Reverse Transcriptase Region Research Articles

Prevalence of HIV-1 pre-treatment drug resistance in a southern province of Iran, 2016-2017.

HIV-1 transmitted drug resistance (TDR) occurs when primary viruses bear drug resistance mutations (DRMs). TDR causes first-line antiretroviral (ARV) therapy (ART) failure and is becoming more pronounced due to the widespread use of ART. With the absence of routine individual-level drug resistance testing, the World Health Organization (WHO) recommends the tracking of TDR mutations and optimizing the first-line ART following pre-treatment drug resistance (PDR) surveys. Here, we report the PDR frequency for the first time in Hormozgan, a southern province of Iran. In this study, 41 blood samples from HIV-1-positive ART-candidate volunteers were collected across the province between April 2016 and March 2017. Phylogenetic analysis of the sequenced protease (PR) and reverse transcriptase (RT) regions showed that 39 out of 41 samples (95%) were CRF35_AD and the two remaining cases were subtype B (2.5%) and C (2.5%). D67G (2.4%), a mutation that reduces susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs) was the only detectable TDR mutation in this population. Two other DRMs, including E138A (9.7%) and V179T (4.9%), which confer resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), were also identified. Although no major protease inhibitor (PI) resistance mutations were detected, the minor mutations L10F and L33F (2.5% each) as well as several highly frequent polymorphic mutations were identified. Our results show a PDR frequency of 17% in infected individuals from Hormozgan, classified further as 2.4% NRTIs and 14.6% NNRTIs. These results suggest that first-line ART should be practiced carefully in Hormozgan province, and alternative regimens may become necessary for all starters.

High prevalence of HIV-1 transmitted drug-resistance mutations from proviral DNA massively parallel sequencing data of therapy-naïve chronically infected Brazilian blood donors.

BackgroundAn improved understanding of the prevalence of low-abundance transmitted drug-resistance mutations (TDRM) in therapy-naïve HIV-1–infected patients may help determine which patients are the best candidates for therapy. In this study, we aimed to obtain a comprehensive picture of the evolving HIV-1 TDRM across the massive parallel sequences (MPS) of the viral entire proviral genome in a well-characterized Brazilian blood donor naïve to antiretroviral drugs.Materials and methodsThe MPS data from 128 samples used in the analysis were sourced from Brazilian blood donors and were previously classified by less-sensitive (LS) or “detuned” enzyme immunoassay as non-recent or longstanding HIV-1 infections. The Stanford HIV Resistance Database (HIVDBv 6.2) and IAS-USA mutation lists were used to interpret the pattern of drug resistance. The minority variants with TDRM were identified using a threshold of ≥ 1.0% and ≤ 20% of the reads sequenced. The rate of TDRM in the MPS data of the proviral genome were compared with the corresponding published consensus sequences of their plasma viruses.ResultsNo TDRM were detected in the integrase or envelope regions. The overall prevalence of TDRM in the protease (PR) and reverse transcriptase (RT) regions of the HIV-1 pol gene was 44.5% (57/128), including any mutations to the nucleoside analogue reverse transcriptase inhibitors (NRTI) and non-nucleoside analogue reverse transcriptase inhibitors (NNRTI). Of the 57 subjects, 43 (75.4%) harbored a minority variant containing at least one clinically relevant TDRM. Among the 43 subjects, 33 (76.7%) had detectable minority resistant variants to NRTIs, 6 (13.9%) to NNRTIs, and 16 (37.2%) to PR inhibitors. The comparison of viral sequences in both sources, plasma and cells, would have detected 48 DNA provirus disclosed TDRM by MPS previously missed by plasma bulk analysis.ConclusionOur findings revealed a high prevalence of TDRM found in this group, as the use of MPS drastically increased the detection of these mutations. Sequencing proviral DNA provided additional information about TDRM, which may impact treatment decisions. The overall results emphasize the importance of continuous monitoring.

Clinical patterns associated with the concurrent detection of anti-HBs and HBV DNA.

