A facile PCR-RFLP method for genotyping of ITPA rs1127354 and rs7270101 polymorphisms.

BACKGROUND: Inosine triphosphate pyrophosphatase (ITPA) gene single nucleotide polymorphisms (SNPs), rs1127354 and rs7270101, may cause a functional impairment in ITPase enzyme, resulting anemia protection in patients with chronic hepatitis C virus (HCV) infection undergoing ribavirin (RBV)-dependent regimens. The main purpose of this study was to provide and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for genotyping of ITPA rs1127354 and rs7270101 polymorphisms in chronic HCV-infected patients. METHODS: In the current study, 100 Iranian patients with chronic hepatitis C were examined and genotyped for ITPA rs1127354 and rs7270101 gene polymorphisms. To genotype rs1127354 and rs7270101 polymorphisms, PCR-RFLP technique and sequencing technique were performed on these samples. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results. RESULTS: The rs1127354 and rs7270101 polymorphisms of ITPA gene were genotyped by PCR-RFLP technique and sequencing simultaneously, and the results of both techniques were 100% concordant in all 100 patients. Both PCR-RFLP and sequencing techniques indicated that the genotypic frequency of rs7270101 was 80% AA, 19% AC and 1% CC, and for rs1127354 was 79% CC, 20% CA and 1% AA, respectively. CONCLUSION: We developed and validated a rapid and inexpensive PCR-RFLP technique for the detection of ITPA rs1127354 and rs7270101 gene polymorphisms.

The ITPA and C20orf194 Polymorphisms and Hematological Changes During Treatment With Pegylated-Interferon Plus Ribavirin in Patients With Chronic Hepatitis C.

BACKGROUND: It has been found that ITPase deficiency is caused by ITPA gene polymorphisms. It was observed that ITPA polymorphisms have impact on hematological changes, including hemoglobin (Hb)-decline during treatment of chronic hepatitis C (CHC) patients with pegylated-interferon (PEG-IFN) plus ribavirin (RBV). OBJECTIVES: This study aimed to assess the effect of ITPA and C20orf194 polymorphisms on hematological changes at week 4 of treatment with PEG-IFN plus RBV in patients with CHC. PATIENTS AND METHODS: In this retrospective study, 168 patients with CHC (56% HCV genotype-1 and 44% HCV genotype-3) under the treatment of PEG-IFN plus RBV were genotyped for rs1127354, rs7270101 and rs6051702 polymorphisms by the polymerase chain reaction-restriction fragment length polymorphism. Hematological changes including Hb-, platelet (Plt)- and white blood cell-decline at week 4 of the treatment were assessed. RESULTS: In univariate analysis, rs1127354 and HCV genotypes were found to influence the Hb-decline at week 4 of the treatment. In multivariate analysis, rs1127354 CA + AA and HCV genotype-3 were found to have a great role on prevention of Hb-decline. Furthermore, rs1127354 and HCV RNA levels were found to influence the Plt-decline at week 4 of the treatment in the univariate analysis. In multivariate analysis, rs1127354 CA + AA and HCV RNA levels less than 600,000 IU/mL were found to be associated with a higher level of Plt-decline. CONCLUSIONS: In patients with CHC, who were treated with PEG-IFN plus RBV, Hb-decline was affected by rs1127354 and HCV genotypes. However, Plt-decline may be altered by rs1127354 and baseline HCV RNA levels.

How Hepatitis C Virus Leads to Hepatocellular Carcinoma: A Network-Based Study.

BACKGROUND: Hepatitis C virus (HCV) has been known as a major cause of hepatocellular carcinoma (HCC) worldwide. However, the distinct molecular mechanisms underlying the effects of HCV proteins on the HCC progression have remained unclear. OBJECTIVES: In the present study, we studied the possible role of HCV in the HCC initiation and invasion using topological analysis of protein-protein interaction (PPI) networks. MATERIALS AND METHODS: After analysis with GEO2R, a PPI network of differentially expressed genes (DEGs) was constructed for both chronic HCV and HCC samples. The STRING and GeneMANIA databases were used to determine the putative interactions between DEGs. In parallel, the functional annotation of DEGs was performed using g: Profiler web tool. The topological analysis and network visualization was carried outperformed using Cytoscape software and the top hub genes were identified. We determined the hub genes-related miRNAs using miRTarBase server and reconstructed a miRNA-Hubgene network. RESULTS: Based on the topological analysis of miRNA-Hubgene network, we identified the key hub miRNAs. In order to identify the most important common sub-network, we aligned two PPI networks using NETAL tool. The c-Jun gene was identified as the most important hub gene in both HCV and HCC networks. Furthermore, the hsa-miR-34a, hsa-miR-155, hsa-miR-24, hsa-miR-744 and hsa-miR-92a were recognized as the most important hub miRNAs with positive correlation in the chronic HCV and HCC samples. Functional annotation of differentially expressed miRNAs (DEMs) using the tool for annotations of human miRNAs (TAM) revealed that there is a considerable overlap between miRNA gene expression profiles of HCV-infected and HCC cells. CONCLUSIONS: Our results revealed the possible crucial genes and miRNAs involved in the initiation and progression of HCC cells infected with HCV.

