Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome.

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are "variants of uncertain significance" (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆GFold) for all DVD missense variants. We find that 5772 VUSs have a large, destabilizing ∆∆GFold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3456 VUSs are likely pathogenic at a probability of 99.0%. Of the 224 genes in the DVD, 166 genes (74%) exhibit one or more missense variants predicted to cause a pathogenic change in protein folding stability. The VUSs prioritized here affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.

Assessing Variants of Uncertain Significance Implicated in Hearing Loss Using a Comprehensive Deafness Proteome.

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6,328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are "variants of uncertain significance" (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆G Fold ) for all DVD missense variants. We find that 5,772 VUSs have a large, destabilizing ∆∆G Fold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3,456 VUSs are likely pathogenic at a probability of 99.0%. These VUSs affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.

Is it Usher syndrome? Collaborative diagnosis and molecular genetics of patients with visual impairment and hearing loss.

Background: Usher syndrome is the most common hereditary syndrome combining deafness and blindness. In the 2017 National Child Count of Children and Youth who are Deaf-Blind, Usher syndrome represented 329 of 10,000 children, but there were also at least 70 other etiologies of deaf-blindness documented. The purpose of this study was to analyze the work-up and ultimate diagnoses of 21 consecutive families who presented to the Genetic Eye-Ear Clinic (GEEC) at the University of Iowa. Our hypothesis was that most families referred to the GEEC would have initial and final diagnoses of Usher syndrome.Materials and Methods: Patients were identified through an IRB approved retrospective chart review of referrals to the GEEC between 2012 and 2019. Details about each patient's history, exam, and clinical and genetic work-up were recorded.Results: From 2012 to 2019, 21 families (25 patients) were referred to the collaborative GEEC. Overall molecular diagnostic rate in this cohort was 14/21 (67%). Evaluation resulted in a change of diagnosis in 11/21 (52%) families. Ultimately, there were eleven unique diagnoses including hereditary, non-hereditary, and independent causes of combined visual impairment and hearing loss. The most common diagnosis was Usher syndrome, which represented 6/21 (29%) families.Conclusions: Providing a correct diagnosis for patients with visual impairment and hearing loss can be challenging for clinicians and their patients, but it can greatly improve clinical care and outcomes. We recommend an algorithm that includes multidisciplinary collaboration, careful clinical evaluation, strategic molecular testing, and consideration of a broad differential diagnosis.

Comprehensive genetic testing in the clinical evaluation of 1119 patients with hearing loss.

Hearing loss is the most common sensory deficit in humans, affecting 1 in 500 newborns. Due to its genetic heterogeneity, comprehensive diagnostic testing has not previously been completed in a large multiethnic cohort. To determine the aggregate contribution inheritance makes to non-syndromic hearing loss, we performed comprehensive clinical genetic testing with targeted genomic enrichment and massively parallel sequencing on 1119 sequentially accrued patients. No patient was excluded based on phenotype, inheritance or previous testing. Testing resulted in identification of the underlying genetic cause for hearing loss in 440 patients (39%). Pathogenic variants were found in 49 genes and included missense variants (49%), large copy number changes (18%), small insertions and deletions (18%), nonsense variants (8%), splice-site alterations (6%), and promoter variants (<1%). The diagnostic rate varied considerably based on phenotype and was highest for patients with a positive family history of hearing loss or when the loss was congenital and symmetric. The spectrum of implicated genes showed wide ethnic variability. These findings support the more efficient utilization of medical resources through the development of evidence-based algorithms for the diagnosis of hearing loss.

Copy number variants are a common cause of non-syndromic hearing loss.

BACKGROUND: Copy 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. METHODS: We 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. RESULTS: Of 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). CONCLUSION: CNVs are an important cause of NSHL and their detection must be included in comprehensive genetic testing for hearing loss.

Mutations in alternative pathway complement proteins in American patients with atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is characterized by acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia, and occurs with an estimated incidence in the USA of 2 per 1,000,000. Disease pathogenesis is related to dysregulation of the alternative pathway (AP) of the complement cascade at the level of the cell membrane secondary to mutations in a number of complement genes including complement factor H (CFH), complement factor H-related 5 (CFHR5), complement factor I (CFI), CD46 (MCP), complement factor B (CFB), complement component 3 (C3) and thrombomodulin (THBD). Since aHUS is rare, mutation rate data in large patient cohorts are scarce. Here we present the first cohort of American patients in whom mutation screening was completed on all genes currently implicated in aHUS. In addition to identifying a number of novel variants, we provide information on the relative frequency of mutations in these genes in an American aHUS population. Twelve percent (12%) of patients carrying disease-associated genetic variants segregated mutations in more than one gene mandating comprehensive genetic testing in the diagnosis and management of these patients.

Factors influencing openness to future smoking among nonsmoking adolescents.

BACKGROUND: To investigate the correlates of youth tobacco use in terms of nonsmoking adolescents' openness to future smoking, a secondary analysis of the 2000 and 2004 Indiana Youth Tobacco Survey (IYTS) was conducted. METHODS: A representative sample of 1416 public high school students in grades 9-12 and 1516 public middle school students in grades 6-8 (71.44% and 72.53% response rates, respectively) were surveyed in 2000, and 3433 public high school students and 1990 public middle school students (63.04% and 65.44% response rates, respectively) were surveyed in 2004. RESULTS: Seventy-four percent of students in 2000 were not open to future smoking and 77% were not open in 2004. The adolescent cohort in 2004 became more exposed to antitobacco messages and less exposed to protobacco messages and environmental tobacco smoke (ETS) compared with their counterpart in 2000. Whereas gender, grade, race/ethnicity, and exposure to antitobacco messages were insignificant predictors for openness to future smoking, exposure to ETS either in homes or in cars was a strong predictor for openness to future smoking (the higher the exposure to ETS, the more open to future smoking) in both unadjusted and adjusted multivariate models. Exposure to protobacco messages had a greater effect on openness to future smoking than exposure to antitobacco messages. The rate of transition from openness to future smoking to tobacco use initiation is higher among white adolescents than among minority adolescents. CONCLUSIONS: More efforts should be made to reduce adolescents' exposure to ETS and protobacco messages.