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Additional file 9: Network S2. CellDesigner diagram as pdf.

Additional file 2: Figure S1. Coverage of the Ras network with directed vs undirected interactions. Figure S2.. Network centrality analysis for hub and non-hub proteins. Figure S3. Crosstalk of the Ras-effector downstream proteins across the 12 effector classes. Figure S4. Shared proteins among the 12 effector classes. Figure S5. Subcellular localization and interaction compatibility. Figure S6. PANTHER GO pathways analysis of the 12 effector classes. Figure S7. Differential enrichment of the SysGO Processes (1) by effector class. Figure S8. Comparison of the enriched class-specific processes with literature reports.

Age-related macular degeneration (AMD) is a complex disease which is characterized by the contribution of a variety of environmental and genetic factors to disease development and progression. In order to elucidate the genetic background of AMD, a broad range of association studies have been conducted, targeting different aspects of disease pathobiology. Those studies revealed associations of genetic variants, epigenetic regulators like microRNAs (miRNA) and also of specific genes with AMD. However, for most of these findings the molecular mechanism of action underlying the association still remains unsolved. Therefore, the present work aimed to investigate how the findings of genetic association studies can lead to the deciphering of molecular mechanisms in order to understand the pathology of AMD and possibly reveal target molecules with regard to precision medicine. In the first subproject, associations of epigenetic regulators, specifically miRNAs, with AMD were investigated. In a mouse model of laser-induced choroidal neovascularization (NV), one of the subtypes of AMD with severe manifestations, three circulating miRNAs with an altered expression in blood were identified after NV induction, namely mmu-miR-486a-5p, mmu-miR-92a-3p and mmu-miR-155-5p. In addition, expression alterations of two of those miRNAs, mmu-miR-486a-5p and mmu-miR-92a-3p, were investigated in ocular tissue of laser-treated mice. Both miRNAs revealed an overexpression in retinal pigment epithelium (RPE)/choroid complex, while none of them was differentially expressed in the neural retina after NV induction. In the second project of this work, genes associated with AMD were investigated regarding potential pleiotropy. In total, 106 genes found by a recent transcriptome-wide association study (TWAS) were analyzed. The analysis revealed that 50 of the investigated genes are located in genomic regions that have already been associated with different human phenotypes by genome-wide association studies (GWAS). Overall, AMD-associated genes were found to be enriched in GWAS loci of neurological diseases, metabolic phenotypes, autoimmune diseases, and phenotypes describing organ functions, in addition to AMD disease itself. Moreover, 23 genes overlapped with GWAS loci of different phenotype groups, thus most likely representing pleiotropic genes. Interestingly, four of those 23 genes did not overlap with a known AMD-associated locus. A locus harboring one of those genes, namely ULK3 at 15q24.1, was further characterized in detail throughout this work. The association of 15q24.1 with AMD was examined using the representative polymorphism rs2168518. Interestingly, the association was mainly observed for the neovascular subtype of AMD and further showed a gender-specific effect. A pleiotropy analysis using UK Biobank data highlighted associations with 15 phenotypes, most of them related to blood pressure. The fact that blood pressure associations of the 15q24.1 locus correspond to the same genetic signal as the AMD association further highlights the importance of the 15q24.1 locus for the neovascular subtype of AMD. Additionally, alterations in gender-specific transcription