The Psychiatric Cell Map Initiative: A Convergent Systems Biological Approach to Illuminating Key Molecular Pathways in Neuropsychiatric Disorders.

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism.

Autism spectrum disorder (ASD) is a complex developmental syndrome of unknown etiology. Recent studies employing exome- and genome-wide sequencing have identified nine high-confidence ASD (hcASD) genes. Working from the hypothesis that ASD-associated mutations in these biologically pleiotropic genes will disrupt intersecting developmental processes to contribute to a common phenotype, we have attempted to identify time periods, brain regions, and cell types in which these genes converge. We have constructed coexpression networks based on the hcASD "seed" genes, leveraging a rich expression data set encompassing multiple human brain regions across human development and into adulthood. By assessing enrichment of an independent set of probable ASD (pASD) genes, derived from the same sequencing studies, we demonstrate a key point of convergence in midfetal layer 5/6 cortical projection neurons. This approach informs when, where, and in what cell types mutations in these specific genes may be productively studied to clarify ASD pathophysiology.

Genomics, convergent neuroscience and progress in understanding autism spectrum disorder.

More than a hundred genes have been identified that, when disrupted, impart large risk for autism spectrum disorder (ASD). Current knowledge about the encoded proteins - although incomplete - points to a very wide range of developmentally dynamic and diverse biological processes. Moreover, the core symptoms of ASD involve distinctly human characteristics, presenting challenges to interpreting evolutionarily distant model systems. Indeed, despite a decade of striking progress in gene discovery, an actionable understanding of pathobiology remains elusive. Increasingly, convergent neuroscience approaches have been recognized as an important complement to traditional uses of genetics to illuminate the biology of human disorders. These methods seek to identify intersection among molecular-level, cellular-level and circuit-level functions across multiple risk genes and have highlighted developing excitatory neurons in the human mid-gestational prefrontal cortex as an important pathobiological nexus in ASD. In addition, neurogenesis, chromatin modification and synaptic function have emerged as key potential mediators of genetic vulnerability. The continued expansion of foundational 'omics' data sets, the application of higher-throughput model systems and incorporating developmental trajectories and sex differences into future analyses will refine and extend these results. Ultimately, a systems-level understanding of ASD genetic risk holds promise for clarifying pathobiology and advancing therapeutics.

Pleiotropy of autism-associated chromatin regulators.

Gene ontology analyses of high-confidence autism spectrum disorder (ASD) risk genes highlight chromatin regulation and synaptic function as major contributors to pathobiology. Our recent functional work in vivo has additionally implicated tubulin biology and cellular proliferation. As many chromatin regulators, including the ASD risk genes ADNP and CHD3, are known to directly regulate both tubulins and histones, we studied the five chromatin regulators most strongly associated with ASD (ADNP, CHD8, CHD2, POGZ and KMT5B) specifically with respect to tubulin biology. We observe that all five localize to microtubules of the mitotic spindle in vitro in human cells and in vivo in Xenopus. Investigation of CHD2 provides evidence that mutations present in individuals with ASD cause a range of microtubule-related phenotypes, including disrupted localization of the protein at mitotic spindles, cell cycle stalling, DNA damage and cell death. Lastly, we observe that ASD genetic risk is significantly enriched among tubulin-associated proteins, suggesting broader relevance. Together, these results provide additional evidence that the role of tubulin biology and cellular proliferation in ASD warrants further investigation and highlight the pitfalls of relying solely on annotated gene functions in the search for pathological mechanisms.

The neurodevelopmental disorder risk gene DYRK1A is required for ciliogenesis and control of brain size in Xenopus embryos.

DYRK1A [dual specificity tyrosine-(Y)-phosphorylation-regulated kinase 1 A] is a high-confidence autism risk gene that encodes a conserved kinase. In addition to autism, individuals with putative loss-of-function variants in DYRK1A exhibit microcephaly, intellectual disability, developmental delay and/or congenital anomalies of the kidney and urinary tract. DYRK1A is also located within the critical region for Down syndrome; therefore, understanding the role of DYRK1A in brain development is crucial for understanding the pathobiology of multiple developmental disorders. To characterize the function of this gene, we used the diploid frog Xenopus tropicalis We discover that Dyrk1a is expressed in ciliated tissues, localizes to ciliary axonemes and basal bodies, and is required for ciliogenesis. We also demonstrate that Dyrk1a localizes to mitotic spindles and that its inhibition leads to decreased forebrain size, abnormal cell cycle progression and cell death during brain development. These findings provide hypotheses about potential mechanisms of pathobiology and underscore the utility of X. tropicalis as a model system for understanding neurodevelopmental disorders.

