MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2.

BACKGROUND: MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2. RESULTS: Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients. CONCLUSION: AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.

Unfurling the functional association between long intergenic noncoding RNAs (lincRNAs) and HPV16-related cervical cancer pathogenesis through weighted gene co-expression network analysis of differentially expressed lincRNAs and coding genes.

Long intergenic noncoding RNAs (lincRNAs) do not overlap annotated coding genes and are located in intergenic regions, as opposed to antisense and sense-intronic lncRNAs, located in genic regions. LincRNAs influence gene expression profiles and are thereby key to disease pathogenesis. In this study, we assessed the association between lincRNAs and HPV16-positive cervical cancer (CaCx) pathogenesis using weighted gene co-expression network analysis (WGCNA) with coding genes, comparing differentially expressed lincRNA and coding genes (DElincGs and DEcGs, respectively) in HPV16-positive patients with CaCx (n = 44) with those in HPV-negative healthy individuals (n = 34). Our analysis revealed five DElincG modules, co-expressing and correlating with DEcGs. We validated a substantial number of such module-specific correlations in the HPV16-positive cancer TCGA-CESC dataset. Four such modules, displayed significant correlations with patient traits, such as HPV16 physical status, lymph node involvement, and overall survival (OS), highlighting a collaborative effect of all genes within specific modules on traits. Using the DAVID bioinformatics knowledgebase, we identified the underlying biological processes associated with these modules as cancer development and progression-associated pathways. Next, we identified the top 10 DElincGs with the highest connectivity within each functional module. Focusing on the prognostic module hub genes, downregulated CTD-2619J13.13 expression was associated with poor patient OS. This lincRNA gene interacted with 25 coding genes of its module and was associated with such biological processes as keratinization loss and keratinocyte differentiation, reflecting severe disease phenotypes. This study has translational relevance in fighting various cancers with high mortality rates in underdeveloped countries.

Correlation of severity & clinical outcomes of COVID-19 with virus variants: A prospective, multicentre hospital network study.

BACKGROUND OBJECTIVES: The clinical course of COVID-19 and its prognosis are influenced by both viral and host factors. The objectives of this study were to develop a nationwide platform to investigate the molecular epidemiology of SARS-CoV-2 (Severe acute respiratory syndrome Corona virus 2) and correlate the severity and clinical outcomes of COVID-19 with virus variants. METHODS: A nationwide, longitudinal, prospective cohort study was conducted from September 2021 to December 2022 at 14 hospitals across the country that were linked to a viral sequencing laboratory under the Indian SARS-CoV-2 Genomics Consortium. All participants (18 yr and above) who attended the hospital with a suspicion of SARS-CoV-2 infection and tested positive by the reverse transcription-PCR method were included. The participant population consisted of both hospitalized as well as outpatients. Their clinical course and outcomes were studied prospectively. Nasopharyngeal samples collected were subjected to whole genome sequencing to detect SARS-CoV-2 variants. RESULTS: Of the 4972 participants enrolled, 3397 provided samples for viral sequencing and 2723 samples were successfully sequenced. From this, the evolution of virus variants of concern including Omicron subvariants which emerged over time was observed and the same reported here. The mean age of the study participants was 41 yr and overall 49.3 per cent were female. The common symptoms were fever and cough and 32.5 per cent had comorbidities. Infection with the Delta variant evidently increased the risk of severe COVID-19 (adjusted odds ratio: 2.53, 95% confidence interval: 1.52, 4.2), while Omicron was milder independent of vaccination status. The independent risk factors for mortality were age >65 yr, presence of comorbidities and no vaccination. INTERPRETATION CONCLUSIONS: The authors believe that this is a first-of-its-kind study in the country that provides real-time data of virus evolution from a pan-India network of hospitals closely linked to the genome sequencing laboratories. The severity of COVID-19 could be correlated with virus variants with Omicron being the milder variant.

Upper respiratory tract microbiome profiles in SARS-CoV-2 Delta and Omicron infected patients exhibit variant specific patterns and robust prediction of disease groups.

