Citations

Abstract Approximately 15% of genetic screens for mutations in BRCA1 and BRCA2 identify Variants of Uncertain Significance (VUS). Primarily missense mutations, VUS are often difficult to interpret, leading to either uncertainty in how to properly counsel a patient or an unnecessary prophylactic surgery. Given the paucity of data for which missenses are classified as truly pathogenic, computational deleterious missense prediction (DMP) algorithms are used to predict whether a mutation is likely deleterious or neutral. Accuracy of DMPs can vary considerably and have only been calibrated on a relatively small number of missense mutations of demonstrable effect on protein function. In this study, the performance of 41 different DMPs was compared to functional data from 455 functionally characterized missense variants in BRCA1 and BRCA2. New optimized thresholds for classifying missense mutations as deleterious are presented for several existing models as well as a newly derived naïve voting method (NVM). The areas under the curve estimates for the NVM approach are between 0.889-0.922, much higher than previous methods. We estimate that the overall pathogenic potential of missense variants to be 6.8% for BRCA1 and 3.2% of BRCA2, but can be as high as 50% depending on protein location. Overall these results provide key insights into how to predict deleterious missense mutations in BRCA1 and BRCA2. Citation Format: Hart SN, Hoskin T, Shimelis H, Feng B, Lindor NM, Monteiro A, Iversen E, Goldgar DE, Suman V, Couch FJ. Optimized prediction of deleterious missense mutations in BRCA1 and BRCA2 genes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-02-03.

Background The rarity of mutations in PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study. Methods We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant. Results For European women, strong evidence of association with breast cancer risk was observed for PALB2 c.1592delT OR 3.44 (95% CI 1.39 to 8.52, p=7.1×10−5), PALB2 c.3113G>A OR 4.21 (95% CI 1.84 to 9.60, p=6.9×10−8) and ATM c.7271T>G OR 11.0 (95% CI 1.42 to 85.7, p=0.0012). We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p≤0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03 (95% CI 1.53 to 6.03, p=0.0006) for African men and CHEK2 c.1312G>T OR 2.21 (95% CI 1.06 to 4.63, p=0.030) for European men. No evidence of association with ovarian cancer was found for any of these variants. Conclusions This report adds to accumulating evidence that at least some variants in these genes are associated with an increased risk of breast cancer that is clinically important.

Abstract Background: While numerous susceptibility loci for epithelial ovarian cancer (EOC) have been identified, few associations have been reported with overall survival. In the absence of common prognostic genetic markers, we hypothesize that rare coding variants may be associated with overall EOC survival and assessed their contribution in two exome-based genotyping projects of the Ovarian Cancer Association Consortium (OCAC). Methods: The primary patient set (Set 1) included 14 independent EOC studies (4,293 patients) and 227,892 variants, and a secondary patient set (Set 2) included six additional EOC studies (1,744 patients) and 114,620 variants. Because power to detect rare variants individually is reduced, gene-level tests were conducted. Sets were analyzed separately at individual variants and by gene, and then combined with meta-analyses (73,203 variants and 13,163 genes overlapped). Results: No individual variant reached genome-wide statistical significance. A SNP previously implicated to be associated with EOC risk and, to a lesser extent, survival, rs8170, showed the strongest evidence of association with survival and similar effect size estimates across sets (Pmeta = 1.1E−6, HRSet1 = 1.17, HRSet2 = 1.14). Rare variants in ATG2B, an autophagy gene important for apoptosis, were significantly associated with survival after multiple testing correction (Pmeta = 1.1E−6; Pcorrected = 0.01). Conclusions: Common variant rs8170 and rare variants in ATG2B may be associated with EOC overall survival, although further study is needed. Impact: This study represents the first exome-wide association study of EOC survival to include rare variant analyses, and suggests that complementary single variant and gene-level analyses in large studies are needed to identify rare variants that warrant follow-up study. Cancer Epidemiol Biomarkers Prev; 25(3); 446–54. ©2016 AACR.