The contribution of de novo coding mutations to autism spectrum disorder.

Whole exome sequencing has proven to be a powerful tool for understanding the genetic architecture of human disease. Here we apply it to more than 2,500 simplex families, each having a child with an autistic spectrum disorder. By comparing affected to unaffected siblings, we show that 13% of de novo missense mutations and 43% of de novo likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding de novo mutations contribute to about 30% of all simplex and 45% of female diagnoses. Almost all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower intelligence quotient (IQ), but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to contributory missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Most of the significance for the latter comes from affected females.

De novo mutations revealed by whole-exome sequencing are strongly associated with autism.

Multiple studies have confirmed the contribution of rare de novo copy number variations to the risk for autism spectrum disorders. But whereas de novo single nucleotide variants have been identified in affected individuals, their contribution to risk has yet to be clarified. Specifically, the frequency and distribution of these mutations have not been well characterized in matched unaffected controls, and such data are vital to the interpretation of de novo coding mutations observed in probands. Here we show, using whole-exome sequencing of 928 individuals, including 200 phenotypically discordant sibling pairs, that highly disruptive (nonsense and splice-site) de novo mutations in brain-expressed genes are associated with autism spectrum disorders and carry large effects. On the basis of mutation rates in unaffected individuals, we demonstrate that multiple independent de novo single nucleotide variants in the same gene among unrelated probands reliably identifies risk alleles, providing a clear path forward for gene discovery. Among a total of 279 identified de novo coding mutations, there is a single instance in probands, and none in siblings, in which two independent nonsense variants disrupt the same gene, SCN2A (sodium channel, voltage-gated, type II, α subunit), a result that is highly unlikely by chance.

No evidence for association of autism with rare heterozygous point mutations in Contactin-Associated Protein-Like 2 (CNTNAP2), or in Other Contactin-Associated Proteins or Contactins.

Contactins and Contactin-Associated Proteins, and Contactin-Associated Protein-Like 2 (CNTNAP2) in particular, have been widely cited as autism risk genes based on findings from homozygosity mapping, molecular cytogenetics, copy number variation analyses, and both common and rare single nucleotide association studies. However, data specifically with regard to the contribution of heterozygous single nucleotide variants (SNVs) have been inconsistent. In an effort to clarify the role of rare point mutations in CNTNAP2 and related gene families, we have conducted targeted next-generation sequencing and evaluated existing sequence data in cohorts totaling 2704 cases and 2747 controls. We find no evidence for statistically significant association of rare heterozygous mutations in any of the CNTN or CNTNAP genes, including CNTNAP2, placing marked limits on the scale of their plausible contribution to risk.

Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation.

The immunoglobulin heavy-chain locus (IGH) encodes variable (IGHV), diversity (IGHD), joining (IGHJ), and constant (IGHC) genes and is responsible for antibody heavy-chain biosynthesis, which is vital to the adaptive immune response. Programmed V-(D)-J somatic rearrangement and the complex duplicated nature of the locus have impeded attempts to reconcile its genomic organization based on traditional B-lymphocyte derived genetic material. As a result, sequence descriptions of germline variation within IGHV are lacking, haplotype inference using traditional linkage disequilibrium methods has been difficult, and the human genome reference assembly is missing several expressed IGHV genes. By using a hydatidiform mole BAC clone resource, we present the most complete haplotype of IGHV, IGHD, and IGHJ gene regions derived from a single chromosome, representing an alternate assembly of ∼1 Mbp of high-quality finished sequence. From this we add 101 kbp of previously uncharacterized sequence, including functional IGHV genes, and characterize four large germline copy-number variants (CNVs). In addition to this germline reference, we identify and characterize eight CNV-containing haplotypes from a panel of nine diploid genomes of diverse ethnic origin, discovering previously unmapped IGHV genes and an additional 121 kbp of insertion sequence. We genotype four of these CNVs by using PCR in 425 individuals from nine human populations. We find that all four are highly polymorphic and show considerable evidence of stratification (Fst = 0.3-0.5), with the greatest differences observed between African and Asian populations. These CNVs exhibit weak linkage disequilibrium with SNPs from two commercial arrays in most of the populations tested.

Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data.

