Genetic Association of DLG5 R30Q with Familial and Sporadic Inflammatory Bowel Disease in Men

Background: The association of DLG5 R30Q with IBD has been replicated in several populations, but is not statistically significant in others. We studied the incidence of DLG5 alleles in a population of IBD patients from Pennsylvania. Methods: DLG5 R30Q (rs1248696) and G1066G (rs1248634) were analyzed with PCR-based RFLP methods in a total of 521 subjects, that included 105 individuals with IBD and 139 without IBD from a familial IBD registry, 107 with sporadic IBD, and 170 unrelated healthy controls. R30Q was further analyzed with SNPlex™ Genotyping System in 473 samples. Results: RFLP genotyping data showed that, DLG5 R30Q was significantly associated with IBD overall (p=0.006), and separately with CD (p=0.009) and UC (p=0.024). The association of R30Q with IBD was entirely due to a male-associated effect (male vs female p=0.015 vs 0.241 (IBD), p=0.024 vs 0.190 (CD), and p=0.019 vs 0.575 (UC)). The frequency of the A allele carriage was elevated in both affected and unaffected members in the familial IBD cohort compared to healthy controls (p=0.037). In the family pedigrees, we observed differences in the expression of IBD in individuals carrying the A allele between families. Conclusions: In the studied population, DLG5 R30Q was associated with all forms of IBD. An elevated presence of the R30Q variant was observed in all members of a familial IBD registry. This association of the R30Q variant with IBD was male-specific.     

Extreme heterogeneity in CARD15 and DLG5 Crohn disease-associated polymorphisms between German and Norwegian populations

The first gene associated with Crohn disease (CD) has been identified as CARD15 (16q12). Three variants, R702W, G908R and 1007fsinsC are strongly and independently associated with the disease. A second gene, conveying a smaller risk for inflammatory bowel disease (IBD), has been identified as DLG5 (10q23). We assess the frequency of the CARD15 SNPs and of the R30Q mutation in DLG5 and their contribution to the development of CD in a cohort of unrelated IBD patients (151 CD, 325 ulcerative colitis (UC)) and healthy controls (236) from South-east Norway (IBSEN cohort). Genotype-based tests of population differentiation using 23 SNPs across CARD15, together with estimates of F(ST), indicated that the German and Norwegian background populations could be differentiated at the CARD15 locus. The Norwegian and German CD samples exhibited particularly strong differentiation at the three predisposing loci and those marking their background haplotype. There were significantly lower frequencies of the CARD15 SNPs and no significant association with CD in the Norwegian samples. Only a marginal association was observed for the subphenotypes ileitis and ileocolitis vs colitis (P=0.048). The population attributable risk percentage (PAR%) for CARD15 variants in the Norwegian cohort is the lowest reported for a European population (1.88%), except Iceland. Similarly, the DLG5 variant showed no association with CD or IBD, however, there was a negative correlation with stricture (P=0.035). The present results are consistent with an emerging pattern of a low frequency of the CARD15 variants in Northern countries where the prevalence of IBD is greatest.     

DLG5 variants contribute to Crohn disease risk in a Canadian population

Variants in the gene encoding the DLG5 scaffolding protein have been reported to be associated with increased risk of inflammatory bowel disease (IBD) and particularly Crohn's disease (Crohn disease; CD). These findings have not been uniformly replicated in follow-up studies. In this study we genotyped a cohort of 402 Canadian CD and 179 ulcerative colitis (UC) patients and 537 healthy controls for three IBD/CD-associated DLG5 variants. Our data reveal that the common DLG5 haplotype (A), which was previously considered protective for IBD, is associated with modest increases in risk for IBD (P=0.02) and CD (P=0.04). The effects of haplotype copy number on risk for IBD were minor, with the odds ratio (ORs) being 1.37 for the heterozygous risk genotype and 1.7 for the homozygous risk genotype. While we were unable to replicate the proposed association between the DLG5 c.113G>A variant and IBD, an association of IBD (P=0.02) and CD (P=0.04) with the rarer c.4136C>A variant was replicated in this cohort. These associations were restricted to the non-Jewish subjects in this cohort and were not detected in the Ashkenazi Jewish population studied here. Within the non-Jewish group, no associations were detected between the DLG5 variants and specific phenotypic features, such as site of disease, and there was no evidence of epistasis between DLG5 and any of the CD-associated CARD15 or SLC22A4/A5 gene variants. Together, the results indicate a role for DLG5 variants in IBD susceptibility and suggest that further studies are warranted to evaluate this role in different IBD populations and to determine the functional pathways that couple DLG5 variants to IBD.     

