Two new studies experimentally demonstrate how ancient genomic
duplications of synaptic genes provided the substrate for diversification that
ultimately expanded vertebrate cognitive complexity.
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 co-expression 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 translational regulation by FMRP and transcriptional co-regulation 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 novel biological framework for investigating the pathophysiology of ASD.
gene networks; systems biology; exome; rare variants; Intellectual disability; human cortical development; gene expression; FMRP; Satb1; MEF2; RNA-seq
Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising extensive histological analysis and comprehensive microarray profiling of ~900 neuroanatomically precise subdivisions in two individuals. Transcriptional regulation varies enormously by anatomical location, with different regions and their constituent cell types displaying robust molecular signatures that are highly conserved between individuals. Analysis of differential gene expression and gene co-expression relationships demonstrates that brain-wide variation strongly reflects the distributions of major cell classes such as neurons, oligodendrocytes, astrocytes and microglia. Local neighbourhood relationships between fine anatomical subdivisions are associated with discrete neuronal subtypes and genes involved with synaptic transmission. The neocortex displays a relatively homogeneous transcriptional pattern, but with distinct features associated selectively with primary sensorimotor cortices and with enriched frontal lobe expression. Notably, the spatial topography of the neocortex is strongly reflected in its molecular topography— the closer two cortical regions, the more similar their transcriptomes. This freely accessible online data resource forms a high-resolution transcriptional baseline for neurogenetic studies of normal and abnormal human brain function.
Neuroscience; Genetics; Genomics; Databases
To understand the emergence of human higher cognition, we must understand its biological substrate—the cerebral cortex, which considers itself the crowning achievement of evolution. Here, we describe how advances in developmental neurobiology, coupled with those in genetics, including adaptive protein evolution via gene duplications and the emergence of novel regulatory elements, can provide insights into the evolutionary mechanisms culminating in the human cerebrum. Given that the massive expansion of the cortical surface and elaboration of its connections in humans originates from developmental events, understanding the genetic regulation of cell number, neuronal migration to proper layers, columns, and regions, and ultimately their differentiation into specific phenotypes, is critical. The pre- and postnatal environment also interacts with the cellular substrate to yield a basic network that is refined via selection and elimination of synaptic connections, a process that is prolonged in humans. This knowledge provides essential insight into the pathogenesis of human-specific neuropsychiatric disorders.
The anatomical and functional architecture of the human brain is largely determined by prenatal transcriptional processes. We describe an anatomically comprehensive atlas of mid-gestational human brain, including de novo reference atlases, in situ hybridization, ultra-high resolution magnetic resonance imaging (MRI) and microarray analysis on highly discrete laser microdissected brain regions. In developing cerebral cortex, transcriptional differences are found between different proliferative and postmitotic layers, wherein laminar signatures reflect cellular composition and developmental processes. Cytoarchitectural differences between human and mouse have molecular correlates, including species differences in gene expression in subplate, although surprisingly we find minimal differences between the inner and human-expanded outer subventricular zones. Both germinal and postmitotic cortical layers exhibit fronto-temporal gradients, with particular enrichment in frontal lobe. Finally, many neurodevelopmental disorder and human evolution-related genes show patterned expression, potentially underlying unique features of human cortical formation. These data provide a rich, freely-accessible resource for understanding human brain development.
Human brain; Transcriptome; Microarray; Development; Gene expression; Evolution
Common genetic variation and rare mutations in genes encoding calcium channel subunits have pleiotropic effects on risk for multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. To gain further mechanistic insights by extending previous gene expression data, we constructed co-expression networks in Timothy syndrome (TS), a monogenic condition with high penetrance for ASD, caused by mutations in the L-type calcium channel, Cav1.2.
To identify patient-specific alterations in transcriptome organization, we conducted a genome-wide weighted co-expression network analysis (WGCNA) on neural progenitors and neurons from multiple lines of induced pluripotent stem cells (iPSC) derived from normal and TS (G406R in CACNA1C) individuals. We employed transcription factor binding site enrichment analysis to assess whether TS associated co-expression changes reflect calcium-dependent co-regulation.
We identified reproducible developmental and activity-dependent gene co-expression modules conserved in patient and control cell lines. By comparing cell lines from case and control subjects, we also identified co-expression modules reflecting distinct aspects of TS, including intellectual disability and ASD-related phenotypes. Moreover, by integrating co-expression with transcription factor binding analysis, we showed the TS-associated transcriptional changes were predicted to be co-regulated by calcium-dependent transcriptional regulators, including NFAT, MEF2, CREB, and FOXO, thus providing a mechanism by which altered Ca2+ signaling in TS patients leads to the observed molecular dysregulation.
