Prevalence of Pathogenic and Potentially Pathogenic Inborn Error of Immunity Associated Variants in Children with Severe Sepsis.

PURPOSE: Our understanding of inborn errors of immunity is increasing; however, their contribution to pediatric sepsis is unknown. METHODS: We used whole-exome sequencing (WES) to characterize variants in genes related to monogenic immunologic disorders in 330 children admitted to intensive care for severe sepsis. We defined candidate variants as rare variants classified as pathogenic or potentially pathogenic in QIAGEN's Human Gene Mutation Database or novel null variants in a disease-consistent inheritance pattern. We investigated variant correlation with infection and inflammatory phenotype. RESULTS: More than one in two children overall and three of four African American children had immunodeficiency-associated variants. Children with variants had increased odds of isolating a blood or urinary pathogen (blood: OR 2.82, 95% CI: 1.12-7.10, p = 0.023, urine: OR: 8.23, 95% CI: 1.06-64.11, p = 0.016) and demonstrating increased inflammation with hyperferritinemia (ferritin [Formula: see text] ng/mL, OR: 2.16, 95% CI: 1.28-3.66, p = 0.004), lymphopenia (lymphocyte count < 1000/µL, OR: 1.66, 95% CI: 1.06 - 2.60, p = 0.027), thrombocytopenia (platelet count < 150,000/µL, OR: 1.76, 95% CI: 1.12-2.76, p = 0.013), and CRP greater than 10 mg/dl (OR: 1.71, 95% CI: 1.10-2.68, p = 0.017). They also had increased odds of requiring extracorporeal membrane oxygenation (ECMO, OR: 4.19, 95% CI: 1.21-14.5, p = 0.019). CONCLUSION: Herein, we describe the genetic findings in this severe pediatric sepsis cohort and their microbiologic and immunologic significance, providing evidence for the phenotypic effect of these variants and rationale for screening children with life-threatening infections for potential inborn errors of immunity.

mRNA and miRNA Profiles of Exosomes from Cultured Tumor Cells Reveal Biomarkers Specific for HPV16-Positive and HPV16-Negative Head and Neck Cancer.

Human papillomavirus (HPV)(+) and HPV(-) head and neck cancer (HNC) cells' interactions with the host immune system are poorly understood. Recently, we identified molecular and functional differences in exosomes produced by HPV(+) vs. HPV(-) cells, suggesting that genetic cargos of exosomes might identify novel biomarkers in HPV-related HNCs. Exosomes were isolated by size exclusion chromatography from supernatants of three HPV(+) and two HPV(-) HNC cell lines. Paired cell lysates and exosomes were analyzed for messenger RNA (mRNA) by qRT-PCR and microRNA (miR) contents by nanostring analysis. The mRNA profiles of HPV(+) vs. HPV(-) cells were distinct, with EGFR, TP53 and HSPA1A/B overexpressed in HPV(+) cells and IL6, FAS and DPP4 in HPV(-) cells. The mRNA profiles of HPV(+) or HPV(-) exosomes resembled the cargo of their parent cells. miR expression profiles in cell lysates identified 8 miRs expressed in HPV(-) cells vs. 14 miRs in HPV(+) cells. miR-205-5p was exclusively expressed in HPV(+) exosomes, and miR-1972 was only detected in HPV(-) exosomes. We showed that HPV(+) and HPV(-) exosomes recapitulated the mRNA expression profiles of their parent cells. Expression of miRs was dependent on the HPV status, and miR-205-5p in HPV(+) and miR-1972 in HPV(-) exosomes emerge as potential discriminating HPV-associated biomarkers.

The transcriptome of the Didelphis virginiana opossum kidney OK proximal tubule cell line.

The OK cell line derived from the kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for D. virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The M. domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the D. virginiana OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the M. domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the M. domestica genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells.

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.

Are genetic variants in the platelet-derived growth factor [beta] gene associated with chronic pancreatitis?

OBJECTIVES: Platelet-derived growth factor [beta] (PDGF-[beta]) is a major signal in proliferation and matrix synthesis through activated pancreatic stellate cells, leading to fibrosis of the pancreas. Recurrent acute pancreatitis (RAP) seems to predispose to chronic pancreatitis (CP) in some patients but not others. We tested the hypothesis that 2 known PDGF-[beta] polymorphisms are associated with progression from RAP to CP. We also tested the hypothesis that PDGF-[beta] polymorphisms in combination with environmental risk factors such as alcohol and smoking are associated with CP. METHODS: Three hundred eighty-two patients with CP (n = 176) and RAP (n = 206) and 251 controls were evaluated. Platelet-derived growth factor [beta] polymorphisms +286 A/G (rs#1800818) seen in 5'-UTR and +1135 A/C (rs#1800817) in first intron were genotyped using single-nucleotide polymorphism polymerase chain reaction approach and confirmed by DNA sequencing. RESULTS: The genotypic frequencies for PDGF-[beta] polymorphisms in positions +286 and +1135 were found to be similar in controls and patients with RAP and CP. There was no difference in genotypic frequencies among RAP, CP, and controls in subjects in the alcohol and smoking subgroups. CONCLUSIONS: Known variations in the PDGF-[beta] gene do not have a significant effect on promoting or preventing fibrogenesis in pancreatitis. Further evaluation of this important pathway is warranted.

