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.

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

PMS2 is one of the DNA-mismatch repair genes included in routine genetic testing for Lynch syndrome and colorectal, ovarian, and endometrial cancers. PMS2 is also included in the American College of Medical Genetics and Genomics' List of Secondary Findings Genes in the context of clinical exome and genome sequencing. However, sequencing of PMS2 by short-read-based next-generation sequencing technologies is complicated by the presence of the pseudogene PMS2CL, and is often supplemented by long-range-based approaches, such as long-range PCR or long-read-based next-generation sequencing, which increases the complexity and cost. This article describes a bioinformatics homology triage workflow that can eliminate the need for long-read-based testing for PMS2 in the vast majority of patients undergoing exome sequencing, thus simplifying PMS2 testing and reducing the associated cost.

Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline ATM sequence variants.

The ClinGen Hereditary Breast, Ovarian and Pancreatic Cancer (HBOP) Variant Curation Expert Panel (VCEP) is composed of internationally recognized experts in clinical genetics, molecular biology and variant interpretation. This VCEP made specifications for ACMG/AMP guidelines for the ataxia telangiectasia mutated (ATM) gene according to the Food and Drug Administration (FDA)-approved ClinGen protocol. These gene-specific rules for ATM were modified from the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP) guidelines and were tested against 33 ATM variants of various types and classifications in a pilot curation phase. The pilot revealed a majority agreement between the HBOP VCEP classifications and the ClinVar-deposited classifications. Six pilot variants had conflicting interpretations in ClinVar and reevaluation with the VCEP's ATM-specific rules resulted in four that were classified as benign, one as likely pathogenic and one as a variant of uncertain significance (VUS) by the VCEP, improving the certainty of interpretations in the public domain. Overall, 28 the 33 pilot variants were not VUS leading to an 85% classification rate. The ClinGen-approved, modified rules demonstrated value for improved interpretation of variants in ATM.