J Sanderson

Novel pharmacogenetic markers for treatment outcome in azathioprine-treated inflammatory bowel disease

Backgroundu2002 Azathioprine (AZA) pharmacogenetics are complex and much studied. Genetic polymorphism in TPMT is known to influence treatment outcome. Xanthine oxidase/dehydrogenase (XDH) and aldehyde oxidase (AO) compete with TPMT to inactivate AZA.

Analysis of single-nucleotide polymorphisms in the interleukin-4 receptor gene for association with inflammatory bowel disease.

Abstractu2002Genetic linkage analysis in families with multiple cases of inflammatory bowel disease (IBD) has mapped a gene which confers susceptibility to IBD to the pericentromeric region of chromosome 16 (IBD1). The linked region includes the interleukin(IL)-4 receptor gene (IL4R). Since IL-4 regulation and expression are abnormal in IBD, the IL4R gene is thus both a positional and functional candidate for IBD1. We screened the gene for single-nucleotide polymorphisms (SNPs) by fluorescent chemical cleavage analysis, and tested a subset of known and novel SNPs for allelic association with IBD in 355 families, which included 435 cases of Crohns disease and 329 cases of ulcerative colitis. No association was observed between a haplotype of four SNPs (val50ile, gln576arg, A3044G, G3289A) and either the Crohns disease or ulcerative colitis phenotypes using the transmission disequilibrium test. There was also no evidence for association when the four markers were analyzed individually. The results indicate that these variants are not significant genetic determinants of IBD, and that the IL4R gene is unlikely to be IBD1. Linkage disequilibrium analyses showed that the val50ile and gln576arg variants are in complete equilibrium with each other, although they are separated by only about 21u2009kilobases of genomic DNA. This suggests that a very dense SNP map may be required to exclude or detect disease associations with some candidate genes.

No association of the NFKB1 promoter polymorphism with ulcerative colitis in a British case control cohort

Recently, Karban and colleagues1 reported an association of a common NFKB1 gene polymorphism, −94ins/delATTG, with ulcerative colitis (UC) in a non-Hispanic, non-Jewish North American population. The deletion was significantly associated with disease in both family based and case control studies: in the combined case control cohort, the allele frequency of −94delATTG (D) was significantly increased in 350 non-Jewish UC cases (45.3%) compared with 802 non-Jewish controls (38.8%, pu200a=u200a0.002). In a recessive model of inheritance, the homozygous (DD) genotype was significantly increased in UC cases (21.4%) compared with controls (14.8%) (pu200a=u200a0.0043), giving an odds ratio of 1.57 for the DD genotype (95% confidence interval 1.14–2.16).nnNuclear factor κB (NFκB) is an important transcription factor implicated in the inflammatory response.2 The NFKB1 gene, which encodes the p105/p50 subunit of the NFκB family of proteins, maps to chromosome 4q24, in a region showing linkage to inflammatory bowel disease3–5; a …

Immunohistochemical analysis of ageing human B and T cell populations reveals an age-related decline of CD8 T cells in spleen but not gut-associated lymphoid tissue (GALT).

It is thought that senescence of the immune system is responsible, at least in part, for many health problems associated with ageing. Previous studies on changes in lymphocyte composition have used flow cytometry to study peripheral blood lymphocytes (PBLs), or cells isolated from rodent tissue, and have yielded conflicting results. We have used immunohistochemistry to determine whether the B and T cells in human tissue from spleen and gut are affected by age. Areas of germinal centre, mantle zone and marginal zone of B cell follicles were measured. In addition, CD4 and CD8 T cells in T cell areas and in B cell follicles were counted. We observed a striking age-related decrease in the proportion of CD8+ T cells in the T cell zones of the spleen. This decrease was not apparent in the T cell population that occupies splenic B cell areas, or in GALT. Further differences, in CD4+ cells, were seen between T cell populations in the T cell zones and those in B cell areas. These findings highlight differences between lymphocyte populations in different lymphoid tissues, and different compartments within each tissue, which may be of importance in future studies of the ageing immune system.

Sequence variation, linkage disequilibrium and association with Crohn's disease on chromosome 5q31

Chromosome 5q31 contains a cluster of genes involved in immune response, including a 250u2009kb risk haplotype associated with Crohns disease (CD) susceptibility. Recently, two functional variants in SLC22A4 and SLC22A5 (L503F and G-207C), encoding the cation transporters OCTN1 and OCTN2, were proposed as causal variants for CD, but with conflicting genetic evidence regarding their contribution. We investigated this locus by resequencing the coding regions of 10 genes in 24 CD cases and deriving a linkage disequilibrium (LD) map of the 27 single nucleotide polymorphisms (SNPs) detected. Ten SNPs representative of the LD groups observed, were tested for CD association. L503F in SLC22A4 was the only nonsynonymous SNP significantly associated with CD (P=0.003), but was not associated with disease in the absence of other markers of the 250u2009kb risk haplotype. Two other SNPs, rs11242115 in IRF1 and rs17166050 in RAD50, lying outside the 250u2009kb risk haplotype, also showed CD association (P=0.019 and P=0.0080, respectively). The RAD50 gene contains a locus control region regulating expression of the Th2 cytokine genes at this locus. Other as yet undiscovered SNPs in this region may therefore modulate gene expression and contribute to the risk of CD, and perhaps of other inflammatory phenotypes.

