Keywords

Introduction

Inherited disorders of hemoglobin, primarily sickle cell disease (SCD) and β-thalassemia are among the most common monogenetic diseases in the world [1]. The importance of these hematological disorders cannot be overstated; it is estimated that 5–7% of the world’s population are carriers of a significant hemoglobin variant [2, 3].

SCD has often been described as the “first molecular disease” following Linus Pauling’s discovery in 1949 that the disorder was due to an abnormal hemoglobin molecule [4]. This landmark report was followed several years later by a crucial elucidation by Vernon Ingram that sickle hemoglobin (HbS) is due to a single amino acid change in the β-globin chain of the hemoglobin molecule [5]. These reports were the first to describe the specific molecular basis for a human disease, and led to a flurry of scientific investigation of normal and abnormal hemoglobin, including several seminal findings describing the genetic basis of the thalassemias [6,7,8,9]. The genetic nature of these hemoglobin disorders has been further defined over the past 50 years, such that now the complete DNA sequences of the normal α- and β-globin gene loci and over 1000 hemoglobin variants are known.

Despite this long history that has led to a detailed understanding of the molecular basis of hemoglobin disorders such as SCD and thalassemia, the global burden of these conditions remains enormous and continues to increase [10, 11]. With the imminent arrival and expansion of cellular and gene therapy technologies, there is no question that SCD and β-thalassemia should be among the highest priorities for these novel and exciting therapies. In this chapter, we will provide an introduction to the global epidemiology, pathophysiology and clinical features of SCD and β-thalassemia, establishing a framework for subsequent discussion of the potential gene and cellular therapies that aim to ameliorate and cure these serious and life-threatening hematological disorders.

Hemoglobin: Structure and Function

Hemoglobin is a protein found abundantly in erythrocytes, with the primary function of oxygen transport; hemoglobin primarily transports oxygen from the lungs to the tissues, and subsequently returns oxygen (in the form of carbon dioxide) back to the lungs. The hemoglobin molecule is a tetramer, consisting of two α-like globin chains (141 amino acids) and two β-like globin chains (146 amino acids). Each polypeptide chain is attached non-covalently to a heme group, which binds oxygen and facilitates gas transport and exchange.

Two tandem α-globin genes (HBA2, HBA1) regulate α-globin production and are located on chromosome 16 along with HBZ, which regulates expression of the embryonic α-like ζ (zeta) globin chains. Each β-globin chain consists of 146 amino acids and is regulated by a single β-globin gene (HBB) located on chromosome 11. HBB is part of a gene cluster on chromosome 11 that regulates expression of four other β-like globin proteins that can pair with α-globin: embryonic ε-globin (HBE1) gene, tandem fetal or γ-globin (HBG2 and HBG1) genes, and the δ-globin (HBD) gene.

The production of globin chains is tightly regulated during ontogeny, with important variations in gene expression occurring throughout normal development and in different disease states. Figure 1.1 illustrates the globin gene clusters and the resulting patterns of globin chain synthesis during normal development. In each setting, two α-like globin chains will normally pair with two β-like globin chains to form a mature hemoglobin heterotetramer. In some instances, however, particularly like those found in thalassemia with quantitatively reduced globin chain production , unpaired globin chains produced in excess can form homotetramers that are unstable and lead to serious clinical consequences.

Fig. 1.1
figure 1

Panel (a) illustrates the patterns of globin chain synthesis during normal development. In each setting, two α-like globin chains will normally pair with two β-like globin chains to form a mature hemoglobin heterotetramer (Adapted from Oliveri NF. NEJM 1999, [104]). Panel (b) illustrates the α- and β-globin gene clusters on chromosomes 11 and 16, respectively

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Types of Hemoglobin

In the early stages of human embryonic development (2–3 weeks of gestation), erythropoiesis occurs primarily in the yolk sac and the dominant globin chains include ζ-globin and ε-globin. The resultant earliest embryonic hemoglobin is thus Hb Gower I (ζ2/ε2) [12]. By the 5th or 6th week of gestation, the primary site of erythrocyte production is the liver and the synthesis of α-globin and γ-globin chains begins to increase, resulting in Hb Portland (ζ2/γ2), Hb Gower II (α2/ε2) [13], and ultimately HbF (fetal hemoglobin, α2γ2) [14]. By 12 weeks of gestation and throughout early infancy, HbF is the predominant hemoglobin within circulating erythrocytes.

The unique characteristics of fetal hemoglobin are important to understand, since HbF has key effects on the pathophysiology of patients with SCD and β-thalassemia, and thus serves as a therapeutic target. HbF has an increased oxygen affinity compared to normal adult hemoglobin (HbA, α2β2), so that HbF has a “left-shifted” oxygen dissociation curve; in the fetus, this allows extraction of oxygen from the mother’s HbA and still provides sufficient tissue oxygenation despite the relatively low oxygen content in the fetal circulation. After birth, coincident with the post-natal increase in ambient oxygen concentration, this increased oxygen affinity is no longer physiologically necessary and HbF production declines.

Toward the end of gestation and continuing over the first 6–12 months of life, the expression of γ-globin genes is reduced in favor of β-globin gene expression. By 6 months of post-natal age, HbA predominates and by 2 years of age, HbF expression is effectively repressed. This important “hemoglobin switch” has been extensively studied and a number of potent genetic regulators have been identified, most notably BCL11A [15,16,17]. The importance and potential therapeutic implications of targeting the hemoglobin switch for the treatment of SCD and β-thalassemia, and will be discussed in more detail in subsequent chapters.

