Testing the interaction between NOD-2 status and serological response to Mycobacterium paratuberculosis in cases of inflammatory bowel disease

In a population-based case-control study we have previously shown that 14% of healthy Manitobans carry one or two mutations in the NOD-2 locus, a gene highly associated with Crohn's disease (CD). The NOD-2 protein is the receptor responsible for recognition of bacterial peptidoglycans, and it is plausible that NOD-2 is involved in the recognition of mycobacteria. Thirty-seven percent of Manitobans with CD had >or=1 NOD-2 mutation, leading to a threefold increased risk of CD for single-mutant carriers and a 30-fold increased risk for double-mutant carriers. In the same population groups, we assessed the seroprevalence for Mycobacterium paratuberculosis and found it to be 35%, with no differences between CD, ulcerative colitis (UC), and controls. Because of high rates of CD and UC in Manitoba, we assessed whether there was an interaction between carrying a NOD-2 mutation and M. paratuberculosis seropositivity. An enzyme-linked immunosorbent assay for serum antibodies to M. paratuberculosis in cattle was adapted for human use. DNA was purified from whole blood. Subjects were genotyped for three NOD-2 variants, G908R, Cins1007fs, and R702W. Multivariate logistic regression analysis showed that NOD-2 gene mutations significantly associated with CD, but M. paratuberculosis serology did not. Furthermore, there was no interaction between NOD-2 mutation status and M. paratuberculosis serology status. For those with the NOD-2 mutation, the likelihood of CD subjects having positive M. paratuberculosis serology was similar to that of controls (odds ratio, 1.31; 95% confidence interval, 0.55-3.11). No interaction could be proven for UC or by combining CD and UC compared to controls. In conclusion, we could not find an interaction between the NOD-2 genotype and M. paratuberculosis serology in relationship to CD or UC.     

Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6. 3%) (P < 0.05). Positive IS900 PCR results occurred more often in U. S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U. S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-gamma tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.     

Classification of inflammatory bowel diseases by means of Raman spectroscopic imaging of epithelium cells

We report on a Raman microspectroscopic characterization of the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC). Therefore, Raman maps of human colon tissue sections were analyzed by utilizing innovative chemometric approaches. First, support vector machines were applied to highlight the tissue morphology (=Raman?spectroscopic?histopathology). In a second step, the biochemical tissue composition has been studied by analyzing the epithelium Raman spectra of sections of healthy control subjects (n=11), subjects with CD (n=14), and subjects with UC (n=13). These three groups exhibit significantly different molecular specific Raman signatures, allowing establishment of a classifier (support-vector-machine). By utilizing this classifier it was possible to separate between healthy control patients, patients with CD, and patients with UC with an accuracy of 98.90%. The automatic design of both classification steps (visualization of the tissue morphology and molecular classification of IBD) paves the way for an objective clinical diagnosis of IBD by means of Raman spectroscopy in combination with chemometric approaches.     

Characterization of genotype-phenotype relationships and stratification by the CARD15 variant genotype for inflammatory bowel disease susceptibility loci using multiple short tandem repeat genetic markers

The classification of ulcerative colitis (UC), Crohn disease (CD), and indeterminate colitis (IC) as forms of inflammatory bowel disease (IBD) is based on clinical, radiological, and histological criteria. The genetic basis of IBD is well founded, and susceptibility loci have been identified on several different chromosomes. We aimed to define genotype-phenotype relationships and interactions with the IBD susceptibility gene CARD15for various IBD susceptibility loci (IBD1, IBD2, IBD5, IBD6, IBD7, and chromosome 4) by characterizing previously described peak LOD score short tandem repeat (STR) markers. The study population consisted of 484 severely affected Caucasian patients with IBD, 144 healthy controls, and 348 nonaffected first-degree relatives of IBD patients. Associations were defined with the use of population- and family-based methodology. Correction for multiple testing was performed with a method based on an experimental false discovery rate. We provide novel evidence to show that IBD2 is involved in susceptibility to IC and terminal ileal CD in this population, with overrepresentation of IBD2 STR D12S83 (GenBank Z16592.1) allele 7 (g.49_60del[CA](6)) in IC (q = 0.038, P = 0.014) and underrepresentation of allele 8 (g.51_60del[CA](5)) in terminal ileal CD (q = 0.038, P = 0.016). The association of IBD2 with IC was confirmed by family-based testing. We also provide novel evidence to show that IBD5 is involved in susceptibility to IC and colonic/ileocolonic CD in this population, with overrepresentation of IBD5 STR D5S1984 (GenBank Z52623.1) allele 5 (g.183_186del[CA](2)) in both IC (q = 0.040, P = 0.005) and colonic/ileocolonic CD (q = 0.040, P = 0.004). Evidence is also given for potential interactions between CARD15and IBD2/IBD5. Other findings include an association of IBD2 with UC, and an association of IBD1 with terminal ileal and colonic/ileocolonic CD.     

