Diverse spectrum of rare deafness genes underlies early-childhood hearing loss in Japanese patients: a cross-sectional, multi-center next-generation sequencing study

Abstract

Background: Genetic tests for hereditary hearing loss inform clinical management of patients and can provide the first step in the development of therapeutics. However, comprehensive genetic tests for deafness genes by Sanger sequencing is extremely expensive and time-consuming. Next-generation sequencing (NGS) technology is advantageous for genetic diagnosis of heterogeneous diseases that involve numerous causative genes.

Methods: Genomic DNA samples from 58 subjects with hearing loss from 15 unrelated Japanese families were subjected to NGS to identify the genetic causes of hearing loss. Subjects did not have pathogenic GJB2 mutations (the gene most often associated with inherited hearing loss), mitochondrial m.1555A>G or 3243A>G mutations, enlarged vestibular aqueduct, or auditory neuropathy. Clinical features of subjects were obtained from medical records. Genomic DNA was subjected to a custom-designed SureSelect Target Enrichment System to capture coding exons and proximal flanking intronic sequences of 84 genes responsible for nonsyndromic or syndromic hearing loss, and DNA was sequenced by Illumina GAIIx (paired-end read). The sequences were mapped and quality-checked using the programs BWA, Novoalign, Picard, and GATK, and analyzed by Avadis NGS.

Results: Candidate genes were identified in 7 of the 15 families. These genes were ACTG1, DFNA5, POU4F3, SLC26A5, SIX1, MYO7A, CDH23, PCDH15, and USH2A, suggesting that a variety of genes underlie early-childhood hearing loss in Japanese patients. Mutations in Usher syndrome-related genes were detected in three families, including one double heterozygous mutation of CDH23 and PCDH15.

Conclusion: Targeted NGS analysis revealed a diverse spectrum of rare deafness genes in Japanese subjects and underscores implications for efficient genetic testing.

Figure 1

Pedigrees of the seven families with hearing loss. Double horizontal bars above a symbol indicate individuals who underwent genetic analysis by targeted next-generation sequencing. Single horizontal bars above a symbol indicate individuals who underwent analysis by Sanger sequencing. A-G denote pedigrees of family 1-7, respectively.

Figure 2

Molecular modeling of ACTG containing the p.G268S mutation. (A) Ribbon model of filamentous actin gamma 1. (B) Magnified ribbon model of filamentous actin gamma 1. Glycine residue 268 is shown in red and indicated by an arrow. Regions in yellow and green indicate the hydrophobic loop (262–274; a) and the corresponding interactive residues (281–289; b), respectively. (C and D) Vertical views of the regions a and b superimposed with predicted surface hydrophobicity in the wild type (C) and the p.G268S mutant (D).

Figure 3

Molecular modeling of MYO7A containing the p.W2160G mutation. (A) Structural motif of myosin 7A. Tryptophan 2160 on the C-terminal 4.1 protein-ezrin-radixin-moesin (FERM) domain is indicated by an arrow. Motor, myosin motor domain; IQ, Isoleucine-glutamine calmodulin-binding motif; CC, coiled-coil domain; MyTH4, myosin tail homology 4 domain; SH3, Src homology 3 domain. (B) Ribbon model of the C-terminal FERM domain consisting of three subdomains (F1, F2, F3) and an MyTH4 domain. (C, D) Magnified ribbon model of the F3 subdomain superimposed with predicted surface hydrophobicity in the wild type (C) and the p.W2160G mutant (D).