title: Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.
abstract: BACKGROUND
  Dent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5'UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.The structural complexity of the CLCN5 5'UTR region has not yet been fully characterized. To date 6 different 5' alternatively used exons--1a, 1b, 1b1 and I-IV with an alternatively spliced exon II (IIa, IIb)--have been described, but their significance and differential expression in the human kidney have not been investigated. Therefore our aim was to better characterize the CLCN5 5'UTR region in the human kidney and other tissues.
  
  
  METHODS
  To clone more of the 5' end portion of the human CLCN5 cDNA, total human kidney RNA was utilized as template and RNA ligase-mediated rapid amplification of cDNA 5' ends was applied.The expression of the different CLCN5 isoforms was studied in the kidney, leucocytes and in different tissues by quantitative comparative RT/PCR and Real--Time RT/PCR.
  
  
  RESULTS
  Eleven transcripts initiating at 3 different nucleotide positions having 3 distinct promoters of varying strength were identified. Previously identified 5'UTR isoforms were confirmed, but their ends were extended. Six additional 5'UTR ends characterized by the presence of new untranslated exons (c, V and VI) were also identified. Exon c originates exon c.1 by alternative splicing. The kidney uniquely expresses all isoforms, and the isoform containing exon c appears kidney specific. The most abundant isoforms contain exon 1a, exon IIa and exons 1b1 and c. ORF analysis predicts that all isoforms except 3 encode for the canonical 746 amino acid ClC-5 protein.
  
  
  CONCLUSIONS
  Our results confirm the structural complexity of the CLCN5 5'UTR region. Characterization of this crucial region could allow a clear genetic classification of a greater number of Dent disease patients, but also provide the basis for highlighting some as yet unexplored functions of the ClC-5 proton exchanger.
authors.affiliation: Laboratory of Histomorphology and Molecular Biology of the Kidney, Department of Medicine DIMED, University of Padova, via Giustiniani, 2, 35128 Padova, (PD), Italy. enrica.tosetto@unipd.it.
authors.forename: Enrica
authors.initials: E
authors.lastname: Tosetto
authors.forename: Alberto
authors.initials: A
authors.lastname: Casarin
authors.forename: Leonardo
authors.initials: L
authors.lastname: Salviati
authors.forename: Alessandra
authors.initials: A
authors.lastname: Familiari
authors.forename: John C
authors.initials: JC
authors.lastname: Lieske
authors.forename: Franca
authors.initials: F
authors.lastname: Anglani
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D018118:Chloride Channels / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D009928:Organ Specificity; D020033:Protein Isoforms / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D018076:DNA, Complementary; D020033:Protein Isoforms