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sTu1945

Seven Non-Cystic Fibrosis-Causing CFTR Variants Are Associated WithChronic Pancreatitis and Define a New Class of Organ-Specific Risk FactorsJessica LaRusch, Michele D. Lewis, Randall Brand, Andres Gelrud, Michael R. O'Connell,David C. Whitcomb

Background: Chronic pancreatitis (CP) describes ongoing pancreatic inflammation frommultiple etiologies. CFTR gene sequencing in small case series of CP identify an excess ofatypical variants that do not cause cystic fibrosis (CF), possibly due to disruption of bicarbon-ate conductance. Methods: Targeted genotyping was performed in 956 pancreatitis cases(CP and recurrent acute pancreatitis, RAP) and 467 unrelated controls for 44 CFTR variantsin the NAPS2 cohort. Association within individual and compound genotypes in casesclassified by etiology and the presence or absence of PRSS1/SPINK1 co-variants was alsoanalyzed. Results: Mutations in CFTR or PRSS1 were not significantly overrepresented amongpatients with an alcoholic etiology, but 7 CFTR variants not clearly associated with CF(R75Q, S1235R, L997F, L967S, R1162L, D1152H and R117H*T7or9) were overrepresentedin cases with non-alcoholic pancreatitis and the effect was amplified by transheterozygousmutations in SPINK1 (OR 1.6 p=0.014; w/ SPINK1 OR 10.1 p=0.0001). These non-CFCFTR variants comprised 60% of CFTR variants among all cases. Of the 190 carriers ofCFTR variants, 54 had multiple mutations in CFTR, SPINK1 or PRSS1; 262 cases carriedat least one genetic variant and 86 carried a high-risk combination of mutations in CFTR,SPINK1 or PRSS1. Conclusions: A new class of non-CF associated CFTR variants may playa major role in the etiopathology of CP. Complex genotypes with CF-associated and non-CF-associated CFTR, and SPINK1 variants were common. This defines a subtype of CPpatients that is distinct from CF, hereditary pancreatitis and alcoholic pancreatitis, and hasimplications for the genetic panel used for testing pancreatitis patients, and in guidingtreatment strategies.

Tu1946

Analysis of 5 TNF-α Promoter Polymorphisms in Acute Pancreatitis (AP) inPittsburgh: No Replication Evidence of Increased Susceptibility to AP but NewEvidence for -1031c and -863a as Risk Alleles for Multi-Organ Failure (Mof)Faraz Bishehsari, Chad T. Toth, Michael R. O'Connell, Georgios I. Papachristou, David C.Whitcomb

Background: Acute pancreatitis (AP) is a complex inflammatory syndrome with variousfactors affecting susceptibility to AP or complications of AP including multi-organ failure(MOF). Tumor necrosis factor-α (TNF-α) is an initial mediator of the systemic inflammatoryresponse syndrome (SIRS), which leads to MOF. At least 5 TNF-α promoter SNPs canregulate TNF-α production and -857 C/T,-308 A/G and -238 A/G have been evaluated insmall AP studies with conflicting results. Our aim was a replications study of earlier findingsand evaluation of the -1031C and -863A alleles, which affects the TNF-α release, for APsusceptibility and MOF. Methods: AP was diagnosed with clinical, biochemical and imagingcriteria. MOF was defined based on persistent (>48 hrs) failure of >2 organ systems includingcardiovascular, pulmonary, and/or renal. DNA was extracted from banked samples and SNPsat -1031 C/T, -863 A/C, -857 C/T,-308 A/G and -238 A/G were tested by DNA sequencing.In total, 612individuals; 211 patients with AP and 401 healthy controls, were analyzed forthe 5 SNPs. Results: Of the 211 patients, 23 (11%) were classified as MOF. None of the variantalleles at the five tested loci were associated with AP. The -1031C allele was significantly morefrequently in patients with MOF (13/23; 56.4% vs. 61/188; 32.4% P<0.03). Likewise, the-863A allele observed more frequently in MOF cases (10/23;43.5% vs. 41/188; 21.8%P<0.03). The two alleles were in linkage disequilibrium with D=0.11. Conclusion: Thisstudy adds to growing evidence that TNF-α promoter SNPs are not associated with APsusceptibility as reported in small early studies. However, in our cohort, the TNF-α enhancing-1031C and -863A alleles significantly increase the risk of progression from localized AP tosystemic inflammation and MOF. Since this is the first report of this biologically plausiblegenetic association, replication studies are warrantedTable 1: Genotypes of TNF-alpha promoter at -1031 and -863 positions in acute pancreatitiswithout MOF (non-MOF AP), compared to the patients with Multiple Organ Failure (MOF).

