Case Report: A Novel Variant c.2262+3A>T of the SCN5A Gene Results in Intron Retention Associated With Incessant Ventricular Tachycardias

Abstract

Objective: Voltage-gated sodium channel Nav1.5 encoded by the SCN5A gene plays crucial roles in cardiac electrophysiology. Previous genetic studies have shown that mutations in SCN5A are associated with multiple inherited cardiac arrhythmias. Here, we investigated the molecular defect in a Chinese boy with clinical manifestations of arrhythmias.Methods: Gene variations were screened using whole-exome sequencing and validated by direct Sanger sequencing. A minigene assay and reverse transcription PCR (RT-PCR) were performed to confirm the effects of splice variants in vitro. Western blot analysis was carried out to determine whether the c.2262+3A>T variant produced a truncated protein.Results: By genetic analysis, we identified a novel splice variant c.2262+3A>T in SCN5A gene in a Chinese boy with incessant ventricular tachycardias (VT). This variant was predicted to activate a new cryptic splice donor site and was identified by in silico analysis. The variant retained 79 bp at the 5′ end of intron 14 in the mature mRNA. Furthermore, the mutant transcript that created a premature stop codon at 818 amino acids [p.(R818*)] could be produced as a truncated protein.Conclusion: We verified the pathogenic effect of splicing variant c.2262+3A>T, which disturbed the normal mRNA splicing and caused a truncated protein, suggesting that splice variants play an important role in the molecular basis of early onset incessant ventricular tachycardias, and careful molecular profiling of these patients will be essential for future effective personalized treatment options.

Case Report: A Novel Non-Canonical Splice Site Variant (c.1638+7T>C) in TRPM6 Cause Primary Homagnesemia With Secondary Hocalcemia

“… The pSPL3 minigene assay in vitro for the splicing variant c.1638+7T>C. (A) PCR fragments were, respectively, cloned into the XhoI and BamHI sites of the pSPL3 vector ( 9 ). The vector mainly produces two transcripts, wildtype composed of exon SD6, an inserted exon14 and exon SA2 (404 bp), and mutant composed only of exon SD6 and SA2 (263 bp).…”

“…In order to generate a minigene mixture, a pSPL3 minigene reporter vector including a conventional expression system with SD6 and SA2 was used as the resultant mRNA transcripts per previous research, as described in our recent study ( 8 , 9 ). Briefly, we generated fragments containing the target exons 14 where the splicing variant was located, and 150 bp of flanking intronic regions with XhoI and BamHI restriction sites.…”

Case report: Functional characterization of a novel CHD7 intronic variant in patients with CHARGE syndrome

“…For instance, it has been used by Van der Klift and others to prove the effects of several mutations in Lynch syndrome finding a high correlation concordance between minigene assays and patient RNA analyses ( Van der Klift et al, 2015 ). In another work, Yin and collaborators used the minigene assay to verify the effect of an intronic variant in the SCN5A gene causing a form of incessant ventricular tachycardias ( Yin et al, 2021 ). These results demonstrate that HEK293 is a reliable cell line to perform minigene assay.…”