Adaptive sequence divergence forged new neurodevelopmental enhancers in humans.

Searches for the genetic underpinnings of uniquely human traits have focused on human-specific divergence in conserved genomic regions, which reflects adaptive modifications of existing functional elements. However, the study of conserved regions excludes functional elements that descended from previously neutral regions. Here, we demonstrate that the fastest-evolved regions of the human genome, which we term "human ancestor quickly evolved regions" (HAQERs), rapidly diverged in an episodic burst of directional positive selection prior to the human-Neanderthal split, before transitioning to constraint within hominins. HAQERs are enriched for bivalent chromatin states, particularly in gastrointestinal and neurodevelopmental tissues, and genetic variants linked to neurodevelopmental disease. We developed a multiplex, single-cell in vivo enhancer assay to discover that rapid sequence divergence in HAQERs generated hominin-unique enhancers in the developing cerebral cortex. We propose that a lack of pleiotropic constraints and elevated mutation rates poised HAQERs for rapid adaptation and subsequent susceptibility to disease.

Mutations in Human Accelerated Regions Disrupt Cognition and Social Behavior.

Comparative analyses have identified genomic regions potentially involved in human evolution but do not directly assess function. Human accelerated regions (HARs) represent conserved genomic loci with elevated divergence in humans. If some HARs regulate human-specific social and behavioral traits, then mutations would likely impact cognitive and social disorders. Strikingly, rare biallelic point mutations-identified by whole-genome and targeted "HAR-ome" sequencing-showed a significant excess in individuals with ASD whose parents share common ancestry compared to familial controls, suggesting a contribution in 5% of consanguineous ASD cases. Using chromatin interaction sequencing, massively parallel reporter assays (MPRA), and transgenic mice, we identified disease-linked, biallelic HAR mutations in active enhancers for CUX1, PTBP2, GPC4, CDKL5, and other genes implicated in neural function, ASD, or both. Our data provide genetic evidence that specific HARs are essential for normal development, consistent with suggestions that their evolutionary changes may have altered social and/or cognitive behavior. PAPERCLIP.

The gut microbiota links disease to human genome evolution.

A large number of studies have established a causal relationship between the gut microbiota and human disease. In addition, the composition of the microbiota is substantially influenced by the human genome. Modern medical research has confirmed that the pathogenesis of various diseases is closely related to evolutionary events in the human genome. Specific regions of the human genome known as human accelerated regions (HARs) have evolved rapidly over several million years since humans diverged from a common ancestor with chimpanzees, and HARs have been found to be involved in some human-specific diseases. Furthermore, the HAR-regulated gut microbiota has undergone rapid changes during human evolution. We propose that the gut microbiota may serve as an important mediator linking diseases to human genome evolution.

Frontotemporal lobar degeneration targets brain regions linked to expression of recently evolved genes.

In frontotemporal lobar degeneration (FTLD), pathological protein aggregation in specific brain regions is associated with declines in human-specialized social-emotional and language functions. In most patients, disease protein aggregates contain either TDP-43 (FTLD-TDP) or tau (FTLD-tau). Here, we explored whether FTLD-associated regional degeneration patterns relate to regional gene expression of human accelerated regions (HARs), conserved sequences that have undergone positive selection during recent human evolution. To this end, we used structural neuroimaging from patients with FTLD and human brain regional transcriptomic data from controls to identify genes expressed in FTLD-targeted brain regions. We then integrated primate comparative genomic data to test our hypothesis that FTLD targets brain regions linked to expression levels of recently evolved genes. In addition, we asked whether genes whose expression correlates with FTLD atrophy are enriched for genes that undergo cryptic splicing when TDP-43 function is impaired. We found that FTLD-TDP and FTLD-tau subtypes target brain regions with overlapping and distinct gene expression correlates, highlighting many genes linked to neuromodulatory functions. FTLD atrophy-correlated genes were strongly enriched for HARs. Atrophy-correlated genes in FTLD-TDP showed greater overlap with TDP-43 cryptic splicing genes and genes with more numerous TDP-43 binding sites compared with atrophy-correlated genes in FTLD-tau. Cryptic splicing genes were enriched for HAR genes, and vice versa, but this effect was due to the confounding influence of gene length. Analyses performed at the individual-patient level revealed that the expression of HAR genes and cryptically spliced genes within putative regions of disease onset differed across FTLD-TDP subtypes.

Massively parallel discovery of human-specific substitutions that alter enhancer activity.

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

A Systematic Review of the Human Accelerated Regions in Schizophrenia and Related Disorders: Where the Evolutionary and Neurodevelopmental Hypotheses Converge.

