33 - 103 Disorders of Hemoglobin

103 Disorders of Hemoglobin

■ ■TREATMENT Treatment of the underlying disorder controls anemia of inflammation, as shown by the effect of anti-IL-6 agents in Castleman’s disease and rheumatoid arthritis. Standard treatment of anemia in CKD is based on ESAs (Chap. 322) with a target hemoglobin established at <11.5 g/dL to avoid cardiovascular complications. Since iron deficiency leads to ESA hyporesponsiveness, iron supplementation is usually required, mostly parenterally, because in advanced CKD high hepcidin levels prevent iron absorption. Intravenous iron is administered at high or refracted doses, ideally to maintain transferrin saturation at 30–40% and ferritin between 200 and 700 μg/L. Iron supplementation may lower the dose of ESA required to maintain the hemoglobin target. An oral alternative to intravenous iron is ferric citrate, which, binding intestinal phosphate, provides iron while reducing phosphate levels. Prolyl hydroxylase inhibitors that stabilize HIF-2α have been approved in some coun­ tries with the aim of replacing with a single oral drug both ESA and iron, since HIF upregulates EPO production as well as duodenal iron absorption. Although these novel antianemic drugs are efficacious, safety concerns still exist for their long-term use. Intravenous iron can be used in cancer-related anemia to reduce the need of transfusions, especially with ferritin <100 μg/L and before elective surgery. ESAs are used in low-risk myelodysplastic syndromes and in patients receiving chemotherapy notably when cancer is deemed uncurable. Intravenous iron supplementation is currently recommended in CHF patients with either ferritin <100 μg/L or transferrin saturation <20% with ferritin of 100–300 μg/L. Nonetheless, there is still some uncertainty as to whether this represents the best definition of iron defi­ ciency in CHF. Novel drugs targeting the hepcidin-ferroportin axis to counteract iron maldistribution are under investigation. Blood transfusions remain the therapeutic option for severe ane­ mia in the ICU, with guidelines increasingly recommending restric­ tive hemoglobin thresholds (7–8 g/dL) for hemodynamically stable patients. Intravenous iron is proposed to minimize the use of trans­ fusions, with some studies reporting improved clinical outcomes, although the risk of infections and hypophosphatemia are of concern in these fragile patients. ANEMIA OF AGING Prevalence of anemia in the elderly is ~10% at the age of 65 years, 25% in people >85 years, and 50% in those affected by multimorbidity, hos­ pitalized, or institutionalized. Anemia is typically mild (hemoglobin 11–12 g/dL) and multifactorial, and its impact is frequently underesti­ mated. Mounting evidence strengthens its negative influence on quality of life, muscle weakness, and the risk of falls and fractures, as well as its independent association with mortality. The adverse effects of anemia are seen in black patients at hemoglobin levels ~1 g/dL lower than in white patients. Approximately one-third of the cases are estimated to be due to nutritional causes, especially iron deficiency; one-third are due to inflammation, including CKD; and one-third remain without a detect­ able origin and are defined “unexplained anemia of aging.” Age-related decline in testosterone levels in males, stem cell exhaustion, and EPO reduction might contribute. Absolute iron deficiency is common, due to poor dietary iron intake, impaired absorption by chronic gastritis, or the use of proton pump inhibitors and blood losses. A gastrointestinal malignancy should always be ruled out in anemic elderly patients, but chronic bleeding may also result from nonmalignant intestinal lesions such as angiodysplasias and the use of antithrombotic drugs for com­ mon disorders such as atrial fibrillation. A recent study has shown that 100 mg of daily aspirin given for primary cardiovascular prevention in elderly subjects was associated with a 20% higher risk of anemia and iron deficiency as compared to placebo. The proposed ferritin cutoff for iron deficiency (45–70 μg/L) is higher than in younger patients. Treatment of iron deficiency may mitigate the negative prognostic implications of anemia. Functional iron deficiency due to overt or subclinical inflam­ mation often contributes, complicating the laboratory diagnosis. Traditionally “bone marrow senescence,” now known as clonal hema­ topoiesis of indeterminate potential (CHIP), is present in >10% of

otherwise-healthy individuals aged 70 years and increases sharply to

60% in the eighties. CHIP is associated with a chronic low-grade inflammatory status that worsens anemia and cardiovascular risk. Patients should be followed up for the risk of developing myelodys­ plastic syndromes (Chap. 107). The unexplained anemia of aging is usually characterized by an EPO level that is lower than expected for the degree of anemia. EPO can increase the hemoglobin level, but it is unclear whether that change has an influence on the hospitalization, frailty, and mortality risks associated with the anemia.

■ ■FURTHER READING Camaschella C: Iron deficiency. Blood 133:30, 2019. Cleland JFG et al: Redefining both iron deficiency and anaemia in cardiovascular disease. Eur Heart J 44:1992, 2023. Galy B et al: Mechanisms controlling cellular and systemic iron homeostasis. Nat Rev Mol Cell Biol 25:133, 2024. Ganz T: Anemia of Inflammation. N Engl J Med 381:1148, 2019. GBD 2021 Anaemia Collaborators: Prevalence, years lived with disability, and trends in anaemia burden by severity and cause, 1990-2021: Findings from the Global Burden of Disease Study 2021. Lancet Haematol 10:e713, 2023. Macdougall I: Anaemia in CKD: Treatment standard. Nephrol Dial CHAPTER 103 Transplant 26:770, 2024. Oyedeji CI et al: How I treat anemia in older adults. Blood 143:205, 2024. Pasricha SR et al: Iron deficiency. Lancet 397:233, 2021. Patel KV et al: Haemoglobin concentration and the risk of death in older adults: Differences by race/ethnicity in the NHANES III follow-up. Br J Haematol 145:514, 2009. Van Doren L, Auerbach M: IV iron formulations and use in adults. Disorders of Hemoglobin Hematol Am Soc Hematol Educ Program 1:622, 2023. Weyand AC et al: Prevalence of iron deficiency and iron-deficiency anemia in US females aged 12-21 years, 2003-2020. JAMA 329:2191, 2023. Vijay G. Sankaran, Martin H. Steinberg

Disorders of Hemoglobin Hemoglobinopathies result from changes in the amino acid sequence of globin; in thalassemia, synthesis of normal globin is insufficient. Together the disorders of hemoglobin compose the most common Mendelian genetic diseases. In addition, they are responsible for most instances of hemolytic anemia. Sickle cell disease and the hemoglobin E (HbE)–associated syndromes are the most prevalent hemoglobin­ opathies; β and α thalassemia are the most prevalent thalassemias. In addition to these common disorders of hemoglobin, rare globin mutations cause hemoglobin instability, cause increased or decreased affinity of hemoglobin for oxygen (O2), and allow spontaneous oxida­ tion of hemoglobin, reducing its O2 transport. O2 transport can also be reduced by exposure to carbon monoxide (CO) and extrinsic oxidizing agents (Table 103-1). HEMOGLOBIN Easy access to erythrocytes to study hemoglobin structure and func­ tion, reticulocytes to examine hemoglobin biosynthesis, and leukocyte DNA to define the mutations of hemoglobin, with the availability of hematopoietic stem and progenitor cells from blood and bone mar­ row, has placed hemoglobin disorders in the forefront of molecular medicine. The biology of hemoglobin provides the background for understanding the pathophysiology of its many genetic and acquired disorders and approaches to their treatment.

TABLE 103-1  Disorders of Hemoglobin I. Hemoglobinopathies—hemoglobin variants with amino acid sequence variants that alter the physical, chemical, or functional properties of hemoglobin A. Common variants with unusual properties

