Macrophage-stimulating protein polymorphism rs3197999 is associated with a gain of function: implications for inflammatory bowel disease

Crohn's disease and ulcerative colitis, the two main types of inflammatory bowel disease (IBD), were reported to be associated with a variety of genetic polymorphisms. A subset of these polymorphisms was identified in both diseases and only three of them were found in primary sclerosing cholangitis (PSC). rs3197999 (Arg689Cys) located in the MST1 gene is one of the most convincingly replicated IBD/PSC-associated polymorphisms but its functional consequences have not been investigated, yet. We expressed both MST1 gene variants (Arg(689) (MSP(wt)) and Cys(689) (MSP(mut)) in a eukaryotic cell system and compared their stimulatory effects on macrophage-like THP-1 cells. Except for the rate of apoptosis that remained unchanged, MSP(mut) significantly increased the stimulatory effect of MSP (macrophage-stimulating protein) on chemotaxis and proliferation by THP-1 cells, indicating a gain of function associated with the Arg689Cys exchange. A broad set of evidence reported previously suggests that pro-inflammatory changes in macrophage function have a major role in the initiation of the inflammatory process in IBD and PSC. Therefore, the gain of function observed with rs3197999 in MST1 might provide a cellular mechanism for the consistent association of this polymorphism with an increased risk for IBD and PSC.     

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.     

Combined segregation and linkage analysis of inflammatory bowel disease in the IBD1 region using severity to characterise Crohn's disease and ulcerative colitis. On behalf of the GISC

Inflammatory bowel disease (IBD) is a chronic relapsing disorder affecting the gastro-intestinal tract and is subdivided into two main subtypes: Crohn's disease (CD) and ulcerative colitis (UC). Although the aetiology of IBD is unknown, a strong genetic susceptibility is suggested and different candidate regions have been identified for both CD and UC. The IBD1 region on chromosome 16 has been confirmed to be important for susceptibility to CD, whereas conflicting evidence has been obtained for UC. We performed a combined linkage and segregation analysis in the identified IBD1 region on a sample of 82 extended families with IBD using a parametric method implemented in the computer program COMDS. This approach allows simultaneous evaluation of linkage while estimating the mode of inheritance and to include severity of the trait to characterise the CD and UC phenotypes. Our results are consistent with the presence of a major gene in the IBD1 region close to D16S408 involved in both UC and CD. Furthermore, our data support evidence that a single mutation in the gene leads more frequently to UC, whereas inheritance of two mutant alleles results in the more severe CD. In our study the IBD1 locus was found to have a major role in IBD predisposition in the Italian population.     

Population‐Specific Susceptibility to Crohn's Disease and Ulcerative Colitis; Dominant and Recessive Relative Risks in the Japanese Population

Crohn's disease (CD), a type of chronic inflammatory bowel disease (IBD), is commonly found in European and East Asian countries. The calculated heritability of CD appears to be higher than that of ulcerative colitis (UC), another type of IBD. Recent genome‐wide association studies (GWAS) have identified more than thirty CD‐associated genes/regions in the European population. In the East Asian population, however, a clear association between CD and an associated gene has only been detected with TNFSF15. In order to determine if CD susceptibility differs geographically, nine SNPs from seven of the European CD‐associated genomic regions were selected for analysis. The genotype frequencies for these SNPs were compared among the 380 collected Japanese samples, which consisted of 212 IBD cases and 168 controls. We detected a significant association of both CD and UC with only the TNFSF15 gene. Analysis by the modified genotype relative risk test (mGRR) indicated that the risk allele of TNFSF15 is dominant for CD, but is recessive for UC. These results suggest that CD and UC susceptibility differs between the Japanese and European populations. Furthermore, it is also likely that CD and UC share a causative factor which exhibits a different dominant/recessive relative risk in the Japanese population.     

Investigation of NOD1/CARD4 variation in inflammatory bowel disease using a haplotype-tagging strategy

