Functional characterization of a novel missense CLCN5 mutation causing alterations in proximal tubular endocytic machinery in Dent's disease
Functional characterization of a novel missense CLCN5 mutation causing alterations in proximal tubular endocytic machinery in Dent's disease
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DOI：
10.1159/000111253
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Saito, Akihiko
中科院分区：
文献类型：
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作者：
Tanuma, Atsuhito;Sato, Hiroyoshi;Saito, Akihiko
背景/目标：核内体氯离子/质子交换基因CLCN 5的突变导致登特病，一种X连锁隐性近端肾小管疾病。发现clcn5基因敲除小鼠的肾脏内吞系统受到影响。然而，丹特氏病患者受损的内吞机制尚未得到彻底调查。方法：对一例日本Dent病患者及其家系的CLCN 5基因进行测序。错义CLCN 5突变的功能丧失表型通过在爪蟾卵母细胞和CHO细胞中的基因表达进行了研究。对肾活检标本进行近端小管内吞机制蛋白、megalin、cubilin和disabled-2（Dab2）的免疫组织化学分析。结果如下：基因组分析显示，在CLCN 5的第333密码子的第一个核苷酸处的一个新的G-到-A转换，导致甘氨酸被精氨酸取代。突变基因在爪蟾卵母细胞中的低效率表达导致氯电流被废除。在DNA转染的CHO细胞中，突变蛋白的N-糖基化受损是明显的。与对照组相比，患者近端小管中megalin、cubilin和Dab2的表达显著降低，并且在某些部分观察到不规则染色。结论：一种新的G333R CLCN 5突变导致Dent病患者megalin、cubilin和Dab2表达缺陷。版权所有（c）2007 S. Karger AG，巴塞尔。
Background/Aims: Mutations of the endosomal chloride/ proton exchanger gene, CLCN5, cause Dent's disease, an X-linked recessive proximal tubular disorder. The renal endocytic system was found to be affected in clcn5 knockout mice. However, the impaired endocytic machinery of Dent's disease patients has not been thoroughly investigated. Methods: The CLCN5 gene was sequenced in a Japanese patient with Dent's disease and his family. The loss-of-function phenotype of the missense CLCN5 mutation was investigated by gene expression in Xenopus oocytes and CHO cells. Immunohistochemical analysis was performed on kidney biopsy specimens for endocytic machinery proteins, megalin, cubilin, and disabled-2 (Dab2) in proximal tubules. Results: Genomic analysis revealed a novel G-to-A transition at the first nucleotide of the 333rd codon of CLCN5, causing a substitution of glycine with arginine. Inefficient expression of the mutant gene in Xenopus oocytes resulted in abolished chloride currents. Impaired N-glycosylation of the mutant protein was evident in the DNA-transfected CHO cells. Proximal tubular expression of megalin, cubilin, and Dab2 was markedly reduced and irregular staining in some portions was observed in the patient compared with controls. Conclusions: A novel G333R CLCN5 mutation caused defective expression of megalin, cubilin, and Dab2 in a patient with Dent's disease. Copyright (c) 2007 S. Karger AG, Basel.