Abstract
Purpose
The aim of this study was to estimate the contribution of deleterious mutations in the RAD51B, RAD51C, and RAD51D genes to invasive epithelial ovarian cancer (EOC) in the population and in a screening trial of individuals at high risk of ovarian cancer.
Patients and Methods
The coding sequence and splice site boundaries of the three RAD51 genes were sequenced and analyzed in germline DNA from a case-control study of 3,429 patients with invasive EOC and 2,772 controls as well as in 2,000 unaffected women who were BRCA1/BRCA2 negative from the United Kingdom Familial Ovarian Cancer Screening Study (UK_FOCSS) after quality-control analysis.
Results
In the case-control study, we identified predicted deleterious mutations in 28 EOC cases (0.82%) compared with three controls (0.11%; P < .001). Mutations in EOC cases were more frequent in RAD51C (14 occurrences, 0.41%) and RAD51D (12 occurrences, 0.35%) than in RAD51B (two occurrences, 0.06%). RAD51C mutations were associated with an odds ratio of 5.2 (95% CI, 1.1 to 24; P = .035), and RAD51D mutations conferred an odds ratio of 12 (95% CI, 1.5 to 90; P = .019). We identified 13 RAD51 mutations (0.65%) in unaffected UK_FOCSS participants (RAD51C, n = 7; RAD51D, n = 5; and RAD51B, n = 1), which was a significantly greater rate than in controls (P < .001); furthermore, RAD51 mutation carriers were more likely than noncarriers to have a family history of ovarian cancer (P < .001).
Conclusion
These results confirm that RAD51C and RAD51D are moderate ovarian cancer susceptibility genes and suggest that they confer levels of risk of EOC that may warrant their use alongside BRCA1 and BRCA2 in routine clinical genetic testing.
INTRODUCTION
Epithelial ovarian cancer (EOC) has a significant heritable component. A woman with a single first-degree relative diagnosed with ovarian cancer has a three-fold increased risk of the disease.1,2 Twin studies suggest that most of the familial clustering results from inherited genetic factors.3 High-penetrance mutations in BRCA1 and BRCA2 are associated with the majority of breast-ovarian cancer syndrome occurrences.4–6 The cumulative estimated risks of ovarian cancer averaged across all possible polygenic risk modifiers by age 70 years are 36% in BRCA1 carriers and 12% in BRCA2 carriers.7
Other ovarian cancer susceptibility genes include the mismatch repair genes MSH6, MSH2, and MLH1,8 which also are associated with colorectal and endometrial cancers. Several common low-penetrance susceptibility alleles conferring relative risks (RRs) of less than 1.5-fold have been found using genome-wide association studies.9–17 The known high-risk susceptibility genes account for approximately 40% of the excess familial risk of EOC,18 whereas rare moderate-risk variants and common low-risk variants contribute less than 5%.15 Identification of additional susceptibility genes that confer RRs greater than 2 could decrease mortality as a result of ovarian cancer through surgical intervention (eg, risk-reducing salpingo-oophorectomy [RRSO]) in at-risk individuals. Recent advances in high-throughput next-generation sequencing technologies have enabled the rapid, targeted analysis of multiple candidate genes in large populations and have recently identified some novel susceptibility genes for ovarian cancer, including RAD51C,19 RAD51D,20 and BRIP1.21 Existing data suggest that the population prevalence of germline mutations in these genes is low, but the published risk estimates (albeit on the basis of small sample sizes) suggest genetic testing of these genes may have clinical utility. RAD51D mutations were associated with a 6.3-fold increase in risk (95% CI, 2.9 to 14),20 whereas BRIP1 mutations were associated with an 8.1-fold increased risk of ovarian cancer (95% CI, 4.7 to 14).21
The aims of this study were to establish the prevalence and penetrance of deleterious mutations in the three interacting double-strand DNA break repair genes RAD51B, RAD51C, and RAD51D.
PATIENTS AND METHODS
Study Participants
The 3,447 confirmed invasive EOC cases and 2,812 unaffected controls were from four population-based ovarian cancer case-control studies (AOC [Australian Ovarian Cancer Study], MAL [Malignant Ovarian Cancer Study], SEA [Studies of Epidemiology and Risk Factors in Cancer Heredity], and UKO [United Kingdom Ovarian Cancer Population Study]), one clinic-based case-control study (MAYO [Mayo Clinic Ovarian Cancer Study]), one familial ovarian cancer series of cases and matched controls from Poland (POC [Poland ovarian cancer study]), and two familial ovarian cancer registries from the United Kingdom and United States (UKR [United Kingdom Familial Ovarian Cancer Registry] and GRR [Gilda Radner Familial Ovarian Cancer Registry]). These studies have been previously described (Table 1 and Appendix Table A1, online only). Forty-three duplicate samples and four RAD51C mutation–positive controls were included for quality control.
Table 1.
Study Patient Cases Sequenced for RAD51B, RAD51C, and RAD51D After Quality-Control Analysis
| Study | Study Abbreviation | No. of Patient Cases | No. of Controls | Total No. of Participants |
|---|---|---|---|---|
| On the basis of patients not selected for family history | ||||
| Australian Ovarian Cancer Study1 | AOC | 413 | 428 | 841 |
| Malignant Ovarian Cancer1 | MAL | 190 | 191 | 381 |
| SEARCH2 | SEA | 1,259 | 1,382 | 2,641 |
| United Kingdom Ovarian Cancer Population Study1* | UKO | 361 | 531 | 892 |
| Mayo Clinic Ovarian Cancer Study2 | MAYO | 912 | 146 | 1,058 |
| Family based | ||||
| Poland family history, Poland ovarian cancer study1† | POC | 89 | 94 | 183 |
| United Kingdom Familial Ovarian Cancer Registry3† | UKR | 48 | — | 48 |
| Gilda Radner Familial Ovarian Cancer Registry3† | GRR | 157 | — | 157 |
| Total of all studies | 3,429 | 2,772 | 6,201 |
Abbreviation: SEARCH, Studies of Epidemiology and Risk Factors in Cancer Heredity.
Only study not screened for BRCA1/BRCA2 mutations.
All patient cases had a family history of ovarian cancer.
Also included were 2,000 unaffected participants enrolled onto the United Kingdom Familial Ovarian Cancer Screening Study (UK_FOCSS).22 Eligible participants were women age ≥ 35, with an estimated lifetime risk of ovarian cancer of ≥ 10% on the basis of a family history of ovarian and/or breast cancer and/or the presence of known predisposing germline gene mutations (BRCA1, BRCA2, and MMR genes) in the family. Volunteers were recruited between June 2002 and September 2010 from 42 United Kingdom regional centers. All participants were tested for BRCA1 and BRCA2 mutations, and carriers were excluded from this study.
All studies had approval from the appropriate ethics committee, and all study participants provided written, informed consent.
Sequencing Library Preparation and Sequencing
We used the 48.48 Fluidigm Access Arrays (Fluidigm, San Francisco, CA) for target sequence enrichment, as described previously8 and according to the manufacturer's protocol. The RAD51 genes were in a panel of 11 genes sequenced in SEA and MAYO and in a panel of six genes in the remaining studies. The results for the other genes have been reported previously8 or are unpublished. Fifty-six primer pairs were designed to cover the exons and splice sites of RAD51B, RAD51C, and RAD51D (Appendix Table A2, online only) with a combined sequencing target of 4 kb. The primer design achieved greater than 95% coverage of the target sequence. Sequencing libraries were quantified by using a KAPA library quantification kit (Kapa Biosystems, Boston, MA) with specific probes for the ends of the adapters according to the manufacturer's protocol. The sequence libraries were sequenced using single-end sequencing on the Illumina GAII (Illumina, San Diego, CA) or paired end sequencing on the Illumina HiScan (Illumina) or Illumina HiSeq 2000 (Illumina) according to the manufacturer's protocol. Each lane sequenced 384 barcoded samples.
