Methodology for Genetic Mutations in CP: Mutations in 3 Genes I

Methods

Methodology for Genetic mutations in CP: Mutations in 3 genes i.e. cationic trypsinogen (PRSS1), CFTR and SPINK1 genes were tested. The controls were healthy age and sex matched persons who had no personal or family history of any pancreatic disease or diabetes.

Genetic Studies: Venous blood samples were collected from patients and controls. DNA was extracted from the peripheral blood of patients by phenol chloroform method. The extracted DNAs were subjected to mutational screening tests for the following gene mutations – (i) cationic trypsinogengenemutation (ii) CFTR genemutation (iii) SPINK1 genemutation.

Cationic trypsinogen genemutation (R122H and N29I mutations):

R122H Mutation: A region (911bp) in exon 3 was amplified by PCR and products were restricted with Afl III (New England Biolabs, Germany) as described earlier (1). G to A mutation at residue 122 of cationic trypsinogen genecreates a restriction site, which is recognized by Afl III. In presence of R122H mutation, 3 bands corresponding to 911bp, 565bp and 346bp are seen whereas only one band (911bp) is seen with the wild type.

N29I Mutation: A region (1018bp) in exon 2 of cationic trypsinogen genewas amplified using allele specific PCR as described by Teich et al (2). In presence of N29I mutation, two bands of 1018bp and 476bp are seen, whereas only one band of 1018bp is seen with the wild type.

CFTR gene mutation: Two common CFTR genemutations, F508del and 3849+10 Kb C>T, and common variant of poly (T) sequence in intron 8 of CFTR genewere analyzed in all the patients. Furthermore, in order to detect other unknown mutations in the CFTR gene, we studied 19 of the 27 exons of the CFTR gene because others and we have found most mutations in these 19 exons (3, 4). Screening for mutations in the CFTR gene was done by single strand conformational polymorphism (SSCP) and heteroduplex analysis (5). If SSCP analysis suggested any band shift, those samples were sequenced for confirming the sequence change (Macrogen Inc, Korea).

Common variant of poly (T) sequence in intron 8: Allele-specific PCR was performed as described by Friedman et al (6) and the PCR products were analyzed on ethidium stained 3% agarose gel, to distinguish the 5T, 7T and 9T alleles.

Intron 19 ( 3849+10 KbC>T) mutation: A region (437bp) in intron 19 was amplified by PCR and the products were restricted with Hph I. Normally, there is one restriction site for this enzyme in the amplified region and the mutation creates an additional site for restriction. Homozygotes give 222, 127 and 88bp products whereas heterozygotes give 222, 127, 88 as well as 349bp products on restriction. Samples negative for this mutation give only 349 and 88bp products (7).

SPINK1 geneN34S mutation: A 595bp region was amplified by PCR and the products were restricted for 2-4 hours at 65C with TaaI (MBI Fermentas, Germany). For wild type allele, complete digestion with TaaI was expected to produce 4 fragments of 20, 137, 139 and 299bp. On agarose gel electrophoresis, the 20bp fragment is hardly visible, whereas 137 and 139bp fragments appear as a single band with increased intensity. When the mutated allele is present, the 299bp fragment is cut, resulting in two bands, sized 109 and 190bp (8).

Results

Supplementary Table 1: Clinicalandgenotypicdetailsofpatientswithidiopathicchronic pancreatitispositiveforeitherSPINK1and/orCFTRgenenucleotidechange

