Atsuhito Tanuma a , Hiroyoshi Sato b, c , Tetsuro Takeda a , Michihiro Hosojima a , Hiroaki Obayashi a , Hitomi Hama a , Noriaki Iino b , Kiyoko Hosaka a , Ryohei Kaseda a , Naofumi Imai a , Mitsuhiro Ueno a , Maya Yamazaki d , Kenji Sakimura d , Fumitake Gejyo a , Akihiko Saito b
20 November 2007
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.