AMD-Associated Genes Encoding Stress-Activated MAPK Pathway Constituents Are Identified by Interval-Based Enrichment Analysis

Purpose To determine whether common DNA sequence variants within groups of genes encoding elements of stress-activated mitogen-activated protein kinase (MAPK) signaling pathways are, in aggregate, associated with advanced AMD (AAMD). Methods: We used meta-regression and exact testing methods to identify AAMD-associated SNPs in 1177 people with AAMD and 1024 AMD-free elderly peers from 3 large-scale genotyping projects on the molecular genetics of AMD. SNPs spanning independent AAMD-associated genomic intervals were examined with a multi-locus-testing method (INRICH) for enrichment within five sets of genes encoding constituents of stress-activated MAPK signaling cascades. Results: Four-of-five pathway gene sets showed enrichment with AAMD-associated SNPs; findings persisted after adjustment for multiple testing in two. Strongest enrichment signals (P = 0.006) existed in a c-Jun N-terminal kinase (JNK)/MAPK cascade (Science Signaling, STKE CMP_10827). In this pathway, seven independent AAMD-associated regions were resident in 6 of 25 genes examined. These included sequence variants in: 1) three MAP kinase kinase kinases (MAP3K4, MAP3K5, MAP3K9) that phosphorylate and activate the MAP kinase kinases MAP2K4 and MAP2K7 (molecules that phosphorylate threonine and tyrosine residues within the activation loop of JNK); 2) a target of MAP2K7 (JNK3A1) that activates complexes involved in transcriptional regulation of stress related genes influencing cell proliferation, apoptosis, motility, metabolism and DNA repair; and 3) NR2C2, a transcription factor activated by JNK1A1 (a drugable molecule influencing retinal cell viability in model systems). We also observed AAMD-related sequence variants resident in genes encoding PPP3CA (a drugable molecule that inactivates MAP3K5), and two genes (TGFB2, TGFBR2) encoding factors involved in MAPK sensing of growth factors/cytokines. Conclusions: Linkage disequilibrium (LD)-independent genomic enrichment analysis yielded associations of AAMD with aggregates of functionally related genes encoding constituents of the JNK MAPK signaling pathway. FDA-approved drugs now exist to target constituents of stress-activated MAPK pathways and may offer reasonable approaches to preventing or treating AAMD

Maternal fish consumption during pregnancy and smoking behavioural patterns

Abstractn-3 Highly unsaturated fatty acids (HUFA), are essential components of neuronal membranes and mediate a range of complex bioactive properties including gene expression, myelination, cell-signalling and dopaminergic function. Deficits inn-3 HUFA have been linked to increased risks for addictive disorders, thus we posited that lower fish consumption would be associated with greater risks for perinatal smoking among 9640 mothers enroled in the Avon Longitudinal Study of Parents and Children. We used univariable and multivariable regression models to examine relationships between self-reported prenatal dietary intakes ofn-3 HUFA-rich foods (fish and shellfish) and maternal smoking; outcomes included cessation and the number of cigarettes smoked per d. Both before and during pregnancy, there was consistent evidence (P&lt;0·001) of protective fish intake–smoking associations; relative to mothers reporting no fish consumption, those who reported some fish consumption (&lt;340 g/week) and high fish consumption (340 g+/week) at 32 weeks of gestation showed lower likelihoods of smoking (adjustedPvalues &lt;0·001). Respective OR for these relationships were 0·87 (95% CI 0·77, 0·97) and 0·73 (95% CI 0·61, 0·86). Although the prevalence of smoking diminished, from a high of 31·6% (pre-pregnancy) to a low of 18·7% (second trimester), the magnitude of fish intake–smoking associations remained stable following adjustment for confounders. These observations suggest that greater fish orn-3 HUFA consumption should be evaluated as an intervention to reduce or prevent smoking in randomised clinical trials.</jats:p

MicroRNA signatures in vitreous humour and plasma of patients with exudative AMD

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and miR-152 in the vitreous humour which was reproducible in plasma. Moreover, miR-146a/miR-106b ratios discriminated patients with NV AMD with an area under the Receiver Operating Characteristic curve (ROC AUC) of 0,977 in vitreous humour and 0,915 in plasma suggesting potential for a blood-based diagnostic. Furthermore, using the AMD Gene Consortium (AGC) we mapped a NV AMD-associated SNP (rs1063320) in a binding site for miR-152-3p in the HLA-G gene. The relationship between our detected miRNAs and NV AMD related genes was also investigated using gene sets derived from the Ingenuity Pathway Analysis (IPA). To our knowledge, our study is the first to correlate vitreal and plasma miRNA signatures with NV AMD, highlighting potential future worth as biomarkers and providing insight on NV AMD pathogenesis

Fenofibrate Inhibits Cytochrome P450 Epoxygenase 2C Activity to Suppress Pathological Ocular Angiogenesis

Neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related-macular-degeneration are major causes of blindness. Fenofibrate treatment in type 2 diabetes patients reduces progression of diabetic retinopathy independent of its peroxisome proliferator-activated receptor (PPAR)α agonist lipid lowering effect. The mechanism is unknown. Fenofibrate binds to and inhibits cytochrome P450 epoxygenase (CYP)2C with higher affinity than to PPARα. CYP2C metabolizes ω-3 long-chain polyunsaturated fatty acids (LCPUFAs). While ω-3 LCPUFA products from other metabolizing pathways decrease retinal and choroidal neovascularization, CYP2C products of both ω-3 and ω-6 LCPUFAs promote angiogenesis. We hypothesized that fenofibrate inhibits retinopathy by reducing CYP2C ω-3 LCPUFA (and ω-6 LCPUFA) pro-angiogenic metabolites. Fenofibrate reduced retinal and choroidal neovascularization in PPARα-/-mice and augmented ω-3 LCPUFA protection via CYP2C inhibition. Fenofibrate suppressed retinal and choroidal neovascularization in mice overexpressing human CYP2C8 in endothelial cells and reduced plasma levels of the pro-angiogenic ω-3 LCPUFA CYP2C8 product, 19,20-epoxydocosapentaenoic acid. 19,20-epoxydocosapentaenoic acid reversed fenofibrate-induced suppression of angiogenesis ex vivo and suppression of endothelial cell functions in vitro. In summary fenofibrate suppressed retinal and choroidal neovascularization via CYP2C inhibition as well as by acting as an agonist of PPARα. Fenofibrate augmented the overall protective effects of ω-3 LCPUFAs on neovascular eye diseases

AAMD-associated sequence variants (<i>P</i>≤0.005)^* resident in genes of the STKE JNK MAPK pathway.

<p>Note: Odds ratios (ORs) and 95% confidence intervals (95% CIs) were computed, respectively, in Cohort 1/2/3 with age-, sex-, and smoking-adjusted logistic regression models comparing 675/227/275 people with advanced AMD (AAMD) to 512/198/314 of their AMD-free peers, aged >65 years at the time of genotyping. Participants from Cohort 1 were examined at the University of Michigan Ann Arbor. Those in Cohort 2 were examined at the University of Pennsylvania in Philadelphia. Those in Cohort 3 were examined at the Mayo Clinic in Rochester, Minnesota. Alleles are listed as minor|major.</p>*<p>Only SNPs significant at <i>P</i>≤0.002 were considered as AAMD-related in the pathway analysis. In most cases dominant models of inheritance (grouping minor allele homozygotes with heterozygotes) yielded strongest relationships – ORs reported here are from analyses using this model, with the exception of that for <i>NR2C2</i> (here the additive model was used). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071239#pone.0071239.s004" target="_blank">Table S3</a> contains allelic frequencies of variants in this table.</p

Annotations of AAMD-associated sequence variants resident in MAPK pathways.

<p>Note: DNase, DNase hypersensitivity cluster; ENH, histone enhancer mark; Int., variant resident in intron; JNK, genes present in STKE JNK MAPK Pathway (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071239#pone-0071239-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071239#pone.0071239.s005" target="_blank">S4</a>); PRO, histone promoter mark; RPE, retinal pigment epithelial cell; SNP, single nucleotide polymorphism; SYN, synonymous coding variant; UTR, untranslated region.</p>*<p>Annotations marked with an asterisk are for qualities of sequence variants in nearly complete linkage disequilibrium (r²≥0.80) with the AAMD-associated variant in the SNP column. Full names of genes represented by gene symbols exist at <a href="http://www.ncbi.nlm.nih.gov/gene" target="_blank">http://www.ncbi.nlm.nih.gov/gene</a>. Information on regulatory features was obtained from HaploReg (<a href="http://www.broadinstitute.org/mammals/haploreg/" target="_blank">www.broadinstitute.org/mammals/haploreg/</a>). Pathway:</p>a<p>KEGG MAPK Signaling Pathway;</p>b<p>BioCarta MAPK Signaling Pathway;</p>c<p>STKE JNK MAPK Pathway;</p>d<p>STKE p38 MAPK;</p>e<p>BioCarta p38 MAPK Pathway.</p

AAMD-associated genes enriching STKE JNK MAPK pathway.

<p>Diagram was generated with Ingenuity Pathway Analysis® software and is based on Johnson GL, Lapadat R, JNK Pathway, <i>Science's STKE</i>, CMP_10827. Unshaded (white) symbols represent genes that were not tested. Symbols shaded in gray represent relationships with <i>P</i>-values >0.002. Symbols shaded in red represent relationships significant at <i>P</i>-values ≤0.002. Full names for symbols representing genes exist at <a href="http://www.ncbi.nlm.nih.gov/gene/" target="_blank">www.ncbi.nlm.nih.gov/gene/</a>. Values beneath symbols are <i>P</i>-values for association computed with meta-analysis on of age-, sex, and smoking-adjusted odds ratios from 3 independent cohorts participating in large-scale genotyping projects on the molecular genetics of AMD (1177 people with AAMD and 1024 of their AMD-free peers).</p