PMC:4067558 / 4121-13232 JSONTXT

{"project":"2_test","denotations":[{"id":"24768552-17322880-2047086","span":{"begin":311,"end":312},"obj":"17322880"},{"id":"24768552-20531469-2047087","span":{"begin":615,"end":617},"obj":"20531469"},{"id":"24768552-20663923-2047087","span":{"begin":615,"end":617},"obj":"20663923"},{"id":"24768552-22843504-2047088","span":{"begin":780,"end":782},"obj":"22843504"},{"id":"24768552-20531469-2047089","span":{"begin":1097,"end":1098},"obj":"20531469"},{"id":"24768552-20663923-2047090","span":{"begin":1118,"end":1120},"obj":"20663923"},{"id":"24768552-22843504-2047090","span":{"begin":1118,"end":1120},"obj":"22843504"},{"id":"24768552-22843504-2047091","span":{"begin":1561,"end":1563},"obj":"22843504"},{"id":"24768552-2745388-2047092","span":{"begin":1908,"end":1910},"obj":"2745388"},{"id":"24768552-7814313-2047093","span":{"begin":1964,"end":1966},"obj":"7814313"},{"id":"24768552-16926617-2047094","span":{"begin":2214,"end":2216},"obj":"16926617"},{"id":"24768552-20202923-2047095","span":{"begin":4381,"end":4383},"obj":"20202923"},{"id":"24768552-21357381-2047096","span":{"begin":4436,"end":4438},"obj":"21357381"},{"id":"24768552-17982118-2047096","span":{"begin":4436,"end":4438},"obj":"17982118"},{"id":"24768552-22589738-2047097","span":{"begin":4498,"end":4500},"obj":"22589738"},{"id":"24768552-20531469-2047098","span":{"begin":5197,"end":5198},"obj":"20531469"},{"id":"24768552-20531469-2047099","span":{"begin":5498,"end":5500},"obj":"20531469"},{"id":"24768552-21552272-2047099","span":{"begin":5498,"end":5500},"obj":"21552272"},{"id":"24768552-21658581-2047100","span":{"begin":6435,"end":6436},"obj":"21658581"},{"id":"24768552-22083728-2047101","span":{"begin":6513,"end":6515},"obj":"22083728"},{"id":"24768552-21681106-2047102","span":{"begin":6627,"end":6629},"obj":"21681106"},{"id":"24768552-21129364-2047103","span":{"begin":7091,"end":7093},"obj":"21129364"},{"id":"24768552-20531469-2047104","span":{"begin":7329,"end":7330},"obj":"20531469"},{"id":"24768552-21170055-2047105","span":{"begin":7708,"end":7710},"obj":"21170055"},{"id":"24768552-21784246-2047106","span":{"begin":7788,"end":7790},"obj":"21784246"},{"id":"24768552-20976243-2047107","span":{"begin":7996,"end":7998},"obj":"20976243"},{"id":"24768552-20531469-2047108","span":{"begin":9072,"end":9073},"obj":"20531469"},{"id":"24768552-21658583-2047109","span":{"begin":9095,"end":9097},"obj":"21658583"},{"id":"24768552-21249183-2047110","span":{"begin":9109,"end":9111},"obj":"21249183"}],"text":"Subjects and Methods\n\nASD Samples\nThe samples were collected as part of the AGP, an international consortium with over 50 sites in North America and Europe. The first phase of the AGP involved examining genetic linkage and chromosomal rearrangements in 1,168 families with at least two ASD-affected individuals.5 In the second phase, we genotyped simplex and multiplex families by using high-resolution microarrays to examine the contribution of rare CNVs and common SNPs to ASD. The second phase was divided in two stages; the results of stage 1, involving the first half of the families, were published in 2010.6,20 In stage 2, we genotyped the remaining families (n = 1,604) for a total of over 2,845 families and performed genome-wide CNV (this study) and association studies.21 Informed consent was obtained from all participants, and all procedures followed were in accordance with the ethical standards on human experimentation of the participating sites. The AGP sample set is a collection of families comprising an affected proband and two parents, as previously described in Pinto et al.6 and Anney et al.20,21 Many of the subjects at the recruiting sites were tested for fragile X syndrome (FXS [MIM 300624]) and assessed for chromosomal rearrangements with karyotype, fluorescence in situ hybridization, or multiplex-ligation-dependent probe amplification (MLPA); subjects with known karyotypic abnormalities, FXS, or other genetic disorders were typically excluded. The main analyses presented here were restricted to subjects of European ancestry.21 All diagnostic, clinical, and cognitive assessments were carried out at each contributing site. All data were gathered at a central coordination site for standardization of data formatting and data quality assurance.\n\nAutism Classification\nAffected AGP participants were classified according to the Autism Diagnostic Observation Schedule (ADOS)22 and the Autism Diagnostic Interview, Revised (ADI-R).23 The ADOS is a semistructured, clinically administered instrument for assessing and diagnosing ASD. The ADI-R is a structured clinical interview conducted with the parents or caregivers; spectrum classification on the ADI-R was based on Risi et al.24 The AGP strict and spectrum classifications are based on both instruments (Table S1A, available online). To meet criteria for strict autism, affected individuals must have an autism classification on both measures, whereas for the spectrum classification, individuals must meet the autism spectrum criteria on both measures or meet criteria for autism on one measure if the other measure was not available or not administered. The mean age of ADI-R assessment was 8 years.\n\nSimplex and Multiplex Classification\nFamily type was classified as simplex, multiplex, or unknown. Simplex families had one known affected individual among the first- to third-degree relatives (cousins only) and included affected monozygotic twins. Multiplex families had at least two first- to third-degree relatives (cousins only) with a validated, clinical ASD diagnosis. All other situations, including instances where a family history of autism was not assessed explicitly, were coded as unknown.