CNVs: harbingers of a rare variant revolution in psychiatric genetics.

The genetic bases of neuropsychiatric disorders are beginning to yield to scientific inquiry. Genome-wide studies of copy number variation (CNV) have given rise to a new understanding of disease etiology, bringing rare variants to the forefront. A proportion of risk for schizophrenia, bipolar disorder, and autism can be explained by rare mutations. Such alleles arise by de novo mutation in the individual or in recent ancestry. Alleles can have specific effects on behavioral and neuroanatomical traits; however, expressivity is variable, particularly for neuropsychiatric phenotypes. Knowledge from CNV studies reflects the nature of rare alleles in general and will serve as a guide as we move forward into a new era of whole-genome sequencing.

Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

Altered human oligodendrocyte heterogeneity in multiple sclerosis.

Oligodendrocyte pathology is increasingly implicated in neurodegenerative diseases as oligodendrocytes both myelinate and provide metabolic support to axons. In multiple sclerosis (MS), demyelination in the central nervous system thus leads to neurodegeneration, but the severity of MS between patients is very variable. Disability does not correlate well with the extent of demyelination1, which suggests that other factors contribute to this variability. One such factor may be oligodendrocyte heterogeneity. Not all oligodendrocytes are the same-those from the mouse spinal cord inherently produce longer myelin sheaths than those from the cortex2, and single-cell analysis of the mouse central nervous system identified further differences3,4. However, the extent of human oligodendrocyte heterogeneity and its possible contribution to MS pathology remain unknown. Here we performed single-nucleus RNA sequencing from white matter areas of post-mortem human brain from patients with MS and from unaffected controls. We identified subclusters of oligodendroglia in control human white matter, some with similarities to mouse, and defined new markers for these cell states. Notably, some subclusters were underrepresented in MS tissue, whereas others were more prevalent. These differences in mature oligodendrocyte subclusters may indicate different functional states of oligodendrocytes in MS lesions. We found similar changes in normal-appearing white matter, showing that MS is a more diffuse disease than its focal demyelination suggests. Our findings of an altered oligodendroglial heterogeneity in MS may be important for understanding disease progression and developing therapeutic approaches.

Frequency and Complexity of De Novo Structural Mutation in Autism.

Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.

Marker chromosome genomic structure and temporal origin implicate a chromoanasynthesis event in a family with pleiotropic psychiatric phenotypes.

Small supernumerary marker chromosomes (sSMC) are chromosomal fragments difficult to characterize genomically. Here, we detail a proband with schizoaffective disorder and a mother with bipolar disorder with psychotic features who present with a marker chromosome that segregates with disease. We explored the architecture of this marker and investigated its temporal origin. Array comparative genomic hybridization (aCGH) analysis revealed three duplications and three triplications that spanned the short arm of chromosome 9, suggestive of a chromoanasynthesis-like event. Segregation of marker genotypes, phased using sSMC mosaicism in the mother, provided evidence that it was generated during a germline-level event in the proband's maternal grandmother. Whole-genome sequencing (WGS) was performed to resolve the structure and junctions of the chromosomal fragments, revealing further complexities. While structural variations have been previously associated with neuropsychiatric disorders and marker chromosomes, here we detail the precise architecture, human life-cycle genesis, and propose a DNA replicative/repair mechanism underlying formation.

Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia.

Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.

Single-nucleus and spatial transcriptomic profiling of human temporal cortex and white matter reveals novel associations with AD pathology.