Sequence Data Analysis
Sequenced reads were demultiplexed with standard Illumina software. We used the Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/)23 for sequencing read alignment against the human genome reference sequence (UCSC hg19; University of California Santa Cruz Genome Reference Consortium; http://genome.ucsc.edu/cgi-bin/hgGateway). The Genome Analysis Toolkit (GATK; https://www.broadinstitute.org/gatk/)24 was used for base quality-score recalibration, local insertion/deletion (indel) realignment, and variant (substitution and indel) discovery. Variants were considered only if they satisfied the set of recommended GATK filters, as described in the GATK best practices guide. ANNOVAR (http://annovar.openbioinformatics.org/en/latest/)25 was used to annotate the sequence variation detected. We used PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/bgi.shtml),26 SIFT (http://sift.bii.a-star.edu.sg/),27 and Provean (http://provean.jcvi.org/protein_batch_submit.php?species=human)28 to predict the function of missense variants. We used MaxEntScan (http://genetics.bwh.harvard.edu/pph2/bgi.shtml)29 to predict the pathogenic potential of possible splicing variants in sequences from 3 base pairs (bp) in the exon to 20 bp in the intron for the 3′ acceptor sites and 3 bp in the exon and 6 bp in the intron for the 5′ donor sites. Variants with a MaxEntScan score that decreased by more than 40% compared with the consensus sequence were assumed to affect splicing.
The alternate allele frequency (Altfreq) for each variant detected in each sample was defined as the fraction of alternative allele reads compared with the total number of reads at that position. We applied thresholds for variant calling, as defined previously8: With a minimum read depth of 15, alternate allele heterozygotes were called if the depth was ≥ 500 and the Altfreq was ≥ 10%; if the depth ranged from 250 to less than 500 and the Altfreq was ≥ 15%; if the depth ranged from 30 to less than 250 and the Altfreq was ≥ 20%; or if the depth ranged from 15 to less than 30 and the Altfreq was ≥ 30%. Samples with fewer than 80% of the target bases covered at a read depth of ≥ 15 (40 controls and 18 cases) were excluded. We defined deleterious variants as those predicted to result in protein truncation (frameshift indels, consensus splice site substitutions, and nonsense substitutions) or those missense mutations that have been previously reported as deleterious on the basis of in vitro analysis19,30 or predicted by MaxEntScan to affect splicing.
Ninety percent of the target sequence bases had read depths ≥ 15. The coverage for the three genes is summarized in Appendix Table A2. Concordance for variants called in the 43 duplicate samples was 100%. Four RAD51C mutation–positive controls also were detected.
Mutation Validation
We visually inspected the sequence alignments for all of the called deleterious variants by using the Integrative Genomics Viewer (Broad Institute, Cambridge, MA; https://www.broadinstitute.org/igv/). We validated all deleterious variants by polymerase chain reaction amplification and Sanger sequencing.31
Statistical Methods
We tested for an association between deleterious mutations and ovarian cancer risk by using unconditional logistic regression adjusted for the country of origin (Australia, Denmark, Poland, the United Kingdom, and the United States). Odds ratios and associated 95% CIs also were calculated with data from the case-control studies that were not family based (AOC, MAL, MAYO, SEA, and UKO).
We estimated the cumulative risk of ovarian cancer with equation 1 by applying the estimated odds ratio (RR) to population incidence data for England from 201132:
 |
See the Data Supplement for a spreadsheet with calculations.
We identified multiple missense variants that have unknown functional effects on the protein. We excluded all missense variants that had a minor allele frequency (MAF) of greater than 1% from additional analyses, because large-scale genome-wide association studies have shown that the RR conferred by a common susceptibility allele are small (RR < 1.3) and thus not detectable by the smaller sample size of this targeted-sequencing study. The statistical power to detect single rare alleles by association, even if they confer larger risk (RR > 2), is still modest. Therefore, we used the rare admixture likelihood (RAML) burden test33 to test for an association on a gene-by-gene basis. The RAML test combines the data for multiple variants and allows for alleles associated with either an increased or a decreased risk. We classified variants with an MAF ≤ 1% into three groups: deleterious variants as defined previously (these were excluded from the RAML analyses); variants predicted to have a damaging effect on protein function by at least two of three prediction tools (SIFT [score ≤ 0.05], PolyPhen-2 [classified as probably damaging/damaging], and Provean [score ≤ −2.5]); and variants with probable benign effects. Only patients who had a call rate greater than 80% for missense variants and variants that had a call rate greater than 80% and genotype frequencies consistent with the Hardy-Weinberg equilibrium (P > 10−5) were included in these analyses.
RESULTS
Deleterious RAD51B, RAD51C, and RAD51D Mutations in Ovarian Cancer Cases and Controls
Sequence data for the coding regions and splice site boundaries of RAD51B, RAD51C, and RAD51D were available for 3,429 invasive EOC cases and 2,772 controls after quality control (Table 1). We identified 135 unique variants, of which eight (5.9%) were frameshift indels, 10 (7.4%) were nonsense substitutions, five (3.7%) were predicted splice site alterations, and 113 (78%) were missense substitutions. Of the 113 missense variants, one (RAD51C 428A>G) was deleterious,30 105 had an MAF less than 1%, and seven (5.1%) had an MAF greater than 1%.
We identified deleterious mutations in two cases for RAD51B, 14 cases and two controls for RAD51C, and 12 cases and one control for RAD51D. Of these, 23 deleterious mutation carriers were identified in 3,135 cases (0.73%) unselected for family history (Table 2 and Appendix Table A3, online only). One case had two deleterious mutations close to each other and in cis (G217X and Q219X) in RAD51D. The prevalence of deleterious mutations was significantly higher (P < .001) in cases (28 of 3,429; 0.82%) than in controls (three of 2,772; 0.11%). Eight deleterious mutations were detected in more than one individual. Three of these (RAD51C 732delT and A428G and RAD51D C898T) were identified in a case and a control. Of the 29 predicted deleterious variants in cases, 22 (76%) were frameshift indels or nonsense variants, six (21%) were splice site substitutions, and one (3.4%) was a missense variant previously reported as deleterious.30
Table 2.
Mutation Carriers Identified in RAD51B, RAD51C, and RAD51D in Ovarian Cancer Patient Cases and Controls
| Mutation Carrier Status | Controls |
Patient Cases |
||||
|---|---|---|---|---|---|---|
| All |
Unselected for Family History |
|||||
| No. | % | No. | % | No. | % | |
| Noncarrier | 2,769 | 99.9 | 3,401 | 99.2 | 3,112 | 99.3 |
| Mutation carrier | ||||||
| Any mutation | 3 | 0.11 | 28 | 0.82 | 23 | |
| RAD51B | 0 | 0 | 2 | 0.06 | 2 | 0.06 |
| RAD51C | 2 | 0.07 | 14 | 0.41 | 10 | 0.32 |
| RAD51D | 1 | 0.04 | 12* | 0.35 | 11* | 0.35 |
One patient case carried two deleterious mutations.
We also evaluated the prevalence of RAD51B, RAD51C, and RAD51D variants in 2,000 individuals from UK_FOCSS. We identified 149 unique variants, of which three (2.0%) were frameshift indels, three (2.0%) were nonsense substitutions, two (1.3%) were predicted splice site alterations, and 141 (95%) were missense substitutions. Thirteen participants carried one of the eight different deleterious mutations in one of these genes (one in RAD51B, seven in RAD51C, and five in RAD51D). The overall prevalence (0.65%) was significantly greater than that of the general population controls (P < .001; Table 3).
Table 3.
