David N. Rider

Telomere length varies by DNA extraction method: implications for epidemiologic research

Background: Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from patients with colorectal cancer. Methods: We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 patients with colorectal cancer and 2,952 healthy controls. DNA was extracted with phenol/chloroform, PureGene, or QIAamp. Results: We observed differences in RTL depending on DNA extraction method (P < 0.001). Phenol/chloroform-extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01–6.54) compared with PureGene-extracted DNA (mean RTL of 0.75; range 0.00–12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02–3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17–0.58) was less than from either PureGene or phenol/chloroform (ranges, 0.04–2.67 and 0.32–2.81, respectively). Conclusions: RTL measured by qPCR from QIAamp-extracted DNA was less than from either PureGene or phenol/chloroform (P < 0.001). Impact: Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL. Cancer Epidemiol Biomarkers Prev; 22(11); 2047–54. ©2013 AACR.

Association of Genetic Variation in Genes Implicated in the β-Catenin Destruction Complex with Risk of Breast Cancer

Aberrant Wnt/β-catenin signaling leading to nuclear accumulation of the oncogene product β-catenin is observed in a wide spectrum of human malignancies. The destruction complex in the Wnt/β-catenin pathway is critical for regulating the level of β-catenin in the cytoplasm and in the nucleus. Here, we report a comprehensive study of the contribution of genetic variation in six genes encoding the β-catenin destruction complex (APC, AXIN1, AXIN2, CSNK1D, CSNK1E, and GSK3B) to breast cancer using a Mayo Clinic Breast Cancer Case-Control Study. A total of 79 candidate functional and tagging single nucleotide polymorphisms (SNP) were genotyped in 798 invasive cases and 843 unaffected controls. Of these, rs454886 in the APC tumor suppressor gene was associated with increased breast cancer risk (per allele odds ratio, 1.23; 95% confidence intervals, 1.05-1.43; Ptrend = 0.01). In addition, five SNPs in AXIN2 were associated with increased risk of breast cancer (Ptrend < 0.05). Haplotype-based tests identified significant associations between specific haplotypes in APC and AXIN2 (P ≤ 0.03) and breast cancer risk. Further characterization of the APC and AXIN2 variants suggested that AXIN2 rs4791171 was significantly associated with risk in premenopausal (Ptrend = 0.0002) but not in postmenopausal women. The combination of our findings and numerous genetic and functional studies showing that APC and AXIN2 perform crucial tumor suppressor functions suggest that further investigation of the contribution of AXIN2 and APC SNPs to breast cancer risk are needed. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2101–8)

Evaluation of candidate stromal epithelial cross-talk genes identifies association between risk of serous ovarian cancer and TERT, a cancer susceptibility "Hot-Spot"

We hypothesized that variants in genes expressed as a consequence of interactions between ovarian cancer cells and the host micro-environment could contribute to cancer susceptibility. We therefore used a two-stage approach to evaluate common single nucleotide polymorphisms (SNPs) in 173 genes involved in stromal epithelial interactions in the Ovarian Cancer Association Consortium (OCAC). In the discovery stage, cases with epithelial ovarian cancer (n = 675) and controls (n = 1,162) were genotyped at 1,536 SNPs using an Illumina GoldenGate assay. Based on Positive Predictive Value estimates, three SNPs—PODXL rs1013368, ITGA6 rs13027811, and MMP3 rs522616—were selected for replication using TaqMan genotyping in up to 3,059 serous invasive cases and 8,905 controls from 16 OCAC case-control studies. An additional 18 SNPs with P per-allele<0.05 in the discovery stage were selected for replication in a subset of five OCAC studies (n = 1,233 serous invasive cases; n = 3,364 controls). The discovery stage associations in PODXL, ITGA6, and MMP3 were attenuated in the larger replication set (adj. P per-allele≥0.5). However genotypes at TERT rs7726159 were associated with ovarian cancer risk in the smaller, five-study replication study (P per-allele = 0.03). Combined analysis of the discovery and replication sets for this TERT SNP showed an increased risk of serous ovarian cancer among non-Hispanic whites [adj. ORper-allele 1.14 (1.04–1.24) p = 0.003]. Our study adds to the growing evidence that, like the 8q24 locus, the telomerase reverse transcriptase locus at 5p15.33, is a general cancer susceptibility locus.

