Genes within De Novo CNVs Cluster in a Gene Network
De novo deletions (52 in European affected subjects) were found to be significantly enriched within each gene set or pathway cluster (for 85 of the 86 gene sets or pathways), as well as across clusters (chi-square test p = 9.8 × 10−9) (Table S12B), prompting us to search for enriched biological functions within de novo events separately. Taking into account our observations of a significant multigene burden in ASD subjects (Figures 3C and 3D), we analyzed de novo events by using NETBAG36 to identify up to two ASD candidate genes per CNV among 102 de novo events in 99 subjects. NETBAG identifies networks of genes under the premise that if genomic regions are perturbed by genetic variants associated with the same phenotype, they will contain genes forming connected clusters. The NETBAG analysis resulted in a network of 113 genes (global cluster p value = 0.02; Figure 4B). Ten genes have been previously implicated in autosomal-dominant or X-linked forms of ASD and ID (UBE3A [MIM 601623], NRXN1, SHANK2, EHMT1, SYNGAP1 [MIM 603384], and SMARCA2 [MIM 600014]) or ID only (ZEB2 [MIM 605802], FLNA [MIM 300017], SKI [MIM 164780], and IKBKG [MIM 300248]). On the basis of cumulative evidence from various sources, an additional 68% (67/98) are likely to affect ASD risk (Tables S14A–S14E); 27/67 of these are either FMRP targets or PSD genes. Compared to all other genes within de novo CNVs (or deletion CNVs only), genes in the network exhibited a significantly higher pHI (Wilcoxon rank sum p = 7.07 × 10−8), and 55% (59/107 [6 without information]) had a pHI > 0.35. A similar NETBAG analysis of de novo CNVs in control subjects did not yield significant results.36
We further characterized the biological processes related to the NETBAG cluster (Tables S14B–S14E; Figures 4B and S3) and found a significant enrichment (false-discovery rate [FDR] < 10%) of genes involved in chromatin and transcription regulation, MAPK signaling, and synaptic signaling and components (Figure 4B). We recapitulated many of the results of our gene-set analysis (Figure 4A), notably for synapse functions and processes, and also identified genes involved in chromatin and transcription regulation. The latter category included a high-risk gene associated with ASD, the chromatin gene CHD2 (MIM 602119), which is affected by a de novo 83 kb deletion in a male with ASD, mild ID, and dysmorphic features including micrognathia and protruding ears. His ASD-affected brother has mild ID, similar dysmorphic features, and epilepsy with onset at age 9 years and carries the same deletion, which removes the first six exons of CHD2. Neither parent carries the deletion, suggesting germline mosaicism (the deletion arose on the paternal chromosome). De novo SNVs in CHD2 have been reported in an ASD subject and in several individuals with a broad spectrum of neurodevelopmental disorders, including ID and epileptic encephalopathy8,49,50 (Figure S6). Two other genes in the chromodomain family have been linked to neurodevelopmental disorders: CHD7 (MIM 608892) in CHARGE syndrome (MIM 214800) and CHD8 (MIM 610528) in ASD. Another example is TRIP12 (MIM 604506), encoding an E3 ubiquitin ligase that can regulate chromatin function to maintain genome integrity (Figure S12). The chromatin and transcription module showed a predominance of genes with a prenatally biased expression profile (Figures 4B and S4).