<i>PRRT2</i> coding variants identified in this study.

<p>Schematic diagram of the <i>PRRT2</i> gene and of the three PRRT2 protein isoforms. Mutation identified in this study are indicated in green (controls and HGDP), orange (controls, HGDP, and patients) and red (patients only).</p

<i>PRRT2</i> coding variants identified in individuals with ASD and controls.

a<p>Variants observed in the human genome diversity panel.</p>b<p>The p.A361_P362del was considered as probably damaging since it affects conserved amino acids of PRRT2.</p>c<p>Odds ratio, confidence intervals (CI) and P values were calculated only for populations from European ancestry using a 2-tailed Fisher exact test.</p

Genetic variability in Europe and Africa for all genes located within the 16p11.2 deletion.

<p>A. Nonsynonymous mutations per nonsynonymous sites (pN) and synonymous mutations per synonymous sites (pS) were estimated using the data from 4300 individuals from European ancestry and 2012 individuals from African ancestry available at the Exome Variant Server (<a href="http://evs.gs.washington.edu/EVS/" target="_blank">http://evs.gs.washington.edu/EVS/</a>). The horizontal lanes correspond to the means of pN and pS for the 27 genes. Difference of pN/pS between Europe and Africa were calculated using a 2-tailed Fisher exact test and the −Log₁₀ P value is indicated. B. Plot of the −Log₁₀ P values obtained for the difference between the pN/pS ratio in Africa and in Europe in relation the ratio (Europe pN/pS)/(Africa pN/pS).</p

Synonymous and nonsynonymous variations of <i>PRRT2</i> in worldwide populations.

a<p>Number of nonsynonymous and synonymous sites for PRRT2 are 668.5 and 231.5, respectively.</p>b<p>For the calculation of the pN/pS for Europe, the value of pS was set to 0.004 (the minimum of 1 synonymous variant observed in 159 individuals).</p

<i>PRRT2</i> variants identified in individuals from worldwide populations.

<p>A total of 961 individuals from the human genome diversity panel (HGDP) were sequenced for all <i>PRRT2</i> exons. The diameter of each circle is proportional to the number of individuals who were sequenced for <i>PRRT2</i>.</p

Chromatograms of the <i>PRRT2</i> A217PfsX8 mutation before and after whole genome amplification.

<p>The chromatograms of <i>PRRT2</i> sequence before (Native DNA) and after whole genome amplification of the DNA from three independent patients using two different protocols GenomiPhi DNA Amplification Kit (WGA-1) or Repli-G Whole Genome Amplification kit (WGA-2). The mutation was not present in the native DNA, but was detected after whole genome amplification.</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

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

<i>SHANK2</i> mutations in patients with ASD.

<p>A. A heterozygous deletion of <i>SHANK2</i> was identified with the Illumina Human 1M-Duo SNP array in a patient with autism (AU038_3). The deletion spans 421 kb on chromosome 11q13.3, covers twelve exons of the human <i>SHANK2</i> and is not present in the parents. Each dot shows Log R Ratio (LRR; in red) and B allele frequency (BAF; in green). QuantiSNP score is represented with a blue line and indicates the deletion size. B. Location of the CNV and sequence variants (from this study and Berkel <i>et al.</i> 2010) along the SHANK2 protein: in red the variations specific to ASD, in orange the variations shared by ASD and controls and in green the variations specific to controls <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002521#pgen.1002521-Berkel1" target="_blank">[26]</a>. The breakpoints of the <i>SHANK2</i> deletion in AU038_3 are represented with a dotted line on the protein. Stars indicate the variants affecting conserved amino acids. C. A total of 40 variants were identified and variants affecting conserved amino acids in other SHANK proteins are enriched in patients with ASD (n_conserved = 12 and n_{non-conserved} = 3) compared with controls (n_conserved = 6 and n_{non-conserved} = 11) (Fisher's exact test 1-sided, P = 0.013, OR = 6.83, 95% IC = 1.19–53.40). D. The percentage of carriers of <i>SHANK2</i> variants in patients with ASD and Controls. Variants affecting a conserved amino acid among the SHANK proteins are enriched in patients with ASD (n_conserved = 29 and n_{non-conserved} = 822) compared with controls (n_conserved = 16 and n_{non-conserved} = 1074) (Fisher's exact test 1-sided, P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). Open squares and filled squares represent the non-conserved and conserved amino acids, respectively. ANK, Ankyrin repeat domain; SH3, Src homology 3 domain; PDZ, postsynaptic density 95/Discs large/zona occludens-1 homology domain; SAM, sterile alpha motif domain; BSR, brain specific region; H, homer binding site; D, dynamin binding site; C, cortactin binding site. The proline-rich region is represented as a horizontal gray line.</p