Both NOD1/CARD4 and NOD2/CARD15 are intracellular pattern-recognition receptors involved in the innate immune response. Germline NOD2/CARD15 variation has a definite effect on susceptibility to Crohn's disease (CD) and phenotype, although this contribution is weak in Scotland and Scandinavia. The NOD1/CARD4 gene lies within the putative inflammatory bowel disease (IBD) locus at 7p14.3. We have assessed, in detail, the influence of germline NOD1/CARD4 variation on IBD susceptibility and phenotype in the Scottish population. Two thousand two hundred and ninety-six subjects, including 356 children with IBD, were involved in parallel case-control and family-based association studies. Nine tagging single-nucleotide polymorphisms (SNPs) were selected based on HapMap data spanning the whole of the NOD1/CARD4 gene. Our case-control SNP analysis was powered to detect an effect size with OR 1.5 for IBD and OR 1.6 for CD. No significant associations were observed between any of nine the NOD1/CARD4 SNPs studied and IBD, CD or ulcerative colitis (UC) (P > 0.05 for all). Haplotype case-control analysis was also negative (P > 0.05 in IBD, CD and UC). Multimarker transmission disequilibrium testing analysis was negative (P > 0.05 in IBD, CD and UC). NOD2/CARD15 variant carriage had no influence on NOD1/CARD4 effect on IBD susceptibility. This study represents the first application of a gene -wide haplotype-tagging approach to assess, in detail, the contribution of NOD1/CARD4 in IBD.     

Sex stratification of an inflammatory bowel disease genome search shows male-specific linkage to the HLA region of chromosome 6

Inflammatory bowel disease (IBD) is a multifactorial disorder, with both genetic and environmental factors contributing to the two clinical phenotypes of Crohn's disease (CD) and ulcerative colitis (UC). The underlying genetic model is thought to involve multiple genes with complex interactions between disease loci, and the NOD2 gene on chromosome 16 has recently been identified as a CD susceptibility locus. Several genome-wide linkage studies have identified candidate regions, but there has been little replication across studies. Here we investigate the role of sex-specific loci in susceptibility to IBD. Linkage data from our previously reported genome search and follow-up study were stratified by the sex of the affected sib pair. Non-parametric linkage analysis was performed using Genehunter Plus. Simulation studies were used to assess the significance of differences in LOD scores between male and female families for each chromosome. Several regions of sex-specific linkage were identified, including existing and novel candidate loci. The major histocompatibility region on chromosome 6p, referred to as IBD3, showed evidence of male-specific linkage with a maximum LOD score of 5.9 in both CD and UC male-affected families. Regions on chromosomes 11, 14 and 18 showed strong evidence of linkage in male-affected families but not in female-affected families. No evidence of sex-specific linkage was found in the IBD1 or IBD2 candidate regions of chromosomes 16 and 12. The existence of sex-specific linkage is further evidence of the complex mechanisms involved in IBD and will facilitate future studies to identify susceptibility genes.     

Association of CARD8 with inflammatory bowel disease in Koreans

Caspase recruitment domain (CARD)-containing protein 8 (CARD8) is a potential candidate risk gene for inflammatory bowel disease (IBD) because of its role as a component of the NALP3 inflammasome and as an inhibitor of nuclear factor-kappa B. Previous studies examining the association of a CARD8 single-nucleotide polymorphism (SNP) (rs2043211, p.Cys10X) with IBD yielded mixed results in Caucasians that may result from interaction with NALP3 or NOD2 (nucleotide-binding oligomerization domain 2) variants. To understand the genetic association between CARD8/NALP3 and IBD in Koreans, we investigated seven CARD8, four NALP3 and four NOD2 SNPs in 650 Crohn's disease (CD), 660 ulcerative colitis (UC) patients and 688 controls from the Korean population. rs2043211 of CARD8 showed significant association with UC (P = 0.011; odds ratio = 1.50, 95% confidence intervals = 1.12-2.00, P = 0.006 under recessive model). In contrast, an SNP in intron 1, rs1972619, was associated with CD only (P = 0.033). None of the NALP3 or NOD2 SNPs was significantly associated with CD or UC in the Korean populations. The stop allele of rs2043211 was associated with higher serum interleukin-1β levels only in female patients with UC (P = 0.027). Our data suggest that CARD8 variants might have roles in the pathogenesis of CD and UC in Koreans.     

Genetic variation in the IGSF6 gene and lack of association with inflammatory bowel disease.

The immunoglobulin superfamily 6 gene (IGSF6) on chromosome 16p11-p12 has been investigated as a positional and functional candidate for inflammatory bowel disease (IBD) susceptibility. Screening of the six exons of IGSF6 for single nucleotide polymorphisms (SNPs) detected four novel SNPs, and validated three of six SNPs listed in the international SNP database (dbSNP). The seven SNPs in IGSF6 formed five distinct linkage disequilibrium groups. There was no evidence for association of the common SNPs with disease in a large cohort of patients with IBD. The novel SNPs and the linkage disequilibrium map will be a useful resource for the analysis of IGSF6 in other immune disorders.     

