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.
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. 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.
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Division of Nephrology, Department of Medical and Surgical Sciences, University of Padua, 35128, Padua, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Forino
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Division of Nephrology, Department of Medical and Surgical Sciences, University of Padua, 35128, Padua, Italy.
authors.forename: Romina
authors.initials: R
authors.lastname: Graziotto
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Division of Nephrology, Department of Medical and Surgical Sciences, University of Padua, 35128, Padua, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Tosetto
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Division of Nephrology, Department of Medical and Surgical Sciences, University of Padua, 35128, Padua, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gambaro
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Division of Nephrology, Department of Medical and Surgical Sciences, University of Padua, 35128, Padua, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: D'Angelo
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Division of Nephrology, Department of Medical and Surgical Sciences, University of Padua, 35128, Padua, Italy. franca.anglani@unipd.it.
authors.forename: Franca
authors.initials: F
authors.lastname: Anglani
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D018118:Chloride Channels / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D040461:Gene Components; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007669:Kidney Calculi / Q000235:genetics; D007962:Leukocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D020121:5' Untranslated Regions; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D017931:DNA Primers; D018076:DNA, Complementary; D022821:RNA Splice Sites; D012333:RNA, Messenger