factor binding sites might contribute to the male specific association with AMD. Noteworthy, the representative variant rs2168518 of the 15q24.1 locus is located in the seed region of a miRNA, hsa-miR-4513, and might therefore contribute to post-transcriptional expression regulation mediated through this miRNA. The impact of hsa-miR-4513 and its seed variant rs2168518 on gene expression regulation was investigated in another project of this work. Overall, 23 target genes of this miRNA were detected and independently validated to be specifically regulated by an allele of the rs2168518 polymorphism in hsa-miR-4513. Moreover, six target genes were exemplary followed up in detail by Western Blot analysis and luciferase reporter assay, whereby three of those genes, namely CD2BP2, CDKN2A and KLF6, consistently revealed allele-specific effects. Finally, publicly available databases were incorporated to investigate medical relevance of hsa-miR-4513 target genes. This in silico analysis highlighted CDKN2A as a promising candidate potentially contributing to AMD pathology. In summary, the results of this work showed that examining genetic associations from different perspectives focusing on different molecular mechanisms, such as pleiotropy or gene expression, can reveal different important aspects of disease pathobiology that may underlie a genetic association. In some circumstances, this can also lead to the identification of several different disease-relevant genes, which, however, can only be identified by a specific research question. Therefore, it is crucial to comprehensively investigate single genetic association signals with a combination of complementary in silico and in vitro analyses to link association data with functional mechanisms. Only by understanding the pathobiology underlying the genetic associations, the development of new therapeutic options for the pathology of AMD will be possible in the future., Die Krankheitsentstehung wie auch die Progression der altersabh��ngigen Makuladegeneration (AMD) wird durch eine Vielzahl an genetischen sowie umweltbedingten Faktoren beeinflusst, was die AMD als eine sogenannte komplexe Erkrankung klassifiziert. Um den Beitrag der Genetik zu untersuchen wurden mehrere genetische Assoziationsstudien durchgef��hrt, die verschiedene Aspekte der Pathobiologie untersucht haben. Unter anderem wurden Assoziationen von genetischen Varianten, epigenetischen Regulatoren wie micro-RNAs (miRNA) und spezifischen Genen mit der AMD beschrieben. Die molekularen Mechanismen, die diesen Assoziationen zu Grunde liegen, sind bisher allerdings kaum bekannt. Aus diesem Grund befasst sich die vorliegende Arbeit mit der Fragestellung wie die Erkenntnisse von genetischen Assoziationsstudien zur Entschl��sselung von molekularen Mechanismen f��hren k��nnen, um die Pathologie der AMD zu verstehen und eventuell Zielmolek��le im Sinne einer Pr��zisionsmedizin offen zu legen. Im ersten Teilprojekt wurden Assoziationen von epigenetischen Regulatoren mit der AMD untersucht, im Speziellen miRNAs. In einem Mausmodell mit Laser-induzierter choroidaler Neovaskularisation (NV), einem Subtyp der AMD mit schwerwiegenden Manifestationen, wurden drei zirkulierende miRNAs identifiziert (mmu-miR-486a-5p, mmu-miR-92a-3p und mmu-miR-155-5p), die nach der Induktion der NV eine ver��nderte Expression im Blut der behandelten Tiere aufwiesen. Zus��tzlich wurde die Expression von zwei dieser miRNAs, mmu-miR-486a-5p und mmu-miR-92a-3p, im Augengewebe von gelaserten M��usen untersucht. Beide miRNAs zeigten nach der Induktion der NV eine erh��hte Expression im retinalen Pigmentepithel (RPE)/Aderhaut Komplex, wohingegen keine der beiden miRNAs eine Expressionsver��nderung in der Netzhaut aufwies. Im zweiten Projekt dieser Arbeit wurden Gene, welche mit der AMD assoziiert sind, auf pleiotrope Effekte hin untersucht. Es wurden insgesamt 106 Gene analysiert, die durch