Ana Vega

Splicing Predictions, Splicing Assays, and Variant Classification Using ACMG/AMP Guidelines: Challenges Observed with BRCA1 and BRCA2 Variants

Abstract Background Germline loss-of-function variants in BRCA1 and BRCA2 are established drivers of hereditary breast and ovarian cancer, often acting through aberrant splicing. However, not all spliceogenic changes are pathogenic, and many variants remain classified as uncertain due to insufficient experimental evidence and challenges in applying the ACMG/AMP variant interpretation framework to splicing alterations. Methods In this study, we examined the splicing outcomes of 17 variants—10 in BRCA1 [c.135-2A>G; c.135-5T>C; c.5074+1G>C; c.5332+2_5332+4del; c.5333-8C>T; c.5335C>G p.(Gln1779Glu); c.302-24_302-22del; c.302-23A>G; c.547+57T>C; c.4096+34C>G] and 7 in BRCA2 [c.-39-5delT; c.67+3A>G; c.425G>A p.(Ser142Asn); c.425G>T p.(Ser142Ile); c.517-13_517-9del; c.681+5G>C; c.67+84_67+85del]—identified in families with suspected hereditary breast and/or ovarian cancer. Depending on sample availability, we assessed splicing either on carrier-derived mRNA or via splicing-reporter minigene assay. Results Eight variants triggered aberrant splicing, while 9 showed no spliceogenic effect. Our findings, combined in some cases with previously published data, allowed us to apply the PVS1_(RNA) criterion at full strength to some variants. For others, residual full-length transcripts or in-frame mis-spliced isoforms precluded full application of PVS1_(RNA). Conclusions Following ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel specifications based on ACMG/AMP guidelines, we classified 4 variants as pathogenic or likely pathogenic, 10 as benign or likely benign, and 3 as uncertain significance. This comprehensive analysis of splicing defects refines the clinical classification of BRCA1 and BRCA2 variants and highlights the value of combining experimental and computational evidence to enhance genetic risk assessment in hereditary cancer.

Ovarian and Breast Cancer Risks Associated With Pathogenic Variants in RAD51C and RAD51D

Abstract Background The purpose of this study was to estimate precise age-specific tubo-ovarian carcinoma (TOC) and breast cancer (BC) risks for carriers of pathogenic variants in RAD51C and RAD51D. Methods We analyzed data from 6178 families, 125 with pathogenic variants in RAD51C, and 6690 families, 60 with pathogenic variants in RAD51D. TOC and BC relative and cumulative risks were estimated using complex segregation analysis to model the cancer inheritance patterns in families while adjusting for the mode of ascertainment of each family. All statistical tests were two-sided. Results Pathogenic variants in both RAD51C and RAD51D were associated with TOC (RAD51C: relative risk [RR] = 7.55, 95% confidence interval [CI] = 5.60 to 10.19; P = 5 × 10-40; RAD51D: RR = 7.60, 95% CI = 5.61 to 10.30; P = 5 × 10-39) and BC (RAD51C: RR = 1.99, 95% CI = 1.39 to 2.85; P = 1.55 × 10-4; RAD51D: RR = 1.83, 95% CI = 1.24 to 2.72; P = .002). For both RAD51C and RAD51D, there was a suggestion that the TOC relative risks increased with age until around age 60 years and decreased thereafter. The estimated cumulative risks of developing TOC to age 80 years were 11% (95% CI = 6% to 21%) for RAD51C and 13% (95% CI = 7% to 23%) for RAD51D pathogenic variant carriers. The estimated cumulative risks of developing BC to 80 years were 21% (95% CI = 15% to 29%) for RAD51C and 20% (95% CI = 14% to 28%) for RAD51D pathogenic variant carriers. Both TOC and BC risks for RAD51C and RAD51D pathogenic variant carriers varied by cancer family history and could be as high as 32–36% for TOC, for carriers with two first-degree relatives diagnosed with TOC, or 44–46% for BC, for carriers with two first-degree relatives diagnosed with BC. Conclusions These estimates will facilitate the genetic counseling of RAD51C and RAD51D pathogenic variant carriers and justify the incorporation of RAD51C and RAD51D into cancer risk prediction models.