Reverse Transcriptase Gene Research Articles

Downregulation and Hypermethylation of GABPB1 Is Associated with Aggressive Thyroid Cancer Features.

Simple SummaryPromoter mutations of the telomerase reverse transcriptase (TERT) gene have been suggested as an oncogenic event in various cancers, including thyroid cancer (TC). GABPB1 is reported to activate TERT gene expression and has been proposed as a cancer therapeutic target. The aim of this study is to explore the fate of TC cells after disruption of GABPB1 and its role in TC. We found that besides the reported oncogenic role of GABPB1 in activating TERT, it also has tumor-suppressive functions in TC. Therefore, targeting GABPB1 for cancer therapy should be cautious since it may counteract its tumor-suppressive functions.Promoter mutations of the telomerase reverse transcriptase (TERT) gene occur frequently in thyroid carcinoma (TC), including papillary (PTC) and anaplastic subtypes (ATC). Given that the ETS family transcription factors GABPA and GABPB1 activate the mutant TERT promoter and induce TERT expression for telomerase activation, GABPB1 has been proposed as a cancer therapeutic target to inhibit telomerase. Here, we sought to determine the role of GABPB1 in TC pathogenesis. In TC-derived cells carrying the mutated TERT promoter, GABPB1 knockdown led to diminished TERT expression but significantly increased invasive potentials in vitro and metastatic potential in a xenograft zebrafish model and altered expression of markers for epithelial-to-mesenchymal transition. GABPB1 expression was downregulated in aggressive TCs. Low GABPB1 expression correlated with its promoter hypermethylation, which in turn was also associated with shorter disease-free survival. Consistently, DNA methylation inhibitors enhanced GABPB1 expression, as observed upon reduced promoter methylation. Our results suggest that GABPB1 is required for TERT expression and telomerase activation, but itself serves as a tumor suppressor to inhibit TC progression. Furthermore, aberrant DNA methylation leads to GABPB1 silencing, thereby promoting TC aggressiveness. Thus, caution is needed if targeting GABPB1 for cancer therapy is considered.

Establishment and characterization of a sheep endometrial epithelial cell line

Endometrial epithelial cells play a significant role in the "dialogue" between the embryo and the mother, but in vitro studies to clarify this are hampered by the limited lifespan of primary cells. As such, it is necessary to develop an in vitro model to study endometrial function. Morphological analysis showed that the pEECs were homogeneous, formed characteristic cobblestone monolayers, and expressed the epithelial cell-specific marker, cytokeratin 18. The isolated and purified cells were transfected with a plasmid encoding human telomerase reverse transcriptase (TERT) gene, pCI-neo-TERT, to establish an immortal endometrial epithelial cell line (iEECs). The transfected cells were cultured with G418 and monoclonal cells were selected for expanded culture. Expression of TERT mRNA was detected by RT-qPCR and protein was quantitated by Western blot. TERT expression was stable and continued to be active with no signs of aging. Assays for cell proliferation and apoptosis indicated higher proliferation and cellular activity in iEECs than pEECs. After stimulated by interferon tau (IFN-τ), both iEECs and pEECs showed similar upregulation levels in all the underlying genes. Taken together, these findings demonstrate that iEECs retained the basic morphology and function of pEECs, providing a robust in vitro model for study of the function of ovine endometrial epithelial cells.

Molecular Mechanisms of Alveolar Epithelial Stem Cell Senescence and Senescence-Associated Differentiation Disorders in Pulmonary Fibrosis.