We applied WGCNA to construct co-expression networks related to neural development and depolarization in iPSC-derived neural cells from TS and control individuals for the first time. These analyses illustrate how a systems biology approach based on gene networks can yield insights into the molecular mechanisms of neural development and function, and provide clues as to the functional impact of the downstream effects of Ca2+ signaling dysregulation on transcription.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-014-0075-5) contains supplementary material, which is available to authorized users.
Purpose of review
A strong male bias in autism spectrum disorder (ASD) prevalence has been observed with striking consistency, but no mechanism has yet to definitively account for this sex difference. Toward the pursuit of a more complete understanding of the biological basis for sex-differential risk, this review explores the current status of epidemiological, genetic, and neuroendocrinological work addressing ASD prevalence and liability in males and females.
Recent studies continue to report a male bias in ASD prevalence, but also suggest that sex differences in phenotypic presentation, including fewer restricted and repetitive behaviors and externalizing behavioral problems in females, may contribute to this bias. Genetic studies demonstrate that females are protected from the effects of heritable and de novo ASD risk variants, and compelling work suggests that sex chromosomal genes and/or sex hormones, especially testosterone, may modulate the effects of genetic variation on the presentation of an autistic phenotype.
ASDs affect females less frequently than males, and several sex-differential genetic and hormonal factors may contribute. Future work to determine the mechanisms by which these factors confer risk and protection to males and females is essential.
autism spectrum disorders; sex differences; hormones; sex chromosomes; genetic liability
The aetiology and pathogenesis of non-genetic forms of frontotemporal dementia (FTD) is unknown and even with the genetic forms of FTD, pathogenesis remains elusive. Given the association between systemic inflammation and other neurodegenerative processes, links between autoimmunity and FTD need to be explored.
To describe the prevalence of systemic autoimmune disease in semantic variant primary progressive aphasia (svPPA), a clinical cohort, and in progranulin (PGRN) mutation carriers compared to neurologically healthy normal controls (NC) and Alzheimer’s disease (AD) as dementia controls.
Academic medical centres.
129 svPPA, 39 PGRN, 186 NC, and 158 AD patients underwent chart review for autoimmune conditions. A large subset of svPPA, PGRN, and NC cohorts underwent serum analysis for tumor necrosis factor α (TNF-α) levels.
Chi-square comparison of autoimmune prevalence and follow up logistic regression.
There was a significantly increased risk of autoimmune disorders clustered around inflammatory arthritides, cutaneous disorders, and gastrointestinal conditions in the svPPA and PGRN cohorts. Elevated TNF-α levels were observed in svPPA and PGRN compared to NC.
svPPA and PGRN are associated with increased prevalence of specific and related autoimmune diseases compared to NC and AD. These findings suggest a unique pattern of systemic inflammation in svPPA and PGRN and open new research avenues for understanding and treating disorders associated with underlying transactive response DNA-binding protein 43 (TDP-43) aggregation.
Autism spectrum disorder (ASD) represents a heterogeneous group of disorders, which presents a substantial challenge to diagnosis and treatment. Over the past decade, considerable progress has been made in the identification of genetic risk factors for ASD that define specific mechanisms and pathways underlying the associated behavioural deficits. In this Review, we discuss how some of the latest advances in the genetics of ASD have facilitated parsing of the phenotypic heterogeneity of this disorder. We argue that only through such advances will we begin to define endophenotypes that can benefit from targeted, hypothesis-driven treatments. We review the latest technologies used to identify and characterize the genetics underlying ASD and then consider three themes—single-gene disorders, the gender bias in ASD, and the genetics of neurological comorbidities—that highlight ways in which we can use genetics to define the many phenotypes within the autism spectrum. We also present current clinical guidelines for genetic testing in ASD and their implications for prognosis and treatment.
