Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

Massively parallel discovery of human-specific substitutions that alter enhancer activity.

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

A Statistical Framework for Mapping Risk Genes from De Novo Mutations in Whole-Genome-Sequencing Studies.

Analysis of de novo mutations (DNMs) from sequencing data of nuclear families has identified risk genes for many complex diseases, including multiple neurodevelopmental and psychiatric disorders. Most of these efforts have focused on mutations in protein-coding sequences. Evidence from genome-wide association studies (GWASs) strongly suggests that variants important to human diseases often lie in non-coding regions. Extending DNM-based approaches to non-coding sequences is challenging, however, because the functional significance of non-coding mutations is difficult to predict. We propose a statistical framework for analyzing DNMs from whole-genome sequencing (WGS) data. This method, TADA-Annotations (TADA-A), is a major advance of the TADA method we developed earlier for DNM analysis in coding regions. TADA-A is able to incorporate many functional annotations such as conservation and enhancer marks, to learn from data which annotations are informative of pathogenic mutations, and to combine both coding and non-coding mutations at the gene level to detect risk genes. It also supports meta-analysis of multiple DNM studies, while adjusting for study-specific technical effects. We applied TADA-A to WGS data of ∼300 autism-affected family trios across five studies and discovered several autism risk genes. The software is freely available for all research uses.

Synaptic, transcriptional and chromatin genes disrupted in autism.

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

Patterns and rates of exonic de novo mutations in autism spectrum disorders.

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

DRAW+SneakPeek: analysis workflow and quality metric management for DNA-seq experiments.

SUMMARY: We report our new DRAW+SneakPeek software for DNA-seq analysis. DNA resequencing analysis workflow (DRAW) automates the workflow of processing raw sequence reads including quality control, read alignment and variant calling on high-performance computing facilities such as Amazon elastic compute cloud. SneakPeek provides an effective interface for reviewing dozens of quality metrics reported by DRAW, so users can assess the quality of data and diagnose problems in their sequencing procedures. Both DRAW and SneakPeek are freely available under the MIT license, and are available as Amazon machine images to be used directly on Amazon cloud with minimal installation. AVAILABILITY: DRAW+SneakPeek is released under the MIT license and is available for academic and nonprofit use for free. The information about source code, Amazon machine images and instructions on how to install and run DRAW+SneakPeek locally and on Amazon elastic compute cloud is available at the National Institute on Aging Genetics of Alzheimer's Disease Data Storage Site (http://www.niagads.org/) and Wang lab Web site (http://wanglab.pcbi.upenn.edu/).

Massively parallel disruption of enhancers active in human neural stem cells.

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders, potentially by altering neural progenitor proliferation. However, the effects of genetic variation within regulatory elements on neural progenitors remain obscure. We use sgRNA-Cas9 screens in human neural stem cells (hNSCs) to disrupt 10,674 genes and 26,385 conserved regions in 2,227 enhancers active in the developing human cortex and determine effects on proliferation. Genes with proliferation phenotypes are associated with neurodevelopmental disorders and show biased expression in specific fetal human brain neural progenitor populations. Although enhancer disruptions overall have weaker effects than gene disruptions, we identify enhancer disruptions that severely alter hNSC self-renewal. Disruptions in human accelerated regions, implicated in human brain evolution, also alter proliferation. Integrating proliferation phenotypes with chromatin interactions reveals regulatory relationships between enhancers and their target genes contributing to neurogenesis and potentially to human cortical evolution.

Ultrafast high-endurance memory based on sliding ferroelectrics.

The persistence of voltage-switchable collective electronic phenomena down to the atomic scale has extensive implications for area- and energy-efficient electronics, especially in emerging nonvolatile memory technology. We investigate the performance of a ferroelectric field-effect transistor (FeFET) based on sliding ferroelectricity in bilayer boron nitride at room temperature. Sliding ferroelectricity represents a different form of atomically thin two-dimensional (2D) ferroelectrics, characterized by the switching of out-of-plane polarization through interlayer sliding motion. We examined the FeFET device employing monolayer graphene as the channel layer, which demonstrated ultrafast switching speeds on the nanosecond scale and high endurance exceeding 1011 switching cycles, comparable to state-of-the-art FeFET devices. These characteristics highlight the potential of 2D sliding ferroelectrics for inspiring next-generation nonvolatile memory technology.

Infantile melanoma--a triple threat: diagnosis and management.

Cases of infantile melanoma are very rare, and only a few have been presented in the literature. Pediatric melanoma can present to the clinician as a "triple threat" of delayed diagnosis resulting in thick lesions at risk for metastasis, histologic uncertainty of diagnosis, and a lack of data guiding regional node management and adjuvant therapy. Melanoma in childhood is an uncommon condition, and it is even more atypical in infants. There is evidence however that it is increasing in frequency. We present 2 cases of infantile melanoma diagnosed before age 1 and multidisciplinary management performed at our institution. One child was diagnosed with melanoma arising within a congenital melanocytic nevus, and the other presented with melanoma from a de novo acquired scalp lesion. The ambiguity surrounding correct pathologic diagnosis of melanoma in this age group, and the tendency for late diagnosis with thicker lesions presents special staging and treatment challenges to the team of specialists involved in the care of these children.

Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens.

CRISPR screens have been used to connect genetic perturbations with changes in gene expression and phenotypes. Here we describe a CRISPR-based, single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC) to link genetic perturbations to genome-wide chromatin accessibility in a large number of cells. In human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 105 chromatin-related genes, generating chromatin accessibility data for ~30,000 single cells. We correlate the loss of specific chromatin remodelers with changes in accessibility globally and at the binding sites of individual transcription factors (TFs). For example, we show that loss of the H3K27 methyltransferase EZH2 increases accessibility at heterochromatic regions involved in embryonic development and triggers expression of genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze changes in nucleosome spacing following the loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, single-cell method for studying the effect of genetic perturbations on chromatin in normal and disease states.

