Integrated systems analysis reveals a molecular network underlying autism spectrum disorders.

Autism is a complex disease whose etiology remains elusive. We integrated previously and newly generated data and developed a systems framework involving the interactome, gene expression and genome sequencing to identify a protein interaction module with members strongly enriched for autism candidate genes. Sequencing of 25 patients confirmed the involvement of this module in autism, which was subsequently validated using an independent cohort of over 500 patients. Expression of this module was dichotomized with a ubiquitously expressed subcomponent and another subcomponent preferentially expressed in the corpus callosum, which was significantly affected by our identified mutations in the network center. RNA-sequencing of the corpus callosum from patients with autism exhibited extensive gene mis-expression in this module, and our immunochemical analysis showed that the human corpus callosum is predominantly populated by oligodendrocyte cells. Analysis of functional genomic data further revealed a significant involvement of this module in the development of oligodendrocyte cells in mouse brain. Our analysis delineates a natural network involved in autism, helps uncover novel candidate genes for this disease and improves our understanding of its molecular pathology

Traumatic Brain Injury Induces Genome-Wide Transcriptomic, Methylomic, and Network Perturbations in Brain and Blood Predicting Neurological Disorders.

The complexity of the traumatic brain injury (TBI) pathology, particularly concussive injury, is a serious obstacle for diagnosis, treatment, and long-term prognosis. Here we utilize modern systems biology in a rodent model of concussive injury to gain a thorough view of the impact of TBI on fundamental aspects of gene regulation, which have the potential to drive or alter the course of the TBI pathology. TBI perturbed epigenomic programming, transcriptional activities (expression level and alternative splicing), and the organization of genes in networks centered around genes such as Anax2, Ogn, and Fmod. Transcriptomic signatures in the hippocampus are involved in neuronal signaling, metabolism, inflammation, and blood function, and they overlap with those in leukocytes from peripheral blood. The homology between genomic signatures from blood and brain elicited by TBI provides proof of concept information for development of biomarkers of TBI based on composite genomic patterns. By intersecting with human genome-wide association studies, many TBI signature genes and network regulators identified in our rodent model were causally associated with brain disorders with relevant link to TBI. The overall results show that concussive brain injury reprograms genes which could lead to predisposition to neurological and psychiatric disorders, and that genomic information from peripheral leukocytes has the potential to predict TBI pathogenesis in the brain

Discovery of noncanonical translation initiation sites through mass spectrometric analysis of protein N termini

Translation initiation generally occurs at AUG codons in eukaryotes, although it has been shown that non-AUG or non-canonical translation initiation can also occur. However, the evidence for noncanonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling (Ribo-seq) studies. Here, using a strategy specifically designed to enrich N termini of proteins, we demonstrate that many human proteins are translated at noncanonical TISs. The large majority of TISs that mapped to 5' untranslated regions were noncanonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). It has been controversial whether the amino acid corresponding to the start codon is incorporated at the TIS or methionine is still incorporated. We found that methionine was incorporated at almost all noncanonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by the available ribosome profiling data. Sequence conservation across species and a higher abundance of noncanonical TISs than canonical ones in some cases suggests that the noncanonical TISs can have biological functions. Overall, this study provides evidence of protein translation initiation at noncanonical TISs and argues that further studies are required for elucidation of functional implications of such noncanonical translation initiation

Identification of putative second genetic hits in schizophrenia carriers of high-risk copy number variants and resequencing in additional samples

Copy number variants (CNVs) conferring risk of schizophrenia present incomplete penetrance, suggesting the existence of second genetic hits. Identification of second hits may help to find genes with rare variants of susceptibility to schizophrenia. The aim of this work was to search for second hits of moderate/high risk in schizophrenia carriers of risk CNVs and resequencing of the relevant genes in additional samples. To this end, ten patients with risk CNVs at cytobands 15q11.2, 15q11.2-13.1, 16p11.2, or 16p13.11, were subjected to whole-exome sequencing. Rare single nucleotide variants, defined as those absent from main public databases, were classified according to bioinformatic prediction of pathogenicity by CADD scores. The average number of rare predicted pathogenic variants per sample was 13.6 (SD 2.01). Two genes, BFAR and SYNJ1, presented rare predicted pathogenic variants in more than one sample. Follow-up resequencing of these genes in 432 additional cases and 432 controls identified a significant excess of rare predicted pathogenic variants in case samples at SYNJ1. Taking into account its function in clathrin-mediated synaptic vesicle endocytosis at presynaptic terminals, our results suggest an impairment of this process in schizophrenia

Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified

Peripheral T-cell lymphomas not otherwise specified (PTCLs/NOS) are rare and aggressive tumours whose molecular pathogenesis and diagnosis are still challenging. The microRNA (miRNA) profile of 23 PTCLs/NOS was generated and compared with that of normal T-lymphocytes (CD4+, CD8+, naive, activated). The differentially expressed miRNA signature was compared with the gene expression profile (GEP) of the same neoplasms. The obtained gene patterns were tested in an independent cohort of PTCLs/NOS. The miRNA profile of PTCLs/NOS then was compared with that of 10 angioimmunoblastic T-cell lymphomas (AITLs), 6 anaplastic large-cell lymphomas (ALCLs)/ALK+ and 6 ALCLs/ALK - . Differentially expressed miRNAs were validated in an independent set of 20 PTCLs/NOS, 20 AITLs, 19 ALCLs/ALK - and 15 ALCLs/ALK+. Two hundred and thirty-six miRNAs were found to differentiate PTCLs/NOS from activated T-lymphocytes. To assess which miRNAs impacted on GEP, a multistep analysis was performed, which identified all miRNAs inversely correlated to different potential target genes. One of the most discriminant miRNAs was selected and its expression was found to affect the global GEP of the tumours. Moreover, two sets of miRNAs were identified distinguishing PTCL/NOS from AITL and ALCL/ALK - , respectively. The diagnostic accuracy of this tool was very high (83.54%) and its prognostic value validated

Proximity Analysis of Native Proteomes Reveals Phenotypic Modifiers in a Mouse Model of Autism and Related Neurodevelopmental Conditions

One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving the identification of high-specificity spatial proteomes. The resulting native proximity proteomes are enriched for human genes dysregulated in the brain of autistic individuals, and reveal proximity interactions between proteins from high-confidence risk genes with those of lower-confidence that may provide new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology

BAZ1B in Nucleus Accumbens Regulates Reward-Related Behaviors in Response to Distinct Emotional Stimuli

ATP-dependent chromatin remodeling proteins are being implicated increasingly in the regulation of complex behaviors, including models of several psychiatric disorders. Here, we demonstrate that Baz1b, an accessory subunit of the ISWI family of chromatin remodeling complexes, is upregulated in the nucleus accumbens (NAc), a key brain reward region, in both chronic cocaine-treated mice and mice that are resilient to chronic social defeat stress. In contrast, no regulation is seen in mice that are susceptible to this chronic stress. Viral-mediated overexpression of Baz1b, along with its associated subunit Smarca5, in mouse NAc is sufficient to potentiate both rewarding responses to cocaine, including cocaine self-administration, and resilience to chronic social defeat stress. However, despite these similar, proreward behavioral effects, genome-wide mapping of BAZ1B in NAc revealed mostly distinct subsets of genes regulated by these chromatin remodeling proteins after chronic exposure to either cocaine or social stress. Together, these findings suggest important roles for BAZ1B and its associated chromatin remodeling complexes in NAc in the regulation of reward behaviors to distinct emotional stimuli and highlight the stimulus-specific nature of the actions of these regulatory proteins

Identification of RNA bound to the TDP-43 ribonucleoprotein complex in the adult mouse brain

