Analysis of red cell membrane protein genes in hereditary spherocytosis

遗传性球形红细胞增多症红细胞膜蛋白基因分析

• 批准号: 07672508
• 负责人: IDEGUCHI Hiroshi
• 金额: $ 1.28万
• 依托单位: Fukuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672508/
• 关键词: Hereditary spherocytosis; Band 3 protein; Gene analysis

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.

我们用SDS-PAGE定量分析了54例日本遗传性球形红细胞增多症（HS）患者中9例的红细胞膜蛋白，发现带3（B3）蛋白减少（约15%）。从外周血白细胞中提取基因组DNA，用内含子引物对B3基因的19个外显子（exon 2 ~ exon 20）进行PCR扩增，然后用非RI单链构象多态性（non-RI SSCP）方法筛选B3突变。结果发现9例B3缺乏症患者存在SSCP异常，并通过DNA测序确定了9个B3突变。移码突变1例（B3-Princeton：Codon 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：密码子837，ACG*GCG）和三例剪接位点突变（B3-博多：内含子15，nt. 1，G*A，B3-Fukuoka III：内含子16，nt. 1，G*A，B3-熊本：内含子19，nt. 620，G*C）。所有病例均为各突变的杂合子。在红细胞膜上未发现突变B3基因衍生的B3蛋白，表明这些突变可能导致mRNA或蛋白质的不稳定性，或可能干扰合成蛋白质向膜的正常掺入。B3缺乏可能导致潜在的细胞骨架蛋白组装减少，脂质双层不稳定和微泡形成，可能产生球形红细胞。

项目成果

井手口 裕: "遺伝性球状赤血球症" 日本臨床. 54. 180-185 (1996)

Yutaka Ideguchi：“遗传性球形红细胞增多症”日本临床杂志 54. 180-185 (1996)。

Hiroshi Ideguchi: Hereditary spherocytosis.Clinical Studies in Medical Biochemistry, (edited by Glew, R.H.& Ninomiya Y.), 2nd edition, Oxford University Press (in press), (1997)

Hiroshi Ideguchi：遗传性球形红细胞增多症。医学生物化学临床研究，（由 Glew，R.H. 编辑）