Disease-specific ACMG/AMP guidelines improve sequence variant interpretation for hearing loss.

PURPOSE: The ClinGen Variant Curation Expert Panels (VCEPs) provide disease-specific rules for accurate variant interpretation. Using the hearing loss-specific American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, the Hearing Loss VCEP (HL VCEP) illustrates the utility of expert specifications in variant interpretation. METHODS: A total of 157 variants across nine HL genes, previously submitted to ClinVar, were curated by the HL VCEP. The curation process involved collecting published and unpublished data for each variant by biocurators, followed by bimonthly meetings of an expert curation subgroup that reviewed all evidence and applied the HL-specific ACMG/AMP guidelines to reach a final classification. RESULTS: Before expert curation, 75% (117/157) of variants had single or multiple variants of uncertain significance (VUS) submissions (17/157) or had conflicting interpretations in ClinVar (100/157). After applying the HL-specific ACMG/AMP guidelines, 24% (4/17) of VUS and 69% (69/100) of discordant variants were resolved into benign (B), likely benign (LB), likely pathogenic (LP), or pathogenic (P). Overall, 70% (109/157) variants had unambiguous classifications (B, LB, LP, P). We quantify the contribution of the HL-specified ACMG/AMP codes to variant classification. CONCLUSION: Expert specification and application of the HL-specific ACMG/AMP guidelines effectively resolved discordant interpretations in ClinVar. This study highlights the utility of ClinGen VCEPs in supporting more consistent clinical variant interpretation.

ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs.

PURPOSE: Proper interpretation of genomic variants is critical to successful medical decision making based on genetic testing results. A fundamental prerequisite to accurate variant interpretation is the clear understanding of the clinical validity of gene-disease relationships. The Clinical Genome Resource (ClinGen) has developed a semiquantitative framework to assign clinical validity to gene-disease relationships. METHODS: The ClinGen Hearing Loss Gene Curation Expert Panel (HL GCEP) uses this framework to perform evidence-based curations of genes present on testing panels from 17 clinical laboratories in the Genetic Testing Registry. The HL GCEP curated and reviewed 142 genes and 164 gene-disease pairs, including 105 nonsyndromic and 59 syndromic forms of hearing loss. RESULTS: The final outcome included 82 Definitive (50%), 12 Strong (7%), 25 Moderate (15%), 32 Limited (20%), 10 Disputed (6%), and 3 Refuted (2%) classifications. The summary of each curation is date stamped with the HL GCEP approval, is live, and will be kept up-to-date on the ClinGen website ( https://search.clinicalgenome.org/kb/gene-validity ). CONCLUSION: This gene curation approach serves to optimize the clinical sensitivity of genetic testing while reducing the rate of uncertain or ambiguous test results caused by the interrogation of genes with insufficient evidence of a disease link.

Correction: ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel.

PURPOSE: Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS: The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS: The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION: Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss.

Due to the high genetic heterogeneity of hearing loss (HL), current clinical testing includes sequencing large numbers of genes, which often yields a significant number of novel variants. Therefore, the standardization of variant interpretation is crucial to provide consistent and accurate diagnoses. The Hearing Loss Variant Curation Expert Panel was created within the Clinical Genome Resource to provide expert guidance for standardized genomic interpretation in the context of HL. As one of its major tasks, our Expert Panel has adapted the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants in HL genes. Here, we provide a comprehensive illustration of the newly specified ACMG/AMP HL rules. Three rules remained unchanged, four rules were removed, and the remaining 21 rules were specified. These rules were further validated and refined using a pilot set of 51 variants assessed by curators and disease experts. Of the 51 variants evaluated in the pilot, 37% (19/51) changed category based upon application of the expert panel specified rules and/or aggregation of evidence across laboratories. These HL-specific ACMG/AMP rules will help standardize variant interpretation, ultimately leading to better care for individuals with HL.

Improving hearing loss gene testing: a systematic review of gene evidence toward more efficient next-generation sequencing-based diagnostic testing and interpretation.

PURPOSE: With next generation sequencing technology improvement and cost reductions, it has become technically feasible to sequence a large number of genes in one diagnostic test. This is especially relevant for diseases with large genetic and/or phenotypic heterogeneity, such as hearing loss. However, variant interpretation remains the major bottleneck. This is further exacerbated by the lack in the clinical genetics community of consensus criteria for defining the evidence necessary to include genes on targeted disease panels or in genomic reports, and the consequent risk of reporting variants in genes with no relevance to disease. METHODS: We describe a systematic evidence-based approach for assessing gene-disease associations and for curating relevant genes for different disease aspects, including mode of inheritance, phenotypic severity, and mutation spectrum. RESULTS: By applying this approach to clinically available hearing loss gene panels with a total of 163 genes, we show that a significant number (45%) of genes lack sufficient evidence of association with disease and thus are expected to increase uncertainty and patient anxiety, in addition to intensifying the interpretation burden. Information about all curated genes is summarized. Our retrospective analysis of 539 hearing loss cases tested by our previous OtoGenomeV2 panel demonstrates the impact of including genes with weak disease association in laboratory wet-bench and interpretation processes. CONCLUSION: Our study is, to our knowledge, the first to highlight the urgent need for defining the clinical validity of gene-disease relationships for more efficient and accurate clinical testing and reporting.Genet Med 18 6, 545-553.

