Variants in CPA1 are strongly associated with early onset chronic pancreatitis

Abstract

Chronic pancreatitis is an inflammatory disorder of the pancreas. We analyzed CPA1, encoding carboxypeptidase A1, in subjects with nonalcoholic chronic pancreatitis (cases) and controls in a German discovery set and three replication sets. Functionally impaired variants were present in 29/944 (3.1%) German cases and 5/3,938 (0.1%) controls (odds ratio (OR) = 24.9, P = 1.5 × 10(-16)). The association was strongest in subjects aged ≤ 10 years (9.7%; OR = 84.0, P = 4.1 × 10(-24)). In the replication sets, defective CPA1 variants were present in 8/600 (1.3%) cases and 9/2,432 (0.4%) controls from Europe (P = 0.01), 5/230 (2.2%) cases and 0/264 controls from India (P = 0.02) and 5/247 (2.0%) cases and 0/341 controls from Japan (P = 0.013). The mechanism by which CPA1 variants confer increased pancreatitis risk may involve misfolding-induced endoplasmic reticulum stress rather than elevated trypsin activity, as is seen with other genetic risk factors for this disease.

Figure 1

Endoplasmic reticulum (ER) stress induced by the p.Asn256Lys CPA1 variant. (A) AR42J rat acinar cells were transfected with the indicated wild-type, mutant or empty adenovirus vectors for 24 h using 4×10⁷ pfu per mL virus concentration. Conditioned media (200 μL) were precipitated with trichloroacetic acid (10% final concentration) and analyzed by SDS-PAGE and Coomassie blue staining. Note the complete lack of secretion of the p. Asn256Lys mutant. The faint band at 40 kDa represents an endogenous protein also found in the medium from cells infected with empty virus. The asterisk indicates the characteristically strong amylase band. See Methods for experimental details. A representative gel of three independent transfections is shown. (B) XBP1 splicing was assessed by RT-PCR and agarose gel electrophoresis with ethidium bromide staining. A representative gel of three independent experiments is shown. (C) Levels for spliced, unspliced and total XBP1 mRNA were measured by quantitative real-time PCR as described in Methods and expressed as fold changes relative to levels measured in cells transfected with empty adenovirus. (D) Quantitative real-time PCR measurement of BiP and calreticulin mRNA was performed as described in Methods and expressed as fold changes relative to levels measured in cells transfected with empty adenovirus. Error bars represent standard deviation (n = 3).