Schizophrenia is a psychiatric disorder that results from genetic and environmental factors interacting and disrupting neurodevelopmental trajectories. Human Accelerated Regions (HARs) are evolutionarily conserved genomic regions that have accumulated human-specific sequence changes. Thus, studies on the impact of HARs in the context of neurodevelopment, as well as with respect to adult brain phenotypes, have increased considerably in the last few years. Through a systematic approach, we aim to offer a comprehensive review of HARs' role in terms of human brain development, configuration, and cognitive abilities, as well as whether HARs modulate the susceptibility to neurodevelopmental psychiatric disorders such as schizophrenia. First, the evidence in this review highlights HARs' molecular functions in the context of the neurodevelopmental regulatory genetic machinery. Second, brain phenotypic analyses indicate that HAR genes' expression spatially correlates with the regions that suffered human-specific cortical expansion, as well as with the regional interactions for synergistic information processing. Lastly, studies based on candidate HAR genes and the global "HARome" variability describe the involvement of these regions in the genetic background of schizophrenia, but also in other neurodevelopmental psychiatric disorders. Overall, the data considered in this review emphasise the crucial role of HARs in human-specific neurodevelopment processes and encourage future research on this evolutionary marker for a better understanding of the genetic basis of schizophrenia and other neurodevelopmental-related psychiatric disorders. Accordingly, HARs emerge as interesting genomic regions that require further study in order to bridge the neurodevelopmental and evolutionary hypotheses in schizophrenia and other related disorders and phenotypes.

Evolution of Gene Regulation in Humans.

As a species, we possess unique biological features that distinguish us from other primates. Here, we review recent efforts to identify changes in gene regulation that drove the evolution of novel human phenotypes. We discuss genotype-directed comparisons of human and nonhuman primate genomes to identify human-specific genetic changes that may encode new regulatory functions. We also review phenotype-directed approaches, which use comparisons of gene expression or regulatory function in homologous human and nonhuman primate cells and tissues to identify changes in expression levels or regulatory activity that may be due to genetic changes in humans. Together, these studies are beginning to reveal the landscape of regulatory innovation in human evolution and point to specific regulatory changes for further study. Finally, we highlight two novel strategies to model human-specific regulatory functions in vivo: primate induced pluripotent stem cells and the generation of humanized mice by genome editing.

Recently evolved human-specific methylated regions are enriched in schizophrenia signals.

BACKGROUND: One explanation for the persistence of schizophrenia despite the reduced fertility of patients is that it is a by-product of recent human evolution. This hypothesis is supported by evidence suggesting that recently-evolved genomic regions in humans are involved in the genetic risk for schizophrenia. Using summary statistics from genome-wide association studies (GWAS) of schizophrenia and 11 other phenotypes, we tested for enrichment of association with GWAS traits in regions that have undergone methylation changes in the human lineage compared to Neanderthals and Denisovans, i.e. human-specific differentially methylated regions (DMRs). We used analytical tools that evaluate polygenic enrichment of a subset of genomic variants against all variants. RESULTS: Schizophrenia was the only trait in which DMR SNPs showed clear enrichment of association that passed the genome-wide significance threshold. The enrichment was not observed for Neanderthal or Denisovan DMRs. The enrichment seen in human DMRs is comparable to that for genomic regions tagged by Neanderthal Selective Sweep markers, and stronger than that for Human Accelerated Regions. The enrichment survives multiple testing performed through permutation (n = 10,000) and bootstrapping (n = 5000) in INRICH (p < 0.01). Some enrichment of association with height was observed at the gene level. CONCLUSIONS: Regions where DNA methylation modifications have changed during recent human evolution show enrichment of association with schizophrenia and possibly with height. Our study further supports the hypothesis that genetic variants conferring risk of schizophrenia co-occur in genomic regions that have changed as the human species evolved. Since methylation is an epigenetic mark, potentially mediated by environmental changes, our results also suggest that interaction with the environment might have contributed to that association.

Functional associations of evolutionarily recent human genes exhibit sensitivity to the 3D genome landscape and disease.

Genome organization is intricately tied to regulating genes and associated cell fate decisions. In this study, we examine the positioning and functional significance of human genes, grouped by their evolutionary age, within the 3D organization of the genome. We reveal that genes of different evolutionary origin have distinct positioning relationships with both domains and loop anchors, and remarkably consistent relationships with boundaries across cell types. While the functional associations of each group of genes are primarily cell type-specific, such associations of conserved genes maintain greater stability across 3D genomic features and disease than recently evolved genes. Furthermore, the expression of these genes across various tissues follows an evolutionary progression, such that RNA levels increase from young genes to ancient genes. Thus, the distinct relationships of gene evolutionary age, function, and positioning within 3D genomic features contribute to tissue-specific gene regulation in development and disease.

