patients were selected for inclusion in this study from different cohorts tested on either family - based wes , targeted resequencing or high - resolution melting analysis ( table 1 ) .
clinical evaluation was performed by at least one expert clinical geneticist . written informed consent for inclusion in the study
was obtained for all patients and consent for the publication of photographs was obtained for patients 1 , 2 , 4 , 5 , 6 and 8 .
pcr was performed using gotaq polymerase ( promega , madison , wi , usa ) on dna from peripheral blood and on cdna from lymphoblastoid cells , using standard protocol . capillary electrophoresis sequencing ( abi 3130 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) was performed using the abi bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , carlsbad , ca , usa ) , following standard protocol .
data was analysed in clc dna workbench ( clc bio , aarhus , denmark ) .
patient 1 was detected in a family - based wes study ( unpublished data ( ch , gv , filip van nieuwerburg , nvda , rfk ) ) .
patient dna was fragmented using covaris m220 focused - ultrasonicator ( covaris , ma , usa ) , followed by truseq dna sample preparation ( illumina inc , san diego , ca , usa ) , enrichment using the seqcap ez human exome library v3.0 kit ( nimblegen , roche , penzberg , germany ) , and sequencing on hiseq 2000 ( illumina inc , san diego , ca , usa ) , all following standard protocols . data analysis was performed using galaxy ( see urls ) .
variants were filtered by variantdb ( see urls , manuscript in preparation ) to exclude variants with ( 1 ) low quality , using thresholds based on correlation between ngs data and snp - chip genotyping ; ( 2 ) intronic or intergenic location , except splice sites ; and ( 3 ) inheritance from the parents .
wes was performed using illumina technology ( illumina inc , san diego , ca , usa ) , and sequence data was returned and analysed using software supplied from oxford gene technology .
presence of reported ( de novo ) mutations were confirmed by an independent technique such as sanger sequencing .
raw sequence data will be uploaded in the european genome - phenome archive ( embl - ebi ) database . patients 7 , 8 and 9 were discovered from a mip based screen of 2743 probands with intellectual disability and/or asd ) .
patient 10 was included from a mip based screen of 2446 autism patients from the simon simplex collection ( ssc ) .
the mip screening and analysis was performed as previously described , and mip probe sequences for adnp are available in oroak et al , 2012 .
192 control chromosomes were screened for the presence of the mutations identified in the 10 patients using hrm .
primers were designed using the hrma assay design module of beacon designer 8.10 ( premier biosoft , ca , usa ) .
hrm was performed on a lightcycler 480 ( roche , penzberg , germany ) with the lcgreen incorporating dye ( idaho technology inc . ,
was identified using the same protocol , as part of the cohort of 148 probands with idiopathic asd , for which microarray analysis did not reveal any abnormalities .
mrna expression was examined by an optimized three - step real - time quantitative pcr assay following the protocol described before . besides adnp itself
, adnp2 was included based on the reported correlation of expression in human brain tissue .
tmpo , ccnc and plagl2 were reported to be significantly downregulated in homozygous adnp knockout mice embryos , while abcf3 was reported to be upregulated in heterozygous adnp knockout mice embryos .
qpcr primers were selected from literature , the rtprimerdb or designed using an in - house automated pipeline ( see urls ) , conforming to requirements of intron - spanning location , no snp content , no dimer formation at the 3 end of the primers , and low amplicon folding , with no folding in primer binding sites .
the amplification efficiency of the different primers was assessed and confirmed to be above 1.85 .
expression values of two cdna syntheses originating from two different rna isolations per patient were compared to the values obtained from eight control individuals .
statistical testing was performed using linear mixed models in order to investigate significant differences in expression levels of the patients compared to controls .
patients were selected for inclusion in this study from different cohorts tested on either family - based wes , targeted resequencing or high - resolution melting analysis ( table 1 ) .
clinical evaluation was performed by at least one expert clinical geneticist . written informed consent for inclusion in the study
was obtained for all patients and consent for the publication of photographs was obtained for patients 1 , 2 , 4 , 5 , 6 and 8 .
pcr was performed using gotaq polymerase ( promega , madison , wi , usa ) on dna from peripheral blood and on cdna from lymphoblastoid cells , using standard protocol .
