The extreme genetic heterogeneity of nonsyndromic hearing loss (NSHL) makes genetic diagnosis expensive and time consuming using available methods. To assess the feasibility of targetenrichment and massively parallel sequencing technologies to interrogate all exons of all genes implicated in NSHL, we tested nine patients diagnosed with hearing loss. Solid-phase (NimbleGen) or solution-based (SureSelect) sequence capture, followed by 454 or Illumina sequencing, respectively, were compared. Sequencing reads were mapped using GSMAPPER, BFAST, and BOWTIE, and pathogenic variants were identified using a custom-variant calling and annotation pipeline (ASAP) that incorporates publicly available in silico pathogenicity prediction tools (SIFT, BLOSUM, Polyphen2, and Align-GVGD). Samples included one negative control, three positive controls (one biological replicate), and six unknowns (10 samples total), in which we genotyped 605 single nucleotide polymorphisms (SNPs) by Sanger sequencing to measure sensitivity and specificity for SureSelect-Illumina and NimbleGen-454 methods at saturating sequence coverage. Causative mutations were identified in the positive controls but not in the negative control. In five of six idiopathic hearing loss patients we identified the pathogenic mutation. Massively parallel sequencing technologies provide sensitivity, specificity, and reproducibility at levels sufficient to perform genetic diagnosis of hearing loss.deafness | genomics | Usher syndrome | diagnostics | next-generation sequencing H ereditary sensorineural hearing loss (SNHL) is the most common sensory impairment in humans (1, 2). In developed countries, two-thirds of prelingual-onset SNHL is estimated to have a genetic etiology, of which ∼70% is nonsyndromic hearing loss (NSHL). Eighty percent of NSHL is autosomal recessive nonsyndromic hearing loss (ARNSHL), ∼20% is autosomal dominant (AD), and the remainder is composed of X-linked and mitochondrial forms (1, 3). To date, 134 deafness loci have been identified, and 32 recessive (DFNB), 23 dominant (DFNA) and 2 X-linked (DFNX) genes have been cloned; 8 genes are associated with both ARNSHL and ADNSHL (4).Establishing a genetic diagnosis of NSHL is a critical component of the clinical evaluation of deaf and hard-of-hearing persons and their families. If a genetic cause of hearing loss is determined, it is possible to provide families with prognostic information, recurrence risks, and improved habilitation options. For persons diagnosed with Usher syndrome, preventative measures including sunlight protection and vitamin therapy can be implemented to minimize the rate of progression of retinitis pigmentosa (5). Most current genetic testing strategies for NSHL rely on a gene-specific Sanger sequencing approach. Because mutations in a single gene, GJB2 (DFNB1), account for up to 50% of ARNSHL in many world populations (6), this approach has changed the evaluation of patients with presumed ARNSHL. However, the mutation frequency in other genes in persons with NSHL in outbred populat...

Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness.

BackgroundCopy number variants (CNVs) are a well-recognized cause of genetic disease; however, methods for their identification are often gene-specific, excluded as ‘routine’ in screens of genetically heterogeneous disorders, and not implemented in most next-generation sequencing pipelines. For this reason, the contribution of CNVs to non-syndromic hearing loss (NSHL) is most likely under-recognized. We aimed to incorporate a method for CNV identification as part of our standard analysis pipeline and to determine the contribution of CNVs to genetic hearing loss.MethodsWe used targeted genomic enrichment and massively parallel sequencing to isolate and sequence all exons of all genes known to cause NSHL. We completed testing on 686 patients with hearing loss with no exclusions based on type of hearing loss or any other clinical features. For analysis we used an integrated method for detection of single nucleotide changes, indels and CNVs. CNVs were identified using a previously published method that utilizes median read-depth ratios and a sliding-window approach.ResultsOf 686 patients tested, 15.2% (104) carried at least one CNV within a known deafness gene. Of the 38.9% (267) of individuals for whom we were able to determine a genetic cause of hearing loss, a CNV was implicated in 18.7% (50). We identified CNVs in 16 different genes including 7 genes for which no CNVs have been previously reported. CNVs of STRC were most common (73% of CNVs identified) followed by CNVs of OTOA (13% of CNVs identified).ConclusionCNVs are an important cause of NSHL and their detection must be included in comprehensive genetic testing for hearing loss.