Genetic association between EPHX1 and Crohn’s disease: population stratification, genotyping error, or random chance?

We read with interest the article by de Jong and colleagues ( Gut 2003; 52 :547–51) reporting studies of genetic associations between DNA polymorphisms in xenobiotic metabolising genes and Crohn’s disease (CD). The authors employed a case control study design to test seven polymorphisms in five candidate genes for disease association. Evidence was found for a significant association of a single nucleotide polymorphism (SNP), Tyr113His (348T>C), in the microsomal epoxide hydrolase 1 gene ( EPHX1 ), with CD. Homozygosity for the T (Tyr 113) allele was significantly higher in cases than in healthy controls (χ2u200a=u200a23.7, p<0.0001, odds ratio 2.9). The observed frequency of the T allele in controls was 41%, which is outside the range of frequencies (58–94%) reported in other control populations (reviewed in de Jong et al ). Its frequency in CD cases was 67%. In view of the strength of reported association, we sought to replicate …

Genetic variation at the chromosome 16 chemokine gene cluster: development of a strategy for association studies in complex disease.

The chemokine gene cluster [CCL22, CX3CL1, CCL17] (previously known as [SCYA22, SCYD1, SCYA17]) is a candidate locus for one of the susceptibility genes for inflammatory bowel disease that are located in the peri‐centromeric region of chromosome 16. Screening for sequence variation at this locus led to the detection of 14 single nucleotide polymorphisms (SNPs). An efficient experimental and computational approach was developed to estimate allele frequencies and pairwise linkage disequilibrium relationships between SNPs at this locus, and to test them for association with inflammatory bowel disease. The 12 common SNPs were assigned to 5 distinct linkage disequilibrium groups. Genotyping of one SNP from each linkage disequilibrium group in a large cohort of families with inflammatory bowel disease did not provide convincing evidence of association with either Crohns disease or ulcerative colitis. We describe an efficient experimental design from SNP screening to association testing. This strategy can be used to test candidate genes for involvement in susceptibility to complex disease.

OC-013 Genetic susceptibility to orofacial granulomatosis

Introduction Orofacial granulomatosis (OFG) is a rare, disfiguring inflammatory disorder of the mouth where a proportion of cases also have intestinal Crohn’s disease (CD). The aetiology remains largely unknown, although there is high prevalence of allergy in OFG with and without CD. Our objective was to investigate whether OFG and CD have shared genetic aetiology or whether OFG is mediated by distinct immune-related genetic susceptibility variants. Method Patients were clinically assessed and determined to either demonstrate isolated oral manifestations (OFG only) or concurrent intestinal CD (CD/OFG). Genomic DNA from 263 patients was genotyped using the Immunochip, a custom Illumina microarray assessing 196,524 genetic variants across multiple immune-related disease loci. Patient data was compared to data for 4,307 population controls from the UKIBD consortium. Statistical analysis was performed using PLINK, a whole genome association analysis program and the R statistical package. Results Analyses revealed two significant associations (p < 2 × 10–6) within the OFG only cohort with single nucleotide polymorphisms (SNPs) on chromosome 11q13.5 near the LRRC32gene (p = 1.6 × 10–9) and on chromosome 6 (p = 3.9 × 10–7) within the MHC class I region. The 11q13.5 locus has previously shown association with atopic conditions and the MHC class I region is implicated in numerous allergic and autoimmune diseases, including CD. In addition, a highly suggestive association was detected from the CD/OFG group on chromosome 5p13 (p = 2.5 × 10–6), a known risk locus for CD. Collectively, these results suggest that OFG is influenced by common variants implicated in allergy and immunity, supporting the link between OFG and allergy. However there may also be some overlap with genetic aetiology for CD. Replication in a larger independent cohort is required to substantiate our findings. Conclusion OFG is likely to be a complex disease mediated by diverse genetic variants, sharing genetic susceptibility with allergic disorders and autoimmune conditions such as CD. Disclosure of interest None Declared.

