Elise Ruark

Germline mutations in RAD51D confer susceptibility to ovarian cancer

Recently, RAD51C mutations were identified in families with breast and ovarian cancer. This observation prompted us to investigate the role of RAD51D in cancer susceptibility. We identified eight inactivating RAD51D mutations in unrelated individuals from 911 breast-ovarian cancer families compared with one inactivating mutation identified in 1,060 controls (P = 0.01). The association found here was principally with ovarian cancer, with three mutations identified in the 59 pedigrees with three or more individuals with ovarian cancer (P = 0.0005). The relative risk of ovarian cancer for RAD51D mutation carriers was estimated to be 6.30 (95% CI 2.86–13.85, P = 4.8 × 10−6). By contrast, we estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59–2.96, P = 0.50). These data indicate that RAD51D mutation testing may have clinical utility in individuals with ovarian cancer and their families. Moreover, we show that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D mutation carriers.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case–control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10−5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10−4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10−9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Germline RAD51C mutations confer susceptibility to ovarian cancer

To the Editor: In 2010, Meindl and colleagues proposed that germline RAD51C mutations confer high risk for breast and ovarian cancer, comparable to BRCA1 and BRCA2 mutations1,2. However, multiple follow-up studies have provided no supportive evidence that RAD51C mutations predispose to breast cancer3–12. Following the original report, we began investigating the role of other RAD51 paralogs in breast and ovarian cancer susceptibility. This led to our recent discovery that germline RAD51D mutations predispose to ovarian cancer13. We identified truncating RAD51D mutations in 8 of 911 familial breast-ovarian cancer pedigrees and 1 of 1,060 population controls. Our analysis of simultaneous association with both breast and ovarian cancer risk showed that RAD51D mutations confer a sixfold increased risk of ovarian cancer (relative risk (RR) = 6.30, 95% confidence interval (CI) = 2.86–13.85; P = 4.8 °— 10-6) but do not affect or cause only a small increase in breast cancer risk (RR = 1.32, 95% CI = 0.59– 2.96; P = 0.50). This result was supported by our analysis of 737 familial breast cancer pedigrees with no ovarian cancer, in which we detected no RAD51D mutations. These findings prompted us to reevaluate the role of RAD51C in cancer susceptibility. We sequenced the full coding region and intronexon boundaries of RAD51C in 1,132 probands from families with a history of ovarian cancer occurring with or without breast cancer, 272 individuals with ovarian cancer from a hospital-based unselected case series and 1,156 population-based controls (Supplementary Tables 1 and 2 and Supplementary Methods). We identified 12 mutations that result in premature protein truncation in cases compared to 1 such mutation in controls (P = 0.009) (Table 1 and Supplementary Fig. 1). Nine mutations were identified among the 1,132 familial cases, and there was a higher prevalence of mutations in families with multiple ovarian cancer cases: 4 mutations were detected in 311 families with 2 or more cases of ovarian cancer, and 2 mutations were detected in the 67 families with 3 or more cases of ovarian cancer. Three mutations were identified among the 272 individuals with ovarian cancer unselected for family history, suggesting that ~1% of ovarian cancer cases harbor germline RAD51C mutations. We also identified a total of 12 nonsynonymous RAD51C variants (Supplementary Table 3). Four variants were identified in cases and controls; only one, c.790G>A, encoding a p.Gly264Ser amino-acid change, showed any evidence of association with cancer (P = 0.02), consistent with other studies2,9,12. Of note, this variant is predicted to be benign by in silico analyses and has limited impact on RAD51C function2. The remaining eight nonsynonymous variants were each identified in a single individual; there was no significant difference in the overall frequency (P = 0.36), position or predicted functional effects of these variants between cases and controls (Supplementary Table 3). These data exemplify the inherent complexities of evaluating the clinical consequences of missense variants (outside simple Mendelian disorders) and underscore why non-truncating and truncating variants should be considered separately. Analyzing controls for specific rare variants detected in cases and concluding that their absence in controls is evidence of pathogenicity can result in over-interpretation of the data. Such findings confirm that the specific variant is rare but can seldom provide conclusive evidence of disease association. Full sequencing of the gene in both cases and controls is a more appropriate analysis, as it allows the spectrum of variants in cases and controls to be directly compared. Functional and conservation data can be useful in the evaluation of variants, but in vitro functional effects do not necessarily imply that the variant has clinical sequelae. Moreover, as we and others have shown (for example, in studies of the breast cancer susceptibility genes BRIP1 and ATM), such an assumption can result in incorrect attribution of pathogenicity14,15. Better information is provided when mutational and functional analyses are equally ascertained in both cases and controls. To estimate the risk associated with RAD51C mutations, we undertook modified segregation analysis, in which we simultaneously modeled the risks of ovarian and breast cancer and incorporated control data and information from the full pedigrees of mutation-positive and mutation-negative families (Supplementary Methods). The relative risk of ovarian cancer for RAD51C mutation carriers was estimated to be 5.88 (95% CI = 2.91–11.88; P = 7.65 × 10-7), which constitutes a >9% cumulative risk by age 80. In contrast, there was no evidence of an association with breast cancer (RR = 0.91, 95% CI = 0.45–1.86; P = 0.8). Thus, the cancer risk estimates for RAD51C mutations were similar to those estimated for RAD51D mutations13. These data are fully consistent with the results presented by Meindl et al. and provide a likely explanation for why Meindl et al. identified RAD51C mutations only in breast cancer cases that had relatives with ovarian cancer and not in 620 familial breast cancer pedigrees without ovarian cancer. As RAD51C

