PSA, VØT and JS have affiliation to Statens Serum Institut. Statens Serum Institut is not a commercial company. It is a public enterprise under the Danish Ministry of Health (please see
Conceived and designed the experiments: SB PSA MF BAN JS UV VA. Performed the experiments: SB JB NP S. Roug JG SYT JBB S. Rashid BKR SA TBO HJH MKT VØT. Analyzed the data: SB PSA MF BAN JS UV VA. Contributed reagents/materials/analysis tools: SB JB NP S. Roug JG SYT JBB S. Rashid BKR SA TBO HJH MKT VØT MF. Wrote the paper: SB PSA UV VA. Drafting the article or revising it critically: SB PSA JB NP S. Roug JG SYT JBB S. Rashid BKR SA TBO HJH MKT VØT MF BAN JS UV VA. Final approval of the version to be published: SB PSA JB NP S. Roug JG SYT JBB S. Rashid BKR SA TBO HJH MKT VØT MF BAN JS UV VA.
The inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), result from the combined effects of susceptibility genes and environmental factors. Polymorphisms in genes regulating inflammation may explain part of the genetic heritage.
Using a candidate gene approach, 39 mainly functional single nucleotide polymorphisms (SNPs) in 26 genes regulating inflammation were assessed in a clinical homogeneous group of severely diseased patients consisting of 624 patients with CD, 411 patients with UC and 795 controls. The results were analysed using logistic regression.
Sixteen polymorphisms in 13 genes involved in regulation of inflammation were associated with risk of CD and/or UC (p≤0.05). The polymorphisms
The biological effects of the studied polymorphisms suggest that genetically determined high inflammatory response was associated with increased risk of CD. The many SNPs found in
Chronic inflammatory bowel diseases (IBDs), Crohn's disease (CD) and ulcerative colitis (UC), are complex diseases that result from the interaction of numerous genetic and environmental factors
Genetic association studies have identified innate immunity as a critical component in the development of IBD. Until now, more than 163 IBD susceptibility polymorphisms have been confirmed, most of which are associated with both CD and UC, by candidate and genome wide association studies (GWAS)
Functional polymorphisms in genes in the inflammatory pathways may explain some of the genetic heritage involved in IBD. The transcription factor NFκB is a central regulator of inflammation. NFκB can be activated by Toll like receptors (TLRs). TLRs recognize pathogen-associated molecular patterns (PAMPs) that are broadly shared by pathogens but distinguishable from host molecules such as bacterial or viral DNA, flagellin or lipopolysaccharide (LPS). The TLRs initiates a kinase cascade that ultimately activates the IKK-complex, which phosphorylates and degrades the NFκB inhibitor IκBα. NFκB is shuttled from the cytosol to the nucleus where it initiates expression of pro- and anti-inflammatory cytokines including TNF-α, IL-6 and IL-10
To identify susceptibility loci we assessed 39 mainly functional polymorphisms in genes involved in inflammation, particular in the NFκB pathway, in a homogeneous Danish cohort of 624 patients with severe CD, 411 patients with severe UC and 795 healthy controls. The candidate gene approach using functional polymorphisms allows interpretation of the underlying biological mechanisms based on increased or decreased gene expression or protein activity.
Functional polymorphisms in genes in the inflammatory pathways involved in regulation of the NFκB pathway (
A prior anti-TNF naïve Danish cohort of patients with IBD was established. In short, blood samples retrieved as part of the routine screening for latent
A candidate gene approach was used with focus on polymorphisms in the TNF-α and NFκB pathways. In addition, polymorphisms in genes which have been shown to be associated with CD and/or UC, polymorphisms in inflammatory cytokines and polymorphisms in
Functional polymorphisms in relevant genes were found by searching pubmed with “polymorphism AND gene-name AND (reporter gene OR luciferase OR ELISA OR enzyme-linked immunosorbent assay OR RT-PCR OR reverse transcriptase PCR OR EMSA OR electrophoretic mobility shift assay OR flow cytometry)”.
For patients with IBD the DNA was extracted from cryopreserved blood clots by using the Maxwell 16 Blood purification kit (Promega) according to the manufacturers' instructions with a median yield of 4.90 µg (range 0.8–25 µg) pr 300 µl total blood
Genotyping of
The 39 genotypes were replicated in 94 randomly selected samples and yielded >99% identical genotypes.
Logistic regression was used to compare genotype distributions among patients with CD, UC and IBD versus healthy controls (
Statistical analyses were performed using STATA version 11 (STATA Corp., Texas, USA).
