Sequencing of the complex CTRB1-CTRB2 locus in chronic pancreatitis.

BACKGROUND: /Objectives: A recent Genome-wide Association Study (GWAS) in alcoholic chronic pancreatitis (ACP) identified a novel association with the CTRB1-CTRB2 (chymotrypsinogen B1, B2) locus, linked to a 16.6 kb inversion that was confirmed in non-alcoholic chronic pancreatitis (NACP). Moreover, recent findings on the function of CTRB1 and CTRB2 suggest a protective role in pancreatitis development. The aim of the present study was to investigate the CTRB1-CTRB2 locus for rare genetic variants associated with chronic pancreatitis (CP). METHODS: We analyzed 134 patients with ACP and 203 patients with NACP and compared them to up to 258 healthy controls. Genotyping was performed with polymerase chain reaction, followed by Sanger sequencing of all exons and the exon-intron-boundaries of CTRB1 and CTRB2. Finally, in silico analyses of the identified variants were conducted. RESULTS: None of the seven rare missense variants or the single 5'-UTR variant in CTRB1 and CTRB2 was associated with ACP or NACP. In silico analysis predicted that variant p. Trp5Leu in CTRB1 and variant c.-4C > T in CTRB2 might alter protein expression and variants p. Asp222His in CTRB1 and p. Ala247Thr in CTRB2 might affect protein function. However, all of these variants were also described in public databases. CONCLUSIONS: The present study did not reveal an association of rare variants in CTRB1 and CTRB2 with ACP or NACP. Although rare missense variants were almost exclusively found in patients, only four variants were predicted to affect protein expression or function. Thus, a major influence of rare variants in the CTRB1-CTRB2 locus on CP development is unlikely.

Protease-Sensitive Pancreatic Lipase Variants Are Associated With Early Onset Chronic Pancreatitis.

OBJECTIVES: Premature activation of the digestive protease trypsin within the pancreatic parenchyma is a critical factor in the pathogenesis of pancreatitis. Alterations in genes that affect intrapancreatic trypsin activity are associated with chronic pancreatitis (CP). Recently, carboxyl ester lipase emerged as a trypsin-independent risk gene. Here, we evaluated pancreatic lipase (PNLIP) as a potential novel susceptibility gene for CP. METHODS: We analyzed all 13 PNLIP exons in 429 nonalcoholic patients with CP and 600 control subjects from Germany, in 632 patients and 957 controls from France, and in 223 patients and 1,070 controls from Japan by DNA sequencing. Additionally, we analyzed selected exons in further 545 patients with CP and 1,849 controls originating from Germany, United States, and India. We assessed the cellular secretion, lipase activity, and proteolytic stability of recombinant PNLIP variants. RESULTS: In the German discovery cohort, 8/429 (1.9%) patients and 2/600 (0.3%) controls carried a PNLIP missense variant (P = 0.02, odds ratio [OR] = 5.7, 95% confidence interval [CI] = 1.1-38.9). Variants detected in patients were prone to proteolytic degradation by trypsin and chymotrypsin. In the French replication cohort, protease-sensitive variants were also enriched in patients with early-onset CP (5/632 [0.8%]) vs controls (1/957 [0.1%]) (P = 0.04, OR = 7.6, 95% CI = 0.9-172.9). In contrast, we detected no protease-sensitive variants in the non-European populations. In the combined European data, protease-sensitive variants were found in 13/1,163 cases (1.1%) and in 3/3,000 controls (0.1%) (OR = 11.3, 95% CI = 3.0-49.9, P < 0.0001). CONCLUSIONS: Our data indicate that protease-sensitive PNLIP variants are novel genetic risk factors for the development of CP.

Genome-wide association study identifies inversion in the CTRB1-CTRB2 locus to modify risk for alcoholic and non-alcoholic chronic pancreatitis.

OBJECTIVE: Alcohol-related pancreatitis is associated with a disproportionately large number of hospitalisations among GI disorders. Despite its clinical importance, genetic susceptibility to alcoholic chronic pancreatitis (CP) is poorly characterised. To identify risk genes for alcoholic CP and to evaluate their relevance in non-alcoholic CP, we performed a genome-wide association study and functional characterisation of a new pancreatitis locus. DESIGN: 1959 European alcoholic CP patients and population-based controls from the KORA, LIFE and INCIPE studies (n=4708) as well as chronic alcoholics from the GESGA consortium (n=1332) were screened with Illumina technology. For replication, three European cohorts comprising 1650 patients with non-alcoholic CP and 6695 controls originating from the same countries were used. RESULTS: We replicated previously reported risk loci CLDN2-MORC4, CTRC, PRSS1-PRSS2 and SPINK1 in alcoholic CP patients. We identified CTRB1-CTRB2 (chymotrypsin B1 and B2) as a new risk locus with lead single-nucleotide polymorphism (SNP) rs8055167 (OR 1.35, 95% CI 1.23 to 1.6). We found that a 16.6 kb inversion in the CTRB1-CTRB2 locus was in linkage disequilibrium with the CP-associated SNPs and was best tagged by rs8048956. The association was replicated in three independent European non-alcoholic CP cohorts of 1650 patients and 6695 controls (OR 1.62, 95% CI 1.42 to 1.86). The inversion changes the expression ratio of the CTRB1 and CTRB2 isoforms and thereby affects protective trypsinogen degradation and ultimately pancreatitis risk. CONCLUSION: An inversion in the CTRB1-CTRB2 locus modifies risk for alcoholic and non-alcoholic CP indicating that common pathomechanisms are involved in these inflammatory disorders.

Common variants in the CLDN2-MORC4 and PRSS1-PRSS2 loci confer susceptibility to acute pancreatitis.

BACKGROUND/OBJECTIVES: Acute pancreatitis (AP) is one of the most common gastrointestinal disorders often requiring hospitalization. Frequent aetiologies are gallstones and alcohol abuse. In contrast to chronic pancreatitis (CP) few robust genetic associations have been described. Here we analysed whether common variants in the CLDN2-MORC4 and the PRSS1-PRSS2 locus that increase recurrent AP and CP risk associate with AP. METHODS: We screened 1462 AP patients and 3999 controls with melting curve analysis for SNPs rs10273639 (PRSS1-PRSS2), rs7057398 (RIPPLY), and rs12688220 (MORC4). Calculations were performed for the overall group, aetiology, and gender sub-groups. To examine genotype-phenotype relationships we performed several meta-analyses. RESULTS: Meta-analyses of all AP patients depicted significant (p-value < 0.05) associations for rs10273639 (odds ratio (OR) 0.88, 95% confidence interval (CI) 0.81-0.97, p-value 0.01), rs7057398 (OR 1.27, 95% CI 1.07-1.5, p-value 0.005), and rs12688220 (OR 1.32, 95% CI 1.12-1.56, p-value 0.001). For the different aetiology groups a significant association was shown for rs10273639 (OR 0.76, 95% CI 0.63-0.92, p-value 0.005), rs7057398 (OR 1.43, 95% CI 1.07-1.92, p-value 0.02), and rs12688220 (OR 1.44, 95% CI 1.07-1.93, p-value 0.02) in the alcoholic sub-group only. CONCLUSIONS: The association of CP risk variants with different AP aetiologies, which is strongest in the alcoholic AP group, might implicate common pathomechanisms most likely between alcoholic AP and CP.