96a2db98-c2e8-477f-885b-4c0167b4d1a1
title
Identification of a novel splice site mutation of  CLCN5  gene and characterization of a new alternative 5' UTR end of ClC-5 mRNA in human renal tissue and leukocytes.
6da56781-9482-4b85-9489-b4ad49010811
abstract
Mutations in the  CLCN5  gene have been detected in Dent's disease and its phenotypic variants (X-linked recessive nephrolithiasis, X-linked recessive hypophosphatemic rickets, and idiopathic low-molecular-weight proteinuria of Japanese children). Dent's disease is a tubular disorder characterized by low-molecular-weight proteinuria, and nephrolithiasis associated with nephrocalcinosis and hypercalciuria. ClC-5 is the first chloride channel for which a definitive role in the trafficking and acidification-dependent recycling of apical membrane proteins has been established. In the course of  CLCN5  SSCP analysis in patients with hypercalciuric nephrolithiasis, we detected a novel  mutation at intron 2 of the CLCN5 gene, a T-to-G substitution, located 17 bp upstream of the AG acceptor site CLCN5  gene, a T-to-G substitution, located 17 bp upstream of the AG acceptor site. To determine the effect of  IVS2-17 T>G  mutation on the correct splicing of intron 2, we studied ClC-5 transcripts in a patient's peripheral blood leukocytes by means of quantitative comparative RT/PCR, and found a new ClC-5 5' UTR isoform characterized by the untranslated exon 1b and by retention of intron 1b. This new isoform--isoform B1--was not correlated with mutation since it was detected also in control leukocytes and in renal tissues of kidney donors, thus confirming its physiological role. By RACE analysis we determined the putative transcriptional start site which is located at intron 1a, 251 nt upstream of the first nucleotide of the untranslated exon 1b. ORF analysis revealed that intron 1b retention in isoform B1 stabilizes the initiation of translation to the AGT at position 297 of the ClC-5 cDNA coding region.