Haplotypes of PADI4 susceptible to rheumatoid arthritis are also associated with ulcerative colitis in the Japanese population

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic inflammatory disorder characterized by intractable inflammation specific to the gastrointestinal tract. The precise etiology of IBD remains unknown. Recently, haplotypes of peptidylarginine deiminase type 4 (PADI4) have been identified as the rheumatoid arthritis (RA)-susceptible gene. PADI4 is located at 1p36, which is one of chromosomal loci susceptible for IBD. Then, we examined whether haplotypes and diplotypes of PADI4 are associated with IBD in the Japanese population. We studied haplotypes of PADI4 in 114 patients with UC, 83 patients with CD, and 200 gender-matched healthy controls by PCR-restriction fragment length polymorphism. Frequencies and distributions of haplotypes and diplotypes were compared statistically between patients and controls by logistic regression analysis. The frequency of haplotype 1 was significantly decreased in patients with UC, compared to that in controls (P=0.037; odds ratio (OR)=0.702). In contrast, the frequency of haplotype 2 in patients with UC was significantly higher than that in controls (P=0.003; OR=1.722). Moreover, of a total of 114 patients with UC, 15 (13.2%) had a diplotype homozygous for haplotype 2, the frequency being significantly higher than in controls (9/200, 4.5%; P=0.008, OR=3.215). Our results indicate that haplotype 1 of PADI4 is associated with non-susceptibility to UC, whereas haplotype 2 is susceptible to UC. Thus, it is likely that PADI4 is one of genetic determinants of UC in the Japanese population.     

Peripheral cell-mediated immune response to mycobacterial antigens in inflammatory bowel disease.

A mycobacterial etiology has been proposed in Crohn's disease (CD). We have sought evidence of increased or modified T lymphocyte immune responses to Mycobacterium tuberculosis and Myco, paratuberculosis in patients with CD (n = 13), compared with ulcerative colitis (UC; n = 17) and controls (n = 17). Peripheral blood cells were cultured with phytohaemagglutinin (positive mitogen control), mycobacterial purified protein derivative (PPD) preparations, lysates, column fractions and whole, heat-killed bacteria. Responses of T cells and T cell subsets were assessed by expression of activation markers (CD25, CD69), coupled with blastogenesis assays (3H-thymidine uptake) and estimates of proliferation. Virtually all patients responded to Myco. paratuberculosis and Myco. tuberculosis antigens. There were no significant differences between patient groups, although there was a very high overall correlation (r = 0.95; P < 0.0001) between responses to the two mycobacterial species. Most of the activation and proliferative responses resided in the CD4+ (T helper) subset. Although up to 15% of CD8+ (suppressor/cytotoxic) cells also became activated, the CD8+ cells did not proliferate subsequently. Cells expressing the alternate gamma delta form of the T cell receptor (TCR gamma delta+) did not activate or proliferate in response to mycobacterial antigens. There were no differences in any of these parameters between patient groups. We conclude that there is no specific increase or alteration in cell-mediated anti-mycobacterial immunity in inflammatory bowel disease (IBD). Thus our data do not support a mycobacterial etiopathology of Crohn's disease.     

