A Family With X-Linked Charcot-Marie-Tooth Disease Type 1: A Case for Reclassifying a Variant of Uncertain Significance in GJB1and Review of the Literature.

Panel-based germline genetic testing of established breast and ovarian cancer predisposition genes is widely used in high-risk clinics to inform on risks of breast and ovarian cancer. While most protein truncating mutations identified in predisposition genes are associated with moderate to high risks of these cancers, variants of uncertain significance (VUS), in the form of missense and intronic variants, have an undefined relevance to cancer. Many hundreds of VUS in BRCA1 and BRCA2 identified by clinical genetic testing over the last 20 years still have uncertain clinical relevance. In addition, VUS in other known predisposition genes are being identified by current panel-testing in up to 10% of individuals without BRCA1 or BRCA2 mutations. These VUS cause problems in cancer risk estimation and clinical management of patients undergoing clinical genetic testing. In an effort to evaluate the clinical significance of VUS in cancer predisposition genes we have developed functional assays that characterize the impact of the variants on the functions of the proteins encoded by the predisposition genes. In particular, a homology directed repair reporter assay providing a quantitative measure of homologous recombination repair of DNA double strand breaks has been developed for characterization of VUS in homologous recombination associated genes. When applied to 13 known pathogenic and 30 known neutral variants from the DNA binding domain of BRCA2, this assay exhibits 100% specificity and 100% sensitivity. Based on these results a model estimating the probability of pathogenicity of each BRCA2 VUS was developed. The HDR assay and probability model has now been applied to 230 BRCA2 VUS identified through genetic testing. Here we identify 80 BRCA2 variants that are deficient in HDR activity. We further combined these results with prior probabilities of pathogenicity derived from the sequence based Align GVGD in silico prediction model to estimate the posterior probability of pathogenicity for each VUS. Our results suggest that in the absence of family based segregation information, results from the HDR functional assay and in silico prediction models can be used to predict the pathogenicity of BRCA2 VUS. The assay has also been used to characterize VUS in the PALB2 and RAD51C predisposition genes, leading to identification of a subset of missense variants that are deficient in HDR repair activity and may be associated with increased risk of breast and ovarian cancer. These results will prove useful for improved risk assessment of carriers of these BRCA2, PALB2 and RAD51C variants and their family members and for validation of other approaches to mutation classification. Citation Format: Fergus J. Couch, Hermela Shimelis, Andreas Schroeder, Chunling Hu, Emily Hallberg, Gary Lipton, Edwin Iversen, Noralane M. Lindor. Characterization of variants of uncertain significance (VUS) in breast and ovarian cancer predisposition genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4685. doi:10.1158/1538-7445.AM2015-4685

Eligibility Criteria and Genetic Testing Results from a High-Risk Cohort for Hereditary Breast and Ovarian Cancer Syndrome in Southeastern Ontario

Variants of uncertain significance (VUSs) are rampant in clinical genetic testing, frustrating clinicians, patients, and laboratories because the uncertainty hinders diagnoses and clinical management. A comprehensive assessment of VUSs across many disease genes is needed to guide efforts to reduce uncertainty. To describe the sources, gene distribution, and population-level attributes of VUSs and to evaluate the impact of the different types of evidence used to reclassify them. This cohort study used germline DNA variant data from individuals referred by clinicians for diagnostic genetic testing for hereditary disorders. Participants included individuals for whom gene panel testing was conducted between September 9, 2014, and September 7, 2022. Data were analyzed from September 1, 2022, to April 1, 2023. The outcomes of interest were VUS rates (stratified by age; clinician-reported race, ethnicity, and ancestry groups; types of gene panels; and variant attributes), percentage of VUSs reclassified as benign or likely benign vs pathogenic or likely pathogenic, and enrichment of evidence types used for reclassifying VUSs. The study cohort included 1 689 845 individuals ranging in age from 0 to 89 years at time of testing (median age, 50 years), with 1 203 210 (71.2%) female individuals. There were 39 150 Ashkenazi Jewish individuals (2.3%), 64 730 Asian individuals (3.8%), 126 739 Black individuals (7.5%), 5539 French Canadian individuals (0.3%), 169 714 Hispanic individuals (10.0%), 5058 Native American individuals (0.3%), 2696 Pacific Islander individuals (0.2%), 4842 Sephardic Jewish individuals (0.3%), and 974 383 White individuals (57.7%). Among all individuals tested, 692 227 (41.0%) had at least 1 VUS and 535 385 (31.7%) had only VUS results. The number of VUSs per individual increased as more genes were tested, and most VUSs were missense changes (86.6%). More VUSs were observed per sequenced gene in individuals who were not from a European White population, in middle-aged and older adults, and in individuals who underwent testing for disorders with incomplete penetrance. Of 37 699 unique VUSs that were reclassified, 30 239 (80.2%) were ultimately categorized as benign or likely benign. A mean (SD) of 30.7 (20.0) months elapsed for VUSs to be reclassified to benign or likely benign, and a mean (SD) of 22.4 (18.9) months elapsed for VUSs to be reclassified to pathogenic or likely pathogenic. Clinical evidence contributed most to reclassification. This cohort study of approximately 1.6 million individuals highlighted the need for better methods for interpreting missense variants, increased availability of clinical and experimental evidence for variant classification, and more diverse representation of race, ethnicity, and ancestry groups in genomic databases. Data from this study could provide a sound basis for understanding the sources and resolution of VUSs and navigating appropriate next steps in patient care.

