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Molecular analysis of the patients with idiopathic low molecular weight proteinuria

特发性低分子蛋白尿患者的分子分析

基本信息

批准号：
08670858
负责人：
IGARASHI Takashi
金额：
$ 1.41万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

Chloride channel 5 protein, CLC5, is highly expressed in the cytosomes and lysosomal membrane of renal proximal tubules which works to keep the intralysosomal pH low. We have indicated the clinical similarity between Japanese idiopathic low molecular weight proteinuria and British Dent's disease, and identified 18 different mutations of CLC5 gene (CLCN5) in 21 families among 29 families with idiopathic low molecular weight proteinuria. Three nonsense mutations, W270X, R648X and R704X, found in 5 Japanese families of the disease was also found in British families with Dent's disease and North American X-linked nephrolithiasis suggesting that those mutations are mutational hot spots in CLCN5. Seven mutations (33%) occurred in exon 8 of CLCN5, and 4 (19%) in exon 11. Heterologous expression of 4 missense mutations, S270R, L278F, R280P and L706P in Xenopus oocytes demonstrated 70%, 100%, 70% and 30% reduction respectively in channel activity when compared with the wild-type. Western blot analysis disclosed that the mutated protein L706P is expressed not only in oocyte membrane but in cytoplasm suggesting that the terminal portion of CLC5 protein might act as an anchoring the protein at the cell membrane. The genotype phenotype correlation analyzed by Man-Whitney test revealed that 1) urine beta2-microglobulin and Ca/Cr are elevated in the patients with CLCN5 mutations than in the controls, 2) urine Ca/Cr is lower in the patients with CLCN5 missense mutations than in the patients with other CLCN5 mutations, and 3) the incidence of nephrocalcinosis is lower in patients with CLCN5 missense mutations than in the patients with other CLCN5 mutations.

氯通道 5 蛋白 (CLC5) 在肾近曲小管的细胞体和溶酶体膜中高度表达，其作用是保持溶酶体内的 pH 值较低。我们指出了日本特发性低分子蛋白尿与英国Dent病的临床相似性，并在29个特发性低分子蛋白尿家系中的21个家系中鉴定出18种不同的CLC5基因（CLCN5）突变。在 5 个日本该疾病家族中发现的三种无义突变 W270X、R648X 和 R704X 在患有 Dent 病和北美 X 连锁肾结石的英国家族中也被发现，表明这些突变是 CLCN5 中的突变热点。 CLCN5 的外显子 8 发生 7 个突变（33%），外显子 11 发生 4 个突变（19%）。爪蟾卵母细胞中 4 个错义突变 S270R、L278F、R280P 和 L706P 的异源表达表明，与野生型相比，通道活性分别降低了 70%、100%、70% 和 30%。 Western blot分析显示突变蛋白L706P不仅在卵母细胞膜中表达，而且在细胞质中表达，表明CLC5蛋白的末端部分可能充当该蛋白在细胞膜上的锚定物。 Man-Whitney 检验分析的基因型表型相关性显示：1）CLCN5 突变患者的尿液 β2 微球蛋白和 Ca/Cr 高于对照组；2）CLCN5 错义突变患者的尿液 Ca/Cr 低于其他 CLCN5 突变患者；3）CLCN5 错义突变患者的尿液 Ca/Cr 低于其他 CLCN5 突变患者；3）CLCN5 突变患者的肾钙质沉着症发生率较低。 CLCN5 错义突变高于具有其他 CLCN5 突变的患者。