Simultaneous detection of anti-HBs and HBV DNA is a rare serological combination and has been described in acute and chronic HBV infection. To scrutinize viral and clinical patterns associated with concurrent detection of anti-HBs and HBV DNA. Simultaneous detection of anti-HBs and HBV DNA was observed in 64/1444 (4.4%) patients treated for HBV infection at the University Hospital of Essen from 2006 to 2016 (8 with acute, 20 with reactivated, and 36 chronic HBV infection). Clinical data and laboratory parameters were analyzed. Regions of the small hepatitis B surface antigen (SHB) and the reverse transcriptase (RT) were sequenced using next generation sequencing (NGS). Among the 64 patients with detectable HBV DNA and anti-HBs, 17 were HBsAg negative (HBsAg[-]), and two had acute liver failure. Patients with acute HBV infection had fewer genotype specific amino acid substitutions in the SHB region than patients with reactivated HBV infection (4 [4.5] vs 9 [16.25], P = 0.043). However, we could observe a significantly higher number of mutations in the a-determinant region when comparing chronically infected patients to patients with acute infection (0 [1] vs 1 [1], P = 0.044). The ratio of nonsynonymous to synonymous mutations (Ka/Ks) was on average >1 for the SHB region and <1 for the RT region. The Ka/Ks ratio (>1) in the SHB region indicates that anti-HBs might have exerted selection pressure on the HBsAg. In three cases the diagnosis of acute HBV infection would have been at least delayed by only focusing on HBsAg testing.

Comparison of error correction algorithms for Ion Torrent PGM data: application to hepatitis B virus

Ion Torrent Personal Genome Machine (PGM) technology is a mid-length read, low-cost and high-speed next-generation sequencing platform with a relatively high insertion and deletion (indel) error rate. A full systematic assessment of the effectiveness of various error correction algorithms in PGM viral datasets (e.g., hepatitis B virus (HBV)) has not been performed. We examined 19 quality-trimmed PGM datasets for the HBV reverse transcriptase (RT) region and found a total error rate of 0.48% ± 0.12%. Deletion errors were clearly present at the ends of homopolymer runs. Tests using both real and simulated data showed that the algorithms differed in their abilities to detect and correct errors and that the error rate and sequencing depth significantly affected the performance. Of the algorithms tested, Pollux showed a better overall performance but tended to over-correct ‘genuine’ substitution variants, whereas Fiona proved to be better at distinguishing these variants from sequencing errors. We found that the combined use of Pollux and Fiona gave the best results when error-correcting Ion Torrent PGM viral data.

Drug resistance mutation profiles of the drug-naïve and first-line regimen-treated HIV-1-infected population of Suzhou, China.

Little is known about the prevalence of drug-resistant mutations in HIV-1-positive individuals in Suzhou, China. To elucidate the transmitted drug resistance (TDR) and acquired drug resistance mutation (ADR) profiles, we collected blood specimens from 127 drug-naïve and 117 first-line drug-treated HIV-1-infected individuals sampled from 2014 to 2016 in Suzhou. We successfully amplified pol fragments from 100 drug-naïve and 20 drug-treated samples. We then determined the drug-resistant mutations to protease (PR) and reverse-transcriptase (RT) inhibitors according to the Stanford drug resistance database. Overall, 11 and 13 individuals had transmitted (drug-naïve group) and acquired (treated group) resistance mutations, respectively. Six transmitted drug-resistant mutations were found, including two mutations (L33F and L76V) in the protease region and four (K70N/E and V179D/E) in the RT region. Only L76V was a major mutation, and K70N/E and V179D/E are known to cause low-level resistance to RT inhibitors. All 13 treated participants who had major drug resistance mutations demonstrated intermediate to high resistance to efavirenz and nevirapine, and six had a treatment duration of less than three months. No major mutations to RT inhibitors were found, implying that the epidemic of transmitted resistance mutations was not significant in this area. Our results suggest that more frequent virus load and drug resistance mutation tests should be conducted for individuals receiving antiretroviral treatment, especially for newly treated patients. Our research provides insights into the occurrence of HIV-1 drug resistance in Suzhou and will help to optimize the treatment strategy for this population.

