scholarly journals In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with Autism risk genes

2019 ◽  
Author(s):  
Xin Jin ◽  
Sean K. Simmons ◽  
Amy X. Guo ◽  
Ashwin S. Shetty ◽  
Michelle Ko ◽  
...  
Keyword(s):  
De Novo ◽  
Disease Risk ◽  
Autism Spectrum ◽  
Specific Gene ◽  
Functional Evaluation ◽  
Loss Of Function ◽  
Risk Genes ◽  
Biologically Relevant ◽  
Relevant Context

AbstractThe thousands of disease risk genes and loci identified through human genetic studies far outstrip our current capacity to systematically study their functions. New experimental approaches are needed for functional investigations of large panels of genes in a biologically relevant context. Here, we developed a scalable genetic screen approach, in vivo Perturb-Seq, and applied this method to the functional evaluation of 35 autism spectrum disorder (ASD) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing brain in utero, and then performed single-cell RNA-Seq in the postnatal brain. We identified cell type-specific gene signatures from both neuronal and glial cell classes that are affected by genetic perturbations, and pointed at elements of both convergent and divergent cellular effects across this cohort of ASD risk genes. In vivo Perturb-Seq pioneers a systems genetics approach to investigate at scale how diverse mutations affect cell types and states in the biologically relevant context of the developing organism.

scholarly journals In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes

Science ◽  
2020 ◽  
Vol 370 (6520) ◽  
pp. eaaz6063 ◽  
Author(s):  
Xin Jin ◽  
Sean K. Simmons ◽  
Amy Guo ◽  
Ashwin S. Shetty ◽  
Michelle Ko ◽  
...  
Keyword(s):  
De Novo ◽  
Disease Risk ◽  
Cell Types ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Risk Genes ◽  
Cellular Effects ◽  
Gene Modules ◽  
Conserved Gene

The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type–specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.

scholarly journals Integrating de novo and inherited variants in over 42,607 autism cases identifies mutations in new moderate risk genes

2021 ◽  
Author(s):  
Xueya Zhou ◽  
Pamela Feliciano ◽  
Tianyun Wang ◽  
Irina Astrovskaya ◽  
Chang Shu ◽  
...  
Keyword(s):  
Genetic Risk ◽  
De Novo ◽  
Meta Analysis ◽  
Neurodevelopmental Disorder ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Moderate Risk ◽  
Full Spectrum ◽  
Risk Genes ◽  
Biological Parents

AbstractDespite the known heritable nature of autism spectrum disorder (ASD), studies have primarily identified risk genes with de novo variants (DNVs). To capture the full spectrum of ASD genetic risk, we performed a two-stage analysis of rare de novo and inherited coding variants in 42,607 ASD cases, including 35,130 new cases recruited online by SPARK. In the first stage, we analyzed 19,843 cases with one or both biological parents and found that known ASD or neurodevelopmental disorder (NDD) risk genes explain nearly 70% of the genetic burden conferred by DNVs. In contrast, less than 20% of genetic risk conferred by rare inherited loss-of-function (LoF) variants are explained by known ASD/NDD genes. We selected 404 genes based on the first stage of analysis and performed a meta-analysis with an additional 22,764 cases and 236,000 population controls. We identified 60 genes with exome-wide significance (p < 2.5e-6), including five new risk genes (NAV3, ITSN1, MARK2, SCAF1, and HNRNPUL2). The association of NAV3 with ASD risk is entirely driven by rare inherited LoFs variants, with an average relative risk of 4, consistent with moderate effect. ASD individuals with LoF variants in the four moderate risk genes (NAV3, ITSN1, SCAF1, and HNRNPUL2, n = 95) have less cognitive impairment compared to 129 ASD individuals with LoF variants in well-established, highly penetrant ASD risk genes (CHD8, SCN2A, ADNP, FOXP1, SHANK3) (59% vs. 88%, p= 1.9e-06). These findings will guide future gene discovery efforts and suggest that much larger numbers of ASD cases and controls are needed to identify additional genes that confer moderate risk of ASD through rare, inherited variants.

scholarly journals Haploinsufficiency of autism candidate gene NUAK1 impairs cortical development and behavior

2018 ◽  
Author(s):  
Virginie Courchet ◽  
Amanda J Roberts ◽  
Peggy Del Carmine ◽  
Tommy L. Lewis ◽  
Franck Polleux ◽  
...  
Keyword(s):  
Social Preference ◽  
De Novo ◽  
Sensorimotor Gating ◽  
Learning Task ◽  
Autism Spectrum ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Cortical Connectivity

