Developmental and temporal characteristics of clonal sperm mosaicism.

Throughout development and aging, human cells accumulate mutations resulting in genomic mosaicism and genetic diversity at the cellular level. Mosaic mutations present in the gonads can affect both the individual and the offspring and subsequent generations. Here, we explore patterns and temporal stability of clonal mosaic mutations in male gonads by sequencing ejaculated sperm. Through 300× whole-genome sequencing of blood and sperm from healthy men, we find each ejaculate carries on average 33.3 ± 12.1 (mean ± SD) clonal mosaic variants, nearly all of which are detected in serial sampling, with the majority absent from sampled somal tissues. Their temporal stability and mutational signature suggest origins during embryonic development from a largely immutable stem cell niche. Clonal mosaicism likely contributes a transmissible, predicted pathogenic exonic variant for 1 in 15 men, representing a life-long threat of transmission for these individuals and a significant burden on human population health.

Getting to the Cores of Autism.

The genetic architecture of autism spectrum disorder (ASD) is itself a diverse allelic spectrum that consists of rare de novo or inherited variants in hundreds of genes and common polygenic risk at thousands of loci. ASD susceptibility genes are interconnected at the level of transcriptional and protein networks, and many function as genetic regulators of neurodevelopment or synaptic proteins that regulate neural activity. So that the core underlying neuropathologies can be further elucidated, we emphasize the importance of first defining subtypes of ASD on the basis of the phenotypic signatures of genes in model systems and humans.

CNVs: harbingers of a rare variant revolution in psychiatric genetics.

The genetic bases of neuropsychiatric disorders are beginning to yield to scientific inquiry. Genome-wide studies of copy number variation (CNV) have given rise to a new understanding of disease etiology, bringing rare variants to the forefront. A proportion of risk for schizophrenia, bipolar disorder, and autism can be explained by rare mutations. Such alleles arise by de novo mutation in the individual or in recent ancestry. Alleles can have specific effects on behavioral and neuroanatomical traits; however, expressivity is variable, particularly for neuropsychiatric phenotypes. Knowledge from CNV studies reflects the nature of rare alleles in general and will serve as a guide as we move forward into a new era of whole-genome sequencing.

Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

Customized de novo mutation detection for any variant calling pipeline: SynthDNM.

MOTIVATION: As sequencing technologies and analysis pipelines evolve, de novo mutation (DNM) calling tools must be adapted. Therefore, a flexible approach is needed that can accurately identify DNMs from genome or exome sequences from a variety of datasets and variant calling pipelines. RESULTS: Here, we describe SynthDNM, a random-forest based classifier that can be readily adapted to new sequencing or variant-calling pipelines by applying a flexible approach to constructing simulated training examples from real data. The optimized SynthDNM classifiers predict de novo SNPs and indels with robust accuracy across multiple methods of variant calling. AVAILABILITYAND IMPLEMENTATION: SynthDNM is freely available on Github (https://github.com/james-guevara/synthdnm). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling.

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.

The effects of common structural variants on 3D chromatin structure.

BACKGROUND: Three-dimensional spatial organization of chromosomes is defined by highly self-interacting regions 0.1-1 Mb in size termed Topological Associating Domains (TADs). Genetic factors that explain dynamic variation in TAD structure are not understood. We hypothesize that common structural variation (SV) in the human population can disrupt regulatory sequences and thereby influence TAD formation. To determine the effects of SVs on 3D chromatin organization, we performed chromosome conformation capture sequencing (Hi-C) of lymphoblastoid cell lines from 19 subjects for which SVs had been previously characterized in the 1000 genomes project. We tested the effects of common deletion polymorphisms on TAD structure by linear regression analysis of nearby quantitative chromatin interactions (contacts) within 240 kb of the deletion, and we specifically tested the hypothesis that deletions at TAD boundaries (TBs) could result in large-scale alterations in chromatin conformation. RESULTS: Large (> 10 kb) deletions had significant effects on long-range chromatin interactions. Deletions were associated with increased contacts that span the deleted region and this effect was driven by large deletions that were not located within a TAD boundary (nonTB). Some deletions at TBs, including a 80 kb deletion of the genes CFHR1 and CFHR3, had detectable effects on chromatin contacts. However for TB deletions overall, we did not detect a pattern of effects that was consistent in magnitude or direction. Large inversions in the population had a distinguishable signature characterized by a rearrangement of contacts that span its breakpoints. CONCLUSIONS: Our study demonstrates that common SVs in the population impact long-range chromatin structure, and deletions and inversions have distinct signatures. However, the effects that we observe are subtle and variable between loci. Genome-wide analysis of chromatin conformation in large cohorts will be needed to quantify the influence of common SVs on chromatin structure.

