A Targeted Genetic Association Study of Epithelial Ovarian Cancer Susceptibility

BACKGROUND: Genome-wide association studies have identified several common susceptibility alleles for epithelial ovarian cancer (EOC). To further understand EOC susceptibility, we examined previously ungenotyped candidate variants, including uncommon variants and those residing within known susceptibility loci. RESULTS: At nine of eleven previously published EOC susceptibility regions (2q31, 3q25, 5p15, 8q21, 8q24, 10p12, 17q12, 17q21.31, and 19p13), novel variants were identified that were more strongly associated with risk than previously reported variants. Beyond known susceptibility regions, no variants were found to be associated with EOC risk at genome-wide statistical significance (p \u3c5x10(-8)), nor were any significant after Bonferroni correction for 17,000 variants (p\u3c 3x10-6). METHODS: A customized genotyping array was used to assess over 17,000 variants in coding, non-coding, regulatory, and known susceptibility regions in 4,973 EOC cases and 5,640 controls from 13 independent studies. Susceptibility for EOC overall and for select histotypes was evaluated using logistic regression adjusted for age, study site, and population substructure. CONCLUSION: Given the novel variants identified within the 2q31, 3q25, 5p15, 8q21, 8q24, 10p12, 17q12, 17q21.31, and 19p13 regions, larger follow-up genotyping studies, using imputation where necessary, are needed for fine-mapping and confirmation of low frequency variants that fall below statistical significance

Estimates of array and pool-construction variance for planning efficient DNA-pooling genome wide association studies

<p>Abstract</p> <p>Background</p> <p>Until recently, genome-wide association studies (GWAS) have been restricted to research groups with the budget necessary to genotype hundreds, if not thousands, of samples. Replacing individual genotyping with genotyping of DNA pools in Phase I of a GWAS has proven successful, and dramatically altered the financial feasibility of this approach. When conducting a pool-based GWAS, how well SNP allele frequency is estimated from a DNA pool will influence a study's power to detect associations. Here we address how to control the variance in allele frequency estimation when DNAs are pooled, and how to plan and conduct the most efficient well-powered pool-based GWAS.</p> <p>Methods</p> <p>By examining the variation in allele frequency estimation on SNP arrays between and within DNA pools we determine how array variance [var(e_array)] and pool-construction variance [var(e_construction)] contribute to the total variance of allele frequency estimation. This information is useful in deciding whether replicate arrays or replicate pools are most useful in reducing variance. Our analysis is based on 27 DNA pools ranging in size from 74 to 446 individual samples, genotyped on a collective total of 128 Illumina beadarrays: 24 1M-Single, 32 1M-Duo, and 72 660-Quad.</p> <p>Results</p> <p>For all three Illumina SNP array types our estimates of var(e_array) were similar, between 3-4 × 10^-4for normalized data. Var(e_construction) accounted for between 20-40% of pooling variance across 27 pools in normalized data.</p> <p>Conclusions</p> <p>We conclude that relative to var(e_array), var(e_construction) is of less importance in reducing the variance in allele frequency estimation from DNA pools; however, our data suggests that on average it may be more important than previously thought. We have prepared a simple online tool, PoolingPlanner (available at <url>http://www.kchew.ca/PoolingPlanner/</url>), which calculates the effective sample size (ESS) of a DNA pool given a range of replicate array values. ESS can be used in a power calculator to perform pool-adjusted calculations. This allows one to quickly calculate the loss of power associated with a pooling experiment to make an informed decision on whether a pool-based GWAS is worth pursuing.</p

Functional Analysis and Fine Mapping of the 9p22.2 Ovarian Cancer Susceptibility Locus.

Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer. SIGNIFICANCE: Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene.See related commentary by Choi and Brown, p. 439

rs495139 in the TYMS-ENOSF1 Region and Risk of Ovarian Carcinoma of Mucinous Histology

Thymidylate synthase (TYMS) is a crucial enzyme for DNA synthesis. TYMS expression is regulated by its antisense mRNA, ENOSF1. Disrupted regulation may promote uncontrolled DNA synthesis and tumor growth. We sought to replicate our previously reported association between rs495139 in the TYMS-ENOSF1 3' gene region and increased risk of mucinous ovarian carcinoma (MOC) in an independent sample. Genotypes from 24,351 controls to 15,000 women with invasive OC, including 665 MOC, were available. We estimated per-allele odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression, and meta-analysis when combining these data with our previous report. The association between rs495139 and MOC was not significant in the independent sample (OR = 1.09; 95% CI = 0.97-1.22; p = 0.15; N = 665 cases). Meta-analysis suggested a weak association (OR = 1.13; 95% CI = 1.03-1.24; p = 0.01; N = 1019 cases). No significant association with risk of other OC histologic types was observed (p = 0.05 for tumor heterogeneity). In expression quantitative trait locus (eQTL) analysis, the rs495139 allele was positively associated with ENOSF1 mRNA expression in normal tissues of the gastrointestinal system, particularly esophageal mucosa (r = 0.51, p = 1.7 x 10(-28)), and nonsignificantly in five MOC tumors. The association results, along with inconclusive tumor eQTL findings, suggest that a true effect of rs495139 might be small.Peer reviewe

rs495139 in the TYMS-ENOSF1 Region and Risk of Ovarian Carcinoma of Mucinous Histology.

Thymidylate synthase (TYMS) is a crucial enzyme for DNA synthesis. TYMS expression is regulated by its antisense mRNA, ENOSF1. Disrupted regulation may promote uncontrolled DNA synthesis and tumor growth. We sought to replicate our previously reported association between rs495139 in the TYMS-ENOSF1 3' gene region and increased risk of mucinous ovarian carcinoma (MOC) in an independent sample. Genotypes from 24,351 controls to 15,000 women with invasive OC, including 665 MOC, were available. We estimated per-allele odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression, and meta-analysis when combining these data with our previous report. The association between rs495139 and MOC was not significant in the independent sample (OR = 1.09; 95% CI = 0.97⁻1.22; p = 0.15; N = 665 cases). Meta-analysis suggested a weak association (OR = 1.13; 95% CI = 1.03⁻1.24; p = 0.01; N = 1019 cases). No significant association with risk of other OC histologic types was observed (p = 0.05 for tumor heterogeneity). In expression quantitative trait locus (eQTL) analysis, the rs495139 allele was positively associated with ENOSF1 mRNA expression in normal tissues of the gastrointestinal system, particularly esophageal mucosa (r = 0.51, p = 1.7 × 10-28), and nonsignificantly in five MOC tumors. The association results, along with inconclusive tumor eQTL findings, suggest that a true effect of rs495139 might be small

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC

Exome genotyping arrays to identify rare and low frequency variants associated with epithelial ovarian cancer risk

Rare and low frequency variants are not well covered in most germline genotyping arrays and are understudied in relation to epithelial ovarian cancer (EOC) risk. To address this gap, we used genotyping arrays targeting rarer protein-coding variation in 8,165 EOC cases and 11,619 controls from the international Ovarian Cancer Association Consortium (OCAC). Pooled association analyses were conducted at the variant and gene level for 98,543 variants directly genotyped through two exome genotyping projects. Only common variants that represent or are in strong linkage disequilibrium (LD) with previously-identified signals at established loci reached traditional thresholds for exome-wide significance (P < 5.0 × 10 (−) (7)). One of the most significant signals (P(all histologies )=( )1.01 × 10 (−) (13);P(serous )=( )3.54 × 10 (−) (14)) occurred at 3q25.31 for rs62273959, a missense variant mapping to the LEKR1 gene that is in LD (r(2 )=( )0.90) with a previously identified ‘best hit’ (rs7651446) mapping to an intron of TIPARP. Suggestive associations (5.0 × 10 (−) (5 )>( )P≥5.0 ×10 (−) (7)) were detected for rare and low-frequency variants at 16 novel loci. Four rare missense variants were identified (ACTBL2 rs73757391 (5q11.2), BTD rs200337373 (3p25.1), KRT13 rs150321809 (17q21.2) and MC2R rs104894658 (18p11.21)), but only MC2R rs104894668 had a large effect size (OR = 9.66). Genes most strongly associated with EOC risk included ACTBL2 (P(AML )=( )3.23 × 10 (−) (5); P(SKAT-o )=( )9.23 × 10 (−) (4)) and KRT13 (P(AML )=( )1.67 × 10 (−) (4); P(SKAT-o )=( )1.07 × 10 (−) (5)), reaffirming variant-level analysis. In summary, this large study identified several rare and low-frequency variants and genes that may contribute to EOC susceptibility, albeit with possible small effects. Future studies that integrate epidemiology, sequencing, and functional assays are needed to further unravel the unexplained heritability and biology of this disease

