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Posttranscriptional RNA modifications by adenosine-to-inosine (A-to-I) editing are abundant in the brain, yet elucidating functional sites remains challenging. To bridge this gap, we investigate spatiotemporal and genetically regulated A-to-I editing sites across prenatal and postnatal stages of human brain development. More than 10,000 spatiotemporally regulated A-to-I sites were identified that occur predominately in 3′ UTRs and introns, as well as 37 sites that recode amino acids in protein coding regions with precise changes in editing levels across development. Hyper-edited transcripts are also enriched in the aging brain and stabilize RNA secondary structures. These features are conserved in murine and non-human primate models of neurodevelopment. Finally, thousands of cis -editing quantitative trait loci (edQTLs) were identified with unique regulatory effects during prenatal and postnatal development. Collectively, this work offers a resolved atlas linking spatiotemporal variation in editing levels to genetic regulatory effects throughout distinct stages of brain maturation. [Display omitted] • Alu editing activity is tightly regulated and increases across neurodevelopment • Spatiotemporally regulated sites in 3′ UTRs and coding regions offer functional insights • Hyper-edited RNA is enriched in the aging brain and stabilizes RNA secondary structures • cis -editing quantitative trait loci have unique regulatory effects in development This resource article illuminates precise regulation of A-to-I editing throughout human brain development. These findings provide an atlas of spatiotemporally and genetically regulated A-to-I sites and their putative functional effects during prenatal and postnatal periods. Additional avenues to dissect the role of RNA editing in neurodevelopment are provided. [ABSTRACT FROM AUTHOR]

Autism spectrum disorders (ASDs) are characterized by phenotypic and genetic heterogeneity. Our analysis of functional networks perturbed in ASD suggests that both truncating and nontruncating de novo mutations contribute to autism, with a bias against truncating mutations in early embryonic development. We find that functional mutations are preferentially observed in genes likely to be haploinsufficient. Multiple cell types and brain areas are affected, but the impact of ASD mutations appears to be strongest in cortical interneurons, pyramidal neurons and the medium spiny neurons of the striatum, implicating cortical and corticostriatal brain circuits. In females, truncating ASD mutations on average affect genes with 50-100% higher brain expression than in males. Our results also suggest that truncating de novo mutations play a smaller role in the etiology of high-functioning ASD cases. Overall, we find that stronger functional insults usually lead to more severe intellectual, social and behavioral ASD phenotypes. [ABSTRACT FROM AUTHOR]

Background: Structural variation (SV) influences genome organization and contributes to human disease. However, the complete mutational spectrum of SV has not been routinely captured in disease association studies. Results: We sequenced 689 participants with autism spectrum disorder (ASD) and other developmental abnormalities to construct a genome-wide map of large SV. Using long-insert jumping libraries at 105X mean physical coverage and linked-read whole-genome sequencing from 10X Genomics, we document seven major SV classes at ~5 kb SV resolution. Our results encompass 11,735 distinct large SV sites, 38.1% of which are novel and 16.8% of which are balanced or complex. We characterize 16 recurrent subclasses of complex SV (cxSV), revealing that: (1) cxSV are larger and rarer than canonical SV; (2) each genome harbors 14 large cxSV on average; (3) 84.4% of large cxSVs involve inversion; and (4) most large cxSV (93.8%) have not been delineated in previous studies. Rare SVs are more likely to disrupt coding and regulatory non-coding loci, particularly when truncating constrained and disease-associated genes. We also identify multiple cases of catastrophic chromosomal rearrangements known as chromoanagenesis, including somatic chromoanasynthesis, and extreme balanced germline chromothripsis events involving up to 65 breakpoints and 60.6 Mb across four chromosomes, further defining rare categories of extreme cxSV. Conclusions: These data provide a foundational map of large SV in the morbid human genome and demonstrate a previously underappreciated abundance and diversity of cxSV that should be considered in genomic studies of human disease. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1158-6) contains supplementary material, which is available to authorized users.

Recent research has uncovered a significant role for de novo variation in neurodevelopmental disorders. Using aggregated data from 9246 families with autism spectrum disorder, intellectual disability, or developmental delay, we show ~1/3 of de novo variants are independently observed as standing variation in the Exome Aggregation Consortium’s cohort of 60,706 adults, and these de novo variants do not contribute to neurodevelopmental risk. We further use a loss-of-function (LoF)-intolerance metric, pLI, to identify a subset of LoF-intolerant genes that contain the observed signal of associated de novo protein truncating variants (PTVs) in neurodevelopmental disorders. LoF-intolerant genes also carry a modest excess of inherited PTVs; though the strongest de novo impacted genes contribute little to this, suggesting the excess of inherited risk resides lower-penetrant genes. These findings illustrate the importance of population-based reference cohorts for the interpretation of candidate pathogenic variants, even for analyses of complex diseases and de novo variation.