RATIONALE: Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown pathogenesis. OBJECTIVE: To determine the contribution of de novo copy number variants (CNVs) in the pathogenesis of sporadic CHD. METHODS AND RESULTS: We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism arrays and whole exome sequencing. Results were experimentally validated using digital droplet polymerase chain reaction. We compared validated CNVs in CHD cases with CNVs in 1301 healthy control trios. The 2 complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either single nucleotide polymorphism array (P=7×10(-5); odds ratio, 4.6) or whole exome sequencing data (P=6×10(-4); odds ratio, 3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (P=0.02; odds ratio, 2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in whole exome sequencing and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q subtelomeric deletions. CONCLUSIONS: We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD.

The developmental transcriptome of the human brain: implications for neurodevelopmental disorders.

PURPOSE OF REVIEW: Recent characterizations of the transcriptome of the developing human brain by several groups have generated comprehensive datasets on coding and noncoding RNAs that will be instrumental for illuminating the underlying biology of complex neurodevelopmental disorders. This review summarizes recent studies successfully utilizing these data to increase our understanding of the molecular mechanisms of pathogenesis. RECENT FINDINGS: Several approaches have successfully integrated developmental transcriptome data with gene discovery to generate testable hypotheses about when and where in the developing human brain disease-associated genes converge. Specifically, these include the projection neurons in the prefrontal and primary motor--somatosensory cortex during mid-fetal development in autism spectrum disorder and the frontal cortex during fetal development in schizophrenia. SUMMARY: Developmental transcriptome data is a key to interpreting disease-associated mutations and transcriptional changes. Novel approaches integrating the spatial and temporal dimensions of these data have increased our understanding of when and where disease occurs. Refinement of spatial and temporal properties and expanding these findings to other neurodevelopmental disorders will provide critical insights for understanding disease biology.

Autism genes converge on microtubule biology and RNA-binding proteins during excitatory neurogenesis.

Recent studies have identified over one hundred high-confidence (hc) autism spectrum disorder (ASD) genes. Systems biological and functional analyses on smaller subsets of these genes have consistently implicated excitatory neurogenesis. However, the extent to which the broader set of hcASD genes are involved in this process has not been explored systematically nor have the biological pathways underlying this convergence been identified. Here, we leveraged CROP-Seq to repress 87 hcASD genes in a human in vitro model of cortical neurogenesis. We identified 17 hcASD genes whose repression significantly alters developmental trajectory and results in a common cellular state characterized by disruptions in proliferation, differentiation, cell cycle, microtubule biology, and RNA-binding proteins (RBPs). We also characterized over 3,000 differentially expressed genes, 286 of which had expression profiles correlated with changes in developmental trajectory. Overall, we uncovered transcriptional disruptions downstream of hcASD gene perturbations, correlated these disruptions with distinct differentiation phenotypes, and reinforced neurogenesis, microtubule biology, and RBPs as convergent points of disruption in ASD.

Autism gene variants disrupt enteric neuron migration and cause gastrointestinal dysmotility.

The comorbidity of autism spectrum disorders and severe gastrointestinal symptoms is well-established, yet the molecular underpinnings remain unknown. The identification of high-confidence large-effect autism risk genes offers the opportunity to identify convergent, underlying biology by studying these genes in the context of the gastrointestinal system. Here we show that the expression of these genes is enriched in human prenatal gut neurons as well as their migratory progenitors, suggesting that the development and/or function of these neurons may be disrupted by autism-associated pathogenic variants, leading to gastrointestinal dysfunction. Here we document the prevalence of gastrointestinal issues in patients with large-effect variants in sixteen of these genes, highlighting dysmotility, consistent with potential enteric neuron dysfunction. Using the high-throughput diploid frog Xenopus tropicalis , we individually target five of these genes ( SYNGAP1, CHD8, SCN2A, CHD2 , and DYRK1A ) and observe disrupted enteric neuronal progenitor migration for each. More extensive analysis of DYRK1A reveals that perturbation causes gut dysmotility in vivo , which can be ameliorated by treatment with a selective serotonin reuptake inhibitor (escitalopram) or a serotonin receptor 6 agonist, identified by in vivo drug screening. This work suggests that atypical development of enteric neurons contributes to the gastrointestinal distress commonly seen in individuals with autism and that increasing serotonin signaling may be a productive therapeutic avenue.

Rare X-linked variants carry predominantly male risk in autism, Tourette syndrome, and ADHD.

