The result did not change when we evaluated the various subcategories of rare X-linked CNVs including exonic, deletions, duplications, size, brain-expressed, or

ASD-associated. We next considered whether the absence of association of rare transmitted CNVs might be a consequence of an inability to differentiate functional from neutral variants. We looked Z-VAD-FMK to pathway analyses to help address this question, reasoning that if the specific genic content of CNVs contributed to disease risk, we would find a greater enrichment of biological pathways in probands compared to their unaffected siblings. We used two gene ontology and pathway analysis tools, MetaCore from GeneGo, Inc. and DAVID (Dennis et al., 2003 and Huang Vemurafenib in vitro et al., 2009), to analyze 1516 genes within CNVs exclusive to probands and 1357 genes exclusive to siblings. The total number and size of rare transmitted CNVs used to determine these gene sets were highly similar in probands and siblings (Figure 5). GeneGo networks identified 22 pathways showing significant enrichment in probands versus only four enriched pathways among siblings. This difference was significant based on 100 permutations of the data set (p = 0.04). DAVID yielded consistent results with 59 pathways enriched in probands and 19 in siblings (p = 0.01, permutation analysis) (Figure 6). For the present study, we elected

to restrict our evaluation of pathways to the general question described here. A manuscript that is in preparation describes a more extensive analysis, focusing on both structural and gene expression data from the SSC. We next examined all rare CNVs in the SSC in light of previously reported findings, comparing our data to the list of ASD regions included in the recent AGP analysis (Pinto et al., 2010). We also considered genes implicated by recent common variant studies, including SEMA5A ( Weiss et al., 2009), MACROD2 (

Anney et al., 2010), CDH9 and CDH10 ( Wang et al., 2009), the MET oncogene Insulin receptor ( Campbell et al., 2006), EN2 ( Gharani et al., 2004), as well as selected schizophrenia loci ( International Schizophrenia Consortium, 2008, McCarthy et al., 2009, Millar et al., 2000, Stefansson et al., 2008, Walsh et al., 2008 and Xu et al., 2008) ( Table 3). We identified multiple regions in which rare transmitted and/or rare de novo events corresponded to previously characterized loci in both ASD and schizophrenia. Finally, we looked for evidence of association for all CNVs in the SSC sample, common or rare, transmitted or de novo, evaluating all high-confidence autosomal CNVs together with all confirmed de novo CNVs. In this instance, we did not use a frequency cutoff to define a set of rare transmitted events. A total of 3667 recurrent regions were identified; 6 showed relative enrichment in probands and 5 showed relative enrichment in siblings. No result reached significance after correction for multiple comparisons (Table S7 and Figure 7C).