The proximal region of chromosome 15 is one of the genomic hotspots for copy number variants ( CNVs). Among the rearrangements observed in this region, CNVs from the interval between the common breakpoints 1 and 2 ( BP1 and BP2) have been reported cosegregating with autism spectrum disorder ( ASD). Although evidence supporting an association between BP1- BP2 CNVs and autism accumulates, the magnitude of the effect of BP1- BP2 CNVs remains elusive, posing a great challenge to recurrence-risk counseling. To gain further insight into their pathogenicity for ASD, we estimated the penetrance of the BP1- BP2 CNVs for ASD as well as their effects on ASD-related phenotypes in a well-characterized ASD sample (n = 2525 families). Transmission disequilibrium test revealed significant preferential transmission only for the duplicated chromosome in probands (20 T:9 NT). The penetrance of the BP1- BP2 CNVs for ASD was low, conferring additional risks of 0.3% (deletion) and 0.8% (duplication). Stepwise regression analyses suggest a greater effect of the CNVs on ASD-related phenotype in males and when maternally inherited. Taken together, the results are consistent with BP1- BP2 CNVs as risk factors for autism. However, their effect is modest, more akin to that seen for common variants. To be consistent with the current American College of Medical Genetics guidelines for interpretation of postnatal CNV, the BP1- BP2 deletion and duplication CNVs would probably best be classified as variants of uncertain significance ( VOUS): they appear to have an impact on risk, but one so modest that these CNVs do not merit pathogenic status. Autism Res 2014, 7: 355-362. © 2014 International Society for Autism Research, Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background De novo loss-of-function (dnLoF) mutations are found twofold more often in autismspectrumdisorder (ASD) probands than their unaffected siblings. Multiple independent dnLoF mutations in the same gene implicate the gene in risk and hence provide a systematic, albeit arduous, path forward for ASD genetics. It is likely that using additional non-genetic data will enhance the ability to identify ASD genes. Methods To accelerate the search for ASD genes, we developed a novel algorithm, DAWN, to model two kinds of data: rare variations from exome sequencing and gene co-expression in the mid-fetal prefrontal and motor-somatosensory neocortex, a critical nexus for risk. The algorithm casts the ensemble data as a hidden Markov random field in which the graph structure is determined by gene co-expression and it combines these interrelationships with node-specific observations, namely gene identity, expression, genetic data and the estimated effect on risk. Results Using currently available genetic data and a specific developmental time period for gene coexpression, DAWN identified 127 genes that plausibly affect risk, and a set of likely ASD subnetworks. Validation experiments making use of published targeted resequencing results demonstrate its efficacy in reliably predicting ASD genes. DAWN also successfully predicts known ASD genes, not included in the genetic data used to create the model. Conclusions Validation studies demonstrate that DAWN is effective in predicting ASD genes and subnetworks by leveraging genetic and gene expression data. The findings reported here implicate neurite extension and neuronal arborization as risks for ASD. Using DAWN on emerging ASD sequence data and gene expression data from other brain regions and tissues would likely identify novel ASD genes. DAWN can also be used for other complex disorders to identify genes and subnetworks in those disorders. [ABSTRACT FROM AUTHOR]

Mice lacking DIX domain containing-1 (DIXDC1), an intracellular Wnt/β-catenin signal pathway protein, have abnormal measures of anxiety, depression and social behavior. Pyramidal neurons in these animals’ brains have reduced dendritic spines and glutamatergic synapses. Treatment with lithium or a Glycogen Synthase Kinase-3 (GSK3) inhibitor corrects behavioral and neurodevelopmental phenotypes in these animals. Analysis of DIXDC1 in over 9,000 cases of autism, bipolar disorder and schizophrenia reveals higher rates of rare inherited sequence-disrupting single nucleotide variants (SNVs) in these individuals compared to psychiatrically-unaffected controls. Many of these SNVs alter Wnt/β-catenin signaling activity of the neurally-predominant DIXDC1 isoform; a subset that hyperactivate this pathway cause dominant neurodevelopmental effects. We propose that rare missense SNVs in DIXDC1 contribute to psychiatric pathogenesis by reducing spine and glutamatergic synapse density downstream of GSK3 in the Wnt/β-catenin pathway.