Abstract 1169: mtTERT promoter as a target for treatment of glioblastoma

Abstract Approximately 86% of GBM tumors exhibit mutation at -124 or -146 bases upstream of the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutation in the promoter region of hTERT impairs repression, leading to overexpression of hTERT; inappropriate hTERT is associated with oncogenesis, tumor maintenance, and resistance to apoptosis. We surveyed long-term glioma cell lines and glioma PDX models for mt-hTERT; mRNA and protein expression of hTERT were assessed by qPCR and western blot. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element and consequent over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the somatic mutations and restores the silencer function of the G-quadruplex thereby reducing hTERT activity. TG-4260 directly decreases the transcription activity of the WT and the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. Finally, TG-4260 significantly inhibits telomerase and shortens telomere length after five days of treatment and induces a senescence-like phenotype. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. Finally, we screened GBM cell models against a novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Citation Format: Saumya R. Bollam, Harshil D. Dhruv, Hyun-Jin Kang, Sen Peng, Vijay Gokhale, Laurence Hurley, Michael Berens. mtTERT promoter as a target for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1169. doi:10.1158/1538-7445.AM2017-1169

Abstract 111: Targeting STAT3 and telomerase for the treatment of colorectal cancer

Abstract There is an increasing evidence of pro-inflammatory cytokines involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-α, induced colorectal cancer cells more active and invasive. Combined treatments of IL-6 and TNF-α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3 and NF-κB physically interacted upon cytokine stimulation. Similarly, STAT1 hetero-dimerized with STAT3 and the binding affinity was enhanced with cytokine treatments. STAT3 bound the promoter region of human telomerase reverse transcriptase (hTERT), and IL-6 and TNF-α stimulation further enhanced this STAT3 binding affinity. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6 and TNF-α induced cancer cell invasiveness and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3 and NF-κB interactions. STAT3 binding to hTERT promoter was inhibited and telomerase activity was decreased with the withaferin A treatments. Taken together, pro-inflammatory cytokines induced-cancer cell invasiveness is mediated by STAT3 regulated mechanism in colorectal cancer cells. Our study suggests the novel natural compound therapy for the metastatic colorectal cancer in clinical settings. Citation Format: Seyung Chung, Quincy Okobi, Debbie Adekoya, Jaydutt Vadgama. Targeting STAT3 and telomerase for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 111. doi:10.1158/1538-7445.AM2017-111

Integrating nested PCR with high-throughput sequencing to characterize mutations of HBV genome in low viral load samples.

Due to the low viral load of hepatitis B virus (HBV) in plasma samples, conventional techniques have limitations to the detection of antiviral resistance mutations. To solve the problem, we developed a fast, highly sensitive, and accurate method to sequence the HBV whole-genome sequencing in plasma samples which had various viral loads from very low to high.Twenty-one plasma samples were collected from patients who were carriers of HBV from the Hangzhou First People's Hospital. Two pairs of conserved, overlapping, nested primers were used to amplify and sequence the whole HBV genome in 8 plasma samples with different viral loads. High-throughput sequencing was performed on Illumina MiSeq platform. Concomitantly, 3 samples were directly sequenced without PCR amplification. We compared amplicon-sequencing with direct sequencing to develop a method for amplifying and characterizing the whole genome of HBV.HBV genome was amplified from all samples and verified by Sanger sequencing, regardless of the viral loads. Sequencing results revealed that only a few reads were mapped to the HBV genome following direct sequencing, while the amplicon-sequencing reads had a good coverage and depth. We identified 50 intrahost single nucleotide variations (iSNVs), 14 of which were low frequency mutations. Interestingly, iSNVs were more common in low viral load samples than in high viral load samples, and mutations in the reverse transcriptase (RT) region were most prevalent.We conclude that amplicon-sequencing is not only a practical method to detect HBV infection with a high sensitivity and accuracy but also enables to detect mutations in the HBV genome in low viral load samples from HBV-infected patients. Thus, our findings provide a new diagnosis method of HBV infection, which is capable of detection of low frequent mutations in low viral load samples.