Background: Racial/ethnic disparities in minority access to genetic testing have perpetuated a higher likelihood of identifying an uncertain result in minority populations. Methods :Patient data was obtained from the Informed Genetics Annotated Patient Registry (iGAP), an IRB-approved multi-center longitudinal, observational study designed to capture genetic and genomic test results and their utilization and impact on treatment practices and outcomes. Patients self-declare race/ethnicity. Genetic panels contained between 1 and 148 genes and variant classification was determined by the performing genetic testing companies and reported as negative, variant of uncertain significance (VUS),likely-pathogenic, or pathogenic. Descriptive statistics were used to assess and compare data of these populations and germline genetic testing results indicating variant of uncertain significance. Results: The three racial/ethnic groups have similar percentages of BRCA1 and BRCA2pathogenic variants. However, Caucasians have considerably more pathogenic variants in lesser penetrant genes [see Table 1]. Conclusions: Racial/ethnic groups vary by volume of lesser penetrant genes with Caucasians having the highest numbers. Correlation by SNP heritage assignment may lead to a better understanding of these differences. Gene NameAshkenazi PV% Askenazi PVs% of PV subjects AshkenaziAshkenazi VUS%VUS Ashkenazi VUS% of subjects VUS AshkenaziAsian% Asian PVs% of PV subjects AsianAsian VUS%VUS Asian mutations% of subjects VUS AsianBlack/African% Black/African PVs% of PV subjectsBlack/AfricanBlack/African VUS%VUS Black/Africanmutations% of subjects VUSBlack/AfricanCaucasian% Caucasian PVs% of PV subjects CaucasianCaucasian VUS% Caucasian Mutations% of subjects VUS CaucasianHispanic% Hispanic PVs% of PV subjects HispanicHispanic VUS% Hispanic Mutations% of subjects VUS HispanicOther% Other PVs% of PV subjects OtherOther RE VUS% Other RE Mutations% of subjects VUS Other#PV Total% Total PV#VUS TotalBRCA20.00%0.00%0.00%0.00%125.00%25.00%24.17%4.26%531.25%31.25%34.62%4.76%3414.47%16.50%122.42%2.44%815.69%18.18%45.26%5.48%211.11%12.50%36.67%6.82%5014.97%24BRCA1327.27%30.00%0.00%0.00%0.00%0.00%36.25%6.38%318.75%18.75%23.08%3.17%177.23%8.25%91.81%1.83%1631.37%36.36%11.32%1.37%211.11%12.50%24.44%4.55%4112.28%17CHEK20.00%0.00%112.50%12.50%0.00%0.00%12.08%2.13%0.00%0.00%0.00%0.00%2711.49%13.11%51.01%1.02%11.96%2.27%0.00%0.00%15.56%6.25%12.22%2.27%298.68%8ATM19.09%10.00%0.00%0.00%0.00%0.00%510.42%10.64%0.00%0.00%812.31%12.70%218.94%10.19%346.85%6.92%35.88%6.82%911.84%12.33%0.00%0.00%36.67%6.82%257.49%59CHEK2/1100delC19.09%10.00%0.00%0.00%0.00%0.00%12.08%2.13%0.00%0.00%0.00%0.00%104.26%4.85%30.60%0.61%23.92%4.55%22.63%2.74%0.00%0.00%0.00%0.00%133.89%6MUTYH0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%114.68%5.34%51.01%1.02%11.96%2.27%22.63%2.74%15.56%6.25%12.22%2.27%133.89%8PALB20.00%0.00%0.00%0.00%0.00%0.00%12.08%2.13%0.00%0.00%0.00%0.00%72.98%3.40%71.41%1.43%35.88%6.82%11.32%1.37%15.56%6.25%12.22%2.27%113.29%10BLM19.09%10.00%0.00%0.00%0.00%0.00%48.33%8.51%0.00%0.00%34.62%4.76%93.83%4.37%112.22%2.24%0.00%0.00%22.63%2.74%0.00%0.00%0.00%0.00%102.99%20MITF0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%83.40%3.88%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%82.40%0NBN0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%11.54%1.59%83.40%3.88%40.81%0.81%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%82.40%5FH327.27%30.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%41.70%1.94%40.81%0.81%0.00%0.00%11.32%1.37%0.00%0.00%0.00%0.00%72.10%5MSH619.09%10.00%112.50%12.50%0.00%0.00%0.00%0.00%0.00%0.00%4.62%4.76%31.28%1.46%132.62%2.65%23.92%4.55%22.63%2.74%15.56%6.25%0.00%0.00%72.10%19MUTYH-Biallelic/Compound Heterozygous0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%52.13%2.43%10.20%0.20%11.96%2.27%0.00%0.00%15.56%6.25%0.00%0.00%72.10%1MUTYH-Monoallelic0.00%0.00%0.00%0.00%0.00%0.00%0.00%0.00%16.25%6.25%0.00%0.00%52.13%2.43%0.00%0.00%11.96%2.27%0.00%0.00%0.00%0.00%0.00%0.00%72.10%0PMS219.09%10.00%112.50%12.50%0.00%0.00%0.00%0.00%16.25%6.25%11.54%1.59%52.13%2.43%51.01%1.02%0.00%0.00%33.95%4.11%0.00%0.00%0.00%0.00%72.10%10Other00.00%0.00%562.50%62.50%375.00%75.00%3164.58%65.96%531.25%31.25%4467.69%69.84%6125.96%29.61%38377.22%78.00%1325.49%29.55%4964.47%67.12%950.00%56.25%3475.56%77.27%9127.25%546Grand Total11PV Ash108VUS Ash84PV Asi448Asi VUS4716PV Bla1665VUS Bla63235PV Cau206496VUS Cau49151PV His4476VUS His7318PV Oth1645VUS Oth44334334 PV Mutations Found738 Citation Format: Peter Beitsch, Chloe Wernecke, Kelly Bontempo, Brenna Bentley, Maureen Graham, Pat Whitworth, Rakesh Patel. Racial and ethnic groups have different clustering of common cancer genes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-19.

