TY - JOUR
T1 - Functional Categorization of BRCA1 Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays
AU - Bouwman, Peter
AU - van der Heijden, Ingrid
AU - van der Gulden, Hanneke
AU - de Bruijn, Roebi
AU - Braspenning, Merel E
AU - Moghadasi, Setareh
AU - Wessels, Lodewyk F A
AU - Vreeswijk, Maaike P G
AU - Jonkers, Jos
AU - Blok, Marinus
AU - Dutch-Belgian VUS workgroup
N1 - ©2020 American Association for Cancer Research.
PY - 2020/9/1
Y1 - 2020/9/1
N2 - PURPOSE: Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway.EXPERIMENTAL DESIGN: Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families.RESULTS: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants.CONCLUSIONS: We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.
AB - PURPOSE: Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway.EXPERIMENTAL DESIGN: Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families.RESULTS: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants.CONCLUSIONS: We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.
KW - MISSENSE VARIANTS
KW - SEQUENCE VARIANTS
KW - BREAST-CANCER
KW - INTEGRATED EVALUATION
KW - SPLICING ANALYSIS
KW - DIRECTED REPAIR
KW - OVARIAN-CANCER
KW - CLASSIFICATION
KW - GUIDELINES
KW - MUTATIONS
U2 - 10.1158/1078-0432.ccr-20-0255
DO - 10.1158/1078-0432.ccr-20-0255
M3 - Article
C2 - 32546644
SN - 1078-0432
VL - 26
SP - 4559
EP - 4568
JO - Clinical Cancer Research
JF - Clinical Cancer Research
IS - 17
ER -