Autism Spectrum Disorder Risk Genes Research Articles

Rho GTPase Regulators and Effectors in Autism Spectrum Disorders: Animal Models and Insights for Therapeutics.

The Rho family GTPases are small G proteins that act as molecular switches shuttling between active and inactive forms. Rho GTPases are regulated by two classes of regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Rho GTPases transduce the upstream signals to downstream effectors, thus regulating diverse cellular processes, such as growth, migration, adhesion, and differentiation. In particular, Rho GTPases play essential roles in regulating neuronal morphology and function. Recent evidence suggests that dysfunction of Rho GTPase signaling contributes substantially to the pathogenesis of autism spectrum disorder (ASD). It has been found that 20 genes encoding Rho GTPase regulators and effectors are listed as ASD risk genes by Simons foundation autism research initiative (SFARI). This review summarizes the clinical evidence, protein structure, and protein expression pattern of these 20 genes. Moreover, ASD-related behavioral phenotypes in animal models of these genes are reviewed, and the therapeutic approaches that show successful treatment effects in these animal models are discussed.

Forecasting risk gene discovery in autism with machine learning and genome-scale data

Genetics has been one of the most powerful windows into the biology of autism spectrum disorder (ASD). It is estimated that a thousand or more genes may confer risk for ASD when functionally perturbed, however, only around 100 genes currently have sufficient evidence to be considered true “autism risk genes”. Massive genetic studies are currently underway producing data to implicate additional genes. This approach — although necessary — is costly and slow-moving, making identification of putative ASD risk genes with existing data vital. Here, we approach autism risk gene discovery as a machine learning problem, rather than a genetic association problem, by using genome-scale data as predictors to identify new genes with similar properties to established autism risk genes. This ensemble method, forecASD, integrates brain gene expression, heterogeneous network data, and previous gene-level predictors of autism association into an ensemble classifier that yields a single score indexing evidence of each gene’s involvement in the etiology of autism. We demonstrate that forecASD has substantially better performance than previous predictors of autism association in three independent trio-based sequencing studies. Studying forecASD prioritized genes, we show that forecASD is a robust indicator of a gene’s involvement in ASD etiology, with diverse applications to gene discovery, differential expression analysis, eQTL prioritization, and pathway enrichment analysis.

Genome-wide, integrative analysis of circular RNA dysregulation and the corresponding circular RNA-microRNA-mRNA regulatory axes in autism.

Circular RNAs (circRNAs), a class of long noncoding RNAs, are known to be enriched in mammalian neural tissues. Although a wide range of dysregulation of gene expression in autism spectrum disorder (ASD) have been reported, the role of circRNAs in ASD remains largely unknown. Here, we performed genome-wide circRNA expression profiling in postmortem brains from individuals with ASD and controls and identified 60 circRNAs and three coregulated modules that were perturbed in ASD. By integrating circRNA, microRNA, and mRNA dysregulation data derived from the same cortex samples, we identified 8170 ASD-associated circRNA-microRNA-mRNA interactions. Putative targets of the axes were enriched for ASD risk genes and genes encoding inhibitory postsynaptic density (PSD) proteins, but not for genes implicated in monogenetic forms of other brain disorders or genes encoding excitatory PSD proteins. This reflects the previous observation that ASD-derived organoids show overproduction of inhibitory neurons. We further confirmed that some ASD risk genes (NLGN1, STAG1, HSD11B1, VIP, and UBA6) were regulated by an up-regulated circRNA (circARID1A) via sponging a down-regulated microRNA (miR-204-3p) in human neuronal cells. Particularly, alteration of NLGN1 expression is known to affect the dynamic processes of memory consolidation and strengthening. To the best of our knowledge, this is the first systems-level view of circRNA regulatory networks in ASD cortex samples. We provided a rich set of ASD-associated circRNA candidates and the corresponding circRNA-microRNA-mRNA axes, particularly those involving ASD risk genes. Our findings thus support a role for circRNA dysregulation and the corresponding circRNA-microRNA-mRNA axes in ASD pathophysiology.