HbA2 (α2δ2) is a minor hemoglobin expressed beginning at about 6 months of age, but only represents 2–3.5% of total hemoglobin, since the δ-globin gene is inefficiently transcribed due to a faulty promoter region [18]. Hb A2 does not have much clinical significance in healthy persons, but is increased in the setting of β-chain imbalance, and thus serves as an important diagnostic feature of β-thalassemia trait.

Classifying the Hemoglobin Disorders

The disorders of hemoglobin can be classified broadly into two distinct categories: quantitative or qualitative disorders. Quantitative disorders include those in which there is an absence or significant decrease in the production in one of the globin chains; these disorders are commonly referred to as the thalassemias. Although not the focus of this chapter, α-thalassemia features quantitative decrease of alpha globin chains, typically the result of gene deletion events resulting in the loss of one or both tandem α-globin genes on chromosome 16. The analogous β-thalassemia most commonly results from point mutations within the HBB promoter, exonic, or intronic sequences; the result is reduced or absent β-globin chain synthesis. In contrast, the sickle cell disorders are qualitative defects of hemoglobin that result from a structural defect in the β-globin chains. The sickle mutation (βS) is by far the most common qualitative hemoglobin disorder , but hundreds of qualitative hemoglobin mutations causing significant clinical sequelae have been described. For the purposes of this chapter, we will focus on the two most common and most pathological hemoglobin disorders: β-thalassemia and sickle cell disease.

β-Thalassemia

Overview and Historical Perspective

In 1925, at the American Pediatric Society Meeting, Detroit pediatrician Thomas Cooley described five children of Italian origin with severe anemia, splenomegaly, a “peculiar appearance” secondary to “yellowish discoloration of the skin and…thickening of the cranial bones” and a blood smear notable for poikilocytosis , anisocytosis, and target cells [19]. Cooley noted that the clinical condition of these children was similar to the chronic anemia previously named “anemia infantum pseudoleucaemica” a generation earlier [20, 21]. The term “thalassemia,” which is derived from the Greek words for sea (thalassa) and blood (haema), was first coined in 1932 by Nobel laureate pathologist George Whipple and his pediatrician colleague William Bradford in their seminal report of autopsy findings in children with Cooley’s anemia, most notably the wide deposition of iron [22, 23]. The genetic basis of thalassemia was further described over the next decade by several investigators who noted both the recessive nature of the disease and the distinction between a “minor” and “major” phenotype [24,25,26].

Epidemiology and Global Burden

β-thalassemia represents one of the world’s most common inherited conditions, with estimates that up to 1.5% of the world’s population are carriers of a pathological β-thalassemia mutation, and over 40,000 infants are born each year with either β-thalassemia major or HbE/β-thalassemia [1, 2]. The β-thalassemia gene mutations originated primarily in the Mediterranean region and extended eastward in a band across the Middle East, India and Southeast Asia, but subsequent migration has resulted in a widespread global distribution.

The geographic origins of the β-globin mutations responsible for β-thalassemia clearly overlap the geographic distribution of malaria endemicity [27], although protection against severe malaria for β-thalassemia carriers has not been demonstrated as clearly as for carriers of the HbS mutation. The genetic mutations leading to β-thalassemia (including HbE), similar to the HbS mutation, are examples of balanced genetic polymorphisms in which the heterozygous state offers a survival advantage, while the homozygous state results in significant morbidity and early mortality [28, 29]. The relative survival advantage conferred by the heterozygous state provides genetic pressure that leads to persistence of these deleterious mutations.

Molecular Basis and Pathophysiology

β-thalassemia includes a heterogeneous group of inherited anemias characterized by defective β-globin synthesis. Over 270 different β-globin gene mutations (mostly point mutations) have been described that result in a reduction or absence of β-globin production [30, 31]. Some mutations , notably gene deletions and nonsense point mutations, are so damaging to β-globin gene expression that there is a total absence of β-globin production (β0-thalassemia), while others result in an incomplete quantitative reduction in β-globin production (β+-thalassemia). The clinical heterogeneity of β-thalassemia is primarily dependent upon the severity of the genetic mutation and the resultant imbalance between α- and β-globin chains. The molecular basis of β-thalassemia will be discussed in further detail in Chap. 2.

Globin gene synthesis is tightly regulated through the series of α- and β-globin (or β-like) genes on chromosomes 16 and 11, respectively. The pathophysiology of β-thalassemia has been well described, and is due not only to insufficient production of β-globin chains, but also the relative excess of α-globin chains that are deleterious when not bound in a hemoglobin tetramer. During fetal development and early infancy, when ɣ-globin is still expressed, any excess α-globin chains can pair with ɣ-globin to produce HbF. When ɣ-globin expression is repressed, the α-chains are in excess without a sufficient number of available β-like globin chains resulting in α:β chain imbalance and a reduction in hemoglobin formation. Excess α-globin chains precipitate within erythrocytes as hemichromes forming reactive oxygen species that are toxic to both developing erythroblasts and mature erythrocytes. This leads to inadequate erythroid differentiation and increased apoptosis of erythrocyte precursors within the marrow, a process referred to as ineffective erythropoiesis. The anemia is only exacerbated further by hemolysis secondary to erythrocyte membrane damage [32, 33].

