Identification of novel exonic variants contributing to hereditary breast and ovarian cancer in west Indian population.

Breast and ovarian cancers are the most common cancer types in females worldwide and in India. Patients with these cancers require an early diagnosis which is essential for better prognosis, treatment and improved patient survival. Recently, the utilization of next-generation sequencing (NGS)-based screening has accelerated molecular diagnosis of various cancers. In the present study, we performed whole-exome sequencing (WES) of 30 patients who had a first or second-degree relative with breast or ovarian cancer and are tested negative for BRCA1/2 or other high and moderate-risk genes reported for HBOC. WES data from patients were analyzed and variants were called using bcftools. Functional annotation of variants and variant prioritization was performed by Exomiser. The clinical significance of variants was determined as per ACMG classification using Varsome tool. The functional analysis of genes was determined by STRING analysis and disease association was determined by open target tool. We found novel variants and gene candidates having significant association with HBOC conditions. The genes identified by exomiser (phenotype score > 0.75) are associated with various biological processes such as DNA integrity maintenance, transcription regulation, cell cycle regulation, and apoptosis. Our findings provide novel and prevalent gene variants associated with the HBOC condition in the West Indian population which could be further studied for early diagnosis and better prognosis of HBOC.

Transcriptome profiling reveals upregulation of benzoate degradation and related genes in Pseudomonas aeruginosa D6 during textile dye degradation.

An upsurge in textile dye pollution has demanded immediate efforts to develop an optimum technology for their bioremediation. However, the molecular mechanism underpinning aerobic decolorization of dyes is still in its infancy. Thus, in the current work, the intricacies of aerobic remediation of textile dyes by Pseudomonas aeruginosa D6 were understood via a transcriptomic approach. The bacterium isolated from the sludge sample of a common effluent treatment plant was able to decolorize 54.42, 57.66, 50.84 and 65.86% of 100 mg L-1 of four different dyes i.e., TD01, TD04, TD05, and TD06, respectively. The maximum decolorization was achieved within six days and thus, the first and sixth day of incubation were selected for transcriptome analysis at the early and late phase of the decolorization, respectively. The expression profiles of all samples were compared to gain insight into the dye-specific response of bacterium and it was found that it behaved most uniquely in the presence of the dye TD01. Several genes critical to core metabolic processes like the TCA cycle, glycolysis, pentose phosphate pathway, translation, cell motility etc. Were found to be overexpressed in the presence of dyes. Interestingly, in response to dyes, the benzoate degradation pathway was significantly upregulated in the bacterium as compared to control (i.e., bacterium without dye). Thus, seven genes contributing to the induction of the same were further studied by RT-qPCR analysis. Overall, the involvement of the benzoate pathway implies the appearance of aromatic intermediates during decolorization, which in turn infers dye degradation.

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis.

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is higher globally than production due to increased incidences of various heart conditions. The main source of streptokinase is various strains of Streptococci. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD gene-mediated post-translational modification of streptolysin, an inhibitor of streptokinase expression through the degradation of FasX small RNA (through CoV/RS), which stabilizes streptokinase mRNA. In order to improve the stability of mRNA and increase the expression of streptokinase, which is inhibited by SagA, we used CRISPR-Cas9 to successfully knockout the SagD gene and observed a 13.58-fold increased expression of streptokinase at the transcript level and 1.48-fold higher expression at the protein level in the mutant strain compared to wild type. We have demonstrated the successful gene knockout of SagD using CRISPR-Cas9 in S. equisimilis, where an engineered strain can be further used for overexpression of streptokinase for therapeutic applications.

Molecular Diagnosis of Muscular Dystrophy Patients in Western Indian Population: A Comprehensive Mutation Analysis Using Amplicon Sequencing.

Muscular Dystrophies (MDs) are a group of inherited diseases and heterogeneous in nature. To date, 40 different genes have been reported for the occurrence and/or progression of MDs. This study was conducted to demonstrate the application of next-generation sequencing (NGS) in developing a time-saving and cost-effective diagnostic method to detect single nucleotide variants (SNVs) and copy number variants (CNVs) in a single test. A total of 123 cases clinically suspected of MD were enrolled in this study. Amplicon panel-based diagnosis was carried out for 102 (DMD/BMD) cases and the results were further screened using multiplex ligation-dependent probe amplification (MLPA). Whilst in the case of LGMD (N = 19) and UMD (N = 2), only NGS panel-based analysis was carried out. We identified the large deletions in 74.50% (76/102) of the cases screened with query DMD or BMD. Further, the large deletion in CAPN3 gene (N = 3) and known SNV mutations (N = 4) were identified in LGMD patients. Together, the total diagnosis rate for this amplicon panel was 70.73% (87/123) which demonstrated the utility of panel-based diagnosis for high throughput, affordable, and time-saving diagnostic strategy. Collectively, present study demonstrates that the panel based NGS sequencing could be superior over to MLPA.

Bacterial line of defense in Dirinaria lichen from two different ecosystems: First genomic insights of its mycobiont Dirinaria sp. GBRC AP01.

