In Vitro Characterization of R14W Mutation in SEC23B, the CDAII Causative Gene

Abstract 2098

Congenital dyserythropoietic anemias (CDAs) designate a group of genetic disorders in which ineffective erythropoiesis is the predominant mechanism of anemia marked by distinct morphologic abnormalities of the majority of erythroblasts in the bone marrow. CDA type II (CDAII) is the most common type of CDA. It is characterized by a recessive model of inheritance, mild to moderate anemia, jaundice, and splenomegaly (Fukuda MN, Glycobiology 1990; Fukuda MN, Clin Haematol 1993), by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance (Schwarz K and Iolascon A. et al., Nat Genet. 2009). The specific hallmark of diagnosis is the presence of the more abundant protein of membrane red cell, band 3, in a hypoglycosilated state; this is thinner and migrated slightly faster than in controls on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Anselstetter V et al., Br J Haematol 1977).

The causative gene of CDAII, SEC23B, is a member of the SEC23 subfamily of the SEC23/SEC24 family, which is involved in vesicle trafficking. The encoded protein has similarity to yeast Sec23p (66.4%) component of COPII, the coat protein complex responsible for vesicle budding from the ER. The function of this gene product has been implicated in cargo selection and concentration.

The SEC23B gene spans approximately 54 kb on human chromosome 20p11.23 and codifies for a protein of 767 aminoacids divided in five functional domains: zink finger, trunk, β-sheet, helical and gelsolin domain. Although most of the SEC23B mutations are sporadic events, 4 mutations (R14W, E109K, R497C, I318T) accounted for more than 50% of mutated alleles. The aim of this study is the in vitro characterization of the R14W mutation, the most frequent variant in Italy, particularly in South of Italy (Russo R et al., Am J Hematol. in press). We used the human erythroleukemia cell line, HEL, as in vitro model because it is more similar to mature red cell. By using in silico tool ESyPred3D Web Server 1.0, we predicted that this aminoacidic substitution alters the zink finger domain 3D structure, when compared to wild type protein. This tool implements homology modeling approach followed by a final analysis with MODELLER release 4 in order to build a 3D model of the submitted protein (Lambert et al, 2002). However, when we transfected the SEC23B-R14W we observed a strong reduction of gene expression in the mutant when compared to SEC23B-wt construct by qRT-PCR. These results have been also confirmed at the protein level. In fact the protein expression of SEC23B-R14W showed a reduction comparable to gene expression respect to SEC23B-wt construct. Immunofluorescence analyses by confocal microscopy, were used for the investigation of the cellular localization of SEC23B-R1W protein and, interestingly, the localization of mutant protein was not changed when compared to that wt. Our data allow us to hypothesize that the mutation R14W gives rise to anomalous protein product quantity, but the protein function would be like not altered.

Our findings demonstrated that the most frequent mutation found in Italy, SEC23B-R14W, results in a reduced half-life of the mutated mRNA, without altering the cellular localization in HEL cell line. SEC23B belongs to a multiproteic compelx that assembles with the others complex proteins in accordance with a specific structure. Each structure establishes a cargo selectivity. Further studies are necessary in order to understand what is the role of SEC23B in selectivity of the cargo in erythroid cells and how its disruption could determines the appearance of the principal pathological phenotype in CDAII patients, for example the hypoglycosilation of band 3.