Recessive GCH1 Deficiency Causing DOPA-Responsive Dystonia Diagnosed by Reported Negative Exome.

An exome sequencing result on a child with atypical gait was reported as negative; follow-up biochemical evaluation and reanalysis led to diagnosis of treatable DOPA-responsive dystonia.

MicroRNA regulation of vascular smooth muscle cells and its significance in cardiovascular diseases.

Cardiovascular disease (CVD) is among the leading causes of death worldwide. MicroRNAs (miRNAs), regulatory molecules that repress protein expression, have attracted considerable attention in CVD research. The vasculature plays a big role in CVD development and progression and dysregulation of vascular cells underlies the root of many vascular diseases. This review provides a brief introduction of the biogenesis of miRNAs and exosomes, followed by overview of the regulatory mechanisms of miRNAs in vascular smooth muscle cells (VSMCs) intracellular signaling during phenotypic switching, senescence, calcification, and neointimal hyperplasia. Evidence of extracellular signaling of VSMCs and other cells via exosomal and circulating miRNAs is also presented. Lastly, current drawbacks and limitations of miRNA studies in CVD research and potential ways to overcome these disadvantages are discussed in detail. In-depth understanding of VSMC regulation via miRNAs will add substantial knowledge and advance research in diagnosis, disease progression, and (or) miRNA-derived therapeutic approaches in CVD research.

Non-coding RNA and lung cancer progression.

Lung cancer (LC) is a major killer disease globally. This situation is further supported by yearly increase in new LC cases and its poor 5-year survival which is less than 15%. Although a large percentage of LC cases have been attributed to smoking, a considerable amount of nonsmokers also develops this disease, thereby suggesting a genetic and/or epigenetic undertone to LC development. Several growth-related genes such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) as well as tumor suppressor genes such as p53 have been implicated in LC pathogenesis and progression. Likewise, the genome only contains approximately 1% of coding regions. Hence, noncoding portion of the genome such as noncoding RNAs (ncRNAs) has been studied and discovered to play a cogent role in LC pathogenesis. More precisely, microRNAs (miRNAs) and long ncRNAs (lncRNAs) have been studied for decades. Posttranscriptional gene modulation function of miRNAs is well established and characterized. Likewise, the antagonizing interaction between lncRNAs and miRNAs had also been proven to further control gene expression during healthy and disease conditions like LC. More recently, renewed attention toward circular RNAs [circular RNAs (circRNAs)] study showed that circRNAs can also sponge miRNAs to modulate gene expressions too. Hence, miRNAs, lncRNAs, and circRNAs seem to function within a circuit to optimally determine which gene is needed to be upregulated or downregulated in biological system. Therefore, this review will discuss important ncRNAs, namely miRNA, lncRNA, and circRNA in LC progression. Paracrine effect of exosomal ncRNA will be also reviewed. In addition, the prospect of these ncRNAs in enhancing better LC treatment will be highlighted as well.

Exosome-shuttled miR-92b-3p from ischemic preconditioned astrocytes protects neurons against oxygen and glucose deprivation.

Ischemic preconditioning (IPC) exerts protective effects against ischemic cerebral injury. In the present study, an in vitro model of cerebral ischemia (oxygen and glucose deprivation, OGD) was established to investigate the neuroprotective mechanism of IPC. We found that conditioned medium (C.M.) from astrocytes rather than neurons nor microglia cell line BV2 exerted neuroprotection. Moreover, exosomes derived from OGD preconditioned astrocytes can be taken up by neurons and attenuated OGD-induced neuron death and apoptosis. High-throughput microRNA (miRNA) sequencing revealed that miR-92b-3p levels in exosomes released from preconditioned astrocytes were increased. Overexpression of miR-92b-3p in neurons with miR-92b-3p mimic achieved the same protective effects as C.M. from astrocytes. Thus, we propose that the mechanism of IPC may associate with astrocytes, and that exosome-mediated miR-92b-3p shuttle from preconditioned astrocytes to neurons participate in these process.

Eomes-expressing T-helper cells as potential target of therapy in chronic neuroinflammation.

