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

Abstract

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.

Mutational Analysis of CLC-5, Cofilin and CLC-4 in Patients with Dent’s Disease

“…Consequently, the t → c transition resulted in skipping of exon 3, and a smaller mRNA transcript. This aberrant processing of pre-mRNA is consistent with that reported in a patient carrying a mutation affecting the g nucleotide at position +1 bp of the donor splice site consensus sequence [27], and in another patient carrying a mutation affecting the a nucleotide at position –2 bp of the acceptor splice site consensus sequence of intron 2 [28]. The mutant pre-mRNA transcript would either lead to mRNA degradation through nonsense-mediated decay [24] or, if translated, to a frameshift with a missense peptide from codon 36 and a premature termination at codon 38.…”

“…2) represent novel CLCN5 mutations, whereas the missense (Ser244Leu and Arg516Trp) and nonsense mutations (Arg347Stop and Arg718Stop) occur in other unrelated patients with Dent’s disease [3,4,5,6, 23, 27,32,33,34,35,36,37]. The total number of CLCN5 mutations now published, including the results of our present study, is 148, and these are scattered throughout the coding region, with no evidence for major mutational hot spots [27, 28, 34,37,38,39,40,41]. Furthermore, there appears to be no correlation between the mutations and phenotypes (fig.…”

Heterogeneity in the processing of CLCN5 mutants related to Dent disease

“…We performed functional, biochemical, and cell-biology analyses in X. laevis oocytes and in HEK293 cells of nine CLCN5 pathogenic missense mutations that had been described before Hoopes et al, 2004;Igarashi et al, 1998;Lloyd et al, 1996;Ramos-Trujillo et al, 2007;Tosetto et al, 2006]. The G260V, Y272C, L278F, G513R, K546E, and W547G mutations are located in helix H, in the loop between helices H and I, in helix I, and in helices O and Q, respectively (Fig.…”

Dent’s disease: clinical features and molecular basis

“…The mutations are scattered through the gene and include missense, nonsense and splice site mutations, insertions, and deletions [8,23,25,[67][68][69][70][71][72][73][74][75]. Approximately half of these mutations truncate the protein and, consequently, predict a complete loss of function.…”

“…The major consequence of Dent's disease is a progressive decrease in glomerular filtration rate (GFR) leading to end-stage renal disease (ESRD). This occurs [2,20,23,30,31,69,73,74,111] between the third and the fifth decades of life in more than two-thirds of affected males [2].…”