The correlations between DNA methylation and polymorphisms in the promoter region of the human telomerase reverse transcriptase (hTERT) gene with postoperative recurrence in patients with thyroid carcinoma (TC)

BackgroundThis study aims at exploring the correlations between DNA methylation and polymorphisms in the promoter region of the human telomerase reverse transcriptase (hTERT) gene and postoperative recurrence in patients with thyroid carcinoma (TC).MethodsA total of 312 patients diagnosed with TC were chosen for the study and categorized into recurrence (n = 75) and non-recurrence (n = 237) groups. The hTERT rs2736100 and rs2736098 polymorphisms were detected by performing polymerase chain reaction-restriction fragment length polymorphism. DNA methylation in the promoter region of hTERT gene was evaluated by pyrosequencing. A telephonic and/or outpatient follow-up was conducted for all patients. The correlations of DNA methylation and polymorphisms in the promoter region of hTERT with postoperative recurrence of TC patients underwent analysis.ResultsThe patient in the recurrence group showed evidently different pathological types and tumor stages in comparison to the non-recurrence group. The GG genotype of hTERT rs2736100 might increase the recurrence risk of TC patients. No correlations between hTERT rs2736098 polymorphisms and recurrence risk were observed. Compared to the TT + TG genotype frequency, the rs2736100 GG genotype frequency increased in patients without multicentricity, patients with extrathyroidal invasion, patients with lymph node metastasis, patients with undifferentiated carcinoma, and patients in the III + IV stage. The recurrence group showed significantly higher DNA methylation level compared to the non-recurrence group. The DNA methylation level was closely associated to tumor stage and lymph node metastasis of TC patients in the recurrence group.ConclusionsThe DNA methylation and rs2736100 polymorphisms in the promoter region of hTERT gene might be in correlation to postoperative recurrence of TC patients.

Ultra-Deep Sequencing Analysis on HIV Drug-Resistance-Associated Mutations Among HIV-Infected Individuals: First Report from the Philippines.

A sharp increase in the number of people living with HIV has been documented in the Philippines. In response, the government has instituted antiretroviral therapy (ART) nationwide through HIV treatment hubs. However, no data presently exist on the status of ART drug-resistance-associated mutations (DRMs). In this study, we aim at analyzing DRM profiles in the Philippines and at providing comprehensive data on DRMs to guide treatment decisions and prevent viral failures. We conducted a cross-sectional study in 119 volunteers who tested positive for HIV from more than 8,000 participants screened for HIV across the nation through the 2013 Integrated HIV Behavioral and Serologic Surveillance (IHBSS) program. Amplicons were generated from plasma RNA by using primers designed to analyze diverse HIV-1 isolates targeting the reverse transcriptase region and sequenced on a 454 ultra-deep sequencing (UDS) platform to assess DRMs. DRMs were defined by using the Stanford HIV drug resistance database, and we found only 2 from 110 evaluable individuals with major HIV variants (>20% prevalence) that were highly resistant to the non-nucleoside reverse transcriptase inhibitor (NNRTI: efavirenz and nevirapine). However, a larger fraction of individuals harbored minority drug-resistant HIV variants (0.5%-20% prevalence) and they were highly resistant to NNRTI nevirapine (89/110), rilpivirine (5/110), and efavirenz (49/110). This study is the first report on the presence of HIV drug resistance in the Philippines and demonstrates the utility of UDS in assisting the detection of HIV minor variants. Monitoring for ART-DRMs will assist in improving HIV management strategies in curtailing the evolving epidemic in the Philippines.

Mutation of the TERT promoter leads to poor prognosis of patients with non-small cell lung cancer.

Mutations in the promoter region of telomerase reverse transcriptase (TERT) and alterations in telomere length (TL) have been the focus of research in various types of cancer. In the present study, the frequency and clinical characteristics of TERT promoter mutations and TL were studied in non-small cell lung cancers (NSCLC). TERT promoter mutations and TL were analyzed in 188 patients using DNA sequencing and the reverse transcription-quantitative polymerase chain reaction, respectively. The TERT promoter mutation rate was observed to be 2.2% (4/188 NSCLC cases), and it was significantly associated with regional lymph node invasion (P<0.001). No significant difference in TL was observed between the patients with and without TERT promoter mutations. TL was decreased in males (P=0.058 vs. females) and smokers (P=0.008 vs. non-smokers). Survival analyses demonstrated poor prognoses for patients with NSCLC with TERT promoter mutations (P<0.001). Multivariate survival analyses demonstrated that TERT promoter mutations were an independent prognostic marker for poor overall survival (P=0.045). The results of the present study demonstrated that TERT promoter mutation was not frequent in NSCLC; however, it may have value as a prognostic marker in NSCLC.

