Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis

Abstract

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

Figure 1

Manhattan plot showing the negative log (base 10) of the p-value for the association of SNP genotype with affection status for all SNPs passing quality control filters and falling within a selected region of the PRSS1-PRSS2 and CLDN2 loci. Regions selected to highlight the most associated SNPs. Squares indicate Stage 1 results, circles for Stage 2, diamonds for combined Stage 1 and 2 data. After accounting for the most highly associated SNP at each locus, no other SNP approached genomewide-significant association.

Figure 2

Expression and localization of claudin-2 in the human pancreas using mouse anti-claudin-2 antibodies based on rs12688220 genotype. A. Western blot of anti-claudin-2 antibody from 3 control samples genotyped at rs12688220 (TT is high risk). The antibody reacts with a protein at ~22-23 kDa, consistent with claudin-2. Samples had inflammation and/or fibrosis on histology of adjacent tissue. α-tubulin, loading control. Blots from all controls are presented in Supplementary Figure 8. B. Anti-claudin-2 staining (brown color) of normal-appearing control tissue localizing to ducts but not to acinar cells (scale bar=50μm). C. Severe chronic pancreatitis from a case with the high-risk (T male or TT female) genotype. Claudin-2 staining localizes to the intralobular duct (Duct), atrophic acini (*), and cells with morphologic appearance of macrophages (arrow)(scale bar=50μm). D. Chronic pancreatitis tissue from a patient with the low-risk genotype (CC or CT) with staining localizing to the duct and granular staining in acinar cells (scale bar = 100 μm). E. Chronic pancreatitis, high-risk genotype with intense staining of acinar cell basolateral membrane (scale bar=100 μm, enlarged in inset, scale bar=10μm). F. Immunofluorescence staining of control human pancreatic tissue claudin-2 staining (red) localizing to the ducts (*) and co-localizing with the macrophage marker CD68 (green, colocalized with red is yellow, arrows. Nuclei stained with Hoechst's dye, blue, scale bar = 100μm).