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Abstract The retinal pigmented epithelium (RPE) constitutes the outer blood‐retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age‐related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal‐side sub‐RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal‐side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age‐related retinal diseases (e.g., AMD).

Inherited retinal dystrophies (IRDs) as well as genetically complex retinal phenotypes represent a heterogenous group of ocular diseases, both on account of their phenotypic and genotypic characteristics. Therefore, overlaps in clinical features often complicate or even impede their correct clinical diagnosis. Deciphering the molecular basis of retinal diseases has not only aided in their disease classification but also helped in our understanding of how different molecular pathologies may share common pathomechanisms. In particular, these relate to dysregulation of two key processes that contribute to cellular integrity, namely extracellular matrix (ECM) homeostasis and inflammation. Pathological changes in the ECM of Bruch’s membrane have been described in both monogenic IRDs, such as Sorsby fundus dystrophy (SFD) and Doyne honeycomb retinal dystrophy (DHRD), as well as in the genetically complex age-related macular degeneration (AMD) or diabetic retinopathy (DR). Additionally, complement system dysfunction and distorted immune regulation may also represent a common connection between some IRDs and complex retinal degenerations. Through highlighting such overlaps in molecular pathology, this review aims to illuminate how inflammatory processes and ECM homeostasis are linked in the healthy retina and how their interplay may be disturbed in aging as well as in disease.

Abstract 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.

Abstract Background Advanced age-related macular degeneration (AMD) is a leading cause of blindness. While around half of the genetic contribution to advanced AMD has been uncovered, little is known about the genetic architecture of early AMD. Methods To identify genetic factors for early AMD, we conducted a genome-wide association study (GWAS) meta-analysis (14,034 cases, 91,214 controls, 11 sources of data including the International AMD Genomics Consortium, IAMDGC, and UK Biobank, UKBB). We ascertained early AMD via color fundus photographs by manual grading for 10 sources and via an automated machine learning approach for > 170,000 photographs from UKBB. We searched for early AMD loci via GWAS and via a candidate approach based on 14 previously suggested early AMD variants. Results Altogether, we identified 10 independent loci with statistical significance for early AMD: (i) 8 from our GWAS with genome-wide significance (P

Abstract Background Inherited pathogenic variants in BRCA1 and BRCA2 are the most common causes of hereditary breast and ovarian cancer (HBOC). The risk of developing breast cancer by age 80 in women carrying a BRCA1 pathogenic variant is 72%. The lifetime risk varies between families and even within affected individuals of the same family. The cause of this variability is largely unknown, but it is hypothesized that additional genetic factors contribute to differences in age at onset (AAO). Here we investigated whether truncating and rare missense variants in genes of different DNA-repair pathways contribute to this phenomenon. Methods We used extreme phenotype sampling to recruit 133 BRCA1-positive patients with either early breast cancer onset, below 35 (early AAO cohort) or cancer-free by age 60 (controls). Next Generation Sequencing (NGS) was used to screen for variants in 311 genes involved in different DNA-repair pathways. Results Patients with an early AAO (73 women) had developed breast cancer at a median age of 27 years (interquartile range (IQR); 25.00–27.00 years). A total of 3703 variants were detected in all patients and 43 of those (1.2%) were truncating variants. The truncating variants were found in 26 women of the early AAO group (35.6%; 95%-CI 24.7 - 47.7%) compared to 16 women of controls (26.7%; 95%-CI 16.1 to 39.7%). When adjusted for environmental factors and family history, the odds ratio indicated an increased breast cancer risk for those carrying an additional truncating DNA-repair variant to BRCA1 mutation (OR: 3.1; 95%-CI 0.92 to 11.5; p-value = 0.07), although it did not reach the conventionally acceptable significance level of 0.05. Conclusions To our knowledge this is the first time that the combined effect of truncating variants in DNA-repair genes on AAO in patients with hereditary breast cancer is investigated. Our results indicate that co-occurring truncating variants might be associated with an earlier onset of breast cancer in BRCA1-positive patients. Larger cohorts are needed to confirm these results.

Abstract Background Ligand-driven modulation of the mitochondrial translocator protein 18 kDa (TSPO) was recently described to dampen the neuroinflammatory response of microglia in a retinal light damage model resulting in protective effects on photoreceptors. We characterized the effects of the TSPO ligand XBD173 in the postischemic retina focusing on changes in the response pattern of the major glial cell types of the retina—microglia and Müller cells. Methods Retinal ischemia was induced by increasing the intraocular pressure for 60 min followed by reperfusion of the tissue in mice. On retinal cell types enriched via immunomagnetic separation expression analysis of TSPO, its ligand diazepam-binding inhibitor (DBI) and markers of glial activation were performed at transcript and protein level using RNA sequencing, qRT-PCR, lipid chromatography-mass spectrometry, and immunofluorescent labeling. Data on cell morphology and numbers were assessed in retinal slice and flatmount preparations. The retinal functional integrity was determined by electroretinogram recordings. Results We demonstrate that TSPO is expressed by Müller cells, microglia, vascular cells, retinal pigment epithelium (RPE) of the healthy and postischemic retina, but only at low levels in retinal neurons. While an alleviated neurodegeneration upon XBD173 treatment was found in postischemic retinae as compared to vehicle controls, this neuroprotective effect of XBD173 is mediated putatively by its action on retinal glia. After transient ischemia, TSPO as a marker of activation was upregulated to similar levels in microglia as compared to their counterparts in healthy retinae irrespective of the treatment regimen. However, less microglia were found in XBD173-treated postischemic retinae at 3 days post-surgery (dps) which displayed a more ramified morphology than in retinae of vehicle-treated mice indicating a dampened microglia activation. Müller cells, the major retinal macroglia, show upregulation of the typical gliosis marker GFAP. Importantly, glutamine synthetase was more stably expressed in Müller glia of XBD173-treated postischemic retinae and homeostatic functions such as cellular volume regulation typically diminished in gliotic Müller cells remained functional. Conclusions In sum, our data imply that beneficial effects of XBD173 treatment on the postischemic survival of inner retinal neurons were primarily mediated by stabilizing neurosupportive functions of glial cells.

