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The Kv8.1 potassium ion channel encoded by the KCNV1 gene is a “silent” subunit whose biological function is unknown. In ALS patient-derived motor neurons carrying SOD1(A4V) and C9orf72 mutations, its expression is highly reduced, yielding increased vulnerability to cell death without a change in motor neuronal firing. Our data suggests that Kv8.1 modulates Kv2 channel function to impact neuronal metabolism and lipid/protein transport pathways, but not excitability.

Funder: National Institute of Health (NIH) DP2-GM123495, DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome���s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) ��� sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.

Despite their widespread use in research, there has not yet been a systematic genomic analysis of human embryonic stem cell (hESC) lines at a single-nucleotide resolution. We therefore performed whole-genome sequencing (WGS) of 143 hESC lines and annotated their single-nucleotide and structural genetic variants. We found that while a substantial fraction of hESC lines contained large deleterious structural variants, finer-scale structural and single-nucleotide variants (SNVs) that are ascertainable only through WGS analyses were present in hESC genomes and human blood-derived genomes at similar frequencies. Moreover, WGS allowed us to identify SNVs associated with cancer and other diseases that could alter cellular phenotypes and compromise the safety of hESC-derived cellular products transplanted into humans. As a resource to enable reproducible hESC research and safer translation, we provide a user-friendly WGS data portal and a data-driven scheme for cell line maintenance and selection.

SUMMARYVariation in the human genome contributes to abundant diversity in human traits and vulnerabilities, but the underlying molecular and cellular mechanisms are not yet known, and will need scalable approaches to accelerate their recognition. Here, we advanced and applied an experimental platform that analyzes genetic, molecular, and phenotypic heterogeneity across cells from very many human donors cultured in a single, shared in vitro environment, with algorithms (Dropulation and Census-seq) for assigning phenotypes to individual donors. We used natural genetic variation and synthetic (CRISPR-Cas9) genetic perturbations to analyze the vulnerability of neural progenitor cells to infection with Zika virus. These analyses identified a common variant in the antiviral IFITM3 gene that regulated IFITM3 expression and explained most inter-individual variation in NPCs’ susceptibility to Zika virus infectivity. These and other approaches could provide scalable ways to recognize the impact of genes and genetic variation on cellular phenotypes.HIGHLIGHTSMeasuring cellular phenotypes in iPSCs and hPSC-derived NPCs from many donorsEffects of donor sex, cell source, genetic and other variables on hPSC RNA expressionNatural genetic variation and synthetic perturbation screens both identify IFITM3 in NPC susceptibility to Zika virusA common genetic variant in IFITM3 explains most inter-individual variation in NPC susceptibility to Zika virus

Down syndrome (DS), driven by an extra copy of chromosome 21 (HSA21), and fragile X syndrome (FXS), driven by loss of the RNA-binding protein FMRP, are two common genetic causes of intellectual disability and autism. Based upon the number of DS-implicated transcripts bound by FMRP, we hypothesize that DS and FXS may share underlying mechanisms. Comparing DS and FXS human pluripotent stem cell (hPSC) and glutamatergic neuron models, we identify increased protein expression of select targets and overlapping transcriptional perturbations. Moreover, acute upregulation of endogenous FMRP in DS patient cells using CRISPRa is sufficient to significantly reduce expression levels of candidate proteins and reverse 40% of global transcriptional perturbations. These results pinpoint specific molecular perturbations shared between DS and FXS that can be leveraged as a strategy for target prioritization; they also provide evidence for the functional relevance of previous associations between FMRP targets and disease-implicated genes.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterised by a progressive loss of motor function. The eponymous spinal sclerosis observed at autopsy is the result of the degeneration of extratelencephalic neurons, Betz cells (ETNs, Cortico-Spinal Motor Neuron). It remains unclear why this neuronal subtype is selectively affected. To understand the unique molecular properties that sensitise these cells to ALS, we performed RNA sequencing of 79,169 single nuclei from cortices of patients and controls. In unaffected individuals, we found that expression of ALS risk genes was significantly enriched inTHY1+-ETNs and not in other cell types. In patients, these genetic risk factors, as well as genes involved in protein homeostasis and stress responses, were significantly induced in a wide collection of ETNs, but not in neurons with more superficial identities. Examination of oligodendroglial and microglial nuclei revealed patient-specific changes that were at least in part a response to alterations in neurons: downregulation of myelinating genes in oligodendrocytes and upregulation of a reactive state connected to endo-lysosomal pathways in microglia. Our findings suggest that the selective vulnerability of extratelencephalic neurons is partly connected to their intrinsic molecular properties sensitising them to genetics and mechanisms of degeneration.Graphical abstract and working modelOur study highlights cell type specific changes in premotor/motor cortex of sporadic ALS patients. Specifically, we identify upregulation of synaptic molecules in excitatory neurons of upper cortical layers, interestingly correlating to hyperexcitability phenotypes seen in patients. Moreover, excitatory neurons of the deeper layers of the cortex, that project to the spinal cord and are most affected by the disease, show higher levels of cellular stresses than other neuronal types. Correspondently, oligodendrocytes transition from a highly myelinating state to a more neuronally engaged state, probably to counteract stressed phenotypes seen in excitatory neurons. At the same time, microglia show a reactive state with specific upregulation of endo-lysosomal pathways.

