Identification of functional tumor necrosis factor-alpha promoter variants associated with Helicobacter pylori infection in the Sudanese population: Computational approach

BACKGROUND Helicobacter pylori(H.pylori)is a ubiquitous bacterium that affects nearly half of the world’s population with a high morbidity and mortality rate.Polymorphisms within the tumor necrosis factor-alpha(TNF-A)promoter region are considered a possible genetic basis for this disease.AIM To functionally characterize the genetic variations in the TNF-A 5’-region(-584 to+107)of Sudanese patients infected with H.pylori using in silico tools.METHODS An observational study was carried out in major public and private hospitals in Khartoum state.A total of 122 gastric biopsies were taken from patients who had been referred for endoscopy.Genomic DNA was extracted.Genotyping of the TNF-A-1030 polymorphism was performed using PCR with confronting two-pair primer to investigate its association with the susceptibility to H.pylori infection in the Sudanese population.Furthermore,Sanger sequencing was applied to detect single nucleotide polymorphisms in the 5’-region(-584 to+107)of TNF-A in H.pylori-infected patients.Bioinformatics analyses were used to predict whether these mutations would alter transcription factor binding sites or composite regulatory elements in this region.A comparative profiling analysis was conducted in 11 species using the ECR browser and multiple-sequence local alignment and visualization search engine to investigate the possible conservation.Also,a multivariate logistic regression model was constructed to estimate odds ratios and their 95%confidence intervals for the association between TNF-A-1030,sociodemographic characteristics and H.pylori infection.Differences were statistically significant if P<0.05.Statistical analyses were performed using Stata version 11 software.RESULTS A total of seven single nucleotide polymorphisms were observed in the TNF-A 5’-region of Sudanese patients infected with H.pylori.Only one of them(T>A,-76)was located at the in silico-predicted promoter region(-146 to+10),and it was predicted to alter transcription factor binding sites and composite regulatory elements.A novel mutation(A>T,+27)was detected in the 5’untranslated region,and it could affect the post-transcriptional regulatory pathways.Genotyping of TNF-A-1030 showed a lack of significant association between-1030T and susceptibility to H.pylori and gastric cancer in the studied population(P=0.1756)and(P=0.8116),respectively.However,a significant association was detected between T/C genotype and H.pylori infection(39.34%vs 19.67%,odds ratio=2.69,95%confidence interval:1.17-6.17,P=0.020).Mammalian conservation was observed for the(-146 to+10)region in chimpanzee(99.4%),rhesus monkey(95.6%),cow(91.8%),domesticated dog(89.3%),mouse(84.3%),rat(82.4%)and opossum(78%).CONCLUSION Computational analysis was a valuable method for understanding TNF-A gene expression patterns and guiding further in vitro and in vivo experimental validation.