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

<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

<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

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

Effect of Gcn5 F304S mutation on <i>Drosophila</i> heart function.

<p>(A-C) M-mode kymographs of 1 day old beating hearts of control flies (<i>yw/Df(3L)</i>; A) and Gcn5^<i>null</i> flies rescued with Gcn5 WT (B) or Gcn5 F304S (C). Scale bar: 1 second. (D-H) High-speed movies of beating hearts were analysed using semi-automated Optical Heartbeat Analysis [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007386#pgen.1007386.ref046" target="_blank">46</a>]. For quantification, 8–19 flies were analyzed. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison for all parameters except arrhythmia index (H), which was analysed using Mann-Whitney-Wilcoxon. For all panels: ns, non significant, *p<0.05 **p<0.01, ***p<0.001, ****p<0.0001 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007386#pgen.1007386.s009" target="_blank">S1 Table</a> for details on transgenic flies).</p

Garland nephrocyte phenotype of <i>hts</i>^<i>null</i> and adducin-αγ rescue mutants.

<p>(A) Kirre and Pyd localization in <i>hts</i>^<i>null</i> and rescue mutant garland nephrocytes. Dissected nephrocytes of the indicated genotypes were stained for Kirre (red) and Pyd, corresponding to Neph1 and ZO-1 in vertebrates, (blue). Arrowheads show areas of cell fusion. Scale bar: 10μm. (B) Quantification of nephrocytes showing a continuous Kirre staining using >9 samples/genotype from 3 independent experiments. Statistical analysis was performed with Kruskal-Wallis with Dunn’s post-test. ns, non significant, *p<0.05, ***p<0.001 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007386#pgen.1007386.s009" target="_blank">S1 Table</a> for details on transgenic flies). (C) Pericardial nephrocytes in adducin-αγ WT and E559Q rescue and control adult flies at 15 days post-eclosion were stained for the differentiation markers Kirre (red) and Pyd (blue). Note that <i>hts</i>^<i>null</i> is lethal at this stage. Scale bar: 30μm. (D) Quantification of the number of pericardial nephrocytes from n>8 samples/genotype in 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s post-test. ns, non significant (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007386#pgen.1007386.s009" target="_blank">S1 Table</a> for details on transgenic flies).</p

Pathogenic genetic variants identified in affected individuals with overlapping syndromes.

<p>Pathogenic genetic variants identified in affected individuals with overlapping syndromes.</p

Number of pericardial nephrocytes in single and double knockdown of <i>hts</i> and <i>Gcn5</i>.

<p>(A-D) <i>Dot-GAL4</i>-mediated knockdown of <i>hts</i> or/and <i>Gcn5</i> in pericardial nephrocytes. Pericardial nephrocytes of adult flies with the Hand-GFP background were observed directly for GFP signal after fixation (A). Immunostaining was performed for the differentiation markers Kirre (red) and Pyd (blue; B). Images are representative of pericardial nephrocytes dissected from adult flies at 3 days post-eclosion. Scale bars: 30μm. Graphs represent quantification of the number of pericardial nephrocytes at 3 days (C) and 15 days post-eclosion (D) using >15 samples/genotype from 3 independent experiments. Statistical analysis was performed with Kruskal Wallis with Dunn’s post-test. For all panels: ns, non significant, ***p<0.001 (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007386#pgen.1007386.s009" target="_blank">S1 Table</a> for details on transgenic flies).</p