De novo mutations in schizophrenia implicate synaptic networks

Abstract

Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.

Extended Data Figure 1. Comparison of sequencing metrics for putative de novo calls and parental singletons

Putative de novo calls (child heterozygous, both parents homozygous reference) were compared with variants observed in only a single parent (“singletons”), in terms of (a) depth of all reads at the variant site [DP = depth], (b) fraction of reads with the alternate allele [AB = allele balance], (c) mapping quality of the reads at the site [MQ], (d) the likelihood of the heterozygous genotype [PL = Phred-scaled likelihood], and (e) the number of other samples in the present study with a non-reference allele at that site [AAC = alternate allele count]. Distributions were calculated for putative de novo variants (red), or grouped by sites of putatively recurrent de novos (orange) when relevant, transmitted singletons (green), and non-transmitted singletons (blue).

Extended Data Figure 2. Metrics for de novo variants across cohorts and trios

a. Rates of recurrence of de novo mutations for tri-nucleotide sequences. For each of 96 possible tri-nucleotide base contexts of single-base mutations (accounting for strand symmetry by reverse complementarity), the number of observed de novo SNV is plotted (sorted by this count). Mutation counts are sub-divided into those not found in external data (red), those found in dbSNP (build 137, green), those found in controls in the parallel exome sequencing study (cyan), and those found both in dbSNP and that study (purple) b. Comparison of on-target heterozygous SNV and indel call rate with putative de novo mutation calls. For each proband, the number of heterozygous SNV and indel calls is compared with the number of putative de novo mutations (child heterozygous, both parents homozygous reference). Probands are colored by sequencing center (see Supplementary Text for differences in exome capture), and 6 trios are noticeable outliers from all others in terms of number of putative de novos. c. Variation in sequencing coverage between and across trios and sequencing centers. For each trio, the number of bases covered by 10 reads or more for each member (marked by ‘x’) and the joint coverage in all 3 members (marked by points) are plotted at corresponding horizontal points; trios are sorted in increasing order of joint coverage and colored by sequencing center (see Supplementary Text). The intersection of each exome capture with the RefSeq coding sequence is marked by respective dotted lines.

Extended Data Figure 3. De novo mutation counts and rates

a. The observed distribution of number of validated RefSeq-coding (see Supplementary Text) de novo mutations found for each trio (N=617) is compared with that expected from a Poisson distribution with a rate equal to the observed mean number of de novos (λ=1.032). b. Deleterious mutation rate inversely correlates with academic performance. Individuals were grouped according to their final school grade (3-6, corresponding to D, C, B, A in the US system, http://www.fulbright.bg/en/p-Educational-System-of-Bulgaria-18/), and the proportion of individuals with one or more de novo loss-of-function (LoF) mutations is plotted. See Supplementary Text for details on linear regression performed to evaluate association; note that 19 samples were removed from this analysis for missing parental age or school grade information, leaving a total of 598 trios.

Extended Data Figure 4. Enrichment of de novo SNVs, indels, and CNVs in genes encoding postsynpatic complexes at glutamatergic synapses

a. Number of de novo mutations in postsynaptic complexes in current study (and genes affected) are shown alongside the most conservative estimate of de novo CNV enrichment from Kirov, et al.. NS = nonsynonymous, LoF = loss-of-function. The NMDAR complex gene set was derived a priori from a published proteomics dataset. To avoid investigator bias, we did not add additional members post hoc, thus omitting genes with de novo mutations and important NMDAR functions; these include GRIN2A, which encodes a subunit of the NMDA receptor itself, and AKAP9 which directly anchors protein complexes involved in signalling at NMDA receptors. p<0.05 are marked in bold. b. - g. 95% credible intervals (CI) for fold-enrichment statistics of de novo mutations in postsynaptic gene sets (corresponding to enrichments in a. above, and as marked) were calculated from the posterior distributions of fold-enrichment (observed-to-expected = O/E) statistic values for individuals in this study. Point estimates of O/E are given in Table 3, and correspond to the distribution modes here. The 95% CI is marked by red vertical lines, and a null effect size (value of 1) is marked by a gray line. Note that LoF mutations in the large PSD set are not significantly enriched, and thus the corresponding CI includes an effect size of 1. All posterior distributions were calculated using dnenrich, as described in the Supplementary Text.

Figure 1. De novo mutations from schizophrenia affect genes in the synapse and genes impacted in other neuropsychiatric diseases

a. Synaptic protein-protein interactions between proteins affected by nonsynonymous de novo mutations in schizophrenia. Interactions were retrieved from the expert-curated lists in the SynSysNet database (http://bioinformatics.charite.de/synsysnet/) and plotted to show their general pre/postsynaptic localization. Genes belonging to various functional sets are as marked, and the 4 genes with LoF mutations are noted with a red outline. Proteins with nonsynonymous de novos had more than expected direct interconnections (p=0.008), which was consistent with more overall connectivity to synaptic proteins as a whole (p=0.005). b. Overlap of genes bearing nonsynonymous (NS) de novo mutations in schizophrenia, autism, and intellectual disability. Overlaps of 6 or fewer genes are listed by name. See Extended Data Table 5 for statistical significance of these overlaps; see Table 2 and text for disease sets.