Alzheimer's disease (AD) is a neurodegenerative disorder with complex pathological manifestations and is the leading cause of cognitive decline and dementia in elderly individuals. A major goal in AD research is to identify new therapeutic pathways by studying the molecular and cellular changes in the disease, either downstream or upstream of the pathological hallmarks. In this study, we present a comprehensive investigation of cellular heterogeneity from the temporal cortex region of 40 individuals, comprising healthy donors and individuals with differing tau and amyloid burden. Using single-nucleus transcriptome analysis of 430,271 nuclei from both gray and white matter of these individuals, we identified cell type-specific subclusters in both neuronal and glial cell types with varying degrees of association with AD pathology. In particular, these associations are present in layer specific glutamatergic (excitatory) neuronal types, along with GABAergic (inhibitory) neurons and glial subtypes. These associations were observed in early as well as late pathological progression. We extended this analysis by performing multiplexed in situ hybridization using the CARTANA platform, capturing 155 genes in 13 individuals with varying levels of tau pathology. By modeling the spatial distribution of these genes and their associations with the pathology, we not only replicated key findings from our snRNA data analysis, but also identified a set of cell type-specific genes that show selective enrichment or depletion near pathological inclusions. Together, our findings allow us to prioritize specific cell types and pathways for targeted interventions at various stages of pathological progression in AD.

A structured evaluation of cryopreservation in generating single-cell transcriptomes from cerebrospinal fluid.

Single-cell transcriptomics allows characterization of cerebrospinal fluid (CSF) cells at an unprecedented level. Here, we report a robust cryopreservation protocol adapted for the characterization of fragile CSF cells by single-cell RNA sequencing (RNA-seq) in moderate- to large-scale studies. Fresh CSF was collected from twenty-one participants at two independent sites. Each CSF sample was split into two fractions: one was processed fresh, while the second was cryopreserved for months and profiled after thawing. B and T cell receptor sequencing was also performed. Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. We find no significant difference in cell type proportions and cellular transcriptomes between fresh and cryopreserved cells. Results were comparable at both sites and with different single-cell sequencing chemistries. Cryopreservation did not affect recovery of T and B cell clonotype diversity. Our CSF cell cryopreservation protocol provides an important alternative to fresh processing of fragile CSF cells.

Disease-specific selective vulnerability and neuroimmune pathways in dementia revealed by single cell genomics.

The development of successful therapeutics for dementias requires an understanding of their shared and distinct molecular features in the human brain. We performed single-nuclear RNAseq and ATACseq in Alzheimer disease (AD), Frontotemporal degeneration (FTD), and Progressive Supranuclear Palsy (PSP), analyzing 40 participants, yielding over 1.4M cells from three brain regions ranging in vulnerability and pathological burden. We identify 35 shared disease-associated cell types and 14 that are disease-specific, replicating those previously identified in AD. Disease - specific cell states represent molecular features of disease-specific glial-immune mechanisms and neuronal vulnerability in each disorder, layer 4/5 intra-telencephalic neurons in AD, layer 2/3 intra-telencephalic neurons in FTD, and layer 5/6 near-projection neurons in PSP. We infer intrinsic disease-associated gene regulatory networks, which we empirically validate by chromatin footprinting. We find that causal genetic risk acts in specific neuronal and glial cells that differ across disorders, primarily non-neuronal cells in AD and specific neuronal subtypes in FTD and PSP. These data illustrate the heterogeneous spectrum of glial and neuronal composition and gene expression alterations in different dementias and identify new therapeutic targets by revealing shared and disease-specific cell states.

Schizophrenia risk conferred by rare protein-truncating variants is conserved across diverse human populations.

Schizophrenia (SCZ) is a chronic mental illness and among the most debilitating conditions encountered in medical practice. A recent landmark SCZ study of the protein-coding regions of the genome identified a causal role for ten genes and a concentration of rare variant signals in evolutionarily constrained genes1. This recent study-and most other large-scale human genetics studies-was mainly composed of individuals of European (EUR) ancestry, and the generalizability of the findings in non-EUR populations remains unclear. To address this gap, we designed a custom sequencing panel of 161 genes selected based on the current knowledge of SCZ genetics and sequenced a new cohort of 11,580 SCZ cases and 10,555 controls of diverse ancestries. Replicating earlier work, we found that cases carried a significantly higher burden of rare protein-truncating variants (PTVs) among evolutionarily constrained genes (odds ratio = 1.48; P = 5.4 × 10-6). In meta-analyses with existing datasets totaling up to 35,828 cases and 107,877 controls, this excess burden was largely consistent across five ancestral populations. Two genes (SRRM2 and AKAP11) were newly implicated as SCZ risk genes, and one gene (PCLO) was identified as shared by individuals with SCZ and those with autism. Overall, our results lend robust support to the rare allelic spectrum of the genetic architecture of SCZ being conserved across diverse human populations.

