Oryzocytosis: A Novel Morphological Variant of Hereditary Elliptocytosis Associated with a Novel Mutation in β-Spectrin (SPTB c154 C>T p.Arg52Trp)

Background We here report a family with a variant hereditary elliptocytosis (HE) characterized by previously undescribed mutations in β-spectrin, band 4.2, and the K+ channel ABCB6. The proband was a 2yo boy who presented for evaluation of elliptocytosis. On peripheral blood smear his red cells were elongated, but unusually small and dense in comparison to a classic elliptocytosis. Osmotic gradient ektacytometry (Osmoscan) showed a mild spherocytosis-like pattern and eosin-5-maleimide (EMA) fluorescence test showed a single population of cells with mildly decreased band 3. We termed the morphological pattern “Oryzocytosis” for the rice grain appearance of the cells Methods The proband, 3 siblings (all under 6 yrs of age), and 2 parents were evaluated. Band 3 content was estimated by EMA staining, and membrane deformability was determined by Osmoscan. The following genes were evaluated by next generation sequencing: ANK1, ACTIN, EPB41, EPB42, SLC4A1, SPTA1, SPTB; Ion channel genes- ABCB6, KCNN4, RHAG PIEZO1 (Fulgent Diagnostics, CA). Subsequently, all family members were tested by Sanger sequencing for the identified mutations. RBC ghosts were isolated and profiled on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on non-denaturing gels for spectrin tetramer:dimer ratios. Results Three gene mutations were found in the proband: SPTB c154 C>T p.Arg52Trp affecting the actin binding domain of β-spectrin, EPB42 c70C>T p.Pro24Ser affecting the band 3 binding domain of band 4.2, and ABCB6 c1694T>C p.Phe565Ser affecting a K+ transporter and cellular hydration status. The EPB42 and ABCB6 mutations were inherited from the father. The mother had the SPTB mutation alone, and the most severe phenotype of any family member, with a history of severe neonatal jaundice, multiple transfusions during adolescence, and splenectomy at age 21. She had elliptocytes on peripheral smear, only mild band 3 deficiency, and a normal Osmoscan. Two children inherited the SPTB mutation and were the only children with clinical manifestations by history. The proband inherited all 3 mutations, and had mild neonatal jaundice; a sibling with SPTB and ABCB6 mutations had a history of multiple episodes of mild jaundice. The RBCs of these 2 children were the least deformable on Osmoscan and demonstrated the greatest band 3 deficiencies. The father had both the EPB42 and ABCB6 mutations and passed them on to a third child. Both the father and that child had morphologic spherocytosis but no clinical sequelae. On testing, both had mild band 3 deficiency but no decrease in cell deformability. They had right shifted curves on Osmoscan indicating an altered tolerable range of osmolality, perhaps due to the ABCB6 mutation. The fourth child inherited only the ABCB6 mutation, but the peripheral smear unfortunately was uninterpretable. All 6 individuals had qualitatively high tetramer:dimer ratios on non-denaturing gels, indicating normal tetramerization. Discussion The most clinically significant mutation in this family appears to be in SPTB near the N-terminal actin binding domain. The discordance between the red cell morphology and the combination of decreased band 3 content and spherocytosis-like pattern on Osmoscan (high Omin (S/V), decreased deformability index (DI), and reduced Ohyper) is puzzling (see Figure 1). The unique trapezoidal pattern of classic HE with a double hump in DI max was not seen, nor was the classic hybrid population of RBCs with low and high band 3 content on EMA. Yet all 3 individuals with an SPTB mutation demonstrated some form of elliptocytic morphology. The determinants of elliptocytic shape are not fully defined, but previously described mutations have involved predominantly the spectrin dimer-dimer interaction site or band 4.1. Determining whether our SPTB mutation alters binding of β-spectrin with the junctional complex containing actin and band 4.1 may be key to further defining the mechanism causing Oryzocytosis. Download : Download high-res image (291KB) Download : Download full-size image Disclosures Ravindranath: Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding.