Rare X-linked variants carry predominantly male risk in autism, Tourette syndrome, and ADHD.

Autism spectrum disorder (ASD), Tourette syndrome (TS), and attention-deficit/hyperactivity disorder (ADHD) display strong male sex bias, due to a combination of genetic and biological factors, as well as selective ascertainment. While the hemizygous nature of chromosome X (Chr X) in males has long been postulated as a key point of "male vulnerability", rare genetic variation on this chromosome has not been systematically characterized in large-scale whole exome sequencing studies of "idiopathic" ASD, TS, and ADHD. Here, we take advantage of informative recombinations in simplex ASD families to pinpoint risk-enriched regions on Chr X, within which rare maternally-inherited damaging variants carry substantial risk in males with ASD. We then apply a modified transmission disequilibrium test to 13,052 ASD probands and identify a novel high confidence ASD risk gene at exome-wide significance (MAGEC3). Finally, we observe that rare damaging variants within these risk regions carry similar effect sizes in males with TS or ADHD, further clarifying genetic mechanisms underlying male vulnerability in multiple neurodevelopmental disorders that can be exploited for systematic gene discovery.

Whole-Brain Image Analysis and Anatomical Atlas 3D Generation Using MagellanMapper.

MagellanMapper is a software suite designed for visual inspection and end-to-end automated processing of large-volume, 3D brain imaging datasets in a memory-efficient manner. The rapidly growing number of large-volume, high-resolution datasets necessitates visualization of raw data at both macro- and microscopic levels to assess the quality of data, as well as automated processing to quantify data in an unbiased manner for comparison across a large number of samples. To facilitate these analyses, MagellanMapper provides both a graphical user interface for manual inspection and a command-line interface for automated image processing. At the macroscopic level, the graphical interface allows researchers to view full volumetric images simultaneously in each dimension and to annotate anatomical label placements. At the microscopic level, researchers can inspect regions of interest at high resolution to build ground truth data of cellular locations such as nuclei positions. Using the command-line interface, researchers can automate cell detection across volumetric images, refine anatomical atlas labels to fit underlying histology, register these atlases to sample images, and perform statistical analyses by anatomical region. MagellanMapper leverages established open-source computer vision libraries and is itself open source and freely available for download and extension. © 2020 Wiley Periodicals LLC. Basic Protocol 1: MagellanMapper installation Alternate Protocol: Alternative methods for MagellanMapper installation Basic Protocol 2: Import image files into MagellanMapper Basic Protocol 3: Region of interest visualization and annotation Basic Protocol 4: Explore an atlas along all three dimensions and register to a sample brain Basic Protocol 5: Automated 3D anatomical atlas construction Basic Protocol 6: Whole-tissue cell detection and quantification by anatomical label Support Protocol: Import a tiled microscopy image in proprietary format into MagellanMapper.

Genome-wide de novo risk score implicates promoter variation in autism spectrum disorder.

Whole-genome sequencing (WGS) has facilitated the first genome-wide evaluations of the contribution of de novo noncoding mutations to complex disorders. Using WGS, we identified 255,106 de novo mutations among sample genomes from members of 1902 quartet families in which one child, but not a sibling or their parents, was affected by autism spectrum disorder (ASD). In contrast to coding mutations, no noncoding functional annotation category, analyzed in isolation, was significantly associated with ASD. Casting noncoding variation in the context of a de novo risk score across multiple annotation categories, however, did demonstrate association with mutations localized to promoter regions. We found that the strongest driver of this promoter signal emanates from evolutionarily conserved transcription factor binding sites distal to the transcription start site. These data suggest that de novo mutations in promoter regions, characterized by evolutionary and functional signatures, contribute to ASD.

De Novo Sequence and Copy Number Variants Are Strongly Associated with Tourette Disorder and Implicate Cell Polarity in Pathogenesis.

We previously established the contribution of de novo damaging sequence variants to Tourette disorder (TD) through whole-exome sequencing of 511 trios. Here, we sequence an additional 291 TD trios and analyze the combined set of 802 trios. We observe an overrepresentation of de novo damaging variants in simplex, but not multiplex, families; we identify a high-confidence TD risk gene, CELSR3 (cadherin EGF LAG seven-pass G-type receptor 3); we find that the genes mutated in TD patients are enriched for those related to cell polarity, suggesting a common pathway underlying pathobiology; and we confirm a statistically significant excess of de novo copy number variants in TD. Finally, we identify significant overlap of de novo sequence variants between TD and obsessive-compulsive disorder and de novo copy number variants between TD and autism spectrum disorder, consistent with shared genetic risk.

An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder.

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.

Nucleation and growth of apatite by a self-assembled polycrystalline bioceramic.

The formation aspects of a polycrystalline self-assembled bioceramic leading to the nucleation of hard-tissue mineral from a supersaturated solution are discussed. Scanning electron imaging and surface-sensitive interrogations of the nucleated mineral indicated the presence of an intermediate amorphous layer encompassing a rather crystalline phase that formed on niobium oxide (Nb(2)O(5)) microstructures. The crystalline phase was identified from Raman spectroscopy as hydroxyapatite (HAP), while the phosphorous-rich amorphous layer is suggested to have the chemical form CaO-P(2)O(5). In addition, the mechanism favoring HAP nucleation is discussed in terms of the (0 0 2) and (0 0 1) diffraction planes of HAP and Nb(2)O(5), respectively. The small mismatch along several lattice dimensions strongly suggests epitaxy as a dominant mode in the heterogeneous nucleation of HAP. Furthermore, the effectiveness of this mode was shown to critically depend on the self-organization of the Nb(2)O(5) microstructures. Because nucleation does not appear to depend solely on the integrity of Nb(2)O(5) crystals, the self-organization of Nb(2)O(5) crystals also contributes significantly to HAP nucleation. Based on our results, we propose the organized arrangement of bioceramic crystals as a new mode for the bioinspiration of hydroxyapatite and other hard-tissue mineral.

Bioactivity of novel self-assembled crystalline Nb2O5 microstructures in simulated and human salivas.

Crystalline, self-assembled niobium oxide microstructures formed in situ via potentiostatic anodization of niobium foil in an HF(aq) electrolyte solution are proposed as exceptional nucleators of Ca-P minerals, including hydroxyapatite. This material was tested for bioactivity through immersion in simulated and pooled human salivas. The simulated saliva formulation was based on mineral content found in stimulated human saliva and has a molar Ca/P ratio of 1:3.7. Oxide microstructures and mineral morphologies were examined using scanning electron microscopy. Differences in the mineral phase and morphology were attributed to the contrasting complexities of the two supersaturated solutions, with proteins and enzymes in human saliva most likely imparting a significant role. Dimensions of the niobium oxide microstructures and mineral deposits were characterized using profilometry. Energy dispersive spectroscopy, x-ray diffraction, Raman spectroscopy and electron microprobe analysis were utilized in identifying the nucleated mineral phases. Nucleation from human saliva resulted in mixed-phase mineral formations including amorphous calcium phosphate and poorly crystalline apatites. On the other hand, mineral nucleation from simulated saliva was more specific to hydroxyapatite. Based on these results, we demonstrate that a crystalline, self-assembled metal oxide is a unique and efficient nucleator of hydroxyapatite and other Ca-P minerals in supersaturated salivary solutions.