Chapter 39: The Congenital Dyserythropoietic Anemias

The term congenital dyserythropoietic anemia (CDA), coined by Heimpel and Wendt,¹ applies to a group of rare hereditary refractory anemias characterized by ineffective erythropoiesis, erythroid hyperplasia, abnormal morphology of erythroid precursors, and secondary accumulation of tissue iron.

Dyserythropoiesis is a result of derangement of the multistep process of normal erythroid maturation caused by a maturation arrest at early and late polychromatic erythroblasts and a consequent reduction of daily red cell production (Fig. 39–1A). Depending on the degree of the cellular defect, variable degrees of anemia can result.

Dyserythropoiesis can be considered a subtype of marrow failure syndromes characterized by monolineage involvement and abnormalities in erythroid precursor cells (see Fig. 39–1B). Microscopic morphologic characterization defines the heterogeneity of these syndromes (Fig. 39–2). Indeed, the appearance of abnormal morphology of erythroid progenitors can be from other hereditary defects (such as thalassemias) and acquired causes (rapid regeneration of marrow, myelodysplasia, etc.), but these are usually readily apparent from the history, blood examination, and laboratory tests in the former and multilineage involvement and the absence of prevalent multinuclearity in the latter. Furthermore, the term congenital could be confusing, as the clinical appearance of hereditary dyserythropoietic anemias can be present during different ages. In addition, it is an archaic residual of older nomenclature and should be replaced by “hereditary” as it has been in diseases such as congenital spherocytosis (now hereditary spherocytosis).

Anemia is usually first noted in infancy or childhood. The life span of circulating erythrocytes is moderately reduced, and the dominant factor in pathogenesis is a large component of ineffective erythropoiesis (intramedullary cell death as a result of precursor cell apoptosis) resulting in anemia of variable severity, normal or slightly elevated reticulocytes, moderate increase in indirect bilirubin, low haptoglobin, and, typically, a gradual increase in ferritin levels. Splenomegaly is common. Congenital dyserythropoietic anemias have been classified into three types—types I, II, and III. In addition, a number of cases that do not fit clearly into any of these three categories have been described.²

The prevalence of CDAs is likely higher than reported as a result of their clinical heterogeneity and diagnostic difficulties, as demonstrated by the fact that the correct diagnosis is often delayed into adulthood.

Merging of information from European registries suggests that the prevalence of CDA I and CDA II in Europe is approximately 0.24 and 0.71 cases/million, respectively, whereas CDA III is even less common.^3,4 In 2011, 712 cases from 614 families were included in the German CDA Registry, whereas in the Italian CDA registry 206 cases from 183 families were enrolled. In the literature, 169 cases from 143 families with CDA I and 454 cases from 356 families with CDA II worldwide have been recorded. Hence, CDA I is approximately one-third as frequent as CDA II. Most reported families were from Western European and Middle Eastern countries, but single cases have been also reported in the United States, India, Japan, and China.

The wide variation of incidence of CDA in Europe could be owing to genetic factors and also to the existence of reference centers for the diagnosis of CDA. However, molecular studies have established, at least for CDA type II, the presence of a founder effect in Europeans for the two most common mutations of the SEC23B gene.^5,6

CLINICAL FEATURES

CDA I is inherited as an autosomal recessive disorder. It can manifest in any year of pediatric age, but may not be diagnosed until adulthood. There is a continuous spectrum between moderate and severe forms; more severe presentations are predictably diagnosed at earlier ages.⁷

Anemia, usually macrocytic, is typically moderate and associated with intermittent jaundice, splenomegaly, and, sometimes, hepatomegaly. Hemoglobin values fluctuate around 9 to 10 g/dL. The patient usually does not require transfusion.^7,8,9

