Recessive mutations of TMC1 associated with moderate to severe hearing loss

Abstract

TMC1 encodes a protein required for the normal function of mechanically activated channels that enable sensory transduction in auditory and vestibular hair cells. TMC1 protein is localized at the tips of the hair cell stereocilia, the site of conventional mechanotransduction. In many populations, loss-of-function recessive mutations of TMC1 are associated with profound deafness across all frequencies tested. In six families reported here, variable moderate-to-severe or moderate-to-profound hearing loss co-segregated with STR (short tandem repeats) markers at the TMC1 locus DFNB7/11. Massively parallel and Sanger sequencing of genomic DNA revealed each family co-segregating hearing loss with a homozygous TMC1 mutation: two reported mutations (p.R34X and p.R389Q) and three novel mutations (p.S596R, p.N199I, and c.1404 + 1G > T). TMC1 cDNA sequence from affected subjects homozygous for the donor splice site transversion c.1404 + 1G > T revealed skipping of exon 16, deleting 60 amino acids from the TMC1 protein. Since the mutations in our study cause less than profound hearing loss, we speculate that there is hypo-functional TMC1 mechanotransduction channel activity and that other even less damaging variants of TMC1 may be associated with more common mild-to-severe sensorineural hearing loss.

Keywords: DFNB7/11; Mechanosensory transduction; Moderate or severe hearing loss; TMC1.

Fig. 1

Pedigrees with audiometric data and mutation data of the families a Pedigrees of TMC1-linked families HLAM02, PHAI-01, HLAI-04, HLAI-14, HLAI-17 and HLAI-27. Solid circles and squares denote affected individuals. The nucleotide variants identified for each family are indicated below the symbols of the participants. Genotyping for family HLA-17 was performed with four microsatellite markers spanning the TMC1 gene on chromosome 9. Distances are according to Rutgers’ combined genetic map. A grey bar is the deafness-linked haplotype. b Pure tone audiograms of affected individuals of the participating families. Age of each affected individual is provided with the symbol key. Some affected individuals (without correlation to age) in these pedigrees have a relatively less severe hearing loss as compared to the other affected individuals in their families. c Electropherograms of TMC1 sequence analyses showing the two missense mutations (p.S596R and p.N199I ) in family HLAI-14, HLAI-27 and a splice site mutation (c.1944+1G>T) in family HLAI-04. The affected codons or the splice site are underlined. The arrow indicates the location of the mutation.

Fig. 2

TMC1 c.1944+1G>T variant with diagrammatic representation of its effect, and conservation analyses of novel missense variants of TMC1. a In wild type splicing our assay detected a product of 537 base pairs after splicing of TMC1 exons 15, 16 and 17 (NM_138691.2). The c.1944+1G>T mutates the wild type donor splice site (gt) to tt. This mutation causes the skipping of exon 16 and a loss of 180 nucleotides from the mature mutant TMC1 mRNA. b Selected region from electropherogram of TMC1 cDNA showing the junction of exon 15-16 (left) after splicing in the wild type. On right is a sequence trace from an affected individual in family HLAI04 who is homozygous for the splice site variant c.1944+1G>T. The use of this site results in splicing of TMC1 exon 15 to exon 17 due to the skipping of exon 16. c CLUSTAL Omega sequence alignment showing the evolutionary conservation of TMC1 residue p.N199 from six vertebrate species. The p.N199 residue is highlighted in gray. d CLUSTAL Omega sequence alignment showing the conservation of TMC1 residue p.S596 across six vertebrate species. The amino acid residue p.S596 is not conserved in amphibians and fish.