Claudia Huebner

Has toll-like receptor 4 been prematurely dismissed as an inflammatory bowel disease gene? Association study combined with meta-analysis shows strong evidence for association.

OBJECTIVES:Published association studies of the TLR4 Asp299Gly polymorphism and inflammatory bowel disease (IBD) in caucasian populations have inconsistent results. We tested two TLR4 variants for association with IBD in the New Zealand caucasian population and assessed the cumulative evidence for association of TLR4 Asp299Gly and IBD.METHODS:The TLR4 Asp299Gly and Thr399Ile polymorphisms were genotyped and tested for case-control frequency differences in a New Zealand white cohort of 389 Crohns disease (CD) patients, 405 ulcerative colitis (UC) patients, and 416 population controls. Meta-analysis using a random effects model was performed to test whether 299Gly carriage was associated with UC, CD, or phenotypes of CD patients.RESULTS:There were no significant allele or genotype frequency differences between cases and controls or between CD phenotypes in our New Zealand data. Meta-analysis did not identify any significant associations between CD phenotypes and 299Gly carriage. However, meta-analysis demonstrated significantly higher 299Gly carrier frequencies in CD patients (odds ratio 1.45, 95% CI 1.11–1.90) and in IBD patients (odds ratio 1.36, 95% CI 1.01–1.84) compared to controls.CONCLUSIONS:The meta-analysis provides evidence that Asp299Gly is associated with CD and IBD in whites. Only the Asp299Gly polymorphism has been consistently genotyped in previous TLR4 studies with IBD patients, therefore other TLR4 variants with stronger associations with IBD may exist. Additional well-powered studies of Asp299Gly and other TLR4 variants are urgently needed.

Genetic factors in chronic inflammation: Single nucleotide polymorphisms in the STAT-JAK pathway, susceptibility to DNA damage and Crohn's disease in a New Zealand population

The Signal Transducers and Activators of Transcription (STAT)-Janus kinase (JAK) pathway controls signal transduction between cell surface receptors and the nucleus. Two members of that pathway, STAT3 and JAK2, enhanced the risk of Crohns disease (CD) in recent genome-wide association studies. We replicated these findings in a New Zealand Caucasian case-control cohort, by genotyping two single nucleotide polymorphisms (SNPs) in STAT3 (rs744166(G>A) and rs3816769(C>T)) and rs10758669(A>C) in JAK2, in 302 CD patients and 382 controls. For STAT3, there was a significant decrease in the frequency of the G allele of rs744166 and the C allele of rs3816769 in CD patients as compared with controls (OR=0.76, 95% CI=0.61-0.95, p=0.013; OR=0.71, 95% CI=0.56-0.89, p=0.003). For the JAK2 rs10758669 polymorphism, the homozygous C/C or heterozygous A/C genotypes increased the risk of having CD as compared with the homozygous A/A (OR=1.76, 95% CI=1.26-2.45 and OR=2.36, 95% CI=1.44-3.86, respectively, p=0.0003). Variant alleles in either gene significantly modified the likelihood of inflammatory disease in a colonic location, and of developing extra-intestinal manifestations. The JAK2 variant also strongly enhanced the risk of ileocolonic disease, with stricturing or ileal/stricturing behaviour, requiring a bowel resection. We further studied a subset of our control population, stratified for JAK2 rs10758669 and/or STAT3 rs3816769 genotype. Carrying either the JAK2 or STAT3 IBD risk allele was associated with significantly enhanced susceptibility to DNA damage, as estimated by comet assays in peripheral blood leukocytes, with or without a subsequent oxidative challenge. That is, both risk alleles enhance genomic instability. The JAK2 SNP is part of a haplotype previously associated with enhanced susceptibility to myeloproliferative neoplasms, but functional consequences of the STAT3 variant had not been previously demonstrated. It will be of interest to follow up CD patients carrying either JAK2 or STAT3 risk alleles for development of further secondary effects, including cancer.