Pulmonary senescence is accelerated by unresolved DNA damage response, underpinning susceptibility to pulmonary fibrosis. Recently it was reported that the SARS-Cov-2 viral infection induces acute pulmonary epithelial senescence followed by fibrosis, although the mechanism remains unclear. Here, we examine roles of alveolar epithelial stem cell senescence and senescence-associated differentiation disorders in pulmonary fibrosis, exploring the mechanisms mediating and preventing pulmonary fibrogenic crisis. Notably, the TGF-β signalling pathway mediates alveolar epithelial stem cell senescence by mechanisms involving suppression of the telomerase reverse transcriptase gene in pulmonary fibrosis. Alternatively, telomere uncapping caused by stress-induced telomeric shelterin protein TPP1 degradation mediates DNA damage response, pulmonary senescence and fibrosis. However, targeted intervention of cellular senescence disrupts pulmonary remodelling and fibrosis by clearing senescent cells using senolytics or preventing senescence using telomere dysfunction inhibitor (TELODIN). Studies indicate that the development of senescence-associated differentiation disorders is reprogrammable and reversible by inhibiting stem cell replicative senescence in pulmonary fibrosis, providing a framework for targeted intervention of the molecular mechanisms of alveolar stem cell senescence and pulmonary fibrosis. Abbreviations: DPS, developmental programmed senescence; IPF, idiopathic pulmonary fibrosis; OIS, oncogene-induced replicative senescence; SADD, senescence-associated differentiation disorder; SALI, senescence-associated low-grade inflammation; SIPS, stress-induced premature senescence; TERC, telomerase RNA component; TERT, telomerase reverse transcriptase; TIFs, telomere dysfunction-induced foci; TIS, therapy-induced senescence; VIS, virus-induced senescence.

An Identification and Characterization of the Axolotl (Ambystoma mexicanum, Amex) Telomerase Reverse Transcriptase (Amex TERT).

The Mexican axolotl is one of the few vertebrates that is able to replace its lost body parts during lifespan. Due to its remarkable regenerative abilities, the axolotl emerged as a model organism especially for limb regeneration. Telomeres and the telomerase enzyme are crucial for regeneration and protection against aging processes and degenerating diseases. Despite its relevance for regeneration, the axolotl telomerase and telomere length have not yet been investigated. Therefore, in the present paper, we reveal the sequence of the axolotl telomerase reverse transcriptase gene (Tert) and protein (TERT). Multiple sequence alignment (MSA) showed the known conserved RT- and TERT-specific motifs and residues found in other TERTs. In addition, we establish methods to determine the Tert expression (RT-PCR) and telomerase activity (Q-TRAP) of adult axolotl and blastema tissues. We found that both differentiated forelimb tissue and regenerating blastema tissue express Tert and show telomerase activity. Furthermore, blastema tissue appears to exhibit a higher Tert expression and telomerase activity. The presence of active telomerase in adult somatic cells is a decisive difference to somatic cells of non-regenerating vertebrates, such as humans. These findings indicate that telomere biology may play a key role in the regenerative abilities of cells.

Role of hTERT rs2736100 in pathological scarring.

Hypertrophic and atrophic scars are the effect of a dysregulated wound-healing process in genetically predisposed individuals. The genetic predisposition has acquired significant attention due to the diverse phenotype of pathological scarring in individuals with a positive personal and family history. Recent studies have identified telomere shortening and decreased hTERT activity in pathological scarring, proposing the rs2736100 variant of human telomerase reverse transcriptase (hTERT) gene as a valuable variant gene candidate. We examined the scarring process in 71 female patients who had undergone Caesarean section and developed hypertrophic and atrophic scars with the objective to investigate the role of single nucleotide polymorphism (SNP) rs2736100 in pathological scarring. Genotyping was performed using RT-PCR and follow-up included the Patient Observer Scar Assessment Scale (POSAS) and SCAR scales. Comparative analysis for mean POSAS value between the check-ups at 3 and 6 months revealed a statistical decreased difference of 1.71 points [95% confidence interval (CI), 0.4-2.89; P=0.01], while SCAR highlighted a decreased difference of 0.670 (95% CI, -0.04-1.38; P=0.055). The C variant allele revealed a borderline statistical value for the risk of developing pathological scarring (OR=1.44; 95% CI, 0.876-1.332: P=0.066). In our study a pre-conceptional body mass index (BMI) >25 kg/m2 was statistically associated with pathological scarring. The Fitzpatrick type 4 phototype displayed an increased frequency for the heterozygous genotype in the current study, and it was demonstrated that dark skin tone was associated with abnormal scar formation. Our study investigated the role of hTERT gene variant rs2736100 in hypertrophic and atrophic scarring in a Caucasian population group. We report a borderline statistically significant value for the variant C allele of hTERT SNP for the risk of developing pathological scarring in female patients that had undergone Caesarean section.