Characteristics of the United Kingdom Familial Ovarian Cancer Screening Study Mutation Carriers
| Gene | Mutation Information |
Proband Characteristic |
Family History |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| No. of Affected First-Degree Relatives |
No. of Affected First- and Second-Degree Relatives |
|||||||||
| cDNA Change | Location | Protein Change | Predicted Effect | Ref. Age, Years | Breast Cancer (age in years) | Ovarian Cancer | Breast Cancer | Ovarian Cancer | Breast Cancer | |
| RAD51B | 854-2A>G | Intron 8 | NA | Splicing | 58 | No | 0 | 0 | 2 | 0 |
| RAD51C | C97T | Exon 1 | Q33X | Nonsense | 31 | No | 1 | 1 | 2 | 1 |
| RAD51C | 158delC | Exon 2 | S53fs | Frameshift deletion | 69 | No | 2 | 0 | 2 | 0 |
| RAD51C | C577T | Exon 4 | R193X | Nonsense | 46 | No | 0 | 0 | 0 | 1 |
| RAD51C | C577T | Exon 4 | R193X | Nonsense | 46 | No | 1 | 0 | 2 | 0 |
| RAD51C | C577T | Exon 4 | R193X | Nonsense | 41 | No | 1 | 0 | 2 | 0 |
| RAD51C | 731delT | Exon 5 | I244fs | Frameshift deletion | 51 | No | 1 | 0 | 3 | 0 |
| RAD51C | 731delT | Exon 5 | I244fs | Frameshift deletion | 64 | Yes (57) | 2 | 0 | 3 | 1 |
| RAD51D | 263 + 1G>A | Intron 3 | NA | Splicing | 53 | No | 1 | 0 | 4 | 0 |
| RAD51D | C556T | Exon 6 | R186X | Nonsense | 62 | No | 1 | 0 | 3 | 2 |
| RAD51D | C556T | Exon 6 | R186X | Nonsense | 62 | Yes (52) | 1 | 0 | 1 | 1 |
| RAD51D | C556T | Exon 6 | R186X | Nonsense | 25 | No | 1 | 0 | 1 | 0 |
| RAD51D | 748delC | Exon 9 | H250fs | Frameshift deletion | 50 | No | 1 | 0 | 1 | 1 |
Abbreviation: Ref., reference.
Ovarian Cancer Risks Associated With RAD51B, RAD51C, and RAD51D Mutations
The odds ratio (adjusted for country of origin) associated with a deleterious mutation in any of the three genes was 8.1 (95% CI, 2.4 to 27; P = .001) for all ovarian cancer subtypes and 9.3 (95% CI, 2.7 to 32; P < .001) for the serous subtype. Gene-specific odds ratios (adjusted for country of origin) for all ovarian cancer subtypes were 5.2 for RAD51C (95% CI, 1.1 to 24; P = .035) and 12 for RAD51D (95% CI, 1.5 to 90; P = .019). Gene-specific odds ratios for the serous subtype were 7.4 for RAD51C (95% CI, 1.6 to 35; P = .011) and 12 for RAD51D (95% CI, 1.5 to 97; P = .021). The estimated average cumulative risks of ovarian cancer by age 50 were 1.3% (95% CI, 0.3% to 6.0%) for RAD51C and 3.0% (95% CI, 0.4% to 21%) for RAD51D. The equivalent risks by age 70 were 5.2% (95% CI, 1.1% to 22%) for RAD51C and 12% (95% CI, 1.5% to 60%) for RAD51D.
Clinicopathologic Characteristics Associated With RAD51B, RAD51C, and RAD51D Mutations
The clinical and histopathologic characteristics of all patient cases are listed in Appendix Table A1. Mutation carriers were more likely than noncarriers to have high-grade serous versus other histologic subtypes (P = .046; Table 4). Eighteen percent of mutation carriers were diagnosed at ages 40 to 49 years, and no mutation carrier was diagnosed with ovarian cancer before age 40 years (Table 4). Carriers of a mutation in any of the RAD51 genes were more likely than noncarriers to have a family history of ovarian cancer, although this difference was not statistically significant (24% v 14%; P = .16 for all genes). The proportion of RAD51C mutation carriers with a family history was higher (36%; P = .021; Table 5). In UK_FOCSS participants, mutation carriers were also more likely than noncarriers to be associated with a family history of ovarian cancer (Table 3); 9 of 13 mutation carriers (69%) compared with 548 of 1,987 noncarriers (28%) had a family history comprising two or more ovarian cancer cases in first- or second-degree relatives (P < .001).
Table 4.
Mutation Status by Age at Disease Onset and Histologic Subtype in Patient Cases With Ovarian Cancer
| Mutation Status | No. (%) of Patients by Age at Diagnosis, Years |
Histology, No. (%) |
|||||
|---|---|---|---|---|---|---|---|
| < 40 | 40-49 | 50-59 | ≥ 60 | Unknown | High-Grade Serous | Other | |
| Noncarrier (n = 3,401) | 165 (4.9) | 514 (15) | 1,073 (32) | 1,642 (48) | 7 (0.2) | 1,786 (53) | 1,615 (47) |
| Mutation carrier (n = 28) | 0 | 5 (18) | 11 (39) | 12 (43) | 0 | 20 (71) | 8 (29) |
| RAD51B (n = 2) | 0 | 0 | 0 | 2 (100) | 0 | 1 (50) | 1 (50) |
| RAD51C (n = 14) | 0 | 4 (29) | 5 (36) | 5 (36) | 0 | 10 (71) | 4 (29) |
| RAD51D (n = 12) | 0 | 1 (8.3) | 6 (50) | 5 (42) | 0 | 9 (75) | 3 (25) |
Table 5.
Mutation Status by First Degree of Family History of Breast and/or Ovarian Cancer in Patient Cases With Ovarian Cancer
| Gene | No. (%) of Patient Cases by Family History |
|||
|---|---|---|---|---|
| No FH (n = 2,307) | OvFH Only (n = 430) | BrFH Only (n = 467) | BrOvFH (n = 29) | |
| Noncarrier | 2,292 (71) | 424 (13) | 463 (14) | 29 (0.90) |
| Mutation carrier | ||||
| Any | 15 (60) | 6 (24) | 4 (16) | 0 |
| RAD51B | 1 (100) | 0 | 0 | 0 |
| RAD51C | 6 (43) | 5 (36) | 3 (21) | 0 |
| RAD51D | 8 (80) | 1 (10) | 1 (10) | 0 |
Abbreviations: BrFH, first degree of family history of breast cancer; BrOvFH, first degree of family history of both ovarian and breast cancer; no FH, no first degree of family history of breast or ovarian cancer; OvFH, first degree of family history of ovarian cancer.
RAD51B, RAD51C, and RAD51D Missense Variants and Ovarian Cancer Risk
We used three bioinformatics tools (SIFT, PolyPhen-2, and Provean) to predict the effects on protein function of 112 missense variants. Thirty missense variants were classified as deleterious by all three tools, 12 missense variants by at least two of three tools, and 15 variants by one of three tools; 55 missense variants were predicted to be neutral by all three tools (Appendix Table A4). For the 38 missense variants with an MAF ≤ 1% and predicted by at least two of three tools to have a functional effect, we compared the relative burden in cases and controls for each gene with the RAML test.33 We found some evidence for an association of the rare missense variation in RAD51C with an increased risk of ovarian cancer for all ovarian cancer subtypes (RAML test P = .029), and the effect was stronger for the serous subtype (RAML test P < .001). We also found some evidence of an association of missense variants in RAD51D with an increased risk of serous ovarian cancer (P = .012). There was little evidence of an association of rare missense variants in RAD51B and RAD51D with all ovarian cancer subtypes or in RAD51B with serous ovarian cancer (P > .05).
DISCUSSION
To our knowledge, this study is the largest population-based ovarian cancer study to date to estimate the prevalence of mutations in the RAD51B, RAD51C, and RAD51D genes. Overall, 0.81% of EOC cases had a mutation in one of these three genes compared with 0.11% in controls. Our data suggest that both RAD51C and RAD51D are ovarian cancer susceptibility genes; however, RAD51B mutations are unlikely to contribute substantially to ovarian cancer risk.
Several other studies have reported on the prevalence of germline genetic variations in these genes (Appendix Table A5). However, for most of these, the ascertainment of cases was complex: several sequenced an affected proband (either breast or ovarian cancer) from a family with multiple cases of breast and/or ovarian cancer. Six studies sequenced RAD51C in ovarian cancer cases unselected for family history,34–39 but only one of these carried out equivalent sequencing of controls.34 Three studies sequenced RAD51D in unselected ovarian cancer cases,36,40,41 but none of these sequenced the whole gene in controls. In these studies, the mutation frequency in cases ranged from 0.4% to 1.1% for RAD51C and 0.8% to 1.1% for RAD51D.