Cytoplasmic inclusions containing TDP-43 are a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. TDP-43 is an RNA binding protein involved in gene regulation through control of RNA transcription, splicing and transport. However, the function of TDP-43 in the nervous system is largely unknown and its role in the pathogenesis of ALS is unclear. The aim of this study was to identify genes in the central nervous system that are regulated by TDP-43. RNA-immunoprecipitation with anti-TDP-43 antibody, followed by microarray analysis (RIP-chip), was used to isolate and identify RNA bound to TDP-43 protein from mouse brain. This analysis produced a list of 1839 potential TDP-43 gene targets, many of which overlap with previous studies and whose functions include RNA processing and synaptic function. Immunohistochemistry demonstrated that the TDP-43 protein could be found at the presynaptic membrane of axon terminals in the neuromuscular junction in mice. In conclusion, the finding that TDP-43 binds to RNA that codes for genes related to synaptic function, together with the localization of TDP-43 protein at axon terminals, suggests a role for TDP-43 in the transport of synaptic mRNAs into distal processes

Cellular reprogramming

DNA-methylatie is een epigenetische modificatie. Dit type van modificaties verandert de genetische informatie zelf niet, maar wijzigt de laag erboven. Er worden chemische modificaties toegevoegd of gewijzigd op het DNA of de histonen (waarrond het DNA gewonden is). DNA-methylatie is die modificatie waarbij een methylgroep wordt aangebracht op het cytosine (C) residue en een CG dincucleotide. Als deze wijziging plaats vindt in een zogenaamd CpG eiland (waar deze CG dinucleotiden dens bij elkaar voorkomen) van de promoter van een gen, wordt dit gen niet meer afgeschreven en zal zijn functie verloren gaan. In dit proefschrift worden verschillende aspecten van DNA-methylatie en het belang ervan in de oncologie belicht. In het eerste onderzoeksdeel hebben we verschillende methodes en algoritmes ontwikkeld om methylatiemerkers te identificeren: genen die specifiek in kanker worden gemethyleerd maar niet in normale weefsels of genen die enkel worden gemethyleerd in bepaalde patiëntsubgroepen (bv. die respons vertonen voor een bepaalde chemotherapie). We hebben een databank en methodes gemaakt om kennis uit de literatuur in het snelgroeiende DNA-methylatie veld te halen. Deze databank (PubMeth) laat toe om het verband na te gaan tussen genen en hun methylatiepatroon in verschillende kanker (sub)types. Daarnaast werden verschillende sorterings- en selectiemethodieken ontwikkeld om zo een prioriteit toe te kennen aan de kandidaat DNA-methylatiemerkers. Al deze methodes werden ontwikkeld om data van genoom-wijde experimenten te kunnen verwerken. Nieuwe methylatiemerkers (gemethyleerd in kanker maar niet in normaal) kunnen worden gebruik bij vroegtijdige detectie van kanker. Er werden verschillende computationele oplossingen voorgesteld: - Relaxation ranking: sorteermethodiek gebaseerd op expressie micro-arrays van primair baarmoederhalskanker patiëntmateriaal en re-expressie experimenten op baarmoederhalskanker cellijnen. Deze methode is gebaseerd op lage expressie in kanker en re-expressie na behandeling met het de-methylerend agens DAC en de histondeacetylase inhibitor TSA. De methode gebruikt geen enkele (arbitrair te kiezen) grenswaarde - De deep approach: er werden verschillende DNA-motieven in de promoterregio van genen geïdentificeerd die discrimineren tussen kankerspecifiek gemethyleerde genen en genen die ook gemethyleerd worden in normale weefsels. - De broad approach maakt gebruik van een genoom-wijde alignering van promoterregio’s. Hieruit blijkt dat gekende methylatiemerkers meer dens geclusterd zijn dan verwacht. - Zowel ‘deep’ als ‘broad’ werden gecombineerd met re-expressie studies in cellijnen van verschillende kankertypes. De combinatie van deze experimentele filter en de computationele aanpak verhoogt de succesratio bij het vinden van kankerspecifiek gemethyleerde genen aanzienlijk. - Er werden ook merkers geïdentificeerd die mogelijks de respons op chemotherapie (platinum) kunnen voorspellen in eierstokkanker. Dit opent de weg naar gepersonaliseerde geneeskunde. De methodiek gebruikt een score-schema gebaseerd op zowel primaire kanker stalen (van zowel platinumgevoelige als resistente patiënten) als re-expressie experimenten op kanker cellijnen. De nieuwe identificatiestrategieën werden gevalideerd op primaire kankerstalen en presteren goed: ten opzichte van