Genetic Variants with Significant Association to Age-Related Macular Degeneration (AMD) and their Role in the Regulation of Gene Expression

Genome-wide association studies (GWAS) have led to the identification of a plethora of risk-associated genetic variants for a multitude of complex diseases. The very first GWAS was performed by Klein et al. in the year 2005 and identified variants in the complement factor H (CFH) gene to be associated with age-related macular degeneration (AMD). AMD is a complex eye disease and one of the most common causes of visual impairments and blindness worldwide. It is widely accepted that environmental factors, especially advanced age and smoking, as well as genetic factors contribute substantially to disease risk. Remarkably, the influence of genetics was estimated to be as high as 40-71 %. However, little is known about AMD aetiology, although the latest GWAS performed by Fritsche et al. (2016) revealed 52 independent signals distributed over 34 loci to be associated with AMD. Most of the AMD-associated variants are located in non-coding intergenic or intronic regions of the genome, where functional annotation presents a major challenge. However, these variants may play an important role in the regulation of gene expression. The aim of this thesis was therefore to examine the pathogenesis of AMD in the context of gene expression regulation. A first approach investigated expression quantitative trait loci (eQTL) in liver tissue. Thus, genotype and gene expression data from four independent studies were combined to enable a comprehensive analysis. All samples and studies underwent an especially developed data processing protocol, which applied stringent filter to exclusively allow the detection of highly valid associations. Altogether 588 samples were included and 7,612 genetically regulated genes (Q-Value < 0.05) have been identified. Remarkably, 15 of these are influenced by AMD-associated variants and a comparative analysis reinforced the notion that the initial complement system and lipoprotein metabolism play a role in AMD pathogenesis. In a second project, the Genotype-Tissue Expression (GTEx) database was explored to extend the initial investigations to a variety of tissues. GTEx contains data on 48 different tissues or cell types available from up to 500 donors. The eQTL analysis enabled a new hypothesis regarding gene expression regulatory effects in one of the most significant AMD-associated loci. It was shown that genetic variants within the ARMS2-HTRA1 locus regulate immune system related genes throughout the whole genome. In addition to the bioinformatics studies, in vitro experiments were conducted to validate the developed hypothesis. First, a large genomic deletion within the ARMS2-HTRA1 locus was introduced to assess potential consequences on the expression of bioinformatical predicted target genes. In a second approach, gene expression within the locus was enhanced by targeted application of transcription activation factors. Nevertheless, both strategies were not able to confirm the generated hypothesis in HEK293T cells in the initial experiments. The next project included the comprehensive analysis of eQTL in 314 healthy retinal tissue samples collected from three independent study sites. Altogether, 9,733 genetically regulated genes (Q-value < 0.05) were identified, which allowed insights in gene expression regulation of exclusively healthy retinal tissues for the very first time. Interestingly, only 7 of 34 AMD-associated loci revealed eQTL effects in retina although one must assume that this tissue is a site of the primary/secondary pathology of AMD Therefore, the last project of this thesis aimed at obtaining a comprehensive view on gene expression regulation in the light of AMD genetics. A transcriptome wide association study (TWAS) was performed, which included the genotypes of 16,144 late-stage AMD cases and 17,832 healthy controls from the International AMD Genomics Consortium (IAMDGC). For all these individuals, gene expression was imputed in 27 tissues and analysed in regard to the respective AMD status. This analysis discovered 106 genes, which expression was found to be associated with AMD genetics in at least one tissue. Regulatory effects on gene expression were identified in 25 of the 34 AMD-associated loci. Taken together, this work revealed that gene expression regulation is common in AMD-associated loci. The identified genes reinforce the notion that systemic processes like the complement system or blood lipid levels seem to be relevant for AMD pathology. Furthermore, expression of genes associated with AMD is not restricted to retinal tissue, but instead is rather ubiquitous suggesting processes underlying AMD pathology to be of systemic nature, although the pathological phenotype occurs in the eye

Pleiotropic Locus 15q24.1 Reveals a Gender-Specific Association with Neovascular but Not Atrophic Age-Related Macular Degeneration (AMD)

Funding This research was funded by the Deutsche Forschungsgemeinschaft (GR5065/1-1) and institutional funds (Titel 77). Acknowledgments All contributing sites and additional funding information for the IAMDGC data are acknowledged in this publication: Fritsche et al. (2016) Nature Genetics 48 134–143, (doi:10.1038/ng.3448); The International AMD Genomics consortium’s web page is: http://eaglep.case.edu/iamdgc_web/, and additional information is available on: http://csg.sph.umich.edu/abecasis/public/amd2015/. GERA data came from a grant, the Resource for Genetic Epidemiology Research in Adult Health and Aging (RC2 AG033067; Schaefer and Risch, PIs) awarded to the Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) and the UCSF Institute for Human Genetics. The RPGEH was supported by grants from the Robert Wood Johnson Foundation, the Wayne and Gladys Valley Foundation, the Ellison Medical Foundation, Kaiser Permanente Northern California, and the Kaiser Permanente National and Northern California Community Benefit Programs. The RPGEH and the Resource for Genetic Epidemiology Research in Adult Health and Aging are described in the following publication, Schaefer C, et al., The Kaiser Permanente Research Program on Genes, Environment and Health: Development of a Research Resource in a Multi-Ethnic Health Plan with Electronic Medical Records, In preparation, 2013. This research has been conducted using the UK Biobank Resource under Application Number 44862. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health (commonfund.nih.gov/GTEx). Additional funds were provided by the NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. Donors were enrolled at Biospecimen Source Sites funded by NCI\Leidos Biomedical Research, Inc. subcontracts to the National Disease Research Interchange (10XS170), Roswell Park Cancer Institute (10XS171), and Science Care, Inc. (X10S172). The Laboratory, Data Analysis, and Coordinating Center (LDACC) was funded through a contract (HHSN268201000029C) to The Broad Institute, Inc. Biorepository operations were funded through a Leidos Biomedical Research, Inc. subcontract to Van Andel Research Institute (10ST1035). Additional data repository and project management were provided by Leidos Biomedical Research, Inc. (HHSN261200800001E). The Brain Bank was supported supplements to University of Miami grant DA006227. Statistical Methods development grants were made to the University of Geneva (MH090941 & MH101814), the University of Chicago (MH090951, MH090937, MH101825, & MH101820), the University of North Carolina—Chapel Hill (MH090936), North Carolina State University (MH101819), Harvard University (MH090948), Stanford University (MH101782), Washington University (MH101810), and to the University of Pennsylvania (MH101822). The datasets used for the analyses described in this manuscript were obtained from dbGaP at http://www.ncbi.nlm.nih.gov/gap through dbGaP accession number phs000424.v8.p2.Peer reviewedPublisher PD

Seed sequence polymorphism rs2168518 and allele-specific target gene regulation of hsa-miR-4513

Acknowledgements We thank Lisa Michaelis and Dr Karolina Plößl (Institute of Human Genetics, University of Regensburg) for excellent technical help and thorough proofreading of the manuscript, respectively. We thank Marina Sauer and Franz-Stephan Attenkofer (Institute of Human Genetics, University of Regensburg) for their support in generating the luciferase reporter vectors. Conflict of Interest statement. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding German Research Foundation (GR5065/1-1 to F.G.); and the Helmut Ecker Foundation (Ingolstadt, Germany) (no. 05/17 to B.H.F.W).Peer reviewedPublisher PD

Assigning Co-Regulated Human Genes and Regulatory Gene Clusters

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 gen

Pleiotropic Locus 15q24.1 Reveals a Gender-Specific Association with Neovascular but Not Atrophic Age-Related Macular Degeneration (AMD)

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

A Circulating MicroRNA Profile in a Laser-Induced Mouse Model of Choroidal Neovascularization

Funding: This research was funded by the Deutsche Forschungsgemeinschaft (GR5065/1-1). Author Contributions: Conceptualization, F.G. and B.H.F.W.; Data curation, T.S.; Formal analysis, P.B., M.K., A.A., and T.S.; Funding acquisition, C.K. and F.G.; Investigation, M.K. and B.H.F.W.; Methodology, C.K. and A.A.;Project administration, B.H.F.W.; Resources, M.K., A.A., T.L., and F.G.; Software, C.K. and T.S.; Supervision, T.L., F.G., and B.H.F.W.; Validation, P.B.; Visualization, C.K.; Writing—original draft, C.K. and P.B.; Writing—review & editing, B.H.F.W. All authors have read and agreed to the published version of the manuscript.Peer reviewedPublisher PD

A mega-analysis of expression quantitative trait loci (eQTL) provides insight into the regulatory architecture of gene expression variation in liver

TS was an awardee of the Roche Internships for Scientific Exchange (RiSE) Programme. The work has been supported in part by institutional funds (TG77) of the Institute of Human Genetics Regensburg and by a grant from the Helmut Ecker Foundation (Ingolstadt, Germany) to BHFW (No. 05/17).Peer reviewedPublisher PD

Vitronectin and Its Interaction with PAI-1 Suggests a Functional Link to Vascular Changes in AMD Pathobiology

The pathogenesis of age-related macular degeneration (AMD), a frequent disorder of the central retina, is incompletely understood. Genome-wide association studies (GWAS) suggest a strong contribution of genomic variation in AMD susceptibility. Nevertheless, little is known about biological mechanisms of the disease. We reported previously that the AMD-associated polymorphism rs704C > T in the vitronectin (VTN) gene influences protein expression and functional aspects of encoded vitronectin, a human blood and extracellular matrix (ECM) protein. Here, we refined the association of rs704 with AMD in 16,144 cases and 17,832 controls and noted that rs704 is carried exclusively by the neovascular AMD subtype. Interaction studies demonstrate that rs704 affects the ability of vitronectin to bind the angiogenic regulator plasminogen activator inhibitor 1 (PAI-1) but has no influence on stabilizing its active state. Western blot analysis and confocal imaging reveal a strong enrichment of PAI-1 in the ECM of cultured endothelial cells and RPE cell line ARPE-19 exposed to vitronectin. Large-scale gene expression of VTN and PAI-1 showed positive correlations and a statistically significant increase in human retinal and blood tissues aged 60 years and older. Our results suggest a mechanism by which the AMD-associated rs704 variant in combination with ageing may contribute to the vascular complications in AMD

Epistatic interactions of genetic loci associated with age-related macular degeneration

The currently largest genome-wide association study (GWAS) for age-related macular degeneration (AMD) defines disease association with genome-wide significance for 52 independent common and rare genetic variants across 34 chromosomal loci. Overall, these loci contain over 7200 variants and are enriched for genes with functions indicating several shared cellular processes. Still, the precise mechanisms leading to AMD pathology are largely unknown. Here, we exploit the phenomenon of epistatic interaction to identify seemingly independent AMD-associated variants that reveal joint effects on gene expression. We focus on genetic variants associated with lipid metabolism, organization of extracellular structures, and innate immunity, specifically the complement cascade. Multiple combinations of independent variants were used to generate genetic risk scores allowing gene expression in liver to be compared between low and high-risk AMD. We identified genetic variant combinations correlating significantly with expression of 26 genes, of which 19 have not been associated with AMD before. This study defines novel targets and allows prioritizing further functional work into AMD pathobiology

A transcriptome-wide association study based on 27 tissues identifies 106 genes potentially relevant for disease pathology in age-related macular degeneration.

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