Frequency spectrum of rare and clinically relevant markers in multiethnic Indian populations (ClinIndb): A resource for genomic medicine in India.

There have been concerted efforts toward cataloging rare and deleterious variants in different world populations using high-throughput genotyping and sequencing-based methods. The Indian population is underrepresented or its information with respect to clinically relevant variants is sparse in public data sets. The aim of this study was to estimate the burden of monogenic disease-causing variants in Indian populations. Toward this, we have assessed the frequency profile of monogenic phenotype-associated ClinVar variants. The study utilized a genotype data set (global screening array, Illumina) from 2795 individuals (multiple in-house genomics cohorts) representing diverse ethnic and geographically distinct Indian populations. Of the analyzed variants from Global Screening Array, ~9% were found to be informative and were either not known earlier or underrepresented in public databases in terms of their frequencies. These variants were linked to disorders, namely inborn errors of metabolism, monogenic diabetes, hereditary cancers, and various other hereditary conditions. We have also shown that our study cohort is genetically a better representative of the Indian population than its representation in the 1000 Genome Project (South Asians). We have created a database, ClinIndb, linked to the Leiden Open Variation Database, to help clinicians and researchers in diagnosis, counseling, and development of appropriate genetic screening tools relevant to the Indian populations and Indians living abroad.

A Case-Control Study Utilizing Red Cell Distribution Width as a Bio-Inflammatory Marker in Pre-eclampsia.

INTRODUCTION: This research was conducted to assess the effectiveness of red cell distribution width (RDW) as an indicator for pre-eclampsia (PE), a condition characterized by elevated blood pressure and the presence of protein in the urine occurring beyond the 20th week of pregnancy. METHODOLOGY: The case-control investigation spanned 10 months, following the acquisition of informed consent and the receipt of ethical clearance. The study sample comprised a total of 70 pregnant women, evenly divided into two groups: 35 cases of PE and 35 normotensive pregnant controls. Both the cases and controls provided 3 ml venous blood samples. The study employed a semi-automated three-part hematological analyzer to establish the baseline RDW for all individuals. RESULTS: This study showed that the individuals with pre-eclampsia had a greater RDW compared to the healthy pregnant women. The observed difference was found to be statistically significant, with a p-value of 0.004. The receiver operating curve (ROC) analysis showed that RDW exhibited significant diagnostic accuracy in differentiating between cases and controls (area under the curve [AUC] = 0.71, P = 0.004) when employing a cut-off value of >= 18.25. The sensitivity was 80% and the specificity was 71.4%. CONCLUSION: In contrast to other indicators of inflammation, RDW is a cost-effective and easily accessible biomarker that can be acquired from routine complete blood counts. It has the potential to be valuable in predicting and diagnosing pre-eclampsia.

Exonized Alu repeats in the 3'UTR of a CYP20A1_Alu-LT transcript act as a miRNA sponge.

OBJECTIVE: Alu repeats have gained huge importance in the creation and modification of regulatory networks. We previously reported a unique isoform of human CYP20A1 i.e. CYP20A1_Alu-LT with 23 Alu repeats exonized in its 9 kb long 3'UTR with 4742 potential binding sites for 994 miRNAs. The role of this transcript was hypothesized as a potential miRNA sponge in primary neurons as its expression correlated with that of 380 genes having shared miRNA sites and enriched in neuro-coagulopathy. This study provides experimental evidence for the miRNA sponge activity of CYP20A1_Alu-LT in neuronal cell lines. RESULTS: We studied the Alu-rich fragment of the CYP20A1_Alu-LT extended 3'UTR with > 10 binding sites for miR-619-5p and miR-3677-3p. Enrichment of the Alu-rich fragment with Ago2 confirmed miRNA association of this transcript. Cloning the fragment downstream of a reporter gene led to a 90% decrease in luciferase activity. Overexpression and knockdown studies revealed a positive correlation between the expression of CYP20A1_Alu-LT and miR-619-5p / miR-3677-3p target genes. GAP43, one of the key modulators of nerve regeneration, was significantly altered by the expression of CYP20A1_Alu-LT. This study, for the first time, provides evidence for a unique regulatory function of exonized Alu repeats as miRNA sponges.

An Alu insertion map of the Indian population: identification and analysis in 1021 genomes of the IndiGen project.

