Kenichi Negoro

Muramyl dipeptide and toll-like receptor sensitivity in NOD2-associated Crohn's disease

Both NOD2 (CARD15) alleles are mutated in roughly 15% of patients with Crohns disease, but functional effects are unclear. We analysed the cytokine response of peripheral blood mononuclear cells to muramyl dipeptide (MDP), the ligand for NOD2. MDP induced little TNFalpha or interleukin 1beta, but strong interleukin-8 secretion. MDP also substantially upregulated secretion of TNFalpha and interleukin 1beta induced by toll-like receptor ligands. These effects were abolished by the most common Crohns NOD2 double mutant genotypes at low nanomolar MDP concentrations, and provide the basis to develop a test of NOD2 functional deficiency. In Crohns disease, there are defects in neutrophil recruitment driven by NOD2 and interleukin 8 and in cross talk between the NOD2 and toll-like receptor pathways, which suggests that the immune system fails to receive an early priming signal.

Analysis of the IBD5 locus and potential gene-gene interactions in Crohn’s disease

Background and aims: Genetic variation in the chromosome 5q31 cytokine cluster (IBD5 risk haplotype) has been associated with Crohn’s disease (CD) in a Canadian population. We studied the IBD5 risk haplotype in both British and Japanese cohorts. Disease associations have also been reported for CARD15/NOD2 and TNF variants. Complex interactions between susceptibility loci have been shown in animal models, and we tested for potential gene-gene interactions between the three CD associated loci. Methods: Family based association analyses were performed in 457 British families (252 ulcerative colitis, 282 CD trios) genotyped for the IBD5 haplotype, common CARD15, and TNF−857 variants. To test for possible epistatic interactions between variants, transmission disequilibrium test analyses were further stratified by genotype at other loci, and novel log linear analyses were performed using the haplotype relative risk model. Case control association analyses were performed in 178 Japanese CD patients and 156 healthy controls genotyped for the IBD5 haplotype. Results: The IBD5 haplotype was associated with CD (p=0.007), but not with UC, in the British Caucasian population. The CARD15 variants and IBD5 haplotype showed additive main effects, and in particular no evidence for epistatic interactions was found. Variants from the IBD5 haplotype were extremely rare in the Japanese. Conclusions: The IBD5 risk haplotype is associated with British CD. Genetic variants predisposing to CD show heterogeneity and population specific differences.

Further Evidence of IBD5/CARD15 (NOD2) Epistasis in the Susceptibility to Ulcerative Colitis

To the Editor: We read with interest the two recent articles describing analyses of the IBD5 (MIM 606348) risk haplotype and inflammatory bowel disease (IBD) in European cohorts (Giallourakis et al. 2003; Mirza et al. 2003). In both European cohorts, the association with the IBD5 risk haplotype and Crohn disease (CD [MIM 266600]) was replicated. Mirza et al. (2003) additionally provided evidence for interaction between IBD5 and CARD15 (NOD2) (MIM 605956) in CD, a finding not seen in the subsequent paper by Giallourakis et al. (2003) or in our own transmission/disequilibrium testing (TDT) analysis (Negoro et al. 2003). Subsequent genotype-phenotype analysis by Giallourakis et al. (2003) found no association between IBD5 and clinical subgroups of patients with CD. In contrast, our group has recently published a case-control study of U.K. whites demonstrating that the association between IBD5 and CD was confined to those individuals with perianal disease (Armuzzi et al. 2003). It would be interesting to know whether association with this particular phenotype was evaluated in the Giallourakis cohort. In 187 trios with ulcerative colitis (UC [MIM 191390]), Giallourakis et al. (2003) also reported a novel association between the IBD5 risk haplotype and UC. This association was most pronounced in those individuals possessing one of the three common CD-associated CARD15 variant alleles, suggesting an epistatic relationship between these replicated CD loci in patients with UC. In our British patients, we observed a similar linkage disequilibrium (LD) pattern across this locus. However, using both TDT (105 transmissions to 124 nontransmissions, P=.24) and case-control studies, we were unable to demonstrate association between the IBD5 risk haplotype and UC (Armuzzi et al. 2003; Negoro et al. 2003). However, stratification of the IBD5 results in UC by CARD15 status was not performed. Following the report by Giallourakis et al. (2003), we therefore stratified our trios to assess the transmissions of IGR2060a_1 (an IBD5 risk haplotype–tagging SNP) from heterozygous parents to affected offspring who also carried at least one CARD15 risk allele, revealing 14 transmissions to 12 nontransmissions (P=.78). The lack of association may reflect a true relationship, or it may be a type I error due to the relatively weak power of this analysis. We therefore genotyped for the three common CARD15 variants and IBD5 haplotype–tagging SNP to assess any epistatic association in a more powerful case-control study of 278 patients with UC (largely independent of the UC trios) and 232 healthy controls (HC). We found a novel association between the CARD15 702Trp variant and UC (table 1). This association was not significant in the IBD5 wild-type homozygotes but became significant in the IBD5 heterozygotes and even more significant in the IBD5 “risk” homozygotes, supporting the theory of an epistatic relation between the IBD5 locus and CARD15 in the susceptibility to UC (table 1). However, there was no such relationship between UC and the 908Arg or Leu1007fsinsC CARD15 alleles (908Arg: HC 0.65%, UC 0.73%, P=.73; Leu1007fsinsC: HC 2.14%, UC 0.74%, P=.057), despite IBD5 stratification (data not shown). We found no particular UC phenotype (need for surgery, age at onset, disease distribution) associated with the CARD15 702Trp allele, with or without IBD5 stratification. Table 1 Case-Control Analysis of CARD15 702Trp Allele Association with UC, Stratified by IBD5 Status We believe that these data confirm the presence of an IBD5/CARD15 epistatic relationship in the susceptibility to UC (although the overall population effect is relatively small [table 1]), in contrast to the non-epistatic relationship between IBD5/CARD15 and CD seen in both our populations and that of Giallourakis et al. (2003). Our data, however, suggest that this epistatic effect is seen exclusively with the 702Trp variant, supporting other data that imply that the 702Trp polymorphism may possess unique properties not shared with the other CARD15 CD-associated variants (Bonen et al. 2003; Rahman et al. 2003; Sugimura et al. 2003). Indeed, our data suggest a trend toward undertransmission/reduced allele frequency of 908Arg/Leu1007fsinsC in UC (table 2 and the paragraph above). Further work is needed to determine whether the epistatic IBD5/CARD15 interaction in UC is a “global” CARD15 phenomenon or, as we have suggested, is restricted to the 702Trp allele. These data support the theory that UC and CD are related polygenic conditions that share some, but not all, susceptibility genes. Table 2 Transmissions versus Nontransmissions in Common CD-Associated CARD15 Variants from the TDT of 244 UC Trios