Conserved Tao Kinase Activity Regulates Dendritic Arborization, Cytoskeletal Dynamics, and Sensory Function in Drosophila.

Dendritic arborization is highly regulated and requires tight control of dendritic growth, branching, cytoskeletal dynamics, and ion channel expression to ensure proper function. Abnormal dendritic development can result in altered network connectivity, which has been linked to neurodevelopmental disorders, including autism spectrum disorders (ASDs). How neuronal growth control programs tune dendritic arborization to ensure function is still not fully understood. Using Drosophila dendritic arborization (da) neurons as a model, we identified the conserved Ste20-like kinase Tao as a negative regulator of dendritic arborization. We show that Tao kinase activity regulates cytoskeletal dynamics and sensory channel localization required for proper sensory function in both male and female flies. We further provide evidence for functional conservation of Tao kinase, showing that its ASD-linked human ortholog, Tao kinase 2 (Taok2), could replace Drosophila Tao and rescue dendritic branching, dynamic microtubule alterations, and behavioral defects. However, several ASD-linked Taok2 variants displayed impaired rescue activity, suggesting that Tao/Taok2 mutations can disrupt sensory neuron development and function. Consistently, we show that Tao kinase activity is required in developing and as well as adult stages for maintaining normal dendritic arborization and sensory function to regulate escape and social behavior. Our data suggest an important role for Tao kinase signaling in cytoskeletal organization to maintain proper dendritic arborization and sensory function, providing a strong link between developmental sensory aberrations and behavioral abnormalities relevant for Taok2-dependent ASDs.SIGNIFICANCE STATEMENT Autism spectrum disorders (ASDs) are linked to abnormal dendritic arbors. However, the mechanisms of how dendritic arbors develop to promote functional and proper behavior are unclear. We identified Drosophila Tao kinase, the ortholog of the ASD risk gene Taok2, as a regulator of dendritic arborization in sensory neurons. We show that Tao kinase regulates cytoskeletal dynamics, controls sensory ion channel localization, and is required to maintain somatosensory function in vivo Interestingly, ASD-linked human Taok2 mutations rendered it nonfunctional, whereas its WT form could restore neuronal morphology and function in Drosophila lacking endogenous Tao. Our findings provide evidence for a conserved role of Tao kinase in dendritic development and function of sensory neurons, suggesting that aberrant sensory function might be a common feature of ASDs.

Integrative analysis of shared genetic pathogenesis by autism spectrum disorder and obsessive-compulsive disorder.

Many common pathological features have been observed for both autism spectrum disorders (ASDs) and obsessive-compulsive disorder (OCD). However, no systematic analysis of the common gene markers associated with both ASD and OCD has been conducted so far. Here, two batches of large-scale literature-based disease–gene relation data (updated in 2017 and 2019, respectively) and gene expression data were integrated to study the possible association between OCD and ASD at the genetic level. Genes linked to OCD and ASD present significant overlap (P-value <2.64e-39). A genetic network of over 20 genes was constructed, through which OCD and ASD may exert influence on each other. The 2017-based analysis suggested six potential common risk genes for OCD and ASD (CDH2, ADCY8, APOE, TSPO, TOR1A, and OLIG2), and the 2019-based study identified two more genes (DISP1 and SETD1A). Notably, the gene APOE identified by the 2017-based analysis has been implicated to have an association with ASD in a recent study (2018) with DNA methylation analysis. Our results support the possible complex genetic associations between OCD and ASD. Genes linked to one disease are worth further investigation as potential risk factors for the other.

Integrated Transcriptome Analyses Revealed Key Target Genes in Mouse Models of Autism.