ABSTRACTEpithelial‐mesenchymal transition (EMT) is a process whereby epithelial cells assume mesenchymal characteristics to facilitate cancer metastasis. However, EMT also contributes to the initiation and development of primary tumors. Prior studies that explored the hypothesis that EMT gene variants contribute to epithelial ovarian carcinoma (EOC) risk have been based on small sample sizes and none have sought replication in an independent population. We screened 15,816 single‐nucleotide polymorphisms (SNPs) in 296 genes in a discovery phase using data from a genome‐wide association study of EOC among women of European ancestry (1,947 cases and 2,009 controls) and identified 793 variants in 278 EMT‐related genes that were nominally (P < 0.05) associated with invasive EOC. These SNPs were then genotyped in a larger study of 14,525 invasive‐cancer patients and 23,447 controls. A P‐value <0.05 and a false discovery rate (FDR) <0.2 were considered statistically significant. In the larger dataset, GPC6/GPC5 rs17702471 was associated with the endometrioid subtype among Caucasians (odds ratio (OR) = 1.16, 95% CI = 1.07–1.25, P = 0.0003, FDR = 0.19), whereas F8 rs7053448 (OR = 1.69, 95% CI = 1.27–2.24, P = 0.0003, FDR = 0.12), F8 rs7058826 (OR = 1.69, 95% CI = 1.27–2.24, P = 0.0003, FDR = 0.12), and CAPN13 rs1983383 (OR = 0.79, 95% CI = 0.69–0.90, P = 0.0005, FDR = 0.12) were associated with combined invasive EOC among Asians. In silico functional analyses revealed that GPC6/GPC5 rs17702471 coincided with DNA regulatory elements. These results suggest that EMT gene variants do not appear to play a significant role in the susceptibility to EOC.

Abstract 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

Abstract Background: Recent bioinformatic analyses and high-throughput sequencing efforts have revealed that RNA editing is a widespread post-transcriptional modification that affects non-coding repetitive elements throughout the genome. This modification, which involves the conversion of adenosine into inosine, is mediated by the action of the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on reports of dysfunctional ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleotide polymorphisms (SNPs) in five ADAR genes (ADAD1, ADAR1, ADAR2, ADAR3, SND1) may contribute to EOC susceptibility, potentially by altering the expression of corresponding genes in ovarian tissues. Methods: Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 healthy controls of European ancestry represented in the international Collaborative Oncological Gene-environment Study, we evaluated the frequency of 7,133 SNPs in 5 RNA editing genes. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) between genotypes and case status under a log-additive genetic model, with adjustment for the first five principal components representing European ancestry. Subgroup analysis was conducted for the four main histologic sub-types of EOC. We also aggregated SNP-level data and conducted gene-level analyses using the Admixture Maximum Likelihood (AML) test and the Sequence-Kernel Association test for the combined effect of common and rare variants (SKAT-CR). Results: Association analysis revealed a top risk-associated SNP (OR (95% CI) = 1.39 (1.17-1.64)) in ADAR3 with P = 1.3 x10−4, 17 additional SNPs in ADAR1, ADAR3, or SND1 with P = 10−4, and 51 SNPs with P = 10−3; this observed number of statistically-significant SNPs was greater than expected. When restricting to the 6,500 invasive serous adenocarcinomas, the magnitude of association strengthened for numerous SNPs. Exploratory analysis for the less common histologic subtypes (endometrioid (n = 1,439), mucinous (n = 696), and clear cell (n = 660)) revealed SNP-level associations (P&lt;10−3) unique to each sub-type. Gene-level analyses based on AML and SKAT-CR revealed that ADAR1 was globally associated (P&lt;0.10) with susceptibility to invasive EOC, with P = 0.024 and P = 0.023, respectively. Expression quantitative trait locus analysis is underway to evaluate associations between genotype and gene expression in EOC and precursor tissues using data from The Cancer Genome Atlas Project and other sources. Conclusions: Preliminary data from this large-scale collaboration suggest that germline variation involving RNA editing genes may influence susceptibility to EOC. This novel finding warrants further investigation, as inherited and acquired alterations affecting RNA editing may serve as important biological mechanisms that promote the development of EOC and possibly other malignancies. Citation Format: Jennifer Permuth-Wey, Brett M. Reid, Y. Ann Chen, Hui-Yi Lin, Alvaro Monteiro, Zhihua Chen, Andrew Berchuck, Georgia Chenevix-Trench, Jennifer Doherty, Simon Gayther, Ellen Goode, Edwin Iversen, Leigh Pearce, Paul D. P. Pharoah, Catherine Phelan, Susan Ramus, Mary Anne Rossing, Joellen Schildkraut, Thomas Sellers, on behalf of the Ovarian Cancer Association Consortium. Inherited variants affecting RNA editing may contribute to ovarian cancer susceptibility. [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 4635. doi:10.1158/1538-7445.AM2015-4635