Association study of TNFSF15 polymorphisms in Japanese patients with inflammatory bowel disease

Tumour necrosis factor superfamily (TNFSF) 15 is a novel member of the TNFSF and its mRNA and protein expression is upregulated in inflammatory bowel disease (IBD), particularly in Crohn’s disease (CD).1,2 Recently, Yamazaki et al performed a large scale case control study using single nucleotide polymorphism (SNP) markers and reported that polymorphisms in TNFSF15 conferred susceptibility to CD in both Japanese and UK populations.3 They also suggested a potential association between a Caucasian ulcerative colitis (UC) cohort and TNFSF15 , but this association was not studied in Japanese patients. To investigate this possible association between TNFSF15 and Japanese UC, and to replicate this association with CD in Japanese, we performed a case control association study in Japanese patients …

Clinical course and longterm prognosis of Japanese patients with Crohn's disease: predictive factors, rates of operation, and mortality.

BackgroundThe purpose of this study was to clarify the long-term course of Crohn’s disease (CD) and predictors of its prognosis in Japan.MethodsThis was a historical cohort study of 276 patients with CD who had been diagnosed between 1965 and 1998. The clinical course was evaluated by the course of the CD score (CCDS) according to the required treatments. The predictive factors were examined by stepwise regression test. The cumulative rates of operation and survival were calculated by the Kaplan-Meier method.ResultsPatients with colitis-type CD had significantly lower annual and cumulative operation rates than those with other types, and showed significantly better progress, estimated by the CCDS, than patients with ileocolitis type. Reliable predictors for the 2- to 5-year clinical course after starting treatment were the CCDS, the presence of laparotomy during the initial year, and onset at age 30 years or more. The predictors for the 6- to 10-year clinical course were the duration of symptoms at diagnosis and onset at age 16 years or less. The predictors for the 11- to 15-year clinical course were the CCDS, the maximum International Organization of the Study of Inflammatory Bowel Disease (IOIBD) assessment score during the first year after starting treatment, and the effectiveness of the initial treatment. Relative survival rates at 5, 10, 15, and 20 years after the onset were 98.9%, 98.1%, 97.7%, and 94.9%, respectively.ConclusionsCD patients with colitis type showed a better clinical course and had significantly different clinical features compared with the patients with ileitis and ileocolitis type. Prediction of the longterm course of CD is possible by using clinical factors during the first year after starting treatment. The relative survival rates in Japanese patients with CD are not different from those seen in Western countries.

