Janette Lamb

Combined Bicarbonate Conductance-Impairing Variants in CFTR and SPINK1 Variants Are Associated With Chronic Pancreatitis in Patients Without Cystic Fibrosis

BACKGROUND & AIMS Idiopathic chronic pancreatitis (ICP) is a complex inflammatory disorder associated with multiple genetic and environmental factors. In individuals without cystic fibrosis (CF), variants of CFTR that inhibit bicarbonate conductance but maintain chloride conductance might selectively impair secretion of pancreatic juice, leading to trypsin activation and pancreatitis. We investigated whether sequence variants in the gene encoding the pancreatic secretory trypsin inhibitor SPINK1 further increase the risk of pancreatitis in these patients. METHODS We screened patients and controls for variants in SPINK1 associated with risk of chronic pancreatitis and in all 27 exons of CFTR. The final study group included 53 patients with sporadic ICP, 27 probands with familial ICP, 150 unrelated controls, 375 additional controls for limited genotyping. CFTR wild-type and p.R75Q were cloned and expressed in HEK293 cells, and relative conductances of HCO(3)(-) and Cl(-) were measured. RESULTS SPINK1 variants were identified in 36% of subjects and 3% of controls (odds ratio [OR], 18.1). One variant of CFTR not associated with CF, p.R75Q, was found in 16% of subjects and 5.3% of controls (OR, 3.4). Coinheritance of CFTR p.R75Q and SPINK1 variants occurred in 8.75% of patients and 0.38% of controls (OR, 25.1). Patch-clamp recordings of cells that expressed CFTR p.R75Q showed normal chloride currents but significantly reduced bicarbonate currents (P = .0001). CONCLUSIONS The CFTR variant p.R75Q causes a selective defect in bicarbonate conductance and increases risk of pancreatitis. Coinheritance of p.R75Q or CF causing CFTR variants with SPINK1 variants significantly increases the risk of ICP.

Serum Immunoglobulin G Fraction 4 Levels in Pancreatic Cancer : Elevations Not Associated With Autoimmune Pancreatitis

CONTEXT Autoimmune pancreatitis is an uncommon, inflammatory disease of the pancreas that presents with clinical features, such as painless jaundice and a pancreatic mass, similar to those caused by pancreatic cancer. Patients with autoimmune pancreatitis frequently have elevated serum immunoglobulin G fraction 4 (IgG4) levels, and their pancreatic tissue may show IgG4-positive plasma cell infiltration. It is imperative to differentiate autoimmune pancreatitis from pancreatic cancer because autoimmune pancreatitis typically responds to corticosteroid treatment. A previous Japanese study reported that serum IgG4 greater than 135 mg/dL was 97% specific and 95% sensitive in predicting autoimmune pancreatitis. OBJECTIVE To prospectively measure serum IgG4 levels in pancreatic cancer patients to ascertain whether increased levels might be present in this North American population. DESIGN We collected blood samples and phenotypic information on 71 consecutive pancreatic cancer patients and 103 healthy controls who visited our clinics between October 2004 and April 2006. IgG4 levels were determined using a single radial immunodiffusion assay. A serum IgG4 level greater than 135 mg/dL was considered elevated. RESULTS Five cancer patients had IgG4 elevation, with a mean serum IgG4 level of 160.8 mg/dL. None of our cancer patients with plasma IgG4 elevation demonstrated evidence of autoimmune pancreatitis. One control subject demonstrated elevated serum IgG4 unrelated to identified etiology. CONCLUSIONS As many as 7% of patients with pancreatic cancer have serum IgG4 levels above 135 mg/dL. In patients with pancreatic mass lesions and suspicion of cancer, an IgG4 level measuring between 135 and 200 mg/dL should be interpreted cautiously and not accepted as diagnostic of autoimmune pancreatitis without further evaluation.

Is the monocyte chemotactic protein-1 -2518 G allele a risk factor for severe acute pancreatitis?