SUMMARYRecently, numerous rare de novo mutations have been identified in children diagnosed with autism spectrum disorders (ASD). However, despite the predicted loss-of-function nature of some of these de novo mutations, the affected individuals are heterozygous carriers, which would suggest that most of these candidate genes are haploinsufficient and/or that these mutations lead to expression of dominant-negative forms of the protein. Here, we tested this hypothesis with the gene Nuak1, recently identified as a candidate ASD gene and that we previously identified for its role in the development of cortical connectivity. We report that Nuak1 is happloinsufficient in mice in regard to its function in cortical axon branching in vitro and in vivo. Nuak1+/− mice show a combination of abnormal behavioral traits ranging from defective memory consolidation in a spatial learning task, defects in social novelty (but not social preference) and abnormal sensorimotor gating and prepulse inhibition of the startle response. Overall, our results demonstrate that Nuak1 haploinsufficiency leads to defects in the development of cortical connectivity and a complex array of behavorial deficits compatible with ASD, intellectual disability and schizophrenia.

scholarly journals Varying intolerance of gene pathways to mutational classes explain genetic convergence across neuropsychiatric disorders

2016 ◽  
Author(s):  
Shahar Shohat ◽  
Eyal Ben-David ◽  
Sagiv Shifman
Keyword(s):  
Gene Expression ◽  
Genome Wide Association Study ◽  
De Novo ◽  
Expression Patterns ◽  
Neuropsychiatric Disorders ◽  
Autism Spectrum ◽  
Specific Gene ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Brain Gene Expression

AbstractGenetic susceptibility to Intellectual disability (ID), autism spectrum disorder (ASD) and schizophrenia (SCZ) often arises from mutations in the same genes, suggesting that they share common mechanisms. We studied genes with de novo mutations in the three disorders and genes implicated by SCZ genome-wide association study (GWAS). Using biological annotations and brain gene expression, we show that mutation class explains enrichment patterns more than specific disorder. Genes with loss of function mutations and genes with missense mutations were enriched with different pathways, shared with genes intolerant to mutations. Specific gene expression patterns were found for each disorder. ID genes were preferentially expressed in fetal cortex, ASD genes also in fetal cerebellum and striatum, and genes associated with SCZ were most significantly enriched in adolescent cortex. Our study suggests that convergence across neuropsychiatric disorders stems from vulnerable pathways to genetic variations, but spatiotemporal activity of genes contributes to specific phenotypes.

scholarly journals Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadja T. Hofer ◽  
Petronel Tuluc ◽  
Nadine J. Ortner ◽  
Yuliia V. Nikonishyna ◽  
Monica L. Fernándes-Quintero ◽  
...  
Keyword(s):  
High Risk ◽  
Neurodevelopmental Disorders ◽  
Developmental Disorder ◽  
De Novo ◽  
Disease Risk ◽  
Neurodevelopmental Disorder ◽  
Autism Spectrum ◽  
De Novo Mutation ◽  
Loss Of Function ◽  
Gain Of Function

Abstract Background There is increasing evidence that de novo CACNA1D missense mutations inducing increased Cav1.3 L-type Ca2+-channel-function confer a high risk for neurodevelopmental disorders (autism spectrum disorder with and without neurological and endocrine symptoms). Electrophysiological studies demonstrating the presence or absence of typical gain-of-function gating changes could therefore serve as a tool to distinguish likely disease-causing from non-pathogenic de novo CACNA1D variants in affected individuals. We tested this hypothesis for mutation S652L, which has previously been reported in twins with a severe neurodevelopmental disorder in the Deciphering Developmental Disorder Study, but has not been classified as a novel disease mutation. Methods For functional characterization, wild-type and mutant Cav1.3 channel complexes were expressed in tsA-201 cells and tested for typical gain-of-function gating changes using the whole-cell patch-clamp technique. Results Mutation S652L significantly shifted the voltage-dependence of activation and steady-state inactivation to more negative potentials (~ 13–17 mV) and increased window currents at subthreshold voltages. Moreover, it slowed tail currents and increased Ca2+-levels during action potential-like stimulations, characteristic for gain-of-function changes. To provide evidence that only gain-of-function variants confer high disease risk, we also studied missense variant S652W reported in apparently healthy individuals. S652W shifted activation and inactivation to more positive voltages, compatible with a loss-of-function phenotype. Mutation S652L increased the sensitivity of Cav1.3 for inhibition by the dihydropyridine L-type Ca2+-channel blocker isradipine by 3–4-fold. Conclusions and limitations Our data provide evidence that gain-of-function CACNA1D mutations, such as S652L, but not loss-of-function mutations, such as S652W, cause high risk for neurodevelopmental disorders including autism. This adds CACNA1D to the list of novel disease genes identified in the Deciphering Developmental Disorder Study. Although our study does not provide insight into the cellular mechanisms of pathological Cav1.3 signaling in neurons, we provide a unifying mechanism of gain-of-function CACNA1D mutations as a predictor for disease risk, which may allow the establishment of a more reliable diagnosis of affected individuals. Moreover, the increased sensitivity of S652L to isradipine encourages a therapeutic trial in the two affected individuals. This can address the important question to which extent symptoms are responsive to therapy with Ca2+-channel blockers.