Reserch progresses in understanding the pathogenicity of multiple sclerosis (MS) in the last couple of decade has enabled us to develop new drug entities available in the clinic. However, we still have not succeeded in preventing conversion from relapsing-remitting MS (RR-MS) to secondary progressive MS (SP-MS) and curing this intractable form of MS. Furthermore, diagnosis is usually retrospective and subjective, relying on gradual worsening of neurological signs/symptoms. This is obviously due to the lack of understanding for the pathogenicity driving disease progression in MS and of reliable biomarkers reflecting the progressive or stationary disease status. Two relevant components are involved in brain pathology of SP-MS, neurodegeneration and inflammation. Neurodegeneration may occur spontaneously in a neuron-intrinsic manner under chronic inflammation, such as glutamate excitotoxicity, mitochondrial/oxidative injury with iron deposit in the brain, and loss of trophic support. Meanwhile, inflammation is usually associated with recurrent relapse and the cumulative infiltration of immune cells, including T cells, B cells, and myeloid cells of peripheral or CNS origin, could ignite the processes of neurodegeneration. Especially, the higher frequency of leptomeningeal follicle-like structures observed in SP-MS patients suggests that immune cells sheltered behind a blood-brain barrier is still active under smoldering CNS inflammation. Recent successes in Ocrelizumab for primary progressive in MS (PP-MS) and Siponimod for SP-MS reappraised the importance of immune cells for pathogenesis progressive MS. Accordingly, our recent comparative analysis between MS and its animal model, experimental autoimmune encephalomyelitis (EAE), raises a new possibility that ectopic expression of eomesodermin (Eomes) in helper T (Th) cells constitutes a previously unappreciated subset of Th cells with cytotoxic potential against neuronal cells. In this review article, I will summarize the mechanisms proposed on pathogenesis of SP-MS and propose a new pathogenic mechanism for neurodegeneration mediated by unique cytotoxic Th cells.

Inter-individual gene variants associated with trabecular bone plasticity: A step forward in the personal genomics of degenerative bone disease.

Continuing tissue destruction in osteoarthrosis is maintained by molecular pathways related to an unbalanced chondrocyte metabolism, the loss of reactive oxygen species (ROS) homeostasis, increase catabolism in a degraded matrix and the limited response to growth factors due to cell aging. Rare deleterious gene variants driving relevant molecular pathways may play a key role in the pathogenesis and genetic control of common diseases and may also influence the common gene variants observed in GWAS. We use molecular profiling technologies based on massive sequencing of genes to interrogate clinical samples for a variety of molecules involved in the pathogenesis pathways of OA and also to derive new insights for drug targeting discovery at an early stage of the disease. By whole-exome sequencing performed in OA patients with extreme phenotypes and in non-related individuals without clinical evidence of OA, the most predominant of the rare gene variants found were non-synonymous single-nucleotide variants (SNV) from exonic DNA regions and with missense functional effects predicting a moderate impact on protein function. A total of 629, 577, and 639 gene variants for the TPF, COA, and ANHNF patients, respectively, were found not to be shared with the 20 non-disease-related individuals. After subtraction of the 306 variants shared among the OA patients, we obtained the individual profiles of 323, 271, and 333 gene variants, for the TPF, COA, and ANHNF patients, respectively. After filtering by the bioinformatics, genetic, and biological criteria established to assess the clinical consequences, comparative analysis of trio sequences using integrative genome visualization tool clearly demonstrate the differences between patients. Analysis of the collagen gene variants identified 78, 20, and 43 genetic collagen variants for the three extreme phenotypes. Rare gene variants encoding for proteins that are less abundant in the trabecular bone matrix, together with those responsible for the control and regulation of bone turnover and plasticity of subchondral trabecular bone, play important roles in OA and help to define the clinical phenotype.

Extracellular vesicles as emerging targets in cancer: Recent development from bench to bedside.

