splenectomy was frequently recommended with the hope These findings suggest that ineffective erythropoiesis could of correcting the anemia; current practice avoids splenecaccount for some of the anemia in this disorder although tomy in all but very selected cases. Splenectomy is usually why this should be is unclear. In children with HbC disease, followed by a slight increase in PCV and by increased numhemoglobin levels and reticulocyte counts were similar to bers of circulating cells containing HbC crystals. Children those in adults and the MCV was between 60 and 80 fL.36 might have a lower prevalence of splenomegaly.36 Sponta+ 0 neous rupture of the spleen has been described in HbC disHbC–␤ thalassemia and ␤ thalassemia have been ease. Increased density of the HbC-containing erythrocytes described, and like HbS-␤ thalassemia, the phenotype is does not impair renal concentrating ability. determined by the ␤ thalassemia mutation. In individuals of African descent, because of the prominence of the “mild” promoter mutations in this population, HbC-␤+ Treatment Recommendations thalassemia has hematological features of ␤ thalassemia trait with microcytosis and either no anemia or very mild As HbC disease is clinically asymptomatic and hematologanemia. HbC levels are 20%–30% and similar to HbS ically benign, no special treatment is needed. Iron should levels in most black patients with HbS–␤+ thalassemia. be avoided because it will not, in the absence of iron lack, repair anemia or microcytosis. Hemolysis is very mild, so Like HbS–␤+ thalassemia in Mediterranean populations, a reasonably nutritious diet should provide sufficient folic ␤+ thalassemia mutations that considerably reduce gene acid, and supplementation is unnecessary. No information expression produce a more severe phenotype approaching exists about hemolysis related pulmonary hypertension in that of HbC–␤0 thalassemia. HbC disease. HbC–␤0 thalassemia can be difficult to distinguish from Parvovirus B19 can transiently interrupt erythropoiesis HbC disease because HbA is absent and HbA2 is not always but because hemolysis is mild, the sequelae of this infection easily measured when HbC is present (Table 21.1). DNA should not be as serious as when it occurs in sickle cell aneanalysis or family studies are required when it is impormia and might not reach the threshold of clinical detection. tant to make the distinction between HbC disease and Patients sometimes have aches and pains and it is not HbC–␤0 thalassemia. Further complicating the separation clear if these can be linked to HbC disease. There is no eviof these disorders is the possibility of ␣ thalassemia coindence that HbC disease is accompanied by a reduction in cidental with HbC disease, which is likely to cause a phelongevity in the Western World, although this might not be notype similar to HbC–␤0 thalassemia. HbC trait has been true in the harsh environmental conditions of the African described with gene deletion hereditary persistence of HbF, Sahel. Hb Lepore, and with ␦␤ thalassemia. All are mild conditions

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HbSC Disease In HbSC disease the erythrocytes contain nearly equal concentrations of HbC and HbS and each hemoglobin component exerts its own special pathological effects that synergistically cause the well-delineated features of HbSC disease. HbSC disease is the neglected stepchild of sickle hemoglobinopathies. Its unique pathophysiology suggests that it could be amenable to treatments that might prevent dense cell formation, yet until very recently, therapeutic trials in HbSC disease have not been done and most often, in clinical trials, its treatment has been lumped with treatment for sickle cell anemia. This might have the dual unfortunate effect of confounding the interpretation of trials for sickle cell anemia and retarding the development of novel treatments for HbSC disease.

Cellular Determinants of Pathophysiology and Severity HbAS and HbSC disease erythrocytes both have far lower concentrations of HbS than cells of sickle cell anemia. Nevertheless, the expectation that because of low HbS concentrations polymerization-induced defects will be attenuated or absent is realized only in HbAS. The reasons why HbAS and HbSC disease differ, which elucidate the pathophysiology of HbSC disease, are discussed in the following sections. HbC Crystals in HbSC Disease. Crystals are the most striking and distinctive feature in circulating red cells of HbSC disease, especially when patients have a normal complement of ␣-globin genes (Figs. 21.7 and 21.8). HbS accelerates the crystallization of HbC in vivo and in vitro.37 HbC crystals are observed in Wright and vital dye– stained smears and in “wet” blood preparations (Fig. 21.7).

Figure 21.8. Several types of red cells found in the blood of HbSC disease patients illustrated by scanning electron microscopy: Folded red cells (Top panel), “pita bread” red cells (middle panel), and highly folded red cell (lower panel) from whole blood of a HbSC disease patient.

Figure 21.7. Erythrocytes in HbSC disease. These cells are characterized by sharp edges, increased red color (particularly in a reticulocyte stained smear), and often by having all the hemoglobin of the cells recruited into the crystal, leaving a ghost of the red cell without cytosolic dissolved hemoglobin. 1 = crystal containing red cell; 2 = “billiard ball” cells; 3 = folded cells. (Lawrence et al., Blood 1991;78: 2104–2112.)

When ␣ thalassemia is present with HbSC disease, typical crystals are absent in some patients. All HbSC disease patients’ red cells exhibit heavily stained conglomerations of hemoglobin that appear marginated with rounded edges in distinction to the straight edged crystals. Both crystals and hemoglobin conglomerations are found in the densest fraction of HbSC disease and represent hemoglobin aggregation distinct from HbS polymer. Regardless of the ␣globin gene haplotype, the blood of HbSC disease patients has additional abnormally shaped cells that are strikingly apparent upon scanning electron microcopy (Fig. 21.8). These bizarre shapes are the product of an increased surface/volume ratio. Crystals, pitted by a normal spleen might

Hemoglobin SC Disease and Hemoglobin C Disorders

Figure 21.9. This graph depicts individual red cell delay times of polymerization in HbSC disease compared with sickle cell anemias and HbAS. Notice that many HbSC cells behave as sickle trait cells, as far as delay times, but that also some of the cells behave as the slowest sickle cell anemia cells. In effect, delay time of HbSC cells is intermediate between sickle cell anemia and sickle cell trait cells, with overlapping.109

remain in the cell in the presence of an enlarged, infarcted and perhaps abnormally functioning spleen. Ratio of HbS to HbC. The ratio of HbS/HbC is approximately 50:50 compared with the 40:60 ratio of HbS/HbA in HbAS. Charge-related differences in ␣␤ dimer assembly account for the lower proportion of HbS in HbAS and HbC in HbC trait. Although HbC has an additional positive charge compared with HbS, both these abnormal variants can compete similarly for ␣-globin chains equalizing the HbS/HbC ratio. Just as HbF is unequally distributed among all cells, there is some evidence that HbC is unequally apportioned among red cells. Using single-cell electrophoresis, the distribution of HbS in HbAS and HbC in HbC trait was found to vary among cells but whether HbS concentration differs among cells in HbSC disease is unknown. HbSC disease is associated with hemolytic anemia and vasoocclusive complications, whereas HbAS is not, a difference that might be attributed to the higher concentration of HbS in HbSC disease. Although the effects of HbA and HbC on HbS polymerization are equivalent, increasing the HbS from 40% to 50% increased the rate of polymerization nearly 15-fold. The duration of the delay time before sickling occurs could play a role in the pathophysiology of HbSC disease. A long delay might allow a cell to move through hypoxic microvasculature before rigidity and rheological incompetence ensue, whereas a shorter delay time could increase a cell’s propensity to induce vasoocclusion (Fig. 21.9). Cell Density, Cation Content, and Density-related Properties. Cation content of HbSC disease red cells is intermediate between normal and HbC disease cells. Oxygenated HbSC disease cells exhibit a volume-stimulated K+ efflux

533 similar to that observed in sickle cell anemia and HbC disease. HbSC disease cells also exhibit a diminished change in cell volume in response to variation of the osmolarity of the suspending medium, which is likely to be due to volume, regulated K+ efflux. Volume-regulated K+ efflux should impact adversely the pathophysiology of disease because any increase of MCHC will aggravate HbS polymerization. The intracellular HbS concentration in HbSC disease is raised to a level where polymerization occurs under physiological conditions. This contrasts with HbAS cells in which polymerization occurs only at high osmolarity and low pH, such as in the renal medulla. Reducing the MCHC in HbSC disease to normal levels of 33 g/dL by osmotically swelling resulted in normalization of many of their polymerizationdependent abnormal properties.38 Among the beneficial effects of cell rehydration were increased hemoglobin oxygen affinity, reduction of viscosity of deoxygenated erythrocyte suspensions, fall in the rate of sickling, and reduction in the deoxygenation-induced K+ leak. At an osmolality of 240 mOsm, where the density distribution of HbSC disease cells most nearly matched normal cells, the cells were found to be biconcave disks. These observations suggest that restoring the density of HbSC cells toward normal might be an efficacious treatment. Analogous to sickle cell anemia, when ␣ thalassemia coexists with HbSC disease fewer dense cells are present. During the course of vasoocclusive crisis in HbSC disease, dense cells decrease just as in sickle cell anemia, suggesting their sequestration.39,40 HbSC disease cells exhibit pathological properties characteristic of both sickle cell anemia and HbC disease cells, and this feature helps explain the greater than anticipated severity of HbSC disease. A morphological analysis of density-separated HbSC disease cells indicated that they became progressively more aberrant in shape as their density increased. The densest fraction of the gradient contained cells with the most abnormal shapes, which were particularly noticeable for their gross pitting and membrane invaginations. HbSC disease cells destined to become extremely dense are likely also to produce crystals as a byproduct of their progressive MCHC increase as the concentration of hemoglobin within crystals is approximately 68 g/dL. Irreversibly sickled cells are rare in HbSC disease and are found in the densest cell fractions (Fig. 21.10). ␣ Thalassemia is associated with a decrease in the dense cell fraction. In contrast with sickle cell anemia, the highest percentage of reticulocytes is found within a fraction of dense cells, although the youngest, or stress reticulocytes, are predominant in the least dense fraction. This suggests that in HbSC disease reticulocytes exit the marrow as low-density cells and become dense within 24 hours, sinking to the next to densest fraction of cells in the blood. Reticulocytes are almost absent from the dense cell fractions that contains the largest proportion of crystal containing cells. Hyperdense reticulocytes migrate to lower

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Figure 21.10. Percoll–Strachan (Larex) density separation of whole blood from normal hemolysate (AA), HbC trait (AC), and three different HbSC disease individuals and one sickle cell anemia patient. Beads: Color-coded density beads to establish the density at different levels of the gradient after centrifugation. See color plates.

densities if Cl− is removed from the media, in concordance with a K:Cl cotransport-mediated phenomena. It seems likely that K:Cl cotransport is responsible for the presence of HbSC disease reticulocytes of unusually high densities.

␤-Globin Gene Cluster Haplotypes in HbSC Disease A Benin haplotype ␤S chromosome was present in 56%, Bantu in 25%, Senegal in 6%, and other haplotypes in 12% of HbSC disease cases.41 No hematological differences among these patients were present.42 Hematological features of individuals with HbSC disease who had Benin or Bantu ␤S chromosomes were similar whether they had a C I or C II haplotype. Most likely, the effect of ␤-globin gene haplotype on the hematological and clinical features of sickle cell anemia is via haplotype-linked elements that affect the levels of HbF. HbF levels in HbSC disease are lower than in sickle cell anemia, probably because there is less hemolysis and bone marrow expansion in HbSC disease. Another speculative consideration is that the ␤C chromosome might not contain the critical genetic elements or polymorphisms that are present in sickle cell anemia and necessary for increased transcription of the ␥ -globin genes (Chapter 27).

CLINICAL FEATURES OF HbSC DISEASE In northern Ghana, Burkina Faso, and western Nigeria, approximately a quarter of the population might have HbSC disease. Centuries of population movements have spread the HbS and HbC genes throughout the world, broadening greatly the distribution of the sickle hemoglobinopathies. HbSC disease has an incidence of approximately 1:833 live births in African Americans.31 All complications that are found in patients with sickle cell anemia have occurred in individuals with HbSC

disease. Nevertheless, most, but not all, of these complications are less frequent and appear at a later age in the course of HbSC disease compared with sickle cell anemia. Hemolysis is less intense so that hemolysis-related complications like leg ulcer, priapism, stroke, pulmonary hypertension, aplastic episodes, and cholelithiasis are less frequent or severe. Disease complications due to blood viscosity, such as osteonecrosis of bone can be as common as in sickle cell anemia; proliferative sickle retinopathy is much more prevalent in HbSC disease than in sickle cell anemia; the mortality of acute chest syndrome might be increased.43 Painful episodes occur at approximately half the frequency as in sickle cell anemia. The lifespan of HbSC disease patients is shortened when compared with control populations, but is longer than in sickle cell anemia. Specific treatments that will prevent complications of this disease are not yet available. In the following sections, the clinical aspects of HbSC disease that differ most from those of sickle cell anemia are discussed. For details of treatment and many complications, which differ little among the genotype of sickle cell disease, the reader should consult Chapters 19, 20, and 29–33.

Hematology and Laboratory Findings HPLC now provides rapid quantitative analysis of hemoglobin fractions and can distinguish HbS, HbC, and many of the variants with which they might be confused (Chapter 28). Blood films can suggest the diagnosis of HbSC disease. Except in the presence of sickle cell–related renal failure or with coincidental medical conditions that can reduce the PCV, patients with HbSC disease do not usually have overt symptoms of anemia. At any age, PCV in HbSC disease is higher than in sickle cell anemia.44 Median values by age and sex for PCV, MCV, platelet count, and bilirubin are shown in Figure 21.11. PCV rises from 31 to 32 in

535

Figure 21.11. PCV, MCV, platelet count, and bilirubin levels in a cross-sectional study of patients with HbSC disease ages 2 years to older than 40 years. PCV, MCV, and reticulocyte counts were available for 586 patients and bilirubin for 467 patients. Box plots show the mean and 25th (bottom of box) and 75th percentile (top of box) of the data.44

536 children aged 2–5 years to approximately 37 in males older than 20 years, a level approximately five points higher than in females with HbSC disease. Many adult men, but fewer adult women, have a normal PCV.44–46 Individuals with high PCV might be at greatest risk for complications such as proliferative retinopathy and osteonecrosis of the femoral head. Leukocyte counts in HbSC disease are normal or only very slightly elevated. Perhaps this is a result of more persistent splenomegaly, reduced hemolysis, and less proliferative activity of the bone marrow. Platelet counts are also lower in HbSC disease than in sickle cell anemia, perhaps for similar reasons. Some individuals with HbSC disease have mild thrombocytopenia, and this can often be related to an enlarged spleen. Leukocytosis, thrombocytosis, and reticulocytosis have been linked to the severity of vasoocclusive disease in sickle cell anemia. Mature sickle cells circulate in the company of leukocytes, platelets, and “stress” reticulocytes that display adhesive ligands that facilitate erythrocyte– endothelial interactions. High granulocyte counts are a risk for mortality in sickle cell anemia. Perhaps some of the milder features of HbSC disease are accounted for by the lower numbers of activated neutrophils and platelets. Bilirubin is usually normal and lactic dehydrogenase (LDH) levels are lower, reflecting mild hemolytic anemia compared with sickle cell anemia. Chromium-51 survival in HbSC disease was 13–26 days and DF 32 P mean lifespan was 29 days. Reticulocytes in HbSC disease ranged from less than 1%–7% supporting the lower hemolytic rate. As in sickle cell anemia, red cell volume was only modestly reduced, whereas plasma volume was more substantially elevated. Red cell survival, plasma volume, and PCV were unrelated to splenomegaly or splenic function. Although hemolysis is only modest, compensation is incomplete. Mild anemia in HbSC disease cannot be entirely accounted for by the rate of red cell destruction because this does not exceed the potential of normal marrow to replace lost cells. In HbSC disease, the PCV and red cell mass average approximately 80%–85% of normal. As hypothesized for sickle cell anemia, it might be that increased P50 and enhanced tissue extraction of oxygen blunts erythropoiesis in HbSC disease. Although hemoglobin-oxygen affinity of hemoglobin isolated from HbSC disease red cells is normal, the whole blood P50 is increased to 32 mm Hg. Serum erythropoietin levels are higher than normal, but lower than found in sickle cell anemia.47 These levels might be inappropriately low for the level of anemia and could also be due to subclinical renal disease or an inappropriate marrow erythroid response. HbSC disease patients usually have HbF levels less than 2% but occasionally the HbF in adults is near 10%. HbF is unlikely to have a major affect on the hematology or clinical features of HbSC disease because of its low level in the majority of patients. In contrast to sickle cell anemia, individuals with HbSC and HbC disease, regardless of

Martin H. Steinberg and Ronald L. Nagel haplotype, have similar G ␥ -globin chain levels despite different degrees of erythropoietic stress. ␣ Thalassemia, a known modulator of sickle cell anemia (Chapter 27), has a minor effect on the hematology of HbSC disease. Although coincident ␣ thalassemia reduces the MCV by approximately 5 fl, there is little change in the hemoglobin concentration or HbF level.48

Growth and Development Growth in HbSC disease is delayed, but less so than in sickle cell anemia. With access to reasonable nutrition, Tanner stages of adult sexual development are achieved at 14–16 and 16–18 years of age in young women and boys, respectively, 1–2 years earlier than in children with sickle cell anemia.49 In some studies, HbSC disease does not appear to affect height, weight, and bone age.50 A study of 298 children with sickle cell anemia and 157 children with HbSC disease revealed that after age 5 years, HbSC disease children had normal growth.

Mortality A 1989 report indicated that 95% of patient’s with HbSC disease in the United States survived to age 20 years.51 These individuals were participants in the Cooperative Study of Sickle Cell Disease (CSSCD) (Chapter 19) and many attended comprehensive sickle cell centers, making it possible that they had more regular and skilled care than the general population of HbSC disease. Mortality in this series was greatest in children aged between 1 and 3 years, and was most often a result of pneumococcal sepsis. Another study, reported in 1990, in which 231 individuals with HbSC disease were compared with 785 sickle cell anemia patients, showed an age-specific death rate (per 100 person-years) up to age 30 years of less than one in HbSC disease compared with two–three in sickle cell anemia. Beyond this age, mortality in all patients increased. Nevertheless, the rate in HbSC disease was less than half that of sickle cell anemia.52 In the largest study of mortality from the United States, the median survival in HbSC disease was 60 years for men and 68 years for women (Fig. 21.12).53 Jamaican children with HbSC disease, diagnosed by cord blood screening, and so unlikely to represent a biased sample, had a 95% chance of surviving to age 2 years. Two hundred eighty-four patients with HbSC disease, more than 80% of whom entered the study as children, were followed over a span of 40 years for nearly 3,000 personyears.48 The median age of death in this study was 37 years and was unaffected by sex. Approximately 10% of patients died, and 60% of these entered the study prior to 1970. No single cause of death predominated. In another study of adults, the median age was 50 years.54 It has been theorized that since these reports, the mortality of sickle cell anemia has fallen in developed countries

Hemoglobin SC Disease and Hemoglobin C Disorders

537

Figure 21.12. Survival curves comparing patients with HbSC disease (A) and sickle cell anemia (B).110

because of the widespread use of prophylactic penicillin, better supportive care for events such as acute chest syndrome and perhaps hydroxyurea. Penicillin prophylaxis is not uniformly used in HbSC disease and studies of hydroxyurea as a treatment for HbSC disease are just starting, so their effects on mortality are not known. There are few data on risk factors for early death in HbSC disease. Median age of onset for irreversible organ failure like stroke, renal failure, and chronic lung disease, that might contribute to early mortality in HbSC disease is 10–35 years later than in sickle cell anemia.52 Even in large series of patients, relatively small numbers of deaths occur in HbSC disease, making it difficult to assess the relationship of HbF, leukocyte count, and prior vasoocclusive episodes such as pain and acute chest syndrome to mortality. It is clear that HbSC disease has a pattern of survival that is different from sickle cell anemia. Increased mortality of HbSC disease is apparent only after age 20 years. Overall prognosis of HbSC disease is, therefore, better than that of sickle cell anemia.

CLINICAL EVENTS Patients with HbSC disease can have all of the problems of sickle cell anemia but usually have a milder clinical course. A survey of 90 Jamaican patients with HbSC disease showed pathology similar to sickle cell anemia, but with generally reduced severity, except for retinal vascular disease. These findings might be a result of reduced hemolysis and higher hemoglobin levels in HbSC disease. In a pediatric cohort, 50% of HbSC patients were found to develop symptoms by age 5 years but 22% remained free of symptoms at age 10 years.55 Approximately 40% of patients were found to have some chronic organ complication with a mean age of onset of approximately 30 years.48 Many individuals with HbSC disease are active in quite strenuous occupations. Some never have the occasion to

seek medical attention for the unique events of sickle cell disease and have their diagnosis established incidental to another medical condition. Rarely, the presenting attribute of HbSC disease is calamitous, such as lifethreatening acute chest syndrome, multiorgan failure, sepsis, or splenic sequestration. Therefore, although sometimes mild and needing little obvious medical supervision, HbSC disease should not be neglected. Patients with HbSC disease should be detected by neonatal screening so that they and their families can be enrolled in programs providing education about the potential complications of this disorder and the needed level of continued monitoring.

Sickle Retinopathy Proliferative sickle retinopathy is perhaps the most typical vasoocclusive complication of HbSC disease (Fig. 21.13). More common in this genotype, where it is found in approximately a third of patients, than in sickle cell anemia where only 3% are affected, the pathology of this entity has been delineated in detail.56,57 Pathophysiology of Sickle Retinopathy. The primary site of vasoocclusion in sickle retinopathy was located at the precapillary level.58 Hairpin loops, a neovascular formation that appeared to result from recanalization of occluded vessels and autoinfarcted preretinal neovascular formations are present. Small pigmented lesions consisting of retinal pigment epithelial cells ensheathing channels that resembled autoinfarcted vessels were also seen. Macular blood flow velocity was compared in 18 patients with sickle cell disease and 45 normal controls and the relation between macular blood flow velocity and red blood cell density was examined.59 Leukocyte velocity in the macular capillaries was negatively associated with a greater range of red cell density, suggesting that sickle red cell density heterogeneity might slow macular capillary blood flow. The distribution and relative levels of components in the fibrinolytic system and growth factors in retina and choroid showed increased plasminogen activator inhibitor-1 immunoreactivity in retinal vessels compared with controls, whereas tissue plasminogen activator localization and immunoreactivity were similar.60 Immunoreactive fibrin was often observed within the lumen of retinal and choroidal vessels and in choroidal neovascularization. Blood vessels containing fibrin generally exhibited elevated plasminogen activator inhibitor-1 immunoreactivity. Von Willebrand factor and basic fibroblast growth factor immunoreactivity were elevated in choriocapillaris and the walls of some retinal vessels. Transforming growth factor– ␤1 (TGF-␤) immunoreactivity was significantly lower in

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Martin H. Steinberg and Ronald L. Nagel

Figure 21.13. Stages of proliferative sickle retinopathy.

sickle cell choriocapillaris than in controls. In chorioretinalpigmented lesions of an HbSC disease patient, basic fibroblast growth factor, TGF-␤1, 2, 3 immunoreactivity was present within migrating retinal pigment epithelial cells. These data suggested that fibrin deposition within retinal and choroidal vessels of sickle cell disease subjects could occur due to elevated plasminogen activator inhibitor–1 activity. Vasoocclusion of choroidal vessels might influence the expression of growth factors in choriocapillaris endothelium, which could stimulate formation of choroidal neovascularization. Fibrosis and gliosis in and near chorioretinal-pigmented lesions, could be stimulated by migrating retinal pigment epithelial cell production of basic fibroblast growth factor and TGF-␤. Pigment epithelium-derived factor (PEDF) is an inhibitor of angiogenesis and a neurotrophic factor in the mammalian eye, and its level is related to retinal neovascularization Using tissue from normal eyes and subjects with sickle cell retinopathy, PEDF was mainly localized to the vitreous condensed at the internal limiting membrane, RPEBruch membrane-choriocapillaris complex and choroidal stroma in normal eyes. In sickle retinopathy, the ratio of PEDF/vascular endothelial growth factor (VEGF) was increased in the nonperfused area of the nonproliferative sickle cell retina and in eyes with proliferative sickle cell retinopathy. Elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations and in feeder vessels of sea fans. Only PEDF was present in nonviable sea fans, suggesting that this might play an important role in inhibiting angiogenesis and inducing the regression of sea fans.61 Neovascularization is a common consequence of retina vasoocclusion. Morphometric analysis in postmortem ocular tissue from subjects with sickle hemoglobinopathies62

showed numerous active and autoinfarcted lesions, representing virtually all stages in preretinal neovascularization. They ranged from single small loops extending from arteries and veins along the retinal surface to the typical complex, elevated sea fan formations. Sea fans developed at hairpin loops and at arteriovenous crossings. There was an average of 5.6 connections between sea fans and retinal vessels; of these, 45% were arteriolar, 52.5% were venular, and 2.6% involved capillaries. Autoinfarction appeared to occur initially within the sea fan capillaries. Preretinal neovascularization in sickle cell retinopathy might arise from both the arterial and venous sides of the retinal vasculature and assume a variety of morphological configurations. Multiple feeding arterioles and draining venules are common, and autoinfarction appears to occur initially at the preretinal capillary level rather than at feeding arterioles. Plasma angiopoietin-1 and angiopoietin-2, soluble Tie-2, VEGF, soluble Flt-1, and von Willebrand factor were measured in patients with HbSC disease or sickle cell anemia who had proliferative and nonproliferative sickle retinopathy. In eight patients who received panretinal laser photocoagulation, plasma was obtained before and 6 months after treatment. Angiopoietin-1 and -2, VEGF, and von Willebrand factor were raised in all sickle cell patient groups compared with controls and only minor changes in were seen after treatment.63 Angiopoietin-2, erythropoietin, VEGF, and soluble Tie-2 and VCAM were elevated suggesting a proangiogenic state. With painful crisis, only VCAM increased.47 Vasoocclusion in the retina has also been studied in transgenic sickle mice and in a rat animal model.64 Retinal vascular occlusions resulted in nonperfused areas of retina and arteriovenous anastomoses. Intra- and extraretinal neovascularization was observed adjacent to nonperfused

Hemoglobin SC Disease and Hemoglobin C Disorders areas. Retinal pigmented lesions were formed by the migration of retinal pigment epithelial cells into sensory retina, often ensheathing choroidal neovascularization. Bilateral chorioretinopathy was present in 30% of animals older than 15 months. These ocular histopathological changes mimicked many aspects of human proliferative sickle cell retinopathy and permitted the detection of choroid abnormalities not previously characterized. Forty-two percent of ␣H ␤S [␤MDD ] (Chapter 12) mice without any proliferative retinopathy had retinal blood vessels containing red cell plugs visualized by endogenous peroxidase activity and lacked luminal horseradish peroxidase (HRP)–reaction product.65 In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion. In areas with normal photoreceptors, the choriocapillaris appeared perfused and HRP-reaction product was present. In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-reaction product, suggesting autoinfarction. Nonperfused retinal and choroidal vessels were observed in mice without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Small foci of HRP loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell–mediated retinal vasoocclusion is an initial event in the chorioretinopathy and outer retinal atrophy. Density separated, labeled HbSC and sickle cell anemia erythrocytes infused into rats were retained in capillaries. Dense cell retention was inversely dependent on pO2 .66 Trapping, not adhesion, appeared to be responsible for retention of sickle cells in the normal retinal vasculature because of preferential retention of dense cells with a lower adherence propensity. Paradoxically, given the prevalence of retinopathy in HbSC disease, there was low retention of HbSC cells. Fractionated sickle erythrocytes were infused into rats that were either hypoxic or were given TNF␣. Sickle erythrocytes were also preincubated with a peptide that inhibits binding of VLA-4 and ␣4␤7 integrins to their ligands. Hypoxia caused retention of dense but not reticulocyte-rich sickle cells. TNF␣ significantly increased retention of all types of sickle cells, and this was inhibited by VLA-4 inhibition or by monoclonal antibodies against fibronectin, a ligand for VLA-4. Similar inhibition of retention was seen in retinal vessels when a VLA-4 inhibitor was used.67 These studies suggested that the mechanisms for retention of sickle cells in retina and choroid appeared identical and was the result of hypoxia-mediated retention of dense red cells and adherence of sickle reticulocytes after cytokine stimulation. TNF␣-stimulated retention of sickle cells in choroid appeared to be mediated by VLA-4, presumably on the surface of some reticulocytes.68 The expression of the adhesion molecules ICAM-1, VCAM-1, and P-selectin, and the distribution and number of polymorphonuclear leukocytes were investigated

539 Table 21.2. Age-related prevalence of proliferative sickle retinopathy in HbSC disease

Age (y)

No. of patients

Percent with retinopathy

0–9 10–14 15–19 20–24 25–29 30–39 >40 Total

47 77 117 81 67 88 56 533

0 6 14 27 60 57 70 32

in sickle cell retinopathy and compared with the normal retina. In cryopreserved postmortem ocular tissue from subjects with sickle cell disease and one control subject, increased ICAM-1, VCAM-1, and P-selectin immunoreactivities were observed in sickle cell subjects and the highest ICAM and P-selectin immunoreactivity was associated with intraretinal vessels adjacent to the preretinal neovascular formation in subjects with proliferative sickle retinopathy. VCAM-1 immunoreactivity was highest in intraretinal vessels adjacent to the newly forming sea fans. Old sea fans had the highest levels of VCAM-1. The increase in adhesion molecule immunoreactivity was paralleled by an increase in intraretinal neutrophils that increased with progression of the disease. These data suggest that adhesion molecule mediated leukocyte adhesion might play a role in sickle cell retinopathy.69 Using a noninvasive method to assess the velocity, adherence, and arterial/venous transit time of normal and sickle erythrocytes in retinal and choroidal vessels, labeled red cells can be observed in the retina of pigmented rats and on the choroid of albino rats. Normal density sickle cells were transiently retained in retinal vessels. Sickle red cells were retained in larger numbers, in some cases for extended periods of time, suggesting that the choroid– capillaris vasoocclusion can play an important role in sickle retinopathy.70 Prevalence. Age-adjusted prevalence of proliferative retinopathy in 533 patients with HbSC disease is shown in Table 21.2. The distinctive “black sea fan,” “black sunburst” and salmon patches typify this disorder and characterize the hemorrhagic, infarctive, proliferative, and resolving lesions of sickle retinopathy. Conjoint effects of a higher PCV, increased cell density, and greater blood viscosity in HbSC disease could account for the higher prevalence of retinopathy in HbSC disease than in sickle cell anemia; it is also possible that the lower HbF levels of HbSC disease play a role. Paradoxically, the high prevalence of proliferative retinopathy in HbSC disease might be an expression of the relative benignity of this genotype. A classification of the peripheral retinal vascular changes in sickle cell

540 disease, based on the appearance of the peripheral retinal vasculature has been proposed in which vessels are either “normal” (Type I) or abnormal (Type IIa or IIb) based on qualitative changes such as irregular vascular margins, capillary stumps, hyper- or hypofluorescence, and capillary bed thinning.71 This classification is based on the observation that proliferative retinopathy develops in response to progressive closure of the vessels of the peripheral retina, and, the recession centripetally, of the margin of retinal perfusion.72,73 Abnormal peripheral retinal vasculature is present in approximately 80% of adults with HbSC disease.74 By age 17 years, the prevalence of abnormal vascular borders is approximately 60%.71 More than 260 patients with sickle cell anemia and 154 with HbSC disease were studied with serial fluorescein angiography and retinal photography, beginning at ages 6–12 years. In the teenage years, each year there is a 6% conversion from a normal to abnormal vascular border. The occurrence of sickle proliferative retinopathy was always associated with an abnormal vascular border. In sickle cell anemia, peripheral retinal vessels are occluded early in the course of disease so that further retinal vascular damage and proliferative lesions cannot develop. The enhanced circulatory competence of the HbSC disease cell could preserve this retinal circulation, permitting the later development of proliferative lesions. If this is true, as hydroxyurea treatment (Chapter 30) makes sickle cell anemia cells more like HbSC disease cells and improves their circulatory competence, the prevalence of proliferative retinopathy could begin to increase in young sickle cell anemia patients treated with this drug. Diagnosis. Sickle retinopathy is recognizable by its different stages, which include nonproliferative lesions and three stages of proliferative lesions including neovascularization, vitreous hemorrhage, and retinal detachment. Whether or not there is an orderly progression from less to more serious stages that precede retinal detachment and blindness or whether catastrophic changes can arise directly from early stages of proliferative retinopathy is not well characterized. Characteristically, following vasoocclusion in the peripheral vessels of the retina – the nonproliferative stages of retinopathy – neovascular malformations called black sea fans grow from the retinal surface into the vitreous. These fragile new vessels can leak blood into the vitreous, at times in amounts sufficient to reduce vision. Organization of vitreal hemorrhages exerts traction on the inelastic retina, causing tears and allowing vitreal fluid to enter the subretinal space. Retinal detachment, a potential consequence of subretinal fluid invasion, can produce blindness. Of 14 patients with HbSC disease who were selected for study because they had nonproliferative lesions or peripheral retinal arteriolar occlusion or arteriovenous anastomoses, 21% developed new sea fans with an average interval of 18 months (range 8–36 months). Based on these rather small numbers of patients, the authors estimated that approximately 14% of young adults with HbSC

Martin H. Steinberg and Ronald L. Nagel Table 21.3. Prevalence of ocular lesions in HbSC disease Ocular lesion

Percent

Nonproliferative Salmon patch Iridescent spot Black sunburst

17 28 62

Proliferative Stage III-Neovascularization Stage IV-Vitreous hemorrhage Stage V-Retinal detachment

45 21 10

disease develop neovascularization each year.75 Complicating the evaluation of retinopathy in HbSC disease is the propensity for spontaneous regression of proliferative lesions due to autoinfarction.76 These authors caution, however, that withholding treatment in the hopes of a spontaneous regression is dangerous because the incidence of spontaneous vitreal hemorrhage increased from 28% to 44% in patients observed for 6–77 months.77 Progression. Patients picked randomly from a clinic population, were enrolled and followed with annual or semiannual detailed ophthalmological examinations.78 Initially, 11 patients had stage III retinopathy and their average duration of follow-up was 7 months. Twelve patients had normal findings or stage I or II disease (Table 21.3). The natural history of proliferative retinopathy in HbSC disease compared with sickle cell anemia in patients who presented with stage III disease is shown in Table 21.4. Forty-eight percent had proliferative disease on initial examination and 74% developed stages III–V disease at final examination; there was little change over time in the prevalence of nonproliferative retinopathy in either HbSC disease or sickle cell anemia patients. In studies from Jamaica, 115 patients with HbSC disease were followed sequentially.79 Efforts were made to ensure that this was a random sample of HbSC disease patients. Proliferative retinopathy was present in 24% of patients at the initial examination, arose de novo during the study in 18%, and was present at the final examination in 40%. The mean age of patients showing progression was 27.7 years. Of 43 individuals, 58 new lesions were found in 18 patients

Table 21.4. Natural history of proliferative retinopathy in sickle cell disease

No. of eyes Duration of observation (mo) Stable disease (%) Regression (%) Progression (%) Vitreous hemorrhage (%) Retinal detachment (%)

Sickle cell anemia

HbSC disease

17 69 59 18 35 6 6

19 77 26 42 58 37 6

Hemoglobin SC Disease and Hemoglobin C Disorders Table 21.5. Evolution of proliferative retinopathy in HbSC disease according to patient age Age (y) Eyes (no.) Progression (%) Regression (%)

Stable (%)

10–19 20–29 30–39 >40

30 23 30 67

10 47 56 24

40 60 43 29

30 17 27 4

younger than 25 years, and the highest risk period was between ages 20 and 24 years. Two thirds of patients with de novo proliferative lesions were aged 15–29 years. Approximately one-third to half of patients had autoinfarction during the course of the study. From ages 10 to 39 years, approximately 7% of individuals in each 5-year age bracket developed proliferative retinopathy per 100 patient-years of observation.80 In follow-up, progressive disease was most frequent between ages 20 and 39 years, and visual loss occurred in only 9% of patients followed for a mean of 4.5 years.81 (Table 21.5) A longitudinal study spanning 20 years was performed in 307 children with sickle cell anemia and 166 children with HbSC disease detected by newborn screening. Participants had annual ophthalmological studies with angiography and angioscopy. Proliferative retinopathy developed in 14 patients with sickle cell anemia and 45 with HbSC disease, unilaterally in 36 patients and bilaterally in 23. The incidence increased with age in both genotypes, with crude annual incidence rates of 0.5 cases per 100 sickle cell anemia and 2.5 cases per 100 HbSC disease patients. By the age of 24–26 years, proliferative retinopathy had occurred in 43% of the subjects with HbSC disease compared with 14% of those with sickle cell anemia. Patients with HbSC disease and unilateral disease had a 17% probability of regression and a 13% probability of progressing to bilateral disease. Those with bilateral had half the chance of regression as those individuals with unilateral disease. Visual loss occurred in only 1 of 82 affected eyes and one required detachment surgery with recovery of normal visual acuity.82 In one study, 30% of patients with HbSC disease had retinopathy with a median age of onset of 28 years.48 Data suggested that the CI ␤C haplotype and Benin ␤S globin gene haplotype carriers with the CI ␤C haplotype had a delayed onset of retinopathy. The numbers of cases were small and a physiological explanation for these observations was not forthcoming. Similarly, coincident ␣ thalassemia appeared to decrease the incidence of retinopathy, but less than 30 cases were examined.

Treatment Recommendations Management of sickle proliferative retinopathy is imperfect. Incipient proliferative disease has been treated with focal or panretinal section photocoagulation to forestall advancing pathology and impaired vision. Some of the

541 earliest comparative trials were inconclusive and questioned the role of photocoagulation, but different techniques of treatment and new technologies now make these results difficult to interpret.83 There are presently no data that conclusively show that prophylactic photocoagulation significantly changes the natural history of retinopathy.78 In a randomized trial of argon laser scatter photocoagulation, 99 eyes were treated and 75 eyes served as controls, with an average follow-up of approximately 45 months.84 There was complete or partial regression of sea fans in 81% of treated eyes and spontaneous regression in 46% of control eyes. New sea fans developed in 34% of treated compared with 41% of control eyes. Using the criteria of visual loss, five control eyes and three treated eyes lost vision during followup, an insignificant difference similar to that seen in an observational study. Also, the incidence of retinal detachment was not reduced. Some data suggest that proliferative retinopathy is most prevalent in individuals with higher hemoglobin levels. Phlebotomy to reduce the hemoglobin concentration to 9–10 g/dL has been advocated as a measure to prevent retinopathy or slow its advancement without evidence of the efficacy of this approach. In summary, proliferative retinopathy occurs often in patients with HbSC disease aged between 15 and 30 years, is progressive, can culminate in visual loss, and does not have a definitive treatment that can eliminate its most severe endpoints. Consultation with ophthalmologists experienced in managing this complication is vital.

Painful Episodes In a given year, more than half of 806 patients with HbSC disease did not have a painful episode, one-third had a single episode of pain and less than 10% had three or more pain episodes.85 This rate was approximately 0.4 episodes per patient-year, less than half the rate in sickle cell anemia. Forty percent of Jamaican patients with HbSC disease had at least one pain episode needing hospitalization; 17% never reported a pain crisis. Approximately 60% of HbSC disease patients in Los Angeles reported at least one pain episode.48

Acute Chest Syndrome Acute chest syndrome, with its high morbidity and appreciable mortality, is seen in approximately 30% of patients with HbSC disease.48 Although this incidence is only 50%–75% that of sickle cell anemia, progression to chronic lung disease is only 0.1 that of sickle cell anemia and the median age of onset is almost one decade later.52,86 The incidence per 100 patient-years in patients of all ages is 5.2. Typical of this condition, the incidence in children age 5 years or younger is twice that of older individuals. Only steady-state leukocyte count was a risk factor for acute chest syndrome in HbSC disease. When compared with

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Martin H. Steinberg and Ronald L. Nagel

C

A

B

D

Figure 21.14. A 52-year-old man with HbSC disease and known osteonecrosis and sickle retinopathy who was never hospitalized for painful episodes; he developed severe pain in his extremities and chest and shortness of breath. The hemoglobin level was approximately 10 g/dL and white blood cell count of 25,000/mL3 . The pain soon became unbearable; he became severely hypoxic and comatose and the hemoglobin concentration and platelet count plummeted. Oxygenation was impossible to maintain and despite rapid use of blood transfusions and supportive care he died within hours of presentation with multiorgan failure. (A) HPLC separation of HbS and HbC. (B) Portion of a DNA sequencing reaction showing compound heterozygosity for HbS and HbC in HBB codon 6. (C) Bone marrow showing necrotic marrow. (D) Lung showing bone marrow emboli in pulmonary arteries.

sickle cell anemia, there were no differences in duration of hospitalization or death rate, but sickle cell anemia patients were more likely to present with severe pain.87 Nevertheless, severe acute chest syndrome events due to embolization of necrotic bone marrow can occur and can terminate in multiorgan failure and death (Fig. 21.14).

Pulmonary Hypertension The first reports of pulmonary hypertension in sickle cell disease considered HbSC disease and sickle cell anemia as a group.88,89 More recently, in an analysis of 43 HbSC patients screened by echocardiography for pulmonary hypertension, the mean age of affected individuals was 52 years. Within this group, anemia, but not markers of hemolysis

such as LDH was associated with the pulmonary hypertension phenotype. As with sickle cell anemia, pulmonary hypertension was associated with a higher risk of death.90

Spleen Splenic function is often preserved in HbSC disease, whereas it is rarely preserved in adults with sickle cell anemia. A positive result of retained splenic function is the reduced incidence of infection with encapsulated bacteria. A negative result is the chance for splenic sequestration crises and splenic infarction to occur in adults. Pocked or “pitted” red cells are found in increased numbers when splenic function declines and is lost, and can be used as a measure of functional asplenia. When pitted red

Hemoglobin SC Disease and Hemoglobin C Disorders cell counts were used to evaluate splenic reticuloendothelial function in HbSC disease, there were 4.9 ± 9.1% pitted cells in this disorder compared with 11.8 ± 7.0% in sickle cell anemia; however pitted cells might not always reflect splenic function. Pitted cells increased with age in sickle cell anemia but did not in HbSC disease suggesting they had stabilized splenic function. Six percent of children with HbSC disease had splenic complications that included acute sequestration crisis, painful infarction, and hemorrhage.91 Similar events can occur in adults with HbSC disease but are rare in adult sickle cell anemia. Slightly over one half of all adults with HbSC disease had splenomegaly, 36% were asplenic, and 12% had normal spleens when evaluated by spleen scanning. When “pit” counts were related to spleen scans, no patient with HbSC disease younger than age 4 years had functional asplenia, although this abnormality was present in 22% of individuals aged 4–12 years and 45% of individuals older than 12 years.92 Coexistent hereditary spherocytosis and HbSC disease were deemed responsible for multiple episodes of splenic sequestration crises in one interesting case report. It was hypothesized that the increased density and reduced deformability of red cells with membrane damage from both hereditary spherocytosis and HbSC disease caused increased splenic trapping and sickling.

543 normally functional spleen. Pneumococcal vaccines should be given and as in sickle cell anemia, parents instructed to seek immediate medical attention for febrile illnesses.

Osteonecrosis The incidence of osteonecrosis in HbSC disease is only slightly lower than in sickle cell anemia with an ageadjusted incidence rate of 1.9/100 patient-years for the hip joints and 1.7/100 patient years for shoulders. Shoulder disease was uncommon in patients younger than age 25 years. Osteonecrosis of the femoral heads was almost as prevalent in HbSC disease as in sickle cell anemia but developed later in life.46,52,95 The estimated age at diagnosis in HbSC disease varied from 36 to 40 years versus 36 years for sickle cell anemia.48,95 In one study, coincident ␣ thalassemia delayed the age of onset by approximately 15 years.48

Leg Ulcers Compared with sickle cell anemia, leg ulcers are infrequent in HbSC disease and found in only approximately 3% of cases.48,96 As in the case of priapism, this could be a result of improved nitric oxide (NO) bioavailability because of reduced hemolysis in HbSC disease compared with sickle cell anemia.

Genitourinary Infection The relative risk of bacteremia is less for HbSC disease patients than for sickle cell anemia patients when correction is made for the total nonhospitalized population at risk, but is much larger than that for the normal population. Gram-negative bacteremia, the systemic infection most commonly found in HbSC disease patients, was less life threatening than the pyogenic bacteremia most commonly found in sickle cell anemia. In children age 4 years and younger, normal “pit” counts suggested that prophylactic penicillin need not be given.92 Two adolescents with functional asplenia were reported to die of pneumococcal sepsis. Fatal pneumococcal sepsis has been described in children age 5 years and younger who were not receiving penicillin but their state of splenic function was unknown.93

Treatment Recommendations Some authorities question the use of prophylactic penicillin in HbSC disease.94 A reasonable approach in children who are most susceptible to pneumococcal infection might be to assess splenic function by “pit” counts, the presence of Howell–Jolly bodies or radionuclide scanning. If splenic function is normal, prophylaxis might be withheld; however, as discussed, the evaluation of splenic function is imperfect. Also, a large spleen does not always equate with a

The pathophysiology of renal lesions in HbSC disease is similar to that in HbAS and sickle cell anemia. Hematuria is often present and appears to be more frequent than in HbAS.2 In HbSC disease, renal concentrating ability is lost at a time intermediate between the loss in sickle cell anemia and HbAS. Maximum concentrating ability in patients with HbSC diseases, mean age 30 years, was 537 mOsm and appeared to decline with age. The two youngest patients, aged 6 and 8 years, had maximally concentrated urines of 640 and 698 mOsms, approximately 60% of normal. In eight children with HbSC disease, mean age 11 years, none had an overnight urine concentration greater than 545 mOsm (normal, 800–1000 mOsm). Seven of eight increased their urine concentration after receiving 1-desamino-8darginine vasopressin, although not to normal.97 Renal function deteriorates with age but chronic renal failure is half as common as in sickle cell anemia (2%– 3%) and its median age of onset is 25 years later.44,48 In one study, 73% of 27 patients had papillary necrosis based on calyceal blunting by intravenous pyelography, but renal function was normal so the clinical significance of this observation is unclear.98 Among 25 deaths in 284 HbSC disease patients, three were due to renal failure.48 Priapism has been estimated to occur in between 3% and 10% of patients with HbSC disease, a rate far lower than in sickle cell anemia.48,99 Among other possible explanations, the reduced hemolysis in HbSC disease compared

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with sickle cell anemia with better NO bioavailability might explain this observation.99

Cerebrovascular Disease Two percent to 3% of HbSC disease patients had a stroke, and 3 deaths due to stroke were noted among 248 patients.48,52 This incidence was three–four times less than in sickle cell anemia. In the CSSCD, 0.8% of individuals with HbSC disease compared with 4% of patients with sickle cell anemia had suffered a stroke at study entry.100

Pregnancy Perinatal mortality in HbSC disease has been reported to vary from 28%–nil in the absence of transfusions and from 0 to 9% when transfusions were given.101 In a heterogeneous collection of patient series with widely different numbers of cases, more recent reports generally had lower mortality. Pregnancy-related complications are higher than in normal controls and not dissimilar to those with sickle cell anemia. The rate of caesarian sections is similar.102 Stress or pregnancy might result in disease symptoms, and the pregnant HbSC disease patient can be as severely affected as the sickle cell anemia patient. In 95 pregnancies in 43 patients with HbSC disease followed from birth, menarche was marginally delayed compared with controls, but the age at first pregnancy was similar. The prevalence of pregnancy-induced hypertension, preeclampsia, antepartum or postpartum hemorrhage did not differ from controls. Sickle-related complications were similar to those in patients with sickle cell anemia with marginally fewer miscarriages, more live deliveries, and higher birthweight.103 Pregnancy and recommendations for its management are discussed in detail in Chapter 19.

HbSC/Hb-G Philadelphia This genotype has a special phenotype because the hybrid molecule, ␣2 G-Philadelphia ␤2 C increases the rate of crystal nucleation compared with native HbC, and HbS enhances this effect in a pathogenetically relevant manner.16 Heterozygotes for the ␤S , ␤C , and the ␣G-Philadelphia genes have abundant circulating intraerythrocytic crystals and increased numbers of folded red cells with a mild clinical course. This phenotype seems to be the result of increased crystallization and decreased polymerization caused by the effects of the ␣G-Philadelphia globin chain on the ␤C and ␤S gene products. Some of the intraerythrocytic crystals in this syndrome, unlike the typical crystal of HbSC disease, are unusually long and thin and resemble sugar cane (Fig. 21.15). A mild clinical course associated with increased crystallization implies that in HbSC disease polymerization of HbS is pathogenically more important than the crystallization induced by ␤C chains.

Figure 21.15. Blood smear directly from finger stick blood from a patient with SC␣G Philadelphia disease. Open arrow, depicts a “sugar cane” crystal, a shape not observed in HbC disease or HbSC disease blood. The black arrows depict red cells with more classic forms of HbC-dependent crystals.16

TREATMENT OF HbSC DISEASE No currently available treatment is unique for HbSC disease. The special pathophysiological features of HbSC disease might in the future allow a direct attack on some basic disease mechanisms such as K:Cl cotransport (Chapter 9 and 31), but these approaches are still under study. Hydroxyurea can effect changes in the HbSC disease erythrocyte that might be independent of any change in HbF. Early trials of hydroxyurea in HbSC disease are discussed in Chapter 30. Transfusions are used in HbSC disease for the same indications as in sickle cell anemia. A study was performed on preoperative transfusion in HbSC disease.104 Eighteen percent of all patients had some postoperative complications. Postoperative events were compared between patients who received preoperative transfusions and those who were untransfused. In patients undergoing intraabdominal surgery, more than a third of untransfused patients had postoperative acute chest syndrome or painful episodes. Although this study was not randomized and both simple and exchange transfusions were used, based on these observations and the larger study of preoperative transfusion in sickle cell anemia,105 preoperative transfusion should be considered in patients with HbSC disease who face procedures with moderate surgical risk. Because some patients with HbSC disease have PCVs that approach normal, in emergent situations such as severe acute chest syndrome or stroke, or when preparing a patient for surgery, exchange transfusions might be the most prudent approach to treatment so as not to unduly raise the PCV and increase blood viscosity.

Hemoglobin SC Disease and Hemoglobin C Disorders Because of the higher PCV, phlebotomy has been considered one approach to treating HbSC disease. In one patient, the PCV rose following splenectomy and the rate of acute painful episodes increased. Following a phlebotomy program to lower the PCV to presplenectomy values and induce iron deficiency, the symptoms regressed.106 A controlled trial of this approach has not been reported.

SUMMARY Positively charged HbC induces cellular changes in HbC trait, HbC disease, and HbSC disease that are a result of cellular dehydration. Although biologically interesting and producing characteristic hematological features in the erythrocyte, only trivial clinical abnormalities are associated with HbC trait and HbC disease. This is not so when HbC is present with HbS. HbSC disease is a clinically important illness with all of the complications of sickle cell anemia, albeit at a reduced rate. An effective treatment would be a major medical advance and clinical trials of agents that can reduce cell density and increase HbF concentration have started. REFERENCES 1. Kan YW, Dozy AM. Evolution of the hemoglobin S and C genes in world populations. Science. 1980;209:388–391. 2. Dacie J. The Haemolytic Anaemias. 3 ed. Edinburgh: Churchill Livingstone; 1988. 3. Labie D, Richin C, Pagnier J, Gentilini M, Nagel RL. Hemoglobins S and C in Upper Volta. Hum Genet. 1984;65: 300–302. 4. Daar S, Hussain HM, Gravell D, Nagel RL, Krishnamoorthy R. Genetic epidemiology of HbS and HbC in Oman: multicentric origin for the ␤S gene. Am J Hematol. 2000; 39–46. 5. Agarwal A, Guindo A, Cissoko Y, et al. Hemoglobin C associated with protection from severe malaria in the Dogon of Mali, a West African population with a low prevalence of hemoglobin S. Blood. 2000;96(7):2358–2363. 6. Modiano D, Luoni G, Sirima BS, et al. Haemoglobin C protects against clinical Plasmodium falciparum malaria. Nature. 2001;414(6861):305–308. 7. Rihet P, Flori L, Tall F, Traore AS, Fumoux F. Hemoglobin C is associated with reduced Plasmodium falciparum parasitemia and low risk of mild malaria attack. Hum Mol Genet. 2004;13(1):1–6. 8. Fairhurst RM, Fujioka H, Hayton K, Collins KF, Wellems TE. Aberrant development of Plasmodium falciparum in hemoglobin CC red cells: implications for the malaria protective effect of the homozygous state. Blood. 2003;101(8):3309– 3315. 9. Mockenhaupt FP, Ehrhardt S, Cramer JP, et al. Hemoglobin C and resistance to severe malaria in Ghanaian children. J Infect Dis. 2004;190(5):1006–1009. 10. Mockenhaupt FP, Ehrhardt S, Burkhardt J, et al. Manifestation and outcome of severe malaria in children in northern Ghana. Am J Trop Med Hyg. 2004;71(2):167–172.

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Martin H. Steinberg and Ronald L. Nagel 107. Boehm CD, Dowling CE, Antonarakis SE, Honig GR, Kazazian HH. Evidence supporting a single origin of the ␤(C)-globin gene in blacks. Am J Hum Genet. 1985;37:771–777. 108. Trabuchet G, Elion J, Dunda O, et al. Nucleotide sequence evidence of the unicentric origin of the ␤C mutation in Africa. Hum Genet. 1991;87:597–601. 109. Eaton WA, Hofrichter J. Sickle cell hemoglobin polymerization. Adv Protein Chem. 1990;40:63–280. 110. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med. 1994;330(23):1639–1644.

22 Sickle Cell Trait Martin H. Steinberg

INTRODUCTION Parents of children with sickle cell anemia seldom have the same disease as their offspring. In 1927, 17 years after the first clinical description of sickle cell anemia, it was discovered that almost 10% of African Americans had erythrocytes that sickled when deoxygenated. With the subsequent identification of HbS, and the observation that patients with sickle cell anemia had predominantly HbS in their hemolysates, whereas their parents’ had both HbS and HbA, the genetics of sickle hemoglobinopathies was characterized and sickle cell trait (HbAS) was defined. Almost 40 years ago, reports of sudden death in military recruits with HbAS triggered a push for mandatory screening for HbAS, denial of military service for some carriers, and insurance coverage cancellation for others. An astounding list of complications of HbAS appeared in the literature. The interpretation of these reports often disregarded the distinction between statistically significant associations and coincidence, and almost all lacked any control comparisons. This chapter reviews what is known, what is presumed, and what is erroneous about the pathogenicity, clinical features, and management of HbAS.

oxygen saturation falls below 60%. Even when HbAS blood is completely deoxygenated, the polymer fraction is only 40% of total hemoglobin. HbAS should therefore be clinically benign, and, despite the multitudinous reports of innumerable complications associated with HbAS, very few are confirmed by careful epidemiological studies.

Diagnosis Indications for detecting HbAS in at risk populations are limited and include genetic counseling, population surveys, evaluating hematuria, planning treatment of hyphema, pregnancy, and perhaps before complicated thoracic surgery. Detecting HbAS requires the quantification HbS and HbA in a hemolysate. This is best accomplished by highperformance liquid chromatography (HPLC) (see Chapter 28), in which the presence and ratio of HbS to HbA are accurately determined. Isoelectric focusing does not provide quantitative data. DNA-based methods definitively detect the presence of both HbS and HbA genes but are usually unnecessary if all one desires to establish is the presence of HbAS. HbAS cannot be diagnosed by medical history, clinical examination, or routine laboratory testing. Neither hematological indices nor peripheral blood film reviews are useful for diagnosis because both are normal in HbAS. With rare exception, the sickle solubility test has no role as a primary screening test or as the sole means of detecting HbS. This test indicates only the presence of HbS and cannot reliably distinguish between HbAS, sickle cell anemia, and HbS–␤+ thalassemia. Table 22.1 shows the prevalence of HbAS in some locations in African and throughout the world. Fetal hemoglobin (HbF) levels are usually normal in HbAS. Carriers of HbAS in whom the HbS gene is on a Senegal or Arab–Indian ␤-globin gene haplotype chromosome (Chapter 27) can have higher HbF levels than other individuals, but they remain within the normal range.2 HbF and F cells (erythrocytes containing HbF) can be increased in individuals with both HbAS and coincident hereditary persistence of fetal hemoglobin (HPFH) due to point mutations in the ␥ -globin gene promoters (Chapters 16 and 17).

PATHOGENESIS HbAS is usually implies simple heterozygosity for the HbS gene (HBB glu6val). Less than half the hemoglobin in the HbAS erythrocyte is HbS; the remainder is mainly HbA. The probability that the mixed hybrid tetramer, ␣2 ␤S ␤A , will enter the polymer phase is only half that of the HbS tetramer, ␣2 ␤S 2 (Fig. 22.1A).1 HbS polymer concentrations sufficient to injure the red cell are prerequisite for the expression of the phenotype of sickle cell disease; in HbAS, high concentrations of HbA preclude clinically significant HbS polymer formation at the oxygen saturation and physiological conditions present in most tissues (Fig. 22.1B). HbS polymer appears in HbAS cells only when

HbS Concentration in HbAS Effects of ␣ Thalassemia The proportion of hemoglobins in the erythrocyte depends on the transcriptional rate of each globin gene, the stability of the mRNA and hemoglobin produced, and the posttranslational assembly and stability of the ␣␤ dimer and hemoglobin tetramer. ␣␤ Dimers rapidly associate into tetramers, and oxyhemoglobin tetramers dissociate into dimers. Hybrid tetramers (␣2 ␤S ␤A ) occur in HbAS. Normally, a slight excess of positively charged ␣ chains are always available to bind 549

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Martin H. Steinberg

Table 22.1. Prevalence of HbAS in world populations Region/country

Prevalence (%)

Africa

1.3–6.3 13.2–24.4 5.1–24.5 2–38 0.8–20 20 (in affected tribal populations) 0.5–1 13 (Eti-Turks)

North Africa West Africa Central Africa East Africa Southern Africa Middle East, Central India Asia, & India Transcaucasia Turkey Iran Saudi Arabia Europe Greece

United States South America Caribbean Central America

Italy (Sicily) Portugal United States Amerindians Brazil

1–60 0–32 (mean =11 in the north) 2–4 8–9 (African Americans) rare 4–8 7–14 (excluding Puerto Rico) 1–20 (dependent on racial origin of population examined)

Data from Africa are summarized from the work of many investigators and adapted from ref. 24.

non-␣ chains. Therefore, the steady-state accumulation of hemoglobin tetramers in the erythrocyte depends predominantly on expression of the non-␣-globin genes. Hemoglobin dimers are assembled by the electrostatic attraction of ␣- and non-␣-globin chains.3 ␤S -Globin chains are more positively charged than ␤A chains, and therefore,

bind ␣ chains less avidly, accounting for HbS levels in heterozygotes (and levels of other positively charged hemoglobin variants also) being less than the 50% expected based on gene dosage alone. HbAS carriers with four ␣-globin genes usually have 40% ± 4% HbS. The percentage of HbS can be altered by an ␣-globin gene mutant, a ␤-globin gene variant trans to the HbS gene (or very rarely in cis), ␤ thalassemia, iron deficiency anemia, and possibly lead poisoning and megaloblastic anemia. HbS concentration in HbAS is trimodally distributed, with peaks at 30%, 35%, and 40%.4 This distribution, with few exceptions, is determined by the number of ␣-globin genes. Mild forms of ␣ thalassemia are extraordinarily common where HbS is prevalent (Chapters 13 and 14). Individuals with HbAS who have ␣ thalassemia exhibit HbA/HbS ratios less than 40%. The ratio of ␣/␤ globin biosynthesis in three families with a proband with HbAS–␣ thalassemia is shown in Figure 22.2. As ␣ thalassemia reduces the pool of available ␣-globin chains, ␤A - and ␤S -globin chains must compete for those available and ␤A chains dimerize twice as effectively with ␣ chains as do ␤S chains. When ␣ gene triplication or quadruplication increases the number of ␣-globin chains, the HbS level increases beyond 40% due to the augmented pool of available ␣-globin.5 These relationships are shown in Table 22.2. Individuals with the -␣4.2 deletion have lower HbS levels than those with the more common -␣3.7 deletion because of the greater ␣-globin chain deficit associated with the former type of ␣ thalassemia (Chapter 13). Homozygotes for the -␣4.2 with HbAS deletion have approximately 20% HbS. Rare individuals with HbAS have only a single ␣-globin gene, or HbH disease.6,7 They have HbS levels of approximately 20%, mild anemia, and marked microcytosis,

Figure 22.1. (A) Solubility of deoxygenated hemoglobin mixtures. Other hemoglobins prevent HbS polymerization by increasing HbS solubility (Csat). The percentage of increase in solubility reflects the likelihood of the other hemoglobin type being included in the deoxy HbS polymer. Neither the ␥ -globin chain of HbF nor the ␦-globin chain of HbA2 is incorporated into the polymer phase, so HbF and HbA2 inhibit polymerization more than do HbA and HbC. From ref. 71, with permission. (B) Effects of HbA and HbF on HbS polymerization. Closed circles indicate fully deoxygenated hemoglobin and open circles indicate fully oxygenated hemoglobin. Polymer is indicated by stacked closed circles. (From ref. 71, with permission.)

Sickle Cell Trait

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Table 22.2. Hematology and percentage of HbS in HbAS with ␣ thalassemia or ␤ thalassemia ␣-Globin genotype

Hemoglobin (g/dL)

MCV (fL)

␣/␤∗ biosynthesis

HbS§ (%)

␣␣␣/␣␣ ␣␣/␣␣ -␣/␣␣␣ -␣/␣␣ ## --␣ /␣␣codon 62 GTG>TG ¶ --/␣␣ -␣/-␣ --/-␣

N N N N–low N 12 ? N–low N 7–10 g/dL

N N N 75–85 73 ? 70–75 50–60

– 1 – 0.85 – ? 0.75 0.50

45 40 40 35 28 ? 30 20–25

65

1.50

10

HbAS with ␤+ Thalassemia in Trans or in Cis 12–13 ␤+ Thalassemia Cis ␤+ Thalassemia in Trans (see Chapter 23)

Modified from ref. 4. N signifies normal levels. ∗ Ratio of radioactivity incorporated into ␣-globin chains vs. ␤-globin chains during incubation of reticulocytes with radioisotopes. The normal ␣/␤ ratio is 1 ± 0.15. Lower ratios suggest the presence of ␣ thalassemia and higher ratios, ␤ thalassemia. ## This single case is associated with a novel frameshift mutation.116 ¶ This genotype has not been reported with HbAS. § Remainder of hemoglobin predominantly HbA. Most values represent means from several studies. Although approximately 50 patients with HbAS and the ␣␣/␣␣ genotype, 40 with the -␣/␣␣ genotype, and 20 with the -␣/-␣ have been studied, far fewer with the other genotypes have been examined, so the data presented should be interpreted with caution. HbS with ␤+ thalassemia assumes a normal ␣-globin genotype.

but no detectable HbH. It might be that in the presence of only a single normal ␤-globin gene, the high affinity of ␣-globin chains for the limited ␤A -globin chains and the instability of a ␤S teramer preclude HbH (␤4 ) formation. Heterozygotes for HbC (HBB glu6lys) and HbE (HBB glu26lys) have similar reductions in the level of the variant hemoglobin when ␣ thalassemia coexists (Chapters 18 and 21). Occasionally, ␣ thalassemia is caused by a point mutation in either the ␣1 or ␣2 gene (HBA1, HBA2) (Chapter 13). When these mutations are present in individuals with

Figure 22.2. ␣/␤A + ␤S biosynthesis in three families in which there was a proband with HbAS–␣ thalassemia. Shown beneath each individual is the ␣/␤A + ␤S biosynthesis ratio. The normal ratio is 0.85–1.25.

HbAS, the level of HbS is likely to be dependent on the suppression of ␣-globin synthesis and the particular ␣-globin gene that is affected, but few data are available. Iron deficiency selectively inhibits the translation of ␣globin mRNA and can produce an acquired form of ␣ thalassemia. Depending on its severity, iron deficiency can mimic HbAS–␣ thalassemia by depressing HbS levels and causing microcytic erythrocytes. In vitro and perhaps in vivo, lead can do the same.

Effects of Variant Hemoglobins, ␤ Thalassemia and HPFH α-Globin variants. HbG Philadelphia (HBA2 or HBA1 asn68lys), an ␣-globin chain variant is the most common hemoglobin variant found with HbAS (Chapter 24). This mutation is usually on a chromosome with a -␣3.7 deletion. Compound heterozygotes with HbAS and HbGPhiladelphia are not anemic and have no hemolysis but might have mild microcytosis. HbG-Philadelphia can be separated from normal hemoglobins by HPLC. When HbAS or HbC trait appears with HbG-Philadelphia, the HPLC and electrophoretic patterns can be confusing as normal ␣-globin chains and ␣G -globin chains combine with non␣-globin chains to form hybrid and normal hemoglobin tetramers. These species can be resolved by HPLC that in contrast to electrophoresis can separate HbS from HbG. Other ␣-globin chain variants can be found with HbAS, and like the example of HbG-Philadelphia, their initial resolution could be puzzling, but, like HbG-Philadelphia with HbAS, they are clinically insignificant. Some of these variants include Hb Memphis (glu23gln), Hb Montgomery (leu48arg) Hb Mexico (gln54glu), Hb Stanleyville-II

552 (pro77arg), Hb Nigeria (ser8cys), HbG-Georgia (pro95leu), and Hb Hopkins-II (his112asp). (The Globin Gene Server http://globin.cse.psu.edu/ provides up-to-date information on hemoglobin variants and thalassemias.) β-Globin Variants. HbS can coexist as a compound heterozygote with many other ␤-globin chain variants, including HbD Ibadan (HBB thr87lys), HbD Iran (HBB glu22gln), HbG San Jose (HBB glu7gly), Hb Osu-Christianbourg (HBB asp52asn), HbE, HbG Galveston (HBB glu43ala), Hb KorleBu (HBB asp73asn; also known as HbG-Accra), and Hb Richmond (HBB asn102lys). The resulting phenotypes depend on the pathogenicity of the ␤-globin variant but usually resemble uncomplicated HbAS (Chapter 23). HbCHarlem (HBB glu6val; asp73asn) migrates like HbC at alkaline pH, is present at a concentration of approximately 40% of the hemolysate, and gives a positive test for sickling (Chapter 23). It can be separated from HbC at acidic pH. HbC-Harlem heterozygotes do not have abnormal hematology or symptoms. βThalassemia, HPFH, Fusion Hemoglobins. Compound heterozygotes with ␤+ thalassemia and HbS synthesize more HbS than HbA because of the reduced expression of the ␤ thalassemia gene. Rarely, a ␤ thalassemia mutation can be cis to the ␤S -globin gene. A C→T mutation at position -88 5
to the ␤-globin gene was found in a HbS gene on a Benin haplotype chromosome.8 Affected patients had a normal ␣-globin genotype with ␣:␤ synthesis consistent with ␤ thalassemia trait. They were mildly anemic with microcytosis, high HbA2 concentration, and HbS levels of 10%–11%. This HbS level is much lower than those found in HbAS with HbH disease (Table 22.2). Point mutations that produce HPFH can be found cis and trans to the HbS gene. They are not associated with a clinical phenotype but can produce confusing HPLC and electrophoretic findings. Depending on the mutation causing HPFH, there can be minor increases in HbF of approximately 5% or major increases that exceed 20% of the total hemoglobin. Individuals with HbAS who inherit the -202 C→T mutation 5
to the A ␥ globin gene have 2%–4% HbF and more than 90% A ␥ chains.9 Mutations associated with these conditions are shown in Table 22.3. The compound heterozygous conditions of HbS-deletion HPFH and HbS– ␦␤ thalassemia are discussed in Chapter 23. Hb Lepore (Chapters 16 and 17) is the product of a poorly expressed fusion gene with ␦- and ␤-globin chain components. Hb Lepore migrates like HbS on electrophoresis at alkaline pH and like HbA at acidic pH but is separable from HbA and HbS by HPLC. Heterozygotes have approximately 10% Hb Lepore and the phenotype of ␤ thalassemia trait. Hb Lepore heterozygotes should not be confused with HbAS or the very rare cases of HbAS–HbH disease or ␤ thalassemia cis to HbAS. The anti-Lepore variants HbP Nilotic (HbP Congo) and Hb Lincoln Park exhibit the same electrophoretic behavior as HbS and form approximately 20% of the hemolysate. Unlike Hb Lepore, they do not cause a thalassemia phenotype. Hb Parchman, only reported once,

Martin H. Steinberg Table 22.3. Nongene deletion hereditary persistence of HbF and HbAS24 ␥ (%)

Mutation

HbF

HbA

HbS

G

-202 C→G 5
G ␥ -202 C→T 5
A ␥ -175 T→C 5
G ␥ -175 T→C 5
A -158 C→T 5
G ␥ in cis

20 2–4 30 40

30 60 30 20

50 40 40 40

100 10 85

migrates electrophoretically like HbS but forms less than 2% of the hemolysate. HbAS has been described with the ␥ ␤ fusion hemoglobin, Hb Kenya (Chapters 16 and 23). Carriers are not anemic but could have mild microcytosis. The hemolysate contains approximately 20% Hb Kenya and 60%–70% HbS; the remainder is HbA2 and HbF.

SYMPTOMATIC “HbAS” With some exceptions, carriers of HbAS do not have the clinical features of sickle cell disease. Nevertheless, sometimes, what appears at first to be HbAS is accompanied by disease symptoms. In addition to the rare conditions discussed, it is formally possible that genetic polymorphisms in the many genes that could affect the phenotype of sickle cell anemia might lead to symptoms in HbAS, but, to date, such associations have not been studied.

Variant Hemoglobins HbS-Antilles (HBB glu6val; val23Ile) migrates like HbS, giving the electrophoretic appearance of HbAS. In contrast to HbAS, HbS-Antilles is associated with sickle cell disease in the heterozygote. Its presence should be considered in patients with HbAS who have hemolytic anemia and vasoocclusive disease (Chapter 23). Heterozygotes have 40%–50% HbS-Antilles, and sickling tests are positive. Erythrocyte damage and symptoms are seen in heterozygotes because the additional val23Ile mutation in HbSAntilles allows this variant to polymerize even when it forms only half of the hemoglobin. A baby girl presented with symptomatic sickle cell disease despite a prior diagnosis of HbAS. One ␤-globin gene contained the HbS mutation and an additional HBB leu68phe mutation. This variant, Hb Jamaica Plain, had severely reduced oxygen affinity, suggesting destabilization of the oxy conformation at ambient partial pressures of oxygen.10

Pyruvate Kinase Deficiency A unique cause of symptomatic HbAS was found with the coexistence of erythrocyte pyruvate kinase deficiency in a carrier of the HbS gene. This patient, a 42-year-old Guinean

Sickle Cell Trait woman, had an HbS level of 44%, packed cell volume of 25, reticulocytes of 203 × 109 /L, cholelithiasis, and a 35year history of hospitalizations approximately five times yearly for painful episodes.11 Erythrocyte density profiles resembled that seen in HbSC disease. The P50 was 41.5 mm Hg (normal ∼25). Severe pyruvate kinase deficiency was present. High 2,3-BPG levels, a consequence of the metabolic block caused by pyruvate kinase deficiency, increased P50 and decreased hemoglobin oxygen affinity allowing HbS polymerization. Intraerythrocytic HbS polymer was inferred by hysteresis – different P50 values during deoxygenation and reoxygenation – of the hemoglobin–oxygen dissociation curves. HbS polymerization under physiological conditions evidently increased cell density, caused hemolytic anemia, and provoked vasoocclusive episodes. Although it would be useful to know in greater detail the clinical features of the vasoocclusive disease in this patient, this case seems to be an exceptional example of a truly symptomatic individual with HbAS. It suggests that metabolic abnormalities can influence the phenotype of HbAS and, by extension, sickle cell disease.

Hereditary Spherocytosis Rarely, HbAS and hereditary spherocytosis coexist.12,13 The dominant phenotype is that of hereditary spherocytosis.14 Patients with this combination have been reported to developed acute splenic sequestration crises and the postsplenectomy specimens showed erythrostasis and sickling. Perhaps these unusual events – sequestration crises are not a feature of HbAS – were the result of the splenomegaly of hereditary spherocytosis and the high mean corpuscular Hb concentration of HbS-containing hereditary spherocytosis erythrocytes.

HEMOGLOBIN VARIANTS MASQUERADING AS HbAS Many variants of the ␤- and ␣-globin chain will migrate like HbS under the conditions of alkaline electrophoresis. Most, but not all, of these variants can be resolved by more sensitive analytical methods such as HPLC, and in principle, all can be distinguished by DNA analysis. None, except the rare variants like HbS Antilles, and Hb Jamaica Plain, with the HbS mutation and other mutations in the same ␤-globin gene, will polymerize. HbG Makassar (HBB glu6ala) cannot be separated from HbS by HPLC, isoelectric focusing, hemoglobin electrophoresis, or globin chain electrophoresis. It is present at a level of approximately 45% and has no pathological consequences.

CLINICAL FEATURES Does HbAS interfere with any physical activities? Is it a risk factor for increased morbidity and mortality? Again, with some exceptions, there is little evidence for

553 the pathogenicity of HbAS under conditions of ordinary living.

Hematology Table 22.2 presents hematological data compiled from various studies of people with HbAS and either normal ␣globin genotype or with ␣ thalassemia or ␤+ thalassemia. In some studies, the packed cell volume in HbAS was slightly reduced but still normal; in others, it did not differ from the packed cell volume in controls.15,16 The disparity might have been due to differences in methodology and the examination of diverse populations with different age ranges. Differences from normal are very small and are unlikely to be clinically significant. Hemolysis is absent in uncomplicated HbAS in which the reticulocyte count and studies of red cell survival were usually normal. The popular Variant IITM HPLC method of hemoglobin variant detection used in the United States is associated with increases in HbA2 when HbS is present because of the coelution of glycosylated HbS.17 A modification of this method, the Variant II HbA2 /HbA1c , improves the reliability of HbA2 measurement in the presence of HbS.18 Gene deletion ␣ thalassemia is associated with very small differences in HbA2 levels that vary according to the numbers of ␣-globin genes.17

Mortality, Rhabdomyolysis, and Sudden Death In diverse cross-sectional, longitudinal, hospital and community-based studies of thousands of subjects with HbAS, carriers did not have excess risk of mortality.19–23 Evidence that Africans with HbAS have increased mortality rates is also lacking, but adequate data are not available.24 Exercise-related sudden death, or near death, in previously healthy, young athletes is always a tragic event. When an athlete dies and has HbAS, a causal relationship is often assumed, and the case reported in the medical literature and lay press. What is lacking, and needed to interpret the clinical significance of these reports and the concerns they engender, is a careful epidemiological study of exertional rhabdomyolysis, associated and not associated with sudden death, in the universe of athletes (90% of deaths in athletes are due to unsuspected cardiovascular disease). Although studies of military recruits showed that death after exertional heat illness and rhabdomyolysis was increased in carriers of HbAS, their incidence of exertional heat illness was not. Reports of sudden death or life-threatening episodes during exercise in HbAS abound and have been extensively reviewed.25 Most deaths were attributed to exertional heat illness, with rhabdomyolysis and idiopathic sudden death accounting for equal numbers of cases. Case reports and small clusters of unexpected sudden death in people with HbAS in special situations – military recruits undergoing basic training and athletes engaging in physically

554 stressful sports – raised the possibility that under extremely rigorous conditions, HbAS could be associated with a higher risk of sudden death.25 Over a span of 5 years, during which 2.1 million recruits underwent basic training in the U.S. Armed Forces, the risk of sudden unexplained death in black recruits with HbAS compared with those without the trait was 27.6.26 Compared with all recruits – blacks without HbAS, and whites – the relative risk was 40. Other HbAS mortality studies suffer from design flaws, principally low power due to small sample size. The risk increased progressively with increasing age from a death rate of 12 per 100,000 at ages 17–18 years to 136 per 100,000 at ages 31–34 years. Perhaps the increasing prevalence of isosthenuria with age in HbAS and the accompanying possibility of dehydration when access to fluids was limited might account for this observation. It was not possible to tell if the fractional percentage of HbS was associated with sudden death. Coincident ␣ thalassemia in individuals with HbAS might be associated with better-preserved urine-concentrating ability.27 Using percentage of HbS as a surrogate for ␣ thalassemia, studies suggested that ␣ thalassemia was underrepresented in individuals with sudden death.25,28 In summary, HbAS is not a risk factor for a higher mortality rate in the general population, and restraint during the rigors of conditioning should eliminate any hazard in special groups.

Treatment Recommendations A prudent course for all exercising individuals includes gradual conditioning, liberal fluid intake, and avoidance of overexertion under all conditions, especially when temperature and humidity are high. When that strategy was adopted by the U.S. military in recruit conditioning and training, the death rate in the HbAS was said to fall to that of the recruit population overall. Unfortunately, the abandonment of this policy was associated with a return to an increased number of deaths in carriers of HbAS.

Thromboembolism Individuals with HbAS might have increased coagulation system activity and a higher prevalence of prothrombotic mutations, but most studies reporting such association are small, poorly controlled, and inconclusive.29,30 In one study, individuals with HbAS had increased levels of d-dimers, thrombin–antithrombin complexes, and prothrombin fragment 1.2 compared with controls.31 The differences between HbAS and controls were small and might be attributable to a few cases with very high values. This study only included 23 individuals with HbAS, nearly half of whom appeared to be atypical with mild anemia and with a hemoglobin concentration of less than 12 g/dL. In contrast to individuals of African descent in whom the factor V Leiden mutation is rare, in Iranians who have

Martin H. Steinberg an HbS gene this prothrombotic mutation appears to be common.32 Two careful studies suggest that middle aged African Americans with HbAS have an increased risk of thromboembolic disease. In a study of 65,000 hospitalized veterans, 2.2% of individuals with HbAS had pulmonary embolism compared with 1.5% of controls, a statistically significant, albeit small, difference.19 This study examined men nearly exclusively, and, the currently used sensitive methods to diagnose pulmonary embolism were not available. A recent case-control study examined 515 hospitalized African Americans with either deep venous thrombosis or pulmonary embolism and compared them with 555 outpatient controls. The prevalence of the HbS allele was 0.070 for cases and 0.032 for controls. The odds of a case having HbAS were approximately twice that of a control, indicating that the risk of venous thromboembolism is increased approximately twofold among individuals with HbAS. The odds ratio for pulmonary embolism was 3.9. The proportion of cases of pulmonary embolism attributable to HbAS was approximately 7%. The prevalence of sickle cell disease was also increased among cases compared with controls, but in most of these cases there was an underlying reason for thromboembolism.33

Treatment Recommendations These studies show that African Americans with HbAS have an increased risk of venous thromboembolic disease. Although it was implied that African Americans should know if they carry HbAS, given its benignity, it is difficult to see how screening the adult population for HbAS would alter the management of thromboembolic disease. The importance of knowing if a patient with thromboembolic disease has HbAS is also unclear as the treatment implications of such knowledge seem nil.

Pregnancy and Contraception It was thought that fertility was increased in women and perhaps in men with HbAS; however, this supposition is not supported by more recent studies.34–37 According to many reports, pregnancy complications are more frequent in HbAS. Some studies suggest that the risks for bacteriuria, pyelonephritis, and urinary tract infection might be increased as much as twofold. In a welldesigned prospective study of 162 pregnancies with HbAS and 1,422 controls, the preeclampsia rate in HbAS was doubled, endometritis occurred more frequently, and gestational age and birth weight were reduced in infants.38 In contrast, a retrospective study, in which HbAS was ascertained by solubility testing, showed no difference in the prevalence of preeclampsia in HbAS and controls.39 A recent retrospective study using data collected from 1991 to 2006 showed that among 36,897 pregnancies, perinatal mortality and preeclampsia were not increased in HbAS.40

Sickle Cell Trait In a recent retrospective cohort study, asymptomatic bacteriuria, acute cystitis, and pyelonephritis had a similar prevalence in HbAS (n = 455) and matched pregnant control patients (n = 448). Although HbAS carriers had significantly higher rates of pyelonephritis, many affected patients had risk factors such as previous pyelonephritis or noncompliance with therapy.41 In a retrospective case-control study, 180 pregnant women with HbAS were compared with controls. Women with HbAS had significantly shorter average duration of pregnancy (233 ± 45 days vs. 255 ± 34 days), lower birth weight infants (2,114 ± 1,093 g vs. 2,672 ± 942 g), and a higher rate of fetal death (9.7% vs. 3.5%). Acute ascending amniotic infection and meconium histiocytosis were noted more frequently.42 The authors speculate that placental infarction led to intrauterine hypoxia. Patients in this study might not represent the African American population overall. They had a high prevalence of comorbid conditions such as hypertension, diabetes, and obesity and the fetal loss rate in the population from which cases and controls were drawn was approximately twice the national average. Also, it was not clear that comorbidities and hematological findings, such as the prevalence of ␣ thalassemia, were similar in HbAS and control pregnancies. In contrast, a retrospective study of more than 5,000 pregnancies showed that HbAS was associated with lower risk of preterm delivery at less than 32 weeks and increased odds of multiple gestations.43 The effect of maternal age on pregnancy outcomes is controversial. Some studies showed a reduced birth weight of infants born to older mothers but others did not. Pregnancy outcomes did not seem to be affected by age. Although some studies reported increased complications of many types, including prematurity and premature rupture of membranes, they often lacked appropriate controls. In Nigerian women, pregnancy outcomes were similar in HbAS and controls and HbAS women had fewer attacks of malaria.44 Iron supplementation to pregnant African women with HbAS is of little benefit, perhaps because of an increased risk of malaria, but those results are inconclusive. Mothers with HbAS were more likely to have HbAS children than those with only HbA.45–47 This maternal segregation distortion could be another means of maintaining a high prevalence of HbAS.

Treatment Recommendations Despite some uncertainties, the available data suggest that HbAS might be associated with increased risks of certain complications of pregnancy, mainly urinary tract infections, low birth weight, and possibly increased fetal loss. These observations suggest that screening for HbAS in at risk pregnancies might be useful if affected individuals are provided a higher level of antenatal care. The scanty literature available does not suggest that any means of contraception is more hazardous in HbAS than in

555 the general population but definitive studies have yet to be reported.

Growth and Development Many investigators have questioned whether HbAS influences growth and development, and although some have found differences in physical and intellectual development, the best evidence suggests that children with HbAS and matched controls are similar. HbAS is not associated with an increased chance of sudden infant death syndrome.48

Exercise Capacity Whether or not athletes with HbAS are at special risk of sudden death and should be treated differently from their peers is a subject of continued debate and the expression of strong opinions without the addition of new definitive studies.49–54 It is questionable whether HbAS affects the physiological responses to exercise or impairs exercise tolerance and performance. Forty-eight people with HbAS, aged 4–21 years, underwent progressive ergometer stress testing at their voluntary maximal performance and the results were compared with 184 controls. Equivocal ischemic changes on electrocardiographic exmination were seen in four individuals with HbAS. The maximum workload and heart rate were lower in the HbAS subjects, but there were no complications of the exercise program.55 One study comparing the response to incremental exercise in people with HbAS and controls revealed no differences in cardiovascular function but did reveal lower blood lactate concentrations in HbAS.56 When anaerobic exercise and exercise metabolism after force-velocity tests were compared in sedentary adults with HbAS and closely matched controls, there were no differences between the groups.57 Under laboratory conditions, when eight HbAS and eight normal individuals were subjected to an incremental exercise test, maximal oxygen uptake and ventilatory thresholds were similar, and no difference in whole blood, plasma, or red blood cell lactate concentrations were found, suggesting that lactate production and clearance is quite similar during exercise. Lactate uptake by HbAS erythrocytes is more rapid during exercise.58 At supramaximal exercise, an increase in red cell rigidity was seen in HbAS, but it is not known if this has any clinical significance.59,60 Concurrent ␣ thalassemia does not effect the oxygen response to exercise in HbAS.61 Before and after basic training, a group of HbAS and control subjects had similar responses to exercise testing.62,63 In some circumstances and among some African tribes, hypoxia at high altitudes appeared to affect performance in vigorous exercise and cause changes in the splenic circulation.64 In other studies at simulated altitude and moderate hypoxic environment, the cardiopulmonary and gas exchange responses of persons with HbAS during brief episodes of exhaustive exercise were comparable to those of

556 controls.65 Acute stressful exercise at an altitude of 1,270 m and a simulated altitude of 2,300 m were not associated with differences in cardiopulmonary and gas exchange between subjects with HbAS and controls. Vigorous arm exercise was associated with sickled cells in effluent venous blood, and the frequency of such cells increased fourfold at simulated altitudes of 4,000 m.66 Performance was unimpaired, and sickled cells were not detected in arterial blood and hemolysis was not detectable. Ventilatory responses to three episodes of heavy exercise was similar in HbAS and controls.49 When athletes with HbAS, with and without coincident ␣ thalassemia, were exercised on a bicycle ergometer and levels of soluble intercellular adhesion molecule–1 and vascular cell adhesion molecule–1 were measured, the HbAS group had higher vascular cell adhesion molecule–1 basal concentrations, and incremental exercise resulted in a significant increase in all subjects. Levels remained elevated in the HbAS group during recovery, and individuals with HbAS–␣ thalassemia had a more rapid return to baseline.67 Many other studies show minimal or no adverse effects of HbAS during exercise.58,68,69 After prolonged intense exercise, prothrombin time, antithrombin III activity, and plasma fibrinogen were similar in individuals with HbAS and controls.70 Black athletes with HbAS are well represented in elite competitive sports. The percentage of African semimarathon runners with HbAS was similar to that of the general population.63

Treatment Recommendations Based on the total body of work on exercise ability and capacity, there is no reason to restrict exercise of any type in persons with HbAS. All individuals engaged in strenuous exercise, trained or untrained, should be properly conditioned, clothed, have unrestricted access to fluids, appropriate rest periods, moderation of the exercise routine when harsh atmospheric conditions like excessive heat and humidity are present, and rapid treatment should symptoms develop. Any restraints applied to carriers of HbAS need not differ from those used to forestall ill effects of exercise in the general population.

Flying and Scuba Diving There are many reports of splenic infarction during flight in unpressurized aircraft at altitudes of 3,000–5,000 m. In the early reports, the diagnosis of HbAS was often made by tests that today would be considered inadequate, and some cases reported could have been instances of HbSC disease or HbS–␤+ thalassemia rather than HbAS. Commercial aircraft are pressurized to 2,500 m and splenic infarct – and any other complication of HbS polymerization – is unlikely to occur under that condition. As of 1996, the US Department of Defense stopped preinduction testing for HbAS

Martin H. Steinberg and as of 2003 HbAS carriers were not restricted from flight or undersea duty. Scuba diving, in which divers breathe compressed air, provides an environment with increased pO2 and poses no known risk to carriers of HbAS.