IMPORTANCE: The role of the upper respiratory tract (URT) microbiome in predicting lung health has been documented in several studies. The dysbiosis in COVID patients has been associated with disease outcomes by modulating the host immune system. However, although it has been known that different SARS-CoV-2 variants manifest distinct transmissibility and mortality rates in human populations, their effect on the composition and diversity of the URT microbiome has not been studied to date. Unlike the older variant (Delta), the newer variant (Omicron) have become more transmissible with lesser mortality and the symptoms have also changed significantly. Hence, in the present study, we have investigated the change in the URT microbiome associated with Delta and Omicron variants and identified variant-specific signatures that will be useful in the assessment of lung health and can be utilized for nasal probiotic therapy in the future.

Identification of HPV16 positive cervical cancer subsets characterized by divergent immune and oncogenic phenotypes with potential implications for immunotherapy.

BACKGROUND: Cervical cancers (CaCx), like many other cancer types, portray high molecular heterogeneity that affects response to therapy, including immunotherapy. In India and other developing countries, CaCx mortality rates are very high because women report to the clinics with advanced cancers in absence of organized screening programs. This calls for implementation of newer therapeutic regimens for CaCx, like immunotherapy, which is again not used commonly in such countries. OBJECTIVE: Therefore, we focused on dissecting tumour immune heterogeneity, if any, identify immune gene-based biomarkers of heterogeneity and subsets of such cancers with the potential for immunotherapy. We also attempted to characterize the cancer-associated phenotypes of such subsets, including viral load, to decipher the relationship of tumour immunogenicity with oncogenicity. METHODS: Employing RNA-seq analysis of 44 HPV16 positive CaCx patients, immune subtypes were identified by unsupervised hierarchical clustering of global immune-gene expression profiles. Proportions of tumor infiltrating immune cells in the tumor milieu were estimated, employing Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), using gene expression data from RNA-seq. The oncogenic phenotypes of the immune subtypes of CaCx were deciphered through differential gene expression (DEGs) and pathway enrichment analysis. Viral load was estimated through TaqMan-based qRT-PCR analysis. RESULTS: Analysis revealed the presence of two immune subtypes of CaCx, A (26/44; 59.09%) and B (18/44; 40.90%). Compared to Subtype-A, Subtype-B portrayed overexpression of immune genes and high infiltration of immune cells, specifically CD8+ T cells (p < 0.0001). Besides, a significant correlation between PD-1 and PD-L1 co-expression among Subtype-B, as opposed to Subtype-A, confirmed the interactive roles of these immune checkpoint molecules in Subtype B. Stepwise discriminant analysis pin-pointed ten immune-genes that could classify 100% of the patients significantly (p < 0.0001) into the two immune subtypes and serve as potential biomarkers of CaCx immunity. Differential gene expression analysis between the subtypes unveiled that Subtype-B was more biologically aggressive than Subtype-A, reflecting loss of structural integrity and promotion of cancer progression. The viral load was significantly lower in Subtype-B (average viral load = 10.74/100 ng of genomic DNA) compared to Subtype-A (average viral load = 14.29/100 ng of genomic DNA). Thus viral load and the ten-gene panel underscore their association with immunogenicity and oncogenicity. CONCLUSION: Our study provides strong evidence that only a subset, about 41% of HPV16 positive CaCx patients in India, portray immune enrichment of the tumor milieu coupled with aggressive phenotypes. Such subtypes are therefore likely to benefit through checkpoint molecule-based or tumor infiltrating lymphocyte-based immunotherapy, which could be a leap forward in tackling aggressive forms of such CaCx in India and other developing countries.

Mapinsights: deep exploration of quality issues and error profiles in high-throughput sequence data.

High-throughput sequencing (HTS) has revolutionized science by enabling super-fast detection of genomic variants at base-pair resolution. Consequently, it poses the challenging problem of identification of technical artifacts, i.e. hidden non-random error patterns. Understanding the properties of sequencing artifacts holds the key in separating true variants from false positives. Here, we develop Mapinsights, a toolkit that performs quality control (QC) analysis of sequence alignment files, capable of detecting outliers based on sequencing artifacts of HTS data at a deeper resolution compared with existing methods. Mapinsights performs a cluster analysis based on novel and existing QC features derived from the sequence alignment for outlier detection. We applied Mapinsights on community standard open-source datasets and identified various quality issues including technical errors related to sequencing cycles, sequencing chemistry, sequencing libraries and across various orthogonal sequencing platforms. Mapinsights also enables identification of anomalies related to sequencing depth. A logistic regression-based model built on the features of Mapinsights shows high accuracy in detecting 'low-confidence' variant sites. Quantitative estimates and probabilistic arguments provided by Mapinsights can be utilized in identifying errors, bias and outlier samples, and also aid in improving the authenticity of variant calls.

