Three linked variants have opposing regulatory effects on isovaleryl-CoA dehydrogenase gene expression.

While genome-wide association studies (GWAS) and positive selection scans identify genomic loci driving human phenotypic diversity, functional validation is required to discover the variant(s) responsible. We dissected the IVD gene locus-which encodes the isovaleryl-CoA dehydrogenase enzyme-implicated by selection statistics, multiple GWAS, and clinical genetics as important to function and fitness. We combined luciferase assays, CRISPR/Cas9 genome-editing, massively parallel reporter assays (MPRA), and a deletion tiling MPRA strategy across regulatory loci. We identified three regulatory variants, including an indel, that may underpin GWAS signals for pulmonary fibrosis and testosterone, and that are linked on a positively selected haplotype in the Japanese population. These regulatory variants exhibit synergistic and opposing effects on IVD expression experimentally. Alleles at these variants lie on a haplotype tagged by the variant most strongly associated with IVD expression and metabolites, but with no functional evidence itself. This work demonstrates how comprehensive functional investigation and multiple technologies are needed to discover the true genetic drivers of phenotypic diversity.

Resolving organoid brain region identities by mapping single-cell genomic data to reference atlases.

Self-organizing tissues resembling brain structures generated from human stem cells offer exciting possibilities to study human brain development, disease, and evolution. These 3D models are complex and can contain cells at various stages of differentiation from different brain regions. Single-cell genomic methods provide powerful approaches to explore cell composition, differentiation trajectories, and genetic perturbations in brain organoid systems. However, it remains a major challenge to understand the heterogeneity observed within and between individual organoids. Here, we develop a set of computational tools (VoxHunt) to assess brain organoid patterning, developmental state, and cell identity through comparisons to spatial and single-cell transcriptome reference datasets. We use VoxHunt to characterize and visualize cell compositions using single-cell and bulk genomic data from multiple organoid protocols modeling different brain structures. VoxHunt will be useful to assess organoid engineering protocols and to annotate cell fates that emerge in organoids during genetic and environmental perturbation experiments.

Organoid single-cell genomic atlas uncovers human-specific features of brain development.

The human brain has undergone substantial change since humans diverged from chimpanzees and the other great apes1,2. However, the genetic and developmental programs that underlie this divergence are not fully understood. Here we have analysed stem cell-derived cerebral organoids using single-cell transcriptomics and accessible chromatin profiling to investigate gene-regulatory changes that are specific to humans. We first analysed cell composition and reconstructed differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. Brain-region composition varied in organoids from different iPSC lines, but regional gene-expression patterns remained largely reproducible across individuals. We analysed chimpanzee and macaque cerebral organoids and found that human neuronal development occurs at a slower pace relative to the other two primates. Using pseudotemporal alignment of differentiation paths, we found that human-specific gene expression resolved to distinct cell states along progenitor-to-neuron lineages in the cortex. Chromatin accessibility was dynamic during cortex development, and we identified divergence in accessibility between human and chimpanzee that correlated with human-specific gene expression and genetic change. Finally, we mapped human-specific expression in adult prefrontal cortex using single-nucleus RNA sequencing analysis and identified developmental differences that persist into adulthood, as well as cell-state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene-regulatory features that are unique to humans.

Detecting ancient positive selection in humans using extended lineage sorting.

Natural selection that affected modern humans early in their evolution has likely shaped some of the traits that set present-day humans apart from their closest extinct and living relatives. The ability to detect ancient natural selection in the human genome could provide insights into the molecular basis for these human-specific traits. Here, we introduce a method for detecting ancient selective sweeps by scanning for extended genomic regions where our closest extinct relatives, Neandertals and Denisovans, fall outside of the present-day human variation. Regions that are unusually long indicate the presence of lineages that reached fixation in the human population faster than expected under neutral evolution. Using simulations, we show that the method is able to detect ancient events of positive selection and that it can differentiate those from background selection. Applying our method to the 1000 Genomes data set, we find evidence for ancient selective sweeps favoring regulatory changes and present a list of genomic regions that are predicted to underlie positively selected human specific traits.

Anti-inflammatory effect of itraconazole in stable allergic bronchopulmonary aspergillosis: a randomized controlled trial.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) complicates chronic asthma and results from hypersensitivity to the fungus Aspergillus fumigatu s, causing an intense systemic immune response and progressive lung damage. OBJECTIVE: We sought to determine whether treatment with the antifungal agent itraconazole reduced eosinophilic airway inflammation in subjects with ABPA. METHODS: A randomized, double-blind, placebo-controlled trial was performed in stable subjects with ABPA (n = 29). Subjects received 400 mg of itraconazole per day (n = 15) or placebo (n = 14) for 16 weeks. All subjects were reviewed monthly with history, spirometry, and sputum induction to measure airway inflammation, serum total IgE and IgG levels to A fumigatu s, and blood eosinophil counts. RESULTS: By using regression analysis in a random-effects model, subjects receiving itraconazole had a decrease in sputum eosinophils of 35% per week, with no decrease seen in the placebo arm (P <.01). Sputum eosinophil cationic protein levels decreased with itraconazole treatment by 42% per week compared with 23% in the placebo group (P <.01). Itraconazole reduced systemic immune activation, leading to a decrease in serum IgE levels (310 IU/mL) compared with levels seen in the placebo group (increase of 18 IU/mL, P <.01) and a decrease in IgG levels to A fumigatu s (15.4 IU/mL) compared with levels seen in the placebo group (increase of 3.7 IU/mL, P =.03). There were fewer exacerbations requiring oral cortico-steroids in those treated with itraconazole compared with in the placebo group (P =.03). CONCLUSION: Itraconazole treatment of subjects with stable ABPA reduces eosinophilic airway inflammation, systemic immune activation, and exacerbations. These results imply that itraconazole is a potential adjunctive treatment for ABPA.