Sex-Differential Gene Expression in Developing Human Cortex and Its Intersection With Autism Risk Pathways.

Background: Sex-differential biology may contribute to the consistently male-biased prevalence of autism spectrum disorder (ASD). Gene expression differences between males and females in the brain can indicate possible molecular and cellular mechanisms involved, although transcriptomic sex differences during human prenatal cortical development have been incompletely characterized, primarily due to small sample sizes. Methods: We performed a meta-analysis of sex-differential expression and co-expression network analysis in 2 independent bulk RNA sequencing datasets generated from cortex of 273 prenatal donors without known neuropsychiatric disorders. To assess the intersection between neurotypical sex differences and neuropsychiatric disorder biology, we tested for enrichment of ASD-associated risk genes and expression changes, neuropsychiatric disorder risk genes, and cell type markers within identified sex-differentially expressed genes (sex-DEGs) and sex-differential co-expression modules. Results: We identified 101 significant sex-DEGs, including Y-chromosome genes, genes impacted by X-chromosome inactivation, and autosomal genes. Known ASD risk genes, implicated by either common or rare variants, did not preferentially overlap with sex-DEGs. We identified 1 male-specific co-expression module enriched for immune signaling that is unique to 1 input dataset. Conclusions: Sex-differential gene expression is limited in prenatal human cortex tissue, although meta-analysis of large datasets allows for the identification of sex-DEGs, including autosomal genes that encode proteins involved in neural development. Lack of sex-DEG overlap with ASD risk genes in the prenatal cortex suggests that sex-differential modulation of ASD symptoms may occur in other brain regions, at other developmental stages, or in specific cell types, or may involve mechanisms that act downstream from mutation-carrying genes.

Males are more commonly diagnosed with autism spectrum disorder than females, and sex differences in brain development may contribute to this difference. Here, we define differences in gene expression patterns between males and females in human prenatal brain tissue from 273 donors to identify 101 genes that are expressed at different levels in males and females and gene sets that show sex-specific expression correlations. Genes with autism-associated DNA variants and genes with altered expression in autism do not preferentially overlap with sex-differential genes, suggesting that sex-differential biology may influence autism risk mechanisms in other brain regions, at other developmental stages, or in specific cell types.

Genomic data resources of the Brain Somatic Mosaicism Network for neuropsychiatric diseases.

Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Chronic alcohol exposure alters gene expression in HepG2 cells.

BACKGROUND: The liver is the primary site of alcohol metabolism and is highly vulnerable to injuries due to chronic alcohol abuse. Several molecular mechanisms, including oxidative stress and altered cellular metabolism, have been implicated in the development and progression of alcoholic liver disease. We sought to gain further insight into the molecular pathogenesis by studying the effects of ethanol exposure on the global gene expression in HepG2 cells. METHODS: HepG2 cells were cultured in the presence or absence of 75 mM ethanol for 9 days, with fresh media daily. Global gene expression changes were studied using Affymetrix GeneChip(®) Human Exon 1.0 ST Arrays. Gene expression differences were validated for 13 genes by quantitative real-time RT-PCR. To identify biological pathways affected by ethanol treatment, differentially expressed genes were analyzed by Ingenuity Pathway Analysis software. RESULTS: Long-term ethanol exposure altered the expression of 1,093 genes (false discovery rate ≤ 3%); many of these changes were modest. Long-term ethanol exposure affected several pathways, including acute phase response, amino acid metabolism, carbohydrate metabolism, and lipid metabolism. CONCLUSIONS: Global measurements of gene expression show that a large number of genes are affected by chronic ethanol, although most show modest effect. These data provide insight into the molecular pathology resulting from extended alcohol exposure.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

Neuronal and glial 3D chromatin architecture informs the cellular etiology of brain disorders.

Cellular heterogeneity in the human brain obscures the identification of robust cellular regulatory networks, which is necessary to understand the function of non-coding elements and the impact of non-coding genetic variation. Here we integrate genome-wide chromosome conformation data from purified neurons and glia with transcriptomic and enhancer profiles, to characterize the gene regulatory landscape of two major cell classes in the human brain. We then leverage cell-type-specific regulatory landscapes to gain insight into the cellular etiology of several brain disorders. We find that Alzheimer's disease (AD)-associated epigenetic dysregulation is linked to neurons and oligodendrocytes, whereas genetic risk factors for AD highlighted microglia, suggesting that different cell types may contribute to disease risk, via different mechanisms. Moreover, integration of glutamatergic and GABAergic regulatory maps with genetic risk factors for schizophrenia (SCZ) and bipolar disorder (BD) identifies shared (parvalbumin-expressing interneurons) and distinct cellular etiologies (upper layer neurons for BD, and deeper layer projection neurons for SCZ). Collectively, these findings shed new light on cell-type-specific gene regulatory networks in brain disorders.

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse brain.

