Genetic correction of Werner syndrome gene reveals impaired pro-angiogenic function and HGF insufficiency in mesenchymal stem cells.

WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN+/+ mesenchymal stem cells (MSCs) exhibited improved pro-angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN-/- MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN-/- MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro-angiogenic function of WS-MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.

Toward a Pathway-Driven Clinical-Molecular Framework for Classifying Autism Spectrum Disorders.

BACKGROUND: The current classification system of neurodevelopmental disorders is based on clinical criteria; however, this method alone fails to incorporate what is now known about genomic similarities and differences between closely related clinical neurodevelopmental disorders. Here we present an alternative clinical molecular classification system of neurodevelopmental disorders based on shared molecular and cellular pathways, using syndromes with autistic features as examples. METHODS: Using the Online Mendelian Inheritance in Man database, we identified 83 syndromes that had "autism" as a feature of disease, which in combination were associated with 69 autism disease-causing genes. Using annotation terms generated from the DAVID annotation tool, we grouped each gene and its associated autism syndrome into three biological pathways: ion transport, cellular synaptic function, and transcriptional regulation. RESULTS: The majority of the autism syndromes we analyzed (54 of 83) enriched for processes related to transcriptional regulation and were associated with more non-neurologic symptoms and co-morbid psychiatric disease when compared with the other two pathways studied. Disorders with disrupted cellular synaptic function had significantly more motor-related symptoms when compared with the other groups of disorders. CONCLUSION: Our pathway-based classification system identified unique clinical characteristics within each group that may help guide clinical diagnosis, prognosis, and treatment. These results suggest that shifting current clinical classification of autism disorders toward molecularly driven, pathway-related diagnostic groups such as this may more precisely guide clinical decision making and may be informative for future clinical trial and drug development approaches.

Functional genomics analysis of Phelan-McDermid syndrome 22q13 region during human neurodevelopment.

Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by varying degrees of intellectual disability, severely delayed language development and specific facial features, and is caused by a deletion within chromosome 22q13.3. SHANK3, which is located at the terminal end of this region, has been repeatedly implicated in other neurodevelopmental disorders and deletion of this gene specifically is thought to cause much of the neurologic symptoms characteristic of PMS. However, it is still unclear to what extent SHANK3 deletions contribute to the PMS phenotype, and what other genes nearby are causal to the neurologic disease. In an effort to better understand the functional landscape of the PMS region during normal neurodevelopment, we assessed RNA-sequencing (RNA-seq) expression data collected from post-mortem brain tissue from developmentally normal subjects over the course of prenatal to adolescent age and analyzed expression changes of 65 genes on 22q13. We found that the majority of genes within this region were expressed in the brain, with ATNX10, MLC1, MAPK8IP2, and SULT4A1 having the highest overall expression. Analysis of the temporal profiles of the highest expressed genes revealed a trend towards peak expression during the early post-natal period, followed by a drop in expression later in development. Spatial analysis revealed significant region specific differences in the expression of SHANK3, MAPK8IP2, and SULT4A1. Region specific expression over time revealed a consistently unique gene expression profile within the cerebellum, providing evidence for a distinct developmental program within this region. Exon-specific expression of SHANK3 showed higher expression within exons contributing to known brain specific functional isoforms. Overall, we provide an updated roadmap of the PMS region, implicating several genes and time periods as important during neurodevelopment, with the hope that this information can help us better understand the phenotypic heterogeneity of PMS.

Barakat syndrome revisited.

Barakat syndrome also known as HDR syndrome (Online Mendelian Inheritance in Man [OMIM] 146255), was first described by Barakat et al. in . It is a rare genetic disorder characterized by the triad of hypoparathyroidism "H," sensorineural deafness "D," and renal disease "R." The defect is caused by deletions in chromosome 10p14 or mutations in the GATA3 gene. Although the syndrome has been phenotypically defined by this triad the literature identifies cases with different components with, or without GATA3 defects making the definition of the syndrome confusing. We analyzed 180 cases and attempted to define the phenotype of the syndrome and suggest guidelines for diagnosis. We suggest that the diagnosis could be confirmed in patients who have all three components, and in those who have two components with a positive family history. GATA3 testing is optional to establish the diagnosis in these patients. The syndrome should be considered in patients with isolated "D" where other causes of "D" have been excluded and those with isolated "R," especially if there is family history of any of these components. In these instances, confirmatory GATA3 testing is indicated to confirm the diagnosis. In patients with nonsurgical "H," where "D" and "R" have been conclusively ruled out GATA3 studies are not needed as none of these patients were shown to be GATA3 haploinsufficient. Only 64.4% of patients in our review had "HDR." Some findings might have not been recognized or may could have appeared later in life, but it is evident that this syndrome is genotypically heterogeneous.