Identification, transmission and genomic characterization of a new member of the family Caulimoviridae causing a flower distortion disease of Rudbeckia hirta

A disease of Rudbeckia hirta (Black-eyed Susan), characterized by severe flower deformation, was observed in Minnesota during 2010–2016. A previously undescribed virus species, named Rudbeckia flower distortion virus (RuFDV, family Caulimoviridae, genus unassigned), was determined to be the causal agent of the disease. Symptoms induced by RuFDV infection resemble those characteristic of phytoplasma-induced diseases, but no phytoplasmas were detected in RuFDV-infected R. hirta. The virus, and the disease were transmitted readily by mechanical inoculation and by the aphid Myzus persicae, but only to R. hirta. Virions of RuFDV are icosahedral, 42–45nm in diameter, and contain a circular 8222bp dsDNA genome containing seven open reading frames (ORFs). The ORFs 2–6 have 28–52% amino acid sequence identity to the movement protein (MP), coat protein (CP), aspartic protease (AP), reverse transcriptase (RT) and RNase H, and translational transactivator (TA) domains of known caulimoviruses. The two remaining ORFs (1 and 7) have no significant amino acid sequence similarity to known viruses. Although the RuFDV ORF 6 is significantly truncated relative to those of other known caulimoviruses, neither this nor the concomitant absence of characteristic virus-encoded cytoplasmic inclusion bodies appears to adversely affect aphid transmission of this virus. Phylogenetic analysis based on the sequence of the RT region revealed no close relationship to known members of the family Caulimoviridae. Based on sequence similarity, genome organization and phylogenetic relatedness, RuFDV appears to be distinct from any currently recognized taxonomic grouping in the family Caulimoviridae.

Molecular Testing of Brain Tumor.

The World Health Organization (WHO) classification of central nervous system (CNS) tumors was revised in 2016 with a basis on the integrated diagnosis of molecular genetics. We herein provide the guidelines for using molecular genetic tests in routine pathological practice for an accurate diagnosis and appropriate management. While astrocytomas and IDH-mutant (secondary) glioblastomas are characterized by the mutational status of IDH, TP53, and ATRX, oligodendrogliomas have a 1p/19q codeletion and mutations in IDH, CIC, FUBP1, and the promoter region of telomerase reverse transcriptase (TERTp). IDH-wildtype (primary) glioblastomas typically lack mutations in IDH, but are characterized by copy number variations of EGFR, PTEN, CDKN2A/B, PDGFRA, and NF1 as well as mutations of TERTp. High-grade pediatric gliomas differ from those of adult gliomas, consisting of mutations in H3F3A, ATRX, and DAXX, but not in IDH genes. In contrast, well-circumscribed low-grade neuroepithelial tumors in children, such as pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and ganglioglioma, often have mutations or activating rearrangements in the BRAF, FGFR1, and MYB genes. Other CNS tumors, such as ependymomas, neuronal and glioneuronal tumors, embryonal tumors, meningothelial, and other mesenchymal tumors have important genetic alterations, many of which are diagnostic, prognostic, and predictive markers and therapeutic targets. Therefore, the neuropathological evaluation of brain tumors is increasingly dependent on molecular genetic tests for proper classification, prediction of biological behavior and patient management. Identifying these gene abnormalities requires cost-effective and high-throughput testing, such as next-generation sequencing. Overall, this paper reviews the global guidelines and diagnostic algorithms for molecular genetic testing of brain tumors.

Altered TERT promoter and other genomic regulatory elements: occurrence and impact.

Study of genetic alterations, inherited or acquired, that increase the risk or drive cancers and many other diseases had remained mostly confined to coding sequences of the human genome. Data from genome wide associations studies, development of the Encyclopedia of DNA Elements (ENCODE), and a spurt in detection of driver somatic mutations have shifted focus towards noncoding regions of the human genome. The majority of genetic variants robustly associated with cancers and other syndromes identified through genome wide studies are located within noncoding regulatory regions of the genome. Genome wide techniques have put an emphasis on the role of three-dimensional chromosomal structures and cis-acting elements in regulations of different genes. The variants within noncoding genomic regions can potentially alter a number of regulatory elements including promoters, enhancers, insulators, noncoding long RNAs and others that affect cancers and various diseases through altered expression of critical genes. With effect of genetic alterations within regulatory elements dependent on other partner molecules like transcription factors and histone marks, an understanding of such modifications can potentially identify extended therapeutic targets. That concept has been augmented by the detection of driver somatic noncoding mutations within the promoter region of the telomerase reverse transcriptase (TERT) gene in different cancers. The acquired somatic noncoding mutations within different regulatory elements are now being reported in different cancers with an increased regularity. In this review we discuss the occurrence and impact of germline and somatic alterations within the TERT promoter and other genomic regulatory elements.

