Butyrophilin-like 2 gene is associated with ulcerative colitis in the Japanese under strong linkage disequilibrium with HLA-DRB1*1502

The human leukocyte antigen (HLA) region has been implicated in the pathogenesis of inflammatory bowel disease (IBD), which is classified into Crohn's disease (CD) and ulcerative colitis (UC). Recently, an association between sarcoidosis and the butyrophilin-like 2 (BTNL2) gene was reported. BTNL2 is located in the HLA region and its messenger RNA is expressed most abundantly in the intestine. In this study, we performed a case-control association study of BTNL2 in the Japanese patients with IBD and performed linkage disequilibrium (LD) analysis between BTNL2 and HLA-DRB1. We analyzed eight polymorphisms selected after direct sequencing and found that none of the polymorphisms were associated with the Japanese CD cohort. In contrast, five polymorphisms were significantly associated with UC, especially three single nucleotide polymorphisms (BTNL2_19, BTNL2_22 and BTNL2_23) were associated as a haplotype. The most frequent haplotype (GGC haplotype) was a low-risk haplotype (P= 0.000052), whereas the other TCT haplotype was a high-risk haplotype (P= 0.0000085). Among the eight polymorphisms, the strongest association with UC was found in BTNL2_19 (OR = 1.92, P= 0.0000035). As expected, the BTNL2_19-T allele showed strong LD with DRB1*1502 (D'= 0.92). When BTNL2_19 was tested as conditional on the DRB1*1502 carrier status, the significant association disappeared, suggesting that the association was because of its strong LD with DRB1*1502. We conclude that BTNL2 does not contribute to the susceptibility to Japanese CD but is associated with Japanese UC because of the strong LD with HLA-DRB1*1502. The strong LD between BTNL2 and HLA-DRB1 raises another issue about the potential role of BTNL2 in other diseases associated with HLA-DRB1.     

Confirmation of the novel association at the BTNL2 locus with ulcerative colitis

Linkage in families and association in population case–control investigations have clearly shown that genes within the major histocompatibility complex region on chromosome 6p are relevant to the susceptibility and pathogenesis of ulcerative colitis (UC) and Crohn's disease. However, identifying the causative variants by fine mapping has not been conclusive. In this study using 58 single nucleotide polymorphisms (SNPs) with 616 UC cases, there was significant association with SNP rs2294881 of the (butyrophilin-like 2) BTNL2 gene with odds ratio (OR) = 2.80, confidence interval (CI) = 1.62–4.84 and P  = 5.69 × 10⁻⁴ ( P _Bonferroni = 3.3 × 10⁻²) and replication of SNP rs9268480. The missense SNP rs2076523 (K196E) showed novel association with a subset of UC cases with colectomy ( n  = 126), OR = 0.25, CI = 0.11–0.58 and P  = 4.42 × 10⁻⁴ ( P _Bonferroni = 2.56 × 10⁻²). These three associated variants within the BTNL2 gene were neither in linkage disequilibrium with each other nor correlated with the SNPs tagging the human leukocyte antigen (HLA)-DRB1*1502 and HLA-DRB1*0301 alleles.     

HLA-B is the best candidate of susceptibility genes in HLA for Japanese ulcerative colitis

Recently, a genome-wide association study for ulcerative colitis (UC) in the UK population was reported, and several susceptibility loci including the human leukocyte antigen ( HLA ) region were identified. The strongest association in the HLA region was found at a 400 kb haplotype block containing HLA-DRB1 . In Japanese population, previous study suggested the association between UC and HLA-B*52 ; however, HLA typing was determined using serotyping with the small sample size. The purpose of this study was to perform an association study in HLA-B by genotyping. A total of 320 patients with UC and 322 healthy controls were recruited in this case–control study. All subjects were Japanese. Genotyping of HLA-B was performed by polymerase chain reaction using a sequence-specific primer. When the allele frequencies were compared, significant associations were found with B*52 [odds ratio (OR) = 3.65, P  = 1.6 × 10⁻¹⁷, P _c = 3.7 × 10⁻¹⁶] and B*4002 (OR = 0.52, P  = 0.00030, P _c = 0.0068). The allele frequency of B*52 was significantly higher in patients diagnosed before 40 years of age than in those diagnosed after 40 years (OR = 1.79, P  = 0.010, P _c = 0.020). A combination association map of Japanese UC using our current and previous studies showed two equal peaks of association on HLA-DRB1 and HLA-B , indicating the possible existence of two casual variants in the HLA region inside and outside the 400 kb block found in UK. We conclude that HLA-B contributes to the susceptibility to Japanese UC, especially cases with younger age of onset. The strength of association for HLA-B was equal to that for HLA-DRB1 in Japanese UC, in contrast to the UK population.     