Genetic variation in the IGSF6 gene and lack of association with inflammatory bowel disease

The immunoglobulin superfamily 6 gene (IGSF6) on chromosome 16p11‐p12 has been investigated as a positional and functional candidate for inflammatory bowel disease (IBD) susceptibility. Screening of the six exons of IGSF6 for single nucleotide polymorphisms (SNPs) detected four novel SNPs, and validated three of six SNPs listed in the international SNP database (dbSNP). The seven SNPs in IGSF6 formed five distinct linkage disequilibrium groups. There was no evidence for association of the common SNPs with disease in a large cohort of patients with IBD. The novel SNPs and the linkage disequilibrium map will be a useful resource for the analysis of IGSF6 in other immune disorders.     

Fine mapping of the IBD1 locus did not identify Crohn disease-associated NOD2 variants: implications for complex disease genetics

Crohn disease (CD) is a chronic relapsing inflammatory condition of the gastrointestinal tract. Recently, polymorphisms in NOD2 (CARD15), a gene mapping to the chromosome 16 IBD1 susceptibility locus, have been associated with susceptibility to CD. One group identified the gene by using classic positional cloning methods. Here, we report linkage and fine mapping analyses using 27 microsatellite markers encompassing the IBD1 susceptibility locus in 131 CD affected sibling pairs, and a simplex family cohort. No evidence for linkage was observed, and microsatellite markers close to NOD2 did not show association. However, significant association was confirmed in 294 CD trios for the NOD2 variants Arg702Trp and Leu1007fsinsC. Our fine mapping study of the IBD1 locus did not enable us to identify NOD2 as a CD gene, despite the presence of association with disease-causing alleles. This study illustrates the difficulties facing microsatellite linkage and linkage disequilibrium mapping methods for identifying disease genes in complex traits.     

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.     

Polymorphisms in multidrug resistance 1 (MDR1) gene are associated with refractory Crohn disease and ulcerative colitis

We used coding and noncoding polymorphisms evenly spaced across the ABCB1/MDR1 gene to perform association analysis in Slovenian patients with inflammatory bowel diseases and to obtain haplotype structure and patterns of linkage disequilibrium (LD) in the MDR1 gene. A disease association study was performed in 307 IBD patients, including 144 patients with ulcerative colitis (UC) and 163 patients with Crohn's disease (CD), and 355 healthy controls. Here we report an association between MDR1 alleles, polymorphisms and haplotypes and refractory CD patients, who do not respond to standard therapy, including patients who develop fistulas. We also report an association with UC and MDR1 polymorphisms in a Slovenian population. Haplotypes significantly associated with diseases were defined by single-nucleotide polymorphisms (SNPs) in exons 12 (1236 C>A), 21(A893S), and 26 (3435 C>T). In addition, two intronic SNPs in LD with the disease haplotype, one in intron 13 (rs2235035) and another in intron 16 (rs1922242), were significantly associated with refractory Crohn (P=0.026, odds ratio (OR) 2.7 and P=0.025, OR 2.8, respectively), as well as with UC (P=0.006, OR 1.8 and P=0.026, OR 1.9, respectively). Our results suggest that MDR1 is a potential target for therapy in refractory CD patients and in patients with UC.     

Increased expression of NKX2.3 mRNA transcribed from the risk haplotype for ulcerative colitis in the involved colonic mucosa

NKX2.3 is a promising candidate for susceptibility genes to inflammatory bowel disease (IBD). The aim of this study was to perform a candidate gene analysis of NKX2.3 in Japanese IBD and to examine how the risk allele (haplotype) affects susceptibility to IBD using allelic expression ratios of NKX2.3 mRNA in the involved colonic mucosa. A total of 344 patients with Crohn's disease (CD), 253 patients with ulcerative colitis (UC), and 243 healthy controls (HCs) were genotyped for 3 tag-single nucleotide polymorphisms (SNPs; rs10883365, rs888208, and rs11596008) around NKX2.3. The allelic expression ratio of NKX2.3-mRNA was examined by TaqMan assay using rs888208 as an allelic (haplotypic) marker. Two SNPs (rs10883365 and rs888208) were significantly associated with UC (p = 7.79 × 10(-4), odds ratio [OR] = 1.54 [95% confidence interval (95% CI) 1.20-1.99], p = 7.70 × 10(-3), OR = 1.41 [95% CI 1.10-1.81], respectively) and 1 SNP (rs10883365) was associated with CD (p = 0.0366, OR = 1.29 [95% CI 1.02-1.63]). Haplotype B formed by the 3 SNPs demonstrated a significant association with UC (p = 6.11 × 10(-4), OR = 1.56 [95% CI 1.21-2.00]). Subgroup analyses indicated that rs10883365 was significantly associated mainly with colonic CD (p = 1.99 × 10(-3), OR = 1.91 [95% CI 1.27-2.88], vs HCs). The allelic expression ratios of NKX2.3 mRNA transcribed from haplotype B (risk haplotype) to haplotype A (the nonrisk haplotype) in the involved mucosa from 10 IBD patients were significantly higher than the allelic ratio of respective genomic DNA (p = 0.00195). We confirmed the association of SNP rs10883365 located in the 5' flanking region of NKX2-3 with Japanese UC and colonic CD and determined the risk haplotype (haplotype B) for UC. The demonstrated allelic expression imbalance supports the idea that the risk haplotype of NKX2.3 confers susceptibility to UC through increasing expression of NKX2.3 mRNA in the colonic mucosa.     