The chronic anemia occurring in β-thalassemia leads to an increase in serum erythropoietin, which is not fully compensated in the setting of ineffective erythropoiesis. Clinical sequelae of ineffective erythropoiesis include massive expansion of the bone marrow erythroid compartment with intramedullary destruction, extramedullary hematopoiesis, splenomegaly, and significantly increased gastrointestinal iron absorption. The degree of globin chain imbalance in β-thalassemia is closely linked to the severity of disease. This is most obvious in the setting of co-inherited α-thalassemia, in which the clinical severity is ameliorated by a closer balance of α- and β-globin chains [34]. In addition to co-inheritance of α-thalassemia, there are additional genetic modifiers of β-thalassemia that directly impact the clinical manifestations of disease, which will be addressed in Chap. 2.

Iron and β-Thalassemia

Dysregulation of iron homeostasis is another important component of the pathophy-siology of β-thalassemia. Ineffective erythropoiesis and the associated upregulated erythropoietic drive result in increased intestinal absorption of iron, causing a wide variety of deleterious consequences including iron deposition into many internal organs with resulting parenchymal dysfunction. Over the past 15 years, the hepatic peptide hormone hepcidin has been firmly established as a key regulator of iron homeostasis. Hepcidin binds to the membrane-bound iron export protein, ferroportin, causing its degradation and subsequent reduction in the export of cellular iron. This reduction in surface ferroportin expression results in decreased iron influx into plasma from the gastrointestinal tract (dietary iron), macrophages (recycled iron), and hepatocytes (stored iron) [35, 36]. When plasma and stored iron levels are high, hepcidin production is increased and inhibits further iron loading by blocking intestinal iron absorption; conversely when iron is needed, hepcidin levels fall and accelerate uptake of intestinal iron.

Erythropoiesis, due to its dependence upon iron, is perhaps the most potent physiological regulator of hepcidin. In an effort to support increased erythropoietic demand, and potentially mediated by erythroferrone [37], hepcidin production is decreased in β-thalassemia and as a result, there is rapid absorption and release of iron into the plasma [38]. The combination of accelerated and ineffective erythropoiesis allows the increased absorption of iron in the gut to continue unabated. Elevated plasma iron, especially labile (non-transferrin-bound) iron, leads to deposition into viscera (particularly the heart, liver, and endocrine organs) with significant tissue damage and organ dysfunction [38]. The mainstay of treatment of β-thalassemia, chronic erythrocyte transfusions, alleviates anemia and reduces the erythropoietic drive, but compounds the hemosiderosis due to the significant iron load from each transfusion. Iron overload , which results from both increased intestinal absorption and from repeated transfusions, remains the most significant cause of morbidity and mortality for patients with β-thalassemia.

Classification and Clinical Manifestations of β-Thalassemia

β-thalassemia is a clinically heterogeneous group of conditions that are typically classified into three groups based on clinical severity: β-thalassemia major, β-thalassemia intermedia, and β-thalassemia minor.

β-Thalassemia Major

The most severe form of β-thalassemia is referred to as β-thalassemia major (TM) , typically the result of inheritance of homozygous (β0/ β0) or compound heterozygous (β0/ β+) mutations that feature minimal (if any) β-globin production and severe α:β chain imbalance, with consequent erythrocyte transfusion dependence. As with other forms of β-thalassemia, however, TM has a variable clinical phenotype dependent upon the specific β-globin gene mutations, other inheritable genetic modifiers and environmental factors.

TM most commonly presents within the first 1–2 years of life due to severe anemia, failure to thrive, and the need for regular blood transfusions. Infants with TM may not develop clinical symptoms in the first 4–6 months of life due to high levels of HbF. However, as the expression of γ-globin is repressed and the absolute amount of fetal hemoglobin declines, hemolytic anemia becomes clinically apparent making the effects of reduced or absent β-globin chains clearly evident. These children can present with anemia, pallor, poor growth, and hepatosplenomegaly due to extramedullary hematopoiesis.

Hematological Manifestations

The defining hematological feature of TM is severe anemia with an inability to maintain a hemoglobin concentration above 7–8 g/dL. Hemolytic anemia leads to the presenting features of TM with pallor, mild jaundice, and without early diagnosis and initiation of chronic transfusion therapy, TM is a lethal condition.

Early initiation of transfusion therapy is critical for normal growth and development and is also protective of significant early organ damage and the aforementioned physical deformations. Hypertransfusion to maintain a hemoglobin concentration above 10 g/dL affords these infants and children an opportunity for normal growth and development [39]. Chronic erythrocyte transfusion therapy should always be combined with aggressive iron surveillance and iron chelation therapy.

Skeletal Manifestations

Ineffective erythropoiesis in TM results in significant extramedullary hematopoiesis that clinically manifests as skeletal abnormalities , particularly in the skull (e.g., frontal bossing, maxillary hyperplasia). Chronic erythrocyte transfusion therapy (approximately every 3–4 weeks) suppresses ineffective erythropoiesis and prevents these types of abnormalities. In addition to the aesthetic complications of bony abnormalities, these patients demonstrate decreased bone mass starting early in life, which leaves them prone to fractures [40]. Routine surveillance of bone mineral density and general bone health (such as vitamin D monitoring) should be performed in all patients with β-thalassemia major.