\n\nDevelopmental Impairment\nCognitive functioning and adaptive function were measured with an appropriate standardized cognitive-testing instrument and the Vineland Adaptive Behavior Scale (VABS),25 respectively. To maximize the available data, we created a developmental-impairment variable by using a hierarchical combination of scores on full-scale, performance, and verbal IQ measures and the VABS composite score. A cutoff of 70 was applied on all measures; subjects who could not complete an IQ assessment because of low functioning or behavior were assigned to the “low” category. In the hierarchy, full-scale IQ (followed by performance IQ, verbal IQ, and finally the VABS composite score) was the preferred measure. For example, a subject with a full-scale IQ \u003c 70 but a performance IQ ≥ 70 was considered positive for developmental impairment. Additionally, subjects missing all IQ information with a “low” VABS composite score were also assigned to the developmental-impairment category.\n\nControl Subjects\nUnrelated control subjects were assembled from three studies in which individuals had no obvious psychiatric history: the Study of Addiction Genetics and Environment (SAGE),26 the Ontario Colorectal Cancer Case-Control Study,27,28 and Health, Aging, and Body Composition (HABC) (Table S1B).29 Samples were genotyped on the same array platforms (Illumina 1M single or duo arrays) as those of ASD subjects and parents and were analyzed with the same quality-control (QC) procedures and CNV analysis pipeline. The control data set used in the primary CNV analysis was composed of 2,640 control individuals of European ancestry (1,241 males and 1,399 females) who passed QC (Table S1B). Secondary analyses included 1,843 subjects from other ancestries (SAGE and HABC non-European control individuals), giving a total of 4,768 control subjects of all ancestries.\n\nData Analysis\nWe performed genotyping and data cleaning, including SNP and intensity QC for CNV detection, as described previously6 to ensure that CNV ascertainment was consistent among affected subjects, parents, and control subjects (see Table S1B for detailed QC steps). Samples not meeting our quality thresholds were excluded.\n\nCNV Analysis\nCNVs were detected with our analytical pipeline of Illumina 1M arrays (v.1 and v.3)6,30 and analyzed for case-control differences in burden with PLINK v.1.0730, R stats, and custom scripts. The p values associated with odds ratios (ORs) were calculated with Fisher’s exact test. Rare de novo CNVs, clinically relevant CNVs, and other selected rare CNVs were validated by at least one method (quantitative PCR, MLPA, and/or long-range PCR). Table S4 shows all validated de novo CNVs. A list of CNV calls passing QC in affected subjects, including all experimentally validated CNVs, is available in Tables S17A, S17B, and S17C.\nSecondary analyses included comparisons of CNV number, length, and intersected gene number between our 102 de novo CNVs identified in affected subjects and the 76 de novo CNVs in control subjects of two published data sets: (1) 17 de novo CNVs identified in 15 unaffected siblings from 872 families with a single ASD-affected offspring and an unaffected sibling from the Simons Simplex Collection4 and (2) 59 de novo CNVs detected in 57 out of 2,623 Icelandic control trios.31\nThe clinical relevance of CNVs was interpreted according to the American College of Medical Genetics guidelines32 irrespective of the subjects’ affected status, and CNVs were classified as pathogenic, uncertain, or benign. Pathogenic CNVs are documented as clinically significant in multiple peer-reviewed publications and databases (e.g., OMIM and GeneReviews), even if the penetrance and the expressivity might be variable.\n\nGene Lists\nIn order to perform burden analyses, we compiled a series of lists:(1) Genes and loci implicated causally in ASD (updated from Betancur),18 all of which have also been implicated in ID, as well as genes and loci implicated in ID, but not yet in ASD (Tables S6A–S6D). Note that the list of genes and loci involved in ASD was updated independently of the data from AGP stage 1;6 thus, genes and loci were included only if there was independent evidence from other studies.\n(2) Highly-brain-expressed genes defined by a log(RPKM [reads per kb per million reads]) \u003e 4.5 by the BrainSpan resource (n = 5,610 genes).\n(3) Functionally characterized control genes not expressed in the brain (log(RPKM) \u003c 1; n = 5,410 genes).\n(4) Postsynaptic density (PSD) genes.33\n(5) Genes found to interact with fragile X mental retardation protein (FMRP).34\n(6) Genes associated with neurological phenotypes compiled from the Human Phenotype Ontology (HPO) and Mammalian Phenotype Ontology (MPO).\n(7) Genes grouped by their probability of haploinsufficiency (pHI)35 into three subgroups: pHI \u003e 0.15 (n = 8,862 genes), pHI \u003e 0.35 (n = 4,136 genes), and pHI \u003e 0.55 (n = 2,214 genes).\n\nOne-Gene- and Multiple-Gene-Hit Burden Analysis\nOne-gene-hit burden analyses were performed with Fisher’s exact test. When considering the possibility that multiple genes within a CNV event or across events in the same subject act in concert to increase risk (i.e., multiple-gene-hit burden), we fit a series of logit models to the data. For the logit model, which is a special case of generalized linear model, log odds of case status (logit) was fit to predictor variables, namely the number of brain-expressed genes (BrainSpan) covered by the CNV and the level of gene expression. To analyze the expression data, we transformed the normalized RPKM value of each gene in the neocortex to log(1+RPKM). All analyses were performed in the statistical package R with the function “glm” and the logit link.\n\nFunctional Enrichment and Network Analyses\nFunctional-enrichment association tests and pathway and network analyses were performed with custom scripts,6 Bioconductor, NETBAG,36 and DAPPLE.37"}