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

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).