Relationship between MN frequency and age for CSA exposed and nonexposed twins.

<p>A significant interaction effect was observed (coefficient [SE] = 0.030 [0.009], <i>P</i> = 0.0006) with the MN level in CSA exposed twins increasing with age while the MN level remained constant across the limited age range evaluated in CSA nonexposed twins.</p

MN frequencies for discordant MZ twin pairs and age-matched controls.

<p>The mean level is indicated by a horizontal bar for controls (mean [SD] 14.2 [9.4]), CSA nonexposed (14.9 [5.6]) and CSA exposed (22.0 [11.3]) twins. CSA exposure status was significant in this combined sample (<i>P</i><0.001), while there was no indication of an additional effect attributable to the familial environment (<i>P = </i>0.406) based on results from generalized mixed-effect models.</p

Lifestyle characteristics and adult psychiatric and substance use disorders in CSA discordant MZ twin pairs.

1<p>indicates pairs discordant for CSA and item where the exposed twin was positive for the item (n₂₁).</p>2<p>indicates pairs discordant for CSA and item where the nonexposed twin was positive for the item (n₁₂).</p>3<p>odds ratio for twin pairs doubly discordant for CSA and item (n₂₁/n₁₂).</p>4<p>prescription and non-prescription use for more than 1 year excluding birth control.</p><p>+/− ∞, value is positive/negative and infinite due to a null value in at least one category.</p>*<p>two-sided P value from exact binomial test.</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

Effect of adducin-αγ E559Q on <i>Drosophila</i> heart function.

<p>(A-C) M-mode of beating 2-week-old control (<i>yw/Df(2R);</i> A), adducin-αγ WT (B) and adducin-αγ E559Q (C) rescue hearts. Scale bar: 1 second. (D-H) High-speed movies of beating adducin-αγ WT, adducin-αγ E559Q rescue and control 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, except for Arrhythmia index (H; n = 8–19, Mann-Whitney-Wilcoxon). 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

Pathogenic genetic variants identified in affected individuals with overlapping syndromes.

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

Identification of homozygous missense mutations in <i>ADD3</i> and <i>KAT2B</i> and effect of <i>ADD3</i> and <i>KAT2B</i> mutations on protein levels in fibroblasts.

<p>(A) Pedigree and segregation status of mutations found in <i>ADD3</i> and <i>KAT2B</i>. Discovery of <i>ADD3</i> mutations in family B and C was facilitated by GeneMatcher [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007386#pgen.1007386.ref044" target="_blank">44</a>]. Half red coloured circles or squares denote patients with neurological defects and half blue coloured symbols denote patients with SRNS and cardiomyopathy. + symbols indicate non-mutated alleles. Mutations and segregation were confirmed by Sanger sequencing. (B) Exon structure of human <i>ADD3</i> cDNA (long isoform NP_058432) and domains of adducin-γ protein. The relative position of <i>ADD3</i> mutations to protein domains and exons are indicated (arrows). All mutations also affect the short isoform of <i>ADD3</i> (NP_001112). Below each mutation, the phylogenetic conservation of the altered amino acid residues is shown. (C) Exon structure of human <i>KAT2B</i> cDNA and domains of KAT2B protein. PCAF-HD, p300/CBP-associated factor homology domain; AT, acetyl transferase domain; B, Bromo domain. The relative position of <i>KAT2B</i> variation to protein domains and exons is indicated (arrow). The phylogenetic conservation of the altered amino acid residue is shown. (D, E) Adducin-γ (D) and KAT2B (E) protein levels in control and patient fibroblasts. Lysates of patient II-3 and II-6 (family A) fibroblasts and age-matched control fibroblasts (Ctrl 1 and 2) were analyzed by western blotting. Results were normalized to the loading control α-tubulin. Each quantification is shown in the lower panel (n = 3 independent experiments, student’s t-test).</p

Expression and localization of Hts and Gcn5 in garland nephrocytes.

<p>(A) Schematic drawing of the localization of garland nephrocytes (GNs) and pericardial nephrocytes (PNs). The garland cells are attached to the proventriculus (PV) whereas the pericardial nephrocytes are lining the heart tube (HT). (B, C) Dissected wild-type (WT) garland nephrocytes were stained for Hts (B; green) and Gcn5 (C; green). At the time of dissection, larvae were in the third instar stage (the same for all other garland nephrocyte stainings). Kirre is in red (B). Nuclei were stained with Hoechst (B,C; blue). Scale bars: 10 μm.</p

Effect of <i>Gcn5</i>/<i>KAT2B</i> variant on histone acetylation and survival of <i>Drosophila</i> nephrocytes.

<p>(A) Acetylated H3K9 in larval garland nephrocytes of <i>Gcn5</i>^<i>null</i> and Gcn5 WT and mutant rescue animals. <i>Dorothy (Dot)-</i>GAL4 (a nephrocyte specific driver) is used in combination with <i>da-</i>GAL4 as the latter shows only minor expression in nephrocytes. Garland nephrocytes of the indicated genotypes were stained for acetylated H3K9 (red) and Hoechst (blue). Scale bar: 5 μm. (B) Pericardial nephrocytes in adult <i>Gcn5</i> rescue mutant flies (7–15 days after eclosion) that express transgenic GFP (green) driven under the <i>Hand</i> promoter (<i>Hand</i>-GFP), specific for nephrocytes and cardiomyoblasts. Dissected pericardial nephrocytes were fixed with PFA and observed directly for GFP signal. Scale bar: 30 μm. (C) Pericardial nephrocytes in adult <i>Gcn5</i> rescue mutants (7–15 days after eclosion). Dissected pericardial nephrocytes of the indicated genotypes were stained for the differentiation markers Kirre (red) and Pyd (blue). Scale bar: 30 μm. (D) Quantification of the pericardial nephrocyte defects found in <i>Gcn5</i>^<i>null</i> rescue mutants (n>13/genotype; 3 independent experiments; Chi-square test). Nephrocytes with abnormal phenotypes included nephrocytes with abnormal distribution, abnormal shape, multinucleated or fragmented nuclei and reduced number of nephrocytes (<20). Phenotype severity was scored as normal (0), medium (1), intermediate (2) and severe (>2). For all panels: ns, non significant, **p<0.01, ***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

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