Genitourinary System Hyposthenuria. The kidney medulla is one of the few sites where under physiological conditions, HbAS erythrocytes are placed in jeopardy. Low pO2 , low pH, and high solute concentration provide a milieu where sickling of even the HbAS erythrocyte can occur.71 Damage to the countercurrent urine-concentrating mechanism and the vasculature of the renal medulla leads to a defect in urine-concentrating ability and hyposthenuria in most adults with HbAS and hematuria in some individuals. These abnormalities might result from microvascular injury in the vasa recta of the renal medulla. A defect in urine acidification, which in sickle cell anemia is related to the severity of the concentration defect, is not seen in HbAS. The polymerization tendency of HbS might determine the extent of the urine-concentrating defect in HbAS.27 To test this hypothesis, urinary-concentrating ability was examined following overnight water deprivation and intranasal arginine vasopressin in HbAS subjects separated into two groups – one with a normal ␣-globin gene complement and one with either heterozygous or homozygous -␣3.7 gene deletion ␣ thalassemia. The ability to concentrate urine was most impaired in subjects with normal ␣-globin genotype and least impaired in subjects with homozygous ␣ thalassemia. Urine osmolality was 882 ± 37 mOsm/kg H2 O in ␣ thalassemia homozygotes and 672 ± 38 mOsm/kg H2 O in the heterozygotes. In all subjects, urinary osmolality correlated linearly and inversely with percentage of HbS. A similar correlation was found between urine-concentrating ability and HbS polymerization tendency at 0.4 oxygen saturation (Fig. 22.3), suggesting that HbS polymer, a function of percentage of HbS, is responsible for the urine-concentrating defect of HbAS. Hematuria. Hematuria occurs episodically in approximately 5% of people with HbAS. Some studies suggest that hematuria is most common at higher HbS levels. Bleeding is more frequent from the left kidney, perhaps because of the anatomy of venous system, and men are affected four times as often as women. Although von Willebrand syndrome has been reported in HbAS, no association has been established between any coagulation abnormality and HbAS hematuria. Papillary necrosis can cause hematuria, sometimes with renal colic. This might be more frequent in HbAS and is perhaps another consequence of sickling in the renal medulla.72,73 Autosomal dominant polycystic kidney disease might be more prevalent and progress to renal failure more rapidly in individuals with HbAS.74

Sickle Cell Trait

557

Medullary Carcinoma. Renal medullary carcinoma, a rare and aggressive tumor, has a unique association with HbAS.75–82 In a retrospective analysis spanning 20 years, 33 black patients, aged 11–40 years, who presented with hematuria and pain had renal medullary carcinoma. Nine patients had proven HbAS, and one had HbSC disease, but all tumor specimens contained sickled cells. The tumors were lobulated with satellite cortical nodules and widespread metastases. Histologically, the tumors had a reticular, yolk sac–like or glandular cystic appearance, with a desmoplastic stroma, hemorrhage, necrosis, neutrophil infiltration, and lymphocyte sheathing (Fig. 22.4). Characteristic findings of renal medullary carcinoma on computed Figure 22.3. Relationship between maximum urine-concentrating ability and polymer fraction at tomography and magnetic resonance imag- 40% oxygen saturation in individuals with HbAS and 4, 3, and 2 ␣-globin genes. (From ref. 27 with ing include an infiltrative renal mass with permission.) associated retroperitoneal adenopathy and caliectasis.83 Chemotherapy was uniformly unsuccessful, can be dangerous in some situations by inducing clot forand survival after diagnosis was approximately 4 months.67 mation and causing obstructive uropathy. The diagnosis of HbAS for 23 of the patients was based Although often recurrent and sometimes producing only on histological findings because diagnostic tests docanemia, hematuria is usually benign and self-limited. Nevumenting HbAS were not available. If HbAS was present ertheless, hematuria could arise from more ominous causes in all 33 patients, the conclusion that the condition prethan a damaged renal medulla. Renal and bladder cancer – disposes to this rare tumor seems inescapable. It has been especially medullary cancer in young patients – stones, hypothesized that the medullary cancers could originate glomerulonephritis, and infection should be excluded durin terminal collecting ducts, where epithelial cell prolifering the evaluation of hematuria. ation has been noted in HbAS. Perhaps rapid cell proliferation and oxygen radicals produced by cell sickling, coupled SPLEEN with preexisting mutations in genes controlling cell growth, predispose to carcinogenesis. In three cases, the ABL gene Young black men with HbAS who resided at altitudes up was amplified.81 Gene expression studies suggested that the to 1,600 m for many years had normal splenic function as tumor signature resembled that of urothelial tumors rather assessed by radionuclide scanning and by the number of than renal cell carcinoma. Genitourinary Lesions Probably Not Caused by HbAS. Other renal or genitourinary system disorders, such as nephrotic syndrome, pyelonephritis, hypotonic bladder, hypertension, renal failure, renal cortical infarction, renal vein thrombosis, and testicular infarction undoubtedly occur in some carriers of HbAS, but there is no evidence of their being caused by HbAS. Priapism, a serious complication of sickle cell anemia, is not associated with HbAS. Renal allograft survival in HbAS is equivalent to that in the general population.

Treatment Recommendations No treatment can reliably stop hematuria in people with HbAS and radical treatments, such as nephrectomy, should be avoided.84 Induced hypotonicity, alkalinization, and epsilon amino caproic acid, an antifibrinolytic agent, have all been used to control severe hematuria but controlled trials have never been reported. Epsilon amino caproic acid

Figure 22.4. Renal medullary carcinoma showing epithelioid cells and spindle cells in a background of fibrosis and small lymphocytes (× 400). Kindly provided by R.S. Figenshau.79

558 red cells with “pits.”85 Splenic infarction can occur in individuals with HbAS when they encounter hypobaric conditions. Splenic infarcts in African Americans with HbAS are rare, or rarely reported. Splenic infarction was not mentioned in a report of African runners with HbAS who competed at altitudes of up to 4,100 m. Especially interesting are reports of splenic infarction in Caucasians with HbAS.86,87 Caucasian men might be overrepresented in reports of altitude-induced splenic syndrome and splenic infarcts at sea level. Twenty of 29 reported cases of splenic syndrome were in Caucasian men.88 Most, but not all, episodes occurred shortly after ascent to approximately 3,000 m. These curious observations have led to much speculation.28 A reasonable supposition is that an African HbS gene within the Caucasian gene pool became isolated from accompanying genes that in Africa evolved along with the HbS gene and modulated its effects. The hypothesized modulating genes could affect the mean corpuscular HbS concentration, which might differ between black and nonblack HbAS carriers. Inconclusive evidence suggests that Caucasian carriers with splenic infarction have a higher HbS concentration than black carriers and that a high mean cell HbS concentration increases the risk of intrasplenic sickling and organ damage. One Caucasian splenic infarction patient had an HbS level of 46.5% and had five ␣-globin genes (Table 22.1). In a report in which both father and son had a splenic infarct, the HbS levels were 41% and 45.6%. In another study, however, HbS levels in a small series of nonblack carriers differed little from those in most black carriers of HbAS. If HbS levels are truly higher in Caucasian carriers, it might be a consequence of the absence of ␣ thalassemia and the reduced cation exchange and lower cation content in Caucasians erythrocytes, a deficit most marked in men (splenic infarction is rarely reported in women), or, differences in other “protective” genes associated with the HbS gene in black patients.

Treatment Recommendations Fear of splenic damage should not dissuade carriers of HbAS from activities at altitude. Physicians should be aware, however, that in this setting, splenic infarction should be considered as a cause of left upper quadrant pain in any racial and ethnic group. Fortunately, most often splenic infarction is a self-limited process and needs no special treatment.

Martin H. Steinberg cellular outflow through the trabecular meshwork and Schlemm canal, raising intraocular pressure. High pressure in the anterior chamber can reduce vascular perfusion pressure elsewhere in the eye, injuring the optic nerve and retina and causing hemorrhage and impairing vision.90 Anterior segment hypoxia and acidosis set in place the vicious circle of erythrostasis typical of sickle cell disease. Secondary open-angle glaucoma can result from poorly treated hyphema. Many ocular abnormalities, including internuclear ophthalmoplegia, transient monocular blindness, vitreous hemorrhage, and optic atrophy have been described in HbAS, but any association with HbAS is likely to be fortuitous.91–93 Chronic open-angle glaucoma is no more common in HbAS than in age- and sex-matched controls. Although proliferative retinopathy, akin to the type known to occur in sickle cell disease has been described in HbAS, in all cases, a known cause of proliferative retinopathy such as diabetes, was present, perhaps predisposing to the development of retinal lesions. Uncomplicated HbAS is not associated with sickle retinopathy.94

Treatment Recommendations Black patients and others at risk of carrying the HbS gene who present with hyphema should be screened for HbAS without delay. A sickle solubility test will do, because it is important at this point to find out only whether HbS is present. Management of hyphema in HbAS is beyond the scope of this chapter; it should be supervised by an ophthalmologist who understands the special problems that can arise when the hyphema and HbAS coexist. Intraocular pressure can be controlled medically in perhaps two thirds of the cases. Anterior chamber hypertension of greater than 24 mm Hg for consecutive 24-hour periods and repeated but transient increases above 30 mm Hg should be reduced by paracentesis, which can be repeated if necessary. Normal pressure during the first 24 hours of observation suggests, but does not establish, that medical treatment will suffice.95

BONES AND JOINTS Reports of osteonecrosis in HbAS make an unconvincing case for their association. Bone infarction has also been described with HbAS, also with no evidence of association beyond coincidence.96

EYE

NERVOUS SYSTEM

In nonhemoglobinopathic individuals, hyphema, or hemorrhage into the anterior chamber, usually clears uneventfully with topical medical therapy. Among individuals with traumatic hyphema, carriers of HbAS might be overrepresented.89 The naturally low pO2 in the anterior chamber causes erythrocyte sickling in HbAS and impedes

Children with HbAS had greater arterial tortuosity on magnetic resonance imaging and magnetic resonance angiography compared with controls, and this was related to the percentage of HbS.97 The authors speculated that this was a sign of mild vasculopathy; however, the clinical implications of this observation are unknown. Cerebral infarcts

Sickle Cell Trait have been reported in HbAS.98 Most of the reports are of one or two cases so that given the ubiquity of both HbAS and stroke, a causal relationship cannot be established. In a retrospective study in Guadeloupe, in 295 patients hospitalized for stroke, HbAS appeared to be more prevalent in individuals with hemorrhagic stroke and less prevalent in individuals with infarctive stroke.99 The truth of the assertion that HbAS is a risk factor for stroke cannot yet be established.98

SURGERY AND ANESTHESIA Untold numbers of patients with HbAS have safely undergone anesthesia and surgery. In a matched-pair analysis of patients with HbAS and control subjects, the frequency of anesthetic, surgical, and postoperative complications was similar; however, most patients were young, and few intraabdominal and thoracic procedures were included. Use of a tourniquet in orthopaedic procedures appears safe in HbAS (and in sickle cell anemia).

Treatment Recommendations Most evidence suggests that anesthetic risk is not increased by HbAS, and the choice of anesthesia need not be limited.100,101 Thus, there is no rationale for routine preoperative screening for HbAS.102 With open heart surgery or complicated intrathoracic surgeries, in which hypoxia might be intrinsic to the procedure, preoperative transfusion was recommended.100,101 Screening for HbAS might be prudent under those circumstances, but there are no well-controlled studies on the preoperative management of intrathoracic surgery in HbAS.

MALARIA The selection pressure from Plasmodium falciparum malaria is expressed primarily in children, and where malaria is endemic, HbAS children have reduced mortality.103,104 HbAS retards the development of cerebral malaria in children aged younger than 5 years, and it reduces their death rate.105 Although the clinical features, haptoglobin levels, parasite counts, and most laboratory measurements are similar in children with and without HbAS, trait carriers are transfused more often. It is estimated that HbAS is almost 90% protective for severe P. falciparum malaria with lesser protection against mild disease.106 When homozygous ␣ thalassemia and HbAS coexist, the protection against P. falciparum malaria afforded by each trait individually was lost, an example of negative epistasis.107 It was suggested that this might be explained by the reduction in HbS concentration caused by ␣ thalassemia or effects on the erythrocyte membrane altering merozoite invasion. Children with HbAS have an imbalanced distribution of immunoglobulin G2 (IgG2) antibodies to merozoite surface

559 protein 2 (MSP2) and a higher frequency of infection with multiple P. falciparum strains. In Gabonese children with HbAS, Fc gamma receptor IIa (CD32) polymorphism and the rate of in vitro invasion of red blood cells from subjects with HbAS and controls with multiple P. falciparum strains were investigated. Lower levels of IgG2 subclass antibodies to MSP2 peptides were independently associated with the Fc gamma receptor IIa-R131 allele and with HbAS, suggesting that IgG3 antibody responses to MSP2 epitopes could be exacerbated by lower IgG2 levels. Longer persistence of ring forms in HbAS might reflect slower multiplication, longer circulation, and enhanced phagocytosis of these nonpathogenic forms and could contribute to the protection against malaria.108 Some aspects of malaria and hemoglobinopathies are discussed in Chapter 26.

OTHER CONDITIONS The grab bag of other diseases reported in individuals with HbAS has been reviewed and new “associations” continue to appear.109 Early reports of impaired pulmonary function in HbAS have been refuted by more carefully controlled studies of diffusing capacity for CO and spirometry at simulated altitudes up to 7,500 m and pulmonary function at rest and after exhaustive exercise in men with HbAS who had been living above 1,600 m for many years. Reports of sickle cell disease–like “crises,” complicated migraine with occlusion of branches of the middle cerebral artery, venous thrombosis, and retroperitoneal fibrosis could not be linked etiologically to HbAS. Leg ulcers have been associated with HbAS in Jamaica. Of 250 patients with chromic leg ulcers, 20% had HbAS, whereas 11% of the general population carried the HbS gene. There are no data to support an increase in leg ulceration in HbAS carriers in temperate climates. Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) deficiency is common in many populations in which HbAS is found. Hemizygotes with the common African type of G6PD deficiency rarely encounter clinical difficulties. In a study of more than 65,000 hospitalized African-American men, nearly 1% had both HbAS and G6PD deficiency. Morbidity from pneumonia and mean age of death were similar for the group with HbAS alone and the group with both HbAS and G6PD deficiency.

TRANSFUSION OF HbAS BLOOD Minor changes distinguish HbAS from normal blood under storage conditions, but transfused HbAS erythrocytes are equivalent to normal cells in safety, effectiveness, and survival. Cryopreserved red cells from individuals with HbAS could hemolyze when deglycerolized but a special deglycerolization protocol can prevent this; thus, when cryopreservation of rare blood types is undertaken, knowing if the donor has HbAS is useful.

560 Table 22.4. Recommendations for counseling and management of HbAS Counseling permits informed family planning Antenatal diagnosis for sickle cell disease is available Occupational choice should be unrestricted Flying in commercial aircraft is safe Unexplained pain should not be ascribed to HbAS Hematuria is usually benign and self-limited but should be carefully evaluated Hyposthenuria is present in most adults with HbAS When strenuous exercise is undertaken, fluid intake should match expected losses Surgical intervention might be needed in hyphema Pregnancy can have complications such as urinary tract infection Surgery is safe Morbidity from most associated diseases is not increased Duration and quality of life are normal Clinically significant anemia is not a complication Microcytosis in HbAS is usually a sign of ␣ thalassemia but should be evaluated.

Blood from donors with HbAS cannot be consistently leukodepleted with any available filter but cell sickling upstream of the filters was not observed.110–112 This might be due to the low pO2 and pH of stored blood and HbS polymerization.113,114 Universal leukodepletion has become the standard in the United States, France, and Canada. Because HbAS carriers are a large fraction of donors in many locales, the loss of their units could seriously reduce blood supplies. Studies have suggested that storage of HbAS whole blood in large-capacity oxygenpermeable bags increases oxygen tension and allows more effective leukoreduction.114 Units from HbAS donors should be avoided when transfusing large amounts of blood into anemic hypoxic neonates.

SCREENING AND COUNSELING Screening for HbAS has limited indications and in most cases should be reserved for individuals likely to carry the HbS gene. The U.S. Armed Forces do not screen before induction but might conduct screening afterward. Although the screening of individuals assigned to flight duty might be prudent to detect asymptomatic individuals with sickle cell disease such as HbS–␤+ thalassemia and HbSC disease, screening the general population for HbAS serves no purpose. Before screening prospective parents, they should be educated about sickle cell disease and HbAS and receive nondirective counseling. Antenatal diagnosis for sickle cell disease is possible in instances in which a pregnancy is at risk for an affected fetus (Chapter 28). A byproduct of neonatal screening for sickle cell disease is the identification of babies with HbAS. The value of finding HbAS in newborns is questionable; nevertheless,

Martin H. Steinberg notifying the parents is reasonable so they can be informed about the implications of the condition and offered further family testing.115 Table 22.4 summarizes an approach for counseling and management of individuals with HbAS.

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562 52. Connes P, Caillaud C, Py G, Mercier J, Hue O, Brun JF. Maximal exercise and lactate do not change red blood cell aggregation in well trained athletes. Clin Hemorheol Microcirc. 2007;36(4):319–326. 53. Le Gallais D, Lonsdorfer J, Bogui P, Fattoum S. Last Word on Point:Counterpoint “Sickle cell trait should/should not be considered asymptomatic and as a benign condition during physical activity.” J Appl Physiol. 2007;103(6):2143. 54. Baskurt OK, Meiselman HJ, Bergeron MF. Sickle cell trait should be considered asymptomatic and as a benign condition during physical activity. J Appl Physiol. 2007;103(6):2142. 55. Alpert BS, Flood NL, Strong WB, Blair JR, Walpert JB, Levy AL. Responses to exercise in children with sickle cell trait. Am J Dis Child. 1982;136:1002–1004. 56. Bile A, Le Gallais D, Mercier B, et al. Blood lactate concentrations during incremental exercise in subjects with sickle cell trait. Med Sci Sports Exerc. 1998;30(5):649–654. 57. Bile A, Le Gallais D, Mercier B, Martinez P, Ahmaidi S, Prefaut C. anaerobic exercise components during the force-velocity test in sickle cell trait. Int J Sports Med. 1996;17:254–258. 58. Fagnete S, Philippe C, Olivier H, Mona MH, Maryse EJ, MarieDominique HD. Faster lactate transport across red blood cell membrane in sickle cell trait carriers. J Appl Physiol. 2006;100(2):427–432. 59. Sara F, Hardy-Dessources MD, Marlin L, Connes P, Hue O. Lactate distribution in the blood compartments of sickle cell trait carriers during incremental exercise and recovery. Int J Sports Med. 2006;27(6):436–443. 60. Connes P, Sara F, Hardy-Dessources MD, et al. Effects of short supramaximal exercise on hemorheology in sickle cell trait carriers. Eur J Appl Physiol. 2006;97(2):143–150. 61. Connes P, Monchanin G, Perrey S, et al. Oxygen uptake kinetics during heavy submaximal exercise: effect of sickle cell trait with or without alpha-thalassemia. Int J Sports Med. 2006;27(7):517–525. 62. Weisman IM, Zeballos RJ, Johnson BD. Cardopulmonary and gas exchange responses to acute strenuous exercise at 1,270 meters in sickle cell trait. Am J Med. 1988;84:377–383. 63. Le Gallais D, Prefaut C, Mercier J, Bile A, Bogui P, Lonsdorfer J. Sickle cell trait as a limiting factor for high-level performance in a semi-marathon. Int J Sports Med. 1994;15:399–402. 64. Thiriet P, Le Hesraan JY, Wouassi D, Bitanga E, Gozal D, Louis FJ. sickle cell trait performance in a prolonged race at high altitude. Med Sci Sports Exerc. 1994;26:914–918. 65. Weisman IM, Zeballos RJ, Johnson BD. Effect of moderate inspiratory hypoxia on exercise performance in sickle cell trait. Am J Med. 1988;84:1033–1040. 66. Martin TW, Weisman IM, Zeballos RJ, Stephson SR. Exercise and hypoxia increase sickling in venous blood from an exercising limb in individuals with sickle cell trait. Am J Med. 1989;87:48–56. 67. Monchanin G, Serpero LD, Connes P, et al. Effects of progressive and maximal exercise on plasma levels of adhesion molecules in athletes with sickle cell trait with or without {alpha}-thalassemia. J Appl Physiol. 2007;102(1):169–173. 68. Samb A, Kane MO, Ba A, et al. Physical performance and thermoregulatory study of subjects with sickle cell trait during a sub-maximal exercise. Dakar Med. 2005;50(2):46–51. 69. Moheeb H, Wali YA, El-Sayed MS. Physical fitness indices and anthropometrics profiles in schoolchildren with sickle cell trait/disease. Am J Hematol. 2007;82(2):91–97.

Martin H. Steinberg 70. Connes P, Tripette J, Chalabi T, et al. Effects of strenuous exercise on blood coagulation activity in sickle cell trait carriers. Clin Hemorheol Microcirc. 2008;38(1):13–21. 71. Embury SH, Hebbel RP, Mohandas N, Steinberg MH. Sickle Cell Disease: Basic Principles and Clinical Practice. 1 ed. New York: Raven Press; 1994. 72. Zadeii G, Lohr JW. Renal papillary necrosis in a patient with sickle cell trait. Am J Soc Nephrol. 1997;8:1034–1039. 73. Lang EK, Macchia RJ, Thomas R, et al. Multiphasic helical CT diagnosis of early medullary and papillary necrosis. J Endourol. 2004;18(1):49–56. 74. Kimberling WJ, Yium JJ, Johnson AM, Gabow PA, MartinezMaldonado M. Genetic studies in a black family with autosomal dominant polycystic kidney disease and sickle-cell trait. Nephron. 1996;72(4):595–598. 75. Adsay NV, DeRoux SJ, Sakr W, Grignon D. Cancer as a marker of genetic medical disease – An unusual case of medullary carcinoma of the kidney. Am J Surg Pathol. 1998;22(2):260– 264. 76. Coogan CL, McKiel CF Jr, Flanagan MJ, Bormes TP, Matkov TG. Renal medullary carcinoma in patients with sickle cell trait. Urology. 1998;51(6):1049–1050. 77. Davidson AJ, Choyke PL, Hartman DS, Davis CJ Jr. Renal medullary carcinoma associated with sickle cell trait; radiologic findings. Radiology. 1995;195:83–85. 78. Davis CJ, Jr., Mostofi FK, Sesterhenn IA. Renal medullary carcinoma: The seventh sickle cell nephropathy. Am J Surg Pathol. 1995;19:1–11. 79. Figenshau RS, Basler JW, Ritter JH, Siegel CL, Simon JA, Dierks SM. Renal medullary carcinoma. J Urol. 1998; 159(3 Pt 1):711–713. 80. Friedrichs P, Lassen P, Canby E, Graham C. Renal medullary carcinoma and sickle cell trait. J Urol. 1997;157:1349. 81. Simpson L, He X, Pins M, et al. Renal medullary carcinoma and ABL gene amplification. J Urol. 2005;173(6):1883– 1888. 82. Hakimi AA, Koi PT, Milhoua PM, et al. Renal medullary carcinoma: the Bronx experience. Urology. 2007;70(5):878–882. 83. Blitman NM, Berkenblit RG, Rozenblit AM, Levin TL. Renal medullary carcinoma: CT and MRI features. AJR Am J Roentgenol. 2005;185(1):268–272. 84. Yang XJ, Sugimura J, Tretiakova MS, et al. Gene expression profiling of renal medullary carcinoma: potential clinical relevance. Cancer. 2004;100(5):976–985. 85. Nuss R, Feyerabend AJ, Lear JL, Lane PA. Splenic function in persons with sickle cell trait at moderately high altitude. Am J Hematol. 1991;37:130–132. 86. Gitschier J, Thompson CB. Non-altitude related splenic infarction in a patient with sickle cell trait. Am J Med. 1989;87:697–698. 87. Lane PA, Githens JH. Splenic syndrome at mountain altitudes in sickle cell trait. it’s occurence in nonblack persons. JAMA. 1985;253:2251–2254. 88. Harkness DR. Sickle cell trait revisited. Am J Med. 1989;87: 30N–34N. 89. Lai JC, Fekrat S, Barron Y, Goldberg MF. Traumatic hyphema in children: risk factors for complications. Arch Ophthalmol. 2001;119(1):64–70. 90. Nasrullah A, Kerr NC. Sickle cell trait as a risk factor for secondary hemorrhage in children with traumatic hyphema. Am J Ophthalmol. 1997;123(6):783–790.

Sickle Cell Trait 91. Sear DA. The morbidity of sickle cell trait: a review of the literature. Am J Med. 1978;64:1021–1036. 92. Leavitt JA, Butrus SI. Internuclear ophthalmoplegia in sickle cell trait. J Neuroophthalmol. 1994;14:49–51. 93. Finelli PF. Sickle cell trait and transient monocular blindness. Am J Ophthalmol. 1976;81:850–851. 94. Nia J, Lam WC, Kleinman DM, Kirby M, Liu ES, Eng KT. Retinopathy in sickle cell trait: does it exist? Can J Ophthalmol. 2003;38(1):46–51. 95. Liebmann JM. Management of sickle cell disease and hyphema. J Glaucoma. 1996;5:271–275. 96. Lally EV, Buckley WM, Claster S. Diaphyseal bone infarctions in a patient with sickle cell trait. J Rheumatol. 1983;10:813– 816. 97. Steen RG, Hankins GM, Xiong X, et al. Prospective brain imaging evaluation of children with sickle cell trait: initial observations. Radiology. 2003;228(1):208–215. 98. Golomb MR. Sickle cell trait is a risk factor for early stroke. Arch Neurol. 2005;62(11):1778–1779. 99. Lannuzel A, Salmon V, Mevel G, Malpote E, Rabier R, Caparros-Lefebvre D. [Epidemiology of stroke in Guadeloupe and role of sickle cell trait]. Rev Neurol (Paris). 1999; 155(5):351–356. 100. Scott-Conner CEH, Brunson CD. The pathophysiology of the sickle hemoglobinopathies and implications for perioperative management. Am J Surg. 1994;168:268–274. 101. Scott-Conner CEH, Brunson CD. Surgery and anesthesia. In: Embury SH, Hebbel RP, Mohandas N, Steinberg MH, eds. Sickle Cell Disease: Basic Principles and Clinical Practice. 1st ed. New York: Lippincott-Raven; 1994:809–827. 102. Crawford MW, Galton S, Abdelhaleem M. Preoperative screening for sickle cell disease in children: clinical implications. Can J Anaesth. 2005;52(10):1058–1063. 103. Hendrickse RG, Hasan AH, Olumide LO, Akinkunmi A. Malaria in early childhood: an investigation of five hundred seriously ill children in whom a “clinical” diagnosis of malaria was made on admission to the children’s emergency room at University College Hospital, Ibadan. Ann Trop Med Parasitol. 1971;65:1–20. 104. Aidoo M, Terlouw DJ, Kolczak MS, et al. Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet. 2002;359(9314):1311–1312.

563 105. Olumese PE, Adeyemo AA, Ademowo OG, Gbadegesin RA, Sodeinde O, Walker O. the clinical manifestations of cerebral malaria among Nigerian children with the sickle cell trait. Ann Trop Paediatr. 1997;17:141–145. 106. Williams TN, Mwangi TW, Wambua S, et al. Sickle cell trait and the risk of Plasmodium falciparum malaria and other childhood diseases. J Infect Dis. 2005;192(1):178– 186. 107. Williams TN, Mwangi TW, Wambua S, et al. Negative epistasis between the malaria-protective effects of alpha(+)thalassemia and the sickle cell trait. Nat Genet. 2005; 37(11):1253–1257. 108. Ntoumi F, Flori L, Mayengue PI, et al. Influence of carriage of hemoglobin AS and the Fc gamma receptor IIa-R131 allele on levels of immunoglobulin G2 antibodies to Plasmodium falciparum merozoite antigens in Gabonese children. J Infect Dis. 2005;192(11):1975–1980. 109. Steinberg MH. Sickle cell trait. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. 1st ed. Cambridge: Cambridge University Press; 2001:811–830. 110. Bodensteiner D. White cell reduction in blood from donors with sickle cell trait. Transfusion. 1994;34(1):84. 111. Leukocyte depletion of whole blood and red cells from donors with hemoglobin sickle cell trait. Transfusion. 1999; 39(Suppl 108S). 112. Schuetz AN, Hillyer KL, Roback JD, Hillyer CD. Leukoreduction filtration of blood with sickle cell trait. Transfus Med Rev. 2004;18(3):168–176. 113. Stroncek DF, Rainer T, Sharon V, et al. Sickle Hb polymerization in RBC components from donors with sickle cell trait prevents effective WBC reduction by filtration. Transfusion. 2002;42(11):1466–1472. 114. Stroncek DF, Byrne KM, Noguchi CT, Schechter AN, Leitman SF. Increasing hemoglobin oxygen saturation levels in sickle trait donor whole blood prevents hemoglobin S polymerization and allows effective white blood cell reduction by filtration. Transfusion. 2004;44(9):1293–1299. 115. Newborn screening for sickle cell disease and other hemoglobinopathies. JAMA. 1987;258:1205–1209. 116. Luo H-Y, Yulong M, Adewoye AH, et al. Two new a thalassemia frame-shift mutations. Hemoglobin. 2007;31:135.

23 Other Sickle Hemoglobinopathies Martin H. Steinberg

INTRODUCTION The Genotypic Complexity of Sickle Cell Disease The sickle hemoglobin gene (HBB glu6val) and ␤ thalassemia alleles are often geographically coincident. Consequently, compound heterozygotes with HbS–␤ thalassemia are commonplace. The incidence of this genotype is dependent on the populations’ ratio of carriers of an HbS gene to carriers of ␤ thalassemia. In the United States, and in Africa where the HbS predominates, sickle cell anemia (homozygous for HBB glu6val) is the prevailing genotype of sickle cell disease. Wherever ␤ thalassemia is more frequent than sickle cell trait (HbAS) – Greece, Italy, and other Mediterranean countries are examples – HbS–␤ thalassemia predominates. Some ␤ thalassemia variants, such as ␦␤ thalassemia and Hb Lepore and gene deletion hereditary persistence of fetal hemoglobin (HPFH), can interact with HbS producing different interesting phenotypes. ␣ Thalassemia is present in 30% of African Americans with sickle cell anemia and might be even more common in some populations from the Middle East and Indian subcontinent. Individuals with sickle cell anemia–␣ thalassemia have distinctive hematological and clinical features. Compound heterozygotes of HbS with other mutant ␣- or ␤-globin genes are also present in any population with a high prevalence of HbS. Depending on the other globin gene mutation, these conditions can resemble the clinically benign HbAS or have a phenotype with hemolytic anemia and vasoocclusive complications. Rare variants, often with intriguing phenotypes, have both the sickle mutation and an additional mutation in the same ␤-globin gene. This chapter will examine the molecular, diagnostic, laboratory, and clinical aspects of compound heterozygous genotypes of sickle cell disease, excluding HbSC disease, which is discussed in Chapter 21. Unless there are special features associated with a genotype discussed in this chapter, its clinical aspects will be covered in chapters on sickle 564

cell anemia (Chapter 19) and HbSC disease (Chapter 21) and in other chapters in which management is discussed (Chapters 20, section VIII, Chapters 29–33). Whatever the hemoglobin genotype responsible for a sickle hemoglobinopathy, the treatment of its complications is nearly always the same. The critical clinical issue is to establish the correct genotypic diagnosis before counseling and when discussing prognosis.

HbS–␤ Thalassemia and HbS–␤ Thalassemia-like Conditions ␤ Thalassemia is classified as ␤0 thalassemia or ␤+ thalassemia (Chapter 16). In the former, the thalassemiacausing mutation totally abolishes expression of the affected gene; in the latter, there is a reduction in ␤-globin gene expression that depends on the thalassemia mutation. Lacking recent blood transfusion, HbA is not present in HbS–␤0 thalassemia; the hemolysate contains only HbS, fetal hemoglobin (HbF), and 4%–6% HbA2 . Therefore, the phenotype of HbS–␤0 thalassemia mimics that seen in sickle cell anemia and is equally variegated. In HbS–␤+ thalassemia, HbA levels range from less than 5%–45% of the hemolysate. Given the primacy of the HbS concentration in determining the phenotype of the resulting disease, individuals with only traces of HbA have a disease resembling sickle cell anemia or HbS–␤0 thalassemia; most patients with HbA levels of 20% or more have a phenotype milder than that of HbSC disease or sickle cell anemia. Because of the confounding influences of genetic modifiers like ␥ -globin gene expression, ␣ thalassemia, and other more obscure modulators of sickle cell disease (Chapter 27), rigid genotype–phenotype correlations are difficult to establish in all instances of HbS–␤ thalassemia. Among the different genotypes of HbS–␤+ thalassemia, higher levels of HbA are usually associated with a milder phenotype and in all types of HbS–␤ thalassemia, as in sickle cell anemia, higher HbF concentrations temper the disease.

Pathophysiology ␤ Thalassemia Mutations in HbS–␤ Thalassemia The pathophysiology of the sickle–␤ thalassemias is similar to the pathophysiology of sickle cell anemia (Chapters 8– 11) with the major determinant of severity being the magnitude of the reduction in ␤A -globin chain production caused by the thalassemia mutation. Different ␤ thalassemia– causing mutations have been described in association with HbS and many of these are shown in Table 23.1. The ␤ thalassemia mutations extant in each population determine the phenotypes of HbS–␤ thalassemia present. HbS– ␤ thalassemia has a different clinical spectrum in different racial and ethnic groups because ␤ thalassemia mutations vary among populations. HbS–␤ thalassemia is most common in selected Italian and Greek populations, in Turks,

Other Sickle Hemoglobinopathies

565

Table 23.1. ␤ Thalassemia mutations in HbS–␤ thalassemia Mutation

PCV/Hemoglobin

HbA

HbF

MCV

Phenotype

␤+ Thalassemia∗ -90 C-T -92 C→T§ -88 C→T -88 C→T in cis to HbS§

11/33 14.5/ 10/31 12/35

? 45 18 90

5 1.4 15 3

75 83 65–70 70

11/32

20

11¶

65–70

0 Lys] in association with Hb S [␤6(A3)Glu-->Val]: characterization, stability, and effects on Hb S polymerization. Hemoglobin. 1993;17:329–343. Ingle J, Adewoye A, Dewan R, et al. Hb Hope [␤136 (H14)Gly-->Asp (GGT-->GAT)]: interactions with Hb S [␤6(A3)Glu-->Val (GAG-->GTG)], other variant hemoglobins and thalassemia. Hemoglobin. 2004;28(4):277–285. Smith CM, Hedlund B, Cich JA, et al. Hemoglobin North Shore: a variant hemoglobin associated with the phenotype of betathalassemia. Blood. 1983;61:378–383. Masiello D, Heeney M, Adewoye AH, Eung SH, Steinberg MH, Chui DHK. HbSE disease; a concise review. Am J Hematol. 2007;82:643–649. Knox-Macaulay HH, Ahmed MM, Gravell D, Al-Kindi S, Ganesh A. Sickle cell-haemoglobin E (HbSE) compound heterozygosity: a clinical and haematological study. Int J Lab Hematol. 2007;29(4):292–301. Higgs DR, Aldridge BE, Lamb J, et al. The interaction of alphathalassemia and homozygous sickle-cell disease. N Engl J Med. 1982;306:1441–1446. Embury SH, Dozy AM, Miller J, et al. Concurrent sickle-cell anemia and alpha-thalassemia: effect on severity of anemia. N Engl J Med. 1982;306:270–274. Embury SH. The interaction of alpha-thalassemia with sickle cell anemia. Hemoglobin. 1988;12:509–517. Steinberg MH, Embury SH. Alpha-thalassemia in blacks: genetic and clinical aspects and interactions with the sickle hemoglobin gene. Blood. 1986;68:985–990. Higgs DR, Clegg JB, Weatherall DJ, Serjeant BE, Serjeant GR. Interaction of the ␣␣␣ globin gene haplotype and sickle haemoglobin. Br J Haematol. 1984;57:671–678. Serjeant BE, Mason KP, Kenny MW, et al. Effect of alpha thalassaemia on the rheology of homozygous sickle cell disease. Br J Haematol. 1983;55:479–486. Embury SH, Backer K, Glader BE. Monovalent cation changes in sickle erythrocytes: a direct reflection of ␣-globin gene number. J Lab Clin Med. 1985;106:75–79. Stevens MC, Maude GH, Beckford M, et al. Alpha thalassemia and the hematology of homozygous sickle cell disease in childhood. Blood. 1986;67:411–414. De Ceulaer K, Higgs DR, Weatherall DJ, Hayes RJ, Serjeant BE, Serjeant GR. alpha-Thalassemia reduces the hemolytic rate in homozygous sickle-cell diseas. N Engl J Med. 1983;309:189– 190. Steinberg MH, Hsu H, Nagel RL, et al. Gender and haplotype effects upon hematological manifestations of adult sickle cell anemia. Am J Hematol. 1995;48(3):175– 181. Steinberg MH, Lu Z-H, Barton FB, et al. Fetal hemoglobin in sickle cell anemia: Determinants of response to hydroxyurea. Blood. 1997;89:1078–1088. Adekile AD, Haider MZ. Morbidity, ␤s haplotype and ␣-globin gene patterns among sickle cell anemia patients in Kuwait. Acta Haematol. 1996;96:150–154. Nolan VG, Wyszynski DF, Farrer LA, Steinberg MH. Hemolysis-associated priapism in sickle cell disease. Blood. 2005;106(9):3264–3267. Kato GJ, McGowan V, Machado RF, et al. Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide

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62.

63.

64.

65.

66.

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585 77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

Powars DR, Chan L, Schroeder WA. Beta S-gene-cluster haplotypes in sickle cell anemia: clinical implications. Am J Pediatr Hematol Oncol. 1990;12(3):367–374. Thomas PW, Higgs DR, Serjeant GR. Benign clinical course in homozygous sickle cell disease: a search for predictors. J Clin Epidemiol. 1997;50:121–126. Lawrence C, Hirsch RE, Fataliev NA, Patel S, Fabry ME, Nagel RL. Molecular interactions between Hb ␣-G Philadelphia, HbC, and HbS: phenotypic implications for SC ␣-G Philadelphia disease. Blood. 1997;90(7):2819–2825. Gu L-H, Wilson JB, Molchanova TP, McKie KM, McKie VC, Huisman THJ. Three sickle cell anemia patients each with a different ␣ chain variant. Diagnostic complications. Hemoglobin. 1993;17:295–301. Witkowska E, Asakura T, Tang D, et al. Compound heterozygote for Hb S and Hb Matsue-Oki ␣75(EF4)Asp->Asn presents with a mild sickle cell disease phenotype. Blood. 1998;92; 525a. ¨ ¨ Altay C, Oner C, Oner R, et al. Genotype-phenotype analysis in HbS-beta-thalassemia. Hum Hered. 1997;47:161– 164. Divoky V, Baysal E, Schiliro G, Dibenedetto SP, Huisman THJ. A mild type of Hb S-␤+ -thalassemia [-92(C-->T)] in a Sicilian family. Am J Hematol. 1993;42:225–226. Gonzalez-Redondo JM, Stoming TA, Lanclos KD, et al. Clinical and genetic heterogeneity in black patients with homozygous ␤-thalassemia from the southeastern United States. Blood. 1988;72:1007–1014. Gonzalez-Redondo JM, Kutlar F, Stoming TA, De Pablos JM, Kilinc Y, Huisman THJ. Hb S(C)␤+ -thalassaemia: different mutations are associated with different levels of normal Hb A. Br J Haematol. 1988;70:85–89. Gonzalez-Redondo JM, Kutlar A, Kutlar F, et al. Molecular characterization of Hb S(C) ␤-thalassemia in American blacks. Am J Hematol. 1991;38:9–14. Huisman TH. Combinations of ␤ chain abnormal hemoglobins with each other or with ␤-thalassemia determinants with known mutations: influence on phenotype. Clin Chem. 1997;43(10):1850–1856. Padanilam BJ, Felice AE, Huisman THJ. Partial deletion of the 5
␤-globin gene region causes ␤0 -thalassemia in members of an American black family. Blood. 1984;64:941– 944. Waye JS, Chui DHK, Eng B, et al. Hb S/␤0 -thalassemia due to the ∼1.4-kb deletion is associated with a relatively mild phenotype. Am J Hematol. 1991;38:108–112. Patterson M, Walker L, Eng B, Waye JS. High Hb A2 ␤-thalassemia due to a 468 bp deletion in a patient with Hb S/␤-thalassemia. Hemoglobin. 2005;29(4):293– 295. Lacan P, Ponceau B, Aubry M, Francina A. Mild Hb S␤+ -thalassemia with a deletion of five nucleotides at the polyadenylation site of the ␤-globin gene. Hemoglobin. 2003;27(4):257–259. Andersson BA, Wering ME, Luo HY, et al. Sickle cell disease due to compound heterozygosity for Hb S and a novel 7.7-kb ␤-globin gene deletion. Eur J Haematol 2007;78(1):82– 85. Steinberg MH, Rosenstock W, Coleman MB, et al. Effects of thalassemia and microcytosis upon the hematological

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Martin H. Steinberg and vaso-occlusive severity of sickle cell anemia. Blood. 1984;63:1353–1360. Serjeant GR, Asfcroft MT, Serjeant BE, Milner PF. The clinical features of sickle-cell ␤ thalassaemia in Jamaica. Br J Haematol. 1973;24:1930–30. Luo HY, Heeney M, Wang WC, et al. Hemoglobinopathies mimicking Hb S/beta-thalassemia: Hb S/S with ␣-

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thalassemia and Hb S/Volga. Am J Hematol. 2006;81(5):361– 365. Charache S, Zinkham WH, Dickerman JD, Brimhall B, Dover GJ. Hemoglobin SC, SS/GPhiladelphia and SOArab disease: diagnostic importance of an integrative analysis of clinical, hematologic and electrophoretic findings. Am J Med. 1977;62:439–446.

SECTION SIX

OTHER CLINICALLY IMPORTANT DISORDERS OF HEMOGLOBIN Martin H. Steinberg

Three chapters discuss rare inherited hemoglobinopathies including unstable hemoglobins, hemoglobins with altered oxygen affinity, hemoglobins easily oxidized, and a miscellaneous group of hemoglobin variants with interesting biological properties, some of which are clinically important. Acquired disorders of hemoglobin can arise from heme iron oxidation due to inherited abnormalities of hemoglobinreducing enzymes or because of exposure to exogenous oxidizing agents. Rare hemoglobinopathies have taught us much about the structure-stability-function relationships of hemoglobin. Hemoglobin mutants have provided the most comprehensive list of mutations of any system in human biology, creating a map for understanding mutation in other genetic loci. Globin gene mutations – these include nearly every class of mutation so far described – provided an early catalog of the possible mechanisms of genetic disease.

An accounting of globin gene mutations in early 2008 listed 1,326 unique mutations (http://globin.cse.psu.edu/). Here, we discuss some rare hemoglobin mutations. As comparatively few globin residues are critical for maintaining the structural integrity and functional utility of the molecule, most hemoglobin mutations are not associated with hematological or clinical abnormalities and so escape detection. Some mutations, although not medically important, illustrate interesting biological and anthropological principles. Abnormal hemoglobins with high or low oxygen affinity, variants that have their heme iron oxidized to the ferric form causing methemoglobinemia (HbM), or hemoglobin variants that are unstable are abnormalities seen rarely by the general physician and infrequently encountered in the practice of hematology. High oxygen affinity hemoglobin variants cause erythrocytosis; low oxygen affinity variants can be accompanied by cyanosis and anemia; HbM variants present with cyanosis. Patients with unstable hemoglobins can have hemolytic anemia that might worsen during infection and when certain drugs are given. Because these uncommon variants are unusual causes of erythrocytosis, cyanosis, and hemolytic anemia, their presence is often unsuspected. When present, their identification can prevent hazardous diagnostic tests, forestall potentially dangerous treatments, permit reassurance of the patients, and allow family counseling. In evaluating these disorders, tests for hemoglobin instability are useful and measurement of the hemoglobin– oxygen dissociation curve is vital when a hemoglobin variant is suspected of causing erythrocytosis or cyanosis. Methemoglobins are suspected by observing an abnormal brown coloration of the blood and spectrophotometric recording of the visible spectrum of the hemolysate. Mutation detection by DNA analysis is the only way of definitively diagnosing rare hemoglobin variants.

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24 Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants of Clinical and Biological Interest

variant hemoglobins are polymorphic because they are genetically neutral and their carriage does not provide a selective advantage. Sometimes, in population isolates, there is a founder effect leading to a higher than expected prevalence of a particular mutation. The geographical distribution of hemoglobin variants in the Americas, Europe, Africa, the former USSR, the Middle East, India, southeast Asia, China, Japan, the Antipodes, and Oceania have been cataloged.2 It is unlikely that new globin mutations that reach polymorphic frequencies will be found. In unexamined population isolates, some new variants present at high frequency might yet be uncovered and sporadic examples of new and already described variants will continue to be unearthed.

Martin H. Steinberg and Ronald L. Nagel Mutation Distribution

INTRODUCTION Mutations of hemoglobin can be polymorphic (>1% of a population), like HbS, HbE, HbC, and the thalassemias, or rare. Our first edition listed 750 unique hemoglobin variants; this number is now more than 1,000.1 In this chapter we address rare mutations. Some are associated with clinical disease; others are interesting solely for the biological principles they illustrate. A current listing of variant human hemoglobins is maintained in the HbVar database at http://globin.cse.psu.edu/, and the journal Hemoglobin (Taylor & Francis, Philadelphia) is a rich source for reports of new variants. Both are invaluable resources for clinicians and investigators with interests in unusual hemoglobin disorders. Globin gene mutations, which include nearly every class of mutation so far described, except trinucleotide repeats and other nucleotide expansions associated with neuromuscular disorders, provided an early catalog of the mutations that can cause genetic disease. Clinically important but rare mutants affect hemoglobin stability causing premature red cell destruction; interfere with normal oxygen binding kinetics producing erythrocytosis; and permit heme iron oxidation, causing cyanosis. Most rare variants have no phenotype and are of biological and diagnostic interest only. Comparatively few globin residues are critical for maintaining the structural integrity and functional performance of the molecule (Chapter 6). Hemoglobin gene mutations are, as a rule, not associated with hematological or clinical abnormalities and escape detection, especially when they are chromatographically silent. Large-scale population screening programs have defined the worldwide prevalence of medically important hemoglobinopathies and thalassemias. Most of the hemoglobin variants known today were discovered as a byproduct of this extensive effort. Unlike some thalassemia mutations and HbS, HbE, and HbC, almost none of these

More than 90% of hemoglobinopathies result from point mutations. Some globin gene codons are associated with as many as seven different mutations; others have one or no described mutations, suggesting both mutational “hot spots” and sites resistant to mutation. Mutations have been described in approximately 75% and 95% of ␣- and ␤-globin gene codons. Multiple different mutations are described in amino acid residues that are functionally important so that a clinical phenotype brings the substitution to clinical attention. For example, seven mutations have been described at ␤99, all associated with high oxygen affinity variants and erythrocytosis. Four of the six variants of ␣141 for which information is available have increased oxygen affinity. Six mutations at ␤92 are all associated with unstable hemoglobins and hemolytic anemia. In contrast, the five mutations described at the ␤22 codon are all stable and functionally normal. It is not known if these sites are particularly prone to mutational events or if we are witnessing a bias of ascertainment in which some these mutations are identified because of their obvious phenotype. Only 20% of the possible globin gene mutations have been described. Clinically silent variants are still likely to escape notice, and many variants that cause a change in the charge of the molecule, simplifying their detection, have been already delineated. Neutral substitutions in which the charge of the molecule is unchanged and a phenotype is not present are rarely detected. Among hemoglobin mutants, ␤-globin gene variants are most frequently described whereas ␣-, ␥ -, and ␦-globin gene variants are reported 65%, 20%, and 10% as often, respectively. This preponderance of ␤-globin gene variants might reflect the fact that with only two ␤-globin genes, the intracellular concentration of ␤-globin variants exceeds that of ␣-globin variants so they are more likely to be clinically apparent (Chapter 13). For example, a high oxygen affinity variant present at 40%–50% total hemoglobin, the level of most ␤-globin gene mutants, is more likely to cause clinically apparent erythrocytosis than a similar ␣-globin 589

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␣1␤2 interface contacts (sliding contact) connecting ␣1␤1 and ␣2␤2 dimers ␣1␤1, ␣2␤2 interface Mutations that reduce 2,3-BPG binding Heme pocket mutations Miscellaneous

gene variant present at the usual level of 20%–25%. Some ␣globin variants might go unnoticed because of their low levels in the blood. Certain ␣-globin mutations are more likely to be lethal because ␣ chains are also essential for the functional hemoglobins of the embryo and fetus (Chapter 7). ␣-Globin chains must also bind three types of non-␣globin chains, ␤-, ␥ -, and ␦-globin chains to form tetramers, increasing their chance of being functionally incompetent if mutated. Very low concentrations of HbA2 and HbF in adults often preclude the detection of an abnormal variant of the ␦ and ␥ -globin genes (Chapter 7). Only rarely do ␥ -globin chain abnormalities have a clinical phenotype in neonates and this usually is due to methemoglobinemia and cyanosis. Structural variants of the embryonic ε- and ␨ -globin chains have not been described.

HEMOGLOBINS WITH ALTERED OXYGEN AFFINITY High Oxygen Affinity Hemoglobins Globin gene mutations can increase the affinity of the hemoglobin molecule for oxygen and cause erythrocytosis transmitted as a dominant trait. Approximately 100 high oxygen affinity variants have been reported.3 Although familial erythrocytosis is a valuable clue to the presence of a high oxygen affinity hemoglobin variant, new mutations cause isolated cases. These ␣- or ␤-globin chain mutations are expressed clinically in the heterozygote; they might be lethal in homozygotes if they affect the ␣-globin chain as this will affect embryonic and fetal hemoglobins. Several homozygotes for ␤-globin high oxygen affinity variants have been described and these individuals might have more severe disease.4–7 High oxygen affinity hemoglobins have also been described with ␤0 thalassemia, and as HbA is not present, they mimic homozygosity for the variant. Pathophysiology. Stabilization of the relaxed (R) state of the hemoglobin tetramer (high oxygen affinity) or destabilization of the tense (T) state (low oxygen affinity) is caused by mutations in critical areas of the globin chain affecting the R-T transition (Table 24.1) (see Chapter 6). The increased avidity for oxygen (low P50 ) of these variants reduces oxygen delivery to tissues stimulating

erythropoietin production and increasing red cell mass. Patients with high oxygen affinity hemoglobins and the erythrocytosis that results from relative tissue hypoxia had normal urine erythropoietin levels but showed increases in erythropoietin when phlebotomized to a normal red cell mass. They might be reasonably compensated for the low P50 by the increased red cell mass and probable increases in tissue blood flow, along with changes in perfusion patterns in selected regions of the body that negate any adverse effect of increased blood viscosity. Oxygen consumption and arterial pO2 are normal but occasionally there is reduced mixed venous pO2 and decreased resting cardiac output. Diagnosis. The diagnosis of high oxygen affinity hemoglobins is suggested by the following: isolated erythrocytosis without accompanying leukocytosis, thrombocytosis and splenomegaly that is typical in polycythemia vera; a family history of erythrocytosis; reduced P50 (partial pressure of oxygen where hemoglobin is half-saturated Fig. 24.1). DNA sequencing defines the mutation. Not unexpectedly, the JAK2 V617F mutation found in most individuals with polycythemia vera is absent in patients with high oxygen affinity hemoglobin variants.8 Determination of the red cell oxygen equilibrium curve (Fig. 24.1) is the benchmark for the diagnosis of erythrocytosis due to high oxygen affinity hemoglobins. The oxygenbinding characteristics of hybrid tetramers (␣2 ␤A ␤var ) are likely to be intermediate between purified HbA and purified variant and the shape of the hemoglobin oxygen dissociation curve can at times be biphasic. Measurement of blood P50 confirms the shift in the hemoglobin–oxygen dissociation curve. Rarely, the whole blood P50 is normal, requiring study of dialyzed purified hemoglobin. An accurate P50 100 (Haldane effect: O2 displaces CO2 from Hb)

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Oxyhemoglobin (% Saturation)

Table 24.1. Sites of globin mutation that are associated with increased oxygen affinity. In addition to single amino acid substitutions, these mutations can include small deletions and insertions of amino acids, reading frameshifts, fusion globins, and elongated globin chains

Martin H. Steinberg and Ronald L. Nagel

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pO2 (mm HG) Figure 24.1. Hemoglobin–oxygen dissociation curve. As the P50 drops (the curve is left-shifted) the affinity of hemoglobin for oxygen increases. An increased P50 decreases hemoglobin–oxygen affinity.

Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants value cannot be “calculated” from pO2 data and directly measuring the saturation of hemoglobin and pO2 is necessary. P50 measurements are not widely available but can be done with several instruments. Both erythrocytes and purified dialyzed hemoglobin will have high oxygen affinity and 2,3-BPG concentrations should be normal, suggesting that altered oxygen affinity is not caused by reduced levels of this heterotopic modulator of hemoglobin function (Chapter 6). When 2,3-BPG mutase deficiency is present, red cell levels of 2,3-BPG will be low and whereas erythrocytes or whole blood will have high oxygen affinity red cells, the oxygen affinity of the purified hemolysate stripped of this intermediate will be normal. In addition to high oxygen affinity hemoglobins, disorders expressed in the erythrocyte that cause isolated erythrocytosis include, 2,3-BPG mutase mutations that reduce the synthesis of this modulator of hemoglobin– oxygen affinity, some instances of methemoglobinemia and chronic carbon monoxide poisoning (Chapter 25). The search for the mutation is initiated by highperformance liquid chromatography (HPLC) analysis of hemoglobin but normal studies do not exclude the diagnosis of a high-affinity hemoglobin that can migrate with HbA. As with all evaluations for abnormal hemoglobins, determining the DNA sequence of the globin genes provides the definitive information. Some examples of high oxygen affinity hemoglobins, illustrating the varying mechanisms and heterogeneous clinical findings seen with these variants, are discussed later. A detailed understanding of the structure of hemoglobin and the importance of each residue in determining function allows a molecular explanation of most of the clinical abnormalities observed. α-Globin Chain Variants. Because of a gene dosage effect, most stable ␣-globin variants form approximately 25% of the total hemoglobin. As a result, the clinical effects of ␣-globin variants are less striking than those of similar ␤-globin variants that usually comprise 50% of total hemoglobin, although coincident thalassemia can modulate the concentration of variant hemoglobins. The first report of a high oxygen affinity hemoglobin was that of a patient aged 81 years with erythrocytosis, an abnormal hemoglobin detected by hemoglobin electrophoresis, and erythrocytes with increased oxygen affinity (Hb Chesapeake; HBA arg92leu). Fifteen members of the proband’s family were similarly affected.9 Hb Chesapeake represented approximately 20% of the total hemoglobin. With a P50 of 19 mm Hg (normal ∼26 mm Hg), Hb Chesapeake produced moderate erythrocytosis. The mutation affected an invariant residue that stabilized the R-state at the ␣1 ␤2 area of contact, making the T conformer less favored. Hb Nunobiki (HBA1 arg141cys) is one of four mutations of this invariant residue, all of which exhibit high oxygen affinity and moderate to mild erythrocytosis. This group of mutations represents an interesting cluster of variants

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that illustrate the effects of different mutations at the same amino acid residue. As a mutant of the 3
HBA1 gene that is expressed to a lesser extent than 5
HBA2 gene, Hb Nunobiki makes up approximately 13% of the hemolysate and is accompanied by only mild erythrocytosis. High oxygen affinity is due to the breaking of the C terminal–C terminal salt bridge indispensable for the stabilization of the T-state, favoring the R-state. β-Globin Chain Variants. All possible single base mutations of the ␤99 site disturbing the ␣1␤2 area of contact have been described and include Hb Kempsey (HBB asp99asn), Hb Yakima (asp99his), Hb Radcliffe (asp99ala), Hb Ypsilanti (asp99tyr), Hb Hotel-Dieu (asp99gly), Hb Chemilly (asp99val), and Hb Coimbra (asp99glu). As expected for stable ␤-globin chain variants, all are present at 40%–50% of the hemolysate, exhibit moderately high oxygen affinity, and are characterized clinically by erythrocytosis. Other properties of this group of mutants include: moderately decreased response to 2,3-BPG with Hbs Kempsey and Radcliffe and slightly decreased response to 2,3-BPG in Hb Hotel Dieu. Hbs Ypsilanti and Radcliffe form stable hybrid tetramers in the hemolysates in which the abnormal ␤ chains coexist with normal ␤ chains. Six of the possible seven mutations of the C-terminal CAC (tyr) codon have also been described. One of them, Hb Cochin-Port Royal (tyr146arg), has nearly normal oxygen affinity but decreased 2,3 BPG interaction and Bohr effect. Three mutations of ␤82 lys have been described: Hb Rahere (lys82thr), Hb Helsinki (lys82met), and Hb Providence (lys82asn). All have moderately high oxygen affinity and moderate erythrocytosis. These mutants have drastically reduced 2,3-BPG binding due to the elimination of one of the normal binding sites for this allosteric effector. Hb Porto Alegre (HBB ser9cys) has high oxygen affinity and a tendency to aggregate (see later) but erythrocytosis is not present. Oligomerization of this mutant diminishes heme–heme interaction and increases the oxygen affinity. Hb Tak (HBB 147[+AC], modified C-terminal sequence 147thr-lys-leu-ala-phe-leu-leu-ser-asn-phe-157tyr-COOH) is elongated by 11 amino acid residues. It forms 40% of the hemolysate and has a very high oxygen affinity with no cooperativity and no allosteric interaction with pH or 2,3-BPG. The C-terminal of the ␤-globin chain is actively involved in the conformational changes of the hemoglobin molecule by stabilizing the T-state. By having these stabilizing interactions disrupted, Hb Tak is totally frozen in the R-state. It is also slightly unstable. Despite these severe functional abnormalities, the heterozygous patient did not have erythrocytosis. The extreme biphasic nature of the hemoglobin–oxygen affinity curve observed in mixtures of Hb Tak and HbA suggested that hybrid tetramer (␣2 ␤A ␤Tak ) formation is absent. The top portion of the oxygen equilibrium curve is normal and begins to be abnormal only below 40% saturation. Because physiological oxygen exchange occurs most commonly above that level of saturation, the

592 tissues might not be hypoxic, removing the stimulus for increased erythropoiesis. Clinical Aspects. Patients with high-affinity hemoglobins and erythrocytosis have a benign clinical course and rarely have complications, apart from a ruddy complexion. Splenomegaly is typically absent. Hemoglobin concentration and packed cell volume are increased variably, and usually only moderately, suggesting that modulation by variations in other genes might affect the physiological response to hypoxia. Some patients with Hb Malmo (HBB his97gln) have been reported to be symptomatic and to benefit from phlebotomy and the transfusion of normal blood, but this is an exception. Many cases of high oxygen affinity hemoglobins are diagnosed during a routine hematological examination or when the family of a proband known to have erythrocytosis is examined. In very limited studies, exercise capacity in the laboratory and the indices of working capacity and cardiac tolerance were similar in patients with high oxygen affinity hemoglobins and controls. It has been suspected that carriers’ of these variants could have enhanced athletic performance under some circumstances and this has lead to the unfortunate and sometimes fatal use of erythropoietin or transfusion to enhance performance in competitive athletics. If high-affinity hemoglobins are diagnosed early, unnecessary invasive diagnostic procedures and inappropriate therapeutic interventions such as cardiac catheterization can be avoided. Patients have received phosphorus-32 treatment based on a mistaken diagnosis of polycythemia vera. Increased morbidity or mortality in mothers or their offspring with high oxygen affinity hemoglobins has not been observed, suggesting that the affinity of the mother’s hemoglobin is irrelevant with respect to oxygen delivery to the fetus. Low ambient pO2 , as in unpressurized airplanes and ascent to altitude, does not represent a risk because high-affinity hemoglobins are avid for oxygen. Hypothetically, carriers would be less prone to “the bends” during deep sea diving because of slower oxygen release during ascension. Treatment. Patients with high oxygen affinity hemoglobins have reasonable compensation for their abnormality with adequate tissue oxygen delivery despite increases in blood viscosity. Intervention is therefore rarely required. Exercise studies before and after phlebotomy in patients with Hb Osler (HBB tyr145asn), a variant with a P50 of 10–11 mm Hg and a hemoglobin concentration of approximately 22 g/dL, did not show impairment after phlebotomy.10 Limited studies have suggested that phlebotomy does not improve exercise performance. Although individuals seldom benefit from phlebotomy, unknown factors might interfere with their normal compensation for high hemoglobin oxygen affinity, and increased blood viscosity might become a burden. Prudence

Martin H. Steinberg and Ronald L. Nagel dictates that before embarking on a regimen of chronic phlebotomy, one should be conservative and review the hematological and physiological findings at 6-month intervals during the first few years after diagnosis. In older patients special attention should be directed to the adequacy of blood flow to the heart and central nervous system.

Variants with Low Oxygen Affinity Hemoglobin variants with reduced affinity for oxygen are in many respects the clinical obverse of high oxygen affinity variants. Expressed in the heterozygotes, with homozygosity likely to be embryonic lethal, these variants, which are far less common than variants with high oxygen affinity, are associated with anemia and at times are accompanied by cyanosis. Pathophysiology. Alterations of critical molecular regions directly involved in the R-T transition result in the stabilization of the T-state or destabilization of the R-state. Low oxygen affinity hemoglobins deliver more oxygen to the tissues per gram of hemoglobin, and this is reflected by an oxygen–hemoglobin dissociation curve shifted toward the right of normal, and an increase in P50 (Fig. 24.1). When hemoglobin has a right-shifted or low affinity curve, the difference between oxygen binding in the lungs at pO2 levels of 100 mm Hg and unloading in the tissues at 40 mm Hg can be twice as great as the differences in a hemoglobin with a normal oxygen equilibrium curve. Patients with moderately right-shifted oxygen equilibrium curve (P50 between 35 and 55 mm Hg) could be anemic. With a further right shift (P50 ∼80) anemia is not present. A right-shifted oxygen equilibrium curve leads to an increase in the synthesis of 2,3-BPG and decrease in its destruction. Diagnosis. Approximately half as many low oxygen affinity variants have been described compared with high oxygen affinity variants. The first report of such a hemoglobin was Hb Kansas (HBB asn102thr) in a patient who presented with asymptomatic cyanosis without anemia or hemolysis. Detection of a low oxygen affinity hemoglobin is part of the differential diagnosis of patients with cyanosis. Before undertaking extensive diagnostic procedures in cases of cyanosis that are not clearly due to cardiovascular or pulmonary disease, obtaining hemoglobin HPLC and measuring blood P50 is advisable. A search for low affinity hemoglobins as an explanation for anemia without cyanosis is less compelling but if other investigations prove fruitless, unexplained normocytic anemia without reticulocytosis might be evaluated by measuring P50 . A simple bedside test to distinguish cyanosis due to low oxygen affinity hemoglobins and cardiopulmonary cyanosis from that of methemoglobinemia, M hemoglobins, and sulfhemoglobinemia (Chapter 25) is to expose blood to pure oxygen. Blood from carriers of low oxygen

Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants affinity hemoglobins or patients with cardiopulmonary disease will turn from purple-greenish to the bright red. Blood of patients with methemoglobinemia, sulfhemoglobinemia, and M hemoglobins will remain abnormally colored. Clinically apparent cyanosis is only observed in carriers of low oxygen affinity variants with greatly rightshifted curves and in whom the variant comprises a substantial portion of the hemolysate. Cyanosis is present from birth in some low oxygen affinity hemoglobins due to ␣-globin chain mutants. In carriers of ␤-globin chain mutants, cyanosis can appear from the middle to the end of the first year of life as ␥ -globin gene expression and HbF synthesis wanes and is replaced by ␤-globin gene expression and HbA. Globin gene sequencing is the sole means of definitive diagnosis Clinical Features. Three low oxygen affinity variants have been described at ␤102. Hb Kansas, the best-studied variant, has a whole blood P50 of approximately 70 mm Hg, decreased cooperativity, and a normal Bohr effect. The ␤102 asn residue is invariant among ␤-globin chains and participates in the only hydrogen bond between asn102 and asp 94 across the ␣1␤2 interface in oxyhemoglobin. This bond is broken when the molecule assumes the Tstate. The new thr residue is incapable of forming this bond and low oxygen affinity results from destabilization of the R conformer. The changes induced by this substitution at the ␣1␤2 interface allow Hb Kansas to dissociate into ␣␤ dimers, the near opposite of the high oxygen affinity Hb Chesapeake. Hb Beth Israel (HBB asn102ser) was found in a patient with cyanosis of the fingers, lips, and nail beds. The P50 was 88 mm Hg and arterial blood was only 63% saturated despite a normal pO2 . The hemolysate also had a low oxygen affinity and a normal Bohr effect. Erythrocyte 2,3-BPG was mildly elevated. The molecular mechanism of reduced oxygen affinity is the same as for Hb Kansas, although the defect might be more disruptive locally as the serine side chain is shorter than that of threonine. Hb Bologna (HBB lys61met) was informative as it was present as a compound heterozygote with ␤0 thalassemia and comprised 90% of the hemolysate. During development, the effects of this mutation were unlikely to be manifest because of high HbF concentrations and adults were neither cyanotic nor anemic despite Hb Bologna forming nearly 50% of the hemolysate and having a P50 of 37.6 mm Hg. Hb Bruxelles (phe42del) is a deletion of the most conserved amino acid residue of hemoglobin. Phenylalanine residues at ␤41 and ␤42 are conserved in all normal mammalian non-␣-globin chains and are indispensable for the structural integrity and oxygen-binding functions of the molecule. From age 4 years, the index case of Hb Bruxelles had severe hemolytic anemia and cyanosis, requiring blood transfusion once. Later in life, her hemoglobin con-

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centration stabilized at 10 g/dL. Reasons for this “switch” of phenotype are unknown. Other mutations of ␤41 and ␤42, which are predominately unstable hemoglobins, are discussed later. In individuals with low oxygen affinity variants, the oxygen saturation measured using pulse oximetry can be spuriously reduced.6,11 Attention should be paid to measurement of blood oxygen saturation when any hemoglobin variant is present. Blood oxygen saturation can be measured using an arterial blood gas analyzer, by pulse oximetry, and by using a CO oximeter. The first method measures blood partial pressure of dissolved oxygen and provides the paO2 and saO2 . The convenient pulse oximeter provides a transcutaneous measure of absorbance at two wavelengths (660 and 940 nm) but is inaccurate when dyshemoglobins, such as methemoglobin (hemoglobin with oxidized [Fe+3 ] heme iron), carboxyhemoglobin, and sulfhemoglobin are present. CO oximetry, the most accurate of all the methods, can be inaccurate in cases of M hemoglobins.12 Oxygen saturation calculated from pH and pO2 should be interpreted with caution as the algorithms used assume normal hemoglobin oxygen affinity, normal 2,3-BPG concentrations, and no dyshemoglobin such as methemoglobin or hemoglobinopathies. CO oximeter reports should include the dyshemoglobin fractions besides the oxyhemoglobin fraction. In cases of an increased methemoglobin fraction, pulse oximeter values trend toward 85%, underestimating the actual oxygen saturation. Hemoglobin M variants can have normal methemoglobin levels and increased carboxyhemoglobin or sulfhemoglobin fractions measured by CO oximetry. Treatment. Treatment for these variants is not needed. The importance of the early diagnosis is to avoid unnecessary work-up and to alleviate concern for the patient and family.

UNSTABLE HEMOGLOBINS Globin chain mutations can cause hemoglobin tetramer instability and intracellular precipitation of its globin subunits. These mutants, sometimes called collectively, congenital Heinz body hemolytic anemia, cause intraerythrocytic precipitates that are detectable by supravital staining, and that appear as globular aggregates called Heinz bodies (Fig. 24.2). The inclusions reduce variably the life of the erythrocyte by binding to the membrane, decreasing cell deformability, and increasing membrane permeability. Erythrocyte enzyme deficiencies can also cause Heinz bodies and hemolysis. More than 135 unstable variants of both the ␤- and ␣-globin chains with widely varying clinical severity have been reported (Fig. 24.3), usually as heterozygotes, although some homozygous cases have been reported. Anemia, reticulocytosis, pigmenturia, and splenomegaly are the major clinical features.13

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Martin H. Steinberg and Ronald L. Nagel Table 24.2. Sites of globin mutation that are associated with unstable hemoglobins. In addition to single amino acid substitutions, these mutations can include small deletions and insertions of amino acids, reading frameshifts, fusion globins, and elongated globin chains Weakening or modification heme–globin interactions Interference with the secondary structure of a globin subunit Interference with the tertiary structure of the subunit Altered subunit interactions interfering with the quaternary structure

in its first two positions. ␣-Helices comprise approximately 70% of the globin subunit and must be folded into a globin motif. Introduction of water into the molecule destroys its stability and this can be caused by substitution of a charged residue, for example, leucine, for a nonpolar residue, like Pathophysiology arginine. Mutations that change the primary structure of globin, Loss of intersubunit contact hydrogen bonds or salt depending on the substitution and its location, can alter bridges in the ␣1␤1 contact area will also reduce stabilthe secondary (␣-helical), tertiary (folding of the globin ity. Dissociation of ␣1␤1 dimers into monomers is norchain) or quaternary (interactions within the hemoglobin mally minimal as it generates methemoglobin and contetramer) structure of hemoglobin by mechanisms shown sequent instability. Dissociation of chains along the ␣1␤1 in Table 24.2. contact generates ␣- and ␤-globin chains that uncoil, loosHeme–globin interactions are vital for oxygen delivery ening their heme–globin interaction and favoring methebut also contribute to molecular stability. For example, moglobin formation. Mutations affecting the ␣1␤1 interintroduction of a charged amino acid residue into the heme face tend to be more unstable than those affecting the ␣1␤2 pocket, a site normally formed by residues with nonpocontact. lar side chains, deletions involving residues that directly Heme loss is inhibited by maintaining heme iron in interact with the heme, mutations other than to a tyrosine the reduced ferrous (Fe2+ ) state by the action of metheresidue of the (F8) proximal histidine or (E7) distal histidine, moglobin reductases and detoxification of oxygen radicals. all cause molecular instability. Therefore, dimerization and the dispersion and precipiDisruption of the secondary structure reduces subunit tation of free heme is minimalized. Hemoglobin dimers solubility and is often a result of the introduction of proline autoxidize and lose heme more readily than tetramers. residue that cannot be accommodated into ␣-helix except Generation of methemoglobin increases the thermoinstability of hemoglobin, suggesting that the His(F8) St Etienne pathways and events accompanying the conVal(FG5) Koln Redondo Djelfa version of ferrous to ferric heme are imporMozhaisk tant for hemoglobin stability. Leu(F4) Leu(F7) Heinz bodies are the product of hemoBoras Sabine Santa Ana globin denaturation. First suggested to be heme-depleted globin chains, these incluCD corner sions were subsequently identified as hemiHelix F Phe(CD1) chromes, derivatives of ferric hemoglobin Hammersmith that have the sixth coordination position Bucuresti occupied by a ligand provided by the globin. HEME Hemichromes are generated when the heme His(E7) Zurich is dissociated from the heme pocket and Bicetre rebinds elsewhere in the globin after the ␣ Val(E4) or the ␤ chains have denatured. Irreversible Cagliari hemichromes are a stage in the formation Phe(E15) Helix E Val(E11) of Heinz bodies. Membranes prepared from Christchurch the red cells of patients with Hb K¨oln (HBB Bristol Lys(E10) Sidney val98met) who have had a splenectomy conToulouse tain aggregates composed of disulfide-linked Figure 24.3. Mutation sites of some unstable hemoglobins. Mutations shown are near the heme spectrin, band 3 (Chapter 9), globin and moiety. Figure 24.2. Heinz bodies. Heinz bodies are the large, single basophilic inclusions inside erythrocytes. Other red cell inclusions represent reticulocytes. (See color plate 24.2.)

Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants

595

Table 24.3. Some hemoglobin variants with elongated globin chains Variant

Mutation

Mechanism

Phenotype

NH2 -Terminal Extension South Florida51

HBB val1met

Doha52

HBB val1glu

Acetylated at the NH2 terminus and appears as HbA1 C 21% in heterozygote, acetylated

Long Island

HBB his2pro

Thionville53

HBA1 val1glu

Initiator met retained, chain extended by 1 residue Initiator met retained, chain extended by 1 residue Initiator met retained, chain extended by 1 residue Initiator met retained, chain extended by 1 residue

Insertions Catonsville54 Za¨ıre Grady Koriyama55

Insertion between ␣37 and ␣38 Insertion between ␣116 and 117 Insertion between ␣118 and 119 Insertion between ␤95 and 96

glu inserted 5 residues inserted 3 residues inserted 5 residues inserted by possible out of frame base pairing. In Hb Gun Hill the same 15-bp segment is deleted

Increased O2 affinity, unstable Normal Normal Severe instability and anemia. Resembles a thalassemic hemoglobinopathy

See Chapter 13

Termination codon mutations

␣ Thalassemia

␤147 (+AC) ␣139 (−A)

2-bp insertion and frameshift 1-bp deletion and frameshift

∼Normal See Table 24.5

COOH-Terminal Extensions ␣-Globin gene termination mutants Tak Wayne

high-molecular-weight complexes composed in part of denatured spectrin. Hemichrome can bind band 3 of the erythrocyte membrane. Decreased deformability of the erythrocyte leads to preferential trapping in the spleen where Heinz bodies are removed. The coincident loss of small amounts of membrane gradually converts discoid cells into spherocytes that are eventually removed from the circulation. Membrane damage might also result from lipid peroxidation and protein cross-linking due to free radical formation that is a result of Fenton chemistry. When the mutation is such that heme dissociates from the abnormal globin chain, as in the example of Hb K¨oln, the partially heme-deficient molecule is susceptible to reversible and irreversible hemichrome formation with subsequent denaturation. Precipitates tend to be pale, and the pattern found during hemoglobin electrophoresis was characterized by multiple bands unless stabilized by the addition of hemin. Dipyrroluria was present, suggesting that free heme was converted to dipyrroles rather than bilirubin. By different mechanisms, hemoglobin chains might be extended beyond their expected length. Small insertions and deletions are caused by slipped mispairing at the replication fork of DNA at the site of direct repeats or inverted repeats. Nonsense to missense mutations at the termination codon, reading frameshifts, failure to cleave the initiator methionine residue from the amino terminus of

No clinical phenotype but appears as HbA1 C 50% in heterozygote, acetylated

globin, amino acid insertions, and combined deletions and insertions have been reported. Although many resulting variants are not associated with hematological abnormalities, some are accompanied by hemolytic anemia that can be severe. Not unexpectedly, distortion of tertiary structure because of chain elongation can provoke different degrees of globin instability. Table 24.3 highlights other variants with extended globin chains. Almost without exception, the deletion of one or more amino acid residues results in an unstable globin. When one or two residues are missing, the clinical phenotype is usually mild with slight anemia. When more than one or two amino acids are deleted the phenotype is apt to be more severe. Hb Bruxelles (HBB phe41 or 42del) is accompanied by severe Heinz body hemolytic anemia. Alpha hemoglobin-stabilizing protein (AHSP) binds free ␣-globin chains protecting them from precipitation (Chapter 4). In vitro studies suggest that the impaired interaction of AHSP with ␣-globin variants where the mutation lies in the molecular sites where ASHP binds ␣ globin might affect the stability of the variant.14,15 In these studies, recombinant Hb Groen Hart (HBA pro119ser), Hb Diamant (HBA pro119leu), and ␣-globin termination mutants had impaired interactions with AHSP. These observations suggest an additional mechanism for unstable ␣-globin variants. Hyperunstable hemoglobins are an uncommon class of variants where the mutation, usually in the third exon of

596 the globin gene, leads often to a truncated globin that is barely detectable or undetectable. These hemoglobins, creating the phenotype of dominant inherited thalassemia, are discussed in Chapter 16. Chronic hemolysis has been associated with dysregulated nitric oxide (NO) biochemistry and reduced NO bioavailability. This subject is discussed in Chapter 11.

Diagnosis Multiple tests are used to diagnose unstable hemoglobins. Patients with unstable hemoglobins might have characteristically dark urine or pigmenturia. This is due to the presence of dipyrroles that are also present in Heinz bodies. The absence of pigmenturia does not exclude the diagnosis of unstable hemoglobin and the severity of the hemolysis is unrelated to pigmenturia. For example, carriers of Hb ¨ K¨oln and Hb Zurich (HBB his63arg) can have pigmenturia but hemolysis can be severe with Hb K¨oln and is usually ¨ very mild with Hb Zurich. The P50 of unstable hemoglobins is quite variable and can be normal, low, or high. This is a result of different mutations variously affecting heme– globin interaction, and the tertiary and quaternary structures of the molecule. Blood Smear and Heinz Body Preparation. Abnormalities of the blood smear are often present but are nonspecific. They might include anisocytosis, basophilic stippling, Howell–Jolly bodies, nucleated red blood cells and microspherocytes. Fragmented cells can appear to have had “a bite” taken from them and are thought to result from the phagocytosis of Heinz bodies during passage of the cell through the spleen. The mean corpuscular hemoglobin concentration can be as low as 25 g/dL because of heme loss or Heinz body formation. Some reported values for reticulocytes might be factitiously high as inclusion bodies are mistaken for reticulocytes. Heinz bodies in circulating red cells are usually found only after splenectomy or during an acute hemolytic episode (Fig. 24.2). Under such circumstances, more than 50% of the cells typically contain one large, spherical inclusion. Heinz body detection requires the incubation of erythrocytes with a supravital stain such as new methylene blue or crystal violet. They appear as single or multiple inclusions of 2 ␮ in diameter or less and often appear membrane attached. Heinz bodies can be found in fresh blood, but usually, incubation for 24 hours without glucose is required for their formation. A normal control should always be run simultaneously. Hemoglobin Stability Tests. The isopropanol test, a good screening test for unstable hemoglobins, can give false positive results when the sample contains more than 5% HbF. In the heat denaturation test, a hemolysate is incubated for 1 or 2 hours at 50◦ C. Hemoglobin instability is suggested by a visible precipitate. Although simple, the results of this test vary because of different concentrations of the

Martin H. Steinberg and Ronald L. Nagel abnormal variant and different temperatures need for their denaturation. A control with a normal, stable hemolysate is vital for correct interpretation. Detection of the Variant Hemoglobin and Mutation Analysis. If clinical and hematological studies suggest an unstable variant, the determination of the molecular defect becomes the final step in diagnosis. Approximately a quarter of unstable hemoglobins are not detectable by commonly used methods of hemoglobin separation, and an unstable variant might be reflected only by a diffuse peak. DNA analysis is the best approach to defining the globin mutation. New mutations are common, thus a family history need not be present.

Clinical Aspects The presence of an unstable hemoglobin should always be prime consideration when hemolytic anemia is present and its cause is not clearly defined. Chronic hemolysis due to unstable hemoglobins can be associated with all of the known complications of hemolysis such as aplastic crisis, jaundice with cholelithiasis, leg ulcers, pulmonary hypertension, splenomegaly, and hypersplenism, along with special features of unstable hemoglobins such as pigmenturia and drug-induced and fever-associated increases in anemia.16,17 Dusky cyanosis has been described in some patients with unstable hemoglobins predisposed to methemoglobin formation. In one case where the ␥ -globin chain of HbF was affected (HbF-Poole; HBG2 trp130gly), hemolytic anemia was present in the newborn but disappeared as the ␥ - to ␤-globin switch was completed. Sometimes, the disease is seen in early childhood; it can be found in adults incidentally or when fever or drug treatment induces hemolysis. Many unstable hemoglobin variants produce mild hemolytic disease with minimal or no anemia in the steady state and a reticulocyte count between 4% and 10%. Splenomegaly might or might not be present. Most patients with mild disease are first seen during a hemolytic crisis induced by drugs or infection. More than one-half of the unstable variants are associated with no hematological abnormality and were detected through screening programs. Although most differences in clinical phenotypes are explained by nature of the amino acid substitution, as in many single gene disorders, other genetic and some environmental factors might modulate the severity of disease. For example, investigation of the basis for intrafamilial vari¨ ation in drug-related hemolysis with Hb Zurich disease suggested that tobacco smoking ameliorated hemolysis, prob¨ ably because the high affinity of Hb Zurich for CO stabilized the hemoglobin tetramer. B19 parvovirus infection can temporarily shut down erythropoiesis, rapidly worsening the anemia and causing aplastic crisis. Anemia might also increase during infection and after treatment with oxidant drugs such as

Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants sulfonamides. The intensity of hemolysis is variable and is dependent on the mutation and fraction of abnormal hemoglobin present. Hb K¨oln, described in multiple kindreds, is the most common unstable hemoglobin and is characterized by anemia, reticulocytosis, splenomegaly, and 10%–25% Hb K¨oln. It is not associated with oxidant drug-induced hemolysis. The independent occurrence of this variant in so many apparently unrelated individuals suggests that the Hb K¨oln mutation, located at a methylated CpG dinucleotide sequence of the ␤-globin gene, can act as a “hotspot” for mutation through the deamination of the methylcytosine nucleotide to form thymine. ¨ Hb Zurich has also been reported often. This variant, forming approximately 25% of the hemolysate, is accompanied by mild anemia exacerbated by oxidant drugs, pigmenturia, and increased affinity for CO. The latter protects the ␤-globin heme group from oxidation and increased instability. Carriers have a special susceptibility to sulfonamide-induced hemolytic crisis. Hb Hasharon (HBA2 asp47his) is another more common variant affecting the ␣-globin chain, found in Ashkenazi Jews, and causing hemolysis in newborns but not in most adults. This variant comprises 15%–20% of the hemolysate and inclusion bodies were not found. Some unstable hemoglobins are linked to ␣- or ␤thalassemia genes: the ␣-chain mutants Hb Suan-Dok (HBA2 leu109arg) and Hb Petah Tikva (HBA ala110asp) coexist in cis with ␣ thalassemia and some ␤-chain mutants like Hb Leiden (HBB glu6 or 7del), Hb Duarte (HBB ala62pro), and HbG-Ferrara (HBB asn57lys) coexist with a ␤0 -thalassemia mutation in trans. Miscellaneous. A 10-year-old girl with hemolytic anemia due to Hb Bristol-Alesha (HBB val68met) had moyamoya and transient ischemic attacks. The authors suggested that chronic hypoxemia might be the cause of occlusive moyamoya in unstable hemoglobinopathies or in hemoglobins with altered oxygen affinity.18

Hyperunstable Hemoglobins Some uncommon globin mutations are hyperunstable. Although these mutants are synthesized in normal amounts, they are unable to form stable tetramers or even dimers and are rapidly catabolized. These variants therefore have features of both an unstable hemoglobins and thalassemias and have been called thalassemic hemoglobinopathies.19 The dominant phenotype is that of severe, dominantly inherited ␤ thalassemia because the effected globin chain fails to accumulate and participate in tetramer formation. These mutations are discussed in Chapters 13 and 16. Treatment. Unstable hemoglobinopathies are generally mild disorders and do not require therapy except for supportive and preventive measures. Administration of folic acid to prevent megaloblastic arrest of erythropoiesis

597

might be warranted, although access to a nutritious diet is probably satisfactory. The possibility of fever-associated hemolysis should be recognized and oxidant drugs should be avoided, including acetaminophen and sulfonamides, especially when history suggests they can provoke hemolysis, and there are other management considerations. Chronic hemolysis is associated with a high incidence of cholelithiasis although this alone is not an indication for surgery. Severe hemolysis raises the question of splenectomy. Undoubtedly, the spleen plays an important pathophysiological role in the destruction of Heinz body containing red cells. This must be balanced with the role of the spleen as a defense against pneumococcal infections early in life and the need for antipneumococcal vaccines and prophylactic penicillin in cases in which splenectomy is performed in childhood. On balance, splenectomy might be beneficial for some severe unstable hemoglobinopathies, and partial correction of the anemia is sometimes achieved. Nevertheless, predicting the response to splenectomy is difficult. Hydroxyurea has been used to stimulate HbF production and help repair anemia in two cases of unstable hemoglobin disease.20

M HEMOGLOBINS Cyanosis is the major clinical feature of amino acid substitutions causing methemoglobinemia. These rare disorders are not noted for their clinical severity; misdiagnosis, and unneeded treatment are their major hazards. Other causes of methemoglobinemia are discussed in Chapter 25.

Pathophysiology In the M (met) hemoglobins, the mutant globin chain creates an abnormal microenvironment for the heme iron, displacing the equilibrium toward the oxidized or ferric (Fe3+ ) state (Fig. 24.4). A combination of Fe3+ and its abnormal coordination with the substituted amino acid generates a visible spectrum that resembles but is clearly different from methemoglobin not due to a globin gene mutation, where the heme iron is oxidized but an associated amino acid substitution is absent (Chapter 25). In the Iwate prefecture of Japan, “black children” had been observed for more than 160 years and this was associated with a brownish colored hemoglobin in the hemolysate of a patient that was eventually characterized as HbM Iwate (HBA his87tyr).21 Four ␤-, two ␣-, and two ␥ -globin HbM variants have been reported. In five of the eight described HbM variants, the mutation involves the substitution of the distal (E7) or proximal (F8) histidine interacting with the heme iron via tyrosine. With HbM Milwaukee (HBB val67glu), the longer side chain of the glutamic acid residue can reach and perturb the heme iron.

598

Martin H. Steinberg and Ronald L. Nagel α M-Boston β M-Saskatoon His E7

Tyr γ F-M Osaka

His F8

Tyr α M-Iwate β M-Hyde Park γ F-M Ft Ripley Figure 24.4. Mutation sites of some hemoglobin M variants.

Some properties of M hemoglobins are shown in Table 24.4. The strength of attachment of ferric heme to globins differs among M hemoglobins. The ␥ -globin gene HbM variants, HbF-M Osaka (HBG2 his63tyr), and HbF-M Fort Ripley (HBG his92tyr) have been associated with neonatal “cyanosis” that disappears with maturation. HbM Milwaukee is the sole example of an M hemoglobin not caused by mutation of the proximal or distal histidine residues but by the nearby ␤67 (E11) val. When this residue is replaced by a residue with an appropriate side chain, as happens with the glutaminyl residue, it perturbs the heme iron and generates an M hemoglobin. Other mutations of that site, such as Hb Bristol (val67asp) or Hb Sidney (val67ala), are unstable or have low affinity but do not lead to heme iron oxidation. X-ray crystallography showed that the carboxylic group of the new glutaminyl residue in Hb Milwaukee occupies the sixth coordination position of the iron, and that the proximal histidine maintains its role as the tenant of the fifth coordinating position, stabilizing the abnormal ferric state of HbM Milwaukee. Oxygen-binding Properties and the R→T Transition of M Hemoglobins. HbM Milwaukee, Hyde Park (HBB his92tyr),

and Hb Boston (HBA his58tyr) all adopt deoxy or deoxy-like conformation upon the deoxygenation of the two normal chains although the abnormal chains cannot off load oxygen. This, and crystallographic, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopy, and 2,3-BPG binding studies affirm that after two heme groups become deoxygenated, the entire molecule adopts the deoxy T conformation. A normal Bohr effect and P50 strongly suggest that in HbM Milwaukee, HbM Saskatoon (HBB his63tyr), and HbM Hyde Park the molecule adopts the R-state when the two normal chains are oxygenated, a finding supported by nuclear magnetic resonance studies of HbM Milwaukee. In contrast, HbM Iwate is in the crystallographic T configuration when its normal heme groups are in the ferric state, explaining its decreased oxygen affinity; the molecule does not shift to the R-state when the normal heme groups are liganded and remains in the low affinity T-state. A similar situation probably exists in HbM Boston, as the habit of the deoxy crystal remains intact after oxygenation, suggesting that no conformational change has occurred that would require a different crystal structure. HbM Saskatoon and HbM Boston have different properties despite the common substitution of the distal histidine because the former variant does not change its conformation when oxygenated, whereas the latter variant does. Why properties of the ␤-globin chains differ from that of the ␣-globin chain when their distal histidine is substituted is unresolved. Iron Oxidation and Spectral Characteristics. In all M hemoglobins, the affected heme groups are stabilized in the ferric state and have an abnormal microenvironment. They exhibit an abnormal visible absorption spectrum that can be easily distinguished from methemoglobin. This characteristic separates these variants from some unstable hemoglobin mutants that also have a tendency to form methemoglobin. Heme iron in the abnormal subunits of the M hemoglobin exhibit abnormally low redox potential. They are oxidized more rapidly by molecular oxygen and are resistant, to a variable degree, to reduction by dithionite. Differences exist in the rate of reduction of the five M hemoglobins with various reductases, including the enzyme active in the normal erythrocyte, NADH-cytochrome

Table 24.4. Hematological features of HbM variants of the ␣- and ␤-globin chains Variant

Percent

Hb (g/dL)

Reticulocytes (%)

P50

Bohr effect

HbM Hyde Park (Milwaukee-2)(␤) HbM Iwate (␣) HbM Boston (␣) HbM Milwaukee (␤) HbM Saskatoon (␤) HbM Chile (␤)

23–32

10–13

4–6

Normal

Present

19 20–30 50 35 17

17 – 14–15 13–16 13.2

– – 1–2 0.8–3.2 1.2

Decreased Decreased Decreased Normal –

Decreased Decreased Present Present –

Methemoglobin

OD Hb M Saskatoon

b5 reductase (CYB5R3). HbM Iwate, HbM Hyde Park, and HbM Boston are not reduced at all; HbM Milwaukee is reduced slowly and HbM Saskatoon is reduced normally by this reductase. These last two variants might be less oxidized in vivo than expected. Full ferric conversion might occur only in vitro, due to the high autoxidation rate of these abnormal hemoglobins. Older red cells might, nevertheless, have fully oxidized abnormal chains consistent with the presence of clinically apparent cyanosis.

