Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals

Copy number variants (CNV) are established risk factors for neurodevelopmental disorders with seizures or epilepsy. With the hypothesis that seizure disorders share genetic risk factors, we pooled CNV data from 10,590 individuals with seizure disorders, 16,109 individuals with clinically validated epilepsy, and 492,324 population controls and identified 25 genome-wide significant loci, 22 of which are novel for seizure disorders, such as deletions at 1p36.33, 1q44, 2p21-p16.3, 3q29, 8p23.3-p23.2, 9p24.3, 10q26.3, 15q11.2, 15q12-q13.1, 16p12.2, 17q21.31, duplications at 2q13, 9q34.3, 16p13.3, 17q12, 19p13.3, 20q13.33, and reciprocal CNVs at 16p11.2, and 22q11.21. Using genetic data from additional 248,751 individuals with 23 neuropsychiatric phenotypes, we explored the pleiotropy of these 25 loci. Finally, in a subset of individuals with epilepsy and detailed clinical data available, we performed phenome-wide association analyses between individual CNVs and clinical annotations categorized through the Human Phenotype Ontology (HPO). For six CNVs, we identified 19 significant associations with specific HPO terms and generated, for all CNVs, phenotype signatures across 17 clinical categories relevant for epileptologists. This is the most comprehensive investigation of CNVs in epilepsy and related seizure disorders, with potential implications for clinical practice

The acyloxy nitroso compounds, NCA and NCP, and the prototypical HNO donor, Angel’s salt, target STAT3.

<p>Chemical structures of (A) 1-Nitrosocyclohexyl acetate, NCA, (B) 1-nitrosocyclohexyl pivalate, NCP, and (C) Angeli’s salt (AS).</p

STAT3 possesses redox-sensitive cysteines.

<p>(A) NCA and NCP block thiolate labeling. Recombinant human STAT3 was treated with vehicle (DMSO), NCA (100 µM) or NCP (100 µM) for 1 h at room temperature and then labeled for 2 h with fluorescein-5-maleimide. Equal amounts of protein were separated by SDS-PAGE and fluorescence in the gel detected (upper panel). To ensure equal loading, Western analysis was done on each fluorescein-labeled sample. Separated proteins on nitrocellulose membranes were probed with a STAT3 antibody and imunoreactive bands quantified using the Li-COR Odyssey infrared imaging system (lower panel). Results shown are representative of 3 independent experiments. (B & C) Oxidation of STAT3 is associated with sulfenic acid formation. Purified recombinant STAT3 was immunoprecipitated and pretreated with 10 mM DTT and then treated with nothing or the oxidant <i>o</i>-IBZ (2.5 mM) for 1 hr at 4°C. Immunoprecipitates were processed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043313#s2" target="_blank">Materials and Methods</a>" to determine sulfenic acid formation (STAT3-SOH). (B) Representative blot. (C) Levels of cysteine-sulfenic acid and STAT3 were quantified by the Li-COR Odyssey Detection System. Treatment with <i>o</i>-IBZ resulted in a significant increase in relative sulfenic acid content. **P<0.01 vs. control, n = 3; paired Student’s t-test.</p

NCP enhances STAT3 glutathionylation and dimerization.

<p>HL-1 cells were treated for 30 min with vehicle (control), 500 µM NCP, 1 mM diamide, or 500 µM NCP and 1 mM diamide together. Cell extracts were prepared. (A) Equal protein amounts of cleared extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3 and glutathionylated protein using a rabbit and mouse antibody, respectively. Immunoreactive bands were detected using Li-COR Odyssey system and secondary antibodies that produced a red (anti-rabbit) or green (anti-mouse) signal. The overlay of the red and green signals produced an orange color. Relative levels of glutathionylated STAT3 were quantified. **P<0.01, 1-way ANOVA and Dunnett’s multiple comparison test (n = 3). (B) Cells were treated as in panel A. Cell extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3, which showed two bands consistent with STAT3 monomers and dimers. The intensity of the higher (dimer) band relative to the lower (monomer) band for each lane was quantified. *P<0.05 and **P<0.01, 1-way ANOVA and Newman–Keuls post-test (n = 3).</p

NCA attenuates LIF-induced gene expression.

