J B A Crusius

Molecular prediction of disease risk and severity in a large Dutch Crohn's disease cohort

Background: Crohn’s disease and ulcerative colitis have a complex genetic background. We assessed the risk for both the development and severity of the disease by combining information from genetic variants associated with inflammatory bowel disease (IBD). Methods: We studied 2804 patients (1684 with Crohn’s disease and 1120 with ulcerative colitis) and 1350 controls from seven university hospitals. Details of the phenotype were available for 1600 patients with Crohn’s disease and for 800 with ulcerative colitis. Genetic association for disease susceptibility was tested for the nucleotide-binding and oligomerisation domain 2 gene (NOD2), the IBD5 locus, the Drosophila discs large homologue 5 and autophagy-related 16-like 1 genes (DLG5 and ATG16L1) and the interleukin 23 receptor gene (IL23R). Interaction analysis was performed for Crohn’s disease using the most associated single nucleotide polymorphism (SNP) for each locus. Odds ratios were calculated in an ordinal regression analysis with the number of risk alleles as an independent variable to analyse disease development and severity. Results: Association with Crohn’s disease was confirmed for NOD2, IBD5, DLG5, ATG16L1 and IL23R. Patients with Crohn’s disease carry more risk alleles than controls (p = 3.85 × 10−22). Individuals carrying an increasing number of risk alleles have an increasing risk for Crohn’s disease, consistent with an independent effects multiplicative model (trend analysis p = 4.25 × 10−23). Patients with Crohn’s disease with a more severe disease course, operations or an age of onset below 40 years have more risk alleles compared to non-stricturing, non-penetrating behaviour (p = 0.0008), no operations (p = 0.02) or age of onset above 40 years (p = 0.028). Conclusion: Crohn’s disease is a multigenic disorder. An increase in the number of risk alleles is associated with an increased risk for the development of Crohn’s disease and with a more severe disease course. Combining information from the known common risk polymorphisms may enable clinicians to predict the course of Crohn’s disease.

A microarray screen for novel candidate genes in coeliac disease pathogenesis

Background and aims: The causative molecular pathways underlying the pathogenesis of coeliac disease are poorly understood. To unravel novel aspects of disease pathogenesis, we used microarrays to determine changes in gene expression of duodenal biopsies. Methods: cDNA microarrays representing 19 200 genes were used to compare gene expression profiles of duodenal biopsies from 15 coeliac disease patients with villous atrophy (Marsh III) and seven control individuals with normal biopsies (Marsh 0). In addition, the specific effect of gluten was studied by comparing the expression profiles of Marsh III lesions of seven patients exposed to gluten with four patients on a gluten free diet. Results: Comparing Marsh III with Marsh 0 lesions identified 109 genes that differed significantly (p<0.001) in expression levels between patients and controls. A large number of these genes have functions in proliferation and differentiation pathways and might be important for correct development of crypt-villous units. Alterations in these pathways may lead to the characteristic hyperplasia and villous atrophy seen in coeliac disease. The analyses also revealed 120 differentially expressed genes (p<0.005) when comparing patients on a gluten free diet with those exposed to gluten. These genes further strengthen our observation of increased cell proliferation in the presence of gluten. Conclusions: Our study provides new candidate genes in the pathogenesis of coeliac disease. Based on our results, we hypothesise that villous atrophy in coeliac disease patients is due to failure in cell differentiation. These genes are involved in pathways not previously implicated in coeliac disease pathogenesis and they may provide new targets for therapy.