599

Hb M Saskatoon

Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants

OD

Clinical Aspects and Diagnosis Clinically, the skin and mucous membranes of HbM carriers have an appearance similar to but not identical with cyanosis, sometimes called pseudocyanosis. Skin and mucosal surfaces are brownish/slate colored, more like methemoglobinemia, but not as slate blue-purple as true cyanosis. This distinction is subtle and might not be apparent without simultaneously comparing the two conditions. Skin reflects hemoglobin molecules with an abnormal ferric heme and abnormal spectrum, because cyanosis is caused by the presence of more than 5 g/dL of deoxyhemoglobin. Pseudocyanosis is present from birth in ␣-globin chain abnormalities and from the middle of the first year of life in the ␤-chain mutants. A mixture of the abnormal pigment and true cyanosis, due to hemoglobin desaturation of the normal chains, is observed in the low oxygen affinity HbM Boston and HbM Iwate. Pseudocyanosis, is not associated with dyspnea or clubbing and carriers have a normal life expectancy. A mild hemolytic anemia and reticulocytosis has been observed in HbM Hyde Park and can be explained by the instability of the hemoglobin induced by partial heme loss. HbM should be considered in all patients with abnormal homogeneous coloration of the skin and mucosa, particularly if pulmonary and cardiac function is normal. The diagnosis can be suspected by observing an abnormal brown coloration of the blood in a tube. To distinguish this coloration from methemoglobin, the addition of KCN to the hemolysate is useful. KCN will turn blood containing methemoglobin red, but has little if any effect on HbM-containing hemolysates. Lack of color conversion with KCN is diagnostic of HbM. A spectrophotometrical recording of the visible spectrum of the hemolysate is critical for the diagnosis. M hemoglobins do not have an absorbance peak at 630–635 nm, which is typical of methemoglobin A (Fig. 24.5). In the presence of HbM, accurate measurement of oxygen saturation and CO hemoglobin is difficult with most instruments. Absorption maxima at different wavelengths of all hemoglobin M variants have been reported. Treatment. Treatment is neither possible nor necessary. A correct diagnosis is most important because this will forestall therapeutic and diagnostic misadventures.

550

650

450

550

650

Figure 24.5. Absorption spectra of HbM Saskatoon. The left panel shows a fresh hemolysate and the right panel, a hemolysate totally oxidized.

RARE MUTATIONS WITHOUT A PHENOTYPE OR WITH MINOR HEMATOLOGICAL CHANGES Posttranslational Modifications Posttranslational modifications of globin are broadly illustrative of the secondary changes possible in all proteins and form an interesting group of hemoglobinopathies. Deamidation. Under certain conditions, mutant asparaginyl residues can be deamidated to aspartate and result in two species of globin caused by a single mutation (Table 24.5).22 Aspartic acid to asparagine mutants could be deamidated, reducing levels of the asparagine-containing globin. Usually, deamidation occurs when a histidine residue is adjacent to the asparagine substitution or when changes in the tertiary structure of a variant globin can bring a histidine into the proximity of an asparagine residue. Other factors favoring asparagine deamidation are glycine, serine, or alanine residues with nonbulky side chains on either side of the asparagine or brought near the asparagine, a basic or acidic residue on one side and a serine or cysteine on the other side of asparagine and peptide chain flexibility. Not all aspartic acid to asparagine mutants are subject to posttranslational deamidation. Two abnormal hemoglobins can be present when variants susceptible to deamidation are analyzed: a native form containing the encoded asparagine and a deamidated form containing aspartic acid. When a variant of this type is present in a compound heterozygote with another variant hemoglobin, a confusing HPLC profile with three hemoglobin bands representing two globins resulting from the asparagine substitution and another from the other ␤-globin gene variant can exist. Oxidation. A young girl with hemolytic anemia was found to have two unstable hemoglobins: Hb Sidney (HBB val67asp) and a new variant called Hb Coventry (HBB leu141del) that formed less than 10% of the hemolysate.

600

Martin H. Steinberg and Ronald L. Nagel Table 24.5. Hemoglobin variants with deamidated asparaginyl residues Variant

DNA mutation

Phenotype

La Roche-sur-Yon (HBB leu81his)

CTC→CAC (?)

Unstable, decreased P50 , mild hemolysis. leu81his facilitates ␤80 asn deamidation

Providence (HBB lys82asn)

AAG→AAC/T (?)

Increased O2 affinity but normal packed cell volume

Redondo (HBB his92asn)

CAC→AAC (?)

Heme loss, unstable, hemolysis, migrates like HbS and HbA2

Osler (HBB tyr145asn)

TAT→AAT

Increased O2 affinity, migrates like HbJ and HbA

J Sardegna (HBA his50asn)

CAC→AAC

Functionally normal. Present in 0.09% of Sardinians

J-Singapore (HBA2 ala79gly)

GCG→GGG

Normal, ∼22%. The HBA2 ala79gly mutation allows deamidation of ␣78 asn56

Wayne (HBA 139 -A)

-A in either HBA2 or HBA1 with 5 codon elongation

Normal hematology, either ∼14% or ∼6% present

As the patient also had HbA, three ␤-globin genes were postulated, one being a ␤␦ gene.23 Another patient with unstable hemoglobin hemolytic anemia was also found with three putative ␤-globin chains: Hb Atlanta (HBB leu75pro), Hb Coventry, and ␤A . These observations, and a third instance of Hb Coventry in a similar clinical setting, suggested that this variant was widespread and only became apparent when present with another unstable hemoglobin. It was proposed that the two mutations were in a single ␤globin gene (HBB leu75pro; leu141del), that was the product of a ␤␦ anti-Lepore gene, the leucine 141 being deleted in the crossover event.24 Two genetic events must have occurred to explain these findings; a de novo ␤75 leu→pro mutation and a sister chromatid exchange in a somatic cell. When DNA techniques were applied to study the structure of the ␤-globin gene cluster and the nucleotide sequence of the ␤-globin genes, only two normal ␤-globin genes were found. The expected leu75pro mutation was found but the codon for ␤141 was intact in genomic DNA and in mRNA. Mass spectroscopy demonstrated the peptide containing ␤141 leucine to contain a novel amino acid of 129 D instead of the 113 D of the leucine peptide and was postulated to be hydroxyleucine. Hydroxyleucine cannot be detected by amino acid analysis so its presence remains conjectural. It was proposed that perturbation of the heme pocket by the leu75pro mutation in the E helix, perhaps via the generation of activated oxygen species, lead to oxidation of some ␤141 leucine residues. This notion provides a parsimonious explanation for the existence of the Hb Coventry anomaly in the same globin chain as Hb Atlanta and other unstable variants.25 Acetylation. Hb Raleigh (HBB val1ala) is acetylated and migrates with HbA1 C.26 Proteins with an NH2 -terminal alanine are often acetylated. Acetylation of a retained N-terminal methionyl occurs when normal cleavage of this

residue malfunctions (see later). Acetylated hemoglobins can be mistaken for Hb A1 C.

N-Terminal Elongated Globins Several mutations of the NH2 -terminal valine of both the ␣- and ␤-globin chain and a HBB his2pro mutation result in a globin elongated by one residue because of the failure to cleave the initiator methionyl (Table 24.3). A hematological phenotype is not present but the NH2 -terminal methionine is acetylated and appears as HbA1 C by some methods of hemoglobin separation.

Interesting ␤-Globin Gene Variants with Point Mutations Hemoglobin G Coushatta (HBB glu22ala). This ␤-globin chain variant has no clinical or hematological manifestations and has been found in geographically separated racial and ethnic groups.27 It is common in China and North American Coushatta Indians and has also been described in Japan, Korea, and Turkey. It is the most common variant hemoglobin described along the Chinese Silk Road. Its presence in Asia, the Old World, and in American Indians raised the question of whether this variant had a unicentric origin and spread to the New World over the ancient Bering land bridge or originated multicentrically in Asia and in Amerindians. When the haplotype of the ␤-globin gene cluster was examined in Louisiana Coushatta Indians and native Chinese who carried Hb G Coushatta, these groups had different haplotypes and ␤-globin gene framework associated with the identical HbG Coushatta mutation. Both the HbG Coushatta mutation (GAA→GCA) and the codon 2 CAC→CAT polymorphism are normal ␦-globin gene sequences, suggesting the possibility of gene conversion. HbG Coushatta had at least two independent origins

Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants

601

Table 24.6. Mutations encoded on HBA2 and HBA1 Variant

HBA2

Genotype

%

HBA1

Genotype

%

J-Paris (ala12asp)

GCC→GAC

␣X ␣/␣␣

24

GCC→GAC

␣␣X /␣␣

20.7

Hekinan (glu27asp)

GAG→GAC

␣X ␣/-␣

27.9

GAG→GAT

␣␣X /␣␣

13.9

G-Philadelphia (asn68lys)∗

AAC→AAA

␣X ␣/␣␣

25.1

AAC→AAG

␣␣/-␣X∗

33.4

J-Broussais (lys90asn)

AAG→AAT

␣X ␣/␣␣

25

AAG→AAC

␣␣X /␣␣␣

18

Manitoba ser102arg)

AGC→CGC

␣X ␣/␣␣

18.7

AGC→AGA

␣␣X /␣␣X

23.9

J-Meerut (ala120glu)

GCG→GAG

␣X α/␣␣

23

GCG→GAG

␣␣X /␣␣

18.4

∗

This variant is usually present on the -␣3.7 rightward deletion chromosome.

that could be due to two separate mutations at codon ␤22, a mutation at this codon and a ␤→␦ gene conversion or two ␤→␦ gene conversion events.28 The ␤-globin gene haplotype predominant in Thailand and in Koreans suggesting yet other possible origins for this ostensibly genetically “neutral” variant.29,30,31 Hemoglobin Vicksburg (HBB leu75del). It was proposed that Hb Vicksburg arose as a stem cell mutation on a ␤+ -thalassemia chromosome.32 When first studied, only Hb Vicksburg was present, then both HbA and Hb Vicksburg were found, and finally, only HbA was present. This suggested that over time there were at least two clones of erythroid progenitors contributing to erythropoiesis.33 A leucine deleted in Hb Vicksburg recalls the origin of Hb Coventry so it remains possible that a technical artifact was responsible for the failure to find a ␤75 leucine residue when Hb Vicksburg was present. Support for the notion of a somatic mutation leading to traces of abnormal hemoglobin comes from studies of the stable variant, Hb Costa Rica (HBB his77arg). Six percent to 8% Hb Costa Rica was found in a young woman but not in any of her relatives. A mutation corresponding to the CAC→CGC transversion expected to code for this variant could not be detected in her genomic DNA. Yet, small amounts of Hb Costa Rica mRNA were present in reticulocytes. Globin gene structure was normal. A somatic mutation causing genetic mosaicism was hypothesized. In follow-up studies in which burst-forming unit erythrocytes were grown from the blood of this patient, only 12%–15% of these erythroid colonies contained both HbA and Hb Costa Rica mRNA, whereas the remainder had mRNA only for HbA. HBB mutations in cis to a ␤+ -thalassemia mutation was the explanation for unusually low levels of HbS and the rare variant, Hb Dhofar (HBB pro58arg).34

Interesting ␣-Globin Gene Variants with Point Mutations Mutations can occur in either or both the 5
HBA2 or the 3
HBA1 genes. Where information is available, mutations are equally divided between both ␣-globin genes.35 Depending on whether they are encoded on the HBA2 or the HBA1 gene, there is a small difference in the levels of stable ␣-globin gene mutants. HBA2 mutants usually form approximately 24% and HBA1 mutants approximately 20% of the hemolysate, a difference explained by the transcription rate of each ␣-globin gene, translational efficiency of the mRNA from each gene, the assembly of ␣␤ dimers, the presence of deleted or triplicated ␣-globin loci, and the presence of nondeletion types of ␣ thalassemia (Chapter 13). Some identical amino acid substitutions encoded on both the ␣1 - and ␣2 -globin genes, often by different mutations, are shown in Table 24.6. Hemoglobin G-Philadelphia (HBA asn68ys). HbG Philadelphia is the most common ␣-globin gene variant in blacks and is found in approximately one in 5,000 African Americans. A clinical phenotype is not present, but because it is usually found with ␣ thalassemia and can be present on many different ␣-globin gene arrangements, its level in the hemolysate varies widely and it has a bimodal or trimodal distribution.36,37 Interactions of HbG-Philadelphia with sickle cell anemia are discussed in Chapter 23. Most often, in the black population, HbG-Philadelphia is present on the -␣3.7 chromosome as an AAC→AAG transversion. It is also found in Italians on the HBA2 gene in a normal ␣-globin gene locus (␣G ␣/␣␣) where the mutation is AAC→AAA.38,39 Different proportions of HbG-Philadelphia found with different ␣-globin gene haplotypes are shown in Table 24.7. In addition to the ␣-globin genotypes shown, it is likely that HbG-Philadelphia will be found in trans to chromosomes with both ␣-globin genes deleted and associated

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Martin H. Steinberg and Ronald L. Nagel

Table 24.7. Levels of HbG-Philadelphia in different ␣-globin genotypes ␣-Globin genotype

% HbG-Philadelphia

␣G ␣/␣␣ -␣G /␣␣ -␣/␣G ␣ -␣/-α G -␣G /-␣G -␣T /-␣G∗ –/-␣G†

25 33 33 50 100 100 HbG+HbH

∗ †

␣T denotes a dysfunctional ␣-globin gene that is not expressed because of a frameshift and new in-phase termination codon. In HbG/HbH disease, both HbH and HbG are present.

with triplicated and quadruplicated ␣-globin loci. In the latter case, the percent of HbG should be lower than usual. Other ␣-globin gene variants present at levels near 50% are probably also present on an ␣ thalassemia-2 chromosome. HbJ-Tongariki (HBA ala115asp) is commonly found in Melanesians and always on a -␣3.7 chromosome. Heterozygotes have approximately 50% and homozygotes, nearly 100% of this variant. This mutation was likely to have occurred on a -␣3.7 thalassemia chromosome that is prevalent in Vanuatuans. Hb Setif (HBA2 asp22tyr) Pseudosickling and Hemoglobin Oligomerization. Only hemoglobin variants with the HBB glu6val mutation polymerize when deoxygenated and cause vasoocclusive disease. Hb Setif, a variant found in North Africa and the Middle East, forms pseudosickled cells in vitro but not in vivo, when it is oxygenated. This is due to the aggregation of Hb Setif.40–44 Pseudosickled Hb Setif cells can be distinguished from sickled HbS-containing cells by many physical characteristics although they have several features in common. The Csat of a 40%/60% hemolysate of Hb Setif/HbA at 290 mOsm was 24 g/dL at 24◦ C. During incubation in NaCl buffer at 450 mOsm, polymer in pseudosickled cells required more than 30 minutes to begin formation; after 24 hours, nearly all cells contained aggregated Hb Setif. In contrast, HbS polymer forms in fractions of a second to seconds. Deoxygenation promotes HbS polymerization but inhibits pseudosickling of Hb Setif cells. Pseudosickled Hb Setif cells have been described as twisted cigars with a monotonous morphology in contrast to the eclectic conformation of sickle cells (Fig. 24.6). In common with HbS erythrocytes, cell deformability decreased with increasing Hb Setif concentrations and increasing osmolality of the suspending buffer, whereas the amount of Hb Setif polymer increased under these conditions. Pseudosickling of Hb Setif is strictly an in vitro phenomenon. Whether Hb Setif was present at levels of less than 20%, attributable to mild instability, or found at concentrations near 40%, because of a coexistent ␣ thalassemia deletion, there was no evidence of hemolysis and vasoocclusive disease. Urine-concentrating ability in carriers of

Hb Setif seemed unimpaired, whereas this is abnormal in sickle cell trait and sickle cell anemia. Hemoglobin Porto Alegre (HBB ser9cys) contains an extra thiol group oriented toward the molecule’s exterior and spontaneously oligomerizes during storage by forming intermolecular disulfide bridges. Although other substitutions introducing a cysteine residue have been described, only Hb Mississippi (HBB ser44cys), Hb Harrow (HBB phe118cys), and Hb Ta-Li (HBB gly83cys) are also known to form low concentrations of oligomers.45 None of these variants are associated with cell sickling. HbI (HBA2 lys16glu). HbI has been encoded on both HBA2 and HBA1 of a single individual and considered an example of concerted evolution (the parallel development of related closely linked genes). In a hematologically normal black woman, 65% of the hemolysate was HbI. Her ␣-globin gene haplotype revealed a normal ␣-globin gene locus and an ␣-globin gene locus with the common -␣3.7 deletion. Genetic studies showed she transmitted to a daughter who had only HbA, the ␣−3.7 deletion, suggesting that HbI was encoded on the normal ␣-globin gene locus. When mRNAs from the HBA1 and HBA2 were selectively isolated and translated in vitro, both encoded a glutaminyl residue at position 16, whereas the HBA1 mRNA also encoded normal mRNA, the product of the chromosome with the ␣3.7 deletion. These observations suggested a gene conversion event between the highly homologous ␣-globin genes. Two of the

Figure 24.6. Pseudosickling of HbSetif erythrocytes by (A) light microscopy and Nomarski optics (B and C) and scanning electron microscopy. (From ref. 43 with permission.)

Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and Other Variants

603

Table 24.8. ␤-Globin gene variants with two amino acid substitutions∗ Variant

Substitutions

Hemoglobins involved

Hematology

Arlington Park HbC-Rothschild HbC-New Cross HbC-Ndjamena

glu6lys; lys95glu glu6lys; trp37arg glu6lys; gly83asp glu6lys; trp37gly

HbC; HbN Baltimore HbC; Hb Rothschild HbC; ? HbC; ?

HbD-Agri HbO-Tibesti

ser9tyr; glu121gln val11ile; glu121lys

Hb Brem-sur-Mer; HbD Punjab Hb Hamilton; HbO Arab

T-Cambodia Corbeil Grenoble Poissy Hb Casablanca Villeparisis Duino Medicine Lake Fannin-Lubbock Masuda Cleveland

glu26lys; glu121gln glu26lys; arg104thr pro51ser; glu52lys gly56arg; ala86pro lys65met: phe122leu his77tyr; asn80ser his92pro; arg104ser val98met; leu32gln val111leu; gly119asp leu114met; gly119asp glu121gln; cys93arg

HbE; Hb D-Los Angeles HbE; Hb Sherwood Forest ?; Hb Osu Christiansborg Hamadan; ? Anakya; Bushey Fukuyama; ?Newcastle; Camperdown ¨ ? Koln; ? Zengcheng; ? D-Los Angeles; Okazaki

Normal Normal Mild instability? Compound heterozygote with HbS causing sickle cell disease ? Compound heterozygote with HbS causing sickle cell disease ? Normal ? erythrocytosis, hemolysis Normal Normal Hemolysis Severe ␤ thalassemia Normal Normal Normal

∗

Variants with the sickle cell mutation and another amino acid substitution are discussed in Chapter 23. Details and references at http://globin.cse.psu.edu.

three ␣-globin genes present in the proband produced HbI, accounting for the very high concentration of the variant. A report of HbI associated with “sickled” cells contains many unknowns, including the structure of the abnormal globin and whether there is some sort of hemoglobin polymer in the cell. Some features of HbI appear to resemble those of Hb Setif. Five stable ␣-globin variants formed by mutations of codon 6, Hb Sarawa (asp6ala), Hb Dunn (asp6asn), Hb Ferndown (asp6val), Hb Woodville (asp6tyr), and Hb Swan River (asp6gly) are all present at a concentration of 10%. Four stable to mildly unstable ␣-globin variants at codon 27 mutations, Hb Fort Worth (glu27gly), Hb Spanish Town (glu27val), Hb Shuanfeng (glu27lys), and Hb Hekinan (glu27asp) are present at concentrations of 4%–14%. In no case has it been established whether the mutant is on HBA2 or HBA1 or whether the ␣-globin gene loci are duplicated. Other than Hb Dunn, whose asparaginyl residue might be deamidated, it is not clear why these variants with uncommonly low blood levels are clustered at these sites.

Two Point Mutations in a Globin Chain Examples of two point mutations in a single ␤-globin chain, where one mutation codes for HbS, are discussed in Chapter 23. ␤-Globin gene variants with two amino acid substitutions in cis are shown in Table 24.8. There are two putative origins for these double-substitution variants: recombination between homologous chromosomes each containing a different mutation and a new mutation in a gene already

containing one mutation. When both mutations are common in a population, like HbC and HbN-Baltimore that in cis characterize Hb Arlington Park, or HbE and HbD-Los Angeles that are found in HbT-Cambodia, then the doubly substituted variant most likely arose by recombination. De novo mutation could explain instances where a rare variant is present along with a polymorphic one, such as HbC or HbE. Occasionally, both mutations are rare. Hb Medicine Lake (␤98 val→met; ␤32 leu→gln) has the phenotype of severe ␤ thalassemia because the ␤-globin chain, containing both the Hb K¨oln and the ␤32 mutation, is likely to be highly unstable and rapidly catabolized (Chapter 13). Lacking a detectable protein, it was not possible to explore directly the properties of the abnormal hemoglobin containing the ␤32 CTG→CAG mutation. It was predicted that because the leu→gln mutation does not alter the charge of the ␤ subunit, the electrophoretic mobility of Hb Medicine Lake might not differ from Hb K¨oln. A hydrophilic glutamine residue contains an uncharged polar side chain that could distort the B helix. Also, perturbation of the adjacent ␤31 leucine that contacts the heme group could result in instability. A definitive assessment of the inheritance of Hb Medicine Lake was not possible, but the most plausible cause of this abnormal hemoglobin was a new mutation in the germ cell of the father or in the proband. Seven variants of HBG1 contain the ile75thr polymorphism (HbF-Sardinia, Chapter 6) and an additional mutation. Curiously, no ␣-globin chain or HBG2 variants with two point mutations have been described.

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Table 24.9. ␤␦ and ␥ ␤ fusion chains Variant

Crossover

Features

Miyada

␤ through 12 ␦ from 12

17% in heterozygotes; normal hematology

P-Congo

␤ through 22 ␦ from 116

Normal hematology

P-Nilotic∗

␤ through 22 ␦ from 50

Normal hematology

P-India

␤ through 87 ␦ from 116

23% in heterozygotes

Kenya

A ␥ through 81 ␤ from 86

6% in heterozygotes; 5% HbF (100%G ␥ ); normal hematology

Hong Kong

within 54 bp of CAP site and exon 1

Protein is identical to ␦ globin. ␤-globin like in 5
untranslated portion. HbA2 16–20%56

Parchman

␦ through 12 ␤ from 22–50 ␦ from 86

1.6%; stable; found in a patient with ␣ thalassemia

∗

Hb Lincoln Park is identical to HbP-Nilotic but is associated with reticulocytosis. It contains a deletion of ␦137 valine and was hypothesized to result from multiple crossing over events. References to variant described before 2000 can be found in http://globin.cse. psu.edu.

and other emergencies without the need for blood typing and cross matching. Free hemoglobins have been difficult to develop as blood substitutes. They must not scavenge NO and form methemoglobin (Chapters 10 and 11), they should overcome the very high oxygen affinity of free hemoglobin, they should not readily dissociate into dimers, and they should be retained in the vasculature.48 None of these conditions have been sufficiently realized to the point of producing a product for late phase clinical trials. A recombinant human ␤-globin variant with gly16asp, glu22ala, and thr87gln was designed as an antisickling protein. The gly16asp substitution increased affinity for ␣globin, whereas the other mutations inhibited deoxyHbS polymerization. This variant binds oxygen cooperatively and has an oxygen affinity that is comparable with HbF. Delay time experiments showed that it is a potent inhibitor of HbS polymerization. When knockout transgenic mice that expressed only this variant were bred with knockout transgenic sickle mice, hematological abnormalities and organ pathology were abolished, suggesting the value of a variant like this in diminishing the pathology of sickle cell disease.49 REFERENCES 1.

Nonthalassemic Fusion Globins Anti-Lepore ␤␦ fusion globins and Hb Kenya, a ␥ ␤ fusion globin, are not usually associated with hematological abnormalities because a normal ␤-globin gene is present on the chromosome along with the hybrid gene. These variants are shown in Table 24.9.

2. 3.

4.

NOVEL COMPOUND HETEROZYGOUS CONDITIONS With the widespread use of hemoglobin screening and increasingly sensitive methods of hemoglobin separation, many interesting but rare compound heterozygous conditions have been described. Although most of these have no hematological abnormalities they could, if not correctly diagnosed, lead to misinformed genetic counseling. As expected, most of these compound heterozygotes include HbS, HbC, and HbE but other interesting combinations have been reported.

5.

RECOMBINANT HEMOGLOBINS

8.

Recombinant hemoglobins can be produced in bacterial and yeast expression systems and in transgenic animals. Hundreds of variants have been engineered and used to study hemoglobin structure, function,46 HbS polymerization, and its inhibition47 and for potential therapeutic purposes.32 As hemoglobin-based blood substitutes, recombinant hemoglobins could provide a shelfstorable oxygen carrying solution for use in surgery, trauma,

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Huisman THJ, Carver MFH, Efremov GD. A Syllabus of Human Hemoglobin Variants. 2nd ed. Augusta: Sickle Cell Anemia Foundation; 1998. Hemoglobin Variants in Human Populations. Boca Raton: CRC Press; 1986. Wajcman H, Galacteros F. Hemoglobins with high oxygen affinity leading to erythrocytosis. New variants and new concepts. Hemoglobin. 2005;29(2):91–106. Williamson D, Beresford CH, Langdown JV, Anderson CC, Green AR. Polycythaemia associated with homozygosity for the abnormal haemoglobin Sherwood Forest (beta 104 (G6)Arg-->Thr). Br J Haematol. 1994;86(4):890–892. Tanphaichitr VS, Viprakasit V, Veerakul G, Sanpakit K, Tientadakul P. Homozygous hemoglobin Tak causes symptomatic secondary polycythemia in a Thai boy. J Pediatr Hematol Oncol. 2003;25(3):261–265. Bruns CM, Thet LA, Woodson RD, Schultz J, Hla KM. Hemoglobinopathy case finding by pulse oximetry. Am J Hematol. 2003;74(2):142–143. Papassotiriou I, Traeger-Synodinos J, Marden MC, et al. The homozygous state for Hb Crete [beta129 (H7) Ala-->Pro] is associated with a complex phenotype including erythrocytosis and functional anemia. Blood Cells Mol Dis. 2005; 34(3):229–234. McClure RF, Hoyer JD, Mai M. The JAK2 V617F mutation is absent in patients with erythrocytosis due to high oxygen affinity hemoglobin variants. Hemoglobin. 2006;30(4):487– 489. Charache S, Weatherall DJ, Clegg JB. Polycythemia associated with a hemoglobinopathy. J Clin Invest. 1966;45(6):813– 822. Charache S, Achuff S, Winslow R, Adamson J, Chervenick P. Variability of the homeostatic response to altered p50. Blood. 1978;52(6):1156–1162.

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Deyell R, Jackson S, Spier S, Le D, Poon MC. Low oxygen saturation by pulse oximetry may be associated with a low oxygen affinity hemoglobin variant, hemoglobin Titusville. J Pediatr Hematol Oncol. 2006;28(2):100–102. Haymond S, Cariappa R, Eby CS, Scott MG. Laboratory assessment of oxygenation in methemoglobinemia. Clin Chem. 2005;51(2):434–444. Williamson D. The unstable haemoglobins. Blood Rev. 1993;7:146–163. Vasseur-Godbillon C, Marden MC, Giordano P, Wajcman H, Baudin-Creuza V. Impaired binding of AHSP to alpha chain variants: Hb Groene Hart illustrates a mechanism leading to unstable hemoglobins with alpha thalassemic like syndrome. Blood Cells Mol Dis. 2006;37(3):173–179. Turbpaiboon C, Limjindaporn T, Wongwiwat W, et al. Impaired interaction of alpha-haemoglobin-stabilising protein with alpha-globin termination mutant in a yeast twohybrid system. Br J Haematol. 2006;132(3):370–373. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. JAMA. 2005;293(13):1653–1662. Gladwin MT. Unraveling the hemolytic subphenotype of sickle cell disease. Blood. 2005;106:2925–2926. Brockmann K, Stolpe S, Fels C, Khan N, Kulozik AE, Pekrun A. Moyamoya syndrome associated with hemolytic anemia due to Hb Alesha. J Pediatr Hematol Oncol. 2005;27(8):436– 440. Thein SL. Structural variants with a ␤-thalassemia phenotype. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. 1st ed. Cambridge: Cambridge University Press; 2001:342–355. Rose C, Bauters F. Hydroxyurea therapy in highly unstable hemoglobin carriers. Blood. 1996;88:2807–2808. Percy MJ, McFerran NV, Lappin TR. Disorders of oxidised haemoglobin. Blood Rev. 2005;19(2):61–68. Wright HT. Sequence and structure determinants of nonenzymatic deamidation of asparagine and glutamine residues in proteins. Protein Eng. 1991;4:283–294. Casey R, Kynoch AM, Lang A, Lehmann H, Nozari G, Shinton NK. Double heterozygosity for two unstable hemoglobins: Hb Sydney (␤67[E11] Val->Ala) and Hb Coventry (␤141[H9] Leu deleted. Br J Haematol. 1978;38:195–209. Moo-Penn W, Bechtel K, Jue D, et al. The presence of hemoglobin S and C Harlem in an individual in the United States. Blood. 1975;46:363–367. Brennan SO, Shaw JG, George PM, Huisman THJ. Posttranslational modification of ␤141 Leu associated with the b75(E19)Leu–>Pro mutation in Hb Atlanta. Hemoglobin. 1993;17:1–7. Moo-Penn W, Bechtel K, Schmidt RM, et al. Hemoglobin Raleigh (␤1 valine->acetylalanine). structural and functional characterization. Biochemistry. 1977;16:4872–4879. Schneider RG, Haggard ME, McNutt CW, Johnson CW, Bowman JE, Barnett DR. Hemoglobin GCoushatta: a new variant in an American Indian family. Science. 1964;143: 197. Li J, Wilson D, Plonczynski M, et al. Genetic studies suggest a multicentric origin of Hb G Coushatta [␤22(B4)Glu∼Ala]. Hemoglobin. 1999;23: 57–67.

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Martin H. Steinberg and Ronald L. Nagel pseudosickling in vitro of hemoglobin Setif-containing erythrocytes. Am J Hematol. 1991;36:131–139. Baudin-Creuza V, Fablet C, Zal F, et al. Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure. Protein Sci. 2002;11(1):129–136. Cheng Y, Shen TJ, Simplaceanu V, Ho C. Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine. Biochemistry. 2002;41(39):11901–11913. Li X, Briehl RW, Bookchin RM, et al. Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids. J Biol Chem. 2002;277(16):13479–13487. Olson JS, Foley EW, Rogge C, Tsai AL, Doyle MP, Lemon DD. NO scavenging and the hypertensive effect of hemoglobinbased blood substitutes. Free Radic Biol Med. 2004;36(6):685– 697. Levasseur DN, Ryan TM, Reilly MP, McCune SL, Asakura T, Townes TM. A recombinant human hemoglobin with antisickling properties greater than fetal hemoglobin. J Biol Chem. 2004;279(26):27518–27524. Hutt PJ, Donaldson MH, Khatri J, et al. Hemoglobin S hemoglobin Osler: A case with 3 ␤ globin chains. DNA sequence (AAT) proves that Hb Osler is ␤ 145 Tyr-->Asn. Am J Hematol. 1996;52:305–309.

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Boissel J-P, Kasper TJ, Shah SC, Malone JI, Bunn HF. Aminoterminal processing of proteins: Hb South Florida a variant with retention of initiator methionine and Na -acetylation. Proc Natl Acad Sci USA. 1985;82:8448–8452. Kamel K, el-Najjar A, Chen SS, Wilson JB, Kutlar A, Huisman THJ. Hb Doha or alpha 2 beta 2[X-N-Met-1(NA1)val->glu] a new beta-chain abnormal hemoglobin observed in a Qatari female. Biochim Biophys Acta. 1985;831:257–260. Vasseur C, Blouquit Y, Kister J et al. Hemoglobin thionville. An ␣-chain variant with a substitution of a glutamate for valine at NA-1 and having an acetylated methionine NH2 terminus. J Biol Chem. 1992;267:12682–12691. Kavanaugh JS, Moo-Penn WF, Arnone A. Accommodation of insertions in helices: The mutation in hemoglobin Catonsville (Pro 37 alpha-Glu-Thr 38 alpha␣) generates a 3(10) --> alpha bulge. Biochemistry. 1993;32:2509–2513. Kawata R, Ohba Y, Yamamoto K, et al. Hyperunstable hemoglobin Korriyama anti-Hb Gun Hill insertion of five residues in the ␤ chain. Hemoglobin. 1988;12:311–321. So CC, Chan AY, Tsang ST, et al. A novel beta–delta globin gene fusion, anti-Lepore Hong Kong, leads to overexpression of delta globin chain and a mild thalassaemia intermedia phenotype when co-inherited with beta(0)-thalassaemia. Br J Haematol. 2007;136(1):158–162.

25 Dyshemoglobinemias Neeraj Agarwal, Ronald L. Nagel, and Josef T. Prchal

Hemoglobin can bind gases other than oxygen (O2 ). These include carbon monoxide (CO) and nitric oxide (NO). Carboxyhemoglobin (COHb) precludes normal O2 transport and is toxic. Nitrosohemoglobin has critical physiological functions discussed in Chapter 10. Normal hemoglobin can be oxidized to methemoglobin and sulfhemoglobin by exogenous agents and these hemoglobin forms can also be found as a result of germline mutations. In aggregate, these modified hemoglobins, referred as dyshemoglobinemias, are the basis of a group of acquired and genetic disorders that are rare but can have serious clinical implications.

METHEMOGLOBINEMIA Methemoglobin is formed when the iron of the heme group is oxidized or converted from the ferrous (Fe2+ ) to the ferric (Fe3+ ) state. The ferric hemes of methemoglobin are unable to reversibly bind O2 . In addition, the presence of ferric heme increases the O2 affinity of the accompanying ferrous hemes in the hemoglobin tetramer.1 This leads to a left shift in the hemoglobin–O2 dissociation curve, which impairs tissue delivery of O2 . Normally, methemoglobin is generated and then reduced physiologically to maintain a very low steady-state blood methemoglobin level of 1% or less of the total hemoglobin. The half-life of methemoglobin is approximately 1 hour if the reductase mechanism is normal.2 Methemoglobinemia occurs when there is imbalance between methemoglobin production and methemoglobin reduction. Methemoglobinemia can have both inherited and acquired causes; hemoglobin oxidation has been recently reviewed.3

Pathophysiology of Methemoglobinemia Production of Methemoglobin. O2 binds the ferrous form of iron present in hemoglobin to form oxyhemoglobin. During this process, one electron is partially transferred from

iron to the bound O2 , forming a ferric–superoxide (Fe+3 O2 ) anion complex.4 At the time of tissue delivery of O2 , some leaves as a superoxide (O2 − ) radical. The partially transferred electron is not returned to the iron moiety, thus leaving the iron in the ferric state and forming methemoglobin (Fig. 25.1). This physiological autooxidation of hemoglobin occurs spontaneously at a slow rate, creating 0.5%–3% methemoglobin per day.5 Oxidation of heme iron can occur by other nonphysiological means. These include 1) reactions with free radicals and endogenous compounds, including hydrogen peroxide (H2 O2 ), NO, O2 - and hydroxyl radical (OH• );6,7 and 2) oxidation by exogenous compounds or their metabolic derivatives. Antioxidant protein 2, the product of the PRDX6 gene, is present in high concentrations in red cells and prevents methemoglobin formation. This member of the peroxiredoxin protein family binds to hemoglobin and prevents spontaneous and oxidant-induced methemoglobin formation. Mutations of this gene or its acquired deficiency are potential candidates responsible for congenital and acquired methemoglobinemia; however, to our knowledge, this has not been yet described.8 Reduction of Methemoglobin. The ferric form of methemoglobin can be reduced to the ferrous form via the following metabolic pathways. 1) Cytochrome b5 reductase (CYB5R3) pathway: Cytochrome b5 reductase (previously termed diaphorase and NADH methemoglobin reductase) is the only physiologically meaningful way of reduction of methemoglobin. Cytochrome b5 reductase, a housekeeping enzyme and a member of the flavoenzyme family of dehydrogenases–electron transferases, is involved in the transfer of electrons from reduced nicotine adenine dinucleotide (NADH) generated by glyceraldehyde-3-phosphate reduction in the glycolytic pathway to cytochrome b5.9,10 In red cells, NAD is reduced to NADH during glycolysis. Flavine adenine dinucleotide (FAD) is a noncovalently bound prosthetic group in the cytochrome b5 reductase, which acts as an acceptor and donor of electrons. NADH donates an electron and reduces FAD to FADH.11–13 In turn, FADH reduces the heme protein of the cytochrome b5, which donates an electron to reduce methemoglobin to ferrous hemoglobin (Fig. 25.2). Besides reducing methemoglobin to hemoglobin by donating an electron, cytochrome b5 also serves as an electron donor in cells with mitochondria. In these cells, a reaction catalyzed by stearyl–coenzyme A (CoA) desaturase transfers electrons from cytochrome b5 to stearyl-CoA in the outer mitochondrial membrane and endoplasmic reticulum.14 These reactions play an important role in fatty acid desaturation and drug metabolism.

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Neeraj Agarwal, Ronald L. Nagel, and Josef T. Prchal

hemoglobinemia, it is not clear which of these forms is most common. Nevertheless, even discounting endemic Navajo, − O2 Athabascan, and Yakutsk congenital methemoglobinemia ++ +++ − +++ Fe − O2 Fe Fe due to cytochrome b5 reductase deficiency, the pub(deoxy Hb) (oxy Hb) (methemoglobin) lished reports of congenital methemoglobinemia due to cytochrome b5 reductase deficiency far exceed cases of Figure 25.1. Generation of methemoglobin from hemoglobin: O2 binds easily HbM. Furthermore, in our experience in a referral laboto the ferrous form of iron present in hemoglobin (Hb Fe2+ ) to form oxyhemoglobin. During this process, one electron is partially transferred from ratory, more than 20 subjects with unrelated hereditary iron to the bound O2 , forming a ferric–superoxide (Fe3+ –O2 ) anion complex. methemoglobinemia were due to cytochrome b5 reductase At the time of tissue delivery of O2 , some of the O2 leaves as a superoxide deficiency compared with only one case of HbM. Only a (O2 − ) radical. The partially transferred electron is not returned to the iron single, well documented family with methemoglobinemia moiety, thus leaving the iron in the ferric state and forming methemoglobin (and other systemic defects) due to cytochrome b5 defi(Hb Fe3+ ). ciency has been reported. Cytochrome b5 Reductase Deficiency. The gene for 2) NADPH-methemoglobin reductase pathway: Norcytochrome b5 reductase (CYB5R3) is on chromosome mally NADPH, an essential coenzyme for this reduc22 and is 31 kb in length, containing nine exons and tase (an electron donor), is generated by glucoseeight introns.24,25 There are more than 403,26–28 mutations 6-phosphate dehydrogenase (G6PD) in the hexose described that result in either type I or type II cytochrome monophosphate (pentose phosphate) shunt. There is b5 reductase deficiency. Although most of the mutants no electron carrier in red blood cells to accept an elechave been found in Caucasians, five unique mutations tron directly from NADPH-methemoglobin reducwere found in Chinese,29 at least three in Thai,30,31 two in tase. This is possible, however, in the presence of the African Americans,32 and one in an Asian Indian.33 The pharmacologically provided electron acceptors such cytochrome b5 reductase mutations that cause endemic as methylene blue or riboflavin that can link electron methemoglobinemia in Yakutsk, Navajo, and Athabascans transfer from NADPH coenzyme of this reductase to have not been defined, due in part to the reluctance of tribal methemoglobin.15–18 Because these electron accepauthorities to consent to studies. An additional mutation of tors are not physiological, NADPH-methemoglobin cytochrome b5 reductase (T116S) is not a disease-causing reductase does not play a role in vivo in the reducmutation but a high-frequency African-specific polymortion of methemoglobin. Nonetheless, this is a very phism that does not cause any appreciable disruption of the important pathway during treatment of toxic methecytochrome b5 reductase secondary structure.34 moglobinemia when the physiological pathway is The cytochrome b5 reductase gene has several potenoverwhelmed (Fig. 25.3). tial transcripts generating multiple isoforms. Differences at the 5
end of rat liver and reticulocyte cDNAs indicate Hereditary Methemoglobinemia the use of alternative promoters for the production of all forms of cytochrome b5 reductase.35 Two putative promotIn 1845, Francois19 reported the first case of congenital ers with different characteristics have been well described. methemoglobinemia in a patient with chronic congenital A constitutive promoter region has similarities with housecyanosis without obvious cardiac or pulmonary disease. A keeping genes as it does not contain a TATA box or CAAT familial incidence of “autotoxic cyanosis” and methemobox but instead contains five GC box sequences (GGGCGG globinemia was later described by Hitzenberger20 and and CCGCCC), representing potential binding sites for the accounts of the early work on this disorder have been transcription factor Sp1.25 The erythroid-specific promoter published.21–23 region contains several possible regulatory elements found There are three types of hereditary methemoglobinemia. in erythroid promoter regions, including a TATA box, CAATTwo, cytochrome b5 reductase deficiency and cytochrome like sequences, two binding sites for the erythroid-specific b5 deficiency, are inherited as autosomal recessive traits. transcription factor GATA-1, and a GT box.36 Two additional The third type, inherited as an autosomal dominant dispromoter regions have been identified but not yet fully order, is caused by globin gene mutations (HbsM), is discharacterized.37 cussed in Chapter 24. Due to the rarity of hereditary metMultiple isoforms of cytochrome b5 reductase are generated from a single gene by ++ Fe NAD+ FADH2 Fe+++ Hemoglobin a combination of alternative promoters and Glycolysis alternative initiation of translation.36–38 One Cb5 Reductase Cb5 isoform, found in nonerythroid cells and Fe+++ NADH Fe++ FAD Methemoglobin reticulocytes but not in mature erythrocytes, is a membrane-associated isoform located on Figure 25.2. Physiological reduction of methemoglobin to hemoglobin: NADH is generated from 3+ 2+ the endoplasmic reticulum membrane and glycolysis and reduces FAD to FADH2 . FADH2 reduces ferric (Fe to ferrous (Fe ) form in the the outer mitochondrial membrane.39,40 This cytochrome b5, which in turn reduces methemoglobin (Fe3+ ) to hemoglobin (Fe2+ ). O2

Dyshemoglobinemias

609 6 Phospho-Gluconate

Hexose Monophosphate G6PD Shunt

NADPH

G6PD

Glucose 6 Phosphate

NADP+

Methemoglobin +++ Fe Methylene blue Hemoglobin ++ Fe Pharmacologic presence

Figure 25.3. Reduction of methemoglobin by methylene blue: NADPH is generated by glucose-6-phosphate dehydrogenase (G6PD) in the hexose monophosphate shunt (pentose phosphate pathway). NADPH, in the presence of pharmacologically supplied electron acceptor such as methylene blue, reduces methemoglobin (Fe3+ ) to hemoglobin (Fe2+ ).

membrane-bound isoform consists of a 275–amino acid hydrophilic moiety that contains the active site and a hydrophobic domain at the N-terminal end that anchors the protein to the membrane.41–43 Two other isoforms are found only in erythroid cells. The isoform involved in methemoglobin reduction in erythroid cells is soluble and consists of the same 275 hydrophilic amino acids found in the ubiquitously expressed membrane-associated isoform.18,44 The other erythroid-specific isoform is membrane-associated with an N-terminal hydrophobic sequence that differs from the sequence found in the ubiquitously expressed isoform.36 Although it contributes only 20%–25% of erythrocyte cytochrome b5 reductase activity in adult humans, it represents a greater proportion in infants.45 In type I cytochrome b5 reductase deficiency (see later) only erythrocytes have decreased enzyme activity. This enzyme is heat labile and presumably is unstable and easily degraded. Thus, although cytochrome b5 reductase is abnormal in all cells in type I deficiency, only mature red cells, which cannot synthesize proteins and replace the enzyme, are significantly affected. Because cytochrome b5 reductase is coded by a single gene, it is hypothesized that type I cytochrome b5 reductase deficiency results from mutations producing an unstable enzyme, whereas mutations in type II deficiency (see later) either affect the catalytic function or cause underproduction of the enzyme, resulting in a generalized decrease in functional cytochrome b5 reductase activity.46 Type I Cytochrome b5 Reductase Deficiency. Most cases of enzymopenic congenital methemoglobinemia are type I, in which the functional deficiency of cytochrome b5 reductase is limited to erythrocytes. Homozygotes or compound heterozygotes47,48 have methemoglobin concentrations of 10%–35% and appear cyanotic. Homozygotes, because of the chronic nature of their methemoglobinemia, are usually physiologically adapted to their disease state and are asymptomatic even with methemoglobin levels up to 40%.49 Headache and easy fatigability are occasionally reported symptoms. Life expectancy is normal and pregnancy is unaffected. Significant compensatory erythrocytosis (polycythemia) is rarely observed. The cyanosis is of cosmetic significance only and can be treated with methylene blue or ascorbic acid, both of which facili-

tate the reduction of methemoglobin through alternate pathways.50 In contrast to the asymptomatic, chronically methemoglobinemic homozygotes (or compound heterozygotes) for type I cytochrome b5 reductase deficiency, heterozygous individuals are at risk for developing acute, symptomatic methemoglobinemia after exposure to exogenous methemoglobin-generating agents. Cohen and colleagues51 report of acute toxic methemoglobinemia in US military personnel receiving malarial prophylaxis in Vietnam is a classic description of this phenomenon. This form of cytochrome b5 reductase deficiency is distributed worldwide but is endemic in the Athabascan Alaskans,52,53 Navajo Indians,54 and Yakutsk natives of Siberia.55,56 The Navajo Indians and the Athabascan Indians of Alaska are known to share a common ancestor; thus, the high frequency of cytochrome b5R deficiency suggests a common origin for all three of these populations. It remains to be determined if the molecular defect resulting in cytochrome b5R deficiency is identical in these populations. In other ethnic and racial groups the defect occurs sporadically. Although cyanosis is difficult to detect due to skin pigmentation, type I deficiency has been reported in two unrelated African American families.57 Point mutations in patients with type I cytochrome b5R deficiency all have been missense mutations, resulting in an amino acid substitution. Most of these mutations are found in the 5
end of the cytochrome b5 reductase gene.58,59 A one mutation, a G to A transition in exon 8 (E212K) in an African American family, occurs in the 3
end.32 As discussed, the T116S polymorphism in exon 5 in African Americans with type I cytochrome b5 reductase deficiency34 is unlikely to be the disease-causing mutation as the amino acid substitution causes no readily appreciable disruption of the enzyme secondary structure and is not associated with methemoglobinemia. Rather, it is one of the most common African-specific polymorphisms known. Type II Cytochrome b5 Reductase Deficiency. Unlike type I mutations that are confined to the red cell, type II mutations are associated with cytochrome b5 reductase deficiency of all cells. Type II cytochrome b5 reductase deficiency represents 10%–15% of cases of enzymopenic congenital methemoglobinemia and has a sporadic

610 distribution. In addition to methemoglobinemia and cyanosis, the affected infants have mental retardation and developmental delay with failure to thrive and typically die during the first year of life.60 Other associated neurological features include microcephaly, opisthotonus, athetoid movements, strabismus, seizures, and spastic quadriparesis. The mechanism resulting in the neurological problems is currently unknown but might be due to abnormal lipid elongation and desaturation in the central nervous system61 or perhaps due to impairment of ferric-iron reduction of other cellular globins.57 Type II mutations have been reported in both the 5
and 3
ends of the cytochrome b5 reductase gene62–68 and are heterogeneous, including deletions, point mutations, splicing site mutations, and premature stop codons. Cytochrome b5 Deficiency. Deficiency of cytochrome b5 is an extremely rare disorder that causes congenital methemoglobinemia. Only one well-documented case of cytochrome b5 deficiency has been described, compared with more than 500 reported cases of cytochrome b5 reductase deficiency,69 and the large number of uncounted subjects affected in the endemic areas. This patient, a product of an Israeli consanguineous marriage, was also a male pseudohermaphrodite. Further analysis revealed that he was homozygous for a splicing mutation in the cytochrome b5 gene, resulting in a premature stop codon and a truncated protein molecule.70 Another family with probable cytochrome b5 deficiency was described prior to the recognition of this entity;71 however, the inheritance pattern in this family was autosomal dominant.

Acquired Methemoglobinemia Most cases of methemoglobinemia are acquired, resulting from increased methemoglobin formation due to exposure to exogenous agents (Table 25.1).72–75 Medication overdose or chemical ingestion causes methemoglobinemia by increasing the production of methemoglobin. Perhaps even more often, standard doses of medication cause methemoglobinemia in either normal individuals or those with partial deficiencies of cytochrome b5 reductase.51,76 Use of multiple drugs together has also been implicated in the development of methemoglobinemia.77 In one study of 138 cases of acquired methemoglobinemia, use of dapsone accounted for 42% of all affected patients, with a mean level of methemoglobin of 7.6% (range 2%– 34%).78 Xylocaine and related compounds appear to be the most common cause of drug-induced acquired methemoglobinemia; the most severe cases were seen after the use of 20% benzocaine spray for topical anesthesia (mean peak methemoglobin level 44%, range 19%–60%). Benzocaine is used during commonly performed endoscopic procedures such as bronchoscopy and endoscopy.79 The estimated incidence in a population undergoing transesophageal echocardiography was 0.115%.80 The molecular mechanism underlying this association has not been

Neeraj Agarwal, Ronald L. Nagel, and Josef T. Prchal Table 25.1. Drugs and agents that might cause methemoglobinemia Anesthetics (local): benzocaine, lidocaine, procaine, prilocaine Antimalarials: chloroquine, primaquine, quinacrine Aniline dyes Chlorates Dapsone Diarylsulfonylureas Doxorubicin Metoclopramide Nitric and nitrous oxide Nitrates and nitrites (amynitrate, isobutyl nitrite, nitroglycerine, sodium and silver nitrate) Nitrobenzenes (shoe and floor polish and in paint solvents) Nitroethane (nail polish remover, propellent, fuel additive) Nitrofurantoin (furadantin) Pyridium (phenazopyridine) Phenacetin (acetaminophen) Phenylhydrazine Rasburicase Sulfonamides (sulfacetamide, sulfamethoxazole, sulfanilamide, sulfapyridine)

elucidated, as previous or subsequent exposure might not be associated with methemoglobinemia. The true incidence of drug-induced methemoglobinemia due to less frequently used agents is not known. Furthermore, it is unknown whether the development of methemoglobinemia is dose related or idiosyncratic. During surgery, methemoglobinemia should be suspected in the presence of clinical “cyanosis,” a normal paO2 and/or the presence of “chocolate brown blood.” Infants, especially those born premature, are particularly susceptible to development of methemoglobinemia because their erythrocytes contain only 50%–60% of adult cytochrome b5 reductase activity.81–83 Although cytochrome b5 reductase levels rise to those of an adult within months of birth, young infants are unusually vulnerable to developing toxic methemoglobinemia following exposure to a number of otherwise relatively harmless medications, local ointments, and dyes used on diapers. This problem can be especially severe upon ingestion of nitrates. Nitrates do not oxidize hemoglobin directly; intestinal bacteria convert the nitrates to nitrites, which then oxidize hemoglobin to methemoglobin. In the United States, formula and food prepared from well water contaminated with nitrates have been implicated in development of methemoglobinemia in infants and children.84–86 The nitrate nitrogen concentration of water should be less than 10 ppm to avoid methemoglobinemia. Methemoglobinemia does not occur in breast-fed infants of mothers who ingest nitrate-contaminated water because nitrates do not concentrate in breast milk. Methemoglobinemia has also been associated with diarrheal illnesses in infants without known exposure to toxins.87,88 The exact mechanism leading to methemoglobinemia is unknown but could be

Dyshemoglobinemias due to metabolic acidosis induced by diarrhea, increased endogenous nitrite production, milk intolerance, or unique bacterial pathogens producing nitrites.89 Other predisposing factors associated with development of methemoglobinemia in infants with diarrhea are low admission weight percentiles, failure to thrive, and diarrhea lasting for more than 7 days.90,91 Administration of over-the-counter local anesthetic for teething has also been reported to cause methemoglobinemia in infants.92 Systemic infection can predispose to methemoglobinemia. NO is a free radical that is synthesized from L-arginine by NO synthases (Chapter 10). During bacteremia and sepsis, proinflammatory cytokines and bacterial lipopolysaccharide stimulate the production of inducible nitric oxide synthase (NOS2) from a variety of cell types including endothelial cells.93–95 Increase in circulating NO leads to increased production of methemoglobin.96,97 Symptomatic methemoglobinemia can also occur as a result of concomitant use of drugs predisposing to methemoglobinemia during infection and inflammation. Methemoglobinemia has been described in a burn patient with necrotizing fasciitis after prolonged application of topical 0.5% silver nitrate solution.98 Methemoglobin production is enhanced by reactions with free radicals and endogenous compounds including H2 O2 . Reduced glutathione (GSH) in red cells helps in decreasing the level of methemoglobin by eliminating free radicals and H2 O2 . Intercellular parasites such as Plasmodium sp. are associated with reduced levels of erythrocyte GSH. A progressive decrease in GSH level has been shown to occur with increasing parasitemia.99 Oxidative stress caused by medications used to treat malaria such as primaquine can also contribute to development of methemoglobinemia. Prophylaxis with primaquine alone, in absence of malarial infection, has been shown to cause mild asymptomatic methemoglobinemia.100

Clinical Features of Methemoglobinemia Most individuals with congenital, chronically elevated methemoglobin concentrations are generally asymptomatic even with methemoglobin levels up to 40% of total hemoglobin.49 Headache and easy fatigability are occasionally reported. The main clinical feature is “cyanosis” or a slateblue color of the skin and mucous membranes, a finding that is due to the different absorbance spectrum of methemoglobin compared with oxyhemoglobin and is of only cosmetic importance. Significant polycythemia (erythrocytosis) is rarely observed.51 Life expectancy is not shortened and pregnancies occur normally. Although cyanosis associated with cytochrome b5 reductase deficiency is readily corrected with methylene blue, this therapy is not effective for methemoglobinemia due to HbM disease. In comparison to the general lack of symptoms in patients with chronic methemoglobinemia, patients with acute acquired methemoglobinemia are symptomatic due

611 to acutely impaired O2 delivery to tissues that does not allow sufficient time for physiological compensation to occur. Early symptoms include headache, fatigue, dyspnea, and lethargy. At higher levels, respiratory depression, altered consciousness, shock, seizures, and death can occur.73 Most individuals presenting with acute acquired methemoglobinemia are not heterozygous for cytochrome b5 reductase deficiency.101 Cyanosis is clinically detected when the absolute concentration of methemoglobin exceeds 1.5 g/dL, which is equivalent to 8%–12% methemoglobin at normal hemoglobin concentrations.49 In contrast, the more common cause of cyanosis, due to decreased hemoglobin O2 saturation, is observed when the absolute level of deoxygenated hemoglobin exceeds 4–5 g/dL, as in severe respiratory failure or cardiac abnormalities due to right-to-left shunts. This form of cyanosis cannot be clinically differentiated from that due to methemoglobinemia, and laboratory analysis is required for its distinction from methemoglobinemia and sulfhemoglobinemia (see later). The general lack of symptoms other than cyanosis, and the normal life expectancy in patients with cytochrome b5 reductase deficiency applies only to the more common type I disease in which the enzymatic deficiency is limited to the red cells. All cells are affected in patients with type II disease who exhibit neurological and developmental abnormalities.60

Diagnosis of Methemoglobinemia Methemoglobinemia can be clinically suspected by the presence of clinical “cyanosis” in the face of normal paO2 . Unlike deoxyhemoglobin, the dark color of the blood in methemoglobinemia does not change with the addition of O2 . Historically pulse oximetry has been considered inaccurate in monitoring O2 saturation in the presence of methemoglobinemia; however, the presence of methemoglobin can be suspected when the O2 saturation as measured by pulse oximetry is significantly less than the O2 saturation calculated from arterial blood gas analysis (“saturation gap”).78,102 The laboratory diagnosis of methemoglobinemia is based on analysis of its absorption spectra, which have peak absorbance at 631 nm. A fresh specimen should always be obtained, as methemoglobin levels tends to increase with storage. The standard method of assaying methemoglobin utilizes a microprocessor-controlled, fixedwavelength cooximeter. This instrument interprets all readings in the 630-nm range as methemoglobin so that false positives can occur in the presence of other pigments including sulfhemoglobin and methylene blue.103,104 Hence, methemoglobin detected by the cooximeter should be confirmed by the specific Evelyn–Malloy method.105 This assay involves the addition of cyanide, which binds to the positively charged methemoglobin, eliminating the peak at 630–635 nm in direct proportion to the methemoglobin

612 concentration. Subsequent addition of ferricyanide converts the entire specimen to cyanomethemoglobin for measurement of the total hemoglobin concentration. Methemoglobin is then expressed as a percentage of the total concentration of hemoglobin. Recently, a new eight-wavelength pulse oximeter, Masimo Rad-57 (the Rainbow-SET Rad-57 Pulse COOximeter, Masimo Inc, Irvine, CA), has been reported to be accurate in measuring carboxyhemoglobin and methemoglobin. The Rad-57 uses eight wavelengths of light instead of the usual two and is thereby able to measure more than two species of human hemoglobin.106 It is approved by the US Food and Drug Administration for the measurement of both carboxyhemoglobin and methemoglobin. In addition to the usual SpO2 value, the Rad-57 displays SpCO and SpMet, which are the pulse oximeter’s estimates of carboxyhemoglobin and methemoglobin percentage levels, respectively. In a study on healthy human volunteers in whom controlled levels of methemoglobin and carboxyhemoglobin were induced, the Rad-57 measured carboxyhemoglobin with an uncertainty of ± 2% within the range of 0%–15% and measured methemoglobin with an uncertainty of 0.5% within the range of 0%–12%.106 Distinguishing the hereditary forms of congenital methemoglobinemia requires interpretation of family pedigrees followed by biochemical analyses. Cyanosis in successive generations suggests the presence of HbM disease, whereas normal parents but possibly affected siblings imply the presence of the autosomal recessive cytochrome b5 reductase deficiency or, rarely, cytochrome b5 deficiency. Incubation of blood with methylene blue distinguishes cytochrome b5 reductase deficiency from HbM disease as this results in the rapid reduction of methemoglobin through the NADPH–flavin reductase pathway in cytochrome b5 reductase deficiency.107–109 Measurement of the level of cytochrome b5 reductase activity, or cytochrome b5 if cytochrome b5 reductase activity is normal, is required to distinguish cytochrome b5 reductase deficiency from cytochrome b5 deficiency; however, the assay for cytochrome b5 is not commercially available. Assays of Enzyme Activity. Types I and II cytochrome b5 reductase deficiency are distinguished by clinical phenotype and analysis of enzymatic activity in erythroid and nonerythroid cells. Reports of decreased cytochrome b5 reductase activity are difficult to compare because several different assays of cytochrome b5 reductase activity, varying in their substrate and in their normal values, have been used.6,47,57,65,66,110,111 These assays also vary in their technical difficulty. The first widely accepted cytochrome b5 reductase activity assay used a difficult to produce and standardize methemoglobin–ferrocyanide complex and its reduction by an enzyme containing tissue homogenate.110 The most rigorous cytochrome b5 reductase enzyme activity is based on partial purification of the

Neeraj Agarwal, Ronald L. Nagel, and Josef T. Prchal enzyme by ultracentrifugation and uses the physiological enzyme substrate (cytochrome b5 prepared by a recombinant DNA technology)42 ; this assay is not readily available and is too complex for nonspecialized research laboratories. The most convenient assay uses readily available ferricyanide111 and easily differentiates type I and type II cytochrome b5 reductase deficiency because patients with type I deficiency have normal enzyme activity in platelets, fibroblasts, Epstein–Barr virus–transformed lymphocytes, and granulocytes whereas, in type II deficiency, the activity in nonerythroid tissues is markedly to moderately decreased.32,57,64,111 Because the enzyme defect is also found in fibroblasts and amniotic cells, analysis of cytochrome b5 reductase activity in cultured amniotic cells for the purpose of prenatal diagnosis of type II disease is possible.112,113 Two families with “type III” deficiency have been described in whom cytochrome b5 reductase activity was allegedly decreased not only in erythrocytes but in also in platelets and leukocytes.114,115 The existence of this entity is difficult to accept because these individuals did not exhibit the neurological abnormalities characteristic of type II deficiency. Reevaluation of one of the patients with a rigorous assay using recombinant cytochrome b5 confirmed sufficient b5 reductase activity in the platelets, leukocytes, and fibroblasts and this analysis was consistent with the presence of type I deficiency.116

Treatment of Methemoglobinemia Treatment of methemoglobinemia depends on the clinical setting. Is the onset acute and due to drugs or other toxic agents or is there congenital life-long methemoglobinemia? All patients with hereditary methemoglobinemia should avoid exposure to aniline derivatives, nitrates, and other agents that could, even in normal individuals, induce methemoglobinemia (Table 25.1). Known heterozygotes for cytochrome b5 reductase deficiency should be similarly counseled.117 Treatment of HbM disease is neither possible nor necessary. Hereditary methemoglobinemia. In most cases, congenital methemoglobinemia results in chronic cyanosis, except when acute methemoglobinemia occurs in heterozygotes for cytochrome b5 reductase deficiency, in which case it should be treated like acquired methemoglobinemia. Treatment of the cyanosis in these individuals is indicated for cosmetic reasons only. Options include methylene blue (100–300 mg/day orally) or ascorbic acid (300– 1,000 mg/day orally in divided doses). Concerns about kidney stone formation with ascorbic acid therapy remain unproven but high-dose therapy can be associated with the theoretical nephrolithiasis risk. Riboflavin (20–30 mg/day) has also been used with some success,118 although clinical experience with its use is very limited. Although effective for the cyanosis, neither methylene blue nor ascorbic acid has any effect on the neurological abnormalities in type II

Dyshemoglobinemias disease. Theoretically, a bone marrow or liver transplant would alleviate these neurological problems if they were due to a problem with circulating fatty acids; however, these approaches have not yet been tested. Acquired Methemoglobinemia. Offending agents in acquired methemoglobinemia should be discontinued (Table 25.1). This condition can be life-threatening when methemoglobin comprises more than 30% of total hemoglobin. Blood transfusion or exchange transfusion might be helpful in patients who are in shock. In lesser degrees of methemoglobinemia, no therapy other than discontinuation of the offending agent(s) might be required. If the patient is symptomatic, which is often the case in deliberate or accidental overdoses or toxin ingestion, specific therapy is indicated. Methylene blue is given intravenously in a dose of 1– 2 mg/kg over 5 minutes. Methylene blue, as mentioned previously, provides an artificial electron acceptor for the reduction of methemoglobin via the NADPH-dependent reductase pathway (Fig. 25.4). The response is usually rapid; the dose can be repeated in hourly, but this is frequently not necessary. Maximum cumulative dose should not exceed 7 mg/kg as overdose can cause dyspnea and chest pain, as well as hemolysis in some susceptible subjects.119,120 Because cooximetry detects methylene blue as methemoglobin, this technique cannot be used to follow the response of methemoglobin levels to treatment with methylene blue. If needed, the more specific Evelyn–Malloy method should be used to discriminate between methemoglobin and methylene blue and to follow response to therapy with methylene blue. Methylene blue should not be administered to patients with G6PD deficiency because the reduction of methemoglobin by methylene blue is dependent on NADPH generated by G6PD (Fig. 25.3). As a result, methylene blue can be ineffective and is potentially dangerous because it has oxidant potential that could further produce hemolysis.121 To avoid these problems, pretreatment screening of populations with a high incidence of G6PD deficiency (e.g., African Americans, patients of Mediterranean descent, and southeast Asians) is reasonable, although not usually practical. If methylene blue is contraindicated, only moderate doses of ascorbic acid, 300–1,000 mg/day orally in divided doses, should be given, as this drug can also cause oxidant hemolysis in G6PD-deficient patients when given in very high doses. Hyperbaric O2 and exchange transfusion have been used with success in severe cases.122 Marked methemoglobinemia can occur after treatment of dermatitis herpetiformis or Pneumocystis carinii infection with dapsone. Cimetidine, used as a selective inhibitor of N-hydroxylation, might be effective in this setting when taken on a regular basis and lowers the methemoglobin level by more than 25%.117,123 Because it works slowly, cimetidine is not helpful for the management of acute symptomatic methemoglobinemia arising from the use of dapsone.

613

A

B

Figure 25.4. CO binding to myoglobin. (A) Binding of CO at the heme group. (B) CO binding to heme is possible because the E helix (and His 64) moves away, making space for the CO molecule without the need for bending.

CARBON MONOXIDE POISONING: CARBOXYHEMOGLOBINEMIA CO, a toxic gas, is unusually dangerous because it is odorless, colorless, and tasteless, increasing the probability of serious and life-threatening accidents when high concentrations are unknowingly present in the environment.124

Epidemiology CO intoxication is one of the most common causes of morbidity due to poisoning in the United States. Although deaths from CO poisoning had decreased in the United States, the total burden (fatal and nonfatal) might not have significantly changed in the past two decades.125 In the United States, CO poisoning results in approximately 40,000

614 emergency department visits per year.126,127 Approximately 500 accidental deaths due to CO poisoning are reported to occur annually in the United States and the number of intentional CO-related deaths is 5–10 times higher.128,129 To examine unintentional, nonfire-related CO exposures, the Centers for Disease Control and Prevention analyzed 2001–2003 data on emergency department visits from the National Electronic Injury Surveillance System All Injury Program and 2001–2002 death certificate data from the National Vital Statistics System.130 This analysis determined that each year, approximately 15,000 U.S. residents’ visit emergency departments for unintentional, non-fire-related CO exposure and approximately 500 die from this exposure. Primary sources of CO were home appliances, and the majority of exposures occurred during the fall and winter months, when persons are more likely to use gas furnaces and heaters. During warmer months, boating activities were another source of exposure. Males were more likely to die from CO poisoning than females, which was consistent with previous findings.131 Death rate was highest among persons aged older than 65 years. The increased death rate in elderly can be attributed to delayed diagnosis because symptoms often resemble those of associated comorbidities.132 The exhaust produced by the typical home-use 5.5-kW generator contains as much CO as that of six idling automobiles.133 A survey of generator use after the 2004 Florida hurricane season reported that 4.6% of persons using generators had used them inside their home or garage. During this 2004 Florida hurricane season (13 August–25 September 2004), there were 167 persons requiring treatment for CO poisoning in 10 hospitals and six deaths were attributed to CO poisoning.134 Although most of the aforementioned causes more often result in acute CO intoxication, cigarette smoking is a common cause of chronic CO intoxication and can increase the COHb level by as much as 15%. By increasing hemoglobin– O2 affinity, COHb can cause erythrocytosis (polycythemia) in smokers. Houses with defective heating exhaust systems and vehicles that leak CO into the passenger compartment, either because of mechanical failure or driving with the rear hatch-door open, are the second most common cause of chronic CO exposure. Occupations that involve a high risk for CO intoxication include garage work with improper ventilation, toll booth attendants, tunnel workers, firefighters, and workers exposed to paint remover, aerosol propellant, or organic solvents containing dichloromethane.135

Pathophysiology of CO Poisoning CO has low solubility in water and is relatively inert because of its extremely high bond enthalpy. Still, it combines with high affinity with the heme of hemoglobin – and with lesser affinities to myoglobin and cytochromes – at the iron core, a site that it shares with O2 .136 Its affinity to cytoglobin and neuroglobin has not yet been studied. Binding of CO to

Neeraj Agarwal, Ronald L. Nagel, and Josef T. Prchal hemoglobin is the basis of its toxicity. An excellent summary of CO chemistry is provided by Bunn and Forget.137 At equilibrium in physiological conditions, CO affinity for hemoglobin is approximately 240 times greater than that of O2 . This very high equilibrium constant is the result of reaction kinetics. Contrary to popular belief, CO reacts more slowly than O2 with the heme of hemoglobin. At 20◦ C and pH 7.0, the “on” rate for CO is 20 mol/L/sec compared with 470 mol/L/sec for O2 . The steric constraints present in the heme pocket make it more difficult for CO to reach the heme group. Indeed, x-ray crystallography and neutron crystal structure showed that the Fe-C-O geometry was not “linear,” but “bent,” an unfavorable position for CO binding.138 Once CO is bound to heme, its “off” rate is only 0.015 mol/L/sec in contrast to 35 mol/L/sec for O2 .136 This extraordinarily slow-release process produces a very high affinity constant of CO for heme and a lifethreatening danger for individuals exposed to high levels of CO. An alternative picture has been proposed of the steric mechanisms involved in the inhibition of CO binding to heme proteins.139 These investigators compared unliganded myoglobin and CO myoglobin at a resolution of ˚ and found perfect linearity of the Fe-C-O complex, 1.15 A, not a “bent” configuration. This geometry was possible because a concerted motion of heme, iron, and helices E and F relieved the steric constraints (Fig. 25.4). Once two molecules of CO are bound to hemoglobin, the molecule switches to the relaxed (R) state, further endangering those exposed to CO, and the two globin chains that remain capable of binding O2 will be in their high-affinity conformation. This high ligand affinity will make more difficult the delivery of O2 to the tissues by the remaining O2 binding sites. As a consequence of this phenomenon, called the Darling– Roughton effect,1 the hemoglobin O2 affinity increases in parallel with increasing CO levels. In the absence of environmental CO, the blood of adults contains approximately 1%–2% COHb. This represents approximately 80% of the total body CO, the remainder probably sequestered in myoglobin and other heme binding proteins. This CO is endogenously produced,140 originating from the degradation of heme by the rate-limiting heme oxygenase–cytochrome P-450 complex, which produces CO and biliverdin. CO levels in expired air have been used as an index of hemolysis, although the difficulty of standardization and the availability of simpler means of assessing red cell destruction have hampered the widespread use of this technique.141,142 Further degradation of biliverdin to bilirubin by biliverdin reductase renders a stoichiometry in which one molecule of hemoglobin, by oxidation of the ␣-methane bridge of the tetrapyrrole ring, generates one molecule of CO and one molecule of biliverdin. Endogenous CO levels differ among individuals. Caloric restriction, dehydration, infancy, and the genetic variations reported in Japanese and Native Americans generate higher endogenous levels of CO. Hemolytic anemia,

Dyshemoglobinemias hematomas, and infection tend to increase CO production up to threefold. Fetuses and newborns have double the normal adult levels of COHb, and levels can be much higher with hemolytic disease of the newborn. Not all endogenous CO is the product of normal metabolism. Drugs such as diphenylhydantoin and phenobarbital, by inducing the cytochrome P-450 complex, increase CO production, as does any drug that causes hemolysis. Endogenous CO production might have important physiological consequences.143 Like its analog, NO, CO can bind to the heme of soluble guanylate cyclase and to the iron/sulfur centers of macrophage enzymes. Guanylate cyclase regulates cyclic guanosine-3
,5
-monophosphate, a second messenger involved in many cellular functions. The exact physiological significance of CO in these reactions remains to be determined. Exogenous sources of CO include atmospheric CO, which is a product of incomplete combustion and oxidation of hydrocarbons, and natural sources.144 High-altitude polycythemia (erythrocytosis) and hypoxemia prolong CO excretion that increases COHb levels and further compounds our imperfect adaptation to high altitude associated with decreased tissue O2 delivery.145 Although the normal adult level of COHb is less than 1% or 2%, this low concentration is rarely found in urban centers because of air pollution. Hemolysis can produce COHb levels of more than 2%. Levels more than 3% must have an exogenous origin, except for rare conditions as occur in carriers of abnormal hemoglobins such as Hb Zurich. The affinity of Hb Zurich for CO is approximately 65 times that of normal hemoglobin.146 The U.S. Environmental Protection Agency considers an acceptable exposure time to be 1 hour to 25 ppm CO or an 8 hours exposure to 9 ppm. This exposure would raise the COHb by 1.5%. Pregnant women and fetuses are particularly at risk,147 because they already have higher levels of COHb. CO readily crosses the placenta and half life of CO in the fetus is as much as five times longer than it is in the mother. This results in approximately 15% higher fetal COHb level than the maternal COHb level.148 The O2 affinity of HbF is shifted to the left149,150 owing to its lack of 2,3-BPG binding, making the Darling–Roughton effect particularly pernicious, yet another reason why cigarette smoking during pregnancy is hazardous to the fetus.

Clinical Features of CO Poisoning Acute intoxication with CO rapidly affects the central and peripheral nervous systems and cardiopulmonary functions. Cerebral edema is common, as are alterations of sensory and peripheral nerve function. CO induces increased permeability in the lung resulting in acute pulmonary edema. Cardiac arrhythmias, generalized hypoxemia, and respiratory failure are the common causes of CO-related death; COHb levels more than 40% are found in these cases. In survivors, considerable neurological deficits might

615 remain. Patients with less severe acute cases have the same type of clinical features as patients with chronic intoxication. Myocardial ischemia, lactic acidosis, convulsions, and coma can sometimes occur. An interesting complication observed several days after the exposure to CO are patches of necrotic skin induced by localized hypoxia. Levels of COHb that can elicit any of these symptoms vary widely among patients. Acute CO intoxication in children151 is responsible for approximately 400 deaths a year, is more severe, and sometimes has unique symptomatology resembling gastroenteritis. Surviving children are more likely to have severe sequelae such as leukoencephalopathy (white cerebral matter destruction), and severe myocardial ischemia.152 Chronic intoxication in adults might result in irritability, nausea, lethargy, headaches, and sometimes a flulike condition. Higher COHb levels produce somnolence, palpitations, cardiomegaly, and hypertension and could contribute to atherosclerosis. Chronic CO poisoning can produce erythrocytosis, the magnitude of which varies with the level of COHb. By increasing red cell production, chronic CO poisoning can mask the mild anemia of acquired or congenital hemolytic disorders.

Diagnosis and Treatment of CO Poisoning CO poisoning is a clinical diagnosis that is confirmed by laboratory testing. Signs and symptoms consistent with CO poisoning in certain circumstances should raise the suspicion of its presence. A higher index of suspicion should attend the simultaneous presentation of multiple patients from the same family or housing complex. The eightwavelength pulse oximeter (Masimo Rad-57) has been reported to be accurate in measuring CO.106 Because CO has a relatively short half-life in vivo, the levels obtained in the emergency setting often do not correlate directly with symptoms. The most important step in the treatment for CO poisoning is prompt removal of patients from the source of CO followed by administering 100% supplemental O2 via a tight-fitting mask. The CO molecule reversibly binds to hemoglobin and is eliminated through the lungs. The serum elimination half-life of CO is 5 hours when breathing room air and 30 minutes with O2 therapy (100% O2 at 3 atm).148 For mild to moderate cases of CO poisoning, which more often happens with chronic intoxication, removing the patient from the source of environmental CO is usually curative. If the COHb level is high, breathing 100% O2 will increase the rate of CO removal. In severe cases of CO poisoning, which more often occur with acute intoxication, after identification and removal of the source of CO, 100% O2 should be administered with cardiac monitoring. Endotracheal intubation should be done in any patient with impaired mental status and other interventions should be dictated by the symptomatology.

616 Hyperbaric O2 , which has complications of its own such as bronchial irritation and pulmonary edema, should be reserved for exceptional cases of CO intoxication. Most often, by the time the patient is brought into a hyperbaric chamber, the simple breathing of 100% O2 has reduced COHb sufficiently to make it unnecessary. Because of conflicting evidence, there is no absolute indication for the use of hyperbaric O2 treatment for patients with CO poisoning. Hyperbaric O2 might be indicated in patients who have obvious neurological abnormalities, have a history of loss of consciousness with their exposure, have cardiac dysfunction, have persistent symptoms despite normobaric O2 , or have metabolic acidosis.153 Locations of hyperbaric chambers throughout the world and in the United States can be found at the Undersea and Hyperbaric Medical Society web site, www.uhms.org under “chamber directory.” Pregnant women exposed to CO are at particularly high risk. CO poisoning is especially dangerous to the fetus because CO readily crosses the placenta and half-life of CO in the fetus is as much as five times longer than it is in the mother. For these reasons treatment with hyperbaric O2 should be carried during pregnancy when the COHb levels exceed 15%. In limited number of studies done on pregnant patients, hyperbaric O2 does not seem to adversely affect the fetus.154,155

SULFHEMOGLOBINEMIA Definition and Pathogenesis Sulfhemoglobin is a modification of the hemoglobin molecule that produces a bright green color because a sulfur atom is incorporated into the porphyrin ring. It has been associated with the use of certain “oxidant” medications, with drug abuse,156–161 with occupational exposure to sulfur compounds,162 and with exposure to polluted air.163 Heme modification by the addition of sulfur is associated with a drastic right shift in the hemoglobin–O2 dissociation curve in the physiologically relevant PO2 range, rendering the molecule totally ineffective for O2 transport.164 Fully sulfurated tetramers seem to have no cooperativity. In most cases, only a fraction of the hemes are modified and full saturation of the unaltered hemes in the lungs and enhanced O2 unloading in tissues would be expected. Because this right shift would ameliorate any decrease in functional hemoglobin mass, sulfuration of hemes might have little physiological significance as long as sufficient proportion of hemoglobin with some unmodified hemoglobin subunits remains. An understanding of the molecular basis of the rightshifted oxygenation curve emerges from isoelectric focusing data. In a clinical sample that contained only 12% modified heme165 the distribution of these hemes among partially sulfurated tetramers resulted in a larger percentage of the tetramers being abnormal. Between 24% and 48% of the hemoglobin tetramers in a clinical sample were

Neeraj Agarwal, Ronald L. Nagel, and Josef T. Prchal abnormal and contributed to the shift of the hemoglobin–O2 dissociation curve.166 The actual effect that these partially modified molecules have on this curve depends on their conformation. Because the half-sulfurated, halfliganded tetramers have an isoelectric point between deoxyhemoglobin and oxyhemoglobin and bind 2,3-BPG under conditions when oxyhemoglobin does not, their conformation must be closer to the T-state (deoxy state) than that of oxyhemoglobin. Whereas in methemoglobinemia some tetramers have subunits fixed in an oxidized R-like state (Darling–Roughton effect),1 in sulfhemoglobinemia some tetramers have subunits fixed in a deoxidized T-like state because they remain unliganded at physiological pO2 . In the former case, a left-shifted oxygenation curve and impaired O2 delivery results, whereas in the latter case, a right-shifted curve and enhanced O2 delivery occur. The slightly higher 2,3-BPG in the blood of patients with sulfhemoglobinemia probably reflects increased binding to the Tlike sulfurated tetramers rather than an actual increase in free 2,3-BPG.166,167

Clinical Presentation, Diagnosis, and Treatment Sulfhemoglobin in concentrations greater than 0.5 g/dL also causes “cyanosis” with a normal paO2 and might be erroneously measured as methemoglobin. Sulfhemoglobin can be distinguished from methemoglobin by its peak absorption at 620 nm, which unlike methemoglobin is not abolished by the addition of cyanide. Sulfhemoglobin and methemoglobin have been reported to coexist in a number of cases of drug-induced acute toxic methemoglobinemia.156–161 The lists of chemicals and drugs reported to produce these syndromes overlap. Acetanilid, usually in the form of Bromo Seltzer, and phenacetin were the main offenders in 62 cases of sulfhemoglobinemia seen at the Mayo Clinic in 1951.157 The aryl hydroxylamine metabolites of these drugs can serve as reducing agents in a cyclic process capable of generating both sulfhemoglobin165 and methemoglobin.168 The origin of the sulfur atom in the former case remains unclear, but both H2 S generated by intestinal flora169 and glutathione170,171 have been suggested. Laboratory assays used for detection of methemoglobinemia are often inadequate to detect sulfhemoglobin, so it is likely that sulfhemoglobinemia is underdiagnosed.172 Although acetanilid was removed from Bromo Seltzer a number of years ago and the Food and Drug Administration removed phenacetin from the United States market, sulfhemoglobinemia is not likely to vanish. Sulfonamides, dapsone, and sulfur-containing ointments are reported offenders162,173,174 and are still widely used. Some drugs reported to produce methemoglobin, including acetaminophen, might be found to also produce sulfhemoglobin when more careful analysis is done. Sulfhemoglobinemia, which has been associated with constipation, possibly because sulfides produced by an expanded intestinal flora when stasis exists, converts methemoglobin

Dyshemoglobinemias to sulfhemoglobin. Treatment of this cause of sulfhemoglobinemia is to treat constipation. Accidents involving occupational exposure to hydrogen sulphide (H2 S) are expected to increase.175 Because exposure of red blood cells to H2 S produces sulfhemoglobin within minutes, it is likely that occupational exposure to H2 S poisoning in industrial societies, particularly emerging ones, will increase. A fatal case of a shrimp fisherman exposed to high levels of ambient H2 S emanating from the dithionite-treated catch has been reported.165 In Alberta, Canada, there were 221 cases of exposure to H2 S over 4 years, with an overall mortality of 6%. Hospital admission was required for 65% of the victims.175 Acute problems were coma, ataxia, and respiratory insufficiency with pulmonary edema. Sulfhemoglobin is seldom suspected in this setting and thus likely underdiagnosed. Increased interest in occupational and environmental pollutants and screening of populations at risk could reveal many more subclinical cases. Although CO2 was thought to be the cause of death in the Lake Nyos African disaster that claimed nearly 2,000 lives as a cloud of toxic gases erupted from the bottom of this tropical lake, H2 S was among the liberated gases that could have modified hemoglobin and contributed to this calamity.176 Significant exposure to H2 S can occur in workers at sulphurous thermal baths. Blood sulfhemoglobin levels has been shown to be a reliable measure of individual exposure to H2 S.177 One case report described the false diagnosis of “methemoglobinemia,” which on further analysis, because of refractoriness to methylene blue, was found to be sulfhemoglobinemia.72 The patient had applied 4 oz of dimethyl sulfoxide to her abdomen to treat interstitial cystitis. Within 24 hours she developed fatigue, cyanosis, and dyspnea with mild exertion. Although sulfhemoglobinemia is probably a relatively nontoxic syndrome in individuals with HbA, it might prove to be a surprisingly toxic syndrome in individuals with HbS, especially compared with methemoglobinemia, which would be expected to ameliorate sickling because of left-shifted hemoglobin dissociation curve associated with methemoglobinemia. The presence of sulfurated subunits will shift the conformation of HbS tetramers toward the unliganded, polymerizing T form, even in the presence of high pO2 , thus leading to polymerization of HbS. Microvascular occlusion could be exacerbated by these tetramers remaining in the polymerizing conformation in both the arterial and venous circulation. This hypothetical scenario has yet to be reported. Congenital sulfhemoglobinemia has been reported but is rare and a fraction of sulfhemoglobin has been described with an unstable hemoglobin.137 Diagnosis of sulfhemoglobinemia is suspected when a patient who is cyanotic with normal to near-normal O2 tension and the clinical and laboratory diagnosis of methemoglobinemia does not respond to therapy with methylene blue. With the eight-wavelength pulse oximeter capable of measuring methemoglobin more accurately, diagnosis

617 of sulfhemoglobinemia will likely be made earlier without requiring waiting for therapy with methylene blue treatment to fail. With equivalent amounts of abnormal pigment, the patient with sulfhemoglobinemia appears more cyanotic than the patient with methemoglobinemia as a result of spectral differences between the pigments, but is less symptomatic as a result of the differences in P50 . This is fortunate, as there is no known treatment analogous to methemoglobin reduction for reconverting sulfhemoglobin to functional hemoglobin; exchange transfusion is the sole therapeutic option, if therapy is needed. In summary, sulfhemoglobin is a green-pigmented protein that can be accurately identified by spectrophotometry and isoelectric focusing. At low concentrations, sulfurated tetramers are shifted toward the deoxy or T form, producing a right shift of the O2 equilibrium curve. For this reason, dyspnea is absent unless the levels of sulfhemoglobin are extraordinarily high. Mild cases that are typically undiagnosed might be revealed in screening studies looking for toxic effects of drugs and environmental pollutants. Awareness of this entity and modern, widely used, simple equipment could allow us to determine the true prevalence of sulfhemoglobinemia.

CONCLUSIONS COHb, methemoglobin, and sulfhemoglobin are present in normal red cells in very low concentrations. Mutations or environmental conditions can increase the concentrations of these liganded or oxidized hemoglobins, producing a dyshemoglobinemia. Although rare, increased levels of dyshemoglobins can be life threatening so that their presence should be identified in a timely fashion to permit effective treatment. The distinction between environmental and genetic dyshemoglobinemia is often blurred because many acquired dyshemoglobins are, in effect, the consequence of both an environmental challenge and a genetic predisposition.

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Dyshemoglobinemias 116. Nagai T, Shirabe K, Yubisui T, et al. Analysis of mutant NADH-cytochrome b5 reductase: apparent “type III” methemoglobinemia can be explained as type I with an unstable reductase. Blood. 1993;81:808. 117. Beutler E. Methemoglobinemia and other causes of cyanosis. In: Beutler E, Lichtman MA, Coller B, Kipps, TJ, eds. Williams’ Hematology. 5th ed. New York: McGraw-Hill; 1995:654. 118. Kaplan J, Chirouze M. Therapy of recessive congenital methaemoglobinemia by oral riboflavin. Lancet. 1978;2:1043. 119. Goluboff N, Wheaton, R. Methylene blue induced cyanosis and acute hemolytic anemia complicating the treatment of methemoglobinemia. J Pediatr. 1961;58:86. 120. Harvey J, Keitt A. Studies of the efficacy and potential hazards of methylene blue therapy in aniline-induced methemoglobinemia. Br J Haematol. 1983;54:29. 121. Rosen P, Johnson C, McGehee WG, et al. Failure of methylene blue treatment in toxic methemoglobinemia: associations with glucose-6-phosphate dehydrogenase deficiency. Ann Intern Med. 1971;75:83. 122. Goldstein G, Doull J. Treatment of nitrite-induce methemoglobinemia with hyperbaric oxygen. Proc Soc Experimental Biology and Medicine. 1971;138:134. 123. Coleman M, Rhodes LE, Scott AK, et al. The use of cimetidine to reduce dapsone-dependent methaemoglobinaemia in dermatitis herpetiformis patients. Br J Clin Pharmacol. 1992;34:244. 124. Vreman H, Mahoney JJ, Stevenson DK. Carbon monoxide and carboxyhemoglobin. Adv Pediatr. 1995;42:303–325. 125. Hampson N. Trends in the incidence of carbon monoxide poisoning in the United States. Am J Emerg Med. 2005;23:838– 841. 126. Hampson N. Emergency department visits for carbon monoxide poisoning in the Pacific northwest. J Emerg Med. 1998;16:695–698. 127. Weaver L. Carbon monoxide poisoning. Crit Care Clin. 1999;15:297–317. 128. Ernst A, Zibrak JD. Carbon monoxide poisoning. N Engl J Med. 1998;339:1603–1608. 129. Centres for Disease Control and Prevention C. Carbon monoxide poisoning from hurricane-associated use of portable generators – Florida 2004. MMWR. 2005;54:697–700. 130. Centers for Disease Control and Prevention C. Unintentional non-fire-related carbon monoxide exposures–United States, 2001–2003. MMWR. 2005;54:36–39. 131. Mott J, Wolfe MI, Alverson CJ, et al. National vehicle emissions policies and practices and declining US carbon monoxide-related mortality. JAMA. 2002;288:988–995. 132. Harper A, Croft-Baker J. Carbon monoxide poisoning: undetected by both patients and their doctors. Age Ageing. 2004;33:105–109. 133. US Environmental Protection Agency Emission facts: Idling vehicle emissions. US Environmental Protection Agency, Washington, DC; 1998 Publication EPA420-F-98-014 1998. 134. Centers for Disease Control and Prevention Carbon monoxide poisoning from hurricane-associated use of portable generators – Florida 2004. MMWR. 2005;54:697–700. 135. Stewart R, Fisher TN, Hosko MJ, et al. Carboxyhemoglobin elevation after exposure to dichloromethane. Science. 1972;176:295–296. 136. Antonini E, Brunori M. Hemoglobin and myoglobin in their reactions with ligands. In: Neuberger A, Tatum EL, eds.

