Likely Pathogenic Variants were Enriched in the Case Cohort
A total of 51 unique rare MS variants (MAF < 0.005) were detected in 65 cases (1.13%) and 134 controls (0.91%) (OR 1.22, 95% CI 0.89–1.65, p = 0.21) (Table 1). Several parameters were used to enrich for potentially pathogenic variants including population frequency, location in known function domains, in silico pathogenicity prediction and tumour phenotype. Consistent with the hypothesis that rare variants are more likely to be deleterious (26), a reduction of the population frequency threshold resulted in increasing odds ratios that reached statistical significance at MAF < 0.0001 (OR 1.87, 95%CI 1.14–3.03, p = 0.01). Similarly, higher CADD and REVEL score thresholds that should enrich for pathogenic variants were associated with higher odds ratios, especially for a REVEL score of > 0.5 (OR 3.95, 95%CI 1.40–12.01, p = 0.006). Two overlapping functional domains are present in the N-terminal third of RAD51C protein (Holliday junction activity: amino acids 1-126; Interaction with RAD51B, RAD51D and XRCC3: amino acids 79–136) and a significant enrichment of MS variants in cases was observed in the interaction domain (OR 10.04, 95%CI 0.99–494.1, p = 0.03), although the number of variants was low (n = 5), resulting in a wide confidence interval.
Table 1
Frequencies of RAD51C missense variants in case and control cohorts according to different filtering criteria to enrich for likely pathogenic variants.
| Groups | Carrier Frequency | Sample Size | p-value | OR (95% CI) |
Case (%) | Control (%) | Case | Control | | |
Rarity | MAF < 0.005 | 65 (1.13%) | 134 (0.91%) | 5734 | 14382 | 0.21 | 1.22 (0.89–1.65) |
MAF < 0.001 | 35 (0.61%) | 56 (0.38%) | 0.05 | 1.57 (1.00-2.44) |
MAF < 0.0001 | 32 (0.56%) | 43 (0.29%) | 0.01 | 1.87 (1.14–3.03) |
In-silico | CADD > 20 | 59 (1.03%) | 125 (0.85%) | 0.29 | 1.19 (0.85–1.63) |
CADD > 25 | 17 (0.30%) | 22 (0.15%) | 0.05 | 1.94 (0.97–3.83) |
REVEL > 0.3 | 17 (0.30%) | 19 (0.13%) | 0.02 | 2.25 (1.10–4.57) |
REVEL > 0.5 | 11 (0.19%) | 7 (0.05%) | 0.006 | 3.95 (1.40-12.01) |
Functional domain | Interaction domain | 4 (0.070%) | 1 (0.01%) | 0.03 | 10.04 (0.99–493.1) |
Holliday domain | 6 (0.10%) | 7 (0.05%) | 0.21 | 2.15 (0.60–7.48) |
Hormone receptor subtype | ER-positive | 23 (1.04%) | 137 (0.93%) | 2209 | 0.64 | 1.09 (0.67–1.71) |
ER-negative | 20 (1.58%) | 1262 | 0.04 | 1.67 (0.99–2.70) |
HER2-positive | 7 (1.21%) | 579 | 0.51 | 1.27 (0.50–2.7)1 |
HER2-negative | 29 (1.20%) | 2426 | 0.27 | 1.26 (0.81–1.89) |
TN | 13 (1.49%) | 871 | 0.11 | 1.58 (0.81–2.80) |
Non-TN | 23 (1.08%) | 2125 | 0.55 | 1.14 (0.70–1.78) |
MAF: Minor Allele Frequency; CADD: Combined Annotation-Dependent Depletion score (34); REVEL: rare exome variant ensemble learner score (35); ER: estrogen receptor; HER2: human epidermal growth factor receptor 2; TN: triple-negative. |
Subgroup analysis based on hormone receptor status was carried out on case subjects where detailed pathology data was available from the Variant in Practice (ViP) study (n = 3,645). Consistent with previous findings for RAD51C LoF carriers, rare MS variants were significantly enriched in the ER-negative breast cancer subgroup (OR 1.67, 95%CI 0.99–2.70, p = 0.04), with a similar but non-significant trend in TNBC cases (OR 1.58, 95%CI 0.81–2.80, p = 0.11).
The distribution and frequency of rare MS variants across RAD51C in the 5,734 cases and 14,382 controls is summarised in Fig. 1. While rare MS variants were distributed across the entire gene, cases showed higher frequencies in the first half of the gene. The position-based odds ratio analysis showed a higher case-control odds ratio for variants located between amino acid positions 82 and 136, coinciding with the interaction domain.
Variants Of Interest Detected In Cases And Controls
Details of the 51 rare RAD51C MS variants identified in this study including case-control numbers, in silico pathogenicity prediction and literature evidence are summarised in Supplementary Table 1. Also included is the reference variant p.Ala126Thr (MAF = 0.0054), a generally-accepted benign variant. All of the variants were very rare (MAF ≤ 0.0001), with the exception of p.Gly264Ser (MAF = 0.0034). Despite the large sample size, most variants were detected in less than three subjects, therefore the frequencies alone were not adequately powered to confirm or refute pathogenicity. The data did, however, suggest that two previously-identified variants, p.Ala126Thr and p.Gly264Ser, do not represent high penetrance alleles. p.Ala126Thr was detected in 68 (1.19%) cases and 133 (0.9%) controls (OR 1.29, p = 0.10), similar to the allele frequency reported in gnomAD database. Similarly, p.Gly264Ser was detected with equal frequencies in cases (n = 30, 0.52%) and controls (n = 80, 0.54%) (OR 0.96, p = 0.92).
