The Phenotypic variability of 16p11.2 distal BP2-BP3 deletion in a transgenerational family and in neurodevelopmentally ascertained samples.

BACKGROUND: We present genomic and phenotypic findings of a transgenerational family consisting of three male offspring, each with a maternally inherited distal 220 kb deletion at locus 16p11.2 (BP2-BP3). Genomic analysis of all family members was prompted by a diagnosis of autism spectrum disorder (ASD) in the eldest child, who also presented with a low body mass index. METHODS: All male offspring underwent extensive neuropsychiatric evaluation. Both parents were also assessed for social functioning and cognition. The family underwent whole-genome sequencing. Further data curation was undertaken from samples ascertained for neurodevelopmental disorders and congenital abnormalities. RESULTS: On medical examination, both the second and third-born male offspring presented with obesity. The second-born male offspring met research diagnostic criteria for ASD at 8 years of age and presented with mild attention deficits. The third-born male offspring was only noted as having motor deficits and received a diagnosis of developmental coordination disorder. Other than the 16p11.2 distal deletion, no additional contributing variants of clinical significance were observed. The mother was clinically evaluated and noted as having a broader autism phenotype. CONCLUSION: In this family, the phenotypes observed are most likely caused by the 16p11.2 distal deletion. The lack of other overt pathogenic mutations identified by genomic sequencing reinforces the variable expressivity that should be heeded in a clinical setting. Importantly, distal 16p11.2 deletions can present with a highly variable phenotype even within a single family. Our additional data curation provides further evidence on the variable clinical presentation among those with pathogenetic 16p11.2 (BP2-BP3) mutations.

Genomic architecture of autism from comprehensive whole-genome sequence annotation.

Fully understanding autism spectrum disorder (ASD) genetics requires whole-genome sequencing (WGS). We present the latest release of the Autism Speaks MSSNG resource, which includes WGS data from 5,100 individuals with ASD and 6,212 non-ASD parents and siblings (total n = 11,312). Examining a wide variety of genetic variants in MSSNG and the Simons Simplex Collection (SSC; n = 9,205), we identified ASD-associated rare variants in 718/5,100 individuals with ASD from MSSNG (14.1%) and 350/2,419 from SSC (14.5%). Considering genomic architecture, 52% were nuclear sequence-level variants, 46% were nuclear structural variants (including copy-number variants, inversions, large insertions, uniparental isodisomies, and tandem repeat expansions), and 2% were mitochondrial variants. Our study provides a guidebook for exploring genotype-phenotype correlations in families who carry ASD-associated rare variants and serves as an entry point to the expanded studies required to dissect the etiology in the ∼85% of the ASD population that remain idiopathic.

Synaptic Dysfunction in Human Neurons With Autism-Associated Deletions in PTCHD1-AS.

BACKGROUND: The Xp22.11 locus that encompasses PTCHD1, DDX53, and the long noncoding RNA PTCHD1-AS is frequently disrupted in male subjects with autism spectrum disorder (ASD), but the functional consequences of these genetic risk factors for ASD are unknown. METHODS: To evaluate the functional consequences of PTCHD1 locus deletions, we generated induced pluripotent stem cells (iPSCs) from unaffected control subjects and 3 subjects with ASD with microdeletions affecting PTCHD1-AS/PTCHD1, PTCHD1-AS/DDX53, or PTCHD1-AS alone. Function of iPSC-derived cortical neurons was assessed using molecular approaches and electrophysiology. We also compiled novel and known genetic variants of the PTCHD1 locus to explore the roles of PTCHD1 and PTCHD1-AS in genetic risk for ASD and other neurodevelopmental disorders. Finally, genome editing was used to explore the functional consequences of deleting a single conserved exon of PTCHD1-AS. RESULTS: iPSC-derived neurons from subjects with ASD exhibited reduced miniature excitatory postsynaptic current frequency and N-methyl-D-aspartate receptor hypofunction. We found that 35 ASD-associated deletions mapping to the PTCHD1 locus disrupted exons of PTCHD1-AS. We also found a novel ASD-associated deletion of PTCHD1-AS exon 3 and showed that exon 3 loss altered PTCHD1-AS splicing without affecting expression of the neighboring PTCHD1 coding gene. Finally, targeted disruption of PTCHD1-AS exon 3 recapitulated diminished miniature excitatory postsynaptic current frequency, supporting a role for the long noncoding RNA in the etiology of ASD. CONCLUSIONS: Our genetic findings provide strong evidence that PTCHD1-AS deletions are risk factors for ASD, and human iPSC-derived neurons implicate these deletions in the neurophysiology of excitatory synapses and in ASD-associated synaptic impairment.