In this study, the mutation frequency in cases unselected for family history was 0.32% for RAD51C and 0.35% in RAD51D. These are likely to be underestimates of the true mutation frequencies. Our next-generation sequencing approach enabled rapid and high-throughput analysis of candidate genes in thousands of samples but did not provide complete coverage of all genes in all samples (mean coverage per sample, 90%). Also, we used polymerase chain reaction–based enrichment of candidate gene coding regions; any deleterious mutations occurring outside these regions (eg, large genomic deletions and rearrangements) would not have been detected. Finally, we did not include missense variants in our prevalence estimates, because we could not be certain of their pathogenicity in the absence of definitive functional assays. However, burden tests for RAD51C and RAD51D variants indicate that rare missense variants that are predicted to disrupt protein function are significantly more prevalent in cases than controls, which suggests that at least a proportion of these variants is deleterious.
High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype, and mutations were more prevalent in patients with HGSOC (1.1%) than in other subtypes (0.49%). This finding, perhaps, is expected, because deficiency of double-strand DNA break repair by homologous recombination as a result of germline mutations in BRCA1 or BRCA2 also is associated with HGSOC.8,42
Although there are similarities in the functional mechanisms associated with the RAD51 genes and BRCA1/BRCA2, the genetic epidemiology suggests there are also differences. For example, BRCA1 and BRCA2 mutations confer risks of both breast and ovarian cancer, but there is little evidence from other studies that RAD51C or RAD51D mutations confer increased risks of breast cancer. The location of truncating mutations in BRCA1/BRCA2 is associated with variable risks of breast and ovarian cancer.43,44 All except two of the predicted truncating mutations identified in RAD51C were located between amino acid 143 and 319 in a functional domain in the C terminus of the protein (residues 79 to 376).45 This domain is important for forming the RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3 complexes. Likewise, all of the deleterious mutations identified in RAD51D were clustered in the C-terminal region (residues 77 to 328), which affects binding to RAD51C and likely impairs double-strand DNA break repair45 (Fig 1).
Fig 1.
Distribution of predicted deleterious variants in RAD51B, RAD51C, and RAD51D. The location of each mutation is shown in the exon structure of the coding sequence. Mutations occurring in multiple individuals are indicated with the number of carriers above the small balloon. Coding regions of all the genes are on the same scale. (*) Deleterious mutation identified (one each in case and control groups). bp, base pair.
Our RR estimate for RAD51D is similar to that reported previously by Loveday et al20 (6.3; 95% CI, 2.9 to 14) on the basis of the analysis of families with multiple cases of ovarian cancer. Our RR estimate for RAD51C is similar to those reported by Pelttari et al35 (6.3; 95% CI, 1.2 to 35) for unselected ovarian cancer. The wide CIs of risk estimates for both genes suggest that caution needs to be applied if the genes are used clinically for genetic risk prediction. In addition, the fact that 18% of ovarian cancers in women carrying RAD51C and RAD51D mutations occurred at younger than 50 years (Table 4) suggests that, if risk estimates were confirmed, offering premenopausal women the option of RRSO should be considered. If clinical testing for RAD51C and RAD51D was approved, women could undergo panel testing for multiple susceptibility genes, and carriers, along with their relatives, could be offered RRSO.
In summary, we estimate that RAD51B, RAD51C, and RAD51D are responsible for approximately one in every 90 high-grade serous EOC occurrences and one in every 120 EOC occurrences. In addition to the benefit of mutation testing of RAD51C and RAD51D for disease prevention, mutation carriers also may be responsive to treatment with poly(ADP-ribose) polymerase inhibitors, which results in synthetic lethality of cells that have mutant homologous recombination or double-strand DNA break repair. This treatment might improve progression-free survival among these patients. Hence, such testing may be useful in patient decision making.
Acknowledgment
We thank all of the study participants who contributed to this study and all of the researchers, clinicians, and technical and administrative staff who made this work possible. In particular, we thank the clinical and scientific collaborators listed at http://www.aocstudy.org/; E. Wozniak, A. Ryan, J. Ford, and N. Balogun (United Kingdom Ovarian Cancer Population Study); C. Pye (United Kingdom Familial Ovarian Cancer Registry); Marie Mack, Craig Luccarini, Caroline Baynes, the SEARCH team, and Eastern Cancer Registration and Information Centre (SEARCH); and Source BioScience laboratories in the United Kingdom at which some of the sequencing was performed.
Glossary Terms
- allele:
an alternative form of a gene (in diploids, one member of a pair) that is located at a specific position on a specific chromosome.
- missense mutation:
a change (mutation) in one nucleotide that results in the coding of a different amino acid.
- penetrance:
the likelihood that a given gene mutation will produce disease. This likelihood is calculated by examining the proportion of people with the particular genetic mutation that show symptoms of disease.
Appendix
Table A1.
Characteristics of the Patients With Ovarian Cancer
| Characteristic | No. (%) of Patients by Study |
No. (%) of Total Patients (N = 3,429) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| AOC (n = 413) | GRR (n = 157) | MAL (n = 190) | MAYO (n = 912) | POC (n = 89) | SEA (n = 1,259) | UKR (n = 48) | UKO (n = 361) | ||
| Mean (range) age at diagnosis, years | 60.1 (23-79) | 49.4 (21-83) | 61.7 (38-80) | 62.5 (23-91) | 51.1 (21-77) | 56.0 (19-74) | 53.0 (24-77) | 61.2 (25-90) | 58.7 (19-91) |
| Morphology | |||||||||
| High-grade serous | 359 (87) | 54 (34) | 137 (72) | 654 (72) | 26 (29) | 341 (27) | 17 (35) | 266 (74) | 1,806 (53) |
| Low-grade serous | 24 (5.8) | 6 (3.8) | 18 (9.5) | 26 (2.9) | 5 (5.6) | 275 (22) | 2 (4.2) | 21 (5.8) | 405 (12) |
| Serous | 14 (3.4) | 34 (22) | 12 (6.3) | 0 | 10 (11) | 0 | 6 (13) | 58 (16) | 151 (4.4) |
| Endometrioid | 3 (0.73) | 19 (12) | 13 (6.8) | 110 (12) | 13 (15) | 214 (17) | 5 (10) | 6 (1.7) | 383 (11) |
| Clear cell | 3 (0.73) | 12 (7.6) | 6 (3.2) | 55 (6.0) | 1 (1.1) | 144 (11) | 2 (4.2) | 2 (0.55) | 225 (6.6) |
| Mucinous | 2 (0.48) | 8 (5.1) | 2 (1.1) | 25 (3.7) | 9 (10) | 116 (9.2) | 3 (6.3) | 1 (0.28) | 166 (4.8) |
| Mixed | 6 (1.5) | 2 (1.3) | 0 | 31 (3.4) | 1 (1.1) | 70 (5.6) | 1 (2.1) | 5 (1.4) | 116 (3.4) |
| Other | 1 (0.24) | 21 (13) | 0 | 11 (1.2) | 24 (27) | 79 (6.3) | 11 (23) | 2 (0.55) | 152 (4.4) |
| Undifferentiated | 1 (0.24) | 1 (0.64) | 2 (1.1) | 0 | 0 | 20 (1.6) | 1 (2.1) | 0 | 25 (0.73) |
| Unknown | 359 (87) | 54 (34) | 137 (72) | 654 (72) | 26 (29) | 341 (27) | 17 (35) | 266 (74) | 1,806 (53) |
| Stage* | |||||||||
| 1 | 17 (4.1) | 0 | 10 (5.3) | 141 (15) | 3 (3.4) | 442 (35) | 2 (4.2) | 27 (7.5) | 642 (19) |
| 2 | 35 (8.5) | 0 | 31 (16) | 51 (5.6) | 2 (2.3) | 115 (9.1) | 2 (4.2) | 71 (20) | 307 (9.0) |
| 3 | 359 (87) | 0 | 149 (78) | 709 (78) | 9 (10) | 431 (34) | 9 (19) | 244 (67) | 1,910 (56) |
| Unknown | 2 (0.48) | 157 (100) | 0 | 11 (1.2) | 75 (84) | 271 (22) | 35 (73) | 19 (5.3) | 570 (17) |
| Grade | |||||||||
| Low | 28 (6.8) | 18 (11) | 42 (22) | 128 (14) | 12 (13) | 411 (33) | 7 (15) | 24 (6.6) | 670 (20) |
| High | 370 (90) | 74 (47) | 135 (71) | 754 (83) | 41 (46) | 670 (53) | 28 (58) | 275 (76) | 2,347 (68) |
| Unknown | 15 (3.6) | 65 (41) | 13 (6.8) | 30 (3.3) | 36 (40) | 178 (14) | 13 (27) | 62 (17) | 412 (15) |
Abbreviations: AOC, Australian Ovarian Cancer Study; GRR, Gilda Radner Familial Ovarian Cancer Registry; MAL, Malignant Ovarian Cancer Study; MAYO, Mayo Clinic Ovarian Cancer Study; POC, Poland Ovarian Cancer Study; SEA, Studies of Epidemiology and Risk Factors in Cancer Heredity; UKO, United Kingdom Ovarian Cancer Population Study; UKR, United Kingdom Familial Ovarian Cancer Registry.