BACKGROUND & AIMS Acute pancreatitis (AP) reflects the intensity of the inflammatory response and is divided into mild AP (MAP) or severe AP (SAP). Monocyte chemotactic protein-1 (MCP-1) gene expression is altered by an A/G polymorphism (-2518), with the G allele increasing MCP-1 production. Our aim was to determine whether the MCP-1 -2518 A/G polymorphism affects the severity of AP. METHODS Seventy-seven consecutive patients and 116 controls were evaluated. The A/G genotype was evaluated by polymerase chain reaction amplification, restriction fragment length polymorphism, and DNA sequencing. MCP-1 serum levels were quantified using a fluorescence bead-based immunoassay. RESULTS Sixty-three of 77 patients had MAP (82%) and 14 of 77 had SAP (18%). Patients with SAP had a significantly greater proportion of the G allele (12 of 14; 86%) than did control subjects (50 of 116; 43%) (odds ratio [OR], 7.9; 95% confidence interval [CI], 1.7-37, P < .003) or MAP patients (29 of 63; 46%) (OR, 7.0; 95% CI, 1.5-34; P < .007). Patients with pancreatitis and AA genotype had a low risk for SAP (OR, .13; 95% CI, .01-.61; P < .003). As predicted by the genotype, the serum MCP-1 levels were significantly higher in the SAP patients when compared with the MAP patients ( P = .002) and they also predicted death. CONCLUSIONS MCP-1 -2518 G allele is a risk factor for severe AP. MCP-1 serum levels, measured early in the course of AP, appear to be an accurate predictor of severity of acute pancreatitis and death.

Angiopoietin-2, a Regulator of Vascular Permeability in Inflammation, Is Associated With Persistent Organ Failure in Patients With Acute Pancreatitis From the United States and Germany

OBJECTIVES:Patients with severe acute pancreatitis (AP) typically develop vascular leak syndrome, resulting in hemoconcentration, hypotension, pulmonary edema, and renal insufficiency. Angiopoietin-1 (Ang-1) and 2 (Ang-2) are autocrine peptides that reduce or increase endothelial permeability, respectively. The aim of this study was to determine whether Ang-1 and/or Ang-2 levels are predictive biomarkers of persistent organ failure (>48 h) and prolonged hospital course.METHODS:Banked serum from 28 patients enrolled in the Severity of Acute Pancreatitis Study at the University of Pittsburgh Medical Center (UPMC) and 58 controls was analyzed for Ang-1 and Ang-2 levels. Separately, serum from 123 patients and 103 controls at Greifswald University (GU), Germany was analyzed for Ang-2 levels. Angiopoietin levels were measured by enzyme-linked immunosorbent assay.RESULTS:In all, 6 out of 28 UPMC patients (21%) and 14 out of 123 GU patients (13%) developed persistent organ failure and were classified as severe AP. Ang-2 was significantly higher on admission in patients who developed persistent organ failure compared with those who did not in UPMC (3,698 pg/ml vs. 1,001 pg/ml; P=0.001) and GU (4,945 pg/ml vs. 2,631 pg/ml; P=0.0004) cohorts. After data scaling, admission Ang-2 levels showed a receiver-operator curve of 0.81, sensitivity 90%, and specificity 67% in predicting persistent organ failure. In addition, Ang-2 levels remained significantly higher in severe AP compared with mild AP patients until day 7 (days 2–4: P<0.005; day 7: P<0.02). Ang-1 levels were not significantly different between mild and severe AP patients on admission.CONCLUSIONS:Elevated serum Ang-2 levels on admission are associated with and may be a useful biomarker of predicting persistent organ failure and ongoing endothelial cell activation in AP.

Low serum adiponectin levels are associated with systemic organ failure in acute pancreatitis.

Objectives: Obesity markedly increases the risk of severe acute pancreatitis (SAP), possibly through the action of adipokines. We tested the hypothesis that serum adiponectin, the primary anti-inflammatory adipokine, is associated with functional polymorphisms in the adiponectin gene (ADIPOQ) and inversely associated with SAP. Methods: Severe AP was defined as the presence of remote organ failure. ADIPOQ polymorphisms rs2241766T>G and rs1501299G>T were evaluated by DNA sequencing. Serum samples were assayed using a Luminex assay (Luminex, Austin, Tex). Results: One hundred thirty-three patients with AP and 94 healthy controls were ascertained. Adiponectin levels were measured in 60 patients with early serum samples (27 patients with mild AP and 33 patients with SAP). Adiponectin levels from days 1 to 3 were inversely correlated with body mass index (BMI) (&rgr; = −0.49; P = 0.002) and were significantly lower for patients with SAP (median, 3.74 &mgr;g/mL) than those with mild AP (6.58 &mgr;g/mL; P = 0.02). Neither ADIPOQ polymorphism affected susceptibility to or severity of AP. A receiver operating characteristics curve using adiponectin levels as the severity predictor provided an area under the curve of 0.75. Conclusions: Serum adiponectin levels in patients with AP are inversely correlated with BMI and organ dysfunction. Further studies are needed to determine whether adiponectin is a marker of low BMI or if it provides significant protection from SAP.

