Histone Acetylome-wide Association Study of Autism Spectrum Disorder.

The association of histone modification changes with autism spectrum disorder (ASD) has not been systematically examined. We conducted a histone acetylome-wide association study (HAWAS) by performing H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) on 257 postmortem samples from ASD and matched control brains. Despite etiological heterogeneity, ≥68% of syndromic and idiopathic ASD cases shared a common acetylome signature at >5,000 cis-regulatory elements in prefrontal and temporal cortex. Similarly, multiple genes associated with rare genetic mutations in ASD showed common "epimutations." Acetylome aberrations in ASD were not attributable to genetic differentiation at cis-SNPs and highlighted genes involved in synaptic transmission, ion transport, epilepsy, behavioral abnormality, chemokinesis, histone deacetylation, and immunity. By correlating histone acetylation with genotype, we discovered >2,000 histone acetylation quantitative trait loci (haQTLs) in human brain regions, including four candidate causal variants for psychiatric diseases. Due to the relative stability of histone modifications postmortem, we anticipate that the HAWAS approach will be applicable to multiple diseases.

A highly conserved program of neuronal microexons is misregulated in autistic brains.

Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide "microexons" display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.

Integrative functional genomic analyses implicate specific molecular pathways and circuits in autism.

Genetic studies have identified dozens of autism spectrum disorder (ASD) susceptibility genes, raising two critical questions: (1) do these genetic loci converge on specific biological processes, and (2) where does the phenotypic specificity of ASD arise, given its genetic overlap with intellectual disability (ID)? To address this, we mapped ASD and ID risk genes onto coexpression networks representing developmental trajectories and transcriptional profiles representing fetal and adult cortical laminae. ASD genes tightly coalesce in modules that implicate distinct biological functions during human cortical development, including early transcriptional regulation and synaptic development. Bioinformatic analyses suggest that translational regulation by FMRP and transcriptional coregulation by common transcription factors connect these processes. At a circuit level, ASD genes are enriched in superficial cortical layers and glutamatergic projection neurons. Furthermore, we show that the patterns of ASD and ID risk genes are distinct, providing a biological framework for further investigating the pathophysiology of ASD.

Broad transcriptomic dysregulation occurs across the cerebral cortex in ASD.

Neuropsychiatric disorders classically lack defining brain pathologies, but recent work has demonstrated dysregulation at the molecular level, characterized by transcriptomic and epigenetic alterations1-3. In autism spectrum disorder (ASD), this molecular pathology involves the upregulation of microglial, astrocyte and neural-immune genes, the downregulation of synaptic genes, and attenuation of gene-expression gradients in cortex1,2,4-6. However, whether these changes are limited to cortical association regions or are more widespread remains unknown. To address this issue, we performed RNA-sequencing analysis of 725 brain samples spanning 11 cortical areas from 112 post-mortem samples from individuals with ASD and neurotypical controls. We find widespread transcriptomic changes across the cortex in ASD, exhibiting an anterior-to-posterior gradient, with the greatest differences in primary visual cortex, coincident with an attenuation of the typical transcriptomic differences between cortical regions. Single-nucleus RNA-sequencing and methylation profiling demonstrate that this robust molecular signature reflects changes in cell-type-specific gene expression, particularly affecting excitatory neurons and glia. Both rare and common ASD-associated genetic variation converge within a downregulated co-expression module involving synaptic signalling, and common variation alone is enriched within a module of upregulated protein chaperone genes. These results highlight widespread molecular changes across the cerebral cortex in ASD, extending beyond association cortex to broadly involve primary sensory regions.

Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

Tropism of SARS-CoV-2 for human cortical astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Author Correction: Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Change history: In this Letter, the labels for splicing events A3SS and A5SS were swapped in column D of Supplementary Table 3a and b. This has been corrected online.

Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing.