31P Survival outcomes in BRCA pathogenetic mutated, variant of unknown significance, and wild type ovarian cancer patients treated with PARP inhibitors

Genetic testing outcome disparities in minority racial and ethnic sub-populations within a hereditary breast and ovarian cancer program serving a diverse, urban-based population (191)

Establishing a genetic diagnosis may lead to major health benefits for the patient and their wider family, but is dependent on the accurate interpretation of test results. The processes of variant interpretation are by their nature imprecise such that the potential for uncertain test results (i.e., variant(s) of uncertain significance [VUS]) are an inevitable consequence of genomic testing. With an increased responsibility for diagnostic testing in the hands of the specialty physician (e.g., endocrinologist) rather than clinical geneticist, it is essential that they are familiar with the possible outcomes of testing including an understanding of the VUS category. While uncertainty is endemic to many aspects of clinical medicine, receiving a VUS result may pose a considerable challenge to both the clinician and the patient. In this article, a framework to support decision-making when confronted with a VUS variant is provided, focusing on the key components of the genetic testing pathway. This highlights the importance of assessing the VUS result in the context of the clinical presentation and genetic testing strategy, the value of multidisciplinary team workingand ensuring good communication with the patient.

Updated recommendations for CFTR carrier screening: A position statement of the American College of Medical Genetics and Genomics (ACMG)

10525 Background: Several groups have described disparities in genetic test results for inherited breast cancer predisposition, with a disproportionate number of variants of unknown significance (VUS) reported in non-Caucasian individuals. These disparities are due in part to the underrepresentation of non-Caucasians in reference databases and clinical genetic testing cohorts. Over the past few years, diversification efforts have been made however, little data exists on how ethnicity- and gene-specific VUS rates have changed over time and whether such disparities have improved or worsened. Methods: We retrospectively reviewed demographic information and test results for individuals who underwent hereditary cancer multigene panel testing between March 2012 and September 2020 at a single laboratory. Individuals who self-reported as African American, Asian, Caucasian, or Hispanic on the test requisition form and whose testing included ATM, BRCA1, BRCA2, CHEK2 and PALB2 (five commonly tested breast cancer predisposition genes with management guidelines) were included in the study (n = 284,130). The frequency of germline variants of unknown significance (VUS) in the five genes was assessed in September 2015 and September 2020. Results: Amongst patients tested between March 2012 and September 2015, 82.8% of the study cohort self-reported as Caucasian and 17.2% were not Caucasian (6.5% African American, 6.0% Hispanic, and 4.6% Asian). The proportion of non-Caucasian individuals in the study cohort increased slightly by September 2020 to 22.8% (77.2% Caucasian, 9.2% African American, 8.4% Hispanic, and 5.3% Asian). Consistent with previous reports, Caucasians had the lowest VUS rate overall in both 2015 and 2020. This was also true at the individual gene level, with the exception of CHEK2. Over time, we observed a relative decrease in VUS rates across all ethnicities. Between 2015 and 2020, the overall VUS rate for the five included genes in non-Caucasian individuals was reduced by 32.0% in non-Caucasians compared to 23.6% in Caucasians. The absolute difference in VUS rate between non-Caucasians and Caucasians decreased from 7.9% in 2015 to 4.5% in 2020. Conclusions: While VUS rates for commonly tested breast cancer predisposition genes remain higher in non-Caucasians relative to Caucasians, our results demonstrate that this gap has been reduced over a five-year time period. These findings may be indicative of efforts by clinicians and laboratories to reduce these disparities. Further studies are necessary to improve the clinical utility of genetic testing in under-represented populations.[Table: see text]