Abstract Background: Cell-type-specific super enhancers, or large clusters of mediator-rich enhancers, are highly transcribed regions of the genome with key functions in the maintenance of cell identity and control. These regulatory elements are easily perturbed and enriched for disease-associated sequence variation. Identification of disease-associated super-enhancers and investigation of their sequence variants may help identify important, biologically relevant associations previously overlooked by genome-wide studies. Thus, we sought to identify variants within ovarian tissue specific super-enhancers and investigate their association with epithelial ovarian cancer (EOC) susceptibility. Methods: We utilized genotype data from a European population of ∼11,000 invasive EOC cases and 22,000 healthy controls from the Ovarian Cancer Association Consortium. A public catalog of 478 super-enhancers, identified by peak histone H3K27ac ChIP-seq signal in ovarian tissue, was used to identify 72,116 variants located within 344 super-enhancer regions. Unconditional logistic regression (log-additive model) was used to assess individual SNP-EOC risk associations. Additionally, we reviewed associations within 500 kb of super-enhancer regions to identify proximal associated variants. When LD structure indicated, we employed conditional analysis to determine independence of the super-enhancer signal. Gene-EOC risk associations were assessed with the Admixture Maximum Likelihood (AML) test and Sequence-Kernel Association Test (SKAT) for the combined effect of common and rare (MAF&lt;0.01) variants. Models were adjusted for population stratification using the first five principal components. To adjust for multiple comparisons, a false discovery rate of 10% was used. Results: The most significant SNP observed was rs147487657 (p = 7.7×10−6) on locus 2q37.3 in the HDAC4 gene and there were an additional 12 SNPs with p&lt;1.0×10−4, although not significant at 10% FDR. Gene-level analyses identified two super-enhancers significantly associated with EOC risk. An enhancer at the previously reported 2q31 locus, containing HOXD8 and HOXD-AS2, associated (SKAT p = 4.3×10−5; AML p&lt;0.001) with the top SNP located within the 5′UTR of HOXD8 transcription factor (rs847163 (T&gt;C); p&lt;2.08×10−5). Conditional analysis did not identify an independent association within the super-enhancer region. The second EOC-associated super-enhancer identified was at locus 1q32 (SKAT p = 0.00052, AML p = 0.001) containing the CSRP1 gene. There were 6 SNPs with p&lt;0.001 and 16 SNPs with p&lt;0.01, all were inversely associated with risk and consistent with the top hit at rs927901 T&gt;C (OR = 0.94; 95% CI: 0.90-0.97; p = 2.1×10−4). Associations varied by histologic subtype, possibly due to variance in disease relevant super-enhancer cell-type.Conclusion: These findings suggest germline variation within super-enhancer regions may contribute to EOC susceptibility Citation Format: Brett M. Reid, Jennifer Permuth-Wey, Y. Ann Chen, Hui-Yi Lin, Alvaro Monteiro, Zhihua Chen, Andrew Berchuck, Georgia Chenevix-Trench, Jennifer Doherty, Simon Gayther, Ellen L. Goode, Edwin Iversen, Leigh Pearce, Paul Pharoah, Catherine Phelan, Susan Ramus, Mary Anne Rossing, Joellen Schildkraut, Thomas Sellers, on behalf of the Ovarian Cancer Association Consortium. Variants within super-enhancer regulatory elements associate with epithelial ovarian cancer risk. [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 4634. doi:10.1158/1538-7445.AM2015-4634