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.

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 gamma-glutamyltransferase 1 (GGT1) gene. This SNP was significantly associated with pancreatic cancer risk in the validation population and the combined dataset (p(allele-based) = 0.019 and p(allele-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 r(2): 0.69) and tagSNP rs2017869 is in strong LD (pairwise r(2): 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. .

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) (rho = -0.49; P = 0.002) and were significantly lower for patients with SAP (median, 3.74 microg/mL) than those with mild AP (6.58 microg/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.

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.

MUTYH exon 7 and 13 mutations associated with colorectal cancer (MAP syndrome) are not commonly associated with sporadic pancreatic cancer.

BACKGROUND: Biallelic MUTYH exon 7 and 13 mutations are associated with a high frequency of somatic K-ras gene guanine to thymine transversion mutations at codon 12 position 1 in MUTYH-associated polyposis patients who have increased risk of colon cancer. The purpose of this study was to determine if a similar association exists between exon 7 and 13 MUTYH mutations and pancreatic cancer. METHODS: Genomic DNA samples from 140 patients with pancreatic cancer and 107 controls were sequenced and analyzed for mutations in each of MUTYH exons 7 and 13. RESULTS: Two patients with pancreatic cancer were identified as heterozygous for a MUTYH Y165C germline mutation. One pancreatic cancer patient was heterozygous for a G382D mutation and an additional patient was heterozygous for a novel missense mutation, L406M. No biallelic mutations were identified in pancreatic cancer or control subjects. CONCLUSION: Despite their association with somatic K-ras mutations and an increased risk of colorectal cancer in MUTYH-associated polyposis patients, MUTYH exon 7 and 13 mutations were not associated with pancreatic cancer in our cohort.

Does the pain-protective GTP cyclohydrolase haplotype significantly alter the pattern or severity of pain in humans with chronic pancreatitis?

BACKGROUND: Pain is often a dominant clinical feature of chronic pancreatitis but the frequency and severity is highly variable between subjects. We hypothesized that genetic polymorphisms contribute to variations in clinical pain patterns. Since genetic variations in the GTP cyclohydrolase (GCH1) gene have been reported to protect some patients from pain, we investigated the effect of the "pain protective haplotype" in well characterized patients with chronic pancreatitis (CP) or recurrent acute pancreatitis (RAP) from the North American Pancreatitis Study 2 (NAPS2). RESULTS: Subjects in the NAPS2 study were asked to rank their pain in one of 5 categories reflecting different levels of pain frequency and severity. All subjects were genotyped at rs8007267 and rs3783641 to determine the frequency of the GCH1 pain-protective haplotype. In Caucasian subjects the frequency of the pain-protective GCH1 haplotype was no different in the control group (n = 236), CP patients (n = 265), RAP patients (N = 131), or in CP patients subclassified by pain category compared to previously reported haplotype frequencies in the general Caucasian population. CONCLUSION: The GCH1 pain-protective haplotype does not have a significant effect on pain patterns or severity in RAP or CP. These results are important for helping to define the regulators of visceral pain, and to distinguish different mechanisms of pain.

Multicenter approach to recurrent acute and chronic pancreatitis in the United States: the North American Pancreatitis Study 2 (NAPS2).

BACKGROUND: Recurrent acute pancreatitis (RAP) and chronic pancreatitis (CP) are complex syndromes associated with numerous etiologies, clinical variables and complications. We developed the North American Pancreatitis Study 2 (NAPS2) to be sufficiently powered to understand the complex environmental, metabolic and genetic mechanisms underlying RAP and CP. METHODS: Between August 2000 and September 2006, a consortium of 20 expert academic and private sites prospectively ascertained 1,000 human subjects with RAP or CP, plus 695 controls (spouse, family, friend or unrelated). Standardized questionnaires were completed by both the physicians and study subjects and blood was drawn for genomic DNA and biomarker studies. All data were double-entered into a database and systematically reviewed to minimize errors and include missing data. RESULTS: A total of 1,000 subjects (460 RAP, 540 CP) and 695 controls who completed consent forms and questionnaires and donated blood samples comprised the final dataset. Data were organized according to diagnosis, supporting documentation, etiological classification, clinical signs and symptoms (including pain patterns and duration, and quality of life), past medical history, family history, environmental exposures (including alcohol and tobacco use), medication use and therapeutic interventions. Upon achieving the target enrollment, data were organized and classified to facilitate future analysis. The approaches, rationale and datasets are described, along with final demographic results. CONCLUSION: The NAPS2 consortium has successfully completed a prospective ascertainment of 1,000 subjects with RAP and CP from the USA. These data will be useful in elucidating the environmental, metabolic and genetic conditions, and to investigate the complex interactions that underlie RAP and CP.

Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population: role of serine protease inhibitor Kazal 1type and alcohol.

AIM: To test the hypothesis that calcium sensing receptor (CASR) polymorphisms are associated with chronic pancreatitis (CP), and to determine whether serine protease inhibitor Kazal 1type (SPINK1) N34S or alcohol are necessary co-factors in its etiology. METHODS: Initially, 115 subjects with pancreatitis and 66 controls were evaluated, of whom 57 patients and 21 controls were predetermined to carry the high-risk SPINK1 N34S polymorphism. We sequenced CASR gene exons 2, 3, 4, 5 and 7, areas containing the majority of reported polymorphisms and novel mutations. Based on the initial results, we added 223 patients and 239 controls to analyze three common nonsynonymous single nucleotide polymorphisms (SNPs) in exon 7 (A986S, R990G, and Q1011E). RESULTS: The CASR exon 7 R990G polymorphism was significantly associated with CP (OR, 2.01; 95% CI, 1.12-3.59; P = 0.015). The association between CASR R990G and CP was stronger in subjects who reported moderate or heavy alcohol consumption (OR, 3.12; 95% CI, 1.14-9.13; P = 0.018). There was no association between the various CASR genotypes and SPINK1 N34S in pancreatitis. None of the novel CASR polymorphisms reported from Germany and India was detected. CONCLUSION: Our United States-based study confirmed an association of CASR and CP and for the first time demonstrated that CASR R990G is a significant risk factor for CP. We also conclude that the risk of CP with CASR R990G is increased in subjects with moderate to heavy alcohol consumption.

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.

The ACE gene I/D polymorphism does not affect the susceptibility to or prognosis of PBC.

BACKGROUND/AIMS: Primary biliary cirrhosis is an autoimmune liver disease that is strongly influenced by poorly defined, complex genetic factors. Alterations of the renin-angiotensin system have been implicated in the pathogenesis of various diseases. A deletion polymorphism of a 287-bp fragment of intron 16 of the angiotensin converting enzyme gene allele results in higher levels of circulating enzyme. Angiotensin converting enzyme deletion genotype has been linked to hypertension and sarcoidosis and has been reported to regulate liver fibrosis in HCV-mediated liver disease. We investigated the frequency of the Angiotensin converting enzyme gene insertion/deletion polymorphism in primary biliary cirrhosis patients. METHODS: 52 biopsy-proven primary biliary cirrhosis patients and 98 healthy controls were evaluated. Angiotensin converting enzyme insertion/deletion polymorphism was detected by polymerase chain reaction amplification of a genomic DNA fragment on intron 16 of the angiotensin converting enzyme gene. Clinical phenotype of primary biliary cirrhosis was verified with positive anti-mitochondrial antibody or M2 antibody, demonstration of cholestatic liver enzymes, and staging of liver biopsy. The differences between these variables among different genotypes were noted. RESULTS: There was no significant difference in genotypes and allele frequency between the primary biliary cirrhosis group and controls. The D allele frequency was 54% in primary biliary cirrhosis cases and 55% in controls (p=ns). Furthermore, there was no significant difference in clinical features between patients with angiotensin converting enzyme-insertion or insertion/deletion genotypes vs. patients with angiotensin converting enzyme-deletion genotype. CONCLUSIONS: In our limited sample, the angiotensin converting enzyme deletion genotype did not make a significant contribution to the pathogenesis or progression of primary biliary cirrhosis.

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.

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.

The MCP-1 -2518 A/G polymorphism is not a susceptibility factor for chronic pancreatitis.

BACKGROUND AND AIMS: Chronic pancreatitis (CP) is an inflammatory process initiated by recurrent acute pancreatitis and characterized by progressive parenchyma destruction and fibrosis. Genetic factors influence susceptibility and modify progression. The monocyte chemotactic protein-1 (MCP-1) -2518 G allele, which modifies the severity of acute pancreatitis, was investigated as a susceptibility factor for CP. METHODS: A genetic association study was performed on 177 CP patients and 116 healthy controls from the NAPS2 Study. The MCP-1 A/G genotype was determined by RFLP and confirmed by DNA sequencing. RESULTS: Compared to the control group the MCP-1 -2518 genotypes were similar: A/A (57% vs. 50%), A/G (34.5% vs. 40%) and G/G (8.5% vs. 10%). These allele frequencies were not statistically different (p = 0.267). CONCLUSIONS: Although the pro-inflammatory chemokine MCP-1 -2518 G allele is a severity factor for AP, it does not significantly alter susceptibility to CP.

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.

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.