Tu1947

Pancreatic and Biliary Secretion Differ in Cystic Fibrosis and Wild-Type PigsAliye Uc, David A. Stoltz, Paula S. Ludwig, Alejandro Pezzulo, Michelle Griffin, MarwaAbu-El-Haija, Maisam Abu-El-Haija, David Meyerholz, Peter J. Taft, Michael J. Welsh

Background: The hallmark feature of pancreatic disease in cystic fibrosis (CF) is the produc-tion of viscous, low-volume and acidic fluid. Because pancreatic function studies in humansare done by sampling the fluid in the jejunum and the pancreatic duct converges with thecommon bile duct (CBD) shortly before opening at the duodenum, it is not known whetherpancreatic or biliary secretions are equally affected in CF. With a pancreatic histopathologysimilar to humans with CF and separate CBD and pancreatic duct openings into the intestine,

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the pig model offers a unique opportunity to examine pancreatic and biliary fluids separately.Hypothesis: Pancreatic and biliary fluid secretion differ in CF and wild type (WT) pigs.Methods: After the animal was anesthetized with inhaled isofluorane, a laparotomy wasdone. Sutures were tied tightly around the duodenum distal to the pylorus and anothersuture was placed 1 cm distal to the pylorus to collect bile. A suture was tied 1 cm distalto the pancreatic duct to collect the pancreatic fluid. FourWT, 3 cystic fibrosis transmembraneconductance regulator (CFTR)-/-, and 2 CFTRΔF508/ΔF508 newborn pigs were studiedshortly after birth. Bile was also sampled from the gallbladder. Fluids were collected atbaseline and 30 min after secretin (0.2 μg/kg IV, ChiRhoClin Inc). pH, volume and proteinconcentrations were measured. Results: Pancreatic fluid volume was 75+29 μl, pH was8.4+0.1 at baseline and 302.5+17 μl and 9.5+0 after secretin in WT pigs. CF pig pancreaticfluid volume was 1.25+0.2 μl, pH was 5.7+0.1 at baseline and 2.25+0.2 μl and 5.4+0.1after secretin (p<0.001 for volume and pH in WT vs. CF pigs). Bile volume was 60.5+12μl, pH was 8.7+0.1 at baseline; 120+8.9 μl and 8.7+0.1 after secretin in WT pigs. Bilevolume was 38+18 ul in CF pigs at baseline and 30+12 ul after secretin; bile pH was8.2+0.05 and did not change after secretin. Bile volume and pH were not different betweenWT and CF pigs. Gallbladder bile pH was 8+0 in WT pigs and 7.4+0.4 in CF pigs (p=ns).Protein concentration in the WT pancreatic fluid was an average of 4.2 mg/ml after secretinvs. 35.1 mg/ml in CF pigs (n=2). Bile protein concentration was an average of 8.6 mg/mlin WT pigs vs. 16.4 mg/ml in CF pigs after secretin (n=2). Conclusion: Pancreatic fluidwas acidic, low in volume and high in protein in CF pigs. Bile volume and pH were notsignificantly different between WT and CF pigs, but bile volume did not increase aftersecretin in CF pigs. The differences between the pancreatic and biliary secretion may haveimportant implications in the pancreaticobiliary disease pathogenesis in CF.

