Three intronic variants altering RNA splicing were identified in the CLCN5 gene by minigene assay

The Dent disease 1 is a rarely inherited renal tubular disease caused by variants in the CLCN5 gene. Increasing evidence suggests that many intronic variants can affect the normal splicing of pre-mRNA by altering various splicing regulatory signals. Therefore, this study aims to provide novel insights into the impact of intronic variants of CLCN5 on pre-mRNA splicing. We analyzed intronic variants of the CLCN5 gene from the Clinvar database, selected four candidate variants using bioinformatics analysis, and identified candidate variants that may alter the splicing of pre-mRNA through minigene assays. Our study revealed that, among four candidate variants, three intronic variants in CLCN5 resulted in splicing alterations(c.603 + 4 A > G, c.727–3 C > A, and c.933 + 3 A > C). Intronic variants c.603 + 4 A > G and c.727–3 C > A induce aberrant splicing that result in partial exon deletions by activating cryptic donor and acceptor splice sites, respectively. In contrast, the c.933 + 3 A > C variant disrupts the classical donor splice site of exon 7, leading to complete exon skipping. To our knowledge, this is a comprehensive study regarding alterations in pre-mRNA of intronic variants in the causal gene CLCN5 of Dent disease. In addition, we propose the importance of assessing the effect of intronic single nucleotide substitutions on CLCN5 at the mRNA level in the absence of patient RNA samples and show that minigenes functional analysis is valuable for assessing the effect of splicing in vitro.