Sr.
No. / Sex / Age at
onset / Aetiology / Diabetes / Pain / Calcification / Pancreatic
ductdilatation / SPINK1 / CFTRgenotype
1 / M / 40 / ICP / + / + / I148T
2 / M / 12 / ICP / - / + / + / N34S/- / T854T
3 / M / 7 / ICP / - / + / T760T
P1290P
4 / M / 19 / ICP / - / + / N34S/-
5 / M / 17 / ICP / - / + / + / + / N34S/N34S / M470V
6 / M / 6 / ICP / - / + / N34S/-
7 / M / 16 / ICP / + / + / N34S/-
8 / M / 8 / ICP / - / + / + / + / N34S/ N34S / M470V
1525-61AG
9 / F / 34 / ICP / + / + / + / + / P1290P
T854T M470V
1525-61AG
10 / M / 15 / ICP / - / + / + / + / N34S/- / M470V
1525-61AG
11 / M / 18 / ICP / + / + / N34S/- / 3601-65CA
12 / M / 28 / ICP / - / N34S/- / T854T
M470V
1525-61AG
1716+77AG
13 / F / 29 / ICP / + / + / + / + / 3601-20TC
3041-92GA T854T
1525-61AG
14 / M / 24 / ICP / - / + / N34S/- / F508del/-
M470V
15 / M / 20 / ICP / - / + / + / + / N34S/ N34S / T854T
1525-61AG
16 / M / 8 / ICP / - / + / + / + / N34S/- / T854T, P1290P
-----
17 / M / 16 / ICP / - / - / + / + / 1716+77A>G
18 / M / 13 / ICP / - / + / N34S/- / 1525-61AG
M470V
19 / F / 20 / ICP / - / + / + / + / N34S/- / 3041-92GA
1525-61AG
20 / F / 17 / ICP / - / + / + / + / N34S/- / 1716+77AG
21 / M / 29 / ICP / - / - / + / + / N34S/- / M470V/M470V
22 / M / 24 / ICP / + / + / + / + / P1290P
M470V
1525-61AG
23 / M / 34 / ICP / - / + / + / + / M470V
24 / M / 35 / ICP / - / + / + / N34S/ N34S / 1716+77AG
3041-92GA
25 / F / 24 / ICP / - / + / + / + / N34S/- / M470V
P1290P
26 / M / 39 / ICP / + / + / + / + / M470V
27 / M / 16 / ICP / - / + / + / + / N34S/- / M470V
28 / M / 20 / ICP / - / + / M470V
1525-61AG
29 / M / 21 / ICP / - / + / 3041-92GA
1716+77AG
30 / M / 15 / ICP / - / + / + / + / 1716+77A>G
31 / M / 40 / ICP / - / + / + / + / T854T/T854T
P1290P
32 / M / 33 / ICP / - / + / + / + / 1716+77A>G
1525-61AG
33 / M / 15 / ICP / - / + / + / + / N34S/- / M470V
1525-61AG
34 / M / 35 / ICP / + / - / + / + / M470V
35 / M / 17 / ICP / - / + / + / + / 3601-65CA
1525-61AG M470V/M470V
36 / F / 30 / ICP / + / + / + / + / N34S/- / 1716+77AG
M470V
1525-61AG
37 / F / 20 / ICP / + / + / + / - / S1235R
T854T
38 / M / 17 / ICP / - / + / - / + / 3041-92GA
1525-61AG
39 / M / 33 / ICP / - / - / + / + / 3601-20TC
P1290P
3601-65CA T854T/T854T
3850-41CG M470V/M470V
40 / M / 29 / ICP / - / + / N34S/-
41 / M / 41 / ICP / + / + / N34S/- / 1525-1GA
M470V
42 / F / 18 / ICP / - / + / + / + / F508del/-
M470V/M470V
43 / M / 10 / ICP / - / + / + / + / N34S/ N34S / 1812-99CT
44 / M / 30 / ICP / - / V456A
1524+4GT
45 / M / 10 / ICP / - / + / + / + / N34S/- / 3041-92GA
M470V
1525-61AG
46 / M / 32 / ICP / - / + / + / + / N34S/- / M470V
47 / M / 31 / ICP / - / + / T854T
M470V
1525-61AG
48 / M / 18 / ICP / - / + / + / + / F508del/-
49 / M / 28 / ICP / - / + / + / 3120+35A>T
50 / M / 17 / ICP / - / + / N34S/- / 3041-92GA
M470V
1525-61AG
51 / F / 7 / ICP / + / + / + / + / N34S/-
52 / M / 15 / ICP / - / + / N34S/-
53 / M / 19 / ICP / - / + / + / + / M470V
1525-61AG
54 / M / 11 / ICP / - / + / + / + / 3041-71GC
55 / F / 31 / ICP / + / + / + / + / N34S/-
56 / M / 17 / ICP / - / + / + / + / S158N
M470V
1525-61AG
57 / M / 13 / ICP / - / + / N34S/-
58 / M / 36 / ICP / - / + / + / + / N34S/-
59 / M / 10 / ICP / - / + / N34S/ N34S / 3041-92GA
60 / M / 14 / ICP / - / + / + / + / N34S/-
61 / M / 19 / ICP / - / + / - / - / N34S/-
62 / F / 23 / ICP / - / + / N34S/-
63 / M / 34 / ICP / - / + / - / - / N34S/N34S
64 / M / 8 / ICP / - / + / N34S/-
65 / M / 25 / ICP / - / + / + / + / N34S/-
66 / F / 26 / ICP / - / + / + / + / N34S/-
67 / F / 39 / ICP / + / + / N34S/ N34S
68 / M / 28 / ICP / + / + / + / + / N34S/-
69 / M / 10 / ICP / - / N34S/-
70 / F / 14 / ICP / - / + / + / + / N34S/-
71 / F / 35 / ICP / + / + / N34S/-
72 / F / 15 / ICP / - / + / - / - / N34S/-
73 / M / 26 / ICP / - / + / - / - / N34S/-

