Using targeted genetic panels to find the causes of neonatal hemolytic anemia

RBC membrane disorders

RBC membrane disorders are relatively common inherited conditions. These can be divided into genetic defects that (1) alter cytoskeleton scaffolding and, consequently, the shape of RBCs; and (2) alter the regulation of RBC volume. The best-known examples of the first group include hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP). The second group is exemplified by hereditary stomatocytosis.

The major determinants of RBC shape and flexibility are the structure of the cytoskeleton and its interaction with the lipid bilayer of the membrane. Adequate amounts of structurally intact proteins, particularly α-spectrin (encoded by SPTA1 ), β-spectrin ( SPTB ), ankyrin-1 ( ANK1 ), band 3, also known as anion exchanger 1 ( SLC4A1 ), and protein 4.2 ( EPB42 ), are essential for normal RBC functioning and lifespan. Mutations in these genes can cause RBC membrane defects. ^, Fig. 8.1 depicts the interaction between RBC membrane proteins and lipid bilayer.

HS has a high prevalence, especially in infants of Northern European ancestry; the frequency may be as high as 1:1000 to 1:3000. It is the most common cause of hereditary HA in the Western population. The pattern of inheritance may be autosomal dominant (AD; 75% of cases) or autosomal recessive (AR; 25% cases). HS is caused by altered “vertical” protein-lipid interactions involving ankyrin (40–65%), band 3 (20–35%), β-spectrin (15–30%), α-spectrin, and protein 4.2 (<5%). HS presenting in AD inheritance pattern is usually caused by mutations in ANK1, SLC4A1, and SPTB. Most ANK1 mutations are point mutations that can suppress the synthesis of ankyrin. Unfortunately, up to 15% to 20% of these mutations occur de novo, and the lack of a family history can cause diagnostic difficulties. As ankyrin is the principal binding site for spectrin on the membrane, its inadequate production results in functional deficiency of the spectrin scaffold. Large deletions within ANK1 gene are rare and may be associated with a larger loss of chromosome 8p11.2 and a contiguous gene syndrome ( FGFR1 , ANK1 , and possibly other genes) clinically manifesting as a combination of Kallmann syndrome and HS.

Mutations in SLC4A1 (band 3) are usually missense or frameshift in nature, affecting both the exons and splice sites. The mutations have a deleterious effect on band 3 expression and produce a mild to moderate HA with an AD pattern of inheritance. SLC4A1 mutations can also cause distal renal tubular acidosis. ^, The AR forms causing severe transfusion-dependent anemia in neonates are usually caused by compound heterozygote mutations in α-spectrin. Since α-spectrin is usually produced in high amounts with considerable reserve, heterozygote mutations are typically not symptomatic. However, two low-expression alleles in α-spectrin need mention ^, ; the α ^LELY (low-expression Lyon) can be seen in up to one third of the normal population and can reduce α-spectrin expression by up to 50%. Neonates with these genetic abnormalities can develop severe transfusion-dependent HPP (discussed later) in conjunction with an HE allele in trans that gradually improves with time . Another low-expression allele, the α ^LEPRA (low-expression PRAgue) is an intronic mutation and can cause a severe form of HS if inherited with a pathogenic SPTA1 allele in trans. Protein 4.2 functions as a binding protein between ankyrin and band 3. Most mutations in the EPB42 gene are missense or nonsense, and cause protein 4.2 deficiency. HS associated with EPB42 mutations is more frequent in the Japanese population and shows AR inheritance and mild to moderate hemolytic anemia.

