Genotype-Phenotype Correlations in Hereditary Elliptocytosis (HE) and Hereditary Pyropoikilocytosis (HPP)

Hereditary elliptocytosis (HE) results from defects in the erythrocyte membrane proteins α-spectrin, β-spectrin or protein 4.1R that weaken the "horizontal" cytoskeletal associations causing decreased mechanical stability and deformability of the red blood cell (RBC). The hallmark of HE is the presence of elliptical RBCs on peripheral blood smears. Diseases within the spectrum of HE include classic HE and hereditary pyropoikilocytosis (HPP).  The traditional diagnosis of HE relies on visualization of elliptocytes on the blood smear, while HPP is characterized by significant poikilocytosis and abnormally shaped RBCs.  In vitro assessment of RBC membrane biomechanical properties using ektacytometry is characteristic. However, this evaluation requires a pure RBC population, which may not be available in patients requiring frequent transfusions and does not predict disease course or severity.

Using a Next-Generation sequencing panel for 12 genes associated with RBC membrane disorders, we identified the causative genetic mutations in 8 patients with clinically diagnosed or suspected HE or HPP (3 with HE and 5 with HPP). We correlated the identified genetic mutations with clinical presentation, blood smear findings, and ektacytometry results.

HE patients: 2 patients with HE had heterozygous mutations in SPTA1 (p.L154_L155insL) resulting in spectrin self-association defect, while the third one had a novel nonsense heterozygous mutation in EPB41 (p.R262*) resulting in defective horizontal interaction between spectrin and actin in the RBC cytoskeleton due to decreased band 4.1R. The same novel mutation was identified in the patient's mother. Both, the patient and the mother, had smear findings and ektacytometry profiles consistent with the diagnosis of HE (Fig. 1A).

HPP patients: 3 out of 5 HPP patients were found to be compound heterozygous for a SPTA1 mutation at the self-association site (p.R28H or p.L154_L155insL) and a low-expression SPTA1 allele (c.6531-12C>T known as α-LELY or Low Expression LYon) which allows for the expanded expression of the HE-causing mutation located in trans, resulting in a more severe phenotype. Of note, two siblings carried the same SPTA1 mutation (p.L154_L155insL); one was heterozygous and had mild HE while the other sibling carried α-LELY in trans to the HE-mutation causing a more severe anemia with poikilocytosis and worsened deformability consistent with HPP (Fig. 1B).  The ektacytometry profile of these two siblings' RBCs demonstrates the role of α-LELY in modifying the phenotype of elliptocytic disorders. A fourth patient with HPP was found to have compound heterozygosity for mutations in SPTA1 (p.L154_L155insL) and SPTB (p.W2024R) both in the self association sites between α- and β-spectrin. The fifth patient was a 3 y.o. boy with transfusion-dependent hemolytic anemia since infancy and therefore his erythrocyte phenotype could not be evaluated.  He was homozygous for a novel mutation in SPTB (p.F2014V); both of his parents were heterozygous for the same mutation.  The parents were asymptomatic but their ektacytometry profiles and blood smears were consistent with HE (Fig. 1C), indicating that this mutation in the b-spectrin dimerization domain alters the α- and β-spectrin interaction causing HE in heterozygous state and HPP in the homozygous state.

Next-Generation sequencing for genetic diagnosis in HE and HPP can confirm or identify the diagnosis and may be helpful to predict the phenotype and clinical severity in these heterogeneous disorders. Combining clinical data, ektacytometry and family studies is important to understand the relevance of new genetic variants to pathology of RBC cytoskeletal disorders, provide insight into the genotype-phenotype correlation, and improve the genetic counseling and clinical care for these disorders.

Figure1. Ektacytometry profiles of : A) a patient and his mother heterozygous for a novel nonsense mutation in EPB41 (p.R262*) causing HE, B) a patient with SPTA1 mutation (p.L154_L155insL) and α-LELY causing HPP compared to a sibling with the same heterozygous mutation in SPTA1 without α-LELY causing HE, C) novel mutation in SPTB (p.F2014V) causing mild HE to heterozygous parents but severe transfusion-dependent anemia in the homozygous child.  Ektacytometry was not performed in the child since he was chronically transfused.