De Novo CNVs and LoF SNVs Converge on Functional Gene Networks
We expanded our analysis to genes altered by both our de novo CNVs (Figure 5A) and de novo LoF SNVs compiled from four exome sequencing studies in ASD.7–10 Eleven genes affected by de novo CNVs (NRXN1, SHANK2, ARID1B, RIMS1 [MIM 606629], TRIP12, SMARCC2 [MIM 601734], DLL1 [MIM 606582], TM4SF19, MLL3 [MIM 606833], PHF2 [MIM 604351], and CSTF2T [MIM 611968]) were found to be altered by de novo LoF SNVs among autism cohorts, and three of them (NRXN1, SHANK2, and RIMS1) were selected by NETBAG (Figure 4B). Of the 11 genes, only TM4SF19 (3q29) belongs to a known locus associated with a recurrent genomic disorder. Despite the limited overlap observed among genes altered by de novo CNVs and LoF SNVs, there is reinforcing evidence of the role of NRXN1 and SHANK2 in ASD (Figure 5A). In addition, RIMS1, altered by both a de novo duplication and a LoF SNV (Figure 5A) and encoding a brain-specific synaptic Rab3a-binding protein, emerges as an ASD candidate gene. RIMS1 has a regulatory role in synaptic-vesicle exocytosis modulating synaptic transmission and plasticity.51 Three other genes affected by de novo CNVs (also picked by NETBAG)—CHD2, SYNGAP1, and SYNCRIP—are also affected by LoF de novo SNVs in ID,50,52 and two (SYNGAP1 and DPYD [MIM 612779]) are altered in schizophrenia.53 CHD2 (discussed above) and SYNGAP1 (encoding a Ras/Rap GTP-activating protein) are both involved in autosomal-dominant ID, ASD, and epilepsy.54
Under the assumption that different genes harboring suspected causative mutations for the same disorder often physically interact, we sought to evaluate protein-protein interactions (PPIs) encoded by genes known to be implicated in ASD and genes affected by rare CNVs or SNVs (data drawn from our de novo CNVs and published de novo ASD LoF SNVs; Figures 5A, 5B, and S5). The union set of 336 unique genes (Table S15) analyzed by DAPPLE37 resulted in a network of direct PPIs encoded by 151 genes (Figure 5A) from each of the three main lists: 54/92 (58.7%) genes involved in ASD, 64/113 (56.6%) de novo CNV genes, and 41/122 (33.6%) de novo LoF SNV genes. The number of direct PPIs was 1.5-fold higher than expected (p < 0.001, Figure 5B), suggesting that many of the de novo CNV or SNV genes and ASD-implicated genes function cooperatively. Overrepresentation analysis identified convergent functional themes related to neuronal development and axon guidance, signaling pathways, and chromatin and transcription regulation. Overall, these findings are consistent among the three different types of analyses shown in Figures 4A, 4B, and 5.
Although 54 genes were previously implicated in ASD, the DAPPLE analysis singled out an additional 97 CNV or SNV high-confidence candidate genes (Figure 5B; Table S16). We found that compared to the 54 ASD-implicated genes, the newly selected 97 CNV or SNV genes had a comparably high pHI (median pHI = 0.58, Figure 6A). This is consistent with the observation that ASD subjects have more deletions with haploinsufficient genes than do controls (Figure 6B). Furthermore, similar to genes with known disease-causing mutations (Figure 6C), those genes have high functional-indispensability scores and a comparable high degree of centrality (i.e., high number of direct neighbors) and number of networks in which they are involved (Figures 6D and 6E). Compared to the genome average, they are also among the top 75% of more-conserved genes (on the basis of Genomic Evolutionary Rate Profiling scores and PhyloP) and are highly expressed in the brain. Interestingly, 39 of the 97 genes are either FMRP-related or PSD genes (of the initial 151 genes identified by DAPPLE, 51 are FMRP interactors and 24 are PSD genes). Thus, despite little overlap in genes, the strong interconnectedness between the resulting networks identifies pathways through which the effects of distinct mutations might converge.