Tu1948

Association of Gamma-Glutamyltransferase 1 Gene (GGT1) PolymorphismsWith Idiopathic Chronic Pancreatitis in the NAPS2 CohortRandall Brand, Jessica LaRusch, Michael M. Barmada, Adam Slivka, Dhiraj Yadav, DavidC. Whitcomb

Background. Recurrent pancreatic injury may lead to chronic inflammation, recognized aschronic pancreatitis (CP) or DNA injury and pancreatic cancer (PC). Since CP patients areat an increased risk for pancreatic cancer development suggests the possibility of sharedgenetic risk factors between these diseases. Recently we conducted a pooling-based genome-wide association study and reported that polymorphisms in the GGT1 gene were associatedwith PC (PMID: 20484958). GGT1 is a membrane-bound protein in the liver, kidney andpancreas and appears to be linked to glutathione trafficking and xenobiotic detoxification.Aim: To determine if the GGT1 variants that increase risk for PC also increase risk for CP,particularly in the subset of patients who were very heavy alcohol users or classified asidiopathic CP (ICP). Methods: The North American Pancreatitis Study (NAPS2) cohort wasstudied. CP cases (n=438) were classified according to the physicians clinic diagnosis ofalcoholic (n=183) or non-alcoholic (n=255) and by strict criteria of very heavy alcohol (>5drinks or 60 grams of ethanol per day, n=99) or idiopathic CP (ICP, n=133). Six commonGGT1 variants (rs2017869, rs4820599, rs5751901, rs8135987, rs1138272, rs1695) wereevaluated using a custom Sequenom MassARRAY iPLEX Gold assay. Data was analyzed bychi square. Results: GGT1 gene polymorphisms were not associated with overall risk forCP, alcoholic including the very heaviest users or non-alcoholic CP. However, with strictphenotyping there was evidence of significant risk for ICP with rs2017869 (OR 1.39, p=0.024), rs5751901 (OR 1.4, p=0.023) and rs8135987 (OR 1.52, p=0.004), and a strongtrend for rs4820599 (p=0.052). These were the same SNPs associated with risk of PC, andrs57510901 is associated with increased serum levels of GGT. Conclusion. These datasuggest that GGT1 gene polymorphisms predispose the pancreas to injury through a GGT1-dependent mechanism. Pancreatic injury linked to GGT1 appears to be a risk factor for ICPas well as PC, and should be further investigated.

Tu1949

Intracellular Signaling Pathway Genes Involved in Pancreatic Adenocarcinoma:A Microarray Tissue AnalysisAndrada Seicean, Ovidiu Balacescu, Roxana Stan-Iuga, Loredana Balacescu, Radu Seicean,Roxana Redis, Ioana Berindan Neagoe, Oliviu Pascu

The pathogenesis of pancreatic ductal adenocarcinoma involves the multi-stage developmentof molecular aberrations affecting signaling pathways that regulate cancer growth and progres-sion. Tissue microarray analysis of pancreatic tumors allows simultaneous assessment ofgenetic disorders, which can lead to identification of biomarkers of poor prognosis. Aim:To characterize the gene expression of pancreatic adenocarcinoma compared to normaltissue from the same patient. Materials and methods. We investigated sixteen samples ofT3 pancreatic adenocarcinoma obtained intraoperatively and compared them to normalpancreatic tissue from the same patients. RNA was extracted and assessed qualitatively andquantitatively, followed by amplification of cDNAusing reverse transcriptase, cRNA synthesis,and hybridization of microarray slides. For each sample 1000 ng of total RNA was available.The overexpressed and underexpressed genes were classified by their known function inthe cell. We selected genes over- or underexpressed three times compared to normal adjacenttissue some described previously in pancreatic pathology, and used RT-PCR for validation.Results: On microarray tissue analysis 41 genes were overexpressed and 402 were undere-xpressed in the pancreatic adenocarcinoma samples. There were selected genes involved intranscription process as ZNF 428, MIXL1, SEPT 1, genes involved in intracellular signalingas FLJ21865, AGRP and genes involved in transmembranar and intracellular transport asCCDC88, UTP14A,VPS11, LLRC21,CHRM3, Marveld3. Validation by qRT-PCR confirmedthe involvement of AGRP and MIXL1 gene in pancreatic adenocarcinoma tissue. Conclusion:Microarray tissue analysis of pancreatic adenocarcinoma showed more underexpressed thanoverexpressed genes. After validation , the overexpressed gene AGRP was shown to be onepossible factor responsible for anorexia and perineural invasion. The possible role in pancre-atic cancer of MIXL1, known to play a role in cellular proliferation and differentiation,should be further clarified.