Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

Enrica Tosetto,Franca Anglani,Leonardo Salviati,John C Lieske,Alberto Casarin,Alessandra Familiari

doi:10.1186/1755-8794-7-41

Abstract

BackgroundDent 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.MethodsTo 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.ResultsEleven 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.ConclusionsOur 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.

Highlights

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
Structure of CLCN5 5′UTR region RACE analysis of CLCN5 5′ cDNA ends in the human kidney detected eleven transcripts initiating at three different nucleotides: - 2407, - 1426 in respect to the ATG initiation codon in exon 2, and at - 660 in respect to the ATG initiation codon in exon III
A transcript initiating at nucleotide - 2407 corresponds to mRNA variant 3, but is extended a further 41 bp in respect to the +1 origin reported in the NCBI reference sequence NM_000084.4

Summary

Introduction

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. Two additional long transcripts due to alternative splicing of exon II and including exons I to IV have been identified (NM_001127899.3: mRNA variant 1 and NM_001127898.3: mRNA variant 2) [5] Both of these transcripts carry the ATG start sequence in exon III, thereby encoding a NH2-terminal extended ClC-5 isoform consisting of 816 amino acids instead of the canonical transcript of 746 amino acids. While analysing 30 CLCN5 negative patients our group identified a nucleotide substitution in the 5′ untranslated exon 1b1 of one individual which appeared disease-causing since it was not detected in 471 X normal chromosomes [10]

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