beschreven priorizatietechnieken is de succesratio verhoogd. Verscheidene nieuwe methylatie biomerkers in verschillende kankertypes (baarmoederhalskanker, eierstok-kanker, hoofd- en nekkanker, neuroblastoom,…) werden succesvol gevalideerd op primaire patiëntenstalen. Vervolgonderzoek op grotere patiëntengroepen zal het mogelijk diagnostische potentieel aantonen. De validatie van sommige studies is momenteel nog lopende. Dit toont de noodzaak aan van snelle en betrouwbare analysetechnieken die geschikt zijn voor validatiedoeleinden. Ten tweede werd er een uniek viraal infectiesysteem gecombineerd met een genoom-wijde methylatiegevoelige detectietechniek om epigenetische ‘herprogrammering’ van humane gastheercellen na infectie met hoge-risico HPV (Humaan Papilloma Virus) te onderzoeken. In dit experi-ment geven we sterke indicaties dat het virus in staat is de gastheercel epigenetisch te wijzigen. Deze hr-HPV virustypes zijn duidelijk geassocieerd met de ontwikkeling van baarmoederhalskanker gezien meer dan 99 % van de patiënten besmet is. Momenteel is het uitstrijkje een wijdverspreide screening methodiek die wordt gebruikt bij vroegdetectie. Onlangs werden er ook vaccins op de markt gebracht die infectie met de meest voorkomende virustypes (ongeveer 80 % van de infecties) moet voorkomen. De vaccinatie zou moeten gebeuren bij meisjes voor het eerste seksueel contact. De ontdekking van biomerkers met hoge specificiteit en sensitiviteit bij baarmoederhalskanker blijft noodzakelijk voor de niet gevaccineerde groep en gezien de vaccins niet tegen alle virustypes bescherming bieden. DNA-methylatiemerkers zijn uitstekende kandida-ten voor vroegdetectie in een screeningsprogramma: ze kunnen op grote schaal en geautomatiseerd gebeuren. Gezien er aangenomen wordt dat verschillende kankertypes verwant zijn met virusinfecties, verhoogt dit onderzoek de kennis bij dit proces en opent dit de weg naar zeer vroege detectie. Momenteel zijn enkel delen van het ‘kanker epigenoom’ bekend. De komst van methodes die grote hoeveelheden data genereren (zoals volgende-generatie sequenering) zou de bestaande kennis in grote mate kunnen laten toenemen. Dit vereist echter grote collecties primaire kankerstalen (liefst nog in verschillende stadia). Een centraal beheerde, goed geannoteerde bibliotheek van patiëntenmateriaal die verschillende kankertypes bevat, zou het onderzoek in een stroomversnelling plaatsen. Bijkomende uitdaging is dat deze technieken het hoogste niveau van precisie hebben (op baseniveau van een enkele DNA-molecule) en tegelijkertijd een hoog aantal stalen verwerken. Dit betekent dat bij het proefopzet sequentiestukken moeten gekozen worden die aangerijkt zijn aan DNA-methylatie of histonmodificaties. De data-analyse strategie moet snel genoeg zijn maar toch nieuwe kennis extraheren uit de terabytes ruwe data die gegenereerd worden. Ons labo werkt zowel op de ontwikkeling van een goed proefopzet als de verwerking van de gegenereerde data.DNA-methylation is an epigenetic modification. These modifications of the DNA do not change the genetic sequence itself, but affect the level ‘above’ it: chemical modifications of the DNA or the histones (where the DNA is wound around) are added or altered. DNA-methylation is the modification where a methyl group is added on cytosine (C) residues in CG dinucleotides. If this takes place in the so-called CpG islands (where CG dinucleotides occur very densely) within the promoter of a gene, this gene is silenced, and will not be transcribed; its function is lost. In this thesis, different aspects of DNA-methylation and its importance in the field of oncology has been dealt with. First, we created different methodologies and algorithms to identify methylation markers: genes that are specifically methylated in cancer but not in normal tissue or genes, methylated in subgroups of patients (e.g. responders of a certain chemotherapy). We built a database and methodologies to discover existing publications in the fast-growing DNA-methylation field. This database (PubMeth) allows to screen which genes are reported methylated in the selected cancer (sub)types and vice versa. Different ranking, sorting and selection techniques were developed to prioritize promising methylation marker candidates. These methodologies were all developed to deal with data from genome-wide approaches. Novel methylation markers (methylated in cancer while not in normal) could be used as early detection markers. Different computational approaches are described here: - Relaxation ranking: ranking strategy