Supplemental table 1: Primers used in the sequencing of the NOD2/CARD15 gene
Exon
Annealing
Amplicon size
temperature(C) (base pairs)
1.
TTGTGCCAGAATTGCTTG
AAGGGTAGAATAAGCTCTGGG
60
503
2.
TCTGAGGCTAGAACCATGG
TGAGGACAAATCAGTCTTGG
57
622
3.
GACCCTTTATTCTGGATGGAAG
CGGTACAGATAATGAGAGTTTGG
60
486
4(1)
TGCTCTCCTATCCCTTCAG
TCAGAGAAGCCCTTGAGG
57
784
4(2)
GAAGTACATCCGCACCGAG
GAAGGCTGCTGTGATCTG
58
690
4(3)
CCAGGCAACTCACCAATG
AAGGGAAGGGATCTGGG
60
667
5,6
CACTTCAGGGATGAATGAAAG
GCATTAGAGAACCCCTGC
58
589
7.
GTCTTCAATGCTTTCTTCCTG
TCTTGTCAAATGGACTCCAG
57
834
8.
AAGTCTGTAATGTAAAGCCAC
CCCAGCTCCTCCCTCTTC
60
281
9.
GAGCACCGCAATCAATTAG
CACTCAATCATCCACCTTTG
60
407
10.
TTCTTTATCCATGAGTTTGGG
CTTTATTGGTTACCTTCACTTC
56
401
11.
GAAGAGAGACGGTTACATTTCAC
CATTCTTCAACCACATCCC
60
522
12(1)
TAAAAACAGCCCTGACTTCC
AATTGTCTTGGGGAACAAAC
60
883
12(2)
ATTCAGAATATTAGTGACCTCAGC
ATGTTGGTCAGGTTGGTC
58
866
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Supplemental table 2: SNPs identified in sequencing of the NOD2/CARD15 gene
SNP SNP description
Patient
rs number
frequency
Exon number / base
change
1
upstream
1
rs5743264
Exon 1 promoter T/C
2
5 UTR(-59)
16
rs5743266
Exon 1 G/A
3
intronic
3
rs2076753
Exon 2 promoter G/T*
4
Ser178Ser
13
rs2067085
Exon 2 C/G
5
Ala211Ala
1
rs5743269
Exon 3 C/T
6
Pro268Ser (SNP 5)
14
rs2066842
Exon 4(1) C/T
7
Arg459Arg (SNP
13
rs2066843
Exon 4(2) C/T
12
rs1861759
Exon 4(2) T/G
1
**
Exon 4(2) C/T
rs2066844
Exon 4 (3)C/T
**
Exon 4 end + 10 bases
6)
8
Arg587Arg (SNP
7)
9
10
Arg702Trp (SNP 8) 5
11
2
A/C
12
Met863Val
2
**
Exon 6 A/G
13
Gly908Arg
2
rs2066845
Exon 8 G/C
4
rs5743291
Exon 9 G/A
10
**
Exon 10 (post exonic
(SNP12)
14
Val955Ile
15
+1) T/A
16
Leu1007fsincC
1
rs2066847
Exon 11 c+/-
2
(SNP13)
17
3 UTR
12
rs3135499
Exon 12(1) A/C
18
3 UTR
12
rs3135500
Exon 12(2) G/A
Legend: * This is described in Lesage35 (and here) as an Exon 2 promoter mutation,
numbered from the ATG in the 2nd exon that was thought to be the start of the
NOD2/CARD15. It is actually in intron one/two. ** No rs number listed in Ensembl
DNA was extracted from whole blood using the salting out technique.48 Direct
sequencing was performed on the 7900 HT sequence detection system (Applied
Biosystems, Foster City, Ca, USA) by the Technical Services section of the MRC
Human Genetics Unit, Edinburgh. 24 Caucasian patients (<16 years at diagnosis) with
CD were studied with the number of patients variants were found in listed in the table.
DNA sequence was analysed using Sequencher v4.5 (Gene Codes Corporation, Ann
Arbor, MI, USA).
3
Supplemental table 3: Demographics of patients involved in the study
Cohort 1
Cohort 2
175/145
137/206
11.08
28.25
IQR (8.55-12.92)
IQR(21.00-42.00)
Current Smoker
7/315 (2.2%)
94/333 (28.2%)
Family history of
107/315 (34.0%)
66/343 (19.2%)
313 (97.8%)
329/331 (99.4%)
Sex (M/F)
Median Age at
diagnosis(y)
IBD
Caucasian (%)
Legend: Cohort 1 320 IBD patients <16 years at diagnosis; Cohort 2 343 CD patients
from adult cohort. Patients were recruited to the study from the three tertiary
paediatric gastroenterology clinics in Scotland and the Western General Hospital,
Edinburgh. A diagnosis of IBD was based on standard criteria.49 Patient data
collection and disease phenotyping was performed as previously described.19;20;22
Informed written consent was obtained from parents and patients prior to participation
in the study. Ethical approval was obtained from the local research ethics committees
in all the participating hospitals.
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Supplemental table 4: Case-control analysis in the different disease groups
Disease
863M/V
p value cf.
955V/I variant
p value cf.
group
carriage
HC
carriage
HC
HC
1/267 (0.37%)
41/253 (16.2%)
CD children
2/207 (0.97%) 0.58
29/202 (14.4%)
0.29
CD adults
5/313 (1.6%)
0.22
36/305 (11.8%)
0.13
Both CD
7/520(1.35%)
0.27
65/507 (12.8%)
0.20
UC
0/80
-
12/78 (15.8%)
0.86
IC
0/30
-
12/29 (41.4%)
0.001
Legend: HC=healthy controls; CD=Crohn’s disease; UC=ulcerative colitis;
IC=indeterminate colitis. The numbers of carriers of the variant form of the SNP
analysed is compared to the number of patients successfully genotyped e.g. there were
273 healthy controls of which 267 were successfully genotyped for the 863M/V SNP.
TaqMan (Applied Biosystems, Foster City, CA, USA) was used to genotype 955V/I
(rs5743291) and 863M/V. Minitab version 13 (Release 13.20, Minitab Inc., State
College, PA, USA) statistical package was used to analyse genotype associations
using chi squared or Fishers exact test where appropriate. A total of 253 healthy
populations controls (101 healthy controls and 152 blood donors) were used for casecontrol analysis.
5