Genome-wide association study of corticobasal degeneration identifies risk variants shared with progressive supranuclear palsy.

Corticobasal degeneration (CBD) is a neurodegenerative disorder affecting movement and cognition, definitively diagnosed only at autopsy. Here, we conduct a genome-wide association study (GWAS) in CBD cases (n=152) and 3,311 controls, and 67 CBD cases and 439 controls in a replication stage. Associations with meta-analysis were 17q21 at MAPT (P=1.42 × 10(-12)), 8p12 at lnc-KIF13B-1, a long non-coding RNA (rs643472; P=3.41 × 10(-8)), and 2p22 at SOS1 (rs963731; P=1.76 × 10(-7)). Testing for association of CBD with top progressive supranuclear palsy (PSP) GWAS single-nucleotide polymorphisms (SNPs) identified associations at MOBP (3p22; rs1768208; P=2.07 × 10(-7)) and MAPT H1c (17q21; rs242557; P=7.91 × 10(-6)). We previously reported SNP/transcript level associations with rs8070723/MAPT, rs242557/MAPT, and rs1768208/MOBP and herein identified association with rs963731/SOS1. We identify new CBD susceptibility loci and show that CBD and PSP share a genetic risk factor other than MAPT at 3p22 MOBP (myelin-associated oligodendrocyte basic protein).

Antisense-mediated Exon Skipping Decreases Tau Protein Expression: A Potential Therapy For Tauopathies.

In Alzheimer's disease, progressive supranuclear palsy, and a number of other neurodegenerative diseases, the microtubule associated protein tau aggregates to form intracellular neurofibrillary tangles and glial tangles, abnormal structures that are part of disease pathogenesis. Disorders with aggregated tau are called tauopathies. Presently, there are no disease-modifying treatments for this disease class. Tau is encoded by the MAPT gene. We propose that reducing MAPT expression and thus the amount of tau protein made could prevent aggregation, and potentially be an approach to treat tauopathies. We tested 31 morpholinos, complementary to the sense strand of the MAPT gene to identify oligonucleotides that can downregulate MAPT expression and reduce the amount of tau protein produced. Oligonucleotides were tested in human neuroblastoma cell lines SH-SY5Y and IMR32. We identified several morpholinos that reduced MAPT mRNA expression up to 50% and tau protein levels up to ~80%. The two most potent oligonucleotides spanned the 3' boundary of exons 1 and 5, masking the 5'-splice sites of these exons. Both morpholinos induced skipping of the targeted exons. These in vitro findings were confirmed in mice transgenic for the entire human MAPT gene and that express human tau protein. These studies demonstrate the feasibility of using modified oligonucleotides to alter tau expression.

Renal neoplasms of childhood.

An abdominal mass in a child in the first year of life is usually of renal origin. The most common renal masses in the perinatal period are nonneoplastic and include hydronephrosis and multicystic dysplastic kidney. With the development of new therapeutic regimens and standardization of treatment protocols, the cure rate for the most common renal neoplasm, Wilms tumor, has risen to more than 90%. This article reviews the role of imaging in the diagnosis, staging, and follow-up of children with renal neoplasms. Pertinent epidemiologic, clinical, and histopathologic considerations are reviewed, and current recommendations for management are presented.

Histological study of osteoid osteoma's blood supply.

The osteoid osteoma is a painful lesion with a special predilection for the femur and tibia of young patients. Although the lesion has been described as richly innervated, its vascular supply has not been critically appraised to date in the pathology literature. To this end, we have undertaken a morphological study of 16 archival cases of osteoid osteoma, focusing primarily on the patterns of vascularization, utilizing traditional histological and immunohistochemical approaches. The study demonstrated that a prominent arterial and arteriolar blood supply was a constant finding within the various zones of soft tissues, skeletal muscle, and bone surrounding the nidus. It also showed that the caliber of the vessels underwent gradual attenuation throughout their centripetal course toward the nidus, where the vessels lost their muscularis as they merged into the capillary network of the nidus. Immunostaining with antibodies to neurofilament and S100 proteins revealed a pattern of innervation that was overall less exuberant than that described in some reports and that was virtually absent from the nidus. Taken together with data reported in the radiological literature, our findings lead us to wonder whether the osteoid osteoma may represent a response to the local stimulation of bony tissue by a primarily aberrant vasculature, a hypothesis that warrants further elucidation using state-of-the-art imaging approaches.

Ultrasound-guided aspiration of the hip in children: a new technique.

Evaluation of the child presenting with an irritable hip often requires aspiration of the hip. There are various methods for doing this procedure. We present a new technique for hip aspiration using high-resolution ultrasound imaging with color Doppler and a needle guide. This technique maximizes chances for a successful aspiration, minimizes risks to the child, avoids radiation exposure, and is easy to do and teach.

Septic arthritis in a patient with juvenile rheumatoid arthritis.

The acute flare of joint inflammation in the child with known juvenile rheumatoid arthritis causes concern primarily regarding the need for additional or modified medical treatment. Acute joint inflammation in an otherwise healthy child creates concern regarding the existence of joint infection. In the early phase of disease, the clinical findings and symptoms of an inflamed joint attributable to juvenile rheumatoid arthritis or infection may be similar and difficult to differentiate from the other. Juvenile rheumatoid arthritis usually is well controlled by medical interventions, however, the initiation of specific treatment is more urgent in children with joint sepsis. The following case report is presented to emphasize the difficulty in evaluation of patients with known juvenile rheumatoid arthritis and coexistent septic arthritis, and to discuss the methods used to differentiate between the two conditions.