Identifying schizophrenia patients who carry pathogenic genetic copy number variants using standard clinical assessment: retrospective cohort study.

BACKGROUND: Copy number variants (CNVs) play a significant role in disease pathogenesis in a small subset of individuals with schizophrenia (~2.5%). Chromosomal microarray testing is a first-tier genetic test for many neurodevelopmental disorders. Similar testing could be useful in schizophrenia. AIMS: To determine whether clinically identifiable phenotypic features could be used to successfully model schizophrenia-associated (SCZ-associated) CNV carrier status in a large schizophrenia cohort. METHOD: Logistic regression and receiver operating characteristic (ROC) curves tested the accuracy of readily identifiable phenotypic features in modelling SCZ-associated CNV status in a discovery data-set of 1215 individuals with psychosis. A replication analysis was undertaken in a second psychosis data-set (n = 479). RESULTS: In the discovery cohort, specific learning disorder (OR = 8.12; 95% CI 1.16-34.88, P = 0.012), developmental delay (OR = 5.19; 95% CI 1.58-14.76, P = 0.003) and comorbid neurodevelopmental disorder (OR = 5.87; 95% CI 1.28-19.69, P = 0.009) were significant independent variables in modelling positive carrier status for a SCZ-associated CNV, with an area under the ROC (AUROC) of 74.2% (95% CI 61.9-86.4%). A model constructed from the discovery cohort including developmental delay and comorbid neurodevelopmental disorder variables resulted in an AUROC of 83% (95% CI 52.0-100.0%) for the replication cohort. CONCLUSIONS: These findings suggest that careful clinical history taking to document specific neurodevelopmental features may be informative in screening for individuals with schizophrenia who are at higher risk of carrying known SCZ-associated CNVs. Identification of genomic disorders in these individuals is likely to have clinical benefits similar to those demonstrated for other neurodevelopmental disorders.

Pathogenicity and functional impact of non-frameshifting insertion/deletion variation in the human genome.

Differentiation between phenotypically neutral and disease-causing genetic variation remains an open and relevant problem. Among different types of variation, non-frameshifting insertions and deletions (indels) represent an understudied group with widespread phenotypic consequences. To address this challenge, we present a machine learning method, MutPred-Indel, that predicts pathogenicity and identifies types of functional residues impacted by non-frameshifting insertion/deletion variation. The model shows good predictive performance as well as the ability to identify impacted structural and functional residues including secondary structure, intrinsic disorder, metal and macromolecular binding, post-translational modifications, allosteric sites, and catalytic residues. We identify structural and functional mechanisms impacted preferentially by germline variation from the Human Gene Mutation Database, recurrent somatic variation from COSMIC in the context of different cancers, as well as de novo variants from families with autism spectrum disorder. Further, the distributions of pathogenicity prediction scores generated by MutPred-Indel are shown to differentiate highly recurrent from non-recurrent somatic variation. Collectively, we present a framework to facilitate the interrogation of both pathogenicity and the functional effects of non-frameshifting insertion/deletion variants. The MutPred-Indel webserver is available at http://mutpred.mutdb.org/.

Frequency and Complexity of De Novo Structural Mutation in Autism.

Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.

SV2: accurate structural variation genotyping and de novo mutation detection from whole genomes.

Motivation: Structural variation (SV) detection from short-read whole genome sequencing is error prone, presenting significant challenges for population or family-based studies of disease. Results: Here, we describe SV2, a machine-learning algorithm for genotyping deletions and duplications from paired-end sequencing data. SV2 can rapidly integrate variant calls from multiple structural variant discovery algorithms into a unified call set with high genotyping accuracy and capability to detect de novo mutations. Availability and implementation: SV2 is freely available on GitHub (https://github.com/dantaki/SV2). Contact: jsebat@ucsd.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Marker chromosome genomic structure and temporal origin implicate a chromoanasynthesis event in a family with pleiotropic psychiatric phenotypes.

