harnessing-power-microrna-cargoes-small-extracellular-vesicles

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic ...

University of salford

Somatic variation in DNA can cause cells to deviate from the preordained genomic path in both disease and healthy conditions. Here, using exome sequencing of paired tissue samples, we show that the normal human brain harbors somatic single base variations measuring up to 0.48% of the total variations. Interestingly, about 64% of these somatic variations in the brain are expected to lead to non-synonymous changes, and as much as 87% of these represent G:C>T:A transversion events. Further, the transversion events in the brain were mostly found in the frontal cortex, whereas the corpus callosum from the same individuals harbors the reference genotype. We found a significantly higher amount of 8-OHdG (oxidative stress marker) in the frontal cortex compared to the corpus callosum of the same subjects (p<0.01), correlating with the higher G:C>T:A transversions in the cortex. We found significant enrichment for axon guidance and related pathways for genes harbouring somatic variations. This could represent either a directed selection of genetic variations in these pathways or increased susceptibility of some loci towards oxidative stress. This study highlights that oxidative stress possibly influence single nucleotide somatic variations in normal human brain.

Human brain harbors single nucleotide somatic variations in functionally relevant genes possibly mediated by oxidative stress.

Editing in microRNAs, particularly in seed can significantly alter the choice of their target genes. We show that out of 13 different human tissues, different regions of brain showed higher adenosine to inosine (A-to-I) editing in mature miRNAs. These events were enriched in seed sequence (73.33%), which was not observed for cytosine to uracil (17.86%) editing. More than half of the edited miRNAs showed increased stability, 72.7% of which had ΔΔG values less than -6.0 Kcal/mole and for all of them the edited adenosines mis-paired with cytosines on the pre-miRNA structure. A seed-editing event in hsa-miR-411 (with A - C mismatch) lead to increased expression of the mature form compared to the unedited version in cell culture experiments. Further, small RNA sequencing of GBM patients identified significant miRNA hypoediting which correlated with downregulation of ADAR2 both in metadata and qRT-PCR based validation. Twenty-two significant (11 novel) A-to-I hypoediting events were identified in GBM samples. This study highlights the importance of specific sequence and structural requirements of pre-miRNA for editing along with a suggestive crucial role for ADAR2. Enrichment of A-to-I editing in seed sequence highlights this as an important layer for genomic regulation in health and disease, especially in human brain.

A-to-I editing in human miRNAs is enriched in seed sequence, influenced by sequence contexts and significantly hypoedited in glioblastoma multiforme.

Clustered miRNAs can affect functioning of downstream pathways due to possible coordinated function. We observed 78-88% of the miR-379/miR-656 cluster (C14MC) miRNAs were downregulated in three sub-types of diffuse gliomas, which was also corroborated with analysis from The Cancer Genome Atlas (TCGA) datasets. The miRNA expression levels decreased with increasing tumor grade, indicating this downregulation as an early event in gliomagenesis. Higher expression of the C14MC miRNAs significantly improved glioblastioma prognosis (Pearson's r = 0.62; p < 3.08e-22). ENCODE meta-data analysis, followed by reporter assays validated existence of two novel internal regulators within C14MC. CRISPR activation of the most efficient internal regulator specifically induced members of the downstream miRNA sub-cluster and apoptosis in glioblastoma cells. Luciferase assays validated novel targets for miR-134 and miR-485-5p, two miRNAs from C14MC with the most number of target genes relevant for glioma. Overexpression of miR-134 and miR-485-5p in human glioblastoma cells suppressed invasion and proliferation, respectively. Furthermore, apoptosis was induced by both miRs, individually and in combination. The results emphasize the tumor suppressive role of C14MC in diffuse gliomas, and identifies two specific miRNAs with potential therapeutic value and towards better disease management and therapy.

Novel internal regulators and candidate miRNAs within miR-379/miR-656 miRNA cluster can alter cellular phenotype of human glioblastoma.

Although role of individual microRNAs (miRNAs) in the pathogenesis of gliomas has been well studied, their role as a clustered remains unexplored in gliomas.

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

Fusion transcripts can contribute to diversity of molecular networks in the human cortex. In this study, we explored the occurrence of fusion transcripts in normal human cortex along with single neurons and astrocytes. We identified 1305 non-redundant fusion events from 388 transcriptomes representing 59 human cortices and 329 single cells. Our results indicate while the majority of fusion transcripts in human cortex are intra-chromosomal (85%), events found in single neurons and astrocytes were primarily inter-chromosomal (80%). The number of fusions in single neurons was significantly higher than that in single astrocytes (p < 0.05), indicating fusion as a possible contributor towards transcriptome diversity in neuronal cells. The identified fusions were largely private and 4 specific recurring events were found both in cortex and in single neurons but not in astrocytes. We found a significant increase in the number of fusion transcripts in human brain with increasing age both in single cells and whole cortex (p < 0.0005 and < 0.005, respectively). This is likely one of the many possible contributors for the inherent plasticity of the adult brain. The fusion transcripts in fetal brain were enriched for genes for long-term depression; while those in adult brain involved genes enriched for long-term potentiation pathways. Our findings demonstrate fusion transcripts are naturally occurring phenomenon spanning across the health-disease continuum, and likely contribute to the diverse molecular network of human brain.

Fusion transcripts in normal human cortex increase with age and show distinct genomic features for single cells and tissues.