BACKGROUND: Genetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain. METHODS: RNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples. RESULTS: In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A, a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories. CONCLUSIONS: Exon usage in SCN1A, SCN2A, SCN3A, and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.

A regulatory variation in OPRK1, the gene encoding the kappa-opioid receptor, is associated with alcohol dependence.

Variations in OPRK1, which encodes the kappa-opioid receptor, are associated with the risk for alcohol dependence. Sequencing DNAs with higher and lower risk haplotypes revealed an insertion/deletion (indel) with a net addition of 830 bp located 1986 bp upstream of the translation start site (1389 bp upstream of the transcription start site). We demonstrated that the upstream region extending from -1647 to -10 bp or from -2312 to -10 bp (relative to the translation start site) could function as a promoter in transient transfection assays. We then determined that the presence of the indel reduced transcriptional activity by half. We used a PCR assay to genotype individuals in 219 multiplex alcohol-dependent families of European American descent for the presence or absence of this indel. Family-based association analyses detected significant evidence of association of this insertion with alcoholism; the longer allele (with the indel), which had lower expression, is associated with higher risk for alcoholism. This indel is, therefore, a functional regulatory variation likely to explain at least part of the association of OPRK1 with alcohol dependence.

Neural Stem Cells Direct Axon Guidance via Their Radial Fiber Scaffold.

Neural stem cells directly or indirectly generate all neurons and macroglial cells and guide migrating neurons by using a palisade-like scaffold made of their radial fibers. Here, we describe an unexpected role for the radial fiber scaffold in directing corticospinal and other axons at the junction between the striatum and globus pallidus. The maintenance of this scaffold, and consequently axon pathfinding, is dependent on the expression of an atypical RHO-GTPase, RND3/RHOE, together with its binding partner ARHGAP35/P190A, a RHO GTPase-activating protein, in the radial glia-like neural stem cells within the ventricular zone of the medial ganglionic eminence. This role is independent of RND3 and ARHGAP35 expression in corticospinal neurons, where they regulate dendritic spine formation, axon elongation, and pontine midline crossing in a FEZF2-dependent manner. The prevalence of neural stem cell scaffolds and their expression of RND3 and ARHGAP35 suggests that these observations might be broadly relevant for axon guidance and neural circuit formation.

Whole-Genome and RNA Sequencing Reveal Variation and Transcriptomic Coordination in the Developing Human Prefrontal Cortex.

Gene expression levels vary across developmental stage, cell type, and region in the brain. Genomic variants also contribute to the variation in expression, and some neuropsychiatric disorder loci may exert their effects through this mechanism. To investigate these relationships, we present BrainVar, a unique resource of paired whole-genome and bulk tissue RNA sequencing from the dorsolateral prefrontal cortex of 176 individuals across prenatal and postnatal development. Here we identify common variants that alter gene expression (expression quantitative trait loci [eQTLs]) constantly across development or predominantly during prenatal or postnatal stages. Both "constant" and "temporal-predominant" eQTLs are enriched for loci associated with neuropsychiatric traits and disorders and colocalize with specific variants. Expression levels of more than 12,000 genes rise or fall in a concerted late-fetal transition, with the transitional genes enriched for cell-type-specific genes and neuropsychiatric risk loci, underscoring the importance of cataloging developmental trajectories in understanding cortical physiology and pathology.

Transcriptome and epigenome landscape of human cortical development modeled in organoids.

Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.

Genome-wide de novo risk score implicates promoter variation in autism spectrum disorder.

Whole-genome sequencing (WGS) has facilitated the first genome-wide evaluations of the contribution of de novo noncoding mutations to complex disorders. Using WGS, we identified 255,106 de novo mutations among sample genomes from members of 1902 quartet families in which one child, but not a sibling or their parents, was affected by autism spectrum disorder (ASD). In contrast to coding mutations, no noncoding functional annotation category, analyzed in isolation, was significantly associated with ASD. Casting noncoding variation in the context of a de novo risk score across multiple annotation categories, however, did demonstrate association with mutations localized to promoter regions. We found that the strongest driver of this promoter signal emanates from evolutionarily conserved transcription factor binding sites distal to the transcription start site. These data suggest that de novo mutations in promoter regions, characterized by evolutionary and functional signatures, contribute to ASD.

Integrative functional genomic analysis of human brain development and neuropsychiatric risks.

To broaden our understanding of human neurodevelopment, we profiled transcriptomic and epigenomic landscapes across brain regions and/or cell types for the entire span of prenatal and postnatal development. Integrative analysis revealed temporal, regional, sex, and cell type-specific dynamics. We observed a global transcriptomic cup-shaped pattern, characterized by a late fetal transition associated with sharply decreased regional differences and changes in cellular composition and maturation, followed by a reversal in childhood-adolescence, and accompanied by epigenomic reorganizations. Analysis of gene coexpression modules revealed relationships with epigenomic regulation and neurodevelopmental processes. Genes with genetic associations to brain-based traits and neuropsychiatric disorders (including MEF2C, SATB2, SOX5, TCF4, and TSHZ3) converged in a small number of modules and distinct cell types, revealing insights into neurodevelopment and the genomic basis of neuropsychiatric risks.

