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

Forino, M.; Graziotto, Romina; Tosetto, Enrica; Gambaro, Giovanni; D’Angelo, Angela; Anglani, Franca

doi:10.1007/s10038-003-0108-1

Cited by 19 publications

(23 citation statements)

References 27 publications

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“…The same expectedsize products were observed in both the blood and the kidney RNA samples (results not shown). Forino et al (2004) have also shown that the CLCN5 splicing patterns in leukocytes and kidney biopsies of normal individuals are the same. We therefore considered that blood cells were a suitable source of RNA to study the effect of CLCN5 mutations on pre-mRNA splicing.…”

Section: Resultsmentioning

confidence: 80%

The Alu insertion in the CLCN5 gene of a patient with Dent’s disease leads to exon 11 skipping

et al. 2005

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Alu sequences are short, interspersed elements that have generated more than one million copies in the human genome. They propagate by transcription followed by reverse transcription and integration, causing mutations, recombination, and changes in pre-mRNA splicing. We have recently identified a 345-bp long Alu Ya5 element inserted in codon 650 within exon 11 of the chloride channel ClC-5 gene (CLCN5) of a patient with Dent's disease. A microsatellite pedigree analysis indicated that the insertion occurred in the germline of the maternal grandfather. Dent's disease is an X-linked renal tubular disorder characterized by low-molecularweight proteinuria, hypercalciuria, nephrolithiasis, and nephrocalcinosis. Here, we found, by RT-PCR amplification of RNA extracted from the patient's blood and subsequent DNA sequencing, that the Alu insertion led to an aberrant splicing of the CLCN5 pre-mRNA that skipped exon 11. Using the ESE finder and RESCUE-ESE Web interfaces, we identified two high-score exonic splicing enhancer (ESE) sequences in the site of insertion. The functional significance of these ESE motifs is suggested by our observation that these sequences are highly conserved among mammal CLCN5 genes. Therefore, we suggest that the Alu insertion causes exon skipping by interfering with splicing regulatory elements.The altered splicing would predict a truncated ClC-5 protein that lacks critical domains for sorting and chloride channel function.

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Section: Resultsmentioning

confidence: 80%

The Alu insertion in the CLCN5 gene of a patient with Dent’s disease leads to exon 11 skipping

et al. 2005

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“…The ideal source of RNA to study the effect of mutations on CLCN5 pre-mRNA splicing would be kidney biopsies from patients. However, as this source is not always available, one can use RNA obtained from lymphocytes, which has been shown to be valid for this type of study (Forino et al 2004;ClaverieMartin et al 2005).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, it has been suggested that, in order to determine the full consequences of a mutation in disease, each mutation should be functionally assessed for its potential effect on splicing (Pagani and Baralle 2004). Until now, only a few CLCN5 mutations affecting pre-mRNA splicing have been described, and only in some cases has the effect been confirmed by mRNA analysis (Morimoto et al 1998;Yamamoto et al 2000;Claverie-Martin et al 2003Forino et al 2004).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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Mutations in the voltage-gated chloride/ proton antiporter ClC-5 gene, CLCN5, are associated with Dent's disease, an X-linked renal tubulopathy. Our interest is to identify and characterize diseasecausing CLCN5 mutations, especially those that alter the splicing of the pre-mRNA. We analyzed the CLCN5 gene from nine unrelated Spanish Dent's disease patients and their relatives by DNA sequencing. Pre-mRNA splicing analysis was performed by RT-PCR. Seven new mutations were identified, consisting of three missense mutations (C219R, F273L, and W547G), one splice-site mutation (IVS-2A > G), one deletion (976delG), and two non-sense mutations (Y140X and W314X). We found that missense mutation W547G also led to increased expression of a new alternative isoform lacking exons 10 and 11 that was expressed in several human tissues. In addition, we describe another novel CLCN5 splicing variant lacking exon 11 alone, which was expressed only in human skeletal muscle. We conclude that missense mutation W547G can also alter the expression levels of a CLCN5 mRNA splicing variant. This type of mutation has not been previously described in the CLCN5 gene. Our results support the importance of a routine analysis at the pre-mRNA level of mutations that are commonly assumed to cause single amino acids alterations.

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“…ClC-5 is expressed in the kidney, particularly in proximal tubular cells and intercalated collecting duct cells. The human CLCN5 gene, spanning about 170 kb of genomic DNA on chromosome Xp11.23/p11.22, consists of 17 exons including 11 coding exons (2-12) and 6 different 5′ alternatively used exons (5′UTR), some remaining untranslated [1-4]. Transcripts including the untranslated exon 1a (NM_000084.4: mRNA variant 3) [2] or 1b (NM_001282163.1: mRNA variant 4) [3] are spliced to exon 2 and contain the start sequence ATG.…”

Section: Introductionmentioning

confidence: 99%

“…Transcripts including the untranslated exon 1a (NM_000084.4: mRNA variant 3) [2] or 1b (NM_001282163.1: mRNA variant 4) [3] are spliced to exon 2 and contain the start sequence ATG. A third mRNA comprises a larger exon 1b and retains intron 1 (exon 1b1) (alternative mRNA variant 4) [4]. Subsequently, two additional long transcripts due to alternative splicing of exon II and including exons I to IV have also been identified (NM_001127899.3: mRNA variant 1 and NM_001127898.3: mRNA variant 2) [5].…”

Section: Introductionmentioning

confidence: 99%

Complexity of the 5′UTR region of the CLCN5gene: eleven 5′UTR ends are differentially expressed in the human kidney

et al. 2014

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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.

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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

Cited by 19 publications

References 27 publications

The Alu insertion in the CLCN5 gene of a patient with Dent’s disease leads to exon 11 skipping

The Alu insertion in the CLCN5 gene of a patient with Dent’s disease leads to exon 11 skipping

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

Complexity of the 5′UTR region of the CLCN5gene: eleven 5′UTR ends are differentially expressed in the human kidney

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