| Research Abstract |
We analyzed red cell membrane proteins quantitatively by SDS-PAGE and found the decrease of band 3 (B3) protein (about 15%) in nine out of 54 Japanese patients with hereditary spherocytosis (HS). Genomic DNAs were extracted from peripheral blood leukocytes and nineteen exons (exon2-exon 20) of B3 gene were PCR (polymerase chain reaction)-amplified using intronic primers, followed by screening B3 mutations by non-RI SSCP (single strand conformation polymorphism) method. As the results, we found abnormal SSCPs in nine patients showing B3 deficiency and determined nine B3 mutations by DNA sequencing. One case was frameshift mutation (B3-Princeton : Codon273-275, insertion of C), five cases missense mutations (B3-Kyoto : Codon490, CGC*TGC,B3-Fukuoka II : Codon492, TGG*CGG,B3-Prague II : Codon760, CGG*CAG,B3-Iizuka : Codon767, GCT*GTT,B3-Tokyo : Codon837, ACG*GCG) and three cases the mutations of splicing sites (B3-Hakata : Intron15, nt. 1, G*A,B3-Fukuoka III : Intron16, nt. 1, G*A,B3-Kumamoto : Intron19, nt. 620, G*C). All cases were a heterozygote of each mutation. The B3 protein derived from mutated B3 gene was not identified on red cell membranes, suggesting that these mutations may cause instability of mRNA or protein, or may disturb normal incorporation of synthesized protein to membranes. The B3 deficiency may cause reduced assembly of underlying cytoskeletal proteins, lipid bilayr destabilization and microvesiculation, presumably generating spherocytic red cells.
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