Patients diagnosed in the neonatal period often have hepatomegaly and jaundice, and may have exhibited intrauterine growth retardation. Spleen size increases with age. Cholelithiasis is a common complication, and jaundice may be aggravated by the coinheritance of A[TA]₇TAA polymorphism in the promoter of the UGT1A1 gene, the main cause of Gilbert syndrome.¹⁰

The disease is occasionally associated with a variety of dysmorphologic features (4 to 14 percent of affected individuals), the most common of which involves the bones of the hand and the foot (syndactyly, hypoplasia of one or several phalanges, presence of supplementary metatarsal bones, and clubfoot) (Fig. 39–3).¹¹ Small stature, almond-shaped blue eyes, hypertelorism, micrognathism, and other abnormalities may also be present.

LABORATORY FEATURES

Anemia is a common finding, usually in the range of 8 to 10 g/dL of hemoglobin. The blood film usually exhibits macrocytosis (mean cell volume [MCV] of approximately 90 to 100 fL), marked poikilocytosis, and occasional nucleated erythrocytes, elliptocytes, and basophilic stippling. The reticulocyte count is inappropriately low for the degree of anemia.^7,8,9 The marrow has intense erythroid hyperplasia. Binucleate polychromatic erythroblasts are evident at a frequency of approximately 3 to 7 percent. A highly specific feature is the presence of chromatin bridges linking two nuclei in more or less incompletely separated polychromatic erythroblasts with 0.6 to 2.8 strands per 100 erythroblasts (see Fig. 39–2A).¹² Ultrastructural abnormalities consist of a spongy (“Swiss cheese”) appearance in the nucleus of up to 60 percent of early and late polychromatic erythroblasts. Nuclei exhibit areas of electrolucency within electron-dense heterochromatin and contain nuclear membrane-lined cytoplasmic intrusions, occasionally with retained cytoplasmic organelles (see Fig. 39–2B).^9,11

GENETICS

The most frequent mutated gene in CDA I patients, CDAN1 (chromosome 15q15.1–15.3), spans 15 kb and is composed of 28 exons.^13,14 The encoded protein, codanin-1, contains 1227 amino acids and it is ubiquitously expressed. Codanin-1 is a cell-cycle-regulated protein. The proximal CDAN1 gene-promoter region appears to be a direct target of transcription factor E2F1, and high levels of codanin-1 are observed in the DNA synthesis (S) phase of the cell mitotic cycle.¹⁵ At mitosis, codanin-1 undergoes phosphorylation and it is excluded from condensed chromosomes. Moreover, codanin-1 binds to Asf1a, a protein involved in chromatin structure dynamics by its role in nucleosome assembly and disassembly.¹⁶

More than 30 unique mutations have been found in the CDAN1 gene, and affected subjects are either homozygotes or compound heterozygotes for the mutation(s) they carry. A genotype–phenotype correlation has not been established. A homozygous patient for null-type mutations has not been described, suggesting that an absence of codanin-1 is lethal. Indeed, CDA I knockout mice embryos die in utero at 6.5 days before erythropoiesis onset, presumably as a result of the critical role of this protein in developmental processes other than erythropoiesis.¹⁷ In approximately 20 percent of families with the CDA I phenotype, CDAN1 mutations were not found.¹⁸ One founder mutation, R1042W, was observed in Bedouins.^2,8,19

A new CDA I causative gene, C15ORF41, has been identified in three unrelated pedigrees of Middle Eastern and Southeast Asian origin by whole-genome sequencing.²⁰ The cellular role of the restriction endonuclease encoded by the C15ORF41 gene is unknown. This protein interacts with Asf1b, the paralogue of Asf1a, which may support the hypothesis that the primary defect in CDA I is confined to DNA replication and chromatin assembly.

In the erythroid lineage during normal maturation, cell division ceases at the polychromatophilic erythroblast stage and cell size is progressively reduced.²¹ Cell mitotic cycle defects in mice result in S-phase arrest, ineffective erythropoiesis, and macrocytosis.²² Based on the probable involvement of CDA I mutated genes in DNA replication and chromatin assembly, it can be speculated that CDA I pathogenesis may involve disruption of the intrinsic connection between cell cycle dynamics and erythroid maturation.

