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

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

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

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 Delineation of the genetic and clinical spectrum of Phelan-McDermid syndrome caused by SHANK3 point mutations

Table S1. Loss of function and de novo missense variants in SHANK3 reported previously. Table S2. Clinical features of individuals with pathogenic or likely pathogenic SHANK3 variants reported in the literature. Table S3. In silico prediction of pathogenicity of missense variants in SHANK3 identified in this study and in the literature. Table S4. Reported truncating and in-frame variants in SHANK3 unlikely to be pathogenic. (XLSX 84 kb

Additional file 2: of Delineation of the genetic and clinical spectrum of Phelan-McDermid syndrome caused by SHANK3 point mutations

Table S5. Descriptive and diagnostic data by patient. Table S6. ASD and intellectual ability classifications in individuals with SHANK3 mutations. Table S7. Language and motor functioning in individuals with SHANK3 mutations. (PDF 132 kb

Novel mutations identified in <i>SLITRK1</i>.

<p>(<b>a</b>) Allelic frequency of novel <i>SLITRK1</i> mutations identified in obsessive-compulsive disorder (OCD) cases. (<b>b</b>) The <i>SLITRK1</i> L63L mutation has been demonstrated by sequencing in one of 762 OC spectrum alleles and in zero of 712 control alleles. (<b>c</b>) The <i>SLITRK1</i> N400I mutation has been demonstrated via sequencing and genotyping in one of 646 OC spectrum alleles and in zero of 2070 control alleles, respectively; <i>^t</i>includes genotyping of 1358 alleles. (<b>d</b>) The <i>SLITRK1</i> T418S mutation has been demonstrated by sequencing in three of 762 OC spectrum alleles and in one of 410 control alleles. (<b>d</b>) Conservation map of <i>SLITRK1</i> region where the three novel mutations were identified (Green – novel variants, gray – evolutionarily conserved regions). (<b>e</b>) A schematic of the <i>SLITRK1</i> protein with the detected variants identified (red – novel variants, gray – previously published variants in Tourette syndrome⁴, black – published variants in trichotillomania⁵. Dotted outline depicts leucine rich repeat (LRR) region 9. Diagram of <i>SLITRK1</i> is available at <a href="http://smart.embl-heidelberg.de" target="_blank">http://smart.embl-heidelberg.de</a>. LRR typ – LRR typical subfamily, LRR CT – LRR C-terminal domain, LRR N-terminal domain; dark blue bar – transmembrane domain.</p

<i>SLITRK1</i> variant N400I fails to induce neurite outgrowth.

<p>(<b>a</b>) LS: Representative images of primary rat E18 hippocampal neurons nucleofected with wildtype <i>SLITRK1</i> or the <i>SLITRK1</i>–N400I variant. RS: Images are also traced to facilitate visualization of thin neurites. Scale bar = 50 µm (<b>b</b>) LS: Representative images of primary mouse E17 cortical neurons nucleofected with wildtype <i>SLITRK1</i> or the <i>SLITRK1</i>–N400I variant. RS: Representative E17 cortical neuron trace. Scale bar = 50 µm (<b>c</b>) The summed total neurite length per hippocampal neuron at 7 <i>div</i> is shown. Each bar on the bar graph represents pooled data of at least 50 neurons per experiment (<i>n</i> = 3). Images are uniformly overexposed to improve neurite visibility. (<b>d</b>) The summed total neurite length per cortical neuron at 3 <i>div</i> is shown. Each bar on the bar graph represents pooled data of at least 14–22 neurons per experiment (<i>n</i> = 2). Statistical significance was assessed using a student's t-test as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070376#s4" target="_blank">Materials and Methods</a>. Error bars are 95% confidence intervals. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Pedigree diagrams of families with <i>SLITRK1</i> variants.

<p>Pedigrees for individuals in whom <i>SLITRK1</i> variants were identified. Each obsessive-compulsive (OC) spectrum proband is labeled with his/her identifier and is designated by a black arrowhead. Individuals affected with an OC spectrum disorder are represented by shaded symbols, with red shading indicating obsessive-compulsive disorder (OCD) and blue shading indicating Tourette syndrome (TS). Psychiatric conditions outside of the OC spectrum are represented by a magenta circle in the center of the symbol. Male family members are represented with squares, females with circles, persons with unspecified gender are diamonds with the number of individuals indicated directly below. All psychiatric pathology is listed under each affected individual. OCD – Obsessive-Compulsive Disorder, TS – Tourette syndrome, BDD – Body Dysmorphic Disorder, GAD – Generalized Anxiety Disorder, MDD – Major Depressive Disorder, N.O.S. – not otherwise specified, PD – Panic Disorder, PTSD – Post-Traumatic Stress Disorder, w/ - with, y/o – years old, ? – psychiatric history is unavailable for the individual.</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