ICP:idiopathicchronicpancreatitis

Supplementary Table 2:ListofhealthycontrolspositiveforSPINK1and/orCFTRvariations:

SrNo. / Sex / SPINK1 / CFTR
1 / M / M470V
1525-61AG
2 / F / M470V
1525-61AG
3 / F / N34S/-
4 / F / M470V
1525-61AG
5 / F / M470V
1525-61AG
6 / M / M470V
7 / F / M470V
1525-61AG
8 / F / M470V
1525-61AG
9 / M / M470V
10 / F / M470V
1525-61AG
11 / M / M470V
1525-61AG
12 / F / N34S/-
13 / F / N34S/-
14 / M / N34S/-

Supplementary Table 3:List of total CFTR gene variants found in this study

Sr. No / Exon/Intron / Nt. change / A.A. change / No. of patients / No. of controls / Type of change and consequence / References
1 / Exon 4 / 575 T>C / I148T / 1 / 0 / M (Pathogenic) / Bozon et al., 199499
2 / Exon 4 / 605 G>A / S158N / 1 / 0 / M (Non pathogenic?) / Present study
3 / Exon 9 / 1499 T>C / V456A / 1 / 0 / ? S.V (? Non-pathogenic; polyphen software - possibly damaging) / Girodon et al., 199910
4 / Intron 9 / 1524+4 G>T / - / 1 / 0 / mRNA splice mutant (Pathogenic) / Present study
5 / Intron 9 / 1525-61 A>G / - / 27 / 8 / S.V (Non-pathogenic) / Ivaschenko et al., 199311
6 / Intron 9 / 1525-1 G>A / - / 1 / 0 / mRNA splice mutant (pathogenic) / Dork et al., 199312
7 / Exon 10 / 1653_1655delCTT / F508del / 3 / 0 / M (pathogenic) / Mathew et al., 198913
8 / Exon 10 / 1540 A/G / M470V / 31 / 10 / S.V (Non-pathogenic) / Kerem et al., 198914
9 / Intron 10 / 1716+77 A>G / - / 7 / 0 / S.V (Non-pathogenic) / Wallace et al., 199610
10 / Intron 11 / 1812-99 C>T / - / 1 / 0 / Unknown / Present study
11 / Exon 13B / 2412 G>A / T760T / 1 / 0 / ? S.V (? Non-pathogenic) / Present study
12 / Exon 14A / 2694 T/C / T854T / 3 / 0 / S.V (Non-pathogenic) / Martinotti et al., 200010
13 / Exon 14 A / 2694 T/G / T854T / 9 / 0 / S.V (Non-pathogenic) / Zielenski et al., 199115
14 / Intron 15 / 3041-92 G>A / - / 8 / 0 / S.V (Non-pathogenic) / Dork et al., 199416
15 / Intron 15 / 3041-71 G>C / - / 1 / 0 / S.V (Non-pathogenic) / Ferec et al., 199117
16 / Intron 16 / 3120+35 A>T / - / 1 / 0 / Unknown / Present study
17 / Intron 18 / 3601-65 C>A / - / 3 / 0 / S.V (Non-pathogenic) / Dork et al., 199118
18 / Intron 18 / 3601-20 T>C / - / 2 / 0 / mRNA splice mutant (Pathogenic) / Kabra et al., 20003
19 / Exon 19 / 3837 T>G / S1235R / 1 / 0 / M (associated with CBAVD) / Cuppens et al., 199319
20 / Intron 19 / 3850-41 C>G / - / 1 / 0 / Unknown / Present study
21 / Exon 20 / 4002 A>G / P1290P / 7 / 0 / S.V (Non-pathogenic) / Ferec et al., 199117, Ivaschenko et al., 199311