There has not yet been a systematic analysis of hESC whole genomes at a single nucleotide resolution. We therefore performed whole genome sequencing (WGS) of 143 hESC lines and annotated their single nucleotide and structural genetic variants. We found that while a substantial fraction of hESC lines contained large deleterious structural variants, finer scale structural and single nucleotide variants (SNVs) that are ascertainable only through WGS analyses were present in hESCs genomes and human blood-derived genomes at similar frequencies. However, WGS did identify SNVs associated with cancer or other diseases that will likely alter cellular phenotypes and may compromise the safety of hESC-derived cellular products transplanted into humans. As a resource to enable reproducible hESC research and safer translation, we provide a user-friendly WGS data portal and a data-driven scheme for cell line maintenance and selection.GRAPHICAL ABSTRACTIN BRIEFMerkle and Ghosh et al. describe insights from the whole genome sequences of commonly used human embryonic stem cell (hESC) lines. Analyses of these sequences show that while hESC genomes had more large structural variants than humans do from genetic inheritance, hESCs did not have an observable excess of finer-scale variants. However, many hESC lines contained rare loss-of-function variants and combinations of common variants that may profoundly shape their biological phenotypes. Thus, genome sequencing data can be valuable to those selecting cell lines for a given biological or clinical application, and the sequences and analysis reported here should facilitate such choices.HIGHLIGHTSOne third of hESCs we analysed are siblings, and almost all are of European ancestryLarge structural variants are common in hESCs, but finer-scale variation is similar to that human populationsMany strong-effect loss-of-function mutations and cancer-associated mutations are present in specific hESC linesWe provide user-friendly resources for rational hESC line selection based on genome sequence

SummaryTens of thousands of genetic variants shape human phenotypes, mostly by unknown cellular mechanisms. Here we describe Census-seq, a way to measure cellular phenotypes in cells from many people simultaneously. Analogous to pooled CRISPR screens but for natural variation, Census-seq associates cellular phenotypes to donors’ genotypes by quantifying the presence of each donor’s DNA in cell “villages” before and after sorting or selection for cellular traits of interest. Census-seq enables population-scale cell-biological phenotyping with low cost and high internal control. We demonstrate Census-seq through investigation of genetic effects on the SMN protein whose deficiency underlies spinal muscular atrophy (SMA). Census-seq quantified and mapped effects of many common alleles on SMN protein levels and response to SMN-targeted therapeutics, including a common, cryptic non-responder allele. We provide tools enabling population-scale cell experiments and explain how Census-seq can be used to map genetic effects on diverse cell phenotypes.Abstract FigureHighlightsCensus-seq reveals how inherited genetic variation affects cell phenotypesGenetic analysis of cellular traits in cell villages of >100 donorsCharacterizing human alleles that shape SMN protein expression and drug responsesDevelopment of protocols and software to enable cellular population genetics

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure and gene regulation. However, the genetic basis of the mammalian DNA replication timing program is poorly understood1–3. To systematically identify genetic regulators of DNA replication timing, we exploited inter-individual variation in 457 human pluripotent stem cell lines from 349 individuals. We show that the human genome’s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs4) – base-pair-resolution sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, to affect replication timing. Analysis of rtQTL locations reveals a histone code for replication initiation, composed of bivalent histone H3 trimethylation marks on a background of histone hyperacetylation. The H3 trimethylation marks are individually repressive yet synergize to promote early replication. We further identify novel positive and negative regulators of DNA replication timing, the former comprised of pluripotency-related transcription factors while the latter involve boundary elements. Human replication timing is controlled by a multi-layered mechanism that operates on target DNA sequences, is composed of dozens of effectors working combinatorially, and follows principles analogous to transcription regulation: a histone code, activators and repressors, and a promoter-enhancer logic.

