2
Supplementary Methods
Resequencing the SPINK1 promoter
French patients
The region spanning -139 to +92 of the SPINK1 genomic sequence (NG_008356.1 was used as the SPINK1 reference sequence; the nucleotide immediately 5' to the A of the ATG translational initiation codon being designated -1.) was analyzed as previously described.7 A ~1.5 kb SPINK1 promoter region spanning -1607 to -44 was sequenced bi-directionally in 213 French patients with idiopathic chronic pancreatitis (ICP) and 550 French controls. The forward and reverse primers used to amplify the SPINK1 promoter were 5’-TCAGCCTCAGAACCTTTGCGCTTA-3’ and 5’-ACTTACCACGTCTCTTCAGAAGC-3’, respectively. PCR was performed using the HotStarTaq™ DNA polymerase (Qiagen, Courtaboeuf, France) with 50 ng genomic DNA in a 20 µL reaction mixture. The PCR program comprised an initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 66°C for 30 s and extension at 72°C for 90 s, and a final extension at 72°C for 10 min. PCR products were purified using ExoSAP-IT (GE Healthcare, Orsay, France) and sequenced using the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Kit v.1.1 (PE Applied Biosystems, Foster City, CA, USA).
German patients
The region spanning -357 to +118 of the SPINK1 genomic sequence was sequenced bi-directionally in 418 German ICP patients and 379 German control subjects using the oligonucleotides 5’-TTTGAGTTCATCTTACAGGTGAG-3’ and 5’-GTGCTTCACAAAGCAACAGGTC-3’ as forward and reverse primers. PCR was performed using 0.5 U AmpliTaq Gold polymerase (Perkin Elmer, Braunschweig, Germany), 400 µM dNTPs, and 0.1 µM each primer in a total volume of 25 µL. Cycle conditions were an initial denaturation for 12 min at 95°C, followed by 40 cycles of 15 s denaturation at 95°C, 30 s annealing at 56°C, 40 s primer extension at 72°C, followed by a final extension for 2 min at 72°C in an automated thermal cycler (Biometra, Göttingen, Germany). The PCR products were incubated with shrimp alkaline phosphatase (USB, Freiburg, Germany) and exonuclease I (USB, Freiburg, Germany) and cycle sequencing was performed using BigDye terminator mix (Applied Biosystems, Darmstadt, Germany). The reaction products were then purified by ethanol precipitation and loaded onto an ABI 3730 Sequencer (Applied Biosystems, Darmstadt, Germany).
Indian patients
The SPINK1 promoter region spanning -644 to -1 was sequenced in both directions in a total of 439 chronic pancreatitis patients, of whom 292 were tropical calcific pancreatitis (TCP) patients and 147 were ICP patients; 316 control individuals of Indian origin were also sequenced. The SPINK1 promoter region was amplified using forward primer 5’-CAAAAGTGGCTCCAACAAG-3’ and reverse primer 5’-GGCTGAAGTTCTGCGTC-3’, respectively. PCR was performed using Thermostable Taq DNA Polymerase (Qiagen) with 50 ng genomic DNA in a 20 µL reaction mix following the manufacturer’s instructions. The PCR conditions comprised an initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 63°C for 30 s and extension at 72°C for 45 s, with a final extension at 72°C for 5 min. PCR products were purified using Millipore MultiScreen® PCRµ96 Filter Plates and sequenced using the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Kit v.1.1 (PE Applied Biosystems, Foster City, CA, USA).
Only three variants [-253T>C, -215G>T and -142T>C] were identified, of which the latter two were identified only in three and two patients respectively. The -253T>C variant was identified in both patients and controls but exhibited a nominal association with TCP [P = 0.0413; OR (TC vs TT) = 0.65 (95% CI, 0.42-1.00)] and a strong association with ICP [P = 0.0133; OR (TC vs TT) = 0.50 (95% CI, 0.28-0.90)]. Given the similar genotype distribution in TCP and ICP, we combined the two datasets.
