we adopted tada ( for  transmission and de novo association ) , a weighted , statistical model integrating de novo , transmitted and case - control variation . tada uses a bayesian gene - based likelihood model including per gene mutation rates , allele frequencies , and relative risks of particular classes of sequence changes . because no aggregate association signal was detected for inherited mis3 variants , they were not included in the analysis . for each gene , variants of each class were assigned the same effect on relative risk . using a prior probability distribution of relative risk across genes for each class of variants , the model effectively weighted different classes of variants in this order : de novo lof > de novo mis3 > transmitted lof , and allowed for a distribution of relative risks across genes for each class . the strength of association was assimilated across classes to produce a gene - level bayes factor ( bf ) with a corresponding false discovery rate or fdr q - value . this framework increases the power compared to use of de novo lof alone ( extended data fig . tada identified 33 autosomal genes with an fdr < 0.1 ( table 1 ) and 107 genes with an fdr < 0.3 ( supplementary tables 2 and 3 and extended data fig . 15 ( 45.5% ) are known asd risk genes ; 11 have been reported previously with mutations in asd patients but were not classed as true risk genes owing to insufficient evidence ( suv420h1 , adnp , bcl11a , and cacna2d3 , cttnbp2 , gabrb3 , cdc42bpb , aph1a , nr3c2 , setd5 , trio ) ; and 7 are completely novel ( ash1l , mll3 , etfb , naa15 , myo9b , mib1 , vil1 ) . adnp mutations have recently been identified in 10 patients with asd and other shared clinical features . two of the newly discovered genes , ash1l and mll3 , converge on chromatin remodeling . mib1 encodes an e3 ubiquitin ligase critical for neurogenesis and is regulated by mir-137 , a microrna that regulates neuronal maturation and is implicated in risk for schizophrenia . when the wes data from genes with fdr < 0.3 were evaluated for the presence of deletion copy number variants ( such cnvs are functionally equivalent to lof mutations ) , 34 cnvs meeting quality and frequency constraints ( supplementary information ) < 0.1 , three contained deletion cnvs mapping to three asd subjects and one parent . of the 74 genes meeting the criterion 0.1  fdr < 0.3 , about a third could be false positives . deletion cnvs were found in 14 of these genes and the data supported risk status for 10 of them ( extended data table 1 , extended data fig . the risk from deletion cnvs , as measured by the odds ratio , is comparable to that from lof snv in cases versus controls or transmission of lof from parents to offspring . inherent in our conception of the biology of asd is the notion that there is variation between genes in their impact on risk : for a given class of variants ( e.g. , lof ) , some genes have large impact , others smaller , and still others have no effect at all . yet mis - annotation of variants , among other confounds , can produce false variant calls in subjects ( supplementary information ) . these confounds can often be overcome by examining the data in a manner orthogonal to gene discovery . for example , females have greatly reduced rates of asd relative to males ( a so - called  female protective effect ) . consequentially , and regardless of whether this is diagnostic bias or biological protection , females have a higher liability threshold , requiring a larger genetic burden before being diagnosed . a corollary is that if a variant has the same effect on autism liability in males as it does in females , that variant will be at higher frequency in female asd cases compared to males . importantly , the magnitude of the difference is proportional to risk as measured by the odds ratio ( or ) ; hence , the effect on risk for a class of variants can be estimated from the difference in frequency between males and females . genes with fdr < 0.1 show profound female enrichment for de novo events ( p=0.005 for lof , p=0.004 for mis3 ) , consistent with de novo events having large impact on liability ( or  20 ; extended data fig . genes with fdr between 0.1 and 0.3 , however , show substantially less enrichment for female events , consistent with a modest impact for lof variants ( or range 2 - 4 , whether transmitted or de novo ) and little to no effect from mis3 variants . the results are consistent with inheritance patterns , lof mutations in fdr < 0.1 genes are rarely inherited from unaffected parents while those in the 0.1 < fdr < 0.3 group are far more often inherited than de novo . by analyzing the distribution of relative risk over inferred asd genes , the estimate relies on the balance of genes with multiple de novo lof mutations versus those with only one : the larger the number of asd genes , the greater proportion that will show only one de novo lof . while there are many more genes to be discovered , many will have a modest impact on risk compared to the genes in table 1 . fdr < 0.3 gene sets are strongly enriched for genes under evolutionary constraint ( p=3.010 , fig . 