Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

NOD2/CARD15 gene mutations in North Algerian patients with inflammatory bowel disease

AIM: To analyse allelic frequency of NOD2 gene variantsand to assess their correlation with inflammatory bowel disease(IBD) in Algeria.METHODS: We studied 132 unrelated patients diagnosed with IBD, 86 with Crohn's disease(CD) and 46 with ulcerative colitis(UC). Data was prospectively collected between January 2011 and December 2013. The demographic and clinical characteristics were recorded for all the patients. A group of 114 healthy unrelated individuals were selected as controls. All groups studied originated from different regions of North Algeria and confirmed the Algerian origin of their parents and grandparents. Informed and written consent was obtained from each of the participants. All individuals were genotyped for the three CDassociated NOD2 variants(p.Arg702 Trp, p.Gly908 Arg and p.Leu1007 fsins C mutations) using the polymerase chain reaction-restriction fragment length polymorphism method. Allele and genotype frequencies in patients and control subjects were compared by χ2 test and Fisher's exact test where appropriate. Odds ratios(OR) and 95% confidence intervals(95%CI) were also estimated. Association analyses were performed to study the influence of these variants on IBD and on clinical phenotypes.RESULTS: The p.Arg702 Trp mutation showed the highest frequency in CD patients(8%) compared to UC patients(2%)(P = 0.09, OR = 3.67, 95%CI: 0.48-4.87) and controls(5%)(P = 0.4, OR = 1.47, 95%CI: 0.65-3.31). In CD patients allelic frequencies of p.Gly908 Arg and p.Leu1007 fsins C variants compared to HC were 3% vs 2%(P = 0.5, OR = 1.67, 95%CI: 0.44-6.34); 2% vs 1%(P = 0.4 OR = 2.69 95%CI: 0.48-14.87 respectively). In UC patients, allelic frequencies of p.Gly908 Arg and p.Leu1007 fsins C variants compared to HC were 1% vs 2%(P = 1, OR = 1.62, 95%CI: 0.17-4.74) and 2% vs 1%(P = 0.32, OR = 0.39, 95%CI: 0.05-2.87). The total frequency of the mutated NOD2 chromosomes was higher in CD(13%), than in HC(8%) and UC(5%). In addition, NOD2 variants were linked to a particular clinical sub-phenotype in CD in this Algerian cohort. As expected, the three NOD2 variants showed a significant association with CD but did not reach statistical significance, despite the fact that the allele frequency of NOD2 variants was in the range found in most of the European populations. This might be due to the non-exposure of the NOD2 carriers to environmental factors, required for the expression of the disease.CONCLUSION: Further analyses are necessary to study genetic and environmental factors in IBD in the Algerian population, using larger patient groups.