Performance Evaluation of the Next‐Generation Sequencing Approach for Molecular Diagnosis of Hereditary Hearing Loss

Abstract

Objective. To evaluate the performance of a next-generation sequencing (NGS)-based targeted resequencing genetic test, OtoSeq, to identify the sequence variants in the genes causing sensorineural hearing loss (SNHL).Study Design. Retrospective study. Setting. Tertiary children's hospital.Subjects and Methods. A total of 8 individuals presenting with prelingual hearing loss were used in this study. The coding and flanking intronic regions of 24 well-studied SNHL genes were enriched using microdroplet polymerase chain reaction and sequenced on an Illumina HiSeq 2000 sequencer. The filtered high-quality sequence reads were mapped to reference sequence, and variants were detected using NextGENe software.Results. A total of 1148 sequence variants were detected in 8 samples in 24 genes. Using in-house developed NGS data analysis criteria, we classified 810 (~71%) of these variants as potential true variants that include previously detected pathogenic mutations in 5 patients. To validate our strategy, we Sanger sequenced the target regions of 5 of the 24 genes, accounting for about 29.2% of all target sequence. Our results showed .99.99% concordance between NGS and Sanger sequencing in these 5 genes, resulting in an analytical sensitivity and specificity of 100% and 99.997%, respectively. We were able to successfully detect single base substitutions, small deletions, and insertions of up to 22 nucleotides.Conclusion. This study demonstrated that our NGS-based mutation screening strategy is highly sensitive and specific in detecting sequence variants in the SNHL genes. Therefore, we propose that this NGS-based targeted sequencing method would be an alternative to current technologies for identifying the multiple genetic causes of SNHL. 1-3 About two-thirds of prelingual-onset SNHL has a genetic etiology, and the rest is due to environmental and unidentified genetic factors. It is estimated that about 70% of patients have nonsyndromic SNHL, and the remaining 30% of patients have syndromic SNHL, where SNHL is accompanied by other physical manifestations.2,3 In the majority of patients, inherited SNHL is monogenic and classified into 4 groups based on the inheritance pattern: autosomal recessive is the most common type, occurring in about 80% of patients, and autosomal dominant accounts for most of the other 20%, whereas X-linked and mitochondrial account for only 1% to 2% of the patients.4-6 Inherited SNHL is genetically heterogeneous, and to date, more than 150 deafness loci have been identified, including 39 autosomal recessive, 25 autosomal dominant, 3 X-linked, and 2 mitochondrial genes. 7Establishing genetic causes of SNHL is important in the clinical management of patients and their families, and thus the continued search for the unidentified genetic causes is of clinical relevance. Candidate genes for syndromic SNHL are determined based on associated symptoms, whereas this approach is not viable for nonsyndromic SNHL as the phenotype caused by most of the genes is indistinguishable. Therefore, screening th...

Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing

“…5,11,12 The findings from our study are concordant with these previous observations but provide novelty due to the more targeted approach compared with whole-exome sequencing 5 and the much greater number of variants (thousands compared with hundreds) analyzed compared with those from all three past reports. Interestingly, our depth-of-coverage requirements appear to have been more stringent than those from the other studies that have examined targeted panels.…”

Genetic Analysis through OtoSeq of Pakistani Families Segregating Prelingual Hearing Loss

“…Using a micro-droplet PCR-based target enrichment followed by NGS (OtoSeq), we screened one affected individual from each of 29 Pakistani families with unique MYO7A, CDH23 and SLC26A4 linkage haplotypes (Figures 1 and 2) for 24 known deafness-causing genes. 28 Unbiased analysis (blinded to linkage data) of the NGS data revealed potential pathogenic mutations in 23 of 29 samples screened. We confirmed these mutations through Sanger sequencing (Figure 3).…”

“…Hence, mutations in cis -acting regulatory or splice elements of MYO7A that are necessary for MYO7A expression in the inner ear and retina would not be detected. Second, the OtoSeq platform has limited resolution in detecting insertions (up to 22 nucleotides), 28 and may have missed complicated mutations like large insertions. Third, the hearing loss segregating in these six families may be spuriously linked to markers at 11q13.5.…”

“…28 Here, we report genetic screening of 34 consanguineous Pakistani families segregating prelingual hearing loss linked to STR markers for MYO7A, CDH23 or SLC26A4 using OtoSeq. We found 24 mutations in 28 of the 34 families including 11 MYO7A alleles not previously reported.…”

Massively Parallel Sequencing for Genetic Diagnosis of Hearing Loss

“…Three studies included formal sensitivity and specificity analysis including two studies which compared MPS to gold-standard Sanger sequencing 8,15 and one study in which MPS was compared to a reference human genome sequence in a publically available HapMap sample 13 . Sensitivity and specificity were both >99% in all three studies when evaluating a total of more than 1,500 genotype calls.…”