Background: Inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are typically associated with a decrease in local pH. Genome-wide association studies (GWAS) identified over 200 non-overlapping single-nucleotide polymorphism (SNP) genetic risk loci for IBD. The proton-sensing G-protein-coupled receptor T-cell death associated gene 8 (TDAG8 or GPR65) has been found to be a genetic risk factor for IBD in recent GWASs. Thereby, the T genotype of the SNP rs8005161 within the GPR65 gene confers increased IBD risk. In response to extracellular acidification GPR65 activates second messengers: cAMP via the Gs signalling pathway or G12/13/Rho signalling. In this study we analyzed the association of the SNP rs8005161 in the IBD cohort of patients with increased IBD risk, and its functional relevance.
Methods: 1138 individuals (591 non-IBD, 203 UC, 344 CD) were genotyped for risk SNPs, GPR65 (rs8005161, rs3742704) and GALC (rs1805078), with Taqman SNP Genotyping Assays. Additionally, more than 2064 IBD patients from the Swiss IBD Cohort Study (SIBDCS) were genotyped by Illumina sequencing. Ten patients with the genotype rs8005161 TT/CT and CC (Wild Type) from the IBD cohort and ten non-IBD controls (CC) were recruited for the functional study. Human CD14+ cells were isolated from blood samples and subjected to an extracellular acidic pH shift (pH 6.6 vs. pH 7.6) in functional assays: cAMP, RhoA GTPase activation.
Results: rs8005161 was more frequent in UC patients (minor allele frequency (MAF) 14.53% vs 10.05% in the non-IBD group), whereas no statistically significant association with IBD, UC or CD was found for the other variants (GPR65 rs3742704, GALC rs1805078) by Taqman genotyping. Sequenced genotype frequency of rare homozygote rs8005161 in the SIBDCS was 1.17%, MAF - 10.3%. No significant differences were observed in the cAMP production between IBD (TT, CT, WT/CC) and non-IBD (WT/CC) genotype carriers upon pH shift from 7.6 to 6.6. However, a decreased activation of GTPase RhoA was seen for IBD rs8005161 (TT) variant carriers after an acidic pH shift.
Conclusions: GPR65 SNP rs8005161 genotyping showed significant association with UC cases. No differences in cAMP signalling in IBD TT/CT/CC subjects compared to healthy CC subjects were observed. In contrast, TT IBD patients showed impaired activation of RhoA upon an acidic pH shift. Our results support the role of GPR65 in the intestinal inflammation in genetically predisposed individuals, emphasizing the link between acid-base homeostasis and IBD pathogenesis.