Human SARS-CoV-2 challenge uncovers local and systemic response dynamics.

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Evidence for 28 genetic disorders discovered by combining healthcare and research data.

De novo mutations in protein-coding genes are a well-established cause of developmental disorders1. However, genes known to be associated with developmental disorders account for only a minority of the observed excess of such de novo mutations1,2. Here, to identify previously undescribed genes associated with developmental disorders, we integrate healthcare and research exome-sequence data from 31,058 parent-offspring trios of individuals with developmental disorders, and develop a simulation-based statistical test to identify gene-specific enrichment of de novo mutations. We identified 285 genes that were significantly associated with developmental disorders, including 28 that had not previously been robustly associated with developmental disorders. Although we detected more genes associated with developmental disorders, much of the excess of de novo mutations in protein-coding genes remains unaccounted for. Modelling suggests that more than 1,000 genes associated with developmental disorders have not yet been described, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of genes associated with developmental disorders.

Accessible Region Conformation Capture (ARC-C) gives high-resolution insights into genome architecture and regulation.

Nuclear organization and chromatin interactions are important for genome function, yet determining chromatin connections at high resolution remains a major challenge. To address this, we developed Accessible Region Conformation Capture (ARC-C), which profiles interactions between regulatory elements genome-wide without a capture step. Applied to Caenorhabditis elegans, ARC-C identifies approximately 15,000 significant interactions between regulatory elements at 500-bp resolution. Of 105 TFs or chromatin regulators tested, we find that the binding sites of 60 are enriched for interacting with each other, making them candidates for mediating interactions. These include cohesin and condensin II. Applying ARC-C to a mutant of transcription factor BLMP-1 detected changes in interactions between its targets. ARC-C simultaneously profiles domain-level architecture, and we observe that C. elegans chromatin domains defined by either active or repressive modifications form topologically associating domains (TADs) that interact with A/B (active/inactive) compartment-like structure. Furthermore, we discover that inactive compartment interactions are dependent on H3K9 methylation. ARC-C is a powerful new tool to interrogate genome architecture and regulatory interactions at high resolution.

Putative cell type discovery from single-cell gene expression data.

We present the Single-Cell Clustering Assessment Framework, a method for the automated identification of putative cell types from single-cell RNA sequencing (scRNA-seq) data. By iteratively applying a machine learning approach to a given set of cells, we simultaneously identify distinct cell groups and a weighted list of feature genes for each group. The differentially expressed feature genes discriminate the given cell group from other cells. Each such group of cells corresponds to a putative cell type or state, characterized by the feature genes as markers. Benchmarking using expert-annotated scRNA-seq datasets shows that our method automatically identifies the 'ground truth' cell assignments with high accuracy.

Dynamic landscape and regulation of RNA editing in mammals.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

Expression Atlas update: from tissues to single cells.

Expression Atlas is EMBL-EBI's resource for gene and protein expression. It sources and compiles data on the abundance and localisation of RNA and proteins in various biological systems and contexts and provides open access to this data for the research community. With the increased availability of single cell RNA-Seq datasets in the public archives, we have now extended Expression Atlas with a new added-value service to display gene expression in single cells. Single Cell Expression Atlas was launched in 2018 and currently includes 123 single cell RNA-Seq studies from 12 species. The website can be searched by genes within or across species to reveal experiments, tissues and cell types where this gene is expressed or under which conditions it is a marker gene. Within each study, cells can be visualized using a pre-calculated t-SNE plot and can be coloured by different features or by cell clusters based on gene expression. Within each experiment, there are links to downloadable files, such as RNA quantification matrices, clustering results, reports on protocols and associated metadata, such as assigned cell types.

Preparation, characterization, and antibacterial activity of tigecycline-loaded, ultrasound-activated microbubbles.