Genetic analysis in Finnish families with inflammatory bowel disease supports linkage to chromosome 3p21

In inflammatory bowel diseases (IBD), certain chromosomal candidate loci have been repeatedly identified by independent studies in different populations. To investigate the contribution of the loci on chromosomes 1, 3, 7, 12, 14, and 16 to the susceptibility of IBD in Finnish population, where the predominant feature is the excess of ulcerative colitis (UC) families compared to Crohn's disease (CD) families, we carried out linkage analyses using 93 Finnish, multiply-affected IBD families. We observed nominal evidence for linkage to chromosome 3p21, consistent with earlier reports. The lod scores peaked at D3S2432, with a maximum two-point lod score of 1.68 (P=0.0027). In addition, we studied whether risk of IBD is associated with functional variants of two positional candidate genes; the chemokine receptor CCR5 gene on chromosome 3p21 and the interleukin-4 receptor alpha-subunit gene (IL4RA) on chromosome 16. We did not find any significant correlation between a 32-bp deletion variant of CCR5 or a single nucleotide change A1902G (Gln576Arg) of IL4RA, and IBD phenotypes, with the exception that in the UC group homozygosity for the G1902 allele of IL4RA was less frequent (0.019 vs 0.049, P=0.038). In conclusion, our study, carried out in a genetically homogenous population, suggests that chromosome 3 may contain a susceptibility gene for IBD.     

Genetic association between CARD9 variants and inflammatory bowel disease was not replicated in a Chinese Han population

Objective: In order to investigate whether CARD9 gene is associated with IBD in Chinese Han population, we replicated 2 SNPs of CARD9 which have been reported to be significantly associated with IBD. Methods: Two SNPs were genotyped using polymerase chain reaction with sequence-specific primers in 288 patients (232 CD patients, 56 UC patients) and 274 controls. Results: The frequencies and distributions of alleles and genotypes of the tested SNPs were analyzed, and no significant differences were found between patients and controls. Conclusions: We observed no significant association between the investigated CARD9 SNPs and the susceptibility of either CD or UC. Further studies with larger sample size focusing on different ethnicities are required to elucidate the correlation between CARD9 and 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.     

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.     

Replication of genetic variation in the MYO9B gene in Crohn's disease

Various genes that may influence the intestinal barrier have been identified, including MAGI2, PARD3, and MYO9B. These genes are associated with inflammatory bowel disease (IBD) in several European studies. A total of 2,049 individuals (656 Crohn's disease [CD], 544 ulcerative colitis [UC], and 849 controls) were genotyped and association studies were performed for 1 single nucleotide polymorphism (SNP) in MAGI2, 1 SNP in PARD3, and 6 SNPs in MYO9B. We reported an association between 3 SNPs in MYO9B and ileal involvement with rs1457092 as the most significant SNP (p = 0.0073, odds ratio [OR] 0.69 [95% confidence interval (95% CI) 0.52-0.90]). The nonsynonymous SNP rs1545620 exhibited a p value of 0.014, OR 0.72 (95% CI 0.55-0.93). MYO9B was not associated with UC. MAGI2 or PARD3 was not associated with IBD. A 6-SNP haplotype block in MYO9B demonstrated association with CD and ileal CD (p = 0.0030 and 0.0065, respectively). These data demonstrate an association of MYO9B with ileal CD; however, there was no association of MAGI2 and PARD3 with IBD. Because the direction of association of MYO9B in this Canadian study was not consistent with European studies, further studies are needed to elucidate the role of MYO9B in IBD.