Functional characterization of a novel missense CLCN5 mutation causing alterations in proximal tubular endocytic machinery in Dent's disease.
OBJECTIVE
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.
Tanuma
Atsuhito
A
Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Sato
Hiroyoshi
H
Takeda
Tetsuro
T
Hosojima
Michihiro
M
Obayashi
Hiroaki
H
Hama
Hitomi
H
Iino
Noriaki
N
Hosaka
Kiyoko
K
Kaseda
Ryohei
R
Imai
Naofumi
N
Ueno
Mitsuhiro
M
Yamazaki
Maya
M
Sakimura
Kenji
K
Gejyo
Fumitake
F
Saito
Akihiko
A
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000328:Adult; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D016466:CHO Cells; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005192:Family Health; D006801:Humans; D007674:Kidney Diseases / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology*; D007687:Kidney Tubules, Proximal / Q000378:metabolism / Q000503:physiopathology*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism; D008297:Male; D020125:Mutation, Missense; D009865:Oocytes; D010375:Pedigree; D010641:Phenotype; D011956:Receptors, Cell Surface / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D014981:Xenopus
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C097503:CLC-5 chloride channel; D018118:Chloride Channels; C102885:DAB2 protein, human; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D011956:Receptors, Cell Surface; D025521:Tumor Suppressor Proteins; C029486:intrinsic factor-cobalamin receptor