T. Walsh

Actionable exomic incidental findings in 6503 participants: challenges of variant classification

Recommendations for laboratories to report incidental findings from genomic tests have stimulated interest in such results. In order to investigate the criteria and processes for assigning the pathogenicity of specific variants and to estimate the frequency of such incidental findings in patients of European and African ancestry, we classified potentially actionable pathogenic single-nucleotide variants (SNVs) in all 4300 European- and 2203 African-ancestry participants sequenced by the NHLBI Exome Sequencing Project (ESP). We considered 112 gene-disease pairs selected by an expert panel as associated with medically actionable genetic disorders that may be undiagnosed in adults. The resulting classifications were compared to classifications from other clinical and research genetic testing laboratories, as well as with in silico pathogenicity scores. Among European-ancestry participants, 30 of 4300 (0.7%) had a pathogenic SNV and six (0.1%) had a disruptive variant that was expected to be pathogenic, whereas 52 (1.2%) had likely pathogenic SNVs. For African-ancestry participants, six of 2203 (0.3%) had a pathogenic SNV and six (0.3%) had an expected pathogenic disruptive variant, whereas 13 (0.6%) had likely pathogenic SNVs. Genomic Evolutionary Rate Profiling mammalian conservation score and the Combined Annotation Dependent Depletion summary score of conservation, substitution, regulation, and other evidence were compared across pathogenicity assignments and appear to have utility in variant classification. This work provides a refined estimate of the burden of adult onset, medically actionable incidental findings expected from exome sequencing, highlights challenges in variant classification, and demonstrates the need for a better curated variant interpretation knowledge base.

Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in middle eastern families

BackgroundIdentification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity.ResultsA custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population.ConclusionsCritical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.

The Founder Mutation MSH2*1906G→C Is an Important Cause of Hereditary Nonpolyposis Colorectal Cancer in the Ashkenazi Jewish Population

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in the mismatch-repair genes. We report here the identification and characterization of a founder mutation in MSH2 in the Ashkenazi Jewish population. We identified a nucleotide substitution, MSH2*1906G-->C, which results in a substitution of proline for alanine at codon 636 in the MSH2 protein. This allele was identified in 15 unrelated Ashkenazi Jewish families with HNPCC, most of which meet the Amsterdam criteria. Genotype analysis of 18 polymorphic loci within and flanking MSH2 suggested a single origin for the mutation. All colorectal cancers tested showed microsatellite instability and absence of MSH2 protein, by immunohistochemical analysis. In an analysis of a population-based incident series of 686 Ashkenazi Jews from Israel who have colorectal cancer, we identified 3 (0.44%) mutation carriers. Persons with a family history of colorectal or endometrial cancer were more likely to carry the mutation than were those without such a family history (P=.042), and those with colorectal cancer who carried the mutation were, on average, younger than affected individuals who did not carry it (P=.033). The mutation was not detected in either 566 unaffected Ashkenazi Jews from Israel or 1,022 control individuals from New York. In hospital-based series, the 1906C allele was identified in 5/463 Ashkenazi Jews with colorectal cancer, in 2/197 with endometrial cancer, and in 0/83 with ovarian cancer. When families identified by family history and in case series are included, 25 apparently unrelated Ashkenazi Jewish families have been found to harbor this mutation. Although this pathogenic mutation is not frequent in the Ashkenazi Jewish population (accounting for 2%-3% of colorectal cancer in those whose age at diagnosis is <60 years), it is highly penetrant and accounts for approximately one-third of HNPCC in Ashkenazi Jewish families that fulfill the Amsterdam criteria.

Abstract AS09: Germline mutations in cancer susceptibility genes in BRCA1 and BRCA2 negative families with ovarian and breast cancer

