Detection of 9 different point mutations of <i>PCDH19</i> in 11 female patients by direct sequencing.

<p>A) Sequence electropherograms of the mutations and the missense variant (c.3319C>G/p.Arg1107Gly) identified in association with the c.859G>T/p.Glu287X nonsense mutation. The mutation nomenclature is based on the <i>PCDH19</i> transcript reference EF676096. Nucleotides are numbered according to the cDNA with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to the journal guidelines (<a href="http://www.hgvs.org/mutnomen" target="_blank">www.hgvs.org/mutnomen</a>). B) Alignment of the regions surrounding the mutations (indicated by an arrow) in orthologous and paralogous proteins, showing the high conservation of each affected amino-acid in vertebrates and in the delta protocadherin paralogous genes.</p

Schematic illustration of the cellular interference mechanism associated with <i>PCDH19</i> mutations.

<p>A) In normal individuals, characterized by a homogeneous population of <i>PCDH19</i>-positive cells, neurons are able to form normal neuronal networks; B) In mutated male patients, hemizygosity leads to a homogeneous population of <i>PCDH19</i>-negative cells; in this condition, neurons preserve the ability to form normal neuronal networks; C) In heterozygous mutated females, random X inactivation leads to the co-existence of two <i>PCDH19</i>-positive and <i>PCDH19</i>-negative cell populations. These two cell populations cause divergent cell sorting and migration (due to attractive or repulsive interactions) and lead to abnormal neuronal networks. Somatic mosaicism in mutated males gives rise to the same pathological situation. The precise mechanisms by which the neuronal networks are altered are still unknown.</p

Clinical characteristics of patients with <i>PCDH19</i> mutations.

<p>2*: patient N 06 1258 is the sister of patient N 06 1257 (index case); PMD = psychomotor development, Nl = normal, F = febrile, unit = unilateral, GTC = generalized tonic-clonic, W-S = words-sentences, abs = absent, AED = anti epileptic drugs.</p

Identification of a deletion encompassing <i>PCDH19</i> in a male patient.

<p>A) Identification of a hemizygous Xq22.1 deletion with a 370 K SNP microarray (Illumina): Y-axes represent Log R ratio (above) and B allele frequency (below); the X-axis indicates the position on the X chromosome. The red line (log R ratio profile) corresponds to the median smoothing series (Beadstudio). B) Analysis of the patient and his mother with CGH microarrays (Nimblegen), showing that the deletion occurred de novo. Indicated genomic positions are based upon the Ensembl Genome Browser. Black horizontal bars (below) represent the gene (<i>PCDH19</i>) and pseudogenes comprised in the deleted region.</p

Pedigrees of the families and segregation analysis of the <i>PCDH19</i> deletion and point mutations.

<p>del/+, m/+ or v/+ denote individuals heterozygous for the deletion, mutation or variant, respectively; +/+ denotes individuals carrying homozygous wild-type alleles. Squares represent males, circles females; filled black symbols: patients diagnosed as having Dravet syndrome; right black half: Cognitive delay or impairment; left grey half: adolescence-onset idiopathic epilepsy. Dots in the middle of the squares indicate unaffected mutation carriers. The arrows indicate the index cases.</p

FISH analysis of the <i>PCDH19</i> deletion in the male patient showing somatic mosaicism in fibroblasts.

<p>(A) Absence of the specific Xq22.1 probe site on metaphase chromosomes in peripheral blood lymphocytes (PBL); (B) In fibroblasts, presence of one hybridization spot in 53% of the cells and absence of signal in the remaining 47%; C) and D) FISH analysis on PBL (C) and fibroblasts (D) of a female control. <i>PCDH19</i>-specific signals (red) are indicated by arrowheads. Magnification ×1000.</p

<i>SHANK</i> variants in patients with ASD and controls.

<p>Coding-sequence variants identified only in patients with ASD (upper panel), shared by patients and controls (lower panel and underlined), and present only in controls (lower panel). Truncating variants are indicated in red. The variants predicted as deleterious or benign are indicated in orange and green, respectively. Coding-sequence variants with a proven <i>in vitro</i> functional impact are indicated with black stars. Conserved domains are represented in color: SPN (yellow), Ankyrin (red), SH3 (orange), PDZ (blue) and SAM (green).</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

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

Summary of the SHANK protein functions and of the main findings obtained for patients with ASD.

<p>The frequency of mutation in patients and control individuals was calculated from the total cohort (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen-1004580-t001" target="_blank">Table 1</a>). The frequency of mutation in patients with normal IQ (IQ>70) and low IQ (IQ<70) were calculated for the patients with available IQ scores (copy-number variants for all SHANK: nASD with IQ>70 = 1 638 & nASD with IQ<70 = 917; SHANK1 coding-sequence variants: nASD with IQ>70 = 354 and nASD with IQ<70 = 278; SHANK2 coding-sequence variants: nASD with IQ>70 = 335 & nASD with IQ<70 = 344; SHANK3 coding-sequence variants: nASD with IQ>70 = 667 & nASD with IQ<70 = 611). The mean IQ and standard deviation was given only for patients carrying truncating or <i>de novo</i> mutations. The black star indicates that Schmeisser et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Bonaglia1" target="_blank">[21]</a> found an increase in NMDA currents, while Won et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Betancur2" target="_blank">[22]</a> found a decrease in NMDA currents in two independent SHANK2 knock-out mice.</p><p>Summary of the SHANK protein functions and of the main findings obtained for patients with ASD.</p