1a , supplementary table 4 ) , consistent with the hypothesis that heterozygous lof mutations in these genes are asd risk factors . indeed over 5% of asd subjects carry de novo lof mutations in our fdr < 0.3 list . we also observed that genes in the fdr < 0.3 list had a significant excess of de novo lof events detected by the largest schizophrenia wes study to date ( p=0.0085 , fig . 1a ) , providing further evidence for overlapping risk loci between these disorders and independent confirmation of the signal in the gene sets presented here . we found significant enrichment for genes encoding mrnas targeted by two neuronal rna - binding proteins : fmrp ( also known as fmr1 ) , mutated or absent in fragile x syndrome ( p=1.2010 , 34 targets , of which 11 are corroborated by an independent data set ) , and , rbfox ( rbfox1/2/3 ) ( p=0.0024 , 20 targets , of which 12 overlap with fmrp ) , with rbfox1 shown to be a splicing factor dysregulated in asd ( fig . these two pathways expand the complexity of the asd neurobiology to post - transcriptional events , including splicing and translation , both of which would sculpt the neural proteome . we found nominal enrichment for human orthologs of mouse genes encoding synaptic ( p=0.031 ) and postsynaptic density ( psd ) proteins ( p=0.046 , fig . enrichment analyses for interpro , smart , or pfam domains ( fdr < 0.05 and a minimum of 5 genes per category ) reveal an overrepresentation of dna / histone - related domains : 8 genes encoding proteins with interpro zinc finger ( znf ) fyve phd domains ( 142 such annotated genes in the genome ; fdr=7.610 ) , and five with pfam su(var)3 - 9 , enhancer - of - zeste ( set ) domains ( 39 annotated in the genome ; fdr=8.210 ) . to implicate additional genes in risk for asd , we use a model called dawn . dawn evokes a hidden markov random field framework to identify clusters of genes that show strong association signal and highly correlated co - expression in a key tissue and developmental context . previous research suggests human mid - fetal prefrontal and motor - somatosensory neocortex is such a critical nexus for risk , thus we evaluated gene co - expression data from that tissue together with tada scores for genes with fdr < 0.3 . because this list is enriched for genes under evolutionary constraint , we generalized dawn to incorporate constraint scores ( supplementary information ) . when ( a ) tada results , ( b ) gene co - expression in mid - fetal neocortex , and ( c ) constraint scores are jointly modeled , dawn identifies 160 genes that plausibly affect risk ( fig . moreover , the model parameter describing evolutionary constraint is an important predictor of clusters of putative risk genes ( p=0.018 ) . a subnetwork obtained by seeding the 160 dawn genes within a high - confidence protein - protein interactome confirmed that the putative genes are enriched for neuronal functions . we kept the largest connected component , containing 95 seed dawn genes , 50 of which were in the fdr < 0.3 gene set . 2 ) . we visualized the enriched pathways and biological functions for each of these clusters on canvases ( extended data fig . many of the previously known asd risk genes fall in cluster c3 , including genes involved in synaptic transmission and cell - cell communication . many tada and dawn genes in this cluster interact tightly with other transcription factors , histone modifying enzymes and dna binding proteins . five tada genes in the cluster c2 are bridged to the rest of the network through mapt , inferred by dawn . the enrichment results for c2 indicate that genes implicated in neurodegenerative disorders could also play a role in neurodevelopmental disorders . to implicate additional genes in risk for asd , we use a model called dawn . dawn evokes a hidden markov random field framework to identify clusters of genes that show strong association signal and highly correlated co - expression in a key tissue and developmental context . previous research suggests human mid - fetal prefrontal and motor - somatosensory neocortex is such a critical nexus for risk , thus we evaluated gene co - expression data from that tissue together with tada scores for genes with fdr < 0.3 . because this list is enriched for genes under evolutionary constraint , we generalized dawn to incorporate constraint scores ( supplementary information ) . when ( a ) tada results , ( b ) gene co - expression in mid - fetal neocortex , and ( c ) constraint scores are jointly modeled , dawn identifies 160 genes that plausibly affect risk ( fig . moreover , the model parameter describing evolutionary constraint is an important predictor of clusters of putative risk genes ( p=0.018 ) . a subnetwork obtained by seeding the 160 dawn genes within a high - confidence protein - protein interactome confirmed that the putative genes are enriched for neuronal functions . we kept the largest connected component , containing 95 seed dawn genes , 50 of which were in the fdr < 0.3 gene set . 