Sequencing Of Tumours From Ms Variant Carriers
Twenty invasive breast tumours and one high grade serous ovarian tumour from 21 cases were sequenced using a targeted gene panel that included all exons and intron boundaries of RAD51C (Table 2). These tumours were from cases that carried one of eight heterozygous candidate variants (p.Gly264Ser, p.Gln143Arg, p.Ile144Thr, p.Arg212His, p.Asp242Asn, p.Ile244Val, p.Arg258His and p.Leu262Val) as well as one homozygous p.Gly264Ser carrier. Of the 20 germline heterozygous carrier tumours, four were found to harbour a second hit through loss of the wild-type allele (loss of heterozygosity, LOH). However, another five had lost the mutant allele while eleven others remained heterozygous. On further investigation, none of the heterozygous cases showed evidence of promoter hyper-methylation or somatic point mutations in RAD51C. Of the eight tumours from heterozygous carriers of the p.Gly264Ser allele, only four showed copy number loss with three of these involving loss of the variant allele. Importantly, both the p.Gly264Ser homozygous carrier and the case with loss of the wild-type allele had HRD scores below those indicative of loss of homologous recombination function (27).
Table 2
Molecular analysis of 21 tumours from RAD51C missense variant carriers.
Sample | Variant | Hormone Receptor/ HER2 Status | Allele status | HRD Score | Promoter Hyper-Methylation | Tp53 Somatic Mutation |
1 | p.Gly264Ser | TN | Germline homozygous | 37 | - | Mutated |
2 | TN | Wild-type loss | 15 | No | Mutated |
3 | TN | Variant loss | 122 | No | Mutated |
4† | TN | Variant loss | N/A | No | N/A |
5 | TN | Heterozygous | 39 | NA | Mutated |
6† | ER-/ HER2- | Variant loss | 43 | No | Mutated |
7 | ER+/HER2- | Heterozygous | 10 | NA | Wild-type |
8 | ER+/HER2- | Heterozygous | 29 | No | Wild-type |
9 | ER+/HER2+ | Heterozygous | 28 | No | Mutated |
10 | p.Glu143Arg | TN | Variant loss | 67 | No | Mutated |
11 | ER-/HER2+ | Heterozygous | 12 | NA | Wild-type |
12 | ER+/HER2- | Heterozygous | 6 | No | Wild-type |
13 | ER+/HER2- | Heterozygous | N/A | No | N/A |
14 | p.Ile144Thr | TN | Wild-type loss | 78 | No | Mutated |
15 | ER+/HER2- | Heterozygous | 18 | No | Wild-type |
16 | p.Arg212His | ER-/HER2+ | Wild-type loss | 47 | No | Mutated |
17 | ER+/HER2- | Heterozygous | 10 | No | Wild-type |
18 | p.Asp242Asn | ER+/HER2+ | Variant loss | 49 | No | Wild-type |
19 | p.Ile244Val | TN | Variant loss | 78 | No | Mutated |
20 | p.Arg258His | OvCa | Wild-type loss | 70 | No | Mutated |
21 | p.Leu262Val | ER+/HER2- | Heterozygous | 28 | No | Wild-type |
All samples were sequenced using targeted panel, with the exception of samples 11 and 20 with whole-exome and samples 4 and 13 with exon-specific Sanger sequencing. HRD: homologous recombination deficiency. |
†carriers are 1st degree related |
Loss of the wild-type allele was identified in two triple-negative tumours carrying p.Ile144Thr and p.Arg212His variants respectively, with both showing high HRD scores, while ER-positive tumours carrying these variants remained heterozygous. An ovarian tumour carrying p.Arg258His also showed LOH and had a high HRD score of 70. Among four tumours sequenced that carried the p.Glu143Arg variant, the one triple-negative case was found to have lost the variant allele, while the one ER-negative and two ER-positive tumours remained heterozygous. All three tumours carrying a germline p.Leu262Val, p.Ile244Val or p.Asp242Asn variant were also showed to remained heterozygous.
Previous studies have shown that breast tumours from individuals carrying a LoF mutation in RAD51C accompanied with loss of the wild-type allele were associated with single base substitution mutational signature 3 (1, 28), and this was assessed for tumours from carriers of candidate MS variants. To achieve the minimum recommended number of somatic mutations (n≥40) for mutational signature estimation, tumours were grouped into those carrying variants of unknown significance (VUS) and those carrying the benign variant p.Gly264Ser (Supplementary Fig. 1). The contribution of signature 3 in tumours carrying a VUS was similar to the tumours carrying the p.Gly264Ser variant. When stratified based on tumour pathology, triple-negative tumours had a higher proportion of signature 3 and higher HRD scores but these were similar in both VUS and benign variant carriers. Whole exome sequencing of a high grade serous ovarian cancer carrying the p.Arg258His variant (case 20) was shown to have lost the wild-type and accompanied by a large proportion of signature 3 and other smaller signatures related to nucleotide excision repair.
Pedigree Segregation Of Ms Variant Carriers
Nine additional family members from seven families, (representing three different variants), were available to examine the segregation of the germline variant detected in the index case (Supplementary Fig. 2). Four of the families carried the p.Gly264Ser variant which was found to be present in two affected first degree relatives (FDR) (ER + BC 43, BC 50), but absent in two affected second degree relatives (ER + BC 38, lobular ER + BC 56) of the respective index cases. The variant p.Gln143Arg was present in a first degree relative diagnosed with TNBC (age 55), ER-positive breast cancer (age 72) and high grade serous ovarian cancer (age 74), while none of the three unaffected FDR, tested from p.Gln143Arg families carried the variant. Finally, the daughter of an index case carrying the p.Gln137Arg variant remained unaffected but is currently only 35 years old.