Protein-Protein Interaction (PPI) network enrichments.

<p>Enrichments are given as the fold change over that expected by chance (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#s4" target="_blank">Materials and Methods</a>).</p

Triplicated mouse model phenotype associations among genes overlapped by sets of <i>de novo</i> CNVs identified in individuals with ASD.

<p>Enrichments are given as the fold change over that expected by chance (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#s4" target="_blank">Materials and Methods</a>).</p

Relationships of mouse model phenotypic terms enriched among genes overlapped by <i>de novo</i> CNVs identified in individuals with ASD.

<p>Relationships between phenotypic terms within the Mammalian Phenotype Ontology are indicated by a blue arrow running from the child term to the parent term. Terms are significant (BH-adjusted <i>p</i><5%) in at least one of 4 sets of <i>de novo</i> CNVs identified in individuals with autism if they are shown with a coloured border (red, dark and light blue). Those terms whose significant enrichment is observed in three independent sets, and thus triplicated, are marked with a boxed letter “T”. Panels A–E show representative clusters of <i>Behaviour/Neurological</i> phenotypic category, while Panel F shows the enriched phenotypes from the <i>Nervous System</i> phenotypic category and Panel G shows representative enrichments from the <i>Hearing/Vestibular/Ear</i> phenotypic category. The number adjacent to the phenotypic terms indicates the rank of that phenotypic term among those phenotypes significantly enriched among a set of 22 disease genes previously implicated in ASD (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#s2" target="_blank">Results</a>).</p

Distinct duplications and deletions of genes whose proteins interact within the <i>ASD-associated</i> network perturb pathways in the same direction.

<p>Genes duplicated within ASD <i>dn</i> CNVs are indicated with green upwards arrows while those deleted are denoted by blue downwards arrows. Previously identified <i>ASD-Implicated</i> genes found to be disrupted in autism patients are denoted with red downwards arrows. The nature of the interactions/regulations between proteins/molecules are shown with different edge types (see in-figure legend). The <i>ASD-associated</i> network (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen-1003523-g002" target="_blank">Figure 2</a>) identifies several deletion/duplication pathway cascades, for example the <i>MAPK3</i> pathway (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#s3" target="_blank">Discussion</a> for additional examples). Here, deletions of the MAPK3 pathway components (i.e. <i>SYNGAP1</i>, <i>SHANK2</i>, <i>KRAS</i>, <i>MAPK3</i>, <i>PAK2</i>, and <i>CREBBP</i>) and duplications of their negative regulators (i.e. <i>FMR1</i>, <i>GDI1</i>, <i>ARHGDIA</i>, <i>CAMK2B</i>, and <i>CAMKK2</i>) found in autistic patients identify converging effects on the MAPK pathway, specifically reduced CREB-dependent transcription <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Webber1" target="_blank">[9]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Muddashetty1" target="_blank">[62]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Chen1" target="_blank">[63]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Hart1" target="_blank">[64]</a>. CREB-dependent transcription has been implicated in neuroadaptation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Levy1" target="_blank">[20]</a>. In addition, increased NO* production leads to the inhibition of <i>MAPK1/3</i> activity <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Raines1" target="_blank">[65]</a>, which fits well with the observed CNV duplications of both <i>NOS1</i> and <i>DLG4</i>, the latter gene promoting recruitment of <i>NOS1 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Nikonenko1" target="_blank">[66]</a>. Similarly, duplication of <i>PRKG1</i>, which is up-regulated by NO* and expresses a product that inhibits IP3 production, is predicted to reduced activation of the calcium-releasing IP3-receptor <i>ITPR1 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003523#pgen.1003523-Ruth1" target="_blank">[67]</a>, which is in turn found to be deleted.</p

Additional file 1 of Integrated Bayesian analysis of rare exonic variants to identify risk genes for schizophrenia and neurodevelopmental disorders

Supplementary Information. This file describes supplementary results, methods, data, figures, and short tables. (PDF 3610 kb

Additional file 2 of Integrated Bayesian analysis of rare exonic variants to identify risk genes for schizophrenia and neurodevelopmental disorders

Supplementary Tables. This file consists of long supplementary tables. (XLSX 13200 KB

Genetic alterations identified in the control subject SWE_Q56_508.

<p>A. <i>SHANK2</i> splice mutation (IVS22+1G>T) detected in a Swedish female control, SWE_Q56_508. The mutation altered the donor splicing site of exon 22 and led to a premature stop in all <i>SHANK2</i> isoforms except for the <i>AF1411901</i> isoform, where it altered the protein sequence (G263V). B. CNVs in the same individual altering <i>LOC339822</i>, <i>SNTG2</i>, <i>PXDN</i> and <i>MYT1L</i>. The two close duplications span 264 kb and 245 kb on chromosome 2 and altered <i>LOC339822</i> and <i>SNTG2</i>, and <i>PXDN</i> and <i>MYT1L</i>, respectively. Dots show the B allele frequency (BAF; in green), Log R ratio (LRR; in red), and QuantiSNP score (in blue). Lower panel: all CNVs listed in the Database of Genomic Variants (DGV) are represented: loss (in red), gain (in blue), gain or loss (in brown). H, homer binding site; D, dynamin binding site; C, cortactin binding site.</p

Scatter plots of the intellectual quotient and the Autism Diagnostic Interview-Revised (ADI-R) scores of the patients with ASD screened for <i>SHANK1-3</i> mutations.