Nonstudent Young Men Put Students at High Risk of HIV Acquisition in Guangxi, China: A Phylogenetic Analysis of Surveillance Data.

BackgroundWe sought to identify students and their sexual partners in a molecular transmission network.MethodsWe obtained 5996 HIV protease and reverse transcriptase gene sequences in Guangxi (165 from students and 5831 from the general populations) and the relevant demographic data. We constructed a molecular transmission network and introduced a permutation test to assess the robust genetic linkages. We calculated the centrality measures to describe the transmission patterns in clusters.ResultsAt the network level, 68 (41.2%) students fell within the network across 43 (8.1%) clusters. Of 141 genetic linkages between students and their partners, only 25 (17.7%) occurred within students. Students were more likely than random permutations to link to other students (odds ratio [OR], 7.2; P < .001), private company employees aged 16–24 years (OR, 3.3; P = .01), private company or government employees aged 25–49 years (OR, 1.7; P = .03), and freelancers or unemployed individuals aged 16–24 years (OR, 5.0; P < .001). At the cluster level, the median age of nonstudents directly linked to students (interquartile range) was 25 (22–30) years, and 80.3% of them had a high school or higher education background. Compared with students, they showed a significantly higher median degree (4.0 vs 2.0; P < .001) but an equivalent median Eigenvector Centrality (0.83 vs 0.81; P = .60).ConclusionsThe tendency of genetic linkage between students and nonstudent young men and their important position in the HIV transmission network emphasizes the urgent need for 2-pronged public health interventions based on both school and society.

Monogenic gene variants in lung transplant recipients with usual interstitial pneumonia.

Aim The prevalence of monogenic disease-causing gene variants in lung transplant recipients with idiopathic pulmonary fibrosis is not fully known. Their impact on clinical outcomes before and after transplantation requires more evidence.Patients and methodsWe retrospectively performed sequence analysis of genes associated with pulmonary fibrosis in a cohort of 23 patients with histologically confirmed usual interstitial pneumonia that had previously undergone double lung transplantation. We evaluated the impact of confirmed molecular diagnoses on disease progression, clinical outcomes and incidence of acute rejection or chronic lung allograft dysfunction after transplantation.Results15 patients out of 23 (65%) had a variant in a gene associated with interstitial lung disease. 11 patients (48%) received a molecular diagnosis, of which nine involved genes for telomerase function. Five diagnostic variants were found in the gene for Telomerase reverse transcriptase. Two of these variants, p.(Asp684Gly) and p.(Arg774*), seemed to be enriched in Finnish lung transplant recipients. Disease progression and the incidence of acute rejection and chronic lung allograft dysfunction was similar between patients with telomere-related disease and the rest of the study population. The incidence of renal or bone marrow insufficiency or skin malignancies did not differ between the groups.ConclusionGenetic variants are common in lung transplant recipients with pulmonary fibrosis and are most often related to telomerase function. A molecular diagnosis for telomeropathy does not seem to impact disease progression or the risk of complications or allograft dysfunction after transplantation.

MPEG-PCL Nanomicelles Platform for Synergistic Metformin and Chrysin Delivery to Breast Cancer in Mice.