OTUD7A Regulates Neurodevelopmental Phenotypes in the 15q13.3 Microdeletion Syndrome.

Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development.

Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder.

We are performing whole-genome sequencing of families with autism spectrum disorder (ASD) to build a resource (MSSNG) for subcategorizing the phenotypes and underlying genetic factors involved. Here we report sequencing of 5,205 samples from families with ASD, accompanied by clinical information, creating a database accessible on a cloud platform and through a controlled-access internet portal. We found an average of 73.8 de novo single nucleotide variants and 12.6 de novo insertions and deletions or copy number variations per ASD subject. We identified 18 new candidate ASD-risk genes and found that participants bearing mutations in susceptibility genes had significantly lower adaptive ability (P = 6 × 10-4). In 294 of 2,620 (11.2%) of ASD cases, a molecular basis could be determined and 7.2% of these carried copy number variations and/or chromosomal abnormalities, emphasizing the importance of detecting all forms of genetic variation as diagnostic and therapeutic targets in ASD.

Genome-wide characteristics of de novo mutations in autism.

De novo mutations (DNMs) are important in Autism Spectrum Disorder (ASD), but so far analyses have mainly been on the ~1.5% of the genome encoding genes. Here, we performed whole genome sequencing (WGS) of 200 ASD parent-child trios and characterized germline and somatic DNMs. We confirmed that the majority of germline DNMs (75.6%) originated from the father, and these increased significantly with paternal age only (p=4.2×10-10). However, when clustered DNMs (those within 20kb) were found in ASD, not only did they mostly originate from the mother (p=7.7×10-13), but they could also be found adjacent to de novo copy number variations (CNVs) where the mutation rate was significantly elevated (p=2.4×10-24). By comparing DNMs detected in controls, we found a significant enrichment of predicted damaging DNMs in ASD cases (p=8.0×10-9; OR=1.84), of which 15.6% (p=4.3×10-3) and 22.5% (p=7.0×10-5) were in the non-coding or genic non-coding, respectively. The non-coding elements most enriched for DNM were untranslated regions of genes, boundaries involved in exon-skipping and DNase I hypersensitive regions. Using microarrays and a novel outlier detection test, we also found aberrant methylation profiles in 2/185 (1.1%) of ASD cases. These same individuals carried independently identified DNMs in the ASD risk- and epigenetic- genes DNMT3A and ADNP. Our data begins to characterize different genome-wide DNMs, and highlight the contribution of non-coding variants, to the etiology of ASD.

Indexing Effects of Copy Number Variation on Genes Involved in Developmental Delay.

A challenge in clinical genomics is to predict whether copy number variation (CNV) affecting a gene or multiple genes will manifest as disease. Increasing recognition of gene dosage effects in neurodevelopmental disorders prompted us to develop a computational approach based on critical-exon (highly expressed in brain, highly conserved) examination for potential etiologic effects. Using a large CNV dataset, our updated analyses revealed significant (P < 1.64 × 10(-15)) enrichment of critical-exons within rare CNVs in cases compared to controls. Separately, we used a weighted gene co-expression network analysis (WGCNA) to construct an unbiased protein module from prenatal and adult tissues and found it significantly enriched for critical exons in prenatal (P < 1.15 × 10(-50), OR = 2.11) and adult (P < 6.03 × 10(-18), OR = 1.55) tissues. WGCNA yielded 1,206 proteins for which we prioritized the corresponding genes as likely to have a role in neurodevelopmental disorders. We compared the gene lists obtained from critical-exon and WGCNA analysis and found 438 candidate genes associated with CNVs annotated as pathogenic, or as variants of uncertain significance (VOUS), from among 10,619 developmental delay cases. We identified genes containing CNVs previously considered to be VOUS to be new candidate genes for neurodevelopmental disorders (GIT1, MVB12B and PPP1R9A) demonstrating the utility of this strategy to index the clinical effects of CNVs.

Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder.