Granulomatous ulcerative colitis: a re-appraisal of the mucosal granuloma in the distinction of Crohn's disease from ulcerative colitis

AIMS: To determine whether the presence and location of giant cells or granulomas in relation to crypts distinguishes between ulcerative colitis and Crohn's disease. METHODS AND RESULTS: Twenty-nine large bowel mucosal biopsy specimens showing giant cells and/or granulomas in a background more typical of ulcerative colitis than Crohn's disease were collected between 1986 and 1996. Each was subject to detailed independent analysis by three histopathologists. Follow-up of the cases was by examination of all previous and subsequent gastrointestinal surgical or biopsy material and by scrutiny of the clinical notes by a gastroenterologist. On the basis of the accumulated histological data 10 of these 29 cases were accorded the diagnosis of ulcerative colitis. In nine of these 10 cases the clinical diagnosis, where known, was in keeping with this and all nine contained only crypt-associated giant cells and/or granulomas. The tenth case contained a solitary free-standing granuloma and clinically the patient had perianal disease, suggesting that the true diagnosis was Crohn's disease. CONCLUSIONS: Isolated giant cells and well-defined epithelioid granulomas distant from crypts do not, as a rule, occur in ulcerative colitis, and hence their presence in a colonoscopic biopsy showing features of chronic inflammatory bowel disease is a strong pointer towards the diagnosis of Crohn's disease. Crypt-associated giant cells and granulomas can occur in ulcerative colitis and in themselves are unreliable features for the discrimination between Crohn's disease and ulcerative colitis.     

Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis

Haem-oxygenase-1 (HO-1) has been shown to exert anti-inflammatory, anti-apoptotic and anti-proliferative effects. We investigated HO-1 expression in patients with inflammatory bowel disease (IBD) and could demonstrate a scattered expression of HO-1 in the intestinal epithelium of severely inflamed colonic mucosa of patients with IBD compared to control specimens such as diverticulitis, suggesting dysregulated expression in IBD. To further analyse potential mechanisms of HO-1 induction in the intestine we employed an in vitro epithelial cell apoptosis model and an experimental colitis model. In vitro induction of HO-1 by the HO-1 inducer cobalt protoporphyrin (CoPP) resulted in a dose-dependent down-regulation of caspase-3 activation in HT-29 cells, indicating an anti-apoptotic function of HO-1 in the intestine. In vivo, preventive HO-1 induction by CoPP in acute dextran sodium sulphate (DSS)-induced colitis led to a significant down-regulation of colonic inflammation (P < 0.01) with a concomitant reduction in interferon (IFN)-gamma - but unaffected interleukin (IL)-10-secretion by isolated mesenteric lymph nodes (P < 0.01). Additionally, TUNEL staining of colonic sections demonstrated fewer apoptotic epithelial cells in the colon of CoPP treated animals. No beneficial effects were observed if HO-1 was induced by CoPP after the onset of acute colitis or in chronic DSS-induced colitis. In conclusion, the data suggest a protective role of HO-1 if it is induced before the onset of inflammation. However, as shown by the lack of effects in established acute or in chronic colitis, the induction of HO-1 may not be a promising approach for the treatment of IBD.     