Genetic mutations are the major pathogenic factor of Autism Spectrum Disorder (ASD). In recent years, more and more ASD risk genes have been revealed, among which there are a group of transcriptional regulators. Considering the similarity of the core clinical phenotypes, it is possible that these different factors may regulate the expression levels of certain key targets. Identification of these targets could facilitate the understanding of the etiology and developing of novel diagnostic and therapeutic methods. Therefore, we performed integrated transcriptome analyses of RNA-Seq and microarray data in multiple ASD mouse models and identified a number of common downstream genes in various brain regions, many of which are related to the structure and function of the synapse components or drug addiction. We then established protein-protein interaction networks of the overlapped targets and isolated the hub genes by 11 algorithms based on the topological structure of the networks, including Sdc4, Vegfa, and Cp in the Cortex-Adult subgroup, Gria1 in the Cortex-Juvenile subgroup, and Kdr, S1pr1, Ubc, Grm2, Grin2b, Nrxn1, Pdyn, Grin3a, Itgam, Grin2a, Gabra2, and Camk4 in the Hippocampus-Adult subgroup, many of which have been associated with ASD in previous studies. Finally, we cross compared our results with human brain transcriptional data sets and verified several key candidates, which may play important role in the pathology process of ASD, including SDC4, CP, S1PR1, UBC, PDYN, GRIN2A, GABRA2, and CAMK4. In summary, by integrated bioinformatics analysis, we have identified a series of potentially important molecules for future ASD research. Autism Res 2020, 13: 352-368. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Abnormal transcriptional regulation accounts for a significant portion of Autism Spectrum Disorder. In this study, we performed transcriptome analyses of mouse models to identify common downstream targets of transcriptional regulators involved in ASD. We identified several recurrent target genes that are close related to the common pathological process of ASD, including SDC4, CP, S1PR1, UBC, PDYN, GRM2, NRXN1, GRIN3A, ITGAM, GRIN2A, GABRA2, and CAMK4. These results provide potentially important targets for understanding the molecular mechanism of ASD.

Association of genes with phenotype in autism spectrum disorder.

Autism spectrum disorder (ASD) is a genetic heterogeneous neurodevelopmental disorder that is characterized by impairments in social interaction and speech development and is accompanied by stereotypical behaviors such as body rocking, hand flapping, spinning objects, sniffing and restricted behaviors. The considerable significance of the genetics associated with autism has led to the identification of many risk genes for ASD used for the probing of ASD specificity and shared cognitive features over the past few decades. Identification of ASD risk genes helps to unravel various genetic variants and signaling pathways which are involved in ASD. This review highlights the role of ASD risk genes in gene transcription and translation regulation processes, as well as neuronal activity modulation, synaptic plasticity, disrupted key biological signaling pathways, and the novel candidate genes that play a significant role in the pathophysiology of ASD. The current emphasis on autism spectrum disorders has generated new opportunities in the field of neuroscience, and further advancements in the identification of different biomarkers, risk genes, and genetic pathways can help in the early diagnosis and development of new clinical and pharmacological treatments for ASD.

Autism spectrum disorder polygenic scores are associated with every day executive function in children admitted for clinical assessment.

Autism spectrum disorder (ASD) and other neurodevelopmental disorders (NDs) are behaviorally defined disorders with overlapping clinical features that are often associated with higher‐order cognitive dysfunction, particularly executive dysfunction. Our aim was to determine if the polygenic score (PGS) for ASD is associated with parent‐reported executive dysfunction in everyday life using the Behavior Rating Inventory of Executive Function (BRIEF). Furthermore, we investigated if PGS for general intelligence (INT) and attention deficit/hyperactivity disorder (ADHD) also correlate with BRIEF. We included 176 children, adolescents and young adults aged 5–22 years with full‐scale intelligence quotient (IQ) above 70. All were admitted for clinical assessment of ASD symptoms and 68% obtained an ASD diagnosis. We found a significant difference between low and high ASD PGS groups in the BRIEF behavior regulation index (BRI) (P = 0.015, Cohen's d = 0.69). A linear regression model accounting for age, sex, full‐scale IQ, Social Responsiveness Scale (SRS) total score, ASD, ADHD and INT PGS groups as well as genetic principal components, significantly predicted the BRI score; F(11,130) = 8.142, P < 0.001, R 2 = 0.41 (unadjusted). Only SRS total (P < 0.001), ASD PGS 0.1 group (P = 0.018), and sex (P = 0.022) made a significant contribution to the model. This suggests that the common ASD risk gene variants have a stronger association to behavioral regulation aspects of executive dysfunction than ADHD risk or INT variants in a clinical sample with ASD symptoms. Autism Res 2020, 13: 207–220. © 2019 International Society for Autism Research, Wiley Periodicals, Inc.Lay SummaryPeople with autism spectrum disorder (ASD) often have difficulties with higher‐order cognitive processes that regulate thoughts and actions during goal‐directed behavior, also known as executive function (EF). We studied the association between genetics related to ASD and EF and found a relation between high polygenic score (PGS) for ASD and difficulties with behavior regulation aspects of EF in children and adolescents under assessment for ASD. Furthermore, high PGS for general intelligence was related to social problems.