Reduced NMDAR1 expression in the Sp4 hypomorphic mouse may contribute to endophenotypes of human psychiatric disorders.

The reduced expression of the Sp4 gene in Sp4 hypomorphic mice resulted in subtle vacuolization in the hippocampus as well as deficits in sensorimotor gating and contextual memory, putative endophenotypes for schizophrenia and other psychiatric disorders. In this study, we examined both spatial learning/memory and hippocampal long-term potentiation (LTP) of Sp4 hypomorphic mice. Impaired spatial learning/memory and markedly reduced LTP were found. To corroborate the functional studies, the expression of N-methyl-D-aspartate (NMDA) glutamate receptors was investigated with both western blot and immunohistochemical analyses. The reduced expression of the Sp4 gene decreased the level of the NR1 subunit of NMDA receptors in Sp4 hypomorphic mice. In human, SP4 gene was found to be deleted sporadically in schizophrenia patients, corroborating evidence that polymorphisms of human SP4 gene are associated with schizophrenia and other psychiatric disorders. Impaired NMDA neurotransmission has been implicated in several human psychiatric disorders. As yet, it remains unclear how mutations of candidate susceptibility genes for these disorders may contribute to the disruption of NMDA neurotransmission. Sp4 hypomorphic mice could therefore serve as a genetic model to investigate impaired NMDA functions resulting from loss-of-function mutations of human SP4 gene in schizophrenia and/or other psychiatric disorders. Furthermore, aberrant expression of additional genes, besides NMDAR1, likely also contributes to the behavioral abnormalities in Sp4 hypomorphic mice. Thus, further investigation of the Sp4 pathway may provide novel insights in our understanding of a variety of neuropsychiatric disorders.

Association of variants in BAT1-LTA-TNF-BTNL2 genes within 6p21.3 region show graded risk to leprosy in unrelated cohorts of Indian population.

Host immune response against Mycobacterium leprae plays an important role in providing resistance to infection and disease progression. Genome-wide linkage and association studies suggest the possibility of multiple risk loci within HLA (6p21.3) region. Any systematic study of relevance within the histocompatibility complex of importance in host immune response would be pertinent because of non-replication of the known loci and unavailable information on some of the unexplored genes and regions. A systematic scan was performed of the selected region involving LTA-TNF-LTB genes within 6p21.3 with a resolution of 1SNP/127 bp; and the SNPs in flanking BAT1, NFKBIL and BTNL2-DRA genes on the basis of their tag status or their presence in promoter/exonic regions with MAF of >5%. Nine SNPs located in BAT1, LTA, TNF genes and BTNL2-DRA interval showed strong association with leprosy susceptibility in two independent sets of North Indian population which was replicated in a geographically distinct East Indian population. Conditional logistic regression showed at least one functional SNP remaining significant in each gene, suggesting an independent role of each of the disease associated SNPs. In vitro reporter assay revealed that two SNPs located at BAT1 promoter and 13 kb upstream to LTA gene affected the transcription factor binding site, hence the gene expression. We unravel the role of unexplored immunologically important genes, BAT1 and BTNL2, in addition to known LTA and TNF genes, and the haplotypes of the significantly associated SNPs therein, to understand susceptibility to the disease, leprosy and its differential severity.

Leprosy and the adaptation of human toll-like receptor 1.

Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae and remains endemic in many parts of the world. Despite several major studies on susceptibility to leprosy, few genomic loci have been replicated independently. We have conducted an association analysis of more than 1,500 individuals from different case-control and family studies, and observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to leprosy (TLR1 I602S, case-control P = 5.7 x 10(-8), OR = 0.31, 95% CI = 0.20-0.48, and HLA-DQA1 rs1071630, case-control P = 4.9 x 10(-14), OR = 0.43, 95% CI = 0.35-0.54). The effect sizes of these associations suggest that TLR1 and HLA-DRB1/DQA1 are major susceptibility genes in susceptibility to leprosy. Further population differentiation analysis shows that the TLR1 locus is extremely differentiated. The protective dysfunctional 602S allele is rare in Africa but expands to become the dominant allele among individuals of European descent. This supports the hypothesis that this locus may be under selection from mycobacteria or other pathogens that are recognized by TLR1 and its co-receptors. These observations provide insight into the long standing host-pathogen relationship between human and mycobacteria and highlight the key role of the TLR pathway in infectious diseases.

Genetic variations and interactions in anti-inflammatory cytokine pathway genes in the outcome of leprosy: a study conducted on a MassARRAY platform.

BACKGROUND: Mycobacterium leprae is the etiologic pathogen that causes leprosy. The outcome of disease is dependent on the host genetic background. METHODS: We investigated the association of 51 single-nucelotide polymorphisms (SNPs) in anti-inflammatory cytokines (IL-10, TGFB1, IL-6, IL-4, and IL-13) and receptors (IL-10RA, IL-10RB, TGFBR1, TGFBR2, IL-6R, IL-4R, IL-5RA, IL-5RB, and IL-13RA1) with susceptibility to leprosy in a case-control study from New Delhi in northern India. This was followed by replication testing of associated SNPs in a geographically distinct and unrelated population from Orissa in eastern India. The functional potential of SNPs was established with in vitro reporter assays. RESULTS: Significant associations (P < .05) were observed for 8 polymorphisms (rs1800871, rs1800872, and rs1554286 of IL-10; rs3171425 and rs7281762 of IL-10RB; rs2228048 and rs744751 of TGFBR2; and rs1800797 of IL-6) with leprosy. This association was replicated for 4 SNPs (rs1554286 of IL-10, rs7281762 of IL-10RB, rs2228048 of TGFBR2, and rs1800797 of IL-6). The interaction study revealed a significantly greater association with leprosy risk than was obtained for any SNP individually. CONCLUSIONS: This study provides an interesting insight on the cumulative polygenic host component that regulates leprosy pathogenesis.

Cell-type-specific cis-eQTLs in eight human brain cell types identify novel risk genes for psychiatric and neurological disorders.

To date, most expression quantitative trait loci (eQTL) studies, which investigate how genetic variants contribute to gene expression, have been performed in heterogeneous brain tissues rather than specific cell types. In this study, we performed an eQTL analysis using single-nuclei RNA sequencing from 192 individuals in eight brain cell types derived from the prefrontal cortex, temporal cortex and white matter. We identified 7,607 eGenes, a substantial fraction (46%, 3,537/7,607) of which show cell-type-specific effects, with strongest effects in microglia. Cell-type-level eQTLs affected more constrained genes and had larger effect sizes than tissue-level eQTLs. Integration of brain cell type eQTLs with genome-wide association studies (GWAS) revealed novel relationships between expression and disease risk for neuropsychiatric and neurodegenerative diseases. For most GWAS loci, a single gene co-localized in a single cell type, providing new clues into disease etiology. Our findings demonstrate substantial contrast in genetic regulation of gene expression among brain cell types and reveal potential mechanisms by which disease risk genes influence brain disorders.

Epigenomic priming of immune genes implicates oligodendroglia in multiple sclerosis susceptibility.

