Exonic mutations and exon skipping: Lessons learned from DFNA5

Abstract

Dysregulation of splicing is a common factor underlying many inherited diseases including deafness. For one deafness-associated gene, DFNA5, perturbation of exon 8 splicing results in a constitutively active truncated protein. To date, only intronic mutations have been reported to cause exon 8 skipping in patients with DFNA5-related deafness. In five families with postlingual progressive autosomal dominant non-syndromic hearing loss, we employed two next-generation sequencing platforms-OtoSCOPE and whole exome sequencing-followed by variant filtering and prioritization based on both minor allele frequency and functional consequence using a customized bioinformatics pipeline to identify three novel and two recurrent mutations in DFNA5 that segregated with hearing loss in these families. The three novel mutations are all missense variants within exon 8 that are predicted computationally to decrease splicing efficiency or abolish it completely. We confirmed their functional impact in vitro using mini-genes carrying each mutant DFNA5 exon 8. In so doing, we present the first exonic mutations in DFNA5 to cause deafness, expand the mutational spectrum of DFNA5-related hearing loss, and highlight the importance of assessing the effect of coding variants on splicing.

Systematic quantification of the anion transport function of pendrin (SLC26A4) and its disease‐associated variants

“…However, it may be possible that some of these exonic single nucleotide variants might primarily affect splicing. To explore this possibility, we performed the in vitro splicing assay (Booth et al, ). Briefly, mini‐gene constructs containing one or two human pendrin exons were created in a pET01 vector for WT and variants and transcribed in HEK293T cells (Supporting Information Material).…”

Novel Loss-of-Function Mutations inCOCHCause Autosomal Recessive Nonsyndromic Deafness

“…Often, if the variant falls outside the traditional splicing window, its impact on splicing is rarely considered. However, several studies have illustrated coding variant's effect on splicing is underappreciated 13,14,41,42 .…”

“…Probands A-C underwent comprehensive genetic testing to screen all known genes implicated in NSHL, common NSHL mimics, and common syndromic forms of deafness using the OtoSCOPE® panel as described [11][12][13][14] . Similarly, a proband from family PKMR266 underwent genetic testing as described 15,16 .…”

“…Bioinformatic and variant analysis were also completed as described [13][14][15] . In brief, after mapping of raw sequence reads and variant calling, variants were annotated and filtered based on quality (depth >5 and call quality >30), minor allele frequency (MAF) <2% in Genome Aggregation Database (gnomAD) 17 and variant effect (missense, nonsense, indel or splice-site).…”

“…In vitro splicing assays were performed as described 13,14,21 . COCH (NM_004086) gene-specific primers were used to amplify exon 5, 7, 8 and 11 using patient genomic DNA and control DNA to obtain the mutant and wildtype allele, respectively.…”