Idiopathic Autism: Cellular and Molecular Phenotypes in Pluripotent Stem Cell-Derived Neurons.

Autism spectrum disorder is a complex neurodevelopmental disorder whose pathophysiology remains elusive as a consequence of the unavailability for study of patient brain neurons; this deficit may potentially be circumvented by neural differentiation of induced pluripotent stem cells. Rare syndromes with single gene mutations and autistic symptoms have significantly advanced the molecular and cellular understanding of autism spectrum disorders; however, in aggregate, they only represent a fraction of all cases of autism. In an effort to define the cellular and molecular phenotypes in human neurons of non-syndromic autism, we generated induced pluripotent stem cells (iPSCs) from three male autism spectrum disorder patients who had no identifiable clinical syndromes, and their unaffected male siblings and subsequently differentiated these patient-specific stem cells into electrophysiologically active neurons. iPSC-derived neurons from these autistic patients displayed decreases in the frequency and kinetics of spontaneous excitatory postsynaptic currents relative to controls, as well as significant decreases in Na+ and inactivating K+ voltage-gated currents. Moreover, whole-genome microarray analysis of gene expression identified 161 unique genes that were significantly differentially expressed in autistic patient iPSC-derived neurons (>twofold, FDR < 0.05). These genes were significantly enriched for processes related to synaptic transmission, such as neuroactive ligand-receptor signaling and extracellular matrix interactions, and were enriched for genes previously associated with autism spectrum disorder. Our data demonstrate aberrant voltage-gated currents and underlying molecular changes related to synaptic function in iPSC-derived neurons from individuals with idiopathic autism as compared to unaffected siblings controls.

The Evolving Diagnostic and Genetic Landscapes of Autism Spectrum Disorder.

The autism spectrum disorders (ASD) are a heterogeneous set of neurodevelopmental syndromes defined by impairments in verbal and non-verbal communication, restricted social interaction, and the presence of stereotyped patterns of behavior. The prevalence of ASD is rising, and the diagnostic criteria and clinical perspectives on the disorder continue to evolve in parallel. Although the majority of individuals with ASD will not have an identifiable genetic cause, almost 25% of cases have identifiable causative DNA variants. The rapidly improving ability to identify genetic mutations because of advances in next generation sequencing, coupled with previous epidemiological studies demonstrating high heritability of ASD, have led to many recent attempts to identify causative genetic mutations underlying the ASD phenotype. However, although hundreds of mutations have been identified to date, they are either rare variants affecting only a handful of ASD patients, or are common variants in the general population conferring only a small risk for ASD. Furthermore, the genes implicated thus far are heterogeneous in their structure and function, hampering attempts to understand shared molecular mechanisms among all ASD patients; an understanding that is crucial for the development of targeted diagnostics and therapies. However, new work is beginning to suggest that the heterogeneous set of genes implicated in ASD may ultimately converge on a few common pathways. In this review, we discuss the parallel evolution of our diagnostic and genetic understanding of autism spectrum disorders, and highlight recent attempts to infer common biology underlying this complicated syndrome.

A Non-inflammatory Role for Microglia in Autism Spectrum Disorders.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction, difficulties with language, and repetitive/restricted behaviors. The etiology of ASD is still largely unclear, but immune dysfunction and abnormalities in synaptogenesis have repeatedly been implicated as contributing to the disease phenotype. However, an understanding of how and if these two processes are related has not firmly been established. As non-inflammatory roles of microglia become increasingly recognized as critical to normal neurodevelopment, it is important to consider how dysfunction in these processes might explain the seemingly disparate findings of immune dysfunction and aberrant synaptogenesis seen in ASD. In this review, we highlight research demonstrating the importance of microglia to the development of normal neural networks, review recent studies demonstrating abnormal microglia in autism, and discuss how the relationship between these processes may contribute to the development of autism and other neurodevelopmental disorders at the cellular level.