eine aktuelle, das gesamte Transkriptom umfassende, Assoziationsstudie (TWAS) gefunden wurden. Bei der Analyse zeigte sich, dass 50 der untersuchten Gene in genomischen Bereichen liegen, die durch Genom-weite Assoziationsstudien (GWAS) bereits mit unterschiedlichen Ph��notypen des Menschen assoziiert wurden. Insgesamt zeigte sich neben der AMD-Erkrankung selbst eine Anreicherung von AMD-assoziierten Genen in GWAS Genorten von neurologischen Erkrankungen, metabolischen Ph��notypen, Autoimmunerkrankungen und Ph��notypen die Organfunktionen beschreiben. Dabei lagen 23 Gene in GWAS Genorten, die eine Assoziation mit mehreren Ph��notypen aufweisen, wodurch diese Gene als potenziell pleiotrop eingeordnet werden k��nnen. Interessanterweise lagen vier der 23 pleiotropen Gene in Genorten, die bisher nicht mit der AMD in Verbindung gebracht wurden. Einer dieser vier Genorte wurde im Verlauf dieser Arbeit ausf��hrlich beschrieben, und zwar 15q24.1, welcher das ULK3 Gen beinhaltet. Die Assoziation des 15q24.1 Genortes mit der AMD wurde anhand des repr��sentativen Polymorphismus rs2168518 untersucht. Interessanterweise war die Assoziation vor allem f��r den neovaskul��ren Subtyp der AMD zu beobachten und zeigte des Weiteren einen geschlechtsspezifischen Effekt. Eine Pleiotropie-Analyse anhand der Daten des UK Biobank Projektes zeigte eine Assoziation von 15q24.1 mit 15 Ph��notypen, wobei die meisten dieser Ph��notypen mit der Regulation des Blutdruckes in Verbindung stehen. Die Tatsache, dass Blutdruck-Assoziationen des 15q24.1 Genortes auf dasselbe genetische Signal wie die AMD-Assoziation zur��ckzuf��hren ist, verdeutlicht noch einmal die Bedeutung des 15q24.1 Genortes f��r den neovaskul��ren Subtyp der AMD. Die spezifische Assoziation von 15q24.1 mit AMD in M��nnern k��nnte wom��glich auf Ver��nderungen in Bindestellen f��r geschlechtsspezifische Transkriptionsfaktoren beruhen. Des Weiteren liegt der rs2168518 Polymorphismus in der Binderegion eines wichtigen post-transkriptionellen Regulators, der miRNA hsa-miR-4513, wodurch er m��glicherweise einen Einfluss auf die durch die miRNA ausgef��hrte Expressionsregulation aus��bt. Daher wurde in einem weiteren Projekt dieser Arbeit der Einfluss des rs2168518 Polymorphismus auf die regulatorischen Eigenschaften von hsa-miR-4513 untersucht. Insgesamt wurden 23 Gene identifiziert und unabh��ngig validiert, die spezifisch durch ein Allel des rs2168518 Polymorphismus in hsa-miR-4513 reguliert werden. Des Weiteren wurden sechs Gene exemplarisch im Detail mittels Western Blot Analyse und Luciferase Reporter Assays untersucht. Drei der untersuchten Gene, CD2BP2, CDKN2A und KLF6, zeigten dabei durchg��ngig Allel-spezifische Effekte. Abschlie��end wurden ��ffentlich zug��ngliche Datenbanken einbezogen, um eine medizinische Relevanz der von hsa-miR-4513 regulierten Gene zu untersuchen. Diese in silico Analyse zeigte, dass CDKN2A ein vielversprechender Kandidat ist, der m��glicherweise zur Pathologie der AMD beitr��gt. Zusammenfassend zeigten die Ergebnisse dieser Arbeit, dass eine Betrachtung von genetischen Assoziationen aus unterschiedlichen Blinkwinkeln mit Fokus auf verschiedene molekulare Mechanismen, wie zum Beispiel Pleiotropie oder Genexpression, unterschiedliche wichtige Aspekte der Krankheitspathobiologie, die einer genetischen Assoziation zu Grunde liegen k��nnen, aufdecken k��nnen. Dies kann unter Umst��nden auch zu der Identifikation von mehreren verschiedenen krankheitsrelevanten Genen f��hren, die aber nur durch eine spezifische Fragestellung identifiziert werden k��nnen. So wird au��erdem verdeutlicht, wie wichtig umfassende und sich erg��nzende in silico und in vitro Analysen sind, um funktionelle Mechanismen hinter den Assoziationssignalen aufzudecken. Nur wenn die Pathobiologie, die genetischen Assoziationen zugrunde liegt, verstanden wird, k��nnen zuk��nftig neue Therapieans��tze f��r die AMD entwickelt werden.