Pooling-Based Genome-Wide Association Study Implicates Gamma-Glutamyltransferase 1 (GGT1) Gene in Pancreatic Carcinogenesis

Background/Aims: Knowledge regarding genetic factors that influence pancreatic cancer risk is currently limited. To identify novel pancreatic cancer susceptibility loci, we conducted a two-stage genome-wide association study. Methods: The Affymetrix® Genome-Wide Human SNP Array 6.0 and DNA pooling were used in the screening stage. Twenty-six single-nucleotide polymorphisms (SNPs) were selected for follow-up. These 26 lead SNPs and additionally selected tagSNPs for the regions around the lead SNPs were evaluated by individual genotyping of the pooling population and an independent validation population. Results: Of the lead SNPs, the strongest association was found with rs4820599 located in the γ-glutamyltransferase 1 (GGT1) gene. This SNP was significantly associated with pancreatic cancer risk in the validation population and the combined dataset (pallele-based = 0.019 and pallele-based = 0.003, respectively). Statistically significant associations were also observed with two GGT1 tagSNPs: rs2017869 and rs8135987. Lead SNP rs4820599 is in high linkage disequilibrium (LD; pairwise r2: 0.69) and tagSNP rs2017869 is in strong LD (pairwise r2: 0.96) with SNP rs5751901, which has been reported to be associated with increased GGT1 serum levels. GGT is expressed in the pancreas and plays a key role in glutathione metabolism. Conclusion: Our results suggest that common variation in the GGT1 gene may affect the risk of pancreatic cancer.

Serum Proteomic Patterns as a Predictor of Severity in Acute Pancreatitis

Background: Approximately 20% of patients with acute pancreatitis (AP) develop a severe and potentially life-threatening course. Serum proteomic pattern analysis for disease diagnosis is a promising novel and rapidly expanding field based on the hypothesis that serum patterns of low molecular mass biomarkers can specifically reflect an underlying organ-specific pathologic state. Aim: To evaluate the potential differences in proteomic profiles between patients with mild and severe AP. Methods: Sera from 21 patients with mild AP and 7 patients with severe AP were analyzed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Samples were profiled in duplicate on IMAC3 ProteinChips® arrays. Results: Of 79 spectral peak clusters (classifiers) detected, 18 had significantly different signal intensities between mild AP (MAP) and severe AP (SAP) sera (p < 0.01; Mann-Whitney U test, averaging for technical replicates) and were considered as potential classifiers in classification and regression tree (CART) analysis. The CART analysis returned simple classification trees consisting of one primary splitter, at 11,720 Da. Training data performance delivered nearly 100% sensitivity and 81% specificity for discrimination of SAP. The next top performing classifier was indicated at 4,283 Da m/z peak. Conclusions: These initial data suggest that serum proteomic profiles contain features that discriminate MAP and SAP. Larger sample sizes will be required for the development and validation of more specific predictive algorithms.

Keratin 8 Mutations Are Not Associated with Familial, Sporadic and Alcoholic Pancreatitis in a Population from the United States

