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

Abstract

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.

Keywords: Genome wide association study; chronic pancreatitis; genetic rearrangement.

Figure 1

Genome-wide association analysis of 1959 cases with alcoholic chronic pancreatitis and 6040 controls derived from population studies and a cohort of alcohol-dependent patients. Genome-wide significance-level threshold (p=5×10^–8) is represented by the black line. Only single-nucleotide polymorphisms that passed quality control are depicted.

Figure 2

Schematic illustration of the CTRB1-CTRB2 locus with the 16.6 kb inversion. The inversion breakpoints lie within the region indicated by the dashed lines. The genomic reference sequence corresponds to the minor allele. The locations of the lead single-nucleotide polymorphism (SNP) rs8055167 and the best tagging SNP rs8048956 are denoted by the empty and black diamond symbols, respectively. Genomic distances are not scaled. Although not shown here, in this locus CTRB2 also harbours a 584 bp deletion variant (allele frequency ~7% in German controls) that eliminates exon 6. CTRB1, chymotrypsin B1 gene; CTRB2, chymotrypsin B2 gene; CTRB1* and CTRB2*, hybrid CTRB1 and CTRB2 genes created by the inversion; E, exon.

Figure 3

Regional association plots for the CTRB1-CTRB2 locus inversion. (A) p Values (–log₁₀) are displayed against single-nucleotide polymorphism (SNP) genomic position (genome build hg19). The inversion is represented by the red line, triangles are genotyped SNPs, circles are imputed SNPs. For calculations, all alcoholic chronic pancreatitis patients and all controls were included. (B) The OR-based regional association plot indicates that the association is driven by the inversion. Here ORs are represented against the genomic position.

Figure 4

Expression and effect of CTRB1 and CTRB2 on trypsinogen activation and degradation. (A) Expression of CTRB1 and CTRB2 mRNA in the pancreas of subjects with different inversion genotypes. Expression ratios of CTRB1 and CTRB2 in pancreatic cDNA samples were determined by real-time polymerase chain reaction using the standard curve method. Results were displayed as box plots showing minimum, first quartile (25%), median, third quartile (75%), maximum and the individual values (black dots). Note that subjects carrying two copies of the major risk allele exhibit a significantly higher CTRB1/CTRB2 expression ratio compared with heterozygous individuals with one major and one minor allele. Significance was calculated with unpaired t-test. (B) Effect of CTRB1 and CTRB2 on the autoactivation of human anionic trypsinogen (PRSS2). Trypsinogen (2 µM) was incubated with 10 nM initial trypsin and 200 nM of the indicated chymotrypsin in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl₂ and 0.05% Tween 20 (final concentrations) at 37°C in 100 µL final volume. At the indicated times, aliquots (2 µL) were withdrawn and trypsin activity was determined using 150 µM N-CBZ-Gly-Pro-Arg-p-nitroanilide substrate. Rate of substrate cleavage is given in mOD/min units measured at 405 nm. Note the lower trypsin activity that develops in the presence of chymotrypsins indicating trypsinogen degradation during activation. Under similar conditions, CTRB1 and CTRB2 had a similar but much smaller effect on the autoactivation of human cationic trypsinogen (PRSS1). (C) Degradation of PRSS2 by CTRB1 and CTRB2. Trypsinogen (1 µM) was incubated with 200 nM of the indicated chymotrypsin and 20 nM SPINK1 trypsin inhibitor in 0.1 M Tris-HCl (pH 8.0) and 25 mM NaCl at 37°C. Reactions were stopped at the indicated times by precipitation of 150 µL aliquots with 10% trichloroacetic acid. Samples were analysed by SDS-PAGE and Coomassie Blue staining. Note the disappearance of the intact trypsinogen band in the CTRB2 incubate. Some of the lower bands correspond to the two chains of autolysed CTRB2. Although not shown, CTRB1 or CTRB2 did not degrade cationic trypsinogen (PRSS1) to a detectable extent. CTRB1, chymotrypsin B1; CTRB2, chymotrypsin B2; PRSS2, anionic trypsinogen.