Gender-stratified analysis of DLG5 R30Q in 4707 patients with Crohn disease and 4973 controls from 12 Caucasian cohorts

Background: DLG5 p.R30Q has been reported to be associated with Crohn disease (CD), but this association has not been replicated in most studies. A recent analysis of gender-stratified data from two case–control studies and two population cohorts found an association of DLG5 30Q with increased risk of CD in men but not in women and found differences between 30Q population frequencies for males and females. Male–female differences in population allele frequencies and male-specific risk could explain the difficulty in replicating the association with CD. Methods: DLG5 R30Q genotype data were collected for patients with CD and controls from 11 studies that did not include gender-stratified allele counts in their published reports and tested for male–female frequency differences in controls and for case–control frequency differences in men and in women. Results: The data showed no male–female allele frequency differences in controls. An exact conditional test gave marginal evidence that 30Q is associated with decreased risk of CD in women (p = 0.049, OR = 0.87, 95% CI 0.77 to 1.00). There was also a trend towards reduced 30Q frequencies in male patients with CD compared with male controls, but this was not significant at the 0.05 level (p = 0.058, OR = 0.87, 95% CI 0.74 to 1.01). When data from this study were combined with previously published, gender-stratified data, the 30Q allele was found to be associated with decreased risk of CD in women (p = 0.010, OR = 0.86, 95% CI 0.76 to 0.97), but not in men. Conclusion: DLG5 30Q is associated with a small reduction in risk of CD in women.

Triallelic Single Nucleotide Polymorphisms and Genotyping Error in Genetic Epidemiology Studies: MDR1 (ABCB1) G2677/T/A as an Example

Accurate measurement of allele frequencies between population groups with differing sensitivities to disease is fundamental to genetic epidemiology. Genotyping errors can markedly influence the biological conclusions of a study. This issue may be especially important now there is increasing recognition of triallelic single nucleotide polymorphisms (SNPs) in the genome and their possible role in diseases like inflammatory bowel disease. For example, the MDR1 (ABCB1) SNP G2677/T/A was, like many other triallelic SNPs, originally described as diallelic. Here, we report a comprehensive analyses of estimated allele frequencies of this SNP in a set of 73 human DNA samples, comparing six commonly used genotyping methods (Applied Biosystems Taqman, Roche LightCycler melting analysis, allelic discrimination PCR, DNA sequencing, Sequenom, and RFLP) from the angle of their error potential. Only Sequenom and DNA sequencing provided accurate measurements, if we had not had prior knowledge of the triallelic nature of this SNP. The other tested methods (with the exception of LightCycler) failed to show any indication of the presence of the rare third A- allele in a diallelic assay. Although most of the errors were due to the inability to detect the third allele, all methods except Sequenom and sequencing produced errors for the detection of the two common alleles G and T (LightCycler, 6 errors; PCR, 4 errors; RFLP, 2 errors; Taqman, 1 error). There is considerable variability in the reported frequencies of the different alleles of the MDR1 G2677/T/A SNP, and the role of this SNP in the etiology of inflammatory bowel disease has been controversial. Our data emphasize the importance of choosing the appropriate method for SNP detection and lead us to suggest that part of the previously reported variation may reflect artifacts associated with the different genotyping methodologies used. The failure to recognize the triallic nature of a SNP may lead to underestimations of real genetic associations. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1185–92)

Genetic analysis of MDR1 and inflammatory bowel disease reveals protective effect of heterozygous variants for ulcerative colitis

Background: Single nucleotide polymorphisms (SNPs) in the multidrug transporter MDR1 have been associated with inflammatory bowel disease (IBD) in different studies. However, the data are highly controversial. Recently, 6 haplotype tagging SNPs (tSNPs), representing the haplotype variations of the MDR1 gene, were identified. The aims of this study were to genotype these variants and correlate them to disease phenotype in New Zealand IBD patients. Materials and Methods: A total of 784 IBD patients and 200 healthy subjects were genotyped for 5 tSNPs and the triallelic MDR1 variant G2677T/A using the Sequenom MassArray platform. Furthermore, the effects of these variants were examined in correlation with phenotypic clinical features. Results: Heterozygous carriers for the variants C1236T, rs2235046 (an SNP in intron 16), and G2677T/A showed a lower risk of developing ulcerative colitis (C1236T: odds ratio [OR] = 0.63, 95% confidence interval [CI] = 0.42‐0.93, P = 0.03; G2677T/A: OR = 0.59, CI = 0.39‐0.89, P = 0.02; and rs2235046: OR = 0.59, 95% CI = 0.38‐0.91, P = 0.009) as compared with homozygotes. None of the analyzed markers were associated with Crohns disease on a genotypic level. Subgroup analysis revealed an association for 2 variants with IBD when stratified for age of onset (C1236T SNP and rs3789243). The MDR1 variant C3435T was associated with disease behavior in CD (OR = 1.45, 95% CI = 1.01‐2.08, P = 0.04), whereas the SNP rs3789243 was found to be associated with pancolitis in UC patients (OR = 1.35, CI = 1.00‐1.82, P = 0.05). Conclusions: The results of our study support the role of MDR1 as a candidate gene for ulcerative colitis. Furthermore, our results suggest the possibility of a heterozygous advantage for certain MDR1 variants for this disease. Inflamm Bowel Dis 2009

