Development of DNA markers using next-generation sequencing approach for molecular authentication of Boerhavia diffusa L. and Tinospora cordifolia (Willd.) Miers.

The adulteration of plants and their materials used in herbal formulations poses a severe health concern. Hence, there is a need to establish a reliable, cost-effective, and robust molecular biomarker to distinguish among species and identify herbal plants and raw drugs from adulterants. The present study used suppressive subtractive hybridization and next-generation sequencing technology to identify novel DNA markers for Boerhavia diffusa L. and Tinospora cordifolia (Willd.) Miers. We identified two primer sets for B. diffusa and one for T. cordifolia. The DNA markers were validated in different accessions of B. diffusa and T. cordifolia and their common adulterants to determine the sensitivity and specificity of developed DNA markers. The designed DNA markers showed 100% sensitivity and specificity in detecting B. diffusa and T. cordifolia from their adulterants. The strategy described here can be extrapolated for developing DNA markers to authenticate other plant species. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03732-7.

Pharmacogenomic considerations for repurposing of dexamethasone as a potential drug against SARS-CoV-2 infection.

Immunomodulatory and analgesic effects of dexamethasone are clinically well established, and this synthetic corticosteroid acts as an agonist of glucocorticoid receptors. Early results of the RECOVERY Trial from the United Kingdom and others suggest certain benefits of dexamethasone against COVID-19 chronic patients. The efforts have been acknowledged by World Health Organization with an interim guideline to use in patients with a severe and critical illness. The inherent genetic variations in genes such as CYP3A5, NR3C1, NR3C2, etc., involved in the pharmacokinetic and pharmacodynamic processes may influence dexamethasone's effects as an anti-inflammatory drug. Besides, the drug may influence transcriptome or metabolic changes in the individuals. In the present review, we summarize the reported genetic variations that impact dexamethasone response and discuss dexamethasone-induced changes in transcriptome and metabolome that may influence potential treatment outcome against COVID-19.

Lay abstract The surge of COVID-19 cases has increased the need for the development of a cure. This has pushed the barriers of the regulatory controls for randomized controlled trials. There has been the usage of immunomodulatory drugs, such as dexamethasone, with promising results in severe COVID-19 patients to reduce mortality. However, there is a need to consider the inherent genetic factors of an individual that may influence the dexamethasone drug's metabolism and action. To understand this, there is a need to evaluate the genes involved in the pharmacokinetics and pharmacodynamic pathways of the drug and study the effects of the drug. This will aid in choosing the right individuals who will benefit from the therapy. Hence, the present review summarized the reported genetic variations that impact dexamethasone drug response.

MirSNPs in clopidogrel metabolism genes predict cardiovascular disease risk: a case-control study and meta-analysis.

Aim: The present study was conducted to decipher the inter-relationship of SNPs and miRNAs involved in pharmacogenomics of clopidogrel on predisposition to cardiovascular diseases (CVDs). Materials & methods: A case-control study was conducted on 410 cases and 386 controls to analyze the association of 13 mirSNPs on CVDs risk. Genotyping was performed by tetra-primer amplification refractory mutation system PCR and validated using Sanger DNA sequencing. miRNA expression analysis was performed using TaqMan assays. A meta-analysis was performed for PON1 rs662 with coronary artery disease. Results & conclusion:PON1 rs662, PON1 rs3917577, CYP3A5 rs15524, COL4A1 rs874204 and PTGIR rs1126510 polymorphisms showed association with CVDs. The miRNA hsa-miR-224-5p showed differential expression in the PON1 rs3917577 GG genotype. The meta-analysis showed the population-specific impact of PON1 rs662 on South Asian and Middle East populations.

SNPs in Sites for DNA Methylation, Transcription Factor Binding, and miRNA Targets Leading to Allele-Specific Gene Expression and Contributing to Complex Disease Risk: A Systematic Review.

INTRODUCTION: The complex genetic diversity among human populations results from an assortment of factors acting at various sequential levels, including mutations, population migrations, genetic drift, and selection. Although there are a plethora of DNA sequence variations identified through genome-wide association studies (GWAS), the challenge remains to explain the mechanisms underlying interindividual phenotypic disparity accounting for disease susceptibility. Single nucleotide polymorphisms (SNPs) present in the sites for DNA methylation, transcription factor (TF) binding, or miRNA targets can alter the gene expression. The systematic review aimed to evaluate the complex crosstalk among SNPs, miRNAs, DNA methylation, and TFs for complex multifactorial disease risk. METHODS: PubMed and Scopus databases were used from inception until May 15, 2019. Initially, screening of articles involved studies assessing the interaction of SNPs with TFs, DNA methylation, or miRNAs resulting in allele-specific gene expression in complex multifactorial diseases. We also included the studies which provided experimental validation of the interaction of SNPs with each of these factors. The results from various studies on multifactorial diseases were assessed. RESULTS: A total of 11 articles for SNPs interacting with DNA methylation, 30 articles for SNPs interacting with TFs, and 11 articles for SNPs in miRNA binding sites were selected. The interactions of SNPs with epigenetic factors were found to be implicated in different types of cancers, autoimmune diseases, cardiovascular diseases, diabetes, and asthma. CONCLUSION: The systematic review provides evidence for the interplay between genetic and epigenetic risk factors through allele-specific gene expression in various complex multifactorial diseases.