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Committees of Central (M20100153) and Southern (S-20120113) Denmark and the Danish Data Protection Agency of Central (RM: J. 2010-41-4719) and Southern (RSD: 2008-58-035) Denmark. The Ethics Committees gave suspension for obtaining written informed consent.
Characteristics of the Danish patients with CD, UC and healthy controls are shown in
Crohns Disease (CD) | Ulcerative Colitis (UC) | Controls | |
(n = 624) | (n = 411) | (n = 795) | |
Male | 272 (44) | 201 (49) | 411 (52) |
Female | 352 (56) | 210 (51) | 384 (48) |
Median (5%–95%) | 37 (20–67) | 42 (20–72) | 43 (23–60) |
Median (5%–95%) | 25 (14–59) | 33 (15–67) | - |
Smokers | 178 (29) | 30 (7) | 207 (26) |
Former smokers | 64 (10) | 86 (21) | 392 (49) |
Never smokers | 156 (25) | 102 (25) | 189 (24) |
Data not available | 226 (36) | 193 (47) | 7 (1) |
Proctitis (E1) | - | 53 (13) | - |
Left side (E2) | - | 183 (45) | - |
Extensive (E3) | - | 134 (33) | - |
Data not available | - | 41 (10) | - |
Colonic (L2) | 208 (33) | - | - |
Ileal (L1) | 172 (28) | - | - |
Ileocolonic (L3) | 210 (34) | - | - |
Data not available | 34 (5) | - | - |
The genotype distributions among the healthy controls deviated from Hardy-Weinberg equilibrium for
The homozygous variant genotype of
After Bonferroni correction for multiple testing both the homozygous and the heterozygous variant genotypes of
The variant allele of the polymorphisms have been shown to increase TLR2 levels (
Gene (SNP) | rs-number | Effect of the SNP | Previously found associations. Disease, genotype, OR (95% CI), p-value | Associations found in this study. Disease, genotype, OR (95% CI), p-value |
−16934 A>T | rs4696480 | Unknown |
ND | IBD: TT, 1.33 (1.01–1.74), p = 0.04 |
C>A | rs11938228 | Unknown |
ND | No association |
C>T | rs1816702 | rs1816702T increase receptor level |
ND | CD: TT, 2.36 (1.08–5.16), p = 0.03 |
597 T>C | rs3804099 | 597C decrease TNF-α, IL-1β & IL-6 level |
ND | No association |
G>A | rs5030728 | Unknown |
ND | No association |
T>C | rs1554973 | Unknown |
ND | UC: TC or CC, 0.67 (0.48–0.94), p = 0.02 |
T>C | rs12377632 | Unknown |
ND | UC: TC or CC, 1.42 (1.01–2.00), p = 0.04 |
1174 C>T | rs5744168 | 1174T (392STOP), decrease TNF-α, IL-1β & IL-6 level |
CD: CT, 0.14 (0.03–0.57), p = 0.002 (Jewish) |
No association |
−1486 T>C | rs187084 | −1486C&1174G decrease expression |
ND | IBD: TC or CC, 1.29 (1.00–1.66), p = 0.05 |
1174 G>A | rs352139 | −1486C&1174G decrease expression |
ND | UC: AA, 0.67 (0.47–0.95), p = 0.03 |
−1625 C>G | rs11465996 | −1625G increase MD-2 & TNF-α level |
ND | UC: CG, 0.74 (0.57–0.96), p = 0.02 |
−159 G>A | rs2569190 | −159AA increase CD14 level |
IBD: GA or AA, 2.95(1.77–4.90),p = 2*10−5 |
No association |
T>C | rs7222094 | rs7222094CC decrease NIK activity |
ND | No association |
163 T>C | rs237025 | 163C increase NFκB1 expression |
ND | No association |
2758 G>A | rs696 | 2758A increase expression |
UC:Extensive colitis (Hungarian) |
UC: GA or AA, 1.28 (1.00–1.65), p = 0.05 |
T>del | rs17103265 | rs17103265del decrease expression |
ND | No association |
−94 ins/del | rs28362491 | −94del decrease p50 subunit expression |
Inconclusive |
CD: Ins/- or -/- , 0.80 (0.65–1.00), p = 0.05 |
−863 C>A | rs1800630 | −863A increase expression |
IBD: AA, 4.82 (2.60–8.96), p = 1*10−4 (Indian) |
Failed to genotype |
−857 C>T | rs1799724 | −857T increase TNF-α level |
Inconclusive |
Failed to genotype |
−308 G>A | rs1800629 | −308A increase expression |
Inconclusive |
UC: GA or AA, 0.75 (0.58–0.98), p = 0.04 |
−238 G>A | rs361525 | −238A decrease expression |
Inconclusive |
No association |
−609 G>T | rs4149570 | −609T increase expression |
ND | CD: TT, 1.41 (1.02–1.94), p = 0.04 |
C>G | rs6927172 | rs6927172G increase expression |