Previous studies demonstrated that Schwann cells (SCs) express distinct motor and sensory phenotypes, which impact the ability of these pathways to selectively support regenerating neurons. In the present study, unbiased microarray analysis was used to examine differential gene expression in denervated motor and sensory pathways in rats. Several genes that were significantly upregulated in either denervated sensory or motor pathways were identified and two secreted factors were selected for further analysis: osteopontin (OPN) and clusterin (CLU) which were upregulated in denervated motor and sensory pathways, respectively. Sciatic nerve transection induced upregulation of OPN and CLU and expression of both returned to baseline levels with ensuing regeneration. In vitro analysis using exogenously applied OPN induced outgrowth of motor but not sensory neurons. CLU, however, induced outgrowth of sensory neurons, but not motor neurons. To assess the functional importance of OPN and CLU, peripheral nerve regeneration was examined in OPN and CLU−/− mice. When compared with OPN+/+ mice, motor neuron regeneration was reduced in OPN−/− mice. Impaired regeneration through OPN−/− peripheral nerves grafted into OPN+/+ mice indicated that loss of OPN in SCs was responsible for reduced motor regeneration. Sensory neuron regeneration was impaired in CLU−/− mice following sciatic nerve crush and impaired regeneration nerve fibers through CLU−/− nerve grafts transplanted into CLU+/+ mice indicated that reduced sensory regeneration is likely due to SC-derived CLU. Together, these studies suggest unique roles for SC-derived OPN and CLU in regeneration of peripheral motor and sensory axons.
Understanding human-specific patterns of brain gene expression and regulation can provide key insights into human brain evolution and speciation. Here, we use next generation sequencing, and Illumina and Affymetrix microarray platforms, to compare the transcriptome of human, chimpanzee, and macaque telencephalon. Our analysis reveals a predominance of genes differentially expressed within human frontal lobe and a striking increase in transcriptional complexity specific to the human lineage in the frontal lobe. In contrast, caudate nucleus gene expression is highly conserved. We also identify gene co-expression signatures related to either neuronal processes or neuropsychiatric diseases, including a human-specific module with CLOCK as its hub gene and another module enriched for neuronal morphological processes and genes co-expressed with FOXP2, a gene important for language evolution. These data demonstrate that transcriptional networks have undergone evolutionary remodeling even within a given brain region, providing a new window through which to view the foundation of uniquely human cognitive capacities.
Induced pluripotent stem cell-derived neurons from patients promise to fill an important niche between studies in humans and model organisms in deciphering mechanisms and identifying therapeutic avenues for neurologic and psychiatric diseases. Recent work begins to tap this potential, and also highlights challenges that must be overcome for it to be fully realized.
Autism spectrum disorder (ASD) is a highly heritable, behaviorally defined, heterogeneous disorder of unknown pathogenesis. Several genetic risk genes have been identified, including the gene encoding the receptor tyrosine kinase MET, which regulates neuronal differentiation and growth. An ASD-associated polymorphism disrupts MET gene transcription, and there are reduced levels of MET protein expression in the mature temporal cortex of subjects with ASD. To address the possible neurodevelopmental contribution of MET to ASD pathogenesis, we examined the expression and transcriptional regulation of MET by a transcription factor, FOXP2, which is implicated in regulation of cognition and language, two functions altered in ASD. MET mRNA expression in the midgestation human fetal cerebral cortex is strikingly restricted, localized to portions of the temporal and occipital lobes. With in the cortical plate of the temporal lobe, the pattern of MET expression is highly complementary to the expression pattern of FOXP2, suggesting the latter may play a role in repression of gene expression. Consistent with this, MET and FOXP2 also are reciprocally expressed by differentiating normal human neuronal progenitor cells (NHNPs) in vitro, leading us to assess whether FOXP2 transcriptionally regulates MET. Indeed, FOXP2 binds directly to the 5′ regulatory region of MET, and overexpression of FOXP2 results in transcriptional repression of MET. The expression of MET in restricted human neocortical regions, and its regulation in part by FOXP2, is consistent with genetic evidence for MET contributing to ASD risk.
Progranulin (GRN) mutations cause frontotemporal dementia (FTD), but GRN’s function in the CNS remains largely unknown. To identify the pathways downstream of GRN, we used weighted gene co-expression network analysis (WGCNA) to develop a systems-level view of transcriptional alterations in a human neural progenitor model of GRN-deficiency. This highlighted key pathways such as apoptosis and ubiquitination in GRN deficient human neurons, while revealing an unexpected major role for the Wnt signaling pathway, which was confirmed by analysis of gene expression data from postmortem FTD brain. Furthermore, we observed that the Wnt receptor Fzd2 was one of only a few genes up-regulated at 6 weeks in a GRN knockout mouse, and that FZD2 reduction caused increased apoptosis, while its upregulation promoted neuronal survival in vitro. Together, these in vitro and in vivo data point to an adaptive role for altered Wnt signaling in GRN deficiency-mediated FTD, representing a potential therapeutic target.