An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder.

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.

Control of species-dependent cortico-motoneuronal connections underlying manual dexterity.

Superior manual dexterity in higher primates emerged together with the appearance of cortico-motoneuronal (CM) connections during the evolution of the mammalian corticospinal (CS) system. Previously thought to be specific to higher primates, we identified transient CM connections in early postnatal mice, which are eventually eliminated by Sema6D-PlexA1 signaling. PlexA1 mutant mice maintain CM connections into adulthood and exhibit superior manual dexterity as compared with that of controls. Last, differing PlexA1 expression in layer 5 of the motor cortex, which is strong in wild-type mice but weak in humans, may be explained by FEZF2-mediated cis-regulatory elements that are found only in higher primates. Thus, species-dependent regulation of PlexA1 expression may have been crucial in the evolution of mammalian CS systems that improved fine motor control in higher primates.

The Cellular and Molecular Landscapes of the Developing Human Central Nervous System.

The human CNS follows a pattern of development typical of all mammals, but certain neurodevelopmental features are highly derived. Building the human CNS requires the precise orchestration and coordination of myriad molecular and cellular processes across a staggering array of cell types and over a long period of time. Dysregulation of these processes affects the structure and function of the CNS and can lead to neurological or psychiatric disorders. Recent technological advances and increased focus on human neurodevelopment have enabled a more comprehensive characterization of the human CNS and its development in both health and disease. The aim of this review is to highlight recent advancements in our understanding of the molecular and cellular landscapes of the developing human CNS, with focus on the cerebral neocortex, and the insights these findings provide into human neural evolution, function, and dysfunction.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

Targeted DNA Sequencing from Autism Spectrum Disorder Brains Implicates Multiple Genetic Mechanisms.

Single nucleotide variants (SNVs), particularly loss-of-function mutations, are significant contributors to autism spectrum disorder (ASD) risk. Here we report the first systematic deep sequencing study of 55 postmortem ASD brains for SNVs in 78 known ASD candidate genes. Remarkably, even without parental samples, we find more ASD brains with mutations that are protein-altering (26/55 cases versus 12/50 controls, p = 0.015), deleterious (16/55 versus 5/50, p = 0.016), or loss-of-function (6/55 versus 0/50, p = 0.028) compared to controls, with recurrent deleterious mutations in ARID1B, SCN1A, SCN2A, and SETD2, suggesting these mutations contribute to ASD risk. In several cases, the identified mutations and medical records suggest syndromic ASD diagnoses. Two ASD and one Fragile X premutation case showed deleterious somatic mutations, providing evidence that somatic mutations occur in ASD cases, and supporting a model in which a combination of germline and/or somatic mutations may contribute to ASD risk on a case-by-case basis.

Variation in the ADH1B proximal promoter affects expression.

The primary pathway of metabolism of dietary alcohol is via its oxidation in liver by alcohol dehydrogenases (ADH). Differences in the ADH enzyme activity or levels of enzyme present could affect the risk for alcoholism. Regulatory variations have been shown to affect the promoter activity and thereby affect the risk for alcoholism. In this study the functional effects of the two SNPs (rs1159918 and rs1229982) in the proximal promoter region of ADH1B that were associated with alcoholism were explored. We examined the effects of five naturally occurring haplotypes on the promoter activity. We observed that a C to A change at rs1229982 increased promoter activity 1.4-fold.

Identification of a FOXA-dependent enhancer of human alcohol dehydrogenase 4 (ADH4).

Human alcohol dehydrogenase 4 (ADH4) is one of the key enzymes involved in the metabolism of alcohol. ADH4 is highly expressed in the liver, and previous studies have revealed several cis-acting elements in the proximal promoter region. In this study we have identified a distal upstream enhancer, 4E, of ADH4. In HepG2 human hepatoma cells, 4E increased the activity of an ADH4 basal promoter by 50-fold. 4E was cell-specific, as no enhancer activity was detected in a human lung cell line, H1299. We have narrowed the enhancer activity to a 565 bp region and have identified multiple liver-enriched transcription factor binding sites in the region. By electrophoretic mobility shift assays, we confirmed binding of FOXA proteins to these sites. Site-directed mutagenesis studies demonstrated that sites 1 and 4 have the biggest effect on enhancer function, and mutations in multiple sites have multiplicative effects. We also studied the effects of three variations in the minimal enhancer region. Two variations had a significant effect on enhancer activity, decreasing the activity to 0.6-fold, while one had small but significant effect. The differences in the functional activity in different haplotypes suggest that this region could play an important role in the risk for alcoholism.