Emergence of a unique SARS-CoV-2 Delta sub-cluster harboring a constellation of co-appearing non-Spike mutations.

Accumulation of diverse mutations across the structural and nonstructural genes is leading to rapid evolution of SARS-CoV-2, altering its pathogenicity. We performed whole genome sequencing of 239 SARS-CoV-2 RNA samples collected from both adult and pediatric patients across eastern India (West Bengal), during the second pandemic wave in India (April-May 2021). In addition to several common spike mutations within the Delta variant, a unique constellation of eight co-appearing non-Spike mutations was identified, which revealed a high degree of positive mutual correlation. Our results also demonstrated the dynamics of SARS-CoV-2 variants among unvaccinated pediatric patients. 41.4% of our studied Delta strains harbored this signature set of eight co-appearing non-Spike mutations and phylogenetically out-clustered other Delta sub-lineages like 21J, 21A, or 21I. This is the first report from eastern India that portrayed a landscape of co-appearing mutations in the non-Spike proteins, which might have led to the evolution of a distinct Delta subcluster. Accumulation of such mutations in SARS-CoV-2 may lead to the emergence of "vaccine-evading variants." Hence, monitoring of such non-Spike mutations will be significant in the formulation of any future vaccines against those SARS-CoV-2 variants that might evade the current vaccine-induced immunity, among both the pediatric and adult populations.

Contrasting Distribution of SARS-CoV-2 Lineages across Multiple Rounds of Pandemic Waves in West Bengal, the Gateway of East and North-East States of India.

The evolution of viral variants and their impact on viral transmission have been an area of considerable importance in this pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We analyzed the viral variants in different phases of the pandemic in West Bengal, a state in India that is important geographically, and compared the variants with other states like Delhi, Maharashtra, and Karnataka, located in other regions of the country. We have identified 57 pango-lineages in 3,198 SARS-CoV-2 genomes, alteration in their distribution, as well as contrasting profiles of amino acid mutational dynamics across different waves in different states. The evolving characteristics of Delta (B.1.617.2) sublineages and alterations in hydrophobicity profiles of the viral proteins caused by these mutations were also studied. Additionally, implications of predictive host miRNA binding/unbinding to emerging spike or nucleocapsid mutations were highlighted. Our results throw considerable light on interesting aspects of the viral genomic variation and provide valuable information for improved understanding of wave-defining mutations in unfolding the pandemic. IMPORTANCE Multiple waves of infection were observed in many states in India during the coronavirus disease 2019 (COVID19) pandemic. Fine-scale evolution of major SARS-CoV-2 lineages and sublineages during four wave-window categories: Pre-Wave 1, Wave 1, Pre-Wave 2, and Wave 2 in four major states of India: Delhi (North), Maharashtra (West), Karnataka (South), and West Bengal (East) was studied using large-scale virus genome sequencing data. Our comprehensive analysis reveals contrasting molecular profiles of the wave-defining mutations and their implications in host miRNA binding/unbinding of the lineages in the major states of India.

Computational prediction of the molecular mechanism of statin group of drugs against SARS-CoV-2 pathogenesis.

Recently published clinical data from COVID-19 patients indicated that statin therapy is associated with a better clinical outcome and a significant reduction in the risk of mortality. In this study by computational analysis, we have aimed to predict the possible mechanism of the statin group of drugs by which they can inhibit SARS-CoV-2 pathogenesis. Blind docking of the critical structural and functional proteins of SARS-CoV-2 like RNA-dependent RNA polymerase, M-protease of 3-CL-Pro, Helicase, and the Spike proteins ( wild type and mutants from different VOCs) were performed using the Schrodinger docking tool. We observed that fluvastatin and pitavastatin showed fair, binding affinities to RNA polymerase and 3-CL-Pro, whereas fluvastatin showed the strongest binding affinity to the helicase. Fluvastatin also showed the highest affinity for the SpikeDelta and a fair docking score for other spike variants. Additionally, molecular dynamics simulation confirmed the formation of a stable drug-protein complex between Fluvastatin and target proteins. Thus our study shows that of all the statins, fluvastatin can bind to multiple target proteins of SARS-CoV-2, including the spike-mutant proteins. This property might contribute to the potent antiviral efficacy of this drug.