Metformin (MET) is a well-known anti-diabetic drug that also has anti-cancer effects. However, high therapeutic doses of MET on cancer cells and the low efficacy of combinatory therapeutic approaches limit its clinical application. Recent studies have shown that chrysin (CHR) can improve the pharmaceutical efficacy of MET by suppressing human telomerase reverse transcriptase (hTERT) and cyclin D1 gene expression. This study aimed to develop different ratios of methoxy poly(ethylene glycol)-b-poly(e-caprolactone) (MPEG-PCL) micelles for breast cancer to co-deliver a synergistic CHR/MET combination. CHR/MET drug-loaded micelles were prepared by modified thin-film hydration.Fourier infrared spectrum, gel permeation chromatography, transmission electron microscopy, and high-performance liquid chromatography were used to evaluate the physicochemical properties of nanostructures. Cell proliferation and cell apoptosis were assessed by MTT and Annexin V-FITC/PI double staining method. The gene expression of hTERT and cyclin D1 was measured by real-time PCR assay. A subcutaneous mouse T47D xenograft model was established to evaluate the in vivo efficiency. When the ratio of MPEG-PCL was 1:1.7, the highest drug loading rate and encapsulation efficiency of CHR (11.31±0.37) and MET (12.22±0.44) were observed. Uniform MPEG-PCL micelles of 51.70±1.91 nm allowed MET to incorporate with CHR, which were co-delivered to breast cancer cells. We demonstrated that CHR/MET co-delivery micelles showed a good synergistic effect on inhibiting proliferation in T47D cells (combination index=0.87) by suppressing hTERT and cyclin D1 gene expression. Compared to the free CHR/MET group, the apoptosis rate on T47D cells by CHR/MET nano-micelles significantly improved from 71.33% to 79.25%. The tumour volume and tumour weight of the CHR/MET group increased more slowly than that of the single-drug treatment group (P<0.05). Compared to the CHR/MET group, the tumour volume and tumour weight of the CHR/MET nano-micelle group decreased by 42% and 59%, respectively. We demonstrated that ratiometric CHR/MET micelles could provide an effective technique for the treatment of breast cancer.

Evaluation of hTERT Gene Expression and Chromosome 7 Copy Number Variation in Anal Squamous Intra-Epithelial Lesions: A Pilot Study.

Background:Akin to cervical squamous intra-epithelial neoplasia (CIN), anal squamous intra-epithelial lesion (a-SIL) is attributed to persistent infection with high-risk human papilloma virus infection. Amplification of human telomerase reverse transcriptase (hTERT) gene and aneuploidy are known to correlate with CIN evolution. It is plausible that the underlying genetic events in a-SIL are similar. We conducted this cross-sectional analytical study with the objective of determining expression of hTERT gene expression and chromosome 7, as marker of cell ploidy in a-SIL.Methods:Conventional anal cytology was performed in 86 adult consenting subjects with history of receptive anal intercourse (RAI) and 4 controls without history of RAI. Cases with a-SIL and controls were subjected to fluorescent in-situ hybridization (FISH) to evaluate hTERT gene and chromosome 7 expression, as marker of cell ploidy. Results were expressed as number of abnormal nuclei (≥3 respective signals), maximum degree of amplification, mean signals/nucleus and proportion of cases showing abnormal nuclei.Results:Twenty cases showed a-SIL; with 15 atypical squamous cells of undetermined significance (ASCUS), 3 low grade squamous intra-epithelial lesion (LSIL) and 2 cases of high-risk cytology. Expression of both hTERT gene and chromosome 7 increased from controls to ASCUS to LSIL with concomitant increase in proportion of cases having abnormal hTERT gene and chromosome 7 expression.Conclusions:Positive association of hTERT gene with a-SIL suggests its possible role in evolution of anal squamous abnormalities. Increase in chromosome 7 also correlated positively with a-SIL. These findings corroborate the similarities between squamous carcinogenesis in CIN and a-SIL.

Telomerase Reverse Transcriptase Promoter Mutations in Human Hepatobiliary, Pancreatic and Gastrointestinal Cancer Cell Lines.

The promoter region of the telomerase reverse transcriptase (TERT) gene is a regulatory element capable of affecting TERT expression, telomerase activity, and telomerase length. Mutations within the TERT promoter region are the most common mutations in many cancers. In this study, we characterized the TERT promoter mutation status in hepatobiliary, pancreatic, and gastrointestinal cancer cell lines. TERT promoter mutation status was assessed by digital PCR in 12 liver cancer, 5 cholangiocarcinoma (CCA), 12 pancreatic cancer, 17 gastrointestinal cancer, and 3 healthy control cell lines. The C228T promoter mutation was detected in 9 liver cancer lines, and the C250T TERT mutation was detected in 1 oesophageal squamous cell carcinoma line. The C228T promoter mutation is specific to liver cancer cell lines among various gastrointestinal cancer cell lines. These data will contribute to future research on the tumorigenic mechanisms and clinical use of digital PCR to detect mutations.