The CREM gene is involved in genetic predisposition to inflammatory bowel disease in the Tunisian population

The identification of susceptibility genes for inflammatory bowel disease (IBD) is key to understanding pathogenic mechanisms. Recently, the results of genetic association studies have highlighted many loci that are shared among several autoimmune diseases. We aimed to study the genetic epidemiology of polymorphisms in specific genes previously associated with other autoimmune diseases, namely the CREM, STAT4, STAT5a, Stat5b, and IRF5 genes. Twelve polymorphisms in the CREM, STAT4, STAT5a, Stat5b, and IRF5 genes were genotyped in a cohort of 107 IBD patients (39 Crohn's disease [CD] and 68 ulcerative colitis [UC]) and 162 controls from southern Tunisia. One CREM single nucleotide polymorphism (SNP) displayed evidence for genetic association with IBD (p=8.7×10(-4), odds ratio [OR]=2.84 [1.58; 5.09]). One STAT4 SNP (p=0.026; OR=1.65 [1.06; 2.58]) exhibited a marginal association with UC but not with CD. No significant association was observed with the SNPs in STAT5a, IRF5, and STAT5b. These results suggest that common variants of the CREM gene are involved in the genetic component conferring general susceptibility to IBD, whereas STAT4 appears to be more specifically associated with UC. This work provides motivation for studies aiming to replicate these findings in larger populations.     

The role of inflammatory bowel disease susceptibility loci in multiple sclerosis and systemic lupus erythematosus

To date, three loci have been validated to confer susceptibility to inflammatory bowel disease (IBD): the CARD15/NOD2 gene, the discs large homolog 5 gene (DLG5), and the IBD5 locus on 5q31 (IBD5). We have explored the possibility that these loci may also be associated with susceptibility to two other chronic inflammatory diseases, multiple sclerosis (MS) and systemic lupus erythematosus (SLE). As the CARD15 risk alleles had previously been assessed in our collection of 496 MS trios, we focused our efforts on the DLG5 risk allele and the IBD5(risk) haplotype (IBD5(risk)) for MS. While there is no evidence of association within our MS sample with either of these polymorphisms, screening of 1027 subjects with SLE suggests that IBD5(risk) may have a modest contribution to disease risk in the subset of SLE subjects without lupus nephritis. In addition, a pooled analysis of existing published and unpublished data in 1305 cases of SLE genotyped for the CARD15 risk alleles suggests that only the CARD15(908R) IBD risk allele may have a strong effect on risk of SLE. Our data, therefore, suggest that both the CARD15 gene and the IBD5 locus may have a role as general susceptibility loci for certain common, genetically complex inflammatory diseases.     

Gene-centric association mapping of chromosome 3p implicates MST1 in IBD pathogenesis