Stages were defined as follows: 1, localized; 2, regional; and 3, distant.
Table A2.
Sequencing Coverage by Gene
| Gene | Accession No. | No. of Coding Exons | Total Coding Length (bp)* | No. of Amplicons Designed | % Coding Sequence* Covered by Design | Mean % Sequence Covered by Read Depth > 15 |
|---|---|---|---|---|---|---|
| RAD51B | NM_133509 | 10 | 1,389 | 23 | 97 | 85 |
| RAD51C | NM_058216 | 9 | 1,339 | 19 | 97 | 93 |
| RAD51D | NM_002878 | 10 | 1,221 | 14 | 95 | 90 |
Abbreviation: bp, base pair.
The sequence also contains 20 bp in the intron for the 3′ acceptor sites and 6 bp in the intron for the donor 5′ sites.
Table A3.
Predicted Deleterious Mutations Found in RAD51B, RAD51C, and RAD51D
| Study and Patient Group | Gene | cDNA Change | Location | Protein Change | Predicted Function | Ref. Age, Years | Ovarian Cancer FH1* | Breast Cancer FH1* | Grade Group | Histology Group |
|---|---|---|---|---|---|---|---|---|---|---|
| Control | ||||||||||
| SEA | RAD51C | 428A>G | Exon 3 | Q143R | Missense | 51 | 0 | 0 | ||
| SEA | RAD51C | 732delT | Exon 5 | I244fs | Frameshift deletion | 56 | 0 | 0 | ||
| AOC | RAD51D | 898C>T | Exon 9 | R300X | Nonsense | 49 | 0 | 1 | ||
| Patient case | ||||||||||
| AOC | RAD51B | 489T>G | Exon 6 | Y163X | Nonsense | 62 | 2 | Serous HG | ||
| SEA | RAD51B | 957G>C | Exon 9 | Q319H | Splicing | 61 | 0 | 0 | 2 | Other |
| SEA | RAD51C | 428A>G | Exon 3 | Q143R | Missense | 49 | 0 | 0 | 1 | Serous LG |
| MAL | RAD51C | 498delT | Exon 3 | V166fs | Frameshift deletion | 52 | 0 | 0 | 2 | Serous HG |
| SEA | RAD51C | 572-1G>T | Intron 3 | Splicing | 54 | 0 | 0 | 2 | Serous HG | |
| POC | RAD51C | 577C>T | Exon 4 | R193X | Nonsense | 60 | Other | |||
| POC | RAD51C | 577C>T | Exon 4 | R193X | Nonsense | 41 | 1 | 2 | Serous HG | |
| AOC | RAD51C | 653_654del | Exon 4 | 218_218del | Frameshift deletion | 64 | 0 | 0 | 2 | Serous HG |
| UKO | RAD51C | 706-2A>G | Intron 4 | Splicing | 65 | 1 | 0 | 2 | Serous HG | |
| UKR | RAD51C | 706-2A>G | Intron 4 | Splicing | 50 | 1 | 2 | Serous HG | ||
| SEA | RAD51C | 732delT | Exon 5 | I244fs | Frameshift deletion | 48 | 0 | 0 | 2 | Serous HG |
| MAYO | RAD51C | 774delT | Exon 5 | R258fs | Frameshift deletion | 55 | 0 | 1 | 2 | Clear cell |
| POC | RAD51C | 905-2delAG | Intron 6 | Splicing | 52 | 1 | Endometrioid | |||
| AOC | RAD51C | 955C>T | Exon 7 | R319X | Nonsense | 74 | 0 | 1 | 2 | Serous HG |
| AOC | RAD51C | 955C>T | Exon 7 | R319X | Nonsense | 40 | 0 | 1 | 2 | Serous HG |
| SEA | RAD51C | 97C>T | Exon 1 | Q33X | Nonsense | 61 | 0 | 0 | 2 | Serous HG |
| UKR | RAD51D | 478C>T | Exon 5 | Q160X | Nonsense | 56 | 1 | 0 | 2 | Serous HG |
| MAL | RAD51D | 564_567del | Exon 6 | 188_189del | Frameshift deletion | 59 | 2 | Serous HG | ||
| MAL | RAD51D | 564_567del | Exon 6 | 188_189del | Frameshift deletion | 76 | 2 | Serous HG | ||
| SEA | RAD51D | 564delT | Exon 6 | T188fs | Frameshift deletion | 59 | 0 | 0 | 2 | Serous HG |
| SEA | RAD51D | 576 + 1G>A | Intron 6 | Splicing | 66 | 0 | 0 | 2 | Endometrioid | |
| UKO | RAD51D | 620C>A | Exon 7 | S207X | Nonsense | 59 | 0 | 0 | 2 | Serous HG |
| SEA | RAD51D | 623dupT | Exon 7 | V208fs | Frameshift insertion | 54 | 0 | 1 | 2 | Serous HG |
| MAYO | RAD51D | 655C>T/649G>T | Exon 7 | G217X/Q219X | Nonsense | 73 | 0 | 0 | 2 | Serous HG |
| SEA | RAD51D | 741_742insTG | Exon 9 | T248_N249delinsX | Nonsense | 56 | 0 | 0 | 2 | Endometrioid |
| SEA | RAD51D | 748delC | Exon 9 | H250fs | Frameshift deletion | 67 | 0 | 0 | 2 | Serous HG |
| SEA | RAD51D | 748delC | Exon 9 | H250fs | Frameshift deletion | 47 | 0 | 0 | 2 | Serous HG |
| SEA | RAD51D | 898C>T | Exon 9 | R300X | Nonsense | 62 | 0 | 0 | 2 | Endometrioid |
Abbreviations: AOC, Australian Ovarian Cancer Study; GRR, Gilda Radner Familial Ovarian Cancer Registry; MAL, Malignant Ovarian Cancer Study; MAYO, Mayo Clinic Ovarian Cancer Study; POC, Poland Ovarian Cancer Study; SEA, Studies of Epidemiology and Risk Factors in Cancer Heredity; Serous HG, high-grade serous; Serous LG, low-grade serous; UKO, United Kingdom Ovarian Cancer Population Study; UKR, United Kingdom Familial Ovarian Cancer Registry.
First degree of family history.
Table A4.