A case report on rare hepatitis B viral subgenotype from a tertiary care center in Chennai

Hepatitis B virus (HBV) is a global health concern with 350 million chronic carriers. With respect to HBV India is classified as an intermediate endemic country. The disease progression may be due to many viral factors including HBV viral load, HBe antigen, genotype, mutations in polymerase gene, and X gene. In this case, the individual was a treatment naïve chronic HBV carrier. The reverse transcriptase gene and X gene were sequenced and mutations were analyzed. The individual had D3 subgenotype. rt80I was identified in reverse transcriptase and A102V in HBx protein. Identification of genotype and mutations in reverse transcriptase/X gene may help in predicting and improving the clinical outcomes.

TERT genetic variability and telomere length as factors affecting survival and risk in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a neoplasm of immature myeloid cells characterized by various cytogenetic alterations. The present study showed that in addition to the FLT3-ITD and NPM1 mutation status, telomere length (TL) and telomerase reverse transcriptase (TERT) gene polymorphisms may affect risk and overall survival (OS) in AML. TL was longer in healthy controls than in AML patients and positively correlated with age in the patients, but not in healthy subjects. TL was found to be independently affected by the presence of the FLT3-ITD mutation. As for the TERT gene polymorphism, AML patients with the TERT rs2853669 CC genotype were characterized by significantly shorter OS than patients carrying the T allele. Another observation in our study is the difference in TL and OS in patients belonging to various risk stratification groups related to the FLT3-ITD and NPM1 mutation status. Patients with adverse risk classification (mutation in FLT3-ITD and lack of mutation in NPM1) presented with the shortest telomeres and significantly worse OS. In conclusion, OS of AML patients appears to be affected by TERT gene variability and TL in addition to other well-established factors such as age, WBC count, or FLT3-ITD and NPM1 mutation status.

The frequency of HIV-1 infection and surveillance drug-resistant mutations determination among Iranians with high-risk behaviors, during 2014 to 2020.

Background and Objectives:Human immunodeficiency virus (HIV) has various transmission routes. Instant antiretroviral therapy (ART) is the recommended treatment for HIV infection. Highly active antiretroviral therapy (HAART) significantly decreases the acquired immunodeficiency syndrome (AIDS) and AIDS-related co-morbidities. Notwithstanding the suitability of HAART, the antiretrovirals (ARVs) have adverse effects and antiretroviral drug resistance mutations are reported among those who receive ARVs. In this survey, the abundance of HIV-1 infection in Iranians with high-risk behaviors, and detection of the surveillance drug-resistant mutations (SDRMs) were evaluated.Materials and Methods:This cross-sectional study was conducted on 250 individuals with high-risk behaviors from September 2014 to February 2020. HIV-1 Ag/Ab in plasma samples was detected using enzyme immunoassay (EIA) kits. The conserved region of HIV-1 was detected in the plasma samples by real-time polymerase chain reaction (PCR) assay. Furthermore, in individuals with positive HIV-1 RNA, HIV-1 viral load testing was performed. After amplification and sequencing of the HIV-1 protease, reverse transcriptase, and integrase genes, surveillance drug resistance mutation (SDRM) and phylogenetic analysis were determined.Results:Out of the 250 participants with high-risk behaviors, six (2.4%) were infected with HIV-1. According to the phylogenetic analysis, the CRF35_AD (83.3% or 5/6) was the dominant subtype, followed by CRF01_AE (16.7% or 1/6). In this research, in none of the HIV-1 infected patients, SDRM for protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and integrase inhibitors (INs) were observed. Nevertheless, in one of the patients, V179L mutation was detected which is a rare non-polymorphic mutation and is listed as a rilpivirine (RPV) -associated resistance mutation.Conclusion:The results of the current survey revealed that 2.4% of people with high-risk behaviors are infected with HIV and the level of drug resistance mutations (DRMs) in these people is very low.