Background and Aims: Genetic predispositions play a major role in the development of chronic pancreatitis. Recently, a mutation in the keratin 8 gene (G62C) was reported to be associated with chronic pancreatitis in Italy. We determined whether mutations in the keratin 8 gene are associated with familial, sporadic and alcoholic recurrent acute or chronic pancreatitis in a population from the United States. Methods: We investigated the relevant genomic region of the keratin 8 gene in 80 patients with familial pancreatitis without a cationic trypsinogen (PRSS1) gene mutation from 52 different families, 21 patients with familial hereditary pancreatitis and a PRSS1 mutation from 20 different families, 126 patients with sporadic pancreatitis without a PRSS1 mutation, 61 patients with alcoholic pancreatitis and 271 controls by direct DNA sequencing. Results: We found the heterozygous G62C mutation in n = 3/80 patients (n = 2/52 patients from different families, 3.8%) with familial pancreatitis without PRSS1 mutation and in n = 3/126 patients (2.4%) with sporadic pancreatitis. We detected an adjacent heterozygous I63V mutation in n = 2/80 patients (n = 2/52 patients from different families, 3.8%) with familial pancreatitis without PRSS1 mutation and in n = 1/61 patients (1.6%) with alcoholic pancreatitis. We found the G62C mutation in n = 2/271 controls (0.7%) and the I63V mutation in n = 2/271 controls (0.7%). There were no statistically significant differences in the genotype frequencies between patients and controls (p > 0.05). Screening of additional available family members revealed that these variants did not segregate with the disease phenotype. There was no statistically significant difference in the frequency of these keratin 8 variants between patients with chronic pancreatitis and controls (p > 0.05). Conclusion: These keratin 8 variants are not associated with familial, sporadic or alcoholic pancreatitis.

Functional Polymorphisms of the GSTT-1 Gene Do Not Predict the Severity of Acute Pancreatitis in the United States

Background/Aim: Acute pancreatitis (AP) is an inflammatory response to pancreatic injury that is clinically classified as mild AP or severe AP, depending on specific criteria. Rahman and colleagues [Gastroenterology 2004;126:1312–1322] reported that genetic variation in the glutathione S-transferase theta-1 gene (GSTT-1) is associated with susceptibility and severity of AP in England. Our aim was to determine whether the same GSTT-1 polymorphism affects the severity of AP in a population from Pittsburgh, Pa., USA. Methods: Ninety-one consecutive patients with AP (19 severe) were prospectively evaluated. The GSTT-1 haplotypes were determined by PCR amplification in all patients and 268 controls. The resulting genotypes were classified as functional (GSTT-1*A/*A or *A/null) and nonfunctional (GSTT-1 null/null) phenotypes. Results: The relative frequencies of functional GSTT-1 phenotypes were similar in subjects with severe AP (15 of 19, 78.9%) and mild AP (61 of 72, 84.7%; p = 0.54) and in the controls (228 of 268, 85.1%; p = 0.66). Furthermore, the GSTT-1 functional and nonfunctional phenotypes were not associated with serum C-reactive protein levels (11.9 vs. 7.3 mg/dl; p = 0.19), interleukin-6 levels (74 vs. 60 pg/ml; p = 0.9), APACHE II scores (7 vs. 9; p = 0.26), or 48-hour Ranson scores (1 vs. 1; p = 0.63). Conclusion: Functional GSTT-1 phenotypes do not correlate with susceptibility to AP or severity of AP in our patient population.

Angiotensin-converting enzyme gene DD genotype neither increases susceptibility to acute pancreatitis nor influences disease severity.

BACKGROUND The renin-angiotensin system (RAS) has been implied in the pathogenesis of various diseases including acute and chronic pancreatitis. Angiotensin-converting enzyme (ACE) is the key enzyme in activating the RAS. Deletion (D)-type polymorphism in the 16th intron of the ACE gene has been associated with higher serum levels of the enzyme. Inhibition of ACE was found to ameliorate acute pancreatitis in animal models suggesting that ACE plays a role in pathogenesis and progression of acute pancreatitis. Objectives were to investigate the occurrence of the ACE insertion/deletion (I/D) polymorphism in acute pancreatitis patients and its association with the severity of the disease. MATERIAL AND METHODS Seventy-nine acute pancreatitis patients and 95 healthy controls were evaluated. Acute pancreatitis cases were grouped as mild or severe according to the Atlanta criteria. MAIN OUTCOME MEASURE The presence of the ACE I/D polymorphism. RESULTS ACE gene I and D allele frequency of patients (44% and 56%) were similar to controls (45% and 55%, respectively). There were no significant differences in severity of pancreatitis between patients with the ACE-insertion or ACE-insertion/deletion versus ACE-deletion genotypes. CONCLUSIONS The ACE gene deletion polymorphism is neither a risk factor for development of acute pancreatitis nor contributes to the severity of disease or development of complications.