A homozygous KAT2B variant modulates the clinical phenotype of ADD3 deficiency in humans and flies.

Recent evidence suggests that the presence of more than one pathogenic mutation in a single patient is more common than previously anticipated. One of the challenges hereby is to dissect the contribution of each gene mutation, for which animal models such as Drosophila can provide a valuable aid. Here, we identified three families with mutations in ADD3, encoding for adducin-γ, with intellectual disability, microcephaly, cataracts and skeletal defects. In one of the families with additional cardiomyopathy and steroid-resistant nephrotic syndrome (SRNS), we found a homozygous variant in KAT2B, encoding the lysine acetyltransferase 2B, with impact on KAT2B protein levels in patient fibroblasts, suggesting that this second mutation might contribute to the increased disease spectrum. In order to define the contribution of ADD3 and KAT2B mutations for the patient phenotype, we performed functional experiments in the Drosophila model. We found that both mutations were unable to fully rescue the viability of the respective null mutants of the Drosophila homologs, hts and Gcn5, suggesting that they are indeed pathogenic in flies. While the KAT2B/Gcn5 mutation additionally showed a significantly reduced ability to rescue morphological and functional defects of cardiomyocytes and nephrocytes (podocyte-like cells), this was not the case for the ADD3 mutant rescue. Yet, the simultaneous knockdown of KAT2B and ADD3 synergistically impaired kidney and heart function in flies as well as the adhesion and migration capacity of cultured human podocytes, indicating that mutations in both genes may be required for the full clinical manifestation. Altogether, our studies describe the expansion of the phenotypic spectrum in ADD3 deficiency associated with a homozygous likely pathogenic KAT2B variant and thereby identify KAT2B as a susceptibility gene for kidney and heart disease in ADD3-associated disorders

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

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

Effect of <i>ADD3</i> and <i>KAT2B</i> mutations on viability and morphology in <i>Drosophila</i>.

<p>(A) Viability for <i>hts</i>^<i>null</i> hemizygous flies and respective rescues with adducin (Add) construct(s) using <i>tubulin-</i>GAL4 (<i>tub></i>). After 48h of egg laying on standard cornmeal/yeast food, viability was calculated as the percentage of hatching adults of the indicated genotype and normalized to the control. The control corresponds to the viable F1 trans-heterozygous flies obtained from the cross between <i>Df(2R)BSC26</i> (harbouring the <i>hts</i> gene) and a non-overlapping deficiency on the same chromosome (<i>Df(2R)247</i>). Quantification is for >100 F1 eclosing flies/genotype/experiment in 5 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post-test. (B) Negative geotaxis assay for one-day-old adult flies. Flies were transferred to a graduated tube, and after tapping, the length climbed in 8 sec was recorded [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007386#pgen.1007386.ref045" target="_blank">45</a>]. Quantification was performed on 6 independent experiments with >38 flies/genotype using one-way ANOVA with Kruskal-Wallis 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). For all panels: ns, non significant, *p<0.05 **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). (C) Viability for <i>Gcn5</i>^<i>null</i> hemizygous flies and respective rescues with Gcn5 and KAT2A/B construct(s) using <i>daughterless-</i>GAL4 (<i>da></i>). Viability was assessed as described in (A). Human <i>KAT2B</i> F307S and S502F mutations correspond to <i>Gcn5</i> F304S and S478F mutations, respectively. Gcn5 S502F variant predicted to be deleterious (PolyPhen-2 score of 0.98) was found on a healthy individual at the homozygous state in our in-house exome database. The control corresponds to the viable F1 trans-heterozygous flies obtained from the cross between <i>Df(3L)sex204</i> (harbouring the <i>Gcn5</i> gene) and a non-overlapping lethal mutant on the same chromosome (<i>CG3103</i>^{<i>0MI0010</i>}). Quantification is for >100 F1 eclosing flies/genotype/experiment in 5 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post-test. (D) Phenotype of Gcn5 WT, Gcn5 F304S and Gcn5 S478F rescue animals. Pictures correspond to adult flies one day post-eclosion and are representative of the defects found in wings (separated wing blades), legs (femur kinking, arrow) and eye (small and mild rough eye). Scale bars: wings: 500μm, legs: 500 μm, eye: 200μm. (E) Quantification of the defects in wings, legs and eyes found in Gcn5 F304S rescue flies is for >100 F1 eclosing flies/genotype. (F) H3K9 acetylation levels of <i>Gcn5</i>^<i>null</i> and Gcn5 WT and F304S rescue animals. Extracted nuclear proteins from 3^rd instar (= late) larvae were analysed by western blotting normalized to non-acetylated Histone 3 (H3). Quantification is shown in the lower panel (n = 3 independent experiments; one-way ANOVA with Bonferroni post-test). For all panels: ns, non significant, *p<0.05 **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

Synergism between ADD3 E659Q and Gcn5 knockdown in nephrocytes and effect of the double knockdown of ADD3 and KAT2B in human cultured podocytes.

<p>(A, B) WT or mutated adducin rescue constructs were expressed in <i>hts</i> and <i>Gcn5 double knockdown</i> pericardial nephrocytes to study the interaction between E659Q mutation and gcn5 loss of function. (A) Immunostaining was performed for Kirre (red) and Pyd (blue). Images are representative of pericardial nephrocytes dissected from adult flies at 3 days post-eclosion. Scale bar: 30μm. (B) Graphs represent quantification of the number of pericardial nephrocytes at the same time point using >20 samples/genotype from 3 independent experiments. Statistical analysis was performed with One-way ANOVA with Dunnett’s post-test. For all panels: **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). (C) Adhesion was assessed using the xCELLigence system (ACEA Biosciences). Cells were plated with complete medium in the E-plate 96. Data obtained were analyzed with the RTCA software. Results are presented as time <i>vs</i>. cell index curve (n = 3 independent experiments; linear regression analysis). (D) Migration was assessed using the Incucyte Scratch wound cell migration assay (Essen Bioscience). Cells were plated with complete medium 48 hours before scratch in ImageLock Plates-96 wells (Essen Bioscience) and images were recorded every 45 minutes after scratch until complete wound closure. Images were analyzed using the Incucyte Zoom software. Results are presented as percentage of wound cell density over time (n = 3 independent experiments; linear regression analysis). For all panels: **p<0.01, ***p<0.001, ****p<0.0001.</p

Clinical phenotype of affected individuals.

<p>Clinical phenotype of affected individuals.</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

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