Actively retrotransposing primate-specific Alu repeats display insertion-deletion (InDel) polymorphism through their insertion at new loci. In the global datasets, Indian populations remain under-represented and so do their Alu InDels. Here, we report the genomic landscape of Alu InDels from the recently released 1021 Indian Genomes (IndiGen) (available at https://clingen.igib.res.in/indigen). We identified 9239 polymorphic Alu insertions that include private (3831), rare (3974) and common (1434) insertions with an average of 770 insertions per individual. We achieved an 89% PCR validation of the predicted genotypes in 94 samples tested. About 60% of identified InDels are unique to IndiGen when compared to other global datasets; 23% of sites were shared with both SGDP and HGSVC; among these, 58% (1289 sites) were common polymorphisms in IndiGen. The insertions not only show a bias for genic regions, with a preference for introns but also for the associated genes showing enrichment for processes like cell morphogenesis and neurogenesis (P-value < 0.05). Approximately, 60% of InDels mapped to genes present in the OMIM database. Finally, we show that 558 InDels can serve as ancestry informative markers to segregate global populations. This study provides a valuable resource for baseline Alu InDels that would be useful in population genomics.

Multiple Alu Exonization in 3'UTR of a Primate-Specific Isoform of CYP20A1 Creates a Potential miRNA Sponge.

Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA-miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3'UTR. CYP20A1_Alu-LT, confirmed by 3'RACE, is an outlier in length (9 kb 3'UTR) and widely expressed. Using publically available data sets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15,928 human cortical neurons. miRanda predicts ∼4,700 miRNA recognition elements (MREs) for ∼1,000 miRNAs, primarily originated within these 3'UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. In total, 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared with random sets of differentially expressed genes (P = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA-mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.

Transcriptome analysis and connectivity mapping of Cissampelos pareira L. provides molecular links of ESR1 modulation to viral inhibition.

Bioactive fractions obtained from medicinal plants which have been used for the treatment of multiple diseases could exert their effects by targeting common pathways. Prior knowledge of their usage could allow us to identify novel molecular links. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira L. (Cipa), used for the treatment of female hormone disorders and fever. Transcriptomic studies on MCF7 cell lines treated with Cipa extract carried out using Affymetrix arrays revealed a downregulation of signatures of estrogen response potentially modulated through estrogen receptor α (ERα). Molecular docking analysis identified 38 Cipa constituents that potentially bind (ΔG < - 7.5) with ERα at the same site as estrogen. The expression signatures in the connectivity map ( https://clue.io/; ) revealed high positive scores with translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is a reported ERα coactivator. Further, gene knockdown experiments revealed that Cipa exhibits antiviral activity in dengue infected MCF7 cells potentially modulated through estrogen receptor 1. This approach reveals a novel pathway involving the ESR1-RPL7 axis which could be a potential target in dengue viral infection.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing.

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

Neutrophil Lymphocyte Ratio (NLR) as a Bio Inflammatory Marker in Pre-eclampsia

Introduction: Pre-eclampsia, a systemic disease unique topregnancy, affects 3–14% of pregnant women. The aim ofthe present study was to evaluate Neutrophil LymphocyteRatio (NLR) as a bio inflammatory marker of pre-eclampsia(PE), a hypertensive disorder of pregnancy characterized byhypertension and proteinuria after 20 weeks of gestation.Material and Methods: This case control study was carriedout over a period of 10 months after informed consent andethical clearance. The study population included 70 pregnantwomen (35 pre-eclampsia cases and 35 normotensive pregnantsubjects as controls). 3 ml venous blood samples were obtainedfrom both the cases and controls. Complete blood count wasdone using semi-automated three part haematology analyserwhich gives the reading of cell counts, NLR was calculatedmanually.Results: The case group (subjects with pre-eclampsia) werefound to have higher neutrophil to lymphocyte ratio (NLR)than that of the control group. This difference was statisticallysignificant (p<0.001). The receiver operating curve (ROC)analysis showed significant diagnostic accuracy of NLR todiscriminate cases and controls (area under the curve [AUC]= 0.73, P < 0.001) at cutoff value of >= 4.86, 68.6% sensitivityand 80% specificity.Conclusion: Unlike many other inflammatory markers, NLRproves to be an inexpensive and readily available biomarkers,obtained from routinely done complete blood counts that maybe useful for prediction and diagnosis of pre-eclampsia.