Endocrine Manifestations

One of the most critical benefits of initiating early transfusion therapy is improved growth and development, which are significantly delayed in untreated TM. Despite the significant and life-saving benefits of chronic transfusion therapy for persons with TM, this therapy presents severe endocrine complications if adequate attention to iron burden and aggressive chelation therapy are not maintained. Endocrine complications are often the first clinical manifestations of iron overload due to iron deposition in the anterior pituitary, which can lead to hypogonadism, growth retardation and short stature, hypothyroidism, hypoparathyroidism, and diabetes mellitus [41]. Routine screening and attention to symptoms related endocrine function are essential for all patients with TM.

Hepatic Manifestations

The excess total body iron characteristic of TM results in significant iron deposition within the liver. Without adequate chelation therapy, hepatic iron deposition begins in the macrophages (Kupffer cells) later moving to the parenchyma and sinusoids, which eventually results in hepatic fibrosis and significant liver dysfunction. Cirrhosis and hepatocellular carcinoma are two serious complications of the chronic hepatitis from longstanding severe iron overload .

Over the past decade, there have been significant improvements in the diagnostic methods of measuring liver iron content. Liver biopsy is now replaced by advanced MRI imaging techniques for quantitative measurement of liver iron content [42]. Non-invasive serial measurements of liver iron content are used to assess the dosing and effectiveness of iron chelation therapy. Frequent discussions with patients to optimize compliance with chelation therapy, however, remain essential to prevent the significant morbidity and even mortality associated with hepatic iron overload.

Cardiac Manifestations

The cardiac complications from iron overload are the most common cause of death for persons with TM. Several studies have demonstrated cardiomyopathy as the cause of approximately 70% of deaths in persons with thalassemia [43,44,45]. The development of effective iron chelating agents has substantially reduced the frequency and severity of fatal cardiomyopathy. Despite these available therapies cardiac dysfunction remains a significant problem when iron chelation therapy is suboptimal. Myocardial iron deposition most commonly results in left ventricular dilatation and systolic dysfunction. It is critical to diagnose and treat myocardial involvement in persons with TM as early as possible, including referral to heart failure specialists as necessary. Similar to the advancements in assessing liver iron content, noninvasive MRI techniques can accurately quantify myocardial iron that and should routinely be performed in all patients with TM.

β-Thalassemia Intermedia

β-thalassemia intermedia (TI) is also a serious blood disorder that requires routine medical care and therapy. The distinction between TI and TM is determined by the clinical severity of the disease. Operationally, patients with TI do not require regular blood transfusions while those with TM do; however, it is possible that a person can initially be diagnosed with TI, but subsequently could become dependent upon transfusions, thereby reclassifying the diagnosis as TM.

The two defining characteristics of TI are that both β-globin gene loci are affected (typically β+/β+ or β0/β+, but occasionally β0/β0 with ameliorating genetic modifiers) and that chronic transfusion therapy is not necessary to maintain an acceptable hemoglobin concentration and reasonable quality of life [46]. A special example of TI is heterozygous HbE with β-thalassemia, found most commonly in persons from Southeast Asia (see Chap. 2). In TI, because patients with TI are often relatively asymptomatic early in life, they often present later in childhood than those with TM.

Despite the transfusion independence, TI is not a benign condition. For the same pathophysiological reasons as described in TM, ineffective erythropoiesis is a key feature of TI and results in chronic hemolytic anemia, hepatosplenomegaly, extramedullary erythropoiesis, and hemosiderosis secondary to increased gastrointestinal iron absorption. Although patients with TI can maintain a hemoglobin concentration of 8–10 g/dL, and do not require chronic transfusions for anemia and its sequelae, regular comprehensive assessments of growth, development, iron burden and organ function are required to ensure that the classification as TI remains appropriate and that they would not benefit from chronic transfusion therapy . Although by definition, patients with TI do not require routine blood transfusions, there are some patients who benefit from intermittent transfusion therapy ; indeed, a growing body of evidence suggests hydroxyurea therapy could play a role in improving hematological parameters and reduce the need for transfusions in patients with TI, or even convert some patients from a diagnosis of TM to TI [47,48,49]. It is important to recognize that TI remains a serious hematological condition, and requires routine clinical surveillance of organ function and liver and cardiac iron content, in order to initiate chelation therapy in a timely manner to prevent the serious long-term health consequences.

β-Thalassemia Minor

The heterozygous β-thalassemia state, also known as β-thalassemia minor or β-thalassemia trait, occurs when one abnormal β-globin mutation leads to ≤50% decrease in β-globin chain production. The typical scenario for β-thalassemia minor is the inheritance of a single β0- or β+-thalassemia mutation. There are no significant clinical manifestations of β-thalassemia minor, but classic hematological features can suggest the diagnosis. Since persons with β-thalassemia minor have a mild imbalance between α- and β-globin chains, some excess α-globin chains combine with δ-globin chains and increase the percentage of hemoglobin A2 above the normal value of 3.5%. Increases in HbA2 to 5–7% are pathognomonic of β-thalassemia trait.

Complete blood counts of persons with β-thalassemia minor demonstrate mild microcytosis, hypochromia, and mild anemia; typical laboratory values include mean corpuscular volume (MCV) 60–75 fL, mean corpuscular hemoglobin (MCH) 22–28 pg, and hemoglobin concentration 10–12 g/dL, respectively. The degree of anemia is dependent upon the specific β-globin gene mutation, but the anemia of β-thalassemia minor is rarely symptomatic or clinically relevant. When considering the diagnosis of β-thalassemia minor, it is important to also investigate the possibility of concomitant iron deficiency anemia, which is extremely common and demonstrates many of the same hematological manifestations [50]. While clues from the Mentzer index can help distinguish these two diagnoses, the diagnosis of β-thalassemia trait is often made after an unsuccessful trial of iron replacement in the setting of mild microcytic anemia. Hemoglobin electrophoresis usually distinguishes these two conditions, although concomitant iron deficiency anemia as well as α-thalassemia can lower HbA2 levels [51].