Imprecision medicine: Systematic gaps in reporting variants of uncertain significance (VUS) and their reclassifications.

Genomic screening of the general adult population: key concepts for assessing net benefit with systematic evidence reviews.

BackgroundVariant interpretation can change over time as new knowledge emerges. Our aim was to determine the frequency and causes of variant reinterpretation on systematic reevaluation in pediatric patients with cardiomyopathy.MethodsOverall, 227 unrelated pediatric patients with cardiomyopathy enrolled in the Heart Centre Biobank harbored a pathogenic/likely pathogenic (P/LP) variant or a variant of uncertain significance (VUS) on clinical genetic testing (2005–2022). Variant pathogenicity was reevaluated using the American College of Medical Genetics and Genomics guidelines. Additional extension cohorts (n=4547, cases) were analyzed to assess variant burden in cases versus controls (gnomAD 4.1.0).ResultsA total of 382 variants (110 P/LP, 272 VUS) in 227 patients were reevaluated. Forty‐nine variants in 49 patients (21.6%) changed classification. Twelve (10.9%) P/LP variants were downgraded to VUS in 14 patients. Leading criteria were high population allele frequency and variant not located in mutational hotspot or critical functional gene domain. Thirty‐seven (13.6%) VUS were upgraded to P/LP in 35 patients. Leading criteria were variant location in mutational hotspot for gene and deleteriousness on in silico prediction. Only 8 reclassified variants had been reported back by the clinical genetic testing laboratory at the time of the study. Ten of the 37 VUS upgraded to P/LP were significantly enriched in cardiomyopathy cases (n=4796) versus controls.ConclusionsOne in 5 patients with cardiomyopathy had a clinically relevant change in variant pathogenicity on systematic reevaluation that would require modifying family clinical screening and cascade genetic testing. These findings underscore the clinical importance of regular variant reinterpretation on follow‐up.

X-linked Charcot-Marie-Tooth Disease type 1(CMT1X) is the second most common form of inherited peripheral neuropathy that is caused by mutations in the gap junction beta-1 (GJB1) gene. Using targeted exome-sequencing, we investigated four CMT families from central-southern China and identified two novel missense variants (p.F31S and p.W44G) and two previously reported variants (p.R220Pfs*23 and p.Y157H) of GJB1. All four probands presented typical early-onset peripheral neuropathy, of which the R220Pfs*23 carrier also had neurologic manifestations in the central nervous system. We then constructed GJB1 expression vectors and performed cell biological analysis in vitro. Expression of FLAG-tagged GJB1 at various time points after transfection revealed evident protein aggregation with both wild-type and mutant forms, indicated with immunostaining and immunoblotting. Detergent-based sequential fractionation confirmed that all mutants were higher expressed and more prone to aggregate than the wild-type, whereas the R220Pfs*23 mutant showed the greatest amount of SDS-soluble multimers and monomers among groups. Moreover, intracellular aggregation probably occurs in the endoplasmic reticulum compartment rather than the Golgi apparatus. Gap junction plaques were present in all groups and were only compromised in frameshift mutant. Further evidence reveals significant intracellular stress granule formation induced by mutated GJB1 and impaired cell viability indicative of cytotoxicity of self-aggregates. Together, our findings demonstrate novel GJB1 variants-induced cell stress and dysfunction and provide insights into understanding the pathomechanisms of GJB1-CMTX1 and other related disorders.

Genetic Counseling and Testing for Pulmonary Arterial Hypertension in the United States

Framework From a Multidisciplinary Approach for Transitioning Variants of Unknown Significance From Clinical Genetic Testing in Kidney Disease to a Definitive Classification