Knowledge of the molecular defects in CDA patients permits detection of carriers for prenatal diagnosis.

TREATMENT, COURSE, AND PROGNOSIS

Severe forms of CDA I may present with hydrops fetalis.²³ Pulmonary hypertension was reported in three Bedouin newborns with CDA I.²⁴ Iron overload as a result of transfusion, hemosiderosis, and/or enhanced iron absorption characteristic of inefficient erythropoiesis is the main concern as patients age. Monitoring for iron overload is required; the following assessments are recommended: (1) annual measurement of serum ferritin concentration, and (2) myocardial T2* magnetic resonance imaging (MRI) and hepatic R2* MRI starting in adolescence (Chap. 43). Inappropriately low serum hepcidin levels could account for iron overload. High levels of s-hemojuvelin (HJV) in patients affected with CDA I, compared with controls, have been documented.²⁵ Hepcidin-to-ferritin ratio was negatively correlated with s-HJV, suggesting that s-HJV may suppress hepcidin.²⁶ Rare patients develop retinal angioid streaks.²⁷

Fertility in patients is not affected. However, fetal anemia during pregnancy in affected women is associated with some morbidity. In some cases, intrauterine transfusions are warranted by the severity of anemia as judged by fetal blood sampling.²³

Transfusions should be avoided whenever possible because of the risk of iron overload. When anemia is mild and does not require transfusion, small-volume regular phlebotomies may be used to decrease body iron. Iron chelation or phlebotomies should be instituted when ferritin levels exceed 500 to 1000 mg/L. Iron chelation may be required for iron overload in those patients who cannot tolerate iron depletion by phlebotomies. Splenectomy is usually not beneficial.^2,9 Cholecystectomy for cholelithiasis is commonly performed.

Interferon-α was once used in a child with hepatitis C and CDA I; it was associated with an increase in hemoglobin level. A 9-year followup showed that the treatment remained effective and, on repeated liver biopsies, iron overload was normalized. In this case, the effective dose of interferon-α was 2 million units twice a week. Pegylated interferon could be used as well, at a dose of 30 mcg/wk.²⁸ The mechanism behind this response is unknown. Allogeneic hematopoietic stem cell transplantation was successful in several transfusion-dependent children.²⁹

CLINICAL AND LABORATORY FEATURES

CDA II is an autosomal, recessively inherited condition in which the severity of anemia varies from mild to severe and in which approximately 7 percent of cases are transfusion-dependent.^2,8,30,31 This disorder becomes variably manifest in infancy, childhood, or adolescence. Very few cases are characterized by clinical manifestations during intrauterine life, but hydrops fetalis caused by severe anemia has been reported.^32,33 More commonly, anemia is mild and, in several cases, diagnosis has been based on the appearance of complications (mainly iron overload) during adulthood.^2,8

Erythrocytes of CDA II patients lyse in acidified serum (Ham test; Chap. 40) because of a naturally occurring immunoglobulin M class antibody that recognizes an antigen present on CDA II red cells but which is absent on normal cells. Thus, the acronym HEMPAS (hemolytic anemia with a positive acidified serum test) is commonly used as a synonym for CDA II. The technical difficulty of this test, and the fact that cross-testing of more than 30 normal sera is needed to obtain a reliable result, has undermined its usefulness.³⁴