Impacts of the G145R Mutation on the Structure and Immunogenic Activity of the Hepatitis B Surface Antigen: A Computational Analysis.

BACKGROUND: Vaccine-escaped hepatitis B virus (HBV) mutations occur within the "a" determinant area, which is located in the major hydrophilic region (MHR) of the hepatitis B surface antigen (HBsAg) protein. It is now well established that the common G145R mutation is highly capable of escaping from HBsAg immune recognition. However, the impacts of this mutation on the structure and immunogenic activity of HBsAg have been poorly investigated. OBJECTIVES: The present study analyzed the effects of the G145R mutation on the structure and immunogenic activity of the HBsAg. MATERIALS AND METHODS: Three-dimensional (3D) structure of HBsAg for both the wild-type and G145R mutant were predicted and refined using several web tools. After quantitative evaluations, the effects of the G145R mutation on the secondary and 3D structures of the HBsAg were investigated. In parallel, the immunogenic activity of the wild-type and mutant HBsAg was also analyzed using a ClusPro docking server as well as the IEDB web tool. Further analyses were performed via molecular dynamics (MD) simulations using the GROMACS v5.0.2 simulation package. RESULTS: The G145R mutation causes a considerable reduction in the immunogenic activity of the HBsAg through a conformational change in the HBsAg antigenic loops. This mutation inserts a new ß-strand in the "a" determinant region of the HBsAg, leading to a reduced binding affinity to its monoclonal antibody, MAb12. The G145R mutation also increased the compactness and stability of the HBsAg by enhancing the rigidity of the "a" determinant. CONCLUSIONS: These data will be beneficial for designing more advanced antibodies for the recognition of the HBsAg in diagnostics. In addition, the results of this study may assist in the design or development of more effective hepatitis B vaccines.

Prevalence of hepatitis delta virus (HDV) infection in chronic hepatitis B patients with unusual clinical pictures.

BACKGROUND: Probably 5% of the HBV carriers have HDV super infection. The risk of fulminant hepatitis, cirrhosis and hepatocellular carcinoma is higher in superinfection than the settings when HBV is alone. OBJECTIVES: The aim of this study was to evaluate the prevalence of HDV in Iranian HBV isolates and to compare their clinical and virological pictures as well as their HDV genetic variations with other worldwide isolates. PATIENTS AND METHODS: 81 carriers with positive results for HBsAg with upper limit ranges of ALT and low or undetectable levels of HBV viral load who did not respond to HBV therapy were selected. After RT amplification of HDV Delta antigen, direct sequencing and phylogenetic study were performed to explore the genotype(s) and nucleotide/amino acid variations. RESULTS: 12 (14.8%) patients had positive results for both HDV RNA and anti-HDV. The mean ALT level was higher in HDV positive patients (75.9 U/ML) than HBV-mono-infected individuals; however, the mean HBV viral load was lower in coinfected patients than HBV-mono-infected patients. Phylogenetically, genotype I was the only detected genotype, and the most closely related isolates were of Turkish, Italian and Mongolian origin. Within the delta Ag, there were 326 nucleotide mutations, of which 111 and 215 were silent and missense, respectively. The total number of amino acid substitution was 148; most were located in known functional/epitopic domains. There was no correlation between the numbers of amino acid mutations, with clinical, virological status of the patients. CONCLUSIONS: HDV should be suspected in HBV carriers with unusual clinical and virological pictures. Relatedness of Iranian HDV isolates to Italian and Turkish sequences proposed a common Caucasian origin for the distribution of HDV genotype I in this ethnic group.