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 ) .
 summarythe genetic architecture of autism spectrum disorder involves the interplay of common and rare variation and their impact on hundreds of genes . 
 using exome sequencing , analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry - matched or parental controls implicates 22 autosomal genes at a false discovery rate ( fdr ) < 0.05 , and a set of 107 autosomal genes strongly enriched for those likely to affect risk ( fdr < 0.30 ) . these 107 genes , which show unusual evolutionary constraint against mutations , incur de novo loss - of - function mutations in over 5% of autistic subjects . 
 many of the genes implicated encode proteins for synaptic , transcriptional , and chromatin remodeling pathways . these include voltage - gated ion channels regulating propagation of action potentials , pacemaking , and excitability - transcription coupling , as well as histone - modifying enzymes and chromatin remodelers , prominently histone post - translational modifications involving lysine methylation / demethylation . 
Transmission and De novo Association
Estimated odds ratios of top genes
Enrichment analyses
Integrating complementary data
Emergent results
Conclusions
Extended Data
Supplementary Material