title: Functional characterization of a novel missense CLCN5 mutation causing alterations in proximal tubular endocytic machinery in Dent's disease.
abstract: 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.
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Atsuhito
authors.initials: A
authors.lastname: Tanuma
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Sato
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Takeda
authors.forename: Michihiro
authors.initials: M
authors.lastname: Hosojima
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Obayashi
authors.forename: Hitomi
authors.initials: H
authors.lastname: Hama
authors.forename: Noriaki
authors.initials: N
authors.lastname: Iino
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Hosaka
authors.forename: Ryohei
authors.initials: R
authors.lastname: Kaseda
authors.forename: Naofumi
authors.initials: N
authors.lastname: Imai
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Ueno
authors.forename: Maya
authors.initials: M
authors.lastname: Yamazaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Sakimura
authors.forename: Fumitake
authors.initials: F
authors.lastname: Gejyo
authors.forename: Akihiko
authors.initials: A
authors.lastname: Saito
mesh_terms: 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
chemical_list: 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