De novo mutations affect risk for many diseases and disorders, especially those with early-onset. An example is autism spectrum disorders (ASD). Four recent whole-exome sequencing (WES) studies of ASD families revealed a handful of novel risk genes, based on independent de novo loss-of-function (LoF) mutations falling in the same gene, and found that de novo LoF mutations occurred at a twofold higher rate than expected by chance. However successful these studies were, they used only a small fraction of the data, excluding other types of de novo mutations and inherited rare variants. Moreover, such analyses cannot readily incorporate data from case-control studies. An important research challenge in gene discovery, therefore, is to develop statistical methods that accommodate a broader class of rare variation. We develop methods that can incorporate WES data regarding de novo mutations, inherited variants present, and variants identified within cases and controls. TADA, for Transmission And De novo Association, integrates these data by a gene-based likelihood model involving parameters for allele frequencies and gene-specific penetrances. Inference is based on a Hierarchical Bayes strategy that borrows information across all genes to infer parameters that would be difficult to estimate for individual genes. In addition to theoretical development we validated TADA using realistic simulations mimicking rare, large-effect mutations affecting risk for ASD and show it has dramatically better power than other common methods of analysis. Thus TADA's integration of various kinds of WES data can be a highly effective means of identifying novel risk genes. Indeed, application of TADA to WES data from subjects with ASD and their families, as well as from a study of ASD subjects and controls, revealed several novel and promising ASD candidate genes with strong statistical support. [ABSTRACT FROM AUTHOR]

SUMMARY Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from FEMFs (female-enriched multiplex families) with severe disease, enhancing the detection of key autism genes in modest numbers of cases. We show the utility of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated delta catenin protein (CTNND2) in FEMFs and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wildtype and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as FEMFs, are of innate value in multifactorial disorders.

There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis. [ABSTRACT FROM AUTHOR]

Loss-of-function variants in the gene SCN2A , which encodes the sodium channel Na V 1.2, are strongly associated with autism spectrum disorder and intellectual disability. An estimated 20%–30% of children with these variants also suffer from epilepsy, with altered neuronal activity originating in neocortex, a region where Na V 1.2 channels are expressed predominantly in excitatory pyramidal cells. This is paradoxical, as sodium channel loss in excitatory cells would be expected to dampen neocortical activity rather than promote seizure. Here, we examined pyramidal neurons lacking Na V 1.2 channels and found that they were intrinsically hyperexcitable, firing high-frequency bursts of action potentials (APs) despite decrements in AP size and speed. Compartmental modeling and dynamic-clamp recordings revealed that Na V 1.2 loss prevented potassium channels from properly repolarizing neurons between APs, increasing overall excitability by allowing neurons to reach threshold for subsequent APs more rapidly. This cell-intrinsic mechanism may, therefore, account for why SCN2A loss-of-function can paradoxically promote seizure. [Display omitted] • Conditional deletion of Na V 1.2 channels increases action potential (AP) excitability • Na V 1.2 regulates somatodendritc excitability, and Na V 1.6 regulates axonal action potential initiation • Lack of Na V 1.2 channels impairs AP repolarization by reducing K V activation • Reduced K V -mediated AP after hyperpolarization increases AP output Loss of sodium channels in individual neurons is expected to reduce neuronal excitability. Spratt and colleagues show that loss of Scn2a -encoded Na V 1.2 channels in mouse prefrontal pyramidal cells can paradoxically increase excitability due to Na V 1.2's distinct role in regulating somatodendritic excitability, independent of action potential electrogenesis in the axon. [ABSTRACT FROM AUTHOR]

Heterozygous variants in SLC6A1 , encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown. [Display omitted] Germline de novo variants in SLC6A1 are a major cause of neurodevelopmental disorders, including seizures. Enrichment for missense variants, many of which are recurrent, suggests a gain-of-function mechanism. However, here, we show that such variants consistently decreased GABA uptake in vitro , supporting a haploinsufficiency mechanism underlying the observed neurodevelopmental phenotypes. [ABSTRACT FROM AUTHOR]

The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3′UTR MPRA library with genomic elements containing variants from autism patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This targeted technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease. A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elements in vivo, using excitatory cortical neurons and striatal medium spiny neurons as test cases. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a common, complex, and highly heritable condition with contributions from both common and rare genetic variations. While disruptive, rare variants in protein-coding regions clearly contribute to symptoms, the role of rare non-coding remains unclear. Variants in these regions, including promoters, can alter downstream RNA and protein quantity; however, the functional impacts of specific variants observed in ASD cohorts remain largely uncharacterized. Here, we analyzed 3600 de novo mutations in promoter regions previously identified by whole-genome sequencing of autistic probands and neurotypical siblings to test the hypothesis that mutations in cases have a greater functional impact than those in controls. We leveraged massively parallel reporter assays (MPRAs) to detect transcriptional consequences of these variants in neural progenitor cells and identified 165 functionally high confidence de novo variants (HcDNVs). While these HcDNVs are enriched for markers of active transcription, disruption to transcription factor binding sites, and open chromatin, we did not identify differences in functional impact based on ASD diagnostic status. [ABSTRACT FROM AUTHOR]