1. HbS—polymerization
2. HbE—reduced biosynthesis
3. HbC—hemoglobin-membrane interaction B. Altered oxygen affinity
4. High affinity—erythrocytosis
5. Low affinity—cyanosis, anemia C. Hemoglobins that oxidize readily
6. Unstable hemoglobins—hemolytic anemia, jaundice
7. M hemoglobins—methemoglobinemia, cyanosis II. Thalassemias—defective biosynthesis of globin chains A. α Thalassemias B. β Thalassemias C. Complex thalassemias III. Hereditary persistence of fetal hemoglobin—persistence of higher than normal levels of HbF into adult life A. Deletions within the HBB cluster—15–30% HbF in heterozygotes, pancellular HbF B. Point mutations in HBG2/1 promoters—5–30% HbF in heterozygotes; PART 4 Oncology and Hematology pancellular or heterocellular HbF IV. Acquired hemoglobinopathies A. Methemoglobin due to toxic exposures B. Sulfhemoglobin due to toxic exposures C. Carboxyhemoglobin D. HbH in erythroleukemia E. Elevated HbF in myelodysplasia ■ ■DEVELOPMENTAL BIOLOGY Successive waves of erythropoiesis direct the synthesis of different hemoglobin molecules that result from sequential activation and silencing of globin genes (Fig. 103-1). Hemoglobin is a tetramer of two pairs of unlike globin polypeptide chains, each containing a tet­ rapyrrole heme group. O2 binds to heme as erythrocytes traverse the lungs and is released in the tissues. Heme is nestled within a protective pocket of each globin subunit. HBB at 11p15.5 HS5 4 3 2 1 LCR HBA at 16pter HbA HS R1-R4 MCS B A FIGURE 103-1  Globin gene clusters and their hemoglobin products during gestation. A. The order of globin genes in the β- and α-globin gene clusters along with their upstream enhancers, the β-globin locus control region (LCR) and α-globin multispecies conserved sequences (MCS), which contain critical regulatory elements. Normal hemoglobin tetramers contain two α-globin chains and two non-α-globin chains. In the example shown, this is adult HbA. The β-globin gene cluster contains an embryonic ε-globin gene (HBE), two nearly identical fetal γ-globin genes (HBG2, HBG1), a major adult β-globin gene (HBB), and a minor adult δ-globin gene (HBD). The α-globin gene cluster contains an embryonic ζ-globin gene (HBZ) and duplicated α-globin genes (HBA2, HBA1) coding for identical proteins. Embryonic hemoglobins include Gower I (ζ2ε2), Gower II (α2ε2), Portland I (ζ2γ2), and Portland II (ζ2β2). Fetal hemoglobin (HbF, α2γ2) production begins at 6–8 weeks’ gestation, peaks during mid-gestation, then falls to <1% of total hemoglobin during the first 6 months of extrauterine life. B. Sites of erythropoiesis and globin synthesized from the yolk sac and the early embryo (months 1–3), the fetus (months 3–9), after delivery (months 9–12), and afterward (adult).

■ ■GLOBIN GENE CLUSTERS Globin is encoded in two nonallelic gene clusters. The β-globin gene cluster is on chromosome 11; the α-globin gene cluster is on chromo­ some 16 (Fig. 103-1). Tetramers of α-like and β-like globins form embryonic, fetal, and adult hemoglobins. Fetal hemoglobin (HbF, α2γ2) production begins at 6–8 weeks’ gestation, peaks during mid-gestation, then falls to <1% of total hemoglobin during the first 6 months of extrauterine life. Adult hemoglobin A (HbA, α2β2) production follows a pattern reciprocal to that of HbF. The hemoglobin composition of normal adults is >95% HbA, ~1% HbF, and 2–3% HbA2 (α2δ2). HbF and HbA2 are not functionally important in normal adults because of their low concentrations. Measuring their levels can provide helpful diagnostic clues for thalassemia and some hemoglobinopathies. Hemo­ globin is subject to posttranslational modifications. Most important clinically is the nonenzymatic glycosylation of HbA forming the adduct HbA1c, which is useful in the management of diabetes mellitus. ■ ■HEMOGLOBIN STRUCTURE All globin polypeptides have similar but not identical primary struc­ tures. α-Globins contain 141 amino acids, and β-like globins have 146 amino acids. This primary structure dictates, according to the constraints of protein folding, the secondary structure of globin into α-helical sections joined by small nonhelical stretches. Each globin chain folds into a tertiary conformation known as the globin fold, whereby charged amino acid residues face the exterior of the molecules while uncharged residues face the hydrophobic interior. The ironcontaining tetrapyrrole heme moiety is protected from oxidation and located between two of the helical segments; O2 loading and unload­ ing occur when heme iron is in its reduced ferrous form. Globin gene mutations affecting critical heme-binding amino acid residues allow iron to be oxidized, forming methemoglobin, which has high O2 affin­ ity and does not release O2 in tissues. Dimers of α- and non-α-globin chains reversibly assemble into the tetrameric quaternary structure. ■ ■HEMOGLOBIN FUNCTION Hemoglobin transports O2 from lungs to tissues and carbon dioxide (CO2) from tissues to lungs. As a nitrate reductase, it releases nitric oxide (NO) from nitrite to promote vasodilation. Oxygen binding is defined by the hemoglobin-O2 dissociation curve. P50 is a point on this sigmoidal curve that indicates the partial pressure of O2 where hemo­ globin is half saturated (Fig. 103-2). Normal P50 is ~26 mmHg; low P50 indicates that hemoglobin has high O2 affinity, decreasing O2 delivery Yolk sac Spleen Bone marrow Liver

Globin synthesis (%) HBG1 HBG2 HBB

HBE1

HBD

Adult

pH Less O2 delivered Oxyhemoglobin 2,3-BPG T°

Percent saturation of hemoglobin

pH More O2 delivered

2,3-BPG P50 T°

Deoxyhemoglobin

Tissue PO2 (mmHg) FIGURE 103-2  Hemoglobin-oxygen dissociation curve. The P50 is influenced by the binding of 2,3-bisphosphoglycerate (2,3-BPG), a product of glycolysis, in the central cavity of hemoglobin, pH, and temperature. The hemoglobin tetramer can bind up to four molecules of oxygen (O2) in the iron-containing sites of the heme molecules. As O2 is bound, 2,3-BPG and carbon dioxide (CO2) are expelled. Salt bridges are broken, and each of the globin molecules changes its conformation to facilitate O2 binding. O2 release to the tissues is the reverse process, with salt bridges being formed and 2,3-BPG and CO2 bound. Deoxyhemoglobin does not bind O2 efficiently until the cell returns to conditions of higher pH, the most important modulator of O2 affinity (Bohr effect). When acid is produced in the tissues, the dissociation curve shifts to the right, facilitating O2 release and CO2 binding. Alkalosis has the opposite effect, reducing O2 delivery. to tissues; high P50 indicates that hemoglobin has low O2 affinity, releas­ ing more O2 to tissues. The conformation of hemoglobin fully saturated with O2 is known as the R or relaxed state; desaturated hemoglobin is in the T or tense state. The transition between T and R states occurs when two or three O2 molecules are bound. Cooperativity describes the progressively more rapid binding of O2 once the first molecule is bound. Hemoglobin variants that decrease P50 are characterized by isolated erythrocytosis as compensation for hypoxia; variants with increased P50 sometimes are accompanied by cyanosis and anemia as hemoglobin becomes unsaturated and O2 delivery is enhanced. Muta­ tions of residues critical for heme binding, R-T transitions, or tetramer stability cause hemoglobinopathies characterized by hemolytic anemia, methemoglobinemia, erythrocytosis and cyanosis. ■ ■GLOBIN GENE SWITCHING The sequential activation and inactivation of globin genes during development is called “hemoglobin switching.” Transcription factors along with epigenetic elements, such as DNA and histone methyltrans­ ferases and demethylases, interact with enhancers “upstream” of the gene clusters that contact globin gene promoters, silencing the embry­ onic and fetal genes. Developmental factors such as RNA-binding fac­ tors and microRNAs also impact hemoglobin switching. β-Globin Gene Switching  HbF reactivation by drugs and gene therapy is a prime therapeutic goal for treating sickle cell disease and β thalassemia, meriting a discussion of the controls of HbF gene silenc­ ing. An upstream enhancer called the β-globin locus control region (LCR) binds erythroid-specific and ubiquitous transcription factors. The LCR interacts directly with globin gene promoters; transcription factors that silence and activate genes also interact with elements of the globin genes. Competition among the β-like genes for the LCR and autonomous silencing of the embryonic and fetal globin genes depends on transcription factors. Silencing, first of the embryonic gene HBE and then of the two fetal genes, HBG2 and HBG1, favors the interaction of the LCR with HBB allowing its expression (Fig. 103-1).