The clinical picture of CDA II includes hemolytic anemia with marrow erythroid expansion, commonly with splenomegaly, hepatomegaly, intermittent jaundice, and cholelithiasis.^30,31 The blood film exhibits moderate to marked anisocytosis and anisochromia and a number of spherocytes. This, along with the patient’s clinical appearance, may lead to confusion of CDA II with hereditary spherocytosis (HS; Chap. 46). However, typically in HS, the reticulocyte count in comparison to hemoglobin level is higher and the serum transferrin receptor level is lower. Moreover, the majority of HS cases are inherited as autosomal dominant and, thus, a parent is likely to have findings of spherocytosis on blood examination, whereas CDA II is invariably an autosomal recessive condition. Despite these differentiating features, CDA II is at times only diagnosed after the failure of splenectomy to normalize anemia when performed for suspected HS. In the marrow, 10 to 30 percent of intermediate and late erythroblasts have two or more nuclei or lobulated nuclei (see Fig. 39–2A). Karyorrhexis (fragmentation of the nucleus) is common. Gaucher-like cells may develop as a result of phagocytosis of erythroblasts by macrophages. Ringed sideroblasts are present in severe forms.¹² Electron microscopy shows structures that have been misnamed as “double membrane” (see Fig. 39–2B). These are cisternae of the endoplasmic reticulum (ER) that run along the red cell plasma membrane inner surface, and which contain ER-specific proteins, as shown by immunochemistry labeling.³⁵ Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblots, reveals the presence of calreticulin, glucose-regulated protein 78, and disulfide isomerase; these are specific for the ER and are not detected in normal individuals.³⁵

The diagnostic hallmark of CDA II is analysis of erythrocyte membrane proteins by SDS-PAGE identifying narrower band size and faster migration of erythrocyte anion transporters (AE1 or band 3) and band 4.5 proteins.^36,37 Increased destruction of red blood cells in CDA II is associated with hypoglycosylation of AE1, which causes clustering of this protein on the red cell surface and contributes to erythrocyte destruction in the spleen.³⁸ Rare patients have been reported without this characteristic SDS-PAGE pattern; it is recommended that these should be classified as CDA II–like conditions.

GENETICS

Pathognomonic hypoglycosylation of AE1 protein is the outcome of the expression of the mutated gene SEC23B.³⁹

Sequencing analysis in 33 patients from 28 unrelated families showed heterogeneous mutations in the SEC23B gene, either in compound heterozygous or homozygous states.^2,8,39 An in vitro model of gene silencing demonstrated that suppression of SEC23B expression recapitulates the cellular defects in SEC23B-silenced cells.³⁹ Knockdown of zebrafish SEC23B also leads to aberrant erythrocyte development.³⁹

SEC23B is a cytoplasmic coat protein (COP) II component involved in the secretory pathway of eukaryotic cells. COPII is a multisubunit complex essential for transport of correctly folded proteins from the ER toward the Golgi apparatus.⁴⁰ This pathway is critical for membrane homeostasis, localization of proteins within cells and secretion of extracellular factors.^40,41

CDA II belongs to COPII-related human genetic disorders.⁴² Alterations in SAR1B, a paralogue of SEC23B, are identified as the cause of chylomicron retention disease (Anderson disease),⁴³ while a specific mutation in the SEC23A gene causes craniolenticulosutural dysplasia (Boyadjiev-Jabs syndrome).⁴⁴ The specificity of the CDA II phenotype seems to be determined by tissue-specific expression of SEC23B versus SEC23A during erythroid differentiation.³⁹ Alternatively, this specificity could be explained by the presence of tissue-specific proteins (such as band 3 in red blood cells) which might require high levels and full function of a specific COPII component to be correctly transported.^42,45

So far, more than 60 different causative mutations have been described worldwide.^2,8 A genotype–phenotype correlation seems to exist. Particularly, compound heterozygosity for missense and nonsense mutations tends to produce more severe clinical presentations than homozygosity or compound heterozygosity for two missense mutations. Homozygosity or compound heterozygosity for two nonsense mutations has not been reported, suggesting it may be lethal.⁴⁶ Sec23b-deficient mice (Sec23b gt/gt) have been generated and are born without anemia but die shortly after birth, with degeneration of secretory organs, including the pancreas and salivary glands.⁴⁷