A perturbed gene network containing PI3K-AKT, RAS-ERK and WNT-β-catenin pathways in leukocytes is linked to ASD genetics and symptom severity.

Hundreds of genes are implicated in autism spectrum disorder (ASD) but the mechanisms through which they contribute to ASD pathophysiology remain elusive. Here, we analyzed leukocyte transcriptomics from 1–4 year-old male toddlers with ASD or typical development from the general population. We discovered a perturbed gene network that includes genes that are highly expressed during fetal brain development and which is dysregulated in hiPSC-derived neuron models of ASD. High-confidence ASD risk genes emerge as upstream regulators of the network, and many risk genes may impact the network by modulating RAS/ERK, PI3K/AKT, and WNT/β-catenin signaling pathways. We found that the degree of dysregulation in this network correlated with the severity of ASD symptoms in the toddlers. These results demonstrate how the heterogeneous genetics of ASD may dysregulate a core network to influence brain development at prenatal and very early postnatal ages and, thereby, the severity of later ASD symptoms.

Dynamical Electrical Complexity Is Reduced during Neuronal Differentiation in Autism Spectrum Disorder.

SummaryNeuronal activity can be modeled as a nonlinear dynamical system to yield measures of neuronal state and dysfunction. The electrical recordings of stem cell-derived neurons from individuals with autism spectrum disorder (ASD) and controls were analyzed using minimum embedding dimension (MED) analysis to characterize their dynamical complexity. MED analysis revealed a significant reduction in dynamical complexity in ASD neurons during differentiation, which was correlated to bursting and spike interval measures. MED was associated with clinical endpoints, such as nonverbal intelligence, and was correlated with 53 differentially expressed genes, which were overrepresented with ASD risk genes related to neurodevelopment, cell morphology, and cell migration. Spatiotemporal analysis also showed a prenatal temporal enrichment in cortical and deep brain structures. Together, we present dynamical analysis as a paradigm that can be used to distinguish disease-associated cellular electrophysiological and transcriptional signatures, while taking into account patient variability in neuropsychiatric disorders.

Exome sequencing of 457 autism families recruited online provides evidence for autism risk genes

Autism spectrum disorder (ASD) is a genetically heterogeneous condition, caused by a combination of rare de novo and inherited variants as well as common variants in at least several hundred genes. However, significantly larger sample sizes are needed to identify the complete set of genetic risk factors. We conducted a pilot study for SPARK (SPARKForAutism.org) of 457 families with ASD, all consented online. Whole exome sequencing (WES) and genotyping data were generated for each family using DNA from saliva. We identified variants in genes and loci that are clinically recognized causes or significant contributors to ASD in 10.4% of families without previous genetic findings. In addition, we identified variants that are possibly associated with ASD in an additional 3.4% of families. A meta-analysis using the TADA framework at a false discovery rate (FDR) of 0.1 provides statistical support for 26 ASD risk genes. While most of these genes are already known ASD risk genes, BRSK2 has the strongest statistical support and reaches genome-wide significance as a risk gene for ASD (p-value = 2.3e−06). Future studies leveraging the thousands of individuals with ASD who have enrolled in SPARK are likely to further clarify the genetic risk factors associated with ASD as well as allow accelerate ASD research that incorporates genetic etiology.