The toll-like receptor 4 (TLR4) Asp299Gly polymorphism is associated with colonic localisation of Crohn's disease without a major role for the Saccharomyces cerevisiae mannan-LBP-CD14-TLR4 pathway

It is with great interest that we read the paper by Frachimont and colleagues ( Gut 2004; 53 :987–92) in which they describe a novel association of the toll-like receptor 4 (TLR4) +896 A>G polymorphism with both Crohn’s disease (CD) and ulcerative colitis (UC), supporting the genetic influence of pattern recognition receptors (PRRs) in triggering inflammatory bowel disease (IBD). PRRs are sensors of pattern associated molecular patterns of microorganisms in the intestinal flora. Independently, we performed a similar study. However, special attention to the presence of anti- Saccharomyces cerevisiae antibody (ASCA) was taken, as Tada and colleagues1 have recently reported that the S cerevisiae mannan-LBP complex is recognised by CD14 on monocytes and signalling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by lipopolysaccharide (LPS). Patients and controls were recruited from the Outpatient Department of Gastroenterology, VU University Medical Centre, Amsterdam, the Netherlands. The group consisted of 112 CD patients and …

Evidence for genetic heterogeneity in IBD : 1. The interleukin-1 receptor antagonist in the predisposition to suffer from ulcerative colitis

Objective: To investigate a polymorphism within intron 2 of the interleukin-1 receptor antagonist (IL-1 ra) gene in a Dutch white population of patients with ulcerative colitis and Crohns disease. Design: Genotype and allele frequencies of the polymorphic region in the IL-1ra gene were determined in 111 unrelated patients with ulcerative colitis, 92 patients with Crohns disease, and 86 healthy controls. Methods: The polymorphic region of the IL-1ra gene was amplified by the poly-merase chain reaction (PCR). The resultant products were analyzed by elec-trophoresis on 2% agarose gels and stained with ethidium bromide. Amplification of the second exon of HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes was performed by PCR, and Dot-blot analysis with biotin-Iabelled sequence-specific oligonucleotide probes was used for HLA typing. The standard perinuclear antineutrophil cytoplasmic antibodies (pANCA) indirect immunofluorescence assay was performed according to the protocol described by the First International Workshop on ANCA. Results: The frequency of allele 2 of the IL-1ra gene in ulcerative colitis (27.0%) and Crohns disease patients (25.5%) did not differ significantly from healthy controls (23.8%). However, the estimate of the relative risk for carriers of allele 2 for ulcerative colitis [odds ratio (OR): 1.35, 95%-confidence interval (CI) from 0.73 to 2.49] was in agreement with a previous British study. The exact test for homogeneity of odds ratios provided no evidence for heterogeneity between both populations (P= 0.35) and therefore confirmed the genetic association between ulcerative colitis and the IL-1ra allele 2 in a different population. Our data confirm that, in ulcerative colitis, the presence of this allele is a genetic marker for severity of the disease. A significant association was demonstrated between the carriership of allele 2 of the IL-1ra gene and the trend over the three localizations in ulcerative colitis (P=0.023). The same approach for Crohns disease when compared with healthy controls did not provide evidence for a similar association. The meta-analysis, based on the British data and our data, yielded: OR = 1.06, 95%-CI from 0.71 to 1.59, and P= 0.767. No association between IL-Ira gene polymorphism, and pANCA and the HLA-DR2 allele was found in ulcerative colitis. Conclusion: The observations confirm that the allele 2 of the IL-1ra gene is a marker for genetic susceptibility and severity in ulcerative colitis and contributes to the definition of the immunogenetic heterogeneity of the disease.

A genome-wide association study of rheumatoid arthritis without antibodies against citrullinated peptides