Additional file 3: Supplementary Figures.

Genome-wide association studies (GWAS) have identified an abundance of genetic loci associated with complex traits and diseases. In contrast, in-depth characterization of an individual genetic signal is rarely available. Here, we focus on the genetic variant rs2168518 in 15q24.1 previously associated with age-related macular degeneration (AMD), but only with suggestive evidence. In a two-step procedure, we initially conducted a series of association analyses to further delineate the association of rs2168518 with AMD but also with other complex phenotypes by using large independent datasets from the International AMD Genomics Consortium (IAMDGC) and the UK Biobank. We then performed a functional annotation with reference to gene expression regulation based on data from the Genotype-Tissue Expression (GTEx) project and RegulomeDB. Association analysis revealed a gender-specific association with male AMD patients and an association predominantly with choroidal neovascularization. Further, the AMD association colocalizes with an association signal of several blood pressure-related phenotypes and with the gene expression regulation of CYP1A1, a member of the cytochrome P450 superfamily of monooxygenases. Functional annotation revealed altered transcription factor (TF) binding sites for gender-specific TFs, including SOX9 and SRY. In conclusion, the pleiotropic 15q24.1 association signal suggests a shared mechanism between blood pressure regulation and choroidal neovascularization with a potential involvement of CYP1A1.

Additional file 2: Supplementary Note.

Significant association signals from genome-wide association studies (GWAS) point to genomic regions of interest. However, for most loci the causative genetic variant remains undefined. Determining expression quantitative trait loci (eQTL) in a disease relevant tissue is an excellent approach to zoom in on disease- or trait-associated association signals and hitherto on relevant disease mechanisms. To this end, we explored regulation of gene expression in healthy retina (n = 311) and generated the largest cis-eQTL data set available to date. Genotype- and RNA-Seq data underwent rigorous quality control protocols before FastQTL was applied to assess the influence of genetic markers on local (cis) gene expression. Our analysis identified 403,151 significant eQTL variants (eVariants) that regulate 3,007 genes (eGenes) (Q-Value < 0.05). A conditional analysis revealed 744 independent secondary eQTL signals for 598 of the 3,007 eGenes. Interestingly, 99,165 (24.71%) of all unique eVariants regulate the expression of more than one eGene. Filtering the dataset for eVariants regulating three or more eGenes revealed 96 potential regulatory clusters. Of these, 31 harbour 130 genes which are partially regulated by the same genetic signal. To correlate eQTL and association signals, GWAS data from twelve complex eye diseases or traits were included and resulted in identification of 80 eGenes with potential association. Remarkably, expression of 10 genes is regulated by eVariants associated with multiple eye diseases or traits. In conclusion, we generated a unique catalogue of gene expression regulation in healthy retinal tissue and applied this resource to identify potentially pleiotropic effects in highly prevalent human eye diseases. Our study provides an excellent basis to further explore mechanisms of various retinal disease etiologies., Author summary The retina is a multilayered and highly specified neural tissue crucial for high-resolution visual perception and spatial orientation. Environmental and genetic insults to the retina result in many blinding diseases, such as age-related macular degeneration or glaucoma. Commonly, many of these diseases are age-related suggesting that minor changes are accumulating over a life-time, with little or no contribution of strong individual effects. Specifically, this is true for genetic factors known to underlie the etiology of complex diseases including the prevalent eye diseases. In our study, we searched for effects on gene expression due to genetic variation using 311 healthy post-mortem retinal tissue samples. We show that 3,007 of the 16,766 genes investigated are regulated in the retina by genetic variations. Of these, 80 genes are potentially associated to one or more of twelve complex eye diseases or retinal traits tested. Interestingly, 10 genes appear to be involved in the development of several eye traits suggesting that cellular mechanisms may act at a common point in the disease process. Consequently, our study provides the basis to further explore retinal disease pathways and is likely to highlight target molecules for future therapeutic applications.