ND | No association |
−3737 G>A | rs4848306 | −3737A decrease transcription |
ND | No association |
−1464 G>C | rs1143623 | rs1143623C decrease IL-1b level |
ND | No association |
−31 T>C | rs1143627 | −31C decrease expression |
No association (Danish) |
No association |
T>C | rs4251961 | rs4251961C decrease IL-1RA level |
ND | No association |
A>G (I50V) | rs1805010 | rs1805010G increase IL-17 level |
ND | No association |
−6331 T>C | rs10499563 | −6331C decrease expression |
ND | No association |
C>T | rs4537545 | rs4537545TT increase IL-6r and IL-6 level but not TNF-α, IL-1RA and CRP level |
ND | CD: TT, 1.73 (1.12–2.66), p = 0.01 |
−592 C>A | rs1800872 | −592A increase expression |
No association (Danish) |
No association |
C>T | rs3024505 | Unknown |
CD: T-allele, 1.12 (1.07–1.17),p = 2*10−14 |
CD: No association; UC: CT or TT, 1.42 (1.10–1.82), p = 0.007 |
197G>A | rs2275913 | 197A increase expression |
ND | No association |
G>A (R381Q) | rs11209026 | rs11209026GG increase IL-17 serum level |
CD: G-allele, 2.66 (2.36–3.00), p = 1*10−64 |
CD: GA or AA, 0.39 (0.26–0.59), p = 1*10−5A, |
874 T>A | rs2430561 | 874A decrease IFN-γ level |
ND | No association |
−509 C>T | rs1800469 | −509T increase expression |
ND | No association |
1858 G>A | rs2476601 | 1858A decrease TNF-α in serum level |
CD: G-allele, 1.26 (1.17–1.37), p = 5*10−9 |
CD: GA or AA, 0.54 (0.41–0.72), p = 2*10−5A, |
C>G (Pro12Ala) | rs1801282 | rs1801282G decrease PPARγ mRNA level, but upregulations MyD88 TLR4, TLR5, TLR9, P65 and TNF-α mRNA levels |
CD:GG, 0.33 (0.12–0.94), p = 0.03 (Hungarian) |
CD: No association; UC: GG, 2.12 (1.01–4.45), p = 0.05 |
C>T | rs4612666 | rs4612666T decrease expression |
ND | No association |
Crude (unadjusted).
Adjusted for age, gender and smoking status.
Function examined by flow cytometry.
Function examined by luciferase reporter assay.
Function examined by enzyme-linked immunosorbent assay (ELISA).
Function examined by reverse transcriptase PCR (RT-PCR).
Function examined by electrophoretic mobility shift assay (EMSA).
ND: not determined.
Thus, polymorphisms associated with higher TLR2 levels (
The homozygous variant genotype of
The homozygous variant genotype of
The variant allele of the polymorphisms have been shown to increase
Thus, polymorphisms associated with increased
The studied polymorphisms all showed the same direction of effect for both diseases except for the polymorphism in
When including all patients (IBD), the homozygous variant genotype of
After Bonferroni correction for multiple testing both the homozygous and the heterozygous variant genotypes of
The variant allele of the polymorphisms have been shown to increase IL-6r and IL-6 levels (
Thus, polymorphisms associated with higher IL-6r and IL-6 levels (
Haplotype analyses of
The
No associations were found for
The
In this study of severely ill patients, 16 functional polymorphisms in 13 genes involved in regulation of inflammation were found to be associated with CD, UC or CD and UC combined (IBD) (
Eleven of the SNPs have not previously been reported as susceptability polymorphisms of CD, UC or IBD (
The biological interpretation indicates that a genetically determined higher activity of the inflammatory genes
This study was unable to confirm the associations between the variant allele of
The variant allele in
No associations were found for
The results in this study should be interpreted with care.
In conclusion, 16 functional SNPs in 13 genes involved in regulation of inflammation were found to be associated with susceptibility of severe CD, UC or IBD. Eleven of the SNPs have not previously been reported as susceptibility polymorphisms of CD, UC or IBD (
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