Clare Turnbull

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.

Breast-Cancer Risk in Families with Mutations in PALB2

BACKGROUND Germline loss-of-function mutations in PALB2 are known to confer a predisposition to breast cancer. However, the lifetime risk of breast cancer that is conferred by such mutations remains unknown. METHODS We analyzed the risk of breast cancer among 362 members of 154 families who had deleterious truncating, splice, or deletion mutations in PALB2. The age-specific breast-cancer risk for mutation carriers was estimated with the use of a modified segregation-analysis approach that allowed for the effects of PALB2 genotype and residual familial aggregation. RESULTS The risk of breast cancer for female PALB2 mutation carriers, as compared with the general population, was eight to nine times as high among those younger than 40 years of age, six to eight times as high among those 40 to 60 years of age, and five times as high among those older than 60 years of age. The estimated cumulative risk of breast cancer among female mutation carriers was 14% (95% confidence interval [CI], 9 to 20) by 50 years of age and 35% (95% CI, 26 to 46) by 70 years of age. Breast-cancer risk was also significantly influenced by birth cohort (P<0.001) and by other familial factors (P=0.04). The absolute breast-cancer risk for PALB2 female mutation carriers by 70 years of age ranged from 33% (95% CI, 25 to 44) for those with no family history of breast cancer to 58% (95% CI, 50 to 66) for those with two or more first-degree relatives with breast cancer at 50 years of age. CONCLUSIONS Loss-of-function mutations in PALB2 are an important cause of hereditary breast cancer, with respect both to the frequency of cancer-predisposing mutations and to the risk associated with them. Our data suggest the breast-cancer risk for PALB2 mutation carriers may overlap with that for BRCA2 mutation carriers. (Funded by the European Research Council and others.).

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.

A genome-wide association study of testicular germ cell tumor

We conducted a genome-wide association study for testicular germ cell tumor (TGCT), genotyping 307,666 SNPs in 730 cases and 1,435 controls from the UK and replicating associations in a further 571 cases and 1,806 controls. We found strong evidence for susceptibility loci on chromosome 5 (per allele OR = 1.37 (95% CI = 1.19–1.58), P = 3 × 10−13), chromosome 6 (OR = 1.50 (95% CI = 1.28–1.75), P = 10−13) and chromosome 12 (OR = 2.55 (95% CI = 2.05–3.19), P = 10−31). KITLG, encoding the ligand for the receptor tyrosine kinase KIT, which has previously been implicated in the pathogenesis of TGCT and the biology of germ cells, may explain the association on chromosome 12.

Variants near DMRT1, TERT and ATF7IP are associated with testicular germ cell cancer.

We conducted a genome-wide association study for testicular germ cell tumor, genotyping 298,782 SNPs in 979 affected individuals and 4,947 controls from the UK and replicating associations in a further 664 cases and 3,456 controls. We identified three new susceptibility loci, two of which include genes that are involved in telomere regulation. We identified two independent signals within the TERT-CLPTM1L locus on chromosome 5, which has previously been associated with multiple other cancers (rs4635969, OR = 1.54, P = 1.14 × 10−23; rs2736100, OR = 1.33, P = 7.55 × 10−15). We also identified a locus on chromosome 12 (rs2900333, OR = 1.27, P = 6.16 × 10−10) that contains ATF7IP, a regulator of TERT expression. Finally, we identified a locus on chromosome 9 (rs755383, OR = 1.37, P = 1.12 × 10−23), containing the sex determination gene DMRT1, which has been linked to teratoma susceptibility in mice.