Quantitative RT-PCR analysis of SPINK1 expression in the COLO-357 cells
Total RNA was extracted from COLO-357 cells with Trizol Reagent (Invitrogen, Cergy Pontoise, France). First-strand cDNA synthesis was performed using oligo(dT) primers (Qiagen, Courtaboeuf, France) and SuperScript® II reverse transcriptase (Invitrogen, Cergy Pontoise, France) in a 20 µL reaction mixture containing 500 ng total RNA. At the end of the RT reaction, RNaseH (Invitrogen, Cergy Pontoise, France) was added to hydrolyze the RNA template.
Primer pairs, targeting either the SPINK1 or the chymotrypsin C (CTRC) cDNA (Table S1), were designed to perform real-time PCR. Real-time PCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen, Courtaboeuf, France) in a 20 µL reaction mixture containing 0.3 µM each primer and 2 µL each cDNA. The PCR program comprised an initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s, and a final extension at 72°C for 10 min. Specific amplification was confirmed by melting curve analysis.
Real-time PCR reactions were performed in triplicate from several cDNA dilutions (no dilution, 1/2, 1/5, 1/10). For each cDNA dilution, means of the cycle threshold values for the SPINK1 and CTRC genes were compared, with the results being shown in Table S2.
Table S1 Primers used for RT-PCR analysis
SPINK1 / 2-3 / Forward: tgggaagagaggccaaatgt / 137
3-4 / Reverse: gatagaagtctggcgtttccga
CTRC / 4-5 / Forward: ctgcatcagcaacacccg / 146
5-6 / Reverse: caatatcattgcgcaacaggag
Table S2 Expression of SPINK1 and CTRC mRNAs in the COLO-357 cells
Dilution / Cycle threshold (SPINK1) / Mean / Cycle threshold (CTRC) / MeanNo dilution / 23.86 / 23.71
No dilution / 23.60 / 23.69 / 23.68 / 23.66
No dilution / 23.62 / 23.58
1/2 / 24.35 / 24.52
1/2 / 24.33 / 24.37 / 24.57 / 24.56
1/2 / 24.43 / 24.60
1/5 / 24.96 / 25.55
1/5 / 24.67 / 24.92 / 25.26 / 25.28
1/5 / 25.12 / 25.03
1/10 / 25.58 / 25.44
1/10 / 25.82 / 25.64 / 25.95 / 25.83
1/10 / 26.06 / 26.10
a -170G/-164G probe or SpC
5’-AGAACCTGGAGGCCAGGCTATGACAC-3’
b
Competitor - - G/G G/G G/G G/G Ir
Labeled G/G probe + + + + + + +
Nuclear extracts - + + + + + +
Supplementary Figure 1 Confirmation of the functional neutrality of the –170G>A and –164G>C variants by EMSA. (a) Sequence of the labeled probe or specific competitor (SpC) probe. (b) EMSAs performed with labeled probes incubated with COLO 357 nuclear extracts in the presence or absence of SpC or irrelevant (Ir) competitor probes. No specific nucleic acid-protein complexes were evident.
Human ------CAGAAT-CTTTGCCTTGCATGTTTCAGGCCCA 31
Chimpanzee ------CAGAAT-CTTTGCCTTGCATGTTTCAGGCCCA 31