Association of DLG5 variants with inflammatory bowel disease in the New Zealand caucasian population and meta-analysis of the DLG5 R30Q variant†

Background: Variants in the DLG5 gene have been associated with inflammatory bowel disease (IBD) in samples from some, but not all populations. In particular, 2 nonsynonymous single‐nucleotide polymorphisms (SNPs), R30Q (rs1248696) and P1371Q (rs2289310), have been associated with an increased risk of IBD, and a common haplotype (called haplotype “A”) has been associated with reduced risk. Methods: We genotyped R30Q, P1371Q, and a haplotype A tagging SNP (rs2289311) in a New Zealand Caucasian cohort of 389 Crohns disease (CD) patients, 406 ulcerative colitis (UC) patients, and 416 population controls. Each SNP was tested for association with disease susceptibility and clinical phenotypes. We also performed a meta‐analysis of R30Q data from published association studies. Results: The haplotype A tagging SNP was associated with reduced risk of IBD at the 0.05 significance level (P = 0.036) with an allelic odds ratio of 0.83 (95% confidence interval [CI]: 0.69–0.99). Association with haplotype A was strongest (odds ratio ≈0.57) in UC patients with familial IBD or extraintestinal manifestations. The R30Q and P1371Q polymorphisms were not significantly associated with UC, CD, or IBD. Analysis of male and female data did not find any gender‐specific associations. Meta‐analysis gave no evidence of association of R30Q with IBD. Conclusions: Meta‐analysis demonstrates that the minor allele of R30Q is not a risk factor for IBD across populations. This study provides some evidence that DLG5 haplotype A is associated with reduced risk of IBD in the New Zealand Caucasian population, but this association will need to be replicated in an independent sample.

Interactions among genes influencing bacterial recognition increase IBD risk in a population-based New Zealand cohort

Bacterial sensing is crucial for appropriate response by the innate and adaptive immune system against invading microorganisms. Single nucleotide polymorphisms (SNPs) in genes involved in bacterial recognition, CARD15 and TLR4, increased the risk of inflammatory bowel disease (IBD) in a New Zealand Caucasian case-control cohort. We now consider the effects of SNPs in CD14, TLR9, and BPI, analyzed individually, in association with one another, and with SNPs in CARD15 or TLR4 in this same population group. SNPs in CD14 (c.-159 C>T), TLR9 (c.-1237T>C) and BPI (c.645A>G) showed no significant allele or genotype frequency differences between IBD cases and controls. Genotype-phenotype mapping reveals an association with BPI and ileocolonic Crohns disease (CD) as well as an association with CD14 and early-onset ulcerative colitis (UC). Genotype interaction analyses using three different statistical approaches provided significant evidence of interaction for the following combinations: CARD15/TLR4 (CD and UC), CARD15/CD14 (CD and UC), CD14/TLR4 (UC only), and CD14/BPI (UC only). A trend for an association between BPI and TLR4 was observed in UC patients, but failed to reach statistical significance. Our findings support the idea of gene-gene interactions for genes involved in closely related pathways (i.e. bacterial detection). There is evidence that carrying two SNPs in genes may lead to statistical significance for genes and SNPs that do not otherwise confirm as risk alleles for disease aetiology when analysed alone.