The aim of this study was to catalog and evaluate response biomarkers correlated with autism spectrum disorder (ASD) symptoms to improve clinical trials. A systematic review of MEDLINE, Embase, and Scopus was conducted in April 2020. Seven criteria were applied to focus on original research that includes quantifiable response biomarkers measured alongside ASD symptoms. Interventional studies or human studies that assessed the correlation between biomarkers and ASD-related behavioral measures were included. A total of 5,799 independent records yielded 280 articles for review that reported on 940 biomarkers, 755 of which were unique to a single publication. Molecular biomarkers were the most frequently assayed, including cytokines, growth factors, measures of oxidative stress, neurotransmitters, and hormones, followed by neurophysiology (e.g., EEG and eye tracking), neuroimaging (e.g., functional MRI), and other physiological measures. Studies were highly heterogeneous, including in phenotypes, demographic characteristics, tissues assayed, and methods for biomarker detection. With a median total sample size of 64, almost all of the reviewed studies were only powered to identify biomarkers with large effect sizes. Reporting of individual-level values and summary statistics was inconsistent, hampering mega- and meta-analysis. Biomarkers assayed in multiple studies yielded mostly inconsistent results, revealing a "replication crisis." There is currently no response biomarker with sufficient evidence to inform ASD clinical trials. This review highlights methodological imperatives for ASD biomarker research necessary to make definitive progress: consistent experimental design, correction for multiple comparisons, formal replication, sharing of sample-level data, and preregistration of study designs. Systematic "big data" analyses of multiple potential biomarkers could accelerate discovery. [ABSTRACT FROM AUTHOR]

Neurodevelopmental neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia, have strong genetic risk components, but the underlying mechanisms have proven difficult to decipher. Rare, high‐risk variants may offer an opportunity to delineate the biological mechanisms responsible more clearly for more common idiopathic diseases. Indeed, different rare variants can cause the same behavioral phenotype, demonstrating genetic heterogeneity, while the same rare variant can cause different behavioral phenotypes, demonstrating variable expressivity. These observations suggest convergent underlying biological and neurological mechanisms; identification of these mechanisms may ultimately reveal new therapeutic targets. At the 2021 Keystone eSymposium "Neuropsychiatric and Neurodevelopmental Disorders: Harnessing Rare Variants" a panel of experts in the field described significant progress in genomic discovery and human phenotyping and raised several consistent issues, including the need for detailed natural history studies of rare disorders, the challenges in cohort recruitment, and the importance of viewing phenotypes as quantitative traits that are impacted by rare variants. [ABSTRACT FROM AUTHOR]

Background: Recent large‐scale initiatives have led to systematically collected phenotypic data for several rare genetic conditions implicated in autism spectrum disorder (ASD). The onset of developmentally expected skills (e.g. walking, talking) serve as readily quantifiable aspects of the behavioral phenotype. This study's aims were: (a) describe the distribution of ages of attainment of gross motor and expressive language milestones in several rare genetic conditions, and (b) characterize the likelihood of delays in these conditions compared with idiopathic ASD. Methods: Participants aged 3 years and older were drawn from two Simons Foundation Autism Research Initiative registries that employed consistent phenotyping protocols. Inclusion criteria were a confirmed genetic diagnosis of one of 16 genetic conditions (Simons Searchlight) or absence of known pathogenic genetic findings in individuals with ASD (SPARK). Parent‐reported age of acquisition of three gross motor and two expressive language milestones was described and categorized as on‐time or delayed, relative to normative expectations. Results: Developmental milestone profiles of probands with genetic conditions were marked by extensive delays (including nonattainment), with highest severity in single gene conditions and more delays than idiopathic ASD in motor skills. Compared with idiopathic ASD, the median odds of delay among the genetic groups were higher by 8.3 times (IQR 5.8–16.3) for sitting, 12.4 times (IQR 5.3–19.5) for crawling, 26.8 times (IQR 7.7–41.1) for walking, 2.7 times (IQR 1.7–5.5) for single words, and 5.7 times (IQR 2.7–18.3) for combined words. Conclusions: Delays in developmental milestones, particularly in gross motor skills, are frequent and may be among the earliest indicators of differentially affected developmental processes in specific genetically defined conditions associated with ASD, as compared with those with clinical diagnoses of idiopathic ASD. The possibility of different developmental pathways leading to ASD‐associated phenotypes should be considered when deciding how to employ specific genetic conditions as models for ASD. [ABSTRACT FROM AUTHOR]

