Single Nucleotide Polymorphisms in IL 4 , OCTN 1 and OCTN 2 Genes in Association with Inflammatory Bowel Disease Phenotypes in a Caucasian Population in Canterbury , New Zealand

Background: The IBD5 (MIM# 606348) chromosomal region on 5q31 contains the genes for organic cation transporters, OCTN1 and OCTN2, and has been associated with susceptibility to adult onset CD. While several studies argue that OCTN1 and OCTN2 are the causal genes, others fail to replicate these results. Methods: From the Canterbury Inflammatory Bowel Disease Project, 388 CD, 405 UC, and 27 indeterminate colitis (IC) participants were genotyped for functional SNPs in OCTN1, OCTN2, and IL4 genes, in comparison with 370 controls. TaqMan® technologies were used to genotype IL4 rs2243250, OCTN1 rs1050152, and OCTN2 rs2631367 according to standard methodologies. The frequencies of variant SNPs in the control population were compared with frequencies in cases, where phenotype data for IBD was classed according to the Montreal classification. Results: For those individuals carrying only the variant allele in IL4 or OCTN1, despite trends towards increased risk of developing IBD, there were not statistically significant differences. There were no other specific effects of this allele in the location of CD, behaviour of UC or CD, or any other differences between controls and patient subgroups in this population group. However, carrying the variant OCTN2 rs2631367 allele significantly increased the risk of ileocolonic CD (OR=1.42, 95% CI=1.02-1.97, p<0.05). Conclusions: Despite earlier reports suggesting that OCTN1 and OCTN2 variants might provide important risk factors for IBD in New Zealand, this larger population-based study failed to confirm the earlier data. SNPs in the IL4 gene did not affect the analysis. We suggest the possibility that strong effects for these variants in these genes seen in some studies could imply gene-environment interactions. INTRODUCTION Inflammatory Bowel Disease (IBD), encompassing both ulcerative colitis (UC) and Crohn’s disease (CD) is a chronic inflammatory disorder of the digestive tract [1]. Especially for CD, familial clustering and concordance of the disease in monozygotic twin pairs confirms a genetic susceptibility that may interact with environmental factors, including diet [2]. Linkage studies have identified several chromosomal regions that have been consistently associated with disease risk [3]. The IBD5 (MIM# 606348) chromosomal region on 5q31 has been associated with susceptibility to adult onset CD in several studies [4, 5]. A 250 kb interval, identified by hierarchical fine-mapping, has identified a risk-associated haplotype containing 11 SNPs that are strongly linked with each *Address correspondence to this author at the Discipline of Nutrition, Faculty of Medical and Health Sciences, Private Bag 92019, University of Auckland, Auckland, New Zealand; Tel: (+) 6493737599, Ext. 86372; Fax: (+) 6493035962; E-mail: l.ferguson@auckland.ac. nz other, and strongly associated with CD, at least in some populations [4, 6-8]. This region contains the sodiumdependent organic cation transporters, OCTN1 and OCTN2 (alternatively called SLC22A4 and SLC22A5) that function in the transport of various xenobiotics. Peltekova and coworkers [9] sequenced five genes in IBD5, to identify ten single nucleotide polymorphisms (SNPs), which they predicted to have functional effects on gene function. These included the polymorphism L503F in exon 9 of OCTN1 (c.1672C/T; rs1050152) that causes an amino acid substitution, and a G to C transversion (-c.207G/C; rs 2631367) in the promoter of OCTN2, which is thought to disrupt a heat shock transcription factor binding element. Although these SNPs and these genes appear implicated in CD risk for certain population groups, the studies do not replicate in other populations, and the exact nature of the causal gene remains elusive and debatable [10-12]. It is noteworthy that there are also a number of important cytokine genes including interleukin 4 (IL-4) immediately adjacent to the 250 kb risk haplotype. The Single Nucleotide Polymorphisms in IL4, OCTN1 and OCTN2 Genes The Open Gastroenterology Journal, 2008, Volume 2 51 590C/T SNP in this gene has been previously associated with autoimmune diseases such as type I diabetes and rheumatoid arthritis, and has been suggested as a possible causal variant in CD [13]. Gründemann et al. [14] have shown that the fungal metabolite, ergothionine (ET) is the main physiological substrate of OCTN1. Taubert et al. [15] demonstrated that the amino acid substitution L503F is a gain of function polymorphism, resulting in a threefold higher substrate affinity and 50% higher initial transport capacity at low levels of ET ( 10μmol/l) in transfected HEK293 cells. A previous Auckland-based study suggested the possibility of an OCTN1 and OCTN2 haplotype associating with CD risk in New Zealand [16]. However, numbers and statistical significance were low, and we have failed to repeat this observation in our own larger Canterbury-based population. MATERIALS AND METHODS Study Participants The cases in this study are a random subset of the Caucasian participants of the Canterbury Inflammatory Bowel Disease Project, which has been described in detail elsewhere [17]. The subset was identified as those who were willing to provide a second DNA sample for the new studies. All subjects consented to collection of peripheral blood for DNA extraction and genotyping in comparison with their clinical data for CD or UC, as defined using standard diagnostic criteria [18]. The Montreal Classification system allowed genotype-phenotype analysis to be performed [19]. 388 CD participants, 405 UC participants, and 27 indeterminate colitis (IC) participants were genotyped for this study, as described in Table 1. All participants self-reported European ancestry, and patients who self-reported having any Maori or other non-Caucasian ancestry are not included. The 370 New Zealand Caucasian controls were selected at random from the electoral roll, comprising 93% of the population over eighteen years of age in Canterbury, New Zealand [20]. Applied Biosystems TaqMan®SNP Genotyping Assay. SNPs were genotyped using the TaqMan MGB diallelic discrimination system. Probes and oligonucleotides were obtained from Applied Biosystems using the Assay-byDesign product (listed in Table 2). The reactions were prepared by using 2x TaqMan Universal Master Mix, 40x SNP Genotyping Assay Mix, DNase-free water, 10 ng genomic DNA in a final volume of 5 ul per reaction. The PCR amplification was performed using the ABI Prism 7900 HT sequence-detector machine under the following conditions: 10 min 95°C enzyme activation followed by 40 cycles at 92°C for 15 sec and 60°C for 1min (annealing/extension). The allelic discrimination results were determined after the amplification by performing an endpoint read. To estimate genotype accuracy approximately 5% of the samples were genotyped in duplicate or triplicate for each of the markers. There were no conflicting duplicate genotypes among the 70 genotypes (60 for IL4) which could be compared to a duplicate genotype. Table 1. Summary of Clinical and Demographic Data for the Set of Caucasian IBD Patients CD n (%) UC n (%) IC n (%)