Confirmation of COL4A6 variants in X-linked nonsyndromic hearing loss and its clinical implications

Abstract

Hearing loss (HL) is one of the most common sensory defects, of which X-linked nonsyndromic hearing loss (NSHL) accounts for only 1-2%. While a COL4A6 variant has been reported in a single Hungarian family with NSHL associated with inner ear malformation, causative role of COL4A6 variants and their phenotypic consequences in NSHL remain elusive. Here we report two families in which we identified a male member with X-linked HL. Each has inherited a rare hemizygous COL4A6 variant from their respective mothers, NM_001287758.1: c.3272 G > C (p.Gly1091Ala) and c.951 + 1 G > C. An in vitro minigene splicing assay revealed that c.951 + 1 G > T leads to skipping of exon 15, strongly suggesting a pathogenic role for this variant in the HL phenotype. The p.Gly1091Ala variant is classified as a variant of unknown significance based on the variant interpretation guidelines. This report provides evidence for variants in the COL4A6 gene resulting in X-linked NSHL. It highlights the importance of in-depth genetic studies in all family members in addition to the proband, especially in multiplex families, to determine the precise etiology of HL.

Fig. 1. Two families with COL4A6 variants and sanger sequencing trace for COL4A6.

A, B Families A and B with their multiplex pedigrees and segregation for variants in GJB2, GJB6, and COL4A6. C Electropherogram for Sanger sequencing of the hemizygous male (A-III:1 and B-II:3), heterozygous mother (A-II:2 and B-I:2), and unaffected father (A-II:1 and B-I:1).

Fig. 2. Schematic of splicing assay for the COL4A6 c.951 + 1G > T variant identified in Family A.

A Depiction of the altered splice acceptor site in intron 15 (TT) which is bypassed. B Verification of pET01-COL4A6 transfection. Wild-type and mutant cDNA harvested and transcribed from pET01-COL4A6 transfected HEK293 cells. Agarose gel shows 200 bp wild-type product that includes exons 15 and 16, and a 150 bp product with exon 15 skipped in the mutant c.951 + 1 G > T lane. C Sanger sequence confirmation of extracted product noted in agarose gel in (B).