The expression and regulation of class II antigens in normal and inflammatory bowel disease peripheral blood monocytes and intestinal epithelium

Elevated constitutive expression of major histocompatibility (MHC) class II antigens occurs in the enterocytes of patients with IBD. It has been suggested that this aberrant expression of class II molecules may play a role in the pathogenesis of IBD. We examined two possible reasons for such a finding. 1) Heightened sensitivity of IBD enterocytes to endogenous gamma interferon (gamma IFN) and 2) enhanced endogenous secretion of gamma interferon by intestinal cells in close proximity to the enterocytes (lamina propria lymphocytes). Constitutive and gamma interferon stimulated HLA-DR and DP density on intestinal epithelial cells (IEC) and peripheral blood monocytes (PBM) from UC patients (IEC n = 13; PBM n = 20), CD patients (IEC n = 14; PBM n = 18) and non-IBD controls (IEC n = 12; PBM n = 20) were measured via flow cytometry (mean channel fluorescence). gamma IFN production by PHA stimulated and unstimulated lamina propria lymphocyte (LPL) cultures of UC patients (n = 11) CD patients (n = 8) and non-IBD controls (n = 11) was measured using a vesicular stomatitis virus/WISH cell bioassay. We found significantly greater gamma IFN secretion by IBD-derived PHA stimulated LPL than from non-IBD stimulated controls (CD = 39.4 +/- 12.4u; UC41.5 +/- 6.8u; NL = 22.4 +/- 8.3u, p less than 0.05) while gamma IFN induced HLA-DR and DP upregulation was no greater in IBD-derived IEC and PBM than in non-IBD controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-10 (IL-10) genotypes in inflammatory bowel disease

Interleukin-10 (IL-10) is an anti-inflammatory cytokine. Its production in humans is under genetic control, and genotype defines high or low producers of this cytokine. This study addresses the hypothesis that idiopathic inflammatory bowel disease (IBD) patients are more likely to have the low IL-10 producer genotype and phenotype. DNA was extracted from blood cells of patients with Crohn's disease (CD) or with ulcerative colitis (UC) for IL-10 genotyping. The frequency of the high IL-10 producer allele (-1082*G) was decreased in the whole IBD group (41% vs. 51%, P = 0.03) and in the UC patients compared with normal controls (37% vs. 51%; P = 0.04). Hence, there appears to be an association between the IL-10 genotypes and IBD. This suggests that individuals genetically predisposed to produce less IL-10 are at a higher risk of developing IBD, in particular, UC.     

Association of caspase-9 and RUNX3 with inflammatory bowel disease

Previous linkage studies have identified a region at 1p36 as the susceptibility locus (IBD7) of inflammatory bowel disease (IBD). The objective of this study was to investigate whether polymorphisms of caspase-9 (CASP9) gene and RUNX3 are associated with IBD susceptibility and clinical phenotypes. We studied 555 Crohn's disease (CD) and 651 ulcerative colitis (UC) patients recruited from a single UK center. A total of 964 healthy Caucasian subjects were recruited as controls from general practitioner well person clinics in Oxfordshire. Fourteen single nucleotide polymorphisms (SNPs) of CASP9 and 11 SNPs of RUNX3 were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (homogenous MassEXTEND, hME, Sequenom?, Sequenom Inc., San Diego, CA). Linkage disequilibrium (LD) and haplotype association analysis were performed using 2ld and phase v2.0 software. No association of individual SNPs of CASP9 or RUNX3 with UC or CD was identified. The rs1052571 of CASP9 was associated with severe UC [P = 0.0034, odds ratio (OR) = 1.957, 95% confidence interval (CI) = 1.240-3.088]. Significant haplotype associations between CASP9 and IBD were identified, while no association of RUNX3 haplotypes with either UC or CD was found. Our findings suggested that CASP9 gene might be another IBD susceptibility gene.     

Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease

Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900. Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis. In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.     

Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll-Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-gamma and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-gamma-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-gamma+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial-lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD-even in an inactive state of disease-exert an increased capacity for IFN-gamma induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-gamma-producing CD8+ lymphocytes.     

