Analysis of red cell membrane protein genes in hereditary spherocytosis

对54例遗传性球形红细胞增多症(HS)患者的红细胞膜蛋白进行了SDS-PAGE定量分析，发现54例日本遗传性球形细胞增多症(HS)患者中有9例出现带3(B3)蛋白减少(约15%)。提取外周血白细胞基因组DNA，用内含子聚合酶链式反应(PCR)扩增B3基因的19个外显子(外显子2-外显子20)，然后用非RI单链构象多态性(SSCP)方法筛查B3突变。作为结果，我们在9例B3缺乏症患者中发现了异常的SSCP，并通过DNA测序确定了9例B3突变。1例移码突变(B3-普林斯顿：密码子273-275，插入C)，5例错义突变(B3-京都：密码子490，CGC*TGC，B3-福冈II：密码子492，TGG*CGG，B3-布拉格II：密码子760，CGG*CAG，B3-Iizuka：密码子767，GCT*GTT，B3-东京：Codon837，ACG*GCG)和3例剪接位点突变(B3-Hakata：内含子15，NT)。1，G*A，B3-Fukuoka III：Intron 16，NT.1，G*A，B3-熊本：内含子19，NT。620，G*C)。所有病例均为每个突变的杂合子。红细胞膜上未检测到由突变的B3基因产生的B3蛋白，提示这些突变可能导致了mRNA或蛋白质的不稳定，或可能干扰了合成蛋白的正常结合。B3缺乏可能导致潜在细胞骨架蛋白组装减少，脂双层不稳定和微泡形成，推测产生球形红细胞。

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.