<p>Mutations in <i>SHANK1-3</i> are associated with a gradient of severity in cognitive impairment. <i>SHANK1</i> mutations were reported in patients without ID (green dots). <i>SHANK2</i> mutations were reported in patients with mild ID (orange dots). <i>SHANK3</i> mutations were found in patients with moderate to severe deficit (red dots). Black dots correspond to the patients enrolled in the PARIS cohort screened for deleterious <i>SHANK1-3</i> mutations (n = 498). In addition to the PARIS cohort <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Pinto1" target="_blank">[8]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>, three patients with a <i>SHANK1</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a> and two patients with a <i>SHANK2</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a> were included in the scatter plot. A high score of the ADI-R is associated with a more severe profile. The threshold of the “Social”, “Verbal”, “Non-Verbal” and “Repetitive Behavior” Scores are 10, 8, 7 and 3, respectively.</p

Characterization of the functional impact of <i>SHANK2</i> mutations in cultured neuronal cells.

<p>A. The colocalization of <i>ProSAP1A/SHANK2</i>-EGFP (postsynaptic marker) and Bassoon (presynaptic marker) indicated that the mutations did not disturb the formation of SHANK2 clusters at excitatory synapses along the dendrites. B. The quantification of synapse density was performed on 20 transfected hippocampal neurons per construct from at least three independent experiments. The majority of the <i>ProSAP1A</i> variants affecting a conserved amino acid among SHANK proteins reduced significantly the synaptic density compared with the variants that affect amino acid non conserved among SHANK proteins (Mann-Whitney U-test: n_WT = 20, n_mut = 20; U_S557N = 82.5, p_S557N = 0.001; U_R569H = 124, p_R569H = 0.04; U_L629P = 149, p_L629P = 0.17; U_V717F = 114, p_V717F = 0.02; U_A729T = 73, p_A729T = 0.000; U_K780Q = 154, p_K780Q = 0.221; U_R818H = 108, p_R818H = 0.012; U_A822T = 154.5, p_A822T = 0.224; U_V823M = 129, p_V823M = 0.056; U_Y967C = 134, p_Y967C = 0.076; U_G1170R = 78, p_G1170R = 0.001; U_R1290W = 142, p_R1290W = 0.121; U_Q1308R = 162, p_Q1308R = 0.314; U_D1535N = 97, p_D1535N = 0.005; U_P1586L = 137, p_P1586L = 0.910; U_L1722P = 79, p_L1722P = 0.001, *p<0.05, **p<0.01, ***p<0.001). <b>C.</b> Effect of the variants on synaptic density. The y-axis represents −log P compared to WT (P obtained with Mann-Whitney test). After Bonferroni correction for 16 tests, only P values<0.003 were considered as significant. Variants represented in red were specific to ASD, in orange were shared by ASD and controls, and in green were specific to the controls. Open circles and filled circles represent non conserved and conserved amino acids, respectively. Prim, primary; second, secondary.</p

Prevalence and meta-analysis of coding-sequence variant studies in ASD.

<p>A. The prevalence and the confidence interval from a set of single coding-sequence variant studies, and the pooled prevalence and the confidence interval of the meta-analysis. The prevalence is indicated by circles in red, pink, purple and black for “ASD all” (all ASD patients), “ASD IQ<70” (patients with ID; IQ<70), “ASD IQ>70” (patients with normal IQ), and “CTRL” (controls), respectively. Three categories are used to study the prevalence of coding-sequence variants in ASD and controls: all or “A” (all mutation), Damaging or “D” (damaging missense mutation; score obtained from polyphen-2), and Truncating or “T” (mutation altering SHANK protein). The plotted circles are proportional to the corresponding sample size. B. Meta-analysis of coding-sequence variant studies altering <i>SHANK</i> genes. For each study, the Odds ratio and confidence interval is given. Each meta-analysis is calculated using inverse variance method for fixed (IV-FEM) and random effects (IV-REM). The statistics measuring heterogeneity (Q, I² and Tau²) are indicated. The number under the scatter plot correspond to independent studies: 1 = “This study”, 2 = “ Sato et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a>”, 3 = “Berkel et al. (2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a>”, 4 = “Leblond et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>”, 5 = “Boccuto et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Boccuto1" target="_blank">[17]</a>”, and 6 = “[This Study and Durand et al. 2007 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>]”, 7 = “[Gauthier et al. (2009–2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier1" target="_blank">[16]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier2" target="_blank">[47]</a>]”, 8 = “Moessner et al. (2007) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Moessner1" target="_blank">[13]</a>”, 9 = “Schaff et al. (2011) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Schaaf1" target="_blank">[35]</a>”. IV, Inverse Variance; FEM, Fixed Effect Method; REM, Random Effect Method; OR, Odds Ratio; CI, Confidence Interval; IQ, Intellectual Quotient; CNV, Copy Number Variant.</p