Spectrin Johannesburg: alpha and beta spectrin defects in a South African kindred with hereditary elliptocytosis

Abstract

Spectrin is the major component of the erythrocyte protein skeleton. Spectrin tetramers, the predominant form on the membrane, consist of self-associated oc/|3 heterodimers. P spectrin binds to the integral band 3 protein via ankyrin. Hereditary elliptocytosis (HE) is caused by skeletal protein defects that destabilise the membrane resulting in altered erythrocyte morphology. Two probands from a white South African HE kindred characterised by reduced spectrin-ankyrin binding, lowered spectrin dimer self-association and diminished spectrin in the membrane were investigated. Control and proband monomers were isolated and reassociated into hybrid spectrins (hSp). hSp and spectrin structural analyses identified an alteration of the a l l domain in the probands’ spectrin between amino acids 916-1046; the mother’s spectrin structure was normal. Functionally, hSp dimers reassociated with a proband a chain showed lowered self-association and reduced ankyrin binding. RT-PCR analysis of the probands’ a l l domain coding sequences from reticulocyte ENA showed that they produced approximately equal amounts of two KNA messages: one with an exon 20 deletion and the other with a 12bp insertion between exons 19 and 20. The mother produced both abnormal and normal KNA messages. Genomic DNA analysis indicated that the probands are homozygous and the mother heterozygous for a T=>G mutation 13bp upstream from the intron 19/exon 20 boundary. The mutation unmasks a cryptic 3’ acceptor splice site resulting in two pre-mRNA splicing events. Firstly, 12bp of intron 19 are spliced in-frame with exon 20 introducing a translation stop codon. This message terminates translation at amino acid 935 and the peptide was not observed in membrane studies. Secondly, the presence of the stop codon causes skipping of exon 20. Translation of this message deletes 30 amino acids in homologous repeat o9 and alters the cell domain structure. No other mutations were found and the spectrin a ll domain deletion therefore has a long range influence over dimer self-association and ankyrin binding. Ankyrin binding assays of spectrin from an individual with hereditary spherocytosis marked by a mild spectrin deficiency showed normal function. This indicated that the spectrin deficiency was not caused by a spectrin binding defect in this individual.