Autism-like social deficit generated by Dock4 deficiency is rescued by restoration of Rac1 activity and NMDA receptor function

Genetic studies of autism spectrum disorder (ASD) have revealed multigene variations that converge on synaptic dysfunction. DOCK4, a gene at 7q31.1 that encodes the Rac1 guanine nucleotide exchange factor Dock4, has been identified as a risk gene for ASD and other neuropsychiatric disorders. However, whether and how Dock4 disruption leads to ASD features through a synaptic mechanism remain unexplored. We generated and characterized a line of Dock4 knockout (KO) mice, which intriguingly displayed a series of ASD-like behaviors, including impaired social novelty preference, abnormal isolation-induced pup vocalizations, elevated anxiety, and perturbed object and spatial learning. Mice with conditional deletion of Dock4 in hippocampal CA1 recapitulated social preference deficit in KO mice. Examination in CA1 pyramidal neurons revealed that excitatory synaptic transmission was drastically attenuated in KO mice, accompanied by decreased spine density and synaptic content of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)- and NMDA (N-methyl-D-aspartate)-type glutamate receptors. Moreover, Dock4 deficiency markedly reduced Rac1 activity in the hippocampus, which resulted in downregulation of global protein synthesis and diminished expression of AMPA and NMDA receptor subunits. Notably, Rac1 replenishment in the hippocampal CA1 of Dock4 KO mice restored excitatory synaptic transmission and corrected impaired social deficits in these mice, and pharmacological activation of NMDA receptors also restored social novelty preference in Dock4 KO mice. Together, our findings uncover a previously unrecognized Dock4-Rac1-dependent mechanism involved in regulating hippocampal excitatory synaptic transmission and social behavior.

Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks.

We performed a comprehensive assessment of rareinherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extensionof previousfindings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

First behavioural assessment of a novel Immp2l knockdown mouse model with relevance for Gilles de la Tourette syndrome and Autism spectrum disorder

Gilles de la Tourette syndrome (GTS) is a neurodevelopmental disorder, which shares some clinical features with Autism spectrum disorder (ASD). The genetic factors relevant to the development of both disorders are yet to be fully understood, however, some genetic association studies have identified inner mitochondrial membrane peptidase subunit 2 (IMMP2L) as a potential risk gene for both GTS and ASD. The impact of Immp2l deficiency on behavioural domains is currently unknown.A new genetic mouse model for Immp2l was developed. Adult heterozygous (HET) and homozygous (HOMO) Immp2l knockdown (Immp2l KD) mice of both sexes were compared to wild type-like (WT) littermates in the open field (OF), social interaction, novel object recognition, marble burying, and prepulse inhibition (PPI). The effect of acute dexamphetamine (2 mg/kg) on OF behaviour was also determined.OF locomotion was significantly higher in HET compared to HOMO male littermates. Male and female HOMO mice were much more sensitive to the locomotor-stimulating effects of dexamphetamine (DEX), whereas only HOMO males exhibited significant increased DEX-induced OF exploration compared to control groups. HOMO females failed to habituate to an acoustic startle stimulus. Furthermore, compared to HOMO females, HET females showed reduced social interaction, and a similar trend was seen in HET males.The Immp2l KD mouse model possesses moderate face validity for preclinical research into GTS and ASD, in particular as dysfunctional dopaminergic neurotransmission appears to be one mechanism leading to disease presentation. The sex-dependent differences observed in most findings reinforce the strong influence of sex in the pathophysiology of GTS and ASD.

Neurological Diseases With Autism Spectrum Disorder: Role of ASD Risk Genes.