IL23R and IL12B SNPs and Haplotypes Strongly Associate with Crohn's Disease Risk in a New Zealand Population

DNA samples from 339 Crohns disease (CD) and 407 randomly selected controls from the Auckland (New Zealand) IBD project, were genotyped for five common single nucleotide polymorphisms in IL-23R (rs11805303, rs7517847, rs1343151, rs11209026, and rs10889677) and two in IL-12B (rs1363670 and rs6887695). While the IL-12B variants did not show an overall association and other IL23R variants led to minor changes in the risk of CD, rs1343151 and/or rs7517847 variants in the IL-23R gene strongly reduced the risk of developing CD at both allelic and genotype levels. A significantly decreased risk of first diagnosis of childhood CD was observed in individuals carrying the A allele of rs1343151, or between 17–40 y in individuals carrying the G allele in rs7517847 of IL-23R. A significantly decreased risk of ileocolonic or structuring disease was observed in individuals carrying the A allele in either rs11209026 or rs1343151, or the G allele in rs7517847 of IL-23R, and when such individuals did develop the disease, they were unlikely to require a bowel resection. Certain haplotypes very strongly modified risk. There was evidence for interactions of IL-23R variants with the NOD2 wild-type (d/d) genotype. Down-regulating the function of the IL-23R gene may decrease CD risk in the normal population.

Food-derived bioactives as potential regulators of the IL-12/IL-23 pathway implicated in inflammatory bowel diseases

The gene-specific modulation of inflammatory cytokines by food bioactives represents a possible approach to the nutritional or pharmaceutical prevention and treatment of inflammatory bowel disease (IBD). There is evidence for a key role of the interleukin-12beta1/23 receptor (IL-12 Rbeta1/23 R) pathway in IBD, and that reduction of the normal expression of the IL-23 R gene may provide a therapeutic target for this disease. The binding of interleukin-23 (IL-23) to its receptor IL-23 R regulates a newly defined effector T-cell subset, Th17 cells, characterised by the production of interleukin-17 (IL-17) and other cytokines, including tumour necrosis factor-alpha (TNF-alpha). In this study we developed an assay that measured IL-17 and TNF-alpha expression after incubation with specific dietary bioactives in the human T-cell Kit 225. It is anticipated that these changes will reflect differences in IL-23 R production, albeit indirectly. The cell line Kit 225 has similarities to Th17 cells, a subset of T cells producing IL-17 and TNF-alpha, and in initial experiments we demonstrated that the cells express both IL-23 receptor subunits, as well as IL-17 and TNF-alpha genes. Upon verification that stimulation of Kit 225 cells with 1ng/mL IL-23 significantly upregulated IL-17 and TNF-alpha gene expression, and IL-17 production, we supplemented cells with selected food bioactives, caffeic acid phenethyl ester (CAPE), epigallocatechin gallate (EGCG), docosahexaenoic acid (DHA), and linoleic acid (LA), and with phorbol myristate acetate (PMA) and sodium salicylate, used as pro-inflammatory and anti-inflammatory controls, respectively. In both unstimulated cells and after IL-23 stimulation, bioactives modulated the pro-inflammatory cytokines involved in IBD, underlining the possible role of foods in this disease. EGCG and DHA, which significantly inhibited both IL-17 and TNF-alpha expression, appeared particularly interesting.

Mushroom intolerance: a novel diet-gene interaction in Crohn's disease.

Carrying a functional single nucleotide polymorphism (L503F, c. 1672 C>T) in the gene for the Na-dependent organic cation transporter (OCTN1), increases the risk of Crohns disease (CD) in some, but not all, populations. Case-control data on New Zealand Caucasians show no differences for CD risk between individuals carrying the L503F OCTN1 C-allele when compared with those carrying the variant T-allele. However, more of the New Zealand CD cases report intolerance to maize and mushrooms than those who report beneficial effects or no differences. The OCTN1 gene encodes a transporter for ergothionine, a fungal metabolite at high levels in mushrooms but not widely common in other dietary items. An inability to tolerate mushrooms showed statistically significant associations with the variant OCTN1 genotype. That is, among those individuals reporting adverse effects from mushrooms, there was a higher frequency of the variant T-allele when compared with the general population, or with CD patients overall. We believe that this is a novel gene-diet association, suggesting that individuals carrying the OCTN1 variant single nucleotide polymorphism may have an enhanced risk of adverse symptoms associated with consuming mushrooms. Nutrigenomic approaches to dietary recommendations may be appropriate in this group.