Central nervous system infectious disease caused by the multidrug-resistant Acinetobacter baumannii (AB) seriously threatens human life in clinic. Tigecycline has good sensitivity in killing AB, but due to its wide tissue distribution and blood-brain barrier, concentration in cerebrospinal fluid is low, therefore, the clinical effect is limited. Herein, we designed micro-bubbled tigecycline, aimed to enhance its anti-MDRAB effects under ultrasound. The lipid microbubbles with different ratios of lipids to drugs (a ratio of 10:1, 20:1, and 40:1) were prepared by the mechanical shaking method. The morphology, zeta potential and particle size of microbubbles were tested to screen out the much better formulation. Encapsulation efficiency and drug loading amount were determined by ultracentrifugation combined with high-performance liquid chromatography. Then the in vitro antibacterial activity against AB was conducted using the selected ultrasound-activated microbubble. Results showed the selected microbubbles with high encapsulation efficiency and good stability. The mechanical shaking method is feasible for preparation of drug-loaded and ultrasound-activated lipid microbubbles. Using 0.2 mg/mL microbubbles, combined with 1 MHz, 2.5 W/cm2 and 1 min of ultrasound exhibited a potent anit-AB in vitro. This study indicates that tigecycline treatment in form of ultrasound-activated microbubble is a promising strategy against AB infections.

Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3S HDAC complex in C. elegans.

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.

Tumor Extracellular pH-Driven Cancer-Selective Artificial Receptor-Mediated Tumor-Targeted Fluorescence Imaging.

Biomarker receptors on cancer cells can sense and recruit extracellular ligands and ligand-conjugated imaging agents/drugs, providing a critical basis upon which to develop an active tumor-targeting strategy. However, such a strategy can be confounded by both the limited number of cancer biomarker receptors and the inherent heterogeneity of cancer cells. Therefore, we herein report a simple strategy to deploy an exogenous physical label on the surface of cancer cells as an artificial receptor (AR) for active tumor targeting. It can be driven by the tumor extracellular acidic microenvironment to insert into the plasma membrane of cancer cells. Our studies demonstrated that an AR could efficiently sense and recruit the extracellular imaging agent Cy5-streptavidin conjugate to cancer cells, cancer cell spheroids, and an in vivo tumor. Based on the easy synthesis and chemical modification diversity of the peptide, our AR holds promise as a novel tumor-targeted strategy.

Stable Caenorhabditis elegans chromatin domains separate broadly expressed and developmentally regulated genes.

Eukaryotic genomes are organized into domains of differing structure and activity. There is evidence that the domain organization of the genome regulates its activity, yet our understanding of domain properties and the factors that influence their formation is poor. Here, we use chromatin state analyses in early embryos and third-larval stage (L3) animals to investigate genome domain organization and its regulation in Caenorhabditis elegans At both stages we find that the genome is organized into extended chromatin domains of high or low gene activity defined by different subsets of states, and enriched for H3K36me3 or H3K27me3, respectively. The border regions between domains contain large intergenic regions and a high density of transcription factor binding, suggesting a role for transcription regulation in separating chromatin domains. Despite the differences in cell types, overall domain organization is remarkably similar in early embryos and L3 larvae, with conservation of 85% of domain border positions. Most genes in high-activity domains are expressed in the germ line and broadly across cell types, whereas low-activity domains are enriched for genes that are developmentally regulated. We find that domains are regulated by the germ-line H3K36 methyltransferase MES-4 and that border regions show striking remodeling of H3K27me1, supporting roles for H3K36 and H3K27 methylation in regulating domain structure. Our analyses of C. elegans chromatin domain structure show that genes are organized by type into domains that have differing modes of regulation.

Large, rare chromosomal deletions associated with severe early-onset obesity.

Obesity is a highly heritable and genetically heterogeneous disorder. Here we investigated the contribution of copy number variation to obesity in 300 Caucasian patients with severe early-onset obesity, 143 of whom also had developmental delay. Large (>500 kilobases), rare (<1%) deletions were significantly enriched in patients compared to 7,366 controls (P < 0.001). We identified several rare copy number variants that were recurrent in patients but absent or at much lower prevalence in controls. We identified five patients with overlapping deletions on chromosome 16p11.2 that were found in 2 out of 7,366 controls (P < 5 x 10(-5)). In three patients the deletion co-segregated with severe obesity. Two patients harboured a larger de novo 16p11.2 deletion, extending through a 593-kilobase region previously associated with autism and mental retardation; both of these patients had mild developmental delay in addition to severe obesity. In an independent sample of 1,062 patients with severe obesity alone, the smaller 16p11.2 deletion was found in an additional two patients. All 16p11.2 deletions encompass several genes but include SH2B1, which is known to be involved in leptin and insulin signalling. Deletion carriers exhibited hyperphagia and severe insulin resistance disproportionate for the degree of obesity. We show that copy number variation contributes significantly to the genetic architecture of human obesity.

Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1.

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man's risk of disease by 10% (OR 1.10 [1.04-1.16], p<2 × 10(-3)), rare X-linked CNVs by 29%, (OR 1.29 [1.11-1.50], p<1 × 10(-3)), and rare Y-linked duplications by 88% (OR 1.88 [1.13-3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2 × 10(-5)). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.

Deleterious- and disease-allele prevalence in healthy individuals: insights from current predictions, mutation databases, and population-scale resequencing.

We have assessed the numbers of potentially deleterious variants in the genomes of apparently healthy humans by using (1) low-coverage whole-genome sequence data from 179 individuals in the 1000 Genomes Pilot Project and (2) current predictions and databases of deleterious variants. Each individual carried 281-515 missense substitutions, 40-85 of which were homozygous, predicted to be highly damaging. They also carried 40-110 variants classified by the Human Gene Mutation Database (HGMD) as disease-causing mutations (DMs), 3-24 variants in the homozygous state, and many polymorphisms putatively associated with disease. Whereas many of these DMs are likely to represent disease-allele-annotation errors, between 0 and 8 DMs (0-1 homozygous) per individual are predicted to be highly damaging, and some of them provide information of medical relevance. These analyses emphasize the need for improved annotation of disease alleles both in mutation databases and in the primary literature; some HGMD mutation data have been recategorized on the basis of the present findings, an iterative process that is both necessary and ongoing. Our estimates of deleterious-allele numbers are likely to be subject to both overcounting and undercounting. However, our current best mean estimates of ~400 damaging variants and ~2 bona fide disease mutations per individual are likely to increase rather than decrease as sequencing studies ascertain rare variants more effectively and as additional disease alleles are discovered.

Genome-wide transcriptome profiling reveals the functional impact of rare de novo and recurrent CNVs in autism spectrum disorders.

Copy-number variants (CNVs) are a major contributor to the pathophysiology of autism spectrum disorders (ASDs), but the functional impact of CNVs remains largely unexplored. Because brain tissue is not available from most samples, we interrogated gene expression in lymphoblasts from 244 families with discordant siblings in the Simons Simplex Collection in order to identify potentially pathogenic variation. Our results reveal that the overall frequency of significantly misexpressed genes (which we refer to here as outliers) identified in probands and unaffected siblings does not differ. However, in probands, but not their unaffected siblings, the group of outlier genes is significantly enriched in neural-related pathways, including neuropeptide signaling, synaptogenesis, and cell adhesion. We demonstrate that outlier genes cluster within the most pathogenic CNVs (rare de novo CNVs) and can be used for the prioritization of rare CNVs of potentially unknown significance. Several nonrecurrent CNVs with significant gene-expression alterations are identified (these include deletions in chromosomal regions 3q27, 3p13, and 3p26 and duplications at 2p15), suggesting that these are potential candidate ASD loci. In addition, we identify distinct expression changes in 16p11.2 microdeletions, 16p11.2 microduplications, and 7q11.23 duplications, and we show that specific genes within the 16p CNV interval correlate with differences in head circumference, an ASD-relevant phenotype. This study provides evidence that pathogenic structural variants have a functional impact via transcriptome alterations in ASDs at a genome-wide level and demonstrates the utility of integrating gene expression with mutation data for the prioritization of genes disrupted by potentially pathogenic mutations.

A Screen for Genomic Disorders of Infertility Identifies MAST2 Duplications Associated with Nonobstructive Azoospermia in Humans.