Autism spectrum disorder (ASD) is frequently comorbid with other neurological disorders such as intellectual disability (ID) or global development delay (GDD) and epilepsy. The pathogenesis of ASD is complex. So far, studies have identified more than 1000 ASD risk genes. Most of them were also reported to relate with other neurological diseases, and only several of them have been confirmed as pathogenic genes for autism. Little is known about the roles of these risk genes in neurological diseases with ASD. In the present study, we recruited a cohort of 158 neurological disorder probands with 163 variants of 48 ASD risk genes. Of these, 50 individuals (31.6%) were diagnosed with ASD. In the ASD patient subset, we identified several rarely reported candidate genes including DOLK, USH2A, and HUWE1. In a comparison of patients with neurological disorders with and without ASD, we found that ID/GDD was frequently comorbid with ASD whereas epilepsy was more common in the non-ASD group. Statistical analyses of all possible risk factors implicated that variants in synaptic genes, especially non-voltage-gated ion channel genes and in transcriptional and chromosome genes were related to ASD, but none of the investigated environmental factors was. Our results are useful for the future diagnosis and prognosis of patients with neurological disorders and emphasize the utility of genetic screening.

Association Between Breastfeeding Initiation and Duration and Autism Spectrum Disorder in Preschool Children Enrolled in the Study to Explore Early Development.

Studies report inconsistent findings on the relationship between ASD and breastfeeding. We explored associations between ASD and breastfeeding initiation (yes/no) and duration (months categorized in tertiles) in the Study to Explore Early Development, a community-based case-control study in six sites in the Unites States. We adjusted for various child and mother demographic and pregnancy factors. Breastfeeding initiation was reported in 85.7% of mothers of children with ASD and 90.6% of mothers of controls. After adjustment, we found no significant difference in breastfeeding initiation (adjusted odds-ratio [aOR]: 0.88 and 95% confidence interval (CI) 0.60-1.28). However, mothers of children with ASD were less likely to report duration of breastfeeding in the high (≥12 months) versus low tertile (<6 months) (aOR and 95% CI: 0.61 [0.45-0.84]) or the middle (6-<12 months) versus low tertile (0.72: 0.54-0.98). The association of ASD and breastfeeding duration was slightly attenuated when the presence of the broader autism phenotype (BAP) in the mother was accounted for, but still remained for the highest tertile. This association does not appear to be totally explained by maternal BAP. We were unable to distinguish whether the difference in duration was due to difficulties breastfeeding children who later develop ASD, other factors not adjusted in our study, or greater ASD risk resulting from shorter breastfeeding duration. Longitudinal studies that compare reasons why mothers stop breastfeeding between ASD and controls and establish a temporal relation between ASD and breastfeeding are needed. Future studies should also evaluate interactions between ASD risk genes and breastfeeding. Autism Research 2019, 12: 816-829. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: In this study, we compared breastfeeding practices between mothers of children with and without autism spectrum disorder (ASD). We found that the percentage of mothers who started breastfeeding was similar between the two groups, but mothers of children with ASD breastfed for a shorter amount of time compared to mothers of children without ASD. Future studies are needed to evaluate the reasons why the duration of breastfeeding was shorter for mothers of children with ASD compared to those without ASD.

A gene expression signature in developing Purkinje cells predicts autism and intellectual disability co-morbidity status

Autism spectrum disorder (ASD) is a complex neurodevelopmental disease whose underpinning molecular mechanisms and neural substrates are subject to intense scrutiny. Interestingly, the cerebellum has emerged as one of the key brain regions affected in ASD. However, the genetic and molecular mechanisms that link the cerebellum to ASD, particularly during development, remain poorly understood. To gain insight into the genetic and molecular mechanisms that might link the cerebellum to ASD, we analysed the transcriptome dynamics of a developing cell population highly enriched for Purkinje cells of the mouse cerebellum across multiple timepoints. We identified a single cluster of genes whose expression is positively correlated with development and which is enriched for genes associated with ASD. This ASD-associated gene cluster was specific to developing Purkinje cells and not detected in the mouse neocortex during the same developmental period, in which we identified a distinct temporally regulated ASD gene module. Furthermore, the composition of ASD risk genes within the two distinct clusters was significantly different in their association with intellectual disability (ID), consistent with the existence of genetically and spatiotemporally distinct endophenotypes of ASD. Together, our findings define a specific cluster of ASD genes that is enriched in developing PCs and predicts co-morbidity status.

