32 research outputs found
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The role of MIR-199A in BAO-induced transgenerational bone deformities in Japanese Medaka
A Thesis Submitted In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in Marine Biology from Texas A&M University-Corpus Christi.Benzo[a]pyrene (BaP), a ubiquitous environmental pollutant and known carcinogen, has been evidenced to cause bone disorders in a transgenerational manner by altering epigenetic mechanisms like microRNAs (miRNA). Transcriptome analysis of bone tissue in the ancestrally BaP-exposed F3 generation indicated a prominent role of miR-199a in the regulation of significantly enriched molecular pathways for bone formation and oxidative stress. To elucidate the function of miR-199a during bone development wildtype (Orange Red) and transgenic (twist:dsred/col10a1:gfp and col10a1:gfp/osx:mCherry) freshwater medaka (Oryzias latipes) were assessed for calcification and cell-subpopulation level changes in response to miR-199a agonist and antagonist microinjections at the onset of vertebra development. To further characterize the role of miR-199a on bone development, mineralization and osteoblast differentiation, the effects of miR199a-3p agonist and antagonist injections (700 pg) at the beginning of bone formation in medaka embryos 3 days post fertilization (dpf) were studied. The area and intensity of calcification were assessed in wild-type medaka. Medaka transgenic lines - twist:dsred/col10a1:gfp and col10a1:gfp/osx:mCherry were used to assess changes during osteoblast differentiation. No impact on the calcified area and bone thickness upon miR-199a-3p agonist or antagonist injection was identified. The spatial distribution and the cellular density of twist+ mesenchymal stem cells (MSCs) were not affected. A significant reduction upon agonist injection was noted in the cellular density of col10+ osteochondro progenitors (OPCs). No change in spatial distribution and density of osx+ premature osteoblast cells (POCs) was seen upon miR-199a-3p treatment. Assessment of bone gene expression levels in 25 dpf old medaka larvae revealed a potential inhibitory effect of miR-199a-3p on bone development. The tissue, cellular and molecular level experiments confirmed the role of miR-199a-3p in bone maturation and osteoblast differentiation. This study provides a novel understanding of miRNA regulation during two critical windows of bone development. The provided data can be used to delineate the effect of BaP exposure on regulating specific miRNAs. Moreover, developmental levels of miR-199a may be useful as a potential biomarker for later life-stage bone impairment, such as osteoporosis development.Marine BiologyCollege of Scienc
Case Report: Application of whole exome sequencing for accurate diagnosis of rare syndromes of mineralocorticoid excess [version 2; referees: 2 approved]
Syndromes of mineralocorticoid excess (SME) are closely related clinical manifestations occurring within a specific set of diseases. Overlapping clinical manifestations of such syndromes often create a dilemma in accurate diagnosis, which is crucial for disease surveillance and management especially in rare genetic disorders. Here we demonstrate the use of whole exome sequencing (WES) for accurate diagnosis of rare SME and report that p.R337C variation in the HSD11B2 gene causes progressive apparent mineralocorticoid excess (AME) syndrome in a South Indian family of Mappila origin
Case Report: Whole exome sequencing reveals a novel frameshift deletion mutation p.G2254fs in COL7A1 associated with autosomal recessive dystrophic epidermolysis bullosa [version 2; referees: 2 approved, 1 approved with reservations]
Dystrophic epidermolysis bullosa simplex (DEB) is a phenotypically diverse inherited skin fragility disorder. It is majorly manifested by appearance of epidermal bullae upon friction caused either by physical or environmental trauma. The phenotypic manifestations also include appearance of milia, scarring all over the body and nail dystrophy. DEB can be inherited in a recessive or dominant form and the recessive form of DEB (RDEB) is more severe. In the present study, we identify a novel p.G2254fs mutation in COL7A1 gene causing a sporadic case of RDEB by whole exome sequencing (WES). Apart from adding a novel frameshift Collagen VII mutation to the repertoire of known mutations reported in the disease, to the best of our knowledge, this is the first report of a genetically characterized case of DEB from India
RNA secondary structure profiling in zebrafish reveals unique regulatory features
Abstract Background RNA is known to play diverse roles in gene regulation. The clues for this regulatory function of RNA are embedded in its ability to fold into intricate secondary and tertiary structure. Results We report the transcriptome-wide RNA secondary structure in zebrafish at single nucleotide resolution using Parallel Analysis of RNA Structure (PARS). This study provides the secondary structure map of zebrafish coding and non-coding RNAs. The single nucleotide pairing probabilities of 54,083 distinct transcripts in the zebrafish genome were documented. We identified RNA secondary structural features embedded in functional units of zebrafish mRNAs. Translation start and stop sites were demarcated by weak structural signals. The coding regions were characterized by the three-nucleotide periodicity of secondary structure and display a codon base specific structural constrain. The splice sites of transcripts were also delineated by distinct signature signals. Relatively higher structural signals were observed at 3’ Untranslated Regions (UTRs) compared to Coding DNA Sequence (CDS) and 5’ UTRs. The 3′ ends of transcripts were also marked by unique structure signals. Secondary structural signals in long non-coding RNAs were also explored to better understand their molecular function. Conclusions Our study presents the first PARS-enabled transcriptome-wide secondary structure map of zebrafish, which documents pairing probability of RNA at single nucleotide precision. Our findings open avenues for exploring structural features in zebrafish RNAs and their influence on gene expression