The disparate phenotypes in mouse and human could result from residual SEC23B function associated with the hypomorphic mutations observed in humans, or, alternatively, might be explained by species-specific functional differences.⁴⁸

TREATMENT, COURSE, AND PROGNOSIS

The clinical course of this condition is quite heterogeneous. Treatment approaches depend on age, severity of phenotype and comorbidity. Most patients have only mild or moderate anemia and do not require medical intervention. Approximately 10 percent of neonates need at least one erythrocyte transfusion, and some remain transfusion-dependent.^8,31 In most adolescents and adults, transfusional needs are limited to aplastic crises, pregnancy, coexistent infections, or major operations.

The more common, moderate forms may only be diagnosed in adult life because of iron overload (Chap. 43) that is consistently observed even in the absence of transfusions.^2,8,49 Patients with severe forms of CDA II may be transfusion-dependent. In some cases, severe phenotypes could be the result of additional genetic abnormalities, such as coinheritance of glucose-6-phosphate dehydrogenase (G6PD) deficiency or thalassemic trait.⁵⁰

The iron overload is associated with high levels of growth differentiation factor 15 (GDF15).⁵¹ However, GDF15 concentrations are significantly lower in CDA II compared to CDA I patients, despite a similar degree of iron overload in both patient groups. It can be speculated that additional signals may determine hepatic hepcidin expression and the degree of iron overload in CDA II.⁵¹

Ferritin levels should be controlled at least annually, even in patients with only mild anemia. Achievement of normal ferritin concentrations is desirable.⁵² Iron chelation should be instituted when ferritin level exceeds 500 to 1000 cg/L (Chap. 43). If phlebotomies are tolerated, this is the preferred treatment. In instances where the patient cannot tolerate phlebotomy, chelating agents may be used.

Cholelithiasis and splenomegaly are common complications. Coinheritance of the UGT1A (TA)7/(TA)7 genotype could account for the increased rate of gallstones.⁵³ Cholelithiasis, which is frequent in all types of CDA, may require cholecystectomy; decision making should follow therapy guidelines for cholelithiasis.⁵⁴ Splenectomy is not universally recommended for CDA II or CDA I; individual decisions should be influenced by transfusion dependency and the presence of a massively enlarged spleen. Generally accepted criteria for splenectomy have not been defined. Splenectomy leads to a moderate, sustained increase in hemoglobin concentrations and decrease of serum bilirubin levels, but it does not prevent iron overload, and hemoglobin levels postsplenectomy generally do not reach normal values.^5,6 In non–transfusion-dependent patients, it is advisable to follow the guidelines for mild cases of HS.⁵⁴

Allogeneic marrow transplantation from an human leukocyte antigen (HLA)-identical sibling has been successful in transfusion-dependent children with very severe CDA II and in one adult with CDA II and β-thalassemia trait.^32,33,55,56

CLINICAL AND LABORATORY FINDINGS

Type III is the least-common form of CDA. This condition, which is dominantly inherited, was initially coined “hereditary benign erythrocytosis.” One dominantly inherited form was reported as early as 1951 in a woman and her three children, in whom 16.0 to 22.7 percent of hematopoietic stem cell erythroblasts were multinucleated. Giant-size erythrocytes were present in the blood.

Most of our knowledge about CDA III stems from a large family from the province of Västerbotten in northern Sweden.⁵⁷ The diagnosis was made in the adults and older children. The spleen was not palpable, nor was iron overload recorded. The large size of this family made it possible to map the responsible gene to 15q22–25.⁵⁸ In addition, a number of sporadic cases of CDA III have been reported.⁵⁹

In another case, a number of stillbirths, including at least one stillborn with hydrops fetalis, were noted in an Indian family in which the mother, who initially required transfusions, became transfusion-independent after splenectomy.⁶⁰