Massively parallel disruption of enhancers active in human neural stem cells.

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders, potentially by altering neural progenitor proliferation. However, the effects of genetic variation within regulatory elements on neural progenitors remain obscure. We use sgRNA-Cas9 screens in human neural stem cells (hNSCs) to disrupt 10,674 genes and 26,385 conserved regions in 2,227 enhancers active in the developing human cortex and determine effects on proliferation. Genes with proliferation phenotypes are associated with neurodevelopmental disorders and show biased expression in specific fetal human brain neural progenitor populations. Although enhancer disruptions overall have weaker effects than gene disruptions, we identify enhancer disruptions that severely alter hNSC self-renewal. Disruptions in human accelerated regions, implicated in human brain evolution, also alter proliferation. Integrating proliferation phenotypes with chromatin interactions reveals regulatory relationships between enhancers and their target genes contributing to neurogenesis and potentially to human cortical evolution.

Rare variation in non-coding regions with evolutionary signatures contributes to autism spectrum disorder risk.

Little is known about the role of non-coding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of non-coding regions: human accelerated regions (HARs), which show signatures of positive selection in humans; experimentally validated neural VISTA enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole-genome analysis of >16,600 samples and >4,900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly contribute, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in VEs near OTX1 and SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved non-coding regions in ASD risk and suggest potential mechanisms of how regulatory changes can modulate social behavior.

The evolutionary origin of psychosis.

Imagination, the driving force of creativity, and primary psychosis are human-specific, since we do not observe behaviors in other species that would convincingly suggest they possess the same traits. Both these traits have been linked to the function of the prefrontal cortex, which is the most evolutionarily novel region of the human brain. A number of evolutionarily novel genetic and epigenetic changes that determine the human brain-specific structure and function have been discovered in recent years. Among them are genomic loci subjected to increased rates of single nucleotide substitutions in humans, called human accelerated regions. These mostly regulatory regions are involved in brain development and sometimes contain genetic variants that confer a risk for schizophrenia. On the other hand, neuroimaging data suggest that mind wandering and related phenomena (as a proxy of imagination) are in many ways similar to rapid eye movement dreaming, a function also present in non-human species. Furthermore, both functions are similar to psychosis in several ways: for example, the same brain areas are activated both in dreams and visual hallucinations. In the present Perspective we hypothesize that imagination is an evolutionary adaptation of dreaming, while primary psychosis results from deficient control by higher-order brain areas over imagination. In the light of this, human accelerated regions might be one of the key drivers in evolution of human imagination and the pathogenesis of psychotic disorders.

Evolutionary relevance of single nucleotide variants within the forebrain exclusive human accelerated enhancer regions.

BACKGROUND: Human accelerated regions (HARs) are short conserved genomic sequences that have acquired significantly more nucleotide substitutions than expected in the human lineage after divergence from chimpanzees. The fast evolution of HARs may reflect their roles in the origin of human-specific traits. A recent study has reported positively-selected single nucleotide variants (SNVs) within brain-exclusive human accelerated enhancers (BE-HAEs) hs1210 (forebrain), hs563 (hindbrain) and hs304 (midbrain/forebrain). By including data from archaic hominins, these SNVs were shown to be Homo sapiens-specific, residing within transcriptional factors binding sites (TFBSs) for SOX2 (hs1210), RUNX1/3 (hs563), and FOS/JUND (hs304). Although these findings suggest that the predicted modifications in TFBSs may have some role in present-day brain structure, work is required to verify the extent to which these changes translate into functional variation. RESULTS: To start to fill this gap, we investigate the SOX2 SNV, with both forebrain expression and strong signal of positive selection in humans. We demonstrate that the HMG box of SOX2 binds in vitro with Homo sapiens-specific derived A-allele and ancestral T-allele carrying DNA sites in BE-HAE hs1210. Molecular docking and simulation analysis indicated highly favourable binding of HMG box with derived A-allele containing DNA site when compared to site carrying ancestral T-allele. CONCLUSION: These results suggest that adoptive changes in TF affinity within BE-HAE hs1210 and other HAR enhancers in the evolutionary history of Homo sapiens might. have brought about changes in gene expression patterns and have functional consequences on forebrain formation and evolution. METHODS: The present study employ electrophoretic mobility shift assays (EMSA) and molecular docking and molecular dynamics simulations approaches.

FTLD targets brain regions expressing recently evolved genes.

