Abstract
BackgroundIn recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants.MethodsWe conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results.ResultsWe successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G).ConclusionWe found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
Highlights
Dent disease is an X-linked genetic disease of tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, and renal calcification [1]
60% of Dent disease cases are caused by mutations in the CLCN5 gene, which are referred to as Dent disease 1 (OMIM#300,009)
In the Human Gene Mutation Database (HGMD) Professional, most reported mutations of the CLCN5 gene are missense or nonsense mutations, followed by deletions and splicing variants located at obvious splice sites within two bases immediately before and after exons (AG-GT)
Summary
Dent disease is an X-linked genetic disease of tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, and renal calcification [1]. Some previously reported suspected splicing variants were outside these regions [1, 3,4,5,6] Cases with such mutations might exhibit splicing abnormality, this has not been proven. There are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. Results We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants
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