based on expression micro-arrays of primary cervical cancer samples and re-expression experiments on cervical cancer cell lines. This methodology is based on low expression in cancer samples and re-expression after treatment with the demethylation agent DAC and the histone de-acetylase inhibitor TSA. The methodology involves no thresholds. - The deep approach: different DNA-patterns in the promoter region of genes were identified that seem to discriminate between cancer-specifically methylated genes and genes that also are me-thylated in normal tissues. - The broad approach makes use of a genome-wide alignment of promoter regions. Apparently, genes described as methylation markers, are more densely clustered together. - Both the deep approach and the broad approach were combined with data from re-expression studies in cancer cell lines. The combination of this experimental filter and the computational approaches drastically improves the success rate in finding cancer-specific methylation markers in various cancer types. - There were also markers discovered, that may be able to predict chemotherapy (platinum) response in ovarian cancer, clearing the roads towards personalized medicine. The methodology uses a score-scheme, based on both primary cancer samples (from patients sensitive and resistant to platinum therapies) as re-expression experiments of ovarium cancer cell lines (both cisplatin resistant and sensitive). The novel identification strategies were validated on primary cancer samples and perform well: the success rate was improved in comparison with other prioritization attempts. Several novel methylation markers were discovered in different cancer types (cervical cancer, ovarian cancer, head-and-neck cancer, neuroblastoma, …) and were validated on primary samples. Follow-up research on larger clinical cohorts will demonstrate their potential diagnostic power. For some studies, the validation effort is still in progress; this illustrates the need for fast and accurate analysis techniques, suitable for validation purposes. Secondly, a unique viral infection system, combined with a ge-nome-wide methylation detection methodology was used to investigate the epigenetic ‘reprogramming’ of human host cells after infection with high-risk HPV (Human Papilloma Virus). In this experiment, we prove that a virus is able to epigenetically program their host cell. The high-risk HPV types are clearly related with the development of cervical cancer as more than 99 % of the patients is infected with such a virus type. For the moment, the cytological Pap-smear screening technique is widespread and used for early detection. Recently, vaccines were developed in order to prevent infection with the most prominent virus types (about 80 % of infections covered). The vaccination strategy must be applied in young girls (before sexual contact). The discovery of cervical cancer biomarkers with high sensitivity and specificity remains necessary for the non-vaccinated group and screening programs remain needed as the vaccines do not cover all hr-HPV virus types. DNA-methylation markers that seem to be related with infection with the virus, are ideal candidates for very early detection in a screening program: large scale analysis can be automated. As it is believed that multiple cancers may occur after viral infec-tion, or at least that these infections plays a key role in the development, this broadens the knowledge in this process and opens ways to very early detection. Currently, only fractions of the cancer epigenome are known. The introduction of methods that generate significantly large amounts of data (such as next generation sequencing) might be able to greatly expand the current knowledge. However, large collections of primary cancer samples (preferentially of different stages) will be needed. A centrally managed, well described and annotated library of patient material containing different cancer types would be extremely beneficial. In addition, the highest level of precision (base pair level of single DNA-molecules) will be reached for a high number of samples at the same time with sequencing techniques. The initial set-up of such an experiment must be chosen so that the sequenced parts of the genome are enriched in DNA-methylation or histone modifications. The data analysis pipeline for the interpretation of the generated data must perform fast and extract new knowledge out of the terabytes of raw data generated. Both the experimental set-up and the downstream data analysis, our laboratory is working on