Identifying and testing candidate genetic polymorphisms in the irritable bowel syndrome (IBS): association with TNFSF15 and TNFα.

OBJECTIVES: The postinfectious irritable bowel syndrome (PI-IBS) suggests that impaired resolution of inflammation could cause IBS symptoms. The authors hypothesised that polymorphisms in genes whose expression were altered by gastroenteritis might be linked to IBS with diarrhoea (IBS-D) which closely resembles PI-IBS. DESIGN: Part 1: 25 healthy volunteers (HVs), 21 patients 6 months after Campylobacter jejuni infection, 37 IBS-D and 19 IBS with constipation (IBS-C) underwent rectal biopsy for gene expression analysis and peripheral blood mononuclear cell cytokine production assessment. Part 2: Polymorphisms in genes whose expression was altered in Part 1 were assessed in 179 HV, 179 IBS-D, 122 IBS-C and 41 PI-IBS. RESULTS: Part 1: Mucosal expression of seven genes was altered in IBS: CCL11, CCL13, Calpain 8 and TNFSF15 increased while NR1D1, GPR161 and GABRE decreased with similar patterns after infection with C jejuni. Part 2: The authors assessed 21 known single nucleotide polymorphisms (SNPs) in these seven genes and one SNP in each of the TNFα and IL-10 genes. Three out of five TNFSF15 SNPs (rs6478108, rs6478109 and rs7848647) showed reduced minor allele frequency (MAF) (0.28, 0.27 and 0.27) in subjects with IBS-D compared with HV (0.38, 0.36 and 0.37; p=0.007, 0.015 and 0.007, respectively) confirming others recent findings. The authors also replicated the previously reported association of the TNFα SNP rs1800629 with PI-IBS which showed an increase in the MAF at 0.30 versus 0.19 for HV (p=0.04). CONCLUSION: IBS-D and PI-IBS patients are associated with TNFSF15 and TNFα genetic polymorphisms which also predispose to Crohn's disease suggesting possible common underlying pathogenesis.

Impaired uptake of serotonin by platelets from patients with irritable bowel syndrome correlates with duodenal immune activation.

BACKGROUND & AIMS: Patients with irritable bowel syndrome with diarrhea (IBS-D) have increased mucosal serotonin (5-hydroxytryptamine [5-HT]) availability, possibly because immune activation reduces activity of the 5-HT transporter (SERT). We investigated the relationship between mucosal and platelet SERT and immune activation of the duodenal mucosa in patients with IBS-D. METHODS: We quantified mucosal intraepithelial lymphocytes (IELs), mast cells, and enterochromaffin cells in blood samples, measured levels of SERT messenger RNA (mRNA) in mucosal samples, and assessed platelet uptake of 5-HT and platelet membrane binding of (3)H-paroxetine in samples from 29 healthy volunteers (HVs), 20 patients with IBS-D, and 20 untreated patients with celiac disease. RESULTS: Patients with IBS-D or celiac disease had increased numbers of IELs and mast cells compared with HVs (both P < .001). Levels of SERT mRNA were reduced in the mucosa of patients with IBS-D or celiac disease and were inversely correlated with numbers of IELs (r = -0.72, P < .0001). Uptake of 5-HT by platelets from patients with IBS-D or celiac disease was reduced (mean, 17.1 ± 3.5 and 28.3 ± 4.1 nmol·min(-1)·mg(-1), respectively) compared with HVs (50.8 ± 8.0 nmol·min(-1)·mg(-1), P < .01 and P = .05, respectively). Binding of paroxetine to membranes of platelets from patients with IBS-D (median [interquartile range], 226 [92-405] fmol/mg protein) was significantly greater than that from HVs (109 [69-175] fmol/mg protein) and correlated inversely with platelet uptake of 5-HT (r = -0.62, P = .03). Tryptase release from incubated biopsy samples was significantly increased in patients with IBS-D (2.2 [0.42-3.5] vs 0.50 [0.25-0.86] ng·mL(-1)·mg(-1) for HVs; P = .03). CONCLUSIONS: Platelet SERT is reduced in IBS-D and associated with reduced levels of SERT mRNA and duodenal immune activation.

