Genetic Diagnosis of Hereditary Hearing Impairment via Next-generation Sequencing

• Main Authors: Yin-Hung Lin, 林盈宏
• Other Authors: 陳沛隆
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/ftcdu4

博士 === 國立臺灣大學 === 基因體暨蛋白體醫學研究所 === 106 === Sensorineural hearing impairment (SNHI) is the most common sensory deficit in humans, affecting about three per 1000 children. More than 60% of these patients have a genetic cause (i.e. hereditary hearing impairment; HHI). HHI is extremely heterogeneous, with approximately 5000 variants in more than 100 genes reported as causative variants to date. Studies across different populations revealed that hotspots screening of common deafness genes (e.g. GJB2, SLC26A4 and the mitochondrial 12S rRNA) using Sanger sequencing could only achieve genetic diagnosis in ~1/3 hearing-impaired families. Recently, the advent of next-generation sequencing (NGS) has revolutionized the clinical evaluation of hearing-impaired patients by making comprehensive genetic testing possible. In this study, we established an NGS-based diagnostic platform for HHI. A capture-based panel was designed to target 159 deafness genes. Sequencing was performed by Illumina MiSeq which generated paired-end reads of 300 nucleotides. We developed an automated bioinformatics pipeline for analyzing the sequencing data. Disease and gene-specific databases have been curated and built to enable automatic matching. We have explored several unsolved problems of genetic basis of HHI as follows: (1) Diagnosis of patients without common deafness-causing variants We subjected 246 hearing-impaired families to our NGS-based diagnostic platform. We identified causative variants in 24 genes in 101 families, yielding a diagnostic rate of 41%. We identified a novel GATA3 pathogenic variant (c.153delT, p.Phe51Leufs*144) in one family. In another family, we identified a novel missense variant of POU4F3 (c.982A>G, p.Lys328Glu) which co-segregated with the deafness phenotype. We performed function studies for these two novel variants and explored the underlying mechanism. (2) Diagnosis of hearing-impaired children with poor cochlear implantation outcomes Cochlear implantation is currently the treatment of choice for children with severe to profound hearing impairment. However, the outcomes with cochlear implants (CIs) vary significantly among recipients. Twelve children with poor CI outcomes (the “cases”) and 30 “matched controls” with good CI outcomes were subjected to the NGS-based diagnosis platform. We identified DFNB59 p.G292R which might be associated with poor CI outcomes. (3) Diagnosis of enlarged vestibular aqueduct patients without bi-allelic SLC26A4 hotspots Enlarged vestibular aqueduct (EVA) is a common inner ear malformation caused mainly by bi-allelic SLC26A4 variants. We performed the NGS-based diagnostic platform in 50 families without confirmative results by screening two SLC26A4 hotspots (c.919-2A>G and p.H723R). We identified bi-allelic SLC26A4 pathogenic variants (including a large SLC26A4 deletion) in 34 families and EYA1 causative variants in two families, yielding a diagnostic rate of 72% (36/50). (4) Diagnosis of hearing-impaired patients with non-confirmative or genotype–phenotype-mismatched GJB2 pathogenic variants A significant percentage of patients segregate only one variant causing autosomal recessive SNHI in the GJB2. In addition, some patients with mild GJB2 variants such as p.V37I develop severe-to-profound SNHI. We performed NGS in 16 patients with mono-allelic GJB2 pathogenic variant and 22 patients with homozygous GJB2 p.V37I who had SNHI of varied severity. We confirmed the genetic diagnosis in seven patients with a GJB2 pathogenic variant. Of these, five patients had causative variants in other deafness genes; one patient had a splice site variant in the second GJB2 allele; and one patient had ~69% mosaicism for GJB2 c.235delC because of mosaic uniparental disomy. One patient homozygous for p.V37I had causative variants in TMC1, which might be responsible for the severe-to-profound SNHI.