Genotype-phenotype correlations in hereditary elliptocytosis and hereditary pyropoikilocytosis

Abstract

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are heterogeneous red blood cell (RBC) membrane disorders that result from mutations in the genes encoding α-spectrin (SPTA1), β-spectrin (SPTB), or protein 4.1R (EPB41). The resulting defects alter the horizontal cytoskeletal associations and affect RBC membrane stability and deformability causing shortened RBC survival. The clinical diagnosis of HE and HPP relies on identifying characteristic RBC morphology on peripheral blood smear and specific membrane biomechanical properties using osmotic gradient ektacytometry. However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions, and does not predict disease course or severity. Using Next-Generation sequencing, we identified the causative genetic mutations in fifteen patients with clinically suspected HE or HPP and correlated the identified mutations with the clinical phenotype and ektacytometry profile. In addition to identifying three novel mutations, gene sequencing confirmed and, when the RBC morphology was not evaluable, identified the diagnosis. Moreover, genotypic differences justified the phenotypic differences within families with HE/HPP.

Keywords: Elliptocytosis; Hemolytic anemia; Mutation; Pyropoikilocytosis; Red blood cell membrane.

Figure 1

Schematic of the erythrocyte cytoskeleton highlighting the spectrin head-to-head self-association region and the protein 4.1R in the junctional complex contributing to spectrin-actin interaction.

Figure 2. Novel EPB41 mutation causing HE

(A) DNA sequencing of patient #1 and his mother with HE revealed a novel heterozygous nonsense mutation C>T in nucleotide 784 of EPB41 gene. This mutation introduces a stop codon in position p.R262 causing decreased production of protein 4.1R. Ektacytometry profile of patient #1 and his mother demonstrating trapezoidal curve with the maximum value of deformability or elongation index (EImax) decreased and shifted to the left, characteristic for HE. (B) Blood smears of patient #1 and his mother demonstrating multiple elliptocytes consistent with the diagnosis of HE. (C) Immunodetection by capillary electrophoresis of protein 4.1R versus band 3 in RBC ghost membrane of the mother of patient #1 compared to normal control. Densitometry of the bands demonstrates a decrease of 4.1R in this case of HE by 40%. (D) Immunoblotting after SDS-PAGE electrophoresis of RBC ghosts of the mother of patient #1 demonstrating normal protein 4.1R with its subcomponents 4.1a and 4.1b compared to normal controls. No truncated forms of protein 4.1R were detected (molecular weight ladder is shown on the left).

Figure 3. Biallelic spectrin mutations causing HPP

(A) HPP due to SPTA1 mutation in trans to α^LELY. Blood smears and ektacytometry profiles of patient #7 with HPP due to p.L154_L155insL mutation in SPTA1 gene in trans to α^LELY allele and her sibling (patient #2) with HE, heterozygous for the same HE-causing mutation, p.L154_L155insL, in trans to a normal SPTA1 allele. Blood smear demonstrates increased poikilocytosis and elliptocytosis and ektacytometry indicates worsened deformability in the patient carrying α^LELY compared to her sibling. (B) HPP due to novel homozygous SPTB mutation. DNA sequencing revealed a homozygous SPTB missense mutation (c.6040T>G, p.F2014V) in patient # 14 with HPP (P); both father (F) and mother (M) were found heterozygous for the same mutation. 3-D structure of the erythrocyte α- and β-spectrin tetramerization domain complex showing the position of the novel p.F2014V mutation (in blue) in relation to the spectrin self-association site. A mutation at this position likely affects the α-β spectrin interaction causing HE in heterozygous state and HPP in the homozygous state. Blood smear of the father shows elliptocytosis, and ektacytometry profiles of both parents demonstrate RBC deformability consistent with mild HE. Ektacytometry was not performed in the child since he was chronically transfused. (C) HPP due to compound SPTA1 and SPTB mutations. 3-D structure of the erythrocyte spectrin tetramerization domain complex showing (in blue) the positions of the mutations p.W2024R in β-spectrin (within the spectrin self-association site) and p.L154_L155insL in α-spectrin (near the spectrin self-association site) in patient # 15. Blood smear and ektacytometry profile of patient # 15 consistent with HPP. The 3-D structures were depicted in Jmol (an open-source Java viewer for chemical structures in 3D (http://www.jmol.org/), using the protein data bank (PDB) file 3lbx (DOI: 10.2210/pdb3lbx/pdb).