Investigation of JAK2, STAT3 and CCR6 polymorphisms and their gene-gene interactions in inflammatory bowel disease

Genome-wide association studies identified many loci associated with the two forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC). Components of the interleukin-23 signalling pathway, such as IL23R, JAK2 and STAT3, have been implicated in both diseases. In addition, emerging evidence supports the role of IL23-driven Th17 cells in inflammation. Here, we studied the susceptibility nature of three components of IL23 signalling and Th17 cell differentiation: JAK2 rs10758669, STAT3 rs744166 and CCR6 rs2301436 initially associated with CD in Hungarian CD and UC patients. A total of 616 unrelated subjects with either form of IBD and 496 healthy controls were genotyped with PCR-RFLP methods. We also tested the genetic interactions of JAK2, STAT3 and CCR6 polymorphisms in a pairwise fashion with regard to disease risk. We could confirm the susceptibility of STAT3 rs744166 TT homozygotes for UC (OR: 1.483, 95% CI: 1.103-1.992, P = 0.009). Data on genetic interaction reveals that the above JAK2 and STAT3 risk alleles contribute to CD susceptibility in combination with each other (OR: 2.218; 95% CI: 1.097-4.487; P = 0.024), while the JAK2 variant shows a tendency to confer UC risk only on a wild-type STAT3 background (OR: 1.997, 95%CI: 0.994-4.009, P = 0.049). Our results may help in understanding how these natural variants contribute to development of IBD through their genetic association.     

Antineutrophil cytoplasmic antibodies, anti-Saccharomyces cerevisiae antibodies, and specific IgE to food allergens in children with inflammatory bowel diseases

Differential diagnosis between ulcerative colitis (UC) and Crohn's disease (CD) is difficult in the initial phases in pediatric patients with inflammatory bowel diseases (IBD). This study was performed to determine the significance of anti-neutrophil cytoplasmic antibodies (ANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA) in IBD. ANCA were specified with regard to their antigenic specifity, significance to the diagnosis, and correlation of titer with the disease activity. The occurrence of food allergy was questioned, too. Serum samples from 44 children with UC (n = 23) or CD (n = 21) and from disease-control children (coeliac disease, n = 21) were analyzed for IgG ANCA, ANCA target antigens, IgA and IgG ASCA, and IgE to food allergens. Results show that ANCA occur more frequently in UC than in CD and disease-control (74, 24, and 10%, respectively). The presence of ANCA does not reflect disease activity. Antigenic specificity does not differ in any group. IgA-ASCA are found more often in patients with CD (76% versus 17% in UC). The testing for both ANCA and ASCA enabled clear-cut differential diagnosis between UC and CD based on the high specificity (ANCA+ ASCA- 92.5% for UC, ANCA- ASCA+ 93.2% for CD). Specific IgE to food allergens were found in 8.7, 14.3, and 23.8% of patients with UC, CD, and coeliac disease, respectively. We conclude that combined testing of ANCA and ASCA represents a valuable tool in the differential diagnosis between UC and CD in pediatric patients, minimizing invasive diagnostic procedures. Monitoring of ANCA, its specificity, and titer determination does not bring more information. Testing for specific IgE to food allergens may be considered in individual patients.     

The functional genetic variation in the PTPN22 gene has a negligible effect on the susceptibility to develop inflammatory bowel disease

The aim of this study was to assess the possible association between the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene 1858C-->T (rs2476601, encoding R620W) polymorphism and inflammatory bowel disease (IBD). Our study population consisted of 1113 IBD [544 ulcerative colitis (UC) and 569 Crohn's disease (CD)] patients and 812 healthy subjects. All the individuals were of Spanish white origin. Genotyping of the PTPN22 gene 1858C-->T polymorphism was performed by real time polymerase chain reaction technology, using TaqMan 5'-allelic discrimination assay. The frequency of the PTPN22 1858T allele in healthy subjects was 6.2% compared with 6.7% in the UC patients and 5.1% in Crohn's patients. No statistically significant differences were observed when the PTPN22 1858C-->T allele and genotype distribution among CD patients, UC patients and healthy controls were compared. These results indicate that the PTPN22 1858C-->T polymorphism does not appear to play a major role in IBD predisposition in our population.     

