Massive parallel sequencing in steroid-resistant nephrotic syndrome (SRNS)

<p><strong>ABSTRACT</strong></p> <p><strong>Introduction</strong>. To date abnormalities in more than 20 genes have been associated with SRNS. Sequencing of all SRNS genes requires ~ 600 PCR amplicons, rendering conventional mutation testing unfeasible due to financial and time constraints. Hence, current screening algorithms usually include only the most common disease genes and/or use preselection according to additional phenotypic criteria. This practice typically allows for mutation detection in ~15% of patients. We have evaluated targeted NGS screening of SRNS patients enrolled in the PodoNet registry who were found negative for mutations in the first-line SRNS-associated genes.</p> <p><strong>Material and Methods</strong>. Molecular analysis of 31 known or plausible SDNS disease genes was performed by NGS using a custom-designed multiplex PCR kit (MASTR FSGS, Multiplicom). The pilot group consisted of 22 patients with 4.7 years median age at disease onset (range 0.5-20 years), positive family history in 68%, parental consanguinity in 18% and chronic renal insufficiency at last observation in 59%.</p> <p><strong>Results.</strong> Mean coverage was 1269x (median 1286, range 929-1826). 18/22 runs had at least 15x read depth covering 99% of the target sequences. One patient was diagnosed with hereditary SRNS due to a previousy described homozygous pathogenic mutation in SMARCAL1 gene. In addition, three novel sequence variants in the genes PLCE1 (homozygous), LAMB2 (homozygous) and WT1 (heterozygous) were detected. In silico studies support their classification as pathogenic, even though the patients do not present the characteristic clinical and/or histopathological features typically reported for patients with mutations in these genes.</p> <p><strong>Conclusions</strong>. Our detection of pathogenic mutations in 4 out of 22 SRNS patients screened negative by conventional selective screening approaches support targeted NGS testing in all SRNS patients, regardless of age at diagnosis, absence of extrarenal manifestations or histological subtype. We anticipate that systematic NGS screening of the SRNS cohorts collected in EURenOmics will allow re-evaluation of mutation incidence rates in SRNS and become the new standard of genetic diagnostics in this condition.</p

Additional file 1: of Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders

Table S1. Detailed information of all patients and controls whose brain samples were used in this study. Table S2. Possibly deleterious variants in known ASD and ID genes. Table S3. DEGs identified in undifferentiated cells. Table S4. DEGs identified in differentiated cells. Table S5. Validation of RNA-Seq using RT-qPCR on Fluidigm array. Table S6. Top 20 cannonical pathways deregulated in undifferentiated cells. Table S7. Top 4 nodes enriched for protein-protein interaction as calculated by ClusterOne Plugin. Table S8. Top 20 cannonical pathways deregulated in the cell cycle modules of differentiated cells. Table S9. Top 20 cannonical pathways deregulated in the cell neuronal modules of differentiated cells. Table S10. Cell type enrichment analysis of DEGs from the Cell Cycle and Neuronal Modules. (XLSX 888 kb

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

Pathogenic genetic variants identified in affected individuals with overlapping syndromes.

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

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

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