Molecular analysis of the patients with idiopathic low molecular weight proteinuria

氯离子通道5蛋白（CLC5）在肾近端小管的胞体和溶酶体膜中高度表达，维持溶酶体内的低pH值。我们发现了日本特发性低分子蛋白尿与英国邓特病的临床相似性，并在29个特发性低分子蛋白尿家族中的21个家族中发现了18种不同的CLC5基因（CLCN5）突变。在5个日本家族中发现的W270X、R648X和R704X三个无义突变，在英国邓特病和北美x连锁肾结石家族中也发现了这些突变，提示这些突变是CLCN5的突变热点。7个突变（33%）发生在CLCN5的外显子8,4个突变（19%）发生在外显子11。异源表达S270R、L278F、R280P和L706P 4种错义突变的非洲爪蟾卵母细胞的通道活性分别比野生型降低70%、100%、70%和30%。Western blot分析发现，突变蛋白L706P不仅在卵母细胞膜中表达，而且在细胞质中也有表达，提示CLC5蛋白的末端可能起到了在细胞膜上锚定蛋白的作用。Man-Whitney检验的基因型表型相关性分析显示：1)CLCN5突变患者尿β 2微球蛋白和Ca/Cr高于对照组，2)CLCN5错义突变患者尿Ca/Cr低于其他CLCN5突变患者，3)CLCN5错义突变患者肾钙化症的发生率低于其他CLCN5突变患者。

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.