Variant Detection in 3' Exons of PMS2 Using Exome Sequencing Data.

PMS2 is one of the mismatch repair genes included in routine genetic testing for Lynch syndrome, colorectal, ovarian, and endometrial cancers. PMS2 is also included in the American College of Medical Genetics and Genomics (ACMG) secondary findings gene list in the context of clinical exome and genome sequencing. However, sequencing of PMS2 by short-read based next generation sequencing (NGS) technologies is complicated by the presence of the pseudogene PMS2CL and often supplemented by long-range based approaches such as long-range polymerase chain reaction (LR-PCR) or long-read based next generation sequencing, which increases the complexity and cost. Here, we described a bioinformatics homology triage workflow that can eliminate the need for long-read based testing for PMS2 for the vast majority of patients undergoing exome sequencing, thus simplifying PMS2 testing and reducing the associated cost.

Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain.

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.