<p>HMEC-1 were pretreated with vehicle (0.04% v/v DMSO) or 100 µM NCA for 1 h and then treated 1 h with 2 ng/mL LIF or vehicle. RNA was extracted, reverse transcribed, and analyzed by real time PCR for (A) ICAM-1 and (B) CEBPD expression. Results were normalized to GAPDH and expressed as the fold-increase over control levels. Values are mean ± SEM for (A) 7 or (B) 9 independent experiments. **P<0.01 and ***P<0.001, 1-way ANOVA and Newman–Keuls post-test.</p

NCA and NCP inhibit LIF-induced STAT3 activation in human microvascular endothelial cells.

<p>HMEC-1 were pretreated for 1 h with vehicle (0.04% v/v DMSO), (A) 100 µM NCP, or (C) 100 µM NCP. Afterwards, cells were dosed for various times with 2 ng/mL LIF. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3 as a loading control. (B and D) Results were quantified and expressed as the ratio of phosphorylated STAT3 to total STAT3. **P<0.01 and ***P<0.001 vs. same time point control (n = 4); 2-way ANOVA and Bonferroni post-test.</p

Effect of NCA on JAK1 activity.

<p>(A) LIFR phosphorylation was modestly decreased by NCA. HMEC-1 cells were pretreated with 100 µM NCA for 1 h followed by treatment with 2 ng/mL LIF for 10 min. Proteins were extracted and LIF receptor immnoprecipitated. Levels of phosphorylated tyrosine (pY) and LIFR were evaluated by Western blotting. (B) Quantification of the immunoblots. Values are mean ± SEM for 3 independent experiments. Columns with the same letter are significantly different from each other. ^b,d,eP<0.05 and ^a,cP<0.001; 1-way ANOVA and Newman–Keuls post-test. (C) NCA did not affect JAK1 catalytic activity. A FRET-based Z′-LYTE Assay (Invitrogen SelectScreen Profiling Service) was performed to assess the effect of various concentrations of NAC (0.51 nM –10 µM) on the catalytic activity of JAK1.</p

Oxidative stress, NCP and diamide alter the Western blot profile of STAT3 under nonreducing conditions.

<p>(A & B) Aliquots of a cleared mouse heart homogenate were incubated for 30 min with vehicle, 500 µM NCP, 1 mM diamide, or 500 µM NCP+1 mM diamide. Samples were processed for SDS-PAGE and Western blot analysis in nonreducing or reducing sample buffer. (A) Membranes were probed for STAT3 using the Li-COR Odyssey detection system. (B) Intensity of the STAT3 band in the nonreduced sample was normalized to the intensity of the band after reduction. ***P<0.001 vs. Control, 1-way ANOVA and Newman–Keuls post-test (n = 3 mouse hearts). (C) Ratio of nonreduced to reduced STAT3 in wild type (WT) and failing (Gaq) mouse hearts. STAT3 levels in mouse myocardial tissue from WT (FVB/N) and heart failure mice (Gaq overexpressing) (n = 3) were determined via immunoblot analysis under nonreducing or reducing (3.75% β-mercaptoethanol (β-ME)) conditions. Protein loads were normalized using the direct blue 71 stained membranes (DB71). *P<0.05 (Student t-test).</p

Effect on NCA on cell proliferation and viability.

<p>HMEC-1 cells were treated for 24 h with nothing (Control), vehicle (0.04% v/v DMSO), or 100 µM NCA. (A) Cell proliferation was assessed as the increase in live cells. (B) Cell viability was assessed by alarmarBlue assay. Results were normalized to control values for alamarBlue reduction. Values are mean ± SEM for (A) 4 and (B) 5 independent experiments, each performed using triplicate dishes of cells per condition. *P<0.05 and **P<0.01 vs. DMSO and Control, respectively; 1-way ANOVA and Newman–Keuls post-test. (C) NCA increased ERK1/2 phosphorylation as assessed by Western analysis. HMEC-1 were not treated (Control) or treated for 30 or 60 min with vehicle (0.04% v/v DMSO) or 100 µM NCA. Values are mean ± SEM for 4 independent experiments. *P<0.01 vs. Control and ^ΦP<0.01 vs. timed vehicle (DMSO); 1-way ANOVA and Newman–Keuls post-test.</p