The transcription factors BCL11A (2p16) and ZBTB7A (19p13) are the major repressors of HbF gene expression. BCL11A, a zinc finger protein that represses HbF genes, binds TGACCA motifs, the most important at position –115 in the promoter of each γ-globin gene. ZBTB7A binds 85 nucleotides upstream of these BCL11A binding sites. Mutations in these binding sites abolish the normal silencing of the HbF genes, leading to one type of the benign condition called hereditary persistence of fetal hemoglobin (HPFH). When binding of either BCL11A or ZBTB7A is disrupted, HBG2 and HBG1 are dere­ pressed. BCL11A single nucleotide variants (SNVs) are common and are thought to underlie a large portion of the interindividual variation in HbF levels. Disruption of the BCL11A regulatory elements or the promotor binding sites for BCL11A and other repressive factors by gene editing in patient hematopoietic stem cells using CRISPR/Cas leads to 30–50% HbF with possible “cure” of sickle cell disease and β thalassemia, with the former strategy receiving U.S. Food and Drug Administration (FDA) approval for both conditions. `-Globin Gene Switching  A less complex switch takes place in the α-globin gene cluster. A regulatory locus of four elements termed R1–R4 is present within introns of the gene NPRL3 that is upstream of HBA2. R1–R4 are critical for α-globin gene expression, as dem­ onstrated by natural deletions causing thalassemia. A developmental switch from embryonic ζ- to adult α-globin gene expression occurs at about 6 weeks’ gestation. Modulation of HbF Level and Haplotypes of the a-Globin Gene Cluster  Variations in three quantitative trait loci (QTL), BCL11A, MYB (6q23), and a locus linked to the HBB cluster (11p15), are associated with HbF variation among normal individuals and patients with sickle cell anemia and β thalassemia. The MYB gene is essential for hematopoiesis and erythroid differentiation. MYB inhibits HbF expression directly by activation of KLF1 and other repressors and indirectly through alteration of the kinetics of erythroid differ­ entiation. The third QTL is marked by a common variant 158 nucleo­ tides upstream of the transcription start site of HBG2. Five common haplotypes associated with the HBB cluster have been defined by its SNVs. Sickle cell anemia patients with the Senegal and Arab-Indian HbS gene-associated haplotypes have the common –158 C-T variant in the HBG2 promoter. They have higher HbF levels than patients with Benin, Bantu, and Cameroon haplotypes. When young, they might have a milder clinical course.

CHAPTER 103 Disorders of Hemoglobin GENERAL ASPECTS OF HEMOGLOBIN DIAGNOSIS α-Globin gene mutations are expressed in the embryo and fetus and persist throughout life; HbF mutations are expressed in the fetus and in the first months of life, vanishing from notice afterward; δ-globin gene mutations are innocuous and usually not detected; β-globin gene mutations can become clinically apparent after the synthesis of HbF dwindles to stable adult levels. With rare exceptions, all disorders of hemoglobin are autosomal recessive or co-dominant disorders; a family history, usually of ane­ mia, a common feature of most symptomatic hemoglobinopathies and thalassemias, is often present. In addition to pallor and jaundice, sple­ nomegaly is often present. A small number of laboratory tests can con­ firm the diagnosis starting with a complete blood count that includes a reticulocyte count with a careful review of a peripheral blood film. A sustained increase in reticulocyte count indicates the presence of hemolytic anemia. Hemoglobin fractionation by high-performance liquid chromatography (HPLC) or capillary electrophoresis, especially when, in addition to the index case, family members are available for study, is often sufficient to confirm a diagnosis at the level of hemoglo­ bin phenotype. DNA sequencing of the globin genes allows definitive diagnosis; available from excellent reference laboratories, it is a prereq­ uisite for genetic counseling. Sickle cell disease and β thalassemia are chronic hemolytic ane­ mias sharing hemolysis-related complications like venous thrombosis, leg ulcers, and pulmonary hypertension. Differences are that only deoxyHbS polymerizes, while ineffective erythropoiesis is the key

pathophysiologic feature of β thalassemia. Both diseases are “cured” by successful allogeneic hematopoietic stem cell transplantation and gene therapy as discussed below.

SICKLE CELL DISEASE Sickle cell disease is a clinical and hematologic phenotype caused by an assortment of genotypes (Table 103-2). Sickle cell anemia, defined as homozygosity for the sickle hemoglobin mutation (α2βS 2; glutamic acid [E] 7 valine [V] GAG-GTG), is the most common of these genotypes, followed by HbSC disease or compound heterozygosity for HbS and HbC (α2βC 2; E 7 lysine [K] GAG-AAG) genes. Many different thalas­ semia mutations contribute to the HbS-β thalassemias. Compound heterozygous genotypes are less common than HbS homozygotes. HbS has been described in compound heterozygotes with many other vari­ ant hemoglobins. Few of these genotypes, other than HbSOArab, HbSE, and HbSDPunjab are symptomatic. ■ ■ORIGIN, SPREAD, AND EPIDEMIOLOGY HbS originated in Africa between 7000 and 22,000 years ago, reaching high frequencies because of the increased genetic fitness of heterozy­ gotes under selective pressure from Plasmodium falciparum. HbS gene haplotypes have a loose association with the severity of disease because each haplotype has a different average level of HbF. In some regions of Africa, India, and the Middle East, nearly half the population have sickle cell trait. Nigeria alone has ~150,000 newborns each year with sickle cell anemia, about one-third of the world’s total newborns; most die before age 5. Coerced and free population movement has spread the HbS gene throughout the world. The HbS carrier, or sickle cell trait, prevalence is 2–15% in emigrant populations; ~100,000 patients in the United States have sickle cell disease. In the United States, death in childhood is rare; the median age of death in patients with sickle cell anemia is in the fifth or sixth decade. PART 4 Oncology and Hematology ■ ■PATHOPHYSIOLOGY Pathophysiologic features of sickle cell disease are summarized in Fig. 103-3. HbS is physiologically like HbA in most respects except it polymerizes when deoxygenated. Contacts between one of the TABLE 103-2  Common Sickle Hemoglobinopathies GENOTYPE CLINICAL ABNORMALITIES Sickle cell trait (HbAS) 8% of African Americans; hematuria, papillary necrosis, hyposthenuria, increased incidence of chronic kidney disease; 2–4 times increased VTE risk;? stroke; splenic infarction at altitude; rhabdomyolysis Sickle cell anemia (HbSS) Vasoocclusion related: pain, acute chest syndrome, osteonecrosis,

splenic infarction Hemolysis related: stroke, pulmonary and systemic vasculopathy, nephropathy, leg ulceration gallstones, priapism HbS-β0 thalassemia Rate of complications similar to HbSS 80–100 (8–11)/60–85 HbS: >75 HbF: 2–15 HbA2: 5–6 HbS-β+ thalassemia Rate of complications about half the rate of HbSS depending on percent HbA 100–140 (10–14)/70–80 HbS: 60–90 HbA: 5–40 HbF: 1–10 HbA2: 5–6 Hemoglobin SC disease (HbSC) Nearly asymptomatic to disease as severe as HbSS; about half the rate of complications as HbSS. Increased risk of retinopathy HbSE Resembles clinically HbS-β+ thalassemia; symptoms delayed; often

Asian/Indian ancestry HbSS-α thalassemia Present in 30% of HbSS; phenocopies HbS-β0 thalassemia because of microcytosis and high HbA2; like HbSS but with fewer strokes and leg ulcers and less pulmonary vascular and renal disease Note: Laboratory values are averages in untreated adults. Abbreviation: VTE, venous thromboembolism.

β7 valine residues of deoxyHbS and specific amino acid residues of β- and α-globin culminate in fascicles of hemoglobin that injure the sickle erythrocyte. A delay occurs between the initiation of polym­ erization and the accumulation of sufficient polymer to damage the cell. It is unclear how much polymer is needed for cell injury, but polymer leads directly and indirectly to the multiple abnormalities of the sickle erythrocyte that generate the pathophysiology of disease. Prominent among these abnormalities are HbS polymer penetration of the membrane causing vesiculation with membrane microparticle release; increased activity of the Gardos, K/CL cotransport, and Psickle channels that dehydrate the cell, increasing mean corpuscular sickle hemoglobin concentration (MC[HbS]C), reducing cellular deform­ ability, and increasing the polymerization potential of HbS; transloca­ tion of amino phospholipids such as phosphatidylserine to the outer leaflet of the membrane; and oxidation of erythrocyte contents. These and other abnormalities lead to the formation of irreversibly sickled cells (ISCs), which are sickle erythrocytes that are forever deformed because of permanent membrane damage regardless of whether HbS remains polymerized. Damaged sickle erythrocytes are responsible for initiating the vasoocclusive, hemolytic, and inflammatory features of the disease shown in Fig. 103-3. ■ ■DIAGNOSIS Although sickle cell disease can appear in any ethnic group, most often it is present in people of African, Middle Eastern, Mediterranean, and Indian descent. The chief presenting symptom is pain. This might be an arthritis-like hand-foot syndrome in young children or the typical acute painful episode in older children and adults. In HbSC disease and HbS-β+ thalassemia, acute vasoocclusive episodes occur at about half the rate as in sickle cell anemia while complications develop later; rarely, patients with these genotypes are asymptomatic. The key elements of laboratory diagnosis are outlined in Table 103-2 showing typical hematologic findings and hemoglobin fractions. Figure 103-4 displays HPLC profiles and blood films in typical patients with sickle cell trait, sickle cell anemia, and HbSC disease. Clinical and basic laboratory diagnosis is sufficient for general management and counseling; genetic counseling and family planning usually require DNA-based diagnosis. HEMOGLOBIN LEVEL, g/L (g/dL)/MCV, fL HEMOGLOBIN FRACTIONS (%) Normal HbA: 60–70 HbS: 30–40 Percent HbS dependent on presence or absence of α thalassemia 70–100 (7–10)/80–100 HbS: >75 HbF: 2–25 HbA2: 3–4 100–140 (10–14)/70–100 HbS: 50 HbC: 50 90–130 (9–13)/65–75 HbS: 65 HbE: 35 HbF: 1–5 80–100 (8–11)/60–85 HbS: >75 HbF: 2–15 HbA2: 4–5

