Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

Abstract

Background: In 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.

Methods: We 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.

Results: We 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).

Conclusion: We 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.

Keywords: CLCN5; In silico; Minigene; Splicing; Variant.

Fig. 1

Schema for the hybrid minigene. The H492 vector contains two cassette exons, A and B, with a multiple cloning site. The H492 vector also contains a cytomegalovirus (CMV) enhancer–promotor and a bovine growth hormone gene (BGH) polyadenylation site. The arrows show the positions of the primers used in the RT-PCR assay

Fig. 2

Electrophoresis results and schematic transcript analysis results from the minigene constructs. The direct sequence is shown in Supplementary Fig.1. a Wild type (WT) exhibited a single band (full) and No. 1 and No. 2 exhibited a single band (23 bp inclusion). b Wild type (WT) and No. 3 exhibited a single band (full). c Wild type (WT) exhibited a single band (full), while No. 4 exhibited double bands (full and exon 4 skipping). d Wild type (WT) exhibited a single band (full), while No. 5 exhibited quadruple bands (7 bp inclusion, 32 bp deletion, 88 bp deletion, and exon 6 skipping). No. 6 exhibited triple bands (32 bp deletion, 88 bp deletion, and exon 6 skipping)

Fig. 3

Schemas of the splicing patterns found in No. 1, No. 2, No. 5, and No. 6. a The splice site variants (c.105G>A and c.105+5G>C) disrupted the original donor site and resulted in the generation of an aberrant donor site that led to a 23 bp insertion. b The splice site variant (c.517−8A>G) disrupted the original acceptor site and resulted in the generation of an aberrant acceptor site that led to a 7 bp insertion, 32 bp deletion, and 88 bp deletion. c The splice site variant (c.517−3C>A) disrupted the original acceptor site and resulted in the generation of an aberrant acceptor site that led to a 32 bp deletion and an 88 bp deletion