CARD15/NOD2 mutational analysis in Japanese patients with Crohn's disease

To the Editor: Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract and is considered to be a complex, multifactorial disease partly determined by a genetic predisposition. Among the putative CD-susceptibility loci, the pericentromeric region of chromosome 16, designated as IBD1, is most widely and consistently confirmed in Western populations (1, 2). Recently, three independent studies, one from the United States (3) and two from Europe (4, 5), have demonstrated that the three main variants (R702W, G908R, 3020insC) of CARD15/NOD2 (CARD15), located on chromosome 16q12, are associated with CD. In Japanese patients with CD, however, a genome-wide linkage analysis has not yet been performed and our collaborative research could not find any of the main variants of CARD15 (6). As our previous collaborative research was limited to

TNFSF15 transcripts from risk haplotype for Crohn's disease are overexpressed in stimulated T cells

TNFSF15 is a susceptibility gene for Crohns disease (CD). It remains to be elucidated how the associated single nucleotide polymorphisms (SNPs) in TNFSF15 affect the susceptibility to CD. Because there are no non-synonymous SNPs in TNFSF15, we speculated that one or more of the SNPs associated with CD may act as cis-regulatory SNPs. To reveal the effects of the SNPs on the transcriptional activity of TNFSF15, we first examined the allelic expression imbalance of TNFSF15 in peripheral blood mononuclear cells (PBMCs). When PBMCs stimulated by phytohemagglutinin (PHA) were examined, the allelic ratio of mRNA transcribed from the risk haplotype to the non-risk haplotype increased, compared with the ratio without stimulation. When peripheral blood T cells and Jurkat cells stimulated by phorbol 12-myristate 13-acetate + ionomycin were examined, an allelic expression imbalance similar to that observed in PBMCs stimulated by PHA was confirmed. The promoter assay in stimulated Jurkat cells showed that the luciferase activity of the promoter region (-979 to +35) of the risk haplotype was significantly higher than that of the non-risk haplotype, and deletion and mutagenesis analysis demonstrated that this difference resulted from the -358T/C SNP. The promoter activity of -358C (risk allele) was higher than that of -358T (non-risk allele) in stimulated T cells. This effect of -358T/C on the transcriptional activity in stimulated T cells may confer susceptibility to CD.

Lack of association between IBD5 and Crohn's disease in Japanese patients demonstrates population-specific differences in inflammatory bowel disease

Objective. Population-specific differences in the genetic susceptibility to inflammatory bowel disease (IBD) are indicated by the fact that Crohns disease (CD) in Japanese patients does not have any of the common CARD15 variants that are associated with CD in Caucasians. Recently, the disease-causing mutation in the IBD5 haplotype was identified. The TC haplotype, composed of L503F in SLC22A4 and −207G/C in SLC22A5 promoters, was reported to alter the function of the organic cation transporter and to be associated with CD in Caucasians. To determine whether the TC haplotype is also associated with IBD in a Japanese population, we genotyped L503F and −207G/C variants in Japanese subjects. Furthermore, we also performed a case-control association study with all representative single nucleotide polymorphisms (SNPs) in IBD5 using previous information of linkage disequilibrium extension reported in Japanese patients to determine whether there were variants in IBD5 specifically associated with IBD in Japanese patients. Material and methods. A total of 758 Japanese individuals, 241 patients with CD, 247 patients with ulcerative colitis (UC) and 270 healthy controls, were analyzed in this study. Genotyping for SNPs was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. Results. We found L503F and −207G/C to be very rare (<1% frequency) in CD, UC and HC in the Japanese population. Furthermore, we also found that none of the representative SNPs in IBD5 was associated with CD or UC in the Japanese subjects. Conclusions. In contrast to Caucasians, IBD5 is not a major component of the susceptibility to IBD in the Japanese population.

Telomere shortening and the clinicopathologic characteristics of human colorectal carcinomas

It has been reported that shortening of telomeres and strong activation of telomerase occur frequently in colorectal carcinomas. In the current study, the authors examined the correlations between the telomere length of colorectal carcinomas and their clinicopathologic characteristics as well as the activity of telomerase to clarify whether telomere length might represent the biologic behavior of tumors and the mode of tumor development.

Changes of faecal microbiota in patients with Crohn's disease treated with an elemental diet and total parenteral nutrition.

BACKGROUND Intestinal microbiota contributes to the pathogenesis of Crohns disease. Elemental diet and total parenteral nutrition are effective therapies for Crohns disease; however, changes of microbiota as a result of both treatments have not been fully elucidated. AIM To elucidate changes of faecal microbiota in Crohns disease patients treated with elemental diet and total parenteral nutrition. METHODS Stool samples were collected from 33 active Crohns disease patients and 17 healthy subjects, and recollected after elemental diet (8 patients) and total parenteral nutrition (9 patients). Terminal restriction fragment length polymorphism analysis of bacterial 16srDNA was performed to evaluate the whole microbiota. Specific quantitative PCR was then used to determine populations of predominant bacterial groups. RESULTS In Crohns disease patients, the number of terminal restriction fragments, which reflects bacterial species, was significantly lower. Populations of total bacteria and Bifidobacterium were significantly lower and the ratio of Enterococcus was higher. The number of terminal restriction fragments was significantly decreased after total parenteral nutrition, but not after elemental diet. Population of Bacteroides fragilis significantly decreased after elemental diet, while population of Enterococcus significantly increased after total parenteral nutrition. CONCLUSION Faecal microbiota in Crohns disease patients was markedly different from healthy subjects. Species diversity was reduced by total parenteral nutrition, but not by elemental diet.