Integrative analysis of genomic and transcriptomic data of normal, tumour, and co-occurring leukoplakia tissue triads drawn from patients with gingivobuccal oral cancer identifies signatures of tumour initiation and progression.

A thickened, white patch - leukoplakia - in the oral cavity is usually benign, but sometimes (in ~9% of individuals) it progresses to malignant tumour. Because the genomic basis of this progression is poorly understood, we undertook this study and collected samples of four tissues - leukoplakia, tumour, adjacent normal, and blood - from each of 28 patients suffering from gingivobuccal oral cancer. We performed multiomics analysis of the 112 collected tissues (four tissues per patient from 28 patients) and integrated information on progressive changes in the mutational and transcriptional profiles of each patient to create this genomic narrative. Additionally, we generated and analysed whole-exome sequence data from leukoplakia tissues collected from 11 individuals not suffering from oral cancer. Nonsynonymous somatic mutations in the CASP8 gene were identified as the likely events to initiate malignant transformation, since these were frequently shared between tumour and co-occurring leukoplakia. CASP8 alterations were also shown to enhance expressions of genes that favour lateral spread of mutant cells. During malignant transformation, additional pathogenic mutations are acquired in key genes (TP53, NOTCH1, HRAS) (41% of patients); chromosomal-instability (arm-level deletions of 19p and q, focal-deletion of DNA-repair pathway genes and NOTCH1, amplification of EGFR) (77%), and increased APOBEC-activity (23%) are also observed. These additional alterations were present singly (18% of patients) or in combination (68%). Some of these alterations likely impact immune-dynamics of the evolving transformed tissue; progression to malignancy is associated with immune suppression through infiltration of regulatory T-cells (56%), depletion of cytotoxic T-cells (68%), and antigen-presenting dendritic cells (72%), with a concomitant increase in inflammation (92%). Patients can be grouped into three clusters by the estimated time to development of cancer from precancer by acquiring additional mutations (range: 4-10 years). Our findings provide deep molecular insights into the evolutionary processes and trajectories of oral cancer initiation and progression. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

dbGENVOC: database of GENomic Variants of Oral Cancer, with special reference to India.

Oral cancer is highly prevalent in India and is the most frequent cancer type among Indian males. It is also very common in southeast Asia. India has participated in the International Cancer Genome Consortium (ICGC) and some national initiatives to generate large-scale genomic data on oral cancer patients and analyze to identify associations and systematically catalog the associated variants. We have now created an open, web-accessible database of these variants found significantly associated with Indian oral cancer patients, with a user-friendly interface to enable easy mining. We have value added to this database by including relevant data collated from various sources on other global populations, thereby providing opportunities of comparative geographical and/or ethnic analyses. Currently, no other database of similar nature is available on oral cancer. We have developed Database of GENomic Variants of Oral Cancer, a browsable online database framework for storage, retrieval and analysis of large-scale data on genomic variants and make it freely accessible to the scientific community. Presently, the web-accessible database allows potential users to mine data on ∼24 million clinically relevant somatic and germline variants derived from exomes (n = 100) and whole genomes (n = 5) of Indian oral cancer patients; all generated by us. Variant data from The Cancer Genome Atlas and data manually curated from peer-reviewed publications were also incorporated into the database for comparative analyses. It allows users to query the database by a single gene, multiple genes, multiple variant sites, genomic region, patient ID and pathway identities. Database URL: http://research.nibmg.ac.in/dbcares/dbgenvoc/.

SARS-CoV-2 mutation 614G creates an elastase cleavage site enhancing its spread in high AAT-deficient regions.