Increase in human immunodeficiency virus 1 diversity and detection of various subtypes and recombinants in north-eastern Brazil.

Diverse human immunodeficiency virus 1 (HIV-1) subtypes and circulating recombinant forms are found in Brazil. The majority of HIV-1 molecular epidemiological studies in Brazil have been conducted in the southern and south-eastern regions of the country, although several recent studies in the north-eastern region have addressed this issue. The objective of this study was to molecularly characterize HIV-1 circulating in Pernambuco, north-eastern Brazil. A total of 64 samples were collected from 2002 to 2003, and another 103 were collected from 2007 to 2009. The protease and partial reverse transcriptase regions of the HIV-1 polymerase-encoding (pol) gene were sequenced, and subtyping, recombination and phylogenetic analyses were performed.Results/Key findings. Subtype B (60.9 %) was found to be predominant, followed by HIV-1 F (31.4 %). Several BF recombinants (4.2 %), and BC and AG recombinants were also identified. The intra-subtype genetic diversity was estimated to be 0.065 (sd±0.004) for HIV-1 B and 0.055 (sd±0.004) for HIV-1 F, reflecting a greater accumulation of mutations in subtype B (P<0.01). More codons were found to be under positive selective pressure in samples collected from 2007 to 2009, from individuals with a T-cell count≥200 cells mm-3 and from women. Coalescence data indicated that the subtype F population has been continuously expanding. HIV-1 shows high genetic diversity in the state of Pernambuco. Thus, additional molecular evaluations of circulating strains will provide a better understanding of the epidemic and may lead to more effective preventive strategies.

Identification of Novel A2/C2 Inter-Genotype Recombinants of Hepatitis B Virus from a Korean Chronic Patient Co-Infected with Both Genotype A2 and C2.

Nearly all cases of Hepatitis B virus (HBV) infections in South Korea have the C2 genotype. Here, we have identified a chronically infected patient who was co-infected with HBV of both the A2 and C2 genotypes by screening 135 Korean chronically infected patients using direct sequencing protocols targeting the 1032-bp polymerase reverse transcriptase (RT) region. Further polymerase chain reaction (PCR)-cloning analysis (22 clones) of the RT showed that this patient had genotype C2 (12 clones), genotype A2 (six clones) and A2/C2 inter-genotype HBV recombinants (four clones). BootScan analysis showed that three of the four recombinants have different types of recombination breakpoints in both the RT and overlapping hepatitis B surface antigen (HBsAg) region. Given the significance of HBsAg as a diagnostic or vaccination target against HBV infection, clinical implications of these identified recombinants should be studied in the future. To our knowledge, this is the first report on A2/C2 inter-genotype HBV recombinants.

Oral Cancer Screening Based on Methylation Frequency Detection in hTERT Gene Using Electrochemical Hybridization Assay via a Multi‐electrode Chip Coupled with Ferrocenylnaphthalene Diimide

AbstractFerrocenylnaphthalene diimide‐based electrochemical hybridization assay via a multi‐electrode chip was applied to detect the methylation frequency in the promoter region of human telomerase reverse transcriptase (hTERT) gene for clinical samples from tissues, local exfoliated oral cells from a lesion, or from entire oral cavity after their methylation specific PCRs. These methylation frequencies were increased with cancer progress as the following order: healthy volunteers, oral leukoplakia as precancerous lesion, and oral squamous cell carcinoma (OSCC). Operating characteristic analysis of the obtained current data doesn't only give excellent discrimination ability of OSCC, but also of oral leukoplakia from healthy volunteers for all samples. Sensitivity and specificity was 95 % and 90 %, respectively, which is a comparable with methods in practical use.

F221Y mutation in hepatitis B virus reverse transcriptase is associated with hepatocellular carcinoma prognosis following liver resection.