Sickle Cell Disease

Overview and Historical Perspective

Sickle cell disease (SCD) refers to a group of inherited hemolytic anemias characterized by the predominance (>50%) of abnormal sickle hemoglobin (HbS, α2βS 2) within the erythrocytes. This operational definition thus distinguishes SCD from sickle cell trait, a typically benign condition where HbS represents approximately 30–40% of the total cellular hemoglobin. SCD is therefore not a single entity, but a constellation of blood diseases that all feature intracellular deoxy-HbS polymerization and subsequent erythrocyte deformation into a sickled shape.

SCD has been described clinically for generations in West Africa with the first documented reports in the 1800s. SCD was first reported in the Western medical literature in 1910 by James Herrick (with a majority of the work done by his intern Ernest Irons), who reported the abnormal “sickle-shaped” red blood cells of a severely anemic Chicago-area dental student born in Grenada [52, 53]. As discussed earlier, the HbS mutation was identified by Linus Pauling in 1949 resulting in SCD being labeled as the first molecular disease. Although these important discoveries are now more than 50–100 years old, the genetic or environmental factors contributing to the phenotypic variability of SCD remains largely unknown.

Over the past 30 years, the management of patients with SCD has greatly improved, due to careful prospective natural history studies in the United States and Jamaica leading to early recognition of clinical complications, widespread use of pneumococcal immunization, and the judicious use of safe blood transfusions. In addition, landmark clinical trials have proven the lifesaving effects of prophylactic penicillin, the efficacy of hydroxyurea therapy for both adults and children, and the importance of transcranial Doppler (TCD) screening and transfusion therapy for the prevention and management of acute stroke [54,55,56,57,58]. Together with other important pivotal research studies, the morbidity and mortality of SCD has been substantially improved, though much work is still needed, particularly in the global setting.

Epidemiology and Global Burden

SCD is one of the most common monogenic diseases in the world, with an estimated 312,000 annual HbSS births globally, most occurring within sub-Saharan Africa [59]. The sickle mutation (βS) in HBB has its origins in sub-Saharan Africa and the Indian subcontinent. The HbS mutation arose independently in at least four regions within sub-Saharan Africa and one that includes the Arab peninsula and Indian subcontinent (Fig. 1.2) [60, 61]. Although each β-globin haplotype has the same sickle mutation, the flanking genetic polymorphisms result in unique phenotypes for each haplotype [62, 63]. For example, the Senegal (SEN) and Arab-Indian (AI) haplotypes are associated with higher fetal hemoglobin (HbF) levels and an overall less severe phenotype, while the Central African Republic (CAR) haplotype is associated with lower HbF levels and a more severe clinical course.

Fig. 1.2
figure 2

The HbS mutation arose independently in at least four regions within sub-Saharan Africa and one that includes the Arab peninsula and Indian subcontinent

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The multicentric geographical origins and subsequent persistence of the deleterious sickle mutation is due to tremendous genetic selective pressure from P. falciparum malaria. Fig. 1.3 demonstrates that the geographic areas with the highest HbS allele frequency are nearly identical to the areas with the highest malaria endemicity [64]. This striking geospatial overlap is the result of the significant protection provided by the heterozygous HbS carrier state against severe and lethal malaria infection. Strong epidemiological evidence has demonstrated that children who inherit a single HbS allele (i.e., have sickle trait, HbAS) have lower parasite burdens and are 50–90% less likely to die from severe malaria than children with HbAA [65]. The homozygous HbSS state also confers relative protection against malaria infection, but because of the baseline severe anemia, the clinical severity and mortality of malaria in children with HbSS is high [66]. This balanced genetic polymorphism results in relative selection for the sickle cell trait within areas of malaria endemicity but unfortunate persistence of SCD.

Fig. 1.3
figure 3

The geographical distribution of the HbS allele is nearly identical to the geographical prevalence of Plasmodium Falciparum malaria, due to the protective effect of the HbAS carrier state. From Piel FB et al. Nature Communications 2010 [105], reprinted with permission from Nature Publishing Group

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Molecular Basis and Pathophysiology

HbS results from a single point mutation, which causes a substitution at the sixth amino acid in the β-globin protein. The sickle mutation results in a change from a hydrophilic residue (glutamic acid) to a hydrophobic one (valine), causing modification of the hemoglobin tetramer such that under low oxygen tension, the normally soluble hemoglobin rapidly polymerizes within the erythrocyte and stretches the cell membrane into a curvilinear shape. This “sickling” event is the sine qua non of SCD; although there are many other facets to the pathophysiology involving erythrocytes (dehydration, hemolysis, and adhesion), leukocytes (adhesion, leukocytosis), and endothelium (damage, dysfunction, and inflammation), erythrocyte sickling marks the primary phenomenon and is uniquely characteristic of this blood disorder.

The erythrocytes in SCD do not circulate in a permanent deformed state, however, but cycle between their sickled and non-sickle shapes as reversibly sickled cells. After repeated cycles, the cell membrane becomes irreversibly sickled. These cells become fragile, are prone to hemolysis, and have a markedly shortened lifespan that leads to a partially compensated hemolytic anemia that is characteristic in SCD.