IMPORTANCE: The use of genome-wide tests to provide molecular diagnosis for individuals with autism spectrum disorder (ASD) requires more study. OBJECTIVE: To perform chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) in a heterogeneous group of children with ASD to determine the molecular diagnostic yield of these tests in a sample typical of a developmental pediatric clinic. DESIGN, SETTING, AND PARTICIPANTS: The sample consisted of 258 consecutively ascertained unrelated children with ASD who underwent detailed assessments to define morphology scores based on the presence of major congenital abnormalities and minor physical anomalies. The children were recruited between 2008 and 2013 in Newfoundland and Labrador, Canada. The probands were stratified into 3 groups of increasing morphological severity: essential, equivocal, and complex (scores of 0-3, 4-5, and ≥6). EXPOSURES: All probands underwent CMA, with WES performed for 95 proband-parent trios. MAIN OUTCOMES AND MEASURES: The overall molecular diagnostic yield for CMA and WES in a population-based ASD sample stratified in 3 phenotypic groups. RESULTS: Of 258 probands, 24 (9.3%, 95%CI, 6.1%-13.5%) received a molecular diagnosis from CMA and 8 of 95 (8.4%, 95%CI, 3.7%-15.9%) from WES. The yields were statistically different between the morphological groups. Among the children who underwent both CMA and WES testing, the estimated proportion with an identifiable genetic etiology was 15.8% (95%CI, 9.1%-24.7%; 15/95 children). This included 2 children who received molecular diagnoses from both tests. The combined yield was significantly higher in the complex group when compared with the essential group (pairwise comparison, P = .002). [table: see text]. CONCLUSIONS AND RELEVANCE: Among a heterogeneous sample of children with ASD, the molecular diagnostic yields of CMA and WES were comparable, and the combined molecular diagnostic yield was higher in children with more complex morphological phenotypes in comparison with the children in the essential category. If replicated in additional populations, these findings may inform appropriate selection of molecular diagnostic testing for children affected by ASD.

Whole-genome sequencing of quartet families with autism spectrum disorder.

Autism spectrum disorder (ASD) is genetically heterogeneous, with evidence for hundreds of susceptibility loci. Previous microarray and exome-sequencing studies have examined portions of the genome in simplex families (parents and one ASD-affected child) having presumed sporadic forms of the disorder. We used whole-genome sequencing (WGS) of 85 quartet families (parents and two ASD-affected siblings), consisting of 170 individuals with ASD, to generate a comprehensive data resource encompassing all classes of genetic variation (including noncoding variants) and accompanying phenotypes, in apparently familial forms of ASD. By examining de novo and rare inherited single-nucleotide and structural variations in genes previously reported to be associated with ASD or other neurodevelopmental disorders, we found that some (69.4%) of the affected siblings carried different ASD-relevant mutations. These siblings with discordant mutations tended to demonstrate more clinical variability than those who shared a risk variant. Our study emphasizes that substantial genetic heterogeneity exists in ASD, necessitating the use of WGS to delineate all genic and non-genic susceptibility variants in research and in clinical diagnostics.

Human fetal oligodendrocyte progenitor cells from different gestational stages exhibit substantially different potential to myelinate.

To investigate age-related intrinsic regulation of the capacity of human fetal oligodendrocyte progenitor cells (OPCs) to myelinate, potential OPCs were selected from 15- to 23-gestational-week (gw) human fetal brain tissue based on the expression of gangliosides--recognized with the monoclonal antibody A2B5, which detects multipotent cells including OPCs--or platelet-derived growth factor receptor α (PDGFRα), an early marker of the oligodendroglial lineage. Cells were either cultured alone or cocultured with rat dorsal root ganglia neurons (DRGNs). When cultured alone, both the A2B5- and PDGFRα-selected cells exhibited age-dependent increases in early to mid-stage lineage markers, including sulfatides (O4 antibody) and the transcription factor Olig2, while the cell death rate correlated negatively with age. In coculture with neurons, cells also expressed the myelin components galactocerebroside (GC) and myelin basic protein (MBP), and ensheathed axons. In DRGN cocultures, A2B5+ cells derived from >19 gw produced more GC+/MBP+ cells compared with the 15-17-week cells. The number of GC+ cells making axonal contacts, and ensheathing axonal segments per cell increased proportionally to gestational age. This age-dependent difference in GC/MBP cell number and capacity to ensheath axons persisted when PDGFRα selection was used to enrich for the number of OPCs in cultures derived from younger ages. Addition of the growth factors brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) enhanced OPC differentiation under all conditions. These findings indicate that intrinsic regulatory mechanisms associated with the chronological age of the donor cells are key variables to assess when considering the myelination capacity of OPCs for cellular replacement therapy.