Variants in CPA1 are strongly associated with early onset chronic pancreatitis.

The medical ethical review committees of all participating study centers approved this study. All study subjects gave informed consent. We enrolled 944 unrelated German individuals with the diagnosis of non-alcoholic chronic pancreatitis and 465 patients with alcohol-related chronic pancreatitis. In the replication study, we investigated 600 unrelated non-alcoholic chronic pancreatitis patients originating from France ( n = 456), the Czech Republic ( n = 21), and Poland ( n = 123). In addition, we also investigated unrelated subjects affected with non-alcoholic chronic pancreatitis from India ( n = 230) and Japan ( n = 247). The diagnosis of chronic pancreatitis was based on two or more of the following findings: presence of a typical history of recurrent pancreatitis, pancreatic calcifications and/or pancreatic ductal irregularities revealed by endoscopic retrograde pancreaticography or by magnetic resonance imaging of the pancreas and/or pathological sonographic findings. Alcoholic chronic pancreatitis was diagnosed in patients who consumed more than 60 g (females) or 80 g (males) of ethanol per day for more than two years. Control subjects were recruited from Germany ( n = 3,938), France ( n = 2,000), the Czech Republic ( n = 235), Poland ( n = 197), India ( n = 264), and Japan ( n = 341). We designed primers complementary to intronic sequences flanking CPA1 exons based on the published nucleotide sequence (GenBank # NT_007933.15 ) ( Supplementary Table 2 ). After PCR amplification, the entire coding region and the exon-intron boundaries were sequenced. All mutations were confirmed with a second independent PCR reaction. In the German laboratories, we performed PCR using 0.75 U AmpliTaq Gold polymerase (Perkin Elmer, Rodgau, Germany), 400 μmol/L deoxynucleoside triphosphates and 0.1 μmol/L primers in a total volume of 25 μL. Cycle conditions were as follows: initial denaturation for 12 min at 95°C; 48 cycles of 20 s denaturation at 95°C, 40 s annealing at 64°C and 90 s primer extension at 72°C; and a final extension step for 2 min at 72°C. PCR products were digested with Antarctic phosphatase (New England Biolabs, Ipswich MA) or shrimp alkaline phosphatase (USB, Santa Clara, CA) and exonuclease I (New England Biolabs, Ipswich MA). Cycle sequencing was performed using BigDye terminator mix (Applied Biosystems, Darmstadt, Germany) with 56° annealing temperature. The reaction products were purified with ethanol precipitation and loaded onto an ABI 3730 or an ABI 3100-Avant fluorescence sequencer (Applied Biosystems). We investigated the functional consequences of CPA1 alterations by transient transfection of HEK 293T cells (#Q401, GenHunter, Nashville, TN) with wild-type and mutant constructs and analyzing the conditioned medium for the amount of proCPA1 protein constitutively secreted using densitometry of stained gels and CPA1 activity after activation with trypsin and CTRC. Construction of the pcDNA3.1(-) human CPA1 expression plasmid has been reported previously 12 . The coding DNA in this plasmid was derived from IMAGE clone #3949850 (GenBank accession BC005279 ), which contains a c.827A>G (p. H276R) alteration. This mistake was corrected by back-mutating Arg276 to His. CPA1 mutants were created by PCR mutagenesis and ligated into the pcDNA3.1(-) expression plasmid. Recombinant adenovirus carrying wild-type CPA1 or the p.N256K mutant was generated by Viraquest (North Liberty, Iowa). Details regarding the construction of the CPA1 splice-site and duplication mutant expression plasmids are provided in the Supplementary Note . HEK 293T cells were cultured in 6-well tissue culture plates (1.5×10 6 cells per well) in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad CA) supplemented with 10% fetal bovine serum, 4 mM glutamine and 1% penicillin/streptomycin at 37°C. Transfections were carried out at 90% confluence, using 10 μL Lipofectamine 2000 (Invitrogen) and 4 μg expression plasmid in 2 mL DMEM final volume. After overnight incubation, cells were washed and the transfection media was replaced with 2 mL OPTI-MEM I Reduced Serum Medium (Invitrogen). The conditioned OPTI-MEM media were harvested after 48 h incubation. AR42J rat pancreatic acinar cells (American Type Culture Collection #CRL-1492) were maintained in DMEM supplemented with 20% fetal bovine serum, 4 mM glutamine and 1% penicillin/streptomycin at 37°C. Prior to transfection, cells were plated in 6-well plates (10 6 cells per well) and were grown in the presence of 100 nM concentration of dexamethasone for 48 h to induce