Association mapping and candidate gene studies within inflammatory bowel diseases (IBD) linkage regions, as well as genome-wide association studies in Crohn's disease (CD) have led to the discovery of multiple risk genes, but these explain only a fraction of the genetic susceptibility observed in IBD. We have thus been pursuing a region on chromosome 3p21–22 showing linkage to CD and ulcerative colitis (UC) using a gene-centric association mapping approach. We identified 12 functional candidate genes by searching for literature cocitations with relevant keywords and for gene expression patterns consistent with immune/intestinal function. We then performed an association study composed of a screening phase, where tagging single nucleotide polymorphisms (SNPs) were evaluated in 1,020 IBD patients, and an independent replication phase in 745 IBD patients. These analyses identified and replicated significant association with IBD for four SNPs within a 1.2 Mb linkage disequilibrium region. We then identified a non-synonymous coding variant (rs3197999, R689C) in the macrophage-stimulating 1 (MST1) gene (P-value 3.62 × 10^–6) that accounts for the association signal, and shows association with both CD and UC. MST1 encodes macrophage-stimulating protein (MSP), a protein regulating the innate immune responses to bacterial ligands. R689C is predicted to interfere with MSP binding to its receptor, suggesting a role for this gene in the pathogenesis of IBD.     

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.     

Association of celiac disease genes with inflammatory bowel disease in Finnish and Swedish patients

Some genetic loci may affect susceptibility to multiple immune system-related diseases. In the current study, we investigated whether the known susceptibility loci for celiac disease (CelD) also associate with Crohn's disease (CD) and/or ulcerative colitis (UC), the two main forms of inflammatory bowel disease (IBD), in Finnish patients. A total of 45 genetic markers were genotyped in a Finnish data set comprising 699 IBD patients and 2482 controls. Single-marker association with IBD and its subphenotypes was tested. A meta-analysis with a Swedish UC data set was also performed. A total of 12 single-nucleotide polymorphisms associated with CD and/or UC (P<0.05). In the subphenotype analysis, rs6974491-ELMO1 (P=0.0002, odds ratio (OR): 2.20) and rs2298428-UBE2L3 (P=5.44 × 10(-5), OR: 2.59) associated with pediatric UC and CD, respectively. In the meta-analysis, rs4819388-ICOSLG (P=0.00042, OR: 0.79) associated with UC. In the subphenotype meta-analysis, rs1738074-TAGAP (P=7.40 × 10(-5), OR: 0.61), rs6974491-ELMO1 (P=0.00052, OR: 1.73) and rs4819388-ICOSLG (P=0.00019, OR: 0.75) associated with familial UC, pediatric UC and sporadic UC, respectively. Multiple CelD risk loci also confer susceptibility for CD and/or UC in the Finnish and Swedish populations. Certain genetic risk variants may furthermore predispose an individual for developing a particular disease phenotype.     

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.     

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.     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Genetic variants and monoallelic expression of surfactant protein-D in inflammatory bowel disease

Surfactant protein-D (SP-D) is expressed on mucosal surfaces and functions in the innate immune response to microorganisms. We studied the genetic association of the two nonsynonymous SP-D single nucleotide polymorphisms (SNPs) rs721917 and rs2243639 in 256 inflammatory bowel disease (IBD) cases (123 CD and 133 UC) and 376 unrelated healthy individuals from an IBD population from Central Pennsylvania. Case-control analysis revealed a significant association of rs2243639 with susceptibility to Crohn's disease (CD) (p= 0.0036), but not ulcerative colitis (UC) (p= 0.883), and no association of rs721917 with CD (p= 0.328) or UC (p= 0.218). Using intestinal tissues from 19 individuals heterozygous for each SNP, we compared allelic expression of these two SNPs between diseased and matched normal tissues. rs2243639 exhibited balanced biallelic (BB) expression; while rs721917 exhibited differential allelic expression (BB 37%, imbalanced biallelic [IB] 45%, and dominant monoallelic [DM] 18%). Comparison of allelic expression pattern between diseased and matched normal tissues, 13 of 19 individuals (14 UC, 5 CD) showed a similar pattern. The six patients exhibiting a different pattern were all UC patients. The results suggest that differential allelic expression may affect penetrance of the SNP rs721917 disease-susceptibility allele in IBD. The potential impact of SP-D monoallelic expression on incomplete penetrance is discussed.     