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.

Abstract The prevalence of germ line mutations in non‐BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95%CI: 2.67–4.94), CDH1 (OR: 17.04, 95%CI: 3.54–82), CHEK2 (OR: 2.93, 95%CI: 2.29–3.75), PALB2 (OR: 9.53, 95%CI: 6.25–14.51), and TP53 (OR: 7.30, 95%CI: 1.22–43.68). NBN germ line mutations were not significantly associated with BC risk (OR:1.39, 95%CI: 0.73–2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple‐negative breast cancer (TNBC) and estrogen receptor (ER)‐negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)‐positive tumors.

Abstract Genome-wide association studies (GWAS) have identified numerous genetic variants in the human genome associated with diseases and traits. Nevertheless, for most loci the causative variant is still unknown. Expression quantitative trait loci (eQTL) in disease relevant tissues is an excellent approach to correlate genetic association with gene expression. While liver is the primary site of gene transcription for two pathways relevant to age-related macular degeneration (AMD), namely the complement system and cholesterol metabolism, we explored the contribution of AMD associated variants to modulate liver gene expression. We extracted publicly available data and computed the largest eQTL data set for liver tissue to date. Genotypes and expression data from all studies underwent rigorous quality control. Subsequently, Matrix eQTL was used to identify significant local eQTL. In total, liver samples from 588 individuals revealed 202,489 significant eQTL variants affecting 1,959 genes (Q-Value

Abstract Background There is no international consensus up to which age women with a diagnosis of triple-negative breast cancer (TNBC) and no family history of breast or ovarian cancer should be offered genetic testing for germline BRCA1 and BRCA2 (gBRCA) mutations. Here, we explored the association of age at TNBC diagnosis with the prevalence of pathogenic gBRCA mutations in this patient group. Methods The study comprised 802 women (median age 40 years, range 19–76) with oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor type 2 negative breast cancers, who had no relatives with breast or ovarian cancer. All women were tested for pathogenic gBRCA mutations. Logistic regression analysis was used to explore the association between age at TNBC diagnosis and the presence of a pathogenic gBRCA mutation. Results A total of 127 women with TNBC (15.8%) were gBRCA mutation carriers (BRCA1: n = 118, 14.7%; BRCA2: n = 9, 1.1%). The mutation prevalence was 32.9% in the age group 20–29 years compared to 6.9% in the age group 60–69 years. Logistic regression analysis revealed a significant increase of mutation frequency with decreasing age at diagnosis (odds ratio 1.87 per 10 year decrease, 95%CI 1.50–2.32, p 10% for women diagnosed below approximately 50 years. Conclusions Based on the general understanding that a heterozygous mutation probability of 10% or greater justifies gBRCA mutation screening, women with TNBC diagnosed before the age of 50 years and no familial history of breast and ovarian cancer should be tested for gBRCA mutations. In Germany, this would concern approximately 880 women with newly diagnosed TNBC per year, of whom approximately 150 are expected to be identified as carriers of a pathogenic gBRCA mutation.

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.

Abstract 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

Human bestrophin-1 (BEST1) is an integral membrane protein known to function as a Ca2+-activated and volume-regulated chloride channel. The majority of disease-associated mutations in BEST1 constitute missense mutations and were shown in vitro to lead to a reduction in mutant protein half-life causing Best disease (BD), a rare autosomal dominant macular dystrophy. To further delineate BEST1-associated pathology in vivo and to provide an animal model useful to explore experimental treatment efficacies, we have generated a knock-in mouse line (Best1Y227N). Heterozygous and homozygous mutants revealed no significant ocular abnormalities up to 2 years of age. In contrast, knock-in animals demonstrated a severe phenotype in the male reproductive tract. In heterozygous Best1Y227N males, Best1 protein was significantly reduced in testis and almost absent in homozygous mutant mice, although mRNA transcription of wild-type and knock-in allele is present and similar in quantity. Degradation of mutant Best1 protein in testis was associated with adverse effects on sperm motility and the capability to fertilize eggs. Based on these results, we conclude that mice carrying the Best1 Y227N mutation reveal a reproducible pathologic phenotype and thus provide a valuable in vivo tool to evaluate efficacy of drug therapies aimed at restoring Best1 protein stability and function.

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.

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.