Targeted massive parallel sequencing: the effective detection of novel causative mutations associated with hearing loss in small families

Abstract

Background: Hereditary hearing loss is one of the most common heterogeneous disorders, and genetic variants that can cause hearing loss have been identified in over sixty genes. Most of these hearing loss genes have been detected using classical genetic methods, typically starting with linkage analysis in large families with hereditary hearing loss. However, these classical strategies are not well suited for mutation analysis in smaller families who have insufficient genetic information.

Methods: Eighty known hearing loss genes were selected and simultaneously sequenced by targeted next-generation sequencing (NGS) in 8 Korean families with autosomal dominant non-syndromic sensorineural hearing loss.

Results: Five mutations in known hearing loss genes, including 1 nonsense and 4 missense mutations, were identified in 5 different genes (ACTG1, MYO1F, DIAPH1, POU4F3 and EYA4), and the genotypes for these mutations were consistent with the autosomal dominant inheritance pattern of hearing loss in each family. No mutational hot-spots were revealed in these Korean families.

Conclusion: Targeted NGS allowed for the detection of pathogenic mutations in affected individuals who were not candidates for classical genetic studies. This report is the first documenting the effective use of an NGS technique to detect pathogenic mutations that underlie hearing loss in an East Asian population. Using this NGS technique to establish a database of common mutations in Korean patients with hearing loss and further data accumulation will contribute to the early diagnosis and fundamental therapies for hereditary hearing loss.

Figure 1

Pedigree diagrams of 8 Korean families with autosomal dominant hearing loss. An asterisk in the upper right corner of the symbol indicates an individual whose genomic DNA was analyzed by targeted sequencing. Co-segregation of the candidate variations detected in the targeted sequencing and the phenotype of hearing loss was confirmed via Sanger sequencing of DNA from the individuals marked by a bar. Squares and circles represent males and females, respectively. Filled symbols represent affected individuals, and slashes indicate deceased individuals.

Figure 2

The identification of 5 non-synonymous variations showing co-segregation with hearing loss in affected families.Top: Visualization of individual sequencing reads covering the mutations in the genes. The actual read depth for these mutated nucleotides ranged between 46× and 442×. Blue and green reads represent the positive and negative strands, respectively, and the red bases represent bases that differ from the reference sequence (black). Middle: Verification of each variation by Sanger sequencing. All 5 of the variations marked by black arrows are single nucleotide substitutions leading to early truncation of the polypeptide or a change in the amino acid. Bottom: A comparison of amino acid sequences of each gene in multiple vertebrate species. The asterisks indicate the amino acid substituted by the mutation. A single line between the aligned amino acids indicates that there is no base in the aligned species, and a double line indicates that the aligned species has one or more bases that cannot be aligned in the gap region. The majority of the mutated amino acids are strongly conserved across the 9 different species shown.