Genetics, environment, and their interactions impact autism spectrum disorder etiology. Smoking is a suspected autism spectrum disorder risk factor due to biological plausibility and high prevalence. Using two large epidemiological samples, we examined whether autism spectrum disorder was associated with prenatal paternal smoking in a Discovery sample (N = 10,245) and an independent Replication sample (N = 29,773). Paternal smoking was retrospectively assessed with questionnaires. Likelihood of having autism spectrum disorder was estimated with the Autism Spectrum Screening Questionnaire at three levels: low (<10), intermediate (10–14), and high (⩾15). Ordinal regression was used to examine the relationship between prenatal paternal smoking and likelihood of having autism spectrum disorder, adjusting for confounders. A total of 36.5% of Discovery sample fathers and 63.3% of Replication sample fathers smoked during the pregnancy period; 7% of the Replication sample smoker fathers smoked during the pre-conception period but quit during pregnancy period. Discovery sample prenatal paternal smoking significantly increased the likelihood of having autism spectrum disorder in their offspring (adjusted odds ratio=1.27). This was confirmed in the Replication sample with adjusted odds ratio of 1.15 among smoking pre-conception period + pregnancy period fathers; 14.4% and 11.1% increased high likelihood of autism spectrum disorder was attributable to prenatal paternal smoking in Discovery sample and Replication sample, respectively. Smoking prevention, especially in pregnancy planning, may decrease autism spectrum disorder risk in offspring. What is Already Known about This Subject: Genetics, (including de novo mutations), environmental factors (including toxic exposures), and their interactions impact autism spectrum disorder etiology. Paternal smoking is a candidate risk for autism spectrum disorder due to biological plausibility, high prevalence, and potential intervention. What This Study Adds: This original study and its replication confirms that paternal factors can substantially contribute to autism spectrum disorder risk for their offspring. It specifically indicates that paternal smoking both before and during pregnancy contributes significantly to autism spectrum disorder risk. Implications for practice, research, or policy: Smoking prevention, especially in pregnancy planning, may decrease autism spectrum disorder risk in offspring. [ABSTRACT FROM AUTHOR]

Background: Genetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain. Methods: RNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples. Results: In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A, a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories. Conclusions: Exon usage in SCN1A, SCN2A, SCN3A, and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general. [ABSTRACT FROM AUTHOR]

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A , which encodes the sodium channel Na V 1.2, and ANK2 , which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how Na V 1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding Na V 1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a +/− conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology. • Ankyrin-B (Ank2) is expressed throughout neocortical pyramidal cell dendrites • Ankyrin-B scaffolds the sodium channel Na V 1.2 (Scn2a) to dendritic membranes • Haploinsufficiency in either Ank2 or Scn2a impairs dendritic excitability • Shared function suggests that dendritic dysfunction contributes to ASD etiology Autism spectrum disorder (ASD) is associated with dysfunction in hundreds of genes. How dysfunction in ASD-associated genes converges on shared biological mechanisms remains an open question. Here, Nelson et al. show that one ASD-associated gene, Ank2 , scaffolds another, Scn2a , in neocortical pyramidal cell dendrites, with shared effects on dendritic integration. [ABSTRACT FROM AUTHOR]

Interactions between genetic variants--epistasis--is pervasive in model systems and can profoundly impact evolutionary adaption, population disease dynamics, genetic mapping, and precision medicine efforts. In this work, we develop a model for structured polygenic epistasis, called coordinated epistasis (CE), and prove that several recent theories of genetic architecture fall under the formal umbrella of CE. Unlike standard epistasis models that assume epistasis and main effects are independent, CE captures systematic correlations between epistasis and main effects that result from pathway-level epistasis, on balance skewing the penetrance of genetic effects. To test for the existence of CE, we propose the even-odd (EO) test and prove it is calibrated in a range of realistic biological models. Applying the EO test in the UK Biobank, we find evidence of CE in 18 of 26 traits spanning disease, anthropometric, and blood categories. Finally, we extend the EO test to tissue-specific enrichment and identify several plausible tissue-trait pairs. Overall, CE is a dimension of genetic architecture that can capture structured, systemic forms of epistasis in complex human traits. [ABSTRACT FROM AUTHOR]