Progranulin; Frontotemporal Dementia; Wnt; Fzd2; WGCNA
Familial idiopathic basal ganglia calcification (IBGC) or Fahr’s
disease is a rare neurodegenerative disorder characterized by calcium deposits
in the basal ganglia and other brain regions, which is associated with
neuropsychiatric and motor symptoms. Familial IBGC is genetically heterogeneous
and typically transmitted in an autosomal dominant fashion. We performed a
mutational analysis of SLC20A2, the first gene found to cause
IBGC, to assess its genetic contribution to familial IBGC. We recruited 218
subjects from 29 IBGC-affected families of varied ancestry and collected medical
history, neurological exam, and head CT scans to characterize each
patient’s disease status. We screened our patient cohort for mutations
in SLC20A2. Twelve novel (nonsense, deletions, missense, and
splice site) potentially pathogenic variants, one synonymous variant, and one
previously reported mutation were identified in 13 families. Variants predicted
to be deleterious cosegregated with disease in five families. Three families
showed nonsegregation with clinical disease of such variants, but retrospective
review of clinical and neuroimaging data strongly suggested previous
misclassification. Overall, mutations in SLC20A2 account for as
many as 41 % of our familial IBGC cases. Our screen in a large series
expands the catalog of SLC20A2 mutations identified to date and
demonstrates that mutations in SLC20A2 are a major cause of
familial IBGC. Non-perfect segregation patterns of predicted deleterious
variants highlight the challenges of phenotypic assessment in this condition
with highly variable clinical presentation.
Basal ganglia calcification; Fahr’s; Genetics; Sequencing; Mutations
Validating the potential pathogenicity of copy number variants (CNVs) identified in genome-wide studies of autism spectrum disorders (ASD) requires detailed assessment of case/control frequencies, inheritance patterns, clinical correlations, and functional impact. Here, we characterize a small recurrent duplication in the annexin A1 (ANXA1) gene, identified by the Autism Genome Project (AGP) study.
From the AGP CNV genomic screen in 2,147 ASD individuals, we selected for characterization an ANXA1 gene duplication that was absent in 4,964 population-based controls. We further screened the duplication in a follow-up sample including 1,496 patients and 410 controls, and evaluated clinical correlations and family segregation. Sequencing of exonic/downstream ANXA1 regions was performed in 490 ASD patients for identification of additional variants.
The ANXA1 duplication, overlapping the last four exons and 3’UTR region, had an overall prevalence of 11/3,643 (0.30%) in unrelated ASD patients but was not identified in 5,374 controls. Duplication carriers presented no distinctive clinical phenotype. Family analysis showed neuropsychiatric deficits and ASD traits in multiple relatives carrying the duplication, suggestive of a complex genetic inheritance. Sequencing of exonic regions and the 3’UTR identified 11 novel changes, but no obvious variants with clinical significance.
We provide multilevel evidence for a role of ANXA1 in ASD etiology. Given its important role as mediator of glucocorticoid function in a wide variety of brain processes, including neuroprotection, apoptosis, and control of the neuroendocrine system, the results add ANXA1 to the growing list of rare candidate genetic etiological factors for ASD.
ANXA1; Autism; Brain homeostasis; Copy number variants; Duplication; Glucocorticoids
Little is known about how changes in DNA methylation mediate risk for human diseases including dementia. Analysis of genome-wide methylation patterns in patients with two forms of tau-related dementia – progressive supranuclear palsy (PSP) and frontotemporal dementia (FTD) – revealed significant differentially methylated probes (DMPs) in patients versus unaffected controls. Remarkably, DMPs in PSP were clustered within the 17q21.31 region, previously known to harbor the major genetic risk factor for PSP. We identified and replicated a dose-dependent effect of the risk-associated H1 haplotype on methylation levels within the region in blood and brain. These data reveal that the H1 haplotype increases risk for tauopathy via differential methylation at that locus, indicating a mediating role for methylation in dementia pathophysiology.