Common genetic contributions to autism spectrum disorder (ASD) reside in risk gene variants that individually have minimal effect sizes. As environmental factors that perturb neurodevelopment also underlie idiopathic ASD, it is crucial to identify altered regulators that can orchestrate multiple ASD risk genes during neurodevelopment. Cytoplasmic polyadenylation element binding proteins 1-4 (CPEB1-4) regulate the translation of specific mRNAs by modulating their poly(A)-tails and thereby participate in embryonic development and synaptic plasticity. Here we find that CPEB4 binds transcripts of most high-confidence ASD risk genes. The brains of individuals with idiopathic ASD show imbalances in CPEB4 transcript isoforms that result from decreased inclusion of a neuron-specific microexon. In addition, 9% of the transcriptome shows reduced poly(A)-tail length. Notably, this percentage is much higher for high-confidence ASD risk genes, correlating with reduced expression of the protein products of ASD risk genes. An equivalent imbalance in CPEB4 transcript isoforms in mice mimics the changes in mRNA polyadenylation and protein expression of ASD risk genes and induces ASD-like neuroanatomical, electrophysiological and behavioural phenotypes. Together, these data identify CPEB4 as a regulator of ASD risk genes.

Chromosome conformation elucidates regulatory relationships in developing human brain.

Three-dimensional physical interactions within chromosomes dynamically regulate gene expression in a tissue-specific manner. However, the 3D organization of chromosomes during human brain development and its role in regulating gene networks dysregulated in neurodevelopmental disorders, such as autism or schizophrenia, are unknown. Here we generate high-resolution 3D maps of chromatin contacts during human corticogenesis, permitting large-scale annotation of previously uncharacterized regulatory relationships relevant to the evolution of human cognition and disease. Our analyses identify hundreds of genes that physically interact with enhancers gained on the human lineage, many of which are under purifying selection and associated with human cognitive function. We integrate chromatin contacts with non-coding variants identified in schizophrenia genome-wide association studies (GWAS), highlighting multiple candidate schizophrenia risk genes and pathways, including transcription factors involved in neurogenesis, and cholinergic signalling molecules, several of which are supported by independent expression quantitative trait loci and gene expression analyses. Genome editing in human neural progenitors suggests that one of these distal schizophrenia GWAS loci regulates FOXG1 expression, supporting its potential role as a schizophrenia risk gene. This work provides a framework for understanding the effect of non-coding regulatory elements on human brain development and the evolution of cognition, and highlights novel mechanisms underlying neuropsychiatric disorders.

Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Autism spectrum disorder (ASD) involves substantial genetic contributions. These contributions are profoundly heterogeneous but may converge on common pathways that are not yet well understood. Here, through post-mortem genome-wide transcriptome analysis of the largest cohort of samples analysed so far, to our knowledge, we interrogate the noncoding transcriptome, alternative splicing, and upstream molecular regulators to broaden our understanding of molecular convergence in ASD. Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes. Our data suggest that SOX5, a transcription factor involved in neuron fate specification, contributes to this reduction in regional differences. We further demonstrate that a genetically defined subtype of ASD, chromosome 15q11.2-13.1 duplication syndrome (dup15q), shares the core transcriptomic signature observed in idiopathic ASD. Co-expression network analysis reveals that individuals with ASD show age-related changes in the trajectory of microglial and synaptic function over the first two decades, and suggests that genetic risk for ASD may influence changes in regional cortical gene expression. Our findings illustrate how diverse genetic perturbations can lead to phenotypic convergence at multiple biological levels in a complex neuropsychiatric disorder.

Genome-wide DNA methylation profiling identifies convergent molecular signatures associated with idiopathic and syndromic autism in post-mortem human brain tissue.

Autism spectrum disorder (ASD) encompasses a collection of complex neuropsychiatric disorders characterized by deficits in social functioning, communication and repetitive behaviour. Building on recent studies supporting a role for developmentally moderated regulatory genomic variation in the molecular aetiology of ASD, we quantified genome-wide patterns of DNA methylation in 223 post-mortem tissues samples isolated from three brain regions [prefrontal cortex, temporal cortex and cerebellum (CB)] dissected from 43 ASD patients and 38 non-psychiatric control donors. We identified widespread differences in DNA methylation associated with idiopathic ASD (iASD), with consistent signals in both cortical regions that were distinct to those observed in the CB. Individuals carrying a duplication on chromosome 15q (dup15q), representing a genetically defined subtype of ASD, were characterized by striking differences in DNA methylationacross a discrete domain spanning an imprinted gene cluster within the duplicated region. In addition to the dramatic cis-effects on DNA methylation observed in dup15q carriers, we identified convergent methylomic signatures associated with both iASD and dup15q, reflecting the findings from previous studies of gene expression and H3K27ac. Cortical co-methylation network analysis identified a number of co-methylated modules significantly associated with ASD that are enriched for genomic regions annotated to genes involved in the immune system, synaptic signalling and neuronal regulation. Our study represents the first systematic analysis of DNA methylation associated with ASD across multiple brain regions, providing novel evidence for convergent molecular signatures associated with both idiopathic and syndromic autism.

