Identification of miR-379/miR-656 (C14MC) cluster downregulation and associated epigenetic and transcription regulatory mechanism in oligodendrogliomas.

INTRODUCTION: Although role of individual microRNAs (miRNAs) in the pathogenesis of gliomas has been well studied, their role as a clustered remains unexplored in gliomas. METHODS: In this study, we performed the expression analysis of miR-379/miR-656 miRNA-cluster (C14MC) in oligodendrogliomas (ODGs) and also investigated the mechanism underlying modulation of this cluster. RESULTS: We identified significant downregulation of majority of the miRNAs from this cluster in ODGs. Further data from The Cancer Genome Atlas (TCGA) also confirmed the global downregulation of C14MC. Furthermore, we observed that its regulation is maintained by transcription factor MEF2. In addition, epigenetic machinery involving DNA and histone-methylation are also involved in its regulation, which is acting independently or in synergy. The post- transcriptionally regulatory network of this cluster showed enrichment of key cancer-related biological processes such as cell adhesion and migration. Also, there was enrichment of several cancer related pathways viz PIK3 signaling pathway and glioma pathways. Survival analysis demonstrated association of C14MC (miR-487b and miR-409-3p) with poor progression free survival in ODGs. CONCLUSION: Our work demonstrates tumor-suppressive role of C14MC and its role in pathogenesis of ODGs and therefore could be relevant for the development of new therapeutic strategies.

Altered expression and editing of miRNA-100 regulates iTreg differentiation.

RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4(+) T cell differentiation. CD4(+) T cells are an integral component of the adaptive immune system. Naïve CD4(+) T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFß1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end.

Recent admixture in an Indian population of African ancestry.

Identification and study of genetic variation in recently admixed populations not only provides insight into historical population events but also is a powerful approach for mapping disease loci. We studied a population (OG-W-IP) that is of African-Indian origin and has resided in the western part of India for 500 years; members of this population are believed to be descendants of the Bantu-speaking population of Africa. We have carried out this study by using a set of 18,534 autosomal markers common between Indian, CEPH-HGDP, and HapMap populations. Principal-components analysis clearly revealed that the African-Indian population derives its ancestry from Bantu-speaking west-African as well as Indo-European-speaking north and northwest Indian population(s). STRUCTURE and ADMIXTURE analyses show that, overall, the OG-W-IPs derive 58.7% of their genomic ancestry from their African past and have very little inter-individual ancestry variation (8.4%). The extent of linkage disequilibrium also reveals that the admixture event has been recent. Functional annotation of genes encompassing the ancestry-informative markers that are closer in allele frequency to the Indian ancestral population revealed significant enrichment of biological processes, such as ion-channel activity, and cadherins. We briefly examine the implications of determining the genetic diversity of this population, which could provide opportunities for studies involving admixture mapping.

De novo copy number variations in candidate genomic regions in patients of severe autism spectrum disorder in Vietnam.

Autism spectrum disorder (ASD) is a developmental disorder with a prevalence of around 1% children worldwide and characterized by patient behaviour (communication, social interaction, and personal development). Data on the efficacy of diagnostic tests using copy number variations (CNVs) in candidate genes in ASD is currently around 10% but it is overrepresented by patients of Caucasian background. We report here that the diagnostic success of de novo candidate CNVs in Vietnamese ASD patients is around 6%. We recruited one hundred trios (both parents and a child) where the child was clinically diagnosed with ASD while the parents were not affected. We performed genetic screening to exclude RETT syndrome and Fragile X syndrome and performed genome-wide DNA microarray (aCGH) on all probands and their parents to analyse for de novo CNVs. We detected 1708 non-redundant CNVs in 100 patients and 118 (7%) of them were de novo. Using the filter for known CNVs from the Simons Foundation Autism Research Initiative (SFARI) database, we identified six CNVs (one gain and five loss CNVs) in six patients (3 males and 3 females). Notably, 3 of our patients had a deletion involving the SHANK3 gene-which is the highest compared to previous reports. This is the first report of candidate CNVs in ASD patients from Vietnam and provides the framework for building a CNV based test as the first tier screening for clinical management.

Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of approximately 40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.

Whole Exome Sequencing Reveals Novel Candidate Genes in Familial Forms of Glaucomatous Neurodegeneration.

Glaucoma is the largest cause of irreversible blindness with a multifactorial genetic etiology. This study explores novel genes and gene networks in familial forms of primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) to identify rare mutations with high penetrance. Thirty-one samples from nine MYOC-negative families (five POAG and four PACG) underwent whole-exome sequencing and analysis. A set of prioritized genes and variations were screened in an independent validation cohort of 1536 samples and the whole-exome data from 20 sporadic patients. The expression profiles of the candidate genes were analyzed in 17 publicly available expression datasets from ocular tissues and single cells. Rare, deleterious SNVs in AQP5, SRFBP1, CDH6 and FOXM1 from POAG families and in ACACB, RGL3 and LAMA2 from PACG families were found exclusively in glaucoma cases. AQP5, SRFBP1 and CDH6 also revealed significant altered expression in glaucoma in expression datasets. Single-cell expression analysis revealed enrichment of identified candidate genes in retinal ganglion cells and corneal epithelial cells in POAG; whereas for PACG families, retinal ganglion cells and Schwalbe's Line showed enriched expression. Through an unbiased exome-wide search followed by validation, we identified novel candidate genes for familial cases of POAG and PACG. The SRFBP1 gene found in a POAG family is located within the GLC1M locus on Chr5q. Pathway analysis of candidate genes revealed enrichment of extracellular matrix organization in both POAG and PACG.