Each human genome includes de novo mutations that arose during gametogenesis. While these germline mutations represent a fundamental source of new genetic diversity, they can also create deleterious alleles that impact fitness. Whereas the rate and patterns of point mutations in the human germline are now well understood, far less is known about the frequency and features that impact de novo structural variants (dnSVs). We report a family-based study of germline mutations among 9,599 human genomes from 33 multigenerational CEPH-Utah families and 2,384 families from the Simons Foundation Autism Research Initiative. We find that de novo structural mutations detected by alignment-based, short-read WGS occur at an overall rate of at least 0.160 events per genome in unaffected individuals, and we observe a significantly higher rate (0.206 per genome) in ASD-affected individuals. In both probands and unaffected samples, nearly 73% of de novo structural mutations arose in paternal gametes, and we predict most de novo structural mutations to be caused by mutational mechanisms that do not require sequence homology. After multiple testing correction, we did not observe a statistically significant correlation between parental age and the rate of de novo structural variation in offspring. These results highlight that a spectrum of mutational mechanisms contribute to germline structural mutations and that these mechanisms most likely have markedly different rates and selective pressures than those leading to point mutations. [ABSTRACT FROM AUTHOR]

Background: The cause of most fetal anomalies is not determined prenatally. Exome sequencing has transformed genetic diagnosis after birth, but its usefulness for prenatal diagnosis is still emerging. Nonimmune hydrops fetalis (NIHF), a fetal abnormality that is often lethal, has numerous genetic causes; the extent to which exome sequencing can aid in its diagnosis is unclear.Methods: We evaluated a series of 127 consecutive unexplained cases of NIHF that were defined by the presence of fetal ascites, pleural or pericardial effusions, skin edema, cystic hygroma, increased nuchal translucency, or a combination of these conditions. The primary outcome was the diagnostic yield of exome sequencing for detecting genetic variants that were classified as either pathogenic or likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics. Secondary outcomes were the percentage of cases associated with specific genetic disorders and the proportion of variants that were inherited.Results: In 37 of the 127 cases (29%), we identified diagnostic genetic variants, including those for disorders affecting the RAS-MAPK cell-signaling pathway (known as RASopathies) (30% of the genetic diagnoses); inborn errors of metabolism and musculoskeletal disorders (11% each); lymphatic, neurodevelopmental, cardiovascular, and hematologic disorders (8% each); and others. Prognoses ranged from a relatively mild outcome to death during the perinatal period. Overall, 68% of the cases (25 of 37) with diagnostic variants were autosomal dominant (of which 12% were inherited and 88% were de novo), 27% (10 of 37) were autosomal recessive (of which 95% were inherited and 5% were de novo), 1 was inherited X-linked recessive, and 1 was of uncertain inheritance. We identified potentially diagnostic variants in an additional 12 cases.Conclusions: In this large case series of 127 fetuses with unexplained NIHF, we identified a diagnostic genetic variant in approximately one third of the cases. (Funded by the UCSF Center for Maternal-Fetal Precision Medicine and others; ClinicalTrials.gov number, NCT03412760.). [ABSTRACT FROM AUTHOR]

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants. [ABSTRACT FROM AUTHOR]

Autism spectrum disorders are a genetically heterogeneous constellation of syndromes characterized by impairments in reciprocal social interaction. Available somatic treatments have limited efficacy. We have identified inactivating mutations in the gene BCKDK (Branched Chain Ketoacid Dehydrogenase Kinase) in consanguineous fatuities with autism, epilepsy, and intellectual disability. The encoded protein is responsible for phosphorylation-mediated inactivation of the E1α subunit of branched-chain ketoacid dehydrogenase (BCKDH). Patients with homozygous BCKDK mutations display reductions in BCKDK messenger RNA and protein, E1α phosphorylation, and plasma branched-chain amino acids. Bckdk knockout mice show abnormal brain amino acid profiles and neurobehavioral deficits that respond to dietary supplementation. Thus, autism presenting with intellectual disability and epilepsy caused by BCKDK mutations represents a potentially treatable syndrome. [ABSTRACT FROM AUTHOR]