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622 156. Evans A, Enzer N, Eder HA, et al. Hemolytic anemia with paroxymal methemoglobnemia and sulfhemoglobinemia. Arch Intern Med. 1950;86:22. 157. Bradenburg R, Smith HL. Sulfhemoglobinemia: a study of 62 clinical cases. Am Heart J. 1951;42:582. 158. Reynolds T, Ware AG. Sulfhemoglobinemia following habitual use of acetanilid. JAMA. 1952;149:538–542. 159. Cumming R, Pollock A. Drug induced sulphaemoglobinemia and Heinz body anemia in pregnancy with involvement of the foetus. Scott Med J. 1967;12:320–322. 160. Kneezel LD, Kitchens CS. Phenacetin-induced sulfhemoglobinemia: Report of a case and review of the literature. Johns Hopkins Med J. 1967;139:175–180. 161. Lambert M, Sonnet J, Mahien P, et al. Delayed sulfhemoglobinemia after acute dapsone intoxication. Clin Toxicol. 1982;19:45–49. 162. Ford R, Shkov J, Akman WV, et al. Deaths from asphyxia among fisherman. PFR: MMWR. 1978;27:309. 163. Madeiros M, Bechara EJ, Naoum PC, et al. Oxygen toxicity and hemoglobinemia in subjects from a highly polluted town. Arch Environ Health. 1983;38:11–15. 164. Carrico R, Blumberg WE, Peisach J J. The reversible binding of oxygen to sulfhemoglobin. J Biol Chem. 1978;253:7212– 7215. 165. Park C, Nagel RL. Sulfhemoglobinemia. Clinical and molecular aspects. N Engl J Med. 1984;310:1579–1584. 166. Park C, Nagel RL, Blumberg WE. Sulfhemoglobin. Properties of partially sulfurated tetramers. J Bio Chem. 1986;261:8805– 8810. 167. Berzofsky J, Peisach J, Blumberg WE. Sulfheme proteins. II. The reversible oxygenation of ferrous sulfmyoglobin. J Biol Chem. 1971;246:7366–7372.

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SECTION SEVEN

SPECIAL TOPICS IN HEMOGLOBINOPATHIES Martin H. Steinberg

Three chapters in this section, Population Genetics and Global Health Burden, Genetic Modulation of Sickle Cell Disease and Thalassemia, and Developments in Laboratory Methods to Detect Hemoglobinopathies, cover diverse subjects. Without endemic malaria, the genes for HbS, HbC, HbE, and the thalassemias would not exist at polymorphic frequencies as the selective pressure from Plasmodium falciparum is so strong. Weatherall and Williams discuss the “Malaria hypothesis,” and the evidence that carriers of thalassemia and HbS, HbC, and HbE are protected from P. falciparum infection, although the mechanisms behind such protection are incompletely understood. The health burden of hemoglobinopathies and thalassemia is great, particularly in developing countries, and it is likely to increase as development proceeds. Many obstacles to providing genetic services are discussed, among them, the prioritization of scant healthcare resources and the problems posed by communicable disease and malnutrition, leaving hemoglobin disorders underserved. Hemoglobinopathy detection is often a part of the evaluation of anemia and microcytosis. It should first be determined, using hemoglobin high-performance liquid chromatography, whether a variant hemoglobin is present, how abundant the variant is, and whether the variant is relevant to the clinical picture. With background information from the patient’s history, studies of informative family members, physical examination, blood counts, and erythrocyte indices, high-performance liquid chromatography can suggest the genotype of sickle cell disease. Thalassemia mutations are multitudinous and only DNA-based studies can pinpoint the genotype of this disease. For purposes of genetic counseling, especially when antenatal diagnosis

is being considered, DNA-based diagnostics are required, although these tests are not required for managing disease complications in an individual. An accurate prognosis is often facilitated by knowing the genotype of the disease. Mutations of the ␤-globin gene usually change only one hemoglobin component and stable variants usually have a concentration of 40%–50%. In contrast, ␣-globin gene variants should produce abnormal major and minor hemoglobins because, with rare exceptions, all hemoglobin molecules contain ␣-globin chains. Mutation of a single ␣-globin gene usually creates variants forming 20%–25% of the total hemoglobin, and a variant minor hemoglobin fraction, but the actual amounts of different hemoglobin types that accumulate can vary. ␥ -Globin gene mutants are important only in the fetus and usually “disappear” postnatally, when ␥ -globin gene expression is nearly extinguished. ␣-Globin chain abnormalities are expressed at birth and can produce signs of disease neonatally. Characteristically, ␤-globin chain mutations are clinically apparent only after the first few months of life, although their mutations can be detected beforehand. Although a prototypical mendelian, single gene disease, the clinical heterogeneity of sickle cell anemia resembles that of a complex multigenic trait. Similarly, genotype– phenotype correlations are often difficult to establish in thalassemia. Modifying genes must affect the pathogenesis of these disorders and modulate their phenotypes. Candidate gene association studies have linked several genes and pathways with some subphenotypes of sickle cell anemia, including stroke, leg ulceration, priapism, pulmonary hypertension, and osteonecrosis. Genome-wide association studies now permit an “unbiased” assessment of all genes that could modulate disease. To be successful, this work requires precise definitions of phenotypes or heritable quantitative traits, samples of sufficient size to provide reasonable power to detect associations, knowledge of linkage disequilibrium patterns in the study populations, the availability of economically feasible high-throughput genotyping platforms, and sophisticated analytical techniques. These conditions have now been largely met, and some results are beginning to be published but this remains a “discovery” process. Although it is still early in this quest, we can envision a time when disease diagnosis might include the use of tests for polymorphic modifier genes that will allow the treating physician to predict the future level of severity and complications likely to occur in an individual. This knowledge would help rationalize the use of antenatal diagnosis and the selection of patients for risky treatments and procedures. It might also allow a prediction of drug response, preclude exposing some patients to toxic therapies, and identify new pathways to which therapy could be targeted.

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26 Population Genetics and Global Health Burden David J. Weatherall and Thomas N. Williams

It is now widely accepted that the hemoglobinopathies are the most common monogenic diseases in humans. In this chapter we discuss the possible reasons for their very high frequency and uneven distribution among the world’s population and describe recent attempts to determine their global health burden and how this might be managed.

FREQUENCY AND DISTRIBUTION Frequency A number of attempts have been made to review or determine the global frequency and annual birth rates of homozygotes or compound heterozygotes for the important inherited disorders of hemoglobin.1–8 Composite data from these reports for the frequency and distribution by World Health Organization (WHO) regions are summarized in Table 26.1 and similar data for the estimated annual birth rate of severe forms of hemoglobinopathy are summarized in Figure 26.1. As we will discuss later in this chapter, such summaries are based on imperfect data and should be viewed with some caution; nevertheless, it is now generally acknowledged that as a group the hemoglobinopathies represent the most common monogenic diseases of humans. Any explanation for the extremely high gene frequencies of the inherited disorders of hemoglobin must take into account a number of unusual features about their world distribution. The high-frequency regions stretch across the tropical belt of the Old World or areas into which there has been a high rate of migration from this region. None of the hemoglobinopathies have been found in the indigenous populations of the New World despite the fact that some of its populations have existed in tropical conditions over thousands of years. Furthermore, the different hemoglobinopathies are unevenly distributed in the tropical belt of the Old World. For example, HbS reaches its highest frequencies in Africa, parts of the Middle East, and the Indian subcontinent but is not observed further east,

whereas HbE occurs at extremely high frequencies in Southeast Asia, Myanmar, Bangladesh, and parts of the eastern borders of the Indian subcontinent, yet does not extend further west (Fig. 26.2), and every population with a high frequency of different forms of thalassemia has its own particular set of mutations. These observations, which suggest that these conditions have arisen de novo with local expansion, are not compatible with older theories about their distribution, which suggested that it reflects massive population movements in an eastward or westward direction.4 As well as the heterogeneous distribution of the hemoglobinopathies, there are certain other generalizations that can be made about their frequency, which also have to be taken into account in any explanation for why these conditions are so common. The sickle cell (HbS) and ␤ thalassemia genes (Figs. 26.2 and 26.3), although they occur at varying frequencies in different populations, rarely are found in more than 20% of the population. The ␣0 thalassemias occur at similar frequencies to the ␤ thalassemias. On the other hand, the ␣+ thalassemias occur at much higher frequencies, achieving up to 70%–80% of the population in some regions (Fig. 26.4) and, similarly, the carrier rates for HbE greatly exceed those of HbS, reaching as high as 70% in some parts of Asia including northern Thailand and Cambodia. Further details of these gene frequencies, together with the appropriate references and data that underline these generalizations are reviewed in more detail elsewhere.4 In short, the hemoglobinopathies only occur at very high frequencies in warm or tropical climates, their distribution is extremely heterogeneous, and the maximum population frequencies that are achieved appear to vary considerably between different forms of thalassemia and structural hemoglobin variants.

Why Do the Hemoglobinopathies Occur at Such a High Frequency? The high-frequency populations for the hemoglobinopathies are mainly those of developing countries or countries that have only recently made the demographic and epidemiological transitions from the effects of extreme poverty. There is considerable evidence that genetic diseases and congenital malformations occur more frequently in countries with a low per capita gross national income for which a number of explanations have been put forward. These include increased rates of consanguinity, increased parental age, population migration, and natural selection.6 Although it is difficult to obtain accurate data, it is clear that consanguineous marriage is still practiced in many parts of the world and may be acceptable to a minimum of 20% of the world’s population.9,10 This practice is especially common throughout the eastern Mediterranean, North Africa, and the Indian subcontinent, and to a lesser extent in parts of South America and subsaharan Africa.9–11 There is clear evidence that the birth prevalence of autosomal 625

626

David J. Weatherall and Thomas N. Williams Table 26.1. Percent carrier frequencies for common hemoglobin disorders, by WHO region (1–7) Region

HbS

HbC

HbE

␤ Thalassemia

␣ 0 Thalassemia

␣ + Thalassemia

Americas Eastern Mediterranean Europe Southeast Asia Africa Western Pacific

1–20 0–60 0–30 40 0

0–10 0–3 0–5 0 0

0–20 0–2 0–20 0–70 0

0–3 2–18 0–19 0–11 0–13

0–5 0–2 1–2 1–30 0

0–40 1–80 0–12 3–40 2–60

Note: Many of these data are derived from small population samples.

recessive diseases is increased by this mechanism in these populations.6 The percentage of pregnant women aged older than 35 years is also high in middle- and low-income countries, although the major consequence of this appears to be an increase in the number of live births of infants with chromosomal defects or developmental abnormalities.6 A large population movement from areas of high frequency for single gene effects also has the effect of introducing these disorders into new populations, as evidenced by the spread of the hemoglobin disorders to the Americas, the Caribbean, and Europe by the slave trade and later migrations. Although all of these mechanisms might undoubtedly have been responsible for increasing the frequency of autosomal recessive disorders and others over time, they cannot account for the remarkably high gene frequencies of the hemoglobinopathies in these populations, and the evidence is now overwhelming that the principle fac-

1600

tor in maintaining these high gene frequencies is natural selection working through the relative protection of heterozygotes for the common hemoglobinopathies against Plasmodium falciparum malaria.

NATURAL SELECTION AND THE HEMOGLOBINOPATHIES; THE MALARIA HYPOTHESIS Historical Background The notion that variation in host response to infection might have a genetic basis is not new;12,13 however, it was not until the late 1940s, and through the remarkable insight of J.B.S. Haldane, that a plausible genetic protective mechanism was first suggested. During the period just after World War II, independent studies in Italy and in Mediterranean immigrants in the

5000

3000 5000 5500 SS 216,000

4000

3000

8000 2000 6000

Figure 26.1. Estimated annual births of hemoglobinopathies. Data from references 2–5. The SS box shows sickle cell disease in Africa; other figures are for ␤ thalassemia. These data are currently being revised and extended; in many cases they may underestimate the birth rates. (From ref. 4.)

Population Genetics and Global Health Burden

HbE

627

HbS Figure 26.2. The world distribution of hemoglobins S and E. (From ref. 4.)

United States highlighted a remarkably high frequency of thalassemia in these populations.14,15 Influenced by the intense interest in human mutation rates following studies of the survivors of the atomic bombs in Hiroshima and Nagasaki, workers on both sides of the Atlantic suggested that this might reflect a high rate of mutation and, because they appeared to be restricted to certain populations, that the mutation rate might differ between different ethnic groups. At the 8th International Congress of Genetics in Stockholm in 1948, Neel and Valentine, to explain the high frequency of thalassemia in immigrant populations in the United States, calculated a mutation rate for thalassemia of 1:2500. Haldane felt that this was unlikely and that these remarkable gene frequencies were more likely to be the result of heterozygote selection. Furthermore, he went on to suggest, “The corpuscles of anemic heterozygotes are smaller than normal, and more resistant to hypertonic solutions. It is at least conceivable that they are also more resistant to attacks by the sporozoa which cause malaria, a disease prevalent in Italy, Sicily and Greece, where the gene is frequent.”16 In essence, what became known as the “malariahypothesis” intimated that diseases like thalassemia can be considered balanced polymorphisms – conditions in which the gene frequency for the advantageous heterozygous

state increases until it is balanced by the loss of disadvantaged homozygotes from the population. Haldane’s great contribution to this field was to encourage geneticists and hematologists to analyze the high frequency of common genetic diseases of the blood as putative polymorphisms of this kind. Although, over the years, the hypothesis has stood the test of time, as we shall see in subsequent sections it has not always been easy to substantiate Haldane’s ideas in human populations. It is beyond the scope of this chapter to review in detail all the population and experimental work that has followed from Haldane’s original idea. In particular, it has not been possible to review the evidence suggesting that polymorphisms other than the hemoglobinopathies have also come under selection by malaria. Readers who wish to explore this aspect of the topic further are referred to several recent reviews.17–19

Malaria in Human Populations Malaria has decimated large populations in the past and is still one of the world’s most common causes of mortality. Currently, close to 50% of the world’s population are at risk of transmission, more than 3 billion people live in malarious areas and the disease causes between 1 and 3 million deaths per year.20 Four different species of

628

David J. Weatherall and Thomas N. Williams IVS-I-110G A COD 39 C T IVS-I-6 T C IVS-I-1 G A IVS-II-745 C G COD 6-A

IVS-I-110G A COD 39 C T IVS-II-1 G A IVS-I-5 G C COD 8-AA COD 44-C

COD 41/42-TTCT COD 17 A T IVS-II-654 C T -28 A G COD 26 G A(HbE) IVS-I-5 G C COD 19 A G

-29 A G -88 C T

IVS-I-5 G C COD 8/9 + G IVS-I-1 G T 619 bp DEL COD 26 G A(HbE)

Figure 26.3. The world distribution and different mutations that cause ␤ thalassemia. Those shown in boxes are milder mutations. Hemoglobin E is included in the milder mutation because of its ␤ thalassemic phenotype. (From ref. 4.)

--MED 1 -15% -α 3.7 Ι αTα --SEA

5 -15% -α 3.7 Ι

-α 4.2 αTα

5 - 40% (-α 3.7 Ι) αTα

60% (-α 3.7) 40 - 80% αTα (-α 3.7 Ι) (-α 3.7 ΙΙ) (-α 4.2)

5 - 80%

-α 4.2 -α 3.7 ΙΙΙ αTα

α+ Thalassaemia α0 Thalassaemia

Figure 26.4. The world distribution of the ␣ thalassemias. The ␣0 thalassemias are only found at high frequencies in Asia and some Mediterranean countries. The ␣+ thalassemias have a much broader distribution. Some of the different subtypes of ␣0 and ␣+ thalassemia are shown. - -SEA indicates the Southeast Asian deletion form of ␣0 thalassemia. -␣4.2 or -␣3.7 relate to different forms of ␣+ thalassemia with their particular deletion sizes, -␣3.7 is further subdivided into types I, II, and III, again depending on the different deletion sizes. ␣T denotes nondeletion ␣ thalassemia. (From ref. 4.)

Population Genetics and Global Health Burden Plasmodium cause human infections: P. falciparum, P. vivax, P. ovale, and P. malariae. Although the majority of deaths are caused by P. falciparum malaria, P. vivax malaria is a common cause of chronic ill health, particularly in children. Infection occurs when a female anopheline mosquito inoculates sporozoites into humans where they mature in the liver and are then released into the blood stream as merozoites. The latter invade red cells, causing both their destruction and also adhesion to the walls of blood vessels. The complex interactions of malarial parasites and red blood cells, that have to be understood for a full appreciation of the complexities of the potential protective effects of the hemoglobin disorders have been reviewed recently.18,19

Hemoglobin S Because, paradoxically, it was the structural hemoglobin variants, notably HbS, for which Haldane’s hypothesis was first explored we will consider this condition first and then discuss how far his hypothesis has stood up in the case of the thalassemias (see Chapter 22). Early studies on the relationship of the high frequency of the HbS gene to P. falciparum malaria are summarized elsewhere.21 In short, analyses of parasite rates and densities in children with the sickle cell trait (HbAS) and nonaffected controls, together with observations of the rarity of sickling in patients with severe malaria and the distribution of the sickle cell gene combined to provide convincing evidence that HbAS offers at least some degree of protection against severe malarial infection. More recent studies in east and west Africa suggest that the greatest impact of HbS is against fatal or severe malaria, that is profound anemia or cerebral malaria, having no discernible effect on infection rates per se.22–24 Indeed, case-control studies suggest that HbS heterozygotes have a level of protection of approximately 60%–80% against the severe complications of malaria. Some progress has also been made in determining the mechanisms of protection of those with HbAS against P. falciparum malaria, although many questions remain unanswered. Early studies demonstrated that in those with HbAS the rate of sickling is significantly greater in parasitized than in unparasitized red cells,25 findings that were later confirmed and extended under more physiological conditions.26 These studies suggested that the parasites cause a “suicidal infection;” because parasitized HbAS red cells sickle and are more likely to be removed from the circulation. At approximately the same time it was also demonstrated independently by two groups that parasitized HbAS cells maintained at low oxygen tension do not support the growth of malaria parasites as effectively as normal cells at similar oxygen tensions.27,28 Later it was suggested that this effect might be due to the increased loss of potassium or water from the HbAS cells.29

629 Recent studies have also been compatible with the early suggestion21 that there might also be an immune component involved in the protection of individuals with HbAS. In a cohort study of mild clinical malaria in Kenya, the protection attributable to HbAS showed a significant increase with age.30 Compatible with these findings, it was demonstrated in two studies that those with HbAS had significantly higher titers of immunoglobulin G antibodies to a variety of malarial antigens than normal children.31,32 Thus although there are a number of feasible mechanisms at both the cellular and immunological levels for protection of HbAS individuals against P. falciparum malaria the precise mechanisms of these complex interactions that result in such a remarkable protective effect remain to be determined.

Hemoglobin C Although early studies were not convincing, more recent work conducted in West Africa indicates that the relatively high frequencies of HbC in that region have been maintained by resistance to P. falciparum malaria (see Chapter 21).33 In this case there is evidence that protection is greater in homozygotes (90%) than in heterozygotes (30%), suggesting that, unlike the sickle cell mutation, HbC may be an example of a transient polymorphism – one that would move to fixation under continued selective pressure. This hypothesis is based on the assumption that, in itself, homozygosity for HbC has no adverse consequences. If this were the case, however, it is difficult to understand why the frequency of HbC is not higher in African populations. Furthermore, the fitness (in the Darwinian sense) of homozygotes for this variant is not completely established; further work will be required to confirm this interesting suggestion. Although a number of early studies suggested that the protective mechanism of HbC might be mediated through the inability of parasites to grow and develop in affected red cells, this mechanism is not supported by studies of parasite densities in vivo (summarized in ref. 19). More recently it has been shown that the expression of PfEMPI, an important parasite-encoded red cell adhesion protein, is reduced in HbC-containing red cells, an effect that is most marked in homozygotes.34,35 It should be remembered, however, that HbC-containing red cells have a number of abnormal properties including rheological abnormalities and increased levels of oxidant stress; clearly, the final story about the definitive protective mechanism remains to be determined.36

Hemoglobin E Because of its extremely high frequency in Asia, HbE has always seemed to be a good candidate for a malariaprotection polymorphism (see Chapter 18). Many of the early population studies that were conducted failed to show a clear relationship between HbE and malaria.4 It is possible that some of these studies may have been bedeviled

630 by the very high frequency of ␣ thalassemia that occurs in many populations with a high frequency of HbE. Recent extended linkage-disequilibrium analyses of the HbE gene suggest that it is of relatively recent origin and must have come under intense selection.37 In keeping with this observation it has been found that the presence of HbE trait is associated with a reduced severity of disease in adults admitted with acute P. falciparum malaria.38 Furthermore, in vitro culture studies using mixtures of normal and variant red cells have shown that those from HbAE individuals, although not those from HbE homozygotes or different forms of ␣ thalassemia, are more resistant to invasion by the parasite.39 Although these findings are suggestive, a major gap in our information about HbE and malaria is the absence of adequate data from case-control studies of the kind that have been so successful in demonstrating the magnitude of the protective effects of HbS and HbC. Thus, although it seems almost certain that HbE is in some way protective against malaria, a great deal more work needs to be done before we will have any clear understanding of the magnitude of the effect or its mechanism.

␣ Thalassemia The frequency of ␣+ thalassemia in the southwest Pacific follows a clinal distribution from north/west to south/east, with the highest frequency in northern Papua New Guinea and the lowest in New Caledonia (see also Chapters 13 and 14). This distribution shows a strong correlation with malarial endemicity; similar relations are not observed with other genetic polymorphisms in this region.40 The possibility that ␣ thalassemia has been introduced from the mainland populations of Southeast Asia, and that its frequency has been diluted as populations moved south, has been largely excluded by finding that the molecular forms of ␣ thalassemia in Melanesia and Papua New Guinea are different from those of the mainland and are set in different ␣-globin gene haplotypes.40 One feature of the distribution of ␣ thalassemia in this region that, on the face of it, does not fit with the malaria hypothesis was that it is also found in Fiji in the west, Tahiti and beyond in the east, and in Micronesian atolls, populations in which malaria has never been recorded. It has been found that a single mutation, which has been previously defined in Vanuatu, accounts for virtually all the cases of ␣+ thalassemia that have so far been described in Polynesia, indicating that the presence of ␣ thalassemia in these nonmalarious areas has almost certainly resulted from population migration rather than selection.41 These population data were augmented by prospective case-control studies in northern Papua New Guinea, where it was found that compared with normal children, the risk of contracting strictly defined severe malaria was 0.4 for ␣+ thalassemia homozygotes and 0.66 for ␣+ thalassemia heterozygotes.42 Similar findings have since been reported

David J. Weatherall and Thomas N. Williams from studies conducted in two African populations.43,44 Although some progress has been made toward an understanding of the mechanisms of malaria protection afforded by ␣ thalassemia, the overall picture is still far from clear. Paradoxically, early studies in Papua New Guinea suggested that very young children with ␣+ thalassemia might be slightly more prone to uncomplicated malaria than normal children.45 Later studies conducted in Vanuatu appear to support this conclusion, showing that the incidence of uncomplicated malaria and the prevalence of splenomegaly, an index of malaria infection, were both significantly higher in children with ␣ thalassemia than in normal children. Moreover, the effect was most marked in the youngest children and in those affected by the nonlethal parasite, P. vivax.46 It was suggested that the early susceptibility to P. vivax, which may reflect the more rapidly turning over red cells of ␣ thalassemic infants,47 could be acting as a natural vaccine by inducing cross-species protection against P. falciparum. These intriguing observations require further study in other populations. There has also been some progress toward determining how protection might be mediated at a cellular level. Overall, there is no evidence for a reduced rate of invasion or growth of P. falciparum in red cells of the genotype -␣/␣␣ or -␣/-␣, the mild forms of ␣ thalassemia that have been shown to be protective by case-control studies. It has been found that these cells consistently bind more malariaimmune globulin than normal red cells.48,49 When infected with parasites, ␣ thalassemic red cells are less able than normal red cells to form rosettes, an in vitro phenomenon whereby uninfected red cells bind to infected cells. It has been demonstrated that complement receptor 1 expression, which is required for rosette formation, is reduced on ␣ thalassemia red cells,50 offering a convincing potential mechanism for reduced rosetting.51,52 In addition, infected ␣ thalassemic red cells are less able than normal red cells to adhere to human umbilical vein endothelial cells.51 Because rosetting and cytoadherence are mechanisms that underlie sequestration of infected red blood cells and are associated with virulence of infection, these observations suggest that malaria protection by ␣ thalassemia might be determined by specific red cell membrane abnormalities that are associated with this condition. In an extensive series of studies to define other membrane components involved, it was suggested that altered red cell membrane band 3 could also be a target for enhanced antibody binding to ␣ thalassemic cells infected with parasites.49 Thus, although there are a number of tantalizing clues as to possible mechanisms of protection against malaria by the milder forms of ␣ thalassemia, a coherent picture of how they fit together remains to be produced.

␤ Thalassemia (Chapters 16 and 17) In their now classic studies conducted in the 1960s, Siniscalco and colleagues53 found that the population

Population Genetics and Global Health Burden frequencies of thalassemia carriers in Sardinia, a considerable number of whom must have been ␤ thalassemia carriers, correlated with altitude. Although malaria was no longer endemic in Sardinia at that time, historically, its incidence had been correlated closely with altitude. Similar correlations were found later in Melanesia.54 In addition, a relatively small case-control study in Northern Liberia suggested that the ␤ thalassemia trait is protective against severe malaria.55 Thus, although a number of other population studies, mostly analyzing parasite rates or densities, failed to show any correlations between ␤ thalassemia and malaria (see ref. 4), such epidemiological data that are available certainly point to a protective effect of the ␤ thalassemia trait (see also Chapters 16 and 17). Another finding in favor of the concept that ␤ thalassemia is a protective polymorphism is the observation that in every country in which this disease is common there is a different set of mutations. Studies of ␤-globin gene haplotypes (Chapter 27) and their relationship to ␤ thalassemia mutations have been particularly interesting in this respect. Unlike the ␣-globin gene haplotypes there is a “hot-spot” for recombination between the 5
and 3
ends of the ␤ gene haplotypes (Chapter 27). Over time there seems to have been admixture between the 5
and 3
haplotypes among human populations but this has not occurred in the case of thalassemia; the thalassemia mutations, which occur in the 3
haplotype, are almost invariably associated with the same 5
haplotype, indicating that in evolutionary terms they are much more recent and that there has not been time for mixing the 5
and 3
ends of haplotypes that carry these mutations (see ref. 4). This suggests a fairly recent selective pressure, possibly approximately 5,000 years, which is in agreement with current estimations of the time that human populations have been exposed to pathogenic forms of Plasmodium (see later). Very little is known about the potential mechanisms of protection against malaria in the case of ␤ thalassemia. In vitro studies have shown that ␤ thalassemic red cells are invaded at the same rate as normal red cells and that the rate of parasite growth is also indistinguishable from normal. One potential explanation is that protection is related to levels of fetal hemoglobin (HbF) that are associated with this condition. There is clear evidence that the rate of decline of HbF levels after birth is delayed in ␤ thalassemia heterozygotes (see ref. 4) and studies conducted in both humans56 and in transgenic mice57 have found defective development of P. falciparum or P. yoelii, respectively, in red cells that contain human HbF. This could provide a mechanism for protection by ␤ thalassemia during the first year of life, but not thereafter (see later section). A more general hypothesis for why ␤ thalassemia heterozygotes might be protected against malaria has also been proposed.58 In short, it is known that these cells are under increased oxidative stress due to globin chain imbalance; it is suggested therefore that the further stress

631 imposed by the parasite might render these cells prone to damage and rapid removal from the circulation, a mechanism reminiscent of that outlined earlier for the protective effect of HbAS.

Epistatic Interactions Between Protective Polymorphisms Because different hemoglobin disorders that offer protection against malaria frequently occur together in the same population it is becoming increasingly important to determine whether they interact with one another and, in particular, the effect that this might have on changing the pattern of susceptibility to malarial infection. The term epistasis is used to describe nonadditive interactions between two or more different genetic loci. A study in East Africa has provided intriguing information that suggests that there is negative epistasis between the HbS and ␣ thalassemia genes with respect to protection against P. falciparum malaria. Although those with HbAS or heterozygous or homozygous for ␣+ thalassemia alone have been found to have a highly significant protection against P. falciparum malaria (see previous sections) this effect was completely nullified in individuals who had HbAS and were homozygous or heterozygous for ␣+ thalassemia.59 Although the numbers involved in this study were relatively small, results from a recent case-control study conducted in Ghana were compatible with the same conclusion.60 Considering current uncertainty about the precise mechanism of protection mediated by either of these variants alone, it is difficult to explain this remarkable epistatic interaction. Phenotypically, the major difference between HbAS individuals and those who, in addition to HbS, carry ␣ thalassemia is that the doubly affected persons have significantly lower levels of HbS in their red cells (see ref. 4). It is possible therefore that protection in HbAS persons may require a critical level of HbS. In this context it should be noted that these doubly affected individuals have reduced mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) values associated with the particular ␣ thalassemia genotype (Chapter 22). In uncomplicated sickle cell trait HbS accounts for approximately 40% of total red cell hemoglobin and the MCH is 30 pg, giving an absolute amount of HbS per cell of approximately 12 pg. On the other hand, in those with the HbAS who are also homozygous for ␣+ thalassemia, the level of HbS is reduced to approximately 25% and the MCH to only 20 pg, giving an absolute level of HbS of only 5 pg per cell; a more than 50% reduction in the absolute amount of HbS per cell could, therefore, have an important effect on the degree of protection enjoyed by individuals of different genetic combinations. It seems very likely that other interactions of this type occur. Apart from their intrinsic interest in the population genetics of these conditions their further study may provide valuable information about the protective mechanisms of some of the hemoglobinopathies.

632 Summary From the data summarized in the previous sections, it is clear that, at least at the population level, there is reasonably convincing evidence that the five major groups of hemoglobin disorders that occur at very high frequencies in different populations are all associated with protection against P. falciparum malaria in the heterozygous, and, in some cases, homozygous states. The clinical and parasitological evidence for protection varies widely; in some cases there appears to be a genuine reduction in parasite densities, whereas in others the effects seem to be mediated almost entirely by protection against the severe complications of malaria. Perhaps it is not surprising that progress has been much slower in trying to determine the protective mechanisms involved. The studies that have been conducted are asking questions of an enormously complex biological system; transmission rates of malaria vary widely, populations differ in whether one type of parasite predominates or whether there are several. There is a wide difference in antigenic strains of parasite and many of the in vitro techniques that have been used are extremely difficult to standardize between different laboratories. Added to this, the cellular pathophysiology of these different hemoglobinopathies, although there might be some features in common, is undoubtedly different. Against this complex background, however, certain generalizations are beginning to appear. For example, a reduction in rosette formation seems to be common to several different hemoglobinopathies, suggesting that changes in the red cell membrane associated with particular differences in antigen presentation may be at least one common pathophysiological mechanism. No doubt more will be found. An even more difficult question, and one posed by the increasing evidence for an additional immune basis to protection, is how the primary protective mechanisms at the level of the red cell might be associated with enhanced immune response. Clearly this question requires a great deal more work. It is beyond the scope of this chapter to discuss the increasing list of protective polymorphisms associated with malaria. They are the subject of several recent reviews.13,17,18 As in the case of the hemoglobinopathies, in the majority of cases the protective mechanisms have not been fully worked out. There is one notable exception however. Epidemiological studies, first in Africa61 and later in Papua New Guinea62 showed a high frequency of the Duffy-negative phenotype in populations in which malaria is common. Further studies suggested that the Duffy blood group antigen might be the receptor for P. vivax. Later studies demonstrated that the Duffy antigen chemokine receptor (DARC) is not expressed on red cells when there is a promoter mutation that alters a GATA-1 binding site. In cells carrying this mutation DARC expression is abolished and therefore DARC-mediated entry of P. vivax is

David J. Weatherall and Thomas N. Williams inhibited. This receptor is expressed predominantly on reticulocytes and young red cells and hence this observation is in keeping with the proposal discussed earlier in this chapter regarding the possible immune basis for protection against ␣ thalassemia, at least in populations where P. vivax is common. The finding of increased susceptibility to both P. vivax and P. falciparum in babies with ␣ thalassemia in Papua New Guinea, possibly because of the relative increase in young red cells in their blood, might allow early immunization against P. vivax and, because there is cross immune response between the two species, later protection against P. falciparum.46 Some other interesting similarities to observations in the hemoglobinopathies are also appearing in relationship to other protective polymorphisms. For example, evidence has been emerging for several years that individuals of blood group O are protected against P. falciparum malaria (reviewed in ref. 63). Interestingly, as in the case of some of the hemoglobinopathies, individuals of blood group O show significantly reduced rosette formation compared with nongroup O individuals. It appears therefore that there could be common pathways among the malariarelated protective polymorphisms, and further work of this kind might help to clarify some of the protective mechanisms involved in the hemoglobinopathies.

POPULATION AND EVOLUTIONARY IMPLICATIONS The picture that is emerging is that the extremely common hemoglobin disorders occur at a high frequency in those parts of the world in which malaria was, or still is, a major cause of mortality. These protective polymorphisms are extremely patchy in their distribution; the HbS mutation occurs across Africa, the Middle East and the eastern side of India, but not further east; the HbE mutation occurs at an extremely high frequency in many of the populations stretching from the eastern part of the Indian subcontinent throughout the rest of Asia. Every high-frequency population for thalassemia has its own mutations and, at least in the case of HbS and HbE, there is evidence that they may have arisen de novo on more than one occasion. These observations are best explained by the occurrence of local mutations with rapid selection; the lack of homogenization of some of these highly protective polymorphisms suggests that exposure of populations to malaria has been, at least in evolutionary terms, a fairly recent event. Although ancestral forms of the malaria species that infect humans arose many millions of years ago, it is likely that P. falciparum arose from its closest ancestor approximately 10 million years ago.64 After approximately 10 million years of development in Africa, modern humans emigrated out of Africa 40,000–100,000 years ago, probably carrying at least some polymorphisms that evolved from selection against P. falciparum.65 It is likely that death due to malaria increased very rapidly between 5,000 and 10,000 years ago with the development of agriculture and

Population Genetics and Global Health Burden settlements that would have greatly facilitated the transmission of the disease by mosquitoes.66 These observations are all compatible with a fairly recent appearance of the malaria-resistant polymorphisms such as the hemoglobinopathies. This interpretation is certainly in keeping with recent studies of haplotype diversity and linkage disequilibrium at the human glucose-6-phosphate dehydrogenase locus.67 These concepts also go some way toward explaining the absence of common hemoglobinopathies in the indigenous populations of the new world. Presumably the selective factors leading to the high frequency of the thalassemias in Asian populations had not been present before their early migrations into the New World. Currently, it is not clear when malaria reached this region, although it has been suggested that it was as recently as the early Spanish conquests. Hence there might not have been time for selection to generate high frequencies of malaria-resistant polymorphisms in these populations. Another interesting aspect of the population genetics of the hemoglobin disorders is how different polymorphic genes interact in the same population to produce present day frequencies.4,21 Mutations like those for HbS and ␤ thalassemia, which are alleles, interact to produce compound heterozygotes, HbS–␤ thalassemia, which can have clinically severe phenotypes. In this case, selection will not only act against those with sickle cell anemia and homozygous ␤ thalassemia, but also to some extent against compound heterozygotes with HbS–␤ thalassemia. Early theoretical analyses of this type of situation21 concluded that a stable equilibrium would be obtained provided that heterozygotes for both genes enjoyed a selective advantage. A consequence of selection acting in this way is that the more advantageous heterozygous state, in this case HbAS, would tend to come under much stronger selection and therefore the HbS and ␤ thalassemia alleles would tend to be mutually exclusive in populations. Although this is not entirely the case, it mirrors broadly the pattern of distribution of these alleles in Africa. In the case of HbE and ␤ thalassemia in many Asian populations, there is an inverse relationship between the frequency of ␤ thalassemia and HbE, although because both are so common there still is a high frequency of compound heterozygotes.

POPULATION DYNAMICS AND IMPLICATIONS FOR THE CONTROL OF THE HEMOGLOBINOPATHIES Population Dynamics In the interests of public health planning a number of attempts have been made to predict the future burden of the inherited disorders of hemoglobin at local, regional, and global scales. As we will discuss later, such predictions suggest that we will be unlikely to see a significant decline in their current frequencies for the foreseeable future and the hemoglobinopathies will therefore assume increasing

633 importance in many countries in development as they go through their demographic transition. The population dynamics of the hemoglobinopathies are related to a number of issues including potential changes in the factors that result in selective advantage, the rates of epidemiological and demographic transitions in the developing countries, population migration, and the potential role of medical intervention. Predicting what might happen to the gene frequencies of the hemoglobinopathies in the face of a reduction in the incidence of malaria is complex and discussed in detail elsewhere.68 In short, even if malaria were eradicated completely it would take many generations before the frequency of these diseases fell significantly. Furthermore, it is still not clear whether relative protection against malaria is the only factor that has maintained the hemoglobinopathies at their present high level. Studies conducted in Papua New Guinea suggested that the ␣ thalassemias might offer protection against the infectious diseases of childhood,42 although this effect has not been seen in other populations.

The Epidemiological Transition and the Frequency of Severe Forms of Hemoglobinopathy During the last decade of the twentieth century, gross domestic product per head in the developing countries grew by 1.6% each year, and the proportion of people living on less than $1 a day fell from 29% to 23%. These statistics are reflected in improvements in the overall health of a number of populations in the developing world and in declines in childhood mortality, most notably in South America and the Caribbean, east Asia and the Pacific, the Mediterranean region, the Middle East and parts of North Africa.6 The consequences of such epidemiological transition for the recognition of the importance of genetic disease in developing countries were shown graphically in Cyprus after World War II. Thalassemia was not known to occur on the island until 1944, at which time the clinical findings in 20 patients were reported.69 This study highlights the difficulty in identifying diseases of this type against the background of chronic malaria and other infections; it was published at the end of an extremely successful program to control mosquito breeding and other improvements in public health. Hence, during this remarkably short period it became clear that there was a high frequency of genetic anemia. By the early 1970s it was estimated that, if no steps were taken to control the disease, in approximately 40 years the blood required to treat all the children with thalassemia would amount to 78,000 U/year, 40% of the population would need to be donors, and the total cost to the health services would equal or exceed the island’s health budget.4 A similar trend is occurring at present throughout many Asian countries and will undoubtedly be repeated in subsaharan Africa as social conditions improve. A more detailed analysis of the effects

634 of both demographic and epidemiological transitions has been summarized recently.70 The potential effects of improvements in the medical management of the hemoglobinopathies on the size of their gene pools is also complex and difficult to predict. In the short term, such improvements have been shown to result in treatment-seeking migration of affected individuals from high-frequency regions to countries with stronger medical services, potentially widening the geographic range of these diseases. Although improved treatments for the more serious forms that allow patients to survive to reproductive age might have a modest effect on their overall population frequencies, the most contentious issue is the so-called dysgenic effect of programs for the control of these disorders by prenatal diagnosis and termination of affected pregnancies. This important issue is discussed in detail elsewhere4,11,68,71 and will only be outlined here. The dysgenic effect of prenatal diagnosis is based on the premise that if most pregnancies that carry severely affected infants were aborted, the gene frequency for the particular disorder will steadily increase in the population. This is because homozygotes are replaced by healthy individuals, two thirds of whom are heterozygous and hence will pass on their genes. This assumes, of course, that couples undergoing prenatal diagnosis will attain the population norm for their final family size. But such evidence as there is suggests that this might not be the case; studies in Cyprus, for example, have shown that approximately 25% of the decrease in thalassemic births is due to limited reproduction.71 Indeed, as elegantly summarized by Bodmer and Cavalli-Sforza, the changes in the size of the thalassemic gene pool that might follow interventions of this kind are likely to be extremely small: “It would seem therefore that the dysgenic effect of medicine is not a real threat. By the time that it may have a clearly perceptive global effect, 200 or 300 years from now – when the incidence of severe genetic disease may have doubled on average – our descendants almost certainly will have discovered simple methods of therapy.”68 There have been few efforts to convert the health burden of the hemoglobin disorders into disability-adjusted life years, the only measure that the international public health community will accept as an approach to assessing the comparative health burden of different diseases. Recent and very preliminary analyses of this kind70 have shown that the thalassemias pose a health burden comparable to some common communicable diseases in Asia; however, they encountered major difficulties because of lack of information about the gene frequency of some of the hemoglobin disorders in particular developing countries. In the future, it will be essential to improve these data and to obtain the help of health economists to assess the true health burden of the hemoglobinopathies. In short therefore, in planning for the future public health measures required for the control and management of the hemoglobinopathies it can be assumed that as the

David J. Weatherall and Thomas N. Williams developing countries continue to pass through the epidemiological transition they will impose an ever increasing burden on health services.

Control and Management Issues The main approaches to the control and clinical management of the hemoglobinopathies are discussed in other chapters. Here, some of the particular problems relating to the control of the hemoglobinopathies are discussed in the light of their global distribution and peculiarities in their population genetics. The development of approaches to the global control and management of genetic disease in general,72 and of the hemoglobin disorders in particular4,73–75 have been discussed in detail recently. The particular problems relating to the control of the hemoglobinopathies reflect their widespread heterogeneity and extremely uneven distribution even over relatively short geographical distances, their high frequency in rural populations of the developing countries, and inadequacies of education and medical services in these populations. These issues raise problems in bioethics, counseling, education, and the provision of medical care that are not encountered in the developed countries. Ethical and Counseling Issues. Ethical issues in genetic research, screening, and testing in developing countries are discussed in detail in a report by the WHO73 and in reports by the Nuffield Council on Bioethics.76,77 They are also considered in a recent review.72 There are major ethnic differences in how the nature of disease is interpreted. In many societies in which the level of education is limited it is viewed as being the action of evil spirits or other external forces, a belief that might be encouraged by visits to local healers. In these circumstances, even the simplest explanations of the nature of genetic disease might be extremely difficult to communicate. In many patriarchal societies, genetic disease raises particular problems for women who are carriers or who have affected children. Despite careful explanation of the mechanisms of inheritance, husbands frequently blame their wives for having a child with a genetic disease, often leading to the break up of the family and to a high rate of suicide. Genetic information may be used to discriminate or stigmatize in the context of social practices, particularly in countries in which arranged marriages are still common. In short, a screening program that has demonstrated that a woman is a carrier for a genetic disease can make her unmarriageable. Children with genetic diseases in developing countries can be severely stigmatized and even ostracized by their communities. Informed consent for any procedure is extremely difficult to establish and in many developing countries there are no regulatory or ethical bodies. All these issues, and many others, need to be taken into account when developing genetic services of any kind in the developing countries. Although the principles of genetic

Population Genetics and Global Health Burden counseling are the same as in the developed countries, their application is quite different and must be developed hand in hand with those who have a full knowledge of the local scene. Indeed, a great deal more research is required about the appreciation of disease in different ethnic groups and how this may be best approached by sensitive counseling. Genetic Services in Developing Countries. There have been several reviews of the problems of developing services for the control of genetic and congenital disorders in the developing countries.6,73,74 Establishing programs for the hemoglobinopathies presents particular problems because this field has never come under the auspices of clinical genetics and usually requires special training in centers with experience of the field for appropriate pediatricians, hematologists, and technical staff. Once these personnel are in place, training of appropriate counselors and the development of public education programs can proceed. Next, based on local social and religious beliefs, decisions need to be made about whether community control will be achieved by prenatal diagnosis or community education alone. At a later stage, prenatal screening programs can be developed. Each country requires one or more centers with expertise in both the laboratory diagnosis and clinical management of the hemoglobinopathies, depending on its size. In the current climate it is extremely difficult to achieve any of these developments in developing countries. Their governments are unwilling or unable to give priority to these conditions because of the many other more pressing problems of communicable disease, often associated with a concomitant increase in their health burdens by a rapidly rising rate of noncommunicable disease. Currently, very few governments in Asia or subsaharan Africa are able to support any form of hemoglobinopathy program and hence without some form of external international help it will be a long time before these diseases come under control in many parts of the world. In a report published by the WHO73 and in a followup article,78 suggestions were made about how the international medical and scientific community could help the developing countries in establishing programs for the control of hemoglobinopathies. These concepts were based in part on the success of the hemoglobin field over the past 30 years in evolving programs for the control of the thalassemias in several developing countries by the development of north/south partnerships, that is a partnership between experts in centers in the developed countries and those in whom the skills were lacking in the developing world. The natural evolution of north/south partnerships is the development of south/south partnerships in which those developing countries that have gained skills in the diagnosis and treatment of genetic disorders can form partnerships with countries in which these skills are lacking. Currently, several of the centers in developing countries that were established by north/south partnerships are taking forward the concept of helping neighboring countries.

635 None of these developments will move forward successfully without some form of external support from international health organizations. Like many of the developing country governments, to date these bodies have prioritized more immediate health problems such as those posed by communicable disease, malnutrition, and dysfunctional healthcare systems and, as a consequence, have shown little interest in the hemoglobinopathies. At the time of writing there does appear to be at least some recognition on their part of the increasing health burden posed by the common inherited disorders of hemoglobin. For example, the next phase of the Global Burden of Disease study will include the hemoglobinopathies in its assessment of the relative roles of different diseases in the overall economic burden of disease.79 REFERENCES 1. 2. 3. 4. 5.

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HbF

27 Genetic Modulation of Sickle Cell Disease and Thalassemia Martin H. Steinberg and Ronald L. Nagel

INTRODUCTION Sickle cell anemia is a typical mendelian, single gene disease. Nevertheless, because of its characteristic phenotypic heterogeneity it resembles a multigenic trait. That is, the mutation in HBB is necessary, but alone insufficient to account for the phenotypic differences among patients, and other genes and the environment are likely to modulate its phenotype. In ␤ thalassemia, and even in HbH disease, genotype–phenotype correlations are also often difficult to establish. Modulation of the phenotypes of these disorders by epistatic and other modifying genes has been a subject of increasing interest. Although studies based on candidate-modulating genes – genes chosen for study on the basis of their possible affects on a phenotype – have started to suggest genes and pathways that might modulate the phenotype of sickle cell anemia, a complete picture of genetic modulators should emerge as genome-wide association studies mature. It is likely that fetal hemoglobin (HbF) concentration, and its distribution among erythrocytes is the major genetic modulator of both sickle cell disease and the ␤ thalassemias. The coincidence of ␣ thalassemia with sickle cell anemia or ␤ thalassemia is another powerful modulatory influence. Individually, other genetic modulators are likely to have small effects, yet together the interactions of modulatory genes (and environmental factors) might have an important influence on morbidity and mortality. In this chapter we will first discuss HbF and the genetic elements and genes that might modulate its levels and then the effects of ␣ thalassemia in sickle cell disease and ␤ thalassemia. Finally we will look at genetic association studies that have attempted to link polymorphisms in many genes with the phenotypes of sickle cell disease and thalassemia. As in other chapters, we will try to summarize and consolidate information available in the first edition of this book and concentrate on more recent data.

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HbF (␣2 ␥ 2 ) is the best-known modulator of sickle cell anemia and ␤ thalassemia. Its effect in sickle cell disease is mediated principally by its ability to inhibit HbS polymerization. In ␤ thalassemia, HbF compensates for the reduced expression of the ␤-globin gene, with the resulting deficit of HbA and severe anemia. HbF concentrations vary considerably among patients with sickle cell disease and ␤ thalassemia, suggesting that there is genetic regulation of ␥ -globin gene (HBG1, HBG2) expression and HbF levels.1–5 Initially, only very high levels of HbF were considered capable of influencing the phenotype of sickle cell anemia. Further epidemiological studies suggested that any increment in HbF was clinically and perhaps therapeutically important.6–8

Haplotypes of the ␤-Globin (HBB ) Genelike Cluster in Sickle Cell Anemia and ␤ Thalassemia An important event for understanding the genetic heterogeneity of sickle cell anemia was the discovery that the ␤S globin gene (HBB glu6val) was in linkage disequilibrium (LD) with a polymorphic site in its 3
flanking region that was identifiable by a Hpa I restriction endonuclease cleavage site (Fig. 27.1). The complexity of this linkage was suggested by the finding that the Hpa I single nucleotide polymorphism (SNP), a recognition site for cleavage of DNA by this restriction endonuclease, was territorially segregated in Atlantic West Africa, Bantu-speaking central Africa and central west Africa, where this SNP was either negatively or positively linked to the HbS mutation (Fig. 27.1). A ␤-globin gene cluster haplotype was based on a series of restriction endonuclease–defined SNPs in and surrounding this gene cluster.9,10 The ␤S -globin gene was present on three different haplotypes, and each haplotype was localized to one of three geographical regions of Africa.11 This suggested that the ␤S gene had at least three origins, with subsequent expansion of the frequency of the abnormal gene in each area of origin (Fig. 27.1). Other independent origins of the HbS mutation occurred in Cameroon (Cameroon haplotype) and in the Indian subcontinent (Arab–Indian haplotype). The origins of the HbS gene in Africa, its expansion within Africa, the role of Plasmodium falciparum in the selection of this gene, its spread throughout the world, and the genetic differences among HbS gene haplotypes have been extensively reviewed.12–18 A C-T polymorphism 158 bp 5
to HBG2 (rs7482144) in carriers of Senegal and Arab–Indian haplotypes is strongly associated with HbF levels. Nevertheless, there is considerable diversity of HbF levels in carriers of this SNP, suggesting that other modulatory elements could have an effect. One study suggested that the ␤-globin gene cluster

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Figure 27.1. Haplotypes of the ␤-like globin gene cluster. (Above) Africa, the Mideast, and India showing regions of greatest frequency of their cognate haplotypes and the fall in this frequency moving outward from the center. Arrows depict possible routes of gene flow. (Below) This group of genes and their associated regulatory elements corresponds to a 60-kb stretch of DNA that contains the ␤-globin gene (HBB ) and other highly homologous globin genes including the G ␥ - and A ␥ -globin genes (HBG2, HBG1, the ␦-globin gene [HBD ], the ε-globin gene [HBE1], and some pseudogenes). It is generally inherited en bloc but a “hot spot” of recombination has been located around the ␺␤-␦ region. Depicted by arrows are also the location of the sites that are polymorphic in human populations and cleaved by several restriction endonucleases. Haplotypes correspond to a set of SNPs identified by the presence or absence of selected restriction endonuclease cleavage sites. Although approximately 20 different haplotypes are found in different ethnic groups, only five–seven of them are frequent.

haplotype, independent of HbF level, is a correlate of survival in hydroxyurea-treated sickle cell anemia patients.19 How this might occur is unclear; other modifying genes or elements might be linked to the ␤-globin like cluster, although to date, a haplotype-associated effect on HbF concentration is the sole known modulating factor. Molecular Characteristics of the African and Indo– European β s -linked Haplotypes. SNPs that define a haplotype are present in a 63-kb stretch of DNA subdivided into a 34-kb 5
, 19-kb 3
, and 9 kb central domain. Many other SNPs are present in these haplotype blocks and some could modulate the expression of the ␥ -globin genes and the accumulation of HbF; however, a mechanism for modulation of HbF gene expression is still lacking. Atypical haplotypes in sickle cell anemia usually result from recombination between common and rare haplotypes. In African Americans, because of their genetic diversity, many atypical haplotypes are found.20 In the central domain, 5
to the ␦-globin gene, is a “hot spot” for recombination. When a 5
subhaplotype, including the ␤-globinlike gene cluster locus control region (LCR, Chapter 5), becomes linked to a ␤S -globin gene by recombination, polymorphisms in the LCR or any region 5
to the site of recom-

bination need not be the same as that linked to the ␤S globin gene in the original haplotype. For example, a 5
Benin subhaplotype from a ␤A chromosome might contain SNPs distinct from those of the 5
Benin subhaplotype linked to the ␤S -globin gene. This is a consequence of the relatively short time the ␤S -globin gene has been linked to the 5
Benin haplotype compared with the ␤A -globin gene. This longer period of evolution allows for many more SNPs to be associated with the LCR. In some atypical haplotypes of African Americans, the 5
subhaplotype is likely to be of Caucasian origin, further uncoupling the ␤S -globin gene from the influence of genetic elements that could have coevolved to maintain high HbF levels. LD between the ␤S -globin gene and the Arab–Indian haplotype was discovered independently in sickle cell anemia patients inhabiting the eastern oasis of Saudi Arabia. This haplotype is also linked to the C-T SNP 5
to HBG2. The Arab–Indian haplotype also has a unique (AT)9 T5 repeat sequence 5
to the ␤ gene and an unusual sequence in HS-2 of the LCR. The (AT)9 T5 motif lies within a negative regulatory region between nucleotides −610 and −490, and binds the protein BP-1, a putative repressor of gene expression, which was postulated to influence the fractional concentration of HbS in sickle cell trait (HbAS).21,22 Nevertheless,

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Figure 27.2. The ␤-like globin gene cluster and the 5
G ␥ -158 C-T SNP. The restriction endonuclease sites defining the ␤-like globin gene cluster are shown, including the 5
G ␥ -158 C-T SNP (rs7482144) (site number 2) that characterizes the Senegal and Arab–India haplotype. Sickle cell anemia patients who are compound heterozygotes for a Senegal and Benin chromosome typically have between 6% and 10% HbF, whereas homozygotes can have up to 20% HbF. These numbers are modulated by age, sex, hydroxyurea treatment, and likely, other genetic differences among patients.

although Indian subjects with HbAS have a lower percentage of HbS than subjects of African descent, this might be a result of ␣ thalassemia or even iron deficiency (Chapter 23). In 5
HS-2 of the Arab–Indian haplotype, the AT repeat region has the unique form (AT)10 ACACATATACGT(AT)12 . How and if these and other polymorphisms modulate HbF and G ␥ -globin chain levels in the Arab–Indian and Senegal haplotypes is unclear. In ␤ thalassemia, different mutations were associated with each haplotype and more than one haplotype was linked to the same thalassemia mutation.

Effects of ␤-Globin Gene Cluster Haplotype on HbF Sickle Cell Anemia. Any effect of the ␤-globin gene cluster haplotype on the phenotype of sickle cell anemia and ␤ thalassemia appears to be dependent on the HbF level associated with a haplotype. This, in turn, is associated with the presence of the 5
HBG2 C-T SNP (rs7482144) and perhaps other cis-acting elements (Fig. 27.2). Other studies have suggested that regions 5
to the ␥ -globin genes have regulatory importance. Multiple point mutations in the promoters of the ␥ -globin genes have been found to modulate ␥ -globin gene expression and cause the nondeletion HPFH phenotype (Chapter 7). Erythroid colonies derived from precursor cells obtained from carriers of HbAS with the Arab–Indian haplotype and normal HbF concentration synthesize more HbF than colonies derived from controls.23,24 Erythroid colony growth in vitro might mimic the perturbed erythropoiesis that occurs with hemolysis, suggesting that hematopoietic stress might be needed for the −158 HBG2 C-T SNP to increase levels of HbF. In normal adults and neonates, the −158 C-T SNP is also associated with small but statistically

significant increases in the synthesis of HbF and G ␥ -globin chains.25,26 The Cameroon haplotype and Mediterranean haplotypes II and VI have in common a high G ␥ :A ␥ ratio but lack the −158 C-T SNP. Haplotype II is linked to ␤ thalassemia and to a 4-bp deletion (AGCA) in the region of a GCA repeat at −124 to −127 bp relative to the A ␥ CAP-site.27 This 4bp deletion has been reported in the promoters of the A ␥ alleles on both ␤A and ␤S chromosomes; no effect on the expression of the A ␥ -globin gene was noted however.28 It was suggested that the 4-bp deletion is a common polymorphism linked to the A ␥ gene (see later). Other polymorphisms that might be associated with HbF level have been called the pre-␥ framework.29 β Thalassemia. Many different mutations cause ␤ thalassemia, making an understanding of the molecular basis of HbF modulation in ␤ thalassemia more difficult. Most studies suggest that the −158 C-T HBG2 SNP is a major modulating factor.28,30,31 One study of polymorphisms related to high HbF suggested that a haplotype of the A ␥ -␦globin intergenic region, the motif (TA)9 N10 (TA)10 in HS-2, and the pre-G ␥ haplotype were sufficient but not necessary for high HbF expression. The genetic determinant(s) of high HbF without HPFH was linked to a specific A ␥ -␦ intergenic haplotype.32 In HbE–␤ thalassemia, the Senegal haplotype but not the (AT)(x)T(y) 5
to the ␤-globin gene was linked to the highHbF phenotype.33 More detailed studies of HbE–␤0 thalassemia examined 67 SNPs within the ␤-globin genelike cluster in two groups of unrelated patients, all of whom had a normal ␣-globin genotype. Approximately 200 patients had mild disease and 300 had severe disease when severity was graded by a clinical scoring system.34 The SNPs associated with disease

Genetic Modulation of Sickle Cell Disease and Thalassemia severity and HbF comprised two distinct LD blocks, one containing the ␤-globin gene and the other extending from the LCR to the ␦-globin gene, and they were separated by a recombination hotspot in the region of the ␤-globin gene promoter. Forty-five SNPs within the interval, including the LCR region and the ␦-globin gene, showed strong association with disease severity. The strongest association was observed with the −158 C-T HBG2 SNP, but this explained only approximately 5% and 8% of the HbF variation in the two groups. Carriers of the T allele were more likely to have a milder disease course and higher level HbF in both the mild and severe patient groups. The T allele was always in cis with the HbE allele. Although it seems likely that the association with disease severity is mediated through the effect on HbF level (severe cases, 32.7% ± 11.7%; mild cases, 39.2% ± 11.6%), these differences are small and a mechanistic explanation for the differences in HbF is still lacking.

Genetic Modulation of HbF HbF expression is regulated by complex interactions among chromosome remodeling activities, transcription factors, genes modulating erythropoiesis, and elements linked to the ␤-globin gene cluster (Chapters 4 and 5). This complexity provides many sites for modulation of HbF level in sickle cell disease and thalassemia. LCR and HbF. With rare exceptions, the only cis-acting SNP consistently associated with HbF levels in sickle cell anemia has been the aforementioned −158 C-T transversion 5
to HBG2. Most studies of the influence of the LCR on HbF production have focused on 5
HS-2, which appears to be the only 5
hypersensitive site that is polymorphic among HbS-associated haplotypes. Because this region probably has some regulatory role in globin gene transcription, it was of interest to see if polymorphisms of this region were associated with phenotypic variation in sickle cell anemia and the results of these studies were previously summarized.13 To date, SNPs in the LCR associated with HbF in sickle cell anemia have yet to be pinpointed. In a sickle cell anemia patient homozygous for the Benin haplotype with a HbF of 21% and with 65% G ␥ -chains, the sequence of HS-2 was characteristic of the Senegal type but the −158 C-T SNP was absent, suggesting a crossover 5
to the G ␥ -globin gene. The Senegal HS-2 is distinguished by the presence of Sp1 and GATA-binding sites and a motif similar to that found 5
to the BP-1–binding site; however, the functional significance of this association is not known. HbF levels in six Benin haplotype homozygotes with a Senegal haplotype HS-2 were between 2.6% and 8.5%, suggesting that this polymorphism alone did not lead to increased levels of HbF of the magnitude often seen in carriers of a Senegal haplotype. Polymorphisms in a tandem repeat of the sequence (TA)x N10–12 (TA)y that contains a Hox2-binding site were examined in 100 patients with sickle cell anemia aged 1–18 years.35 Nearly 8% of chromosomes had a discordance

641 in the HS-2 tandem repeat that was not characteristic of the haplotype.36,37 A region between −1445 and −1225 5
to the promoter of the G ␥ -globin gene was found to vary among haplotypes. Senegal–Benin chromosomes associated with modest HbF levels had a likely breakpoint for recombination upstream of −1500 bp 5
to G ␥ -globin gene promoter. In contrast, when a high HbF was present with the Senegal– Benin chromosome recombination, the breakpoint was 3
to position −369 to −309 in the G ␥ -globin gene promoter. In HbAS with a Benin haplotype, when the normal chromosome had the HS-2 (TA)9 N12 (TA)10 structure, HbF (0.9%) and F cells (8.3%) were approximately twice as high as with the presence of other configurations for this region.38 These data suggest that HbF is influenced by elements 3
to HS-2 and 5
to the ␥ -globin gene promoter. F-cell numbers in Benin haplotype HbAS carriers were more strongly associated with the (TA)9 N12 (TA)10 configuration of HS-2 of ␤A chromosomes than the −158 C-T SNP.39 These subjects did not have hemolytic anemia so the results are less likely to reflect differential F cell survival and more likely to estimate HbF production. Although other cis-acting elements, for example, in additional phylogenetically conserved regions of the LCR outside the core sequences of its constituent hypersensitive sites, might partake in the regulation of ␥ -globin gene transcription; definitive associations have not yet been found.13 The activity of constructs containing variant HS-2 enhancers derived from HbS chromosomes was studied to examine the functional effects of these polymorphisms.40 A relationship of reporter gene activity with HbF and the ␤-globin gene cluster haplotype was not found. In a multiplex assay permitting simultaneous analysis of three polymorphic cis-acting elements spanning 53 kb of the ␤-globin gene cluster, concordance between polymorphic alleles in ␥ - and ␤-globin gene promoters was identified. SNPs in HS2 of the LCR were found juxtaposed to atypical cis alleles in the ␥ -globin gene promoter. Analysis of many such hybrid haplotype chromosomes suggested that polymorphisms in the ␥ -globin gene promoter exerted the dominant influence on HbF level in sickle cell disease.5 β-Globin Gene Silencer. Located −530 bp 5
to the ␤globin gene is an AT-rich region with the core structure (AT)x (T)y , which is polymorphic and linked to the ␤-globin gene cluster haplotype. This element has been proposed as a ␤-globin gene silencer that might influence the expression of the ␤-globin gene by variably binding a putative repressor protein, BP-1, depending on the (AT)x (T)y composition.41 BP-1 is a member of the homeobox gene family and the Distal-less subfamily, genes important in early development. BP-1 protein can repress the ␤-globin gene promoter and in the human erythroid cell line MB02; its expression decreases upon induction of the ␤globin gene.21 By modulating erythropoiesis, BP-1 might also effect HbF production.22 The Arab–Indian motif has a higher affinity for BP-1 than the Bantu haplotype motif. This could be reflected

642 at the protein level by less HbS in Indian carriers of the ␤S gene and a normal ␣-globin genotype, when compared with blacks with HbAS, however, this interpretation, as discussed in Chapter 22, is not clear-cut. (AT)9 (T)5 , along with the −158 C-T polymorphism, can be associated with high HbF in some homozygous ␤ thalassemia patients. In these patients, the (AT)9 (T)5 motif was associated with approximately 10 g/dL HbF, whereas (AT)7 (T)7 was accompanied by approximately 5 g/dL of HbF. Four–base pair Deletion Linked to the A γ Gene. Only the Cameroon haplotype ␤S chromosome is associated with an AGCA deletion at nucleotides −222 to −225 5
to the ␤-globin gene and is always linked to the A ␥ T allele. The effects of this deletion on ␥ -globin gene expression are controversial and unlikely to affect carriers of other haplotypes. In a study of sickle cell anemia in which a Cameroon haplotype was trans to a typical HbS haplotype, HbF levels, packed cell volume (PCV) and mean corpuscular volume was similar whatever the haplotype in trans.20 It was also suggested that the 4-bp deletion was associated with decreased expression of not only the A ␥ T globin gene, but also of the G ␥ -globin gene in cis. Studies in ␤ thalassemia in which the 4-bp deletion was present also suggested decreased G ␥ gene expression in cis to the A ␥ T allele. Together, these results implicate the region of the 4bp deletion as a possible cis-acting element that augments the expression of both ␥ -globin genes when combined with a trans-acting factor.42 G γ Gene 5
Regulatory Region. Approximately 1.65–1.15 kb 5
to the G ␥ gene lies an area of 0.5 kb proposed to be another region that potentially has a regulatory role in ␥ -globin gene expression.29 Designated the pre-G ␥ framework, this region was found to have four polymorphic variants that, like most other cis-acting sequences with possible regulatory roles, are linked to the ␤-gene cluster haplotype. This region contains four GATA-1–binding sites and Sp1 and CRE protein–binding domains. Strongest protein binding was associated with the Senegal pre-G ␥ framework and the Benin haplotype–linked pre-G ␥ enhancer activity was sevenfold lower than the Bantu and Senegal type preG ␥ framework. The physiological significance of these findings remains unclear. A SNP, GATA→GAGA, in a GATA site that is in a putative silencing element at nucleotide −567 5
of the G ␥ globin gene promoter was associated with increased HbF in two otherwise normal individuals. This mutation alters a GATA-1–binding motif to a GAGA sequence. DNA–protein binding assays showed that this GATA motif was capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of G ␥ -globin gene promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nucleotides −675 and −526. In addition, the T→G mutation at the GATA motif increased the promoter activity by two- to threefold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of ␥ -globin gene expression. This GATA motif appears to

Martin H. Steinberg and Ronald L. Nagel have a functional role in silencing ␥ -globin gene expression in adults. The T→G mutation in this motif disrupts GATA1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up regulation of ␥ globin gene expression.43 The functional importance of this site was supported by studies in transgenic adult ␤-YAC mice, where it was shown that during definitive erythropoiesis, ␥ -globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the −566/−567 GATA sites of both ␥ -globin gene promoters. Chromatin immunoprecipitation assays showed that GATA-1, FOG-1, and Mi2 were recruited to the −566 or the −567 GATA sites of the ␥ -globin gene promoters when ␥ -expression was low, but not when these genes were being expressed.44 A C-T SNP in 3
HS1 was associated with increased HbF in 11 ␤ thalassemia intermedia patients lacking other explanations for their mild phenotype.45 This report is difficult to interpret because the ␤ thalassemia mutations in these cases were diverse and two individuals had HbF levels less than 10%. Polymorphisms in trans-acting Regulatory Elements Regulating HbF Expression. It has been estimated that the −158 SNP 5
to HBG2 and other postulated haplotype-associated effects on HbF account for less than 25% the variability of HbF in sickle cell anemia. This suggests that trans-acting regulatory elements exert control over HbF levels. Putative trans-acting elements modulating HbF include four quantitative trait loci (QTLs). These are the F cell production locus, a QTL at Xp22 associated with F cell number;4 a QTL at 6q22.3-23.2 associated with F cell numbers first described in an extended Asian–Indian family;1,2,46 a QTL at chromosome 8q that appeared to interact with the −158 C → T SNP;47 and a QTL at 2p16.1, identified in a genome-wide association study of healthy adults that mapped to BCL11A, a zinc-finger protein, which accounted for 15% of F cell variance.48 These elements were first associated with F cells or HbF in individuals with ␤ thalassemia trait or those who did not have a hemoglobinopathy. Further examination of some of these QTLs was undertaken to identify the genes and polymorphisms associated with modulation of HbF in sickle cell anemia and ␤ thalassemia. A candidate gene screening study in patients with sickle cell anemia showed associations with HbF and SNPs in phosphodiesterase 7 (PDE7B), microtubule-associated protein 7 (MAP7 ), mitogen-activated protein kinase kinase kinase 5 (MAP3K5 ), and peroxisomal biogenesis factor 7 (PEX7 ). These genes abut but were not within 6q 23.2.3 It had been hypothesized that variable expression of a collection of linked genes of functional interdependence might be associated with certain phenotypes,49 and long-range LD, as seen in this QTL, has been reported for other chromosomal regions.50 To refine further the functional importance of the 6q QTL direct sequencing of five protein-coding genes, ALDH8A1, HBS1L, MYB, AHI1, and PDE7B, within the

Genetic Modulation of Sickle Cell Disease and Thalassemia 1.5-Mb candidate interval of 6q23 was done but failed to detect mutations that could be associated with HbF modulation.51 The expression profile of these genes in cultured erythroid cells of healthy adults with non-gene deletion hereditary persistence of HbF (HPFH) found that two genes, MYB and HBS1L, were down regulated. Transfection of K562 cells with cDNA of MYB and HBS1L showed that overexpression of only MYB inhibited ␥ -globin gene expression. Low levels of MYB were associated with low cell expansion and accelerated erythroid differentiation, suggesting that differences in the intrinsic levels of MYB might account for some variation in adult HbF levels by its effect on the cell cycle.52 In northern European families, polymorphisms within and 5
to HBS1L were strongly associated with F cell levels, accounting for 17.6% of the F cell variance. Although mRNA levels of HBS1L and MYB in erythroid precursors are positively correlated, only HBS1L expression correlated with high F cells, suggesting that HBS1L variants modulate HbF.53 A QTL at chromosome 8q appeared to interact with the −158 C-T SNP to modulate HbF levels in the same Asian– Indian family in which the 6q QTL was first discovered.2,47 In more than 870 dizygotic twins, effects of the 8q QTL on HbF were also conditional on the genotype of the −158 HBG2 C-T SNP.54 Panels of haplotype tagging SNPs in the ␤-globin genelike cluster and in QTLs on chromosomes 8q and Xp were genotyped in two independent sickle cell anemia patient groups to study their association with baseline HbF levels. In one group of 327 individuals, three SNPs in TOX (thymus high mobility protein; 8q12.1) were associated with HbF. Three additional SNPs in TOX showed significant association in a second group of 987 individuals. Joint analysis of all SNPs and covariates confirmed the association with TOX and identified SNPs potentially associated with HbF in two genes in the Xp22.2-p22.3 QTL. SNPs in TOX and a few other genes were associated with the HbF response to hydroxyurea treatment in sickle cell anemia.55 TOX belongs to a conserved high-mobility group box protein family that binds the minor groove of DNA. Eight hundred and fifty SNPs in 320 candidate genes in the ␤-globin genelike cluster, QTL on chromosomes 6q, 8q, and Xp and other candidate genes were studied in a group of 1,518 adults and children with sickle cell anemia and validated in an independent group of 211 adults. HbF concentration was modeled as a continuous variable with values in a finite interval by using a novel Bayesian approach. In individuals aged 24 years or older, five SNPs in TOX, two SNPs in the ␤-globin genelike cluster, two SNPs in the Xp QTL, and one SNP in chromosome 15q22 were associated with HbF. Four SNPs in 15q22 were associated with HbF only in the larger patient sample. When patients younger than 24 years were examined, additional genes, including four with roles in nitric oxide (NO) metabolism, were associated with HbF level. These observations raise the possibility that different genes might modulate the rate of decline of HbF and the final HbF levels in sickle cell anemia.56

643 The results of this analysis confirmed work where more traditional analytical approaches showed associations of SNPs in TOX, GPM6B, and the ␤-globin genelike cluster with HbF levels. Included in the 15q22-21 interval are MAP2K1, SMAD3, and AQP9. None of these genes have a known connection to HbF synthesis or erythropoiesis, and this region is not a known QTL associated with HbF. Genome-wide association studies focused on HbF concentration in normal individuals and patients with ␤ thalassemia trait, ␤ thalassemia intermedia, and sickle cell anemia, have been reported.57 Six SNPs in BCL11A (2p16.1) were first found to be associated with F cells in 179 normal adults58 with the highest and lowest 5% of F cell numbers. BCL11A, is a highly conserved zinc-finger protein, and codes for a transcription factor containing three C2H2type zinc finger motifs, a proline rich region, and an acidic domain.48 Similar associations of SNPs in BCL11A were found in Sardinian ␤ thalassemia patients and by focused genotyping, in more than 1,200 patients with sickle cell anemia. In the ␤ thalassemia patients the C allele of SNP rs1188686 was associated with higher HbF in the general population and in patients with thalassemia intermedia compared with thalassemia major. With genome-wide association studies of 113 parents of Thai HbE-␤ thalassemia patients and 255 unrelated African Americans with sickle cell anemia, and by focused genotyping of 250 parents of ␤ thalassemia major patients from Hong Kong, association of HbF and F cells were found with SNP rs766432, one of the same SNPs of BCL11A previously reported. In the patients with sickle cell anemia, homozygotes for the C allele of SNP rs766432 had an average of 7% HbF compared with 3% HbF in patients homozygous for the A allele. These findings in at least four different populations are consistent with an aboriginal BCL11A variant in Yorubans that is highly conserved among ancestral populations, even as the haplotype blocks containing this gene have diverged. Together, these observations suggest that possible functional motifs responsible for modulating HbF level or F cells might reside within or immediately adjacent to a 3-kb region bounded by rs1427407 and rs4671393 in intron 2 of BCL11A. A small guanosine triphosphate (GTP)-binding protein, secretion-associated and RAS-related (SAR1A) protein is inducible by hydroxyurea and might play a pivotal role in induction of ␥ -globin gene expression via its role in erythroid maturation. Polymorphisms in the SAR1A promoter were associated with differences HbF levels or the HbF response to hydroxyurea in patients with sickle cell anemia. Three SNPs in the upstream 5
untranslated region (−809 C-T, −502 G-T and −385 C-A) were significantly associated with the HbF response in sickle cell anemia patients treated with hydroxyurea and four SNPs (rs2310991, −809 C-T, −385 C-A and rs4282891) were significantly associated with the change in absolute HbF level after 2 years of treatment.59,60 Effects of β-Globin Gene Cluster Haplotype on the Phenotype of Sickle Cell Disease. Generally, a haplotype is

644 associated with characteristic hematological and clinical findings, although there is considerable heterogeneity within any haplotype. For example, carriers of the HbS gene on Senegal or Arab–Indian haplotype usually have the highest HbF level and PCV and the mildest clinical course. Individuals with Bantu haplotypes have the lowest HbF level and PCV and the most severe clinical course. Carriers of the Benin haplotype have intermediate features (reviewed in ref. 13). Studies of small numbers of African patients with sickle cell anemia and different ␤-globin gene cluster haplotypes who had distinct hematological characteristics first suggested that haplotype could be a marker for the phenotypic heterogeneity of sickle cell anemia. In most of Africa, the environmental, nutritional, and infectious obstacles make it difficult to distinguish the role of haplotype in modulating the course of disease, but clinical differences have been noted. It appears that Africans with the Senegal haplotype fare better than those with other common haplotypes, but there are too few clinical data to permit dogmatism.61 Most of the detailed and larger studies of the clinical and hematological effects of haplotype in sickle cell anemia have been in regions where the HbS gene arrived by gene flow, and after many years of genetic admixture, patients in such regions are usually haplotype heterozygotes, which complicates interpretation of the association of haplotype with phenotype. Reports of the clinical and hematological effects of haplotype in sickle cell anemia should be interpreted carefully because often few patients were studied, the patient’s ages differed among series, clinical events might not have been sharply defined, and distinctions between haplotype homozygotes and heterozygotes were often not clearly drawn. In longitudinal studies from the United States, the Senegal haplotype was associated with fewer hospitalizations and painful episodes.62,63 An effect of the Senegal haplotype on reducing episodes of acute chest syndrome was of marginal significance. The Bantu haplotype was associated with the highest incidence of organ damage, and renal failure was strongly associated with this haplotype.64 Both sex and haplotype affect HbF levels in sickle cell anemia. PCV was higher in males with Benin and Bantu haplotypes. Among carriers with a Senegal haplotype, the PCV in males and females was equal and females had the highest HbF. Females with the Senegal haplotype and high HbF can have less hemolysis and therefore higher PCV.65 Most work suggests that the Arab–Indian haplotype is also associated with milder disease although vasoocclusive events do occur.66–69 Lacking a reasonable hypothesis about how the haplotype of the ␤-globin gene cluster could modify disease severity, other than via an effect on HbF, it seems most reasonable to conclude that the effect of haplotype on the phenotype of disease is mediated through a cis-acting effect on HbF.

Martin H. Steinberg and Ronald L. Nagel EFFECTS OF ␣ THALASSEMIA IN SICKLE CELL DISEASE AND ␤ THALASSEMIA Sickle Cell Disease Approximately a third of patients with sickle cell anemia have coincidental ␣ thalassemia (Chapter 23). These individuals have less hemolysis, higher PCV, lower mean corpuscular volume, and lower reticulocyte counts (see Table 23.9, Chapter 23). Coincident ␣ thalassemia results in longer erythrocyte lifespan because of the reduction of dense and rigid red cells (Table 23.8, Chapter 23). ␣ Thalassemia decreased the risk of organ failure in carriers of a Bantu haplotype. In Jamaicans, the absence of ␣ thalassemia coupled with a high HbF presaged benign disease.70 When phenotypes of sickle cell anemia were clustered into two or three phenotype groups, neither ␣ thalassemia nor the ␤-globin gene cluster haplotype appeared to influence the clinical events defining the groups.71 The benefits and liabilities afforded by the presence or absence of ␣ thalassemia in sickle cell anemia is very likely to be due to its effects on hemolysis. Recent work suggests that concurrent ␣ thalassemia reduces the incidence of stroke, priapism, leg ulceration, and pulmonary hypertension but has little effect or increases the chances of developing osteonecrosis, acute chest syndrome, and painful episodes.72–75 As discussed in Chapter 11, hemolysis is associated with a proliferative vasculopathy due to decreased bioavailable NO.

␤ Thalassemia Concurrent ␣ thalassemia or the presence of ␣-globin gene duplications modifies the phenotype of ␤ thalassemia. As the pathophysiology of ␤ thalassemia is largely determined by imbalance globin chain synthesis (Chapter 17), concurrent ␣ thalassemia, by reducing the accumulation of ␣-globin chains, tends to balance the deficit in ␤-globin chains and causes a milder disease. A sufficient ␣-globin gene deficit can cause a thalassemia intermedia phenotype in homozygotes or compound heterozygote for severe ␤-thalassemia mutations where the expected phenotype would be thalassemia major. Conversely, extra ␣-globin genes, as seen with chromosomes containing triplicated or quadruplicated ␣-globin loci, by increasing the imbalance in ␣:␤ synthesis, can convert a heterozygote for ␤ thalassemia into a symptomatic thalassemia intermedia.76,77 One might hypothesize that with an improvement in the ␣:␤ globin synthesis ratio and reduced hemolysis, hemolysis-related complications of ␤ thalassemia, which also include pulmonary hypertension, leg ulcers, and perhaps priapism, might also be reduced; however, there are no data on this subject. Role of α-Hemoglobin Stabilizing Protein. ␣-Hemoglobin stabilizing protein (ERAF, 16p11.2), by binding free ␣globin chains, prevents their proteolysis and preserves their ability to form ␣:␤ dimers.78 In ␤ thalassemic mice that

Genetic Modulation of Sickle Cell Disease and Thalassemia

645 hemolysis and NO dysregulation

coagulation inflammation/oxidant injury, .02, cytokines

NOS, ICAMS,VCAMS, integrins, selectins

HbF, alpha thalassemia, membrane phospholipids, adhesion molecules, cation transporters

Figure 27.3. Some facets of sickle cell disease pathobiology that might be genetically modulated. (See color plate 27.3.)

are also deficient in ␣-hemoglobin–stabilizing protein, the phenotype of the ␤ thalassemia was more severe.79 The role of ␣-hemoglobin–stabilizing protein as a modulator of ␤ thalassemia is less conclusive, with no relationship found between haplotypes of ␣-hemoglobin– stabilizing protein and the severity of HbE–␤ thalassemia.80,81 In a study of more than 100 healthy individuals, expression of ␣-hemoglobin–stabilizing protein was measured. Among six common variants of this protein, four were strongly associated with its expression. In nine anemic patients with heterozygous ␤ thalassemia who also had a triplicated ␣-globin locus, one variant of ␣-hemoglobin– stabilizing protein was more common than expected, suggesting that variation in the protein could contribute to some heterogeneity in ␤ thalassemia.82

ifying or epistatic genes that potentially affect the pathogenesis of sickle cell anemia and modulate the phenotype of disease include: mediators of hemolysis; vascular remodeling; inflammation; oxidant injury; NO biology; vasoregulation; cell–cell interaction; blood coagulation and hemostasis; growth factors; cytokines and receptors; and transcriptional regulators (Fig. 27.3). These genes have modifying affects independent of effecting HbS polymerization, the mechanism by which both HbF and ␣ thalassemia impact the phenotype of disease (Fig. 27.4). Polymorphisms have been found in candidate genes that might affect the phenotype of sickle cell disease (Table 27.1).83

HEMOGLOBIN A2 HbA2 , a tetramer of ␣-and ␦-globin chains (Chapter 7), impairs the polymerization of HbS to the same extent as the ␥ -globin chain of HbF. HbA2 has the advantage of being evenly distributed in all red cells, whereas HbF is sequestered in F cells. The naturally low level of HbA2 makes it an inconsequential contributor to the total hemoglobin concentration but there are instances when the level of HbA2 is increased far beyond its usual values. Some exceptionally high HbA2 levels are the result of deletions that remove the ␤-globin gene promoters. When HbA2 and HbF levels are high, their combination might modulate sickle cell disease and cause a milder phenotype, but few patients with these genotypes are available to establish conclusively its phenotype (Chapter 23). With these rare exceptions, it is unlikely that variation in HbA2 level affects the phenotype of sickle cell anemia.

MODULATION OF THE PHENOTYPE OF SICKLE CELL ANEMIA Genetic Polymorphisms as Predictors of Disease Severity The diversity of sickle cell anemia cannot be explained solely by HbF and ␣-globin gene–linked modulation. Mod-

Figure 27.4. The relationships between ␤-like globin gene cluster haplotypes and clinical features of sickle cell anemia. PCV connotes packed cell volume. The clinical features include events such as osteonecrosis, acute chest syndrome, renal failure, and other common disease complications. These associations of haplotype and phenotype in sickle cell anemia are only generalities, and within each haplotype group there is considerable heterogeneity whose cause is unknown.168

646

Martin H. Steinberg and Ronald L. Nagel Table 27.1. Genetic polymorphisms affecting some phenotypes of sickle cell anemia and ␤ thalassemia Phenotype

Gene/SNP marker

References 91 92 92 92 92 92 96 94,95 72 104–106, 165, 166

Pulmonary hypertension Cholelithiasis Priapism Leg ulcers Bacteremia

VCAM1/ G1238C VCAM1/T-1594C IL4R/S503P TNFA/G-308S LDLR/NcoI +/− ADRB2/Q/27E AGT/AG repeats HLA genes Multiple genes MTHFR/C677T BMP6, ANXA2 NOS3/T-786C NOS1/AAT repeats TGFBR3, SMAD1, KL, NRCAM, SMAD3, SMAD7 PIK3CG TGF␤/BMP pathway genes UGT1A/promoter repeats KL, TGFBR3, AQP1, ITGAV KL, TEK, TGF␤/BMP pathway genes TGF␤/BMP pathway genes

116 122, 123, 125 101, 102 74 107

␤ Thalassemia Cholelithiasis Iron overload Bone disease Cardiovascular disease

UGT1A HFE COl1A1, VDR APOE

see text 151, 152 156–159 160

Sickle cell anemia Stroke

Osteonecrosis Acute chest syndrome

117 167 121

Other genetic variants, some mentioned in the text, have been studied and have shown no associations.

A unifying theme emerging from these studies is that polymorphisms in genes of the transforming growth factor–␤/ bone morphogenetic protein (TGF␤/BMP) pathway appear involved in several subphenotypes of disease and could reflect the hitherto unappreciated role of this very large pathway in the pathobiology of disease.84 The first approaches to understanding how genetic polymorphism might modulate the phenotype of sickle cell anemia were based on the selection of candidate genetic modifiers. A candidate gene–based approach is limited by the imagination needed to define likely candidates and alone cannot provide a complete picture of the genetic heterogeneity that must account for the complex pathobiology of any disease. In some studies, picking the “wrong” SNP or choosing insufficient numbers of SNPs in large genes might have lead to false-negative results. Nonsynonymous coding region SNPs are an obvious first choice to examine, but they are the least common. Recent studies suggest that by affecting protein folding, even synonymous SNPs might modulate a genes’ function,85 and genome-wide association studies have yield surprising results in other diseases in which the strongest associations of genotype with phenotype have occurred in gene-poor regions.86 SNPs that effect gene expression might be even more important, but it is difficult to identify all of these by in silico analysis.

Genome-wide association studies are just beginning and have the promise of unbiased interrogation of the entire genome for an association with a phenotype. Copy number variants can also be assessed with the available technology. Analytical methods for genetic association studies are evolving, and dealing with the problem of false-positive results can be vexing when more than 1 million SNPs can be genotyped across the genome of thousands of cases.87 Other issues that must be considered in genome-wide association studies include: LD with causative polymorphisms; interpretation of associations with SNPs without an obvious function; gene–gene interaction; gene–environment interaction; and precise phenotype definition. Painful Episodes. Acute painful episodes are the major clinical event in sickle cell disease. These episodes are one measure of disease severity, and a predictor of early death in adults.6 The rate of painful episodes varies widely among patients; highest pain rates are found in patients with high PCV and low HbF. In addition to HbF concentration and a possible role of ␣ thalassemia, a genetic basis for the heterogeneous distribution of painful episodes among patients has not been described. Case-control studies are problematic because nearly all patients will have pain, therefore, finding genes that modify the risk of pain will be difficult. SNPs modulating HbF levels in sickle cell anemia

Genetic Modulation of Sickle Cell Disease and Thalassemia were genotyped in the CSSCD patients and a smaller number of patients from Brazil. SNPs in BCL11A, HBS1L-MYB and the C-T polymorphism 158 bp 5
to HBG2 (rs7482144) were strongly associated with HbF and the rate of painful episodes.88 Patient response to opioid analgesics varies considerably, and the efficacy of these drugs is known to depend on genetic variability of their catabolic enzymes and receptors.89 How this might affect the treatment of the sickle cell acute painful episode is unknown. Stroke. Most genetic association studies in sickle cell anemia have examined the stroke phenotype (Chapter 19). A family predisposition to stroke in sickle cell disease suggested that inherited modulation of this phenotype was possible.90 Among the genes associated with stroke in sickle cell anemia, two alleles of vascular adhesion molecule–1 (VCAM1), G1238C in the coding region of immunoglobulin domain 5, and, T-1594C, an intronic SNP, had an association with stroke. The coding region SNP was protective, whereas the intronic SNP predisposed to smallvessel stroke.91,92 Preliminary studies also suggested that theVCAM1 G1238C SNP was protective for developing high transcranial Doppler flow velocity, which is a strong predictor of stroke in children (Chapter 19).93 VCAM1 is approximately 19,000 bp long and has nine exons and at least 200 SNPs. Clearly, choosing the “right” SNP is not trivial and an association is likely to identify LD. Six SNPs in the intercellular adhesion molecule–1 and CD 36 genes (ICAM1; CD36) were not associated with stroke.91 When stroke was subdivided into large- and smallvessel disease based on imaging studies, SNPs in the interleukin-4 receptor gene (IL4R; nonsynonymous coding region, S503P) predisposed to large vessel stroke, whereas TNF␣ gene (TNFA; noncoding G308A) and ␤-adrenergic receptor 2 (ADRB2; nonsynonymous coding region, Q27E) SNPs were protective. In the small-vessel stroke group, a low-density lipoprotein receptor (LDLR; untranslated region) SNP was protective. Homozygosity for the combination of TNFA -308 GG and the IL4R 503P heterozygosity was associated with a strong predisposition to large-vessel stroke.92 The endothelium and vascular response to regulators might distinguish small from large blood vessels but a continuum must exist, making the pathophysiological basis of these observations enigmatic. Human leukocyte antigen (HLA) genes might be risk factors for vascular disease. In sickle cell anemia patients with cerebral infarction, the HLA DRB1∗ 0301 and ∗ 0302 alleles increased the risk of stroke and the DRB1∗ 1501 protected from stroke. DQB1∗ 0201, in LD with DRB1∗ 0301, was associated with stroke and DQB1∗ 0602, in LD with DRB1∗ 1501, was protective. HLA genotyping was performed in patients with large-vessel stroke and with small-vessel stroke.94,95 In the small-vessel stroke group, HLA DPB1∗ 0401 was associated with stroke, whereas DPB1∗ 1701 was protective. In the large-vessel stroke patients, DPB1∗ 0401 was associated with susceptibility, and DPB1∗ 1701 was associated with

647 a trend toward protection. Also, HLA-A∗ 0102 and A∗2612 caused susceptibility to stroke and A∗3301 was protective. Other genes have been studied in the stroke phenotype. These include the C1565T mutant of the platelet glycoprotein IIIa (ITPG3) gene, angiotensinogen (AGT ), cystathionine B synthase (CBS; 278thr 68-bp insertion), cholesterol ester transfer protein (CETP; −628A), apolipoprotein C III (APOC3; −641A) the C677T polymorphism in 5,10-methylenetetrahydrofolate reductase (MTHFR) the plasminogen activator inhibitor–1 gene (PAI1), TGF␤ receptor 3 (TGFBR3), and adenine cyclase 9 (ADCY9).96–99 These studies were all equivocal or negative; however, the sample size was usually insufficient for a definitive result. It seems unlikely that a still undiscovered gene polymorphisms in any single gene will have a dominant effect on a phenotype of disease. To examine the interactions among genes and their SNPs and to develop a prognostic model for stroke in sickle cell anemia, a bayesian network was developed to analyze SNPs in candidate genes in 1,398 unrelated patients with sickle cell anemia (Fig. 27.5).72 SNPs in 11 genes and four clinical variables, including ␣ thalassemia and HbF, interacted in a complex network of dependency to modulate the risk of stroke. This network of interactions included three genes, BMP6, TGFBR2, and TGFBR3 with a functional role in the TGF␤/BMP pathway and P-selectin (SELP). The model was validated in a different population by predicting the occurrence of stroke in unrelated individuals with 98.2% accuracy, predicting the correct outcome for all stroke patients, and for 98% of the nonstroke patients. This gave a 100% true positive rate, a 98.14% true negative rate, and a predictive accuracy of 98.2%. As traditional analytical methods are often inadequate for the discovery of the genetic basis of complex traits in large association studies, bayesian networks are a promising approach. The predictive accuracy of this stroke model is a step toward the development of prognostic tests that are better able to identify patients at risk for stroke. The association in the general population of genes like SELP and genes in the TGF␤ pathway with stroke suggest that predisposition to stroke might be shared by both sickle cell anemia patients and stroke victims overall. Gene expression studies have also contributed to understanding predisposition to stroke.100 When individuals at risk for stroke, estimated based on the presence of circle of Willis disease or history of stroke, were compared with controls, transcripts in genes of inflammation-related pathways expressed in blood-outgrowth endothelial cells were most strongly associated with stroke or predisposition to stroke. Priapism. SNPs in 44 candidate genes were examined for their association with priapism in 148 patients with sickle cell anemia with priapism and controls who had not developed priapism. Polymorphisms in Klotho (KL) showed an association with priapism by genotypic and haplotype analyses.101 KL has a role in NO biochemistry and directly or indirectly promotes endothelial NO production.