Genetic studies to discover common variants associated with epithelial ovarian cancer risk and variation in age of natural menopause

Background. Epithelial ovarian cancer (EOC) and age of natural menopause (ANM) are two complex traits impacting women’s health. ANM is also an important EOC risk factor. Insight into genetic factors influencing EOC and ANM could provide novel entry points for understanding EOC pathogenesis, and the normal process of ovarian aging. Methods. A two-stage genome-wide association study (GWAS) design using DNA pooling in Stage 1 was used to discover single nucleotide polymorphisms (SNPs) associated with histology-specific EOC risk, and population-specific variation in ANM. SNP-trait associations discovered in Stage 1 of these GWAS were replicated in two different consortia; EOC association in the Ovarian Cancer Association Consortium (OCAC), and ANM associations in the ReproGen Consortium. Results. Eight subtype-specific SNP-EOC associations discovery in Stage 1 of the EOC GWAS were replicated (unadjusted P <0.05) in the datasets of the OCAC: 4 in the mucinous subtype, 2 in the endometrioid and clear cell (ENCC) subtypes combined, and 2 in the low-malignancy potential (LMP) serous subtype. These associations did not achieve genome-wide significance (P < 5x10⁻⁸). However, several of the loci implicated by these SNPs harbour attractive candidate genes for ovarian cancer biology, including the mucinous locus harbouring RAD51B, the ENCC locus harbouring GRB10, and the LMP serous locus harbouring BPIL2/C22orf26. The GWAS of ANM performed in Iranian women revealed one SNP-trait association exclusive to this population (rs10140275; unadjusted P=4.0x10⁻⁴) and one shared with the European population (rs10840211). In the replication of European GWAS findings in Iranian women one SNP at the 20p12.3 locus was replicated (rs16991615, unadjusted P=0.02). SNPs tagging the 19q13.42 locus narrowly missed replication in our dataset (rs1172822, unadjusted P= 0.08). Conclusion. Eight SNP-EOC associations warrant further replication and/or fine-mapping in samples with reliable tumour information. These SNPs tag loci harbouring candidate genes involved in DNA repair, the PI3K/AKT pathway, and immune function, suggesting these pathways and processes may be important to the etiology of the rarer EOC subtypes. The generality of the European ANM GWAS findings in established in another human population (20p12.3). Further, one genetic variant influencing ANM in the Iranian population but not the European population is reported.Medicine, Faculty ofMedical Genetics, Department ofGraduat

rs495139 in the TYMS-ENOSF1 region and risk of ovarian carcinoma of mucinous histology

Thymidylate synthase (TYMS) is a crucial enzyme for DNA synthesis. TYMS expression is regulated by its antisense mRNA, ENOSF1. Disrupted regulation may promote uncontrolled DNA synthesis and tumor growth. We sought to replicate our previously reported association between rs495139 in the TYMS-ENOSF1 3′ gene region and increased risk of mucinous ovarian carcinoma (MOC) in an independent sample. Genotypes from 24,351 controls to 15,000 women with invasive OC, including 665 MOC, were available. We estimated per-allele odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression, and meta-analysis when combining these data with our previous report. The association between rs495139 and MOC was not significant in the independent sample (OR = 1.09; 95% CI = 0.97-1.22; p = 0.15; N = 665 cases). Meta-analysis suggested a weak association (OR = 1.13; 95% CI = 1.03-1.24; p = 0.01; N = 1019 cases). No significant association with risk of other OC histologic types was observed (p = 0.05 for tumor heterogeneity). In expression quantitative trait locus (eQTL) analysis, the rs495139 allele was positively associated with ENOSF1 mRNA expression in normal tissues of the gastrointestinal system, particularly esophageal mucosa (r = 0.51, p = 1.7 × 10−28), and nonsignificantly in five MOC tumors. The association results, along with inconclusive tumor eQTL findings, suggest that a true effect of rs495139 might be small