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      Four Additional CLCN5 Exons Encode a Widely Expressed Novel Long CLC-5 Isoform but Fail to Explain Dent’s Phenotype in Patients without Mutations in the Short Variant

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          Background: Dent’s disease is caused by mutations in the CLCN5 gene coding for the chloride channel CLC-5. However, sequencing of CLCN5 exonic regions in some patients presenting with low-molecular-weight proteinuria and hypercalciuria – the hallmarks of Dent’s disease – failed to identify causative mutations. Aim: Given the observation that some species harbour a CLCN5 mRNA encoding an extended CLC-5 aminoterminus compared with the so far known human form, we worked on the presumption that an orthologous (longer) CLCN5 transcript is also present in humans and that our patients may have mutations herein. Methods: Extensive databank mining, reverse transcription polymerase chain reaction (RT-PCR) and automated sequencing were used in the search for novel CLCN5 transcripts. The human CLCN5 gene was investigated in 7 patients out of five families by direct automated sequencing of PCR-amplified DNA products. Results: Two new human CLCN5 transcripts expressed in kidney and various other tissues could be identified. These arise from a novel site of transcription initiation, alternative splicing and the use of four additional CLCN5 exons. If being translated, both these mRNAs would lead to an enlarged CLC-5 protein consisting of 816 amino acids by adding 70 aminoterminal residues to the so far known 746-amino-acid-long isoform. Sequence analysis of the henceforward 17 CLCN5 exons revealed no mutation in the patients with a phenotype resembling Dent’s disease. Conclusions: Despite the identification of further targets to explain Dent’s disease, the molecular defect in our patients remains to be elucidated. Hence, their phenotype may be explained by mutations that affect so far unknown regulating elements of the CLCN5 gene or another gene(s), probably encoding CLC-5 accessory protein(s).

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          Functional characterization of renal chloride channel, CLCN5, mutations associated with Dent'sJapan disease.

          The annual urinary screening of Japanese children above three years of age has identified a progressive renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrocalcinosis, and this represents a variant of Dent's disease. Hitherto, 12 mutations of the X-linked renal specific chloride channel, CLCN5, have been reported in the Dent'sJapan variant. To further identify such CLCN5 mutations and to define the structure-function relationships of this channel, we have investigated five unrelated, non-consanguinous Japanese families with this disorder. Leukocyte DNA from probands was used with CLCN5 primers for PCR amplification of the coding region, and the DNA sequences of the products determined. Functional studies were performed by expressing the mutants in Xenopus oocytes. Five CLCN5 mutations consisting of two nonsense (R648X and R704X), two missense (S270R and L278F) and one acceptor splice site mutation (ag-->cg) in intron 4 were identified. The missense and splice site mutations represent novel abnormalities. Heterologous expression in Xenopus oocytes of wild-type and the missense mutants demonstrated that the mutations, which were translated, either abolished or markedly reduced chloride conductance. These results expand the spectrum of CLCN5 mutations associated with this renal disorder and provide insight into possible structure-function relationships. For example, both the missense mutations are located within a short putative loop between two transmembrane domains, and our results suggest that this region may have an important functional role in the regulation of channel activity.
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            Clinical and genetic studies of CLCN5 mutations in Japanese families with Dent's disease.

            Dent's disease is an X-linked renal tubular disorder that is characterized by low molecular weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. The disease is caused by inactivation of a renal chloride channel gene, CLCN5, that encodes a 746-amino acid protein with 12 to 13 transmembrane domains. The Japanese variant of Dent's disease has been observed to be less severe, and we have investigated two unrelated Japanese families for CLCN5 mutations. Six patients from two unrelated families were studied. Leukocyte DNA from probands was used with CLCN5-specific primers for polymerase chain reaction (PCR) amplification of the coding region and exon-intron boundaries, and the DNA sequences of the products were determined to identify abnormalities in the gene. RNA extracted from the kidney, leukocytes, or urine sediments was used to characterize further the effects of the identified mutations. beta2-microglobulinuria was detected in five patients, hypercalciuria in two patients, nephrolithiasis in three patients (2 of whom were females), and one 51-year-old man had renal failure. Two novel CLCN5 mutations consisting of an a to g transition at the invariant ag acceptor splice site of intron 5 and an intragenic deletion that encompassed the region between intron 3 and intron 6 were identified. The acceptor splice site mutation led to the utilization of two alternative cryptic splice sites in exon 6 that resulted in a frameshift or skipping of the exon 6. The deletional mutation, which resulted in a loss of exons 4, 5, and 6, is predicted to lead to a loss of domains 1 through 4. Both mutations predict truncated chloride channels that are likely to result in a functional loss. The observations of renal failure in one male and nephrolithiasis in two females represent important new findings in this Japanese variant of Dent's disease that is associated with CLCN5 mutations. In addition, our study is the first to demonstrate the use of urinary sediment cells and renal tissue for the detection of CLCN5 transcript abnormalities. These results help to expand the spectrum of CLCN5 mutations associated with Dent's disease.
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              Chloride channels are different.


                Author and article information

                Kidney Blood Press Res
                Kidney and Blood Pressure Research
                S. Karger AG
                29 July 2003
                : 26
                : 3
                : 176-184
                aDepartment of Clinical Biochemistry, University of Bonn, Bonn, Germany; bDepartment of Paediatrics, University of Marburg, Marburg, Germany; cDepartment of Paediatrics and Adolescence, Oulu, Finland, and dChildren’s Hospital Medical Center, University of Bonn, Bonn, Germany
                71883 Kidney Blood Press Res 2003;26:176–184
                © 2003 S. Karger AG, Basel

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                Figures: 4, Tables: 2, References: 38, Pages: 9
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