Autism spectrum disorder (ASD) is a complex disorder with an unclear aetiology and an estimated global prevalence of 1%. However, studies of ASD in the Vietnamese population are limited. Here, we first conducted whole exome sequencing (WES) of 100 children with ASD and their unaffected parents. Our stringent analysis pipeline was able to detect 18 unique variants (8 de novo and 10 ×-linked, all validated), including 12 newly discovered variants. Interestingly, a notable number of X-linked variants were detected (56%), and all of them were found in affected males but not in affected females. We uncovered 17 genes from our ASD cohort in which CHD8, DYRK1A, GRIN2B, SCN2A, OFD1 and MDB5 have been previously identified as ASD risk genes, suggesting the universal aetiology of ASD for these genes. In addition, we identified six genes that have not been previously reported in any autism database: CHM, ENPP1, IGF1, LAS1L, SYP and TBX22. Gene ontology and phenotype-genotype analysis suggested that variants in IGF1, SYP and LAS1L could plausibly confer risk for ASD. Taken together, this study adds to the genetic heterogeneity of ASD and is the first report elucidating the genetic landscape of ASD in Vietnamese children.

Genetic landscape of autism spectrum disorder in Vietnamese children.

RNA editing is a post-transcriptional modification, which can provide tissue-specific functions not encoded in DNA. Adenosine-to-inosine is the predominant editing event and, along with cytosine-to-uracil changes, constitutes canonical editing. The rest is non-canonical editing. In this study, we have analysed non-canonical editing of microRNAs in the human brain. We have performed massively parallel small RNA sequencing of frontal cortex (FC) and corpus callosum (CC) pairs from nine normal individuals (post-mortem). We found 113 and 90 unique non-canonical editing events in FC and CC samples, respectively. More than 70% of events were in the miRNA seed sequence-implicating an altered set of target mRNAs and possibly resulting in a functional consequence. Up to 15% of these events were recurring and found in at least three samples, also supporting the biological relevance of such variations. Two specific sequence variations, C-to-A and G-to-U, accounted for over 80% of non-canonical miRNA editing events-and revealed preferred sequence motifs. Our study is one of the first reporting non-canonical editing in miRNAs in the human brain. Our results implicate miRNA non-canonical editing as one of the contributing factors towards transcriptomic diversity in the human brain.

Human Brain Shows Recurrent Non-Canonical MicroRNA Editing Events Enriched for Seed Sequence with Possible Functional Consequence.

Breast cancer is the most frequently diagnosed cancer globally and is one of the most important contributors to cancer-related deaths. Earlier diagnosis is known to reduce mortality, and better biomarkers are needed. MiRNA clusters often co-express and target mRNAs in a coordinated fashion, perturbing entire pathways; they thus merit further exploration for diagnostic or prognostic use. MiR-379/656, at chromosome 14q32, is the second largest miRNA cluster in the human genome and implicated in various malignancies including glioblastoma, melanoma, gastrointestinal tumors and ovarian cancer highlighting its potential importance. In this study, we focus on the diagnostic and prognostic potentials of MiR-379/656 in breast cancer and its molecular subtypes.

Diagnostic and Prognostic Potential of MiR-379/656 MicroRNA Cluster in Molecular Subtypes of Breast Cancer.

Whole-Exome Sequencing of Vitiligo Lesions Indicates Lower Burden of Somatic Variations: Implications in Risk for Nonmelanoma Skin Cancers.

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 -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  and  from POAG families and in  and  from PACG families were found exclusively in glaucoma cases.  and  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  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.

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

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.

Article	Year
Human brain harbors single nucleotide somatic variations in functionally relevant genes possibly mediated by oxidative stress. F1000Research\| PubMed ID: 28149503	2016
A-to-I editing in human miRNAs is enriched in seed sequence, influenced by sequence contexts and significantly hypoedited in glioblastoma multiforme. Scientific reports\| PubMed ID: 28550310	2017
Novel internal regulators and candidate miRNAs within miR-379/miR-656 miRNA cluster can alter cellular phenotype of human glioblastoma. Scientific reports\| PubMed ID: 29769662	2018
Identification of miR-379/miR-656 (C14MC) cluster downregulation and associated epigenetic and transcription regulatory mechanism in oligodendrogliomas. Journal of neuro-oncology\| PubMed ID: 29931616	2018
Fusion transcripts in normal human cortex increase with age and show distinct genomic features for single cells and tissues. Scientific reports\| PubMed ID: 31992760	2020
Genetic landscape of autism spectrum disorder in Vietnamese children. Scientific reports\| PubMed ID: 32193494	2020
Human Brain Shows Recurrent Non-Canonical MicroRNA Editing Events Enriched for Seed Sequence with Possible Functional Consequence. Non-coding RNA\| PubMed ID: 32498345	2020
Diagnostic and Prognostic Potential of MiR-379/656 MicroRNA Cluster in Molecular Subtypes of Breast Cancer. Journal of clinical medicine\| PubMed ID: 34575183	2021
Whole-Exome Sequencing of Vitiligo Lesions Indicates Lower Burden of Somatic Variations: Implications in Risk for Nonmelanoma Skin Cancers. The Journal of investigative dermatology\| PubMed ID: 36535361	2023
Whole Exome Sequencing Reveals Novel Candidate Genes in Familial Forms of Glaucomatous Neurodegeneration. Genes\| PubMed ID: 36833422	2023
De novo copy number variations in candidate genomic regions in patients of severe autism spectrum disorder in Vietnam. PloS one\| PubMed ID: 38451970	2024

Arijit Mukhopadhyay

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