Coding SNPs in hsa-miR-1343-3p and hsa-miR-6783-3p target sites of CYP2C19 modulates clopidogrel response in individuals with cardiovascular diseases.

AIMS: To investigate the impact of microRNA target SNPs (mirSNPs) and their interaction with miRNAs on important drug-metabolizing enzymes, transporters and target genes for prediction of clopidogrel drug response in cardiovascular disease individuals. MAIN METHODS: A prospective cross-sectional study was conducted on 292 individuals undergoing clopidogrel drug therapy. All the enrolled participants were administered 300 mg loading dose followed by 75 mg dose of maintenance therapy. Platelet aggregations were measured before administration of the loading dose and 2 h post fifth day dose of clopidogrel maintenance therapy. Clopidogrel carboxylic acid metabolite from plasma and urine were analyzed post maintenance therapy using the RP-HPLC method. Genotyping of mirSNP's shortlisted through in silico analysis was performed by tetra ARMS PCR and validated by Sanger DNA sequencing. The levels of selected miRNAs were estimated by the TaqMan-PCR assay. Functional validation of mirSNPs was performed in HepG2 cells after transfecting with the selected gene and miRNA mimics. Protein expressions were analyzed by western blot. KEY FINDINGS: 23% of enrolled individuals showed resistance to clopidogrel therapy. Out of 13 mirSNP's analyzed, CYP2C19 rs4244285 was associated with clopidogrel drug resistance and clopidogrel carboxylic acid metabolite in urine and plasma. hsa-miR-1343-3p and hsa-miR-6783-3p levels were significantly high in individuals with CYP2C19 rs4244285 mutant genotype and these miRNAs down-regulated the protein expression of CYP2C19. SIGNIFICANCE: We demonstrated the role of coding mirSNP (rs4244285) in the regulation of the CYP2C19 gene through miRNAs and its implications to clopidogrel drug response prediction in the Indian population.

Aberrant DNA methylation and miRNAs in coronary artery diseases and stroke: a systematic review.

Coronary artery disease (CAD) and ischemic stroke are the two most predominant forms of cardiovascular diseases (CVDs) caused by genetic, epigenetic and environmental risk factors. Although studies on the impact of 'epigenetics' in CVDs is not new, its effects are increasingly being realized as a key regulatory determinant that may drive predisposition, pathophysiology and therapeutic outcome. The most widely studied epigenetic risk factors are regulated by DNA methylation and miRNA expression. To keep pace with growing developments and discoveries, a comprehensive review was performed using Pubmed, Science Direct and Scopus databases to highlight the role of DNA methylation and miRNAs in CAD and stroke subjects. Network analysis was performed using ClueGO software and miRTargetLink database. We identified 32 studies of DNA methylation on CAD and stroke, of which, 6 studies showed differences in global DNA methylation, 10 studies reported the genome-wide difference in DNA methylation and 16 studies demonstrated altered DNA methylation at 14 candidate loci. The network analysis showed positive regulation of nitric oxide biosynthetic process, homocysteine metabolic process and negative regulation of lipid storage. About, 155 miRNAs were associated with CAD, stroke and related phenotypes in 83 studies. Interestingly, mir-223 hypomethylation and altered expression were associated with cerebral infarction and stroke. The target prediction for 18 common miRNAs between CAD and stroke showed strong interaction with SP3 and SP1 genes. This systematic review addresses the present knowledge on DNA methylation and miRNAs in CAD and stroke, whose abnormal regulation has been implicated in etiology or progression of the diseases.

In silico characterization of functional single nucleotide polymorphisms of folate pathway genes.

Folate metabolism genes are pivotal to critical biological processes and are related to several conditions, including developmental, cognitive, and cardiovascular anomalies. A systematic catalog of genetic polymorphisms in protein coding regions, regulatory transcription factor binding sites, and miRNA binding sites associated with folate pathway genes may contribute to personalized medicine. We performed a comprehensive computational survey of single nucleotide polymorphisms (SNPs) of folate pathway genes to highlight functional polymorphisms in the coding region, transcription factor binding sites, and miRNAs binding sites. Folate pathway genes were searched through PubMed and Kyoto Encyclopedia of Genes and Genomes pathway databases. SNPs were identified and characterized using the University of California, Santa Cruz genome browser and SNPnexus tool. Functional characterization of nonsynonymous SNPs (nsSNPS) was performed using bioinformatics tools, and common deleterious nsSNPs were identified. We identified 48 genes of folate pathway containing 287 SNPs in the coding regions. Out of these SNPs, rs5742905, rs45511401, and rs1801133 were predicted to be deleterious through four different bioinformatics tools. Three-dimensional structures of two proteins with and without deleterious nsSNPs were predicted by SWISSPDB viewer and SuperPose. Besides, a total of 237 SNPs was identified in transcription factor binding sites using the Genomatix software suite and six miRNA target site SNPs using miRNASNP. This systematic and extensive in silico analysis of functional SNPs of folate pathway may provide a foundation for future targeted mechanistic, structure-function, and genetic epidemiological studies.