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.

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.

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.

TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation.

Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs mechanistically is unclear. Here, we found that midnolin promoted the destruction of many nuclear proteins, including transcription factors encoded by the immediate-early genes. Diverse stimuli induced midnolin, and its overexpression was sufficient to cause the degradation of its targets by a mechanism that did not require ubiquitination. Instead, midnolin associated with the proteasome via an α helix, used its Catch domain to bind a region within substrates that can form a ß strand, and used a ubiquitin-like domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation.

Hidden protein-altering variants influence diverse human phenotypes.

Structural variants (SVs) comprise the largest genetic variants, altering from 50 base pairs to megabases of DNA. However, SVs have not been effectively ascertained in most genetic association studies, leaving a key gap in our understanding of human complex trait genetics. We ascertained protein-altering SVs from UK Biobank whole-exome sequencing data (n=468,570) using haplotype-informed methods capable of detecting sub-exonic SVs and variation within segmental duplications. Incorporating SVs into analyses of rare variants predicted to cause gene loss-of-function (pLoF) identified 100 associations of pLoF variants with 41 quantitative traits. A low-frequency partial deletion of RGL3 exon 6 appeared to confer one of the strongest protective effects of gene LoF on hypertension risk (OR = 0.86 [0.82-0.90]). Protein-coding variation in rapidly-evolving gene families within segmental duplications-previously invisible to most analysis methods-appeared to generate 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.

Sestrin mediates detection of and adaptation to low-leucine diets in Drosophila.

Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine1. Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation2,3, but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn-/-) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (SesnL431E) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient.

Genome-wide association study of more than 40,000 bipolar disorder cases provides new insights into the underlying biology.

Bipolar disorder is a heritable mental illness with complex etiology. We performed a genome-wide association study of 41,917 bipolar disorder cases and 371,549 controls of European ancestry, which identified 64 associated genomic loci. Bipolar disorder risk alleles were enriched in genes in synaptic signaling pathways and brain-expressed genes, particularly those with high specificity of expression in neurons of the prefrontal cortex and hippocampus. Significant signal enrichment was found in genes encoding targets of antipsychotics, calcium channel blockers, antiepileptics and anesthetics. Integrating expression quantitative trait locus data implicated 15 genes robustly linked to bipolar disorder via gene expression, encoding druggable targets such as HTR6, MCHR1, DCLK3 and FURIN. Analyses of bipolar disorder subtypes indicated high but imperfect genetic correlation between bipolar disorder type I and II and identified additional associated loci. Together, these results advance our understanding of the biological etiology of bipolar disorder, identify novel therapeutic leads and prioritize genes for functional follow-up studies.

Complement genes contribute sex-biased vulnerability in diverse disorders.

Many common illnesses, for reasons that have not been identified, differentially affect men and women. For instance, the autoimmune diseases systemic lupus erythematosus (SLE) and Sjögren's syndrome affect nine times more women than men1, whereas schizophrenia affects men with greater frequency and severity relative to women2. All three illnesses have their strongest common genetic associations in the major histocompatibility complex (MHC) locus, an association that in SLE and Sjögren's syndrome has long been thought to arise from alleles of the human leukocyte antigen (HLA) genes at that locus3-6. Here we show that variation of the complement component 4 (C4) genes C4A and C4B, which are also at the MHC locus and have been linked to increased risk for schizophrenia7, generates 7-fold variation in risk for SLE and 16-fold variation in risk for Sjögren's syndrome among individuals with common C4 genotypes, with C4A protecting more strongly than C4B in both illnesses. The same alleles that increase risk for schizophrenia greatly reduce risk for SLE and Sjögren's syndrome. In all three illnesses, C4 alleles act more strongly in men than in women: common combinations of C4A and C4B generated 14-fold variation in risk for SLE, 31-fold variation in risk for Sjögren's syndrome, and 1.7-fold variation in schizophrenia risk among men (versus 6-fold, 15-fold and 1.26-fold variation in risk among women, respectively). At a protein level, both C4 and its effector C3 were present at higher levels in cerebrospinal fluid and plasma8,9 in men than in women among adults aged between 20 and 50 years, corresponding to the ages of differential disease vulnerability. Sex differences in complement protein levels may help to explain the more potent effects of C4 alleles in men, women's greater risk of SLE and Sjögren's syndrome and men's greater vulnerability to schizophrenia. These results implicate the complement system as a source of sexual dimorphism in vulnerability to diverse illnesses.

Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain.

The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in 690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.

Using Droplet Digital PCR to Analyze Allele-Specific RNA Expression.

Genome-wide association studies have discovered thousands of common alleles that associate with human phenotypes and disease. Many of these variants are in non-protein-coding (regulatory) regions and are believed to affect phenotypes by modifying gene expression. In any organism with a diploid genome, such as humans, measuring the expression of each allele of a gene provides a well-controlled way to identify allelic influences on that gene's expression. Here, we describe a protocol for precisely measuring the allele-specific expression of individual genes. This method targets the nucleotide differences between the two alleles of a gene within an individual and measures the "allelic skew," the extent to which one allele is expressed more than the other. We cover the design of effective assays, the optimization of reactions, and the interpretation of the resulting data.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

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.

Schizophrenia risk from complex variation of complement component 4.

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

Endothelial Cells from Capillary Malformations Are Enriched for Somatic GNAQ Mutations.

BACKGROUND: A somatic mutation in GNAQ (c.548G>A; p.R183Q), encoding Gαq, has been found in syndromic and sporadic capillary malformation tissue. However, the specific cell type containing the mutation is unknown. The purpose of this study was to determine which cells in capillary malformations have the GNAQ mutation. METHODS: Human capillary malformation tissue was obtained from 13 patients during a clinically indicated procedure. Droplet digital polymerase chain reaction, capable of detecting mutant allelic frequencies as low as 0.1 percent, was used to quantify the abundance of GNAQ mutant cells in capillary malformation tissue. Six specimens were fractionated by fluorescence-activated cell sorting into hematopoietic, endothelial, perivascular, and stromal cells. The frequency of GNAQ mutant cells in these populations was quantified by droplet digital polymerase chain reaction. RESULTS: Eight capillary malformations contained GNAQ p.R183Q mutant cells, two lesions had novel GNAQ mutations (p.R183L and p.R183G), and three capillary malformations did not have a detectable GNAQ p.R183 mutation. Mutant allelic frequencies ranged from 2 to 11 percent. Following fluorescence-activated cell sorting, the GNAQ mutation was found in the endothelial but not the platelet-derived growth factor receptor-ß-positive cell population; mutant allelic frequencies were 3 to 43 percent. CONCLUSION: Endothelial cells in capillary malformations are enriched for GNAQ mutations and are likely responsible for the pathophysiology underlying capillary malformation.

Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.

Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.

A rapid molecular approach for chromosomal phasing.

Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11-116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).

Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA.

OBJECTIVES: To test the hypothesis that somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS). STUDY DESIGN: We used next generation sequencing, droplet digital polymerase chain reaction, and single molecule molecular inversion probes to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Children's Hospital who had an isolated LM (n = 17), KTS (n = 21), fibro-adipose vascular anomaly (n = 8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n = 31) from Seattle Children's Hospital. RESULTS: Most individuals from Boston Children's Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), fibro-adipose vascular anomaly (5/8), and congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (31/33) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ∼ 80% of cases. Seventy-four percent of patients with LM from Seattle Children's Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells. CONCLUSIONS: Somatic PIK3CA mutations are the most common cause of isolated LMs and disorders in which LM is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism because the abundance of mutant cells in a malformed tissue can be low.

Genetic variation in human DNA replication timing.

Genomic 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.