Systems biology and gene networks in neurodevelopmental and neurodegenerative disorders.

Genetic and genomic approaches have implicated hundreds of genetic loci in neurodevelopmental disorders and neurodegeneration, but mechanistic understanding continues to lag behind the pace of gene discovery. Understanding the role of specific genetic variants in the brain involves dissecting a functional hierarchy that encompasses molecular pathways, diverse cell types, neural circuits and, ultimately, cognition and behaviour. With a focus on transcriptomics, this Review discusses how high-throughput molecular, integrative and network approaches inform disease biology by placing human genetics in a molecular systems and neurobiological context. We provide a framework for interpreting network biology studies and leveraging big genomics data sets in neurobiology.

Sex-chromosome dosage effects on gene expression in humans.

A fundamental question in the biology of sex differences has eluded direct study in humans: How does sex-chromosome dosage (SCD) shape genome function? To address this, we developed a systematic map of SCD effects on gene function by analyzing genome-wide expression data in humans with diverse sex-chromosome aneuploidies (XO, XXX, XXY, XYY, and XXYY). For sex chromosomes, we demonstrate a pattern of obligate dosage sensitivity among evolutionarily preserved X-Y homologs and update prevailing theoretical models for SCD compensation by detecting X-linked genes that increase expression with decreasing X- and/or Y-chromosome dosage. We further show that SCD-sensitive sex-chromosome genes regulate specific coexpression networks of SCD-sensitive autosomal genes with critical cellular functions and a demonstrable potential to mediate previously documented SCD effects on disease. These gene coexpression results converge with analysis of transcription factor binding site enrichment and measures of gene expression in murine knockout models to spotlight the dosage-sensitive X-linked transcription factor ZFX as a key mediator of SCD effects on wider genome expression. Our findings characterize the effects of SCD broadly across the genome, with potential implications for human phenotypic variation.

Integrative network analysis reveals biological pathways associated with Williams syndrome.

BACKGROUND: Williams syndrome (WS) is a neurodevelopmental disorder that has been attributed to heterozygous deletions in chromosome 7q11.23 and exhibits a variety of physical, cognitive, and behavioral features. However, the genetic basis of this phenotypic variability is unclear. In this study, we identified genetic clues underlying these complex phenotypes. METHODS: Neurobehavioral function was assessed in WS patients and healthy controls. Total RNA was extracted from peripheral blood and subjected to microarray analysis, RNA-sequencing, and qRT-PCR. Weighted gene co-expression network analysis was performed to identify specific alterations related to intermediate disease phenotypes. To functionally interpret each WS-related module, gene ontology and disease-related gene enrichment were examined. We also investigated the micro (mi)RNA expression profiles and miRNA co-expression networks to better explain the regulation of the transcriptome in WS. RESULTS: Our analysis identified four significant co-expression modules related to intermediate WS phenotypes. Notably, the three upregulated WS-related modules were composed exclusively of genes located outside the 7q11.23 region. They were significantly enriched in genes related to B-cell activation, RNA processing, and RNA transport. BCL11A, which is known for its association with speech disorders and intellectual disabilities, was identified as one of the hub genes in the top WS-related module. Finally, these key upregulated mRNA co-expression modules appear to be inversely correlated with a specific downregulated WS-related miRNA co-expression module. CONCLUSIONS: Dysregulation of the mRNA/miRNA network involving genes outside of the 7q11.23 region is likely related to the complex phenotypes observed in WS patients.

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.

Specific responses of human hippocampal neurons are associated with better memory.