Spectrum of large copy number variations in 26 diverse Indian populations: potential involvement in phenotypic diversity.

Copy number variations (CNVs) have provided a dynamic aspect to the apparently static human genome. We have analyzed CNVs larger than 100 kb in 477 healthy individuals from 26 diverse Indian populations of different linguistic, ethnic and geographic backgrounds. These CNVRs were identified using the Affymetrix 50K Xba 240 Array. We observed 1,425 and 1,337 CNVRs in the deletion and amplification sets, respectively, after pooling data from all the populations. More than 50% of the genes encompassed entirely in CNVs had both deletions and amplifications. There was wide variability across populations not only with respect to CNV extent (ranging from 0.04-1.14% of genome under deletion and 0.11-0.86% under amplification) but also in terms of functional enrichments of processes like keratinization, serine proteases and their inhibitors, cadherins, homeobox, olfactory receptors etc. These did not correlate with linguistic, ethnic, geographic backgrounds and size of populations. Certain processes were near exclusive to deletion (serine proteases, keratinization, olfactory receptors, GPCRs) or duplication (homeobox, serine protease inhibitors, embryonic limb morphogenesis) datasets. Populations having same enriched processes were observed to contain genes from different genomic loci. Comparison of polymorphic CNVRs (5% or more) with those cataloged in Database of Genomic Variants revealed that 78% (2473) of the genes in CNVRs in Indian populations are novel. Validation of CNVs using Sequenom MassARRAY revealed extensive heterogeneity in CNV boundaries. Exploration of CNV profiles in such diverse populations would provide a widely valuable resource for understanding diversity in phenotypes and disease.

Nuclear morphology and c-Jun N-terminal kinase 1 expression differentiate serum-starved oxidative stress signalling from hydrogen peroxide-induced apoptosis in retinal neuronal cell line.

Oxidative stress induced by serum starvation and H2O2 exposure, both triggers apoptosis in retinal neuronal cell line RGC-5 (retinal ganglion cell-5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC-5 under these conditions. Sub-confluent cells were treated either with H2O2 or maintained in SFM (serum-free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC-5 by 72 h serum starvation and 500 M H2O2 exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H2O2 treatment. Serum starvation did not alter JNK1 (c-Jun N-terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho-JNK) was evident. Conversely, H2O2 exposure blocked the expression and activation of JNK1 to phospho-JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC-5 cells may follow different signalling pathways when challenged with serum starvation and H2O2.

miRvar: A comprehensive database for genomic variations in microRNAs.

microRNAs are a recently discovered and well studied class of small noncoding functional RNAs. The regulatory role of microRNAs (miRNAs) has been well studied in a wide variety of biological processes but there have been no systematic effort to understand and analyze the genetic variations in miRNA loci and study its functional consequences. We have comprehensively curated genetic variations in miRNA loci in the human genome and established a computational pipeline to assess potential functional consequences of these variants along with methods for systematic curation and reporting of variations in these loci. The data is made available on the Leiden Open (source) Variation Database (LOVD) platform at http://genome.igib.res.in/mirlovd to provide ease of aggregation and analysis and is open for community curation efforts.

Evaluation of the antimicrobial potential of two flavonoids isolated from limnophila plants.

The antimicrobial potential of two bioflavonoids, i.e., 5,7-dihydroxy-4',6,8-trimethoxyflavone (1) and 5,6-dihydroxy-4',7,8-trimethoxyflavone (2), isolated from Limnophila heterophylla Benth. and L. indica (Linn.) Druce (Scrophulariaceae), respectively, were evaluated against the microbial strains Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Alternaria solani, and Candida albicans. Compounds 1 and 2 exhibited moderate but broad antimicrobial activities against both Gram-positive and Gram-negative bacteria and also against the fungal pathogens. Moreover, the mechanism of action of 1 and 2 on the cellular functions or structures of some of the microorganisms was studied. Compound 1 showed a bactericidal effect against E. coli and S. aureus (MICs of 200 and 250 µg/ml, resp.), while compound 2 was found to effectively kill B. subtilis by cell lysis. The growth of A. solani and C. albicans was inhibited by compounds 1 and 2, respectively. The effects of the flavonoids on the cellular structures and the carbohydrate metabolic pathways were studied by scanning electron microscopy (SEM) of the treated cells and by assessing the specific activity of key enzymes of the pathways, respectively. At sublethal doses, they enhanced the activity of gluconeogenic fructose bisphosphatase, but decreased the activity of phosphofructokinase and isocitrate dehydrogenase, the key enzymes of the EmbdenMeyerhofParnas pathway and the tricarboxylic acid cycle, respectively.