Progressive supranuclear palsy (PSP) and frontotemporal dementia (FTD) are two neurodegenerative diseases linked, at the pathologic and genetic level, to the microtubule associated protein tau. We studied epigenetic changes (DNA methylation levels) in peripheral blood from patients with PSP, FTD, and unaffected controls. Analysis of genome-wide methylation patterns revealed significant differentially methylated probes in patients versus unaffected controls. Remarkably, differentially methylated probes in PSP vs. controls were preferentially clustered within the 17q21.31 region, previously known to harbor the major genetic risk factor for PSP. We identified and replicated a dose-dependent effect of the risk-associated H1 haplotype on methylation levels within the region in independent datasets in blood and brain. These data reveal that the H1 haplotype increases risk for tauopathy via differential methylation, indicating a mediating role for methylation in dementia pathophysiology.
ataxia; cerebellum; PDYN; SCA23; MSA; multiple system atrophy
Human neural progenitors from a variety of sources present new opportunities to model aspects of human neuropsychiatric disease in vitro. Such in vitro models provide the advantages of a human genetic background, combined with rapid and easy manipulation, making them highly useful adjuncts to animal models. Here, we examined whether a human neuronal culture system could be utilized to assess the transcriptional program involved in human neural differentiation and in modeling some of the molecular features of a neurodevelopmental disorder, such as autism. Primary normal human neuronal progenitors (NHNPs) were differentiated into a post-mitotic neuronal state through addition of specific growth factors and whole-genome gene expression was examined throughout a time course of neuronal differentiation. After four weeks of differentiation, a significant number of genes associated with autism spectrum disorders (ASD) are either induced or repressed. This includes the ASD susceptibility gene neurexin 1, which showed a distinct pattern from neurexin 3 in vitro, and which we validated in vivo in fetal human brain. Using weighted gene co-expression network analysis (WGCNA), we visualized the network structure of transcriptional regulation, demonstrating via this unbiased analysis that a significant number of ASD candidate genes are coordinately regulated during the differentiation process. Since NHNPs are genetically tractable and manipulable, they can be used to study both the effects of mutations in multiple ASD candidate genes on neuronal differentiation and gene expression in combination with the effects of potential therapeutic molecules. These data also provide a step towards better understanding of the signaling pathways disrupted in ASD.
Model system; neuropsychiatric disease; pharmacogenomics; high-throughput drug screen; neurodevelopment
Autism spectrum disorders (ASDs) are male-biased and genetically heterogeneous. While sequencing of sporadic cases has identified de novo risk variants, the heritable genetic contribution and mechanisms driving the male bias are less understood. Here, we aimed to identify familial and sex-differential risk loci in the largest available, uniformly ascertained, densely genotyped sample of multiplex ASD families from the Autism Genetics Resource Exchange (AGRE), and to compare results with earlier findings from AGRE.
From a total sample of 1,008 multiplex families, we performed genome-wide, non-parametric linkage analysis in a discovery sample of 847 families, and separately on subsets of families with only male, affected children (male-only, MO) or with at least one female, affected child (female-containing, FC). Loci showing evidence for suggestive linkage (logarithm of odds ≥2.2) in this discovery sample, or in previous AGRE samples, were re-evaluated in an extension study utilizing all 1,008 available families. For regions with genome-wide significant linkage signal in the discovery stage, those families not included in the corresponding discovery sample were then evaluated for independent replication of linkage. Association testing of common single nucleotide polymorphisms (SNPs) was also performed within suggestive linkage regions.
We observed an independent replication of previously observed linkage at chromosome 20p13 (P < 0.01), while loci at 6q27 and 8q13.2 showed suggestive linkage in our extended sample. Suggestive sex-differential linkage was observed at 1p31.3 (MO), 8p21.2 (FC), and 8p12 (FC) in our discovery sample, and the MO signal at 1p31.3 was supported in our expanded sample. No sex-differential signals met replication criteria, and no common SNPs were significantly associated with ASD within any identified linkage regions.
With few exceptions, analyses of subsets of families from the AGRE cohort identify different risk loci, consistent with extreme locus heterogeneity in ASD. Large samples appear to yield more consistent results, and sex-stratified analyses facilitate the identification of sex-differential risk loci, suggesting that linkage analyses in large cohorts are useful for identifying heritable risk loci. Additional work, such as targeted re-sequencing, is needed to identify the specific variants within these loci that are responsible for increasing ASD risk.
Male brain; Sex differences; Intermediate phenotype; Linkage analysis; Association; AGRE
In this issue of Neuron, two papers (Lim et al. 2013, Yu et al. 2013) use whole exome sequencing (WES) to elucidate the contribution of inherited variation to the risk for autism by leveraging the increased penetrance of homozygous and compound heterozygous rare variants in autosomes and hemizygous rare variants in the X chromosome of males. Together, they expand our knowledge about the genetic architecture of ASD, verify previously identified genes, and identify novel mutations that will guide the discovery of the critical biological processes disrupted in autism.
Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of β-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information–based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer’s disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.
spinocerebellar ataxia; cerebellum; C9ORF72