HbS polymer

Triplet codon T 7 Glu Valine residue GAG HbS solution HbS polymer N Oxygenated Deoxygenated Hemolysis HbS cell Cell heterogeneity FIGURE 103-3  Pathophysiology of sickle cell disease. HbS is in solution when oxygenated but reversibly polymerizes when deoxygenated. Polymerization is dependent on the 30th power of hemoglobin concentration. In the sickle cell, this means that small changes in hemoglobin concentration or cell hydration can have large effects on polymerization. Polymerization begins seconds to minutes following deoxygenation. Erythrocyte deformation, or sickling, is initially reversible, but after an undetermined number of cell sickling events, the cell becomes irreversibly deformed. These are known as irreversibly sickled cells (ISCs). Their membrane is permanently damaged, although depending on their oxygen (O2) content, HbS could be in solution. Sickle erythrocytes lead to the clinical and laboratory phenotypes of disease. Sickle cells interact with endothelial cells and other blood cells, occluding flow in small and sometimes large vessels and causing the many complications thought to be a result of vasoocclusion. Sickle cells also live <20 days (normal ~120 days) hemolyzing intra- and extravascularly. Intravascular hemolysis depletes haptoglobin and hemopexin while liberating heme, arginase, and other danger-associated molecular patterns (DAMPs) into the blood. This scavenges nitric oxide (NO), activates platelets and endothelium, reduces antioxidant activity, causes vasoconstriction, and is proinflammatory. ■ ■COMPLICATIONS Complications of sickle cell disease can be grouped into those that likely are a consequence of the related entities of sickle vasoocclusion and those due to intravascular hemolysis (Fig. 103-3). Complications associated with vasoocclusion seem to respond best to induction of HbF. Some complications of disease are presented in Table 103-3. Acute Painful Episodes  Characterized by unprovoked severe pain in extremities or the torso that is often symmetrical and stereo­ typical for each patient and that usually requires treatment with strong opioids in the emergency department, acute painful episodes are the most common acute events in sickle cell disease. They are the chief cause of concern for patients, most of whom have them at some time in their life. Their frequency varies; most patients have one to two episodes a year; some rarely have them; others are hardly ever without them. Acute painful episodes last days to weeks. Pain in sickle cell dis­ ease can also be chronic from osteonecrosis, osteoporosis, or leg ulcers. Chronic and acute pain can overlap. Pain can also be induced by the opioids. Most of the time, patients have some degree of pain that does not reach the intensity of the acute episode. This can be treated with oral opioids dispensed monthly. No diagnostic test can confirm or refute the presence of an acute pain episode whose cause is uncommonly identified. Physical examination is not often useful diagnostically. Some patients will have pain on pressure over an affected area, perhaps accompanied by swelling; mild fever is common. Often a 1–2 g/dL decrease in hemoglobin level and a modest increase in the leukocyte count are noted. The presence of ISCs and

Vasoocclusion R RBC ISC EC NO NO synthase NO –

CHAPTER 103 Arginine NO Arginase Ornithine Citruline Disorders of Hemoglobin the reticulocyte count are of no diagnostic value. Drastic decreases in hemoglobin and platelet levels with more extreme leukocytosis can por­ tend development of severe acute chest syndrome or multiorgan failure. Some patients die suddenly shortly after admission for an acute painful episode. The cause of this sudden unexpected death is usually unknown; among the possibilities are arrhythmias and pulmonary embolism. Admitting patients to monitored beds or using continuous pulse oximetry for the first 48–72 h of hospitalization might prevent some of these deaths and help identify early acute chest syndrome that follows within 72 h in about a quarter of admissions for acute pain. After searching for possible precipitants such as infection or dehydra­ tion and treating these appropriately, the foundation of treatment is the proper dosing of opioid analgesics. By the time a patient presents at the emergency department or clinic requesting treatment, they have usually tried nonsteroidal anti-inflammatory drugs (NSAIDs) and oral opioids. In most patients, relief of pain requires intravenous opioids. Many patients are opioid tolerant, requiring higher than usual doses for satisfactory relief. Dosing should not be on an “as-needed” schedule; patient-controlled analgesia or frequent fixed doses of opioids with res­ cue doses for breakthrough pain are the preferred means of treatment, with frequent assessments to ensure pain relief without excessive seda­ tion. Adjunctive treatment includes incentive spirometry to forestall pulmonary complications, maintaining hydration with half-normal saline with care not to overhydrate, prophylaxis for thromboembolism, and antihistamines and laxatives to counter expected side effects of opioids. Unless hypoxia is present, supplemental O2 is unnecessary. NSAIDs have little value in patients receiving intravenous opioids.

45.0 45.0 37.5 37.5 30.0 30.0 15.0 % 22.5 22.5 E 2.14 A2 3.64 15.0 1.20 1.36 1.67 7.5 7.5 4.51 2.33 0.0 0.0

HbF HbS HbA HbA2 HbS PART 4 Oncology and Hematology FIGURE 103-4  Diagnosis of sickle cell disease. A. From left to right, high-performance liquid chromatography separation in sickle cell trait, sickle cell anemia, and

HbSC disease. Beneath each chromatogram, the individual protein peaks are identified. B. Left: Dense, elongated, and pointed cells are the irreversibly sickled cells characteristic of the sickle cell anemia and sickle cell-β0 thalassemia. Target cells and nucleated red cells are also present. Right: Target cells, cells with squared ends of HbC crystals, cells folded like tacos, and contracted microspherocytes are typical of HbSC disease. (Source: B [right]: Reproduced with permission from American Society of Hematology.) TABLE 103-3  Complications of Sickle Cell Disease COMPLICATION INCIDENCE, DIAGNOSIS, AND FEATURES TREATMENT Priapism ~30% of males; can be episodic and short duration (stuttering); severe episodes can cause impotence; associated with markers of hemolysis Stroke and silent infarction 10–15% of all cases; infarction in early childhood into adulthood; hemorrhagic in adults; neurocognitive abnormalities in adults even without apparent stroke; associated with markers of hemolysis Gallstones/surgery ~40% of patients; bilirubin levels and stones related to polymorphisms of UGT1A; in surgery requiring general anesthesia, simple preoperative transfusion to a hemoglobin of 10 g/dL is recommended Hepatic disease

80% of patients have hepatomegaly; intrahepatic cholestasis can have bilirubin ~100 mg/dL; viral hepatitis, iron overload, RBC sequestration, extrahepatic cholestasis also contribute Nephropathy ~30% of adults age >30 years; hyperfiltration in children, renal failure in adults; early albuminuria, later nephrotic-range proteinuria; associated with markers of hemolysis Lung/pulmonary hypertension Restrictive disease; asthma common; 5–10% have pulmonary hypertension by right heart catheterization; 30% have increased TRV that portends poor prognosis; associated with markers of hemolysis Retinopathy 30% in HbSC disease, 3% in HbSSa; develops in peripheral retina; vitreous hemorrhage and retinal detachment can cause blindness Acute anemic episodes B19 parvovirus infection, folic acid deficiency, splenic sequestration, delayed hemolytic transfusion reaction with destruction of transfused and sometimes autologous red cells Multiorgan failure Can accompany severe acute chest syndrome; often confused with sepsis and can coexist with sepsis; CNS liver, muscle, lung, kidney affected Pregnancy Screening both partners for hemoglobin disorders with risk counseling is critical component of family planning aSickle cell anemia (HbSS). Abbreviations: ACE, angiotensin-converting enzyme; CNS, central nervous system; ICU, intensive care unit; NSAIDs, nonsteroidal anti-inflammatory drugs; RBC, red blood cell; TRV, tricuspid regurgitant jet velocity.

