Detection of single‐nucleotide polymorphisms in the intron 9 region of the nucleotide oligomerization domain‐1 gene in ulcerative colitis patients of North India

Abstract

The nucleotide-binding oligomerization domain-1 (NOD1) gene encodes a pattern recognition receptor that senses pathogens. NOD1/caspase recruitment domain (CARD4) signaling leads to the activation of nuclear factor-kB, and plays an important role in innate immunity. Certain polymorphisms and mutations in NOD1/CARD4 might result in a dysfunctional innate immune response during bacterial recognition, which might have direct implications in inflammatory bowel disease (IBD) pathogenesis. We carried out a systemic analysis for the presence of polymorphic variants in the intron 9 region of the leucine-rich repeat (LRR) domain encompassing the exon-intron boundaries of the NOD1 gene. To detect unknown single-nucleotide polymorphisms, we used the denaturing high-performance liquid chromatography (DHPLC) screening technique and validated our data by restriction fragment length polymorphism and direct sequencing. Genotype and allele frequencies showed significant differences in their distribution. The mutations discriminating alleles in the intron 9 region of the LRR domain of the NOD1 gene were correctly predicted by DHPLC technique and statistically verified in IBD and non-IBD individuals. Of the seven mutations detected, only four showed a significant association with disease activity. Mutations detected earlier in the exon 6 region of NOD1 were also used for the haplotype analysis. The GTTG haplotype was found to be significantly overrepresented in ulcerative colitis (UC) patients, as compared to the controls (P = 3.3726E(-6) ). Our study has revealed a polymorphism association in the LRR domain of the NOD1 gene with the severity of UC disease. This might be due to disruption of the LRR region critical for NOD1-mediated bacterial sensing. A gene-wide, haplotype-based approach shows that GTTG haplotype carriers are overrepresented in UC patients, and that could increase the risk of the disease.