Maaike P G Vreeswijk

Evidence for pathogenicity of BRCA2 c.8351G>A p.(Arg2784Gln) and the challenges in classification of pathogenic variants with reduced penetrance

Background The BRCA2 c.8351G>A p.(Arg2784Gln) variant has long been classified as a variant of uncertain significance (VUS) due to conflicting evidence used in variant classification. This study aims to clarify its pathogenicity and associated risks for breast and ovarian cancer. Methods This study was conducted by the international Evidence-based Network for the Interpretation of Germline Mutant Alleles consortium. We collected data from 29 informative families with this variant. Co-segregation likelihood ratios (LRs) were calculated using the full-likelihood method to assess pathogenicity, and cancer risks were estimated with modified segregation analysis. Results Co-segregation analysis using a grid search across scaled penetrance levels for BRCA2 truncating variants yielded the strongest evidence in favour of pathogenicity, with LR maximised at approximately 20% of full penetrance (LR=11.026). Furthermore, estimated breast cancer risks were markedly higher for early onset breast cancer; women diagnosed at <50 years had a HR of 4.5, compared with a HR of 1.65 for women diagnosed at ≥50 years. The estimated lifetime risks were 25% for breast cancer and 6% for ovarian cancer. Evidence of pathogenicity was also supported by the presence of the variant allele in two patients with Fanconi anaemia. Conclusions Our results indicate that BRCA2 c.8351G>A p.(Arg2784Gln) has a disease-causing effect, with reduced penetrance, similar to other pathogenic variants in moderate risk breast cancer genes such as ATM and CHEK2 . We also provide risk-adapted recommendations for clinical management. Importantly, one should be aware of a reduced penetrance as the underlying reason for conflicting results among pieces of evidence used for variant classification.

Variants of uncertain clinical significance in hereditary breast and ovarian cancer genes: best practices in functional analysis for clinical annotation

Germline DNA tests to identify pathogenic variants in genes linked to hereditary breast and ovarian cancer susceptibility have become widely available. However, the clinical utility of genetic testing depends on reliable evidence-based classification of sequence variants. Determination of pathogenicity traditionally relies on painstaking pedigree-based segregation analyses. However, the rapid increase in usage of germline DNA tests has led to the discovery of a large number of variants of uncertain clinical significance (VUS). For most VUS there is insufficient information for segregation analysis and therefore assessment of functional consequences is increasingly being used to support clinical annotation. Functional assays need to be accurate, robust, and reproducible to be used for clinical purposes. Here we use the lessons learned from

Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12

Abstract Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell–based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. Significance: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.

Performance of a RAD51-based functional HRD test on paraffin-embedded breast cancer tissue

Abstract Purpose BRCA-deficient breast cancers (BC) are highly sensitive to platinum-based chemotherapy and PARP inhibitors due to their deficiency in the homologous recombination (HR) pathway. However, HR deficiency (HRD) extends beyond BRCA-associated BC, highlighting the need for a sensitive method to enrich for HRD tumors in an alternative way. A promising approach is the use of functional HRD tests which evaluate the HR capability of tumor cells by measuring RAD51 protein accumulation at DNA damage sites. This study aims to evaluate the performance of a functional RAD51-based HRD test for the identification of HRD BC. Methods The functional HR status of 63 diagnostic formalin-fixed paraffin-embedded (FFPE) BC samples was determined by applying the RAD51-FFPE test. Samples were screened for the presence of (epi)genetic defects in HR and matching tumor samples were analyzed with the RECAP test, which requires ex vivo irradiated fresh tumor tissue on the premise that the HRD status as determined by the RECAP test faithfully represented the functional HR status. Results The RAD51-FFPE test identified 23 (37%) of the tumors as HRD, including three tumors with pathogenic variants in BRCA1/2. The RAD51-FFPE test showed a sensitivity of 88% and a specificity of 76% in determining the HR-class as defined by the RECAP test. Conclusion Given its high sensitivity and compatibility with FFPE samples, the RAD51-FFPE test holds great potential to enrich for HRD tumors, including those associated with BRCA-deficiency. This potential extends to situations where DNA-based testing may be challenging or not easily accessible in routine clinical practice. This is particularly important considering the potential implications for treatment decisions and patient stratification.