Confirmation of association of IRGM and NCF4 with ileal Crohn's disease in a population-based cohort

Genome-wide association studies have identified PHOX2B, FAM92B, IRGM and NCF4 as candidate susceptibility factors for ileal Crohn's disease (CD). Here we sought to determine whether these genes were also associated with ileal CD in New Zealand Caucasians, as well as with ileocolonic CD, colonic CD and ulcerative colitis (UC). A total of 507 CD patients, 475 UC patients and 576 controls were genotyped for the single nucleotide polymorphisms rs16853571 (PHOX2B), rs4821544 (NCF4), rs13361189 and rs4958847 (IRGM), and rs8050910 (FAM92B). NCF4 and IRGM were significantly associated with ileal CD (P-value(rs4821544)=0.0090, odds ratio (OR)=1.425, 95% confidence interval (CI): 1.092-1.859; P-value(rs13361189)=0.0017, OR=1.942, 95% CI: 1.274-2.959; P-value(rs4958847)=0.0022, OR=1.767, 95% CI: 1.224-2.558), but not with other forms of inflammatory bowel disease (IBD). No association of PHOX2B or FAM92B with IBD was detected. Our study has demonstrated that IRGM and NCF4 are ileal-specific CD susceptibility factors in New Zealand Caucasians.     

Inducible and endothelial nitric oxide synthase genes polymorphism in inflammatory bowel disease

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.     

Evidence of linkage of the inflammatory bowel disease susceptibility locus on chromosome 16 (IBD1) to ulcerative colitis.

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) of unknown aetiology which are characterised by chronic inflammation of the gastrointestinal tract. Epidemiological studies suggest the presence of a genetic component in the aetiology of both CD and UC. A susceptibility gene for Crohn's disease has recently been mapped to the pericentromeric region of chromosome 16 (IBD1), and this finding has been replicated in two subsequent studies. Although CD and UC are distinct clinical entities, the fact that both disorders occur in a significant proportion of families with multiple cases of IBD suggests that overlapping sets of susceptibility genes may be involved. We have addressed this question for IBD1 by typing eight microsatellite markers from the locus in 70 kindreds affected with either UC only or with both UC and CD and analysing the data for linkage by both non-parametric and parametric methods. Evidence for linkage was detected in families affected with only UC, with a mean proportion of 0.70 affected sib pairs sharing alleles identical by descent at D16S3136 (p=0.01), and a peak non-parametric linkage score of 2.02 at D16S3120 with the GENEHUNTER program (p=0.02). The estimated sib relative risk attributable to IBD1 in these families was 1.46. Surprisingly, no evidence of linkage was detected in the families affected with both UC and CD (p>0.2). The data suggest that IBD1 may also contribute to susceptibility to ulcerative colitis, and that it is likely to be located in the 12 cM interval between D16S419 and D16S409.     

Mannan-binding lectin (MBL) gene polymorphisms in ulcerative colitis and Crohn's disease

The inflammatory bowel diseases (IBD), Crohn's disease (CD), and ulcerative colitis (UC), are complex multifactorial traits involving both environmental and genetic factors. Mannan-binding lectin (MBL) plays an important role in non-specific immunity and complement activation. Point mutations in codons 52, 54 and 57 of exon 1 of the MBL gene are associated with decreased MBL plasma concentrations and increased susceptibility to various infectious diseases. If these MBL mutations could lead to susceptibility to putative IBD-etiological microbial agents, or could temper the complement-mediated mucosal damage in IBD, MBL could function as the link between certain microbial, immunological and genetic factors in IBD. In this study, we investigated the presence of the codon 52, 54 and 57 mutations of the MBL gene in 431 unrelated IBD patients, 112 affected and 141 unaffected first-degree relatives, and 308 healthy control individuals. In the group of sporadic IBD patients (n = 340), the frequency of the investigated MBL variants was significantly lower in UC patients when compared with CD patients (P = 0.01) and with controls (P = 0.02). These results suggest that MBL mutations which decrease the formation of functional MBL could protect against the clinical development of sporadic UC, but not of CD. This could be explained by the differential T-helper response in both diseases.