45.0 37.5 30.0 22.5 3.65 15.0 3.61 2.20 2.33 F 1.14 7.5 2.43 A2 4.51 4.67 A2 5.17 0.0

HbS HbC Many therapies including α-adrenergic agonists, stilbesterol; consult urology for treatment, which is time-critical Transcranial Doppler screening in children ages 2–16; transfusion for at-risk patients; hydroxyurea If asymptomatic, usually let be; otherwise, laparoscopic cholecystectomy Exchange transfusion for intrahepatic cholestasis; transplant for end-stage liver failure Screen for microalbuminuria by age 10 years; avoid NSAIDs; use ACE inhibitors or receptor antagonists for albuminuria; erythropoietin for symptomatic anemia; dialysis or transplant for renal failure Consult expert pulmonologist; screen yearly by echocardiography measurement of TRV Screen annually starting at age 10 tears with fluorescein angiography; laser photocoagulation for proliferative disease RBC transfusion if symptomatic; splenectomy if more than 1 or 2 episodes of sequestration; anti-parvovirus IgM positive in acute infection, IgG in past infection Exchange transfusion, ICU support All pregnancies are “high risk”; transfuse if sickle cell events increase, if previous miscarriage, multiple fetuses

Acute Chest Syndrome  This pneumonia-like illness is the second most frequent acute sickle cell–related event. It occurs in >50% of patients, often more than once. Acute chest syndrome can be mild, especially in children, in whom it can result from viral infection, or devastating, where multiple lobes of the lung are affected with severe hypoxia, multiorgan failure, and death. Chest pain, cough, fever, and hypoxia and a pulmonary infiltrate on chest x-ray are the major diag­ nostic criteria. The etiology includes in situ thrombosis, emboli, any type of infection, and postoperative hypoventilation. Management in adults is dictated by the severity of the episode and the need for supple­ mental oxygen. Patients who are hypoxic and febrile can be admitted directly to the intensive care unit. Antibiotics are almost always used in these patients even though a causative bacterium is not often cultured. Supplemental O2 is given for an O2 saturation <95%. Overhydration and excessive opioids can compound dyspnea and hypoxia. Hypoxic patients who are febrile with leukocytosis with more than a trivial infiltrate on x-ray are transfused. In the severely ill patient, exchange transfusion, if possible, is the preferred modality. However, if transfu­ sion of the severely ill patient is indicated and hours are needed to arrange red cell exchange, simple or top-up transfusion should be started first. Simple transfusions also suffice for less severely affected patients. Most patients survive acute chest syndrome, but in the most severe cases, often caused by embolization of necrotic bone marrow, death can be rapid even with prompt and proper treatment. Thrombo­ cytopenia, leukocyte counts >20,000/dL, and rapidly developing acute anemia often portend severe acute chest syndrome with its possibility of acute respiratory distress syndrome and multiorgan failure. Asthma is very common in patients with sickle cell disease. Some adults have chronic lung disease with reduced diffusing capacity for CO that could be a sequela of acute chest syndrome. Osteonecrosis  This painful and sometimes crippling complica­ tion that most often affects hips bilaterally occurs in about half of all patients with sickle cell anemia and is also common in HbSC disease; shoulders are less often affected. Beginning with chronic pain that can become severe, loss of function, especially in the hips, is often the final stage. Magnetic resonance imaging (MRI) can detect the earliest stages, whereas x-ray is less sensitive. Physical therapy and NSAIDs provide some initial relief; oral opioids are sometimes required. Joint replacement can restore lost mobility while relieving pain. Life span of prosthetic joints is finite, so surgery should be delayed as long as mobil­ ity is satisfactory and pain tolerable. Leg Ulcers  The incidence of leg ulcers is highly dependent on geography and hemoglobin genotype. They are far less common in HbSC disease and HbS-β+ thalassemia than in sickle cell anemia and HbS-β0 thalassemia. In temperate climates, 10–20% of patients are affected; tropical and subtropical areas have an incidence rate up to 75%; ulcers rarely occur in patients from the Middle East. Leg ulcers can be small and superficial or deep and encompass most of the lower leg. They can be extraordinarily painful. Long-standing, recurrent large ulcers are difficult to treat. Wet-to-dry dressings and Unna boots are reasonable choices for initial treatment. ■ ■SICKLE CELL TRAIT (CARRIERS, OR SIMPLE HETEROZYGOSITY FOR THE HBS GENE) Carriers of sickle cell trait outnumber patients with the disease by 25 to 1. Although testing for sickle cell disease is part of most perinatal cord blood screening programs, counseling and follow-up of detected carriers are imperfect, so adolescents and adults can be unaware they carry sickle cell trait. Counseling carriers about the complications of sickle cell trait and their likelihood of having offspring with sickle cell disease is essential. Carriers should be counseled prior to participation in sports because of the risk, albeit small, of sudden death from heatrelated exertional rhabdomyolysis. Optimal hydration before and dur­ ing exercise can prevent most episodes of heat-related illness. Usually a benign condition with a normal life expectancy, some complications are shown in Table 103-2.

■ ■TREATMENT, SCREENING, COUNSELING, AND ANTENATAL DIAGNOSIS Patients should, if possible, be referred to a sickle cell center for initial consultation, institution of therapy, and follow-up. Cooperation among primary care providers, hematologists, and other specialists provides the best preventive care and management of complications. The fre­ quency at which a patient is seen depends on their therapeutic regimen and complications.

Remarkable changes in the treatment landscape have occurred with the promise of even greater benefits from new curative approaches. The following discussion focuses on treatment to prevent the complications of disease. Hydroxyurea  Hydroxyurea is the standard of care for all patients with sickle cell anemia and HbS-β0 thalassemia regardless of symptoms. Although in some symptomatic patients with HbSC disease, its benefits in this genotype are understudied. The major mechanism of action of hydroxyurea is to induce high levels of HbF. Hydroxyurea increases HbF unevenly among red cells (heterocellularly), so some cells have greater protection from HbS polymerization than others. When started in adults, where the average baseline HbF is ~5%, HbF increases to ~10%. Nevertheless, pain and acute chest syndrome are reduced by about half, hemoglobin concentration increases by ~1 g/dL, and after 17.5 years of follow-up, mortality was reduced by 49%. In contrast, all young children respond robustly to hydroxyurea. When started at <1 year of age at a dose of ~27 mg/kg, HbF levels were 33.3 ± 9.1% and hemoglobin concentration was 10.1 ± 1.3 g/dL. Acute events were markedly reduced with little toxicity. Based on these and other studies in high- and low-resource countries, unless there is a contraindication, hydroxyurea should be given to all infants with sickle cell anemia and HbS-β0 thalassemia starting at 9 months of age at a dose of ~20 mg/kg

and titrated to the maximal tolerated dose based on neutrophil and platelet counts. CHAPTER 103 Disorders of Hemoglobin Voxelotor  Voxelotor increases the affinity of the hemoglobin mol­ ecule for O2. At a dose of 1500 mg daily, hemoglobin concentration increased ~1 g/dL in 59% of patients with a reduction in the biomark­ ers of hemolysis. Its effects on acute vasoocclusive events are unclear. Many questions remain about the long-term effects of voxelotor. Less hemolysis reduces the propensity for stroke, nephropathy, pulmonary hypertension, leg ulcers, and priapism. Will voxelotor be accompanied by these long-term benefits? Could the high O2 affinity of a modi­ fied hemoglobin be harmful for some patients? The answers to these important questions require further study. Crizanlizumab  Downstream effects of HbS polymerization include adhesive interactions among endothelial cells, leukocytes, platelets, and erythrocytes. P-selectin is one molecule involved in these interactions; blocking selectins prevents sickle cell–endothelial cell adhesion. Crizanlizumab, a P-selectin-blocking monoclonal antibody given intravenously every month, reduced acute painful episodes by ~45%. Hemolysis was unaffected. A follow-up trial failed to replicate the results of the rigorous study that led to FDA approval. l-Glutamine  The mechanism of action of this agent, presumed to be the reduction of oxidative stress in sickle erythrocytes, is unsettled. A phase 3 placebo-controlled trial showed that l-glutamine was asso­ ciated with a 25% reduction in painful episodes and 33% reduction in hospitalization. There is little consensus regarding how these recently approved drugs should be integrated into treatment with hydroxyurea. The effects of voxelotor and crizanlizumab appear to be additive to those of hydroxyurea. Voxelotor can be added to hydroxyurea if the benefits of hydroxyurea alone are insufficient, as they are in most adults. If both hydroxyurea and voxelotor are taken at effective doses and acute vasoocclusive complications continue, crizanlizumab might then be added. The dropout rates in the crizanlizumab and l-glutamine trials were ~35%, so adherence to these therapeutics could be problematic.