Viral load as a risk factor of reverse transcriptase inhibitor drug resistance mutation in antiretroviral-treated people living with HIV/AIDS

Background The human immunodeficiency virus type 1 (HIV-1) is a major contagion faced by the population of Indonesia. The success of antiretroviral treatment (ART) is threatened by the emergence of drug resistance mutations (DRM). The aim of this study was to determine the association between CD4 count, CD4 count changes, viral load, adherence to therapy, and therapy history in the presence of DRM in people living with HIV/AIDS (PLWHA). MethodsThis was a cross-sectional study involving 269 adults who underwent antiretroviral (ARV) therapy for at least 6 months. The frequencies of DRM and polymorphisms were measured by partial amplification of the reverse transcriptase (RT) gene using RT-nested PCR on samples with viral loads of &gt;1000 copies/mL. Sequencing was performed using the Sanger method, and edited by BioEdit. The edited sequences were submitted to http://hivdb.stanford.edu for DRM determination. Respondents’ medical data, CD4 count, viral load, and DRM were analyzed by simple and multiple logistic regression. ResultsThe multiple logistic regression analysis showed a significant association of CD4 count (aOR=12.47; 95% CI: 1.45 -107.39; p=0.023) and viral load at the time of study (aOR=29.56; 95% CI: 3.47-251.52; p=0.002) with the presence of DRM in respondents. ARV substitution history was not associated with the presence of DRM. There were 17 respondents (6.3%) carrying HIV-1 DRM, with M184V/I (11 sequences) as the most frequent pattern of NRTI resistance, and K103 (9 sequences) as that of NNRTI resistance. ConclusionThis study demonstrated that viral load at the time of the study was the most influential determinant factor for the presence of DRM in PLWHA.

MiR-138-5p Suppresses Cell Growth and Migration in Melanoma by Targeting Telomerase Reverse Transcriptase.

Recent evidence suggests the existence of a miRNA regulatory network involving human telomerase reverse transcriptase gene (hTERT), with miR-138-5p playing a central role in many types of cancers. However, little is known about the regulation of hTERT expression by microRNA (miRNAs) in melanocytic tumors. Here, we investigated the effects of miR-138-5p in hTERT regulation in melanoma cells lines. In vitro studies demonstrated higher miR-138-5p and lower hTERT messenger RNA (mRNA) expression in human epidermal melanocytes, compared with melanoma cell lines (A2058, A375, SK-MEL-28) by quantitative polymerase chain reaction (qPCR) observing a negative correlation between them. A2058 melanoma cells were selected to be transfected with miR-138-5p mimic or inhibitor. Using luciferase assay, hTERT was identified as a direct target of this miRNA. Overexpression of miR-138-5p detected by Western blot revealed a decrease in hTERT protein expression (p = 0.012), and qPCR showed a reduction in telomerase activity (p < 0.001). Moreover, suppressions in cell growth (p = 0.035) and migration abilities (p = 0.015) were observed in A2058-transfected cells using thiazolyl blue tetrazolium bromide and flow cytometry, respectively. This study identifies miR-138-5p as a crucial tumor suppressor miRNA involved in telomerase regulation. Targeting it as a combination therapy with immunotherapy or targeted therapies could be used in advanced melanoma treatment; however, more preclinical studies are necessary.

TERT Promoter Mutations and rs2853669 Polymorphism: Useful Markers for Clinical Outcome Stratification of Patients With Oral Cavity Squamous Cell Carcinoma.

ObjectiveTo date, no useful prognostic biomarker exists for patients with oral squamous cell carcinoma (OCSCC), a tumour with uncertain biological behaviour and subsequent unpredictable clinical course. We aim to investigate the prognostic significance of two recurrent somatic mutations (-124 C>T and -146 C>T) within the promoter of telomerase reverse transcriptase (TERT) gene and the impact of TERT single nucleotide polymorphism (SNP) rs2853669 in patients surgically treated for OCSCC.MethodsThe genetic frequencies of rs2853669, -124 C>T and -146 C>T as well as the telomere length were investigated in 144 tumours and 57 normal adjacent mucosal (AM) specimens from OCSCC patients.ResultsForty-five tumours harboured TERT promoter mutations (31.3%), with -124 C>T and -146 C>T accounting for 64.4% and 35.6% of the alterations respectively. Patients with -124 C>T TERT promoter mutated tumours had the shortest telomeres in the AM (p=0.016) and showed higher risk of local recurrence (hazard ratio [HR]:2.75, p=0.0143), death (HR:2.71, p=0.0079) and disease progression (HR:2.71, p=0.0024) with the effect being potentiated by the co-occurrence of T/T genotype of rs2853669.Conclusion-124 C>T TERT promoter mutation as well as the T/T genotype of the rs2853669 SNP are attractive independent prognostic biomarkers in patients surgically treated for OCSCC, with the coexistence of these genetic variants showing a synergistic impact on the aggressiveness of the disease.