648

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Figure 27.5. A bayesian network describing the associations of SNPs in candidate genes with the likelihood of developing nonhemorrhagic stroke in sickle cell anemia.72 (See color plate 27.5.)

EDN1.9

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THALASSEMIA

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Genetic Modulation of Sickle Cell Disease and Thalassemia A strong association was also found between the prevalence of priapism, the severity of hemolysis, and the presence of ␣ thalassemia.73 Of approximately 200 men older than 18 years, 83 had a history of priapism. A candidate gene study in this population failed to show an association of priapism with SNPs in KL, nevertheless, there were associations with SNPs in TGFBR3, AQP1 and the adhesion molecule, ITGAV.102 Further study of this population, which included 186 men with a mean age of 32.4 years, found that a single coding SNP in F13A1, the Factor XIII gene was associated with priapism with an odds ratio of 2.43 for individuals with C/C compared with the C/G genotype.103 Osteonecrosis. Osteonecrosis is found in nearly half of all adults with sickle cell anemia. It is one of the socalled vasoocclusive/viscosity-associated phenotypes and it appears to be increased by concurrent ␣ thalassemia (Chapter 19). The C677T polymorphism in MTHFR was found in 36% of adults with sickle cell disease and osteonecrosis but only 13% of controls.94 Nevertheless, other reports failed to confirm this association (reviewed in ref. 78). The C1565T SNP in ITPB3 and a polymorphism in PAI1 were not associated with sickle osteonecrosis.105 These studies all examined small numbers of patients and few SNPs. When sickle cell anemia patients with osteonecrosis were compared with controls, individuals with osteonecrosis had a higher prevalence of coincident ␣ thalassemia, and there were no differences in PCV or HbF levels. Significant associations were observed with SNPS in seven genes (BMP6, TGFBR2, TGFBR3, EDN1, ERG, KL, and ECE1). Additional SNPs, equally distributed within the gene, were typed in all seven genes and a significant association with many SNPs in KL and BMP6 was found.106 Of the 18 SNPs typed in KL, 10 were significantly associated with osteonecrosis. Most of these SNPs were located in the 20-kb region, representing the first half of the first KL intron and were in LD with each other. SNPs in BMP6 (5/14) and ANXA2 (6/13) were also associated with osteonecrosis; however, these SNPS were distributed throughout the intronic and 3
untranslated regions of the genes, and disease-associated SNPs tended to be in LD. The distribution of haplotypes of all three genes was significantly different among cases and controls. These genes might play a role in bone metabolism. Among its many functions, KL is a glycosyl hydrolase that participates in a negative regulatory network of the vitamin D endocrine system. BMPs, including BMP6, are secreted proteins structurally related to TGF␤ and activins and are involved in bone formation and development. ANXA2, a member of the calcium-dependent phospholipid-binding protein family, is involved in osteoblast mineralization. Lipid rafts containing annexin-2 seem important for alkaline phosphatase activity in bone. The actual mechanisms by which variants of these genes predispose sickle cell patients to vascular complications are unknown. Leg Ulcers. Sickle cell leg ulcers are related to hemolysis and associated with SNPs in genes that could affect sickle

649 vasoocclusion. Leg ulcer patients had lower PCV and higher levels of lactate dehydrogenase, bilirubin, aspartate aminotransferase, and reticulocytes than controls. Age-adjusted comparisons showed that sickle cell anemia–␣ thalassemia was more frequent among controls than cases, suggesting that the likelihood of having leg ulcers was related to the intensity of hemolysis, which is modulated by ␣ thalassemia. In candidate gene association studies, associations were found with SNPs in KL, TEK, and several genes in the TGF␤/BMP signaling pathway. The TEK receptor tyrosine kinase is involved in angiogenesis, whereas the TGF␤/BMP signaling pathway modulates wound healing and angiogenesis, among its other functions.74 Bacteremia. Infection, bacteremia, and sepsis are common events in sickle cell anemia. SNPs in candidate genes have been associated with an increased risk of sepsis in other diseases. In a case-control study, bacteremia in sickle cell anemia was associated with SNPs and haplotypes of genes of the TGF␤/BMP pathway such as BMP6, TGFBR3, BMPR1A, SMAD6, and SMAD3. The TGF␤/BMP pathway might play an important role in immune function.107 Renal Disease. Patients with sickle cell anemia associated with a Bantu haplotype are more likely to develop renal failure and other vasoocclusive complications, perhaps because these individuals have the lowest levels of HbF.64,108 SNPs in selected candidate genes are also associated with glomerular filtration rate. When the estimated glomerular filtration rate was used as a phenotype of sickle nephropathy, and tagging SNPs in approximately 70 genes of the TGF␤/BMP pathway were studied, four SNPs and three haplotypes in BMPR1B, a BMP receptor gene, yielded statistically significant associations. The TGF␤/BMP pathway has been associated with the development of diabetic nephropathy, which has some features in common with sickle cell nephropathy.109 Pulmonary Disease. Pulmonary hypertension or pulmonary vascular disease has emerged as an important risk factor for premature death in patients with sickle cell anemia, but its genetic basis has only recently started to be studied.110,111 Pulmonary hypertension is likely to be modulated by the effects of genes that control NO and oxidant radical metabolism, cell–cell interaction, vasculogenesis, and vasoreactivity. For example, mutations in BMP receptor 2 (BMPR2) and other genes have been associated with both familial and idiopathic pulmonary hypertension.112–114 A study of patients with sickle cell pulmonary hypertension compared with controls found that SNPs in BMPR2 and ADCY6 were associated with this phenotype, and followup studies suggested that SNPs in TGFBR3 were associated with this phenotype.115 Further work by this group involved the study of 297 SNPs in 49 candidate genes in 111 patients screened for pulmonary hypertension, and evidence of an association was primarily identified for genes in the TGF␤/BMP superfamily, including ACVRL1, BMPR2, and BMP6.116 BMP6 SNP 449853, associated with pulmonary hypertension, was also associated with stroke

650 and bacteremia in studies of totally different patients, supporting these associations.72,107 Acute chest syndrome is a common vasoocclusive complication with, significant morbidity and mortality rates.111 Both an increased and decreased risk of having acute chest syndrome were associated with a T786C SNP in the endothelial NO synthase gene (NOS3); one small study did not replicate either of these observations.117–119 Exhaled NO levels were reduced in patients with acute chest syndrome compared with controls, and this was associated with the number of AAT repeats in intron 20 of NOS1.120 In follow-up studies, the ATT repeat polymorphism in NOS1 was associated with acute chest syndrome only in patients without asthma (itself, a risk factor for acute chest syndrome).119 In this study of 134 children, 64% had at least one acute chest syndrome, and 36% had asthma. This study examined a small number of patients. Also, the relationship between the number of NOS1 ATT repeats and risk of acute chest syndrome was an unusual curvilinear one. The association was modest, at best, and a plausible reason why this polymorphism should be important only in acute chest syndrome patients without asthma is not obvious. A T8002C SNP in the endothelin-1 gene (EDN1) was associated with an increased risk of acute chest syndrome in 173 children in whom sickle cell anemia was detected at birth and patients followed longitudinally.118 All of these studies examined small numbers of cases and controls; most were limited to children, and few SNPs were examined. To deal with these issues, a candidate gene association study of acute chest syndrome was performed using data from 1,422 individuals with sickle cell anemia. Because the etiology and clinical course of acute chest syndrome in patients younger than 4 years is different from that in older individuals, the population was dichotomized into pediatric (aged ≤ 5 years) and older children and adults (aged > 5 years).121 There were 170 acute chest syndrome cases and 884 controls in the group younger than 5 years and 388 cases and 819 controls in older group. Using time to first event in an age-, sex-, leukocyte-, reticulocyte-, and platelet count–adjusted analysis, and controlling for the false discovery rate, two SNPs were found to be significantly associated with acute chest syndrome in both patient groups. These SNPs were in TGFBR3 and in an unknown gene in LD with SMAD7, which is also in the TGF␤ pathway. Additional SNPs significantly associated with acute chest syndrome in younger cases were in PIK3CG, a member of the PI3/PI4 kinase family involved in cell–cell adhesion. Six additional SNPs were associated with acute chest syndrome in older patients were found in SMAD1, KL, NRCAM, and SMAD3. The NOS1 ATT repeat polymorphism, EDN1, and NOS3 SNPs discussed previously were not examined in this study. Hyperbilirubinemia and Gallstones. Promoter polymorphisms in the uridine diphosphate–glucuronosyltransferase 1A (UGT1A) gene are associated with unconjugated hyperbilirubinemia and Gilbert syndrome. Children with

Martin H. Steinberg and Ronald L. Nagel sickle cell disease had a significantly higher mean bilirubin level if they carried the 7/7 UGT1A genotype compared with the wild-type 6/6 or 6/7 genotypes; patients with the 7/7 genotype were more likely to have had a cholecystectomy. This suggested that symptomatic cholelithiasis is more common in carriers of this genotype.122,123 Steady-state bilirubin levels are also influenced by the presence of ␣ thalassemia and the HbF level.124,125 The 7/7 and 7/8 genotype were risk factors for symptomatic gallstones only in older patients with sickle cell disease, and although coincident ␣ thalassemia was associated with less hemolysis, it did not compensate for the UGT1A promoter polymorphism.126,127 ␣ Thalassemia, regardless of the UGT1A genotype, was associated with reduced serum bilirubin levels.125 Erythrocyte Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency and the Endothelium. G6PD deficiency is common in sickle cell anemia. Studies of the phenotype of combined G6PD deficiency and sickle cell anemia have given disparate results. In a multiinstitutional study, however, G6PD deficiency was not associated with differential survival, reduced hemoglobin levels, increased hemolysis, more pain crises, septic episodes, or a higher incidence of acute anemic episodes.128 Using DNA-based methods to detect unequivocally the GdA- allele of G6PD, it was reported that the frequencies of GdA− and of the normal GdB and GdA+ genes were identical in patients with sickle cell anemia and controls.129 Blood counts were similar in patients with and without G6PD deficiency, although the hemoglobin concentration was lower in sickle cell anemia with the GdA− gene. The prevalence of GdA− did not change with age. Although there is clearly little, if any, modulation of the phenotype of sickle cell anemia by coincident G6PD deficiency, perhaps the “right” phenotype has not been studied. Patients with sickle cell anemia have impaired flowdependent and independent vasodilation.130 This might be a consequence of intravascular hemolysis, heme scavenging of NO with decreased NO bioavailability, and oxidant stress (Chapter 11). Adequate availability of G6PD is needed to maintain both NO levels and preserve the proper redox milieu. It has been proposed that a G6PD-deficient phenotype could be present in critical vascular tissues in G6PDdeficient individuals and perhaps even in sickle cell disease patients with a normal G6PD genotype.131 In sickle cell disease, flux through the pentose phosphate pathway is reduced.132 G6PD activity in sickle cell disease might be inadequate to maintain intracellular GSH levels, producing in effect, a functional G6PD-deficient state.133 This same relative deficiency might also occur in endothelium and play a role in the endothelium-related pathophysiology of disease. It has been hypothesized that the hyperaldosteronism of sickle cell anemia might impair vascular reactivity by decreasing endothelial G6PD activity.134 Sickle erythrocyte adherence to endothelium is likely to be an important component of the pathophysiology of

Genetic Modulation of Sickle Cell Disease and Thalassemia disease (Chapter 8). One determinant of this interaction is red cell adhesion to laminin, which increases in response to adrenaline stimulation of ␤2 -adrenergic receptors and adenylate cyclase. Polymorphisms of the ␤2 -adrenergic receptor (ADRB2) and adenylate cyclase (ADCY6) genes were associated with increased adhesion, suggesting that this and other features of the interaction of sickle erythrocytes with endothelial cells could be genetically modulated.135 Compound Phenotypes and Integrated Measure of Disease Severity. Few studies have combined disease complications and laboratory variables to seek associations with polymorphisms in candidate genes. One small study of patients with histories of stroke, acute chest syndrome, osteonecrosis, and priapism showed that patients with complications had a significantly higher frequency of the HPA-5b allele compared with controls.136 In this small study, an individual needed only a single complication to be included. Most events were osteonecrosis, and only four individuals had more than a single phenotype. To produce an integrated disease phenotype, a Bayesian network model was developed that described the complex associations of 25 clinical and laboratory variables, deriving a score to define disease severity as the risk of death within 5 years137 (Chapter 19). The genetic basis of severity was examined using this score as a phenotype and studying the association of 795 SNPs in 320 candidate genes by using a Bayesian test that compared the genotype distribution among cases with mild and severe disease and calculated the probability that the two distributions were different in 741 HbS homozygotes older than 18 years of age.138 Positive associations were validated in a smaller independent patient sample. Among the SNPs in genes associated with disease severity, some associations, like TGFBR3, confirmed previous findings in stroke and pulmonary hypertension in patients with sickle cell anemia, and support the speculation that dysregulation of the TGF␤/BMP signaling pathway might play a major role in the modulation of disease severity. Other genes in which SNPs were associated with severity, such as ECE1 and KL, are expressed in endothelium or modulate endothelial function. Some associated genes play a less obvious role in the pathobiology of disease, such as HAO2 and TOX, but were strongly associated with sickle cell disease severity and with normal aging. It seems likely that increased oxidative stress and the relentless progression of vasculopathy in sickle cell anemia cause accelerated tissue damage, and this might be modulated by a set of genes similar to those involved in the normal aging process. As the results of unbiased genome-wide association studies are added to capture polymorphisms not included in candidate gene studies, a predictive network with even greater reliability than one using only clinical and laboratory variables might be developed. To begin this process, 684 mild and severe sickle cell anemia patients, based their severity score, were compared with 877 centenarians and 1,850 younger controls and each SNP was tested for

651 association with the traits of severe or less severe sickle cell anemia and exceptional longevity. The analysis identified 140 SNPs in more than 50 genes and some intergenic regions that showed robust and consistent associations, a number more than twice than expected by chance. Among the most ”significant” associations were genes with putative roles in blood circulation, coronary artery disease, type 2 diabetes, triglyceride, and glucose metabolism and sudden death. SNPs in HAO2 and MAP2K, associated with both sickle cell disease severity and exceptional longevity in candidate gene studies, were also associated in the genome-wide association studies. These data further suggest that common metabolic pathways are likely to influence the chance of developing complications of mendelian and multigenic diseases and the likelihood of achieving exceptional longevity.139 This might explain the commonality of genes whose SNPs are associated with the vascular complications of sickle cell anemia, arteriosclerosis and diabetes. A new paradigm suggests that hitherto unexpected genetic differences modulate a limited number of pathways that form a common route toward determining good health and disease. Duffy red cell glycoproteins (Fya and Fyb ) have been implicated in the clearance of inflammatory cytokines. Duffy negative individuals that include approximately 70% of African Americans might clear these cytokines less efficiently. In adults with sickle cell anemia, end-organ damage and overall disease severity, assessed by a composite score reflecting the presence or absence of injury to a group of organs, were compared between Duffy-positive and -negative patients, as assessed by a SNP (rs2814778) in the promoter of the DARC gene. Sixty-five percent of Duffynegative patients had chronic organ damage and 32% had proteinuria, statistically significant differences from Duffypositive cases.140

GENETIC MODULATION OF THE PHENOTYPIC DIVERSITY OF ␤ THALASSEMIA Genetic Polymorphisms as Predictors of Disease Severity Very little is known about the modifiers of the phenotype in ␤ thalassemia other than the roles played by HbF level and ␣ thalassemia.30 Hyperbilirubinemia and Gallstones. As with sickle cell anemia, bilirubin levels are modified by promoter polymorphisms of UGT1A.141–146 Iron Loading. Part of the phenotype of ␤ thalassemia is the tendency for iron loading as a result of chronic hemolytic anemia, increased iron absorption, and blood transfusion. Hereditary hemochromatosis – most often a result of a C282Y mutation in HFE, a gene regulating iron metabolism – can also be found in rare cases of ␤ thalassemia and might increase iron loading and its consequences.147 However, in a larger study of patients with ␤ thalassemia, there was no association of the C282Y HFE

652 SNP with iron overload.148,149 Concurrent ␤-thalassemia trait can increase iron loading in classic hereditary hemochromatosis.150 Homozygosity for the H63D SNP in HFE has been associated with increased serum ferritin in Italian carriers of ␤ thalassemia trait. In northern Indian ␤ thalassemia carriers, this SNP was not associated with iron loading.151–153 In the Chinese, classic HFE mutations are even less common than in Italians; thus it is unlikely that polymorphisms of this gene would influence iron loading in ␤ thalassemia. Mutations in the transferrin receptor gene (TFR2) can cause hemochromatosis. Two polymorphisms of TFR2 were not associated with ferritin level or transferrin saturation in Chinese patients with transfusion-dependent ␤ thalassemia or in nontransfusion-dependent ␤-thalassemia intermedia.154 Bone Disease. Bone disease and osteoporosis are causes of morbidity in severe ␤ thalassemia, and bone mass is regulated by many genes.155 In severe ␤ thalassemia, a G-T SNP in the collagen type ␣1 gene (COL1A1) was associated with osteoporosis and in patients with ␤-thalassemia major and intermedia, an intronic SNP in VDR, the vitamin D (1,25dihydroxyvitamin D3) receptor gene, was associated with a reduction in lower spine bone mineral density.156–159 SNPs in TGFB1 were not associated with osteoporosis.158 Both studies examined a small number of cases and controls and very few SNPs, so these results must be replicated in larger patient samples. Cardiovascular Disease. Heart failure, a result of iron loading, is the common cause of death in severe ␤ thalassemia. Decreased antioxidant activity of the apolipoprotein E (APOE ) 4 allele was postulated as a genetic risk factor for the development of left ventricular failure in homozygous ␤ thalassemia. Greek patients with ␤ thalassemia were grouped into 1) patients without heart disease, 2) patients with left ventricular failure, and 3) patients with left ventricular dilation and normal systolic function. Patients with normal hearts had an APOE 4 allele frequency similar to that of normal controls. Patients with left ventricular failure had a significantly higher frequency of APOE 4 than the controls, suggesting that this allele can represent a genetic risk factor for the development of heart failure.160 Two SNPs in the estrogen receptor gene (ESR1) were examined in pre- and postpubertal, well-treated, homozygous ␤ thalassemia patients. Individuals lacking an Xba I restriction site polymorphism had higher body mass index, triglycerides, and blood pressure. The authors suggested that this SNP might influence nutrition and could be considered an additional risk factors for later cardiac disease.161

GENETIC MODULATION OF THE PHENOTYPIC DIVERSITY OF HbH DISEASE Heterogeneity of ␣-globin gene mutations leads to phenotypic diversity of HbH disease.162 If little is known about

Martin H. Steinberg and Ronald L. Nagel the modulation of phenotype in ␤ thalassemia, even less is known about the other modulators of phenotype in HbH disease or Hb Bart’s hydrops fetalis caused by identical ␣globin gene mutations. As might be expected, polymorphism of the UGT1A promoter affects the bilirubin concentration and the likelihood of cholelithiasis.163 Iron overload is a cause of disability in HbH disease even in the absence of regular blood transfusion. When polymorphisms of TFR2 and HFE were tested for their association with iron overload in HbH disease, iron loading was similar in patients with and without these mutations. It was concluded that excess iron in HbH disease is most likely a result of increased absorption.164 REFERENCES 1. Garner C, Mitchell J, Hatzis T, Reittie J, Farrall M, Thein SL. Haplotype mapping of a major quantitative-trait locus for fetal hemoglobin production, on chromosome 6q23. Am J Hum Genet. 1998;62(6):1468–1474. 2. Garner CP, Tatu T, Best S, Creary L, Thein SL. Evidence of genetic interaction between the beta-globin complex and chromosome 8q in the expression of fetal hemoglobin. Am J Hum Genet. 2002;70(3):793–799. 3. Wyszynski DF, Baldwin CT, Cleves MA, et al. Polymorphisms near a chromosome 6q QTL area are associated with modulation of fetal hemoglobin levels in sickle cell anemia. Cell Mol Biol. 2004;50(1):23–33. 4. Chang YPC, Maier-Redelsperger M, Smith KD, et al. The relative importance of the X-linked FCP locus and ␤-globin haplotypes in determining haemoglobin F levels: A study of SS patients homozygous for ␤S haplotypes. Br J Haematol. 1997;96:806–814. 5. Ofori-Acquah SF, Lalloz MR, Serjeant G, Layton DM. Dominant influence of gamma-globin promoter polymorphisms on fetal haemoglobin expression in sickle cell disease. Cell Mol Biol. 2004;50(1):35–42. 6. Platt OS, Thorington BD, Brambilla DJ, et al. Pain in sickle cell disease-rates and risk factors. N Engl J Med. 1991;325:11– 16. 7. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med. 1994;330(23):1639–1644. 8. Steinberg MH, Barton F, Castro O, et al. Effect of hydroxyurea on mortality and morbidity in adult sickle cell anemia: risks and benefits up to 9 years of treatment. JAMA. 2003;289(13):1645–1651. 9. Orkin SH, Antonarakis SE, Kazazian HH Jr. Polymorphism and molecular pathology of the human beta-globin gene. Prog Hematol. 1983;13:49–73. 10. Antonarakis SE, Kazazian HH Jr, Orkin SH. DNA polymorphism and molecular pathology of the human globin gene clusters. Hum Genet. 1985;69:1–14. 11. Pagnier J, Mears JG, Dunda-Belkhodja O, et al. Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa. Proc Natl Acad Sci USA. 1984;81:1771–1773. 12. Nagel RL. Origins and dispersion of the sickle gene. In: Embury SH, Hebbel RP, Mohandas N, Steinberg MH, eds. Sickle Cell Disease: Basic Principles and Clinical Practice. 1st ed. New York: Raven Press; 1994;353–380.

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from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy. Biochim Biophys Acta. 1993;1182(2):179–188. Amer J, Ghoti H, Rachmilewitz E, Koren A, Levin C, Fibach E. Red blood cells, platelets and polymorphonuclear neutrophils of patients with sickle cell disease exhibit oxidative stress that can be ameliorated by antioxidants. Br J Haematol. 2006;132(1):108–113. Leopold J, Dam A, Maron BA, et al. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity. Nat Med. 2007;13:189–197. Eyler CE, Jackson T, Elliott LE, et al. Beta(2)-adrenergic receptor and adenylate cyclase gene polymorphisms affect sickle red cell adhesion. Br J Haematol. 2008;141:105– 108. Castro V, Alberto FL, Costa RN, et al. Polymorphism of the human platelet antigen-5 system is a risk factor for occlusive vascular complications in patients with sickle cell anemia. Vox Sang. 2004;87(2):118–123. Sebastiani P, Nolan VG, A, et al. Predicting severity of sickle cell disease. Blood. 2007;110:2727–2735. Sebastiani P, Wang L, Perls T, et al. A repertoire of genes modifying the risk of death in sickle cell anemia. Blood. 2007;110:52a. Sebastiani P, Timofeev N, Hartley SH, et al. Genome-wide association studies suggest shared polymorphisms are associated with severity of sickle cell anemia and exceptional longevity. Blood. 2008;112:1446a. Afenyi-Annan A, Kail M, Combs MR, et al. Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease. Transfusion. 2008;48:917–924. Galanello R, Melis MA, Podda A, et al. Deletion deltathalassemia: the 7.2 kb deletion of Corfu ␦␤-thalassemia in a non-beta-thalassemia chromosome. Blood. 1990;75:1747– 1749. Galanello R, Perseu L, Melis MA, et al. Hyperbilirubinaemia in heterozygous beta-thalassaemia is related to coinherited Gilbert’s syndrome. Br J Haematol. 1997;99(2):433– 436. Galanello R, Piras S, Barella S, et al. Cholelithiasis and Gilbert’s syndrome in homozygous beta-thalassaemia. Br J Haematol. 2001;115(4):926–928. Sampietro M, Lupica L, Perrero L, et al. The expression of uridine diphosphate glucuronosyltransferase gene is a major determinant of bilirubin level in heterozygous betathalassaemia and in glucose-6-phosphate dehydrogenase deficiency. Br J Haematol. 1997;99(2):437–439. Huang YY, Huang CS, Yang SS, Lin MS, Huang MJ, Huang CS. Effects of variant UDP-glucuronosyltransferase 1A1 gene, glucose-6-phosphate dehydrogenase deficiency and thalassemia on cholelithiasis. World J Gastroenterol. 2005;11(36):5710–5713. Au WY, Cheung WC, Chan GC, Ha SY, Khong PL, Ma ES. Risk factors for hyperbilirubinemia and gallstones in Chinese patients with ␤ thalassemia syndrome. Haematologica. 2003;88(2):220–222. Rees DC, Luo LY, Thein SL, Singh BM, Wickramasinghe S. Nontransfusional iron overload in thalassemia: Association with hereditary hemochromatosis. Blood. 1997;90(8):3234– 3236.

Genetic Modulation of Sickle Cell Disease and Thalassemia 148. Cappellini MD, Fargion SR, Sampietro M, Graziadei G, Fiorelli G. Nontransfusional iron overload in thalassemia intermedia: Role of the hemochromatosis allele. Blood. 1998;92(11):4479–4480. 149. Borgna-Pignatti C, Solinas A, Bombieri C, et al. The haemochromatosis mutations do not modify the clinical picture of thalassaemia major in patients regularly transfused and chelated. Br J Haematol. 1998;103(3):813– 816. 150. Piperno A, Mariani R, Arosio C, et al. Haemochromatosis in patients with ␤-thalassaemia trait. Br J Haematol. 2000;111(3):908–914. 151. Melis MA, Cau M, Deidda F, Barella S, Cao A, Galanello R. H63D mutation in the HFE gene increases iron overload in beta-thalassemia carriers. Haematologica. 2002;87(3):242– 245. 152. Merryweather-Clarke AT, Pointon JJ, Shearman JD, Robson KJ. Global prevalence of putative haemochromatosis mutations. J Med Genet. 1997;34(4):275–278. 153. Garewal G, Das R, Ahluwalia J, Marwaha RK. Prevalence of the H63D mutation of the HFE in north India: its presence does not cause iron overload in beta thalassemia trait. Eur J Haematol. 2005;74(4):333–336. 154. Ma ES, Lam KK, Chan AY, Ha SY, Au WY, Chan LC. Transferrin receptor-2 polymorphisms and iron overload in transfusion independent ␤-thalassemia intermedia. Haematologica. 2003;88(3):345–346. 155. Uitterlinden AG, van Meurs JB, Rivadeneira F, Pols HA. Identifying genetic risk factors for osteoporosis. J Musculoskelet Neuronal Interact. 2006;6(1):16–26. 156. Wonke B, Jensen C, Hanslip JJ, et al. Genetic and acquired predisposing factors and treatment of osteoporosis in thalassaemia major. J Pediatr Endocrinol Metab. 1998;11(Suppl 3):795–801. 157. Pollak RD, Rachmilewitz E, Blumenfeld A, Idelson M, Goldfarb AW. Bone mineral metabolism in adults with ␤-thalassaemia major and intermedia. Br J Haematol. 2000; 111(3):902–907.

657 158. Perrotta S, Cappellini MD, Bertoldo F, et al. Osteoporosis in ␤-thalassaemia major patients: analysis of the genetic background. Br J Haematol. 2000;111(2):461–466. 159. Arisal O, Deviren A, Fenerci EY, et al. Polymorphism analysis in the COLIA1 gene of patients with thalassemia major and intermedia. Haematologia. 2002;32(4):475–482. 160. Economou-Petersen E, Aessopos A, Kladi A, et al. Apolipoprotein E epsilon 4 allele as a genetic risk factor for left ventricular failure in homozygous beta-thalassemia. Blood. 1998;92(9):3455–3459. 161. Ferrara M, Matarese SM, Borrelli B, et al. Impact of excess weight and estrogen receptor gene polymorphisms on clinical course of homozygous beta thalassemia. Hematology. 2005;10(5):407–411. 162. Chen FE, Ooi C, Ha SY, et al. Genetic and clinical features of hemoglobin H disease in Chinese patients. N Engl J Med. 2000;343(8):544–550. 163. Au WY, Cheung WC, Hu WH, et al. Hyperbilirubinemia and cholelithiasis in Chinese patients with hemoglobin H disease. Ann Hematol. 2005;84(10):671–674. 164. Chan V, Wong MS, Ooi C, et al. Can defects in transferrin receptor 2 and hereditary hemochromatosis genes account for iron overload in HbH disease? Blood Cells Mol Dis. 2003;30(1):107–111. 165. DeCastro L, Rinder HM, Howe JG, Smith BR. Thrombophilic genotypes do not adversely affect the course of sickle cell disease (SCD). Blood. 1998;92:161a. 166. Zimmerman SA, Howard TA, Whorton MR, Rosse WF, Ware RE. The A312G polymorphism in ␣-fibrinogen is associated with stroke and avascular necrosis in patients with sickle cell anemia. Blood. 1998;92:36b. 167. Sullivan KJ, Kissoon N, Duckworth LJ, et al. Low exhaled nitric oxide and a polymorphism in the NOS I gene is associated with acute chest syndrome. Am J Respir Crit Care Med. 2001;164(12):2186–2190. 168. Powars D, Hiti A. Sickle cell anemia: ␤S gene cluster haplotypes as genetic markers for severe disease expression. Am J Dis Child. 1993;147:1197–1202.

heterotetramers have dissociated. Heterotetramers can be detected by conventional separation techniques if oxygen is rigorously excluded because exchange is greatly slowed in deoxyhemoglobin.1

28 Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders Mary Fabry and John M. Old

INTRODUCTION Hemoglobinopathy detection is often a part of the evaluation of anemia, hemolysis, microcytosis, cyanosis, or erythrocytosis. For this purpose, protein (hemoglobin)-, cellular-, and DNA-based approaches to the detection of variant hemoglobins and thalassemias are available. Diagnostic details can be found in each disease-specific chapter, whereas in the following pages we focus on the available methods and their strengths and weaknesses. Characterization of mutant hemoglobins and thalassemias described throughout this book takes place in different contexts: large newborn screening laboratories that need to identify positively the most common mutants; general hematology laboratories that most often encounter common hemoglobinopathies and thalassemias; and reference or research laboratories that can detect rare mutant globin genes. Approaches that are necessary in one setting might not be practical in others. Normal adult blood contains predominantly HbA (␣2 ␤2 ) and small amounts of HbF (␣2 ␥ 2 ) and HbA2 (␣2 ␦2 ). After synthesis, monomeric globin chains form ␣/non-␣ dimers that do not dissociate under physiological conditions. In the presence of oxygen, hemoglobin tetramers rapidly dissociate into very low concentrations of dimers that can then form new tetramers.1,2 This implies that when more than one ␣- or non-␣-chain is present, the predominant form in the red cell will be the heterotetramer (for example, in red cells of HbSC disease, the dominant species will be ␣2 ␤S ␤C , and ␣2 ␤S ␥ heterotetramers form when HbS is present with high levels of HbF (Fig. 28.1). Most hemoglobin separation techniques detect only the homotetramer because the migratory properties of the individual dimers are similar to those of the homotetramers (identical ␣␤ subunits); hence, following dissociation of the heterotetramer into dimers, the two dimers will move apart toward the region where the respective homotetramers are found and recombine. This process will continue until all 658

PROTEIN- AND CELLULAR-BASED METHODS USED TO IDENTIFY AND STUDY HEMOGLOBIN DISORDERS Sample Preservation, Preparation, and Laboratory Safety Analytical techniques depend on the starting materials, which include hemoglobin in dried filter paper blots. Anticoagulated whole blood should be refrigerated and processed within 48 hours. Hemoglobin is best stored as red cells in plasma because the red cell contains methemoglobin reductase, which will convert methemoglobin to deoxyhemoglobin. Plasma glucose and albumin stabilize the red cell and its membrane and ensure that enzyme activity will be maintained. With prolonged storage, methemoglobin will be generated and less stable mutant hemoglobins might be selectively lost. Hemoglobin for analysis is usually prepared by hypotonic lysis with shaking. Care must be taken that all cells undergo lysis. In sickle cell disease, red cells are heterogeneous in their properties with different hemoglobin compositions (Chapter 7) and can be particularly resistant to lysis. Long-term storage of hemoglobin samples requires first washing the cells in isotonic saline to remove plasma proteins and then immersing them in liquid nitrogen or a freezer at −135◦ C or below. Hemoglobin stored at higher temperatures gradually loses heme, becomes insoluble, and can form degradation products that complicate analysis. Safety of personnel working in hematology laboratories is a major consideration with the primary risks of hepatitis and human immunodeficiency virus. All human samples should be regarded as a potential source of infection. The first line of defense is care in sample handling: avoiding aerosol formation; wearing gloves; and observing precautions to avoid transferring material from gloves to other surfaces that may be touched by exposed skin. For more detailed discussion of laboratory safety, see the Occupational Safety and Health Agency web site (www.osha.gov).

Electrophoresis Electrophoresis was once the most widely used method for initial detection of variant hemoglobins but has been supplanted by high-performance liquid chromatography (HPLC) as the most useful screening approach. Proteins are composed of amino acids that bear side chains that can ionize. The pH at which a side chain is half ionized is its pK. Proteins are amphoteric; that is, they bear both positively and negatively charged side chains, and the net charge will depend on the pH of the solution and the pKs of its amino acids. Because proteins are charged

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Figure 28.1. Hemoglobin is a tetramer of two ␣␤ dimers that do not dissociate under physiological conditions; however, the tetramers rapidly dissociate and reassociate, generating very low concentrations of dimers under physiological conditions. Mixing two hemoglobins, represented by black and white, rapidly leads to an equilibrium mixture in which the original homotetramers and the heterotetramer exist in a ratio of 1:2:1.

molecules, they will migrate in an electrical field and the relative speed and direction will depend on the sign and magnitude of the net charge. At pH 8.6, all human hemoglobins will have a net negative charge and will migrate in an electrical field toward the positive pole or anode. Mutations that do not alter the charge may be “silent” and not detectable by electrophoresis; however, interaction with the matrix (such as cellulose acetate or agar) can also affect the rate of migration. Most electrophoretic methods separate hemoglobin tetramers, but only tetramers composed of two identical ␣-non-␣ dimers, or homotetramers, are seen at the end of the separation process. Because many different hemoglobins migrate similarly under a given set of conditions, electrophoresis is usually performed at two different pHs and on two different supporting mediums, but this method is insensitive to many rare variants. The usual choice is cellulose acetate electrophoresis at pH 8.4 and citrate agar electrophoresis at pH 6.0.3 On citrate agar, in addition to the net charge, there is some selective interaction with the substrate, which further aids in resolution of some hemoglobins. This is based on the interaction of the central cavity of the hemoglobin with agaropectin in agar.4 In all cases, a set of reference hemoglobins, usually HbA, A2 , F, S, and C should be included with each run. Electrophoresis can also be performed on separated globin chains if denaturing conditions are used.5–7 Another approach that sometimes allows separation of overlapping hemoglobins, one of which contains cysteine, is treatment of the sample with cystamine.8 Most laboratories use commercially prepared kits that contain the hemolyzing agent, buffers, support media, a means for applying multiple samples, and a stain. The

sample is lysed and applied to the cathodic or negative side and an electrical field is applied. Gels can be stained with protein- or hemoglobin-specific stains, or the color of the hemoglobin itself can be used for visualization. Quantification of hemoglobin fractions by densitometry is possible but inaccurate when a hemoglobin fraction is present at less than 5%.

Isoelectric Focusing, Capillary Isoelectric Focusing, and Capillary Electrophoresis Isoelectric focusing (IEF) is capable of much higher resolution than the electrophoretic techniques. If a molecule has two or more ionizable groups, at least one of which has a pK in the acidic range and another in the basic range, then there will be a pH at which the net charge is zero. Proteins and amino acids all have a pH at which the net charge is zero, which is called the isoelectric point (pI) where there is no net movement in the presence of an externally applied electrical field. To separate proteins based on their pIs, a stable pH gradient must be created. This is achieved by applying a set of ampholytes with pIs that cover the range of pIs of the proteins that are to be separated on a support matrix. During the initial period after the current is applied, both the ampholytes and the proteins to be separated move as the pH gradient is formed. If a protein molecule finds itself on the acidic side of its pI it will migrate to the cathode, and if it finds itself on the basic side of its pI, it will migrate toward the anode (hence the term isoelectric focusing). Sharp bands of individual proteins are thus formed. With optimization, methods using free ampholytes can resolve two proteins with a pI difference of approximately 0.01 pH unit (Fig. 28.2).9 Even higher resolution can

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Figure 28.2. Separation of various mutant hemoglobins by IEF. A precast agarose gel (Resolve Systems, Wallac Inc., Akron, OH) was used at 75 mA, 1,200 V, 14◦ C, for 90 minutes. Present on the gel are: lane 1, HbA–HbN Baltimore; lanes 2, 6, 8, 12, 20, 23, HbAS; lane 3 HbA–HbD Punjab; lane 5 normal newborn; lanes 7, 18 controls (HbA2 , HbS, HbF, HbA, HbJ Baltimore, HbI Texas); lane 16 ␤ thalassemia trait; lane 19 HbA–Hb Hope; lane 21 HbE trait; lane 24 HbC trait. (Gel contributed by H. Wajcman, Director of Research INSERM, Hopital Henri Mondor, Paris, France.)

be attained if the ampholytes are covalently bound to the matrix and the pH gradient is preformed by casting the gel with a two-vessel gradient mixer, thus preforming a stable pH gradient before the protein is applied. This type of gel is capable of resolution of proteins with pIs differing by approximately 0.001 pH units.10,11 The most commonly used IEF technique is application of multiple samples to a commercially prepared thin-layer gel, which precludes automated technology. Capillary IEF is performed by using pH gradients in fine capillary tubes. Available commercial systems require small sample size, have good resolving power, are rapid and sensitive, allow automated data analysis, and provide quantitative information. Capillary zone electrophoresis is another alternative in which hemoglobin molecules migrate through a capillary filled with a salt buffer near physiological conditions. There are no matrix interactions to introduce complexities or alter reproducibility, and the system has the potential for automation.12,13 Capillary electrophoresis combined with mass spectrometry has been applied to analysis of proteins present in single cells.14,15 A wide variety of detection systems have been used, including conventional UV/visible detection similar to that used by HPLC instruments,16 absorption imaging,17 and mass spectrometry.18

High-performance Liquid Chromatography The techniques described so far depend primarily on the ionization (pKs) of the amino acid side chains of intact hemoglobin molecules and their migration in an electrical field. In HPLC, ionic and hydrophobic interactions of the sample with the supporting matrix are the basis of separation. The sample is applied to the top of the column under conditions wherein it interacts strongly with the matrix. The proteins are then eluted with a developing solution (buffer) of gradually increasing strength, until all of the proteins are eluted. In cation and anion exchange chromatography, the properties of the developing solution that are varied are pH and ionic strength (salt concentration) and in reverse phase chromatography, the hydrophobicity (organic solvent) content is also varied. Hemoglobin

may be separated as the intact tetramer or, under denaturing conditions, the individual globin chains can be separated. Human hemoglobins usually have a relatively small number of possible homotetramers and analysis of the intact tetramer generally yields readily interpretable results. HPLC is currently the standard for the initial evaluation of hemoglobin variants and thalassemia. It is rapid, automated, capable of resolving most of the common and many uncommon variants, and provides reliable measurement of hemoglobin fractions such as HbA2 and HbF (see later). Dedicated clinical systems based on HPLC separation of tetrameric hemoglobin with very rapid-automated sampling are in widespread use and allow preprogramed detection of hemoglobin variants (see Fig. 23.7, Chapter 23). Hemoglobin from transgenic mice – because of the possible formation of human – mouse chimeric dimers and hence a wide variety of homotetramers – and less common human variants yield a more readily interpretable chromatogram when denaturing conditions are used and the isolated globin chains are detected (Fig. 28.3). In most systems the pumps and the detector are computer controlled and the area under each detectable peak is automatically calculated. Very small internal diameters increase column resolution and decrease the amount of sample required at the cost of increased time per sample.19 Under the appropriate conditions HPLC readily separates hemoglobin variants that cannot be resolved by other means. The equipment used for HPLC is expensive, sophisticated, and difficult to maintain compared with that used for electrophoresis or IEF. HPLC screening techniques have been developed in which individual samples can be run in approximately 3 minutes with very high reproducibility.

Mass Spectrometry Mass spectrometry is rarely used for clinical purposes and DNA-based studies have largely supplanted its use for definitive detection of rare variants. This method can start with a hemolysate and return the molecular weights of individual globin chains to within 1 atomic mass unit (amu). This level of accuracy allows detection of posttranslational

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Figure 28.3. IEF and HPLC of hemoglobins from several transgenic mice expressing human HbS and mouse globins. The bands representing homotetramers composed of dimers of mouse ␣ and human ␤S chains, mouse ␣ and mouse ␤ chains, human ␣ and human ␤S chains, and human ␣ and mouse ␤ chains were separated by IEF, isolated from the gel, and separated by denaturing HPLC to allow identification of the tetramers present in the IEF gel. (From Fabry et al., 1992 Proc Natl Acad Sci, USA 89;1250–1254, with permission.)

modification20 and verification of mutant and recombinant hemoglobins, and in contrast to electrophoretic techniques, mass spectrometry can separate similarly charged samples. Individual bands or peaks from IEF or HPLC can also serve as the starting sample. Requiring a few picomoles of protein, hemoglobin is an ideal molecule for mass spectrometry because it has a favorable size, an adequate number of protonation sites, distinct mass differences between mutants, and few posttranslational modifications.21 Mass spectrometry combined with capillary electrophoresis has been applied to analysis of proteins present in single or small numbers of red cells.15 This method is based on the principle that charged particles moving through a magnetic field will undergo deflection proportional to the charge/mass ratio. Particles with the smallest charge/mass ratio are the least affected. The sample is first converted to gaseous ions and an accelerating voltage is applied. The particles are focused through a slit into a highly evacuated area, where they are subjected to the magnetic field that causes them to separate. The most important methods of ionization are electrospray ionization (ESI),22 and matrix-assisted laser desorption (MALDI). ESI is most suitable for intact globin chain analysis and MALDI is the method of choice for peptide identification. ESI allows mass spectrometers to be interfaced with HPLC and capillary IEF devices, which frequently allows immediate positive identification of the separated species and is

also useful for identification of small peaks that could represent derivatives or degradation products.23,24 Mass spectrometry can be used for complete sequencing of proteins based on identification of fragments that can be produced classically by proteolytic digestion and then exposed to MALDI. The abnormal fragments can be identified, and in many cases, the mutation deduced. With a few exceptions, whole blood hemolysates can be subjected to digestion without further separation. Separation is necessary when the variant is present at a low level, a mass difference of 1 D is suspected, or high levels of HbF are present. In these cases the sample can be purified by IEF or HPLC prior to digestion.26 Alternatively, the protein can be fragmented in the spectrometer itself without resorting to wet chemistry and the fragments produced in the spectrometer can be used to identify the portion bearing the mutant animo acid.23–28

Hemoglobin F HbF (Chapter 7) in adult blood is normally is less than 1%– 2% of total hemoglobin. It is unevenly distributed among red cells. Cells with high levels of HbF are called F cells and, in sickle cell disease, F cells are seldom found among the most dense cells. This implies that when either HbF or F cells are measured, care must be taken to avoid biasing the sample. Careful mixing into the suspension of any cells that

662 have settled at the bottom of the tube is sufficient; however, automated systems for resuspending normal red cells might not be sufficient for blood from patients with any of the sickle hemoglobinopathies. Several methods may be used for quantitation of HbF, including alkaline denaturation, electrophoresis, or IEF followed by densitometry, disposable minicolumns, and HPLC. HbF is more resistant to denaturation by alkaline conditions than is HbA. In the alkaline denaturation method, potassium hydroxide is added to a known concentration of hemoglobin and, after a predetermined time interval, the reaction is stopped by adding a known volume of ammonium sulfate, which lowers the pH and precipitates the denatured hemoglobin that is removed by filtration. The concentration of hemoglobin remaining in solution is then determined. Two versions of the alkaline denaturation protocol exist: that of Betke et al.,29 which is reliable for HbF less than 10%–15%, and thereafter systematically underestimates the percentage of HbF, and that of Jonxis and Visser,30 which is only accurate when HbF is greater than 10%. HPLC allows accurate quantitation of HbF and is currently the most widely used method.31,32 It is accurate for samples with levels of HbF down to approximately 0.5% and has no upper limit of detection. Some hemoglobin components that are present at low concentrations elute in the position of HbF under some HPLC conditions (Chapter 7). Electrophoresis or IEF followed by densitometry are less reliable than HPLC for HbF quantification. For detection of very low levels of HbF radioimmunoassay,33,34 and enzyme-linked immunosorbent assay (ELISA)35 are alternative techniques. Because HbF is almost always unevenly distributed among erythrocytes – an exception is some types of hereditary persistence of HbF (HPFH) – immunological techniques using flow cytometry that allow the determination of F cells are also useful and can provide a reasonable surrogate for whole blood HbF levels.36 Measuring F cells is discussed later.

Hemoglobin A2 HPLC systems that detect many hemoglobin variants are the current standard for measuring HbA2 levels. Electrophoresis can separate HbA2 from HbA and HbF, enabling accurate measurement of this minor hemoglobin component but this requires elution from gels or membranes because densitometric scanning is inaccurate.45,46 Acid agar gel electrophoresis does not resolve HbA2 from HbA. Refrigeration and freezing may reduce the percentage of HbA2 in stored hemolysates. The differential elution of HbA2 in minicolumns is reliable, rapid, and inexpensive.37 HbA2 cannot be separated from hemoglobin variants with similar positive charges, such as HbC and HbE, by electrophoresis or many column-chromatographic methods. In the company of abnormal globins that contain only a single additional positive charge, such as HbS, the HbA2 level has been reported to be higher than normal but

Mary Fabry and John M. Old appropriate separation methods can circumvent this overestimation of HbA2 . Recent improvement allows reasonably accurate measurement of HbA2 by HPLC even in the presence of HbS as in sickle cell trait (HbAS) or sickle cell anemia, although the level of HbA2 reported is slightly higher than normal because of the coelution of minor HbS fractions (Chapter 7). HbE, and Hb Lepore have very similar retention time to HbA2 , and these hemoglobins cannot be positively separated. HbC and Hb O-Arab also cannot be definitively distinguished by cation HPLC that affords excellent resolution of HbA2 from HbS and HbC. HbA2 can be measured immunologically33,38 with high specificity. The levels obtained correlate well with the more traditional methods of measurement. Capillary IEF has been used to measure HbA2 in normal subjects and in those with ␤ thalassemia trait, HbAS, and sickle cell anemia.39,40 Accurate measurements are possible, although in one study HbA2 levels in HbAS and sickle cell anemia were slightly elevated compared with normal. Nevertheless, they were clearly separated from ␤ thalassemia trait. Glycosylated forms of HbA2 , analogous to the minor components of HbA, are present and can be quantified by HPLC and IEF. Like HbA1c , these glycohemoglobins are elevated in poorly controlled diabetics (Chapter 7).

Functional Properties of Hemoglobin P50 and Other Measurements of Oxygen Saturation The partial pressure, at which hemoglobin is half saturated with oxygen, or P50 , is an important property of hemoglobin in solution and in the red cell. P50 is extremely sensitive to the presence of 2,3-bisphosphoglycerate (2,3-BPG), inorganic phosphate or other phosphates, pH (Bohr effect), CO2 , anions, and, in the case of HbS, polymer formation. In dilute solutions, the P50 of HbA and HbS are equal. In the red cell and in plasma, HbA has a P50 of 25 ± 2 mm Hg when equilibrated with gases containing 5% CO2 . The increase in P50 (or decrease in oxygen affinity) is primarily due to the effect of 2,3-BPG on the oxygen affinity of hemoglobin. P50 of HbS in the red cell is higher, ranging from 25 to 45 mm Hg. HbS polymerization increases P50 in proportion to the amount of polymer formed because polymer has a lower P50 .41 With HbA, the association curve (Hb → HbO2 ) is identical to the dissociation curve (HbO2 → Hb); there is no hysteresis. The P50 in blood samples with HbS depends on whether equilibrium with polymer has been reached, that is, whether polymer formation or polymer melting is complete before the next level of oxygenation is tested. The delay time for polymer formation is dependent to the 30th power of the deoxyhemoglobin concentration.42 In the absence of equilibrium there is hysteresis and the P50 for the association and dissociation curves will differ. Changing the pO2 at a slower rate will bring the two curves closer together.

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders The most common measurements of P50 are based on measurement of oxygen association curves. Two parameters are simultaneously measured: pO2 , which is continuously varied, and the percentage of hemoglobin, which is saturated with oxygen. In these measurements, the hemoglobin is first fully deoxygenated and then oxygen is gradually reintroduced, whereas the pO2 is measured with an oxygen electrode. At the same time, hemoglobin saturation with oxygen is measured optically. The Hemoscan (no longer manufactured) used equilibrated hemoglobin or whole blood samples between layers of gaspermeable membranes and allowed measurement of whole blood P50 in plasma. The Hemox-Analyzer (TCS Medical Products) measures percentage of oxyhemoglobin with a dual-wavelength spectrophotometer at 560 nm, which is sensitive to the transition between deoxyhemoglobin and oxyhemoglobin, and at 570 nm, at which the absorption is sensitive to the hemoglobin concentration but insensitive to the oxygenation state. Dual-wavelength systems cannot discriminate between oxyHb and several other forms of hemoglobin such as CO, NO, metHb, or sulfHb, which can lead to systematic errors (Chapter 25). Blood gas analyzers measure the pH, pO2 , and pCO2 for samples of whole blood and then calculate hemoglobin saturation with oxygen and a number of other parameters, based on the normal P50 . This will systematically overestimate the percentage of oxyhemoglobin present in the blood of sickle cell disease patients and any other hemoglobinopathies that result in elevated P50 . Similarly, a low P50 such as is found for HbF will result in systematic underestimation of the percentage of oxyhemoglobin. The presence of met- and carbonmonoxy hemoglobin (CO-Hb) are not accounted for in these measurements. A CO-Oximeter (Instrumentation Laboratories, Lexington, MA) uses multiple wavelengths to measure the hemoglobin concentration, percentage oxyhemoglobin, hemoglobin, percentage metHb, and percentage CO-Hb and can be used in conjunction with blood gas analyzers to give a more accurate, but still incomplete, picture of hemoglobin saturation. The 2,3-BPG content of the red cell is needed to evaluate the significance of P50 measurements because altered P50 might be the result of a hemoglobin abnormality or altered 2,3-BPG content. If a hemoglobin with a P50 different from that of HbA is present (such as HbS or HbF), the hemoglobin saturation cannot be obtained from a blood gas analyzer, which uses the P50 of HbA to calculate hemoglobin saturation from the measured pO2 . A useful test for patient monitoring is measurement of the arterial hemoglobin saturation with oxygen that can be measured in vivo by use of a pulse oximeter. The pulse oximeter estimates arterial hemoglobin saturation by measuring the light absorbance of pulsating vascular tissue at two wavelengths. The relationship between measured light absorbance and saturation was developed empirically and is built into the oximeter software.

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The presence of methemoglobin and CO-Hb will lead to systematic overestimation of oxygen saturation by pulse oximetry (Chapter 25).

Detection and Estimation of Polymer Formation in Red Cells and Solutions Containing HbS Solubility The insolubility of deoxyHbS in high-phosphate solutions is the basis of a rapid nonquantitative test that when combined with the results of HPLC provides a positive identification of sickling hemoglobin variants. Solubility tests should never be used for definitive diagnosis. If they are performed correctly, they can identify the presence of a sickling hemoglobin variant (Chapter 23). This could be clinically important when rapidly knowing if HbS is present is imperative, but the need for this knowledge is uncommon.

CSAT The solubility of a substance is the concentration of that substance in equilibrium with a condensed phase that may be crystalline, polymer, or particulate. In the case of HbS, the solubility is important because it allows estimation of the percentage of the HbS content of the red cell that will be converted to polymer under fully deoxygenated conditions. The concentration of deoxyHbS in equilibrium with the polymer phase (CSAT ) is useful for characterizing sickle hemoglobins that contain a second mutation and for characterizing mixtures of HbS with other hemoglobins. Several methods are used. One method uses quasiphysiological conditions and the other relies on high phosphate to precipitate sickle hemoglobins. Formation of protein crystals and polymers is extremely sensitive to the nature and concentration of the counter ions present. Either the crystal structure itself or the rate of nucleation can be affected. In the low-phosphate method,43 hemoglobin is concentrated versus 0.1 M potassium phosphate buffer and deoxygenated, first by alternating vacuum and nitrogen, and then by adding enough sodium dithionite solution to give a final concentration equal to three times the heme concentration. The samples are transferred anaerobically to CSAT tubes filled with paraffin oil, incubated overnight in a nitrogen atmosphere, and centrifuged for 2 hours at 35,000 rpm with purified HbS used as a control. The supernatants are removed anaerobically, and concentrations and deoxy pHs are determined; solubility is expressed in grams per deciliter of HbS in equilibrium with the polymer. This is regarded as the reference method. Another method is based on the turbidity of a microparticulate suspension that is formed when HbS is introduced into a solution at pH 7.0 and 27◦ C with a highphosphate concentration that has been deoxygenated with dithionite.44 A third alternate approach uses 50 mM

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phosphate in the presence of 70 kD dextran to decrease polymer solubility.45 Because solubility is determined by the residues in the contact area between the tetramers, this method offers an advantage over the high-phosphate method, in which the insoluble phase does not have a fiber structure. Finally, a method that takes advantage of the observation that polymerization of HbS shifts the P50 to the right and that this shift is proportional to the amount of polymer formed is available. Correlation of the CSAT determined by P50 with that determined by the centrifugation method has been shown to be good for a wide range of samples46 and has been applied to suspensions of red cells.47 In the high-phosphate method, the pH dependence of solubility is opposite to that found in the minimum gelling concentration method.48

method is more expensive and is sensitive to all forms of human hemoglobin, including HbF. Lactate dehydrogenase (LDH) is released from the red cell on hemolysis. There are five isoforms of LDH that come from red cell, heart, kidney, lymphoid cells, platelets, liver, and skeletal muscle. If only LDH isoforms 1 and 2 are measured, the sources are narrowed to red cell, heart, and kidney. A high correlation between serum LDH and plasma heme measured by ELISA exists in sickle cell anemia.52 Absence of an accepted standard of LDH measurement makes it difficult to compare values between different laboratories, making agreement problematic on a single upper limit of LDH that would identify all cases of high plasma hemoglobin.

Plasma Hemoglobin

Complete Blood Count

The importance of cell free or plasma hemoglobin in sickle cell disease and other hemolytic anemias has recently been emphasized49,50 (Chapter 11). In plasma, due to the low hemoglobin concentration, all hemoglobin will be dissociated into less stable dimers, and oxidation will result in conversion to methemoglobin with potential loss of heme. The first consideration for measurement of plasma hemoglobin is avoiding artifacts due to technique in blood draws or sample preparation. This is particularly so in sickle cell disease because of the greater fragility of sickle erythrocytes. Small bore needles, rapid flow, and vortexing should be avoided. Red cells and plasma should be separated promptly. In recent studies,50 blood was collected from artery or vein by using large-bore catheters (18 gauge). The first 3 mL of blood was discarded, and the blood then slowly drawn into heparinized syringes. Blood was spun at 750 G at 4◦ C for 10 minutes without braking, and the plasma was removed. Plasma was then spun at 14,000 G at 4◦ C for 10 minutes to eliminate residual erythrocytes and platelets. Several techniques can be applied to measure cell-free hemoglobin: For relatively high concentrations, plasma can be diluted 1:1 with Drabkin reagent. A more sensitive technique is use of tetramethylbenzidine.51 Tetramethylbenzidine is noncarcinogenic and more sensitive than benzidine. Samples can be read at 600 nm, at room temperature, and concentrations can be calculated using the slope of the sample and the slope of a calibrator. This method is sensitive to heme in all forms: heme in hemoglobin, bound to albumin, and heme in haptoglobin and hemopexin. An ELISA can also be used.52 Both Drabkin reagent and tetramethylbenzidine are inexpensive and do not require complex instrumentation and they are sensitive to heme in all forms. This can lead to overestimation of plasma hemoglobin, but because heme or heme proteins bound to albumin, haptoglobin, or hemopexin are usually present at a relatively low concentration, this might not be a major drawback. The ELISA

Blood for a complete blood count (CBC) should be slowly drawn into an anticoagulant, usually EDTA. Prompt but gentle mixing is needed to avoid clotting. This is particularly important for patients with low packed cell volume (PCV) because the amount of anticoagulant may be only marginally sufficient. Red cells from patients with hemoglobinopathies can be fragile, and narrow gauge needles, rapid flow, or violent mixing should be avoided. Whole blood can be stored refrigerated or on wet ice and used within 24 hours and shipped overnight on wet ice. To avoid hemolysis, freezer packs should not be used and should never be placed in direct contact with tubes containing whole blood. Special care may be needed to resuspend whole blood from patients with sickle cell disease because these cells may adhere to each other and resist resuspension.

CHARACTERIZING THE RED CELL

Manual Methods Although red cell indices are determined by automated methods in most laboratories, manual methods are still used sometimes. The ratio of hemoglobin to PCV can be used to calculate a mean corpuscular hemoglobin concentration (MCHC) that might be more reliable than that determined by automated measurements. The most accurate way to measure hemoglobin concentration is by the cyanmethemoglobin method, which is insensitive to oxygenation and pH. Hemoglobin is oxidized to methemoglobin by ferricyanide and then interacts with cyanide to form cyanomethemoglobin and its absorbance at 540 nm is read in a spectrophotometer. A dedicated microhematocrit centrifuge that generates a force of 12,000 G and microhematocrit tubes with or without heparin are used to estimate PCV. For samples from patients with high PCV, and those with sickle cell anemia or hereditary spherocytosis, an additional centrifugation might be required. If MCHC is to be calculated from hemoglobin and hematocrit, multiple measurements should be made.

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders Automated Methods Current automated CBC methods measure properties of single cells flowing through a detector.53,54 Two general types of detection are currently used: Coulter-type detectors based on impedance and high-frequency conductivity, and detectors based on optical properties of the cell. Most instruments measure several variables for a large number of cells and present results as an average value; the averaging of samples with red cell heterogeneity, therefore, such as those from sickle cell anemia or hereditary spherocytosis, might yield values that seem normal. All instruments count particles, estimate their size, and hemolyze the cells and measure hemoglobin. The results of the direct measurements (hemoglobin, red blood cell count, white blood cell count, and platelet count) are the most accurate values, but failure to hemolyze all cells can lead to error in hemoglobin concentration in hemoglobinopathies such as sickle cell disease. Failure to resuspend completely all cells in samples of blood that have been stored for several hours can also lead to errors in hemoglobin and PCV. Because identification of erythrocytes and platelets is based on their size, fragmentation might lead to misclassification. Most systems measure hemoglobin by the cyanmethemoglobin method. Coulter detectors are based on the observation that the electrical conductivity of cells is lower than that of saline solutions and measurement of the impedance across a small orifice with cells flowing through allows cell size (mean corpuscular volume [MCV]) to be estimated. Factors that lead to deviations are cellular asymmetry and loss of an intact cell membrane, both of which may be features in samples with abnormal red cells. Cell volume measurements using light scattering (Mie principle) are based on the observation that the intensity of light scattered at small angles in the forward direction is proportional to cell size. Two major factors lead to deviations: cell shape and variation in refractive index, which is related to hemoglobin concentration. The first problem has been approached by sphering the cells at constant volume, and the second problem has been addressed directly by measuring at two wavelengths, which allows intracellular hemoglobin concentration and refractive index to be estimated. Incomplete sphering is a problem for some samples such as those from sickle cell disease patients. PCV, mean corpuscular hemoglobin (MCH), MCHC, and red cell distribution width (RDW) and other functions that provide information on dense cells are calculated from measured variation in red cell size. For most instruments, the least reliable measurement is the MCHC, which is calculated from the whole blood hemoglobin concentration divided by the mean cell volume times the number of cells. Because the calculation requires three measurements in most instruments, the error from all of them enters into the calculation; the exception to this is the Bayer Advia machine, which measures intracellular hemoglobin concentration directly as cell hemoglobin concentration mean (CHCM).

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The major advantages of automated methods are speed, low manpower requirements, and high precision. Comparisons of available instruments have been made.53–57 Automated systems have high reproducibility; however, the accuracy is sensitive to errors in calibration that systematically affect the results so that absolute values may vary between instruments. All systems generate flags to call attention to unusual values, which may be taken either as indications of pathology or a need to examine the sample more closely. In general all instruments performed all tests satisfactorily with the exception of estimation of MCHC, which was the least reliable. Analyzers using the Mie principle, by measuring density of individual red cells, are able to generate histograms of variation in cell density (cell hemoglobin distribution) of individual red cells in a given blood sample (see later). This is useful for following changes in the distribution of dense red cells in sickle cell anemia.

Reticulocyte Count Reticulocyte counts, along with PCV, are useful for evaluating hematopoiesis and hemolysis. Reticulocytes contain remnants of mRNA; they continue to synthesize hemoglobin for 1–2 days in the marrow and for another day after release into the circulation. The reticulocyte count may be elevated even in cases where hemoglobin levels are normal, and, in these cases, can serve as a more sensitive indicator of errors in either production or destruction of red cells. The peripheral blood of normal adults has less than 2% reticulocytes, but the count varies with the method. If the individual is under hematopoietic stress, the peripheral blood can contain stress reticulocytes, cells prematurely released from the bone marrow, or even nucleated erythrocytes. All reticulocyte counts are based on the presence of mRNA in the cell. In the manual method, the cells are stained with new methylene blue, which precipitates RNA, rendering it visible. The amount and distribution of the precipitate allow the degree of immaturity of the reticulocyte to be estimated, and stress reticulocytes have a characteristic appearance. After staining, the cells are spread on a microscope slide and reticulocytes are counted as a percentage of all red cells. Automated reticulocyte counts use fluorescence-stained cells that are read in a flow cytometer, which may be either dedicated to reticulocyte counting or part of a larger automated system. Red cells are usually discriminated from white cells and platelets by size, and a maximum fluorescence gate is usually set that may exclude nucleated red cells. The relative age of reticulocytes can be estimated by the degree of fluorescence. One drawback of these methods is that staining with the most frequently used reagent, thiazole orange, requires approximately 30 minutes incubation and variation in the chosen time of incubation can affect the final reticulocyte count. Based on the intensity of the reaction between the dye Oxazine 750 and reticulocyte mRNA, reticulocytes can be classified as low-, medium-, or high-staining intensity

666 reticulocytes. An increase in the number of high-staining intensity reticulocytes indicates the presence of stress reticulocytes. Reticulocytes can also be stained with a labeled antibody to the transferrin receptor.58 Bayer Advia counters can also measure reticulocyte volume (MCVr), reticulocyte hemoglobin concentration (CHCMr), and total reticulocyte hemoglobin (retHb), ratio of total hemoglobin to retHb (Hb/retHb), absolute reticulocyte count, mature red cell hemoglobin (rbcHb), rbcHb/retHb ratio, and numbers of erythrocytes with MCHC more than a chosen value. From the absolute reticulocyte count and the CHr, the retHb is calculated, which is expressed in grams per liter, the hemoglobin content of all reticulocytes.59 The ratio of rbcHb to retHb defines the ratio between the hemoglobin contained in mature red cells and in the reticulocytes. Under steady-state conditions, erythrocyte survival can be estimated indirectly from the ratio of hemoglobin contained in mature red cells and in reticulocytes. A reduction in retHb and a concomitant increase in rbcHb/retHb ratio provide indirect evidence for prolonged red cell survival. Changes in MCVr can provide a much more rapid indication of response of patients to folate or vitamin B12 deficiency.60

Measures of Cell Heterogeneity Most automated counters produce an indication of red cell heterogeneity called the RDW, which is calculated from the measured variation in red cell size by a nonlinear formula that magnifies heterogeneity. Increased reticulocyte count will increase RDW and a correlation between the RDW and percentage of cells in the densest part of density gradient separations has also been shown.61 Bayer Advia counters also compute a hemoglobin distribution width that is based on the variation of the CHCM, which is directly determined for each cell. Typically hemoglobin distribution width is a much smaller number than RDW. MCHC has distinct characteristics in many hemoglobinopathies. Small increases in MCHC occur when normal erythrocytes are deoxygenated and this can be visually demonstrated on continuous density gradients.62,63 Hemoglobin concentration is directly proportional to cell density, but is not necessarily correlated with cell volume unless cells from the same source are compared at different osmolarities. For example, the red cells of patients with HbE are microcytic but have a narrow range of densities identical to that of individuals with HbA.64 Red cells from normal adults have an MCHC of 33 ± 1.5 g/dL by conventional measurements; however, more sensitive measurements show that there are consistent differences between males and females and between African Americans and Caucasians. Several hemoglobinopathies have altered MCHC with a narrow range of densities. For example, in ␦␤ thalassemia trait the cells are uniformly less dense by approximately 2 g/dL and in homozygous HbC disease, the cells are uniformly denser by approximately 4 g/dL. In

Mary Fabry and John M. Old other diseases, such as sickle cell disease, hereditary spherocytosis, and some of the thalassemias, there is a broad but characteristic distribution of red cells that include both high- and low-density red cells. MCHC is the least reliable of all automated measurements and is frequently considered uninformative, so alternative methods are used to analyze and isolate red cells according to density. These can be broken into three broad classes: methods using microhematocrit tubes filled with materials of different densities, methods that rely on discontinuous or layered density of supporting media for separation, and techniques that rely on continuous density gradients. All methods take advantage of the fact that cells will move under the influence of centrifugal force until they find a region of density the same as that of the cell – continuous gradients – or stop when they encounter a region of higher density – at the boundaries in discontinuous gradients. Discontinuous gradients based on a number of substances have been used in density-based separation. The most successful of these methods are based on substances that are nontoxic to the cell, have a low osmolarR ity, neutral pH, and low viscosity. Phthalate esters, Percoll
, R R R


Hypaque , Stractan , (Larex ), and others have all been used for red cell separations. Danon and coworkers65 devised a simple method of generating density profiles and isolating cells of defined density by using mixtures of phthalate esters that cover the density range of red cells. A disadvantage of this method is that the separated cells are not viable for physiological studies. Stractan (arabinopolygalactan, now marketed as Larex) is a high-molecular-weight product made from the bark of the larch tree. It is physiologically benign and has a low osmotic contribution, which allows the ionic content of solutions to be adjusted freely.66 A disadvantage of discontinuous gradients is that they usually have four or fewer densities that can either mask relatively large changes in density of cells at the upper boundary between density levels, or exaggerate small changes in cells at the lower boundary. Percoll is a commercially available product that spontaneously generates a continuous density gradient when centrifuged. It is composed of silica particles coated with polyvinyl pyrrolidone with range of sizes whose rate of sedimentation by centrifugation force is determined by size. The density at any depth in the tube is determined by the number and size of the particles. For this reason the density profile of Percoll gradients depends on the duration of centrifugation, viscosity (which is temperature dependent), and G force applied. The density range of Percoll is suitable for separation of white cells and needs to be increased R before it can be applied to red cells. Renografin
and Percoll can be combined to produce a continuous density gradient capable of separation of red cells by their density.67 A disadvantage of Renografin is its high osmolarity, which results in a final osmolarity of the mixture of more than

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders

Figure 28.4. Percoll–Larex continuous density gradient. Red cell density is directly proportional to the MCHC. The least dense (lowest MCHC) cells are at the top of the gradient and the highest density cells are at the bottom. This technique provides a method for directly visualizing the distribution of red cell density in a sample of whole blood. The example shows a continuous density gradient with density marker beads and red cells from patients with normal hemoglobin (AA), HbC trait (AC), HbC disease (CC), sickle cell anemia (SS), HbS-O Arab, and hereditary spherocytosis (HS). (See color plate 28.4.)

360 mOsm. Stractan and Percoll can produce continuous density gradients with physiological pH, osmolarity, and ionic composition.68 These gradients are sensitive to very small changes in red cell density and can be used both preparatively and analytically. Assignment of density is based on position relative to density marker beads and on measured MCHC of cells isolated from defined positions in the gradient. An example of the power of separation of Percoll–Larex gradients is given in Figure 28.4, in which the red cell density distributions of several common hemoglobinopathies are compared. All separations based on red cell density are sensitive to the pH and osmolarity of the solutions because these variables will change the MCHC of the cells. Sedimentation through the interface may depend on red cell deformability,69 leading to systematic underestimation of the density of poorly deformable cells. Stractan (Larex) is tedious to prepare, but cells isolated from these gradients are suitable for transport measurements and other protocols requiring physiologically intact cells.

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When stained with hematoxylin–eosin F cells are stained; in gene deletion HPFH all erythrocytes are uniformly stained. Development of monoclonal antibodies to HbF in several laboratories allowed a more quantitative detection of HbF. Cells are first lightly fixed and the membrane permeabilized with detergents and/or organic solvents. After fixation and permeabilization, the cells are stained with antibody and may be observed either by microscopy or by flow cytometry70–72 (Fig. 28.5). Early measurements used immunodiffusion to create a ring of precipitated HbF antibody around the positive cells. Fluorescent labels can be attached directly to the antibodies and cells observed by fluorescence microscopy.70 Monoclonal antibodies against several hemoglobins including HbF are commercially available. These antibodies can be used for immunofluorescent labeling of F cells in fixed smears on slides, including archival samples.70

Globin Chain Synthesis Incorporation of labeled amino acids into intact cells was first reported more than 50 years ago. Imbalance of synthesis of ␣- versus non-␣-globin chains is the fundamental definition of thalassemia. Globin-chain synthesis can be measured in blood reticulocytes and marrow erythroid precursors, although the results are not always equivalent. The most convenient means of chain separation is

F Cells With the exception of some forms of HPFH (Chapter 16), HbF is not uniformly distributed among red cell. Erythrocytes with detectable amounts of HbF are called F cells (Chapter 7), and these can be detected by the acid elution method and flow cytometry. The Kleihauer and Betke method relies on resistance of precipitated HbF to acid elution. Cells are spread on a slide, fixed, and then incubated with citric acid–phosphate buffer. HbF remains precipitated while all other hemoglobins are eluted from the cell.

Figure 28.5. Comparison of distribution of ␥ -globin chains in red cells by fluorescence-activated cell sorting analysis using fluorescein isothiocyanate– labeled antibody specific for human ␥ -globin chains (supplied by Thomas Campbell of EG&G Wallac). In this case, the examples are two different types of transgenic mice expressing exclusively human hemoglobin and either a low level of ␥ globin (panel A) or a high level of ␥ globin (panel B). Left panels: fluorescence intensity versus cell size; right panels: number of cells versus fluorescence intensity. Note the presence of two populations of red cells that represent non-F cells and F cells (high fluorescence intensity).

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Figure 28.6. Chain synthesis as measured by incorporation of radioactive leucine followed by separation of globin chains by denaturing reverse-phase HPLC. In this case the sample studied was from a transgenic mouse expressing human ␣, ␤S , and ␤S-Antilles as well as mouse ␣- and ␤-globins.

reverse-phase HPLC.73 Fractions containing globin chains are collected, and the radioactivity incorporated is counted (Fig. 28.6). The efficiency of labeling can be greatly improved if the percentage reticulocytes is enriched by a process such as layering whole blood on a discontinuous Stractan gradient and collecting the lightest fraction that contains the most reticulocytes. Chain synthesis is still the gold standard when trying to elucidate the mechanism(s) by which synthesis of ␥ -globin is enhanced by butyrate in sickle cell disease.74,75

Hemoglobin S Polymerization, Rate of Sickling, and Percentage of HbS Polymer When the concentration of deoxyHbS exceeds the CSAT , polymer is formed. Polymer formation does not occur immediately because, prior to polymer formation, nucleation must occur, and because the nucleus consists of several hemoglobin molecules, there is a very high concentration dependence (Chapter 6). The time between deoxygenation and the onset of polymer formation is called the delay time76 that has one of the highest concentration dependencies in biology77 and may range from values of less than a microsecond at high hemoglobin concentrations to several minutes at low concentrations. The distortion of the red cell known as sickling is the result of intracellular polymer formation and can be used as an indirect endpoint for polymer formation. Two methods have been used to measure the rate of sickling: manual mixing and continuous flow. In the manual and continuous flow methods, oxygenated red cells are mixed with buffered sodium dithionite, which reacts with the oxygen and, at predetermined intervals, cells are fixed with isotonic formalin; the number of cells that have sickled are counted, and the results plotted as a function of time. The earliest recorded time point for continuous mixing was at 25 msec. Because

Mary Fabry and John M. Old samples of blood from sickle cell disease patients can contain appreciable numbers of cells with unusual shapes, the number of deformed cells can be counted in a control sample and subtracted from the total of sickled cells. This method is capable of showing that cells from patients with HbSC disease have a stronger response to reduced osmolarity than cells from sickle cell anemia patients and can detect retardation of polymer formation by antisickling agents. Polymer formation can occur much more rapidly than the mixing time in these methods and methods capable of directly studying polymerization in solutions and intact cells have been devised. These more quantitative methods consist of two parts: 1) rapid generation of high concentrations of nonpolymerized deoxyHbS, and 2) a sensitive, rapid readout. Two methods have been used to generate high concentrations of deoxyhemoglobin: temperature jump and laser photolysis. Readout methods have included optical birefringence, turbidity, light scattering, water proton line width and relaxation time, and differential interference contrast. The temperature jump method makes use of the negative temperature dependence of HbS solubility and is very simple, but requires a minimum of approximately 10 seconds to reach thermal equilibrium and has a resolution of approximately 1 second. In addition, the highest hemoglobin concentration that can be attained at 0◦ C without polymer is approximately 30 g/dL. The majority of sickle cells have a MCHC exceeding this value. Because of these limitations a laser photolysis method was developed that allows direct measurement of polymer formation with very high time resolution both in solutions and in red cells. Using laser photolysis of CO-Hb, deoxyHb is produced followed by detection of polymer formation by light scattering. The principle of this measurement is the observation that CO can be completely dissociated from hemoglobin by exposure to a focused laser beam at 514.5 nm, yielding deoxyhemoglobin and that this deoxyhemoglobin can be maintained with continuous photolysis with minimal heating. Polymerization can then be monitored by the method of choice such as light scattering. New insights into nucleation, fiber growth, and factors affecting red cell shape have been obtained using differential interference contrast and birefringence.78,79

Clinical Evaluation of Hemoglobinopathies and Thalassemias using Protein-based and Cellular Diagnostics When clinical evaluation, family history, or the results of a screening or routine blood count raise the possibility of a hemoglobinopathy or thalassemia, the first approach to diagnosis lies in the further evaluation of the erythrocyte indices, such as MCV or MCH, followed by expert examination of a peripheral blood film. The reticulocyte count, when persistently elevated is highly suggestive of

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders hemolysis. As discussed in individual disease-specific chapters, peripheral blood smear morphology can provide clues to the diagnosis of most of the common hemoglobinopathies and thalassemia. No single hemoglobin analytical procedure available in most clinical laboratories can distinguish definitively all common and clinically important variant hemoglobins, and rare variants can be especially difficult to resolve. The highest levels of hemoglobin-based diagnostics are limited to reference laboratories. The ␤ thalassemias rely on quantitation of HbA2 levels to detect heterozygotes, and some ␣ thalassemias are characterized by HbH inclusion bodies. Quantitation of HbF is important for the diagnosis of thalassemia syndromes and HPFH. If a hemoglobin variant is detected, the major clinical questions to be resolved are, what is the nature and abundance of the variant, and is it relevant to the clinical condition? In general, mutations of the ␤-globin gene usually cause only a single abnormal hemoglobin fraction, and if stable (Chapter 24), forms half of the total hemoglobin. In contrast, ␣-globin chain variants cause alterations in all hemoglobins, as all hemoglobins contain an ␣-globin chain. Usually, mutation of one ␣-globin gene creates variants forming 20%–25% of the total HbA, and proportionally similar fractions of the total HbA2 and HbF. ␥ -Globin variants will alter a single major hemoglobin during fetal and neonatal life, but after the first year of life will alter only the small residual amount of HbF, and as a result, are rarely detected. Mutants of the ␦-globin gene will alter only the HbA2 band. The actual amounts of different hemoglobin types that accumulate in vivo depend on the expression of each globin gene, the stability of the monomer, dimer and tetramer, and on the posttranslational assembly of ␣␤ dimers (Chapter 7). Not all variant hemoglobins are resolvable using the commonly available protein-based means of detection, and a definitive diagnosis of rare variants is usually not possible with these methods. Therefore, rather than attempt the questionable, when definitive diagnosis is called for, it is best to move to DNA-based diagnostics.

DNA-BASED DIAGNOSIS OF THE HEMOGLOBIN DISORDERS Hemoglobinopathies were the first genetic diseases to be characterized at the molecular level and consequently have been used as a prototype for the development of new techniques of mutation detection. Many different PCRbased techniques can be used to detect the known globin gene mutations, including dot blot analysis, reverse dot blot analysis, the amplification refractory mutation system (ARMS), denaturing gradient gel electrophoresis (DGGE), mutagenically separated polymerase chain reaction (MSPCR), gap-PCR, and restriction endonuclease (RE) analysis. Each method has its advantages and disadvantages and the

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particular one chosen by a laboratory for the diagnosis of point mutations depends not only on the technical expertise available in the diagnostic laboratory but also on the type and variety of the mutations likely to be encountered in the individuals being screened. The spectrum of ␣- and ␤-globin gene mutations is different for each population, and many mutations are regionally specific. Although more than 1,200 different globin gene alleles have been identified, mutation analysis is simplified by the fact that each country has only a few common alleles. Thus, a strategy is required for mutation diagnosis that involves knowledge of the ethnic group of the patient under study, the gene frequencies of the mutations in the ethnic group of the patient, and the appropriate method of mutation detection for those mutations. The following is an overview of the diagnostic methods currently being used worldwide for DNA-based diagnosis, including their use in antenatal diagnosis, and we describe strategies and the methods used in one large national laboratory for the diagnosis of mutations in an ethnically heterogeneous population. The approach used in ethnically homogeneous populations will be different and more focused.

SOURCES OF DNA Blood DNA is normally prepared from 5 to 10 mL of peripheral blood that is anticoagulated with heparin or preferably EDTA. The DNA can be isolated by the standard method of phenol–chloroform extraction and ethanol precipitation or by using one of several commercial kits based on salt extraction, protein precipitation etc. Sufficient DNA is obtained for both molecular analysis and subsequent storage in a DNA bank at −20◦ C.80 If DNA banking is not required, a much smaller quantity of blood may be used for just PCR techniques. Mutation analysis may be performed by simply adding 1 ␮L of boiled whole blood to the PCR mixture.81

Amniotic Fluid DNA can be prepared from amniotic fluid cells directly or after culturing. It is prudent to split an amniotic fluid sample into a sample to be sent for culturing and a sample for direct DNA analysis. It takes 2–3 weeks to grow amniocytes to confluence in a 25-mL flask, but culturing has the advantage that a large amount of DNA is obtained (the yield from such a flask has varied from 15 to 45 ␮g, enough DNA for all types of analyses). This provides a backup for failure with a direct analysis or material for confirmation in case of maternal contamination problems. A diagnosis can be made using DNA from the noncultivated cell sample in most cases. Approximately 2 ␮g of DNA is obtained from 7 mL of amniotic fluid and this is sufficient for all PCRbased methods of analysis. The method of DNA preparation

670 for both cultured and noncultivated cells is essentially the same as that for chorionic villi.80

Mary Fabry and John M. Old and ␤ thalassemia.88 The approach has proved subject to technical difficulties and is costly and time consuming and so is not widely applicable.

Chorionic Villi The two main approaches to chorionic villus sampling, ultrasound-guided transcervical aspiration and ultrasound guided transabdominal sampling, provide good-quality samples of chorionic villi for fetal DNA diagnosis. Sufficient DNA is normally obtained for both PCR and Southern blot analysis of the globin genes. In 200 chorionic villus sampling DNA diagnoses, the average yield of DNA was 46 ␮g and only in one instance was less than 5 ␮g obtained.80 The main technical problem with this source of fetal DNA is the risk of contamination with maternal DNA, which arises from the maternal decidua that is sometimes obtained along with the chorionic villi. Careful dissection and removal of the maternal decidua with the aid of a phase-contrast microscope, yields pure fetal DNA samples, as shown by a report of 457 first trimester diagnoses for ␤ thalassemia in Italians, without any misdiagnoses.82 Maternal contamination can be ruled out in most cases by the presence of one maternal and one paternal allele following the amplification of highly polymorphic repeat markers. Originally, variable number tandem repeat (VNTR) polymorphic markers were amplified and analyzed by gel electrophoresis and ethidium bromide staining,83 but now most laboratories have switched to using short tandem repeat markers or microsatellite polymorphic markers analyzed on a DNA sequencing machine.84 The risk of misdiagnosis from maternal DNA contamination might be further reduced by the preparation and analysis of DNA from a cleaned single villus frond;85 however, following a case in which a single frond appeared to give a different sickle cell genotype compared with the remaining chorionic villus sampling material, this techniques was abandoned.

Fetal Cells in Maternal Blood Fetal cells have long been known to be present in the maternal circulation and they provide an attractive noninvasive approach to prenatal diagnosis, but attempts to isolate the fetal cells as a source of fetal DNA by using immunological methods and cell sorting have had only moderate success in providing a population of cells pure enough for fetal DNA analysis. Until recently, analysis of fetal cells in maternal blood could only be applied for the prenatal diagnosis of ␤ thalassemia in women whose partners carried a different mutation, as reported for the diagnosis of Hb Lepore.86 The development of the technique of isolation of single nucleated fetal erythrocytes by micromanipulation under microscopic observation87 has permitted the analysis of both fetal genes in single cells from maternal blood. This approach has now been used successfully for prenatal diagnosis in two pregnancies at risk for sickle cell anemia

Fetal DNA in Maternal Plasma The analysis of fetal DNA in maternal plasma is a simpler and more robust procedure than the analysis of DNA in fetal nucleated red cells in maternal blood as no enrichment process is involved.89 Cells are removed from the plasma by simple centrifugation and then the DNA in the plasma can be purified by standard methods.90 Fetal DNA has been detected in as little as 10 mL of maternal plasma at 11–17 weeks gestation and is cleared very rapidly from the maternal plasma postpartum.91 The technique is being used for the prenatal diagnosis of sex-linked disease and fetal RhD blood group type. The approach can only be used to detect the paternally inherited mutation and thus for ␤ thalassemia it is limited in that it is only potentially applicable to couples in whom the paternal mutation is different than the maternal mutation. It has been used for the prenatal exclusion of ␤ thalassemia major in eight fetuses at risk, using allele-specific primers for the detection of the CD 41/42 (-CTTT) mutation by real-time PCR92 and the detection of homozygous ␣0 thalassemia.93 Further development of the method to detect both maternal and paternal linked polymorphisms might allow the technique to exclude maternal ␤ thalassemia mutations.94

Preimplantation Diagnosis Preimplantation genetic diagnosis represents a state-of-theart procedure that allows at risk couples to have diseasefree children without the need to terminate affected pregnancies. PCR-based diagnostic methods can be potentially applied for preimplantation genetic diagnosis using three types of cells: polar bodies from the oocyte/zygote stage, blastomeres from cleavage stage embryos, and trophectoderm cells from blastocysts.95 Although the technique requires a combined expertise in both reproductive medicine and molecular genetics, a small number of centers around the world can now perform this procedure for hemoglobin disorders, resulting in the birth of more than 50 healthy children. Preimplantation diagnosis has been used successfully for both ␣96,97 and ␤ thalassemia.98,99 The approach is especially useful for couples for whom religious or ethical beliefs will not permit the termination of pregnancy, and for couples who have already had one or more therapeutic abortions. Nevertheless, preimplantation genetic diagnosis is technically challenging, multistep, and expensive. The PCR protocol must be able to detect accurately the required genotype in single cells, be optimized to minimize PCR failure and avoid the problem of allele drop out, which could lead to misdiagnosis. Protocols designed to monitor the

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders occurrence of allele drop out include multiplex PCR to detect both alleles that contribute to the genotype, such as DGGE, single-strand conformation analysis and real time PCR.100 The birth of a healthy unaffected baby depends not only on an accurate diagnosis, but also on the success of each of the multiple stages of the assisted reproduction procedure. Overall, the success rate of the procedure is only 20%–30% and thus this approach is not likely to be used routinely for the monitoring of pregnancies at risk for hemoglobin disorders. One specific use of this approach is to allow the birth of a normal child who is HLA identical to an affected sibling, thus permitting a possible cure by stem cell transplantation.20

Table 28.1. Globin gene disorder deletions diagnosable by gap-PCR Disorder

Deletion mutation

Distribution

␣0 Thalassemia

--SEA --MED -(␣)20.5 --FIL --THAI -␣3.7 -␣4.2 290-bp deletion 532-bp deletion 619-bp deletion 1393-bp deletion 1605-bp deletion 3.5-kb deletion 10.3-kb deletion 45-kb deletion Hb Lepore Spanish Sicilian Vietnamese Macedonian/Turkish Indian Chinese HPFH1 HPFH2 HPFH3 Hb Kenya

Southeast Asia Mediterranean Mediterranean Philippines Thailand Worldwide Worldwide Turkey, Bulgaria Africa India, Pakistan Africa Croatia Thailand India Philippines, Malaysia Mediterranean, Brazil Spain Mediterranean Vietnam Macedonia, Turkey India, Bangladesh Southern China Africa Ghana India Africa

␣+ Thalassemia ␤0 Thalassemia (␦␤)0 Thalassemia

DNA DIAGNOSIS OF ␣ THALASSEMIA ␣ Thalassemia is almost always a result of mutations affecting either one ␣-globin gene (␣+ thalassemia) or both ␣globin genes on the same chromosome (␣0 thalassemia) (Chapter 13). The majority of the mutations are gene deletions but some point mutations in one of the two ␣-globin genes resulting in ␣+ thalassemia have been described. The deletion breakpoints of the seven most common deletion alleles have been determined, and these deletions can be diagnosed quickly by the technique known as gapPCR (Table 28.1). The remainder of the deletion alleles used to be diagnosed by Southern blot analysis,101 but now are diagnosed by the technique called multiplex ligation– dependent probe amplification (MLPA).102

Diagnostic Strategy ␣0 Thalassemia is found in mainly patients of Mediterranean or Southeast Asian in origin. Although ␣0 thalassemia has been described in patients of Asian Indian or African origin, it is extremely uncommon, and patients with the phenotype of ␣0 thalassemia trait usually have the genotype of homozygous ␣+ thalassemia. ␣+ Thalassemia can reach high gene frequencies in parts of Africa and Asia, with the -␣3.7 deletion being the predominant mutation in African, Mediterranean, and Asian individuals and the -␣4.2 being more common in Southeast Asian and the Pacific islands populations. The strategy for screening is based on ethnic origin of the individual, although PCR now makes it easy to screen for all the common deletion mutations in any individual. In multiethnic countries like the United Kingdom and United States the ethnic origin of carriers of ␣ thalassemia include individuals of African, Indian, Pakistani, Chinese, Southeast Asian, Greek Cypriot, and Turkish Cypriot descent and occasionally members of the indigenous population. In this environment, screening is first done for the -␣3.7 -kb and -␣4.2 -kb ␣+ thalassemia deletions by multiplex gap-PCR. If indicated by the MCH, MCV, or suspected HbH disease, screening is next done for ␣0 thalassemia deletions by multiplex gap-PCR for either the Mediterranean or

671

(A ␥ ␦␤)0 Thalassemia

HPFH

Southeast Asian deletions according to an individuals’ ethnic origin. If negative results are still obtained, screening for nondeletion mutations by selective DNA sequencing of both ␣-globin genes for suspected ␣+ thalassemia carriers and screening, for other deletions by MLPA analysis in suspected ␣0 thalassemia carriers can also be done. All prenatal diagnoses for ␣0 thalassemia are carried out by both gapPCR and MLPA.

