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Contains fulltext : 218949.pdf (Publisher’s version ) (Open Access) Genome-wide association studies (GWAS) for late stage age-related macular degeneration (AMD) have identified 52 independent genetic variants with genome-wide significance at 34 genomic loci. Typically, such an approach rarely results in the identification of functional variants implicating a defined gene in the disease process. We now performed a transcriptome-wide association study (TWAS) allowing the prediction of effects of AMD-associated genetic variants on gene expression. The TWAS was based on the genotypes of 16,144 late-stage AMD cases and 17,832 healthy controls, and gene expression was imputed for 27 different human tissues which were obtained from 134 to 421 individuals. A linear regression model including each individuals imputed gene expression data and the respective AMD status identified 106 genes significantly associated to AMD variants in at least one tissue (Q-value < 0.001). Gene enrichment analysis highlighted rather systemic than tissue- or cell-specific processes. Remarkably, 31 of the 106 genes overlapped with significant GWAS signals of other complex traits and diseases, such as neurological or autoimmune conditions. Taken together, our study highlights the fact that expression of genes associated with AMD is not restricted to retinal tissue as could be expected for an eye disease of the posterior pole, but instead is rather ubiquitous suggesting processes underlying AMD pathology to be of systemic nature.

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.

Elucidating the role of genetic variation in the regulation of gene expression is key to understanding the pathobiology of complex diseases which, in consequence, is crucial in devising targeted treatment options. Expression quantitative trait locus (eQTL) analysis correlates a genetic variant with the strength of gene expression, thus defining thousands of regulated genes in a multitude of human cell types and tissues. Some eQTL may not act independently of each other but instead may be regulated in a coordinated fashion by seemingly independent genetic variants. To address this issue, we combined the approaches of eQTL analysis and colocalization studies. Gene expression was determined in datasets comprising 49 tissues from the Genotype-Tissue Expression (GTEx) project. From about 33,000 regulated genes, over 14,000 were found to be co-regulated in pairs and were assembled across all tissues to almost 15,000 unique clusters containing up to nine regulated genes affected by the same eQTL signal. The distance of co-regulated eGenes was, on average, 112 kilobase pairs. Of 713 genes known to express clinical symptoms upon haploinsufficiency, 231 (32.4%) are part of at least one of the identified clusters. This calls for caution should treatment approaches aim at an upregulation of a haploinsufficient gene. In conclusion, we present an unbiased approach to identifying co-regulated genes in and across multiple tissues. Knowledge of such common effects is crucial to appreciate implications on biological pathways involved, specifically when a treatment option targets a co-regulated disease gene.

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.

Over the last 15 years, genome-wide association studies (GWAS) have greatly advanced our understanding of the genetic landscape of complex phenotypes. Nevertheless, causal interpretations of GWAS data are challenging but crucial to understand underlying mechanisms and pathologies. In this review, we explore to what extend the research community follows up on GWAS data. We have traced the scientific activities responding to the two largest GWAS conducted on age-related macular degeneration (AMD) so far. Altogether 703 articles were manually categorized according to their study type. This demonstrates that follow-up studies mainly involve “Review articles” (33%) or “Genetic association studies” (33%), while 19% of publications report on findings from experimental work. It is striking to note that only three of 16 AMD-associated loci described de novo in 2016 were examined in the four-year follow-up period after publication. A comparative analysis of five studies on gene expression regulation in AMD-associated loci revealed consistent gene candidates for 15 of these loci. Our random survey highlights the fact that functional follow-up studies on GWAS results are still in its early stages hampering a significant refinement of the vast association data and thus a more accurate insight into mechanisms and pathways.

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.