Extracellular vesicles (EVs) have emerged as important players of cancer initiation and progression through cell-cell communication. They have been recognized as critical mediators of extracellular communications, which promote transformation, growth invasion, and drug-resistance of cancer cells. Interestingly, the secretion and uptake of EVs are regulated in a more controlled manner than previously anticipated. EVs are classified into three groups, (i) exosomes, (ii) microvesicles (MVs), and (iii) apoptotic bodies (ABs), based on their sizes and origins, and novel technologies to isolate and distinguish these EVs are evolving. The biologically functional molecules harbored in these EVs, including nucleic acids, lipids, and proteins, have been shown to induce key signaling pathways in both tumor and tumor microenvironment (TME) cells for exacerbating tumor development. While tumor cell-derived EVs are capable of reprogramming stromal cells to generate a proper tumor cell niche, stromal-derived EVs profoundly affect the growth, resistance, and stem cell properties of tumor cells. This review summarizes and discusses these reciprocal communications through EVs in different types of cancers. Further understanding of the pathophysiological roles of different EVs in tumor progression is expected to lead to the discovery of novel biomarkers in liquid biopsy and development of tumor specific therapeutics. This review will also discuss the translational aspects of EVs and therapeutic opportunities of utilizing EVs in different cancer types.

Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs.

Metabolic changes within the cell and its niche affect cell fate and are involved in many diseases and disorders including cancer and viral infections. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS). KSHV latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, these miRNAs are responsible for inducing the Warburg effect in infected cells. Here we identify a novel mechanism enabling KSHV to manipulate the metabolic nature of the tumour microenvironment. We demonstrate that KSHV infected cells specifically transfer the virus-encoded microRNAs to surrounding cells via exosomes. This flow of genetic information results in a metabolic shift toward aerobic glycolysis in the surrounding non-infected cells. Importantly, this exosome-mediated metabolic reprogramming of neighbouring cells supports the growth of infected cells, thereby contributing to viral fitness. Finally, our data show that this miRNA transfer-based regulation of cell metabolism is a general mechanism used by other herpesviruses, such as EBV, as well as for the transfer of non-viral onco-miRs. This exosome-based crosstalk provides viruses with a mechanism for non-infectious transfer of genetic material without production of new viral particles, which might expose them to the immune system. We suggest that viruses and cancer cells use this mechanism to shape a specific metabolic niche that will contribute to their fitness.

Unexplained early onset epileptic encephalopathy: Exome screening and phenotype expansion.

Early onset epileptic encephalopathies (EOEEs) represent a significant diagnostic challenge. Newer genomic approaches have begun to elucidate an increasing number of responsible single genes as well as emerging diagnostic strategies. In this single-center study, we aimed to investigate a cohort of children with unexplained EOEE. We performed whole-exome sequencing (WES), targeting a list of 137 epilepsy-associated genes on 50 children with unexplained EOEE. We characterized all phenotypes in detail and classified children according to known electroclinical syndromes where possible. Infants with previous genetic diagnoses, causative brain malformations, or inborn errors of metabolism were excluded. We identified disease-causing variants in 11 children (22%) in the following genes: STXBP1 (n = 3), KCNB1 (n = 2), KCNT1, SCN1A, SCN2A, GRIN2A, DNM1, and KCNA2. We also identified two further variants (in GRIA3 and CPA6) in two children requiring further investigation. Eleven variants were de novo, and in one paternal testing was not possible. Phenotypes were broadened for some variants identified. This study demonstrates that WES is a clinically useful screening tool for previously investigated unexplained EOEE and allows for reanalysis of data as new genes are being discovered. Detailed phenotyping allows for expansion of specific gene disorders leading to epileptic encephalopathy and emerging sub-phenotypes.

Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload.

UNLABELLED: To identify polymorphisms associated with variability of iron overload severity in HFE-associated hemochromatosis, we performed exome sequencing of DNA from 35 male HFE C282Y homozygotes with either markedly increased iron stores (n = 22; cases) or with normal or mildly increased iron stores (n = 13; controls). The 35 participants, residents of the United States, Canada, and Australia, reported no or light alcohol consumption. Sequencing data included 82,068 single-nucleotide variants, and 10,337 genes were tested for a difference between cases and controls. A variant in the GNPAT gene showed the most significant association with severe iron overload (P = 3 × 10(-6) ; P = 0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of twenty-two participants with severe iron overload had glyceronephosphate O-acyltransferase (GNPAT) polymorphism p.D519G (rs11558492; 15 heterozygotes, one homozygote). No control participant had this polymorphism. To examine functional consequences of GNPAT deficiency, we performed small interfering RNA-based knockdown of GNPAT in the human liver-derived cell line, HepG2/C3A. This knockdown resulted in a >17-fold decrease in expression of the messenger RNA encoding the iron-regulatory hormone, hepcidin. CONCLUSION: GNPAT p.D519G is associated with a high-iron phenotype in HFE C282Y homozygotes and may participate in hepcidin regulation.