scholarly journals CHD8 Regulates Cellular Homeostasis and Neuronal Function Genes Across Multiple Models of CHD8 Haploinsufficiency

2018 ◽  
Author(s):  
A. Ayanna Wade ◽  
Kenneth Lim ◽  
Rinaldo Catta-Preta ◽  
Alex S. Nord
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Chromatin Remodeling ◽  
De Novo ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Cell Functions ◽  
High Affinity

ABSTRACTThe packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Recent sequencing of patient mutations has linked de novo loss-of-function mutations to chromatin remodeling factors with specific, causal roles in neurodevelopmental disorders. Characterizing cellular and molecular phenotypes arising from haploinsufficiency of chromatin remodeling factors could reveal convergent mechanisms of pathology. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeling factor gene and has among the highest de novo loss-of-function mutations rates in patients with autism spectrum disorder (ASD). Mutations to CHD8 are expected to drive neurodevelopmental pathology through global disruptions to gene expression and chromatin state, however, mechanisms associated with CHD8 function have yet to be fully elucidated. We analyzed published transcriptomic and epigenomic data across CHD8 in vitro and in vivo knockdown and knockout models to identify convergent mechanisms of gene regulation by CHD8. We found reproducible high-affinity interactions of CHD8 near promoters of genes necessary for basic cell functions and gene regulation, especially chromatin organization and RNA processing genes. Overlap between CHD8 interaction and differential expression suggests that reduced dosage of CHD8 directly relates to decreased expression of these genes. In addition, genes important for neuronal development and function showed consistent dysregulation, though there was a reduced rate and decreased affinity for CHD8 interactions near these genes. This meta-analysis verifies CHD8 as a critical regulator of gene expression and reveals a consistent set of high affinity CHD8 interaction targets observed across human and mouse in vivo and in vitro studies. Our findings highlight novel core functions of CHD8 and indicate direct and downstream gene regulatory impacts that are likely to be associated with neuropathology underlying CHD8-associated neurodevelopmental disorder.

scholarly journals Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

2020 ◽  
Author(s):  
Jasmin Morandell ◽  
Lena A. Schwarz ◽  
Bernadette Basilico ◽  
Saren Tasciyan ◽  
Armel Nicolas ◽  
...  
Keyword(s):  
Neuronal Migration ◽  
Motor Coordination ◽  
De Novo ◽  
Critical Role ◽  
Autism Spectrum ◽  
Cytoskeletal Proteins ◽  
Inhibitory Neurons ◽  
Loss Of Function ◽  
Proteomic Approach

AbstractDe novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). Here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 haploinsufficient mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3 mutant brain displays cortical lamination abnormalities due to defective neuronal migration and reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal columnar organization, Cul3 haploinsufficiency is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level, employing a quantitative proteomic approach, we show that Cul3 regulates cytoskeletal and adhesion protein abundance in mouse embryos. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neuronal cells results in atypical organization of the actin mesh at the cell leading edge, likely causing the observed migration deficits. In contrast to these important functions early in development, Cul3 deficiency appears less relevant at adult stages. In fact, induction of Cul3 haploinsufficiency in adult mice does not result in the behavioral defects observed in constitutive Cul3 haploinsufficient animals. Taken together, our data indicate that Cul3 has a critical role in the regulation of cytoskeletal proteins and neuronal migration and that ASD-associated defects and behavioral abnormalities are primarily due to Cul3 functions at early developmental stages.