Small supernumerary marker chromosomes (sSMC) are chromosomal fragments difficult to characterize genomically. Here, we detail a proband with schizoaffective disorder and a mother with bipolar disorder with psychotic features who present with a marker chromosome that segregates with disease. We explored the architecture of this marker and investigated its temporal origin. Array comparative genomic hybridization (aCGH) analysis revealed three duplications and three triplications that spanned the short arm of chromosome 9, suggestive of a chromoanasynthesis-like event. Segregation of marker genotypes, phased using sSMC mosaicism in the mother, provided evidence that it was generated during a germline-level event in the proband's maternal grandmother. Whole-genome sequencing (WGS) was performed to resolve the structure and junctions of the chromosomal fragments, revealing further complexities. While structural variations have been previously associated with neuropsychiatric disorders and marker chromosomes, here we detail the precise architecture, human life-cycle genesis, and propose a DNA replicative/repair mechanism underlying formation.

When loss-of-function is loss of function: assessing mutational signatures and impact of loss-of-function genetic variants.

MOTIVATION: Loss-of-function genetic variants are frequently associated with severe clinical phenotypes, yet many are present in the genomes of healthy individuals. The available methods to assess the impact of these variants rely primarily upon evolutionary conservation with little to no consideration of the structural and functional implications for the protein. They further do not provide information to the user regarding specific molecular alterations potentially causative of disease. RESULTS: To address this, we investigate protein features underlying loss-of-function genetic variation and develop a machine learning method, MutPred-LOF, for the discrimination of pathogenic and tolerated variants that can also generate hypotheses on specific molecular events disrupted by the variant. We investigate a large set of human variants derived from the Human Gene Mutation Database, ClinVar and the Exome Aggregation Consortium. Our prediction method shows an area under the Receiver Operating Characteristic curve of 0.85 for all loss-of-function variants and 0.75 for proteins in which both pathogenic and neutral variants have been observed. We applied MutPred-LOF to a set of 1142 de novo vari3ants from neurodevelopmental disorders and find enrichment of pathogenic variants in affected individuals. Overall, our results highlight the potential of computational tools to elucidate causal mechanisms underlying loss of protein function in loss-of-function variants. AVAILABILITY AND IMPLEMENTATION: http://mutpred.mutdb.org. CONTACT: predrag@indiana.edu.

FOXP1-related intellectual disability syndrome: a recognisable entity.

BACKGROUND: Mutations in forkhead box protein P1 (FOXP1) cause intellectual disability (ID) and specific language impairment (SLI), with or without autistic features (MIM: 613670). Despite multiple case reports no specific phenotype emerged so far. METHODS: We correlate clinical and molecular data of 25 novel and 23 previously reported patients with FOXP1 defects. We evaluated FOXP1 activity by an in vitro luciferase model and assessed protein stability in vitro by western blotting. RESULTS: Patients show ID, SLI, neuromotor delay (NMD) and recurrent facial features including a high broad forehead, bent downslanting palpebral fissures, ptosis and/or blepharophimosis and a bulbous nasal tip. Behavioural problems and autistic features are common. Brain, cardiac and urogenital malformations can be associated. More severe ID and NMD, sensorineural hearing loss and feeding difficulties are more common in patients with interstitial 3p deletions (14 patients) versus patients with monogenic FOXP1 defects (34 patients). Mutations result in impaired transcriptional repression and/or reduced protein stability. CONCLUSIONS: FOXP1-related ID syndrome is a recognisable entity with a wide clinical spectrum and frequent systemic involvement. Our data will be helpful to evaluate genotype-phenotype correlations when interpreting next-generation sequencing data obtained in patients with ID and/or SLI and will guide clinical management.

From de novo mutations to personalized therapeutic interventions in autism.

The high heritability, early age at onset, and reproductive disadvantages of autism spectrum disorders (ASDs) are consistent with an etiology composed of dominant-acting de novo (spontaneous) mutations. Mutation detection by microarray analysis and DNA sequencing has confirmed that de novo copy-number variants or point mutations in protein-coding regions of genes contribute to risk, and some of the underlying causal variants and genes have been identified. As our understanding of autism genes develops, the spectrum of autism is breaking up into quanta of many different genetic disorders. Given the diversity of etiologies and underlying biochemical pathways, personalized therapy for ASDs is logical, and clinical genetic testing is a prerequisite.

Modelling schizophrenia using human induced pluripotent stem cells.

Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80-85%. Although post-mortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD, the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear. To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons (Supplementary Fig. 1). SCZD hiPSC neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of SCZD hiPSC neurons identified altered expression of many components of the cyclic AMP and WNT signalling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic loxapine. To date, hiPSC neuronal pathology has only been demonstrated in diseases characterized by both the loss of function of a single gene product and rapid disease progression in early childhood. We now report hiPSC neuronal phenotypes and gene expression changes associated with SCZD, a complex genetic psychiatric disorder.

Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia.

Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.

Mapping copy number variation by population-scale genome sequencing.

Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.