SARS-CoV-2 was first reported from China. Within three months, it evolved to 10 additional subtypes. Two evolved subtypes (A2 and A2a) carry a non-synonymous Spike protein mutation (D614G). We conducted phylodynamic analysis of over 70,000 SARS-CoV-2 coronaviruses worldwide, sequenced until July2020, and found that the mutant subtype (614G) outcompeted the pre-existing type (614D), significantly faster in Europe and North-America than in East Asia. Bioinformatically and computationally, we identified a novel neutrophil elastase (ELANE) cleavage site introduced in the G-mutant, near the S1-S2 junction of the Spike protein. We hypothesised that elevation of neutrophil elastase level at the site of infection will enhance the activation of Spike protein thus facilitating host cell entry for 614G, but not the 614D, subtype. The level of neutrophil elastase in the lung is modulated by its inhibitor α1-antitrypsin (AAT). AAT prevents lung tissue damage by elastase. However, many individuals exhibit genotype-dependent deficiency of AAT. AAT deficiency eases host-cell entry of the 614G virus, by retarding inhibition of neutrophil elastase and consequently enhancing activation of the Spike protein. AAT deficiency is highly prevalent in European and North-American populations, but much less so in East Asia. Therefore, the 614G subtype is able to infect and spread more easily in populations of the former regions than in the latter region. Our analyses provide a molecular biological and evolutionary model for the higher observed virulence of the 614G subtype, in terms of causing higher morbidity in the host (higher infectivity and higher viral load), than the non-mutant 614D subtype.

Whole-exome analyses of congenital muscular dystrophy and congenital myopathy patients from India reveal a wide spectrum of known and novel mutations.

BACKGROUND AND PURPOSE: Congenital muscular dystrophies (CMDs) and congenital myopathies (CMs) are a group of genetically and clinically heterogeneous degenerative primary muscle disorders with onset at birth or during infancy. Due to vast heterogeneity, clinical examination and protein-based analyses often fail to identify the genetic causes of these diseases. The aim of this study was to genetically diagnose a cohort of 36 difficult-to-diagnose CMD and CM cases of Indian origin using next-generation sequencing methods. METHODS: Whole-exome sequencing (WES) was performed to identify pathogenic mutations in previously reported CMD and CM-related genes using variant calling and stringent variant filtration process. Subsequently, in silico homology modelling and molecular dynamics simulations (MDS) studies were undertaken for a number of novel and missense variants. RESULTS: A total of 33 and 21 rare and deleterious mutations were identified in 28 genes previously reported in CMD and CM based on OMIM, ClinVar and Orphanet, respectively. We could accurately diagnose 54% patients (n = 12/22) in the CMD group and 35% patients (n = 5/14) in the CM group. Furthermore, MDS studies for mutations located in LMNA, LAMA2 and RYR1 suggest that the wild-type proteins are more stable than their mutant counterparts, implying a potential mechanism of pathogenesis. CONCLUSION: The WES findings led us to identify reported as well as novel variants for the first time in Indian patients with CMD and CM. This allowed us to achieve an accurate genetic diagnosis, which was difficult using conventional diagnostic tools. Transferring these WES findings to clinical practice will help guide clinical care of the affected patients and inform genetic counselling.

Application of Random Forest and data integration identifies three dysregulated genes and enrichment of Central Carbon Metabolism pathway in Oral Cancer.

BACKGROUND: Studies of epigenomic alterations associated with diseases primarily focus on methylation profiles of promoter regions of genes, but not of other genomic regions. In our past work (Das et al. 2019) on patients suffering from gingivo-buccal oral cancer - the most prevalent form of cancer among males in India - we have also focused on promoter methylation changes and resultant impact on transcription profiles. Here, we have investigated alterations in non-promoter (gene-body) methylation profiles and have carried out an integrative analysis of gene-body methylation and transcriptomic data of oral cancer patients. METHODS: Tumor and adjacent normal tissue samples were collected from 40 patients. Data on methylation in the non-promoter (gene-body) regions of genes and transcriptome profiles were generated and analyzed. Because of high dimensionality and highly correlated nature of these data, we have used Random Forest (RF) and other data-analytical methods. RESULTS: Integrative analysis of non-promoter methylation and transcriptome data revealed significant methylation-driven alterations in some genes that also significantly impact on their transcription levels. These changes result in enrichment of the Central Carbon Metabolism (CCM) pathway, primarily by dysregulation of (a) NTRK3, which plays a dual role as an oncogene and a tumor suppressor; (b) SLC7A5 (LAT1) which is a transporter dedicated to essential amino acids, and is overexpressed in cancer cells to meet the increased demand for nutrients that include glucose and essential amino acids; and, (c) EGFR which has been earlier implicated in progression, recurrence, and stemness of oral cancer, but we provide evidence of epigenetic impact on overexpression of this gene for the first time. CONCLUSIONS: In rapidly dividing cancer cells, metabolic reprogramming from normal cells takes place to enable enhanced proliferation. Here, we have identified that among oral cancer patients, genes in the CCM pathway - that plays a fundamental role in metabolic reprogramming - are significantly dysregulated because of perturbation of methylation in non-promoter regions of the genome. This result compliments our previous result that perturbation of promoter methylation results in significant changes in key genes that regulate the feedback process of DNA methylation for the maintenance of normal cell division.

Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility.

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S2 domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in SD domain) and G1124V (in S2 subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird's eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.

A novel hotspot and rare somatic mutation p.A138V, at TP53 is associated with poor survival of pancreatic ductal and periampullary adenocarcinoma patients.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is a cancer of the exocrine pancreas and 5-year survival rates remain constant at 7%. Along with PDAC, Periampullary Adenocarcinoma (PAC) accounts for 0.5-2% of all gastrointestinal malignancies. Genomic observations were well concluded for PDAC and PACs in western countries but no reports are available from India till now. METHODS: Targeted Next Generation Sequencing were performed in 8 (5 PDAC and 3 PAC) tumour normal pairs, using a panel of 412 cancer related genes. Primary findings were replicated in 85 tumour samples (31 PDAC and 54 PAC) using the Sanger sequencing. Mutations were also validated by ASPCR, RFLP, and Ion Torrent sequencing. IHC along with molecular dynamics and docking studies were performed for the p.A138V mutant of TP53. Key polymorphisms at TP53 and its associated genes were genotyped by PCR-RFLP method and association with somatic mutations were evaluated. All survival analysis was done using the Kaplan-Meier survival method which revealed that the survival rates varied significantly depending on the somatic mutations the patients harboured. RESULTS: Among the total 114 detected somatic mutations, TP53 was the most frequently mutated (41%) gene, followed by KRAS, SMAD4, CTNNB1, and ERBB3. We identified a novel hotspot TP53 mutation (p.A138V, in 17% of all patients). Low frequency of KRAS mutation (33%) was detected in these samples compared to patients from Western counties. Molecular Dynamics (MD) simulation and DNA-protein docking analysis predicted p.A138V to have oncogenic characteristics. Patients with p.A138V mutation showed poorer overall survival (p = 0.01). So, our finding highlights elevated prevalence of the p53p.A138V somatic mutation in PDAC and pancreatobiliary PAC patients. CONCLUSION: Detection of p.A138V somatic variant in TP53 might serve as a prognostic marker to classify patients. It might also have a role in determining treatment regimes. In addition, low frequency of KRAS hotspot mutation mostly in Indian PDAC patient cohort indicates presence of other early drivers in malignant transformation.

Study of Caspase 8 mutation in oral cancer and adjacent precancer tissues and implication in progression.

It is hypothesized that same driver gene mutations should be present in both oral leukoplakia and cancer tissues. So, we attempted to find out mutations at one of the driver genes, CASP8, in cancer and adjacent leukoplakia tissues. Patients (n = 27), affected by both of cancer and adjacent leukoplakia, were recruited for the study. Blood and tissue DNA samples were used to identify somatic mutations at CASP8 by next generation sequencing method. In total, 56% (15 out of 27) cancer and 30% (8 out of 27) leukoplakia tissues had CASP8 somatic mutations. In 8 patients, both cancer and adjacent leukoplakia tissues, located within 2-5 cm of tumor sites, had identical somatic mutations. But, in 7 patients, cancer samples had somatic mutations but none of the leukoplakia tissues, located beyond 5cm of tumor sites, had somatic mutations. Mutated allele frequencies at CASP8 were found to be more in cancer compared to adjacent leukoplakia tissues. This study provides mutational evidence that oral cancer might have progressed from previously grown leukoplakia lesion. Leukoplakia tissues, located beyond 5cm of cancer sites, were free from mutation. The study implies that CASP8 mutation could be one of the signatures for some of the leukoplakia to progress to oral cancer.

Analysis of RNA sequences of 3636 SARS-CoV-2 collected from 55 countries reveals selective sweep of one virus type.