A genome-wide map of circular RNAs in adult zebrafish
Circular RNAs are a new addition to the growing list of diverse species of RNAs that are formed by covalent linked 3' and 5' end forming a closed loop structure. Circular RNAs are characteristically resistant to exonuclease treatment and are relatively stable to linear transcripts. Circular RNAs are formed by alternate splicing mechanism but do not follow the canonical order of exons. Backsplice junctions are unique to circRNAs. CircRNAs are shown to possess potential to act as miRNA sponges and control transcription of mRNAs. CircRNAs are also reported as biomarkers for the disease like Alzheimer's, Parkinson's and cancer. A huge number of circRNA transcripts have been identified in model organisms including C.elegans, mouse, Drosophila as well as human. But there are no circular RNAs reported in zebrafish that is a very good model to study developmental stages, cardiovascular and blood-related disorders. In order to use zebrafish as a model organism and study the role of circRNAs in disease, we have used in-house generated RNA-sequencing data for five tissues including blood, brain, muscle, gills and heart. We discarded the reads mapped contiguously and full length over reference genome and identified back-splice junctions for putative circRNA transcripts. We identified a total of 3428 circRNA junctions out of which 78% were tissue specific. We validated 22 selected candidates for 5 tissues based on literature significance. We quantitatively analysed 5 tissue-enriched candidates using Real-time PCR. We also observed that major proportion of circRNAs is originating from protein coding loci. These circRNAs could be used to further study their role in hematopoietic and cardiovascular diseases
Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia [version 2; referees: 2 approved]
X-linked agammaglobulinemia (XLA) is an extremely rare inherited primary immunodeficiency characterized by recurrent bacterial infections, decrease in number of mature B cells and low serum immunoglobulins. XLA is caused by mutations in the gene encoding Bruton's tyrosine kinase. We report a case of a young Indian boy suspected to have XLA. Immunophenotyping was performed for the affected child using CD20, CD19 and CD3 antibodies. Whole exome sequencing was performed using trio-based approach. The variants were further analyzed using capillary sequencing in the trio as well as maternal grandmother. Initial immunophenotyping in the affected child showed decreased count of CD19+ B cells. To strengthen the clinical findings and confirm the diagnosis of XLA, we performed whole exome sequencing. Our analysis identified a novel frameshift insertion (c.1325dupT) in the BTK gene, which was further validated by Sanger sequencing. Our approach shows the potential in using whole exome sequencing to pinpoint the molecular lesion, enabling timely diagnosis and genetic counseling, and potentially offering prenatal genetic testing for the family
Familial hypertrophic cardiomyopathy - identification of cause and risk stratification through exome sequencing
Background: Hypertrophic Cardiomyopathy (HCM) with variable clinical presentations and heterogeneity is the common cause of sudden cardiac death. Genetic diagnosis is challenging in these complex diseases but exome sequencing as a genetic diagnostic tool provides explainable results. Methods: In a familial Hypertrophic Cardiomyopathy with multigenerational inheritance with apparent phenotype, had a history of sudden death and severe arrhythmia followed by implantation of Implantable cardioverter defibrillator (ICD). Exome sequencing (100×) trailed by effective filtering steps for exome variants on the basis of different parameters, segregated variants are prioritized for the disease and further clinical relevance are evaluated for the variants. Results: A rare causal variant in troponin-T gene (TNNT2, NM_000364.3;c.274C > T;p.Arg92Trp) is identified, shared by only affected members, absent in unaffected members and also in 200 unrelated control chromosomes. TNNT2 mutation act as a driver mutation but mutations in other disease-related genes, KCNMB1, LPL, APOE and other biochemical factors provides risk stratification within affected family members. Conclusion: This study contributes to the role of “rare variants” in complex disease phenotypes and heterogeneity within family and the necessity of whole exome targeted approaches in complex cardiomyopathy, which are known to harbor private mutations
Case Report: Whole exome sequencing identifies variation c.2308G>A p.E770K in RAG1 associated with B- T- NK+ severe combined immunodeficiency [version 2; referees: 2 approved, 1 not approved]
Severe combined immunodeficiency is a large clinically heterogeneous group of disorders caused by a defect in the development of humoral or cellular immune responses. At least 13 genes are known to be involved in the pathophysiology of the disease and the mutation spectrum in SCID has been well documented. Mutations of the recombination-activating genes RAG 1 and RAG 2 are associated with a range of clinical presentations including, severe combined immunodeficiency and autoimmunity. Recently, our understanding of the molecular basis of immune dysfunction in RAG deficiency has improved tremendously with newer insights into the ultrastructure of the RAG complex. In this report, we describe the application of whole exome sequencing for arriving at a molecular diagnosis in a child suffering from B- T- NK+ severe combined immunodeficiency. Apart from making the accurate molecular diagnosis, we also add a genetic variation c.2308G>A p.E770K to the compendium of variations associated with the disease