2 ) . we visualized the enriched pathways and biological functions for each of these clusters on canvases ( extended data fig . many of the previously known asd risk genes fall in cluster c3 , including genes involved in synaptic transmission and cell - cell communication . many tada and dawn genes in this cluster interact tightly with other transcription factors , histone modifying enzymes and dna binding proteins . five tada genes in the cluster c2 are bridged to the rest of the network through mapt , inferred by dawn . the enrichment results for c2 indicate that genes implicated in neurodegenerative disorders could also play a role in neurodevelopmental disorders . amongst critical synaptic components found mutated in our study are voltage - gated ion channels involved in fundamental processes including propagation of action potentials ( e.g. , nav1.2 channel ) , neuronal pacemaking , and excitability - transcription coupling ( e.g. , cav1.3 channel ) ( fig . we identified , 4 lof and 5 mis3 variants in scn2a ( nav1.2 ) , 3 mis3 in cacna1d ( cav1.3 ) , 2 lof in cacna2d3 ( -auxiliary subunits of l - type voltage - gated ca channels , including cav1.3 ) . remarkably , three de novo mis3 variants in scn2a hit residues mutated in homologous genes in patients with other syndromes , including brugada syndrome ( scn5a ) or epilepsy disorders ( scn1a ) ( p.r379h and p.r937h ) . these arginines , as well as the threonine mutated in p.t1420 m , cluster to the p - loops forming the ion selectivity filter , in proximity of the inner ring ( deka motif ) ( fig . because homologous channels mutated in these arginines do not conduct inwards na currents , p.r379h and p.r937h might have similar effect . two de novo cacna1d variants ( p.g407r and p.a749 g ) hit positions proximal to residues mutated in patients with primary aldosteronism and neurological deficits ( fig . the reported mutations interfere with channel activation and inactivation . amongst variants found in cases , p.a59v maps to the nscate domain , also important for ca - dependent inactivation , while p.s1977l and p.r2021h co - cluster in the c - terminal proline - rich domain , the site of interaction with shank3 , a key psd scaffolding protein . mutations in rims1 and rimbp2 , which can associate with cav1.3 , were found in our cohort ( but with an fdr.0.3 ) . chromatin remodeling involves histone - modifying enzymes ( encoded by histone modifier genes , hmgs ) and chromatin remodelers (  readers ) that recognize specific histone post - translational modifications ( ptms ) and orchestrate their effects on chromatin . our gene set is enriched in hmgs ( 9 hmgs out of 152 annotated in histome , fisher 's exact test , p=2.210 ) . histone - lysine n - methytransferase activity ( 5 genes out of 41 so annotated ; fdr=2.210 ) highlights this as a prominent pathway . lysines on histones 3 and 4 can be mono- , di- , or tri - methylated , providing a versatile mechanism for either activation or repression of transcription . of 107 tada genes , five are set lysine methyltransferases , four are jumonji ( jmjc ) lysine demethylases , and two are readers ( fig . rbfox1 co - isolates with h3k4me3 , and our dataset is enriched in targets shared by rbfox1 and h3k4me3 ( p=0.0166 , fig . 1a , supplementary table 4 ) . some de novo missense variants targeting these genes map to functional domains ( extended data fig . , we extended our analyses in search of additional de novo variation in the cases of the case - control sample . by sequencing complete parent - child trios for many chd8 variants , five variants were found to be de novo , two of which affect essential splice sites and cause loss of function by exon skipping or activation of cryptic splice sites in lymphoblastoid cells ( fig . 3b ) . given the role of hmgs in transcription , we reasoned that tada genes might be interconnected through transcription  routes  . we searched for a connected network ( seeded by 9 tada hmgs ) in a transcription factor interaction network ( chea ) . we found that 46 tada genes are directly interconnected in a 55-gene cluster ( extended data fig . 8) ( p=0.002 ; 1,000 random draws ) , for a total of 69 when including all known hmgs ( fig . 4 ) ( p=0.001 ; 1,000 random draws ) . examining the human gene mutation database we found that the 107 tada genes included 21 candidate genes for intellectual disability , 3 for epilepsy , 17 for schizophrenia , 9 for congenital heart disease and 6 for metabolic disorders ( fig . 5 ) . complementing earlier reports , asd subjects show a clear excess of de novo lof mutations over expectation , with a pile - up of such events in a handful of genes . while this handful has a large effect on risk , most asd genes have much smaller impact . this gradient emerges most strikingly from the contrast of risk variation in male and female asd subjects . unlike some earlier studies , but consistent with expectation , the data also show clear evidence for effect of de novo missense snv on risk ; for risk generated by lof variants transmitted from unaffected parents ; and for the value of case - control design in gene discovery . indeed , by integrating data on de novo , inherited and case control variation , the yield of asd gene discoveries was doubled over what would be obtained from a count of de novo lof alone . almost uniformly asd