Multiple sclerosis (MS) is characterized by a targeted attack on oligodendroglia (OLG) and myelin by immune cells, which are thought to be the main drivers of MS susceptibility. We found that immune genes exhibit a primed chromatin state in single mouse and human OLG in a non-disease context, compatible with transitions to immune-competent states in MS. We identified BACH1 and STAT1 as transcription factors involved in immune gene regulation in oligodendrocyte precursor cells (OPCs). A subset of immune genes presents bivalency of H3K4me3/H3K27me3 in OPCs, with Polycomb inhibition leading to their increased activation upon interferon gamma (IFN-γ) treatment. Some MS susceptibility single-nucleotide polymorphisms (SNPs) overlap with these regulatory regions in mouse and human OLG. Treatment of mouse OPCs with IFN-γ leads to chromatin architecture remodeling at these loci and altered expression of interacting genes. Thus, the susceptibility for MS may involve OLG, which therefore constitutes novel targets for immunological-based therapies for MS.

CellMixS: quantifying and visualizing batch effects in single-cell RNA-seq data.

A key challenge in single-cell RNA-sequencing (scRNA-seq) data analysis is batch effects that can obscure the biological signal of interest. Although there are various tools and methods to correct for batch effects, their performance can vary. Therefore, it is important to understand how batch effects manifest to adjust for them. Here, we systematically explore batch effects across various scRNA-seq datasets according to magnitude, cell type specificity, and complexity. We developed a cell-specific mixing score (cms) that quantifies mixing of cells from multiple batches. By considering distance distributions, the score is able to detect local batch bias as well as differentiate between unbalanced batches and systematic differences between cells of the same cell type. We compare metrics in scRNA-seq data using real and synthetic datasets and whereas these metrics target the same question and are used interchangeably, we find differences in scalability, sensitivity, and ability to handle differentially abundant cell types. We find that cell-specific metrics outperform cell type-specific and global metrics and recommend them for both method benchmarks and batch exploration.

muscat detects subpopulation-specific state transitions from multi-sample multi-condition single-cell transcriptomics data.

Single-cell RNA sequencing (scRNA-seq) has become an empowering technology to profile the transcriptomes of individual cells on a large scale. Early analyses of differential expression have aimed at identifying differences between subpopulations to identify subpopulation markers. More generally, such methods compare expression levels across sets of cells, thus leading to cross-condition analyses. Given the emergence of replicated multi-condition scRNA-seq datasets, an area of increasing focus is making sample-level inferences, termed here as differential state analysis; however, it is not clear which statistical framework best handles this situation. Here, we surveyed methods to perform cross-condition differential state analyses, including cell-level mixed models and methods based on aggregated pseudobulk data. To evaluate method performance, we developed a flexible simulation that mimics multi-sample scRNA-seq data. We analyzed scRNA-seq data from mouse cortex cells to uncover subpopulation-specific responses to lipopolysaccharide treatment, and provide robust tools for multi-condition analysis within the muscat R package.

Tau Pathology Drives Dementia Risk-Associated Gene Networks toward Chronic Inflammatory States and Immunosuppression.

To understand how neural-immune-associated genes and pathways contribute to neurodegenerative disease pathophysiology, we performed a systematic functional genomic analysis in purified microglia and bulk tissue from mouse and human AD, FTD, and PSP. We uncover a complex temporal trajectory of microglial-immune pathways involving the type 1 interferon response associated with tau pathology in the early stages, followed by later signatures of partial immune suppression and, subsequently, the type 2 interferon response. We find that genetic risk for dementias shows disease-specific patterns of pathway enrichment. We identify drivers of two gene co-expression modules conserved from mouse to human, representing competing arms of microglial-immune activation (NAct) and suppression (NSupp) in neurodegeneration. We validate our findings by using chemogenetics, experimental perturbation data, and single-cell sequencing in post-mortem brains. Our results refine the understanding of stage- and disease-specific microglial responses, implicate microglial viral defense pathways in dementia pathophysiology, and highlight therapeutic windows.