A sequence of SVA retrotransposon insertions in ASIP shaped human pigmentation.

Retrotransposons comprise about 45% of the human genome1, but their contributions to human trait variation and evolution are only beginning to be explored2,3. Here, we find that a sequence of SVA retrotransposon insertions in an early intron of the ASIP (agouti signaling protein) gene has probably shaped human pigmentation several times. In the UK Biobank (n = 169,641), a recent 3.3-kb SVA insertion polymorphism associated strongly with lighter skin pigmentation (0.22 [0.21-0.23] s.d.; P = 2.8 × 10-351) and increased skin cancer risk (odds ratio = 1.23 [1.18-1.27]; P = 1.3 × 10-28), appearing to underlie one of the strongest common genetic influences on these phenotypes within European populations4-6. ASIP expression in skin displayed the same association pattern, with the SVA insertion allele exhibiting 2.2-fold (1.9-2.6) increased expression. This effect had an unusual apparent mechanism: an earlier, nonpolymorphic, human-specific SVA retrotransposon 3.9 kb upstream appeared to have caused ASIP hypofunction by nonproductive splicing, which the new (polymorphic) SVA insertion largely eliminated. Extended haplotype homozygosity indicated that the insertion allele has risen to allele frequencies up to 11% in European populations over the past several thousand years. These results indicate that a sequence of retrotransposon insertions contributed to a species-wide increase, then a local decrease, of human pigmentation.

Transcriptome-wide characterization of genetic perturbations.

Single cell CRISPR screens such as Perturb-seq enable transcriptomic profiling of genetic perturbations at scale. However, the data produced by these screens are often noisy due to cost and technical constraints, limiting power to detect true effects with conventional differential expression analyses. Here, we introduce TRanscriptome-wide Analysis of Differential Expression (TRADE), a statistical framework which estimates the transcriptome-wide distribution of true differential expression effects from noisy gene-level measurements. Within TRADE, we derive multiple novel, interpretable statistical metrics, including the "transcriptome-wide impact", an estimator of the overall transcriptional effect of a perturbation which is stable across sampling depths. We analyze new and published large-scale Perturb-seq datasets to show that many true transcriptional effects are not statistically significant, but detectable in aggregate with TRADE. In a genome-scale Perturb-seq screen, we find that a typical gene perturbation affects an estimated 45 genes, whereas a typical essential gene perturbation affects over 500 genes. An advantage of our approach is its ability to compare the transcriptomic effects of genetic perturbations across contexts and dosages despite differences in power. We use this ability to identify perturbations with cell-type dependent effects and to find examples of perturbations where transcriptional responses are not only larger in magnitude, but also qualitatively different, as a function of dosage. Lastly, we expand our analysis to case/control comparison of gene expression for neuropsychiatric conditions, finding that transcriptomic effect correlations are greater than genetic correlations for these diagnoses. TRADE lays an analytic foundation for the systematic comparison of genetic perturbation atlases, as well as differential expression experiments more broadly.

Single-nucleus sequencing reveals enriched expression of genetic risk factors in extratelencephalic neurons sensitive to degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by a progressive loss of motor function linked to degenerating extratelencephalic neurons/Betz cells (ETNs). The reasons why these neurons are selectively affected remain unclear. Here, to understand the unique molecular properties that may sensitize ETNs to ALS, we performed RNA sequencing of 79,169 single nuclei from cortices of patients and controls. In both patients and unaffected individuals, we found significantly higher expression of ALS risk genes in THY1+ ETNs, regardless of diagnosis. In patients, this was accompanied by the induction of genes involved in protein homeostasis and stress responses that were significantly induced in a wide collection of ETNs. Examination of oligodendroglial and microglial nuclei revealed patient-specific downregulation of myelinating genes in oligodendrocytes and upregulation of an endolysosomal reactive state in microglia. Our findings suggest that selective vulnerability of extratelencephalic neurons is partly connected to their intrinsic molecular properties sensitizing them to genetics and mechanisms of degeneration.

Genetic drivers and cellular selection of female mosaic X chromosome loss.