Introduction Rheumatoid arthritis (RA) patients can be classified based on presence or absence of anticitrullinated peptide antibodies (ACPA) in their serum. This heterogeneity among patients may reflect important biological differences underlying the disease process. To date, the majority of genetic studies have focused on the ACPA-positive group. Therefore, our goal was to analyse the genetic risk factors that contribute to ACPA-negative RA. Methods We performed a large-scale genome-wide association study (GWAS) in three Caucasian European cohorts comprising 1148 ACPA-negative RA patients and 6008 controls. All patients were screened using the Illumina Human Cyto-12 chip, and controls were genotyped using different genome-wide platforms. Population-independent analyses were carried out by means of logistic regression. Meta-analysis with previously published data was performed as follow-up for selected signals (reaching a total of 1922 ACPA-negative RA patients and 7087 controls). Imputation of classical HLA alleles, amino acid residues and single nucleotide polymorphisms was undertaken. Results The combined analysis of the studied cohorts resulted in identification of a peak of association in the HLA-region and several suggestive non-HLA associations. Meta-analysis with previous reports confirmed the association of the HLA region with this subset and an observed association in the CLYBL locus remained suggestive. The imputation and deep interrogation of the HLA region led to identification of a two amino acid model (HLA-B at position 9 and HLA-DRB1 at position 11) that accounted for the observed genome-wide associations in this region. Conclusions Our study shed light on the influence of the HLA region in ACPA-negative RA and identified a suggestive risk locus for this condition.

Defining the contribution of the HLA region to cis DQ2-positive coeliac disease patients

The major genetic susceptibility to coeliac disease is contributed by the human leukocyte antigen (HLA) region. The primary association is with the HLA-DQ2 molecule, encoded by the DQA1*05 and DQB1*02 alleles, which is expressed by over 90% of patients. The aim of our study was to perform an extensive scan of the entire HLA region to determine whether there is evidence for the presence of additional HLA susceptibility genes for coeliac disease in the Dutch population, acting independently of DQ2. In all, 16 microsatellite markers and the DQA1 and DQB1 genes were genotyped in simplex cis DQ2-positive coeliac disease families and cis DQ2-positive control families. Allele frequencies of markers on phase-known DQ2-positive haplotypes transmitted to patients were compared to a combined group of DQ2-positive nontransmitted and control haplotypes, thereby controlling for the DQ2 contribution. No significant differences at any of the marker loci were detected, suggesting that DQ2 is the major HLA risk factor for coeliac disease. Individuals homozygous for DQ2 or heterozygous for DQA1*05-DQB1*02/DQA1*0201-DQB1*02 were found to be at five-fold increased risk for development of coeliac disease (P<10−8). This risk seems to be conferred by the presence of a second DQB1*02 allele next to one DQA1*05-DQB1*02 haplotype, independently of the second DQA1 allele.

LINKAGE AND ASSOCIATION STUDIES OF IL1B AND IL1RN GENE POLYMORPHISMS IN PREECLAMPSIA

Objective: To determine whether preeclampsia is either associated with or linked to two polymorphisms in the IL1B gene (IL1B- TaqI and IL1B- 511) and one polymorphism in the IL1RN gene (IL1RN- IVS2). Methods: Genotyping was performed in 150 affected sib-pair families and 104 healthy Dutch blood donors. Genotype and allele frequencies as well as allelic associations were assessed in three groups of unrelated women from these 150 families; 133 with either eclampsia, preeclampsia or the haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, 101 with preeclampsia only, and 63 with HELLP syndrome only. These frequencies were compared to those in controls. Frequencies of transmitted and nontransmitted haplotypes, inferred from the three polymorphisms, were compared. Allele sharing between affected siblings from all 150 families was assessed by means of multipoint nonparametric affected sib-pair analyses. Results: No significant differences in genotype and allele frequencies were found between the unrelated study groups and controls. No allelic associations were apparent, nor were there differences in frequencies of transmitted and nontransmitted haplotypes within affected families. Excess allele sharing for any of the three polymorphic markers was absent in affected sib-pairs. Conclusions: None of the IL1B and IL1RN polymorphisms provided evidence for either association or linkage with the risk for (pre)eclampsia/HELLP syndrome, preeclampsia only or HELLP syndrome only.

Signal transducer and activator of transcription 6 gene G2964A polymorphism and inflammatory bowel disease.