Diagnosis of Deletion Mutations by PCR The two most common ␣+ thalassemia gene deletions, the -␣3.7 and -␣4.2 alleles, together with 5 ␣0 thalassemia deletion genes, the --FIL , --THAI , --MED , -(␣)20.5 , and the --SEA alleles can be diagnosed by gap-PCR81,103–106 (Table 28.1). Gap-PCR is the simplest of amplification techniques, using two primers complimentary to the sense and antisense strand of the DNA regions that flank the deletion. Amplified product is only obtained from the ␣ thalassemia deletion allele as the primers are located too far apart on the normal DNA sequence for successful amplification. Therefore, the normal allele (␣␣) is detected by amplifying across

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Mary Fabry and John M. Old

Figure 28.7. Prenatal diagnosis of the --MED ␣0 thalassemia allele by using gap-PCR. The amplification products after agarose gel electrophoresis and ethidium bromide staining are shown as follows: track 1, maternal DNA; track 2, paternal DNA; track 3, normal DNA; and tracks 4 and 5, different concentrations of chorionic villi DNA. The diagram below shows the location of the --MED deletion with respect to the ␣-globin gene cluster. Primers A and B span the deletion and amplify --MED allele to give a 650-bp product; primers B and C span one breakpoint and amplify the normal allele to give a 1000-bp product.

one of the breakpoints, using one primer complimentary to the deleted sequence and one to the normal sequence. An example of the use of gap-PCR for the prenatal diagnosis of the Mediterranean ␣0 thalassemia mutation --MED is illustrated in Figure 28.7. Gap-PCR provides a quick diagnostic test for ␣0 thalassemia trait but requires careful application for prenatal diagnosis. Prenatal diagnosis results by gap-PCR should be confirmed by MLPA analysis. Amplification of sequences in the ␣-globin gene cluster is technically more difficult than that of the ␤-globin gene cluster, requiring more stringent conditions for success due to the higher GC content of the ␣-globin gene cluster.107 For a more reliable diagnosis of the carrier state by gap-PCR, amplification of the GC-rich ␣-globin locus can be improved by using betaine and dimethyl sulfoxide in the PCR reaction. These agents enhance the reaction by disrupting the base pairing of the GC-rich region and lead to the destabilization of the secondary structure by making the GC and AT base pairs equally stable in the DNA duplex. The application of these agents with redesigned primers has lead to the development of multiplex assays to detect heterozygosity and homozygosity of the seven deletion mutations.81,108 Experience has shown that great care is still needed in interpreting the results, as some tests still result occasionally in unpredictable reaction failure and allele drop out. Testing several different dilutions of a DNA sample can help resolve this problem, as the inhibition causing allele drop out can be diluted out.

Other approaches have been developed to provide quick, simple, rapid, accurate, and cost effective methods of screening for the deletion mutations. These include the use of real-time quantitative PCR analysis for the Southeast Asian ␣0 thalassemia deletion in Taiwan,109 the use of denaturing HPLC to diagnose the 4.2-kb ␣+ thalassemia deletion gene in Chinese individuals,110 real-time quantitative PCR to detect the Southeast Asian ␣0 thalassemia deletion,93 and the use of an oligonucleotide microarray to detect the Southeast Asian ␣0 thalassemia deletion and the -␣3.7 -kb and -␣4.2 -kb ␣+ thalassemia deletions.111,112 The most useful recent development for the diagnosis of deletion mutations is MLPA.102 In this method, two sets of 35 probes have been developed to detect all known deletions located in the ␣-globin gene cluster on chromosome 16p13.3. This method will detect rare and novel forms of deletional ␣ thalassemia that cannot be diagnosed by gapPCR and provides an excellent back-up screening method for the common deletion mutations. In some cases, MLPA can replace Southern blotting for the routine diagnosis of ␣0 thalassemia deletions.

Diagnosis of Point Mutations by PCR The nondeletion ␣+ thalassemia mutations can be identified by PCR techniques following the selective amplification of the ␣-globin genes.113 This technique allows the amplified product from each ␣-globin gene to be analyzed for the expected known mutation according to the ethnic

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders origin of the patient or for the gene to be sequenced to identify new mutations. Selective amplification followed by DNA sequence analysis is a reasonable approach for the diagnosis of all nondeletion ␣+ thalassemia mutations. Several of the nondeletion ␣+ thalassemia mutations create or destroy a restriction enzyme site and can be analyzed for by restriction enzyme digestion of the amplified product. For example, Hb Constant Spring mutation can be diagnosed by Mse I digestion.114 In theory, any technique for the direct detection of point mutations such as allele specific oligonucleotide hybridization or allele specific priming can be used for the diagnosis of nondeletion ␣+ thalassemia mutations. No simple strategy to diagnose all the known mutations has been developed however. The only published approach to date is a complex strategy involving the combined application of the indirect detection methods of DGGE and single-strand conformation analysis, followed by direct DNA sequencing.115

DNA DIAGNOSIS OF ␤ THALASSEMIA Although more than 200 different ␤-globin gene mutations have been associated with the phenotype of thalassemia, only approximately 30 mutations are found in at risk groups at a frequency of 1% or greater (as listed in Table 28.2), and thus just a small number account for the majority of the mutations worldwide.116 All of the mutations are regionally specific and the spectrum of mutations has now been determined for most at risk populations.117 Each population has been found to have just a few of the common mutations together with a larger and more variable number of rare mutations. This makes it easy to screen for ␤ thalassemia mutations in most cases if the ethnic origin of the patient is known.

Diagnostic Strategy The strategy for identifying ␤ thalassemia mutations in most diagnostic laboratories is to screen for the common ones first using a PCR-based technique that allows the detection of multiple mutations simultaneously. This approach will identify the mutation in more than 90% of cases. Further screening for the possible rare mutation will identify the defect in most of the remaining cases. Mutations remaining unknown after this second screening are characterized by direct DNA sequence analysis. The ␤globin gene can be amplified simultaneously in three sections of approximately 500 bp in length for direct DNA sequence analysis using an automatic DNA sequencer. Alternatively, the site of the mutation can be localized first by the application of a nonspecific detection method such as DGGE and then the mutation identified using just one sequencing reaction. Although a bewildering variety of PCR techniques have been described for the molecular diagnosis of point mutations, most diagnostic laboratories are using one or more of the techniques described herein.

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Panels of primers for the detection of the common mutations in each ethnic group can be developed by the primer-specific amplification method known as ARMS. Panels for the common Mediterranean, Asian Indian, Chinese, and African mutations are available along with a panel for the most common silent or normal HbA2 ␤ thalassemia mutations. Rare or novel mutations are then diagnosed by DNA sequence analysis. Prenatal diagnosis results for ␤ thalassemia are confirmed by a second method, either by restriction enzyme analysis of amplified product (RE-PCR), or more usually, by DNA sequence analysis.

Allele-specific Oligonucleotide PCR The hybridization of allele-specific oligonucleotide (ASOs) probes to amplified genomic DNA bound to a nylon membrane in the form of dots was the first PCR method to be developed. This method, known as dot blotting, is based on the use of two oligonucleotide probes for each mutation, one complimentary to the mutant DNA sequence and the other complimentary to the normal ␤-globin gene sequence at the same position. The probes are usually 5
end-labeled with 32 P-labeled deoxynucleoside triphosphates, biotin, or horseradish peroxidase. The genotype of the DNA sample is diagnosed by observing the presence or absence of a hybridization signal from the mutation specific and normal probes. The technique has been applied in many laboratories with great success, especially for populations with just one common and a small number of rare mutations, as in the example of Sardinia.118 When screening for a large number of different mutations, this method becomes limited by the need for separate hybridization and washing step for the detection of each mutation. To overcome this problem of screening for multiple mutations, the technique of reverse dot blotting has been developed in which the roles of the oligonucleotide probe and the amplified genomic DNA are reversed.119 Unlabeled oligonucleotide probes complementary to the mutant and normal DNA sequences are fixed to a nylon membrane strip in the form of dots or slots. Amplified genomic DNA, labeled by either the use of end-labeled primers or the internal incorporation of biotinylated deoxyuridine triphosphate, is then hybridized to the filter. This allows multiple mutations to be tested in one hybridization reaction. It has been applied to the diagnosis of ␤ thalassemia mutations in Mediterranean individuals,120 African Americans,121 and Thais,122 using a two-step procedure with one nylon strip for the common and another for the less common mutations. Reverse hybridization screening is the only technique for the diagnosis of ␤ thalassemia mutations to have been developed commercially with some success and there are currently there are two competing systems on the market. One uses a strip with oligonucleotide probes for eight common Mediterranean mutations affixed together with probes to detect HbS and HbC mutations in amplified DNA. Another uses nucleotides complementary to mutant and

674

Mary Fabry and John M. Old Table 28.2. The distribution of the common ␤ thalassemia mutations expressed as percentage gene frequencies of the total number of thalassemia chromosomes studied Mediterranean Mutation -88 (C→T) -87 (C→G) -30 (T→A) -29 (A→G) -28 (A→G) CAP + 1 (A→C) CD5 (-CT) CD6 (-A) CD8 (-AA) CD8/9 (+G) CD15 (G→A) CD16 (-C) CD17 (A→T) CD24 (T→A) CD30 (G→A) CD30 (G→C) CD39 (C→T) CD41/42 (-TCTT) CD71/72 (+A) IVSI-1 (G→A) IVSI-1 (G→T) IVSI-5 (G→C) IVSI-6 (T→C) IVSI-110 (G→A) IVSII-1(G→A) IVSII-654 (C→T) IVSII-745 (C→G) 619-bp deletion Others

Indian

Italy

Greece

Turkey

0.4

1.8

1.2 2.5

Pakistan

Chinese India

China

Thailand

0.8

African African-American 21.4

1.9 11.6

60.3 4.9

1.7 0.4

1.2 2.9 0.6

0.8 0.6 7.4 28.9 3.5 1.3

12.0 0.8 1.7

0.8 10.5

24.7 7.9

40.1

4.3

17.4

13.6

0.9 3.5

0.9

7.9

13.7

8.2 26.4

6.6 48.5

3.5 38.6 12.4

46.4 2.3

2.5

4.9

15.7

8.9

6.8

7.9

2.5

16.3 29.8 1.1

7.4 43.7 2.1

17.4 41.9 9.7

3.5

7.1

2.7

4.1

2.2

9.7

23.3 0.5

13.3 0.9

10.6

CD = Codon; IVS = intervening sequence.

normal sequences immobilized in the wells of a microplate and thus is more complex, requiring a dedicated microplate incubator, washer, and reader. Two kits, one for eight common Mediterranean mutations and one for the eight most common Southeast Asian ␤-thalassemia mutations, including HbE, are available.

Oligonucleotide Microarrays The principle of reverse dot blotting has been brought up to date by the development of microarrays for the simultaneous detection of multiple ␤ thalassemia mutations. Several groups have now published details of a DNA chip platform that has been used to genotype ␤ thalassemia carriers and patients.112,123 The approach of tagged singlebased extension and hybridization to glass or flow-through

arrays has been developed for the detection of 17 ␤-globin gene mutations124 and a similar approach of arrayed primer extension has been used to detect 23 mutations.125 It is not clear whether these state-of-the-art methods will be economically viable and replace conventional techniques in the future. This will depend on the market for thalassemia mutation chips, especially for the diagnosis of mutations in populations with just one or two very common mutations that can be easily screened for by rapid low-technology methods and for which the additional screening capacity on the chip would be redundant.

Primer-specific Amplification Different diagnostic methods have been developed based on the principle of primer-specific amplification, where

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Figure 28.8. ARMS-PCR screening of a DNA sample for seven common Mediterranean mutations. The gel shows alternating tracks containing the amplification products from DNA of a patient with ␤ thalassemia trait and those from control DNA for each of the seven mutations (labeled 1–7). The mutations screened for are: 1, IVSI-110 (G→A); 2, IVSI-1 (G→A); 3, IVSI-6 (T→C); 4, codon 39 (C→T); 5, codon 6 (-A); 6, IVSII-1 (G→A); 7, IVSII-745 (C→G); as shown in the diagram of the ␤ gene. In the first 12 tracks, the control primers D and E (Table 28.4) were used (producing an 861-bp fragment), and in the last 2 tracks, the G ␥ -Hin d III RFLP control primers were used (producing a 323-bp fragment).

a perfectly matched PCR primer is much more efficient in annealing and directing primer extension than a mismatched primer. The most widely used method is ARMS, in which a primer will only permit amplification to take place when it perfectly matches the target DNA sequence at the 3
terminal nucleotide.126 The target DNA is amplified using a common primer and either of two allele specific primers, two complimentary to the mutation to be detected (the ␤ thalassemia primer) and the other complimentary to normal DNA sequence at the same position. The method provides a quick screening assay that does not require any form of labelling as the amplified products are visualized by agarose gel electrophoresis and ethidium bromide staining. ARMS primers were first developed for the screening and prenatal diagnosis of ␤ thalassemia mutations in the Asian Indian and Cypriot populations in the United Kingdom.127 Subsequently, ARMS primers have been designed to screen for the common mutations of all ethnic groups.128 Figure 28.8 shows the results of a screening a Cypriot individual with ␤ thalassemia trait for seven common mutations by ARMS-PCR. Details of the mutation-specific and normal sequence-specific primers used to diagnose the common ␤ thalassemia mutations are presented in Tables 28.3 and 28.4. ARMS-PCR is currently the main approach for mutation detection and prenatal diagnosis for ␤ thalassemia, HbE–thalassemia, and the sickling disorders in the United Kingdom. The technique has been established in nations such as India because of its rapidity and economy, making realistic for the first time the development of a prenatal diagnostic service in developing countries.129 Other variations on the primer-specific amplification theme are multiplex ARMS, competitive oligonucleotide priming (COP)–PCR and mutagenetically separated (MS)PCR. More than one mutation can be screened for at

the same time in a single PCR reaction by multiplexing the ARMS primers provided that they are coupled with the same common primer.130 Fluorescent labeling of the common primer allows the sizing of the amplification products on an automated DNA fragment analyzer.131 If the normal and mutant ARMS primers for a specific mutation are coamplified in the same reaction they compete with each other to amplify the target sequence. This technique is called COP and requires the two ARMS primers to be labeled differently. Fluorescent labels permit a diagnosis to be made by means of a color complementation assay.132 A variation of this method is to use ARMS primers that differ in length instead of the label. The primers compete with each other to produce fragments that can be distinguished simply by agarose gel electrophoresis. Normal, heterozygous and homozygous DNAs are diagnosed by simple analysis of the presence or absence of the two products. This technique, called MS-PCR, has been applied to the prenatal diagnosis of ␤ thalassemia in Taiwan.133

Restriction Enzyme PCR This is a useful but limited technique because very few ␤ thalassemia mutations create or abolish a restriction endonuclease site and generate diagnosable products (Table 28.5). The presence or absence of the enzyme recognition site is determined from the pattern of fragments of digested fragments after agarose or polyacrylamide gel electrophoresis. Mutations that do not naturally create or abolish restriction sites may be diagnosed by the technique of amplification created restriction sites. This method uses primers that are designed to insert new bases into the amplified product to create a restriction enzyme recognition site adjacent to the mutation sequence. This technique

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Mary Fabry and John M. Old Table 28.3. Primer sequences used for the detection of common mutations for ␤ thalassemia and ␤-globin chain variants by the allele-specific priming technique Mutation

Oligonucleotide sequence

Second primer

Product size (bp)

␤-Thalassemia -88 (C→T) -87 (C→G) -30 (T→A) -29 (A→G) -28 (A→G) CAP + 1 (A→G) CD5 (-CT) CD6 (-A) CD8 (-AA) CD8/9 (+G) CD15 (G→A) CD16 (-C) CD17 (A→T) CD24 (T→A) CD30 (G→A) CD30 (G→C) CD39 (C→T) CD41/42 (-TCTT) CD71-72 (+A) IVSI-1 (G→A) IVSI-1 (G→T) IVSI-5 (G→C) IVSI-6 (T→C) IVSI-110 (G→A) IVSII-1 (G→A) IVSII-654 (C→T) IVSII-745 (C→G)

TCACTTAGACCTCACCCTGTGGAGCCTCAT CACTTAGACCTCACCCTGTGGAGCCACCCG GCAGGGAGGGCAGGAGCCAGGGCTGGGGAA CAGGGAGGGCAGGAGCCAGGGCTGGGTATG AGGGAGGGCAGGAGCCAGGGCTGGGCTTAG ATAAGTCAGGGCAGAGCCATCTATTGGTTC TCAAACAGACACCATGGTGCACCTGAGTCG CCCACAGGGCAGTAACGGCAGACTTCTGCC ACACCATGGTGCACCTGACTCCTGAGCAGG CCTTGCCCCACAGGGCAGTAACGGCACACC TGAGGAGAAGTCTGCCGTTACTGCCCAGTA TCACCACCAACTTCATCCACGTTCACGTTC CTCACCACCAACTTCAGCCACGTTCAGCTA CTTGATACCAACCTGCCCAGGGCCTCTCCT TAAACGTGTCTTGTAACCTTGATACCTACT TAAACCTGTCTTGTAACCTTGATACCTACG CAGATCCCCAAAGGACTCAAAGAACCTGTA GAGTGGACAGATCCCCAAAGGACTCAACCT CATGGCAAGAAAGTGCTCGGTGCCTTTAAG TTAAACCTGTCTTGTAACCTTGATACCGAT TTAAACCTGTCTTGTAACCTTGATACCGAAA CTCCTTAAACCTGTCTTGTAACCTTGTTAG TCTCCTTAAACCTGTCTTGTAACCTTCATG ACCAGCAGCCTAAGGGTGGGAAAATAGAGT AAGAAAACATCAAGGGTCCCATAGACTGAT GAATAACAGTGATAATTTCTGGGTTAACGT∗ TCATATTGCTAATAGCAGCTACAATCGAGG∗

A A A A A A A B A B A B B B B B B B C B B B B B B D D

684 683 626 625 624 597 528 207 520 225 500 238 239 262 280 280 436 439 241 281 281 285 286 419 634 829 738

␤-variants ␤S CD6 (A→T) ␤C CD6 (G→A) ␤E CD26 (G→A) ␤D-Punjab CD121 (G→C) ␤D-Iran CD22 (G→C)

CCCACAGGGCAGTAACGGCAGACTTCTGCA CCACAGGGCAGTAACGGCAGACTTCTCGTT TAACCTTGATACCAACCTGCCCAGGGCGTT TCTGTGTGCTGGCCCATCACTTTGGCAAGC CAACCTGCCAGGGCCTCACCACCAACATG

B B B E B

207 206 236 250 255

The above primers are coupled as indicated with primers A, B, C, or D. A: CCCCTTCCTATGACATGAACTTAA B: ACCTCACCCTGTGGAGCCAC C: TTCGTCTGTTTCCCATTCTAAACT D: GAGTCAAGGCTGAGAGATGCAGGA E: GGCAGAATCCAGATGCTCAAGGCCCTTC F: CAATGTATCATGCCTCTTTGCACC The control primers used for all the above mutation-specific ARMS primers except the two marked ∗ are primers D plus F. For IVSII-654(C→T) and IVSII-745(C→G), the G ␥ -Hin d III RFLP primers (listed in Table 28.6) are used as control primers.

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders

677

Table 28.4. Primer sequences used for the detection of normal DNA sequence by the allele-specific priming technique

Mutation

Oligonucleotide sequence

Second primer

Product size (bp)

-88 (C→T) -87 (C→G) CD5 (-CT) CD6 (-A) CD8 (-AA) CD8/9 (+G) CD15 (G→A) CD30 (G→C) CD39 (C→T) CD41/42 (-TCTT) IVSI-1 (G→A) IVSI-1 (G→T) IVSI-5 (G→C) IVSI-6 (T→C) IVSI-110 (G→A) IVSII-1 (G→A) IVSII-654 (C→T) IVSII-745 (C→G)

TCACTTAGACCTCACCCTGTGGAGCCACTC CACTTAGACCTCACCCTGTGGAGCCACCCC CAAACAGACACCATGGTGCACCTGACTCCT CACAGGGCAGTAACGGCAGACTTCTCCTCA ACACCATGGTGCACCTGACTCCTGAGCAGA CCTTGCCCCACAGGGCAGTAACGGCACACT TGAGGAGAAGTCTGCCGTTACTGCCCAGTA TAAACCTGTCTTGTAACCTTGATACCTACC TTAGGCTGCTGGTGGTCTACCCTTGGTCCC GAGTGGACAGATCCCCAAAGGACTCAAAGA TTAAACCTGTCTTGTAACCTTGATACCCAC GATGAAGTTGGTGGTGAGGCCCTGGGTAGG CTCCTTAAACCTGTCTTGTAACCTTGTTAC AGTTGGTGGTGAGGCCCTGGGCAGGTTGGT ACCAGCAGCCTAAGGGTGGGAAAATACACC AAGAAAACATCAAGGGTCCCATAGACTGAC GAATAACAGTGATAATTTCTGGGTTAACGC TCATATTGCTAATAGCAGCTACAATCGAGC

A A A B A B A B A B B A B A B B D D

684 683 528 207 520 225 500 280 299 439 281 455 285 449 419 634 829 738

␤-variants CD6 (A→T) ␤E CD26 (G→A)

AACAGACACCATGGTGCACCTGACTCGTGA TAACCTTGATACCAACCTGCCCAGGGCGTC

A B

527 236

S

See Table 28.3 legend for details of primers A–D and control primers.

has been applied to the detection of Mediterranean ␤ thalassemia mutations.134

mutations and are worth considering as alternative diagnostic approaches for point mutations.

Other Methods for Point Mutations

Gap-PCR and MLPA

Many other techniques for the diagnosis of known ␤-globin gene point mutations have been published, including the use of denaturing high-performance liquid chromatography (DHPLC), the DNA ligase reaction, minisequencing, real-time PCR and multiplex primer extension technology. For example, DHPLC has been used for the analysis of polymorphic duplexes created by allele-specific priming,135 the analysis of five common Southeast Asian mutations by multiplex minisequencing,136 multiplex primer extension analysis for 10 Taiwanese mutations137 and the most common Chinese mutations,138 and the screening for 11 most common Greek mutations.139 Real-time PCR quantification and melting curve analysis using LightCycler technology have been used to provide rapid genotyping for a panel of the 10 most frequent Greek mutations100 and six Lebanese mutations.140 The DNA ligase method has been updated by the development of a novel piezoelectrical method for detection of a single base mutation in codon 17 of the ␤-globin gene using nano-gold-amplified DNA probes.141 All provide rapid and accurate genotyping of the common

Deletions in the ␤-globin gene cluster are detected by gapPCR and/or MLPA analysis. Table 28.1 lists the deletions detectable by gap-PCR. Small deletion mutations in the ␤-globin gene sequence may be detected by PCR using two primers complimentary to the sense and antisense strand in the DNA regions that flank the deletion.142–149 For large deletions, amplified product using flanking primers is obtained only from the deletion allele because the distance between the two primers is too great to amplify normal DNA. As is the case with the ␣ thalassemia deletions, the normal allele may be detected by amplifying sequences spanning one of the breakpoints, using a primer complimentary to the deleted sequence and one complimentary to flanking DNA.

PCR Methods for Unknown Mutations A number of techniques have been applied for the characterization of ␤-thalassemia mutations without prior knowledge of the molecular defect. The most widely used

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Mary Fabry and John M. Old

Table 28.5. ␤-thalassemia mutations detectable by RE-PCR Position

Mutation

Ethnic group

Affected site

-88

C→T

African/Asian Indian

+Fok I

-87

C→G

Mediterranean

-Avr II

-87

C→T

Italian

-Avr II

-87

C→A

African/Yugoslavian

-Avr II

-86

C→G

Lebanese

-Avr II

-86

C→A

Italian

-Avr II

-29

A→G

African/Chinese

+Nla III

+43 to +40 (-AAAC)

Chinese

+Dde I

Initiation CD

T→C

Yugoslavian

-Nco I

Initiation CD

T→G

Chinese

-Nco l

Initiation CD

A→G

Japanese

-Nco I

CD 5

(-CT)

Mediterranean

-Dde I

CD 6

(-A)

Mediterranean

-Dde I

CD 15

(-T)

Asian Indian

+Bgl I

CD 17

A→T

Chinese

+Mae I

CD 26

G→T

Thai

-Mnl I

CD 26

G→A

Southeast Asian

-Mnl I

CD 27

G→T

Mediterranean

-Sau 96 I

CD 29

C→T

Lebanese

-Bsp M I

CD 30

G→C

Tunisian/African

-Bsp M I

CD 30

G→A

Bulgarian

-Bsp M I

IVSI-1

G→A

Mediterranean

-Bsp M I

IVSI-1

G→T

Asian Indian/Chinese

-Bsp M I

IVSI-2

T→G

Tunisian

-Bsp M I

IVSI-2

T→C

African

-Bsp M I

IVSI-2

T→A

Algerian

-Bsp M I

IVSI-5

G→A

Mediterranean

+Eco R V

IVSI-6

T→C

Mediterranean

+Sfa N I

IVSI-116

T→G

Mediterranean

+Mae I

IVSI-130

G→C

Turkish

-Dde I

IVSI-130

G→A

Egyptian

-Dde I

CD 35

C→A

Thai

-Acc I

CD 37

G→A

Saudi Arabian

-Ava II

Czechoslovakian

-Ava II

CD 38/39

(-C)

CD 37/8/9

(-GACCCAG) Turkish

-Ava II

CD 39

C→T

+Mae I

Mediterranean

CD 43

G→T

Chinese

-Hinf I

CD 47

(+A)

Surinamese

-Xho I

CD 61

A→T

African

-Hph I

CD 74/75

(-C)

Turkish

-Hae III

CD 121

G→T

Polish, French, Japanese -Eco R I

IVSII-1

G→A

Mediterranean

-Hph I

IVSII-4,5

(-AG)

Portuguese

-Hph I

IVSII-745

C→G

Mediterranean

+Rsa I

of these methods is DGGE, which allows the separation of DNA fragments differing by a single base change according to their melting properties.150 The technique involves the electrophoresis of double-stranded DNA fragments through a linearly increasing denaturing gradient until the lowest melting temperature domain of the fragment denatures, creating a branched molecule that effectively becomes stationary in the gel matrix. DNA fragments differing by 1 bp in the low melting temperature domain have different melting temperatures and can be separated in most but not all cases. The ␤-globin gene is amplified in segments using five–seven pairs of primers, one of each pair having a GC-rich sequence added to it to create a high melting domain. Heterozygous DNA creates four bands, two heteroduplexes of normal and mutant sequence and two homoduplexes, one of normal sequence and the other of mutant sequence. DGGE has been used for prenatal diagnosis of ␤ thalassemia in India151 and also for the analysis of point mutations resulting in ␦ thalassemia and nondeletion HPFH.152,153 Another approach by heteroduplex analysis is using nondenaturing gel electrophoresis. Unique heteroduplex patterns can be generated for each mutation by annealing an amplified target DNA fragment with an amplified heteroduplex generator molecule, a synthetic oligonucleotide of approximately 130 bases in length containing deliberate sequence changes or identifiers at known mutation positions.154 Other methods such as mismatch cleavage, single-strand conformation polymorphism, and protein truncation test have been used to detect unknown mutations but they have not been applied specifically to the diagnosis of hemoglobinopathies or thalassemias. These techniques simply pinpoint the presence of a mutation or DNA polymorphism in the amplified target sequence. Sequencing of the amplified product can then be performed manually or automatically to identify the localized mutation. This can now be done very efficiently with an automated DNA sequencing machine and fluorescence detection technology. Although it is clear that many novel detection methods have been developed and used, many large laboratories prefer direct DNA sequencing of amplified product as the primary method of identifying rare or unknown ␤ thalassemia mutations. Once a rare or novel mutation has been identified through DNA sequencing, the DNA sample can be used as a control for the development of ARMS primers to provide a more rapid and cheaper screening of further cases.155

␤-Globin Gene Haplotype Analysis At least 18 restriction fragment length polymorphisms (RFLPs) have been characterized within the ␤-globin gene cluster.156 Conveniently, most of these RFLP sites are nonrandomly associated with each other and they combine to

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders produce just a handful of haplotypes157 (Chapter 27). In particular, they form a 5
cluster from the ε-globin gene to the ␦-globin gene and a 3
cluster around the ␤-globin gene, with a relative hot spot for meiotic recombination in between.158 Each ␤ thalassemia mutation is strongly associated with just one or two common haplotypes, and haplotype analysis has been used extensively to study the origin of globin gene mutations found in different ethnic groups159 (Chapter 27). The ␤-globin gene cluster haplotype normally consists of five RFLPs located in the 5
cluster (Hind II/ε-gene; Hind III/G ␥ -gene; Hind III/A ␥ -gene; Hind II/3
␺ ␤; and Hind II/5
␺ ␤) and two RFLPs in the 3
cluster (Ava II/␤-gene; BamH I/␤-gene). All the RFLPs can be easily analyzed by RE-PCR except the BamH I polymorphism, for which a Hinf I RFLP located just 3
to the ␤-globin gene is used instead, because these two RFLPs have been found to exist in linkage disequilibrium.160 The primer sequences together with the sizes of the fragments generated are listed in Table 28.6. Primers for three other useful RFLPs are also listed: an Ava II RFLP in the ␺ ␤-gene; a Rsa I RFLP located just 5
to the ␤-globin gene; and the G ␥ -Xmn I RFLP C→T polymorphism at position −158 5
to the G ␥ -globin gene that acts as a nondeletion HPFH allele under conditions of erythropoietic stress, raising the HbF level in patients with ␤ thalassemia or sickle cell anemia (Chapters 27 and 30).

DNA DIAGNOSIS OF ␦␤ THALASSEMIA, Hb LEPORE, AND HPFH The ␦␤ thalassemia, Hb Lepore, and HPFH deletion mutations were characterized originally by restriction endonuclease mapping and Southern blotting, but gap-PCR is now used for the diagnosis of the common mutations (Table 28.1). This technique is useful for the diagnosis of Hb Lepore,86 six ␦␤-thalassemia alleles, and three HPFH deletion mutations161 and has also been used for the diagnosis of Hb Kenya. It provides a useful and simple screening method for distinguishing HPFH from ␦␤ thalassemia in Asian Indian, African and Mediterranean individuals. Those testing negative and carrying novel or rare ␦␤ thalassemia, ε␥ ␦␤ thalassemia, and HPFH deletion mutations can now be examined by MLPA analysis.102 Fifty probes have been developed to cover a region of 500 kb of the ␤-globin gene cluster, including the locus control region. This enables all large deletions to be identified, including the ε␥ ␦␤ thalassemias, that leave the ␤-globin genes intact and are not easily detected by conventional techniques.

DNA DIAGNOSIS OF ABNORMAL HEMOGLOBINS More than 1,000 hemoglobin variants have been described to date, although only a few are clinically important and require routine diagnosis by DNA-based methods. These variants are HbS, HbC, HbE, HbD-Punjab, and HbO-Arab.

679

The mutations for these five abnormal hemoglobins can be diagnosed by a variety of methods.

Sickle Hemoglobin The HbS mutation destroys the recognition site for three restriction enzymes, MnI I, Dde I, and Mst II. Mst II was used for detection of the ␤S allele by Southern blot analysis because it cuts infrequently around the ␤-globin gene producing large DNA fragments. For PCR, the enzyme Dde I is the enzyme of choice (Fig. 28.9). The primer sequences are listed in Table 28.7. The ␤S mutation can also be detected by a variety of other PCR-based techniques such as ASO/dot blotting or ARMS. Prenatal diagnosis of sickle cell disease should always be done using two methods, ARMS-PCR and Dde I PCR, with careful attention being paid to the intensity of the stained bands on electrophoresis. The ratio of intensity between the mutant band and the control or normal bands must be identical to those of all the control samples. Fainter bands indicate possible maternal DNA contamination; stronger bands may indicate PCR product contamination.

Hemoglobin C The HbC mutation does not abolish the recognition site for Dde I or Mst II because the mutation occurs at a nonspecific nucleotide in the enzyme recognition sequences. Thus another method such as ASO/dot blotting or the ARMS technique must be used. The primer sequences used for the ARMS method are included in Table 28.3.

Hemoglobin D-Punjab and Hemoglobin O-Arab The mutations giving rise to the abnormal variants HbD Punjab (HBB glu121gln) and HbO-Arab (HBB glu121lys) both abolish an EcoR I site at codon 121. Mutation detection is conducted very simply by RE-PCR using EcoR I. The primer sequences used for this approach are listed in Table 28.7. This assay does not distinguish the two variants, so this test must be combined with hemoglobin HPLC. HbD-Punjab can be detected by ARMS (Table 28.3).

Hemoglobin E The HbE mutation abolishes a Mnl I site and may be diagnosed by amplification of exon 1 and restriction enzyme analysis. The primer sequences used for this approach are listed in Table 28.7. The HbE mutation may also be diagnosed easily using ASO probes or ARMS primers (Tables 28.3 and 28.4).

Other Variants A definitive identification of the other abnormal variants requires either DNA sequencing or analysis of the amino

680

Mary Fabry and John M. Old

Table 28.6. Primers used for the analysis of ␤-globin gene cluster RFLPs

RFLP and Primer Sequences 5
–3


Product size (bp)

Coordinates on GenBank sequence U01317

Absence of site (bp)

Presence of site (bp)

Annealing temperature (◦ C) 55◦

Hin d II/ε TCTCTGTTTGATGACAAATTC AGTCATTGGTCAAGGCTGACC

760

18652–18672 19391–19411

760

315 445 55◦

Xmn I /G␥ AACTGTTGCTTTATAGGATTTT AGGAGCTTATTGATAACCTCAGAC

657

33862–33883 34495–34518

657

455 202

Hin d III /G␥ AGTGCTGCAAGAAGAACAACTACC CTCTGCATCATGGGCAGTGAGCTC

326

35677–35700 35981–36004

326

235 91

65◦

65◦

Hin d III /A␥ ATGCTGCTAATGCTTCATTAC TCATGTGTGATCTCTCAGCAG

635

Hin d II /5
␺ ␤ TCCTATCCATTACTGTTCCTTGAA ATTGTCTTATTCTAGAGACGATTT Ava II /␺ ␤ Sequence as for Hind 5
␺ ␤ RFLP Hin d II /3
␺ ␤ GTACTCATACTTTAAGTCCTAACT TAAGCAAGATTATTTCTGGTCTCT Rsa I /␤ AGACATAATTTATTAGCATGCATG CCCCTTCCTATGACATGAACTTAA

40357–40377 40971–40991

635

327 308 55◦

795

46686–46709 47457–47480

795

691 104

795

46686–46709 47457–47480

795

440 355

55◦

55◦ 913

49559–49582 50448–50471

913

479 434 55◦

1200

61504–61527 62680–62703

411 plus 694 & 95

330 & 81 plus 694 & 95 65◦

Ava II /␤ GTGGTCTACCCTTGGACCCAGAGG TTCGTCTGTTTCCCATTCTAAACT

328

62416–62439 62720–62743

328

228 100

Hin f I /␤ GGAGGTTAAAGTTTTGCTATGCTGTAT GGGCCTATGATAGGGTAAT

474

63974–64001 64429–64447

320 plus 154

213 & 107 plus 154

55◦

Table 28.7. Oligonucleotide primers for the detection of ␤S , ␤E , ␤D Punjab, and ␤0 Arab mutations as RFLPs Mutation and affected RE site

Primer sequences 5
–3


Annealing temperature (◦ C)

␤S CD6 (A→T) (Loses Dde I site)

ACCTCACCCTGTGGAGCCAC GAGTGGACAGATCCCCAAAGGACTCAAGGA

␤E CD26 (G→A) (Loses Mnl I site)

Product size, bp

Absence of site, bp

Presence of site, bp

65 65

443

386/67

201/175/67

ACCTCACCCTGTGGAGCCAC GAGTGGACAGATCCCCAAAGGACTCAAGGA

65 65

443

231/89/56/35/33

171/89/60/35/33

␤D Punjab CD121 (G→C) (Loses Eco R I site)

CAATGTATCATGCCTCTTTGCACC GAGTCAAGGCTGAGAGATGCAGGA

65 65

861

861

552/309

␤0 Arab CD121 (G→A) (Loses Eco R I site)

CAATGTATCATGCCTCTTTGCACC GAGTCAAGGCTGAGAGATGCAGGA

65 65

861

861

552/309

Laboratory Methods for Diagnosis and Evaluation of Hemoglobin Disorders

681

Figure 28.9. The diagnosis of the HbS gene mutation by Dde I digestion of amplified DNA. The gel shows Dde I–digested fragments of: HbAS individuals (tracks 2, 3, and 5); normal HbA individual (track 6); and sickle cell anemia (track 4). Track 1 contains ␾X174 – Hae III DNA markers. The primers used are listed in Table 28.7. The Dde I site 5
to the one at codon 6 (marked by the dotted arrow) is a rare polymorphic site caused by the sequence change G→A at position −83 to the ␤-globin gene. When present the fragment of 175 bp is cleaved to give 153-bp and 27-bp fragments as shown in track 2.

acid change by mass spectrometry, although a few have had other quick diagnostic tests developed, such as the ARMS technique for the ␣-chain variant HbQ-India162 and HbDIran (Table 28.3). DNA sequence analysis can be performed in the same manner as that described for the identification of ␣-chain and ␣ thalassemia point mutations, or more simply through the use of just one sequencing reaction, by sequencing cDNA produced by the reverse transcription of globin mRNA.84 For the future, the ultimate goal of the identification of all known and unknown hemoglobin variants, by a DNA chip containing every possible codon mutation is within technological reach, but again, this attractive “one stop” method of total variant identification might never find a market as most variants have no clinical significance and identification is often of academic interest only.

ANTENATAL DIAGNOSIS An antenatal screening program for detection of at risk couples should be able to identify by hematological and DNA testing the majority of carrier states for ␣ thalassemia, ␤ thalassemia, ␦␤ thalassemia, and common variant hemoglobins such as HbS, HbC, HbD-Punjab,

HbO-Arab, and Hb Lepore.163 Strategies for antenatal screening depend on the programs’ context. In multiethnic countries such as the United Kingdom or United States, two strategies are applied; one based on universal screening in high prevalence areas for thalassemia or a hemoglobinopathy and one based on ethnic origin of the woman and her partner in low prevalence areas. For example, in the United States, neonatal screening for the HbS gene is universal. The screening algorithms and guidelines for referral of patients for DNA analysis in the United Kingdom can be found on the NHS Sickle Cell and Thalassaemia Screening Programme web site (http://www.kclphs.org.uk/haemscreening). PCR-based techniques provide a quick and relatively simple method for the prenatal diagnosis of homozygous ␣0 thalassemia, ␤ thalassemia, and sickle cell disease. The techniques have proven to be reliable and accurate as long as careful attention is given to all potential diagnostic pitfalls and best-practice guidelines are followed.164,165 These include using two different mutation detection methods whenever possible and excluding maternal DNA contamination by the analysis of VNTR or STR polymorphisms. Kits for profiling 12 or more STR markers simultaneously by

682 using an ABI or Beckman DNA sequencer are now available and recommended for checking maternal contamination of fetal DNA.84

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686 134. Linderman R, Hu SP, Volpato F, Trent RJ. (1991) Polymerase chain reaction (PCR) mutagenesis enabling rapid non-radioactive detection of common ␤ thalassaemia mutations in Mediterraneans. Br J Haematol. 1991;78:100–104. 135. Webster MT, Wells RS, Clegg JB. Analysis of variation in the human beta-globin gene cluster using a novel DHPLC technique. Mutat Res. 2002;501:99–103. 136. Yip SP, Pun SF, Leung KH, Lee SY. Rapid, simultaneous genotyping of five common Southeast Asian beta-thalassemia mutations by multiplex minisequencing and denaturing HPLC. Clin Chem. 2003;49:1656–1659. 137. Su YN, Lee CN, Hung CC, et al. Rapid detection of beta-globin gene (HBB) mutations coupling heteroduplex and primerextension analysis by DHPLC. Hum Mutat. 2003;22:326–336. 138. Wu G, Hua L, Zhu J, Mo QH, Xu XM. Rapid, accurate genotyping of beta-thalassaemia mutations using a novel multiplex primer extension/denaturing high-performance liquid chromatography assay. Br J Haematol. 2003;122:311–316. 139. Bournazos SN, Tserga A, Patrinos GP, Papadakis MN. A versatile denaturing HPLC approach for human beta-globin gene mutation screening. Am J Hematol. 2007;82:168–170. 140. Naja RP, Kaspar H, Shabakio H, Chakar N, Makhoul Nj, Zalloua PA. Accurate and rapid prenatal diagnosis of the most frequent East Mediterranean beta-thalassemia mutations. Am J Hematol. 2004;75:220–224. 141. Pang L, Li J, Jiang J, Shen G, Yu R. DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach. Anal Biochem. 2006;358: 99–103. 142. Craig JE, Kelly SJ, Barnetson R, Thein SL. Molecular characterisation of a novel 10.3 kb deletion causing ␤-thalassaemia with unusually high Hb A2. Br J Haematol. 1992;82:735–744. 143. Dimovski AJ, Efremove DG, Jankovic L, Plaseska D, Juricic D, Efremov GD. A ␤0 thalassaemia due to a 1605 bp deletion of the 5
␤-globin gene region. Br J Haematol. 1993;85:143–147. 144. Faa V, Rosatelli MC, Sardu R, Meloni A, Toffoli C, Cao A. A simple electrophoretic procedure for fetal diagnosis of ␤-thalassaemia due to short deletions. Prenatal Diag. 1992;12:903–908. 145. Waye JS, Cai S-P, Eng B, et al. High hemoglobin A2 ␤0 thalassaemia due to a 532 bp deletion of the 5
␤-globin gene region. Blood. 1991;77:1100–1103. 146. Waye JS, Eng B, Hunt JA, et al. Filipino ␤-thalassaemia due to a large deletion: identification of the deletion endpoints and polymerase chain reaction (PCR)-based diagnosis. Hum Genet. 1994;94:530–532. 147. Old JM, Petrou M, Modell B, Weatherall Prenatal Diag. D. J. Feasibility of antenatal diagnosis of ␤-thalassaemia by DNA polymorphisms in Asian Indians and Cypriot populations. Br J Haematol. 1984;57:255–263. 148. Thein SL, Hesketh C, Brown KM, Anstey AV, Weatherall DJ. Molecular characterisation of a high A2 ␤ thalassemia by direct sequencing of single strand enriched amplified genomic DNA. Blood. 1989;73:924–930. 149. Lynch JR, Brown JM, Best S, Jennings MW, Weatherall DJ. Characterisation of the breakpoint of a 3.5 kb deletion of the ␤-globin gene. Genomics. 1991;10:509–511. 150. Losekoot M, Fodde R, Harteveld CL, Van Heeren H, Giordano PC, Bernini LF. Denaturing gradient gel electrophoresis and

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direct sequencing of PCR amplified genomic DNA: a rapid and reliable diagnostic approach to beta thalassaemia. Br J Haematol. 1991;76:269–274. Gorakshaker AC, Lulla CP, Nadkarni AH, et al. Prenatal diagnosis of ␤-thalassemia using denaturing gradient gel electrophoresis among Indians. Hemoglobin. 1997;21:421–435. Gottardi E, Losekoot M, Fodde R, Saglio G, Camaschella C, Bernini LF. Rapid identification of denaturing gradient gel electrophoresis of mutations in the ␥ -globin gene promoters in non-deletion type HPFH. Br J Haematol. 1992;80:533–538. Papadakis M, Papapanagiotou E, Loutradi-Anagnostou A. Scanning methods to identify the molecular heterogeneity of ␦-globin gene especially in ␦-thalassemias: detection of three novel substitutions in the promoter region of the gene. Hum Mutat. 1997;9:465–472. Savage DA, Wood NAP, Bidwell JL, Fitches A, Old JM, Hui KM. Detection of ␤-thalassaemia mutations using DNA heteroduplex generator molecules. Br J Haematol. 1995;90:564– 571. Old JM, Khan SH, Verma I, et al. A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction. Hemoglobin. 2001;25:397–407. Kazazian HH Jr, Boehm CD. Molecular basis and prenatal diagnosis of ␤-thalassemia. Blood. 1988;72:1107–1116. Antonarakis SE, Boehm CD, Diardina PJV, Kazazian HH Jr. Non-random association of polymorphic restriction sites in the ␤-globin gene cluster. Proc Natl Acad Sci USA. 1982;79: 137–141. Chakravarti A, Buetow KH, Antonarakis SE, Waber PG, Boehm CD, Kazazian HH. Non-uniform recombination within the human ␤-globin gene cluster. Am J Hum Genet. 1984;36:1239–1258. Varawalla NY, Fitches AC, Old JM. Analysis of beta-globin gene haplotypes in Asian Indians: origin and spread of beta-thalassaemia on the Indian subcontinent. Hum Genet. 1992;90:443–449. Semenza GL, Dowling CE, Kazazian HH Jr. Hinf I polymorphisms 3
to the human ␤ globin gene detected by the polymerase chain reaction (PCR). Nucl Acids Res. 1989;17:2376. Craig JE, Barnetson RA, Prior J, Raven JL, Thein SL. Rapid detection of deletions causing ␦ ␤ thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification. Blood. 1994;83:1673–1682. Abraham R, Thomas M, Britt R, Fisher C, Old J. Hb Q-India; an uncommon variant diagnosed in three Punjabi patients with diabetes is identified by a novel DNA analysis test. J Clin Pathol. 2003;56:296–299. Old JM. Screening and genetic diagnosis of haemoglobinopathies. Scand J Clin Lab Invest. 2006;66:1–16. The Globin Gene Disorder Working Party of the BCSH General Haematology Task Force. Guidelines for the fetal diagnosis of globin gene disorders. J Clin Pathol. 1994;47:199–204. Old JM. Best practice recommendations. In: Prevention of Thalassaemias and Other Haemoglobin Disorders. Vol 2. Nicosia, Cyprus: Thalassaemia International Federation; 2005:1–16.

SECTION EIGHT

NEW APPROACHES TO THE TREATMENT OF HEMOGLOBINOPATHIES AND THALASSEMIA Martin H. Steinberg

The treatment of hemoglobin disorders is evolving and clinical trials of many new agents are underway. Hydroxyurea is used to increase fetal hemoglobin (HbF) levels, stem cell transplantation has the potential for cure, and a larger repertory of iron chelators might make long-term transfusion more feasible. In this section of five chapters, three cover clinically available treatments, discussing in detail aspects of HbF induction, blood transfusion with iron chelation, and stem cell transplantation. One chapter focuses on innovative treatment approaches that remain, at the time of writing, investigative. Treatments include antioxidants, statins, antiinflammatory agents, transport channel inhibitors, antiadhesive agents, and therapeutic methods of increasing nitric oxide bioavailability. The first patients have been treated in a gene therapy trial in which lentiviral vectors containing therapeutic ␤-like globin genes are used to counter the results of the sickle or ␤ thalassemia mutation, and a final chapter brings this field up to date. Transfusions are not innocuous and are complicated by alloimmunization, the transmission of unsuspected viral diseases, and iron overload. Controlled, randomized trials of the utility of transfusions for specific complications are sparse. When transfusion is contemplated, expert opinion, with its pitfalls, is relied on in most instances. Usually, it is unclear if simple transfusion or exchange transfusion yields superior results for sickle cell disease complications such as stroke in children or the acute chest syndrome. Strong personal feelings among clinicians regarding the method of transfusion make the chance of definitive clinical trials dim. Since our last edition, efficacious new oral iron chelators have been approved in many countries. Deferiprone might have special benefit for the iron-overloaded heart, but it is not approved for use in the United States. Deferasirox, an oral agent, has efficacy similar to desferrioxamine, the standard for chelating agents. If long-term experience shows that the toxicities of deferiprone and deferasirox are manageable, they will be major adjuncts in dealing with transfusion-induced iron overload.

Stem cell transplantation has been used successfully in sickle cell anemia and ␤ thalassemia. Pioneers in the development of transplantation for sickle cell disease and ␤ thalassemia discuss criteria for patient selection, conditioning regimens, complications and their management, and the most recent results of transplantation. Transplantation is a dynamic field and its future includes the use of cord blood stem cells, inducing stable mixed chimerism, and new possibilities for immunosuppression. Stem cell transplantation, if successful, provides the sole cure for ␤ thalassemia and sickle cell disease, but with the risk, albeit small, of transplant-related death. The efficacy of hydroxyurea has been proven in a randomized controlled trial in which its use was associated with a nearly 50% reduction in the incidence of pain crisis, acute chest syndrome, frequency of hospitalization, and the use of blood transfusion. Moreover, during a 10-year followup period that was no longer randomized or controlled, mortality appeared to be reduced by 40% and toxicity was minor. This cytotoxic agent could have other therapeutic mechanisms in addition to inducing HbF with effects on erythrocytes and vasculature. Studies of hydroxyurea in very young children, in whom therapeutic benefits should be greatest, are nearing completion and the results awaited eagerly. In children, the HbF response is greater than seen in adults with levels near 20% achieved after 6 months– 1 year of treatment and with minimal short-term toxicity. Early interruption of the vasoocclusive and hemolytic processes in young patients might also prevent damage to the central nervous system, lungs, kidneys, and bones that end in neurocognitive damage, pulmonary hypertension, disability, and premature death. We do not yet know the long-term negative effects of hydroxyurea – is it mutagenic, carcinogenic, or leukemogenic? Whether or not the incidence of neoplasia is increased is not known, but in seriously ill patients, small risks are likely to be dominated by the benefits of treatment. In the very young, in whom this agent is likely to be used for decades one must consider longer-term toxicities. In HbSC disease, hydroxyurea appeared to reduce hemolysis but a clinical a trial of this agent in HbSC has not been done. Studies of butyrate given by short-term pulses have shown remarkable increases in HbF that could be synergistic with those of hydroxyurea. Butyrate was the first agent whose effects could be mediated by epigenetic mechanisms, and work on orally available agents of this general class of drug is continuing. Finally, decitabine, a less toxic analog of 5-azacytidine, the drug that began the effort to induce HbF synthesis, has shown promising results in sickle cell anemia, and development of an oral form is progressing. The principles of gene therapy approaches to sickle cell disease and ␤ thalassemia are discussed along with the current problems of this approach, foremost among them, how to achieve safe, stable and robust transgene expression. 687

688 Until cures of disease by stem cell transplantation or gene therapy become feasible and widely applicable, drug treatment will remain in the forefront. A single agent that will prevent or reverse the disease pathophysiology seems an unlikely possibility, except for the still unachievable goal of an agent that will increase HbF to sufficient concentrations in each sickle erythrocyte to totally prevent HbS polymerization and its consequences. Any other class of

Martin H. Steinberg drug used alone is unlikely to be sufficient. Trials of combined treatments should be undertaken and include one or more HbF-inducing agent and drugs that target facets of pathophysiology other than HbS polymerization. One can imagine clinical trials that combine agents that increase nitric oxide bioavailability, reduce intercellular interactions, reduce hemolytic anemia, and are antiinflammatory used along with HbF-inducing drugs.

active lives. Their satisfaction with the benefits of hypertransfusion therapy was tempered by a growing appreciation of the problem of transfusional iron overload and the need for iron chelation therapy.

29 Hemoglobin Level and Physiological Parameters

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease Janet L. Kwiatkowski and John B. Porter

TRANSFUSION Thalassemia Major (Cooley Anemia) Eighty years ago, Dr. Thomas Cooley and his colleagues at the Children’s Hospital of Michigan administered blood transfusions to children with a newly recognized clinical entity whose features included severe anemia, splenomegaly, and peculiar facies.1 Five years later, Cooley’s name had become indelibly associated with the disease, and transfusions were administered along with ferrous carbonate, ultraviolet rays, and extract of pituitary gland.2 Not surprisingly, given the state of crossmatching at the time, the response to transfusion was poor. Another 30 years elapsed before improvements in blood banking and recognition of the benefits of a higher hemoglobin level came together to initiate the era of modern transfusion therapy for thalassemia major.

Transfusion Programs In 1963, the results of an evaluation of 35 children with thalassemia major, aged 12 years or younger, whose pretransfusion hemoglobin levels fell into three categories – 4.0– 5.9 g/dL, 6.0–7.9 g/dL, or 8.0–9.9 g/dL – were described.3 Children in the highest hemoglobin group had better linear growth, less enlargement of the liver and spleen, less facial and skull bony abnormalities, fewer fractures, and less cardiomegaly than children in the two lowest hemoglobin groups. Two patients who received regular red cell transfusions to maintain their hemoglobin level above 10 g/dL at all times were also reported.4 More than 40 years later, this regimen – hypertransfusion – remains the standard of care for the treatment of thalassemia major. General guidelines for hypertransfusion therapy are presented in Tables 29.1 and 2. By 1968, several reports confirmed the benefits of hypertransfusion5–7 and that patients were leading normal,

The target pretransfusion hemoglobin level varies among thalassemia centers, but usually falls between 9.0 and 10.5 g/dL. The goal is to select a hemoglobin level that achieves the important physiological benefits while minimizing the rate of iron accumulation. In regard to the first issue, no significant difference in appearance, cardiac size, occurrence of splenomegaly, or feeling of well-being has been identified with higher or lower hemoglobin levels in a hypertransfusion regimen. Because some of the complications of thalassemia might be related to erythroid hyperplasia in the bone marrow, the level of bone marrow suppression achieved by different target hemoglobin levels could have clinical significance. Both ferrokinetic studies and measurements of soluble serum transferrin receptor levels have demonstrated an inverse relationship between mean pretransfusion hemoglobin level and marrow erythroid activity.8–10 When anemia is severe – hemoglobin levels 4–7 g/dL – the marrow erythroid activity increases to 10 times normal. One important clinical consequence is impaired bone metabolism with osteoporosis;11 however, when the selected mean pretransfusion hemoglobin level is 9 g/dL rather than 10.5 g/dL, erythroid activity increases only threefold,9 and this level of erythroid expansion is associated with little or no impairment of bone metabolism.11 Thus, no significant physiological difference between target pretransfusion hemoglobin levels of 9 and 10.5 g/dL is known at this time.

Hemoglobin Level and Blood Requirements The relationship between target hemoglobin level, blood requirements and rate of iron accumulation in patients with thalassemia major is controversial. In 166 splenectomized and nonsplenectomized patients, transfusion requirements remained constant at mean transfusion hemoglobin levels of 10–14 g/dL (equivalent to pretransfusion hemoglobin levels of ∼8–12 g/dL).12 A subsequent expansion of this study to include 392 patients confirmed the earlier findings and concluded that the maintenance of higher hemoglobin levels in transfusion programs for thalassemia major did not necessitate a higher blood requirement.13 Additional supportive evidence came from a study of “supertransfusion,” in which maintenance of pretransfusion hematocrits of 27% and 35% required similar amounts of blood.14 Nevertheless, several studies found that maintenance of higher hemoglobin levels requires more blood. Transfusion requirements in 14 patients were directly proportional to the mean hemoglobin levels and nearly doubled 689

690

Janet L. Kwiatkowski and John B. Porter

Table 29.1. General guidelines for transfusion. Therapy for thalassemia major Variable

Range

Pretransfusion Hb level Blood product Amount of blood Interval between transfusions

9–10.5 g/dL Leukoctye-depleted red cells 1–3 units 2–4 wk

between 9.6 and 13.4 g/dL.15 Among 3,468 patients, transfusion requirements in 1,985 were proportional to mean hemoglobin level in both splenectomized and nonsplenectomized individuals.16 Only one large study is reported in which the annual transfusion requirement has been measured repeatedly in the same patients under two different transfusion regimens.17 From 1982 to 1986, when the mean pretransfusion hemoglobin level of 32 patients was 11.3 g/dL, the mean annual transfusion requirement was 137 mL/kg17 (Table 29.3). Following modification of the transfusion regimen in 1987–1992, the mean transfusion hemoglobin level fell to 9.4 g/dL, and the mean blood requirement decreased to 104 mL/kg. Of note, the change in target hemoglobin level from 11.3 to 9.4 g/dL was associated with a reduction in serum ferritin levels from a mean level of 2,448 to 1,187 ␮g/L. The improvement in overall iron status at the lower hemoglobin level might have been a combined effect of decreased transfusional iron loading, enhanced desferrioxamine-induced iron excretion, and maintenance of an acceptable level of gastrointestinal iron absorption.

Beginning Transfusion Therapy The decision to begin chronic transfusion therapy in an infant or young child with homozygous ␤ thalassemia is based on a combination of laboratory and clinical data. The specific molecular defect may provide general, albeit imperfect, prognostic clues, and the course of an affected sibling might also be helpful. Steady-state hemoglobin levels that remain below 7 g/dL on at least two measurements are one indication for transfusion, particularly in children, but this is not the only consideration. Clinical factors and other laboratory findings in addition to the hemoglobin level will determine whether a chronic hemolytic anemia or

Table 29.2. Estimation of blood requirements according to hematocrit of donor unit Hematocrit of packed red cell units (%)

Amount of packed red cells to raise Hb by 1 g/dL (mL/kg)

60 65 70 75

3.7 3.5 3.2 3.0

Table 29.3. Effect of target hemoglobin level on transfusion requirements in thalassemia

Years

Mean pretransfusion Hb level (g/dL)

Mean transfusion requirement (mL/kg/y)

Median serum ferritin (␮g/L)

1981–1986 1987–1992

11.31 ± 0.5 9.4 ± 0.4

137 ± 26 104 ± 23

2,280 1,004

From ref. 17.

a chronic transfusion program better serves the interests of the patient. For example, the development of Cooley facies, growth retardation, pathological fractures, or persistent fatigue is a strong indication for transfusion, even when the hemoglobin level is 8 g/dL. On the other hand, for a patient with none of these problems despite a lower hemoglobin level, the marginal benefits of transfusions might be outweighed by the associated risks. Clinicians should regularly assess growth, and in conjunction with the family, the overall well-being of the child. In addition, the nucleated red cell count and/or other laboratory indicators of the degree of erythropoiesis such as soluble transferrin receptor levels should be monitored because the consequences of excessive bone marrow expansion could be as important as those of anemia in prompting regular transfusions. Later initiation of transfusion therapy might delay the need for iron chelation therapy until an age at which side effects are less common. Earlier initiation of transfusion therapy might prevent irreversible facial changes and lower the risk of alloimmunization. When the indications for transfusion therapy are equivocal but a decision is made to proceed, the treatment should be reevaluated if the patient later undergoes splenectomy. Sometimes, removal of the spleen makes the difference between transfusion dependency and nondependency.

Choice of Blood Product Patients with thalassemia should receive leukocytereduced red blood cells to decrease or eliminate febrile reactions. The reduction of white cells to less than 5 × 106 per unit of red cells could carry the additional benefit of delaying sensitization to human leukocyte antigens in patients who might later be candidates for bone marrow transplantation. Filtration of the blood product, before storage, or at the time of administration, is the method most commonly used for leukocyte reduction. Prestorage leukocyte reduction is usually more efficient than bedside filtration.18 Prestorage leukocyte reduction might also decrease cytokine accumulation in the blood during storage, further diminishing the chance of a febrile reaction. One study has suggested that prestorage leukocyte reduction has the additional advantage of reducing the proliferation of Yersinia enterocolitica in blood products

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease inoculated with the organism.19 Because infection with Y. enterocolitica can be a serious event in patients with iron overload on chelation therapy, decreasing the likelihood of its transmission from asymptomatic donors might reduce the risk of one of the more serious infectious complications of thalassemia. Whether leukocyte reduction removes prions from potentially infected units remains under investigation.20 Even after 42 days of storage, red cell recovery 24 hours after transfusion is 75% or greater.21 Little information is available, however, regarding the survival of these red cells after this time. Earlier studies showed that the survival of red cells stored in acid-citrate-dextrose for 28 days was reduced in comparison with red cells stored for 14 days.22 Although this difference is not important for patients with acute or one-time indications for transfusions, it might have a marked effect on the overall blood requirements of patients receiving long-term transfusion. Until more is known about the effect of storage time on posttransfusion red cell survival, it seems prudent to use red cells less than 14–21 days old for patients with thalassemia major.

Frequency and Amount of Transfusion When using donor units with a hematocrit of 75%, transfusion of 10–15 mL of red cells per kilogram body weight at 3- or 4-week intervals usually maintains a minimum hemoglobin level of 9.0–10.5 g/dL. New additive solutions have reduced the hematocrit of packed red cells from 75%–80% to 55%–65%, and larger volumes of the blood product are therefore necessary to achieve the same target hemoglobin level. This can pose problems for small children or for older patients with heart disease, and in these instances, hemoconcentration of the donor units can be used. Although intervals between transfusions of 3–4 weeks accommodate the school or work schedule of most patients with thalassemia major, shorter intervals would more closely mimic the physiological situation by reducing fluctuations in hemoglobin level. Moreover, mathematical modeling suggests that maintenance of a pretransfusion hemoglobin level of 9 g/dL with transfusions every 2 weeks rather than every 4 weeks would reduce overall blood requirements by 20%.23 In a large group of splenectomized patients, however, a shorter interval of 2 weeks between transfusions had no measurable effect on transfusion requirements in comparison with an interval of 3 or 4 weeks.24 Other conditions might increase the frequency of transfusion or the amount of blood needed to maintain the target hemoglobin level. For example, transfusion requirements rise during pregnancy in women with ␤ thalassemia major.25,26 Patients undergoing treatment of hepatitis C infection with ribavirin need additional blood due to druginduced hemolysis; transfusion requirements rise by a median of approximately 40% (range 25%–136%), compared with pretreatment transfusion requirements.27,28

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Transfusion requirements also appear to show seasonal variation. In patients from four thalassemia centers, pretransfusion hemoglobin levels were significantly lower in the summer months in all centers except the one where monthly temperatures varied the least.29 Possible explanations include hemodilution from expanded plasma volume in the patients and lower hemoglobin levels in donor blood in the summer months.

Young Red Cells Several groups have evaluated the effect of young red cell (neocyte) transfusions on overall blood requirements, and therefore on the rate of iron loading. The theoretical benefit of this modified blood product lies in the assumption that although all red cells contribute the same amount of iron upon senescence, younger red cells from the donor will circulate longer in the recipient before their iron is released. Thus, enhancement of the blood product for younger donor red cells should reduce the transfusion requirements and the rate of iron loading. Studies in animals have confirmed that age-dependent separation of red cells by density gradient centrifugation has a marked effect on red cell survival.30 Both simple centrifugation of single donor units and neocyte enhancement during continuous centrifugation apheresis of the donor have yielded red cells with younger estimated mean ages, and in labeled erythrocyte survival studies, longer half-lives than conventional units.10,31–34 Several factors have impeded the clinical application of young red cell transfusions. In particular, preparation of neocytes by continuous flow centrifugation is costly, and trials using neocytes prepared from single donor units have been disappointing. In three studies, the reduction in the rate of iron loading was only 13%– 20%, and both the preparation costs and donor exposures were significantly higher in transfusion regimens in which neocytes were used.35–38

Partial Exchange Transfusion Partial exchange transfusion, whether performed manually or by erythrocytapheresis, decreases the net blood requirement by combining the administration of donor red cells with the removal of red cells from the patient. The rationale for the use of partial exchange transfusion to prevent complications of sickle cell disease is straightforward. Donor HbA-containing cells directly replace endogenous HbS-containing cells, and because the goal of lowering the HbS level is largely independent of the total hemoglobin level, little or no net gain of red cells occurs. The goal of transfusion therapy for thalassemia major differs from that for sickle cell disease. The critical outcome in thalassemia is maintaining a particular hemoglobin level. Although the rationale for the use of partial exchange transfusion for this purpose is less obvious, some studies support this approach in thalassemia major.39 Seventeen patients

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Janet L. Kwiatkowski and John B. Porter Table 29.4. Comparison of simple transfusion and red cell exchange transfusion in thalassemia

Simple transfusion Red cell exchange transfusion

Mean pretransfusion Hb level (g/dL)

Mean posttransfusion Hb level (g/dL)

Mean transfusion interval (d)

Transfusion requirement level (mL/kg/d)

9.7 9.6

14.2 14.5

35.7 50.8

0.41 0.29

From ref. 39.

with thalassemia, and one each with sickle cell disease and Diamond–Blackfan anemia, underwent regular partial exchange transfusion for 6–7 months. Continuous flow centrifugation was used, and returned blood was enriched for younger red cells. In comparison with simple transfusion, the net rate of iron loading fell by 29% (Table 29.4).39 The mean interval between transfusions increased from 36 to 51 days. Serum ferritin levels decreased by 20% during partial exchange transfusion therapy in contrast with an increase of 12% during a comparable period of conventional transfusion therapy. The authors noted the disadvantages of a 40%–60% increase in exposure to donor units and a 1.5–2.0fold increase in transfusion-associated costs. Subsequently, the investigators reported the successful extension of this program without neocyte enrichment.40 A preliminary report describing a prospective clinical trial that utilized a crossover design to compare simple transfusion to partial exchange transfusion confirmed these findings. In 16 patients with thalassemia major, automated partial exchange transfusion decreased net red cell requirements, and therefore, transfusional iron loading, by a mean of 29% in comparison with simple transfusion, although with considerable interpatient variability in response.41 Overall, donor blood exposure increased 2.1– 3.7-fold. Age-dependent separation of pretransfusion blood samples by density gradient analysis showed an increase in younger red cells during partial exchange transfusion compared with simple transfusion. This suggests that the benefit of partial exchange transfusion in thalassemia might be derived primarily from the replacement of previously administered donor red cells with new donor red cells that have a younger mean age. By reducing the rate of new iron accumulation, this method of transfusion could enhance the overall management of transfusional iron overload in some patients with ␤ thalassemia.

Sickle Cell Disease Transfusions are used for both the acute management of complications of sickle cell disease and for chronic therapy to prevent the development or progression of complications. One goal of transfusion in sickle cell disease is to raise the hemoglobin level to improve oxygen carrying capacity. A second goal involves lowering the proportion of HbS-containing erythrocytes to reduce complications

related to vasoocclusion and hemolysis. In some situations, such as an acute anemic event related to parvovirus B19 infection (Chapter 19), the former goal predominates; in other situations, such as severe acute chest syndrome, the two goals transfusion therapy overlap. Still for other indications, such as preparation for surgery while receiving general anesthetic, the benefits of transfusion therapy cannot be attributed with certainty to one goal or the other.

Choice of Blood Product The same considerations of used in selecting a blood product in thalassemia also apply to sickle cell disease and leukocyte reduction is important. Selection of blood with a shorter storage time is as important for chronically transfused patients with sickle cell disease as it is for patients with thalassemia. Extended red cell antigen typing prior to the first transfusion is particularly important in sickle cell disease because, as discussed later, alloimmunization is common. The use of the original antigen profile to determine accurately the specificity of new antibodies and to eliminate the possibility of an autoantibody helps to guide the choice of an appropriate blood product for future transfusions. In chronically transfused patients, in whom the presence of donor red cells can interfere with serological testing, DNA typing of blood groups can aid in accurate determination of the patient’s true red cell antigen genotype.42 The choice of the best blood product to use for patients with sickle cell disease has been controversial.43 Generally, most investigators and clinicians agree that at a minimum, in addition to matching ABO and D, partial antigen matching for C, E, and Kell – the antigens most frequently associated with alloimmunization and delayed hemolytic transfusion reactions in sickle cell disease – should be used when selecting red cell units.43–45 When this transfusion approach was used in a multicenter primary stroke prevention trial, 8% of children receiving chronic transfusions developed a clinically significant alloantibody.46 The rate of new red cell alloantibodies or autoantibodies was only 0.5% per unit, compared with a rate of approximately 3% per unit in prior studies.46–49 Several investigators have advocated the use of blood matched for multiple red cell antigens for patients with sickle cell disease.49,50 This approach can be particularly useful when the racial composition of

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease the donor and recipient pool differ as they do in the United States and elsewhere where certain blood group antigens are common in donors but rare in recipients. A prospective study showed that the initiation of complete matching of donor blood for the Rh, Kell, Kidd, and Duffy systems with less stringent matching for other minor groups resulted in a 10-fold reduction in the rate of new alloantibody formation per unit.50 Because of the high cost associated with extended antigen matching, other investigators have proposed using this approach only after patients have developed one or two alloantibodies.51,52 Units donated by African Americans have an eightfold greater likelihood of antigen identity than units from predominantly Caucasian donor pools.53 Programs to increase blood donations by African Americans have been introduced to increase the availability of phenotypically matched blood products for chronically transfused patients with sickle cell disease.54,55 Such a program has been successful in providing racially matched (as well as C, E, and Kell antigen matched) red cell units for the majority of transfusions to children with sickle cell disease in a large urban sickle cell disease program.54 Another strategy to reduce alloimmunization is to recruit a limited number of dedicated donors to provide the majority of red cell units used for chronically transfused patients with sickle cell disease, thereby limiting donor exposure.55 The longterm effect of such programs on alloimmunization rates remains to be seen. Patients with sickle cell disease in chronic transfusion programs generally should not be given blood from donors with sickle cell trait. As discussed later, the target of chronic transfusion programs is a reduction in the percentage of endogenous HbS-containing cells to a particular level, and this is measured in the laboratory by the relative amounts of HbA (from the donor) and HbS (from the recipient). Although not harmful to the patient, the administration of sickle cell trait blood obscures the laboratory assessment of the proportion of the patient’s HbS-containing cells that remain in the circulation.

Transfusion Methods Simple Transfusion In the acute setting, red cells often are administered as a simple transfusion, although exchange transfusion also is used emergently for complications such as stroke and severe acute chest syndrome. When administering red cells to relieve anemia, the physician must first determine whether there is accompanying hypovolemia. If the blood volume is reduced, as with acute splenic sequestration, sufficient red cells to raise the hemoglobin concentration to the desired level can be given without concern about circulatory overload. In contrast, in a more slowly developing anemia such as an aplastic episode or with advancing renal disease, the patient’s blood volume is maintained,

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and the volume of administered red cells must be calculated to limit the risk of congestive heart failure. One simple approach is to use the hemoglobin level to determine the volume of the transfusion product. For example, if the hemoglobin level is 3 g/dL, the volume of packed red cells is 3 mL/kg of body weight. In most clinical situations, even a small rise in the hemoglobin level is sufficient to address the immediate problem. As noted, however, the use of newer additive solutions has reduced the hematocrit of donor red cell units, complicating the balance between raising the hemoglobin concentration and preventing volume overload; a diuretic might be helpful in this situation. A further consideration when utilizing simple transfusions in the acute setting is that the posttransfusion hemoglobin level should not exceed 10 g/dL because higher levels can lead to hyperviscosity.56,57 Simple transfusion also is frequently used in chronic transfusion programs. In general, the administration of 10– 15 mL/kg of red cells every 3 or 4 weeks will maintain the pretransfusion HbS level at less than 30%–50%. The target pretransfusion HbS level is variable, but generally a value of less than 30% is used for primary stroke prevention and for secondary stroke prevention at least for the first few years after the event.58,59 A switch from a target HbS of 30%–50% after 3 or 4 years of more aggressive transfusion for secondary stroke prevention is used in some centers (see later).60 A target HbS level of 50% often is used for the prevention of severe chronic pain and acute chest syndrome, although studies to determine the optimal HbS level to manage these complications are lacking. The main benefit of targeting a higher HbS level is the reduction in red cell transfusion requirements, which limits iron loading.60

Exchange Transfusion Exchange transfusion can be performed either manually or by an automated process, erythrocytapheresis. A single partial exchange transfusion is useful for the rapid reduction of HbS levels in the patient with sickle cell disease who has an acute complication such as stroke. Partial exchange transfusion can decrease the HbS level below 30% in less than 2 hours without volume overload or hyperviscosity that would accompany repeated simple transfusions for this purpose.61,62 In comparison with simple transfusion, manual or automated partial exchange transfusion decreases the net amount of blood needed to lower HbS levels by combining the administration of donor red cells with the removal of the patient’s red cells.60,63–65 This has proven to be a valuable tool in the management of iron overload in patients with sickle cell disease in long-term transfusion programs for prevention of stroke or other complications. New iron accumulation is stopped or greatly reduced. Limitations to exchange transfusion include greater donor exposure, higher cost, less widespread availability due to the need for special expertise, and the need for large intravenous access.63,64

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Janet L. Kwiatkowski and John B. Porter Table 29.5. Indications for transfusion in sickle cell disease

Episodic transfusion

Chronic transfusion

∗

Generally Accepted

Controversial

Stroke Aplastic episode Splenic sequestration Acute chest syndrome Acute multiorgan failure Retinal artery occlusion Prior to surgery with general anesthesia – moderate- to high-risk procedure∗ Stroke Abnormal transcranial Doppler ultrasound Recurrent acute chest syndrome Recurrent severe vasoocclusive pain Pulmonary hypertension

Priapism Prior to surgery with general anesthesia – low risk procedure∗

Recurrent priapism Pregnancy Silent cerebral infarcts∗ Recurrent splenic sequestration Leg ulcers

Currently under study.

Clinical Application of Transfusion Therapy in Sickle Cell Disease Specific complications of sickle cell disease and their treatment are discussed in Chapter 19. The following focuses on the data for and against transfusion therapy for selected disease complications. Particular attention is devoted to randomized, prospective studies of transfusion therapy, but few such studies are available. The specific clinical indications for transfusion therapy are divided into episodic and chronic therapy and evidence-based recommendations are made when possible (Table 29.5).

Indications for Episodic Transfusions Transient Aplastic Episode Patients with sickle cell disease can develop profound anemia in association with transient red cell aplasia caused by infection with parvovirus B19. Furthermore, cerebrovascular events, acute chest syndrome, splenic sequestration, and vasoocclusive pain episodes can occur in association with parvovirus B19 infection.66,67 Transfusion of red cells is indicated to relieve the severe anemia and to prevent or treat organ complications. When the anemia is milder and the need for a transfusion is uncertain, evidence of imminent bone marrow recovery such as nucleated red cells in the peripheral blood or the reappearance of reticulocytes might favor waiting. The HBB genotype of the patient might help predict the eventual need for transfusion: Individuals with sickle cell anemia and HbS–␤0 thalassemia generally maintain a lower baseline hemoglobin level and, thus, often develop more severe anemia with an aplastic episode. Nonetheless, patients with HbSC disease or HbS–S ␤+ thalassemia also can require transfusion, so knowledge of the genotype cannot substitute for close clinical and labora-

tory monitoring. For example, in a retrospective study of 84 patients with aplastic episodes, 72% received transfusion therapy, including 85% of those with sickle cell anemia, 57% with HbSC disease, and 14% with other genotypes.66 If red cells transfusion is necessary, the slow administration of one or two transfusions of small volume, raising the hemoglobin by 2–3 g/dL over 4–8 hours, is usually sufficient to improve the clinical condition until spontaneous recovery occurs.

Acute Splenic Sequestration Sudden, massive enlargement of the spleen with pooling of large amounts of blood within the organ can cause profound anemia and hypovolemia. Transfusion therapy carries two benefits: restoration of intravascular volume and improvement in oxygen carrying capacity. Acute splenic sequestration is one of the few complications of sickle cell disease in which rapid administration of red cells is not only safe but can also be lifesaving. Occasionally, transfusion with Rh- and ABO-specific, non-crossmatched blood might be necessary to prevent death from shock and severe anemia. Hypovolemia makes consideration of the volume of the transfused red cells less important; however, small amounts of blood are usually sufficient to reverse the process of sequestration, with subsequent release of trapped blood back into the circulation, which further improves the intravascular volume and the hemoglobin level.

Acute Stroke Red cell transfusion to lessen the anemia, reduce tissue hypoxia, and reduce the percentage of HbS is the mainstay of treatment for acute infarctive stroke, and red cells should be administered as quickly as possible to patients who develop this complication. Manual or automated exchange

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease transfusion, when available, often is used initially. The goal is to reduce the percentage of HbS to less than 30% of the total hemoglobin and to raise the hemoglobin level to approximately 10 g/dL. No prospective studies have been undertaken to compare simple with exchange transfusion. A multicenter, retrospective study found a fivefold higher incidence of stroke recurrence in children who were treated acutely with simple transfusion compared with those treated with exchange transfusion.68 Nonetheless, a simple transfusion to raise the hemoglobin level to no higher than 10 g/dL should be given if exchange transfusion cannot be performed within a few hours of presentation.

Acute Chest Syndrome The use of red cell transfusions in the management of acute chest syndrome can cause a rapid improvement in oxygenation.69 Both simple and exchange transfusion have been used for this indication. In one multicenter study, no significant difference between simple and exchange transfusion in improvement in oxygen levels in patients with acute chest syndrome was seen, although transfusion method was at the treating physician’s discretion and, thus, subject to bias.69 Given the lack of randomized, controlled trials comparing these two transfusion methods, management usually is based on the patient’s clinical condition and physician preference. Simple transfusions often are used as first-line treatment with the administration of 1–2 U to raise the hemoglobin level to no greater than 10 g/dL. In more severe cases, such as patients requiring mechanical ventilation or those developing a worsening clinical condition despite simple transfusion, or for patients with a high baseline hemoglobin level that precludes simple transfusion, exchange transfusion should be considered. Case reports and uncontrolled series describe rapid clinical and radiological improvement and higher paO2 levels after exchange transfusion.70–73 In the largest series to date, 35 patients with acute chest syndrome and clinical deterioration underwent exchange transfusion.74 Thirty-two of the 35 patients, including five of seven on assisted ventilation, had a rapid and dramatic improvement in oxygenation and other clinical parameters, leading the investigators to recommend that exchange transfusion be considered for patients with diffuse pulmonary involvement. Almost half of all acute chest syndromes follow hospitalization, usually for acute painful episodes.69 Early transfusion might prevent the development of acute chest syndrome in high-risk patients. Such an approach is preferable because it could help prevent lung injury and associated long-term complications. Elevated secretory phospholipase A2 levels, particularly in association with fever, predict an increased risk of acute chest syndrome in patients admitted with vasoocclusive pain75 . In a pilot study, patients with sickle cell disease admitted with pain who developed fever and elevated phospholipase A2 levels

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Table 29.6. Effect of goal of perioperative transfusions on outcome

Complications Serious or life-threatening postoperative complications Acute chest syndrome Fever or infection Painful crisis Neurological event Death Transfusion-related complications New antibody Hemolysis

Group 1 (HbS100 ng/mL) with a negative chest radiograph were randomized to receive a single transfusion of approximately 10 mL/kg or to standard care.76 Five of eight (63%) patients randomized to standard care developed acute chest syndrome compared with none of seven patients randomized to receive transfusions. A multicenter study currently is under design to study further the benefit of red cell transfusions in preventing acute chest syndrome.

Perioperative Management The use of transfusions to prevent complications of general anesthetics and surgery is particularly controversial. To reduce the high risk of perioperative complications, some have favored simple transfusions to correct the anemia, whereas others have favored exchange transfusion to decrease the HbS level. Uncontrolled studies of these approaches yielded perioperative complication rates of 0%–20%, and a very low death rate.58,77–88 Other investigators argued that careful attention to hydration and oxygenation rather than administration of red cells produced an equally successful outcome, at least in minor surgical procedures.82,89–92 Partial resolution of the role of perioperative transfusion therapy came from a large, multiinstitutional, randomized study of two approaches.93 Patients in group 1 received simple or exchange transfusions to reduce the HbS level below 30% and to raise the total hemoglobin level to 10 g/dL; patients in group 2 received simple transfusions to raise the total hemoglobin level to 10 g/dL, irrespective of the HbS level. The type of transfusion therapy did not affect the rate of minor or major perioperative complications other than those related to transfusion itself (Table 29.6).93 Acute chest syndrome occurred in 11% of patients in group 1 and 10% of patients in group 2. Both deaths occurred in group 1. The reduction in HbS level in group 1 required twice as much donor blood as the increase

696 in hemoglobin level in group 2. As a result, patients in group 1 had a significantly higher rate of alloimmunization. Although this study randomly assigned patients only to one of two transfusion arms, the investigators concurrently followed a group of patients who, for different reasons, did not undergo transfusion.93 In a subanalysis of patients undergoing cholecystectomy, the most common surgical procedure, the rate of sickling-related complications was higher in the patients who did not undergo transfusion than in those receiving either of the two transfusion protocols.94 Acute chest syndrome occurred in 19% of the 37 nontransfused patients, and two patients died. In other series of patients who did not routinely receive transfusions preoperatively, retrospective analyses have demonstrated complication rates of 13%–26%.82,85,95 Adverse events generally were more common in patients undergoing major surgery or emergency surgery. Questions and controversies remain regarding optimal transfusion practices for patients undergoing surgery. Given the lack of randomization to a no-transfusion treatment arm, it remains unclear if transfusion truly is superior or if meticulous supportive care without transfusion could be equally effective, at least for some surgical procedures. For example, success has been reported using a regimen without preoperative transfusion, but with supportive care measures including continuous positive airway pressure postoperatively in patients with sickle cell disease undergoing cholecystectomy.92 The benefit of routine preoperative transfusion in those undergoing low-risk surgical procedures also is unclear. In one retrospective study of 34 minor surgical procedures, such as myringotomy, strabismus repair, and dental procedures, performed in children without preoperative transfusion, minor complications were seen in 15% of cases and neither acute chest syndrome nor severe complications developed.89 The value and goal of preoperative transfusions in patients with HbSC disease is not established. One multicenter study in which transfusions were administered at the physicians’ discretion showed a 20% incidence of sickle cell complications with moderate-risk procedures in patients with HbSC disease who did not receive preoperative transfusion compared with none in transfused cases;96 no difference was seen between the two groups for low-risk procedures. Currently available data also do not address some issues such as the management of a patient with sickle cell disease who maintains a steady-state hemoglobin level of 10 g/dL or a patient receiving hydroxyurea with good hematological response, and transfusion strategies in these instances vary among sickle cell centers. Given the lack of a robust randomized clinical trial comparing transfusion to no transfusion in the preoperative period, considerable variability in clinical practice exists. In a recent prospective study involving 31 hospitals in Great Britain, among 114 patients with sickle cell disease who underwent surgery, 43% were not treated with preoperative transfusion, whereas 39% received simple transfusion,

Janet L. Kwiatkowski and John B. Porter and 23% underwent exchange transfusion.97 Although for hip surgery and otorhinolaryngological procedures, exchange transfusion and simple transfusion, respectively, were used in most cases, for other surgical procedures such as cholecystectomy, preoperative transfusion practices varied widely. In multivariate analysis, moderate/high-risk surgery was the only variable associated with postoperative complications and transfusion was not associated with a lower risk of complications. A randomized, multicenter study has been designed to compare surgical complication rates between groups randomized to receive or not to receive preoperative transfusions, which might provide better guidance for optimal management of the patient with sickle cell disease who needs surgery. Based on current data, preoperative transfusion to raise the hemoglobin level to 10 g/dL generally appears to be equally as effective as a transfusion program to reduce the HbS below 30%. With either approach, acute chest syndrome remains a substantial risk, especially in patients with a history of lung disease who are undergoing a high-risk operation. Transfusions before minor, elective procedures with short anesthesia times might convey no advantage in patients without chronic lung disease or other significant complications.

Vasoocclusive Pain Episode The value of transfusion therapy in the management of acute painful episode is uncertain. Partial exchange transfusion to reduce the HbS below 50% improved the clinical condition and reduced the duration of hospitalization in eight patients.98 No subsequent study has confirmed or disproved these old but intriguing findings. In general, the use of red cell transfusion for the management of acute painful episodes is not indicated; however, acute painful episodes occasionally may progress to catastrophic multiorgan failure associated with a falling hemoglobin level. In 17 such episodes, transfusion therapy rapidly and dramatically improved the encephalopathy, urine output, oxygenation. and general clinical condition.99

Priapism Transfusion therapy for the treatment of acute, prolonged episodes of priapism is also controversial. No randomized, controlled studies exploring the use of this therapeutic modality exist and several small, uncontrolled studies of simple and exchange transfusion in the treatment of priapism have yielded inconsistent results.100–109 In one recent case series, among six patients treated with exchange transfusion the procedure was “very” effective in five and partially effective in one patient,109 whereas in another report of seven patients treated with red cell exchange, only one patient improved partially.103 Overall, no difference between the results with simple and exchange transfusion is readily discernible, aside from descriptions of

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease individual patients who had immediate detumescence or relief of pain during erythrocytapheresis. Importantly, neurological events, including headache, seizures, and strokes, have occurred in a small but worrisome number of patients within 1–14 days of simple or exchange transfusion for priapism (ASPEN syndrome).104,110–112 Whether the risk of such events is greater after transfusion therapy for priapism than other complications of sickle cell disease is uncertain. The limited benefit of transfusion therapy for priapism and the possible risk of neurological sequelae should be carefully considered in managing this acute problem.

Indications for Chronic Transfusion Therapy Stroke Although transfusion therapy has not been studied in a randomized controlled clinical trial for prevention of recurrent stroke, data from several case series113–115 and two multicenter retrospective studies59,116 support its beneficial effect. Regular red cell transfusions to maintain the HbS level below 30% reduce the risk of recurrent infarctive stroke from approximately 70%117 to 10%–20%.59,116 This finding has led to the recommendation to initiate chronic transfusion therapy after a first stroke in children with sickle cell disease. Although the value of transfusion in preventing recurrent stroke is clear, the optimal duration of therapy is uncertain. Most recurrences in nontransfused patients are in the first 3 years after the initial event,117 suggesting that lowering the HbS level is particularly important during this period. This view is supported by the recurrence of stroke in seven of 10 patients who stopped transfusion therapy after 1 or 2 years118 and in four of 10 patients who stopped after 1–4 years.119 The benefits of longer transfusion therapy are debatable. In one study, stopping transfusions after periods of 5–12 years resulted in a 50% recurrence rate within 1 year,120 whereas in another study, stopping transfusions in nine patients after a mean duration of 6 years resulted in no recurrences during the next 3–18 years.121 Six of the nine patients began hydroxyurea therapy at a median of 4 years after discontinuing transfusions, which might have affected stroke risk. At present, most pediatric sickle cell centers continue transfusion therapy indefinitely for the prevention of recurrent stroke. This therapy, however, frequently ends when patients transfer to adult centers. The determination of the optimal duration of transfusion therapy for prevention of recurrent stroke awaits a prospective, randomized trial that avoids the pitfall of selection bias and that accounts for the potential impact of transfusions administered for other purposes. Given that the major reason for discontinuing transfusions is the concern about iron overload, an alternative to stopping therapy is to modify the transfusion program to reduce the rate of iron accumulation. In a study of nine patients with no recurrence of stroke and no

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Table 29.7. Effect of transfusion therapy on prevention of first stroke in children with sickle cell disease and abnormal transcranial doppler ultrasonograph

Patient-months Transfusion (n = 63) No Transfusion (n = 67)

Cerebral infarctions

Intracerebral hematoma

1,321

1

0

1,229

10

1

From ref. 123.

progressive neurological deterioration during at least 4 years of simple transfusion therapy, a subsequent increase in the pretransfusion HbS level from 30% to 50% reduced blood requirements, and thus the rate of iron loading by approximately 30% without raising the risk of recurrent infarctive stroke.60 A second study supported the safety and benefits of this modified simple transfusion program.122 Even greater reductions in net blood requirements have been achieved with manual or automated exchange transfusions (Table 29.7).60,63,64 Novel transfusion programs that prevent recurrent stroke and reduce the risk of iron overload and new oral chelation treatment options could increase the clinician’s level of comfort with long-term therapy.

Abnormal Transcranial Doppler Ultrasonography The screening technique of transcranial Doppler ultrasonography (TCD) now allows the identification of children who are at high risk of developing a first stroke, creating the potential for intervention before overt neurological injury occurs (Chapter 19). In the Stroke Prevention Trial in Sickle Cell Anemia (STOP) 63 children with abnormal TCD studies were randomly assigned to receive regular transfusions to maintain the percentage of HbS less than 30%, and 67 to receive standard care without transfusions.58 Transfusions were associated with a significantly lower risk of stroke (Table 29.7),123 which led to early termination of the trial. In the years following the publication of the STOP results, the rate of first stroke in children in California with sickle cell disease fell from a baseline of 0.88 per 100 patient-years to 0.17 per 100 patient-years, suggesting a tremendous impact of TCD screening and transfusion therapy on preventing this devastating complication.124 A follow-up study, Optimizing Primary Stroke Prevention in Sickle Cell Anemia (STOP II), was designed to assess whether transfusions could be discontinued in children with a history of abnormal TCD whose TCD studies normalized while receiving transfusions, suggesting a reversion to low-risk of stroke.125 Children considered to have more severe vasculopathy as demonstrated by moderate to severe vessel stenosis detected by brain magnetic resonance angiography were excluded. Among the 41 children

698 who discontinued transfusions, 14 had a reversion to abnormal TCD studies and an additional two children developed stroke; no TCD reversions or strokes occurred in the 38 children who continued transfusions. This highly significant finding led to the current recommendation to continue transfusion therapy indefinitely for primary stroke prevention. Modified transfusion programs that allow the HbS level to rise to 50% or higher have not been studied in this clinical setting and thus the target pretransfusion HbS level is usually kept at less than 30%.

Silent Cerebral Infarcts Given that silent infarcts occur commonly in sickle cell anemia, often progress over time, and are associated with neurocognitive abnormalities and a higher risk of overt stroke126–129 effective treatments are needed. Transfusion therapy has been suggested as a treatment option for patients with silent infarcts who demonstrate neurocognitive abnormalities,46 but the benefit of such therapy is currently unknown. In the STOP study, among the 37% of randomized subjects who had silent infarcts, those receiving transfusions had a significantly lower risk of progression of silent infarcts than those receiving standard care.130 Whether transfusions will prevent silent infarct progression and neurological complications in children without abnormal TCD remains to be determined. A pilot study showed that transfusion therapy for children with silent infarcts is feasible131 and a larger, randomized, controlled trial assessing the effect of transfusions on the prevention of neurological complications in children with silent infarcts is currently underway.132

Pregnancy Transfusions are commonly used in the management of acute complications such as toxemia, severe acute chest syndrome, exacerbation of anemia, stroke, acute renal failure, or when emergency surgery is needed in pregnant women with sickle cell disease.133 Greater controversy surrounds the use of prophylactic transfusion therapy during pregnancy.134–142 Assessment of the effectiveness of this approach is hindered by differing transfusion protocols and by retrospective analyses. Even if one limits the additional confounding effect of general improvements in obstetrical care by considering only those studies reported in the past three decades, conclusions differ widely. For example, in a retrospective review of 101 women with sickling disorders treated between 1981 and 1990 at a single institution, the investigators concluded that the early (mean 19 weeks) introduction of partial exchange transfusions to maintain the HbS below 50% resulted in fewer episodes of pain, a lower incidence of pneumonia, a reduction in preterm deliveries, a lower perinatal death rate, and fewer low-birth-weight infants.138 In contrast, two retrospective British studies covering similar periods found no beneficial effect of prophylactic transfusions on either maternal or

Janet L. Kwiatkowski and John B. Porter fetal outcome.136,143 One study used partial exchange transfusions from the first or second trimester but no consistent target hemoglobin level or HbS percentage,136 whereas the other used simple transfusion to reduce the HbS level below 25% and raise the hemoglobin to 11 g/dL.143 The most informative data regarding the role of prophylactic transfusions during pregnancy in women with sickle cell disease come from a multicenter, prospective, randomized, controlled study.137 Thirty-six women received prophylactic simple or partial exchange transfusions beginning before 28 weeks of gestation to maintain the hemoglobin level between 10 and 11 g/dL and to reduce the HbS level to less than 35%. An equal number of women received transfusions only for medical or obstetrical emergencies. Although women receiving prophylactic transfusions had fewer painful episodes and a lower cumulative incidence of sickle cell disease–related complications, the two treatment groups did not differ in obstetrical complications or perinatal outcome, which was high at 10%. Even without a clear benefit of transfusions on perinatal outcome, the lower rate of sickle cell complications could be enough to prompt some physicians to treat pregnant patients with transfusions.

Recurrent Acute Chest Syndrome and Pulmonary Hypertension The role of transfusion therapy for acute chest syndrome might extend to the prevention of recurrences in patients with severe or repeated episodes. This long-term application is predicated on the finding that recurrent acute chest syndrome might be immediately life threatening or contribute to chronic pulmonary failure, pulmonary hypertension, and cor pulmonale.144,145 In a retrospective study of 17 children receiving chronic transfusion therapy, 13 for stroke and four for recurrent pain, the rate of hospitalizations for acute chest syndrome was reduced significantly from 0.7 per year before treatment to 0.04 per year on transfusion therapy.146 These findings were confirmed in an analysis of children participating in the STOP trial: The hospitalization rate for acute chest syndrome was significantly lower in the transfusion group compared with the standard care group (4.8 vs. 15.3 per 100 patient-years).128 A single retrospective study evaluated the effect of chronic transfusions in children treated for the primary indication of recurrent or severe acute chest syndrome.147 In that study, transfusions were effective in preventing recurrences of acute chest syndrome. The incidence of acute chest syndrome fell significantly from 1.3 per year before transfusions to 0.1 per year during treatment. Chronic transfusions did not reduce the severity of acute chest syndrome in those who developed recurrent episodes. The effect of transfusion therapy on late pulmonary outcomes remains unknown. Although studies of red cell transfusions for the management of pulmonary hypertension are lacking, this treatment might be beneficial for some patients who develop this complication. Potential benefits

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease of transfusion include improved oxygen carrying capacity and reduction in the amount of HbS. Maintaining a low HbS level should reduce hemolysis, a factor thought to be central to the pathogenesis of pulmonary hypertension in sickle cell disease.148,149 In addition, transfusions might prevent other complications of sickle cell disease such as acute chest syndrome and pain, which can exacerbate pulmonary hypertension. Further studies are needed to determine the role of transfusions in the management of pulmonary hypertension.

Recurrent Splenic Sequestration Short-term chronic transfusion therapy might be beneficial in some infants with recurrent acute splenic sequestration to reduce the risk of recurrence and delay the need for splenectomy until the age of 1–2 years. Given, however, that functional asplenia develops in children with sickle cell anemia and HbS–␤0 thalassemia when they are 6–12 months old,150 it is unclear if deferring surgical asplenia adds much benefit in the protection against invasive infection. One study of 130 Jamaican patients with sickle cell disease who underwent splenectomy showed no increased risk of bacteremia or death compared with nonsplenectomized control subjects.151 The study group included 46 young children who underwent splenectomy at a median age of 2.3 years for recurrent splenic sequestration. With the routine use of penicillin prophylaxis and the availability of the pneumococcal conjugate 7-valent vaccine, the benefit of postponing splenectomy in young children is even less clear. Furthermore, recurrences of splenic sequestration occur even while receiving regular transfusions to maintain the HbS below 30% and recurrences are frequent when the transfusion program is interrupted or completed.152 Thus, the role of a short course of prophylactic transfusions for the prevention of recurrent splenic sequestration remains unproven.

Recurrent Painful Episodes Five percent of patients with sickle cell disease have three– 10 episodes of intense pain resulting in hospitalization each year.153 No study to date has directly addressed the value of transfusion therapy in reducing the physical and social consequences severe pain; however, the experience gained from transfusion therapy for prevention of primary or recurrent stroke demonstrates that reduction of the HbS level to 30% reduces the rate of vasoocclusive pain episodes seven–10-fold, suggesting that such an approach could be beneficial.146,154 Randomized, controlled clinical trials are needed to address the effect of transfusion therapy on recurrent or unusually severe pain.

Other Proposed Indications Long-term transfusion therapy might help prevent recurrent episodes of priapism.104 Case reports and uncontrolled

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series also have suggested a possible therapeutic role for red cell transfusions in the management of other complications of sickle cell disease such as leg ulcers, avascular necrosis, and persistent hematuria. In some of these descriptions the benefit of transfusion has appeared to be very dramatic, but specific recommendations must await more extensive clinical trials.

Other Approaches New approaches to the management of sickle cell disease might redefine the role of transfusion therapy. For example, hydroxyurea therapy is an alternative to transfusions for the treatment of recurrent painful episodes and acute chest syndrome. In addition, a strategy of switching from transfusion therapy to hydroxyurea treatment for secondary stroke prevention showed promising results in a pilot study of 35 children,155 and a larger, multicenter study is currently underway. Bone marrow transplantation is being offered with increasing frequency as an alternative to transfusion therapy for patients with stroke or other severe or recurrent complications. These advances, as well as progress in the general management of pregnancy, general anesthetics, and other medical and surgical conditions, will require regular reevaluation of the effectiveness and safety of transfusions.

Other Hemoglobinopathies Patients with unstable hemoglobins might need occasional red cell transfusions during exacerbations of anemia. If the unstable hemoglobin has an increased oxygen affinity, the decision to transfuse reaches a higher level of complexity.156 In this situation, the chronic hemolysis due to the unstable hemoglobin blocks the usual compensatory erythrocytosis in response to the hemoglobin’s abnormally increased oxygen affinity. The resultant “normal” hemoglobin level may have only one-half of the normal oxygen delivery capacity. Simple transfusions to achieve normal oxygen delivery at the time of surgery or acute illnesses might raise the hemoglobin to unacceptably high levels. Alternatively, exchange transfusion carries the dual benefits of improving oxygen delivery while maintaining a normal total hemoglobin level, and replacing the unstable hemoglobin variant with HbA.

COMPLICATIONS OF TRANSFUSION THERAPY Most adverse consequences of transfusion therapy for thalassemia, sickle cell disease, and other hemoglobinopathies are independent of the underlying blood disorder, although they might occur with differing frequency in each of these diseases because of environmental or other reasons. The following sections focus on complications that are particularly problematic for patients with hemoglobinopathies receiving long-term transfusions. A detailed discussion of other complications such as febrile or allergic reactions and

700 acute hemolytic transfusion reactions can be found in textbooks of transfusion medicine. Iron overload, the leading and most complex clinical problem of long-term transfusion therapy, is discussed in full in the second major section of this chapter.

Alloimmunization Thalassemia Alloantibodies have been detected in 3%–37% of patients with thalassemia.24,157–163 Variations among the protocols practiced at different centers are likely due to several factors. Alloimmunization rates are generally higher when there is a greater discordance of red cell antigen frequencies between donors and recipients. For example, the prevalence of alloimmunization was 20.8% among thalassemia patients of Asian descent treated at a northern California center where donors are primarily Caucasian,158 whereas lower rates of 5%–10% were reported in more homogeneous populations.157,159 The degree of antigen matching of blood products also affects alloimmunization rates. Antibodies are most commonly directed against antigens of the Rh and Kell systems.159–161 In the northern Californian thalassemia population, the alloimmunization rate was only 3% among patients receiving Rh- and Kell-matched blood, compared with 33% in those treated with nonphenotypically matched blood.158 Extension of antigen matching beyond the Rh and Kell systems might not be helpful.165 Immune factors, related to both the recipient and to the blood product preparation, could also impact alloimmunization rate. For example, splenectomy might increase the risk of alloimmunization, whereas prestorage leukocyte depletion can lower the rate because leukocytes release potentially immunomodulatory cytokines during storage.158,161 The age at which transfusion therapy is initiated in thalassemia major has a consistent effect on the rate of alloimmunization. In two studies from Greece, alloimmunization occurred more than twice as often in children beginning transfusion therapy after age 3 years160 and four times as often in children beginning transfusion therapy after 1 year of age.164 In 110 alloimmunized patients in Greece and Italy, alloantibodies developed in fewer than 3% of children who began transfusion therapy in the first year of life, but did occur in more than 15% of children who received their first transfusion after age 4 years.24 Because alloimmunization by itself is rarely a significant clinical problem in thalassemia, these findings of the influence of age might not have a substantial impact on the decision to initiate or delay transfusion therapy. In some children, however, receiving regular transfusions, alloantibodies are accompanied by red cell autoantibodies, presenting additional and often serious clinical problems. This unusual but severe complication is worthy of consideration in deciding at what age to initiate transfusion therapy in the child with a marginal hemoglobin level or clinical status.

Janet L. Kwiatkowski and John B. Porter Sickle Cell Disease Red cell alloimmunization occurs in 3%–76% of patients with sickle cell disease who have been transfused.46,47,49,50,157,165–169 In the Cooperative Study of Sickle Cell Disease, the overall rate of alloimmunization in 1,814 patients transfused before or after study entry was 18.6%.167 In a subanalysis of 604 patients who had no alloantibodies at study entry and subsequently underwent transfusion, the investigators found a significantly higher incidence of alloimmunization in patients with sickle cell anemia in comparison with patients with other sickling disorders. A relationship between age and risk of alloimmunization exists in sickle cell disease as it does in thalassemia. Patients with sickle cell disease who received their first transfusion at age 10 years or older had a 20.7% rate of alloimmunization compared with 9.6% in individuals first transfused when aged younger than 10 years. Alloimmunization rose continuously with an increasing number of transfusions. Patients first transfused at a young age consistently required more transfusions to become alloimmunized than those first transfused at older ages. Red cell antigens causing alloimmunization in patients with sickle cell disease most commonly belong to the Rh, Kell, Duffy, and Kidd systems. Antibodies to E, C, K, Jkb , and Fya account for approximately 80% of clinically significant alloantibodies, reflecting the differences in red cell phenotype between blood donors who are predominantly Caucasian and patients with sickle cell disease who are predominantly of African ancestry.49,167 The red cell alloimmunization rate was 76% among patients with sickle cell disease who underwent transfusion in the United Kingdom compared with a rate of only 2.6% in patients transfused in Jamaica, where donors and recipients are more genetically similar.166 Similarly, the rate of alloimmunization among previously transfused Caucasians with sickle cell disease in Italy is only 5.4%.170 Red cell autoantibodies also can develop in patients with thalassemia or sickle cell disease, often in association with alloantibodies.171–173 The mechanism underlying the formation of autoantibodies is unclear, but possible causes include antigenic changes in the erythrocyte membrane or more generalized immune dysregulation. Erythrocyte autoantibodies occur in 5%–8% of multiply transfused children with sickle cell disease but cause hemolysis in only approximately 2%.46,169,171 Alloimmunization has consequences that range from additional work and expense in the blood bank to severe hemolysis. Delayed hemolytic transfusion reactions pose a particularly important problem for patients with sickle cell disease.47,52,174–176 In two prospective studies, delayed hemolytic transfusion reactions occurred in 38% and 44% of newly alloimmunized patients with sickle cell disease.49,93 Delayed hemolytic transfusion reactions arise when an alloantibody is not present in sufficient quantity to be detected by direct or indirect antiglobulin testing or

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease when the antibody is directed against a low-frequency antigen that is not present on the screening cells used for the indirect antiglobulin test.177 On reexposure to the offending antigen, an anamnestic response occurs, and hemolysis begins in 3–10 days. Fever, and back, leg, or abdominal pain can accompany the delayed hemolysis, mimicking a vasoocclusive episode.176,178 In addition to destruction of the transfused cells, the patient’s HbS-containing cells also can be destroyed, leading to profound anemia.173,176,179 Reticulocytopenia might occur and the direct antiglobulin test also can be negative, further complicating the diagnosis. Treatment with steroids, intravenous immunoglobulin, and erythropoietin might be helpful; further transfusions, even using apparently compatible blood, should be carefully considered in light of the risk of life-threatening hemolysis.176,179

Transfusion-transmitted Infections Because patients with thalassemia major and regularly transfused patients with sickle cell disease usually receive 12–50 U of red cells each year, they are at higher risk than most blood recipients for transfusion-transmitted infections such as hepatitis and human immunodeficiency virus (HIV). Rates of seropositivity for hepatitis C have varied from 12% in patients with thalassemia who received treatment in the United Kingdom to 91% in those who were treated in Italy.180,181 Serological testing of donated blood for hepatitis C, which became available in developed countries in the early 1990s, has reduced the risk of acquiring this infection from transfusion. In a 2004 report from the Thalassemia Clinical Research Network, 35% of 334 North American patients with thalassemia had evidence of hepatitis C exposure.182 Among children aged younger than 5 years, only 5% had evidence of hepatitis C exposure compared with 70% of patients aged 25 years and older. Antibodies to hepatitis C have been detected in 23%–30% of repeatedly transfused patients with sickle cell disease.183,184 Nucleic acid testing for hepatitis C, implemented in most developed countries by 2000, is expected to reduce greatly the risk of transfusion-transmitted hepatitis C virus. Viral hepatitis might have particularly grave clinical consequences in regularly transfused patients with hemoglobinopathies because iron overload aggravates the liver damage and can diminish the response to antiviral therapy.185,186 New cases of transfusion-transmitted hepatitis B are rare in the United States as a result of vaccination programs and blood screening. High rates of hepatitis B infection continue to occur in thalassemia patients in some parts of the world,187,188 mostly attributable to late or no vaccination. A recent study of children with thalassemia in India infected with hepatitis B found a number of mutations in viral DNA regions involved in reactivity to antihepatitis B surface antibody, raising concern that such mutations might contribute to vaccine failures.189 This intriguing finding deserves further study.

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The risk of transfusion-transmitted HIV infection in patients with thalassemia and sickle cell disease has varied with time and geographical location. In 1984, when donor testing was still unavailable, 12% of patients with thalassemia at one center in New York were seropositive for HIV.190 In contrast, in a 1987 study of 3,633 patients with thalassemia in Europe, only 1.56% were seropositive for HIV.191 Following the introduction of routine donor screening programs and serological testing for HIV by enzymelinked immunosorbent assay, new seroconversions became extremely rare in many countries.192,193 In a more report from the Thalassemia Clinical Research Network, 2% of North American patients with thalassemia major were positive for HIV.182 The current risk of acquiring HIV from transfusion is less than 1 in 2 million.194 In countries with a high prevalence of HIV coupled with inadequate blood safety programs, the risk of HIV infection in repeatedly transfused children with sickle cell disease remains high.195 Iron overload, as assessed by serum ferritin level, might contribute to the progression of HIV infection in patients with thalassemia.196 The iron chelator desferrioxamine, on the other hand, might have a dose-dependent inhibitory effect on disease progression.197 This observation is supported by in vitro studies of the effect of chelators on HIV infectivity. Both desferrioxamine and deferiprone have an inhibitory effect on HIV-1 replication198 and more recently, the newer oral chelator, deferasirox has been shown to inhibit HIV-1 transcription.199 Red cell transfusions infrequently transmit bacterial infections.200 Yersinia enterocolitica, the organism responsible for seven of the eight fatal red cell–transmitted bacterial infections reported to the Food and Drug Administration between 1986 and 1991, deserves special attention. This contaminant represents a particular problem for chronically transfused patients because iron overload and the iron chelator desferrioxamine enhance its pathogenicity. Y. enterocolitica enters the blood supply when blood donation coincides with asymptomatic bacteremia, making efforts at prevention difficult. Evaluating febrile transfusion reactions or persistent unexplained fevers in regularly transfused patients with hemoglobinopathies should include careful consideration of systemic or localized Y. enterocolitica infection.

Hypersplenism and Increased Transfusion Requirements A sustained increase in transfusion requirements is an indication for splenectomy in patients with thalassemia major.201 When the annual blood requirement exceeds 200 mL of packed red cells (hematocrit 75%) per kilogram body weight, a minimum reduction of at least 20% usually follows removal of the spleen. The role of splenectomy in reducing transfusion requirements might be less important in patients in whom chelation therapy is working well, and should be weighed against potential complications such as postsplenectomy sepsis and thrombosis. Moreover, other

702 causes of increased blood requirements, such as alloimmunization or different preparation of donor units, should be sought before removing the spleen for this purpose. Regular red cell transfusions can have the unintended consequence of reversing partial splenic atrophy in children and adults with sickle cell disease. As the newly restored spleen enlarges, transfusion requirements sometimes increase substantially.202,203 In such cases, removal of the spleen reduces the need for donor blood and therefore the accumulation of iron by 39%–51%. Careful monitoring of spleen size and annual transfusion requirements assists in identifying patients with hypersplenism who might benefit from surgical intervention.

Hypertension and Encephalopathy Hypertension, accompanied or followed shortly by headache, or changes in mental status and seizures, is an unusual but potentially devastating complication of transfusion therapy in thalassemia, especially when the hemoglobin is raised from very low levels to normal or high levels.204,205 This constellation of findings also has been reported after transfusion in sickle cell disease.111,206 The cause is unknown. Autopsy findings in six cases showed brain edema and congestion, gross or microscopic hemorrhage, and microdissecting aneurysms.207 No underlying vascular disease distinguished these patients from those without encephalopathy. Similar symptoms of hypertension, seizures, and mental status changes accompanied by reversible T2 hyperintensities on brain magnetic resonance imaging (MRI), constitutes a syndrome known as reversible posterior leukoencephalopathy. This has also have been reported in children with sickle cell disease and severe acute chest syndrome treated with erythrocytapheresis.208 Complaints of headache or weakness after transfusion should prompt careful evaluation for neurological abnormalities including cerebral hemorrhage; hypertension, if present, should be treated aggressively.

IRON OVERLOAD AND CHELATION THERAPY Pathophysiology of Iron Overload Rates of Iron Loading The average iron content of a healthy human is 40–50 mg/kg of body weight, with 30 mg/kg present in hemoglobin. Approximately 4 mg/kg is found in muscle myoglobin and 2 mg/kg in iron-containing enzymes. The remaining body iron is in the storage form of ferritin or hemosiderin, predominantly in liver, spleen and bone marrow. Mean iron stores are 769 mg in men and 323 mg in women.209,210 A healthy individual absorbs approximately 10% of the dietary iron, or 1–2 mg daily. This intake is matched by insensible losses through exfoliation of skin, urinary tract, and gut mucosal cells together with

Janet L. Kwiatkowski and John B. Porter gastrointestinal and menstrual loss of red blood cells. Humans have a limited capacity to modulate iron absorption and no physiological mechanism for excreting excess iron. Iron can accumulate as a consequence of increased gastrointestinal absorption or from repeated red cell transfusions. In thalassemia major, iron loading predominantly derives from blood transfusion but excess iron absorption might also contribute. A unit of red cells, processed from 420 mL of donor blood, contains approximately 200 mg of iron (0.47 mg iron/mL of whole donor blood or 1.16 mg iron/mL of pure red cells). Splenectomized patients with thalassemia major received the red cells derived from approximately 300 mL/kg/year of whole blood per (range 200–400 mL whole blood/kg)211 to maintain a mean hemoglobin level of 12 g/dL. This volume of transfused blood is equivalent to 0.4 mg/kg of transfused iron daily or 28 mg of iron in an adult weighing 70 kg. Iron loading varies greatly both within a given diagnosis and among diagnoses.212,213 In thalassemia major with a mean loading rate of 0.4 mg/kg/day212 there is considerably variability with approximately 20% of patients receiving less than 0.3 mg/kg/day, approximately 60% receiving 0.3– 0.5 mg/kg/day, and a further 20% more than 0.5 mg/ kg/day. The transfusion requirements in nonsplenectomized patients are generally higher than splenectomized patients and might contribute to an increased rate of iron loading. In one study of patients on hypertransfusion regimens who required more than 250 mL/kg/year of packed red cells, splenectomy decreased the annual iron loading by an average of 39%.214 Average transfusion requirements in nonspelenectomized thalassemia major patients are 0.43 mg/kg/day compared with 0.33 mg/kg/day in splenectomized patients.212 In general, hypertransfusion decreases the rate of splenic enlargement,215 and the early introduction of a hypertransfusion regimen might diminish the extent to which the spleen contributes to an increased blood transfusion requirement.211 The contribution of increased iron absorption to iron loading in hemoglobin disorders is variable depending on the underlying condition. In thalassemia major, gastrointestinal iron absorption accounted for a further 1–4 mg daily of net iron loading216 in a splenectomized adult patient who weighed 70 kg. In thalassemia intermedia, blood transfusion is intermittent or absent, and the increased iron loading is mainly a consequence of excess iron absorption. The amount of excess iron absorption depends on the degree of ineffective erythropoiesis, the extent of erythroid expansion, and the severity of the anemia, all of which are highly variable in thalassemia intermedia. In one study, patients with thalassemia intermedia absorbed 26%–73% of food iron.217 Patients with this form of thalassemia absorbed 60% (range 17%–90%) of a 5-mg dose of ferrous sulfate, whereas healthy controls absorbed 10% (5%–15%).217 In another study, absorption varied from 20% to 75% in HbE–␤ thalassemia and correlated

0.5

24% 59% 17%

0.6 0.5

SS Exchange

0.1

SS Transfusion

0.2

MDS

0.3

Thal. Major

0.4

DBA

Transfusional Iron loading (mg/kg/day)

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease

0 Figure 29.1. Rates of transfusional iron loading by diagnosis. (Original figure derived from data in refs. 212, 225, 246, 453.)

with plasma iron turnover, transferrin saturation, and liver iron concentration.218 Iron absorption in thalassemia intermedia can thus be as much as five–10 times normal, or 0.1 mg/kg/day.219 In sickle cell disease, iron overload does not occur in the absence of blood transfusion.220 Indeed, a variable proportion of untransfused patients might be iron deficient.221–223 Repeated simple blood transfusions will inevitably lead to iron overload, but can mitigate iron deficiency.60,224 The average rate of transfusional iron loading was less in sickle cell disease (0.22 mg/kg/day)225 than in thalassemia major.212 Figure 29.1 shows the relative rates of iron accumulation from transfusion measured in clinical trials involving desferrioxamine and deferasirox. Various exchange transfusion rates were highest in Diamond– Blackfan anemia (DBA) and lowest in sickle cell disease. These rates affect the chelation doses that are typically required for each condition.212,213

Distribution of Excess Iron The tissue distribution and clinical manifestations of transfused iron are influenced by the mode and rate of iron loading and by the absolute levels of tissue iron within the body. Increased iron absorption leading to a predominantly parenchymal distribution of iron loading is a feature of genetic hemochromatosis and is also found in many conditions associated with ineffective erythropoiesis.218,226–228 Postmortem examination of patients with thalassemia major showed a striking variability in iron concentrations among different tissues.229 In thalassemia major in the pre– chelation era, iron was found at high concentrations in liver, heart, and endocrine glands with very little present in striated muscle229 and none in the brain and nervous tissue. In the absence of chelation therapy, siderosis in liver macrophages and hepatocytes correlates with the

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number of units of blood transfused, with age, and with liver iron concentration.230 With more advanced iron overload or in splenectomized patients, hepatocyte deposition predominates.230 Recent evidence in iron-loaded mice suggests that uptake of nontransferrin-bound iron (NTBI) into tissue expressing L-type calcium channels can account for this uneven distribution of iron overload and in particular the uptake into heart and endocrine tissues.231 Another factor that could influence iron distribution in transfusional iron overload is hepcidin synthesis by the liver. Under relatively normal levels of iron loading, hepcidin is upregulated by increments in iron loading causing decreased iron absorption from the gut and decreased iron release from macrophages by secondary down regulation of ferroportin.232 This process could occur through increments in transferrin saturation with progressive iron loading.233 There is evidence, however, that hepcidin synthesis is downregulated by ineffective erythropoiesis as seen in thalassemia234–236 and at very high levels of iron loading in the liver,237 possibly through production of reactive oxygen species. This would lead to an increased propensity to distribute iron to the heart and endocrine tissues in conditions associated with high levels of ineffective erythropoiesis and also when liver iron levels reach a point at which increased damage through reactive oxygen species production is seen. Increased alanine aminotransferase values238 and progression of fibrosis239 are associated with liver iron concentrations above approximately 15–20 mg/g dry weight. These levels of liver iron loading are also associated with an increased likelihood of myocardial iron deposition in patients who have not undergone intensive chelation treatment.240,241 In transfusional iron overload, cardiac iron is preferentially distributed to the ventricular compared with atrial myocardium or conducting tissue.240 In nonthalassemic patients in the pre–chelation era, increased myocardial iron was found at postmortem in approximately 30% of patients after 50–75 U of blood, in 60% of patients who received 65–200 U of blood and in all patients who had received 200–300 U of blood.240 Ventricular myocardial iron distribution is uneven, being maximal in the subepicardium, intermediate in the subendocardial region and papillary muscles, and least in the middle third of the ventricular myocardium. Focal areas of fibrosis can also be seen in papillary muscles or ventricles.240 The uneven distribution of iron makes endocardial biopsy an unreliable tool for assessing cardiac iron concentration and iron-mediated cardiac damage.242,243 The use of cardiac MRI to estimate myocardial iron has added to the understanding of factors affecting myocardial iron uptake and removal with chelation therapy. In adult patients with myelodysplastic syndrome who have not undergone chelation therapy, increased myocardial iron was demonstrated by MRI using signal intensity ratios to skeletal muscle, after 75 U of blood or when the liver iron concentration exceeded 400 ␮mol/g (∼ 23 mg/g dry wt.).241 Correlations between

704 liver iron concentration and myocardial iron and between serum ferritin and myocardial iron were seen prior to initiation of chelation in these patients: After 6 months of chelation therapy with desferrioxamine, these correlations were still present but less strong in the case of serum ferritin and myocardial iron. With chronic chelation therapy in thalassemia major, a clear relationship between liver iron and cardiac iron is no longer seen,244 most likely because the rate of iron removal from the liver by chelation therapy exceeds that from the heart.245 With modern chelation regimes, as many as 45% of thalassemia major patients still show evidence of myocardial iron loading on MRI.246 Iron loading in the heart can occur after multiple transfusions in a variety of other conditions such as Diamond–Blackfan anemia and sideroblastic anemia.246 In sickle cell disease, multiple transfusions are less likely to result in myocardial iron deposition,246,247 perhaps as a result of lower levels of plasma NTBI compared with thalassemia patients matched for body iron load.248

Mechanisms of Iron Overload Toxicity In the absence of iron overload, iron is unavailable to participate in the generation of harmful free radicals by its binding to physiological ligands such as transferrin. When iron overload develops, these protective mechanisms become saturated, and NTBI249 and increased quantities of redox-active low-molecular-weight intracellular iron250 are available to participate in the generation of harmful free radicals. NTBI species are likely to be heterogeneous, consisting of iron citrate monomers, oligomers and polymers and protein-bound forms.251,252 In experimental models, NTBI is rapidly taken up by hepatocytes253 and by myocytes.254 Although the most important effects of NTBI result from its pattern of uptake and subsequent iron accumulation in tissues expressing appropriate receptors,231 plasma NTBI (or subtractions of it) can generate lipid peroxidation through free radical formation,255 and depletion of plasma antioxidants is associated with increased NTBI levels.256 In cultured heart cells, species of NTBI are taken up at 200 times the rate of transferrin iron and generate free radicals, lipid peroxidation, organelle dysfunction, and abnormal rhythmicity.254,257 A free radical is defined as any species capable of independent existence that contains one or more unpaired electrons. The products formed sequentially during the reduction of molecular oxygen (O2 ) to water are superoxide (O2 -), hydrogen peroxide (H2 O2 ) and the hydroxyl radical (·OH), the reduction of the latter resulting in the formation of water. The hydroxyl radical has a great affinity for electrons and will oxidize all substances within its immediate vicinity diffusion radius of 2.3 nm.258 H2 O2 is relatively stable and nontoxic by itself. It is, however, an important precursor of ·OH requiring the availability of catalytic trace elements such as iron, copper, or cobalt. Iron is particularly important because it is present at sufficient concentrations

Janet L. Kwiatkowski and John B. Porter in tissues and because of the favorable redox potential of the Fe2+/ Fe3+ couple (between +0.35 and -0.5 V). The oxidation of Fe2+ to Fe3+ , with the concomitant generation of the ·OH from water, is referred to as the Fenton reaction. H2 O2 + Fe2+ → OH− + HO
+ Fe3+ (Fenton reaction)# Within cells are a number of physiological scavengers of toxic oxygen products such as superoxide dismutase, which dismutates O2 - to H2 O2 , and catalase, which scavenges H2 O2 . Tissue damage depends on the relative rates of formation and scavenging of toxic free radicals. ·OH can damage proteins, DNA, and membrane lipids by peroxidation. Particularly important is the initiation of peroxidation of lipid (Lp) by hydrogen abstraction:259 LpH + HO
→ Lp
+ H2O. Because iron is concentrated in hepatocyte lysosomes in both primary and secondary iron overload, these organelles are particularly susceptible to lipid peroxidation.260,261 The resulting lysosomal fragility262 is directly proportional to the degree of iron overload.263 In cultured heart cells, iron increased lysosomal instability and caused direct damage to mitochondria.257,264 Lipid peroxidation and damage to organelles and lysosomal fragility might lead to apoptotic cell death265–267 but might also encourage fibrogenesis. Iron-induced aldehyde lipid peroxidation products such as malondialdehyde268 and others269 have been shown to promote collagen gene expression and fibrogenesis in cultured fibroblasts and perisinusoidal stellate (Ito) cells. Fibrogenesis is also associated with autocrine production of transforming growth factor–␤ (TGF␤) in stellate cells270 and increased mRNA levels of TGF␤ and procollagen al(I) have been observed in a model of iron and alcohol induced fibrogenesis.271 Increased TGF␤ expression and malondialdehyde protein adducts have also been reported in hepatocytes and sinusoidal cells of patients with genetic hemochromatosis,272 suggesting that iron overload increases both lipid peroxidation and TGF-β gene expression, which together might promote hepatic injury and fibrogenesis. In other tissues, similar mechanisms are likely to be involved, although the concentration and distribution of intracellular iron clearly differs among tissues: Tissues that have a high mitochondrial activity could be particularly susceptible to iron toxicity.273 A scheme for iron-induced cell injury is shown in Figure 29.2.

Clinical Consequences of Transfusional Iron Overload The consequences of transfusional iron overload and the effects of chelation treatment on these consequences are best described in thalassemia major, in which transfusion typically begins in the first few years of life and transfusional iron loading occurs at a rapid rate. In other forms of transfusional iron overload, the pathological effects of transfusional iron overload are less well documented.

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease

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desferrioxamine infusions in the late 1970s277 (Fig. 29.3). Risk factors for the development of fatal cardiomyopathy include late commencement of chelation therapy,276,279 failure Oxidative Stress to maintain serum ferritin below 2,500 ␮g/L over a period of a decade or more,277,279–283 failure to control iron concentration below 15 Lipid peroxidation mg/g dry weight,276,282 poor compliance with chelation therapy,276,279,281 and a fall in ejection fraction below reference ranges.279 Development of left ventricular dysfunction is increasingly likely when a myocardial T2∗ values fall Organelle damage below 20 m sec244 and especially if T2∗ values TGFβ eg lysosomes, mitochondria are less than 8 msec.284 Hypogonadism commonly occurred in 55% of patients aged more than 12 years285 and Lysosomal fragility Collagen synthesis led to disturbances of growth and sexual matenzyme leakage uration. The frequency of this complication is falling in progressive birth cohorts treated from an early age with desferrioxamine, so Cell death Fibrosis that at age 20 years, 65% of patients born between 1970 and 1974 had evidence of hypogonadism, and this fell to 14% in 1980–1984 Figure 29.2. Mechanisms of liver damage in iron overload.277 birth cohorts277 (Table 29.8). Hypogonadism is typically secondary to anterior pituitary dysfunction (hypoThalassemia Major gonadotrophic hypogonadism), and hence in many female In thalassemia major, if chelation treatment is withheld, thalassemics with secondary amenorrhea,286 induction of death from iron-induced cardiac failure is common from ovulation and successful pregnancy are often achievable.287 274–276 the second decade on. By contrast, in conditions The effects of iron damage to the anterior pituitary with such as genetic hemochromatosis in which iron loading consequent hypogonadotrophic hypogonadism will clearly is slower, cirrhosis is a common presenting feature, usube more devastating if transfusion begins early in life and ally in the fourth and fifth decades, and commonly leads damage occurs before growth has ceased and full sexual to death from liver failure or hepatocellular cancer. Cirrhomaturity has been achieved. sis is also a common feature in thalassemia major, being Other complications such as diabetes, hypothypresent in 50% of patients at postmortem examination, parroidism,288 and hypoparathyroidism are seen in a variticularly if chronic infective hepatitis (e.g., hepatitis C) is able proportion of patients, and the frequency of these also present. Although cirrhosis was a relatively uncomcomplications is also falling with improved chelation. mon cause of death in thalassemia major275 as patients live longer with improved chelation, cirrhosis and hepaSurvival in birth cohorts with DFO tocellular carcinoma277 are likely to become increasingly 85-97 80-84 common problems. Liver fibrosis can develop early in the 1.00 75-79 course of thalassemia major, even in the absence of infec70-74 tive hepatitis. For example, fibrosis has been observed 278 as early as 3 years after starting transfusion. Fibrosis 0.75 correlates with age, the number of units of blood trans65-69 fused, and with liver iron concentration.230 In these stud0.50 ies there was an exponential increase in hepatic fibrosis 60-64 with increasing liver iron concentrations. The concentration of liver iron at which fibrosis progresses was examined 0.25 p < 0.00005 in ex-thalassemic patients who had received a successful bone marrow transplant.239 In the absence of active hepA 0.00 atitis C, fibrosis only progressed when liver iron concentrations exceeded 16-mg/g dry weight. 0 5 10 15 20 25 30 Age (years) The most common cause of death in thalassemia major remains iron-induced cardiomyopathy but this has Figure 29.3. Survival by birth cohort in desferrioxamine-treated thalassemia been reduced since the introduction of subcutaneous major patients.277 Survival probability

Free iron

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Janet L. Kwiatkowski and John B. Porter

Table 29.8. Falling complications since the introduction of DFO chelation therapy patients with thalassemia major born after 1960, n = 977

Death at 20 y Hypogonadism Diabetes Hypothyroidism

Birth 1970–1974∗

Birth 1980–1984†

5% 64.5% 15.5% 17.7%

1% 14.3% 0.8% 4.9%

∗

DFO intramuscularly, 1975 DFO subcutaneously, 1980. In 1995, 121 patients switched to DFP (censored at this time).

†

The frequency of diabetes at age 20 years has fallen from 16% to 0.8% between birth cohorts from 1970–1974 to 1980–1984277 (Table 29.8). In optimally treated patients who receive more than 260 infusions of desferrioxamine each year, these complications are absent at 15 years of follow-up.281 Low levels of adrenal androgen secretion with normal glucocorticoid reserve have also been reported.289 Other features of iron overload include skin pigmentation, arthropathy, ascorbic acid deficiency and osteoporosis.219 Infections are the second most frequent cause of death in thalassemia, occurring particularly commonly in younger age groups.275 Postsplenectomy sepsis due to encapsulated organisms is an important contributor,290 but iron overload also appears to play a central role in increasing susceptibility to infection. Reports of infection with Y. enterocolitica, Vibrio vulnificus, Listeria monocytogenes, Escherichia coli, Candida sp. and Klebsiella can be found.291–294 The increased infective risk partly results from the saturation of transferrin with iron, depriving the body of an important mechanism to withhold this nutrient from bacteria. Defective neutrophil and macrophage function,295–297 presumably secondary to the effects of excess iron, might also be important. Some bacteria such as Yersinia are able to utilize iron from iron complexes of chelators.298 This is particularly likely if the chelator is a naturally occurring siderophore such as desferrioxamine. The relevance of the rate of iron loading to the clinical manifestations of iron overload is further illustrated by the contrasting complications of excessive iron in thalassemia major and thalassemia intermedia. Thalassemia intermedia patients have different transfusion requirements.218 Because of the slower buildup of iron, damaging levels of iron loading typically develop later than in thalassemia major and might not affect pituitary function until the third or fourth decade or beyond. Therefore, in thalassemia intermedia, growth and sexual development are typically unaffected by iron overload per se, and, when present, are more likely to be the consequences of ineffective erythropoiesis and anemia.

Sickle Cell Disease In sickle cell disease, in which regular blood transfusions to correct anemia are usually not needed, iron overload only

develops in individuals who receive repeated blood transfusions to prevent certain complications. In the minority of patients who receive repeated transfusions sufficient to cause significant iron overload, clinical consequences begin later than in thalassemia major, and thus effects on growth and sexual development are relatively uncommon. The use of the serum ferritin to estimate iron loading is problematic in sickle cell disease. The ferritin level is disproportionately increased in relation to iron loading for several weeks after a painful sickle episode299 and often correlates poorly with hepatic iron concentration,300 although under carefully controlled conditions a correlation is seen.301 Transfusion-induced iron loading in the liver301 with fibrosis and cirrhosis are seen.302–304 Portal fibrosis develops quickly: after 21 months of transfusion without chelation, fibrosis was found in the livers of approximately a third of patients; after 4 years of transfusion, with variable chelation, fibrosis was still present in a third of patients with cirrhosis and in one of 29 patients in the absence of hepatitis C infection.304 The fibrosis score correlates with liver iron concentration303 but develops at widely varying hepatic iron concentrations of 9–38 mg/g dry weight in the absence of hepatitis C infection.305 Postmortem studies have found that cirrhosis was present in nearly half of patients who died with severe liver siderosis.306 Postmortem studies also show clear evidence of iron deposition in the heart in heavily transfused patients;240,306 however, MRI evidence suggests that the propensity for myocardial iron deposition246,247,307 is less than in other forms of transfusional iron overload. A variety of endocrine disturbances have been reported in sickle cell disease,308,309 including hypothyroidism310 and increased pancreatic deposition of iron.311 If sickle cell disease and thalassemia major patients matched for liver iron are compared, the incidence of heart disease, gonadal failure, and endocrine disturbances appears to be less in sickle cell disease patients aged younger than 20 years compared with thalassemia major.308 Sickle cell disease patients typically begin transfusion later than those with thalassemia, and the rate of transfusion is on average lower, so that without carefully controlled studies the significance of these differences is unclear. At similar levels of iron loading, plasma NTBI values are lower in sickle cell disease than in thalassemia,248,312 and this could account for decreased iron distribution to organs such as the heart.247,248,307 Lower plasma NTBI values could result from the existence of a chronic inflammatory state in sickle cell disease312 that would depress NTBI though increased hepcidin synthesis.

Other Transfusion-dependent Anemias In multitransfused patients with myelodysplastic disease and a mean transfusional load of 120 U, there was evidence for multiple endocrine disorders including impaired glucose tolerance, decreased gonadotrophins, and impaired ACTH reserve.313 A functional impact of myocardial iron deposition was suggested by one study in which heart

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease failure was the cause of death in 51% of patients.314 Among 705 patients followed for 2 years or until death, cardiac comorbidities were seen in 79% of chronically transfused patients, in only 54% of nontransfused patients, and in 42% of a control population.315 Furthermore overall survival was significantly worse in iron overloaded myelodysplastic disease patients compared with untransfused patients;316,317 an independent effect of iron loading on survival is uncertain without prospective studies.

Monitoring of Iron Overload Liver Iron Concentration Liver iron concentration (LIC) is a key measure of iron overload because its value relates to body iron stores in a well-defined mathematical relationship: body iron stores in mg/kg = 10.6 x the LIC (in mg/g dry weight).318 Normal LIC values are up to 1.8-mg/g dry weight and levels up to 7 mg/g dry weight are seen in some nonthalassemic populations without apparent adverse effects.319 In principle, iron balance with a given chelation regime can be calculated from the change in LIC over time, provided the iron input from transfusion is known. Change in LIC over a given time can also be used to calculate the efficiency of a chelator (i.e. the proportion of administered drug that is excreted in an iron bound form). Thus the change in LIC over a period of time period (6 months–1 year) is useful way to assess the efficiency and effectiveness of an iron chelator regime. Measurement of LIC also has prognostic and pathophysiological significance. Sustained LIC values more than approximately 15–20 mg/g dry weight over a period of time predict an increased risk of liver fibrosis progression239 and liver function abnormalities.238 In patients not receiving chelation therapy, similar LIC values predict an increased risk of myocardial iron deposition240,241 and iron-induced cardiomyopathy.276,282 Intensive chelation therapy reduces liver iron faster than cardiac iron so that a simple relationship between LIC and estimated heart iron values is often not seen once long-term or intensive chelation therapy has been used.245 Patients might develop heart failure after iron has been removed from the liver because excess iron remains in the heart.320 Although low LIC in previously highly iron overloaded patients does not necessarily guarantee low heart iron concentrations, sustained high levels of LIC has clearly poor prognostic significance. The use of LIC to follow the progress of transfusional iron overoad is becoming easier because noninvasive approaches to measurement are now more widely available. Liver biopsy to measure iron overload has the obvious disadvantage that it is invasive and carries a small risk of bleeding but in expert hands, using ultrasound guidance, no mortality was seen in more than 1,000 biopsies, with an overall complication rate of 0.5%.321 Although liver iron is somewhat uneven in distribution,322 this is not a serious problem provided a sample of 1 mg dry weight (4 mg wet or

707

∼ 2.5 cm in length) is obtained.318,323 The presence of cirrhosis could increase the unevenness of iron distribution and hence the accuracy of LIC measurement.323 In expert hands, with established methodology,324 and with samples of adequate size, and in the absence of cirrhosis the coefficient of variation on duplicate specimens is only 6.6%.325 A problem in comparing values between studies is that the measurement of wet and dry weights of samples varies considerably among laboratories. Paraffinembedded biopsy material can be used to measure LIC324 but a recent multicenter study has shown that the ratio of wet/dry weight differs using this approach when compared with using fixed nonembedded tissue, being higher in paraffin-embedded tissue (5.9) than vacuum-dried fresh samples (3.8).326 In many clinics worldwide, the regular measurement of LIC by biopsy is organizationally difficult. Furthermore, many patients who have been monitored successfully for many years by using the serum ferritin level are reluctant to undergo yearly liver biopsies. On the other hand, liver biopsy yields additional information about inflammation and fibrosis that might be particularly important in monitoring the activity of hepatitis C or evaluating experimental chelation regimens. Because of the increased use of LIC to monitor treatment, there is a need to harmonize both the techniques used to measure LIC and the units in which it is expressed. For example, some laboratories weigh the wet samples but use a conversion factor from wet to dry weight and express results in the latter units. A variety of conversion factors from wet to dry weight have been used by different centers. The implementation of an internationally agreed on standard and methodology would be helpful to the interpretation of studies on outcome of chelation treatment. For these reasons, a noninvasive method to measure LIC that is reliable, standardized, and widely available would be highly desirable. Noninvasive measurement of LIC has been possible for many years by using superconducting quantum interface devices (SQUIDs) but these are expensive and only available in four centers worldwide (west and east coast of the United States, Germany, and Italy) and have been calibrated using different approaches. Excellent correlations with LIC values measured by biopsy were reported in hemochromatosis patients (r = 0.98)327 but less impressive correlations were obtained in thalassemia,328 possibly because of the diverse methodology for LIC biopsy measurements. Standardization between these devices has been problematic.329 In recent multicenter trials using SQUID, significant differences in SQUID measurements were obtained from different centers.213 The current development of room temperature handheld susceptometric devices would have wider applications. Several MRI techniques for measuring LIC are now available,330,331 relying on the general principle that tissue iron exerts a paramagnetic effect on surrounding tissues that affects the relaxation time of molecules excited by the application of a magnetic field. There is now a standardR ized and validated MRI method (FerriScan
) in which there

708 is a linear relationship between the measured value (R2) and LIC by biopsy over a clinically useful range.330 Further advantages of this method are that it uses standard MRI equipment that is available in most hospitals, can be used with little extra training of local staff, and analysis of data acquired locally is performed at a central facility.

Use and Limitations of Serum Ferritin Although LIC predicts body iron and this can now be measured noninvasively, the practical advantages of having a blood test for monitoring iron overload that can be repeated with each clinic visit are obvious. Serum ferritin fits many of the requirements as it broadly correlates with iron loading and can be performed frequently and inexpensively. There are limitations to its use and interpretation: Serum ferritin is increased not only by increased body iron stores but also as a result of tissue damage and inflammation; levels are depressed by ascorbate deficiency. Unlike tissue ferritin, serum ferritin is predominantly iron free, being secreted in glycosylated form by macrophages proportional to their iron content, up to values of approximately 3,000 ␮g/L.332 Above this value, iron-rich ferritin tends to leak from hepatocytes so that responses to treatment at levels more than 3,000 ␮g/L might occur at a different rate compared with values below 3,000 ␮g/L.333 The relationship between serum ferritin and body iron stores can vary with the chelator being used, and it is important to define such a relationship for each chelation regime so that informed dose adjustments can be made.334 Serum ferritin is usually measured by immunoassay with reference to an agreed international standard; however, ‘kits’ for measuring serum ferritin have been optimized for identifying iron deficiency so care must be taken at high ferritin values to ensure that dilutions allow measurements within the linear range of the assay. In normal subjects335 and in genetic hemochromatosis336 a good correlation exists between iron mobilizable stores by quantitative phlebotomy and serum ferritin. With transfusional iron overload in the pre–chelation era, a correlation of serum ferritin with the number of units transfused in thalassemic children was present,337 but this correlation was reduced in adults.332 Serum ferritin also correlates with the LIC; in children with thalassemia major, this correlation was present both in those receiving and not receiving chelation therapy (r = 0.75).337 In patients with HbE–␤ thalassemia and HbH disease, a correlation of r = 0.82 was noted between the log of serum ferritin and liver iron measure by SQUID.218 A very weak correlation between serum ferritin and LIC was seen in a diverse group of patients, many of whom had liver disease (r = 0.11). When the serum ferritin was corrected for liver inflammation, by dividing by the aspartate aminotransferase, the correlation was much closer (r = 0.92).338 More recent studies, although confirming the general relationship between serum ferritin and LIC, have emphasized that this is not close enough in multiply transfused thalassemia patients,

Janet L. Kwiatkowski and John B. Porter many of whom have hepatitis C, to give a clinically reliable prediction of LIC.339,340 In one study, variation in body iron stores accounted for only 57% of the variability in plasma ferritin.301 The relationship between serum ferritin and iron stores is similar in thalassemia major and sickle cell disease301 provided serum values are taken several weeks away from a vasoocclusive episode,224 but in thalassemia intermedia, serum ferritin tends to underestimate the amount of iron overloading.234 Depression of serum ferritin occurs in ascorbate deficiency. Subclinical but biochemically demonstrable ascorbate deficiency is common in iron-overloaded patients256,341 due to the rapid oxidation of ascorbic acid in the presence of redox active iron.342 In ascorbate-deficient patients, the correlation between serum ferritin and LIC was 0.39, whereas in ascorbate-replete patients it was 0.77341 . Repletion of ascorbate status increased transferrin saturation and serum ferritin rose in approximately half the treated patients. In an individual patient with iron overload, long-term control of serum ferritin, at least with desferrioxamine therapy, has prognostic significance. If the ferritin is maintained below 2,500 ␮g/L on a long-term basis, this is associated with a significantly lower risk of iron-mediated cardiac disease and death.277,280–282,343 Maintenance of a serum ferritin of 1,000 ␮g/L might be associated with additional advantages.277 Serum ferritin has also been used as means of modifying the dose of chelation treatment, based on experience with desferrioxamine therapy in which toxicity due to this agent is more likely in the context of low serum ferritin levels. Dose modification algorithms have been constructed for desferrioxamine under circumstances of falling ferritin values;340,344 however, the basis for dose modification with other chelation regimens has yet to be established.

Monitoring of the Heart Iron-mediated cardiomyopathy remains the most common cause of death in thalassemia major;277 therefore, the identification of patients at the greatest risk is of paramount importance, so that effective intensification of therapy can be introduced. Long-term quantitative sequential measurement of left ventricular ejection fraction in thalassemia major has shown that a fall below reference values indicated a 35-fold increased risk of cardiac failure and death with a median interval to progression of 3.5 years.343 Longitudinal monitoring of heart function is best achieved using a method that can be standardized and is not operator dependent. In the hands of an expert, echocardiography is a useful tool for visualizing heart function but it is difficult to quantify left ventricular ejection fraction reproducibly over time. Mulitgated acquisition scanning is well suited to this purpose as it has excellent reproducibility and less operator dependence, but MRI techniques are now widely available and measurement of left ventricular ejection fraction

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease function can now be combined with measurement of heart iron in a single visit. Little or no role exists for measuring cardiac iron by biopsy because of the uneven distribution of cardiac iron.240,242,243 MRI can be used to estimate tissue iron because iron overload results in shortening of the T1 and T2 tissue relaxation times on which MRI is based and thus leads to a reduction in signal intensity. The decrease in the intensity of spin echo images with iron overload derives from decrements in the T2 relaxation times.345,346 This shortening is due mainly to the paramagnetic properties of ferritin iron.346,347 The problem in measuring heart iron was that levels were considerably lower than in the liver and the heart is rapidly moving. Early approaches lacked sensitivity over a wide enough linear range but several MRI methods have now been developed that are applicable to the heart.241,244 Myocardial T2∗ is a gradient echo method that is now the most widely used to estimate myocardial iron and has been found to be highly reproducible.348 The T2∗ gradient echo sequence was first used in imaging of iron deposits in the brain349 but is particularly applicable to myocardial imaging because a short acquisition time is possible, which is advantageous when measuring a moving tissue. T2 is related to T2∗ by the formula 1/T2∗ = 1/T2 + 1/T’, where T2 is the tissue relaxation and T’ is the magnetic inhomogeneity of the tissue being analyzed. As the T2∗ becomes shorter implying higher tissue iron concentrations, there is an increased chance of left ventricular function being adversely affected.244 In one study, the left ventricular ejection fraction was decreased in 10% of patients with T2∗ less than 20 msec, 18% of patients with T2∗ values between 8 and 10 msec, 38% of those with T2∗ of 6 msec, and 70% at 4 msec.284 Myocardial iron deposition has been found by using this technique in as many as half of adult patients with thalassemia major and in a variety of other forms of transfusional iron overload.246 Myocardial T2∗ can be used to identify patients most at risk of myocardial decompensation, which can occur suddenly over a period of a few days, and allow consideration of intensification of chelation therapy in those most at risk prior to decompensation.

Monitoring for the Clinical Effects Iron Overload The earliest consequence of iron overload in transfused children is hypogonadotrophic hypogonadism, which although not fatal, has severe consequences for growth, sexual development, fertility, and bone health.286 Close monitoring of growth and sexual development in children is therefore vital so that chelation can be intensified before irreversible effects ensue. In older patients, regular monitoring for the cardiological, endocrinological, and growth and developmental effects of iron overload is an essential part of management of iron overload. For example, monitoring for impaired glucose tolerance might identify patients at most risk of developing diabetes. Monitoring for hypothyroidism and hypoparathyroidism is also advisable,

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Table 29.9. Monitoring for iron toxicity from transfusion Observation

Frequency

Iron intake rate Chelation dose and frequency Growth and sexual development Liver function Sequential ferritin GTT, thyroid, Ca metab. Liver iron Heart function Heart iron (T2∗ )

Each transfusion 3 monthly 6 monthly children 3 monthly 3 monthly Yearly in adults Yearly Yearly Yearly

Expense

particularly in adults. As there is no clear evidence that damage to these tissues can be reversed, prevention of these complications by adequate control of body iron at all times is the optimal strategy. A scheme for such monitoring is outlined in Table 29.9 showing that monitoring will tend to be predicated by the availability of local resources.

Monitoring Tissue Iron in Other Organs MRI can be used to visualize iron in other organs affected by iron loading such as the anterior pituitary and pancreas. MRI of the pituitary shows a correlation between anterior pituitary size and biochemical markers of anterior pituitary function.350 More recently T2∗ MRI has been used to assess iron in the anterior pituitary,351,352 but prospective studies are required to determine how changes in pituitary T2∗ relate to biochemical evidence of disturbed gonadotrophin production. Attempts have been made to use MRI to relate changes in pancreatic function to iron deposition and a possible correlation between MRI signal and pancreatic exocrine but not endocrine function was found.353,354

Urine Iron Excretion Measurement of urinary iron has been used with desferrioxamine monotherapy, with deferiprone monotherapy, and with combinations of the two drugs. Because all iron is excreted by the fecal route with deferasirox, urinary iron measurement has not been used with this chelator. A relationship between 24-hour urinary iron excretion following intramuscular desferrioxamine and iron stores was recognized soon after the introduction of the chelator.355 Later it was suggested that 24-hour urinary iron excretion could be used to titrate an individual’s therapy with desferrioxamine by “tailoring the dose”356 as the dose at which the urinary iron excretion plateaus varies considerably.356 Several limitations to this approach exist. First, there are practical difficulties for many families in obtaining reliable sequential 24-hour urine collections outside of a hospital setting. Second, there is considerable day-to-day variability in urinary iron excretion when taking desferrioxamine, even under controlled conditions of collection, and this might be compounded by variable daily ascorbate

710 intake.356 Finally, the proportion of urinary iron excretion relative to fecal iron excretion is not constant. The urinary iron excretion decreases following blood transfusion and suppression of erythropoiesis and is proportionately less at higher doses of desferrioxamine.357 Whereas fecal iron excretion contributes to at least half of desferrioxamineinduced iron excretion there is little fecal excretion with deferiprone, making urine iron determination a potentially useful tool. When the two drugs are used in combination, however, changes in urinary iron excretion without reference to total iron removal or the fecal excretion are uninterpretable.

Other Markers of Iron Overload Plasma transferrin saturation increases with increasing iron loading but is also affected by inflammation,358 hepcidin synthesis,359 and degree of erythropoiesis,360 making its use in the management of transfusional iron overload of limited value. NTBI is present when transferrin becomes saturated in iron overload249 and sometimes before.361,362 Plasma NTBI negatively correlates with evidence of antioxidant depletion.256 NTBI is highly labile, being only partially removed with chelation therapy and returning rapidly after elimination of the chelator.363 NTBI is also affected by factors other than iron overload. For example, fewer ironloaded thalassemia intermedia patients appear to have higher NTBI levels, which rebound faster after cessation of iron chelation than thalassemia major patients with higher levels of iron loading. This suggests that ineffective erythropoiesis may influence plasma NTBI.364 Plasma NTBI is heterogeneous, consisting of iron citrate of varying molecular weights and loosely protein bound iron species.252 Total plasma NTBI can be measured by a number of methods.365,366 An assay has been developed for a redox active subfraction that has been termed labile plasma iron.367 Assays for NTBI have variable reference ranges but generally correlate.365 An assay measuring a labile subfraction, the component capable of accelerating oxidation of a fluorophore, termed labile plasma iron,367 is convenient for measuring in the presence of chelators. Progressive removal of this subfraction has been seen with deferasirox,368 consistent with the notion that continuous chelation minimizes exposure to NTBI species.364 The application of NTBI or labile plasma iron as a way of tailoring chelation therapy on an individual patient basis has yet to be determined. How hepcidin levels can be used to guide chelation management is also unclear.233,369

Objectives of Chelation Therapy Iron Balance The primary objective of chelation therapy is to balance iron excretion with intake so that tissue iron never reaches damaging levels. If this strategy is successfully applied,

Janet L. Kwiatkowski and John B. Porter iron-mediated tissue damage is prevented. Unfortunately many patients either start treatment too late or fail to maintain effective exposure to iron chelation so that rescue chelation therapy is needed. Rescue therapy has two objectives: to detoxify iron pools promoting acute tissue damage and to remove excess storage iron from affected tissues. The first objective can be achieved rapidly in some circumstances, for example by inducing rapid reversal of heart failure before much storage iron has been removed. The second goal of removing storage iron can take months or years especially in tissue not designed to store iron such as the heart.245 Because of the difficulty in removing established storage iron overload from heart, pituitary, thyroid, and pancreas, a long-term strategy to prevent primary iron loading is preferable. At lower levels of iron loading, chelation therapy might lead to removal of essential metals required for physiological functions. In this instance, over chelation too early in the iron loading process could lead to unwanted toxicities.370 Iron chelation requires that iron loading and excretion be balanced and that the intensity of chelation is matched to the level and rate of iron loading.

Maintenance of Safe Tissue Iron Concentrations How the age iron loading begins, its rate, and how the absolute levels of iron loading contribute to tissue damage or determine the distribution of iron overload are incompletely understood. Thus there is debate about what constitutes “safe” levels of body iron burden. Good evidence supports the notion that control of serum ferritin (mainly iron free), liver iron (iron-rich ferritin and hemosiderin), and myocardial T2∗ visible iron (iron-rich storage iron) has prognostic significance. This does not mean that these forms of iron are the direct cause of tissue damage however, but rather that these are valuable markers for potential iron-mediated problems. Both biopsy and MRI techniques measure storage iron rather than the small fraction of labile iron that is directly toxic to cells.264,371,372 The slow accumulation of iron in heterozygotes for hereditary hemochromatosis is typically not associated with liver damage, even though LIC values can approach 7 mg/g dry weight.319 Conversely, liver fibrosis might develop in transfused and nontransfused,373 iron-overloaded patients at or around these levels.303,304 Progressive liver damage and abnormal liver tests in the absence of viral hepatitis are associated with LIC values of more than 16 mg/g dry weight239 and similar sustained values over a decade or more are associated with an increased risk of cardiomyopathy in thalassemia major.276 Yet we do not yet understand the relationship between myocardial iron load and LIC, particularly in patients undergoing chelation therapy. In the pre–chelation era, or in patients undergoing only light chelation therapy, there appears to be a relationship between myocardial iron loading and LIC, with myocardial iron appearing after approximately 75 U of blood in

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease

711

Cellular interactions of iron chelators

Transferrin iron

Ferritin (c) LVDCC

Nontransferrin Iron (a)

Plasma

Bile Labile Iron (b)

Storage Fe

Lysosomal

d degradation Labile Fe

Fe

Free-radical generation

Fe

Functional Iron proteins

Organelle damage LVDCC = L-type voltage-dependent calcium channel.

Fe Fe

Although it can take months or years to remove all the excess iron from iron-loaded subjects, a well-designed chelator should ideally decrease the iron-mediated damage rapidly and until all the excess storage iron has been removed. To do this, potentially harmful labile iron pools must be removed rapidly and rendered harmless246 (Fig. 29.4). As iron pools are constantly being turned over, continuous exposure to chelation is theoretically desirable, both in terms of maximizing the efficiency of chelation therapy and minimizing the exposure to harmful free radicals generated by labile iron species. Clinical data supporting the concept of detoxification of iron before storage iron has been changed significantly are seen in studies with intravenous desferrioxamine in which improvement in liver tests is rapid and precedes changes in T2∗ reflecting storage iron.246

Iron Pools Available for Chelation Labeling of iron pools both in experimental animals374,375 and in humans376 suggest that only a small proportion of total body iron is available for chelation at any moment. This is broadly related to two iron turnover pools: red cell catabolism in macrophages (1 in Fig. 29.5): hemosiderin and ferritin catabolism in hepatocytes (2 in Fig. 29.5). This has a fundamental bearing on the strategies adopted for chelation therapy because it follows that escalation of chelation doses will have a finite impact on increasing iron

Hepatocyte

Fe

Macrophage

Fe Fe Fe

Feces

Detoxification of Harmful Iron Species

2

Red cell catabolism

Fe Fe Fe Fe

Kidney

Fe

Figure 29.4. Mechanism of chelator action at cellular level.246

adults (15–20 mg/g dry weight with LIC). Together, the data suggest that it would seem wise to commence chelation therapy before LIC values approach 15 mg/g dry weight, probably at a time when LIC values exceed approximately 7 mg/g dry weight, and to aim to maintain LIC values lower than 7 mg/g dry weight. Whether the maintenance of LIC values lower than 7 mg/g dry weight at all times will prevent myocardial iron loading requires prospective evaluation for which studies are currently ongoing.

Fe

1

Fe

Urine

Figure 29.5. Major chelatable iron pools and excretion pathways.249

excretion. The reason for this can be appreciated from consideration of both extracellular and intracellular iron turnover.

Extracellular Iron The major turnover of body iron, amounting to some 20– 30 mg/day in healthy individuals, is through the plasma compartment from the breakdown of effete red cells in the reticuloendothelial system. Iron is released onto transferrin for subsequent delivery to erythroid precursors in the bone marrow.377 This can increase up to sevenfold when hemolysis and ineffective erythropoiesis are pronounced, as in thalassemia intermedia. Considerable evidence both in animal studies375 and in humans376 indicates that iron derived from the breakdown of hemoglobin in macrophages is chelated directly by desferrioxamine and other compounds such as DTPA before binding to transferrin378 (Fig. 29.4). Whether this interception occurs extracellularly or within the macrophages is uncertain, but in vitro studies suggest the former is more likely with desferrioxamine.379 This will presumably vary depending on rates of access of the chelator in question to intracellular iron pools in macrophages. Studies using selective 59 Fe cell–labeling techniques in iron-overloaded rats374,375,380 and ferrokinetic data in humans376 suggest that most urinary iron excretion with desferrioxamine is derived from catabolized red cells (1 in Fig. 29.5). Although other chelators used for the treatment of iron overload are excreted though different pathways than desferrioxamine the sources of chelatable iron are essentially the same. Thus with deferasirox, animal studies show that although nearly all iron bound to the chelator is excreted in feces, much of this is derived from red cell breakdown.381 Deferiprone-bound iron is excreted almost entirely in urine but there is evidence for chelation of iron both from red cell breakdown and from parenchymal stores.382

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Janet L. Kwiatkowski and John B. Porter Table 29.10. Characteristics of approved chelating agents

Denticity Molecular weight Iron (III) binding (pM) Lipid solubility Charge free ligand Charge iron complex Absorption route Max. plasma levels Elimination Route of iron excretion Metabolism

Deferiprone

Desferrioxamine

Deferasirox

Bidentate 175 19.9 High Neutral Neutral Oral 90–450 ␮M Rapid Urine Inactive glucuronide

Hexadentate 560 26.6 Low Positive Positive Parenteral 7 ␮M (total) Very rapid Urine + Feces Active metabolites

Tridentate 373 22.5 High Negative (1-) Negative (3-) Oral 20 ␮M Slow Feces Active glucuronide

Another potential source of chelatable iron outside cells is transferrin-bound iron (Fig. 29.4). Although this is quantitatively very small (∼ 4 mg) at any moment, approximately 20–30 mg pass through this pool every day after iron is released from reticuloendothelial cells. In principle, iron chelators are in competition with transferrin for iron released by reticuloendothelial cells. Because the concentration of transferrin in plasma exceeds that of desferrioxamine, deferasirox, or deferiprone, except at transient peak concentrations, and because the stability constant of transferrin for iron is comparable to that of these chelators, iron binds effectively to transferrin even when chelators are being used. Furthermore, once plasma iron has bound to transferrin it is virtually unavailable for chelation by desferrioxamine.380 Hydroxypyridinones can remove iron from transferrin but the concentrations required to do so at a significant rate make this an unlikely mode of action in vivo. For example, at 100 ␮M concentrations of deferiprone, approximately a quarter of the iron is removed from 90% saturated transferrin (50 ␮M) and only 12% for 30% saturated transferrin after 24 hours.383 It must also be remembered that iron can be donated to transferrin by bidentate hydroxypyridin-4-ones but not by desferrioxamine at clinically relevant concentrations of transferrin and chelator.383 This is because the iron complexes of bidentate chelators are inherently less stable than those of hexadentate ligands such as desferrioxamine. Thus, because the plasma concentrations following orally administered chelators such as deferiprone are only transiently above 100 ␮M384 (Table 29.10), donation of iron to transferrin might be as likely as iron removal, particularly when transferrin is not saturated in the steady state. Another important potentially chelatable extracellular iron pool is NTBI (Fig. 29.4). Total NTBI values do not usually exceed approximately 10 ␮M364 but the speciation is heterogeneous. These forms are not all equally available for rapid chelation.252 Iron citrate polymers are more slowly accessed by chelators than monomers and dimers.252 In plasma of iron-overloaded patients at clinically relevant concentrations, not all NTBI is removed by desferrioxamine.363 The component of NTBI that is

rapidly accessed by desferrioxamine has been termed desferrioxamine-chelatable iron.385 Only the subfraction of NTBI capable of redox cycling, so-called labile plasma iron, is rapidly available for chelation.386 The contribution of chelation of NTBI to iron balance has not been determined, but removal of NTBI by chelation therapy is potentially important for iron detoxification. NTBI is only partly removed by chelation with desferrioxamine and rebounds rapidly after cessation of an intravenous infusion364 or subcutaneous infusion of chelator.363 Thus, with conventional nightly subcutaneous desferrioxamine infusion, NTBI can enter tissues during the day.363 Using the labile plasma iron assay, it was found that there is also rapid rebound of labile plasma iron at night with daytime use of deferiprone, but that when deferiprone is given three times a day with desferrioxamine at night, 24-hour coverage can be achieved.367 A similar degree of 24-hour coverage from labile plasma iron can be achieved with once daily deferasirox because of its longer plasma half-life.368

Intracellular Iron The major source of chelatable intracellular iron derives from the continuous turnover of intracellular ferritin and to a lesser extent hemosiderin (source b in Fig. 29.4 and source 2 in Fig. 29.5). Iron chelators can be considered as interacting potentially with three intracellular iron pools. These are the labile “transit” iron pool or (pool a), which is generally of low molecular weight, finite, and rapidly chelatable; the storage iron pool of hemosiderin and ferritin (pool b), which is relatively slowly chelatable but larger at any moment than pool a; and the functional iron pools (pool c), which are iron-containing molecules essential for normal cellular function (e.g., hemoglobin, myoglobin, enzymes such as lipoxygenase and ribonucleotide reductase). Pool a is in dynamic exchange with the other two pools. The goal of chelation therapy is to chelate pool a without affecting the important functions of iron in pool c (Fig. 29.4). Although some of pool b may be depleted directly, this is relatively slow because chelators cannot remove iron from ferritin cores at a clinically useful rate.

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease Transit iron can theoretically be chelated at any point during its uptake into cells by receptor-mediated endocytosis of transferrin, liberation from transferrin in the acidic endosome, or egress from the endosome via divalent metal ion transporter 1 (SLC11A2).387 Labile chelatable intracellular iron has been demonstrated in a variety of cells including reticulocytes, marrow cells, intestinal epithelial cells, blood leukocytes, alveolar macrophages, and hepatocytes, although evidence has been largely indirect and the nature of the pool is uncertain.371,388–391 It is clear that iron entering the cell, either from transferrin or by other mechanisms, becomes transiently chelatable before incorporation into ferritin.392 Early studies in Chang cells showed approximately 20% of cellular iron was present as a nonheme, nonferritin soluble form after 24-hour incubation393 and was rapidly chelatable with EDTA, desferrioxamine, or transferrin.394 Quantification of this pool from tissue homogenate iron extracted by desferrioxamine showed a correlation with an ultrafilterable fraction bound to lowmolecular-weight ligand(s).395 Studies using the fluorescent probe calcein in K562 cells suggested that the labile intracellular iron pool is present at 0.4 ␮M with an estimated transit time of 24 hours.390 Similar values have been obtained with rat hepatocytes labeled with 59 FeCl3 and analyzed for desferrioxamine-chelatable 59 Fe396 and using electron paramagnetic resonance in K562 cells.397 The magnitude of the labile iron pool is assumed to be sensed by a cytosolic iron-responsive protein so that increments in labile plasma iron increase ferritin mRNA translation and decrease transferrin receptor mRNA stability,398 thereby having homeostatic effects on labile plasma iron concentrations. When cells become heavily iron loaded or the rate of uptake of NTBI into cells exceeds an as-yet undetermined rate, the sequestrating capacity of cellular ferritin is exceeded,390 leading to an expanded labile plasma iron that is an obvious target of intracellular iron chelation therapy by drugs that are able to cross the cytoplasmic membrane. The same properties of chelators that favor mobilization of intracellular iron399 such as lipid solubility, also favor chelation of the labile intracellular pool400–402 . Thus hydroxpyridinones access labile iron pools more rapidly than desferrioxamine372,397,400,403 the latter taking approximately 4 hours, whereas hydroxpyridinones may take only a few minutes. Deferasirox appears to access labile intracellular iron rapidly and have access to iron pools within organelles.372,402 With respect to pool b, iron can be mobilized from ferritin by desferrioxamine in vitro but the rate is less than 0.5%/hour, even when desferrioxamine is present in 15-fold molar excess.404 Furthermore, acidic pH values, such as those present in the lysosome, do not appear to enhance this rate.405 Bidentate hydroxypyridinones can gain access to the ferritin core directly by virtue of their smaller size.406 At neutral pH values, iron mobilization from rat ferritin is still only 1%/hour even in the presence of 15fold chelator excess.405 Thus access by chelators to ferritin

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iron will occur at useful rates in vivo only when this protein is being degraded by proteolysis with the subsequent release of iron to the labile intracellular transit pool. It has been estimated that ferritin is turned over intracellularly with a half-life of approximately 72 hours407 and that proteolysis is predominantly lysosomal rather than cytosolic.408 With respect to pool c, it is clear that the physicochemical properties of chelators have a major influence on their rate of access to iron. Desferrioxamine, by virtue of its hexadentate structure, relatively high molecular weight (Table 29.10), and its hydrophilicity compared with the clinically studied hydroxypyridinones, accesses intracellular iron and iron within organelles more slowly than hydroxypyridinones.400,409 Furthermore, interaction with key enzymes in pool c such as lipoxygenase410 and ribonucleotide reductase411 is significantly slower for desferrioxamine than hydroxypyridinones. Because of the pivotal role of ribonucleotide reductase in DNA synthesis and cell proliferation, its rapid inhibition by hydroxypyridinones is a putative mechanism for the leukopenia and marrow hypoplasia associated with these agents.411–413

Constraints of Chelator Design To be unavailable to participate in the generation of free radicals, iron must be fully coordinated at each of its six ligand-binding sites. If any of these remain partially coordinated, iron may participate in the Fenton reaction, resulting in lipid peroxidation with organelle and cell damage. The design of a chelator is crucial to preventing these events. In general, hexadentate ligands, which have six coordination sites and hence bind iron in a 1:1 ratio, scavenge iron at low chelator concentrations more efficiently and are more stable in their iron complexed forms than bidentate or tridentate chelators (Fig. 29.6). The latter chelator classes have two or three iron-coordinating sites per ligand, respectively, and therefore require three or two chelating molecules, respectively, to coordinate iron (III) completely. EDTA, which only coordinates one free electron, does not diminish the reactivity of iron salts in the Fenton reaction and indeed might catalyze such reactions.259 By contrast the hexadentate desferrioxamine and physiological ligands such as lactoferrin and transferrin that surround the iron more completely are powerful inhibitors of lipid peroxidation in several systems.414 The iron–chelate complexes of bidentate hydroxypyridinones, being less stable than the hexadentate desferrioxamine, can generate free radicals and damage cell membranes with increased lipid peroxidation and loss of cell viability, particularly if the chelators have high lipid solubility.406 The effect can be “designed out” by synthesizing hydroxypyridinones with high pM (-log of the uncoordinated metal [iron] concentration calculated at pH 7.4, 10-␮M ligand concentration and 1-␮M iron[III] values).415 Deferasirox, a tridentate chelator, is inherently less stable than ferrioxamine but is unlikely to participate in redox cycling of iron because Fe deferasirox

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Janet L. Kwiatkowski and John B. Porter

Deferasirox (DFS) Tridentate

Desferrioxamine (DFO) Hexadentate

O O

O O

Fe O

O O

Deferiprone (DFP) Bidentate

O

Fe

OO O

O O

O O

Fe

O O

O

Figure 29.6. Denticity of iron chelation.213

is a very weak oxidizing agent, making its reduction under physiological conditions unlikely.416 Hexadentate chelators tend to have the advantage over bidentate and tridentate ligands of a greater ability to scavenge iron at low concentrations, a lesser potential to redistribute iron and a slower access to iron in pool c. Conversely, lower molecular weight bidentate and tridentate ligands have a potential to be absorbed from the gut and to access intracellular iron pools more rapidly. It is difficult to design hexadentate molecules with molecular weights less than 400, thus severely limiting their absorption from the gastrointestinal tract.417 Consequently no hexadentate chelators have yet been identified with sufficient oral bioavailability for clinical use. Many bidentate and tridentate chelators have sufficiently low molecular weights for acceptable oral bioavailability. To minimize the inherent disadvantages of their lower denticity, novel chelators with greater stability of the iron–chelate complex are being sought. Clinically useful iron chelators should possess a sufficiently high iron binding (stability) constant409,417 and have specificity of iron binding over other essential metals like zinc and copper. A more clinically relevant expression of iron binding than the stability constant is the pM (Table 29.10). This measure takes into account the tendency of bidentate chelators to dissociate at low concentrations and is a more useful indicator of the ability of a chelator to scavenge iron at low chelator concentrations. Chelators with high pM values are therefore desirable. The distribution of chelators to different tissues and subcellular compartments will also inevitably affect their toxicity. Ideally a compound should have low penetration of the central nervous system, where adverse effects have been observed with desferrioxamine (see later) and should have a high extraction of iron from liver cells where iron is present in high concentrations.417,418 The rate at which chelators gain access to intracellular iron pools is determined by their lipid solubility, charge, shape, and molecular weight.401,409,419 Once within cells, the structure of iron chelators also determines the rate of interaction with key iron-containing enzymes in pool 3. Larger and less lipophilic molecules tend to interact with intracellular metalloenzymes more slowly than small lipid soluble molecules.403,411,420

Finally, the pharmacokinetics and metabolism of an iron chelator are critical to its success. The metabolism of chelators has been shown to have a key bearing on their efficacy and toxicity.421–424 As the majority of body iron is not directly chelatable, increasing the chelator dose might not have a proportionately increased effect on iron excretion while increasing the risk of toxicity disproportionately due to an excess of free chelator. This relative unavailability limits the efficiency of chelation therapy, where chelation efficiency refers to the proportion of the drug that ends up being excreted in the iron-bound form. In iron-overloaded humans, the efficiency of desferrioxamine is approximately 13%, deferiprone is approximately 4%, and deferasirox is approximately 27%.425,426 The relatively low efficiency of deferiprone is likely to be explained not only by the rapid metabolism to forms that do not bind iron but also by its rapid elimination.427 Conversely, the long plasma halflife of deferasirox contributes to the higher efficiency of this drug.428 Thus iron excretion with deferasirox429 as with desferrioxamine363,430 is directly proportional to the area under the curve of the drugs. A further advantage of slow chelator elimination is that 24-hour protection from labile iron species in plasma or within cells can be achieved.364,367

CHELATION THERAPY WITH DESFERRIOXAMINE Historical Perspective Desferrioxamine, a naturally occurring hexadentate siderophore derived from Streptomyces pilosus, was discovered by chance in 1960 during work on isolation of the iron-containing antibiotic ferrimycin from Streptomyces griseoflavus. Following its isolation, the iron-bound form, ferrioxamine B, was initially investigated as a potential iron donor for iron-deficient subjects, but it was excreted intact in the urine without losing or exchanging its iron. The notion that an iron-free ferrioxamine, desferrioxamine, might be used to chelate excess iron was supported by clinical studies.431 Desferrioxamine when first given to thalassemia major patients as single intramuscular injections increased urinary iron excretion in a dose-dependent manner, proportional to iron stores.432 Once daily intramuscular injections of 500 mg, 6 days a week in thalassemia major children over a period of 7 years, stabilized liver iron at approximately 3% dry weight and also stabilized liver fibrosis compared with untreated comparators.325 Twenty-four-hour intravenous infusions produced more urinary iron than the same dose given as an intramuscular bolus.433 The same group and others subsequently showed that 24-hour subcutaneous desferrioxamine infusions resulted in urinary iron excretion nearly equivalent to that achieved with intravenous infusions.433,434 Urinary iron excretion was similar giving the same dose over 12 rather than 24 hours, and this was generally achieved iron balance.356,435 Metabolic iron balance studies revealed that between 30% and 50% of total

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease iron excretion with desferrioxamine was in the feces357 and that the proportion increased with ascending desferrioxamine doses and following suppression of erythropoiesis by blood transfusion.357 Nightly 8–12-hour subcutaneous infusions of desferrioxamine gradually became standard practice. The use of ascorbate, 2–3 mg/kg daily, on the same day as desferrioxamine administration increased urinary iron excretion,357 but doses in excess of this were possibly associated with cardiac toxicity.436 In the 1980s progressively larger doses of desferrioxamine were tried, often by the intravenous route, in attempts to reverse massive iron overload or to reverse cardiac failure.437–439 Although some of these objectives were achieved, significant toxicity from desferrioxamine began to be reported, most noticeably retinal440 and auditory toxicity.441 Later, effects on bone and growth were reported in children.442,443 From these and other studies, a “standard” dosing regimen for desferrioxamine has emerged, aimed at balancing the beneficial effects of chelation with unwanted effects of excessive dosing. It must be made clear however that some aspects of “standard therapy” recommendations, such a when to begin and the dose recommended to maximize growth potential, have been arrived at by empirical retrospective analysis rather than prospective randomized trials.

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plasma, clearance is almost exclusively renal because ferrioxamine is not cleared by the liver. In renal disease, levels of ferrioxamine may therefore accumulate; however, ferrioxamine is highly stable and does not redistribute iron significantly within the body. With 24-hour infusion, the duration of protection from NTBI and labile plasma iron is continuous, but plasma levels still rarely exceed 10 ␮M at conventional doses.364 Metabolism of the iron-free drug but not the iron complex occurs within hepatocytes, so that an increase in metabolites indicates a decrease in the availability of chelatable iron.363,446 Fecal iron excretion as ferrioxamine is almost entirely due to intrahepatic iron chelation, whereas urinary iron is mainly derived from plasma iron turnover380 (Fig. 29.5). Uptake of desferrioxamine into hepatocytes results in chelation of cytosolic and possibly lysosomal iron to form ferrioxamine that is then excreted in the bile.374 At conventional doses approximately a third of the iron is excreted through this route into the feces, and this amount increases with higher desferrioxamine doses,447 reflecting a greater proportion of intrahepatic chelation.

Evidence of Efficacy of Desferrioxamine Effects on Iron Balance and Liver Iron

Chemistry and Pharmacology of Desferrioxamine The hexadentate structure of desferrioxamine necessitates a relatively high-molecular-weight structure of 560 (or when administered clinically as the mesylate, 657) that limits its absorption from the gut, and therefore only parenteral routes of administration are realistic. Its hexadentate structure helps desferrioxamine to scavenge Fe3+ particularly at low concentrations of iron, as evidenced by the high pM (Table 29.10). A hexadentate structure and high pM also contribute to stabilization of the iron complex, and iron redistribution or partial iron complexation is insignificant. Other metals are bound with a much lower affinity; only the chelation of aluminum has clinical significance. Desferrioxamine has been successfully used to treat aluminum overload in renal dialysis patients at doses of 5– 10 mg/kg/week.444 Entry of desferrioxamine into most cell types and into subcellular compartments is retarded by two mechanisms, its relatively high molecular weight and its highly hydrophilic nature.400,411 in hepatocytes, however, there appears to be a facilitated uptake mechanism.363 The free drug is positively charged, as is the iron complex. This latter property accounts for the slow egress of the iron complex from cells.411 Elimination of desferrioxamine from plasma is fast, with an initial half-life of 0.3 hours and a terminal half-life of 3 hours.445 With an intravenous infusion of 50 mg/kg/day, mean steady-state concentrations of 7.4 ␮M are achieved445 (Table 29.10). Desferrioxamine and its major metabolites are cleared by the liver and the kidney in their iron-free forms. Once iron is bound to form ferrioxamine in the

Iron balance can be estimated from metabolic balance studies with measurement of iron excretion in urine and feces and iron input in the diet357 or by measuring the change in LIC over time and relating this change to the Angelucci formula, which relates LIC to total body iron stores.318 Early studies suggested that iron balance, based on urine iron alone, could be achieved at a dose of approximately 30 mg/kg/day when given as a 12-hour infusion.356 In patients not previously receiving chelation therapy, the quantity of urine iron excreted correlated with transferrin saturation,448 the number of transfusions,448 and the dose of desferrioxamine356,432,448 Fecal excretion contributes a further 30%–50% to total desferrioxamine-induced iron output.357 Formal metabolic iron balance studies suggested that daily 12-hour infusions at 30 mg/kg could achieve iron balance in thalassemia major, particularly if oral ascorbic acid was supplemented at the equivalent of 2–3 mg/ kg/day.357 Intramuscular desferrioxamine was more effective at stabilizing liver iron at 6 years follow-up in thalassemia major.325 Using subcutaneous infusions of 40 mg/kg, more maintained liver iron at safe levels.449–452 Intensification of therapy by combining subcutaneous and intravenous therapy can normalize liver iron levels.438 Surprisingly, detailed studies of the dose effect of desferrioxamine on LIC and hence on iron balance have only become available recently as a byproduct of prospective 1-year comparisons with deferasirox, in which changes in LIC over a 1-year period were measured in 290 patients receiving sliding scale doses of desferrioxamine between 25 mg/kg/day

716 and 60 mg/kg/day 5 nights a week.453 These studies have revealed the critical importance of dose and transfusion rate on iron balance.212 Importantly at doses of desferrioxamine used in many recent studies, and at average transfusional iron loading rates of 0.3–0.5 mg/kg/day, negative iron balance was achieved in only half of patients prescribed 40 mg/kg/day 5 days per week. When doses of 50 mg/kg or higher were given, 86% of patients were in negative iron balance, even at high transfusional rates of 0.5 mg/kg/day or higher.212 The proportions of patients in iron balance might be even less during routine clinical where compliance is likely to be poorer.

Effects on Serum Ferritin Dose-dependent reductions of serum ferritin with desferrioxamine have been recognized for several decades, and the value of maintaining serum ferritin at less than 2,500 ␮g/L has been linked to protection from heart disease and with survival.280 Formal trials relating changes in serum ferritin to dosage have been scarce. Recent studies comparing desferrioxamine with other chelators have added some important information about its effects on ferritin. In interpreting these findings it is critical to know what treatment patients received prior to the formal study as a change in ferritin is unlikely if patients simply continue the same dosage of drug. In a prospective study of 290 patients with thalassemia major mainly treated with desferrioxamine prior to the trial and followed for 1 year, those receiving a daily dose of 40 mg/kg five times per week had mean decreased serum ferritin of approximately 360 ␮g/L, whereas a dose of 50 mg/kg decreased serum ferritin by approximately 1,000 ␮g/L.453 These are average effects, and dose should be increased if the transfusional iron intake exceeds the average.