Rhesus monkey ------CAGAAT-GTTTGCCTTGCATGTTTCAGGCCCA 31
Cow ------TTGCAC-ATTTACCTAGGAAGGCCTAGGGCAT 31
Dog ------TCTGACAGAAT-CTTGGCCTTGAATATTCCAGGCCTG 36
Rat AAACCTGAGAGGATGTTGGGAAT-CAGTTAACTTGCTTTTCCTGGCAGAATTTAGGTCCA 59
Mouse ------GGATGTCAGGAATGCAGCTAATTTGCTTTTCCTGGAAGAATTTGGGTCCA 50
** *** * * ** *
-215G>A/T -170G>A -164G>C
Human CCTGGCTCCTTTCACCTTTCTTACACAGGTGACATTC-CCAGAACCTGGAGGCCAGGCTA 90
Chimpanzee CCTGGCTCCTTTCACCTTTCTTACACAGGTGACATTC-CCAGAACCTGGAGGCCAGGCTA 90
Rhesus monkey CCTGGCTCCTTTCACCTTTCTTACGCAGGTGACATTC-CCAGAACCTGGAGGCCAGGCTA 90
Cow TCCT-CTCCTA-CACCTTTCTTACACTGGTGGTGTGC-CCCAAACCTGGAGGACAGGCTA 88
Dog CACTGTTTCTT-TCACTTTCTTATGCAGATA-CATTC-CCGAAACATGG-GGCCAGGCTA 92
Rat ATCT-TAACTGCCCTTTTTCCTACCTAGGTAACATTTTCCCCAGCCTGGAGGCCA----- 113
Mouse AAGT-TTTCTGCCCTTCTTCCTACCTAGGTTACATTTTCCCCAGCCTGGAGGCCA----- 104
** *** ** * * * ** * * *** ** **
-147A>G -142T>C
Human TGACACAGAGTCAATCAATAACCAGGGAGATCTGTGATATAGCCCAGTAGGTGGGGCCTT 150
Chimpanzee CGACACAGAGTCAATCAATAACCAGGGAGATCTGTGATATAGCCCAGTAGGTGGGGCCTT 150
Rhesus monkey CAACACAGAGTCAATCAATAACCAGGGAGATCTATGATATAGCCCAGTAGGTGGGGCCTT 150
Cow TACTACGGAGTCAATCAATAACCAAGGAGATCTGTGACGCAGAGCAGGAGGTGGAGACTG 148
Dog TGCTACAGAGTCAATCAGTCACCAA--AGATCCATGA--CAAGGCAGGAGGTGGAGCCTG 148
Rat ----ACAAAGTCAATCAATAACCAGAGATACCTATTATAGGGCACAGTGGGT------161
Mouse ----CACAAGTCAATCAATAACCAAAGATACCTATTATAGGGCACAGTGGGTGGAGCCTG 160
********* * **** * * * * * *** ***
-81C>T -53C>T
Human GCTGCC---ATCTGCCATATGACCCTTCCAGTCCCAGGCTTCTGAAGAGACGTGGTAAGT 207
Chimpanzee GCTGCC---ATCTGCCATATGACCCTTCCAGTCCCAGGCTTCTGAAGAGACGTGGTAAGT 207
Rhesus monkey GCTGTC---GTCTGCCATATGACCCTTCCAGTCCCAGGCTTCTGAAGAGACGTGTTAAGT 207
Cow GAAGCCCACGCACACTATATGACCCTTCCAGTCCCAGGGTTCCAGAGCGAAGTG-TAAGT 207
Dog GGTGGCCACCCTGACTATATAACTCTTCCAGTCTCAGGGTTCTGAAGAGAAGTGGTTAGT 208
Rat -ATTACCA-TCTGCCTATATGAC-----CACTCCTCAGTTTCTGAAGAGAAGCA-----C 209
Mouse TATTACCA-TCTGCCTATATGAC-----CACTCCTCAGTTTCTGAAGAGAAGCA-----C 209
* * **** ** ** ** * *** ** ** *
-41G>A -22C>T -7T>G
Human GCGGTGCAGTTTTCAACTGACCTCTGGACGCAGAA-CTTCAGCCATGAAGGTAA 260
Chimpanzee GCGGTGCAGTTTTCAACTGACCTCTGGACGCAGAA-CTTCAGCCATGAAGGTAA 260
Cow TCGGTGCAGTTTTCGACTGAGCTCTAGAACCAGCA-TTTCAGCCATGAAGGTGG 260
Dog TCAAT--AGTTTTCAACTGAGCTCTGGAAGCAGAACTTTCAGCTATGAAGGTAA 260
Rhesus monkey GCAGTGCAGTTTTCAACTGACCTCTGGACGCAGAA-TTTCAGCCATGAAGGTAA 260
Rat CCTGCACAGTTCTT--CTGAGTTTTGGACCTAGGT-CTACAACCATGAAGGTAG 260
Mouse CCTGTATAGTTCTT--CTGGCTTTTGCACCCAGAT-CTTCGACAATGAAGGTGG 260
* **** * *** * * * ** * * * ********
Supplementary Figure 2 Evolutionary conservation of the HNF1-binding site (shaded in grey) across mammalian species.