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. 2018 Mar;39(3):433-440.
doi: 10.1002/humu.23384. Epub 2018 Jan 11.

Exonic Mutations and Exon Skipping: Lessons Learned From DFNA5

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Free PMC article

Exonic Mutations and Exon Skipping: Lessons Learned From DFNA5

Kevin T Booth et al. Hum Mutat. .
Free PMC article

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.

Keywords: DFNA5; RNA-splicing; deafness; exon-skipping; non-syndromic hearing loss.

Conflict of interest statement

Conflict of Interest: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
ADNSHL pedigrees showing segregation of novel mutations in DFNA5 with the deafness phenotype. Filled symbols denote affected individuals; red and bold and blue and bold represent the DFNA5 and USH2A mutant alleles, respectively; grey filled symbols indicate individuals with USH2A. Audiograms were obtained using pure tone audiometry with air conduction from frequencies from 250 Hz to 8,000 Hz. (“O”, OtoSCOPE; “W”, Whole Exome Sequencing; A-C: Pedigrees for family CDS-6824, CDS-7393 and L-8700115, respectively)
Figure 2
Figure 2
ADNSHL pedigrees segregating recurrent mutations in DFNA5. Filled symbols denote affected individuals; red and bold represent the DFNA5 mutant allele segregating with the HL. Audiograms were obtained using pure tone audiometry with air conduction from frequencies from 250 Hz to 8,000 Hz. (“W”, Whole Exome Sequencing; A: family 11330, B: family 10490 with serial audiograms for individual IV.11)
Figure 3
Figure 3
Mini-gene splicing analysis and schematic representation of DFNA5 exon 8 variants and splicing factor binding. A: Gel electrophoresis of wild-type DFNA5 exon 8 and the c.1102C>G, c.1183G>A, c.1154C>T and c.991-2A>G variants and empty pET01 vector. The inclusion of exon 8 results in a 438bp product and its exclusion result in a 245bp band. B: Novel and previously described variant alignment to DFNA5 exon 8 and its flanking introns. Novel variants are on bottom and previously described variants are on top. Italics and underline denote previously identified variants also identified in this study. C: Predicted binding sites of splicing factors to the novel variants in this study. Values between parentheses represent predicted splicing signal, wild-type black and mutant red.

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