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1 , and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease. • ∼19,000 enhancers defined in nine regions of the developing human telencephalon • Chromatin dynamics correlate with sequence motifs and spatiotemporal gene expression • Identified cortical layer-specific enhancers and validated a layer 5 FEZF2 enhancer • Genetic variants from patients alter activity of an enhancer for an autism risk gene A high-resolution atlas of regulatory elements driving regional, temporal, and laminar gene expression programs in the developing human telencephalon reveals enhancers and genetic variants regulating human disease genes. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is strongly associated with de novo gene mutations. One of the most commonly affected genes is SCN2A. ASD-associated SCN2A mutations impair the encoded protein Na V 1.2, a sodium channel important for action potential initiation and propagation in developing excitatory cortical neurons. The link between an axonal sodium channel and ASD, a disorder typically attributed to synaptic or transcriptional dysfunction, is unclear. Here we show that Na V 1.2 is unexpectedly critical for dendritic excitability and synaptic function in mature pyramidal neurons in addition to regulating early developmental axonal excitability. Na V 1.2 loss reduced action potential backpropagation into dendrites, impairing synaptic plasticity and synaptic strength, even when Na V 1.2 expression was disrupted in a cell-autonomous fashion late in development. These results reveal a novel dendritic function for Na V 1.2, providing insight into cellular mechanisms probably underlying circuit and behavioral dysfunction in ASD. • Protein truncation in SCN2A /Na V 1.2 bears considerable autism spectrum disorder risk • Na V 1.2 governs action potential initiation in immature neocortical pyramidal cells • Mature Scn2a +/− neurons have impaired dendritic excitability and excitatory synapses • Cell-autonomous loss of Na V 1.2 in mature cells alone impairs synaptic function Haploinsufficiency in the gene SCN2A , which encodes the sodium channel Na V 1.2, has strong autism association. Spratt et al. show that Scn2a contributes to dendritic excitability in mature neocortical pyramidal cells, where its loss impairs excitatory synaptic function and plasticity. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) that affects approximately 4% of males and 1% of females in the United States. While causes of ASD are multi-factorial, single rare genetic variants contribute to around 20% of cases. Here, we report a case series of seven unrelated probands (6 males, 1 female) with ASD or another variable NDD phenotype attributed to de novo heterozygous loss of function or missense variants in the gene LARP1 (La ribonucleoprotein 1). LARP1 encodes an RNA-binding protein that post-transcriptionally regulates the stability and translation of thousands of mRNAs, including those regulating cellular metabolism and metabolic plasticity. Using lymphocytes collected and immortalized from an index proband who carries a truncating variant in one allele of LARP1, we demonstrated that lower cellular levels of LARP1 protein cause reduced rates of aerobic respiration and glycolysis. As expression of LARP1 increases during neurodevelopment, with higher levels in neurons and astrocytes, we propose that LARP1 haploinsufficiency contributes to ASD or related NDDs through attenuated metabolic activity in the developing fetal brain., Competing Interests: Declaration of interests S.J.S. receives research funding from BioMarin Pharmaceutical. M.L. is an employee and shareholder of Invitae Corp. I.M.W. is an employee of GeneDx, LLC. S.P.B. is a founder and director of RNA Guardian, Ltd.; a patent holder of WO1999062548A9 and WO2016075455A1; has an advisory committee membership to UCB; and has provided consultancy to Simbec Orion, Theolytics, Oxford Drug Discovery, and Ellipses., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis., Competing Interests: Competing interests statement:J.L.R. is cofounder, stockholder, and currently on the scientific board of Neurona, a company studying the potential therapeutic use of interneuron transplantation. J.L.R has stock in Neurona.

Recent advances in gene therapy, particularly for single-gene disorders (SGDs), have led to significant progress in developing innovative precision medicine approaches that hold promise for treating conditions such as primary hydrocephalus (CH), which is characterized by increased cerebrospinal fluid (CSF) volumes and cerebral ventricular dilation as a result of impaired brain development, often due to genetic causes. CH is a significant contributor to childhood morbidity and mortality and a driver of healthcare costs. In many cases, prenatal ultrasound can readily identify ventriculomegaly as early as 14-20 weeks of gestation, with severe cases showing poor neurodevelopmental outcomes. Postnatal surgical approaches, such as ventriculoperitoneal shunts, do not address the underlying genetic causes, have high complication rates, and result in a marginal improvement of neurocognitive deficits. Prenatal somatic cell gene therapy (PSCGT) promises a novel approach to conditions such as CH by targeting genetic mutations in utero, potentially improving long-term outcomes. To better understand the pathophysiology, genetic basis, and molecular pathomechanisms of CH, we conducted a scoping review of the literature that identified over 160 published genes linked to CH. Mutations in L1CAM, TRIM71, MPDZ, and CCDC88C play a critical role in neural stem cell development, subventricular zone architecture, and the maintenance of the neural stem cell niche, driving the development of CH. Early prenatal interventions targeting these genes could curb the development of the expected CH phenotype, improve neurodevelopmental outcomes, and possibly limit the need for surgical approaches. However, further research is needed to establish robust genotype-phenotype correlations and develop safe and effective PSCGT strategies for CH., (© 2024 The Author(s). Prenatal Diagnosis published by John Wiley & Sons Ltd.)

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner., (© 2024. The Author(s).)

