LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy.

Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial-associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+ ) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta-b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2-mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA-mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.

IndiGenomes: a comprehensive resource of genetic variants from over 1000 Indian genomes.

With the advent of next-generation sequencing, large-scale initiatives for mining whole genomes and exomes have been employed to better understand global or population-level genetic architecture. India encompasses more than 17% of the world population with extensive genetic diversity, but is under-represented in the global sequencing datasets. This gave us the impetus to perform and analyze the whole genome sequencing of 1029 healthy Indian individuals under the pilot phase of the 'IndiGen' program. We generated a compendium of 55,898,122 single allelic genetic variants from geographically distinct Indian genomes and calculated the allele frequency, allele count, allele number, along with the number of heterozygous or homozygous individuals. In the present study, these variants were systematically annotated using publicly available population databases and can be accessed through a browsable online database named as 'IndiGenomes' http://clingen.igib.res.in/indigen/. The IndiGenomes database will help clinicians and researchers in exploring the genetic component underlying medical conditions. Till date, this is the most comprehensive genetic variant resource for the Indian population and is made freely available for academic utility. The resource has also been accessed extensively by the worldwide community since it's launch.

Long Non Coding RNA Based Regulation of Cerebrovascular Endothelium.

The rapid and high throughput discovery of long non coding RNAs (lncRNAs) has far outstripped the functional annotation of these novel transcripts in their respective cellular contexts. The cells of the blood brain barrier (BBB), especially the cerebrovascular endothelial cells (CVECs), are strictly regulated to maintain a controlled state of homeostasis for undisrupted brain function. Several key pathways are understood in CVEC function that lead to the development and maintenance of their barrier properties, the dysregulation of which leads to BBB breakdown and neuronal injury. Endothelial lncRNAs have been discovered and functionally validated in the past decade, spanning a wide variety of regulatory mechanisms in health and disease. We summarize here the lncRNA-mediated regulation of established pathways that maintain or disrupt the barrier property of CVECs, including in conditions such as ischemic stroke and glioma. These lncRNAs namely regulate the tight junction assembly/disassembly, angiogenesis, autophagy, apoptosis, and so on. The identification of these lncRNAs suggests a less understood mechanistic layer, calling for further studies in appropriate models of the blood brain barrier to shed light on the lncRNA-mediated regulation of CVEC function. Finally, we gather various approaches for validating lncRNAs in BBB function in human organoids and animal models and discuss the therapeutic potential of CVEC lncRNAs along with the current limitations.

Analysis of the potential impact of genomic variants in global SARS-CoV-2 genomes on molecular diagnostic assays.

An epidemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus diseases (C0VID-19) initially reported in Wuhan, China has rapidly emerged into a global pandemic affecting millions of people worldwide. Molecular detection of SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) forms the mainstay in screening, diagnosis and epidemiology of the disease. Since the virus evolves by accumulating base substitutions, mutations in the viral genome could possibly affect the accuracy of RT-PCR-based detection assays. The recent availability of genomes of SARS-CoV-2 isolates motivated us to assess the presence and potential impact of variations in target sites of the oligonucleotide primers and probes used in molecular diagnosis. We catalogued a total of 132 primer or probe sequences from literature and data available in the public domain. Our analysis revealed that a total of 5862 unique genetic variants mapped to at least one of the 132 primer or probe binding sites in the genome. A total of 29 unique variants were present in ≥ 1% of genomes from at least one of the continents (Asia, Africa, Australia, Europe, North America, and South America) that mapped to 36 unique primers or probes binding sites. Similarly, a total of 27 primer or probe binding sites had cumulative variants frequency of ≥ 1% in the global SARS-CoV-2 genomes. These included primers or probes sites which are used worldwide for molecular diagnosis as well as approved by national and international agencies. We also found 286 SARS-CoV-2 genomic regions with low variability at a continuous stretch of ≥ 20bps that could be potentially used for primer designing. This highlights the need for sequencing genomes of emerging pathogens to enable evidence-based policies for development and approval of diagnostics.

Pharmacogenomic landscape of COVID-19 therapies from Indian population genomes.

Aim: Numerous drugs are being widely prescribed for COVID-19 treatment without any direct evidence for the drug safety/efficacy in patients across diverse ethnic populations. Materials & methods: We analyzed whole genomes of 1029 Indian individuals (IndiGen) to understand the extent of drug-gene (pharmacogenetic), drug-drug and drug-drug-gene interactions associated with COVID-19 therapy in the Indian population. Results: We identified 30 clinically significant pharmacogenetic variants and 73 predicted deleterious pharmacogenetic variants. COVID-19-associated pharmacogenes were substantially overlapped with those of metabolic disorder therapeutics. CYP3A4, ABCB1 and ALB are the most shared pharmacogenes. Fifteen COVID-19 therapeutics were predicted as likely drug-drug interaction candidates when used with four CYP inhibitor drugs. Conclusion: Our findings provide actionable insights for future validation studies and improved clinical decisions for COVID-19 therapy in Indians.