HE and HPP are caused by altered “horizontal” protein-protein interactions in the RBC cytoskeleton negatively affecting RBC deformability and producing misshapen erythrocytes: ovalocytes or elliptocytes in HE and numerous nonspecific poikilocytes in HPP. From a clinical and pathogenetic perspective, HE and HPP are viewed as two entities on a continuous spectrum, ranging from asymptomatic with a mild phenotype in HE to severe anemia, hemolysis, and transfusion dependency in HPP. ^, The key abnormality is in formation of α-β spectrin heterodimers or their self-association into tetramers. The principal genes involved in HE/HPP are SPTA1, SPTB, or EPB41. SPTA1 and SPTB mutations account for 60% to 75% of these patients. ^,

SPTA1 gene mutations are classically missense, affecting the dimer-tetramer self-association site. Because α-spectrin chains are produced in excess, an isolated heterozygous pathogenic mutation in SPTA1 is unlikely to produce a clinical condition. However, a compound heterozygosity with another pathogenetic mutation or, what happens more frequently, with a common SPTA1 low expression allele with decreased α-spectrin production, can result in an overt HPP. SPTB mutations are seen in approximately 30% of HE patients. The effect of these mutations also alters the association of spectrin heterodimers to tetramers. Alterations of protein 4.1 due to EPB41 mutations result in its quantitative or qualitative deficiencies. The types of mutations vary, ranging from point mutations to large deletions. The heterozygous state is asymptomatic, but homozygotes can experience a severe disease. Characteristically, the elliptocytes in patients with mutated EPB41 are numerous (almost 100% of RBCs) and slender. ^{, ,}

RBC volume disorders are caused by an inability to regulate water and ion transport through the membrane, which introduces overhydration or dehydration of the cell. Thus the conditions in this group are broadly divided into overhydrated and dehydrated stomatocytosis. Dehydrated stomatocytosis (hereditary xerocytosis [HX]) is more prevalent and presents as a HA of variable degree and unexplained iron overload. The most commonly mutated gene in HX is PIEZO1, which encodes for a mechanically activated cation channel. The pathogenic PIEZO1 mutations are usually gain-of-function. Fig. 8.2 depicts the peripheral smear and osmotic gradient ektacytometry result from a patient with HS. A few patients with HX have mutations in KCNN4 that encodes the Gardos channel. Overhydrated stomatocytosis is a very rare disorder with only approximately 100 patients described worldwide. Heterozygous mutations in the Rh-associated glycoprotein ( RHAG ) are commonly associated with overhydrated stomatocytosis.

Inherited hemoglobin defects

Abnormalities of globin chains are broadly divided into quantitative (thalassemias) and qualitative (hemoglobinopathies or variant hemoglobins). Most hemoglobin variants are clinically benign and asymptomatic, but some hemoglobinopathies and thalassemias can present as NHA. In a normal neonate, RBCs contain predominantly Hb F (α ₂ γ ₂ ) at approximately 70% to 80%, followed by Hb A (α ₂ β ₂ ) at 20%, and Hb A2 (α ₂ δ ₂ ) at 0.5% to 1%. Due to a relatively low amount of β-globin chains, both β-thalassemia and β-chain variants do not manifest in neonates. However, development of anemia with or without hemolysis at about 6 months of age should raise a suspicion of a β-globin chain disorder. Individuals with α-thalassemia demonstrate a wide spectrum of genotypes and clinical presentations, ranging from asymptomatic individuals with or without changes in the blood counts (usually associated with a silent carrier state, -α/αα), to varying degrees of anemia and hemolysis (like those with Hb H disease, --/-α). Neonates with Hb H disease show increased Hb Bart (γ ₄ ), which is switched to Hb H (β ₄ ) at a later age. Some variant hemoglobins, including the α- and γ-chain variants, may result in NHA due to low stability. One example is the Hb Hasharon, an α-chain variant (HBA2:c.142G>C) ; the point mutation introduces an amino acid substitution in the α-chain that weakens its interaction with a normal γ-globin chain. The unstable hemoglobin hybrid comprises 14% to 19% of the total hemoglobin. Such γ-globin variants can present with NHA and hyperbilirubinemia, but these clinical manifestations resolve by 4 to 6 months of age when γ-globin is replaced by β-globin. ^,