(A) HPP due to SPTA1 mutation in trans to α LELY. No truncated forms of protein 4.1R were detected (molecular weight ladder is shown on the left). (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. Densitometry of the bands demonstrates a decrease of 4.1R in this case of HE by 40%. (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. (B) Blood smears of patient #1 and his mother demonstrating multiple elliptocytes consistent with the diagnosis of HE. 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. This mutation introduces a stop codon in position p.R262 causing decreased production of protein 4.1R. (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. The typical HE ektacytometry curve is trapezoidal with decreased RBC deformability, while a larger decrease in deformability is noted in HPP ( Figures 2 and and3) 3). The resulting ektacytometry curve reflects biomechanical properties of the RBCs including osmotic fragility, surface-to-volume ratio, cytoskeleton flexibility, and cytoplasmic viscosity. In ektacytometry, the deformability of the patient's RBCs is assessed based on their laser diffraction pattern while they are subjected to a defined value of shear stress and an increasing osmotic gradient. Ektacytometry is an objective reference technique that can aid in the diagnosis of RBC membrane disorders. The clinical diagnosis of HE and HPP relies on identifying abnormal RBC morphology on peripheral blood smear (elliptocytes, poikilocytosis and fragmented RBCs), and identifying characteristic membrane biomechanical properties using osmotic gradient ektacytometry. 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. SPTA1 and SPTB mutations can be within or near the spectrin self-association domains disrupting the stability of spectrin tetramer, or away from the spectrin head-to head self-association site, mostly affecting residues critical for interactions between spectrin helices or between spectrin and ankyrin. HE/HPP disease is caused by mutations in SPTA1 and SPTB genes, causing qualitative defects of α- and β-spectrin respectively, and in EPB41 gene causing quantitative or qualitative defects of protein 4.1R. Defects in the spectrin-protein 4.1R-actin complex weaken the “horizontal” cytoskeletal associations causing decreased mechanical stability and deformability of erythrocytes. Binding of the spectrin tetramer with actin at the junctional complex is mediated by protein 4.1R and is essential for RBC membrane stability ( Figure 1). Spectrin, the primary RBC cytoskeleton protein, is composed of α-β heterodimers assembled in antiparallel fashion into flexible rods which self-associate head-to-head (each head composed by the N-terminal region of α-spectrin and the C-terminal region of β-spectrin) to form tetramers. Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are genetically and phenotypically heterogeneous hemolytic anemias that result from mutations in the genes encoding the red blood cell (RBC) cytoskeleton proteins α-spectrin, β-spectrin, or protein 4.1R. Moreover, genotypic differences justified the phenotypic differences within families with HE/HPP. In addition to identifying three novel mutations, gene sequencing confirmed and, when the RBC morphology was not evaluable, identified the diagnosis. 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. However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions, and does not predict disease course or severity. 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. The resulting defects alter the horizontal cytoskeletal associations and affect RBC membrane stability and deformability causing shortened RBC survival. 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).
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