Mosaic loss of the X chromosome (mLOX) is the most common clonal somatic alteration in leukocytes of female individuals1,2, but little is known about its genetic determinants or phenotypic consequences. Here, to address this, we used data from 883,574 female participants across 8 biobanks; 12% of participants exhibited detectable mLOX in approximately 2% of leukocytes. Female participants with mLOX had an increased risk of myeloid and lymphoid leukaemias. Genetic analyses identified 56 common variants associated with mLOX, implicating genes with roles in chromosomal missegregation, cancer predisposition and autoimmune diseases. Exome-sequence analyses identified rare missense variants in FBXO10 that confer a twofold increased risk of mLOX. Only a small fraction of associations was shared with mosaic Y chromosome loss, suggesting that distinct biological processes drive formation and clonal expansion of sex chromosome missegregation. Allelic shift analyses identified X chromosome alleles that are preferentially retained in mLOX, demonstrating variation at many loci under cellular selection. A polygenic score including 44 allelic shift loci correctly inferred the retained X chromosomes in 80.7% of mLOX cases in the top decile. Our results support a model in which germline variants predispose female individuals to acquiring mLOX, with the allelic content of the X chromosome possibly shaping the magnitude of clonal expansion.

Kynurenic acid inflammatory signaling expands in primates and impairs prefrontal cortical cognition.

Cognitive deficits from dorsolateral prefrontal cortex (dlPFC) dysfunction are common in neuroinflammatory disorders, including long-COVID, schizophrenia and Alzheimer's disease, and have been correlated with kynurenine inflammatory signaling. Kynurenine is further metabolized to kynurenic acid (KYNA) in brain, where it blocks NMDA and α7-nicotinic receptors (nic-α7Rs). These receptors are essential for neurotransmission in dlPFC, suggesting that KYNA may cause higher cognitive deficits in these disorders. The current study found that KYNA and its synthetic enzyme, KAT II, have greatly expanded expression in primate dlPFC in both glia and neurons. Local application of KYNA onto dlPFC neurons markedly reduced the delay-related firing needed for working memory via actions at NMDA and nic-α7Rs, while inhibition of KAT II enhanced neuronal firing in aged macaques. Systemic administration of agents that reduce KYNA production similarly improved cognitive performance in aged monkeys, suggesting a therapeutic avenue for the treatment of cognitive deficits in neuroinflammatory disorders.

Key Roles of CACNA1C/Cav1.2 and CALB1/Calbindin in Prefrontal Neurons Altered in Cognitive Disorders.

Importance: The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. Objective: To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. Design, Setting, and Participants: The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Main Outcomes and Measures: Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Results: Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by ß1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or ß1-adrenoceptor antagonist protected working memory from stress. Conclusions and Relevance: The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

Modeling interpretable correspondence between cell state and perturbation response with CellCap.

Single-cell transcriptomics, in conjunction with genetic and compound perturbations, offers a robust approach for exploring cellular behaviors in diverse contexts. Such experiments allow uncovering cell-state-specific responses to perturbations, a crucial aspect in unraveling the intricate molecular mechanisms governing cellular behavior and potentially discovering novel regulatory pathways and therapeutic targets. However, prevailing computational methods predominantly focus on predicting average cellular responses, disregarding the inherent response heterogeneity associated with cell state diversity. In this study, we present CellCap, a deep generative model designed for the end-to-end analysis of single-cell perturbation experiments. CellCap employs sparse dictionary learning in a latent space to deconstruct cell-state-specific perturbation responses into a set of transcriptional response programs. These programs are then utilized by each perturbation condition and each cell at varying degrees. The incorporation of specific model design choices, such as dot-product cross-attention between cell states and response programs, along with a linearly-decoded latent space, underlay the interpretation power of CellCap. We evaluate CellCap's model interpretability through multiple simulated scenarios and apply it to two real single-cell perturbation datasets. These datasets feature either heterogeneous cellular populations or a complex experimental setup. Our results demonstrate that CellCap successfully uncovers the relationship between cell state and perturbation response, unveiling novel insights overlooked in previous analyses. The model's interpretability, coupled with its effectiveness in capturing heterogeneous responses, positions CellCap as a valuable tool for advancing our understanding of cellular behaviors in the context of perturbation experiments.

CSMD1 regulates brain complement activity and circuit development.

Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.

A concerted neuron-astrocyte program declines in ageing and schizophrenia.