Catalog of Missense Mutations Found in RAD51B, RAD51C, and RAD51D
| Gene and Variant Type | Chromosome | Position | cDNA | Exon | Protein | SIFT* | PolyPhen-2† | Provean‡ | Score§ | No. of Controls‖ | No. of Patient Cases¶ |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Common variant (MAF ≥ 1%; n = 7) | |||||||||||
| RAD51B | 14 | 68352648 | 515T>G | Exon 6 | L172W | 1 | 1 | 0 | 2 | 64 | 69 |
| RAD51B | 14 | 68353893 | 728A>G | Exon 7 | K243R | 1 | 1 | 0 | 2 | 57 | 82 |
| RAD51B | 14 | 69061259 | 1094C>G | Exon 11 | P365R | 0 | 1 | 0 | 1 | 119 | 148 |
| RAD51C | 17 | 56772522 | 376G>A | Exon 2 | A126T | 0 | 0 | 0 | 0 | 31 | 37 |
| RAD51C | 17 | 56798128 | 859A>G | Exon 6 | T287A | 1 | 1 | 1 | 3 | 44 | 63 |
| RAD51D | 17 | 33433487 | 494G>A | Exon 6 | R165Q | 0 | 0 | 0 | 0 | 536 | 780 |
| RAD51D | 17 | 33430313 | 698A>G | Exon 8 | E233G | 0 | 1 | 1 | 2 | 90 | 124 |
| Potentially deleterious rare variant (n = 38)# | |||||||||||
| RAD51B | 14 | 68331751 | 347A>G | Exon 5 | Q116R | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51B | 14 | 68331826 | 422T>A | Exon 5 | I141N | 1 | 1 | 1 | 3 | 1 | 0 |
| RAD51B | 14 | 68331829 | 425A>G | Exon 5 | D142G | 1 | 1 | 1 | 3 | 1 | 0 |
| RAD51B | 14 | 68352608 | 475C>T | Exon 6 | R159C | 1 | 1 | 1 | 3 | 0 | 2 |
| RAD51B | 14 | 68352609 | 476G>A | Exon 6 | R159H | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51B | 14 | 68352686 | 553T>G | Exon 6 | C185G | 0 | 1 | 1 | 2 | 1 | 1 |
| RAD51B | 14 | 68353814 | 649A>G | Exon 7 | R217G | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51B | 14 | 68878170 | 883G>A | Exon 9 | A295T | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51B | 14 | 68878171 | 884C>T | Exon 9 | A295V | 1 | 0 | 1 | 2 | 1 | 0 |
| RAD51C | 17 | 56770081 | 77A>T | Exon 1 | K26M | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56770084 | 80T>C | Exon 1 | L27P | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56772417 | 271C>T | Exon 2 | L91F | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56772481 | 335G>T | Exon 2 | G112V | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56772540 | 394A>C | Exon 2 | T132P | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56772543 | 397C>A | Exon 2 | Q133K | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56774068 | 419T>G | Exon 3 | V140G | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56774134 | 485G>A | Exon 3 | G162E | 1 | 1 | 1 | 3 | 1 | 0 |
| RAD51C | 17 | 56774146 | 497T>G | Exon 3 | V166G | 1 | 0 | 1 | 2 | 0 | 1 |
| RAD51C | 17 | 56780662 | 677T>C | Exon 4 | L226P | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56787260 | 746G>A | Exon 5 | R249H | 0 | 1 | 1 | 2 | 1 | 0 |
| RAD51C | 17 | 56787349 | 835G>C | Exon 5 | A279P | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51C | 17 | 56809885 | 1006A>C | Exon 8 | T336P | 1 | 0 | 1 | 2 | 0 | 1 |
| RAD51D | 17 | 33446607 | 26G>C | Exon 1 | C9S | 1 | 0 | 1 | 2 | 2 | 5 |
| RAD51D | 17 | 33445598 | 185C>T | Exon 3 | S62L | 1 | 0 | 1 | 2 | 1 | 0 |
| RAD51D | 17 | 33445581 | 202G>A | Exon 3 | G68S | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33434138 | 349T>A | Exon 5 | C117S | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33434081 | 406G>C | Exon 5 | D136H | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33433490 | 491T>C | Exon 6 | L164P | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33433488 | 493C>T | Exon 6 | R165W | 1 | 1 | 1 | 3 | 1 | 0 |
| RAD51D | 17 | 33433448 | 533T>G | Exon 6 | M178R | 1 | 0 | 1 | 2 | 1 | 1 |
| RAD51D | 17 | 33430511 | 629C>T | Exon 7 | A210V | 1 | 1 | 1 | 3 | 0 | 2 |
| RAD51D | 17 | 33430487 | 653G>A | Exon7 | G218D | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33430296 | 715C>T | Exon 8 | R239W | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33428338 | 785C>T | Exon 9 | P262L | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33428330 | 793G>A | Exon 9 | G265R | 1 | 1 | 1 | 3 | 2 | 0 |
| RAD51D | 17 | 33428309 | 814C>T | Exon 9 | P272S | 1 | 1 | 1 | 3 | 1 | 0 |
| RAD51D | 17 | 33428300 | 823C>T | Exon 9 | R275W | 1 | 1 | 1 | 3 | 0 | 1 |
| RAD51D | 17 | 33428015 | 944G>A | Exon 10 | G315E | 1 | 0 | 1 | 2 | 1 | 0 |
| Probably benign rare variant (n = 67) | |||||||||||
| RAD51B | 14 | 68290285 | 25G>A | Exon 2 | V9M | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51B | 14 | 68290324 | 64C>T | Exon 2 | H22Y | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51B | 14 | 68292196 | 100T>C | Exon 3 | S34P | 0 | 1 | 0 | 1 | 1 | 0 |
| RAD51B | 14 | 68292283 | 187A>G | Exon 3 | K63E | 0 | 0 | 0 | 0 | 1 | 1 |
| RAD51B | 14 | 68301803 | 205G>A | Exon 4 | G69R | 0 | 0 | 0 | 0 | 2 | 0 |
| RAD51B | 14 | 68301820 | 222G>T | Exon 4 | R74S | 0 | 0 | 1 | 1 | 1 | 0 |
| RAD51B | 14 | 68301824 | 226G>A | Exon 4 | A76T | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51B | 14 | 68301830 | 232T>C | Exon 4 | F78L | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51B | 14 | 68301863 | 265G>A | Exon 4 | A89T | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51B | 14 | 68301872 | 274G>A | Exon 4 | E92K | 0 | 0 | 0 | 0 | 1 | 1 |
| RAD51B | 14 | 68301894 | 296C>T | Exon 4 | A99V | 1 | 0 | 0 | 1 | 0 | 1 |
| RAD51B | 14 | 68331763 | 359T>C | Exon 5 | M120T | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51B | 14 | 68331840 | 436G>A | Exon 5 | A146T | 0 | 1 | 0 | 1 | 1 | 1 |
| RAD51B | 14 | 68352659 | 526A>G | Exon 6 | K176E | 1 | 0 | 0 | 1 | 1 | 0 |
| RAD51B | 14 | 68352672 | 539A>G | Exon 6 | Y180C | 0 | 0 | 0 | 0 | 20 | 39 |
| RAD51B | 14 | 68353784 | 619G>T | Exon 7 | V207L | 0 | 0 | 0 | 0 | 17 | 22 |
| RAD51B | 14 | 68353913 | 748T>G | Exon 7 | S250A | 0 | 0 | 0 | 0 | 0 | 2 |
| RAD51B | 14 | 68878147 | 860C>A | Exon9 | S287Y | 1 | 0 | 0 | 1 | 0 | 1 |
| RAD51B | 14 | 68878180 | 893A>G | Exon 9 | N298S | 0 | 1 | 0 | 1 | 0 | 1 |
| RAD51B | 14 | 68878224 | 937C>G | Exon 9 | L313V | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51B | 14 | 68934949 | 1018G>C | Exon 10 | E340Q | 0 | 0 | 0 | 0 | 2 | 0 |
| RAD51B | 14 | 68934959 | 1028T>C | Exon 10 | V343A | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51B | 14 | 69061225 | 1060C>G | Exon 11 | Q354E | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51B | 14 | 69061226 | 1061A>C | Exon 11 | Q354P | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51B | 14 | 69061228 | 1063G>A | Exon 11 | A355T | 0 | 0 | 0 | 0 | 13 | 21 |
| RAD51C | 17 | 56770011 | 7G>A | Exon 1 | G3R | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56770018 | 14C>T | Exon 1 | T5M | 1 | 0 | 0 | 1 | 1 | 0 |
| RAD51C | 17 | 56770036 | 32A>G | Exon 1 | Q11R | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56770131 | 127C>T | Exon 1 | P43S | 0 | 0 | 1 | 1 | 0 | 1 |
| RAD51C | 17 | 56772345 | 199G>A | Exon 2 | E67K | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56772359 | 213T>A | Exon 2 | N71K | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51C | 17 | 56772390 | 244C>A | Exon 2 | H82N | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56772398 | 252G>T | Exon 2 | K84N | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56772504 | 358A>G | Exon 2 | T120A | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51C | 17 | 56774057 | 408G>A | Exon 3 | M136I | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51C | 17 | 56774080 | 431T>C | Exon 3 | I144T | 0 | 0 | 1 | 1 | 1 | 0 |
| RAD51C | 17 | 56774142 | 493A>T | Exon 3 | M165L | 0 | 0 | 0 | 0 | 1 | 2 |
| RAD51C | 17 | 56774155 | 506T>C | Exon 3 | V169A | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51C | 17 | 56774158 | 509T>G | Exon 3 | V170G | 0 | 0 | 1 | 1 | 1 | 1 |
| RAD51C | 17 | 56774170 | 521C>G | Exon 3 | T174S | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56774214 | 565G>A | Exon 3 | G189R | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56780592 | 607A>G | Exon 4 | N203D | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56780605 | 620A>G | Exon 4 | H207R | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56787298 | 784T>G | Exon 5 | L262V | 0 | 0 | 0 | 0 | 1 | 4 |
| RAD51C | 17 | 56787304 | 790G>A | Exon 5 | G264S | 0 | 0 | 1 | 1 | 19 | 23 |
| RAD51C | 17 | 56798141 | 872A>T | Exon 6 | D291V | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51C | 17 | 56801448 | 952G>A | Exon 7 | D318N | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56801452 | 956G>A | Exon 7 | R319Q | 0 | 0 | 0 | 0 | 1 | 1 |
| RAD51C | 17 | 56811513 | 1061C>T | Exon 9 | A354V | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51C | 17 | 56811542 | 1090A>G | Exon 9 | S364G | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51D | 17 | 33446566 | 67C>T | Exon 1 | H23Y | 0 | 0 | 0 | 0 | 1 | 1 |
| RAD51D | 17 | 33446143 | 131G>A | Exon 2 | G44D | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51D | 17 | 33446143 | 131G>C | Exon 2 | G44A | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51D | 17 | 33445575 | 208G>A | Exon 3 | D70N | 0 | 0 | 1 | 1 | 0 | 1 |
| RAD51D | 17 | 33434132 | 355T>C | Exon 5 | C119R | 0 | 0 | 0 | 0 | 2 | 2 |
| RAD51D | 17 | 33434093 | 394G>A | Exon 5 | V132I | 1 | 0 | 0 | 1 | 0 | 2 |
| RAD51D | 17 | 33433451 | 530A>G | Exon 6 | Q177R | 0 | 0 | 0 | 0 | 2 | 0 |
| RAD51D | 17 | 33433447 | 534G>C | Exon 6 | M178I | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51D | 17 | 33433446 | 535C>G | Exon 6 | L179V | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51D | 17 | 33433413 | 568G>A | Exon 6 | A190T | 0 | 0 | 0 | 0 | 0 | 2 |
| RAD51D | 17 | 33428370 | 753A>G | Exon 9 | I251M | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51D | 17 | 33428279 | 844G>A | Exon 9 | E282K | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51D | 17 | 33428261 | 862G>C | Exon 9 | G288R | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51D | 17 | 33428251 | 872G>A | Exon 9 | R291H | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51D | 17 | 33428245 | 878C>T | Exon 9 | A293V | 0 | 0 | 0 | 0 | 1 | 0 |
| RAD51D | 17 | 33428037 | 922A>G | Exon 10 | M308V | 0 | 0 | 0 | 0 | 0 | 1 |
| RAD51D | 17 | 33428022 | 937A>G | Exon 10 | T313A | 0 | 0 | 0 | 0 | 1 | 0 |
SIFT: 0, tolerated; 1, not tolerated.
PolyPhen-2: 0, benign/possibly damaging; 1, probably damaging.
Provean: 0, neutral; 1, deleterious.
Score: number of algorithms (SIFT/PolyPhen-2/Provean) that predict deleterious effect of the missense variant.
No. of times the variant was identified in controls.
No. of times the variant was identified in controls.
At least two of three prediction algorithms predict deleterious effect on protein function.
Table A5.
Reported Targeted Sequencing on RAD51 Genes
| Study and Location by Gene | No. of Patients Analyzed |
No. (%) of RAD51 Mutations Identified |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Total | BC | BC/OC | OC | uOC | Controls* | Total | BC | BC/OC | OC | uOC | Controls* | |
| RAD51C | ||||||||||||
| Germany19 | 1,100 | 620 | 480 | 0 | 0 | 480 + 2,432† | 6 (0.5) | 6 (1.25) | 0 | 0 | 0 | |
| Untied States (Zheng et al)‡ | 92 | 0 | 92 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Canada (Akbari et al)‡ | 454 | NS | NS | NS | 0 | 0 | 0 | NS | NS | NS | 0 | |
| Finland35 | 2,747 | 130 + 2,061† | 139 | 8 | 409† | 2,086† | 8 (0.3) | 0 | 2 (1.4) | 2 (25) | 4† (1.0) | 2† (0.1) |
| Finland and Sweden38 | 1,704 | 1,105† | 35 | 0 | 232 + 332† | 871† | 2 (0.1) | 0 | 1 (2.8) | 0 | 1 (0.4) + 0† | 0† |
| United States (Clague et al)‡ | 286 | 133 | 34 | 119 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Australia37 | 1,655 | 1,053 | 314 | 21 | 267 | 427 | 3 (0.2) | 0 | 1 (0.3) | 1 (4.8) | 1 (0.4) | 0 |
| The Netherlands and Canada (De Leeneer et al)‡ | 351 | 0 | 239 | 112 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Spain30 | 785 | 485 | 300 | 0 | 0 | 500 | 5 (0.6) | 1 (0.2) | 4 (1.3) | 0 | 0 | 0 |
| United States (Lu et al)‡ | 192 | 157 | 35 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| United Kingdom34 | 1,404 | 0 | 1,102 | 30 | 272 | 1,156 | 12 (0.9) | 0 | 8 (0.7) | 1 (3.3) | 3 (1.1) | 1 (0.09) |
| France (Coulet et al)‡ | 117 | 0 | 82 | 35 | 0 | 0 | 3 (2.6) | 0 | 2 (2.4) | 1 (2.9) | 0 | 0 |
| Germany (Schnurbein et al)‡ | 825 | 500 | 325 | 0 | 0 | 0 | 2 (0.3) | 1 (0.2) | 1 (0.3) | 0 | 0 | 0 |
| United States36 | 367 | 0 | 0 | 0 | 367 | 0 | 3 (0.82) | 0 | 0 | 0 | 3 (0.82) | 0 |
| Spain (Blanco et al)‡ | 516 | 410 | 89 | 17 | 0 | 0 | 3 (0.6) | 1 (0.24) | 2 (2.2) | 0 | 0 | 0 |
| This study§ | 3,429 | 0 | 0 | 294 | 3,135 | 2,772 | 14 (0.41) | 0 | 0 | 4 (1.4) | 10 (0.32) | 2 (0.07) |
| Total | 16,024 | 6,654 | 3,266 | 636 | 5,014 | 10,724 | 61 | 3 | 27 | 9 | 22 | 5 |
| Total fully sequenced | 524 | 4,273 | 4,903 | 9 (0.017) | 18 (0.004) | 3 (0.0006) | ||||||
| RAD51D | ||||||||||||
| United Kingdom20 | 1,648 | 737 | 911 | 0 | 0 | 1,060 | 0 | 8 (0.88) | 0 | 0 | 1 (0.09) | |
| Canada and Belgium (Osher et al)‖ | 175 | 0 | 175 | 0 | 0 | 0 | 1 (0.57) | 0 | 1 (0.57) | 0 | 0 | 0 |
| Finland (Pelttari et al)‖ | 2,200 | 95 + 297 | 541† | 1,287† | 2 | 3 (0.55) | 0 | |||||
| United Kingdom40 | 1,305 | 741 | 303 | 16 | 245 | 466 | 2 | 0 | 0 | 0 | 2 (0.82) | 0 |
| Spain (Gutierrez-Enriquez et al)‖ | 713 | 171 | 491 | 51 | 4 (0.81) | |||||||
| United States36 | 367 | 0 | 0 | 0 | 367 | 0 | 4 | 0 | 0 | 0 | 4 (1.1) | 0 |
| This study§ | 3,429 | 0 | 0 | 294 | 3,135 | 2,772 | 1 (0.34) | 11 (0.35) | 1 (0.036) | |||
| Total fully sequenced | 361 | 3,747 | 4,298 | 5 (1.4) | 17 (0.45) | 2 (0.046) | ||||||
Abbreviations: BC, breast cancer case proband from breast cancer familial study; BC/OC, breast and/or ovarian cancer proband from breast and/or ovarian cancer family; NS, not specified; OC, ovarian cancer proband from ovarian cancer family; uOC, ovarian cancer cases not selected based on family history.