TERT Rare Variants in Myeloid Neoplasia: Lack of Clinical Impact or Role as Risk Alleles

▪Dyskeratosis congenita is a prototypic inherited telomeropathy. Telomere length (TL) shortening has also been described in aplastic anemia (AA) and attributed in occasional patients to the presence of mostly heterozygous germline alterations in telomerase machinery genes (most commonly in the reverse transcriptase gene, TERT). Among various heterozygous variants described, certain TERT variants associated with shortened TL (e.g. H412Y and A202T) showed later a higher prevalence in general population questioning their pathogenicity and role as risk alleles. Nevertheless, shortened TL has been shown to correlate with poor outcomes in AA and myelodysplastic syndromes (MDS) along with a higher progression rate to acute myeloid leukemia, and increased non-relapse mortality after HSCT. A high prevalence (41/1514; 2.7%) of non-recurrent germline TERT rare variants, has been found in patients with MDS undergoing HSCT (Reilly et al Blood 2021) without any clinically apparent diagnosis of a telomere biology disorder. Despite their classification as VUS, these alterations were shown to result in impairment of telomere elongation in vitro and reduced TL in vivo. Clinically, VUS carriers were also characterized by younger age at MDS presentation (median 52 years) and shorter survival due to a higher incidence of non-relapse mortality.We hypothesized that should such rare variants (traditionally thought as VUS) represent founder lesions and constitute risk alleles, one would expect to find some recurrence of these lesions in other cohorts of patients with myeloid neoplasia (MN). To test this hypothesis we screened a large meta-analytic cohort of MN patients (n=2560) including our patients (The Cleveland Clinic Foundation and MLL-Munich Leukemia laboratory) and a public series (The BEAT AML Master Trial and TCGA among others) for the presence of TERT coding variants. When whole genome sequencing was available (n=1432) TelomereHunter tool was used to estimate TL in carriers of TERT rare variants and wild-type (WT) cohorts.Overall, we identified 73 TERT coding variants. Based on maximum gnomAD population allele frequency (AF) <0.001 and ACMG and pathogenicity scores according to the classifiers of VarSome (https://varsome.com), we found 37/2560 (1.4%) variants which were categorized as rare and VUS. Of note, only 6 patients harbored established TERT pathogenic variants while the rest were common and VUS. Alterations occurred in the RTD (52%), TRBD-Linker region (29%) and CTE domain (19%). When compared to the aforementioned HSCT cohort described by Reilly et al, TERT rare VUS had a lower frequency and did not overlap with any of the VUS reported in this study, except for 2 patients harboring one variant each in amino acid locations 741 and 1041. Assessment of TL, possible in 12 patients carrying TERT rare variants, did not reveal differences compared to TERT WT cases. Interestingly, none of these rare variants were also observed in a control cohort of patients with AA (n=268).In terms of clinical features, MN patients with TERT rare variants harbored more somatic hits in DNMT3A and SF3B1 (26% each), and FLT3 (15%) with a diverse spectrum of additional molecular lesions, including germline VUS of other genes (4 patients harbored concomitant VUS in BRCA1, FANCG, and SAMD9, with another patient carrying a likely benign SAMD9L variant) (Fig1). When compared to WT counterparts, patients harboring TERT rare variants had a similar median age at MN diagnosis (70 vs. 66, P= 0.38) and similar survival outcomes (P= 0.96). Importantly, HSCT outcomes were ascertained only in 7 cases because of the real-life nature of our cohort which compiles MN patients of any subtype and age, thereby not biased by transplant referral like in the aforementioned study focusing only on MDS patients undergone HSCT. Finally, principal causes of death were primary disease (63%), presence of another malignancy (19%) and infections (13%) and, of note, no patient died of non-infectious pulmonary causes.In sum, our study, while confirming a relatively high (albeit not significant vs. controls) combined TERT VUS population burden in MN, also strongly indicates that rare TERT VUS are non-recurrent. Therefore, they cannot be considered risk alleles individually nor can their biological (combined or individual) impact in terms of non-transplant outcomes, irrespective of their in vitro functional effects. [Display omitted] DisclosuresCarraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Takeda: Other: Independent review committee; Astex: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy.