scholarly journals Dissecting Autism Genetic Risk Using Single-cell RNA-seq Data

2020 ◽  
Author(s):  
Siying Chen ◽  
Xueya Zhou ◽  
Eve Byington ◽  
Samuel L. Bruce ◽  
Haicang Zhang ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Sequencing ◽  
De Novo ◽  
Expression Patterns ◽  
Autism Spectrum ◽  
Supervised Machine Learning ◽  
Specific Gene ◽  
Rna Seq ◽  
Risk Genes ◽  
Sequencing Studies

AbstractAutism spectrum disorder (autism) is a condition with strong but heterogenous genetic contribution. Recent exome and genome sequencing studies have uncovered many new risk genes through de novo variants. However, a large fraction of enrichment of de novo variants observed in cases are not accounted for by known or candidate risk genes, suggesting that the majority of risk genes are still unknown. Here we hypothesize that autism risk genes share a few common cell-type specific gene expression patterns during brain development, and such information can be quantified to improve statistical power of detecting new risk genes. We obtained large-scale single-cell RNA-seq data from human fetal brain collected through a range of developmental stages, and developed a supervised machine-learning approach “A-risk” (Autism risk), to predict the plausibility of autism risk genes across the genome. Using data from recent exome sequencing studies of autism, A-risk achieves better performance in prioritizing de novo variants than other methods, especially for genes that are less intolerant of loss of function variants. We stratified genes based on A-risk and mutation intolerance metrics to improve estimation of priors in extTADA and identified 71 candidate risk genes. In particular, CLCN4, PRKAR1B, and NR2F1 are potentially new risk genes with further support from neurodevelopmental disorders. Expression patterns of both known and candidate risk genes reveals the important role of deep-layer excitatory neurons from adult human cortex in autism etiology. With the unprecedented revolution of single-cell transcriptomics and expanding autism cohorts with exome or genome sequencing, our method will facilitate systematic discovery of novel risk genes and understanding of biological pathogenesis in autism.

scholarly journals Identification and validation of novel candidate risk genes in endocytic vesicular trafficking associated with esophageal atresia and tracheoesophageal fistulas

2021 ◽  
Author(s):  
Guojie Zhong ◽  
Priyanka Ahimaz ◽  
Nicole A. Edwards ◽  
Jacob J. Hagen ◽  
Christophe Faure ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Congenital Anomalies ◽  
De Novo ◽  
Simulation Analysis ◽  
Loss Of Function ◽  
Developmental Defects ◽  
Risk Genes ◽  
Significant Burden ◽  
Background Mutation Rate ◽  
Complex Cases

Esophageal atresias/tracheoesophageal fistulas (EA/TEF) are rare congenital anomalies caused by aberrant development of the foregut. Previous studies indicate that rare or de novo genetic variants significantly contribute to EA/TEF risk, and most individuals with EA/TEF do not have pathogenic genetic variants in established risk genes. To identify novel genetic contributions to EA/TEF, we performed whole genome sequencing of 185 trios (probands and parents) with EA/TEF, including 59 isolated and 126 complex cases with additional congenital anomalies and/or neurodevelopmental disorders. There was a significant burden of protein altering de novo coding variants in complex cases (p=3.3e-4), especially in genes that are intolerant of loss of function variants in the population. We performed simulation analysis of pathway enrichment based on background mutation rate and identified a number of pathways related to endocytosis and intracellular trafficking that as a group have a significant burden of protein altering de novo variants. We assessed 18 variants for disease causality using CRISPR-Cas9 mutagenesis in Xenopus and confirmed 13 with tracheoesophageal phenotypes. Our results implicate disruption of endosome-mediated epithelial remodeling as a potential mechanism of foregut developmental defects. This research may have implications for the mechanisms of other rare congenital anomalies.

scholarly journals UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology

2019 ◽  
Vol 116 (25) ◽  
pp. 12500-12505 ◽  
Author(s):  
Jie Wang ◽  
Sen-Sen Lou ◽  
Tingting Wang ◽  
Rong-Jie Wu ◽  
Guangying Li ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Dendritic Spine ◽  
Isotope Labeling ◽  
Neurodevelopmental Disorder ◽  
Critical Role ◽  
Motor Impairment ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Pp2a Activity

Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am−/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11–13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am−/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am−/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.

Sign in / Sign up

Export Citation Format

Share Document