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.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Anglani F, Murgia A, Bresin S, Bernardi P, Clementi M, Tenconi R (1993) A new disease-causing mutation in the GAP-related domain of the NF1 gene. Hum Mol Genet 2(7):1057–1059
Brudno M, Gelfand MS, Spengler S, Zorn M, Dubchak I, Conboy JG (2001) Computational analysis of candidate intron regulatory elements for tissue-specific alternative pre-mRNA splicing. Nucleic Acids Res 29(11):2338–2348
Càceres JF, Komblihtt AR (2002) Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Genet 18(4):186–193
Ceol M, Forino M, Gambaro G, Sauer U, Schleicher ED, D’Angelo A, Anglani F (2001) Quantitation of TGF-beta 1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with nuclease protection assay and in situ hybridization. J Clin Lab Anal 15(4):215–222
Del Prete D, Forino M, Gambaro G, D’Angelo A, Anglani F (1998) Comparative kinetic RT/PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens. Exp Nephrol 6(6):563-567
Devuyst O, Christie PT, Courtoy PJ, Beauwens R, Takker RV (1999) Intra-renal and subcellular distribution of the human chloride channel, ClC-5, reveals a pathophysiological basis for Dent’s disease. Hum Mol Genet 8(2):247–257
Fisher SE, Black GCM, Lloyd SE, Hatchwell E, Wrong O, Thakker RV, Craig IW (1994) Isolation and partial characterization of a chloride channel gene which is expressed in kidney and is a candidate for Dent’s disease (an X-linked hereditary nephrolithiasis). Hum Mol Genet 3(11):2053-2059
Fisher S, Van Bakel I, Lloyd SE, Pearce SHS, Thakker RV, Craig IW (1995) Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis). Genomics 29:598–606
Gunther W, Luchow A, Cluzeaud F, Vandewalle A, Jentsch TJ (1998) ClC-5, the chloride channel mutated in Dent’s disease, colocalizes with the proton pump in endocytotically active kidney cells. Proc Natl Acad Sci USA 95:8075–8080
Gunther W, Piwon N, Jentsch TJ (2003) The ClC-5 chloride channel knock-out mouse an animal model for Dent’s disease. Eur J Physiol 445:456–462
Hayama A, Uchida S, Sasaki S, Marumo F (2000) Isolation and characterization of the human CLC-5 chloride channel gene promoter. Gene 261:355–364
Holcik M, Sonenberg N, Korneluk RG (2000) Internal ribosome initiation of translation and the control of cell death. Trends Genet 16(10):469–473
Jackson RJ, Kaminski A (1995) Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond. RNA1(10):985–1000
Jentsch TJ, Stein V, Weinreich F, Zdebik AA (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82(2):503–568
Kozak M (2001) New Ways of Initiating translation in eukariotes?. Mol Cell Biol 21(6):1899–1907
Lim LP, Burge CB (2001) A computational analysis of sequence features involved in recognition of short introns. Proc Natl Acad Sci USA 98(20):11193–11198
Lloyd SE, Gunther W, Pearce SHS, Thomson A, Bianchi ML, Bosio M, Craig IW, Fisher SE, Scheinman SJ, Wrong O, Jentsch TJ, Thakker RV (1997) Characterization of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders. Hum Mol Genet 6(8):1233–1239
Luyckx V, Goda FO, Mount DB, Nishio T, Hall A, Hebert SC, Hammod TG, Yu ASL (1998) Intrarenal and subcellular localization of rat CLC5. Am J Physiol 275(5Pt2):F761–F769
Morrish BC, Rumsby MG (2002) The 5’ untranslated region of protein kinase C delta directs translation by an internal ribosome entry segment that is most active in densely growing cells and during apoptosis. Mol Cell Biol 22(17):6089–6099
Nissim-Rafinia M, Kerem B (2002) Splicing regulation as a potential genetic modifier. Trends Genet 18(3):123–127
Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ (2000) ClC-5 Cl- channel disruption impairs endocytosis in a mouse model for Dent’s disease. Nature 408:369–373
Sakamoto H, Sado Y, Naito I, Kwon T, Inoue S, Endo K, Kawasaki M, Uchida S, Nielsen S, Sasaki S, Marumo F (1999) Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase. Am J Physiol 277(6):F957-F965
Sayer JA, Simmons NL (2002) Urinary stone formation: Dent’s disease moves understanding forward. Exp Nephrol 10:176–181
Shalev A, Blair PJ, Hoffmann SC, Hirshberg B, Peculis BA, Harlan DM (2002) A proinsulin gene spice variant with increased translation efficiency is expressed in human pancreatic islet. Endocrinology 143(7):2541–2547
Thakker RV (2000) Molecular pathology of renal chloride channels in Dent’s disease and Bartter’s syndrome. Exp Nephrol 8:351–360
Thanaraj TA, Clark F (2001) Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions. Nucleic Acids Res 29(12):2581–2593
Van der Velden AW, Thomas AAM (1999) The role of 5’ untranslated region of an mRNA in translation regulation during development. Int J Biochem Cell Biol 31:87–106
Wang SS, Devuyst O, Courtoy PJ, Wang X, Wang H, Wang Y, Takker RV, Guggino S, Guggino WB (2000) Mice laking renal chloride channel, CLC-5, are a model for Dent’s disease, a nephrolithiasis disorder associated with defective receptor-mediated endocytosis. Hum Mol Genet 9(20):2937–2945
Acknowledgements
This work was supported by grants from Ricerca Regionale Sanitaria Finalizzata 1999–2001 (no. 798/03/98) and from Ministero dell’Istruzione, dell’Università e della Ricerca 2002–2004 (no. 2002062925–003).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Forino, M., Graziotto, R., Tosetto, E. et al. 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. J Hum Genet 49, 53–60 (2004). https://doi.org/10.1007/s10038-003-0108-1
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1007/s10038-003-0108-1
Keywords
This article is cited by
-
Genetics and phenotypic heterogeneity of Dent disease: the dark side of the moon
Human Genetics (2021)
-
Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1
Clinical and Experimental Nephrology (2020)
-
Complexity of the 5′UTR region of the CLCN5gene: eleven 5′UTR ends are differentially expressed in the human kidney
BMC Medical Genomics (2014)
-
Association between OPG, RANK and RANKL gene polymorphisms and susceptibility to acute coronary syndrome in Korean population
Journal of Genetics (2012)
-
A missense mutation in the chloride/proton ClC-5 antiporter gene results in increased expression of an alternative mRNA form that lacks exons 10 and 11. Identification of seven new CLCN5 mutations in patients with Dent’s disease
Journal of Human Genetics (2007)