Lichens have been widely studied for their symbiotic properties and for the secondary metabolites production by its fungal symbiont. Recent molecular studies have confirmed coexistence of bacteria along with the fungal and algal symbionts. Direct nucleic acid study by -omics approaches is providing better insights into their structural and functional dynamics. However, genomic analysis of individual members of lichen is difficult by the conventional approach. Hence, genome assembly from metagenome data needs standardization in the eukaryotic system like lichens. The present study aimed at metagenomic characterization of rock associated lichen Dirinaria collected from Kutch and Dang regions of Gujarat, followed by genome reconstruction and annotation of the mycobiont Dirinaria. The regions considered in the study are eco-geographically highly variant. The results revealed higher alpha diversity in the dry region Kutch as compared to the tropical forest associated lichen from Dang. Ascomycota was the most abundant eukaryote while Proteobacteria dominated the bacterial population. There were 23 genera observed only in the Kutch lichen (KL) and one genus viz., Candidatus Vecturithrix unique to the Dang lichen (DL). The exclusive bacterial genera in the Kutch mostly belonged to groups reported for stress tolerance and earlier isolated from lithobionts of extreme niches. The assembled data of KL & DL were further used for genome reconstruction of Dirinaria sp. using GC and tetra-pentamer parameters and reassembly that resulted into a final draft genome of 31.7 Mb and 9556 predicted genes. Twenty-eight biosynthesis gene clusters were predicted that included genes for polyketide, indole and terpene synthesis. Association analysis of bacteria and mycobiont revealed 8 pathways specific to bacteria with implications in lichen symbiosis and environment interaction. The study provides the first draft genome of the entire fungal Dirinaria genus and provides insights into the Dirinaria lichen metagenome from Gujarat region.

Mutational Landscape for Indian Hereditary Breast and Ovarian Cancer Cohort Suggests Need for Identifying Population Specific Genes and Biomarkers for Screening.

BACKGROUND: Breast and ovarian cancers are the most prevalent cancers and one of the leading causes of death in Indian women. The healthcare burden of breast and ovarian cancers and the rise in mortality rate are worrying and stress the need for early detection and treatment. METHODS: We performed amplicon sequencing of 144 cases who had breast/ovarian cancer disease (total 137 cases are patients and seven are tested for BRCA1/2 carrier) Using our custom designed gene panel consisting of 14 genes, that are associated with high to moderate risk of breast and ovarian cancers. Variants were called using Torrent Variant Caller and were annotated using ThermoFisher's Ion Reporter software. Classification of variants and their clinical significance were identified by searching the variants against ClinVar database. RESULTS: From a total of 144 cases, we were able to detect 42 pathogenic mutations in [40/144] cases. Majority of pathogenic mutations (30/41) were detected in BRCA1 gene, while (7/41) pathogenic mutations were detected in BRCA2 gene, whereas, (2/41) pathogenic mutations were detected in TP53 gene and BRIP1, PALB2, and ATM genes respectively. So, BRCA genes contributed 88.09% of pathogenic mutations, whereas non-BRCA genes contributed 11.91% of pathogenic mutations. We were also able to detect 25 VUS which were predicted to be damaging by in silico prediction tools. CONCLUSION: Early detection of cancers in the Indian population can be done by genetic screening using customized multi-gene panels. Indications of our findings show that in the Indian population, apart from the common BRCA genes, there are other genes that are also responsible for the disease. High frequency mutations detected in the study and variants of uncertain significance predicted to be damaging by in silico pathogenicity prediction tools can be potential biomarkers of hereditary breast and ovarian cancer in Indian HBOC patients.

Development of PR genes panel for screening aphid-tolerant cultivars in Brassica juncea.

The exorbitant yield loss incurred by Indian farmers every year (10-90%) in rapeseed-mustard (Brassica juncea) is chiefly attributed to the progressive infestation of mustard fields by Lipaphis erysimi (Kalt.), a major insect pest belonging to the family of Homoptera. Currently there are no successful tolerant cultivars developed by conventional means in Brassica juncea with systemic plant responses in the form of direct or indirect defenses against aphid attack. Lack of specific methods for screening large numbers of genotypes required in breeding for selection of tolerant cultivars in mustard is one of the main causes of slow progress in developing resistant varieties of Brassica juncea. Traditional phenotype-based breeding has to be augmented with recent molecular approaches for potential genotype selection and cultivar development in Brassica juncea. In current study a pathogen-responsive gene panel was developed which could be used for expression-assisted breeding program in mustard for selection of tolerant types against aphid infestation, minimizing the huge crop losses suffered by farmers every year.

A snapshot of microbial communities from the Kutch: one of the largest salt deserts in the World.

Here we present the first report on the taxonomic diversity of the microbial communities of the saline desert of the Great Rann of Kutch, Gujarat, India, using a metagenomic approach. Seven samples, differing in salinity levels and covering different seasons, were analysed to investigate the dynamics of microbial communities in relation to salinity and season. Metagenomic data generated using whole metagenome sequencing revealed that despite its very high salinity (4.11-30.79 %), the saline desert's microbiota had a rich microbial diversity that included all major phyla. Notably, 67 archaeal genera, representing more than 60 % of all known archaeal genera, were present in this ecosystem. A strong positive correlation (0.85) was observed between the presence of the extremely halophilic bacterium Salinibacter and salinity level. Taxonomic and functional comparisons of the saline desert metagenome with those of other publicly available metagenomes (i.e. sea, hypersaline lagoon, solar saltern, brine, hot desert) was carried out. The microbial community of the Kutch was found to be unique yet more similar to the sea biomes followed by hypersaline lagoon.