Classification of Sickle Cell Disease

SCD encompasses a variety of genotypes, including both homozygous HbSS disease and compound heterozygous sickling conditions such as HbSC and HbSβ-thalassemia. Sickle cell anemia (SCA) is a term that refers to the most severe forms of SCD, such as HbSS or HbSβ0-thalassemia. Table 1.1 provides representative blood counts and hemoglobin electrophoresis patterns that are commonly observed in persons with various forms of SCD.

Table 1.1 Laboratory findings and clinical phenotype for common sickle genotypes (hematological parameters represent typical median values)
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Clinical Manifestations of Sickle Cell Disease

Although primarily a disorder of hemoglobin and red blood cells, SCD is a systemic disorder that has an effect upon all organ systems because of the importance of hemoglobin in tissue oxygenation. There are certain clinical manifestations that present early in childhood, while there are others that do not typically develop until adolescence or early adulthood. There is also wide clinical heterogeneity among the different SCD genotypes, with SCA (most commonly due to Hb SS and Hb Sβ0 thalassemia) having a more clinically severe phenotype but with extensive variability among individual patients. Currently, there are no reliable ways to predict which clinical symptoms will happen to which patients at which point in their life. The discussion in this chapter will focus mostly on the clinical manifestations of SCA, but will also briefly summarize the unique clinical complications seen in the other types of SCD.

Hematologic Manifestations

There is significant variability in the hematologic parameters for persons with SCA, but patients tend to maintain their own stable baseline hemoglobin concentration, which typically ranges from 6 to 9 g/dL. In addition to moderate to severe anemia, persons with SCA typically have significant reticulocytosis and elevated white blood cell, absolute neutrophil, and platelet counts due to chronic inflammation. The hematologic abnormalities of HbSC and HbS β+thalassemia are similar to, though not as severe as, those for patients with SCA and are outlined in Table 1.1 [67,68,69].

Vaso-Occlusive Manifestations

Vaso-occlusive painful crisis (VOC) or vaso-occlusive event (VOE) is the most common cause for hospital admission in adults with SCD . Dactylitis is an example of a vaso-occlusive painful event and is often the first clinical manifestation of SCA; it occurs in very young patients as the result of the infarction of bone marrow within the small bones, with an intense local inflammatory response in the surrounding tissues. Clinically, infants and toddlers present with acute and painful swelling of the dorsum of their hands and/or feet. In older children and adult patients, infarcts often manifest as acute painful episodes that are localized to a very specific site such as the sternum, ribs, arms or legs. Swelling may or may not be present and imaging is not always diagnostic.

Pain is a very common and even daily complication of SCA and can be difficult to manage. Accurate quantification and evaluation of pain can be challenging, as there may be no accurate measures other than patient report. Unfortunately, it is not uncommon for patients with SCA to be accused of misrepresenting their pain, or reporting pain as part of drug-seeking behavior. This is an important misperception among clinical providers who do not recognize how frequent and severe sickle cell pain can be.

Infectious Manifestations

Due to the splenic dysfunction and several other abnormalities in innate immunity, patients with SCD have a significantly increased risk of serious and life-threatening infections. Invasive bacterial infections from encapsulated organisms (e.g., Streptococcus pneumoniae, Haemophilus influenza type b, and Salmonella species) are the most common and severe infections, particularly for young children with SCD. Without early diagnosis of SCD allowing for initiation of vaccination and antibiotic prophylaxis, these infections can result in early mortality for young children with SCD. The combination of diagnosis by newborn screening and early interventions such as vaccinations and penicillin prophylaxis has significantly reduced the early mortality of SCD in the US and Europe [70, 71].

Transient aplastic crisis is another common and serious infectious complication of SCA seen most commonly in young children. Children typically present with fever, non-specific signs and symptoms of a viral illness, significant pallor and lethargy. It is most commonly associated with parvovirus B19, but can occur in association with other viral conditions. Parvovirus specifically targets erythroid progenitor cells in the bone marrow and effectively shuts down erythropoiesis for approximately 5–7 days. In healthy children, this temporary decrease in erythrocyte production is not clinically apparent, but for patients with SCA, who have 10–15% turnover in their RBC volume each day, clinical complications are apparent and severe. Dramatic reticulocytopenia is the hallmark laboratory finding that distinguishes this condition from anemia due to other infections or acute splenic sequestration crisis. With significant symptomatic anemia and reticulocytopenia, patients with SCA who develop aplastic crisis commonly require a transfusion of packed red blood cells to maintain an adequate hemoglobin level until reticulocytopenia resolves. Once the viremia is cleared and reticulocytes recover, patients have measurable IgG antibodies to parvovirus and are considered immune against subsequent infection.

Neurological Manifestations

Cerebrovascular disease contributes significantly to the morbidity and mortality of SCA, and can range from poor school performance to overt clinical stroke. The dramatically increased risk of stroke for children with SCA compared to otherwise healthy children has been well documented. In the era before TCD screening, the risk of stroke by age 20 years for children with SCA was 11% and increased to 24% by age 45 years [72]. In the US Cooperative Study of Sickle Cell Disease, ischemic strokes were more common in patients less than 20 years of age and for those greater than 30 years of age, while hemorrhagic events were more common between the ages of 20 and 30 years [72].