Background Non-syndromic hearing loss (NSHL) is the most common sensory impairment in humans. Until recently its extreme genetic heterogeneity precluded comprehensive genetic testing. Using a platform that couples targeted genomic enrichment (TGE) and massively parallel sequencing (MPS) to sequence all exons of all genes implicated in NSHL, we test 100 persons with presumed genetic NSHL and in so doing establish sequencing requirements for maximum sensitivity and define MPS quality score metrics that obviate Sanger validation of variants. Methods We examined DNA from 100 sequentially collected probands with presumed genetic NSHL without exclusions due to inheritance, previous genetic testing, or type of hearing loss. We performed TGE using post-capture multiplexing in variable pool sizes followed by Illumina sequencing. We developed a local Galaxy installation on a high performance-computing cluster for bioinformatics analysis. Results To obtain maximum variant sensitivity with this platform 3.2–6.3 million total mapped sequencing reads per sample are required. Quality score analysis showed that Sanger validation is not required for 95% of variants. Our overall diagnostic rate was 42% but varied by clinical features from 0% for persons with asymmetric hearing loss to 56% for persons with bilateral autosomal recessive NSHL. Conclusions These findings will direct the use of TGE and MPS strategies for genetic diagnosis for NSHL. Our diagnostic rate highlights the need for further research on genetic deafness focused on novel gene identification and an improved understanding of the role of non-exonic mutations. The unsolved families we have identified provide a valuable resource to address these areas.

Objective To evaluate the use of new genetic sequencing techniques for comprehensive genetic testing for hearing loss. Data Sources Articles were identified from PubMed and Google Scholar databases using pertinent search terms. Review Methods Literature search identified 30 studies as candidates that met search criteria. Three studies were excluded and eight studies were found to be case reports. 20 studies were included for review analysis including seven studies that evaluated controls and 16 studies that evaluated patients with unknown causes of hearing loss; three studies evaluated both controls and patients. Conclusions In the 20 studies included in review analysis, 426 control samples and 603 patients with unknown causes of hearing loss underwent comprehensive genetic diagnosis for hearing loss using massively parallel sequencing. Control analysis showed a sensitivity and specificity > 99%, sufficient for clinical use of these tests. The overall diagnostic rate was 41% (range 10% to 83%) and varied based on several factors including inheritance and pre-screening prior to comprehensive testing. There were significant differences in platforms available in regards to number and type of genes included and whether copy number variations were examined. Based on these results, comprehensive genetic testing should form the cornerstone of a tiered approach to clinical evaluation of patients with hearing loss along with history, physical exam, and audiometry and can determine further testing that may be required, if any. Implications for Practice Comprehensive genetic testing has become the new standard of care for genetic testing for patients with sensorineural hearing loss.

Background Up to 7% of patients with severe-to-profound deafness do not benefit from cochlear implantation. Given the high surgical implantation and clinical management cost of cochlear implantation (> $1 million lifetime cost), prospective identification of the worst performers would reduce unnecessary procedures and healthcare costs. Because cochlear implants bypass the membranous labyrinth but rely on the spiral ganglion for functionality, we hypothesize that cochlear implant (CI) performance is dictated in part by the anatomic location of the cochlear pathology that underlies the hearing loss. As a corollary, we hypothesize that because genetic testing can identify sites of cochlear pathology, it may be useful in predicting CI performance. Methods 29 adult CI recipients with idiopathic adult-onset severe-to-profound hearing loss were studied. DNA samples were subjected to solution-based sequence capture and massively parallel sequencing using the OtoSCOPE® platform. The cohort was divided into three CI performance groups (good, intermediate, poor) and genetic causes of deafness were correlated with audiometric data to determine whether there was a gene-specific impact on CI performance. Results The genetic cause of deafness was determined in 3/29 (10%) individuals. The two poor performers segregated mutations in TMPRSS3, a gene expressed in the spiral ganglion, while the good performer segregated mutations in LOXHD1, a gene expressed in the membranous labyrinth. Comprehensive literature review identified other good performers with mutations in membranous labyrinth-expressed genes; poor performance was associated with spiral ganglion-expressed genes. Conclusions Our data support the underlying hypothesis that mutations in genes preferentially expressed in the spiral ganglion portend poor CI performance while mutations in genes expressed in the membranous labyrinth portend good CI performance. Although the low mutation rate in known deafness genes in this cohort likely relates to the ascertainment characteristics (postlingual hearing loss in adult CI recipients), these data suggest that genetic testing should be implemented as part of the CI evaluation to test this association prospectively.

By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.

BackgroundThere is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.ResultsA total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.ConclusionsThe CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.