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a relationship between people's cortical neurons and cortical astrocytes. We used single-nucleus RNA sequencing to analyse the prefrontal cortex of 191 human donors aged 22-97 years, including healthy individuals and people with schizophrenia. Latent-factor analysis of these data revealed that, in people whose cortical neurons more strongly expressed genes encoding synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the synaptic neuron and astrocyte program (SNAP). In schizophrenia and ageing-two conditions that involve declines in cognitive flexibility and plasticity1,2-cells divested from SNAP: astrocytes, glutamatergic (excitatory) neurons and GABAergic (inhibitory) neurons all showed reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy people of similar age, may underlie many aspects of normal human interindividual differences and may be an important point of convergence for multiple kinds of pathophysiology.

Protein-altering variants at copy number-variable regions influence diverse human phenotypes.

Copy number variants (CNVs) are among the largest genetic variants, yet CNVs have not been effectively ascertained in most genetic association studies. Here we ascertained protein-altering CNVs from UK Biobank whole-exome sequencing data (n = 468,570) using haplotype-informed methods capable of detecting subexonic CNVs and variation within segmental duplications. Incorporating CNVs into analyses of rare variants predicted to cause gene loss of function (LOF) identified 100 associations of predicted LOF variants with 41 quantitative traits. A low-frequency partial deletion of RGL3 exon 6 conferred one of the strongest protective effects of gene LOF on hypertension risk (odds ratio = 0.86 (0.82-0.90)). Protein-coding variation in rapidly evolving gene families within segmental duplications-previously invisible to most analysis methods-generated some of the human genome's largest contributions to variation in type 2 diabetes risk, chronotype and blood cell traits. These results illustrate the potential for new genetic insights from genomic variation that has escaped large-scale analysis to date.

High-dimensional phenotyping to define the genetic basis of cellular morphology.

The morphology of cells is dynamic and mediated by genetic and environmental factors. Characterizing how genetic variation impacts cell morphology can provide an important link between disease association and cellular function. Here, we combine genomic sequencing and high-content imaging approaches on iPSCs from 297 unique donors to investigate the relationship between genetic variants and cellular morphology to map what we term cell morphological quantitative trait loci (cmQTLs). We identify novel associations between rare protein altering variants in WASF2, TSPAN15, and PRLR with several morphological traits related to cell shape, nucleic granularity, and mitochondrial distribution. Knockdown of these genes by CRISPRi confirms their role in cell morphology. Analysis of common variants yields one significant association and nominate over 300 variants with suggestive evidence (P < 10-6) of association with one or more morphology traits. We then use these data to make predictions about sample size requirements for increasing discovery in cellular genetic studies. We conclude that, similar to molecular phenotypes, morphological profiling can yield insight about the function of genes and variants.

Concerted neuron-astrocyte gene expression declines in aging and schizophrenia.

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a striking relationship between people's cortical neurons and cortical astrocytes. We used single-nucleus RNA-seq to analyze the prefrontal cortex of 191 human donors ages 22-97 years, including healthy individuals and persons with schizophrenia. Latent-factor analysis of these data revealed that in persons whose cortical neurons more strongly expressed genes for synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the Synaptic Neuron-and-Astrocyte Program (SNAP). In schizophrenia and aging - two conditions that involve declines in cognitive flexibility and plasticity 1,2 - cells had divested from SNAP: astrocytes, glutamatergic (excitatory) neurons, and GABAergic (inhibitory) neurons all reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy persons of similar age, may underlie many aspects of normal human interindividual differences and be an important point of convergence for multiple kinds of pathophysiology.

BCFtools/liftover: an accurate and comprehensive tool to convert genetic variants across genome assemblies.

MOTIVATION: Many genetics studies report results tied to genomic coordinates of a legacy genome assembly. However, as assemblies are updated and improved, researchers are faced with either realigning raw sequence data using the updated coordinate system or converting legacy datasets to the updated coordinate system to be able to combine results with newer datasets. Currently available tools to perform the conversion of genetic variants have numerous shortcomings, including poor support for indels and multi-allelic variants, that lead to a higher rate of variants being dropped or incorrectly converted. As a result, many researchers continue to work with and publish using legacy genomic coordinates. RESULTS: Here we present BCFtools/liftover, a tool to convert genomic coordinates across genome assemblies for variants encoded in the variant call format with improved support for indels represented by different reference alleles across genome assemblies and full support for multi-allelic variants. It further supports variant annotation fields updates whenever the reference allele changes across genome assemblies. The tool has the lowest rate of variants being dropped with an order of magnitude less indels dropped or incorrectly converted and is an order of magnitude faster than other tools typically used for the same task. It is particularly suited for converting variant callsets from large cohorts to novel telomere-to-telomere assemblies as well as summary statistics from genome-wide association studies tied to legacy genome assemblies. AVAILABILITY AND IMPLEMENTATION: The tool is written in C and freely available under the MIT open source license as a BCFtools plugin available at http://github.com/freeseek/score.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Sibling chimerism among microglia in marmosets.