Long-term Effects on Survival Because no other chelation treatment was available when desferrioxamine was introduced, the pattern of survival in progressive birth cohorts gives a clear indication of its impact on survival as well as its cardioprotective effects. The impact of desferrioxamine on overall survival began to emerge in the 1980s454 but the full impact was clearly documented only later.275–277,280,281 Only 70% of patients born before 1970, and hence prior to the modern era of iron chelation, survived to age 20 years compared with 89% of patients born after 1970 who received effective chelation treatment from an early age.455 In nearly 1,000 Italian patients born between 1970 and 1974, mortality was 5% by age 20 years and this fell to 1% in cohorts born between 1980 and 1984 when desferrioxamine was widely used.277 The age of starting treatment has been shown to be a key factor in outcome,276,277,343 although the optimal age has not been assessed in prospective studies. Compliance with nightly infusions is key to long-term survival. Life table

Janet L. Kwiatkowski and John B. Porter analysis shows that patients who comply well with treatment can have a 100% survival rate to age 25 years, whereas survival for patients who comply poorly is only 32%.276 For patients who administer subcutaneous infusions of desferrioxamine more than 250 times a year, survival to age 30 years is 95%. If the frequency of infusion falls below 250 times a year, or approximately five times a week, survival to age 30 years is only 12%.281 The environment in which patients are treated has an important effect on compliance and survival. In thalassemic patients treated at a single thalassemia center, 83% of patients survived to age 40 years, and all compliant patients born after 1975 survived to age 25 years.320 The effect of compliance on survival has not been reported with other chelators. The provision of comprehensive care that delivers the necessary practical and psychological lifelong support to patients on chronic transfusion is therefore vital.

Effects on the Heart Because iron-induced cardiomyopathy remains the most common cause of death in thalassemia major, the effects of desferrioxamine on heart disease and survival will be considered together. Perhaps the most persuasive evidence for the cardioprotective effects of desferrioxamine is the ability to reverse preexisting cardiomyopathy. Improvement in left ventricular function after 1 year of treatment was first noted in a small group of thalassemia patients with subclinical heart disease in response to intensive subcutaneous desferrioxamine456 and was sustained with long-term follow-up.439 This effect was confirmed in other studies.457 Evidence for reversal of clinically overt heart failure in thalassemia major was first shown in three of five patients treated with intravenous doses of desferrioxamine of up to 200 mg/kg/day.437 Later work using a discontinuous, high-dose regimen of 6–12 g of desferrioxamine over 12 hours daily458 showed improvement in symptoms and echocardiographic parameters in one patient with congestive cardiac failure and another with a severe ventricular arrhythmia. Other experience with discontinuous therapy has been variable. No improvement in the cardiac status of two patients was seen at 100 mg/kg over 8–12 hours459 but improvements were observed in eight thalassemic patients followed-up for 6.5 years while using high-dose administration via an indwelling central venous line for 8–10 hours daily.460 Experience with continuous 24-hour therapy has been more consistent, having the theoretical advantages of continuous removal of NTBI364 and of reestablishing good compliance.333,461 In one study, four patients were treated with continuous 24-hour therapy with improvement in left ventricular ejection fraction in two and reversal in atrial fibrillation in a third.457 In nine other patients, not only did continuous 24-hour infusions produce superior urinary iron excretion compared with an equivalent subcutaneous 12-hour regimen, but the intravenous regimen encouraged

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease excellent compliance.461 Longer-term studies have established that continuous intravenous therapy is a safe, lifesaving therapeutic option in the management of highrisk thalassemia: 25 intravenous devices were inserted into 17 patients over a 16-year period, and desferrioxamine, usually at 50 mg/kg/day, was infused continuously over 24 hours, 6–7 days a week.333 Resting ejection fraction improved significantly from 36%–49% in seven of nine patients with previously documented deterioration in left ventricular function. This occurred in some cases within a few days of starting treatment and therefore cannot be attributed to normalization of iron stores but to the depletion of a limited toxic labile iron pool. Atrial fibrillation was reversed in all six patients in whom this was present within 12 months; in one patient, cardioversion occurred within 5 days without the need for conventional antiarrhythmic drugs. Drug toxicity was limited to a single early case of reversible retinopathy in a patient with preexisting diabetes mellitus who had been on an initial dose of 80 mg/kg/day. Long-term follow-up showed 62% survival at 13 years in those with demonstrable cardiac disease. Deaths occurred in patients whose compliance was not maintained. Large doses such as those initially used to reverse heart failure and that were associated with severe retinal problems437,440 might not be necessary and doses of 50–60 mg/kg/day could be sufficient.333 The risk of overdosing can be minimized by adjusting the dose as the ferritin falls, maintaining the therapeutic index of desferrioxamine to serum ferritin ratio of 0.025 as recommended for conventional subcutaneous desferrioxamine treatment.333 Reversal of left ventricular dysfunction similar to that achieved with continuous intravenous therapy has also been shown with 24-hour subcutaneous therapy,343 although this is demanding and only suitable for some patients. With the increasing use of T2∗ to estimate myocardial iron, there is now evidence that desferrioxamine, when given at sufficient doses and with sufficient frequency and duration can improve myocardial T2∗ . Patients with severe myocardial iron overload and a mean T2∗ less than 6 msec, with advanced myocardial dysfunction, when given continuous desferrioxamine infusions at 50 mg/kg/day or more showed improved T2∗ values of 3 msec over 1 year.245 During this same observation period, liver iron levels were nearly normalized and left ventricular function normalized within 3 months of treatment, suggesting that heart function can be improved by continuous desferrioxamine even when heart storage iron remains high. It might take several years, however, to normalize heart iron when T2∗ values begin less than 10 msec.320 It might not be necessary to use intravenous treatment to improve myocardial T2∗ values if myocardial iron loading is only mild: improvement in cardiac T2∗ even at low, intermittent doses has been confirmed in two prospective randomized studies,462,463 even in people on a previous similar treatment regime. Using subcutaneous treatment at relatively low doses of 35 mg/kg patients with baseline T2∗ values between 8 and 20 msec showed

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improvement in T2∗ of 1.8 msec over 1 year;462 doses of 40– 50 mg/kg 5 days a week lead to an improvement of 3 msec over 1 year.464

Other Long-term Effects on Morbidity Other effects of desferrioxamine therapy include: improvement in liver fibrosis,325 decreased severity of hypogonadism,465 improved glucose tolerance,466 and decreased incidence of diabetes,276 and hyperparathyroidism.277 The onset of glucose intolerance is delayed and glucose intolerance can be improved466 by the timely use of desferrioxamine. Hypothyroidism might also be reversed.469 Introduction of subcutaneous infusions of desferrioxamine before age 10 years significantly reduces gonadal dysfunction, with improvement in pubertal status and growth.465 Concomitant improvement in fertility has also been seen, although secondary amenorrhea is still common.286 Unlike heart failure,467,468 once advanced endocrine dysfunction has developed, reversal has not been documented. Intramuscular desferrioxamine at relatively modest doses stabilizes hepatic fibrosis and there is no progression over an 8-year period in patients receiving subcutaneous infusions of desferrioxamine in the absence of histological evidence of active hepatitis.451

Tolerability of Desferrioxamine General Most of the toxic effects of desferrioxamine are dose related; effects on growth, skeletal changes, audiometric, and retinopathic effects are more likely at higher doses of the drug. Toxicity from desferrioxamine in thalassemia major is very unlikely at doses up to 40 mg/kg/day. The risk of toxicity at any given dose is greater in patients with low levels of iron loading than in those with high levels of iron loading so that as serum ferritin falls, particularly below 1000 ␮g/L, it is wise to consider reducing the dose.344,470 It is clear that some unwanted effects such a those on growth and bone development are mostly applicable to children in whom special care must be taken to avoid doses of greater than 40 mg/kg/day. These patients should be monitored particularly carefully with the consideration of dose lowering as iron levels fall.

Injection Site Reactions Local mild reactions can occur with skin reddening and soreness at the site of subcutaneous infusions. These are often caused by desferrioxamine being reconstituted above the recommended concentration of 10%. Increasing the volume of water used to dilute the desferrioxamine can substantially decrease reactions. On occasions when local reactions remain a problem, the addition of 5–10 mg hydrocortisone to the desferrioxamine solution can help.

718 Effects on Growth and Bone Although desferrioxamine usually improves growth in thalassemia major by decreasing iron overload, excessive amounts might cause growth retardation. The risk factors are, age younger than 3 years at commencement of treatment, higher doses of desferrioxamine,442 and lower levels of iron overload. It is advisable to monitor height velocity as well as sitting and standing height twice yearly and adjust dosing as necessary. A quick resumption in growth follows reduction in desferrioxamine dosing without the need to stop treatment.443 Rickets-like bone abnormalities have been described in association with decreased growth442 and radiographic abnormalities of the distal ulnar, radial, and tibial metaphases appear to be associated features. Vertebral growth retardation or milder changes involving vertebral demineralization and flatness of vertebral bodies have also been noted in patients receiving chelation therapy.281 It might be advisable to undertake regular surveillance for the toxic effects of desferrioxamine on bone471 with annual radiological assessment of the thoracolumbar–sacral spine and the forearm and knees and to reduce the dose of the chelator if significant changes are noted.

Renal and Auditory Toxicity Retinal and optic nerve disturbances, sometimes associated with pigmentary retinal changes, were originally described in patients receiving 125 mg/kg/day440 and are rare at currently recommended doses. Other abnormalities that have been linked to excessive use of desferrioxamine include blurred vision, loss of central vision, night blindness, and optic neuropathy.441 The risk might be higher in patients with diabetes or other factors affecting the blood–retinal barrier,472 so these groups should be monitored more carefully by using electroretinographic techniques. Desferrioxamine therapy should be withdrawn until symptoms partially or fully resolved and then resumed at a reduced dose with close monitoring by electroretinography. High-frequency sensorineural hearing loss was initially described in approximately 25% of patients on high-dose desferrioxamine regimens.441 This complication might be reversible if diagnosed early.344 The risk is greatest in patients with low degrees of iron overload receiving high doses of desferrioxamine. By keeping the therapeutic index344 below 0.025 and by monitoring audiometry regularly, the risks can be minimized. It is advisable to perform audiometry before starting treatment and approximately once a year during treatment.

Infections There is an increased risk of Yersinia infection in iron overload, and this risk increases further with desferrioxamine treatment as Yersinia does not make a natural siderophore and uses iron from ferrioxamine to facilitate its

Janet L. Kwiatkowski and John B. Porter growth.473,474 Patients who present with diarrhea, abdominal pain, or fever should stop taking desferrioxamine until Yersinia infection can reasonably be excluded by appropriate stool samples, blood cultures, and serological testing. If Yersinia infection is proven or seriously suspected, desferrioxamine should be withheld until the infection has been eliminated by antibiotic treatment. Prolonged treatment with an antibiotic such a ciprofloxacin is occasionally necessary to prevent recurrence, but it is rarely necessary to withhold desferrioxamine after the initial infection has been treated. Very rarely, other infections such as Pneumocystis carinii 475 and mucormycosis 476 have been associated with desferrioxamine. In any patient with undiagnosed fever it is wise to consider stopping the desferrioxamine until the cause is identified.

Miscellaneous Effects Generalized reactions such as fever, muscle aches, and arthralgia occur rarely. True systemic allergic reactions are uncommon but can include anaphylaxis. Some patients can be successfully desensitized using published procedures,477,478 Renal impairment, characterized by a reduction in the glomerular filtration rate, has been reported in occasional patients given high doses479 and a clinically significant decrease in glomerular filtration rate occurred in 40% of patients receiving subcutaneous desferrioxamine that was reversible on its discontinuation.480 A fatal acute respiratory distress syndrome–like syndrome has been described in patients with acute iron poisoning given 15 mg/kg/hour desferrioxamine infusions for periods in excess of 24 hours; lower doses and shorter infusions are recommended for acute iron poisoning.481 Pulmonary injury with fibrosis has also been reported in patients with chronic iron overload receiving doses of 10–22 mg/kg/hour.482 In noniron-overloaded patients, the use of desferrioxamine potentiated the action of prochlorperazine, a phenothiazine derivative, leading to reversible coma in two patients.483 Lens opacities, observed rarely in patients receiving high doses of desferrioxamine, improved when the chelator was withdrawn.437 In a recent randomized study comparing desferrioxamine with deferasirox, five of 290 patients randomized to desferrioxamine without lens opacities at baseline appeared to develop them over a 1-year period. In the context of the knowledge that clinically significant lens opacities are uncommon in patients on long-term desferrioxamine, these findings need interpreting with caution. Thrombocytopenia has been observed with desferrioxamine in two patients on renal dialysis.484 It is important to avoid a sudden bolus infusion of desferrioxamine in patients receiving intravenous treatment through an indwelling catheter or during blood transfusions because this can lead to nausea, vomiting, hypotension with acute collapse,485 or even transient aphasia.486

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease General Treatment Recommendations for Thalassemia Major Standard Therapy Guidelines aim to achieve a balance between the unwanted effects of under or over chelation, and advise what is most practical for the patient. Standard current practice for commencing desferrioxamine in ␤ thalassemia major is to begin at the age of 3 years or when the ferritin reaches 1,000 ␮g/L, whichever is sooner, and not to exceed 30 mg/kg until response to this regimen can be assessed. Although it is clear that the introduction of desferrioxamine at excessive doses or too early increase the risks of toxicity, the optimal dosing and timing have not been the subjects of prospective, randomized trials, and recommendations are therefore to some extent empirically based. Delay in treatment until after age 3 years has been recommended because of observations of poor growth and metaphyseal dysplasia when started earlier.442,471,487–489 Conversely, delay in starting treatment increases the risk of iron-mediated toxicity as hepatic fibrosis associated with liver iron loading has been reported in children as young as age 3 years.278,490 It is possible that very low doses given from an earlier age could prevent accumulation of liver iron without concomitant desferrioxamine toxicity, but there are presently no data that address this directly. Furthermore, failure to start treatment sufficiently early increases the risk of growth retardation due to the well-established effects of iron overload on growth and sexual development. Downward dose adjustment is advisable with young age and low levels of iron loading. In patients younger than age 5 years, doses in excess of 35 mg/kg/day might be inadvisable. Ferritin can be used as an approximate indicator of falling iron levels, and doses of desferrioxamine should be reduced accordingly keeping the therapeutic index less than 0.025 (mean daily dose in mg/kg, divided by the serum ferritin in ␮g/L).344,370 If liver iron quantitation is available, a scheme for dose adjustment has been suggested.340 Liver iron determinations are unlikely to be available more frequently than yearly, even in centers performing these measurement routinely, therefore, ferritin measurements will still be useful in dose adjustment. Upward dose adjustment should be considered in patients with rates of transfusional iron intake greater than 0.5 mg/kg/day. Doses of 50–60 mg/kg/day will be necessary for negative iron balance in approximately half of these patients.212 Even at intermediate transfusion rates between 0.3 and 0.5 mg/kg/day, approximately a quarter of all patients will require doses higher than 50 mg/ kg/day to achieve negative iron balance. Doses less than 35 mg/kg/day result in positive iron balance even in patients with a transfusional iron loading rate of less than 0.3 mg/kg/day.212 Therefore, in adults, doses above 40 mg/kg/day and typically 50–60 mg/kg/day are often necessary. Ascorbate supplementation of 2–3 mg/kg/day is

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another useful way of increasing iron excretion and it is recommended for patients who are stable and not in heart failure or do not have an arrhythmia on the days that desferrioxamine is given. Patients with mild myocardial iron loading and T2∗ of 10–20 msec can also benefit from modest increments in desferrioxamine dose or frequency. With T2∗ less than 10 seconds or with left ventricular dysfunction, a greater degree of intensification is required.

Rescue Therapy For patients with acute heart failure, continuous desferrioxamine infusion is recommended. This is usually most conveniently achieved by diluting desferrioxamine in 500 mL of saline and infusing through a peripheral vein. For longer-term infusions, once the patient has been stabilized, an indwelling line is usually necessary, although 24-hour infusion has been successfully achieved by the subcutaneous route in selected cases.343 Doses in excess of 50– 60 mg/kg/day have been used successfully but increase the risk of pulmonary482 and retinal toxicity.440 Complications associated with the use of central venous access lines include infection and thrombosis.333 To limit the thrombotic complications of central venous access lines, close monitoring and prophylactic anticoagulant therapy have been suggested. Intensification, as described previously, has also been successfully used in very severe iron overload without myocardial dysfunction or as a way of reestablishing compliance with chelation therapy with good long-term outcome if compliance is maintained.333 With evidence of moderately increased myocardial loading or T2∗ of 10–20 msec, improvement in these measures might be achieved by increasing the dose or duration of exposure or compliance with subcutaneous desferrioxamine above that given for standard therapy. With severe myocardial iron loading and T2∗ less than10 msec, but without heart failure, intensification of therapy is necessary and can include increasing the dose and/or duration of desferrioxamine exposure without245,343 or with the addition of deferiprone.

Methods of Delivery for Desferrioxamine The mode of delivery of standard desferrioxamine is critical to compliance and hence the success of therapy. It is usually infused at night via a thin subcutaneous needle inserted into the abdomen, arm, or lateral thigh region, which is connected to a portable pump over 8–12 hours, five–seven times per week at a daily dose of 20–60 mg/kg. The infusion site needs to be rotated nightly and a solution infused should not damage tissues. The most widely delivery system has been the battery-operated syringe driver, delivering between 10 and 30 mL, depending on the manufacturer. These are best suited to subcutaneous infusions for 8–12 hours daily, but some can also be used for intravenous infusions. Lighter or smaller systems have been developed that might be better suited to continuous

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Janet L. Kwiatkowski and John B. Porter Deferasirox

Mean total body iron excretion ± SD, mg Fe/kg/d

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Average transfusional iron intake in MDS12 Average transfusional iron intake in SCD11

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Figure 29.7. Dose–response comparison of DFO and DFX.212

infusion. Some battery-operated pumps can take 50–100 mL of desferrioxamine solution, allowing continuous intravenous treatment for up to 1 week. An alternative is to use disposable pumps that expel fluid under the pressure produced by filling an expandable balloon.491 These devices are light and silent, making them attractive to patients who need continuous infusions, but disposable pumps are expensive. Recently introduced mechanical delivery devices and gas-operated systems are also lighter and more compact than conventional syringe driver models. Subcutaneous bolus injections have been evaluated as an alternative approach to chelation therapy if pumps are unavailable or impractical. Studies suggest that iron excretion with twice daily subcutaneous injections of desferrioxamine might be comparable to that observed with the same total dose given as an 8-hour subcutaneous infusion.492,493 In a randomized comparison of twice daily bolus injections and subcutaneous infusion, similar decrements of serum ferritin and approximate liver iron values were seen in both groups of thalassemia patients.494 This approach might be an alternative when pumps are not available and when patients are prepared to inject twice daily.

Use of Desferrioxamine in Sickle Cell Disease and Thalassemia Intermedia Sickle Cell Disease Patients with sickle cell disease who receive long-term transfusion therapy to prevent the complications will eventually require iron chelation therapy unless exchange

transfusions are used in preference to simple transfusions. A variety of chronic anemias associated with iron loading, in which regular transfusion is not necessary but erythropoietic reserve might be marginal, such as pyruvate kinase deficiency, sideroblastic anemias, and thalassemia intermedia, may also benefit from chelation therapy if venesection is not feasible. The optimal dose to achieve iron balance can be referred to the rate of iron loading from transfusion that can easily be calculated from the number of blood units given over a given time period. On the basis of the transfusional loading rate, the optimal dose can be referred to a graph212 relating dose to iron balance. Thus the dose of desferrioxamine to obtain iron balance will on average be lower in sickle cell disease (20 mg/kg/day) than in thalassemia major (40 mg/kg/day) (see Fig. 29.7). Although in sickle cell disease, iron distribution to endocrine organs and to the heart is less common than in thalassemia major, chelation therapy is still necessary to maintain iron balance and decrease the risks of iron overload. Iron balance with desferrioxamine and the proportions of fecal and urinary iron excretion appear similar to those seen in thalassemia syndromes.495 The rate of iron loading varies considerably between patients and depending whether simple transfusion or erythrocytapheresis is used. In a recent multicenter study the average rate of transfusional iron loading was less in sickle cell disease (0.22 mg/kg/day).225 The use of ferritin for dose adjustment is more problematic in sickle cell disease as discussed previously. LICs above 7 mg/g dry weight are a clear indication for starting treatment because the risk of liver fibrosis increases above this value,304 which will typically be

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease exceeded after 21 months of simple transfusion.303,304 Maximal doses should not exceed those given in thalassemia syndromes, and careful monitoring for toxicity is advisable.

Thalassemia Intermedia In thalassemia intermedia, iron overload might result from increased iron absorption up to five times the normal rate217,218 or from sporadic transfusions. Serum ferritin tends to underestimate the degree of iron overloading234 so that liver iron determination is particularly helpful in planning the optimal regime. In general, because of the lack of regular transfusion, negative iron balance can be achieved at relatively modest doses or frequency of chelation. By referring to the dose response for other diseases (Fig. 29.7), a mean daily dose of desferrioxamine at 10 mg/kg/day would maintain iron balance or put patients into negative iron balance. In practice, iron can be decreased steadily and safely by giving standard doses (40 mg/kg) of desferrioxamine twice or three times a week in such patients, thus achieving mean daily doses of 10–20 mg/kg/day. Because of the discordance of serum ferritin from body iron burden in both thalassemia intermedia and sickle cell disorders, it might be advisable to quantitate liver iron before making a decision to initiate chelation therapy and to repeat liver iron quantitation at regular intervals during treatment.

ORAL CHELATION THERAPY Although desferrioxamine is highly efficacious, complying with long-term administration has been problematic for an important subset of patients limiting its effectiveness. This has driven the development of orally absorbed chelators. Inconveniently, the very properties that encourage oral absorption also increase the access of chelators to iron or other metal pools that are necessary for normal physiological functions. Many promising compounds have been discarded because preclinical animal toxicology or clinical evaluation has shown too narrow a margin between the therapeutic and the toxic effects. Two orally absorbed iron chelators are now in widespread clinical use: deferiprone and deferasirox.

Deferiprone Historical Perspective The hydroxypyridinones were first described in 1982.496 Initial clinical trials were conducted without the backing of a major pharmaceutical company and showed promising effects on urinary iron excretion in myelodysplasia and patients with transfusional iron overload.497,498 Animal toxicology demonstrated a dose-dependent and time-dependent suppression of the bone marrow, both in iron overloaded and nonoverloaded states.419,499,500

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Several cases of agranulocytosis were also reported in humans.501–503 Other effects in animal models, such as redistribution of iron from the liver to the heart and liver fibrosis, were later reported with the closely related hydroxypyridine, CP94, in an iron-overloaded gerbil model.504,505 Thymic atrophy and teratogenic effects at doses close to the effective clinical therapeutic range were later identified.506 Further development led to trials to establish the efficacy of deferiprone and the frequency of complications, but the original efficacy study was discontinued following disagreements between the pharmaceutical company and clinical investigators.340 The results of a 4-year toxicity trial have been reported.507 More recently, prospective randomized studies have been undertaken, particularly focusing on the cardioprotective effects of the drug.462 Deferiprone was licensed as second-line therapy for transfusional iron overload in Europe in 1999 but is not licensed in North America. The drug is licensed in India under the trade name Kelfer.

Chemistry and Pharmacology Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one) (L1, CP20, Kelfer, Ferriprox) is a member of the family of 3-hydroxypyrid-4-one bidentate chelators496 that bind to iron in a 3:1 ratio. It is less hydrophilic than desferrioxamine with approximately one-third of the molecular weight (139). Although the stability constant for iron (III) is approximately six orders of magnitude higher than desferrioxamine, the pM of 20 is lower than that of desferrioxamine at 26.6.418 As a consequence, iron (III) coordination is most efficient at high concentrations of the chelator but at concentrations of 1 ␮M iron and 10 ␮M chelator, desferrioxamine will scavenge iron more efficiently. The lower stability at lower concentrations of drug also increases the potential to redistribute iron within the body508 and leads to the potential formation of incomplete 1:1 and 1:2 iron–chelate complexes at low chelator concentrations. Such incomplete complexes have the potential to participate in the generation of hydroxyl radicals.406 Unlike desferrioxamine, which is positively charged both in its iron free and complexed forms, deferiprone and its 3:1 iron complex are both neutrally charged. This neutral charge, and the less hydrophilic nature of the iron chelator and its complex, encourages a rapid diffusion of the free chelator into cells and its iron complex out of cells. Thus, unlike desferrioxamine, which slowly accumulates within cells at concentrations above those in the plasma with ferrioxamine egressing only slowly, the iron complex of deferiprone egresses relatively quickly.363,400,411 Following oral administration, absorption is rapid with the drug appearing in plasma within 5–10 minutes of ingestion. High concentrations are achieved at commonly prescribed doses, reaching levels in excess of 300 ␮M after oral ingestion of a 50 mg/kg dose.384,427 These levels are 5– 10 times higher than infused desferrioxamine445,509 (Table 29.10). In vitro studies in myocytes show that these high

722 concentrations, together with a more rapid access to intracellular iron, might favor the scavenging of chelatable iron by deferiprone.402 It has been suggested that at clinically relevant desferrioxamine concentrations of 10 ␮M, the effects of desferrioxamine on preventing and reversing cardiac pathology might result more from prevention of iron uptake into myocytes than from rapid iron removal.402 With deferiprone, however, these high levels are short-lived with an elimination t1/2 of 1.52 hours,427 and if given three times daily, are punctuated by low levels between doses and negligible nocturnal levels. The drug is metabolized in hepatocytes to the inactive glucuronide, which is the predominant form recovered in the urine.384,510 In contrast to desferrioxamine, iron excretion with deferiprone is almost exclusively in the urine.495,509,511 The rapid inactivation of deferiprone in the liver by glucuronidation421 might explain the relatively less impressive effect of deferiprone on liver iron removal compared with desferrioxamine,426 but in other cells such as myocytes this would be less of a limitation and could explain why deferiprone appears to be more effective in the heart than the liver. In contrast, the highly hydrophilic nature and high molecular weight will tend to retard access to intracellular iron pools,372,400,403 and the positive charge of the iron complex will retard egress from cells.400,411

Evidence of Efficacy of Deferiprone Evidence of efficacy with deferiprone has been based on the effects of the drug on iron excretion, serum ferritin, LIC, and more recently the iron-induced cardiomyopathy.

Effects on Iron Excretion and Balance Early observations demonstrated that urinary iron excretion in response to deferiprone 75 mg/kg was comparable to that with desferrioxamine infused subcutaneously over 8–12 hours at a dose of 40–50 mg/kg.509 Urinary iron excretion correlated with the serum ferritin and desferrioxamine-induced urinary iron excretion.509 Formal iron balance studies showed that total iron excretion at 75 mg/kg was 62% of that achieved with subcutaneous desferrioxamine at 50 mg/kg given over 8 hours.495 Urinary iron excretion did not change significantly in a group of patients receiving 75 mg/kg/day with a mean follow-up of 39 months.512 Dose equivalence for desferrioxamine and deferiprone has not been determined in iron balance studies.

Effects on Serum Ferritin Many early small nonrandomized studies have examined the effects of deferiprone on serum ferritin. Ferritin values fell in the most heavily iron overloaded subjects with serum ferritin of 5,000 ␮g/L and more,502 but in patients with levels below 2,500 ␮g/L levels did not typically fall. In a study

Janet L. Kwiatkowski and John B. Porter in India, serum ferritin fell from initially very high levels by an average of more than 3,500 ␮g/L over 20 months.513 In a Canadian study of 21 patients given a dose of 75 mg/kg/day, the serum ferritin declined from 3,975 to 2,546 ␮g/L after a mean follow-up of 3 years.339 Changes were most marked in patients starting with high ferritin values, whereas in those patients starting with ferritin values below 2,500 ␮g/L there was no significant further change. In a study from London, there was no overall change in serum ferritin in 26 patients treated for 3 years at 75 mg/kg (initial value 2,937 ␮g/L, final value 2,323 ␮g/L).512 In 52 patients from Turin with a median pretreatment ferritin value of 1,826 ␮g/L, there was no significant change in the serum ferritin level after 2 years of therapy with deferiprone at a dose of 75 mg/kg/day.514 Randomized prospective comparisons of desferrioxamine and deferiprone have been undertaken more recently with a combined total 235 patients.462,515–518 Due to differences in previous treatments, baseline ferritin values, and dosing regimes, pooled comparisons were not particularly revealing. Nevertheless, statistically significant decrease in serum ferritin in favor of desferrioxamine was seen at 6 months516,517 with no difference at 12 months.515

Effect on Liver Iron Relatively few prospective studies have examined changes in liver iron. Early studies measured LIC after periods of treatment but not before, making interpretation of results difficult. For example, hepatic iron was above 15 mg/g dry weight in 10 of 17 patients (58%) in a 2–4-year follow-up study and below 7 mg/g dry weight in only two patients.512 In another study of 52 previously well chelated patients treated for 2 years at 75 mg/kg/day, the hepatic iron concentration increased in 69% of patients by a mean of 43%514 and mean values had increased by 56% from 5.1 to 8.0 mg/g dry weight after 3 years. In a nonrandomized report with 7–8-year follow-up of seven patients, there was an “unexplained resurgence” of serum ferritin after 4–5 years, associated with a concomitant increase in liver iron in three.519 In 21 patients given deferiprone, LICs were followed prospectively initially for 3.1 years;339 in 10 previously suboptimally chelated patients, LICs fell from 23 mg/g dry weight to 11 mg/g dry weight (P < 0.005) with values above 15 mg/g dry weight in only two patients. In 11 patients in whom desferrioxamine had been effectively taken prior to use of deferiprone, hepatic iron concentrations remained below 15 mg/g dry weight; at 4.6 years, hepatic iron was above 15 mg/g dry wt in seven of 18 patients (39%).452 A combined systematic analysis of several randomized studies in a total of 143 subjects has been reported.518 The patient selection, dosing, and scheduled period of followup varied considerably between studies, as did the findings. In a Canadian study, at 33 months of treatment at 75 mg/kg, a mean increase in liver iron of 5 mg/g dry weight in 18 patients was noted with deferiprone compared with a 1 mg/g dry weight increase with desferrioxamine.520 In an

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease Italian study, a comparable decrease in LIC was seen with both deferiprone (21 patients) and with desferrioxamine (15 patients) at 30 months.515 In a 1-year study of other Italian patients given 100 mg/kg daily, a mean decrease of 0.93 mg/g dry weight was seen with deferiprone (27 patients) compared with a decrease of 1.54 mg/g dry weight with desferrioxamine, at a dose of 43 mg/kg five–seven times a week (30 patients).462 In a small 6-month randomized study, where baseline LIC values were higher than in the aforementioned studies, a greater decrease in LIC was reported with deferiprone (75 mg/kg) (6.6 mg/g dry weight, n = 6) than with desferrioxamine (30–60 mg/kg 5–7 days/ week) (2.9 mg/g dry weight).517 As with ferritin findings, patients with initially high LICs might respond better than those with lower values. Apparent loss of response with time could also reflect changes in compliance with longer periods of observation. Other factors that might account for the variable response between studies are differences in the rate of drug inactivation by glucuronidation or differences in transfusion rates.

Effects on the Heart Initial data of the effects on myocardial dysfunction, myocardial iron, and the prevalence of heart disease gave conflicting results, most likely reflecting the retrospective nature of patient allocation and differences in prestudy chelation histories. More recently prospective studies have demonstrated a beneficial effect on both heart function and myocardial T2∗ , particularly at higher doses. An early study showed continued cardiac mortality in 51 thalassemia patients who previously were poorly chelated with desferrioxamine. Four died of cardiac causes512 leading to the authors to conclude that in the face of preexisting severe iron overload, deferiprone cannot reliably protect patients from cardiomyopathic mortality. In a large retrospective analysis of 532 Italian thalassemia major patients, nine died of heart failure while being treated with deferiprone.521 On the other hand, a retrospective study of survival in 157 Italian patients treated with deferiprone between 1995 and 2003 for a median of 4.3 years reported significantly better survival in this group than in 369 patients treated with desferrioxamine. As patients were not randomized to desferrioxamine or to deferiprone the possibility of selection bias cannot be excluded. Furthermore the dropout rate of 31% was particularly high in deferiprone-treated patients. Another Italian study compared cardiac complications and survival over 6 years in 54 deferiprone-treated patients with 75 retrospectively allocated desferrioxamine-treated patients.522 Three deaths occurred in the desferrioxamine group and none in the deferiprone-treated group; however, the three deaths were in patients who started chelation at a late age. Furthermore, five patients in the desferrioxamine group had New York Heart Association class II–IV cardiac disease at baseline compared with only one in the deferiprone group.

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Another retrospective analysis of 539 thalassemic patients from Cyprus born after 1960 and followed from 1980 to 2004 showed an increased trend in cardiac mortality between 1980 and 2000 with a subsequent decline after 2000, which the authors attributed to increased deferiprone use.523 A possible beneficial effect of deferiprone on myocardial T∗ was first suggested524 in a retrospective nonrandomized study of 15 patients treated with deferiprone compared with 30 desferrioxamine-treated patients. Desferrioxamine patients were retrospectively matched for serum ferritin from a large group of 160 patients.524 The authors concluded that the lower incidence of reduced T2∗ in the deferiprone group was likely to be due to deferiprone treatment rather than any unintentional selection bias resulting from retrospective allocation of comparator groups. To clarify the apparent inconsistencies between the aforementioned studies, prospective studies are needed. Two studies examining the effect of deferiprone monotherapy have been published. In one prospective randomized trial, 71 patients treated with deferiprone at conventional doses of 75 mg/kg/day were compared with 73 patients treated with standard-dose desferrioxamine. After 1 year, both groups showed a similar and significant improvement in cardiac nuclear MRI signal.515 By contrast, in a prospective randomized study,462 61 thalassemic patients with moderate cardiac siderosis and T2∗ 8–20 msec were randomized to continue on desferrioxamine at an average dose of 43 mg/kg/day or deferiprone at a higher than usual mean dose of 92 mg/kg/day. In the standard-dose desferrioxamine group, the T2∗ improved 13%, whereas improvement was 27% in the deferiprone group. Although the ejection fraction was in the normal range at baseline in both groups, it improved significantly in the deferipronecompared with the desferrioxamine-treated patients (3.1% vs. 0.3%). Taken together, these studies suggest that highdose deferiprone might be more effective at improving myocardial T2∗ than standard-dose intermittent desferrioxamine 5 days a week, but at standard doses of deferiprone this advantage is more difficult to show. If patients have evidence of shortened T2∗ values, particularly with T2∗ values less than 10 msec, standard doses of desferrioxamine or deferiprone are not recommended; either high-dose deferiprone or intensified desferrioxamine by continuous infusion is recommended. An alternative approach, discussed later, would be to combine the two treatments.

Unwanted Effects of Deferiprone Neutropenia and Agranulocytosis Agranulocytosis was initially reported in 3%–4% of patients treated with deferiprone, and mild neutropenia occurred in an additional 4%.502,525 Neutropenia can last from 4–124 days.501,526,527 In 532 thalassemic patients treated for a total of 1,154 patient-years, the rates of agranulocytosis

724 and neutropenia were 0.43 and 2.08 per 100 patientyears, respectively.521 In a study designed to establish the frequency of agranulocytosis defined as neutrophils 0.0–0.5 × 109 /L, only one of 187 patients (0.5%) developed agranulocytosis.527 Nine patients (4.8%) developed milder neutropenia (absolute neutrophil count 0.5–1.5 × 109 /L). No additional cases of agranulocytosis occurred, but seven new cases of mild neutropenia developed during the next 3 years of treatment.507 The lower incidence of agranulocytosis compared with some earlier estimates suggests that this might be decreased by monitoring the white count weekly and stopping therapy in a timely manner. There are very few data on evaluation of deferiprone in children aged younger than 6 years, and a study of 44 patients showed no cases of agranulocytosis occurred, but thrombocytopenia was seen in 45% 3 months–1 year after starting deferiprone and was reversible on cessation of treatment.528 As this was not a randomized study, the significance of this observation is difficult to gauge and further studies are needed in this age group. The mechanism of agranulocytosis is unclear. It has been suggested that this occurs though a dosedependent inhibition of DNA synthesis and ribonucleotide reductase411 or possibly though a zinc-depletion mechanism.529 Alternatively the effect in humans might be idiosyncratic; however, several observations do not fit this mechanism. First, bone marrow hypoplasia is dose dependent in animal studies,413 which is not typical of an idiosyncratic response and dose–response studies have not been conducted in humans. Agranulocytosis might appear as a late event after more than 1 year of therapy and can be preceded by previous episodes of neutropenia,521 a pattern also not characteristic of idiosyncratic reactions. The involvement of other hematopoietic lineages in both animal studies413,419 and humans528,530,531 would also be more consistent with a general effect on hematopoietic progenitors. Milder forms of neutropenia might be related to hypersplenism and intercurrent infections rather than to drug toxicity.532

Janet L. Kwiatkowski and John B. Porter with the duration of treatment and increased from 6% at 1 year to 13% at 4 years.507 In a pooled analysis, fluctuations in liver tests were reported in 44% of patients.525 Accelerated liver fibrosis was reported in a randomized study comparing deferiprone(n = 19) with desferrioxamine-treated patients (n = 20) over 3.5 years,452 and some progression of liver fibrosis was also found in other reports.519,534 No progression was seen in 34 of 187 patients with available biopsies who were treated for 3.5 years535 or in a randomized 1-year study with desferrioxamine.515 None of these studies were designed to evaluate progression of liver fibrosis, and interpretation of the findings is limited by a number of factors such as unclear hepatitis C mRNA status,452 variations in the duration of treatment,534 failure to record baseline liver fibrosis,512,519 and lack of clarity about changes in LIC.535 Thus long-term prospective data comparing liver fibrosis with desferrioxamine has not been reported.518 Small decreases in plasma zinc532 and cases of zinc deficiency525 not requiring cessation of therapy have been reported.

Recommended Dosing Formal safety studies have only been conducted at 75 mg/kg in three divided doses and this is the usual dose given. Published tolerability data at 100 mg/kg, a dose also licensed in the European Union, are confined to a single 1year prospective study in 32 patients.462 As no excess toxicity was reported, it might be reasonable to increase the dose in otherwise unresponsive patients. Nevertheless it is not known whether tolerability issues such as neutropenia and agranulocytosis increase at doses above 75 mg/kg/day. Few data are available in young children, and this is reflected in the licensing in the European Union, which is confined to patients older than 6 years.

Combined Chelation Therapy: Deferiprone and Desferrioxamine Pharmacology

Other Unwanted Effects The largest prospective clinical study designed to characterize the safety profile of deferiprone included 187 patients.532 Nausea and vomiting were relatively common in the first year of therapy, occurring in 24% of patients, with abdominal pain in 14%, and arthralgia in 13%. Analysis after 4 years of treatment demonstrated that additional gastrointestinal symptoms were infrequent but the overall dropout rate increased from 15% after 1 year to 55% at 4 years.507 Arthralgia and arthropathy vary greatly among studies, with an incidence 30%–40%513,533 in the developing world and less than 5% in Italy.521 The higher iron load in the patients from the developing world might increase the risk of iron redox cycling by incomplete chelate complexes in the joints. The risk of arthropathy appears to increase

When control of iron load or iron distribution is inadequate with monotherapy, the combined use of two chelators might be useful. In principle, two chelators can be given simultaneously or sequentially and the pharmacological implications differ. The use of more than one chelator, often referred to as mixed-ligand therapy,536 is well established.536,537 There is often a marked synergism of metal removal when a small kinetically labile ligand is combined with a larger hexadentate chelator. Typical examples are nitrilotriacetate/desferrioxamine for iron removal, penicillamine/DTPA for copper removal538 and salicylic acid/EDTA for plutonium removal.536 Mixed-ligand therapy with deferiprone and desferrioxamine relies on the principle of the low-molecularweight bidentate deferiprone rapidly accessing chelatable

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease iron pools unavailable to desferrioxamine and subsequently “shuttling” the chelated iron onto a desferrioxamine “sink.”382,539,540 Although this might increase net iron excretion, it could also increase unwanted effects due to shuttling from key metalloenzymes. If chelators are given sequentially, for example desferrioxamine by night and deferiprone by day, the main effect will be to provide a greater period of chelation exposure, increasing the protection time without significant shuttling of metals and with less risk of interactive toxicities. For example whereas monotherapy with desferrioxamine at night or deferiprone during the day only provides intermittent decrements in labile plasma iron, by alternating desferrioxamine at night with deferiprone by day decreases in labile iron can be obtained. Although deferasirox could also be used together with other chelators, at this time experience is very limited so that the discussion of combined treatment will be limited to desferrioxamine and deferiprone.

Clinical Regimens Assessed in Combined Therapy Many combinations have been given with various degrees of pharmacological overlap, even within a given study. Combined treatment has often been given as a way of helping patients who comply poorly with desferrioxamine monotherapy or have a poor response with deferiprone monotherapy to achieve more exposure to chelation. For example, a patient with inadequate ferritin or liver iron control on deferiprone given as 75 mg/kg/day in three divided doses would take additionally desferrioxamine two–three times a week. Another group that has been considered for mixed treatment is high-risk cases with particularly low myocardial T2∗ values or with very high levels of iron overload. The regimens used have tended to have a greater degree of overlap of chelators. For patients with cardiac dysfunction, the additional value of combined therapy over intensive desferrioxamine treatment is presently under investigation in a randomized trial. The effects of combined therapy in controlled trials have recently been included in a systematic review518 in which the considerable variability in responses between studies has been attributed to the diverse nature of the regimens used and differences in the prestudy treatments.

Effects on Serum Ferritin and Liver Iron Several prospective randomized studies have now compared the use of various combined treatments with that of desferrioxamine or deferiprone monotherapy. In a small study, no significant difference in serum ferritin was seen between standard desferrioxamine monotherapy 5 nights a week compared with 7 days of deferiprone and 2 nights of desferrioxamine.541 In another study, effects on ferritin and liver iron were similar with desferrioxamine monotherapy 5 nights a week compared with deferiprone 7 days a week plus 2 nights of desferrioxamine.542 In a study in which

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desferrioxamine was given five times a week in the combination arm, the serum ferritin and liver T2∗ decreased more in the combined group of desferrioxamine five times a week plus deferiprone 7 days a week than with desferrioxamine monotherapy five times a week.463 Together, these studies suggest that deferiprone given 7 days a week combined with desferrioxamine 2 nights a week will have similar effects on serum ferritin as desferrioxamine monotherapy given 5 nights but a greater effect than with deferiprone monotherapy at 75 mg/kg/day. When patients were randomized to one of three regimens, monotherapy of deferiprone or desferrioxamine or to combined deferiprone 7 days a week with desferrioxamine 2 nights a week, the greatest ferritin decrease was seen with desferrioxamine monotherapy and the least with deferiprone monotherapy.516 A similar three-armed study is in broad agreement with these findings. Desferrioxamine monotherapy 5 nights a week was compared with deferiprone monotherapy 7 days a week or the same treatment plus desferrioxamine twice a week.543 This 1-year study measured LIC at baseline and at the end of the study, allowing calculation of iron balance and total iron excretion based on the known transfusion rate. The combination therapy (n = 8) produced a significantly larger decrease in LIC and serum ferritin and a higher total iron excretion that deferiprone monotherapy (n = 12) and a similar effect to desferrioxamine monotherapy (n = 12). Decrease in LIC was only seen in 42% of patients on deferiprone monotherapy but in 88% of patients on combined treatment. These studies suggest that control of iron balance with monotherapy is more likely to be achieved with desferrioxamine than with deferiprone. Combined therapy with daily deferiprone plus desferrioxamine at least 2 nights a week is roughly equivalent to desferrioxamine given five times a week.

Effect on the Heart Initial observational studies with a variety of combined regimens suggested possible beneficial effects on myocardial T2∗ or on heart function. In 79 patients treated with deferiprone 7 days a week with a variable desferrioxamine regimen, improvement in left ventricular ejection fraction was found compared with the previous noncompliant desferrioxamine monotherapy.544 In another observational study, 42 patients given deferiprone 7 days a week plus desferrioxamine 2–6 nights a week over 3–4 years showed improvement in the left ventricular shortening.545 More recently, a randomized controlled study of 65 patients with moderate heart iron loading and T2∗ 8–20 msec was undertaken comparing monotherapy with standard desferrioxamine five times a week with the same desferrioxamine treatment plus deferiprone 7 days a week.463 Despite the relative conservative monotherapy desferrioxamine regimen, T2∗ improved 3 msec in 1 year but this increased to 6 msec with combined treatment. Although all patients had left ventricular ejection fraction of more than 56%

726 at baseline, left ventricular ejection fraction increased by approximately 2.5% in the combination arm and 0.5% in the monotherapy arm. In another small randomized study in patients with normal left ventricular ejection fraction at baseline, no difference was found with combination treatment after 1 year or with deferiprone monotherapy, but a small decrease was found with desferrioxamine monotherapy.543 Interestingly, desferrioxamine was given only twice a week, whereas in the study in which an improvement in left ventricular ejection fraction was seen, desferrioxamine was given five times a week in the combination arm.463 Prospective studies in patients with abnormal left ventricular ejection fraction are needed but until such data are available, it is recommended that desferrioxamine is given with or without deferiprone for the maximum practicable duration when patients have evidence of decreased left ventricular function.

Safety of Combined Treatment Formal safety data on combined treatment are limited. In general, alternating regimens are less likely to be an issue for toxicity compared with regimens in which chelation is simultaneous or overlapping. A meta-analysis of the incidence of agranulocytosis with combined regimens compared with deferasirox monotherapy suggested that the risk might be increased several-fold, although the numbers of evaluable patients are small (Macklin, IND submission to FDA, 2004); the increased incidence appeared to occur mostly in those regimens in which the drugs were administered simultaneously. In a recently reported prospective study, one case of agranulocytosis and two of neutropenia were seen at 1 year in the combination arm containing 32 patients.463 No excess in arthropathy was seen in the combination arm and no new tolerability issues that were not recognized with monotherapy have been reported.

Deferasirox (ICL670, Exjade) Historical Perspective Deferasirox is a member of a new class of tridentate (Fig. 29.6) iron-selective synthetic chelators, the bishydroxyphenyl-triazoles. Unlike the clinical development of desferrioxamine and deferiprone that were predominantly clinician led, deferasirox has been taken through a development program that complies with modern regulatory processes involving over 1,000 patients in phase I– IV studies. Preregistration phase I-III trials were designed to last 1 year but many have been extended to 3 years or more. The drug is now licensed for the treatment of transfusional iron overload, including children older than 2 years in most countries. The long-term tolerability profile and longer-term effects on complications of iron overload are still being evaluated.

Janet L. Kwiatkowski and John B. Porter Chemistry and Pharmacology The tridentate structure with 2:1 chelator:iron binding results in an iron complex coordinated by four oxygen and two nitrogen atoms with the chemical formula: 4-[3,5bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid and a molecular weight of 373. The stability of this complex as estimated by the pM values is intermediate between desferrioxamine and deferiprone (Table 29.10). Deferasirox is lipophilic but highly protein bound in plasma, which appears to confer good tissue penetration with faster mobilization of tissue iron than desferrioxamine.546 In culture systems, mobilization of myocyte iron appears to be efficient.381,402 This has been confirmed in studies with iron-overloaded gerbils.547 The drug has low solubility in water and is given once daily as an oral suspension. Pharmacokinetics were examined in the first phase I study of single oral doses of deferasirox involving 24 patients with thalassemia major. In this randomized, double-blind study, patients received single oral doses of dispersible tablets ranging from 2.5 to 80 mg/kg.428 Deferasirox was absorbed promptly and was detectable in the blood for 24 hours. A plasma half-life of 11–19 hours was found, supporting once-daily oral administration. The area under the curve (AUC) at 0–24 hours and Cmax of deferasirox increased nearly proportionally with the dose. At doses between 10 and 40 mg/kg/day the AUC of the iron complex of deferasirox was approximately 20%–30% of that of the iron free drug. A later study showed that with once daily repeated dosing at 20 mg/kg, peak plasma levels reach a mean of 80 ␮M with trough values of 20 ␮M (Table 29.10).548 The urinary excretion of deferasirox and its iron complex was less than 0.1% of the dose. A further study of using single doses of 14 C deferasirox showed that metabolism occurred at several sites with glucuronidation in the liver.549 Elimination of this and other metabolites was predominantly fecal. It was concluded that the final elimination process of deferasirox, the Fe-complex, and metabolites was by hepatobiliary anion transport and that the metabolic drug interaction potential via cytochrome p450 enzymes was low.

Effects on Iron Balance, LIC, and Ferritin Initial iron balance studies entailed formal metabolic balance studies. Twenty-four patients randomly allocated to one of three doses or to placebo showed that excretion averaged 0.13, 0.34, and 0.56 mg/kg/day at deferasirox doses of 10, 20, and 40 mg/kg/day, respectively, predicting equilibrium or negative iron balance at daily doses of 20 mg and above.429 The fraction of iron in the urine was less than 6% of total iron elimination with the majority occurring in the feces. These studies also showed that iron excretion in each patient was linearly related to exposure to the drug as measured by the AUC.

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease Iron balance over 1 year was assessed in a series of studies which used the Angelucci principle,318 relating LIC to body iron stores, together with the measured transfusion rate to calculate net iron balance in a range of conditions associated with transfusional iron overload. The pivotal phase III study in 586 thalassemia major patients aged 2–53 years compared changes in LIC, serum ferritin, and net iron balance in 290 patients treated with desferrioxamine with 296 patients treated with deferasirox453 In this and other similar studies in sickle cell disease225 and other rare anemias213 the dosing regimen chosen was conservative, with the dose chosen on the basis of baseline LIC obtained by biopsy or by SQUID. Although this was an understandable precaution to avoid overchelation in patients with a low iron load (LIC 0.5 mg/kg/day) transfusion rates.212 At 30 mg/kg, 96% of patients with a low iron-loading rate were in negative iron balance but at intermediate and high transfusional loading rates, the proportion fell to 80% of patients.212

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nary clinical studies. Cell culture402 and animal studies548,592 suggested that deferasirox would be effective at accessing myocardial iron. Although studies on myocardial iron estimation were not included as part of the phase II and III trials, preliminary data obtained during these studies in 27 patients treated at doses of 10–30 mg/kg/day showed an improved myocardial T2∗ at 1 year from 18.7 to 23 msec and improvement was significant for both patients with baseline T2∗ values below and above 20 msec.464,550 Prospective studies are ongoing to examine the effects of deferasirox on myocardial iron as measured by T2∗ . Preliminary results in one of these studies have been reported.551 Eighteen patients receiving 30 mg//kg for 6 months showed a significant mean improvement in myocardial T2∗ from 9.5 to 11.2 msec, with improvement in 14 of 18 patients. Randomized comparison of the effects with those of desferrioxamine is also in progress. No data on patients with preexisting myocardial dysfunction are yet available.

Unwanted Effects and their Management In the key registration phase III study, deferasirox was well tolerated453 with 5.7% discontinuing drug over 1 year. Common adverse events were generally mild, including transient gastrointestinal events in 15% and skin rash in 11%. The drug is now licensed in European Union and the United States for use in children aged 2 years and older with transfusional iron overload. Follow up data in the five core phase II/III studies are now at a median of 3.5 years with no evidence of new or progressive toxicities.552 Understandably, it is too early yet to allow any statements on the impact of deferasirox on survival. Recommended patient monitoring includes monthly creatinine and liver function and annual auditory and ophthalmic examinations, including slit-lamp examination and funduscopy.

Gastrointestinal Disturbances Gastrointestinal disturbances such as diarrhea, constipation, nausea, or vomiting were often self-limiting but in persistent cases were managed by dose modification or by changing the timing of the dose from morning to evening.

Skin Rash Mild to moderate skin rashes were usually managed by temporary dose reductions followed by subsequent return to the therapeutic dose. Severe skin rashes were usually managed by dose interruptions followed by gradual reintroduction at a smaller dose. Occasional cases of angioedema have been seen for which cessation of drug is recommended.

Renal Effects Effects on the Heart Thus far, experience of the effects of deferasirox on the heart are confined to cell culture, animal data, and prelimi-

Dose-dependent increases in serum creatinine of a third or more above baseline were observed in 38% of patients. These generally remained within the normal range and

728 never exceeded two times the upper limit of normal. Increases in creatinine typically occurred within a few weeks of starting or increasing therapy, were not progressive, and reversed or stabilized with dose adjustment when necessary. In older myelodysplastic syndrome patients in whom baseline creatinine values were just below the upper limit of normal, some patients increased values above normal but these were managed by dose modification. No cases of progressive increments above the normal range were seen in these studies. Proteinuria has been described with deferasirox but is also seen in thalassemia major patients who are not receiving chelation therapy553 and in sickle cell disease, and therefore baseline testing for proteinuria should be performed before starting treatment. Monthly testing for proteinuria is recommended. If positive, quantitation might be helpful and if this exceeds 1 mg/mL creatinine on more than one occasion treatment should be temporarily withheld or adjusted.

Liver Function In general the alanine aminotransferase values were observed to fall as LIC values fell with treatment. Increased liver enzymes judged to be related to deferasirox were observed in two patients in the core studies.453 Outside core studies, other cases of transaminitis have been seen that respond to dose reduction or drug cessation. Monthly liver monitoring is therefore recommended.

Effects in Children In five phase II and III studies, approximately half of the 703 patients were children younger than 16 years and included children as young as age 2 years. With follow-up at approximately 3 years, no adverse effects on growth or skeletal development have been found and no specific tolerability issues for children have been identified. Evaluation of pediatric patients has shown that growth and development proceeded normally while on deferasirox, lending support to its use in very young patients.

Janet L. Kwiatkowski and John B. Porter Recommended Dosing Regimen Deferasirox is taken as a suspension in 100–200 mL of water, orange, or apple juice stirred with a nonmetallic utensil. The relationship between dose and iron balance has been studied in large studies over a 1-year period. A decision needs to be made at the time of therapy whether iron balance alone is sufficient or whether negative iron balance is desirable. Recommended starting dose for iron balance is 20 mg/kg, but as can be seen from Figure 29.7, the required dose can be adjusted upward or downward depending on the iron-loading rate. In patients with low transfusion rates of less than 0.3 mg/kg/day a daily dose of 20 mg/kg should be sufficient to maintain iron balance, whereas the doses for intermediate (0.3–0.5) and high (>0.5) loading rates are 25 mg and 30 mg/kg, respectively.212 Response rates in myelodysplastic syndrome, rare anemias,213 and sickle cell disease225 are consistent with those in thalassemia with respect to dose dependency of LIC and ferritin response. The effective dose in milligram/kilogram is approximately half that obtained with subcutaneous desferrioxamine five times per week. In the first 4 weeks after starting deferasirox, weekly serum creatinine and liver function tests are advisable and repeated monthly thereafter. If the dose is adjusted upward, it is advisable to monitor these same variables for 4 weeks after dose increments. Studies are ongoing at doses up to 40 mg/kg/day to assess the safety and efficacy of such doses for the small proportion of patients who fail to respond to lower doses. The proportion of patients with ferritin values less than 1,000 ␮g/L in extension studies up to 3 years is falling, but this has not been associated with increased toxicity.554 At 42 months of deferasirox treatment, 25% of patients had ferritin levels less than 1,000 ␮g/L, and some patients achieve levels less than 500 ␮g/L, below which the drug is not licensed. To avoid a stop-go approach, it is sensible to reduce the dose slightly as values fall below 1,000 ␮g/L so ferritin can be kept between 500 and 1,000 ␮g/L without stopping treatment.

Future Perspectives with Chelation Therapy Other Effects Deafness, neurosensory deafness, and hypacusis were reported as adverse events irrespective of drug relationship in thalassemia major patients in the 1-year core trial in eight patients on deferasirox and seven on desferrioxamine.453 Cataracts or lenticular opacities were reported as adverse events irrespective of drug relationship in two patients on deferasirox and five on desferrioxamine. Overall the tolerability profile compared favorably with desferrioxamine.453 The results of the 4-year extension will be useful in interpreting the significance of these findings. Importantly, no drug-related agranulocytosis was observed in thalassemia or sickle cell disease patients from the core studies.225,453

The immediate future for chelation therapy is likely to be exciting as results of prospective studies become available with monotherapy and combined therapies using the currently available iron chelators. With three chelators now licensed in many countries, there is considerable scope for assessing how best to use these in combination if monotherapy fails. Unless this is done in a systematic way, however, the true risks and benefits are likely to remain unclear. New chelators continue to be synthesized and evaluated555 but have yet to reach beyond phase II of clinical evaluation. Intravenous depot desferrioxamine is an interesting concept that still requires formal evaluation beyond phase I clinical studies.556 The prospective evaluation of MRI to characterize iron distribution and its

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease relationship to transfusional iron loading and to the relative effects of different chelation regimens will be valuable in defining how best to use the tools now available for managing transfusional iron overload.

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485. Modell B. Advances in the use of iron-chelating agents for the treatment of iron overload. Prog Haematol. 1979;11:267– 312. 486. Dickerhoff R. Acute aphasia and loss of vision with desferrioxamine overdose. J Pediatr Hematol Oncol. 1987;9:287– 288. 487. Olivieri NF, Koren G, Matsui D, et al. Reduction of tissue iron stores and normalization of serum ferritin during treatment with the oral iron chelator L1 in thalassemia intermedia. Blood. 1992;79(10):2741–2748. 488. Rodda CP, Reid ED, Johnson S, Doery J, Matthews R, Bowden DK. Short stature in homozygous beta-thalassaemia is due to disproportionate truncal shortening. Clin Endocrinol. 1995;42(6):587–592. 489. Piga A, Facello O, Gaglioti G, Pucci A, Pietribiasi F, Zimmerman A. No progression of liver fibrosis in thalassaemia major during deferiprone or desferrioxamine iron chelation. Blood. 1998;92 (Supple 2)(10):21b. (Abstract) 490. Berkovitch M, Collins AF, Papadouris D, et al. Need for early, low-dose chelation therapy in young children with transfused homozygous beta thalassemia. Blood. 1993;82:359a. 491. Araujo A, Kosaryan M, MacDowell A, et al. A novel delivery system for continuous desferrioxamine infusion in transfusional iron overload. Br J Haematol. 1996;93(4):835–837. 492. Jensen PD, Heickendorff L, Pedersen B, et al. The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload. Br J Haematol. 1996;94(2):288–299. 493. Borgna-Pignatti C, Cohen A. Evaluation of a new method of administration of the iron chelating agent deferoxamine. J Pediatr. 1997;130(1):86–88. 494. Yarali N, Fisgin T, Duru F, et al. Subcutaneous bolus injection of deferoxamine is an alternative method to subcutaneous continuous infusion. J Pediatr Hematol Oncol. 2006;28(1):11– 16. 495. Collins AF, Fassos FF, Stobie S, et al. Iron-balance and doseresponse studies of the oral iron chelator 1,2- dimethyl-3hydroxypyrid-4-one (L1) in iron-loaded patients with sickle cell disease. Blood. 1994;83(8):2329–2933. 496. Hider R, Kontoghiorghes G, Silver J. Pharmaceutical Compositions. UK Patent GB 2118176A, 1982. 497. Kontoghiorghes GJ, Aldouri MA, Hoffbrand AV, et al. Effective chelation of iron in beta thalassaemia with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one. Br Med J. 1987;295(6612):1509–1512. 498. Kontoghiorghes GJ, Aldouri MA, Sheppard L, Hoffbrand AV. 1,2-dimethyl-3-hydroxypyrid-4-one, an orally active chelator for treatment of iron overload. Lancet. 1987b;1:1294–1295. 499. Porter J, Hoyes K, Abeysinghe R, Huehns E, Hider R. Animal Toxicology of iron chelator L1. Lancet. 1989;2:156. (Letter) 500. Porter JB, Hoyes KP, Abeysinghe RD, Brooks PN, Huehns ER, Hider RC. Comparison of the subacute toxicity and efficacy of 3-hydroxypyridin-4-one iron chelators in overloaded and nonoverloaded mice. Blood. 1991;78(10):2727–2734. 501. Hoffbrand A, Bartlett A, Veys P, O’Connor N, Kontoghiorghes G. Agranulocytosis and thrombocytopenia in patient with Blackfan-Diamond anaemia during oral chelator trial. Lancet. 1989;2:457. 502. al-Refaie FN, Wickens DG, Wonke B, Kontoghiorghes GJ, Hoffbrand AV. Serum non-transferrin-bound iron in betathalassaemia major patients treated with desferrioxamine and L1. Br J Haematol. 1992;82(2):431–436.

Transfusion and Iron Chelation Therapy in Thalassemia and Sickle Cell Disease 503. al-Refaie FN, Veys PA, Wilkes S, Wonke B, Hoffbrand AV. Agranulocytosis in a patient with thalassaemia major during treatment with the oral iron chelator, 1,2-dimethyl3-hydroxypyrid-4-one. Acta Haematol. 1993;89(2):86– 90. 504. Carthew P, Dorman BM, Edwards RE, Francis JE, Smith AG. A unique rodent model for both cardiotoxic and heptatotoxic effects of prolonged iron overload. Lab Invest. 1993;69:217– 222. 505. Carthew P, Smith AG, Hider RC, Dorman B, Edwards RE, Francis JE. Potentiation of iron accumulation in cardiac myocytes during the treatment of iron overload in gerbils with the hydroxypyridinone iron chelator CP94. Biometals. 1994;7(4):267–71. 506. Berdoukas V, Bentley P, Frost H, Schnebli HP. Toxicity of oral iron chelator L1. Lancet. 1993;341(8852):1088. 507. Cohen AR, Galanello R, Piga A, De Sanctis V, Tricta F. Safety and effectiveness of long-term therapy with the oral iron chelator deferiprone. Blood. 2003;102(5):1583–1587. 508. Pippard MJ, Pattanapanyssat K, Tiperkae J, Hider RC. Metabolism of the iron chelates of desferrioxamine and hydroxypyridinones in the rat. In: Proceedings of the European Iron Club. Budapest, Hungary; 1989:55. 509. Olivieri NF, Koren G, Hermann C, et al. Comparison of oral iron chelator L1 and desferrioxamine in iron-loaded patients. Lancet. 1990;336(8726):1275–1279. 510. Lange R, Lameijer W, Roozendaal KL, Kersten M. Pharmaceutical analysis and pharmacokinetics of the oral iron chelator 1,2-dimethyl-3-hydroxypyridi-4-one (DMHP). In: Proceedings of 4th International Conference on Oral Chelation. Limasol, Cyprus; 1993. 511. Kontoghiorghes G, Sheppard L, Barr J, et al. Iron Balance studies with the oral chelator 1,2,dimethyl-3hydroxypyridin-4-one. Br J Haematol. 1988;69:129a. 512. Hoffbrand AV, Al-Refaie F, Davis B, et al. Long-term trial of deferiprone in 51 transfusion-dependent iron overloaded patients. Blood. 1998;91(1):295–300. 513. Agarwal MB, Gupte SS, Viswanathan C, et al. Long-term assessment of efficacy and safety of L1, an oral iron chelator, in transfusion dependent thalassaemia: Indian trial. Br J Haematol. 1992;82(2):460–466. 514. Longo F, Fischer R, Engelbert R, Nielsen P, Sachetti L, Piga A. Iron balance in thalassemia patients treated with deferiprone. Blood. 1998;92(Suppl 1):235a. 515. Maggio A, D’Amico G, Morabito A, et al. Deferiprone versus deferoxamine in patients with thalassemia major: a randomized clinical trial. Blood Cells Mol Dis. 2002;28(2):196– 208. 516. Gomber S, Saxena R, Madan N. Comparative efficacy of desferrioxamine, deferiprone and in combination on iron chelation in thalassemic children. Indian Pediatr. 2004;41(1):21– 27. 517. Ha SY, Chik KW, Ling SC, et al. A randomized controlled study evaluating the safety and efficacy of deferiprone treatment in thalassemia major patients from Hong Kong. Hemoglobin. 2006;30(2):263–274. 518. Roberts D, Brunskill S, Doree C, Williams S, Howard J, Hyde C. Oral deferiprone for iron chelation in people with thalassaemia. Cochrane Database Syst Rev. 2007(3):CD004839. 519. Tondury P, Zimmermann A, Nielsen P, Hirt A. Liver iron and fibrosis during long-term treatment with deferiprone in

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744 538. Jackson GE, May PM, Williams DR. The action of chelating agents in the removal of copper from ceruloplasmin: an in vitro study. FEBS Lett. 1978;90(1):173–177. 539. Grady R, Giardina P. Iron Chelation: Rationale for Combination Therapy. Florida: The Saratoga Group; 2000. 540. Giardina PJ, Grady RW. Chelation therapy in betathalassemia: an optimistic update. Semin Hematol. 2001; 38(4):360–366. 541. Mourad FH, Hoffbrand AV, Sheikh-Taha M, Koussa S, Khoriaty AI, Taher A. Comparison between desferrioxamine and combined therapy with desferrioxamine and deferiprone in iron overloaded thalassaemia patients. Br J Haematol. 2003;121(1):187–189. 542. Galanello R, Kattamis A, Piga A, et al. A prospective randomized controlled trial on the safety and efficacy of alternating deferoxamine and deferiprone in the treatment of iron overload in patients with thalassemia. Haematologica. 2006;91(9):1241–1243. 543. Aydinok Y, Ulger Z, Nart D, et al. A randomized controlled 1-year study of daily deferiprone plus twice weekly desferrioxamine compared with daily deferiprone monotherapy in patients with thalassemia major. Haematologica. 2007;92(12):1599–1606. 544. Origa R, Bina P, Agus A, et al. Combined therapy with deferiprone and desferrioxamine in thalassemia major. Haematologica. 2005;90(10):1309–1314. 545. Kattamis A, Ladis V, Berdousi H, et al. Iron chelation treatment with combined therapy with deferiprone and deferioxamine: a 12-month trial. Blood Cells Mol Dis. 2006;36(1):21– 25. 546. Nick H, Acklin P, Lattmann R, et al. Development of tridentate iron chelators: from desferrithiocin to ICL670. Curr Med Chem. 2003;10:1065–1076. 547. Wood JC, Otto-Duessel M, Aguilar M, et al. Cardiac iron determines cardiac T2∗ , T2, and T1 in the gerbil model of iron cardiomyopathy. Circulation. 2005;112:535–543.

Janet L. Kwiatkowski and John B. Porter 548. Piga A, Galanello R, Forni GL, et al. Randomized phase II trial of deferasirox (Exjade, ICL670), a once-daily, orallyadministered iron chelator, in comparison to deferoxamine in thalassemia patients with transfusional iron overload. Haematologica. 2006;91:873–880. 549. Porter J, Waldmeier F, Bruin G, et al. Pharmacokinetics, metabolism and elimination of the iron chelator drug ICL670 in patients, following single oral doses of 1000 mg [14C]labelled drug at steady state. Blood. 2003;102:5b: Abstract 3720. 550. Eleftheriou P, Tanner M, Pennell D, Porter J. Response of myocardial T2∗ to oral deferasirox monotherapy for 1 year in 29 patients with transfusion-dependent anaemias; a subgroup analysis. Haematologic. 2006;91 (suppl):Abstract 999. R reduces car551. Wood J, Thompson A, Paley C, et al. Exjade
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, ICL670) in the management of blood transfusion-induced iron overload: results after a median reatment duration of 3.5 years. Submitted 2008. 553. Ong-ajyooth L, Malasit P, Ong-ajyooth S, et al. Renal function in adult beta-thalassemia/HB E disease. Nephron. 1998:78:156–161. 554. Cappellini M, Vinchinsky E, Ford J, Rabault B, Porter J. EvalR , ICL670) therapy in patients uation of deferasirox (Exjade
with transfusional iron overload who achieve serum ferritin (SF) ≤1000 ng/ml in long-term studies. Session type: Blood. 2007;110 16b:Abstract 3795. 555. Donovan JM, Plone M, Dagher R, Bree M, Marquis J. Preclinical and clinical development of deferitrin, a novel, orally available iron chelator. Ann N Y Acad Sci. 2005;1054:492– 449. 556. Dragsten PR, Hallaway PE, Hanson GJ, Berger AE, Bernard B, Hedlund BE. First human studies with a high-molecularweight iron chelator. J Lab Clin Med. 2000;135:57–65.

30 Induction of Fetal Hemoglobin in the Treatment of Sickle Cell Disease and ␤ Thalassemia Yogen Saunthararajah and George F. Atweh

INTRODUCTION The beneficial effects of high levels of fetal hemoglobin (HbF) in sickle cell disease and ␤ thalassemia have been recognized for many years. In 1948, Watson et al.1 noted that newborns with sickle cell disease do not suffer from the clinical complications of their disease until HbF declines to adult levels after the first 6 months of life. Later, it was shown that the majority of patients with sickle cell disease from some regions of Saudi Arabia2 and India3 who co-inherit another genetic determinant associated with high HbF levels, have a very mild sickling disorder. More recently, the Cooperative Study of Sickle Cell Disease, a large multicenter study of the natural history of sickle cell disease (see Chapter 19) demonstrated an inverse correlation between HbF levels and the frequency of painful crises and early death.4,5 These clinical and epidemiological observations are supported by laboratory studies that demonstrate a sparing effect of HbF on polymerization of deoxyhemoglobin S.6 Similarly, in ␤ thalassemia, an increase in the synthesis of fetal ␥ -globin chains may decrease the imbalance between ␣- and non-␣-globin chains and ameliorate the severity of the anemia. This appreciation of the beneficial effects of HbF in sickle cell disease and ␤ thalassemia stimulated a great deal of interest in the development of therapeutic agents to increase HbF production in patients with these disorders. In this chapter, we discuss the preclinical and clinical development of the different classes of pharmacological inducers of HbF production including DNA hypomethylating agents such as 5-azacytidine and decitabine, cytotoxic agents such as hydroxyurea, and inhibitors of histone deacetylase such as butyrate.

DNA HYPOMETHYLATING AGENTS Gene expression is a process that is regulated to an important degree by the packaging of DNA around histones

and by methylation of deoxycytosines that precede deoxyguanines (CpG) in the promoters of genes. The DNA methylation patterns that regulate the expression of cell type– specific genes are established by DNA methyltransferase (DNMT) enzymes that recognize and methylate target deoxycytosines. Generally, methylation in the promoter of a tissue-specific gene is associated with nonexpression of the gene. Approximately 65% of all CpG dinucleotides in vertebrate DNA are methylated at the cytosine residue by the action of DNA DNMT enzymes. As would be expected, ␥ -globin gene promoter DNA is relatively unmethylated at developmental stages when it is expressed, and methylated in adult bone marrow erythroid cells when the genes are not expressed.7 This suggests that DNA hypomethylating agents might reactivate the expression of the silenced ␥ globin gene. The related compounds 5-azacytidine and decitabine (Fig. 30.1) are cytosine analogs first synthesized more than 40 years ago. Like other nucleoside analogs, they were originally viewed as cytotoxic chemotherapy agents and drug regimens were based on the maximum tolerated dose. It was only with the emergence of the field of epigenetics that inhibition of DNA methylation, reactivation of gene expression, and induction of cellular differentiation were recognized as actions of decitabine (5-azacytidine exerts its DNA hypomethylating effect by being converted to decitabine by ribonucleotide reductase).8 Importantly, the DNA hypomethylating effect is seen at low and very well-tolerated doses of the drugs.9 The specific mechanism by which decitabine depletes DNMT in dividing cells explains why low doses of decitabine can hypomethylate DNA without causing cytotoxicity whereas higher doses are toxic: The mechanism of action of decitabine begins with decitabine phosphorylation by deoxycytosine kinase (DCK) and incorporation into the replicating genome, just like natural deoxycytidine. DNMT is covalently trapped as it tries to methylate the 5-N position of the triazene ring of DNA-incorporated decitabine. This covalent trapping/ modification depletes cellular DNMT and decreases DNA methylation. At high decitabine doses, hydrolytic cleavage at DNMT–CpG complexes overwhelms the cellular repair machinery, damaging the replicating genome to an extent that results in cell death (cytotoxicity). Lower doses do not overwhelm the cellular repair machinery, and although temporary growth arrest (cytostatic effect) might occur, cell division ultimately continues but with depleted DNMT, DNA hypomethylation, and consequently altered gene expression. 5-Azacytidine, but not decitabine, incorporates into RNA and inhibits protein synthesis. On a mole for mole basis, decitabine is a more potent inhibitor of DNMT than 5-azacytidine. The ␥ -globin gene in baboons is structurally and functionally similar to that of human beings. Prompted by the association of ␥ -globin gene promoter methylation with its developmental silencing, the ability of 5-azacytidine to induce HbF synthesis in baboons was studied. 745

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Yogen Saunthararajah and George F. Atweh NH 2

NH 2

NH2

4 5

N

3

N

N

N

N

2 HO

6

5′

HO N

O

4′

1

O

N

O

HO N

O

O

1′ 3′

2′

OH

OH

A

OH

B

OH

C

Figure 30.1. Structure of natural deoxycytidine (A), decitabine (B), and 5-azacytidine (C). Decitabine, a deoxynucleotide, is only incorporated into DNA and is a more specific and potent DNA-hypomethylating agent than 5-azacytidine, which is also incorporated into RNA.

5-Azacytidine (injected into anemic baboons at a dose of 2–4 mg/kg/d 5 d/wk for 2 wk) increased HbF levels up to 70% of total hemoglobin (HbF 70%).11 This effect was achieved at doses of the drug that caused mild neutropenia, but did not seem to change RBC or platelet production. The results in baboons led to a number of clinical studies in patients with sickle cell disease and ␤ thalassemia.12–14 In two patients with sickle cell disease treated with 5-azacytidine at a dose of 2 mg/kg/day for 5 days, HbF levels increased to 22% without substantial toxicity.13 The percentage of F cells (Chapter 7) increased up to 80%. There was also improvement in surrogate clinical markers, denoted by decreased irreversibility of sickle cells, dense cells, and indirect bilirubin.14,15 5-Azacytidine treatment of three patients with end-stage ␤ thalassemia also produced large increases in total hemoglobin (3–5 g/dL).16 In these patients with ␤ thalassemia, the total hemoglobin increases consist of HbF. Two of these patients, treated for 30 months (1–2 mg/kg/d, 4 d/wk, 1 time/mo), remained transfusion independent for the duration of treatment. Despite these very promising early results, further trials with 5-azacytidine were curtailed due to concerns regarding potential carcinogenicity. These concerns were largely based on a study conducted by Carr et al.17 in the Fisher rat testicular cancer model, which suggested that 5-azacytidine caused tumors in 31 of 70 rats treated with the drug. The study received criticism because the incidence of testicular cancer in the control population of Fisher rats was 0%, which is not in accordance with the usual rate (∼20%) of spontaneous testicular cancer development in these rats.18 Another controversy was whether HbF induction resulted from DNA hypomethylation induced by 5azacytidine, or cytotoxic effects. If cytotoxicity was the mechanistic basis for HbF induction, then other cytostatic/ cytotoxic agents such as the well-tolerated and orally available ribonucleotide reductase inhibitor hydroxyurea might also be effective. This led to studies of hydroxyurea in

patients with sickle cell disease and thalassemia, culminating in the Multicenter Study of Hydroxyurea (MSH) in patients with symptomatic sickle cell anemia. The MSH conclusively demonstrated that hydroxyurea reduced the frequency of acute painful episodes, acute chest syndrome, and transfusions.19 The historical efficacy of 5-azacytidine in HbF reactivation suggested that DNA-hypomethylating agents could still have a role in the management of sickle cell disease. Decitabine holds two apparent advantages over 5-azacytidine: 1) the majority of preclinical studies suggest chemoprevention of cancer rather than carcinogenicity, and 2) decitabine is exclusively incorporated into DNA (5-azacytidine is also incorporated into RNA) and is a more potent DNA-hypomethylating agent. Therefore, studies are being pursued that focus on the use of decitabine to reactivate HbF synthesis in patients with symptomatic sickle cell disease. In the first study, eight patients were treated with decitabine at low doses (compared with doses of decitabine used in previous cancer trials) of 0.15 mg/kg–0.30 mg/kg given 5 days per week for 2 weeks.9 Five of these patients did not have increases in HbF with hydroxyurea treatment (hydroxyurea levels documented compliance), two were low responders to hydroxyurea, and one patient was not in the MSH. The average ␥ -globin synthesis relative to non–␣globin synthesis prior to therapy was 3.2% and increased to 13.7% after treatment. HbF increased from 3.6% to 13.5%, whereas F cells increased from 21% to 55%. The HbF content per F cell increased from 17% to 24%. Hydroxyurea treatment increased HbF levels in these patients from 2.4% to 2.6%. With decitabine treatment, however, the average HbF increased to 12.7%, and total hemoglobin increased by 1 g/dL in six of eight patients. The only toxicity was transient neutropenia. These studies suggested that the optimal daily dose of decitabine is 0.2 mg/kg, producing greater increases in HbF without much increased

Induction of Fetal Hemoglobin in the Treatment of Sickle Cell Disease and ␤ Thalassemia risk of neutropenia compared with decitabine 0.15 mg/kg/ day. A follow-up study examined whether increased HbF and total hemoglobin levels could be maintained without toxicity with repeated cycles administered every 6 weeks over a 36-week period.20 In six sickle cell anemia patients, the average HbF during the last 20 weeks of treatment was 13.9% ± 2.8% (mean ± standard deviation [SD]) compared with a baseline HbF of 3.1% ± 2.8%. Maximum HbF levels were 18.45% ± 4.5% (mean ± SD). Maximum F cell percentages ranged between 58% and 87%. The average hemoglobin value increased from 7.2 g/dL to 8.8 g/dL (mean ± SD). Maximum hemoglobin values were 9.7 ± 0.5 g/dL. The neutrophil nadirs between weeks 16 and 36 were all less than 1,500/␮L, occurred at approximately 5– 6 weeks of each treatment cycle, and rapidly recovered. Interestingly, a peak in the platelet count mirrored the neutrophil count nadir. With the exception of neutropenia, there were no other toxicities. In a subsequent study, the objectives were to 1) evaluate whether decitabine is safe and effective if given by a more practical subcutaneous route; 2) assess whether weekly administration could produce cumulative and stable increases in HbF and total hemoglobin levels; 3) measure additional surrogate clinical endpoints (the pathophysiology of vasoocclusive crises in sickle cell disease involves erythrocyte adhesion to endothelium, endothelial damage, and activation of coagulation and inflammatory cascades; therefore, red cell adhesion to thrombospondin and laminin, markers of endothelial damage, coagulation system activation, and inflammatory activity were measured); and 4) confirm that, at the low doses being used, decitabine hypomethylates DNA without typical chemotherapy-associated myelotoxicity. In addition to hematological parameters, bone marrow morphology, and DNA methylation changes were assessed.21 Decitabine was administered subcutaneously at 0.2 mg/ kg, one–three times/week in two cycles of 6 weeks each, with a 2-week interval between cycles. Eight patients were studied who either did not respond to or did not tolerate hydroxyurea therapy. All patients demonstrated statistically significant increases in HbF (± SD) from 6.5% ± 1.4% to 20.4% ± 2.0%, and F cells from 38.1% ± 7.6% to 71.4% ± 6.5% with treatment. Three hydroxyurea nonresponding patients had lower HbF levels at baseline but demonstrated a rate of increase in HbF similar to that seen in five hydroxyurea responders. The only toxicity noted was neutropenia, which coincided with increases in the platelet count of up to 860 × 109 /L. Total hemoglobin increased from 7.6 ± 1 g/dL to 9.6 ± 0.9 g/dL. Both the absolute reticulocyte count and total bilirubin decreased during treatment. The reticulocyte count correlated inversely with total hemoglobin, suggesting that the reticulocyte decrease resulted from decreased hemolysis. There were significant improvements in a spectrum of surrogate clinical endpoints that measure the activity of vasoocclusive pathophysiology: 1)

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RBCs from patients demonstrated decreased adhesion to the matrix molecules thrombospondin and laminin; 2) D-dimer levels, which are a measure of coagulation system activity, decreased; and 3) soluble vascular adhesion molecule–1 and von Willebrand Factor propeptide levels, which are measures of endothelial damage, decreased. A number of observations suggest that the mechanism by which decitabine produces HbF elevations is DNA hypomethylation. DNA hypomethylation per se is not necessarily cytotoxic,10 suggesting that low doses of decitabine might hypomethylate DNA without inducing cytotoxicity. The low doses of decitabine used in the clinical trials and in vitro studies were sufficient to produce DNA hypomethylation.22 Platelet counts increased during therapy, concurrent with a downward trend in neutrophil and monocyte counts. Although there was a decrease in reticulocyte counts during therapy, there was a strong inverse correlation between the decreasing reticulocyte counts and increasing hemoglobin levels, suggesting that the decreased reticulocyte count resulted from decreased hemolysis. Serial bone marrow aspirate specimens obtained during therapy did not demonstrate a decrease in marrow spicule cellularity. Instead, there was an increase in megakaryocyte numbers and a decrease in the myeloid/ erythroid ratio (an increase in the proportion of erythroid cells). In vitro studies with decitabine treatment of human CD34+ hematopoietic cells were consistent with the in vivo results, demonstrating an increase in the proportion of mixed colonies (erythroid and myeloid) formed relative to pure granulocyte–macrophage colonies. Thus, decitabineinduced hypomethylation in the ␥ -globin gene promoter could contribute to the increased HbF with treatment, but hypomethylation at other genes might also be important because the change in pattern of hematopoietic differentiation could be contributing to the increased HbF. With regard to pharmacological reactivation of HbF, decitabine has a number of advantages when compared with hydroxyurea. All patients treated to date have had clinically significant increases in HbF. The large increases in HbF could translate into more clinical benefits and potentially some protection from organ complications such as pulmonary hypertension and stroke.23 The noncytotoxic mechanism of action of decitabine might facilitate prolonged administration and durable responses. Decitabine could potentially synergize with other epigenetically active agents, such as histone deacetylase inhibitors.24 Presently, there are no data regarding the clinical effectiveness of decitabine in sickle cell disease. The trials conducted to date, which have measured surrogate clinical endpoints such as HbF levels, red cell adhesion, coagulation system activation, and endothelial damage, indicate the promise of this drug as a treatment for sickle cell disease. Extended phase II/III studies evaluating clinical endpoints such as crisis frequency, quality of life, and survival will be required to demonstrate definitively a clinical benefit. As a further incentive to such studies, off-label treatment

748 of patients with severe sickle cell disease on a chronic basis produced notable clinical improvement.25 Although most preclinical studies show negligible mutagenicity and a chemopreventive rather than carcinogenic effect and no unusual cytogenetic abnormalities have been reported in the many patients with myelodysplastic syndrome treated with decitabine thus far, further studies involving chronic dosing of patients with sickle cell disease or thalassemia should attempt to measure the risks of inducing DNA mutations and chromosome instability, perhaps in comparison to the current standard of care, hydroxyurea, which has a similar risk profile. Decitabine is likely to be teratogenic and could have effects on male fertility; these concerns are significant because many of the target patient population are of reproductive age.

HYDROXYUREA Interestingly, controversy about the mechanism of activation of fetal ␥ -globin gene expression by 5-azacytidine led to the identification of hydroxyurea as a second therapeutic agent that can stimulate HbF production. Some investigators hypothesized that 5-azacytidine increases HbF production by accelerating erythroid cell differentiation rather than by hypomethylation of DNA.26 To support this hypothesis, experiments were performed to show that hydroxyurea, an S-phase–specific chemotherapeutic agent without DNA-demethylating activity, can also increase HbF production in phlebotomized monkeys27 and in patients with sickle cell disease.28 These interesting observations, however, did not completely resolve the controversy about the mechanism of action of 5-azacytidine or decitabine because these agents appear to be more potent inducers of HbF production than hydroxyurea. Moreover, decitabine was shown to increase HbF levels in sickle cell patients who were resistant to hydroxyurea, suggesting that the two drugs have different mechanisms of action.9 Nonetheless, hydroxyurea had some significant advantages, it is an oral agent with a relatively good safety profile and it was already in wide use for the treatment of myeloproliferative disorders. Thus, it quickly replaced 5-azacytidine as the agent of choice for stimulating HbF production in patients with sickle cell disease.

Clinical Use of Hydroxyurea The clinical use of hydroxyurea was discussed in detail in the first edition of this book and elsewhere more recently.29–32 Hydroxyurea was first used in a number of small, nonrandomized clinical trials that confirmed its HbF-inducing activity in sickle cell disease.33–34 These studies provided the proof of principle and led to the identification of an effective dose schedule for the use of this drug in the treatment of patients with sickle cell disease. These phase I/II studies were followed by a large randomized, placebo-controlled

Yogen Saunthararajah and George F. Atweh MSH that had well-defined clinical efficacy endpoints.19 This study was terminated prematurely when interim analysis showed a significant reduction in the frequency of crises and acute chest syndrome in the hydroxyureatreated group compared with the control group. There was also a reduction in the frequency of blood transfusions in the hydroxyurea-treated group but no changes in the frequency of stroke or death during the 2.5 years of study.19 The effects of hydroxyurea on quality of life were mixed but most evident in patients with the highest HbF response to treatment.35 On the basis of the controlled clinical trial, hydroxyurea became the first HbF-inducing drug to be approved by the Food and Drug Administration for the treatment of sickle cell anemia, and as a result, hydroxyurea is now widely used in the United States and Europe. Hydroxyurea should be used in all adults when indications for this treatment are present (Table 30.1). Unfortunately, for complex and poorly understood reasons, only a fraction of patients who might benefit from treatment receive it. In follow-up studies to the MSH, cumulative mortality was reduced nearly 40% and a favorable result was related to the ability of the drug to increase HbF and reduce painful episodes and acute chest syndrome.36 Children have a more robust HbF response to hydroxyurea than do adults.31,32,37–41 In a study of more than 100 children who received maximal drug doses, HbF increased to almost 20% and the treatment effects were sustained for 7 years without clinically important toxicity.42 Some have proposed using hydroxyurea for secondary and primary prevention of stroke in children, and recent studies suggest that hydroxyurea, by a mechanism still unknown, can in some individuals reduce transcranial Doppler flow rates into the normal range.43–47 This issue is now being studied in a controlled clinical trial. The clinical benefits of hydroxyurea in HbSC disease are unknown, although the drug does have effects on the HbSC disease erythrocyte.48,49 This topic is also just beginning to be studied in a therapeutic trial. Although hydroxyurea was associated with reduced mortality in follow-up of the MSH, patients still die of complications of this disease while receiving treatment. In a single-center analysis of the 34 patients who died due to sickle cell disease–related causes, acute chest syndrome was the cause of death in 35%. The deceased and surviving patients did not differ significantly in average hydroxyurea dose or HbF response but were older when hydroxyurea was first started, were more anemic, and had significantly higher serum blood urea nitrogen and serum creatinine levels. They might represent a subgroup of older patients, possibly with more severe disease and organ damage who would benefit from earlier treatment.50 Preventing the usual HbF to HbS switch in gene expression early in childhood, before HbF levels decline to subtherapeutic concentrations and sickle vasculopathy results, should “cure” sickle cell anemia. Studies using hydroxyurea beginning in the first year of life are underway and focus on

Induction of Fetal Hemoglobin in the Treatment of Sickle Cell Disease and ␤ Thalassemia

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Table 30.1. A method for treating patients with sickle cell anemia with hydroxyurea. Special caution should be exercised in patients with compromised renal or hepatic function and when patients are habituated to narcotics. Contraception should be practiced by both men and women because hydroxyurea is a teratogen and its effects in pregnancy are unknown. After a stable and nontoxic dose of hydroxyurea is reached, blood counts may be done at 4- to 8-week intervals. Granulocytes should be ≥2,000/mm3 , platelets ≥80,000/mm3 . A fall in hemoglobin level (in most patients who respond, the hemoglobin level increases slightly) and absolute reticulocyte count to