Signal transducer and activator of transcription 6 (STAT6) is a key transcription factor involved in interleukin 4 (IL‐4) and IL‐13‐mediated Th2 response. The STAT6 gene is located on chromosome 12q13.3–14·1 (IBD2 region) and is therefore a positional and functional candidate gene for study in inflammatory bowel disease. We investigated the G2964A polymorphism in the 3′ untranslated region of the STAT6 gene in Dutch patients with inflammatory bowel disease and healthy controls. The G2964A polymorphism in the STAT6 gene was genotyped in 141 unrelated Dutch Caucasian patients with ulcerative colitis, 183 patients with Crohns disease and 173 healthy individuals by PCR and the amplification‐created restriction site method. Patients with Crohns disease were classified according to the Vienna classification and the patients with ulcerative colitis were classified with the age at onset, extent of disease and colectomy. We did not find significant differences in genotype and allele frequencies of the G2964A polymorphism in the STAT6 gene between ulcerative colitis, Crohns disease and healthy controls. Subgroups of the patients with Crohns disease classified according to the Vienna classification and those with ulcerative colitis classified according to age of onset, disease extension and colectomy did not differ in the distribution of this polymorphism. The STAT6 G2964A gene polymorphism is not involved in the overall susceptibility or in determining the phenotype of IBD.

The role of the shared epitope in arthralgia with anti-cyclic citrullinated peptide antibodies (anti-CCP), and its effect on anti-CCP levels

OBJECTIVES Patients presenting with both arthralgia and antibodies to cyclic citrullinated peptide (anti-CCP) have an increased risk of developing rheumatoid arthritis (RA). To further characterise this patient group and shed more light on its relationship with clinically manifest early arthritis and established RA, an immunogenetic and serological analysis was performed. METHODS In a group of 111 patients with anti-CCP-positive arthralgia, anti-CCP levels and shared epitope (SE) status were determined. Data were compared with 125 and 128 patients with anti-CCP-positive early arthritis and established RA respectively. RESULTS In patients with anti-CCP-positive arthralgia, the frequency of SE allele positivity is significantly lower when compared with anti-CCP-positive early arthritis and established RA (58% vs 80%, and 58% vs 92%, respectively, both p<0.001). Median anti-CCP levels were higher in the group of patients with SE-positive arthralgia compared with the group of patients with SE-negative arthralgia (p = 0.02). Median anti-CCP levels were similar in the groups of patients with SE-positive arthralgia and arthritis. CONCLUSIONS The lower frequency of SE positivity in patients with arthralgia compared with patients with RA indicates that, compared with patients who were SE positive, patients who were SE negative as a group go through a longer arthralgia phase, or alternatively have a lower risk for transition from anti-CCP positive arthralgia to RA. Furthermore, the present results suggest that in this early stage the effect of the SE on disease risk may be mediated through higher anti-CCP levels.

Susceptibility to ankylosing spondylitis: no evidence for the involvement of transforming growth factor beta 1 (TGFB1) gene polymorphisms.

Background: Genetic factors are thought to be crucial in the pathogenesis of ankylosing spondylitis. Transforming growth factor β1 (TGFβ1) is a multifunctional cytokine that plays a key role in inflammation. Two functional single nucleotide polymorphisms (SNPs) in the TGFB1 gene have been described: TGFB1 T869C and TGFB1 G915C. Objective: To determine whether these SNPs contribute to ankylosing spondylitis susceptibility or its disease characteristics. Methods: Genomic DNA was isolated from the peripheral blood of 134 patients with ankylosing spondylitis and 194 healthy blood donors. All subjects were unrelated and of white Dutch ethnicity. The diagnosis of ankylosing spondylitis was made according to the modified New York criteria. The TGFB1 T869C and TGFB1 G915C SNPs were genotyped by a polymerase chain reaction–single strand conformation polymorphism haplotyping method. Results: No significant differences were found between patients and controls in genotype, allele, and haplotype frequencies or in the carrier rate of the rare alleles of the TGFB1 T869C and TGFB1 G915C SNPs. Conclusions:TGFB1 T869C and TGFB1 G915C SNPs are not major factors in the susceptibility to ankylosing spondylitis or its disease characteristics.