Hepatitis B virus (HBV) reverse transcriptase (RT) is encoded by the polymerase gene in the reverse transcriptase region, which overlaps with the S gene. The association between mutations of HBV RT and the pathobiological features of hepatocellular carcinoma (HCC) remain to be elucidated. The present study aimed to examine mutations in this region of the HBV genome and its clinical significance. Briefly, HBV total DNA was extracted from 84 pairs of HCC tumor tissue and corresponding adjacent non‑tumor tissue samples. The RT/S regions (nt130‑1161) were amplified and sequenced using the Sanger method, and associations between RT mutations and the clinical characteristics of patients with HCC were analyzed. Finally, 27 and 29 mutations with frequencies >5% were identified in the RT and S regions, respectively. The rtF221Y variation and a tumor size >8cm were found to be independent risk factors for the postoperative recurrence of HCC, with hazard ratios of 2.345 (95% CI, 1.391‑3.953; P=0.001) and 1.838 (95% CI, 1.069‑3.161; P=0.028), respectively. rtF221Y was also an independent risk factor for poor overall survival rates (HR=2.557; 95% CI, 1.344‑4.866; P=0.004). The mutation of R122K in the HBV S protein was closely associated with tumor recurrence (P<0.001). As a result, rtF221Y was identified as a risk factor for poor prognosis and may be a potential viral marker for predicting prognosis in HCC.

HBV quasispecies composition in Lamivudine-failed chronic hepatitis B patients and its influence on virological response to Tenofovir-based rescue therapy

The present study sought to evaluate the structure of HBV quasispecies in Lamivudine (LMV)-failed chronic hepatitis B (CHB) patients and its impact in defining the subsequent virological responses to Tenofovir (TDF)-based rescue-therapy. By analyzing HBV clones encompassing reverse transcriptase (RT) and surface (S) region from LMV-failed and treatment-naïve CHB patients, we identified 5 classical and 12 novel substitutions in HBV/RT and 9 substitutions in immune-epitopes of HBV/S that were significantly associated with LMV failure. In silico analysis showed spatial proximity of some of the newly-identified, mutated RT residues to the RT catalytic centre while most S-substitutions caused alteration in epitope hydrophobicity. TDF administration resulted in virological response in 60% of LMV-failed patients at 24-week but non-response in 40% of patients even after 48-weeks. Significantly high frequencies of 6 S-substitutions and one novel RT-substitution, rtH124N with 6.5-fold-reduced susceptibility to TDF in vitro, were noted at baseline in TDF non-responders than responders. Follow-up studies depicted greater evolutionary drift of HBV quasispecies and significant decline in frequencies of 3 RT and 6 S-substitutions in responder-subgroup after 24-week TDF-therapy while most variants persisted in non-responders. Thus, we identified the HBV-RT/S variants that could potentially predict unfavorable response to LMV/TDF-therapy and impede immune-mediated viral clearance.

Detection of Aberrant TERT Promoter Methylation by Combined Bisulfite Restriction Enzyme Analysis for Cancer Diagnosis

Aberrant CpG dinucleotide methylation in a specific region of the telomerase reverse transcriptase (TERT) promoter is associated with increased TERT mRNA levels and malignancy in several cancer types. However, routine screening of this region to aid cancer diagnosis can be challenging because i) several established methylation assays may inaccurately report on hypermethylation of this particular region, ii) interpreting the results of methylation assays can sometimes be difficult for clinical laboratories, and iii) use of high-throughput methylation assays for a few patient samples can be cost prohibitive. Herein, we describe the use of combined bisulfite restriction enzyme analysis (COBRA) as a diagnostic tool for detecting the hypermethylated TERT promoter using invitro methylated and unmethylated genomic DNA as well as genomic DNA from four melanomas and two benign melanocytic lesions. We compare COBRA with MassARRAY, a more commonly used high-throughput approach, in screening for promoter hypermethylation in 28 formalin-fixed, paraffin-embedded neuroblastoma samples. COBRA sensitively and specifically detected samples with hypermethylated TERT promoter and was as effective as MassARRAY at differentiating high-risk from benign or low-risk tumors. This study demonstrates the utility of this low-cost, technically straightforward, and easily interpretable assay for cancer diagnosis in tumors of an ambiguous nature.