Autism spectrum disorder (ASD), Tourette syndrome (TS), and attention-deficit/hyperactivity disorder (ADHD) display strong male sex bias, due to a combination of genetic and biological factors, as well as selective ascertainment. While the hemizygous nature of chromosome X (Chr X) in males has long been postulated as a key point of "male vulnerability", rare genetic variation on this chromosome has not been systematically characterized in large-scale whole exome sequencing studies of "idiopathic" ASD, TS, and ADHD. Here, we take advantage of informative recombinations in simplex ASD families to pinpoint risk-enriched regions on Chr X, within which rare maternally-inherited damaging variants carry substantial risk in males with ASD. We then apply a modified transmission disequilibrium test to 13,052 ASD probands and identify a novel high confidence ASD risk gene at exome-wide significance (MAGEC3). Finally, we observe that rare damaging variants within these risk regions carry similar effect sizes in males with TS or ADHD, further clarifying genetic mechanisms underlying male vulnerability in multiple neurodevelopmental disorders that can be exploited for systematic gene discovery.

Genome-wide Association Study Points to Novel Locus for Gilles de la Tourette Syndrome.

BACKGROUND: Tourette syndrome (TS) is a childhood-onset neurodevelopmental disorder of complex genetic architecture and is characterized by multiple motor tics and at least one vocal tic persisting for more than 1 year. METHODS: We performed a genome-wide meta-analysis integrating a novel TS cohort with previously published data, resulting in a sample size of 6133 individuals with TS and 13,565 ancestry-matched control participants. RESULTS: We identified a genome-wide significant locus on chromosome 5q15. Integration of expression quantitative trait locus, Hi-C (high-throughput chromosome conformation capture), and genome-wide association study data implicated the NR2F1 gene and associated long noncoding RNAs within the 5q15 locus. Heritability partitioning identified statistically significant enrichment in brain tissue histone marks, while polygenic risk scoring of brain volume data identified statistically significant associations with right and left thalamus volumes and right putamen volume. CONCLUSIONS: Our work presents novel insights into the neurobiology of TS, thereby opening up new directions for future studies.

Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience.

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.

Enhancing WNT Signaling Restores Cortical Neuronal Spine Maturation and Synaptogenesis in Tbr1 Mutants.

Tbr1 is a high-confidence autism spectrum disorder (ASD) gene encoding a transcription factor with distinct pre- and postnatal functions. Postnatally, Tbr1 conditional knockout (CKO) mutants and constitutive heterozygotes have immature dendritic spines and reduced synaptic density. Tbr1 regulates expression of several genes that underlie synaptic defects, including a kinesin (Kif1a) and a WNT-signaling ligand (Wnt7b). Furthermore, Tbr1 mutant corticothalamic neurons have reduced thalamic axonal arborization. LiCl and a GSK3ß inhibitor, two WNT-signaling agonists, robustly rescue the dendritic spines and the synaptic and axonal defects, suggesting that this could have relevance for therapeutic approaches in some forms of ASD.

De Novo Damaging DNA Coding Mutations Are Associated With Obsessive-Compulsive Disorder and Overlap With Tourette's Disorder and Autism.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a debilitating neuropsychiatric disorder with a genetic risk component, yet identification of high-confidence risk genes has been challenging. In recent years, risk gene discovery in other complex psychiatric disorders has been achieved by studying rare de novo (DN) coding variants. METHODS: We performed whole-exome sequencing in 222 OCD parent-child trios (184 trios after quality control), comparing DN variant frequencies with 777 previously sequenced unaffected trios. We estimated the contribution of DN mutations to OCD risk and the number of genes involved. Finally, we looked for gene enrichment in other datasets and canonical pathways. RESULTS: DN likely gene disrupting and predicted damaging missense variants are enriched in OCD probands (rate ratio, 1.52; p = .0005) and contribute to risk. We identified 2 high-confidence risk genes, each containing 2 DN damaging variants in unrelated probands: CHD8 and SCUBE1. We estimate that 34% of DN damaging variants in OCD contribute to risk and that DN damaging variants in approximately 335 genes contribute to risk in 22% of OCD cases. Furthermore, genes harboring DN damaging variants in OCD are enriched for those reported in neurodevelopmental disorders, particularly Tourette's disorder and autism spectrum disorder. An exploratory network analysis reveals significant functional connectivity and enrichment in canonical pathways, biological processes, and disease networks. CONCLUSIONS: Our findings show a pathway toward systematic gene discovery in OCD via identification of DN damaging variants. Sequencing larger cohorts of OCD parent-child trios will reveal more OCD risk genes and will provide needed insights into underlying disease biology.