Association of the cysteinyl leukotriene receptor 1 gene with atopy in the British 1958 birth cohort.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) play an important role in the pathophysiology of many allergic inflammatory disorders. However, data on the contribution of genetic variability of the cysteinyl leukotriene receptor 1 gene (CYSLTR1) in asthma and atopy remain conflicting. OBJECTIVE: We investigated the association of polymorphisms of interest located at this locus and allergic disease prevalence in a national population with an established DNA archive, the British 1958 birth cohort. METHODS: The British 1958 birth cohort comprises all persons born in Britain during 1 week in 1958. Asthma, wheezy bronchitis, and wheezing were ascertained by interview at ages 7, 11, 16, 23, 33, and 42 years. At age 44 to 45 years, serum total circulating IgE levels were measured and atopy was defined as a serum total IgE level of greater than 30 kU/L and specific IgE levels to 1 or more of dust mite, cat fur, and mixed grass of greater than 0.3 kU/L. DNA samples from 8018 participants were genotyped for 2 variants of the CYSLTR1 promoter (Xq13-Xq21). RESULTS: The rare polymorphism C > T (rs7066737) was not associated with any of the phenotypes studied. The common promoter polymorphism A > G (rs2806489) was not associated with total IgE levels or the prevalence or age of onset of asthma, wheezy bronchitis, or wheeze. However, the wild-type allele A was significantly associated with atopy in female subjects (chi(2) = 8.30, P = .004), although not in male subjects (P = .841). CONCLUSIONS: These data suggest that a CYSLTR1 polymorphism previously shown to affect the gene transcription in vitro might influence the risk of atopy in the female white population with suggestive evidence of heterozygote vigor.

Functional polymorphism and differential regulation of CYSLTR1 transcription in human airway smooth muscle and monocytes.

Cysteinyl leukotrienes play an important role in the pathophysiology of many inflammatory disorders, including asthma. The aim of this study was to characterize the mechanisms underlying transcriptional regulation of the human cysteinyl leukotriene receptor 1 (hCYSLTR1) gene. 5'RACE was performed on human airway smooth muscle (HASM) and peripheral blood mononuclear cells. A1128-bp region of the hCYSLTR1 main putative promoter was screened for polymorphisms by sequencing of 48 individuals. Luciferase reporter gene assays were performed using fragments of the core promoter (232 bp to 1128 bp) in HASM and THP1 cells. Three hCYSLTR1 transcripts were found, one representing 90% of all messenger RNA identified. The genomic location of the transcription start sites suggested there are two putative hCYSLTR1 promoters. The majority of the transcriptional activity of the main putative promoter was detected between -232 and -679 bp. Four singlenucleotide polymorphisms in strong linkage disequilibrium were found in the region studied: -561 (rs7066737), -642 (rs2806489), -781 (rs2637204), and -940 (rs321029), with three haplotypes observed. In THP1 cells, the G allele (-642) caused a twofold decrease in luciferase expression compared to the Aallele. These data suggest that the majority of hCYSLTR1 transcripts in HASM and monocytes arise from a single promoter located immediately upstream of the 5\' untranslated region, although rarer transcripts can also occur. This study also raises the possibility that cell-type-dependent differences in transcriptional activity caused by the presence of specific haplotypes within the main CYSLTR1 promoter may be a predictor of disease risk or treatment response.

Alternative promoter use and splice variation in the human histamine H1 receptor gene.

Upstream gene structure and mRNA expression of the human histamine H1 receptor gene was investigated in cells relevant to the pathogenesis of asthma, (primary cultured human airway smooth muscle (HASM) cells, primary cultured human bronchial epithelial cells and bronchial epithelial cell line [BEAS2B]), and other tissues known to express histamine H1 receptors (placenta and brain). Splice variation of the 5' terminal exons gave three separate locations for novel promoters upstream of the detected transcription start sites. Further splice variants in the 5' untranslated region were also observed. Transient transfections of promoter/luciferase constructs showed these regions directed expression in HASM cells and BEAS2B cells. Polymorphism screening of the major regulatory regions identified a number of novel single-nucleotide polymorphisms. Expression of splice variants was confirmed by real-time PCR assays. Results showed one 5' terminal exon splice variant, comprising exons B/K, expressed preferentially in all tissues. Interestingly, the other 5' terminal exon splice variants showed tissue-specific patterns of expression, with variant F/K expressed negligibly (0.1%) in HASM cells, but accounting for 19.3% and 8.3% of total expression in BEAS2B cells and differentiated human bronchial epithelial cells, respectively. Splice variant A/K was second most highly expressed in differentiated human bronchial epithelial cells (23%), whereas its expression in BEAS2B and HASM cells was 1.7% and 4.4%, respectively. These data suggest the use of alternative promoters directing human H1 receptor gene expression, both within and between cell types.