Recently, there has been increasing body of literature describing the frequency and clinical significance of silent cerebral infarcts (SCI) . SCI is a common neurologic finding in SCA, affected up to one-third of young patients; by definition, neuroimaging (most commonly MRI) reveals parenchymal lesions that are not associated with focal neurological deficits. The term “silent” is a misnomer since SCI has been associated with cognitive and intellectual deficits [73], and is a recognized risk factor for overt stroke [74, 75]. Recently the randomized controlled Silent Cerebral Infarct Transfusion (SIT) Trial demonstrated that for children with documented SCI, regular blood transfusions can help prevent further cerebrovascular disease including more SCI, transient ischemic attack, and stroke [76]. Last year, a multi-center trial demonstrated the efficacy of hydroxyurea to prevent stroke in children with abnormal TCD velocities [77], thus offering a useful alternative to lifelong blood transfusions for children with high risk for primary stroke.

Cardiac Manifestations

There are many cardiac findings and complications associated with SCD, although a distinct sickle cell cardiomyopathy has not yet been clearly defined. Many of the common findings, including cardiac flow murmurs and cardiomegaly, are often secondary to the chronic anemic state. Common clinical symptoms include fatigue and dyspnea. One study documented over half of the pediatric participants met the New York Heart Association Class I criteria for functional cardiac disease [78]. A more recent study, which evaluated sudden death that occurs in SCA patients, identified the abnormal heart rhythm of pulseless electrical activity arrest [79] while autonomic dysfunction may also be present [80,80,]. Ongoing research is needed to better define the sickle cell cardiomyopathy and to identify effective treatment modalities for this important cause of morbidity and mortality for adults with SCA. Recently a new model of restrictive cardiomyopathy due to fibrosis, which manifests as disastolic dysfunction, has been proposed that may help explain much of the sickle cell cardiac complications [81].

Pulmonary Manifestations

Pulmonary manifestations are among the most frequent and severe complications for both adults and children with SCD and collectively are associated with increased mortality. Both acute and chronic pulmonary complications can occur in patients with SCD, and seem to be exacerbated in the presence of the clinical diagnosis of asthma [82, 83].

Acute chest syndrome (ACS) is the most common cause of hospitalization and death among patients with SCA [84]. ACS is defined by the presence of new onset respiratory symptoms, fever, elevated leukocyte count, and associated radiographic changes representing a severe and often rapidly progressive inflammatory process in the lung. This clinical event can be triggered by many different causes, including infection, infarction, and fat embolism, although a clear etiologic trigger is not always found [84]. Regardless of its origin, ACS is a life-threatening condition for both children and adults with SCD, and a high index of suspicion must be maintained to initiate early interventions to prevent worsening respiratory symptoms and more serious complications. It is also important to recognize that ACS often develops while a patient is hospitalized for another sickle cell complication, most commonly vaso-occlusive pain. Initiation of incentive spirometry and aggressive pulmonary toilet should be routinely employed for all patients with SCD admitted to the hospital.

Pulmonary hypertension (PH) is a serious and potentially life-threatening complication that can develop in a subset of adults with SCD. Some investigators estimate that 6–11% of patients meet the diagnostic criteria for PH and have a significantly increased risk of early mortality [85]. The American Thoracic Society recently released official clinical practice guidelines regarding the diagnosis, risk stratification and management of PH in SCD [85], although the newer NHLBI evidence-based guidelines recommend evaluation and referral only for symptomatic patients [69]. Right heart catheterization (RHC) is the gold standard for the diagnosis of PH , but there is limited use of RHC in SCD due to the risks of anesthesia for this invasive procedure. In lieu of RHC, tricuspid regurgitant jet velocity from echocardiography is often used to identify elevated right-sided pulmonary pressures, despite having a low positive predictive value for PH due to concomitant cardiac complications [86]. Although a consensus algorithm for screening patients with SCD has not been established, RHC-proven diagnosis of PH appears to be a strong risk factor for early mortality and should be managed aggressively.

Splenic Manifestations

The spleen is one of the earliest organs to sustain damage in the young child with SCD. Its milieu is favorable to sickling, due to the slow circulation and hypoxic conditions within the splenic pulp. Most children with SCA begin a process known as “auto-infarction” of the spleen within the first 2 years of life, due to recurrent damage to the splenic microvasculature by the abnormal sickled red blood cells; this results in functional asplenia with significantly increased susceptibility to infections, particularly by encapsulated bacterial organisms. In patients with SCA, the spleen may be palpable over the first 2–3 years of life due to trapping of sickled erythrocytes, but this does not indicate normal immunological or filtrative function.

Over time, the spleen in SCA is usually no longer palpable once this autoinfarction occurs. In patients with less severe sickle genotypes, however, particularly in HbSC disease, splenomegaly often persists into adulthood. Some adolescents and young adults with HbSC disease develop hematological abnormalities (e.g., worsening anemia, thrombocytopenia, and leukopenia) from hypersplenism and require splenectomy in order to improve their blood counts. Splenic infarction can also occur in patients with persistent splenomegaly.