The autistic brain in the context of normal neurodevelopment.

The etiology of autism spectrum disorders (ASDs) is complex and largely unclear. Among various lines of inquiry, many have suggested convergence onto disruptions in both neural circuitry and immune regulation/glial cell function pathways. However, the interpretation of the relationship between these two putative mechanisms has largely focused on the role of exogenous factors and insults, such as maternal infection, in activating immune pathways that in turn result in neural network abnormalities. Yet, given recent insights into our understanding of human neurodevelopment, and in particular the critical role of glia and the immune system in normal brain development, it is important to consider these putative pathological processes in their appropriate normal neurodevelopmental context. In this review, we explore the hypothesis that the autistic brain cellular phenotype likely represents intrinsic abnormalities of glial/immune processes constitutively operant in normal brain development that result in the observed neural network dysfunction. We review recent studies demonstrating the intercalated role of neural circuit development, the immune system, and glial cells in the normal developing brain, and integrate them with studies demonstrating pathological alterations in these processes in autism. By discussing known abnormalities in the autistic brain in the context of normal brain development, we explore the hypothesis that the glial/immune component of ASD may instead be related to intrinsic exaggerated/abnormal constitutive neurodevelopmental processes such as network pruning. Moreover, this hypothesis may be relevant to other neurodevelopmental disorders that share genetic, pathologic, and clinical features with autism.

Shared Pathways Among Autism Candidate Genes Determined by Co-expression Network Analysis of the Developing Human Brain Transcriptome.

Autism spectrum disorder (ASD) is a neurodevelopmental syndrome known to have a significant but complex genetic etiology. Hundreds of diverse genes have been implicated in ASD; yet understanding how many genes, each with disparate function, can all be linked to a single clinical phenotype remains unclear. We hypothesized that understanding functional relationships between autism candidate genes during normal human brain development may provide convergent mechanistic insight into the genetic heterogeneity of ASD. We analyzed the co-expression relationships of 455 genes previously implicated in autism using the BrainSpan human transcriptome database, across 16 anatomical brain regions spanning prenatal life through adulthood. We discovered modules of ASD candidate genes with biologically relevant temporal co-expression dynamics, which were enriched for functional ontologies related to synaptogenesis, apoptosis, and GABA-ergic neurons. Furthermore, we also constructed co-expression networks from the entire transcriptome and found that ASD candidate genes were enriched in modules related to mitochondrial function, protein translation, and ubiquitination. Hub genes central to these ASD-enriched modules were further identified, and their functions supported these ontological findings. Overall, our multi-dimensional co-expression analysis of ASD candidate genes in the normal developing human brain suggests the heterogeneous set of ASD candidates share transcriptional networks related to synapse formation and elimination, protein turnover, and mitochondrial function.

MicroRNA-29b/Tet1 regulatory axis epigenetically modulates mesendoderm differentiation in mouse embryonic stem cells.

Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.

GermlncRNA: a unique catalogue of long non-coding RNAs and associated regulations in male germ cell development.

Spermatogenic failure is a major cause of male infertility, which affects millions of couples worldwide. Recent discovery of long non-coding RNAs (lncRNAs) as critical regulators in normal and disease development provides new clues for delineating the molecular regulation in male germ cell development. However, few functional lncRNAs have been characterized to date. A major limitation in studying lncRNA in male germ cell development is the absence of germ cell-specific lncRNA annotation. Current lncRNA annotations are assembled by transcriptome data from heterogeneous tissue sources; specific germ cell transcript information of various developmental stages is therefore under-represented, which may lead to biased prediction or fail to identity important germ cell-specific lncRNAs. GermlncRNA provides the first comprehensive web-based and open-access lncRNA catalogue for three key male germ cell stages, including type A spermatogonia, pachytene spermatocytes and round spermatids. This information has been developed by integrating male germ transcriptome resources derived from RNA-Seq, tiling microarray and GermSAGE. Characterizations on lncRNA-associated regulatory features, potential coding gene and microRNA targets are also provided. Search results from GermlncRNA can be exported to Galaxy for downstream analysis or downloaded locally. Taken together, GermlncRNA offers a new avenue to better understand the role of lncRNAs and associated targets during spermatogenesis. Database URL: http://germlncrna.cbiit.cuhk.edu.hk/

Modulation of microRNA expression in human lung cancer cells by the G9a histone methyltransferase inhibitor BIX01294.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of their target genes at the post-transcriptional level. In cancer cells, miRNAs, depending on the biological functions of their target genes, may have a tumor-promoting or -suppressing effect. Treatment of cancer cells with inhibitors of DNA methylation and/or histone deacetylation modulates the expression level of miRNAs, which provides evidence for epigenetic regulation of miRNA expression. The consequences of inhibition of histone methyltransferase on miRNA expression, however, have not been thoroughly investigated. The present study examined the expression pattern of miRNAs in the non-small cell lung cancer cell line, H1299 with or without treatment of BIX01294, a potent chemical inhibitor of G9a methyltransferase that catalyzes the mono-and di-methylation of the lysine 9 residue of histone H3. By coupling microarray analysis with quantitative real-time polymerase chain reaction analysis, two miRNAs were identified that showed consistent downregulation following BIX01294 treatment. The results indicate that histone H3 methylation regulates miRNA expression in lung cancer cells, which may provide additional insight for future chemical treatment of lung cancer.

Long noncoding RNAs in spermatogenesis: insights from recent high-throughput transcriptome studies.

Spermatogenesis is a complex developmental process in which undifferentiated spermatogonia are differentiated into spermatocytes and spermatids through two rounds of meiotic division and finally giving rise to mature spermatozoa (sperm). These processes involve many testis- or male germ cell-specific gene products that undergo strict developmental regulations. As a result, identifying critical, regulatory genes controlling spermatogenesis provide the clues not only to the regulatory mechanism of spermatogenesis at the molecular level, but also to the identification of candidate genes for infertility or contraceptives development. Despite the biological importance in male germ cell development, the underlying mechanisms of stage-specific gene regulation and cellular transition during spermatogenesis remain largely elusive. Previous genomic studies on transcriptome profiling were largely limited to protein-coding genes. Importantly, protein-coding genes only account for a small percentage of transcriptome; the majority are noncoding transcripts that do not translate into proteins. Although small noncoding RNAs (ncRNAs) such as microRNAs, siRNAs, and Piwi-interacting RNAs are extensively investigated in male germ cell development, the role of long ncRNAs (lncRNAs), commonly defined as ncRNAs longer than 200 bp, is relatively unexplored. Herein, we summarize recent transcriptome studies on spermatogenesis and show examples that a subset of noncoding transcript population, known as lncRNAs, constitutes a novel regulatory target in spermatogenesis.

Telomerase protects werner syndrome lineage-specific stem cells from premature aging.

Werner syndrome (WS) patients exhibit premature aging predominantly in mesenchyme-derived tissues, but not in neural lineages, a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here, we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging, we differentiated iPSCs to mesenchymal stem cells (MSCs) and neural stem/progenitor cells (NPCs). We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs, but not in NPCs. We postulate this "aging" discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC, whereas inhibition of telomerase sensitized NPCs to DNA damage. Our findings unveil a role for telomerase in the protection of accelerated aging in a specific lineage of stem cells.

Altered glial marker expression in autistic post-mortem prefrontal cortex and cerebellum.