Genetic epidemiology of autoinflammatory disease variants in Indian population from 1029 whole genomes.

BACKGROUND: Autoinflammatory disorders are the group of inherited inflammatory disorders caused due to the genetic defect in the genes that regulates innate immune systems. These have been clinically characterized based on the duration and occurrence of unprovoked fever, skin rash, and patient's ancestry. There are several autoinflammatory disorders that are found to be prevalent in a specific population and whose disease genetic epidemiology within the population has been well understood. However, India has a limited number of genetic studies reported for autoinflammatory disorders till date. The whole genome sequencing and analysis of 1029 Indian individuals performed under the IndiGen project persuaded us to perform the genetic epidemiology of the autoinflammatory disorders in India. RESULTS: We have systematically annotated the genetic variants of 56 genes implicated in autoinflammatory disorder. These genetic variants were reclassified into five categories (i.e., pathogenic, likely pathogenic, benign, likely benign, and variant of uncertain significance (VUS)) according to the American College of Medical Genetics and Association of Molecular pathology (ACMG-AMP) guidelines. Our analysis revealed 20 pathogenic and likely pathogenic variants with significant differences in the allele frequency compared with the global population. We also found six causal founder variants in the IndiGen dataset belonging to different ancestry. We have performed haplotype prediction analysis for founder mutations haplotype that reveals the admixture of the South Asian population with other populations. The cumulative carrier frequency of the autoinflammatory disorder in India was found to be 3.5% which is much higher than reported. CONCLUSION: With such frequency in the Indian population, there is a great need for awareness among clinicians as well as the general public regarding the autoinflammatory disorder. To the best of our knowledge, this is the first and most comprehensive population scale genetic epidemiological study being reported from India.

Role of Escherichia coli endopeptidases and dd-carboxypeptidases in infection and regulation of innate immune response.

The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan recycling can also produce peptidoglycan fragments capable of activating an innate immune response in host. To investigate how these proteins in Enterobacteriaceae play a role to elicit/evade innate immune responses during infections, we deleted certain endopeptidases and dd-carboxypeptidases from Escherichia coli CS109 and studied the viability of these mutants in macrophages. The ability of infected macrophages to exert oxidative killing, express surface activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses were elevated in macrophages infected with dd-carboxypeptidase mutants but reduced for endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated in macrophages infected with both mutant types. Overall, we have shown, under normal conditions endopeptidases have a tendency to elicit the immune response but their effect is suppressed by the presence of dd-carboxypeptidases. Conversely, DD-carboxypeptidases, normally, tend to reduce immune responses, as their deletions enhanced the same in macrophages. Therefore, we conclude that the roles of endopeptidases and dd-carboxypeptidases are possibly counter-active in wild-type cells where either class of enzymes suppresses each other's immunogenic properties rendering overall maintenance of low immunogenicity that helps E. coli in evading the host immune responses.

A genome-wide map of circular RNAs in adult zebrafish.

Circular RNAs (circRNAs) are transcript isoforms generated by back-splicing of exons and circularisation of the transcript. Recent genome-wide maps created for circular RNAs in humans and other model organisms have motivated us to explore the repertoire of circular RNAs in zebrafish, a popular model organism. We generated RNA-seq data for five major zebrafish tissues - Blood, Brain, Heart, Gills and Muscle. The repertoire RNA sequence reads left over after reference mapping to linear transcripts were used to identify unique back-spliced exons utilizing a split-mapping algorithm. Our analysis revealed 3,428 novel circRNAs in zebrafish. Further in-depth analysis suggested that majority of the circRNAs were derived from previously well-annotated protein-coding and long noncoding RNA gene loci. In addition, many of the circular RNAs showed extensive tissue specificity. We independently validated a subset of circRNAs using polymerase chain reaction (PCR) and divergent set of primers. Expression analysis using quantitative real time PCR recapitulate selected tissue specificity in the candidates studied. This study provides a comprehensive genome-wide map of circular RNAs in zebrafish tissues.

Methods to Study Long Noncoding RNA Expression and Dynamics in Zebrafish Using RNA Sequencing.

Long noncoding RNAs (lncRNAs) belong to a class of RNA transcripts that do not have the potential to code for proteins. LncRNAs were largely discovered in the transcriptomes of human and several model organisms, using next-generation sequencing (NGS) approaches, which have enabled a comprehensive genome scale annotation of transcripts. LncRNAs are known to have dynamic expression status and have the potential to orchestrate gene regulation at the epigenetic, transcriptional, and posttranscriptional levels. Here we describe the experimental methods involved in the discovery of lncRNAs from the transcriptome of a popular model organism zebrafish (Danio rerio). A structured and well-designed computational analysis pipeline subsequent to the RNA sequencing can be instrumental in revealing the diversity of the lncRNA transcripts. We describe one such computational pipeline used for the discovery of novel lncRNA transcripts in zebrafish. We also detail the validation of the putative novel lncRNA transcripts using qualitative and quantitative assays in zebrafish.