HLA-DRB1基因多态性与汉族人溃疡性结肠炎的相关性研究

目的：研究HLA—DRB1基因多态性与汉族人溃疡性结肠炎（UC）的相关性，以期发现UC的易感基因。方法：采用序列特异性引物聚合酶链反应的方法对汉族72例UC患者和314例正常对照者HLA-DRB1基因分型。结果：UC患者携带DRB1＊07等位基因的频率较正常对照组高（19．4％vs9．2％，P=0．0229，OR=2．372，95％CI：1．181～4．766），但经Bonferroni校正后Pc〉0．05，差异无显著性。在临床亚型分析中，全结肠炎组和无肠外表现组的DRB1＊07的频率明显增高。在中重度组中DRB1＊07和DRB1＊14的频率显著增高，轻度组的DRB1＊16的频率明显增高，而携带DRBl＊03的5例患者在临床上均表现为轻度，这些差异与正常对照组相比均有统计学意义。且在轻度组中的DRB1＊16的频率同样明显高于在中重度组的频率。结论：在汉族人群中，HLA-DRB1＊07与全结肠炎、无肠外表现以及中重度UC呈正相关，HLA—DRB1＊14和HLA-DRB1＊16分别与中重度和轻度UC呈正相关，而HLA-DRB1＊03与中重度UC呈负相关，提示HLA—DRB1等位基因与中国汉族人群的UC临床表型有关。     

Mucosal inflammatory cytokine production by intestinal biopsies in patients with ulcerative colitis and Crohn's disease

Chronic inflammation in inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis) may be attributed partly to increased secretion of inflammatory cytokines. The aim of this study was to investigate simultaneously the spontaneous release patterns of tumor necrosis factor-alpha (TNF-), interleukin-1-beta (IL-1), and interleukin-6 (IL-6) by organ cultures of inflamed mucosa from IBD patients. Organ cultures of involved IBD mucosa spontaneously produced increased amounts of TNF-, IL-1, and IL-6 compared to normal mucosa. The patterns of cytokine release between Crohn's disease and ulcerative colitis organ cultures were not significantly different. Increased inflammatory cytokine production by lamina propria mononuclear cells (LPMCs) and mucosa treated with EDTA suggests that these cytokines originate mainly from LPMCs. These results confirm the role of inflammatory cytokines in IBD and shed a new light on the role of TNF- in IBD.     

Identification of a novel HLA-DRB1 allele, DRB1*116502, by sequence-based typing in an African-American individual

The novel allele HLA-DRB1*116502 differs from HLA-DRB1*110201 by nucleotide substitutions at codon 59 (GAG→GAC) and 93 (CGG→AGG).     

Autoimmunity in ulcerative colitis (UC): a predominant colonic mucosal B cell response against human tropomyosin isoform 5

We set out to examine if the IgG-producing cells in the colonic mucosa in UC are committed to tropomyosin isoform 5 (hTM5), a putative autoantigen in UC. Lamina propria mononuclear cells (LPMC) were isolated from colonoscopic biopsy specimens from recto-sigmoid and proximal colon. Twenty-three patients with UC, eight with Crohn's colitis (CC), and 10 non-inflammatory bowel disease (non-IBD) controls were included. The ELISPOT assays were used to quantify lamina propria B cells producing total immunoglobulin (IgA, IgG, IgM), IgG, IgA, as well as IgG against hTM5 isoform. The median value of percentage of total IgG-producing lymphocytes was similar in UC (12%) and CC (11%), but was significantly (P < 0.0002) higher than non-IBD controls (6%). However, in UC, but not in CC and non-IBD, a large number of lamina propria B cells produced IgG against hTM5 (median values: UC 42%, CC 2.5%, non-IBD 0%). This difference in UC when compared with CC and non-IBD was highly significant (P < 0.00001). Twenty-one of 23 (91%) patients with UC had percentage of anti-hTM5 IgG-producing immunocytes more than 2 s. d. above the mean for non-UC patients. In UC but not in CC and non-IBD controls, the increased number of IgG-producing cells are largely committed to produce IgG against hTM5-related epitope(s).     