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Objectives: Germline mutations in cancer susceptibility genes other than BRCA1 and BRCA2 ( BRCA1/2 ) are found in approximately 6% of women with ovarian, fallopian tube, or primary peritoneal cancer. Our objective was to sequence BRCA1/2 -negative ovarian cancer patients with a family history of ovarian or breast cancer to identify inherited mutations that may explain the familial risk. Methods: We used a targeted capture, massively parallel sequencing test called BROCA on ovarian cancer probands with a family history of ovarian or breast cancer, or a personal history of breast cancer. BROCA testing included all known breast and ovarian cancer genes. Only clear loss of function mutations were included. 118 probands were ascertained from a gynecologic oncology tissue bank or outside referrals and provided informed consent. A family history of ovarian cancer was defined as having a first or second degree relative with ovarian cancer. A family history of breast cancer was defined as having a first or second degree relative with pre-menopausal breast cancer, or 2 or more regardless of menopausal status. Subjects were only included in one category. Results: Of 118 ovarian cancer probands, 22 (18.6%) were found to carry deleterious mutations in non- BRCA1/2 cancer susceptibility genes. 8/29 (27.6%) ovarian cancer patients with a personal history of breast cancer had mutations in 7 genes (2 CHEK2 , 2 RAD51D , 1 BRIP1 , 1 TP53 , 1 ATM , and one with both PALB2 and PMS2 ). This included mutations found in 2/5 (40%) who also had a family history of ovarian cancer and 4/10 (40%) who also had a family history of breast cancer. 38 patients had a family history of ovarian cancer with no personal history of breast cancer; 9/38 (23.7%) had mutations in 5 genes (3 BRIP1 , 3 RAD51C , 1 RAD51D , 1 TP53 , and 1 ATM ). Finally, 5/51 (9.8%) ovarian cancer patients with a family history of breast cancer and no personal history of breast cancer had mutations in 5 genes (1 MSH6 , 1 FAM175A , 1 NBN , 1 PALB2 , and 1 CHEK2 ). Conclusions: Germline mutations in DNA-repair genes are present in a substantial fraction of BRCA1/ 2-negative ovarian cancer patients with a personal or family history suggestive of inherited disease. These women may benefit from multiplex gene testing. The detection of inherited mutations in these women may be useful to identify the risk of other cancers, to inform family members of possible risk, and to direct therapy by suggesting candidates for PARP inhibitor therapy. Citation Format: B. Norquist, M. Harrell, T. Walsh, J. Mandell, S. Bernards, K. Agnew, M. Lee, K. Pennington, M.C. King, E. Swisher. Germline mutations in cancer susceptibility genes in BRCA1 and BRCA2 negative families with ovarian and breast cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS09.

Abstract A12: Mutations in homologous recombination genes and response to treatment in GOG 218: An NRG Oncology Study.

Objective: Bevacizumab is an anti-vascular endothelial growth factor monoclonal antibody with activity in ovarian cancer. GOG 218 was a phase III, randomized trial in advanced primary ovarian, fallopian tube, and peritoneal carcinoma (collectively, OC), examining the role of adding bevacizumab to a regimen of q21 day carboplatin and paclitaxel. Our objective was to examine whether mutations in homologous recombination (HR) genes affect response to treatment. Methods: We sequenced germline (from blood) and/or somatic (from neoplastic tissue) DNA from 1195 women enrolled in GOG 218 using the targeted capture and multiplex sequencing assay BROCA-HR, focusing on 16 DNA repair genes. Defects in HR were defined as germline or somatic mutations in genes predicted to affect HR, including BRCA1, BRCA2, and others. Proportional hazards models were used to provide estimates of relative hazards for progression free survival (PFS) and overall survival (OS) adjusted for clinical characteristics. Results: Of 1195 women with OC, germline or somatic mutations were identified in 147 (12.3%) in BRCA1, 78 (6.5%) in BRCA2, and 81 (6.8%) in other, non-BRCA HR genes. Total mutation frequency (all genes combined) in those with high-grade serous histology was 26.9% (261/971), but this was not significantly higher than the mutation frequency in endometrioid histology (10/42, 23.8%), clear cell histology (6/28, 21.4%), or unspecified carcinoma (20/90, 22.2%). Mutation frequency also did not differ by disease site. Median PFS and OS by group were as follows: BRCA1: 15.6 and 53.7 months, BRCA2: 21.6 and 75.2 months, non-BRCA HR: 16 and 56 months, and no mutation: 12.6 and 42 months. Adjusting for study treatment, stage, residual disease, and initial performance status, hazards for progression and death compared to those without mutations were significantly lower for those with mutations, specifically with BRCA1 mutations (hazard ratio (HR) 0.80, 95% CI 0.66 – 0.97, p=0.02 for PFS; HR 0.75, 95% CI 0.60 – 0.95, p=0.02 for OS), with BRCA2 mutations (HR 0.52, 95% CI 0.40 – 0.67, p Conclusions: Women with OC with either germline or somatic mutations affecting HR have significantly longer PFS and OS than those without mutations. Disease site and histology were not predictive of mutation status. The effect of mutation status on response by treatment arm will be reported at presentation. Citation Format: Barbara M. Norquist, Mark F. Brady, Maria I. Harrell, Tom D. Walsh, Ming K. Lee, Suleyman Gulsuner, Sarah S. Bernards, Silvia Casadei, Robert A. Burger, Susan A. Davidson, Robert S. Mannel, Paul A. DiSilvestro, Heather A. Lankes, Nilsa C. Ramirez, Mary Claire King, Michael J. Birrer, Elizabeth M. Swisher. Mutations in homologous recombination genes and response to treatment in GOG 218: An NRG Oncology Study. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A12.