In frontotemporal lobar degeneration (FTLD), pathological protein aggregation is associated with a decline in human-specialized social-emotional and language functions. Most disease protein aggregates contain either TDP-43 (FTLD-TDP) or tau (FTLD-tau). Here, we explored whether FTLD targets brain regions that express genes containing human accelerated regions (HARs), conserved sequences that have undergone positive selection during recent human evolution. To this end, we used structural neuroimaging from patients with FTLD and normative human regional transcriptomic data to identify genes expressed in FTLD-targeted brain regions. We then integrated primate comparative genomic data to test our hypothesis that FTLD targets brain regions expressing recently evolved genes. In addition, we asked whether genes expressed in FTLD-targeted brain regions are enriched for genes that undergo cryptic splicing when TDP-43 function is impaired. We found that FTLD-TDP and FTLD-tau subtypes target brain regions that express overlapping and distinct genes, including many linked to neuromodulatory functions. Genes whose normative brain regional expression pattern correlated with FTLD cortical atrophy were strongly associated with HARs. Atrophy-correlated genes in FTLD-TDP showed greater overlap with TDP-43 cryptic splicing genes compared with atrophy-correlated genes in FTLD-tau. Cryptic splicing genes were enriched for HAR genes, and vice versa, but this effect was due to the confounding influence of gene length. Analyses performed at the individual-patient level revealed that the expression of HAR genes and cryptically spliced genes within putative regions of disease onset differed across FTLD-TDP subtypes. Overall, our findings suggest that FTLD targets brain regions that have undergone recent evolutionary specialization and provide intriguing potential leads regarding the transcriptomic basis for selective vulnerability in distinct FTLD molecular-anatomical subtypes.

Proteins with amino acid repeats constitute a rapidly evolvable and human-specific essentialome.

Protein products of essential genes, indispensable for organismal survival, are highly conserved and bring about fundamental functions. Interestingly, proteins that contain amino acid homorepeats that tend to evolve rapidly are enriched in eukaryotic essentialomes. Why are proteins with hypermutable homorepeats enriched in conserved and functionally vital essential proteins? We solve this functional versus evolutionary paradox by demonstrating that human essential proteins with homorepeats bring about crosstalk across biological processes through high interactability and have distinct regulatory functions affecting expansive global regulation. Importantly, essential proteins with homorepeats rapidly diverge with the amino acid substitutions frequently affecting functional sites, likely facilitating rapid adaptability. Strikingly, essential proteins with homorepeats influence human-specific embryonic and brain development, implying that the presence of homorepeats could contribute to the emergence of human-specific processes. Thus, we propose that homorepeat-containing essential proteins affecting species-specific traits can be potential intervention targets across pathologies, including cancers and neurological disorders.

Rare variation in noncoding regions with evolutionary signatures contributes to autism spectrum disorder risk.

Little is known about the role of noncoding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of noncoding regions: Human Accelerated Regions (HARs), which show signatures of positive selection in humans; experimentally validated neural Vista Enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole genome analysis of >16,600 samples and >4900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in a VE near SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved noncoding regions in ASD risk and suggest potential mechanisms of how changes in regulatory regions can modulate social behavior.

Revisiting Schizophrenia from an Evolutionary Perspective: An Association Study of Recent Evolutionary Markers and Schizophrenia.

The persistence of schizophrenia in human populations at a high prevalence and with a large heritability estimate despite reduced fertility and increased mortality rate is a Darwinian paradox. This may be likely if the genomic components that predispose to schizophrenia are also advantageous for the acquisition of important human traits, such as language and cognition. Accordingly, an emerging group of genomic markers of recent evolution in humans, namely human accelerated regions (HARs), since our divergence from chimpanzees, are gaining importance for neurodevelopmental disorders, such as schizophrenia. We hypothesize that variants within HARs may affect the expression of genes under their control, thus contributing to disease etiology. A total of 49 HAR single nucleotide polymorphisms (SNPs) were prioritized from the complete repertoire of HARs (n = 2737) based on their functional relevance and prevalence in the South Asian population. Test of association using 2 independent schizophrenia case-control cohorts of north Indian ethnicity (discovery: n = 930; replication: n = 1104) revealed 3 SNPs (rs3800926, rs3801844, and rs764453) from chromosome 7 and rs77047799 from chromosome 3 to be significantly associated (combined analysis: Bonferroni corrected P < .002-.000004). Of note, these SNPs were found to alter the expression of neurodevelopmental genes such as SLC25A13, MAD1L1, and ULK4; a few from the HOX gene family; and a few genes that are implicated in mitochondrial function. These SNPs may most likely alter binding sites of transcription factors, including TFCP2, MAFK, SREBF2, E2F1, and/or methylation signatures around these genes. These findings reiterate a neurodevelopmental basis of schizophrenia and also open up a promising avenue to investigate HAR-mediated mitochondrial dysfunction in schizophrenia etiology.