Blood films from these patients show macrocytes, occasional extremely large forms (gigantocytes), and poikilocytes. Patients are generally asymptomatic, with no or moderate anemia, mild jaundice, and, commonly, cholelithiasis. The reticulocyte count is typically less than 3 percent.^57,61 Marrow shows marked erythroid hyperplasia, with large multinucleate erythroblasts with large, lobulated nuclei, and giant multinucleate erythroblasts with up to 12 nuclei (see Fig. 39–2A). On electron microscopy, clefts within heterochromatin, autophagic vacuoles, iron-laden mitochondria, and myelin figures in the cytoplasm have been reported.⁶²

GENETICS

The causative mutation was found to be 2747C>G (P916R) in the KIF23 gene.⁶³ The same mutation was also found in CDA III patients from an American family without any known relation to the Swedish kindred.⁶³ KIF23 encodes a kinesin-superfamily molecule, mitotic kinesin-like protein 1 (MKLP1), a mitotic protein essential for cytokinesis.^64,65 MKLP1 interacts with Arf6 (adenosine diphosphate (ADP)-ribosylation factor 6), ultimately forming an extended β-sheet that interacts with the membrane surface at the cleavage furrow. The Arf6–MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.⁶⁴ Knockdown of Arf6 results in binucleated and multinucleated cells, a hallmark of CDA III erythroblasts.⁶⁶ In knockdown and rescue experiments with in vitro cell lines, cytokinesis failure and binucleated cells were seen more frequently with the P916R mutant than wild-type GFP-MKLP1, indicating that the P916R mutation impairs the function of MKLP1 in cytokinesis.⁶³

COURSE AND PROGNOSIS

In spite of an apparently benign course, CDA III is prone to various long-term complications, including intravascular hemolysis, increased risk of myeloma and other monoclonal gammapathies,⁶⁷ and development of angioid streaks.⁶⁸

A number of cases of CDA that do not have specific features of types I, II, or, to some extent, III disease have been reported.^{69,70,71,72,73,74} Classification has been proposed based largely on cell morphology.⁷⁵ In addition, several genes have been associated with CDA variants, including mutations in the GATA1 and KLF1 genes, which are critical for development of specific blood cell lineages (outlined in Table 39–1).^{69,70,71,72,73,74,76}

Alterations in the erythroid hematopoietic transcription factor KLF1 gene are associated with CDA type IV,⁷⁷ characterized by severe hemolytic anemia, elevated fetal hemoglobin, and deficiency of erythroid proteins CD44 and aquaporin 1 (see Table 39–1).⁷⁸

Additionally, syndromes have been described in which CDA accounts only for one feature of a syndrome phenotype (see Table 39–1). For instance, Majeed syndrome, is comprised of chronic recurrent multifocal osteomyelitis, inflammatory dermatosis, and CDA. The responsible gene is LPIN2 (18p11.31), encoding lipin 2, an ER-phosphatidate phosphatase.⁷⁹ Additionally, dyserythropoiesis associated with exocrine pancreatic insufficiency and calvarial hyperostosis resulting from COX4I2 gene mutations has been described in two Arab families.⁸⁰ Mevalonate kinase deficiency (MKD) resulting from a missense mutation in the MVK gene and showing morphologic marrow cell abnormalities similar to CDA II has also been reported.⁸¹

Congenital dyserythropoietic anemias may be confused with thalassemias and other hemolytic anemias. Marked anisocytosis, including a normomacrocytic component, and low or moderate reticulocyte count out of keeping with the degree of anemia will point to a diagnosis of CDA. Indeed, CDAs are a subtype of marrow failure syndromes characterized by ineffective erythropoiesis and heterogeneous, type-specific morphologic abnormalities in erythroid precursor cells (see Fig. 39–1). Thus, marrow examination is essential if family history is unrevealing. The marrow, if needed, is quite different than in other types of hemolytic anemia or the thalassemias as multinuclearity of erythroblasts is not a feature of the latter two types of anemia.