Since the cytogenetic identification of azoospermia factor regions 40 years ago, the Y chromosome has dominated research on the genetics of male infertility. We hypothesized that hotspots of structural rearrangement, which are dispersed across the genome, may mediate rare, recurrent copy number variations (CNVs), leading to severe infertility. We tested this hypothesis by contrasting patterns of rare CNVs in 970 Han Chinese men with idiopathic nonobstructive azoospermia and 1661 ethnicity-matched controls. Our results strongly support our previous claim that sperm production is modulated by genetic variation across the entire genome. The X chromosome in particular was enriched for loci modulating spermatogenesis--rare X-linked deletions larger than 100 kb were twice as common in patients compared with controls (odds ratio [OR] = 2.05, P = 0.01). At rearrangement hotspots across the genome, we observed a 2.4-fold enrichment of singleton CNVs in patients (P < 0.02), and we identified 117 testis genes, such as SYCE1, contained within 47 hotspots that may plausibly mediate genomic disorders of fertility. In our discovery sample we observed 3 case-specific duplications of the autosomal gene MAST2, and in a replication phase we found another 11 duplications in 1457 patients and 1 duplication in 1590 controls (P < 5 × 10(-5), combined data). With a large, polygenic genetic basis, new ways of establishing the pathogenicity of rare, large-effect mutations will be needed to fully reap the benefit of genome data in the management of azoospermia.

A common X-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphic autism.

We recently reported a deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene in a proband with autism. TMLHE maps to the X chromosome and encodes the first enzyme in carnitine biosynthesis, 6-N-trimethyllysine dioxygenase. Deletion of exon 2 of TMLHE causes enzyme deficiency, resulting in increased substrate concentration (6-N-trimethyllysine) and decreased product levels (3-hydroxy-6-N-trimethyllysine and γ-butyrobetaine) in plasma and urine. TMLHE deficiency is common in control males (24 in 8,787 or 1 in 366) and was not significantly increased in frequency in probands from simplex autism families (9 in 2,904 or 1 in 323). However, it was 2.82-fold more frequent in probands from male-male multiplex autism families compared with controls (7 in 909 or 1 in 130; P = 0.023). Additionally, six of seven autistic male siblings of probands in male-male multiplex families had the deletion, suggesting that TMLHE deficiency is a risk factor for autism (metaanalysis Z-score = 2.90 and P = 0.0037), although with low penetrance (2-4%). These data suggest that dysregulation of carnitine metabolism may be important in nondysmorphic autism; that abnormalities of carnitine intake, loss, transport, or synthesis may be important in a larger fraction of nondysmorphic autism cases; and that the carnitine pathway may provide a novel target for therapy or prevention of autism.

Phenotype-specific effect of chromosome 1q21.1 rearrangements and GJA5 duplications in 2436 congenital heart disease patients and 6760 controls.

Recurrent rearrangements of chromosome 1q21.1 that occur via non-allelic homologous recombination have been associated with variable phenotypes exhibiting incomplete penetrance, including congenital heart disease (CHD). However, the gene or genes within the ~1 Mb critical region responsible for each of the associated phenotypes remains unknown. We examined the 1q21.1 locus in 948 patients with tetralogy of Fallot (TOF), 1488 patients with other forms of CHD and 6760 ethnically matched controls using single nucleotide polymorphism genotyping arrays (Illumina 660W and Affymetrix 6.0) and multiplex ligation-dependent probe amplification. We found that duplication of 1q21.1 was more common in cases of TOF than in controls [odds ratio (OR) 30.9, 95% confidence interval (CI) 8.9-107.6); P = 2.2 × 10(-7)], but deletion was not. In contrast, deletion of 1q21.1 was more common in cases of non-TOF CHD than in controls [OR 5.5 (95% CI 1.4-22.0); P = 0.04] while duplication was not. We also detected rare (n = 3) 100-200 kb duplications within the critical region of 1q21.1 in cases of TOF. These small duplications encompassed a single gene in common, GJA5, and were enriched in cases of TOF in comparison to controls [OR = 10.7 (95% CI 1.8-64.3), P = 0.01]. These findings show that duplication and deletion at chromosome 1q21.1 exhibit a degree of phenotypic specificity in CHD, and implicate GJA5 as the gene responsible for the CHD phenotypes observed with copy number imbalances at this locus.