| Pancreatic cancer is one of the leading causes of malignancy-related death in the world. Germline mutations in some genes that cause hereditary syndromes are highly predisposed to familial pancreatic cancer. However, genetic susceptibility to sporadic pancreatic cancer is largely uncovered. Copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) have been recognized to be associated with susceptibility to certain complex diseases including cancer. In the present study, we examined the associations between CNVs and SNPs and susceptibility to pancreatic ductal adenocarcinoma using a combined genome-wide and candidate genes strategy.Whole-genome germline CNVs were first explored in25patients with pancreatic cancer and25controls using Affymetrix Genome-Wide Human SNP Array5.0and the promising CNVs were validated by real-time qualitative PCR in1027cases with pancreatic cancer and1031controls. The associations between CNVs and risk of pancreatic cancer were indicated by odds ratios (ORs) and95%confidence intervals (CIs) computed by logistic regression model. We found that CNVR2966.1located on chromosome6ql3was significantly associated with pancreatic cancer risk, with the OR being1.31(95%CI=1.08-1.60; P=0.007) for the1-copy genotype compared with the2-copy genotype. CNVR2966.1was found to be a10379-bp nucleotide deletion/insertion within the uniform boundaries of chromosome6:74648791-74659169. Luciferase reporter gene assays revealed an active regulator in CNVR2966.1, which was demonstrated by circular chromosome conformation capture assays to physically interact with the upstream functional sequence of CDKN2B. CDKN2B transcription levels in pancreatic tissues were therefore significantly higher in individuals with2copies of CNVR2966.1than in those with low copy number of CNVR2966.1.To examine SNPs for susceptibility to pancreatic cancer, we conducted a two-stage genome-wide association study (GWAS) that included981cases and1991controls in the first stage followed by a second stage (2603cases and2877controls) and the report has been published (Wu et al, Nat Genet2012). To further analyze the GWAS data, we employed an approach based on candidate genes whose roles in pancreatic cancer have been well-known. We investigated the associations between susceptibility to pancreatic cancer and SNPs which were directly genotyped and well-imputed within10candidate gene loci and their10kb upstream regions (TP53, KRAS, CDKN2A, SMAD4, MAP2K4, BRCA2, MEN1, STK11, PALLD, and APC). The associations with P<0.05and false positive report probability<0.20in the first stage were selected to replicate in the second stage. The function of significant SNPs was examined by biochemical assays. We identified two new pancreatic cancer susceptibility loci, rs11571836located in the BRCA23'-untranslated region was significantly associated with increased pancreatic cancer risk (OR=1.30,95%CI=1.14-1.47; P=7.64×10-5) in a recessive manner. rs12939944located in the MAP2K4intron was significantly associated with decreased risk (OR=0.82,95%CI=0.74-0.91; P=1.08×10-4) in a dominant manner. Functional analyses showed that the rs11571836G allele was associated with lower luciferase activity and BRCA2transcript compared with the rs11571836A allele. For rs12939944polymorphism, individuals with the CC or TC genotype had lower MAP2K4RNA level in pancreatic tissues than those with the TT genotype.In conclusion, our results demonstrate for the first time that CNVR2966.1on6p13and SNPs rs11571836in BRCA2and rs12939944in MAP2K4are genetic susceptibility factors for the development of pancreatic cancer. These genetic variants might either function as long-range regulator or affect transcription regulation of the genes.
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