genes show large constraint against variation , a feature we exploit to implicate other genes in risk . three critical pathways for typical development are damaged by risk variation : ( 1 ) chromatin remodeling , ( 2 ) transcription and splicing , and , ( 3 ) synaptic function . chromatin remodeling controls events underlying the formation of neural connections , including neural neurogenesis and neural differentiation , and relies on epigenetic marks as histone ptms . here we provide extensive evidence for hmgs and readers in sporadic asd , implicating specifically lysine methylation and extending the mutational landscape of the emergent asd gene chd8 to missense variants . risk variation also hits multiple classes and components of synaptic networks , from receptors and ion channels to scaffolding proteins . because a wide set of synaptic genes is disrupted in idiopathic asd , it seems reasonable to conjecture that altered chromatin dynamics and transcription , induced by disruption of relevant genes , leads to impaired synaptic function as well . de novo mutations in asd , intellectual disability and schizophrenia cluster to synaptic genes , and synaptic defects have been reported in models of these disorders . integrity of synaptic function is essential for neural physiology , and its perturbation could represent the intersection between diverse neuropsychiatric disorders . dna was obtained , and exomes were captured and sequenced . after variant calling qc was performed : duplicate subjects and incomplete families were removed ; and subjects with extreme genotyping , de novo , or variant rates were removed . following cleaning , 3,871 subjects with asd remained . de novo and transmission / non - transmission were obtained for trio data ( published de novo from 825 trios were incorporated ) . this path led to the tada analysis , which found 33 asd risk genes with q < 0.1 ; and 107 with q < 0.3 . the multiple lof test ( red ) is a restricted version of tada that uses only the de novo lof data . tada ( blue ) models de novo lof , de novo mis3 , lof variants transmitted / not transmitted and lof variants observed in case / control samples . the sample size ( n ) indicates either ( i ) n trios , for which we record de novo and transmitted variation , or ( ii ) n trios , for which we record only de novo events , plus n cases and n controls . left panel , variants in affected subjects ; right panel , unaffected subjects . for the counts , we only focus on de novo lof and mis3 variants , transmitted / un - transmitted and case / control lof variants . these variant counts are normalized by the length of coding regions of each gene and sample size of each dataset ( |trio|+|case| for left panel , |trio|+|control| for the right panel ) . the deletions are displayed in red if with unknown inheritance , in grey if inherited , and in black in un unaffected subjects . frequency of de novo ( dn ) and transmitted ( tr ) variants per sample in males ( black ) and females ( white ) for genes with q < 0.1 ( upper panel ) , q < 0.3 ( central panel ) , or all tada genes ( lower panel ) . the p values were determined by a one - tailed permutation test ( * p < 0.5 ; * * p < 0.01 ; * * * p < 0.01 ) . p - values using mouse - genome - informatics / mammalian - phenotype ( mgi - mp , blue ) , kyoto encyclopedia of genes and genomes pathways ( kegg , red ) , and gene ontology biological processes ( go , yellow ) are indicated . mis3 are in black , lof in red , and variants identified in other disorders in grey ( fig . 5 ) . jmjc , jumonji c domain ; jmjn , jumonji n domain ; jmjc , phd , plant homeodomain ; arid , at - rich interacting domain ; set , su(var)3 - 9 , enhancer - of - zeste , trithorax domain ; fyr n , fy - rich n - terminal domain ; fyr c , fy - rich c - terminal domain ; pwwp , pro - trp - trp - pro domain ; hmg , high mobility group box ; aws , associated with set domain ; bromo , bromodomain ; bah , bromo adjacent homology . edges indicate transcription regulator ( source node ) and its gene targets ( target node ) based on chea network . count of deletion copy number variants , inferred from sequence , for asd subjects and those unaffected by asd . number of subjects and family status : 849 asd without family information ; 1467 asd subjects in families ; 2766 unaffected parents ; 319 unaffected siblings of asd subjects ; 373 unaffected subjects without family information . no parents in this count were affected ; 7 parents in the study were affected , none carried a cnv reported in the table and these subjects did not enter the calculation . tr - asd = transmitted to asd subject from carrier parent ; nt = parent a carrier but cnv not transmitted to affected child ; tr - not - asd = parent transmits a cnv to an unaffected child . to compute the odds ratio we count the number a of affected carriers ,  b unaffected carriers ( including parents ) ,  c affected subjects who do not have the cnv , and one parent transmits the cnv to an affected and unaffected offspring ; to obtain the total count of controls with a cnv , subtract one . both genes are hit by the same cnv ( 1 asd and 2 unaffected subjects ) .