Supplemental table 1: Primers used in the sequencing of the NOD2/CARD15 gene

Exon / Annealing temperature(C) / Amplicon size
(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 frequency / rs number / 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 6) / 13 / rs2066843 / Exon 4(2) C/T
8 / Arg587Arg (SNP 7) / 12 / rs1861759 / Exon 4(2) T/G
9 / 1 / ** / Exon 4(2) C/T
10 / Arg702Trp (SNP 8) / 5 / rs2066844 / Exon 4 (3)C/T
11 / 2 / ** / Exon 4 end + 10 bases A/C
12 / Met863Val / 2 / ** / Exon 6 A/G
13 / Gly908Arg (SNP12) / 2 / rs2066845 / Exon 8 G/C
14 / Val955Ile / 4 / rs5743291 / Exon 9 G/A
15 / 10 / ** / Exon 10 (post exonic +1) T/A
16 / Leu1007fsincC (SNP13) / 1 / rs2066847 / Exon 11 c+/-
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).

Supplemental table 3: Demographics of patients involved in the study

Cohort 1 / Cohort 2
Sex (M/F) / 175/145 / 137/206
Median Age at diagnosis(y) / 11.08
IQR (8.55-12.92) / 28.25
IQR(21.00-42.00)
Current Smoker / 7/315 (2.2%) / 94/333 (28.2%)
Family history of IBD / 107/315 (34.0%) / 66/343 (19.2%)
Caucasian (%) / 313 (97.8%) / 329/331 (99.4%)

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 GeneralHospital, 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.

Supplemental table 4: Case-control analysis in the different disease groups

Disease group / 863M/V carriage / p value cf. HC / 955V/I variant carriage / p value cf. 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 genotypede.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 case-control analysis.

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