Identification of nine new susceptibility loci for testicular cancer, including variants near DAZL and PRDM14

Testicular germ cell tumor (TGCT) is the most common cancer in young men and is notable for its high familial risks. So far, six loci associated with TGCT have been reported. From genome-wide association study (GWAS) analysis of 307,291 SNPs in 986 TGCT cases and 4,946 controls, we selected for follow-up 694 SNPs, which we genotyped in a further 1,064 TGCT cases and 10,082 controls from the UK. We identified SNPs at nine new loci (1q22, 1q24.1, 3p24.3, 4q24, 5q31.1, 8q13.3, 16q12.1, 17q22 and 21q22.3) showing association with TGCT (P < 5 × 10−8), which together account for an additional 4–6% of the familial risk of TGCT. The loci include genes plausibly related to TGCT development. PRDM14, at 8q13.3, is essential for early germ cell specification, and DAZL, at 3p24.3, is required for the regulation of germ cell development. Furthermore, PITX1, at 5q31.1, regulates TERT expression and is the third TGCT-associated locus implicated in telomerase regulation.

Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Mutations in CEP57 cause mosaic variegated aneuploidy syndrome

Using exome sequencing and a variant prioritization strategy that focuses on loss-of-function variants, we identified biallelic, loss-of-function CEP57 mutations as a cause of constitutional mosaic aneuploidies. CEP57 is a centrosomal protein and is involved in nucleating and stabilizing microtubules. Our findings indicate that these and/or additional functions of CEP57 are crucial for maintaining correct chromosomal number during cell division.

Predisposition gene identification in common cancers by exome sequencing: insights from familial breast cancer

The genetic component of breast cancer predisposition remains largely unexplained. Candidate gene case–control resequencing has identified predisposition genes characterised by rare, protein truncating mutations that confer moderate risks of disease. In theory, exome sequencing should yield additional genes of this class. Here, we explore the feasibility and design considerations of this approach. We performed exome sequencing in 50 individuals with familial breast cancer, applying frequency and protein function filters to identify variants most likely to be pathogenic. We identified 867,378 variants that passed the call quality filters of which 1,296 variants passed the frequency and protein truncation filters. The median number of validated, rare, protein truncating variants was 10 in individuals with, and without, mutations in known genes. The functional candidacy of mutated genes was similar in both groups. Without prior knowledge, the known genes would not have been recognisable as breast cancer predisposition genes. Everyone carries multiple rare mutations that are plausibly related to disease. Exome sequencing in common conditions will therefore require intelligent sample and variant prioritisation strategies in large case–control studies to deliver robust genetic evidence of disease association.

Implementing rapid, robust, cost-effective, patient-centred, routine genetic testing in ovarian cancer patients

Advances in DNA sequencing have made genetic testing fast and affordable, but limitations of testing processes are impeding realisation of patient benefits. Ovarian cancer exemplifies the potential value of genetic testing and the shortcomings of current pathways to access testing. Approximately 15% of ovarian cancer patients have a germline BRCA1 or BRCA2 mutation which has substantial implications for their personal management and that of their relatives. Unfortunately, in most countries, routine implementation of BRCA testing for ovarian cancer patients has been inconsistent and largely unsuccessful. We developed a rapid, robust, mainstream genetic testing pathway in which testing is undertaken by the trained cancer team with cascade testing to relatives performed by the genetics team. 207 women with ovarian cancer were offered testing through the mainstream pathway. All accepted. 33 (16%) had a BRCA mutation. The result informed management of 79% (121/154) women with active disease. Patient and clinician feedback was very positive. The pathway offers a 4-fold reduction in time and 13-fold reduction in resource requirement compared to the conventional testing pathway. The mainstream genetic testing pathway we present is effective, efficient and patient-centred. It can deliver rapid, robust, large-scale, cost-effective genetic testing of BRCA1 and BRCA2 and may serve as an exemplar for other genes and other diseases.

Germline mutations in the PAF1 complex gene CTR9 predispose to Wilms tumour

Wilms tumour is a childhood kidney cancer. Here we identify inactivating CTR9 mutations in 3 of 35 Wilms tumour families, through exome and Sanger sequencing. By contrast, no similar mutations are present in 1,000 population controls (P<0.0001). Each mutation segregates with Wilms tumour in the family and a second mutational event is present in available tumours. CTR9 is a key component of the polymerase-associated factor 1 complex which has multiple roles in RNA polymerase II regulation and is implicated in embryonic organogenesis and maintenance of embryonic stem cell pluripotency. These data establish CTR9 as a Wilms tumour predisposition gene and suggest it acts as a tumour suppressor gene.

A genome-wide association study identifies susceptibility loci for Wilms tumor

Wilms tumor is the most common renal malignancy of childhood. To identify common variants that confer susceptibility to Wilms tumor, we conducted a genome-wide association study in 757 individuals with Wilms tumor (cases) and 1,879 controls. We evaluated ten SNPs in regions significantly associated at P < 5 × 10−5 in two independent replication series from the UK (769 cases and 2,814 controls) and the United States (719 cases and 1,037 controls). We identified clear significant associations at 2p24 (rs3755132, P = 1.03 × 10−14; rs807624, P = 1.32 × 10−14) and 11q14 (rs790356, P = 4.25 × 10−15). Both regions contain genes that are plausibly related to Wilms tumorigenesis. We also identified candidate association signals at 5q14, 22q12 and Xp22.