Abbreviations: Nt.: Nucleotide, M: Mutation, A.A.: Amino acid, S.V: Sequence variation, Unk: Unknown phenotypic consequence;

Supplementary Table 4: Clinicalandgenotypicdetailsofpatientswithalcoholicchronic pancreatitispositiveforeitherSPINK1and/orCFTRgenenucleotidechange

Sr. No. / Sex / Ageat onset / Aetiology / Diabetes / Pain / Calcification / Pancreatic
duct dilatation / SPINK1 / CFTRgenotype
1 / M / 33 / ACP / + / + / + / + / 1525-61AG
2 / M / 42 / ACP / - / + / - / - / P1290P
3 / M / 27 / ACP / - / + / - / + / P1290P
3041-71GC
1525-61AG
4 / M / 37 / ACP / + / + / + / + / 1716+77AG
6 / M / 26 / ACP / - / + / N34S/- / T854T
M470V
1525-61AG
5 / M / 35 / ACP / - / + / - / + / T854T
6 / M / 40 / ACP / - / + / + / + / N34S/-
7 / M / 29 / ACP / - / + / + / + / N34S/-
8 / M / 48 / ACP / + / + / - / - / N34S/-
9 / M / 36 / ACP / - / + / 1716+77AG
M470V
1525-61AG
10 / M / 40 / ACP / - / + / + / + / N34S/-
11 / M / 50 / ACP / - / + / - / - / N34S/-
12 / M / 33 / ACP / - / + / N34S/-

ACP:alcoholicchronic pancreatitis

Supplementary Table 5: Clinicalandgenotypicdetailsofpatients withhereditaryor familial chronicpancreatitispositivefor eitherSPINK1and/orCFTRgenenucleotide change

Sr.
No. / Sex / Ageat
onset / Aetiology / Diabetes / Pain / Calcification / Pancreatic
ductdilatation / SPINK1 / CFTRgenotype / PRSS!mutation
1 / M / 32 / HP / - / R122H+
2 / F / 35 / HP / - / + / + / + / N34S/- / 1525-61A>G / R122H+
3 / M / 8 / HP / - / + / + / + / -
4 / M / 7 / HP / - / + / + / + / -
5 / M / 27 / HP / + / + / + / + / M470V
1525-61AG / -
6 / F / 10 / HP / - / + / + / + / N34S/- / M470V
1525-61AG / -
7 / M / 51 / HP / + / + / + / + / -
8 / M / 48 / FCP / + / + / + / + / -
9 / M / 25 / FCP / - / + / + / + / --
10 / M / 19 / FCP / - / + / + / + / -
11 / M / 37 / FCP / - / + / + / + / N34S/- / -
12 / M / 22 / FCP / - / + / + / + / -
13 / M / 22 / FCP / - / + / + / + / N34S/- / --
14 / F / 29 / FCP / - / + / + / + / -
15 / M / 45 / FCP / - / + / + / + / N34S/- / -

HP:hereditarypancreatitis;FCP:familialchronicpancreatitis

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