A synonymous variant in MYO15A enriched in the Ashkenazi Jewish population causes autosomal recessive hearing loss due to abnormal splicing.

Nonsyndromic hearing loss is genetically heterogeneous. Despite comprehensive genetic testing, many cases remain unsolved because the clinical significance of identified variants is uncertain or because biallelic pathogenic variants are not identified for presumed autosomal recessive cases. Common synonymous variants are often disregarded. Determining the pathogenicity of synonymous variants may improve genetic diagnosis. We report a synonymous variant c.9861 C > T/p.(Gly3287=) in MYO15A in homozygosity or compound heterozygosity with another pathogenic or likely pathogenic MYO15A variant in 10 unrelated families with nonsyndromic sensorineural hearing loss. Biallelic variants in MYO15A were identified in 21 affected and were absent in 22 unaffected siblings. A mini-gene assay confirms that the synonymous variant leads to abnormal splicing. The variant is enriched in the Ashkenazi Jewish population. Individuals carrying biallelic variants involving c.9861 C > T often exhibit progressive post-lingual hearing loss distinct from the congenital profound deafness typically associated with biallelic loss-of-function MYO15A variants. This study establishes the pathogenicity of the c.9861 C > T variant in MYO15A and expands the phenotypic spectrum of MYO15A-related hearing loss. Our work also highlights the importance of multicenter collaboration and data sharing to establish the pathogenicity of a relatively common synonymous variant for improved diagnosis and management of hearing loss.

Prenatal cytogenomic identification and molecular refinement of compound heterozygous STRC deletion breakpoints.

Here, we report the prenatal detection of a compound heterozygous deletion at chromosome 15q15.3 by clinical chromosomal microarray (CMA) testing that included the CATSPER2 male infertility gene. However, given the low resolution of CMA at this homologous locus, it was unclear if the neighboring STRC hearing loss gene was also affected. Therefore, we developed a novel allele-specific PCR strategy, which narrowed the proximal breakpoint of the maternally inherited deletion to a 310 bp interval that was 440 bp upstream from the STRC transcription start site.

Analysis of intragenic USH2A copy number variation unveils broad spectrum of unique and recurrent variants.

Given that all forms of Usher syndrome (USH) present with hearing loss in advance of retinal disease, the syndromic nature of the disorder is rarely appreciated when critical management decisions are being made. As a result, molecular diagnostics are crucial in guiding the management of USH patients. While 11 genes have been associated with USH, the USH2A gene is one of the largest contributors. Approximately 20% of suspected USH probands that undergo USH2A sequencing at our laboratory receive an inconclusive result due to the identification of a monoallelic disease-causing variant in USH2A. Many studies suggest that intragenic deletions and duplications represent an important USH2A variant type that can be missed by sequencing assays if supplemental algorithms or testing methods are not applied. To gain a comprehensive view of the contribution of USH2A CNVs to USH, we conducted prospective and retrospective screening in 700 hearing loss probands. Fourteen individuals with 11 unique USH2A CNVs are reported, including one pathogenic multi-exon duplication. Additionally, we mapped deletion breakpoints and performed a meta-analysis of USH2A CNVs to evaluate recurrence and underlying mechanisms. This analysis revealed breakpoint grouping within three introns, raising the possibility of CNV-susceptible regions within the gene. Overall, our data highlight the diversity of pathogenic CNVs in this gene, demonstrating that the comprehensive, high-resolution USH2A CNV analysis methods employed here are essential components of clinical genetic testing for USH.

Curating Clinically Relevant Transcripts for the Interpretation of Sequence Variants.

Variant interpretation depends on accurate annotations using biologically relevant transcripts. We have developed a systematic strategy for designating primary transcripts and have applied it to 109 hearing loss-associated genes that were divided into three categories. Category 1 genes (n = 38) had a single transcript; category 2 genes (n = 33) had multiple transcripts, but a single transcript was sufficient to represent all exons; and category 3 genes (n = 38) had multiple transcripts with unique exons. Transcripts were curated with respect to gene expression reported in the literature and the Genotype-Tissue Expression Project. In addition, high-frequency loss-of-function variants in the Genome Aggregation Database and disease-causing variants in ClinVar and the Human Gene Mutation Database across the 109 genes were queried. These data were used to classify exons as clinically significant, insignificant, or of uncertain significance. Interestingly, 6% of all exons, containing 124 reportedly disease-causing variants, were of uncertain significance. Finally, we used exon-level next-generation sequencing quality metrics generated at two clinical laboratories and identified a total of 43 technically challenging exons in 20 different genes that had inadequate coverage and/or homology issues that might lead to false-variant calls. We have demonstrated that transcript analysis plays a critical role in accurate clinical variant interpretation.