Agarwal, Mukesh K.

Background Missense (amino-acid changing) variants found in cancer predisposition genes often create difficulties when clinically interpreting genetic testing results. Although bioinformatics has developed approaches to predicting the impact of these variants, many of these approaches have not been readily applicable in the clinical setting. Bioinformatics approaches for predicting the impact of these variants have not yet found their footing in clinical practice because 1) interpreting the medical relevance of predictive scores is difficult; 2) the relationship between bioinformatics “predictors” (sequence conservation, protein structure) and cancer susceptibility is not understood. Methodology/Principal Findings We present a computational method that produces a probabilistic likelihood ratio predictive of whether a missense variant impairs protein function. We apply the method to a tumor suppressor gene, BRCA2, whose loss of function is important to cancer susceptibility. Protein likelihood ratios are computed for 229 unclassified variants found in individuals from high-risk breast/ovarian cancer families. We map the variants onto a protein structure model, and suggest that a cluster of predicted deleterious variants in the BRCA2 OB1 domain may destabilize BRCA2 and a protein binding partner, the small acidic protein DSS1. We compare our predictions with variant “re-classifications” provided by Myriad Genetics, a biotechnology company that holds the patent on BRCA2 genetic testing in the U.S., and with classifications made by an established medical genetics model [ 1 ]. Our approach uses bioinformatics data that is independent of these genetics-based classifications and yet shows significant agreement with them. Preliminary results indicate that our method is less likely to make false positive errors than other bioinformatics methods, which were designed to predict the impact of missense mutations in general. Conclusions/Significance Missense mutations are the most common disease-producing genetic variants. We present a fast, scalable bioinformatics method that integrates information about protein sequence, conservation, and structure in a likelihood ratio that can be integrated with medical genetics likelihood ratios. The protein likelihood ratio, together with medical genetics likelihood ratios, can be used by clinicians and counselors to communicate the relevance of a VUS to the individual who has that VUS. The approach described here is generalizable to regions of any tumor suppressor gene that have been structurally determined by X-ray crystallography or for which a protein homology model can be built.

AbstractThe assessment of the influence of many rare BRCA2 missense mutations on cancer risk has proved difficult. A multifactorial likelihood model that predicts the odds of cancer causality for missense variants is effective, but is limited by the availability of family data. As an alternative, we developed functional assays that measure the influence of missense mutations on the ability of BRCA2 to repair DNA damage by homologous recombination and to control centriole amplification. We evaluated 22 missense mutations from the BRCA2 DNA binding domain (DBD) that were identified in multiple breast cancer families using these assays and compared the results with those from the likelihood model. Thirteen variants inactivated BRCA2 function in at least one assay; two others truncated BRCA2 by aberrant splicing; and seven had no effect on BRCA2 function. Of 10 variants with odds in favor of causality in the likelihood model of 50:1 or more and a posterior probability of pathogenicity of 0.99, eight inactivated BRCA2 function and the other two caused splicing defects. Four variants and four controls displaying odds in favor of neutrality of 50:1 and posterior probabilities of pathogenicity of at least 1 × 10−3 had no effect on function in either assay. The strong correlation between the functional assays and likelihood model data suggests that these functional assays are an excellent method for identifying inactivating missense mutations in the BRCA2 DBD and that the assays may be a useful addition to models that predict the likelihood of cancer in carriers of missense mutations. [Cancer Res 2008;68(9):3523–31]