Environmental Epigenetics In Autism Spectrum Disorder

The developing brain is highly sensitive to environmental exposures during the prenatal period. Evidence from neuroimaging, teratogen exposure case studies, and very early postnatal abnormalities demonstrate that the in utero exposure window is critical to risk of autism spectrum disorder (ASD). Genetic factors also contribute to ASD risk and large discovery efforts have identified important variants and structural changes. Studies of genetics or exposures in isolation to date explain a relatively small proportion of ASD risk. Epigenetics, including DNA methylation, represent a powerful gene-environment interaction mechanism. Prenatal exposures to toxicants such as cigarette smoke and heavy metals, or to nutritional supplements are associated with changes in DNA methylation measured in infants at birth. In an autism enriched-risk birth cohort we examine the roles of DNA methylation in multiple tissues and with environmental exposures. In the Early Autism Risk Longitudinal Investigation (EARLI), families with a child with ASD were recruited early in a subsequent pregnancy. Biological and exposure measures were obtained throughout pregnancy, including genome-wide DNA methylation measures in cord blood and placental tissues. Extensive neurophenotyping was performed until age three when ASD diagnoses were made. We show that the genomic locations of cord blood DNA methylation sites associated with ASD diagnosis are enriched for ASD risk genes. We further demonstrate that pregnancy exposures, such as to folic acid, result in global DNA methylation changes at birth, and this relationship is sensitive to maternal genotype. DNA methylation measures at birth may be ASD risk biomarkers, integrating genetic susceptibility and reflecting early life exposures. Environmental epigenetic epidemiological findings may have implications for ASD etiology.

Identifying Genomic Variations in Monozygotic Twins Discordant for Autism Spectrum Disorder Using Whole-Genome Sequencing

Autism spectrum disorder (ASD) presents a set of childhood neurodevelopmental disorders with impairments in social communication and restricted, repetitive, and stereotyped patterns of behavior. Here, based on the whole-genome sequencing (WGS) data of three monozygotic twins discordant for ASD, we explored multiple patient-specific genetic variations and prioritized a list of ASD risk genes. Our results identified DVMT (discordant variation in monozygotic twin) observed in at least two twin pairs, including 14,310 SNPs, 2,425 indels, and 16,735 CNVs, referring to a total of 2,174 genes, and 37 of these were covered by all three types of variations. Gene ontology (GO) enrichment analysis of biological processes for 2,174 genes showed that the majority of these genes were related to neurodevelopmental processes. In addition, functional network analysis showed that there was a strong functional relevance between 37 genes covered by all three types of variations. In conclusion, for the first time, we conducted a comprehensive scan of genomic differences between monozygotic twins discordant for ASD, providing researchers with in-depth directions. It may also provide effective strategies for clinical treatment of individuals affected by ASD.

Neonatal Tbr1 Dosage Controls Cortical Layer 6 Connectivity

An understanding of how heterozygous loss-of-function mutations in autism spectrum disorder (ASD) risk genes, such as TBR1, contribute to ASD remains elusive. Conditional Tbr1 deletion during late mouse gestation in cortical layer 6 neurons (Tbr1layer6 mutants) provides novel insights into its function, including dendritic patterning, synaptogenesis, and cell-intrinsic physiology. These phenotypes occur in heterozygotes, providing insights into mechanisms that may underlie ASD pathophysiology. Restoring expression of Wnt7b largely rescues the synaptic deficit in Tbr1layer6 mutant neurons. Furthermore, Tbr1layer6 heterozygotes have increased anxiety-like behavior, a phenotype seen ASD. Integrating TBR1 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data from layer 6 neurons and activity of TBR1-bound candidate enhancers provides evidence for how TBR1 regulates layer 6 properties. Moreover, several putative TBR1 targets are ASD risk genes, placing TBR1 in a central position both for ASD risk and for regulating transcriptional circuits that control multiple steps in layer 6 development essential for the assembly of neural circuits.