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes 1 . Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome 2 . We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64&#95;65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5' splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide., (© 2024. The Author(s).)

Importance: Autism spectrum disorder (ASD) is a neurodevelopmental disorder more prevalent in males than in females. The cause of ASD is largely genetic, but the association of genetics with the skewed sex ratio is not yet understood. To our knowledge, no large population-based study has provided estimates of heritability by sex., Objective: To estimate the sex-specific heritability of ASD., Design, Setting, and Participants: This was a population-based, retrospective analysis using national health registers of nontwin siblings and cousins from Sweden born between January 1, 1985, and December 31, 1998, with follow-up to 19 years of age. Data analysis occurred from August 2022 to November 2023., Main Outcomes and Measures: Models were fitted to estimate the relative variance in risk for ASD occurrence owing to sex-specific additive genetics, shared environmental effects, and a common residual term. The residual term conceptually captured other factors that promote individual behavioral variation (eg, maternal effects, de novo variants, rare genetic variants not additively inherited, or gene-environment interactions). Estimates were adjusted for differences in prevalence due to birth year and maternal and paternal age by sex., Results: The sample included 1 047 649 individuals in 456 832 families (538 283 males [51.38%]; 509 366 females [48.62%]). Within the entire sample, 12 226 (1.17%) received a diagnosis of ASD, comprising 8128 (1.51%) males and 4098 (0.80%) females. ASD heritability was estimated at 87.0% (95% CI, 81.4%-92.6%) for males and 75.7% (95% CI, 68.4%-83.1%) for females with a difference in heritability estimated at 11.3% (95% CI, 1.0%-21.6%). There was no support for shared environmental contributions., Conclusions and Relevance: These findings suggest that the degree of phenotypic variation attributable to genetic differences (heritability) differs between males and females, indicating that some of the underlying causes of the condition may differ between the 2 sexes. The skewed sex ratio in ASD may be partly explained by differences in genetic variance between the sexes.

Many autism spectrum disorder (ASD)-associated genes act as transcriptional regulators (TRs). Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify the regulatory targets of ARID1B, BCL11A, FOXP1, TBR1, and TCF7L2, ASD-associated TRs in the developing human and mouse cortex. These TRs shared substantial overlap in the binding sites, especially within open chromatin. The overlap within a promoter region, 1-2,000 bp upstream of the transcription start site, was highly predictive of brain-expressed genes. This signature was observed in 96 out of 102 ASD-associated genes. In vitro CRISPRi against ARID1B and TBR1 delineated downstream convergent biology in mouse cortical cultures. After 8 days, NeuN+ and CALB+ cells were decreased, GFAP+ cells were increased, and transcriptomic signatures correlated with the postmortem brain samples from individuals with ASD. We suggest that functional convergence across five ASD-associated TRs leads to shared neurodevelopmental outcomes of haploinsufficient disruption., Competing Interests: Declaration of interests J.L.R.R. is cofounder and stockholder, and currently on the scientific board, of Neurona, a company studying the potential therapeutic use of interneuron transplantation. S.J.S. receives research funding from BioMarin Pharmaceutical. M.W.S. is a consultant to BlackThorn and ArRett Pharmaceuticals. L.L. is a stockholder and employee of Invitae., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Artificial Intelligence(AI)-based solutions for Gleason grading hold promise for pathologists, while image quality inconsistency, continuous data integration needs, and limited generalizability hinder their adoption and scalability., Methods: We present a comprehensive digital pathology workflow for AI-assisted Gleason grading. It incorporates A!MagQC (image quality control), A!HistoClouds (cloud-based annotation), Pathologist-AI Interaction (PAI) for continuous model improvement, Trained on Akoya-scanned images only, the model utilizes color augmentation and image appearance migration to address scanner variations. We evaluate it on Whole Slide Images (WSI) from another five scanners and conduct validations with pathologists to assess AI efficacy and PAI., Results: Our model achieves an average F1 score of 0.80 on annotations and 0.71 Quadratic Weighted Kappa on WSIs for Akoya-scanned images. Applying our generalization solution increases the average F1 score for Gleason pattern detection from 0.73 to 0.88 on images from other scanners. The model accelerates Gleason scoring time by 43% while maintaining accuracy. Additionally, PAI improve annotation efficiency by 2.5 times and led to further improvements in model performance., Conclusions: This pipeline represents a notable advancement in AI-assisted Gleason grading for improved consistency, accuracy, and efficiency. Unlike previous methods limited by scanner specificity, our model achieves outstanding performance across diverse scanners. This improvement paves the way for its seamless integration into clinical workflows., (© 2024. The Author(s).)