Transfusion  Transfusions are overutilized and underutilized. Major indications for transfusion include severe symptomatic anemia; treatment and prevention of stroke; increasing hemoglobin level to ~10 g/dL before surgery requiring general anesthesia; and severe acute chest syndrome. Sometimes transfusions are given during pregnancy when there is a history of complications or fetal loss. Transfusions should usually be avoided in acute pain episodes and for repair of stable chronic anemia. Automated red cell exchange transfusion is preferred in acute stroke, severe acute chest syndrome, or multiorgan failure or when chronic transfusions are planned. Expert guidelines recom­ mended extended red cell antigen profiling, if possible, before the first transfusion and antigen matching for Rh (C, E or C/c, E/e) and K anti­ gens in addition to ABO/RhD. Complications of transfusion include hyperviscosity, alloimmunization (which occurred in 18.6% of patients transfused between 1979 and 1984 and 27.3% of patients transfused between 2001 and 2011), iron overload, and delayed hemolytic transfu­ sion reactions with hyperhemolysis.

Stem Cell Transplantation  Given the excellent results of human leukocyte antigen (HLA)–identical related donor transplants, which have an event-free survival of >95%, this option might be extended to all patients with a suitable donor. Unfortunately, only 15% of patients have a fully matched donor. New approaches to haploidentical trans­ plants are improving event-free survival in these patients. PART 4 Oncology and Hematology Gene Therapy  Two forms of ex vivo gene therapy are approved for sickle cell disease. Both use mobilized autologous CD34+ stem cells and increase levels of a hemoglobin that inhibits HbS polymerization. In one approach, an HbA gene containing the βT87Q mutation respon­ sible for the antipolymerization effects of HbF is introduced into stem cells via a lentiviral vector. In the second, the major enhancer of the HbF repressor, BCL11A, is disrupted using CRISPR/Cas9 gene editing. In both approaches, following myeloablative conditioning, engineered cells are reinfused and engraft. Both treatments have resulted in near pancellular distribution of 30–50% HbF or HbAT87Q, reduced hemoly­ sis, and total hemoglobin concentrations of >12 g/dL, with nearly total prevention of acute vasoocclusive events. Long-term safety and cure rate will take many more years of follow-up to establish. Preventive Measures and Screening  Cord blood screening for sickle cell disease is done in many countries and all 50 states. Affected patients are then directed to clinics that can initiate early preventive care. In childhood, transcranial Doppler screening beginning at age 2 years and repeated annually until age 16 years, prophylactic penicillin (125 mg for children younger than 3 years; 250 mg for children 3 years and older) twice daily until age 5 years, and vaccination with pneumo­ coccal vaccines are the main measures to prevent stroke and invasive pneumococcal infection. Folic acid, 1 mg daily, is given to prevent megaloblastic erythropoiesis; it is probably unnecessary in people with nutritious diets. All women planning pregnancy should be screened for disorders of hemoglobin by blood counts, erythrocyte indices, and HPLC analysis of hemoglobin. Individuals with HbS or β thalassemia trait should have their partners tested. Only then is it possible to know the risks of a fetus having sickle cell disease (Table 103-2). Antenatal diagnosis using chorionic villus sampling or cell free DNA testing is widely available. THALASSEMIA Thalassemia is caused by reduced accumulation of either α- or β-globin chains causing a relative excess of the unaffected chain. Unbalanced globin synthesis is the hallmark of thalassemia and the proximate cause of its pathophysiology; unpaired globin chains damage the developing erythroblast. Like the HbS mutation and many other red cell traits, thalassemia reached polymorphic levels in tropical and subtropical populations because heterozygotes are protected from severe forms of P. falciparum infection. Estimates are that 1–5% of the world’s popula­ tion carries a thalassemia mutation; in some locales, most people have a thalassemia mutation. These mutations can affect any globin gene, but clinically, β and α thalassemia are the most important. With nearly 500 unique thalassemia-causing mutations (www.globin.bx.psu.edu)

that can interact with each other and with hemoglobinopathies, thal­ assemia syndromes are remarkably diverse. Where resources permit and the mutation is known, genetic counseling can be provided and antenatal diagnosis is possible. HbE (β27 glu-lys) is a common variant whose biosynthesis is reduced because the site of the mutation alters its mRNA processing. Its reduced biosynthesis leads to a deficit of βE-globin chains and features of β thal­ assemia. Hemoglobin Constant Spring is caused by a mutation of the termination codon of HBA2 that leads to the synthesis of an elongated α-globin chain that is unstable and suboptimally synthesized. This variant therefore behaves as an α thalassemia variant. a THALASSEMIA ■ ■EPIDEMIOLOGY Once known as Mediterranean anemia, because of the concentration of cases in Italy, Greece, and other countries bordering the Mediter­ ranean Sea, or as Cooley’s anemia after the physician first describ­ ing cases, β thalassemia is common in most areas of the world where malaria was endemic, including the Mediterranean region, Asia, and the Middle East. Effective programs of screening, counseling, and antenatal diagnosis have reduced the birth of new cases in a number of regions. About 40,000 β thalassemia patients are born yearly. In the United States, there are ~1000 cases of severe β thalassemia. ■ ■CLASSIFICATION β0 Thalassemia mutations totally prevent the accumulation of any globin from the affected gene; β+ thalassemia mutations cause minor or moderate reductions in β-globin synthesis. β Thalassemia major and β thalassemia intermedia are now categorized as transfusiondependent and non-transfusion-dependent based on the number and frequency of transfusions required to sustain a good quality of life. Pathophysiology  Single nucleotide changes are the most common β thalassemia mutations, but gene deletions also occur. A partial listing of the classes of mutations causing β thalassemia include mutations in the promoter elements affecting gene transcription causing mild and sometimes silent β+ thalassemia; mutations in the junctions between exons and introns that affect mRNA processing causing β0 and β+ thal­ assemia; introduction of alternative splice sites into introns or exons usually causing β+ thalassemia; 3′ end-processing sequence mutations preventing RNA polyadenylation leading to mild or silent β+ thalas­ semia; mutations preventing initiation of translation causing β0 thal­ assemia; and introduction of stop codons that prematurely terminate translation (nonsense mutations) producing reading frameshifts and resulting in truncated globin mRNA and β0 thalassemia. In addition, rare causes of β thalassemia have been identified that are unlinked from the β globin locus and caused by mutations in general transcrip­ tion regulators, such as SUPT5H and TFIIH, or erythroid transcription factors like GATA1. In β thalassemia, the deficit in β-globin chain synthesis allows α-globin chains to accumulate in excess. Without a non-α-globin chain partner in dimer and tetramer formation, unpaired α-globin chains are unstable, cannot form a tetramer, and precipitate within the developing erythroblast, causing membrane lipid oxidation and dam­ age. The predominant cause of anemia is intramedullary destruction of erythroid precursors, known as ineffective erythropoiesis. Reduced deformability and phosphatidyl serine exposure also cause extra- and intravascular hemolysis of those erythrocytes that gain entrance into the circulation. In poorly treated β thalassemia, severe anemia leads to bone marrow expansion; hepatosplenomegaly; iron accumulation in liver, heart, and endocrine organs; pulmonary hypertension; and thromboembolic disease. Frightening pictures of children with severe β thalassemia permeate the literature. These examples of near-terminal disease should be rel­ egated to history because treatment with transfusion and iron chelation can prevent their occurrence, hematopoietic stem cell transplantation can “cure” patients who have suitable donors, and efficacious gene therapies are now approved.

■ ■DIAGNOSIS Heterozygous β thalassemia, also known as β thalassemia trait and β thalassemia minor, has mild or no anemia but microcytic/hypochro­ mic erythrocytes with minimal or no increase in reticulocyte count. After recognizing these hematologic abnormalities and excluding iron deficiency, finding an elevated level of HbA2 and perhaps HbF by HPLC is sufficient to establish this diagnosis. The hematologic char­ acteristics of this heterozygous carrier state are listed in Table 103-4. Sometimes, the spleen is enlarged. Before genetic counseling and ante­ natal diagnosis are considered after carrier identification by red cell indices and quantitation of HbA2, the thalassemia-causing mutation should be identified. Sequencing is the key to preventing homozygotes or compound heterozygotes with transfusion-dependent thalassemia. The more severe forms of β thalassemia are hemolytic anemias with hypochromia, microcytosis, reticulocytosis, marked anisocytosis, and poikilocytosis with variable numbers of circulating nucleated red cells (Fig. 103-5). ■ ■COMPLICATIONS Complications of severe β thalassemia are many. They are a consequence of chronic hemolytic anemia, chronic transfusion, and iron loading. Increased iron absorption is especially common in non-transfusiondependent thalassemia. Most complications, listed in Table 103-5, develop because of either inadequate blood transfusion and/or poor iron chelation and iron loading. Even when chelation is optimized, some complications attributable to iron toxicity will develop. Many complications have complex and multifactorial etiologies. Iron stores are estimated by serum ferritin levels; MRI is the most widespread means of noninvasively measuring iron accumulation in liver and heart. ■ ■MANAGEMENT, SCREENING, COUNSELING, AND ANTENATAL DIAGNOSIS Heterozygote screening and counseling couples at risk for affected fetuses, with antenatal diagnosis, if needed, is an effective preven­ tive approach. Severe thalassemia should be dealt with in specialized centers where these and other services are available and managed by a team led by a hematologist experienced with this disease with help from endocrinologists, cardiologists, transfusion medicine specialists, and social services. Transfusion and Iron Chelation  Transfusion every 2–4 weeks with a goal pretransfusion hemoglobin concentration of 9–10.5 g/dL TABLE 103-4  β Thalassemias HEMOGLOBIN