BACKGROUND & OBJECTIVES: SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) is evolving with the progression of the pandemic. This study was aimed to investigate the diversity and evolution of the coronavirus SARS-CoV-2 with progression of the pandemic over time and to identify similarities and differences of viral diversity and evolution across geographical regions (countries). METHODS: Publicly available data on type definitions based on whole-genome sequences of the SARS-CoV-2 sampled during December and March 2020 from 3636 infected patients spread over 55 countries were collected. Phylodynamic analyses were performed and the temporal and spatial evolution of the virus was examined. RESULTS: It was found that (i) temporal variation in frequencies of types of the coronavirus was significant; ancestral viruses of type O were replaced by evolved viruses belonging to type A2a; (ii) spatial variation was not significant; with the spread of SARS-CoV-2, the dominant virus was the A2a type virus in every geographical region; (iii) within a geographical region, there was significant micro-level variation in the frequencies of the different viral types, and (iv) the evolved coronavirus of type A2a swept rapidly across all continents. INTERPRETATION & CONCLUSIONS: SARS-CoV-2 belonging to the A2a type possesses a non-synomymous variant (D614G) that possibly eases the entry of the virus into the lung cells of the host. This may be the reason why the A2a type has an advantage to infect and survive and as a result has rapidly swept all geographical regions. Therefore, large-scale sequencing of coronavirus genomes and, as required, of host genomes should be undertaken in India to identify regional and ethnic variation in viral composition and its interaction with host genomes. Further, careful collection of clinical and immunological data of the host can provide deep learning in relation to infection and transmission of the types of coronavirus genomes.

Epigenomic dysregulation-mediated alterations of key biological pathways and tumor immune evasion are hallmarks of gingivo-buccal oral cancer.

BACKGROUND: Gingivo-buccal oral squamous cell carcinoma (OSCC-GB) is the most common cancer among men in India and is associated with high mortality. Although OSCC-GB is known to be quite different from tongue cancer in its genomic presentation and its clinical behavior, it is treated identically as tongue cancer. Predictive markers of prognosis and therapy that are specific to OSCC-GB are, therefore, required. Although genomic drivers of OSCC-GB have been identified by whole exome and whole genome sequencing, no epigenome-wide study has been conducted in OSCC-GB; our study has filled this gap, and has discovered and validated epigenomic hallmarks of gingivobuccal oral cancer. METHODS: We have carried out integrative analysis of epigenomic (n = 87) and transcriptomic (n = 72) profiles of paired tumor-normal tissues collected from OSCC-GB patients from India. Genome-wide DNA methylation assays and RNA-sequencing were performed on high-throughput platforms (Illumina) using a half-sample of randomly selected patients to discover significantly differentially methylated probes (DMPs), which were validated on the remaining half-sample of patients. RESULTS: About 200 genes showed significant inverse correlation between promoter methylation and expression, of which the most significant genes included genes that act as transcription factors and genes associated with other cancer types. Novel findings of this study include identification of (a) potential immunosuppressive effect in OSCC-GB due to significant promoter hypomethylation driven upregulation of CD274 and CD80, (b) significant dysregulation by epigenetic modification of DNMT3B (upregulation) and TET1 (downregulation); and (c) known drugs that can reverse the direction of dysregulation of gene expression caused by promoter methylation. CONCLUSIONS: In OSCC-GB patients, there are significant alterations in expression of key genes that (a) regulate normal cell division by maintenance of balanced DNA methylation and transcription process, (b) maintain normal physiological signaling (PPAR, B cell receptor) and metabolism (arachidonic acid) pathways, and (c) provide immune protection against antigens, including tumor cells. These findings indicate novel therapeutic targets, including immunotherapeutic, for treatment of OSCC-GB.

Lymph node metastasis in oral cancer is strongly associated with chromosomal instability and DNA repair defects.

Oral squamous cell carcinoma (OSCC) is highly prevalent in south and southeast Asia. Many (30-50%) OSCC patients develop lymph node metastasis (LNM), which is the most important prognostic factor in OSCC. To identify genomic correlates of LNM, we compared exome sequences and copy number variation data of blood and tumor DNA from highly contrasting subgroups of patients to reduce false inferences-(i) patients with LNM and (ii) patients with late stage disease but without LNM. We found that LNM is associated with (i) specific hotspot somatic mutations in TP53 and CASP8; (ii) rare nonsilent germline mutations in BRCA2 and FAT1; (iii) mutations in mito-G2/M and nonhomologous end joining (NHEJ) pathways; (iv) recurrent deletion of genes for DNA repair by homologous recombination; and (v) chromosomal instability. LN+ patients with NHEJ pathway mutations have longer disease-free survival. Five genomic features have a high predictive value of LNM.