Margaret Warren-Perry

Genome-wide association study identifies five new breast cancer susceptibility loci

Breast cancer is the most common cancer in women in developed countries. To identify common breast cancer susceptibility alleles, we conducted a genome-wide association study in which 582,886 SNPs were genotyped in 3,659 cases with a family history of the disease and 4,897 controls. Promising associations were evaluated in a second stage, comprising 12,576 cases and 12,223 controls. We identified five new susceptibility loci, on chromosomes 9, 10 and 11 (P = 4.6 × 10−7 to P = 3.2 × 10−15). We also identified SNPs in the 6q25.1 (rs3757318, P = 2.9 × 10−6), 8q24 (rs1562430, P = 5.8 × 10−7) and LSP1 (rs909116, P = 7.3 × 10−7) regions that showed more significant association with risk than those reported previously. Previously identified breast cancer susceptibility loci were also found to show larger effect sizes in this study of familial breast cancer cases than in previous population-based studies, consistent with polygenic susceptibility to the disease.

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

BRCA1 testing should be offered to individuals with triple-negative breast cancer diagnosed below 50 years

Background:Triple-negative (TN) tumours are the predominant breast cancer subtype in BRCA1 mutation carriers. Recently, it was proposed that all individuals below 50 years of age with TN breast cancer should be offered BRCA testing. We have evaluated the BRCA1 mutation frequency and the implications for clinical practice of undertaking genetic testing in women with TN breast cancer.Methods:We undertook BRCA1 mutation analysis in 308 individuals with TN breast cancer, 159 individuals from unselected series of breast cancer and 149 individuals from series ascertained on the basis of young age and/or family history.Results:BRCA1 mutations were present in 45 out of 308 individuals. Individuals with TN cancer <50 years had >10% likelihood of carrying a BRCA1 mutation in both the unselected (11 out of 58, 19%) and selected (26 out of 111, 23%) series. However, over a third would not have been offered testing using existing criteria. We estimate that testing all individuals with TN breast cancer <50 years would generate an extra 1200 tests annually in England.Conclusion:Women with TN breast cancer diagnosed below 50 years have >10% likelihood of carrying a BRCA1 mutation and are therefore eligible for testing in most centres. However, implementation may place short-term logistical and financial burdens on genetic services.

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.

Gene-gene interactions in breast cancer susceptibility

There have been few definitive examples of gene-gene interactions in humans. Through mutational analyses in 7325 individuals, we report four interactions (defined as departures from a multiplicative model) between mutations in the breast cancer susceptibility genes ATM and CHEK2 with BRCA1 and BRCA2 (case-only interaction between ATM and BRCA1/BRCA2 combined, P = 5.9 × 10(-4); ATM and BRCA1, P= 0.01; ATM and BRCA2, P= 0.02; CHEK2 and BRCA1/BRCA2 combined, P = 2.1 × 10(-4); CHEK2 and BRCA1, P= 0.01; CHEK2 and BRCA2, P= 0.01). The interactions are such that the resultant risk of breast cancer is lower than the multiplicative product of the constituent risks, and plausibly reflect the functional relationships of the encoded proteins in DNA repair. These findings have important implications for models of disease predisposition and clinical translation.

Multi-stage genome-wide association study identifies new susceptibility locus for testicular germ cell tumour on chromosome 3q25

Recent genome-wide association studies (GWAS) and subsequent meta-analyses have identified over 25 SNPs at 18 loci, together accounting for >15% of the genetic susceptibility to testicular germ cell tumour (TGCT). To identify further common SNPs associated with TGCT, here we report a three-stage experiment, involving 4098 cases and 18 972 controls. Stage 1 comprised previously published GWAS analysis of 307 291 SNPs in 986 cases and 4946 controls. In Stage 2, we used previously published customised Illumina iSelect genotyping array (iCOGs) data across 694 SNPs in 1064 cases and 10 082 controls. Here, we report new genotyping of eight SNPs showing some evidence of association in combined analysis of Stage 1 and Stage 2 in an additional 2048 cases of TGCT and 3944 controls (Stage 3). Through fixed-effects meta-analysis across three stages, we identified a novel locus at 3q25.31 (rs1510272) demonstrating association with TGCT [per-allele odds ratio (OR) = 1.16, 95% confidence interval (CI) = 1.06-1.27; P = 1.2 × 10(-9)].

Mutation and association analysis of GEN1 in breast cancer susceptibility

GEN1 was recently identified as a key Holliday junction resolvase involved in homologous recombination. Somatic truncating GEN1 mutations have been reported in two breast cancers. Together these data led to the proposition that GEN1 is a breast cancer predisposition gene. In this article we have formally investigated this hypothesis. We performed full-gene mutational analysis of GEN1 in 176 BRCA1/2-negative familial breast cancer samples and 159 controls. We genotyped six SNPs tagging the 30 common variants in the transcribed region of GEN1 in 3,750 breast cancer cases and 4,907 controls. Mutation analysis revealed one truncating variant, c.2515_2519delAAGTT, which was present in 4% of cases and 4% of controls. We identified control individuals homozygous for the deletion, demonstrating that the last 69 amino acids of GEN1 are dispensable for its function. We identified 17 other variants, but their frequency did not significantly differ between cases and controls. Analysis of 3,750 breast cancer cases and 4,907 controls demonstrated no evidence of significant association with breast cancer for six SNPs tagging the 30 common GEN1 variants. These data indicate that although it also plays a key role in double-strand DNA break repair, GEN1 does not make an appreciable contribution to breast cancer susceptibility by acting as a high- or intermediate-penetrance breast cancer predisposition gene like BRCA1, BRCA2, CHEK2, ATM, BRIP1 and PALB2 and that common GEN1 variants do not act as low-penetrance susceptibility alleles analogous to SNPs in FGFR2. Furthermore, our analyses demonstrate the importance of undertaking appropriate genetic investigations, typically full gene screening in cases and controls together with large-scale case–control association analyses, to evaluate the contribution of genes to cancer susceptibility.