BACKGROUND: The cellular mechanism(s) underlying autism spectrum disorders (ASDs) are not completely understood, but ASDs are thought to ultimately result from disrupted synaptogenesis. However, studies have also shown that glial cell numbers and function are abnormal in post-mortem brain tissue from autistic patients. Direct assessment of glial cells in post-mortem human brain tissue is technically challenging, limiting glial research in human ASD studies. Therefore, we attempted to determine if glial cell-type specific markers may be altered in autistic brain tissue in a manner that is consistent with known cellular findings, such that they could serve as a proxy for glial cell numbers and/or activation patterns. METHODS: We assessed the relative expression of five glial-specific markers and two neuron-specific markers via qRT-PCR. We studied tissue samples from the prefrontal cortex (PFC) and cerebellum of nine post-mortem autistic brain samples and nine neurologically-normal controls. Relative fold-change in gene expression was determined using the ΔΔCt method normalized to housekeeping gene ß-actin, with a two-tailed Student's t-test P <0.05 between groups considered as significant. RESULTS: Both astrocyte- and microglial-specific markers were significantly more highly expressed in autistic PFC as compared to matched controls, while in the cerebellum only astrocyte markers were elevated in autistic samples. In contrast, neuron-specific markers showed significantly lower expression in both the PFC and cerebellum of autistic patients as compared to controls. CONCLUSIONS: These results are in line with previous findings showing increased glial cell numbers and up-regulation of glial cell gene expression in autistic post-mortem brain tissue, particularly in the PFC, as well as decreased number of neurons in both the PFC and cerebellum of autistic patients. The concordance of these results with cell-level studies in post-mortem autistic brain tissue suggests that expression of glial cell-type specific markers may serve as a useful alternative to traditional cellular characterization methods, especially when appropriately-preserved post-mortem tissue is lacking. Additionally, these results demonstrate abnormal glial-specific gene expression in autistic brains, supporting previous studies that have observed altered glial cell numbers or activation patterns in ASDs. Future work should directly assess the correlation between cell-type specific marker levels and cell number and activation patterns.

Detection of epigenetic alterations using tiling arrays.

The epigenetic status of cancer cells is a consequence of the neoplastic transformation of their normal counterpart. Epigenetic changes directly influence gene expression and chromatin organization, which consequently leads to escape from the tumor-suppression mechanisms. Global mapping for specific epigenetic modifications (e.g., DNA methylation) of the entire genome is required to reveal epigenetic hotspots associated with a cancer type/stage. DNA tiling arrays may be applied for genome-wide analysis of different epigenetic marks. Tiling arrays are high-density DNA microarrays that can be custom-made to survey regions of interest (e.g., gene promoters) or permit whole-genome analysis. To identify the genomic alterations associated with testicular cancers we used tiling arrays to profile their methylome. We successfully identified numerous epigenetically modified loci that arose as a consequence of tumor progression.

Mapping genomic features of tiling microarray data by TileMapper.

The recent revolution of genomics techniques has allowed the detection of various sequence features and biological variations on whole-genome scale. However, these high-resolution data present significant challenges for experimental biologists to understand and analyze. The conventional way is to use genome browsers to locate and visualize regions of interest. But it lacks user-friendly data mining functionality. Here we present a protocol that allows rapid annotation of genomic coordinate data by using TileMapper. Interesting biological annotations from large-scale genomic data, such as transcriptome analysis, chromatin immunoprecipitation on chip, or methyl-DNA immunoprecipitation (MeDIP) studies generated from the tiling microarrays and other platforms, could be analyzed without requiring computational skills. The outputs are saved in tabulated format, which permit flexible and simple processing in spreadsheet software, or to be exported to other pipelines for subsequent analysis.

Sex-biased gene expression in the developing brain: implications for autism spectrum disorders.

Autism spectrum disorders affect significantly more males than females. Understanding sex differences in normal human brain development may provide insight into the mechanism(s) underlying this disparity; however, studies of sex differences in brain development at the genomic level are lacking. Here, we report a re-analysis of sex-specific gene expression from a recent large transcriptomic study of normal human brain development, to determine whether sex-biased genes relate to specific mechanistic processes. We discovered that male-biased genes are enriched for the processes of extracellular matrix formation/glycoproteins, immune response, chromatin, and cell cytoskeleton. We highlight that these pathways have been repeatedly implicated in autism and demonstrate that autism candidate genes are also enriched for these pathways. We propose that the overlap of these male-specific brain transcriptional modules with the same pathways in autism spectrum disorders may partially explain the increased incidence of autism in males.