Somatic inactivation of breast cancer predisposition genes in tumors associated with pathogenic germline variants.

BACKGROUND: Breast cancers (BCs) that arise in individuals heterozygous for a germline pathogenic variant in a susceptibility gene, such as BRCA1 and BRCA2, PALB2, and RAD51C, have been shown to exhibit biallelic loss in the respective genes and be associated with triple-negative breast cancer (TNBC) and distinctive somatic mutational signatures. Tumor sequencing thus presents an orthogonal approach to assess the role of candidate genes in BC development. METHODS: Exome sequencing was performed on paired normal-breast tumor DNA from 124 carriers of germline loss-of-function (LoF) or missense variant carriers in 15 known and candidate BC predisposition genes identified in the BEACCON case-control study. Biallelic inactivation and association with tumor genome features including mutational signatures and homologous recombination deficiency (HRD) score were investigated. RESULTS: BARD1-carrying TNBC (4 of 5) displayed biallelic loss and associated high HRD scores and mutational signature 3, as did a RAD51D-carrying TNBC and ovarian cancer. Biallelic loss was less frequent in BRIP1 BCs (4 of 13) and had low HRD scores. In contrast to other established BC genes, BCs from carriers of CHEK2 LoF (6 of 17) or missense (2 of 20) variant had low rates of biallelic loss. Exploratory analysis of BC from carriers of LoF variants in candidate genes such as BLM, FANCM, PARP2, and RAD50 found little evidence of biallelic inactivation. CONCLUSIONS: BARD1 and RAD51D behave as classic BRCA-like predisposition genes with biallelic inactivation, but this was not observed for any of the candidate genes. However, as demonstrated for CHEK2, the absence of biallelic inactivation does not provide definitive evidence against the gene's involvement in BC predisposition.

Estimating the proportion of pathogenic variants from breast cancer case-control data: Application to calibration of ACMG/AMP variant classification criteria.

For genes with reliable estimates of disease risk associated with loss-of-function variants, case-control data can be used to estimate the proportion of variants of typical risk effect for defined groups of variants, of relevance for variant classification. A calculation was derived for a maximum likelihood estimate of the proportion of pathogenic variants of typical effect from case-control data and applied to rare variant counts for ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C, and RAD51D from published breast cancer studies: BEACCON (5770 familial cases and 5741 controls) and breast cancer risk after diagnostic sequencing (60,466 familial and population-based cases and 53,461 controls). There was significant evidence of pathogenic variants among rare noncoding variants, in particular deeper intronic variants, for BRCA1 (13%, p = 8.3 × 10-7 ), BRCA2 (6%, p = 0.016) and PALB2 (13%, p = 0.001). The estimated proportion of pathogenic missense variants varied markedly between genes, generally with enrichment in familial cases, for example, 9% for BRCA2 versus 60%-90% for CHEK2. Stratifying missense variants by position indicated that, for most genes, location within a functional domain significantly predicted pathogenicity, whereas location outside domains provided robust evidence against pathogenicity. Our approach provides novel insights into the spectrum of pathogenic variants of specific breast cancer genes and has wider application to inform gene-focused specifications of American College of Medical Genetics and Genomics (ACMG)/Association of Molecular Pathology (AMP) codes for variant curation.

Integration of tumour sequencing and case-control data to assess pathogenicity of RAD51C missense variants in familial breast cancer.

While protein-truncating variants in RAD51C have been shown to predispose to triple-negative (TN) breast cancer (BC) and ovarian cancer, little is known about the pathogenicity of missense (MS) variants. The frequency of rare RAD51C MS variants was assessed in the BEACCON study of 5734 familial BC cases and 14,382 population controls, and findings were integrated with tumour sequencing data from 21 cases carrying a candidate variant. Collectively, a significant enrichment of rare MS variants was detected in cases (MAF < 0.001, OR 1.57, 95% CI 1.00-2.44, p = 0.05), particularly for variants with a REVEL score >0.5 (OR 3.95, 95% CI 1.40-12.01, p = 0.006). Sequencing of 21 tumours from 20 heterozygous and 1 homozygous carriers of nine candidate MS variants identified four cases with biallelic inactivation through loss of the wild-type allele, while six lost the variant allele and ten that remained heterozygous. Biallelic loss of the wild-type alleles corresponded strongly with ER- and TN breast tumours, high homologous recombination deficiency scores and mutational signature 3. Using this approach, the p.Gly264Ser variant, which was previously suspected to be pathogenic based on small case-control analyses and loss of activity in in vitro functional assays, was shown to be benign with similar prevalence in cases and controls and seven out of eight tumours showing no biallelic inactivation or characteristic mutational signature. Conversely, evaluation of case-control findings and tumour sequencing data identified p.Ile144Thr, p.Arg212His, p.Gln143Arg and p.Gly114Arg as variants warranting further investigation.

Investigation of monogenic causes of familial breast cancer: data from the BEACCON case-control study.

Breast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2, including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon-intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM, PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN. An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10-9) and missense (OR 1.27, p = 3.96 × 10-73) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2-4 and individually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1, WRN, PARP2, CTH and CDK9. The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.

Combined Tumor Sequencing and Case-Control Analyses of RAD51C in Breast Cancer.

BACKGROUND: Loss-of-function variants in RAD51C are associated with familial ovarian cancer, but its role in hereditary breast cancer remains unclear. The aim of this study was to couple breast tumor sequencing with case-control data to clarify the contribution of RAD51C to hereditary breast cancer. METHODS: RAD51C was sequenced in 3080 breast cancer index cases that were negative in BRCA1/2 clinical tests and 4840 population-matched cancer-free controls. Pedigree and pathology data were analyzed. Nine breast cancers and one ovarian cancer from RAD51C variant carriers were sequenced to identify biallelic inactivation of RAD51C, copy number variation, mutational signatures, and the spectrum of somatic mutations in breast cancer driver genes. The promoter of RAD51C was analyzed for DNA methylation. RESULTS: A statistically significant excess of loss-of-function variants was identified in 3080 cases (0.4%) compared with 2 among 4840 controls (0.04%; odds ratio = 8.67, 95% confidence interval = 1.89 to 80.52, P< .001), with more than half of the carriers having no personal or family history of ovarian cancer. In addition, the association was highly statistically significant among cases with estrogen-negative (P <. 001) or triple-negative cancer (P < .001), but not in estrogen-positive cases. Tumor sequencing from carriers confirmed bi-allelic inactivation in all the triple-negative cases and was associated with high homologous recombination deficiency scores and mutational signature 3 indicating homologous recombination repair deficiency. CONCLUSIONS: This study provides evidence that germline loss-of-function variants of RAD51C are associated with hereditary breast cancer, particularly triple-negative type. RAD51C-null breast cancers possess similar genomic and clinical features to BRCA1-null cancers and may also be vulnerable to DNA double-strand break inducing chemotherapies and poly ADP-ribose polymerase inhibitors.