Single Nucleotide Polymorphisms in the TP53 Region and Susceptibility to Invasive Epithelial Ovarian Cancer

The p53 protein is critical for multiple cellular functions including cell growth and DNA repair. We assessed whether polymorphisms in the region encoding TP53 were associated with risk of invasive ovarian cancer. The study population includes a total of 5,206 invasive ovarian cancer cases (2,829 of which were serous) and 8,790 controls from 13 case-control or nested case-control studies participating in the Ovarian Cancer Association Consortium (OCAC). Three of the studies performed independent discovery investigations involving genotyping of up to 23 single nucleotide polymorphisms (SNP) in the TP53 region. Significant findings from this discovery phase were followed up for replication in the other OCAC studies. Mixed effects logistic regression was used to generate posterior median per allele odds ratios (OR), 95% probability intervals (PI), and Bayes factors (BF) for genotype associations. Five SNPs showed significant associations with risk in one or more of the discovery investigations and were followed up by OCAC. Mixed effects analysis confirmed associations with serous invasive cancers for two correlated (r(2) = 0.62) SNPs: rs2287498 (median per allele OR, 1.30; 95% PI, 1.07-1.57) and rs12951053 (median per allele OR, 1.19; 95% PI, 1.01-1.38). Analyses of other histologic subtypes suggested similar associations with endometrioid but not with mucinous or clear cell cancers. This large study provides statistical evidence for a small increase in risk of ovarian cancer associated with common variants in the TP53 region.

Genetic variation within the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways as risk factors for venous thromboembolism

Summary.  Background: Venous thromboembolism (VTE) is highly heritable (estimated heritability [h2] = 0.62) and likely to be a result of multigenic action. Objective: To systematically test variation within genes encoding for important components of the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways for an independent association with VTE. Methods: Non‐Hispanic adults of European ancestry with objectively‐diagnosed VTE, and age‐ and sex‐ matched controls, were genotyped for 13 031 single nucleotide polymorphisms (SNPs) within 764 genes. Analyses (n = 12 296 SNPs) were performed with plink using an additive genetic model and adjusted for age, sex, state of residence, and myocardial infarction or stroke. Results: Among 2927 individuals, one or more SNPs within ABO, F2, F5, F11, KLKB1, SELP and SCUBE1 were significantly associated with VTE, including factor (F) V Leiden, prothrombin G20210A, ABO non‐O blood type, and a novel association with ABO rs2519093 (OR = 1.68, P‐value = 8.08 × 10−16) that was independent of blood type. In stratified analyses, SNPs in the following genes were significantly associated with VTE: F5 and ABO among both genders and LY86 among women; F2, ABO and KLKB1 among FV Leiden non‐carriers; F5, F11, KLKB1 and GFRA1 in those with ABO non‐O blood type; and ABO, F5, F11, KLKB1, SCUBE1 and SELP among prothrombin G20210A non‐carriers. The ABO rs2519093 population‐attributable risk (PAR) exceeded that of FV Leiden and prothrombin G20210A, and the joint PAR of FV Leiden, prothrombin G20210A, ABO non‐O and ABO rs2519093 was 0.40. Conclusions: Anticoagulant, procoagulant, fibrinolytic and innate immunity pathway genetic variation accounts for a large proportion of VTE among non‐Hispanic adults of European ancestry.

Inherited Determinants of Ovarian Cancer Survival

Purpose: Due to variation of outcome among cases, we sought to examine whether overall survival in ovarian cancer was associated with common inherited variants in 227 candidate genes from ovarian cancer–related pathways including angiogenesis, inflammation, detoxification, glycosylation, one-carbon transfer, apoptosis, cell cycle regulation, and cellular senescence. Experimental Design: Blood samples were obtained from 325 women with invasive epithelial ovarian cancer diagnosed at the Mayo Clinic from 1999 to 2006. During a median follow-up of 3.8 years (range, 0.1-8.6 years), 157 deaths were observed. Germline DNA was analyzed at 1,416 single nucleotide polymorphisms (SNP). For all patients, and for 203 with serous subtype, we assessed the overall significance of each gene and pathway, and estimated risk of death via hazard ratios (HR) and 95% confidence intervals (CI), adjusting for known prognostic factors. Results: Variation within angiogenesis was most strongly associated with survival time overall (P = 0.03) and among patients with serous cancer (P = 0.05), particularly for EIF2B5 rs4912474 (all patients HR, 0.69; 95% CI, 0.54-0.89; P = 0.004), VEGFC rs17697305 (serous subtype HR, 2.29; 95% CI, 1.34-3.92; P = 0.003), and four SNPs in VHL. Variation within the inflammation pathway was borderline significant (all patients, P = 0.09), and SNPs in CCR3, IL1B, IL18, CCL2, and ALOX5 which correlated with survival time are worthy of follow-up. Conclusion: An extensive multiple-pathway assessment found evidence that inherited differences may play a role in outcome of ovarian cancer patients, particularly in genes within the angiogenesis and inflammation pathways. Our work supports efforts to target such mediators for therapeutic gain. Clin Cancer Res; 16(3); 995–1007