differentiation. Infections with adenovirus were performed using 4×10 7 plaque forming units (pfu) per mL final adenovirus concentrations in a total volume of 1 mL OPTI-MEM in the presence of dexamethasone (100 nM final concentration). Enzymatic activity of CPA1 was determined after activation with trypsin and chymotrypsin C (CTRC) using the N-[4-methoxyphenylazoformyl]-L-phenylalanine substrate 17 , with minor modifications of our previously published conditions 12 . The CPA1 activity measured in the conditioned medium of transfected cells is referred to as “apparent activity” and reflects the combined effects of the variants on secreted proCPA1 levels, proteolytic degradation during activation and catalytic activity of the activated CPA1. To activate proCPA1, an aliquot (20 μL) of conditioned medium was supplemented with 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl 2 , 0.05% Tween 20, 100 nM human cationic trypsin and 50 nM human CTRC (final concentrations in 40 μL final volume) and was incubated at 37 °C for 60 min. CPA1 activity was then measured by adding 50 μL assay buffer (0.1 M Tris-HCl (pH 8.0), 1 mM CaCl 2 , 0.05% Tween 20) and 10 μL substrate (60 μM final concentration) to the activation mix. The decrease in absorbance was followed at 350 nm for 2 min. Rates of substrate cleavage were calculated from fits to the initial linear portion of the curves and were expressed as percent of the wild-type rate, which was set to 100%. The wild-type activity corresponded to 116 ± 34 mOD·min -1 (average ± S.D.), which equals to 262 ± 77 nM·s -1 substrate cleavage rate. Secreted proCPA1 protein levels in the conditioned medium were determined by SDS-PAGE and densitometry. An aliquot (200 μL) of the medium was precipitated with trichloroacetic acid (10% final concentration), the precipitate was recovered by centrifugation, dissolved in 20 μL Laemmli sample buffer containing 100 mM DTT (final concentration), and heat-denatured at 95 °C for 5 min. Electrophoretic separation was performed on 15% SDS-PAGE mini gels in standard Tris-glycine buffer and gels were stained with Brilliant Blue R-250. Quantitation of bands was carried out with the GelDocXR+ gel documentation system and Image Lab 3.0 software (Bio-Rad, Hercules, CA). To study ER stress, we generated recombinant adenovirus carrying either wild-type proCPA1 or the p.Asn256Lys mutant, infected AR42J rat pancreatic acinar cells (#CRL-1492, American Type Culture Collection [ATCC], Manassas, VA) and measured ER stress markers as described below. Total RNA was extracted from AR42J cell lysates using RNeasy mini kit (Qiagen, Valencia, CA). RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). X-box binding protein 1 (XBP1) splicing was studied by PCR using a primer set that flanked the spliced region and amplified both spliced and unspliced forms ( Supplementary Table 3 ). PCR was carried out using the Taq DNA Polymerase kit (Qiagen) with the following conditions: 10 min initial denaturation at 95°C followed by 35 cycles of 30 sec denaturation at 95°C, 30 sec annealing at 52°C, 30 sec extension at 72°C and a final extension at 72°C for 5 min. The PCR products were resolved on 2% agarose gels and stained with ethidium bromide. Quantification of mRNA expression was performed by real time PCR (7500 Real Time PCR System, Applied Biosystems). XBP1 expression was measured with SYBR Green (PCR Master Mix, Applied Biosystems) using different primer sets for the spliced, unspliced and total mRNA ( Supplementary Table 3 ). Levels of immunoglobulin-binding protein (BiP) and calreticulin mRNA were determined using TaqMan primers (rat BiP, Rn00565250_m1; rat calreticulin, Rn00574451_m1) with TaqMan Universal PCR Mastermix (Applied Biosystems, Carslbad, CA). Real time PCR conditions were as follows: 2 min equilibration at 50°C, 10 min denaturation and enzyme activation at 95°C followed by 40 two-step cycles of 15 sec at 95°C and 60 sec at 60°C. Gene expression was quantitated using the comparative C T method (ΔΔC T method). Threshold cycle (C T ) values were determined using the 7500 System Sequence Detection Software 1.3. Expression levels of target genes were first normalized to the GAPDH internal control gene (ΔC T ) and then to expression levels measured in cells infected with empty adenovirus (ΔΔC T ). Results were expressed as fold changes calculated with the formula 2 -ΔΔCT . The significance of the differences between mutation frequencies in affected individuals and controls were tested by two-tailed Fisher’s Exact Test. Additional odds ratios were calculated using SAS/STAT software (v 9.1) and GraphPad Prism (v 4.03).