Rewiring of human neurodevelopmental gene regulatory programs by human accelerated regions.

Human accelerated regions (HARs) are the fastest-evolving regions of the human genome, and many are hypothesized to function as regulatory elements that drive human-specific gene regulatory programs. We interrogate the in vitro enhancer activity and in vivo epigenetic landscape of more than 3,100 HARs during human neurodevelopment, demonstrating that many HARs appear to act as neurodevelopmental enhancers and that sequence divergence at HARs has largely augmented their neuronal enhancer activity. Furthermore, we demonstrate PPP1R17 to be a putative HAR-regulated gene that has undergone remarkable rewiring of its cell type and developmental expression patterns between non-primates and primates and between non-human primates and humans. Finally, we show that PPP1R17 slows neural progenitor cell cycle progression, paralleling the cell cycle length increase seen predominantly in primate and especially human neurodevelopment. Our findings establish HARs as key components in rewiring human-specific neurodevelopmental gene regulatory programs and provide an integrated resource to study enhancer activity of specific HARs.

Systems Level Analysis and Identification of Pathways and Key Genes Associated with Delirium.

Delirium is a complex pathophysiological process, and multiple contributing mechanisms have been identified. However, it is largely unclear how the genes associated with delirium contribute and which of them play key roles. In this study, the genes associated with delirium were retrieved from the Comparative Toxicogenomics Database (CTD) and integrated through a protein-protein interaction (PPI) network. Delirium-associated genes formed a highly interconnected PPI subnetwork, indicating a high tendency to interact and agglomerate. Using the Molecular Complex Detection (MCODE) algorithm, we identified the top two delirium-relevant network modules, M1 and M5, that have the most significant enrichments for the delirium-related gene sets. Functional enrichment analysis showed that genes related to neurotransmitter receptor activity were enriched in both modules. Moreover, analyses with genes located in human accelerated regions (HARs) provided evidence that HAR-Brain genes were overrepresented in the delirium-relevant network modules. We found that four of the HAR-Brain genes, namely APP, PLCB1, NPY, and HTR2A, in the M1 module were highly connected and appeared to exhibit hub properties, which might play vital roles in delirium development. Further understanding of the function of the identified modules and member genes could help to identify therapeutic intervention targets and diagnostic biomarkers for delirium.

Mechanistically Distinct Pathways of Divergent Regulatory DNA Creation Contribute to Evolution of Human-Specific Genomic Regulatory Networks Driving Phenotypic Divergence of Homo sapiens.

Thousands of candidate human-specific regulatory sequences (HSRS) have been identified, supporting the hypothesis that unique to human phenotypes result from human-specific alterations of genomic regulatory networks. Collectively, a compendium of multiple diverse families of HSRS that are functionally and structurally divergent from Great Apes could be defined as the backbone of human-specific genomic regulatory networks. Here, the conservation patterns analysis of 18,364 candidate HSRS was carried out requiring that 100% of bases must remap during the alignments of human, chimpanzee, and bonobo sequences. A total of 5,535 candidate HSRS were identified that are: (i) highly conserved in Great Apes; (ii) evolved by the exaptation of highly conserved ancestral DNA; (iii) defined by either the acceleration of mutation rates on the human lineage or the functional divergence from non-human primates. The exaptation of highly conserved ancestral DNA pathway seems mechanistically distinct from the evolution of regulatory DNA segments driven by the species-specific expansion of transposable elements. Genome-wide proximity placement analysis of HSRS revealed that a small fraction of topologically associating domains (TADs) contain more than half of HSRS from four distinct families. TADs that are enriched for HSRS and termed rapidly evolving in humans TADs (revTADs) comprise 0.8-10.3% of 3,127 TADs in the hESC genome. RevTADs manifest distinct correlation patterns between placements of human accelerated regions, human-specific transcription factor-binding sites, and recombination rates. There is a significant enrichment within revTAD boundaries of hESC-enhancers, primate-specific CTCF-binding sites, human-specific RNAPII-binding sites, hCONDELs, and H3K4me3 peaks with human-specific enrichment at TSS in prefrontal cortex neurons (P < 0.0001 in all instances). Present analysis supports the idea that phenotypic divergence of Homo sapiens is driven by the evolution of human-specific genomic regulatory networks via at least two mechanistically distinct pathways of creation of divergent sequences of regulatory DNA: (i) recombination-associated exaptation of the highly conserved ancestral regulatory DNA segments; (ii) human-specific insertions of transposable elements.