Tobacco habituated and non-habituated subjects exhibit different mutational spectrums in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common non-skin cancer in the world. Tobacco chewing is implicated with most of the cases of HNSCC but this type of cancer is increasing in non-tobacco chewers as well. This study was instigated to provide comprehensive variant and gene-level data in HNSCC subjects of the Indian population and fill the gap in the literature on comparative assessment of gene mutations in cancer subjects with a habit of tobacco and those without any habit using targeted amplicon sequencing. We performed targeted Amplicon sequencing of 409 tumor suppressor genes and oncogenes, frequently mutated across many cancer types, including head and neck. DNA from primary tumor tissues and matched blood was analyzed for HNSCC patients with a habit of tobacco and those without any habit. PDE4DIP, SYNE1, and NOTCH1 emerged as the highly mutated genes in HNSCC. A total of 39 candidate causal variants in 22 unique cancer driver genes were identified in non-habitual (WoH) and habitual (WH) subjects. Comparison of genes from both the subjects, showed seven unique cancer driver genes (KIT, ATM, RNF213, GATA2, DST, RET, CYP2C19) in WoH, while WH showed five (IL7R, PKHD1, MLL3, PTPRD, MAPK8) and 10 genes (SETD2, ATR, CDKN2A, NCOA4, TP53, SYNE1, KAT6B, THBS1, PTPRT, and FGFR3) were common to both subjects. In addition to this NOTCH1, NOTCH2, and NOTCH4 gene were found to be mutated only in habitual subjects. These findings strongly support a causal role for tobacco, acting via PI3K and MAPK pathway inhibition and stimulation of various genes leading to oncogenic transformations in case of tobacco chewers. In case of non-tobacco chewers it appears that mutations in the pathway affecting the squamous epithelial lineage and DNA repair genes lead to HNSCC. Somatic mutation in CYP2C19 gene in the non-habitual subjects suggests that this gene may have a tobacco independent role in development and progression of HNSCC. In addition to sharing high mutation rate, NOTCH gene family was found to be mutated only in habitual sample. Further, presence of mutated genes not earlier reported to be involved in HNSCC, suggest that the Indian sub-continent may have different sets of genes, as compared to other parts of the world, involved in the development and progression of HNSCC.

Metagenomics of petroleum muck: revealing microbial diversity and depicting microbial syntrophy.

Present study attempts in revealing taxonomic and functional diversity of microorganism from petroleum muck using metagenomics approach. Using Ion Torrent Personal Genome Machine, total of 249 Mb raw data were obtained which was analysed using MG-RAST platform. The taxonomic analysis revealed predominance of Proteobacteria with Gammaproteobacteria as major class and Pseudomonas stutzeri as most abundant organism. Several enzymes involved in aliphatic and aromatic hydrocarbon degradation through both aerobic and anaerobic routes and proteins related to stress response were also present. Comparison of our metagenome with the existing metagenomes from oil-contaminated sites and wastewater treatment plant indicated uniqueness of this metagenome taxonomically and functionally. Based on these results a hypothetical community model showing survival and syntrophy of microorganisms in hydrocarbon-rich environment is proposed. Validation of the metagenome data was done in three tiers by validating major OTUs by isolating oil-degrading microbes, confirmation of key genes responsible for hydrocarbon degradation by Sanger sequencing and studying functional dynamics for degradation of the hydrocarbons by the muck meta-community using GC-MS.

Complete mitogenome of Asiatic lion resolves phylogenetic status within Panthera.

BACKGROUND: The origin, evolution and speciation of the lion, has been subject of interest, debate and study. The present surviving lions of the genus Panthera comprise of eight sub-species inclusive of Asiatic lion Panthera leo persica of India's Gir forest. Except for the Asiatic lion, the other seven subspecies are found in different parts of Africa. There have been different opinions regarding the phylogenetic status of Panthera leo, as well as classifying lions of different geographic regions into subspecies and races. In the present study, mitogenome sequence of P. leo persica deduced, using Ion Torrent PGM to assess phylogeny and evolution which may play an increasingly important role in conservation biology. RESULTS: The mtDNA sequence of P. leo persica is 17,057 bp in length with 40.8% GC content. Annotation of mitogenome revealed total 37 genes, including 13 protein coding, 2 rRNA and 22 tRNA. Phylogenetic analysis based on whole mitogenome, suggests Panthera pardus as a neighbouring species to P. leo with species divergence at ~2.96 mya. CONCLUSION: This work presents first report on complete mitogenome of Panthera leo persica. It sheds light on the phylogenetic and evolutionary status within and across Felidae members. The result compared and evaluated with earlier reports of Felidae shows alteration of phylogenetic status and species evolution. This study may provide information on genetic diversity and population stability.