Association between the rs2736100 polymorphisms of telomerase reverse transcriptase gene and digestive cancers: A meta-analysis.

The conclusions on the association between the rs2736100 polymorphisms of telomerase reverse transcriptase (TERT) gene polymorphism and digestive cancers risk are still debated. This meta-analysis was conducted to update the association between the TERT rs2736100 polymorphisms and the risk of digestive cancers. The association investigations were identified from PubMed and Cochrane Library, and eligible studies were included and synthesized using the meta-analysis method. Eight case-control studies were included in this meta-analysis for associating TERT rs2736100 gene polymorphism and digestive cancer susceptibility. Pooled odds ratio with 95% confidence interval was calculated using a fixed or random-effects model. Overall, no evidence has shown that the TERT rs2736100 polymorphism was associated with the susceptibility to digestive cancers. Besides, stratified analysis with ethnicity also indicated no significant association between TRET rs2736100 and the risk of digestive cancers under all genetic models in both Asian and Caucasian populations were observed. According to the meta-analysis, TERT rs2736100 polymorphism might be unrelated to digestive cancer susceptibility. Evidence with adequate sample size is still needed.

Genetic Diversity and Acquired Drug Resistance Mutations Detected by Deep Sequencing in Virologic Failures among Antiretroviral Treatment Experienced Human Immunodeficiency Virus-1 Patients in a Pastoralist Region of Ethiopia.

PurposeThis study was conducted to investigate the drug resistance mutations and genetic diversity of HIV-1 in ART experienced patients in South Omo, Ethiopia.Patients and MethodsA cross-sectional study conducted on 253 adult patients attending ART clinics for ≥6 months in South Omo. Samples with VL ≥1000 copies/mL were considered as virological failures (VF) and their reverse transcriptase gene codons 90–234 were sequenced using Illumina MiSeq. MinVar was used for the identification of the subtypes and drug resistance mutations. Phylogenetic tree was constructed by neighbor-joining method using the maximum likelihood model.ResultsThe median duration of ART was 51 months and 18.6% (47/253) of the patients exhibited VF. Of 47 viraemic patients, the genome of 41 were sequenced and subtype C was dominant (87.8%) followed by recombinant subtype BC (4.9%), M-09-CPX (4.9) and BF1 (2.4%). Of 41 genotyped subjects, 85.4% (35/41) had at least one ADR mutation. Eighty-one percent (33/41) of viraemic patients harbored NRTI resistance mutations, and 48.8% (20/41) were positive for NNRTI resistance mutations, with 43.9% dual resistance mutations. Among NRTI resistance mutations, M184V (73.2%), K219Q (63.4%) and T215 (56.1%) complex were the most mutated positions, while the most common NNRTI resistance mutations were K103N (24.4%), K101E, P225H and V108I 7.5% each. Active tuberculosis (aOR=13, 95% CI= 3.46–29.69), immunological failure (aOR=3.61, 95% CI=1.26–10.39), opportunistic infections (aOR=8.39, 95% CI= 1.75–40.19), and poor adherence were significantly associated with virological failure, while rural residence (aOR 2.37; 95% CI: 1.62–9.10, P= 0.05), immunological failures (aOR 2.37; 95% CI: 1.62–9.10, P= 0.05) and high viral load (aOR 16; 95% CI: 5.35 51.59, P <0.001) were predictors of ADR mutation among the ART experienced and viraemic study subjects.ConclusionThe study revealed considerable prevalence of VF and ADR mutation with the associated risk indicators. Regular virological monitoring and drug resistance genotyping methods should be implemented for better ART treatment outcomes of the nation.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs’ pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.