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.

The identification of convergent phenotypes in different models of psychiatric illness highlights robust phenotypes that are more likely to be implicated in disease pathophysiology. Here, we utilize human iPSCs harboring distinct mutations in DISC1 that have been found in families with major mental illness. One mutation was engineered to mimic the consequences on DISC1 protein of a balanced translocation linked to mental illness in a Scottish pedigree; the other mutation was identified in an American pedigree with a high incidence of mental illness. Directed differentiation of these iPSCs using NGN2 expression shows rapid conversion to a homogenous population of mature excitatory neurons. Both DISC1 mutations result in reduced DISC1 protein expression, and show subtle effects on certain presynaptic proteins. In addition, RNA sequencing and qPCR showed decreased expression of UNC5D, DPP10, PCDHA6, and ZNF506 in neurons with both DISC1 mutations. Longitudinal analysis of neurite outgrowth revealed decreased neurite outgrowth in neurons with each DISC1 mutation, which was mimicked by UNC5D knockdown and rescued by transient upregulation of endogenous UNC5D. This study shows a narrow range of convergent phenotypes of two mutations found in families with major mental illness, and implicates dysregulated netrin signaling in DISC1 biology.

While a mutation in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS), much remains to be learned concerning the function of the protein normally encoded at this locus. To elaborate further on functions for C9ORF72, we used quantitative mass spectrometry-based proteomics to identify interacting proteins in motor neurons and found that its long isoform complexes with and stabilizes SMCR8, which further enables interaction with WDR41. To study the organismal and cellular functions for this tripartite complex, we generated Smcr8 loss-of-function mutant mice and found that they developed phenotypes also observed in C9orf72 loss-of-function animals, including autoimmunity. Along with a loss of tolerance for many nervous system autoantigens, we found increased lysosomal exocytosis in Smcr8 mutant macrophages. In addition to elevated surface Lamp1 (lysosome-associated membrane protein 1) expression, we also observed enhanced secretion of lysosomal components—phenotypes that we subsequently observed in C9orf72 loss-of-function macrophages. Overall, our findings demonstrate that C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity as well as negatively regulating lysosomal exocytosis—processes of potential importance to ALS.

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.

To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.

Abnormalities of brain connectivity and signal transduction are consistently observed in individuals with schizophrenias (SZ). Underlying these anomalies, convergent in vivo, post mortem, and genomic evidence suggest abnormal oligodendrocyte (OL) development and function and lower myelination in SZ. Our primary hypothesis was that there would be abnormalities in the number of induced pluripotent stem (iPS) cell-derived OLs from subjects with SZ. Our secondary hypothesis was that these in vitro abnormalities would correlate with measures of white matter (WM) integrity and myelination in the same subjects in vivo, estimated from magnetic resonance imaging. Six healthy control (HC) and six SZ iPS cell lines, derived from skin fibroblasts from well-characterized subjects, were differentiated into OLs. FACS analysis of the oligodendrocyte-specific surface, glycoprotein O4, was performed at three time points of development (days 65, 75, and 85) to quantify the number of late oligodendrocyte progenitor cells (OPCs) and OLs in each line. Significantly fewer O4-positive cells developed from SZ versus HC lines (95% CI 1.0: 8.6, F1,10 = 8.06, p = 0.02). The difference was greater when corrected for age (95% CI 5.4:10.4, F1,8 = 53.6, p 1,9 = 4.3, p = 0.07), reaching significance for mature OLs at day 85 in culture (r = 0.70, p

SUMMARYTo discover novel genetic risk factors underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry matched controls. We observed a significant excess of ultra-rare and rare protein-truncating variants (PTV) among ALS cases, which was primarily concentrated in constrained genes; however, a significant enrichment in PTVs does persist in the remaining exome. Through gene level analyses, known ALS genes, SOD1, NEK1, and FUS, were the most strongly associated with disease status. We also observed suggestive statistical evidence for multiple novel genes including DNAJC7, which is a highly constrained gene and a member of the heat shock protein family (HSP40). HSP40 proteins, along with HSP70 proteins, facilitate protein homeostasis, such as folding of newly synthesized polypeptides, and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of degraded proteins can occur leading to aberrant protein aggregation, one of the pathological hallmarks of neurodegeneration.