Chimerism happens rarely among most mammals but is common in marmosets and tamarins, a result of fraternal twin or triplet birth patterns in which in utero connected circulatory systems (through which stem cells transit) lead to persistent blood chimerism (12-80%) throughout life. The presence of Y-chromosome DNA sequences in other organs of female marmosets has long suggested that chimerism might also affect these organs. However, a longstanding question is whether this chimerism is driven by blood-derived cells or involves contributions from other cell types. To address this question, we analyzed single-cell RNA-seq data from blood, liver, kidney and multiple brain regions across a number of marmosets, using transcribed single nucleotide polymorphisms (SNPs) to identify cells with the sibling's genome in various cell types within these tissues. Sibling-derived chimerism in all tissues arose entirely from cells of hematopoietic origin (i.e., myeloid and lymphoid lineages). In brain tissue this was reflected as sibling-derived chimerism among microglia (20-52%) and macrophages (18-64%) but not among other resident cell types (i.e., neurons, glia or ependymal cells). The percentage of microglia that were sibling-derived showed significant variation across brain regions, even within individual animals, likely reflecting distinct responses by siblings' microglia to local recruitment or proliferation cues or, potentially, distinct clonal expansion histories in different brain areas. In the animals and tissues we analyzed, microglial gene expression profiles bore a much stronger relationship to local/host context than to sibling genetic differences. Naturally occurring marmoset chimerism will provide new ways to understand the effects of genes, mutations and brain contexts on microglial biology and to distinguish between effects of microglia and other cell types on brain phenotypes.

A marmoset brain cell census reveals regional specialization of cellular identities.

The mammalian brain is composed of many brain structures, each with its own ontogenetic and developmental history. We used single-nucleus RNA sequencing to sample over 2.4 million brain cells across 18 locations in the common marmoset, a New World monkey primed for genetic engineering, and examined gene expression patterns of cell types within and across brain structures. The adult transcriptomic identity of most neuronal types is shaped more by developmental origin than by neurotransmitter signaling repertoire. Quantitative mapping of GABAergic types with single-molecule FISH (smFISH) reveals that interneurons in the striatum and neocortex follow distinct spatial principles, and that lateral prefrontal and other higher-order cortical association areas are distinguished by high proportions of VIP+ neurons. We use cell type-specific enhancers to drive AAV-GFP and reconstruct the morphologies of molecularly resolved interneuron types in neocortex and striatum. Our analyses highlight how lineage, local context, and functional class contribute to the transcriptional identity and biodistribution of primate brain cell types.

Comparative transcriptomics reveals human-specific cortical features.

The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.

Repeat polymorphisms underlie top genetic risk loci for glaucoma and colorectal cancer.

Many regions in the human genome vary in length among individuals due to variable numbers of tandem repeats (VNTRs). To assess the phenotypic impact of VNTRs genome-wide, we applied a statistical imputation approach to estimate the lengths of 9,561 autosomal VNTR loci in 418,136 unrelated UK Biobank participants and 838 GTEx participants. Association and statistical fine-mapping analyses identified 58 VNTRs that appeared to influence a complex trait in UK Biobank, 18 of which also appeared to modulate expression or splicing of a nearby gene. Non-coding VNTRs at TMCO1 and EIF3H appeared to generate the largest known contributions of common human genetic variation to risk of glaucoma and colorectal cancer, respectively. Each of these two VNTRs associated with a >2-fold range of risk across individuals. These results reveal a substantial and previously unappreciated role of non-coding VNTRs in human health and gene regulation.

Schizophrenia-associated somatic copy-number variants from 12,834 cases reveal recurrent NRXN1 and ABCB11 disruptions.

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)-present in some but not all cells-remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e-4), with recurrent somatic deletions of exons 1-5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5' deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk.