Metadata for all participants.

<p>Nine cohorts of typical individuals and one cohort of individuals with Asperger syndrome were recruited. Values are reported as mean ± standard deviation. BMI (body mass index, in kg/m²), WC (waist circumference, in centimetres).</p

Analytes significantly elevated in females.

<p>All analytes with a meta-analysis p-value of less than 0.05 after adjustment for the false discovery rate are shown. Analytes are ordered by biological function.</p

Sex-specific effects in networks for Asperger syndrome.

<p>Top networks of the cluster of molecules associated with energy production, fatty acid metabolism, and hormone levels from Ingenuity Pathway Knowledgebase software. Individual molecules are coloured according to significant sex difference in controls. Molecules with significant sex-disease interactions from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051504#pone.0051504-Schwarz2" target="_blank">[34]</a> are circled. A2M (alpha 2 macroglobulin), ADIPOQ (adiponectin), RAGE (receptor for advanced glycosylation end products), ANGPT2 (angiopoietin 2), ARNT (aryl hydrocarbon receptor nuclear translocator), CRP (C-reactive protein), ENA-78 (epithelial derived neutrophil activating protein 78), EDNRB (endothelin receptor type B), EPO (erythropoietin), ERK (extracellular-signal-regulated kinase), FCER1A (Fc fragment of IgE, high affinity I, receptor for alpha polypeptide), GAB1 (GRB2-associated binding protein 1), GCLC (glutamate-cysteine ligase, catalytic subunit), IL17F (interleukin-17F), Jnk (c-Jun N-terminal kinase), SCF (stem cell factor), LEP (leptin), LHB (luteinizing hormone beta polypeptide), Mapk (mitogen-activated protein kinase), NFkB (complex) (nuclear factor of kappa light polypeptide gene enhancer in B-cells), NOX3 (NADPH oxidase 3), P38 MAPK (P38 mitogen-activated protein kinase), PRKAA1 (protein kinase, AMP-activated, alpha 1 catalytic subunit), PRKAA2 (protein kinase, AMP-activated, alpha 2 catalytic subunit), PSMD4 (proteasome (prosome, macropain) 26S subunit, non-ATPase, 4), SMPD2 (sphingomyelin phosphodiesterase 2, neutral membrane (neutral sphingomyelinase)), TFF3 (trefoil factor 3), AGT (angiotensinogen), APOA1 (apolipoprotein AI), COL18A1 (collagen, type XVIII, alpha 1), CXCL11 (chemokine (C-X-C motif) ligand 11), BLC (B lymphocyte chemoattractant), MIG (monokine induced by gamma interferon), Fcgr2 (Fc gamma R2), FDX1 (ferredoxin 1), FDXR (ferredoxin reductase), FSH (follicle stimulation hormone), GCLC (glutamate-cysteine ligase, catalytic subunit), GH (growth hormone), GHR (growth hormone receptor), SGOT (serum glutamic oxaloacetic transaminase), KIM-1 (kidney injury molecule 1), HSD3B2 (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2), IL6 (interleukin-6), ITK (IL2-inducible T-cell kinase), LDLR (low density lipoprotein receptor), LPL (lipoprotein lipase), LSS (lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase)), MSMO1 (methylsterol monooxygenase 1), NLRP12 (NLR family, pyrin domain containing 12), NPPB (brain natriuretic peptide), NR1H4 (nuclear receptor subfamily 1, group H, member 4), OSMR (oncostatin M receptor), PI3K (complex) (phosphoinositide-3-kinase), SCARB1 (scavenger receptor class B, member 1), TBG (thyroxine binding globulin), SST (somatostatin), Stat5a/b (signal transducer and activator of transcription a/b), TCR (T-cell receptor), TNF-alpha (tumor necrosis factor-alpha), TNFRSF11B (tumor necrosis factor receptor superfamily, member 11b), VWF (von Willebrand factor).</p

Sex differences of composite variables summarizing analyte clusters.

<p>Values for typical individuals were pooled across nine cohorts; values for Asperger syndrome participants were calculated from cohort 10. The x-axis shows the difference between composite values that reflect the average molecular levels in males and females. Horizontal bars indicate 95% confidence intervals of the difference between sexes.</p

Analytes significantly elevated in males.

<p>All analytes with a meta-analysis p-value of less than 0.05 after adjustment for the false discovery rate are shown. Analytes are ordered by biological function.</p

PCA plots showing individual molecules.

<p>A) Assignment of analyte clusters to biological functions; B) Sex differences as determined by meta-analysis. Colouring indicates significance (q<0.05). The grey area indicates analytes that show no significant sex differences. Plots were generated using data from cohort 7, in which 167 analytes were measured in 162 typical controls (81 females, 81 males).</p

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

Characterization of CNVs in three patients carrying a <i>de novo</i> deletion of <i>SHANK2</i>.

<p>Paternally or maternally inherited CNVs are indicated by squares and circles, respectively. <i>De novo</i> CNVs are indicated by stars. Deletions and duplications are indicated in red and blue, respectively. CNVs hitting exons or only introns are filled with grey and white, respectively. Squares and circles within star represent <i>de novo</i> CNV of paternal or maternal origin; circles within squares represent CNV inherited by father or mother. ABCC6, ATP-binding cassette, sub-family C, member 6 pseudogene 2; ADAM, ADAM metallopeptidase; AMY1, amylase (salivary); AMY2A, amylase (pancreatic); ARHGAP11B, Rho GTPase activating protein 11B; CAMSAP1L1, calmodulin regulated spectrin-associated protein 1-like 1; CHRNA7, cholinergic receptor, nicotinic, alpha 7; CNTN4, contactin 4; CTNNA3, catenin (cadherin-associated protein), alpha 3; CYFIP1, cytoplasmic FMR1 interacting protein 1; DUSP22, dual specificity phosphatase 22; GALM, galactose mutarotase; GCNT2, glucosaminyl (N-acetyl) transferase 2; GOLGA, golgi autoantigen, golgin subfamily a; GSTT1, glutathione S-transferase theta 1; HLA-DRB, major histocompatibility complex, class II, DR beta; LAMA4, laminin, alpha 4; NIPA, non imprinted in Prader-Willi/Angelman syndrome; NLGN1, neuroligin 1; NME7, non-metastatic cells 7; OR, olfactory receptor; PCDHA, protocadherin alpha; RFPL4B, ret finger protein-like 4B; RHD, Rh blood group, D antigen; SFMBT1, Scm-like with four mbt domains 1; SHANK2, SH3 and multiple ankyrin repeat domains 2; SMC2, structural maintenance of chromosomes 2; TNS3, tensin 3; TUBGCP5, tubulin, gamma complex associated protein 5; UGT2B17, UDP glucuronosyltransferase 2 family, polypeptide B17.</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

Genomic structure, isoforms, and expression of human <i>SHANK2</i>.

<p>A. Genomic structure of the human <i>SHANK2</i> gene. Transcription of <i>SHANK2</i> produces four main mRNA from three distinct promoters: <i>SHANK2E</i> (<i>AB208025</i>), <i>ProSAP1A</i> (<i>AB208026</i>), <i>ProSAP1</i> (<i>AB208027</i>) and <i>AF141901</i>. There are three translation starts: in exon 2 for <i>SHANK2E</i>, in exon1b for <i>ProSAP1A</i>, and in exon1c for <i>ProSAP1</i> and <i>AF141901</i>; and two independent stop codons: in exon 22b for <i>AF141901</i> and in exon 25 for <i>SHANK2E</i>, <i>ProSAP1A</i> and <i>ProSAP1</i>. Conserved domains of protein interaction or protein binding site are represented in color: ANK (red), SH3 (orange), PDZ (blue) and SAM (green), H (pink), D, (dark blue) and C (purple). Black stars identify the alternative spliced exons (‘brain-specific exons’ in turquoise: 19, 20 and 23). B. RT-PCRs of <i>SHANK2</i> isoforms on RNA from different human control tissues (Clontech), and different brain regions of four controls (2 males and 2 females). The amplified regions specific to each isoform of <i>SHANK2</i> are indicated by gray boxes. C. Alternative splicing of human <i>SHANK2</i>; exons 19, 20 and 23 are specific to the brain. ANK, ankyrin; SH3, Src homology 3; PDZ, PSD95/DLG/ZO1; SAM, sterile alpha motif; He, heart; Li, liver; B, brain; SM, skeletal muscle; Pl, placenta; K, kidney; Lu, lung; Pa, pancreas; FC, frontal cortex; Hi, hippocampus; TC, temporal cortex; T, thalamus; OC, occipital cortex; Ce, cerebellum; Cx, whole cortex; BLCL, B lymphoblastoid cell lines; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BSR, brain specific region; H, homer binding site; D, dynamin binding site; C, cortactin binding site. The ages of the two males and the two females studied were 74, 42, 55, and 36 years with a post-mortem interval of 10, 21, 24, and 2 h, respectively.</p