A population of human hippocampal neurons has shown responses to individual concepts (e.g., Jennifer Aniston) that generalize to different instances of the concept. However, recordings from the rodent hippocampus suggest an important function of these neurons is their ability to discriminate overlapping representations, or pattern separate, a process that may facilitate discrimination of similar events for successful memory. In the current study, we explored whether human hippocampal neurons can also demonstrate the ability to discriminate between overlapping representations and whether this selectivity could be directly related to memory performance. We show that among medial temporal lobe (MTL) neurons, certain populations of neurons are selective for a previously studied (target) image in that they show a significant decrease in firing rate to very similar (lure) images. We found that a greater proportion of these neurons can be found in the hippocampus compared with other MTL regions, and that memory for individual items is correlated to the degree of selectivity of hippocampal neurons responsive to those items. Moreover, a greater proportion of hippocampal neurons showed selective firing for target images in good compared with poor performers, with overall memory performance correlated with hippocampal selectivity. In contrast, selectivity in other MTL regions was not associated with memory performance. These findings show that a substantial proportion of human hippocampal neurons encode specific memories that support the discrimination of overlapping representations. These results also provide previously unidentified evidence consistent with a unique role of the human hippocampus in orthogonalization of representations in declarative memory.

Spatiotemporal dynamics of the postnatal developing primate brain transcriptome.

Developmental changes in the temporal and spatial regulation of gene expression drive the emergence of normal mature brain function, while disruptions in these processes underlie many neurodevelopmental abnormalities. To solidify our foundational knowledge of such changes in a primate brain with an extended period of postnatal maturation like in human, we investigated the whole-genome transcriptional profiles of rhesus monkey brains from birth to adulthood. We found that gene expression dynamics are largest from birth through infancy, after which gene expression profiles transition to a relatively stable state by young adulthood. Biological pathway enrichment analysis revealed that genes more highly expressed at birth are associated with cell adhesion and neuron differentiation, while genes more highly expressed in juveniles and adults are associated with cell death. Neocortex showed significantly greater differential expression over time than subcortical structures, and this trend likely reflects the protracted postnatal development of the cortex. Using network analysis, we identified 27 co-expression modules containing genes with highly correlated expression patterns that are associated with specific brain regions, ages or both. In particular, one module with high expression in neonatal cortex and striatum that decreases during infancy and juvenile development was significantly enriched for autism spectrum disorder (ASD)-related genes. This network was enriched for genes associated with axon guidance and interneuron differentiation, consistent with a disruption in the formation of functional cortical circuitry in ASD.

RBFOX1 regulates both splicing and transcriptional networks in human neuronal development.

RNA splicing plays a critical role in the programming of neuronal differentiation and, consequently, normal human neurodevelopment, and its disruption may underlie neurodevelopmental and neuropsychiatric disorders. The RNA-binding protein, fox-1 homolog (RBFOX1; also termed A2BP1 or FOX1), is a neuron-specific splicing factor predicted to regulate neuronal splicing networks clinically implicated in neurodevelopmental disease, including autism spectrum disorder (ASD), but only a few targets have been experimentally identified. We used RNA sequencing to identify the RBFOX1 splicing network at a genome-wide level in primary human neural stem cells during differentiation. We observe that RBFOX1 regulates a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins. Downstream alterations in gene expression define an additional transcriptional network regulated by RBFOX1 involved in neurodevelopmental pathways remarkably parallel to those affected by splicing. Several of these differentially expressed genes are further implicated in ASD and related neurodevelopmental diseases. Weighted gene co-expression network analysis demonstrates a high degree of connectivity among these disease-related genes, highlighting RBFOX1 as a key factor coordinating the regulation of both neurodevelopmentally important alternative splicing events and clinically relevant neuronal transcriptional programs in the development of human neurons.

Transcriptional profiling in microglia across physiological and pathological states identifies a transcriptional module associated with neurodegeneration.

Microglia are the resident immune cells of the central nervous system and are involved in brain development, homeostasis, and disease. New imaging and genomics technologies are revealing microglial complexity across developmental and functional states, brain regions, and diseases. We curated a set of publicly available gene expression datasets from human microglia spanning disease and health to identify sets of genes reflecting physiological and pathological microglial states. We also integrated multiple human microglial single-cell RNA-seq datasets in Alzheimer's disease (AD), multiple sclerosis (MS), and Parkinson's disease, and identified a distinct microglial transcriptional signature shared across diseases. Analysis of germ-line DNA identified genes with variants associated with AD and MS that are overrepresented in microglial gene sets, including the disease-associated transcriptional signature. This work points to genes that are dysregulated in disease states and provides a resource for the analysis of diseases in which microglia are implicated by genetic evidence.