Synaptic, transcriptional and chromatin genes disrupted in autism

Abstract

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

Extended Data Figure 1. Workflow of the study

The workflow began with 16 sample sets, as listed in Supplementary Table 1. DNA was obtained, and exomes were captured and sequenced. After variant calling QC was performed: duplicate subjects and incomplete families were removed; and subjects with extreme genotyping, de novo, or variant rates were removed. Following cleaning, 3,871 subjects with ASD remained. Analysis proceeded separately for SNVs and indels, and CNVs. De novo and transmission/non-transmission were obtained for trio data (published de novo from 825 trios^,- were incorporated). This path led to the TADA analysis, which found 33 ASD risk genes with q < 0.1; and 107 with q < 0.3. CNV were called in 2,305 ASD subjects.

Extended Data Figure 2. Expected number of ASD genes discovered as a function of sample size

The Multiple LoF test (red) is a restricted version of TADA that uses only the de novo LoF data. TADA (blue) models de novo LoF, de novo Mis3, LoF variants transmitted/not transmitted and LoF variants observed in case/control samples. The sample size (N) indicates either (i) N trios, for which we record de novo and transmitted variation, or (ii) N trios, for which we record only de novo events, plus N cases and N controls.

Extended Data Figure 3. Heat map of the numbers of variants used in TADA analysis from each dataset in genes with q < 0.3

Left panel, variants in affected subjects; right panel, unaffected subjects. For the counts, we only focus on de novo LoF and Mis3 variants, transmitted/un-transmitted and case/control LoF variants. These variant counts are normalized by the length of coding regions of each gene and sample size of each dataset (|trio|+|case| for left panel, |trio|+|control| for the right panel).

Extended Data Figure 4. Genome browser view of the CNV deletions identified in ASD affected subjects

The deletions are displayed in red if with unknown inheritance, in grey if inherited, and in black in un unaffected subjects. Deletions in parents are not shown. For deletions within a single gene, all splicing isoforms are shown.

Extended Data Figure 5. Frequency of variants by gender

Frequency of de novo (DN) and transmitted (TR) variants per sample in males (black) and females (white) for genes with q < 0.1 (upper panel), q < 0.3 (central panel), or all TADA genes (lower panel). The P values were determined by a one-tailed permutation test (*P < 0.5; **P < 0.01; ***P < 0.01).

Extended Data Figure 6. Enrichment terms for the four clusters identified by protein-protein interaction network

P-values using Mouse-Genome-Informatics/Mammalian-Phenotype (MGI-MP, blue), Kyoto Encyclopedia of Genes and Genomes pathways (KEGG, red), and Gene Ontology biological processes (GO, yellow) are indicated.

Extended Data Figure 7. De novo variants in SET lysine methyltransferases and JmjC lysine demethylases

Mis3 are in black, LoF in red, and variants identified in other disorders in grey (Fig. 5). JmjC, Jumonji C domain; JmjN, Jumonji N domain; JmjC, PHD, plant homeodomain; ARID, AT-rich interacting domain; SET, Su(var)3-9, Enhancer-of-zeste, Trithorax domain; FYR N, FY-rich N-terminal domain; FYR C, FY-rich C-terminal domain; PWWP, Pro-Trp-Trp-Pro domain; HMG, high mobility group box; AWS, associated with SET domain; Bromo, bromodomain; BAH, bromo adjacent homology.

Extended Data Figure 8. Transcription regulation network of TADA genes only

Edges indicate transcription regulator (source node) and its gene targets (target node) based on ChEA network.

Figure 1. ASD genes in synaptic network

a. Enrichment of 107 TADA genes in: FMRP targets from two independent datasets and their overlap; RBFOX targets; RBFOX targets with predicted alterations in splicing; RBFOX and H3K4me3 overlapping targets; genes with de novo mutations in schizophrenia; human orthologues of Genes2Cognition mouse synaptosome or PSD genes; constrained genes; and, genes encoding mitochondrial proteins (as a control). Red bars indicate empirical P-values. b. Synaptic proteins encoded by TADA genes. c. De novo Mis3 variants in Na_v1.2 (SCN2A). The four repeats (I-IV) with P-loops, the EF-hand, and the IQ domain are shown, as are the four amino acids (DEKA) forming the inner ring of the ion selectivity filter. d. Relevant variants in Ca_v1.3 (CACNA1D). Part of the channel is shown, including helices one and six (S1 and S6) for the I-IV domains, NSCaTE motif, EF-hand domain, pre-IQ, IQ, PCRD, DCRD, proline-rich region, and PDZ-binding motif.

Figure 2. ASD genes in neuronal networks

Protein-protein interaction network created by seeding TADA and DAWN predicted genes. Only intermediate genes that are known to interact with at least two TADA and/or DAWN genes are included. Four natural clusters (C1-C4) are demarcated with black ellipses. All nodes are sized based on degree of connectivity.

Figure 3. ASD genes in chromatin remodeling

a. TADA genes cluster to chromatin remodeling complexes. Amino terminals of histones H3, H4 and part of H2A, are shown. Lysine methyltransferases add methyl groups, while lysine demethylases remove them. b. De novo Mis3 and LoF variants in CHD8. The box shows the outcome of RT-PCR and Sanger sequencing in lymphoblastoid cells for two newly identified de novo splice-site variants. The first mutation hits an acceptor splice site (red arrow), causing the activation of a cryptic splice site (red box), a four-nucleotide deletion, frame shift and a premature stop. The second mutation hits a donor splice site (red arrow), causing exon skipping, frame shift and a premature stop.

Figure 4. Transcription regulation network of TADA genes

Edges indicate transcription regulator (source node) and its gene targets (target node) based on ChEA network; interactions among only HMGs are ignored.

Figure 5. Involvement in disease of ASD genes

Venn diagram to visualize the overlap in disease involvement for the TADA genes.