Amino-acid substitution in alpha-spectrin commonly coinherited with nondominant hereditary spherocytosis.

Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, alpha-spectrin Bughill or alpha(BH), that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the alphaII domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the alpha-spectrin gene (GCT-->GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The alpha(BH) variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the alpha(BH) variant appeared to be homozygous for the alpha(BH) variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the alpha(BH) allele and a second, presumably abnormal, alpha-spectrin gene. These results suggest that, in these 6 patients, the second alpha-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of alpha-spectrin. The pattern of transmission of the alpha(BH) allele in certain families suggests that the alpha(BH) amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized alpha-spectrin gene defect that itself is a cause of ndHS.

A nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis.

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

Analysis of a PstI polymorphism of the human erythrocyte band 3 gene (EPB3).

The genomic structure of a 1.5 kb region near the 5' end of the human erythrocyte band 3 gene (EPB3) was determined and the location of a two-allele PstI polymorphism in this region identified. Using this information, a polymerase chain reaction (PCR)-based assay was designed for rapid, simple identification of the PstI polymorphism.