SUMMARY Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders., In Brief Nehme et al. combine two strong neuralizing factors (transcription factor programming and small molecule patterning) to generate human excitatory neurons from stem cells. They further undertake single-cell and reporter gene approaches to select highly differentiated neurons with increased functionality, augmenting their utility in the modeling of nervous system disorders.

SummaryGenomic DNA replicates in a choreographed temporal order that impacts the distribution of mutations along the genome. We show here that DNA replication timing is shaped by genetic polymorphisms that act in cis upon megabase-scale DNA segments. In genome sequences from proliferating cells, read depth along chromosomes reflected DNA replication activity in those cells. We used this relationship to analyze variation in replication timing among 161 individuals sequenced by the 1000 Genomes Project. Genome-wide association of replication timing with genetic variation identified 16 loci at which inherited alleles associate with replication timing. We call these “replication timing quantitative trait loci” (rtQTLs). rtQTLs involved the differential use of replication origins, exhibited allele-specific effects on replication timing, and associated with gene expression variation at megabase scales. Our results show replication timing to be shaped by genetic polymorphism and identify a means by which inherited polymorphism regulates the mutability of nearby sequences.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.

Genetic drift is a mechanism of population divergence that is important in the evolution of plants and animals but is thought to be rare in free-living microorganisms because of their typically large population sizes and unrestricted means of dispersal. We used both phylogenetic and insertion sequence (IS) element analyses in hyperthermophilic archaea of the genus Pyrococcus to test the hypothesis that genetic drift played an important role in the diversification of these microorganisms. Multilocus sequence typing of a collection of 36 isolates of Pyrococcus, from different hydrothermal systems in the Pacific Ocean and the Mediterranean Sea, revealed that Pyrococcus populations from different geographic locations are genetically differentiated. Analysis of IS elements in these isolates exposed their presence in all individuals of only one geographically isolated lineage, that of Vulcano Island in the Mediterranean Sea. Detailed sequence analysis of six selected IS elements in the Vulcano population showed that these elements cause deleterious genomic alterations, including inactivation of gene function. The high frequency of IS elements in the sampled population together with their observed harmful effects in the genome of Pyrococcus provide molecular evidence that the Vulcano Island population of Pyrococcus is geographically isolated and that those genetic mobile elements have been brought up to high frequency by genetic drift. Thus, genetic drift resulting from physical isolation should be considered as a factor influencing differentiation in prokaryotes.

Sensory information may be represented in the brain by stereotyped mapping of axonal inputs or by patterning that varies between individuals. In olfaction, a stereotyped map is evident in the first sensory processing centre, the olfactory bulb (OB), where different odours elicit activity in unique combinatorial patterns of spatially invariant glomeruli. Activation of each glomerulus is relayed to higher cortical processing centres by a set of ∼20-50 'homotypic' mitral and tufted (MT) neurons. In the cortex, target neurons integrate information from multiple glomeruli to detect distinct features of chemically diverse odours. How this is accomplished remains unclear, perhaps because the cortical mapping of glomerular information by individual MT neurons has not been described. Here we use new viral tracing and three-dimensional brain reconstruction methods to compare the cortical projections of defined sets of MT neurons. We show that the gross-scale organization of the OB is preserved in the patterns of axonal projections to one processing centre yet reordered in another, suggesting that distinct coding strategies may operate in different targets. However, at the level of individual neurons neither glomerular order nor stereotypy is preserved in either region. Rather, homotypic MT neurons from the same glomerulus innervate broad regions that differ between individuals. Strikingly, even in the same animal, MT neurons exhibit extensive diversity in wiring; axons of homotypic MT pairs diverge from each other, emit primary branches at distinct locations and 70-90% of branches of homotypic and heterotypic pairs are non-overlapping. This pronounced reorganization of sensory maps in the cortex offers an anatomic substrate for expanded combinatorial integration of information from spatially distinct glomeruli and predicts an unanticipated role for diversification of otherwise similar output neurons.