PTH-067 An evaluation study of lactobacillus brevis cd2 in orofacial granuolomatosis

Introduction A small proportion of patients with Crohn’s disease develop orofacial granulomatosis (OFG), a rare, chronic disfiguring condition of unknown aetiology affecting the oral mucosa and perioral region. Previous studies have suggested that alterations in the oral microbiota may be involved in the pathogenesis of OFG. CD2 is a novel probiotic containing Lactobacillus breviswhich has anti-inflammatory properties, primarily via reduced arginine availability. Previous studies have shown that CD2 reduces oral inflammation in chemotherapy-induced oral mucositis, Behcet’s disease and recurrent aphthous stomatitis. Our aim was to evaluate the tolerability and efficacy of CD2 lozenges in reducing oral inflammation in patients with active OFG. Method This was a single-centre prospective open-label observational study. Patients were recruited from a specialist OFG clinic between February and August 2014. Patients with active OFG received an eight week course of CD2 lozenges taken four times per day. Patients were reviewed before and after treatment and disease activity assessed by Oral disease activity score (ODAS), Visual analogue scale (VAS) of oral soreness and Global physician assessment (GPA). Results 28 patients were recruited with 4 patients withdrawing (3 non-compliance, 1 of which had severe learning difficulties; 1 re-classified as aphthous stomatitis). 7 patients failed follow-up leaving 17 patients (9 males) available for final analysis. The median age was 35 years (range 18–70 years) with 5/17 patients diagnosed with concurrent intestinal Crohn’s disease. Before treatment, the median ODAS was 12 (range 2–36), median VAS was 50% (range 0–90%), with 9/17 patients classified as having mild disease, 2/17 moderate and 6/17 severe. Post treatment, the median ODAS was 12 (range 1–34), median VAS was 20% (range 0–70%), with 9/14 patients had mild disease, 7/14 moderate and 1/14 severe. The reduction in the mean ODAS at 2 months was: 15.5–11.9 = 3.6 (p = 0.044). The mean improvement in oral soreness measured by VAS was: 44%–26% = 18% (p–0.003). There were no adverse events, however one patient discontinued treatment due to severe diarrhoea which resolved upon CD2 cessation. Conclusion CD2 appears to be safe, well-tolerated and of benefit in reducing oral inflammation and oral soreness in active OFG. Given the marked improvement in oral soreness, CD2 may have a role in symptom relief in other oral inflammatory conditions. Based on these results, a larger double-blind prospective study is recommended. The treatment shows promise as an adjunct to dietary therapy and an alternative to systemic immunosuppression. Disclosure of interest None Declared.

PTU-043 Genetic polymorphism in the multidrug resistance-5 gene is associated with non-response to azathioprine treatment in inflammatory bowel disease: Abstract PTU-043

Introduction Multidrug resistance (MDR) genes encode transmembrane ATP-dependent pumps, otherwise known as the ATP-binding cassette subfamily-b (ABCB) transporters responsible for removing xenobiotics, particularly drugs, from cells. Thiopurine metabolites are exported from cells by MDR-4 and MDR-5. Altered ABCB pump activity has been linked to treatment resistance in other contexts. Methods Using Taqman® real-time PCR genotyping assays, a prospective cohort of 192 patients receiving azathioprine for IBD was genotyped for three coding region SNPs in MDR4 and three in MDR5, (all of which occur commonly in the Caucasian population). The SNPs tested were in MDR4: 175C→T, 711A→T and 1954A→G and in MDR5: 2T→C, 343A→G and 1573G→A. Association between genotype and clinical outcome on azathioprine treatment (complete response, non-response or withdrawal due to side-effects) was sought. Association tests were performed under a dominant model, using Fishers Exact test. No correction for multiple testing has been applied. Results The SNP MDR-5u2005c.343A→G was associated with a lack of clinical response to thiopurine treatment p=0.02, OR 2.43, 95%CI 1.14 to 5.17. Moreover, when analysed alongside other known markers of non-response to thiopurine treatment, (TPMT activity >35u2005pmol/h/mgHb and AOX 3404A→G) increasing numbers of markers increased their chance of non-response, p=0.0001 (χ2 for trend). No other SNP had a significant effect on clinical response and no SNP was associated with ADRs. Conclusion This study raises the possibility that MDR polymorphism could be important in thiopurine pharmacogenetics and warrants further study in other cohorts. Reliable markers of non-response to thiopurines could be incorporated into a panel of pharmacogenetic markers to inform the selection and dosing of immunomodulatory drugs, offering each individual the best chance of achieving early effective disease control Abstract 043. Abstract PTU-043 Association between SNPs and clinical outcome SNP CR with SNP NR with SNP p Value OR 95% CI Number (%) Number (%) MDR-4u2005c.1954A→G 10/76 (13) 7/45 (16) 0.79 1.22 0.42 to 3.5 MDR-4u2005c.711A→T 26/76 (34) 13/43 (30) 0.69 0.83 0.37 to 1.86 MDR-4u2005c.175C→T 21/75 (28) 13/43 (30) 0.83 1.11 0.49 to 2.54 MDR-5u2005c.1573G→A 24/74 (32) 15/44 (34) 1.0 1.08 0.49 to 2.38 MDR-5u2005c.2T→C 21/76 (28) 18/45 (40) 0.17 1.75 0.80 to 3.81 MDR-5u2005c.343A→G 29/76 (38) 27/45 (60) 0.02* 2.43 1.14 to 5.17 CR, Complete responders; NR, Non-responders.