Acute splenic sequestration crisis (ASSC) is a common and serious complication that occurs most commonly in young children with SCA during the first 2 years of life. Due to the persistence of an enlarged spleen, ASSC can occur later in childhood and even in adulthood for persons with HbSC disease. ASSC typically presents with a clinical picture of severe anemia that should not be mistaken for transient aplastic crisis, since the management is quite different. Laboratory findings in both settings are notable for significant reduction in hemoglobin concentration, but with ASSC there is an increased reticulocyte count and increased numbers of circulating nucleated red blood cells, since the marrow is unaffected. Physical exam in ASSC is notable for pallor and tender splenomegaly that may extend beyond the umbilicus. If untreated, ASSC can be fatal, so it is important to educate parents about the importance of palpating the spleen regularly and seeking medical attention immediately for splenic enlargement that may portend a life-threatening episode of ASSC . Children with ASSC often require hospitalization and urgent erythrocyte transfusion due to anemia and hypovolemia, but transfusion should be provided carefully in small volumes (e.g., 5 mL/kg) due to the risk of an “overshoot phenomenon” that results from auto-transfusion of sequestered erythrocytes, with potential hyperviscosity and serious neurovascular complications. After recovery, at least half of the children with ASSC will have a recurrence, and many of those will require splenectomy.

Hepatobiliary Manifestations

Due to the chronic hemolysis that occurs in SCD, the development of pigmented gallstones is a very common occurrence, often beginning in childhood. Asymptomatic gallstones do not necessarily require any acute intervention, but symptomatic gallstones and acute cholecystitis benefit from elective cholecystectomy . In most centers, patients with asymptomatic gallstones will also receive surgical intervention, due to the risks of performing emergent surgery in a patient unprepared with preoperative transfusions. Transmission of viral hepatitis from the blood supply is an uncommon occurrence in the United States due to rigorous screening methods, but for older patients or those receiving blood products from regions of the world with potentially unsafe blood supplies, a suspicion for the possibility of viral hepatitis should be raised. In addition to gallstones, there are a number of other rarer hepatobiliary manifestations of SCD , including hepatic crisis, intrahepatic cholestasis, and hepatic sequestration.

Renal Complications

SCD is associated with many renal complications that lead to significant morbidity and mortality within the SCD population. Increased glomerular filtration and the inability to appropriately concentrate the urine are renal manifestations that begin at a very early age [87]. Albuminuria and proteinuria are among the more severe early indications of significant renal organ damage, and may precede progression to more significant end-stage renal disease. Sickle nephropathy can progress to significant renal dysfunction and renal failure. Among adults with SCD, sickle nephropathy is an important contributor to morbidity and mortality and recently losartan was found to reduce urinary albumin excretion [88].

Skeletal Complications

Avascular necrosis (AVN) , or osteonecrosis, is a severe complication of SCD , occurring in up to 50% of patients with SCD [89]. AVN most commonly occurs in the femoral or humeral head and results in limited range of motion, as well as chronic debilitating pain. Early and aggressive recognition and management of AVN is essential to prevent a more chronic and refractory painful condition. It is notable that AVN is a common complication for patients with HbSC disease, likely due to a higher hemoglobin concentration and hyperviscosity.

Other Complications

Due to the critical role of hemoglobin in delivering oxygen to all organs, SCD can affect nearly every organ in the body, with a long list of acute and chronic disease manifestations. These clinical complications will not be discussed in detail here but include growth and pubertal delay, priapism, retinopathy, and chronic leg ulcers [90,91,92,93,94,95].

Sickle Cell Disease Treatment Options

The treatment of SCD has advanced dramatically over the past 30 years, although most therapeutic options are limited to countries with adequate health resources. As discussed earlier, early diagnosis by newborn screening allows timely initiation of lifesaving interventions, including immunizations and prophylactic penicillin therapy. Blood transfusion (either simple packed erythrocytes or exchange transfusion) is an effective treatment for many acute complications of SCD including stroke, acute chest syndrome, splenic sequestration crisis, and aplastic crisis. Chronic transfusion programs, in which patients received blood transfusion on a regular basis, have become the standard of care for both primary and secondary stroke prevention [96, 97].

Over the past two decades, hydroxyurea has been demonstrated to be both safe and effective for the prevention of many complications of SCA. The new NHLBI sickle cell guidelines include strong recommendations for the use of hydroxyurea in adults with SCA, and also recommend that hydroxyurea now be offered to all children with SCA regardless of clinical symptoms [69]. Hydroxyurea is proven to reduce the frequency and severity of painful crises, acute chest syndrome, and the need for transfusion and hospitalization, and a growing body of evidence suggests that hydroxyurea may also prevent or delay chronic organ damage and even improve patient survival [58, 68, 98,99,100,101,102].

Summary

Sickle cell disease and β-thalassemia are common and life-threatening hematological conditions with a large global burden. Over the past century, tremendous scientific discoveries have solved the molecular and genetic basis of these conditions, elucidated the clinical spectrum of disease, and identified potential therapeutic targets. Despite these significant scientific advances, however, effective therapies remain limited and quality of life remains poor for millions of affected persons. Blood transfusion remains the primary treatment by which to manage the acute and chronic complications of SCD and β-thalassemia. Hydroxyurea is now established as the only other effective disease-modifying therapy, especially for SCD, but is not yet prescribed to most patients. Despite the proven merits of blood transfusions and hydroxyurea for the clinical management of these conditions, neither therapeutic option cures the underlying hematological disorder. Hematopoietic stem cell transplantation is the only “curative” treatment option, but transplantation is neither widely available nor affordable, and concerns remain about transplant-related morbidity and mortality [103]. Newer cellular and gene therapies are emerging, and these exciting possibilities are now under investigation in careful clinical trials, leading to real hope for curative therapy for SCD and β-thalassemia in the near future.