Ovarian Cancer Risk Associated with Inherited Inflammation-Related Variants

The importance of inflammation pathways to the development of many human cancers prompted us to examine the associations between single-nucleotide polymorphisms (SNP) in inflammation-related genes and risk of ovarian cancer. In a multisite case-control study, we genotyped SNPs in a large panel of inflammatory genes in 930 epithelial ovarian cancer cases and 1,037 controls using a custom array and analyzed by logistic regression. SNPs with P < 0.10 were evaluated among 3,143 cases and 2,102 controls from the Follow-up of Ovarian Cancer Genetic Association and Interaction Studies (FOCI) post-GWAS collaboration. Combined analysis revealed association with SNPs rs17561 and rs4848300 in the interleukin gene IL1A which varied by histologic subtype (P(heterogeneity) = 0.03). For example, IL1A rs17561, which correlates with numerous inflammatory phenotypes, was associated with decreased risk of clear cell, mucinous, and endometrioid subtype, but not with the most common serous subtype. Genotype at rs1864414 in the arachidonate 5-lipoxygenase ALOX5 was also associated with decreased risk. Thus, inherited variation in IL1A and ALOX5 seems to affect ovarian cancer risk which, for IL1A, is limited to rarer subtypes. Given the importance of inflammation in tumorigenesis and growing evidence of subtype-specific features in ovarian cancer, functional investigations will be important to help clarify the importance of inherited variation related to inflammation in ovarian carcinogenesis.

Functional Polymorphisms in the TERT Promoter Are Associated with Risk of Serous Epithelial Ovarian and Breast Cancers

Genetic variation at the TERT-CLPTM1L locus at 5p15.33 is associated with susceptibility to several cancers, including epithelial ovarian cancer (EOC). We have carried out fine-mapping of this region in EOC which implicates an association with a single nucleotide polymorphism (SNP) within the TERT promoter. We demonstrate that the minor alleles at rs2736109, and at an additional TERT promoter SNP, rs2736108, are associated with decreased breast cancer risk, and that the combination of both SNPs substantially reduces TERT promoter activity.

Nucleotide Excision Repair Pathway Polymorphisms and Pancreatic Cancer Risk: Evidence for role of MMS19L

Background: Nucleotide excision repair is a vital response to DNA damage, including damage from tobacco exposure. Single nucleotide polymorphisms (SNP) in the nucleotide excision repair pathway may encode alterations that affect DNA repair function and therefore influence the risk of pancreatic cancer development. Methods: A clinic-based case-control study in non-Hispanic white persons compared 1,143 patients with pancreatic adenocarcinoma with 1,097 healthy controls. Twenty-seven genes directly and indirectly involved in the nucleotide excision repair pathway were identified and 236 tag-SNPs were selected from 26 of these (one had no SNPs identified). Association studies were done at the gene level by principal components analysis, whereas recursive partitioning analysis was utilized to identify potential gene-gene and gene-environment interactions within the pathway. At the individual SNP level, adjusted additive, dominant, and recessive models were investigated, and gene-environment interactions were also assessed. Results: Gene level analyses showed an association of the MMS19L genotype (chromosome 10q24.1) with altered pancreatic cancer risk (P = 0.023). Haplotype analysis of MMS19L also showed a significant association (P = 0.0132). Analyses of seven individual SNPs in this gene showed both protective and risk associations for minor alleles, broadly distributed across patient subgroups defined by smoking status, sex, and age. Conclusion: In a candidate pathway SNP association study analysis, common variation in a nucleotide excision repair gene, MMS19L, was associated with the risk of pancreatic cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(4):1295–302)

Candidate Gene Analysis Using Imputed Genotypes: Cell Cycle Single-Nucleotide Polymorphisms and Ovarian Cancer Risk

Polymorphisms in genes critical to cell cycle control are outstanding candidates for association with ovarian cancer risk; numerous genes have been interrogated by multiple research groups using differing tagging single-nucleotide polymorphism (SNP) sets. To maximize information gleaned from existing genotype data, we conducted a combined analysis of five independent studies of invasive epithelial ovarian cancer. Up to 2,120 cases and 3,382 controls were genotyped in the course of two collaborations at a variety of SNPs in 11 cell cycle genes (CDKN2C, CDKN1A, CCND3, CCND1, CCND2, CDKN1B, CDK2, CDK4, RB1, CDKN2D, and CCNE1) and one gene region (CDKN2A-CDKN2B). Because of the semi-overlapping nature of the 123 assayed tagging SNPs, we performed multiple imputation based on fastPHASE using data from White non-Hispanic study participants and participants in the international HapMap Consortium and National Institute of Environmental Health Sciences SNPs Program. Logistic regression assuming a log-additive model was done on combined and imputed data. We observed strengthened signals in imputation-based analyses at several SNPs, particularly CDKN2A-CDKN2B rs3731239; CCND1 rs602652, rs3212879, rs649392, and rs3212891; CDK2 rs2069391, rs2069414, and rs17528736; and CCNE1 rs3218036. These results exemplify the utility of imputation in candidate gene studies and lend evidence to a role of cell cycle genes in ovarian cancer etiology, suggest a reduced set of SNPs to target in additional cases and controls. (Cancer Epidemiol Biomarkers Prev 2009;18(3):935–44)