(g/dL)/MCV (fL) HEMOGLOBIN FRACTIONS (%) CLINICAL FEATURES CLASSIFICATION β-Thalassemia trait 100–140 (10–14)/60–80 HbA: 94 HbF:1–2 HbA2: 4–6 Non-transfusion-dependent β thalassemia (thalassemia intermedia) 70–120 (7–12)/65–80 HbA: 60–90 HbF: 10–40 HbA2: 4–6 20–40 (2–4)/50–80 HbA: 0–5 HbF: 90–100 HbA2: 2–5 Transfusion-dependent β thalassemia (Thalassemia major) HbE-β thalassemia 50–80 (5–8)/60–70 HbE: 50–70 HbF: 30–50 110–120 (11–12)/65–75 HbA: 70 HbF: 7–13 HbA2: 2 δβ Thalassemia and Hb Lepore Gene deletion hereditary persistence of fetal hemoglobin (HPFH) 120–140 (12–14)/75–85 HbA: 70 HbF: 15–30 HbA2: 2 Note: Laboratory results are averages in adults.

FIGURE 103-5  α Thalassemia intermedia. Target cells and marked variation in cell size and shape but with general hypochromia and microcytosis characterize the blood film. A lymphocyte is shown for size comparison. to suppress ineffective erythropoiesis, coupled with iron chelation to prevent the accumulation of excess toxic iron that accompanies trans­ fusion, has prevented the development of cardiomyopathy and endo­ crinopathies while extending life to at least 50 years. When to begin transfusions, whether partial exchange transfusion is preferable to simple transfusion, and the choice of blood product require consulta­ tion with experts. To be effective, transfusions and iron chelation must be started early, be uninterrupted, and continue lifelong. Older patients who did not have the advantage of effective chelation are more likely to develop multiple disease-related morbidities such as osteoporosis, endocrinopathies, liver disease, and renal failure. Two orally effective chelating agents, deferasirox and deferiprone, and one intravenous chelator, deferoxamine, are available. CHAPTER 103 Disorders of Hemoglobin Improving Ineffective Erythropoiesis  Luspatercept is a fusion protein containing the extracellular domain of human activin type IIB receptor and the Fc domain of human IgG. By binding transforming growth factor β superfamily ligands and reducing Smad2/3 signaling, luspatercept enhances late-stage erythropoiesis. Given subcutaneously, 1 mg/kg every 3 weeks, it was associated with a 33% reduction in trans­ fusion requirements. Heterozygosity for β+ or β0 thalassemia mutations; “silent” carriers can have normal HbA2 and red cell indices. Defined by infrequent or no transfusion requirement; caused by many different genotypes including homozygosity for “mild” β+ mutations, combinations of β and α thalassemia, homozygous β thalassemia with high HbF-producing capacity, and many others. Iron loading, thromboembolic disease, and pulmonary hypertension are major clinical events. Caused by many different genotypes including homozygosity and compound heterozygosity for β0 and β+ mutations, combinations of β and α thalassemia; transplantation curative; iron chelation required. Common in Southeast Asian populations; in some parts of the world, the most prevalent severe thalassemia; in HbE-β0 thalassemia, only HbE and HbF are found; in HbE-β+ thalassemia, HbA is present. Transfusion dependence depends in part on the thalassemia mutation. Rare; deletions removing the δ- and β-globin genes cause δβ thalassemia; Lepore hemoglobins are fusion globin chains; values are for heterozygotes; homozygotes have 100% HbF with hemoglobin 10–11 g/dL. Rare; large deletions removing the δ- and β-globin genes; values are for heterozygotes; homozygotes, who are asymptomatic, have 100% HbF without anemia.

TABLE 103-5  Complications of a Thalassemia COMPLICATION INCIDENCE, DIAGNOSIS, AND FEATURES Growth retardation Most often a feature of delayed or inadequate transfusions but can occur in well-transfused children. Delayed puberty; secondary amenorrhea 50% and 25%, respectively. Splenomegaly Can trap 1–40% of red blood cell volume; increases plasma volume, worsening heart failure. Splenectomy indicated when transfusion requirement to maintain ideal hemoglobin increases. Prophylactic penicillin after splenectomy. Heart Due to chronic anemia, heightened sensitivity to iron toxicity, thromboembolic pulmonary hypertension, other causes. Progresses through stages to congestive failure and arrhythmias. Assessed by T2* on magnetic resonance imaging (MRI). The available chelating agents might have differential effects on different measure of cardiac function and can be used in combination. Leg ulcers Common in thalassemia intermedia. Hepatic disease Fibrosis progressing to cirrhosis is related to hepatic iron concentration that can be monitored by MRI. Hepatitis also plays a role. Lung disease/ pulmonary hypertension Fibrosis, chronic thromboembolic disease, restrictive pathophysiology, intravascular hemolysis, and reduced nitric oxide bioavailability. PART 4 Oncology and Hematology Thromboembolism Multifactorial etiology, including platelet activation, red cell–endothelial interactions, thrombocytosis, endothelial activation, splenectomy. Endocrinopathies Diabetes, hypothyroidism, hypoparathyroidism, adrenal insufficiency; hypogonadism; hypothalamic-pituitary axis might be especially sensitive to iron. Bone disease Caused by bone marrow expansion, severe iron loading, hypogonadism; osteoporosis in ~50% of patients, even those well treated. Extramedullary hematopoietic masses are a feature of thalassemia intermedia. Infections Transfusion associated; linked to iron overload (Yersinia); malaria. Hematopoietic Stem Cell Transplantation  There is consensus that patients with available donors should be offered transplantation because of the difficulty of lifelong transfusion and chelation and its imperfect efficacy. Quality of life in successfully transplanted patients TABLE 103-6  Thalassemias-GLOBIN GENE ARRANGEMENT HEMOGLOBIN LEVEL,

g/L (g/dL)/MCV (fL) CLINICAL FEATURES CLASSIFICATION 120–150 (12–15)/65–80 The chromosome with one deleted α gene (—α/) is called α+ thalassemia (α thalassemia-2); the chromosome with both deleted α genes is α0 thalassemia (α thalassemia-1); non–gene deletion α thalassemias (αT) often have a more severe phenotype. α-Thalassemia trait −α/αα −α/−α − −/αα αTα/αα Hemoglobin H disease − −/−α αTα/− − αTα/αTα 50–120 (5–12)/60–70 Mild to moderate anemia depending on genotype; non–gene deletion forms of α thalassemia can produce severe HbH disease. Hb Bart’s hydrops fetalis −−/−− Fatal in utero or at birth with rare survivors. Hydrops can also result from combinations of gene deletion and non–gene deletion α thalassemia. α Thalassemia/intellectual disability syndromes (ATR-16) (ATR-X) − −/αα or − −/−α in ATR-16 αα/αα in ATR-X αα/αα Mutations in ATRX; striking male predominance. Hematologic findings of HbH disease. α Thalassemia with myelodysplasia (ATMDS) Note: Laboratory values are averages in adults. αα/denotes the chromosome with two intact α-globin genes; –α/chromosome with one α-globin gene deleted;

– –/chromosome with both α-globin genes deleted; αT represents non–gene deletion α thalassemia caused by point mutations. The –α/chromosome, referred to as α+ or α thalassemia-2, most often has a deletion of 3.7 kb of DNA (–α3.7) or 4.2 kb of DNA (–α4.2) that leaves a single α-globin gene intact. The chromosome where both α-globin genes are deleted (– –/) is called α0 thalassemia or α thalassemia-1. These chromosomes are caused by different-sized deletions that are usually named after their regions of highest frequency such as -SEA, -MED, -FI, and -THAI.

