P-214 Determining the Role of TCF-4 in the Genetic Susceptibility of Crohn’s Disease of the Pouch

Abstract

Crohn’s disease of the pouch is an unfortunate inflammatory complication among ulcerative colitis patients who have undergone restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) that drastically reduces quality of life. While this is a complex disease with numerous contributory factors, it is believed that genetic mutations can make certain people susceptible to CD of the pouch. The aim of our study was to evaluate whether the rs3814570 SNP found in the promoter region of TCF-4 is associated with CD of the pouch and whether this specific SNP contributes to decreased DEFA5 gene expression. DNA was isolated from blood samples and rs3814570 SNP genotyping was carried out by PCR amplification followed by restriction enzyme digest and visualization by gel electrophoresis. DEFA5 gene expression was evaluated by isolating total RNA from pouch biopsies and carrying out real time qRT-PCR. Odds ratios were calculated to detect association between the rs3814570 SNP and CD of the pouch versus normal pouch controls. Mean Ct values were calculated for DEFA5 gene expression and normalized to GADPH endogenous control and one-way ANOVA was done to detect for association of specific TCF-4 genotypes and DEFA5 expression. Subjects with 1 copy of the rs3814570 SNP T allele (heterozygous) or 2 copies of the T-allele (minor allele homozygous) had OR (95% CI) of 1.02 (0.59–1.76) and 1.67 (0.71–3.89), respectively when tested for association with CD of the pouch compared to normal pouch controls. Major allele homozygous (CC) had a DEFA5 mean Ct value (Std Error) of -3.28 (0.13), heterozygous (CT) -1.87 (0.14) and minor allele homozygous (TT) -1.44 (0.24) with one-way ANOVA showing F (2, 108) = 44.7, P < 0.001. Our study is consistent with other studies that found that having 1 or 2 copies of the TCF-4 rs3814570 T allele is associated with decreased DEFA5 expression. There is no significant association with rs3814570 and CD of the pouch, which could either be real or could be due to an inadequate sample size and power to detect an association at the SNP level.