DICER1 syndrome: clarifying the diagnosis, clinical features and management implications of a pleiotropic tumour predisposition syndrome

Background Constitutional DICER1 mutations were recently reported to cause familial pleuropulmonary blastoma (PPB). Aim To investigate the contribution and phenotypic spectrum of constitutional and somatic DICER1 mutations to cancer. Methods and results The authors sequenced DICER1 in constitutional DNA from 823 unrelated patients with a variety of tumours and in 781 cancer cell lines. Constitutional DICER1 mutations were identified in 19 families including 11/14 with PPB, 2/3 with cystic nephroma, 4/7 with ovarian Sertoli–Leydig-type tumours, 1/243 with Wilms tumour (this patient also had a Sertoli–Leydig tumour), 1/1 with intraocular medulloepithelioma (this patient also had PPB), 1/86 with medulloblastoma/infratentorial primitive neuroectodermal tumour, and 1/172 with germ cell tumour. The inheritance was investigated in 17 families. DICER1 mutations were identified in 25 relatives: 17 were unaffected, one mother had ovarian Sertoli–Leydig tumour, one half-sibling had cystic nephroma, and six relatives had non-toxic thyroid cysts/goitre. Analysis of eight tumours from DICER1 mutation-positive patients showed universal retention of the wild-type allele. DICER1 truncating mutations were identified in 4/781 cancer cell lines; all were in microsatellite unstable lines and therefore unlikely to be driver mutations. Conclusion Constitutional DICER1 haploinsufficiency predisposes to a broad range of tumours, making a substantial contribution to PPB, cystic nephroma and ovarian Sertoli–Leydig tumours, but a smaller contribution to other tumours. Most mutation carriers are unaffected, indicating that tumour risk is modest. The authors define the clinical contexts in which DICER1 mutation testing should be considered, the associated tumour risks, and the implications for at-risk individuals. They have termed this condition ‘DICER1 syndrome’. Accession numbers The cDNA Genbank accession number for the DICER1 sequence reported in this paper is NM_030621.2.

Genetic Predisposition to Breast Cancer: Past, Present, and Future

In recent years, our understanding of genetic predisposition to breast cancer has advanced significantly. Three classes of predisposition factors, categorized by their associated risks of breast cancer, are currently known. BRCA1 and BRCA2 are high-penetrance breast cancer predisposition genes identified by genome-wide linkage analysis and positional cloning. Mutational screening of genes functionally related to BRCA1 and/or BRCA2 has revealed four genes, CHEK2, ATM, BRIP1, and PALB2; mutations in these genes are rare and confer an intermediate risk of breast cancer. Association studies have further identified eight common variants associated with low-penetrance breast cancer predisposition. Despite these discoveries, most of the familial risk of breast cancer remains unexplained. In this review, we describe the known genetic predisposition factors, expound on the methods by which they were identified, and consider how further technological and intellectual advances may assist in identifying the remaining genetic factors underlying breast cancer susceptibility.

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

Meta-analysis identifies four new loci associated with testicular germ cell tumor

We conducted a meta-analysis to identify new susceptibility loci for testicular germ cell tumor (TGCT). In the discovery phase, we analyzed 931 affected individuals and 1,975 controls from 3 genome-wide association studies (GWAS). We conducted replication in 6 independent sample sets comprising 3,211 affected individuals and 7,591 controls. In the combined analysis, risk of TGCT was significantly associated with markers at four previously unreported loci: 4q22.2 in HPGDS (per-allele odds ratio (OR) = 1.19, 95% confidence interval (CI) = 1.12–1.26; P = 1.11 × 10−8), 7p22.3 in MAD1L1 (OR = 1.21, 95% CI = 1.14–1.29; P = 5.59 × 10−9), 16q22.3 in RFWD3 (OR = 1.26, 95% CI = 1.18–1.34; P = 5.15 × 10−12) and 17q22 (rs9905704: OR = 1.27, 95% CI = 1.18–1.33; P = 4.32 × 10−13 and rs7221274: OR = 1.20, 95% CI = 1.12–1.28; P = 4.04 × 10−9), a locus that includes TEX14, RAD51C and PPM1E. These new TGCT susceptibility loci contain biologically plausible genes encoding proteins important for male germ cell development, chromosomal segregation and the DNA damage response.