Insights into Pancreatic Cancer Etiology from Pathway Analysis of Genome-Wide Association Study Data

<div><h3>Background</h3><p>Pancreatic cancer is the fourth leading cause of cancer death in the U.S. and the etiology of this highly lethal disease has not been well defined. To identify genetic susceptibility factors for pancreatic cancer, we conducted pathway analysis of genome-wide association study (GWAS) data in 3,141 pancreatic cancer patients and 3,367 controls with European ancestry.</p><h3>Methods</h3><p>Using the gene set ridge regression in association studies (GRASS) method, we analyzed 197 pathways identified from the Kyoto Encyclopedia of Genes and Genomes database. We used the logistic kernel machine (LKM) test to identify major contributing genes to each pathway. We conducted functional enrichment analysis of the most significant genes (<em>P</em><0.01) using the Database for Annotation, Visualization, and Integrated Discovery (DAVID).</p><h3>Results</h3><p>Two pathways were significantly associated with risk of pancreatic cancer after adjusting for multiple comparisons (<em>P</em><0.00025) and in replication testing: neuroactive ligand-receptor interaction, (<em>Ps</em><0.00002), and the olfactory transduction pathway (<em>P</em> = 0.0001). LKM test identified four genes that were significantly associated with risk of pancreatic cancer after Bonferroni correction (<em>P</em><1×10⁻⁵): <em>ABO, HNF1A, OR13C4,</em> and <em>SHH.</em> Functional enrichment analysis using DAVID consistently found the G protein-coupled receptor signaling pathway (including both neuroactive ligand-receptor interaction and olfactory transduction pathways) to be the most significant pathway for pancreatic cancer risk in this study population.</p><h3>Conclusion</h3><p>These novel findings provide new perspectives on genetic susceptibility to and molecular mechanisms of pancreatic cancer.</p></div

Genes significantly associated with pancreatic cancer (<i>P</i><0.01 in LKM) in all 197 pathways analyzed in this study.

<p>Genes significantly associated with pancreatic cancer (<i>P</i><0.01 in LKM) in all 197 pathways analyzed in this study.</p

GRASS analysis of significant pathways in PanScan1 and PanScan2 dataset.

<p>GRASS analysis of significant pathways in PanScan1 and PanScan2 dataset.</p

Pathways significantly associated with pancreatic cancer risk.

<p>Note: All 6 pathways were significant after Bonferroni correction (<i>P</i><2.50×10⁻⁴).</p>a<p>“No. of SNPs” refers to the actual number of SNPs; “No. of eigenSNPs” refers to the number of uncorrelated linear combinations of SNPs used in GRASS analysis.</p>b<p><i>P</i> value obtained from GRASS analysis based on 50,000 permutations.</p>c<p>Genes associated with pancreatic cancer with <i>P</i><0.001, identified using the logistic kernel machine (LKM) method.</p

Ingenuity biologic systems map of the top 81 genes (<i>P</i><0.05 in LKM) of the six pathways that are significantly associated with risk of pancreatic cancer.

<p>The solid line and dashed line, respectively, shows the direct and indirect interactions between genes.</p

Genes significantly associated with pancreatic cancer in (A) the Fc epsilon RI signaling pathway; (B) long-term depression; (C) MODY; (D) olfactory transduction; (E) neuroactive ligand-receptor interaction pathway; and (F) vascular smooth muscle contraction pathways.

<p>Genes with –log10(<i>P</i>) <1 were not included in the plots. For clarity, not all genes are labeled. For details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046887#pone.0046887.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046887#pone.0046887.s003" target="_blank">S3</a>.</p

Levels of <i>APEX1</i> mRNA expression by <i>APEX1</i> Asp148Glu genotypes in Epstein-Barr virus-transformed lymphoblastoid cell lines derived from 270 Hapmap individuals.

<p>Compared with individuals carrying the <i>APEX1-</i>148Asp/Asp genotype, those carrying the <i>APEX1</i>-148Asp/Glu or Glu/Glu genotypes had higher levels of <i>APEX1</i> mRNA expression (Asp/Asp vs. Asp/Glu: <i>P</i> = 0.0003; Asp/Asp vs. Glu/Glu: <i>P</i> = 0.037). Furthermore, the trend test for this increased effect of the <i>APEX1-</i>148 Glu allele on the <i>APEX1</i> expression was statistically significant (<i>P</i>_trend = 0.002). The box represents the interquartile range, which contains 50% of the values. The lower and the upper edges of the box plot are the first quartile (25th percentile) and the third quartile (75th percentile), respectively. The line across the box indicates the median value. The ends of the vertical lines extend to a maximum of 1.5 times the interquartile. In the box plots outliers are marked as dots, which are more than 1.5-fold the box length away from the upper or lower edge of the box. (One individual’s genotyping data were unavailable.).</p

Distribution of DNA adducts in cultured peripheral lymphocytes from 706 cancer-free non-Hispanic white subjects by selected variables.

a<p>The median level of BPDE-induced DNA adducts.</p>b<p><i>P</i> value was obtained using the Wilcoxon two-sample test for the genotype of one or two copies of the minor allele compared with zero copies of the minor allele in each SNP.</p>c<p><i>P</i>_trend value was obtained using the non-parametric analysis of variance test for the trend among genotypes.</p

Genotype-phenotype correlation between genotypes of BER SNPs and levels of BPDE-induced-DNA adducts in cultured peripheral lymphocytes from 706 cancer-free non-Hispanic white subjects.

a<p>The median level of BPDE-induced DNA adducts.</p>b<p><i>P</i> value was obtained using theWilcoxon two-sample test for the genotype of one or two copies of the minor allele compared with zero copies of the minor allele in each SNP.</p>c<p><i>P</i>_trend value was obtained using the non-parametric analysis of variance test for the trend among genotypes.</p

Typical Immunohistochemical staining pattern for TGF-βR2 and SMAD4 in pancreatic adenocarcinoma tumor tissues.

<p>A: Positive nuclear expression of TGF-βR2 in a moderately differentiated ductal adenocarcinoma of the pancreas (Magnification: 10×40). B: Positive nuclear expression of SMAD4 in moderately differentiated ductal adenocarcinoma of the pancreas (Magnification: 10×40).</p