Integration of tumour sequencing and case\u2013control data to assess pathogenicity of RAD51C missense variants in familial breast cancer

Belle W X Lim,Simone M Rowley,Simone Mcinerny,Na Li,Magnus Zethoven,Rodney J Scott,Erica K Sloan,Ian G Campbell,Ella R Thompson,Lisa Devereux,Paul A James

doi:10.1038/s41523-021-00373-y

Abstract

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.

Highlights

Protein-truncating variants in RAD51C predispose to high-grade serous ovarian cancer (HGSOC) and triple-negative (TN) breast cancer (BC), and when these cancers occur in carriers of truncating variants they exhibit biallelic inactivation[1,2,3]
To further investigate the potential pathogenicity of candidate variants, we exploited the fact that RAD51C appears to conform to Knudson’s “two-hit” hypothesis, and performed tumour sequencing from variant carriers to assess for biallelic inactivation and associated homologous recombination deficiency (HRD)
We have previously demonstrated the utility of this reproach for RAD51C loss of function (LoF) variants by revealing the presence of biallelic inactivation in the form of loss of heterozygosity (LOH) in TN BCs that was associated with high

Summary

INTRODUCTION

Protein-truncating variants in RAD51C predispose to high-grade serous ovarian cancer (HGSOC) and triple-negative (TN) breast cancer (BC), and when these cancers occur in carriers of truncating variants they exhibit biallelic inactivation[1,2,3]. BC case–control studies to date have identified potentially predisposing RAD51C MS variants, such as p.Gly264Ser[4,5,6], p.Gln143Arg[7,8] and pArg258His[9,10], while target protein and cellular assays have suggested functional impact and pathogenicity of variants including p.Cys135Tyr and p. To further investigate the potential pathogenicity of candidate variants, we exploited the fact that RAD51C appears to conform to Knudson’s “two-hit” hypothesis, and performed tumour sequencing from variant carriers to assess for biallelic inactivation and associated homologous recombination deficiency (HRD). Case–control analysis data were combined with tumour sequencing, in silico prediction tools, and pedigree segregation to assess the pathogenicity of RAD51C MS variants

RESULTS

METHODS

CODE AVAILABILITY