Plant exomics: concepts, applications and methodologies in crop improvement.

Molecular breeding has a crucial role in improvement of crops. Conventional breeding techniques have failed to ameliorate food production. Next generation sequencing has established new concepts of molecular breeding. Exome sequencing has proven to be a significant tool for assessing natural evolution in plants, studying host pathogen interactions and betterment of crop production as exons assist in interpretation of allelic variation with respect to their phenotype. This review covers the platforms for exome sequencing, next generation sequencing technologies that have revolutionized exome sequencing and led toward development of third generation sequencing. Also discussed in this review are the uses of these sequencing technologies to improve wheat, rice and cotton yield and how these technologies are used in exploring the biodiversity of crops, providing better understanding of plant-host pathogen interaction and assessing the process of natural evolution in crops and it also covers how exome sequencing identifies the gene pool involved in symbiotic and other co-existential systems. Furthermore, we conclude how integration of other methodologies including whole genome sequencing, proteomics, transcriptomics and metabolomics with plant exomics covers the areas which are left untouched with exomics alone and in the end how these integration will transform the future of crops.

Exosomes: a novel pathway of local and distant intercellular communication that facilitates the growth and metastasis of neoplastic lesions.

Normal and diseased cells release bilayered membrane-bound nanovesicles into interstitial spaces and into bodily fluids. A subgroup of such microvesicles is called exosomes and is described in blood as 30 to 100 nm in diameter and as spherical to cup-shaped nanoparticles with specific surface molecular characteristics (eg, expression of the tetraspanins CD9, CD81, and CD63). Extracellular microvesicles provide local signals (eg, autocrine and paracrine) and distant endocrine signals to cells via the transfer of their contents, which include signal proteins, lipids, miRNAs, and functional mRNAs. Exosomes and related microvesicles also aid cells in exporting less-needed molecules and potentially harmful molecules, including drugs; in the case of neoplasia, the export of chemotherapeutic drugs may facilitate cellular chemoresistance. Cancers have adapted the exosome and related microvesicles as a pathway by which neoplastic cells communicate with each other (autocrine) and with nonneoplastic cells (paracrine and endocrine); via this pathway, cancer suppresses the immune system and establishes a fertile local and distant environment to support neoplastic growth, invasion, and metastases. Because exosomes mirror and bind to the cells from which they arise, they can be used for delivery of drugs, vaccines, and gene therapy, as biomarkers and targets. We review how exosomes and related extracellular microvesicles facilitate the progression and metastases of cancers and describe how these microvesicles may affect clinical care.

Exome sequencing reveals new causal mutations in children with epileptic encephalopathies.

PURPOSE: The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses. METHODS: We performed WES (mean coverage approximately 40×) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing. KEY FINDINGS: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures. SIGNIFICANCE: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.

Customisation of the exome data analysis pipeline using a combinatorial approach.

The advent of next generation sequencing (NGS) technologies have revolutionised the way biologists produce, analyse and interpret data. Although NGS platforms provide a cost-effective way to discover genome-wide variants from a single experiment, variants discovered by NGS need follow up validation due to the high error rates associated with various sequencing chemistries. Recently, whole exome sequencing has been proposed as an affordable option compared to whole genome runs but it still requires follow up validation of all the novel exomic variants. Customarily, a consensus approach is used to overcome the systematic errors inherent to the sequencing technology, alignment and post alignment variant detection algorithms. However, the aforementioned approach warrants the use of multiple sequencing chemistry, multiple alignment tools, multiple variant callers which may not be viable in terms of time and money for individual investigators with limited informatics know-how. Biologists often lack the requisite training to deal with the huge amount of data produced by NGS runs and face difficulty in choosing from the list of freely available analytical tools for NGS data analysis. Hence, there is a need to customise the NGS data analysis pipeline to preferentially retain true variants by minimising the incidence of false positives and make the choice of right analytical tools easier. To this end, we have sampled different freely available tools used at the alignment and post alignment stage suggesting the use of the most suitable combination determined by a simple framework of pre-existing metrics to create significant datasets.