Figure S1. Analysis of the historical biogeography of E. verrucosa based on a total dataset of 143 sequences (excluding the GenBank sequences). (DOCX 304 kb)

Table S4. Analysis of pairwise genetic differentiation in a total dataset of 143 specimens of Eriphia verrucosa (excluding the twelve sequences from GenBank). (DOCX 17 kb)

Table S2. Pattern of assignment of the twelve Cox1 sequences of Eriphia verrucosa (retrieved from GenBank) to the detected haplotypes in this study. (DOCX 11 kb)

Table S3. Geographic distribution of the 30 haplotypes of E. verrucosa recorded at the 15 defined populations within the East Atlantic Ocean and Mediterranean Sea. (DOCX 16 kb)

Table S1. Estimation of genetic diversity parameters in Atlantic and Mediterranean specimens of Eriphia verrucosa, based on two analyzed datasets (including and excluding retrieved sequences from GenBank). (DOCX 13 kb)

Background: Age-related macular degeneration (AMD) is a common condition of vision loss with disease development strongly influenced by environmental and genetic factors. Recently, 34 loci were associated with AMD at genome-wide significance. So far, little is known about a genetic overlap between AMD and other complex diseases or disease-relevant traits. Methods: For each of 60 complex diseases/traits with publicly available genome-wide significant association data, the lead genetic variant per independent locus was extracted and a genetic score was calculated for each disease/trait as the weighted sum of risk alleles. The association with AMD was estimated based on 16,144 AMD cases and 17,832 controls using logistic regression. Results: Of the respective disease/trait variance, the 60 genetic scores explained on average 4.8% (0.27-20.69%) and 16 of them were found to be significantly associated with AMD (Q-values < 0.01, p values from < 1.0 x 10(-16) to 1.9 x 10(-3)). Notably, an increased risk for AMD was associated with reduced risk for cardiovascular diseases, increased risk for autoimmune diseases, higher HDL and lower LDL levels in serum, lower bone-mineral density as well as an increased risk for skin cancer. By restricting the analysis to 1824 variants initially used to compute the 60 genetic scores, we identified 28 novel AMD risk variants (Q-values < 0.01, p values from 1.1 x 10(-7) to 3.0 x 10(-4)),known to be involved in cardiovascular disorders, lipid metabolism, autoimmune diseases, anthropomorphic traits, ocular disorders, and neurological diseases. The latter variants represent 20 novel AMD-associated, pleiotropic loci. Genes in the novel loci reinforce previous findings strongly implicating the complement system in AMD pathogenesis. Conclusions: We demonstrate a substantial overlap of the genetics of several complex diseases/traits with AMD and provide statistically significant evidence for an additional 20 loci associated with AMD. This highlights the possibility that so far unrelated pathologies may have disease pathways in common.

In recent years, high-throughput discovery of macromolecular protein structures and complexes has played a major role in advancing a more systems-oriented view of protein interaction and signaling networks. The design of biological systems often employs structural information or structure-based protein design to successfully implement synthetic signaling circuits or for rewiring signaling flows. Here, we summarize the latest advances in using structural information for studying protein interaction and signaling networks, and in synthetic biology approaches. We then provide a perspective of how combining structural biology with engineered cell signaling modules--using additional information from quantitative biochemistry and proteomics, gene evolution, and mathematical modeling--can provide insight into signaling modules and the general design principles of cell signaling. Ultimately, this will improve our understanding of cell- and tissue-type-specific signal transduction. Integrating the quantitative effects of disease mutations into these systems may provide a basis for elucidating the molecular mechanisms of diseases. The work was supported by the EU (PROSPECTS, grant agreement number 201648, and PRIMES, grant agreement number 278568)