Association of interleukin-18 gene single-nucleotide polymorphisms with susceptibility to inflammatory bowel disease

Interleukin-18 (IL-18) is believed to be one of the most important cytokines in the pathogenesis of inflammatory bowel disease (IBD). The aim of the study was to clarify the significance of single-nucleotide polymorphisms (SNPs) at the 5'-end of the IL-18 gene in the development of IBD. DNA was obtained from peripheral blood of 99 patients with ulcerative colitis (UC), 79 patients with Crohn's disease (CD), and 102 healthy controls. All participants were Japanese. SNPs at -656G/T, -607C/A, -137G/C, +113T/G, and +127C/T were determined by means of direct sequencing, and a genetic association with IBD was examined. The frequencies of the G allele at +113 and the T allele at +127 were significantly higher in patients with CD and UC compared with controls. The differences in allelic frequencies were more striking in patients with CD than in patients with UC, and at position +127 than at position +113. The haplotype estimation, according to the E-M algorithm, suggested that TACGT is closely associated with IBD, especially with CD. It was concluded that SNPs at the 5'-end of IL-18 gene might be closely related to the etiology of IBD.     

Genetic analysis in Italian families with inflammatory bowel disease supports linkage to the IBD1 locus--a GISC study.

Epidemiological studies suggest that inherited factors influence susceptibility to inflammatory bowel disease (IBD), and some candidate loci have been described. In order to verify whether the same loci are responsible for predisposition to IBD in our population, we carried out a linkage study in a series of 58 Italian families with Crohn's disease (CD) and ulcerative colitis (UC). HLA-DQ alleles, motilin gene, and 34 microsatellites flanking the previously described loci on chromosomes 3, 6, 7, 12 and 16 were analysed by non-parametric linkage analysis in 16 and 23 families with CD and UC, respectively, and in 19 families where CD and UC coexisted. Non parametric analysis using GENEHUNTER yielded maximum NPL scores for marker D16S408 in all IBD families combined (2.71, P = 0.003), for marker D16S419 in CD (1.97, P = 0.026) and for marker D16S514 in UC families (2.44, P = 0.007). These markers map in the previously described IBD1 region. No significant linkage was found for markers of chromosomes 3, 6, 7 and 12. The present study performed in a Southern European population provides additional support for the conclusion with the IBD1 locus has a clear role in the genetic susceptibility to IBD.     

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.     

HLA class II haplotype associations with inflammatory bowel disease in Jewish (Ashkenazi) and non-Jewish caucasian populations

Ulcerative colitis (UC) and Crohn's disease (CD) are the clinical entities comprising idiopathic inflammatory bowel disease (IBD). Previous studies on the association of IBD and human leukocyte antigen (HLA) class II genes suggested a role for HLA in this disease. Here we present HLA class II (DRB1, DQB1, DQA1, DPB1) allele and haplotype distributions determined using the polymerase chain reaction and sequence-specific oligonucleotide probe methods. A total of 578 UC and CD Caucasian patients and controls from Jewish (Ashkenazi) and non-Jewish populations was examined. Our previously reported association of DR1-DQ5 with CD was attributable to DRB1*0103. A dramatic association with IBD and the highly unusual DRB1*0103-DQA1*0501-DQB1*0301 haplotype (OR = 6.6, p = 0.036) was found. The more common DR1 haplotype, DRB1*0103-DQA1*0101-DQB1*0501, was also associated with IBD (OR = 3.1, p = 0.014), a result suggesting that interaction between DR and DQ may determine the extent of disease risk. Our previously reported association of DR2 with UC was attributable to DRB1*1502 (OR = 2.6, p = 0.006). At the DPB1 locus, a significant association of DPB1*0401 with CD was observed for the combined populations (OR = 1.85, p = 0.007). These observations indicate that some class II alleles and haplotypes confer susceptibility to both UC and CD, implying common immunogenetic mechanisms of pathogenesis, while others confer risk to only one of these diseases, and illustrate the value of DNA HLA typing in disease susceptibility analyses.     

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.