Unaffected controls.
The subset was not fully sequenced but underwent genotyping for mutations detected previously.
RAD51C study references: Zheng et al: Breast Cancer Res Treat 124:857-861, 2010; Akbari et al: Breast Cancer Res 12:404, 2010; Clague et al: PLoS One 6:e25632, 2011; De Leeneer et al: Breast Cancer Res Treat 133:393-398, 2012; Lu et al: Fam Cancer 11:381-385, 2012; Coulet et al: Clin Genet 83:332-336, 2013; Schnurbein et al: Breast Cancer Res 15:R120, 2013; Blanco et al: Breast Cancer Res Treat 147:133-143, 2014.
In the United States, United Kingdom, Australia, Denmark, and Poland.
RAD51D study references: Osher et al: Br J Cancer 106:1460-3, 2012; Pelttari et al: J Med Genet 49:429-432, 2012; Gutierrez-Enriquez et al: Int J Cancer 134:2088-2097, 2014.
Support information appears at the end of this article.
Written on behalf of the Australian Ovarian Cancer Study Group and the Ovarian Cancer Association Consortium.
Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
Authors' disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article.
Support
Supported by the Cancer Councils of New South Wales, Victoria, Queensland, South Australia and Tasmania, Australia; the Cancer Foundation of Western Australia; Cancer Research UK (Grants No. C315/A2621, C490/A10119, C490/A10124, C490/A16561, C490/A6187, C1005/A12677, C1005/A6383, and C1005/A7749); the Danish Cancer Society (Grant No. 94 222 52); the Eve Appeal (the Oak Foundation); the Fred C. and Katherine B. Andersen Foundation; the Mermaid I project; the National Institutes of Health (Grants No. P30CA15083, P30CA016056, P50CA136393, R01CA122443, R01CA178535, R01CA61107, R01CA152990, and R01CA086381); the National Health and Medical Research Council of Australia (Grants No. ID400413 and ID400281); the Pomeranian Medical University; Queensland Cancer Fund; Roswell Park Cancer Institute Alliance Foundation; the United Kingdom Department of Health; the United Kingdom National Institute for Health Research Biomedical Research Centres at the University of Cambridge and at the University College London Hospitals; and the US Army Medical Research and Material Command (Grant No. DAMD17-01-1-0729). J.H. was funded by a United Kingdom Medical Research Council CASE industrial partnership PhD studentship. I.J.J. holds an NIHR senior investigator award.
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Disclosures provided by the authors are available with this article at www.jco.org.
AUTHOR CONTRIBUTIONS
Conception and design: Honglin Song, Susan J. Ramus, Usha Menon, Ian J. Jacobs, Simon A. Gayther, Paul D.P. Pharoah
Financial support: Susan J. Ramus, Ellen Goode
Provision of study materials or patients: Susan J. Ramus, Adam Rosenthal, Aleksandra Gentry-Maharaj, Jan Lubiński, Anna Jakubowska, Ellen L. Goode
Collection and assembly of data: Honglin Song, Ed Dicks, Susan J. Ramus, Maria P. Intermaggio, Jane Hayward, Christopher K. Edlund, David Conti, Patricia Harrington, Lindsay Fraser, Susan Philpott, Christopher Anderson, Adam Rosenthal, Aleksandra Gentry-Maharaj, David D. Bowtell, Kathryn Alsop, Mine S. Cicek, Julie M. Cunningham, Brooke L. Fridley, Jennifer Alsop, Mercedes Jimenez-Linan, Estrid Høgdall, Claus K. Høgdall, Allan Jensen, Susanne Krüger Kjaer, Jan Lubiński, Tomasz Huzarski, Anna Jakubowska, Jacek Gronwald, Samantha Poblete, Shashi Lele, Lara Sucheston-Campbell, Kirsten B. Moysich, Kunle Odunsi, Ellen L. Goode, Usha Menon, Ian J. Jacobs, Paul D.P. Pharoah
Data analysis and interpretation: Honglin Song, Ed Dicks, Susan J. Ramus, Jonathan P. Tyrer, Christopher K. Edlund, Adam Rosenthal, Brooke L. Fridley, Usha Menon, Ian J. Jacobs, Paul D.P. Pharoah
Manuscript writing: All authors
Final approval of manuscript: All authors
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Contribution of Germline Mutations in the RAD51B, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population
The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc.
Honglin Song
No relationship to disclose
Ed Dicks
No relationship to disclose
Susan J. Ramus
No relationship to disclose
Jonathan P. Tyrer
No relationship to disclose
Maria P. Intermaggio
No relationship to disclose
Jane Hayward
No relationship to disclose
Christopher K. Edlund
Patents, Royalties, Other Intellectual Property: BioRealm
David Conti
Consulting or Advisory Role: BioRealm
Patricia Harrington
No relationship to disclose
Lindsay Fraser
No relationship to disclose
Susan Philpott
No relationship to disclose
Christopher Anderson
No relationship to disclose
Adam Rosenthal
Honoraria: Fujirebio Diagnostics
Consulting or Advisory Role: Myriad Genetics
Travel, Accommodations, Expenses: Fujirebio Diagnostics
Aleksandra Gentry-Maharaj
No relationship to disclose
David D. Bowtell
No relationship to disclose
Kathryn Alsop
Consulting or Advisory Role: AstraZeneca (Inst)
Mine S. Cicek
No relationship to disclose
Julie M. Cunningham
No relationship to disclose
Brooke L. Fridley
No relationship to disclose
Jennifer Alsop
No relationship to disclose
Mercedes Jimenez-Linan
No relationship to disclose
Estrid Høgdall
No relationship to disclose
Claus K. Høgdall
No relationship to disclose
Allan Jensen
No relationship to disclose
Susanne Krüger Kjaer
No relationship to disclose
Jan Lubiński
No relationship to disclose
Tomasz Huzarski
No relationship to disclose
Anna Jakubowska
No relationship to disclose
Jacek Gronwald
No relationship to disclose
Samantha Poblete
No relationship to disclose
Shashi Lele
No relationship to disclose
Lara Sucheston-Campbell
No relationship to disclose
Kirsten B. Moysich
No relationship to disclose
Kunle Odunsi
Research Funding: iTeos (Inst)
Ellen L. Goode
No relationship to disclose
Usha Menon
Stock or Other Ownership: Abcodia
Ian J. Jacobs
Employment: Abcodia
Patents, Royalties, Other Intellectual Property: ROC Algorithm
Simon A. Gayther
No relationship to disclose
Paul D.P. Pharoah
No relationship to disclose
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