capillary electrophoresis sequencing ( abi 3130 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) was performed using the abi bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , carlsbad , ca , usa ) , following standard protocol .
data was analysed in clc dna workbench ( clc bio , aarhus , denmark ) .
patient 1 was detected in a family - based wes study ( unpublished data ( ch , gv , filip van nieuwerburg , nvda , rfk ) ) .
patient dna was fragmented using covaris m220 focused - ultrasonicator ( covaris , ma , usa ) , followed by truseq dna sample preparation ( illumina inc , san diego , ca , usa ) , enrichment using the seqcap ez human exome library v3.0 kit ( nimblegen , roche , penzberg , germany ) , and sequencing on hiseq 2000 ( illumina inc , san diego , ca , usa ) , all following standard protocols .
variants were filtered by variantdb ( see urls , manuscript in preparation ) to exclude variants with ( 1 ) low quality , using thresholds based on correlation between ngs data and snp - chip genotyping ; ( 2 ) intronic or intergenic location , except splice sites ; and ( 3 ) inheritance from the parents .
wes was performed using illumina technology ( illumina inc , san diego , ca , usa ) , and sequence data was returned and analysed using software supplied from oxford gene technology .
presence of reported ( de novo ) mutations were confirmed by an independent technique such as sanger sequencing .
raw sequence data will be uploaded in the european genome - phenome archive ( embl - ebi ) database .
patients 7 , 8 and 9 were discovered from a mip based screen of 2743 probands with intellectual disability and/or asd ) .
patient 10 was included from a mip based screen of 2446 autism patients from the simon simplex collection ( ssc ) .
the mip screening and analysis was performed as previously described , and mip probe sequences for adnp are available in oroak et al , 2012 .
192 control chromosomes were screened for the presence of the mutations identified in the 10 patients using hrm .
primers were designed using the hrma assay design module of beacon designer 8.10 ( premier biosoft , ca , usa ) .
hrm was performed on a lightcycler 480 ( roche , penzberg , germany ) with the lcgreen incorporating dye ( idaho technology inc . ,
was identified using the same protocol , as part of the cohort of 148 probands with idiopathic asd , for which microarray analysis did not reveal any abnormalities .
mrna expression was examined by an optimized three - step real - time quantitative pcr assay following the protocol described before . besides adnp itself
, adnp2 was included based on the reported correlation of expression in human brain tissue .
tmpo , ccnc and plagl2 were reported to be significantly downregulated in homozygous adnp knockout mice embryos , while abcf3 was reported to be upregulated in heterozygous adnp knockout mice embryos .
qpcr primers were selected from literature , the rtprimerdb or designed using an in - house automated pipeline ( see urls ) , conforming to requirements of intron - spanning location , no snp content , no dimer formation at the 3 end of the primers , and low amplicon folding , with no folding in primer binding sites .
the amplification efficiency of the different primers was assessed and confirmed to be above 1.85 .
expression values of two cdna syntheses originating from two different rna isolations per patient were compared to the values obtained from eight control individuals .
statistical testing was performed using linear mixed models in order to investigate significant differences in expression levels of the patients compared to controls .
 despite a high heritability , a genetic diagnosis can only be established in a minority of patients with autism spectrum disorder ( asd ) , characterized by persistent deficits in social communication and interaction and restricted , repetitive patterns of behavior , interests or activities1 . 
 known genetic causes include chromosomal aberrations , such as the duplication of the 15q11 - 13 region , and monogenic causes , such as the rett and fragile x syndromes . 
 the genetic heterogeneity within asd is striking , with even the most frequent causes responsible for only 1% of cases at the most . 
 even with the recent developments in next generation sequencing , for the large majority of cases no molecular diagnosis can be established 2 - 7 . here , we report 10 patients with asd and other shared clinical characteristics , including intellectual disability and facial dysmorphisms caused by a mutation in adnp , a transcription factor involved in the swi / snf remodeling complex . 
 we estimate this gene to be mutated in at least 0.17% of asd cases , making it one of the most frequent asd genes known to date . 
Methods
Patients
Sanger sequencing
Whole-exome sequencing (WES)
Molecular inversion probes (MIPs)
High-resolution melting (HRM)
Real-time quantitative PCR
Supplementary Material