Reliability of rectal biopsy in distinguishing between chronic inflammatory bowel disease and acute self-limiting colitis

Aims : In order to assess the validity of previously proposed criteria for the differentiation of chronic inflammatory bowel disease (CIBD) from acute self-limiting colitis (ASLC) all rectal biopsies were reported by a single histopathologist with a long-term gastrointestinal interest over a 4.5-year period. Methods and results : The presence or absence of distorted crypt architecture, increased lymphocytes and plasma cells, villous mucosal architecture, granulomata, basal lymphoid aggregates, basal giant cells and Paneth cell metaplasia was noted for each biopsy. The definite presence of any of the above features, with the exception of intramucosal granulomata, was regarded as indicative of CIBD. Eighteen months later all available case notes were examined and the presenting clinical symptoms and working clinical diagnosis extracted. The final diagnosis, histopathological diagnosis and the presence or absence of any of the above histopathological features were correlated and the positive predictive value of each histopathological feature was calculated. A correct diagnosis of either CIBD or ASLC was made in 80 of 84 and 29 of 31 cases, respectively. Conclusions : Villous mucosal architecture and Paneth cell metaplasia were found to be specific features of CIBD. Distorted crypt architecture, basal lymphoid aggregates and plasma cell infiltration of the lamina propria were also useful features but strict definition of these features is required and discussed. Intramucosal epithelioid granulomas were identified in eight cases of CIBD and four cases of ASLC. In association with ruptured crypts intramucosal granulomas are not specific features of Crohn's colitis.     

Differentiation between ulcerative colitis and Crohn's disease by a quantitative immunohistochemical evaluation of T lymphocytes,neutrophils, histiocytes and mast cells

Mucosal biopsy criteria has limited validity in terms of discrimination between ulcerative colitis (UC) and Crohn's disease (CD). The aim of this study was to set up quantitative immunohistochemical criteria, with a special focus on inflammatory cell distribution within individual specimens and throughout the large bowel. Quantitative evaluation was performed for the density of CD8+, CD45RO+, neutrophil elastase+, CD68+ and mast cell tryptase+ cells in affected and unaffected mucosa taken from 41 patients with UC and 61 patients with CD. Each slide was examined at the highest and lowest density fields, which were further divided into the upper and deeper half of mucosa. Multiple logistic regression analysis using 51 features as independent variables constructed a predictive equation finding the probability of UC (PUC), and the diagnostic categories were subsequently defined based on a receiver-operating characteristic curve. The analysis disclosed five significant features suggesting UC; these implied intense infiltration of CD8+ and mast cell tryptase+ cells, diffuse infiltration of neutrophil elastase+ and CD68+ cells, and continuous infiltration of CD45RO+ cells. The criteria consisted of three diagnostic categories, 'suggestive of UC (PUC > or = 0.7)', 'indeterminate (0.3 < PUC < 0.7)', and 'suggestive of CD (PUC < or = 0.3)'; the criteria had values for sensitivity and specificity exceeding 95%. The immunohistochemical criteria distinguishing UC from CD may help to confirm the diagnosis in patients with ambiguous endoscopic and histological diagnosis.     

Novel alleles at the HLA-DRB1 and -DQB1 loci

Twelve novel human leukocyte antigen class II alleles are described; eight DRB1 alleles and four DQB1 alleles. Nine of the variants are single nucleotide substitutions from their most homologous allele, of which six result in amino acid changes (DRB1*0459, *1156 and *1522; DQB1*0205, *0320 and *0321) and three are silent substitutions (DRB1*030105 and *040304, and DQB1*030104). The remaining alleles (DRB1*0906, *1464 and *1468) differ from their most similar alleles by two to three nucleotide substitutions which alter one to two amino acids.     

Chemokines in the Inflammatory Bowel Diseases

Ulcerative colitis and Crohn's disease are characterized by chronic intestinal inflammation. Intestinal bacteria initiate the activation of intestinal inflammatory processes, which are mediated by proinflammatory cytokines and chemokines. In inflammatory bowel disease, intestinal inflammation is not downregulated, in part due to defective or absent inhibitory processes. Studies to date have demonstrated that IL-8, MCP-1, and ENA-78 are highly expressed in the intestinal mucosa in areas of active Crohn's disease and ulcerative colitis. Neutrophils and macrophages in the inflamed intestine synthesize and secrete large amounts of chemokines in patients with inflammatory bowel disease. Increased chemokine expression has also been observed in epithelial cells, endothelial cells, and smooth muscle cells. Future trials of specific agents capable of inhibiting chemokine synthesis and secretion or blocking chemokine–chemokine receptor interaction will be important to study in patients with ulcerative colitis and Crohn's disease.     

人类白细胞抗原新等位基因DRB1*1219的确认及其序列分析

目的 鉴定分析1个白血病患者家庭HLA-DRB1位点1个新等位基因.方法 应用PCR-序列特异性引物及Luminex DNA杂交流式分型技术进行HLA分型,发现1个与HLA-DRB1*120201相关的未知基因.应用DNA测序技术进行鉴定分析并与已知序列比对分析.结果 先证者DRB1位点有1个等位基因的核苷酸序列与所有已知基因序列均不相同,与同源性最高的DRB1*120201相比,第2外显子第341位核苷酸碱基发生了C→T,结果导致相应85位密码子编码的丙氨酸变为缬氨酸.结论 测序表明被测样本含有1个HLA-DRB1新等位基因,被世界卫生组织HLA因子命名委员会正式命名为HLA-DRB1*1219(序列号EJ374889).

Abstract：

Objective To identify a novel HLA DRB1 allele in a Chinese leukemia family. Methods A new HLA-DRB1 allele was initially detected by polymerase chain reaction-sequence specific primer and unusual reaction pattern by Luminex RSSO, then DNA sequencing was performed to identify the sequence of the novel allele. Results The DNA sequencing revealed the presence of the new allele which differs from the closest macthing HLA- DRB1 * 120201 by a single nucleotide substitution at position (341 C→T in exon 2),resulting in an amino acid change from Ala to Val at coden 85. Conclusion A novel allele was confirmed by DNA sequencing and has been designated HLA-DRB1 * 1219 by the WHO Nomenclature Committee.     

A general autoimmunity gene (PTPN22) is not associated with inflammatory bowel disease in a British population

A single-nucleotide polymorphism (C1858T) causing an amino acid substitution (R620W) in the lymphoid protein tyrosine phosphatase gene PTPN22 has been implicated in type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, juvenile idiopathic arthritis and Hashimoto's thyroiditis, thus revealing a general role for this gene in autoimmune disease. We investigated the association of the C1858T variant in an additional autoimmune disease population by performing a case-control study of 514 British individuals with inflammatory bowel disease (IBD) [294 with Crohn's disease (CD) and 220 with ulcerative colitis (UC)] and 374 normal controls. No significant differences in genotype or allele frequencies were observed between IBD, CD or UC and controls, indicating that PTPN22 does not influence risk of IBD.     

MICA, MICB and C1_4_1 polymorphism in Crohn's disease and ulcerative colitis

MICA and MICB belong to a multicopy gene family located in the major histocompatibility complex (MHC) class I region near the HLA-B gene. They encode for MHC class I molecules, which are induced by stress factors like infection, heat shock or neoplastic transformation and which are mainly expressed on gastrointestinal epithelium. They are recognized by gammadelta T lymphocytes and natural killer (NK) cells. Additionally they are located within a linkage region on chromosome 6p around HLA-B and TNFalpha. Thus the polymorphic MICA and MICB genes are excellent candidate genes for providing the genetic background of inflammatory bowel disease. A strong association of allele A6 of the MICA exon 5 trinucleotide microsatellite polymorphism with ulcerative colitis has been found in Japanese patients. Therefore, we have analysed the MICA exon 5 polymorphism, the MICB intron 1 dinucleotide polymorphism and in addition the tetranucleotide polymorphism C1_4_1, which is located between the MICA gene and the HLA-B gene, in patients of Caucasoid origin with Crohn's disease (n=94) and ulcerative colitis (n=94). In this study we could not find any associations of particular alleles of the MICA, MICB and C1_4_1 polymorphisms with Crohn's disease or ulcerative colitis. We could also not discover any associations of specific two-point or three-point haplotypes with these diseases. Thus it is unlikely that the MICA and MICB genes are involved in causing susceptibility for inflammatory bowel disease, although it cannot be excluded that a weak association could be identified in a larger patient sample.