exceeds that in patients treated with transfusion and chelation. Trans­ plantation from matched sibling donors is curative in >80% of all cases. Unfortunately, only a third of patients have matched donors. The best results are in the youngest patients who have been effectively chelated and received fewer transfusions. Graft failure, graft rejection, graft-versus-host disease, and a mortality of 5–20% depending on risk factors are the major drawbacks of this procedure. Results of haploi­ dentical and unrelated donor transplants are improving but lag those of matched sibling donors. Gene Therapy  The same gene therapy approaches approved for sickle cell disease are approved for transfusion-dependent β thalas­ semia (see Sickle Cell Disease). CRISPR/Cas editing to downregulate BCL11A has resulted in increases in total hemoglobin ≥12 g/dL and HbF ≥10 g/dL, leading to transfusion independence in >90% of 52 patients aged between 12 and 35 years with transfusion-dependent β thalassemia. Results of lentiviral-mediated HbAT87Q additive gene therapy were best with non-β0/β0 genotypes, although some individuals with β0/β0 genotypes could be effectively treated as higher viral titers were used in subsequent gene therapy protocols. ` THALASSEMIA In some respects, the obverse of β thalassemia, clinically consequential α thalassemia is less common than severe β thalassemia. α Thalas­ semia is most often found in Asian populations and is usually caused by deletion of α-globin genes rather than point mutations. ■ ■EPIDEMIOLOGY Carriers of the most common α thalassemia chromosomes (Table 103-6) are found in 5–80% of people from tropical and subtropical regions of Africa, the Middle East, Asia, and Melanesia. About 30% of African Americans carry the common –α3.7 chromosome that contains a single functional α-globin gene. HbH disease, the chief clinically important α thalassemia, is most prevalent in southern China and Southeast Asia. Estimates are that in Thailand ~3500 patients with severe α thalas­ semia are born yearly. Pregnancies affected by hemoglobin (Hb) Bart’s hydrops fetalis occur mainly in southern China and southeastern Asia. ■ ■CLASSIFICATION Each normal chromosome 16 contains two α-globin genes; normal diploid individuals have four α-globin genes. A classification of inher­ ited α thalassemia, as summarized in Table 103-6, is based on the number of functional α-globin genes. If one or two α-globin genes are missing or poorly expressed, these people have α thalassemia ATR-16: Large deletions and rearrangements in chr16p. ATR-X: No α-globin gene deletion or mutation, ATRX mutations, X-linked.

trait. Their hematologic abnormalities are almost always trivial. HbH disease is usually caused by deletion or malfunction of three α-globin genes. Hb Bart’s hydrops fetalis fetuses have no normally functioning α-globin genes. Hundreds of different-sized deletions and rarer point mutations affect the production of α-globin and the magnitude of imbalanced globin synthesis. Because of this mutational complexity, many different variations of the common α thalassemia syndromes are found. ■ ■PATHOPHYSIOLOGY Reduced accumulation of α-globin leaves non-α-globins unpaired and unable to participate in the formation of functional hemoglobin tetramers. In the fetus, absent or reduced synthesis of α-globin allows unpaired γ-globin chains, part of the HbF tetramer, to form γ4 or Hb Bart’s; in adults, when γ-globin synthesis is mostly silenced, unpaired β-globin chains, lacking a suitable partner to form HbA, tetramerize as β4 or HbH. Both Hb Bart’s and HbH have very high O2 affinity and do not unload O2 in tissues; HbH is also unstable. Severe anemia in Hb Bart’s hydrops fetalis is a result of absent normal hemoglobin and inef­ fective erythropoiesis; in HbH disease, unstable HbH leads to oxidative membrane damage with extravascular hemolysis in the spleen and ineffective erythropoiesis. ■ ■DIAGNOSIS Microcytosis/hypochromia with nearly normal hemoglobin concen­ trations, in the absence of iron deficiency and the increased level of HbA2 that is diagnostic of β thalassemia, is sufficient for a presumptive diagnosis of α thalassemia trait. When genetic counseling is needed and antenatal diagnosis contemplated, the molecular basis of the pre­ sumed α thalassemia is required. HbH disease, which is usually due to compound heterozygosity for one chromosome with both α-globin genes deleted and one chromosome with only a single α-globin gene, is defined by the hematologic findings shown in Table 103-6 along with varying levels of reticulocytosis. At birth, when hemoglobin is separated by HPLC, 20–30% Hb Bart’s is present; in adults, traces to 40% HbH are present along with residual Hb Bart’s in some cases. HbH inclusions can be induced in some red cells after incubation and stain­ ing with brilliant cresyl blue. Hemoglobin composition in Hb Bart’s hydrops fetalis is predominantly Hb Bart’s with some Hb Portland if the deletion removing α-globin genes preserves the ζ-globin gene. ■ ■COMPLICATIONS HbH disease is very heterogeneous because of the different combina­ tions of genotypes that can cause this phenotype. Generally, when non–gene deletion mutants, such as Hb Constant Spring, contribute TABLE 103-7  HbC, HbE, and Rare Hemoglobinopathies CLASSIFICATION CLINICAL ABNORMALITIES HbC trait 2% of African Americans; target cells; no disease Normal HbC: 30–40 HbA2: 2–3 HbC disease Target cells; HbC crystals; mild reticulocytosis; splenomegaly HbE trait 50% incidence in some Asian populations; a few target cells; clinically normal HbE disease No hemolysis; 20–80% target cells;

no splenomegaly High O2 affinity hemoglobins Isolated erythrocytosis; often familial; no splenomegaly; no JAK2V617F mutation Low O2 affinity hemoglobins Asymptomatic mild anemia; cyanosis 100–140 (10–14) ~50% variant Unstable hemoglobins Pigmenturia; hemolysis; reticulocytosis; splenomegaly M hemoglobins Some have mild hemolysis; few symptoms 100–140 (10–14)/80–90 20–50% variant depending on gene affected Note: Laboratory values are averages in adults. As noted for HbAS, the amount of HbC and HbE in heterozygotes depends on the number of α-globin genes.

to the genotype, the disease is more severe, and intermittent or regular transfusions are necessary. In the most common − −/−α genotype, mean hemoglobin in adults is ~11 g/dL. Hepatosplenomegaly, jaun­ dice, thalassemic bone changes in the face, and growth impairment are seen in 20–50% of cases, depending on the underlying genotype. Iron loading occurs but is not the severe problem it is in β thalassemia. Pregnancy in these patients should be considered high risk and man­ aged accordingly. Mothers of infants with Hb Bart’s hydrops fetalis have a history of stillbirth and develop preeclampsia, polyhydramnios, and antepartum hemorrhage and have difficult labor and delivery. Intra­ uterine transfusion of the fetus is possible.

■ ■MANAGEMENT, SCREENING, COUNSELING, AND ANTENATAL DIAGNOSIS When planning families, couples from regions where α thalassemia is common who have red cell indices that suggest the possibility of carrying an α thalassemia gene should have genetic counseling based on DNA analysis of their globin genes. Iron should be avoided in noniron-deficient individuals with α thalassemia trait and microcytosis. Transfusions are not usually needed in HbH disease. Nevertheless, depending on the genotype of disease, transfusions might be necessary especially when anemia becomes more severe, for example, with acute anemic episodes or pregnancy. Iron stores should be checked periodi­ cally by measuring serum ferritin or MRI; chelation does not appear to be needed. CHAPTER 103 Hb Bart’s hydrops fetalis is best prevented by screening couples at risk and antenatal diagnosis. Intrauterine therapy and perinatal intensive care have permitted survival of some infants with Hb Bart’s hydrops fetalis. As growth retardation affects ~40% and neurodevel­ opmental delay is present in 20% of survivors, prevention is the best approach. Disorders of Hemoglobin OTHER HEMOGLOBINOPATHIES OF CLINICAL IMPORTANCE (TABLE 103-7) More than 1500 mutations affecting hemoglobin structure have been described (www.globin.bx.psu.edu). Most are clinically silent. HbC and HbE are common. HbC is found in people of African descent and HbE in South China and Southeast Asia. Heterozygotes for HbC and HbE are unaffected clinically. Even individuals homozygous for these muta­ tions, where the variant hemoglobin comprises >90% of the hemoly­ sate, are clinically well with very mild anemia and microcytosis. The major importance of these variants is the interaction of HbC with HbS and HbE with β thalassemia, as outlined in Tables 103-2 and 103-4. A definitive diagnosis for all rare variants depends on DNA analysis. HEMOGLOBIN LEVEL,

g/L (g/dL)/MCV, fL HEMOGLOBIN FRACTIONS (%) 100–130 (10–13)/60–70 HbC: >95 HbF: 2–4 HbA2: 2–3 120–140 (12–14)/80–90 HbE: 27–31 HbF: 1 HbA2: 3 100–120 (10–12)/65–75 HbE: 85–95 HbF: 3–7 HbA2: 3 150–200 (15–20) Variants in α- and β-globin genes; patients are heterozygotes; ~25–50% variant 90–140 (9–14)/70–90 20–35% variant; rare hyperunstable variants can be undetectable and have the phenotype of thalassemia