QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Mitochondrial and Cellular Function in Fibroblasts, Induced Neurons, and Astrocytes Derived from Case Study Patients: Insights into Major Depression as a Mitochondria-Associated Disease.

The link between mitochondria and major depressive disorder (MDD) is increasingly evident, underscored both by mitochondria's involvement in many mechanisms identified in depression and the high prevalence of MDD in individuals with mitochondrial disorders. Mitochondrial functions and energy metabolism are increasingly considered to be involved in MDD's pathogenesis. This study focused on cellular and mitochondrial (dys)function in two atypical cases: an antidepressant non-responding MDD patient ("Non-R") and another with an unexplained mitochondrial disorder ("Mito"). Skin biopsies from these patients and controls were used to generate various cell types, including astrocytes and neurons, and cellular and mitochondrial functions were analyzed. Similarities were observed between the Mito patient and a broader MDD cohort, including decreased respiration and mitochondrial function. Conversely, the Non-R patient exhibited increased respiratory rates, mitochondrial calcium, and resting membrane potential. In conclusion, the Non-R patient's data offered a new perspective on MDD, suggesting a detrimental imbalance in mitochondrial and cellular processes, rather than simply reduced functions. Meanwhile, the Mito patient's data revealed the extensive effects of mitochondrial dysfunctions on cellular functions, potentially highlighting new MDD-associated impairments. Together, these case studies enhance our comprehension of MDD.

The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice.

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.

Polarized Desmosome and Hemidesmosome Shedding via Small Extracellular Vesicles is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

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).

Targeted sequencing and in vitro splice assays shed light on ABCA4-associated retinopathies missing heritability.

The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.

Genetic Risk Score Analysis Supports a Joint View of Two Classification Systems for Age-Related Macular Degeneration.

Purpose: The purpose of this study was to evaluate the utility of combining the Clinical Classification (CC) and the Three Continent age-related macular degeneration (AMD) Consortium Severity Scale (3CACSS) for classification of AMD. Methods: In two independent cross-sectional datasets of our population-based AugUR study (Altersbezogene Untersuchungen zur Gesundheit der Universität Regensburg), we graded AMD via color fundus images applying two established classification systems (CC and 3CACSS). We calculated the genetic risk score (GRS) across 50 previously identified variants for late AMD, its association via logistic regression, and area under the curve (AUC) for each AMD stage. Results: We analyzed 2188 persons aged 70 to 95 years. When comparing the two classification systems, we found a distinct pattern: CC "age-related changes" and CC "early AMD" distinguished individuals with 3CACSS "no AMD"; 3CACSS "mild/moderate/severe early AMD" stages, and distinguished CC "intermediate AMD". This suggested a 7-step scale combining the 2 systems: (i) "no AMD", (ii) "age-related changes", (iii) "very early AMD", (i.e. CC "early"), (iv) "mild early AMD", (v) "moderate early AMD", (vi) "severe early AMD", and (vii) "late AMD". GRS association and diagnostic accuracy increased stepwise by increased AMD severity in the 7-step scale and by increased restriction of controls (e.g. for CC "no AMD without age-related changes": AUC = 55.1%, 95% confidence interval [CI] = 51.6, 58.6, AUC = 62.3%, 95% CI = 59.1, 65.6, AUC = 63.8%, 95% CI = 59.3, 68.3, AUC = 78.1%, 95% CI = 73.6, 82.5, AUC = 82.2%, 95% CI = 78.4, 86.0, and AUC = 79.2%, 95% CI = 75.4, 83.0). A stepwise increase was also observed by increased drusen size and area. Conclusions: The utility of a 7-step scale is supported by our clinical and GRS data. This harmonization and full data integration provides an immediate simplification over using either CC or 3CACSS and helps to sharpen the control group.

Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

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 from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of 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 exosomes 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 exosome release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in exosome content, including basal-side specific desmosome and hemidesmosome shedding via exosomes. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases, (e.g., AMD) and broadly from blood-CNS barriers in other neurodegenerative diseases.

Prevalence of Pathogenic Germline Variants in Women with Non-Familial Unilateral Triple-Negative Breast Cancer.

Introduction: International guidelines recommend genetic testing for women with familial breast cancer at an expected prevalence of pathogenic germline variants (PVs) of at least 10%. In a study sample of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC), we have previously shown that women with TNBC diagnosed before the age of 50 years but without a family history of breast or ovarian cancer (sTNBC) meet this criterion. The present study investigates the PV prevalence in BRCA1, BRCA2, and nine additional cancer predisposition genes in an extended sTNBC study sample including a cohort of women with a later age at sTNBC diagnosis. Patients and Methods: In 1,600 women with sTNBC (median age at diagnosis: 41 years, range 19-78 years), we investigated the association between age at diagnosis and PV occurrence in cancer predisposition genes using logistic regression. Results: 260 sTNBC patients (16.2%) were found to have a PV in cancer predisposition genes (BRCA1: n = 170 [10.6%]; BRCA2: n = 46 [2.9%], other: n = 44 [2.8%]). The PV prevalence in women diagnosed between 50 and 59 years (n = 194) was 11.3% (22/194). Logistic regression showed a significant increase in PV prevalence with decreasing age at diagnosis (OR 1.41 per 10 years younger age at diagnosis; 95% confidence interval: 1.21-1.65; p < 0.001). The PV prevalence predicted by the model was above 10% for diagnoses before the age of 56.8 years. Conclusion: Based on the data presented, we recommend genetic testing by gene panel analysis for sTNBC patients diagnosed before the age of 60 years. Due to the still uncertain estimate for women with sTNBC diagnosed above the age of 60 years, further studies are needed.

The extracellular microenvironment in immune dysregulation and inflammation in retinal disorders.

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.

Systems genomics in age-related macular degeneration.

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors.

The RS1 gene on Xp 22.13 encodes retinoschisin which is known to directly interact with the retinal Na/K-ATPase at the photoreceptor inner segments. Pathologic mutations in RS1 cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in young males. To further delineate the retinoschisin-Na/K-ATPase complex, co-immunoprecipitation was performed with porcine and murine retinal lysates targeting the ATP1A3 subunit. This identified the voltage-gated potassium (Kv) channel subunits Kv2.1 and Kv8.2 as direct interaction partners of the retinal Na/K-ATPase. Colocalization of the individual components of the complex was demonstrated at the membrane of photoreceptor inner segments. We further show that retinoschisin-deficiency, a frequent consequence of molecular pathology in XLRS, causes mislocalization of the macromolecular complex during postnatal retinal development with a simultaneous reduction of Kv2.1 and Kv8.2 protein expression, while the level of retinal Na/K-ATPase expression remains unaffected. Patch-clamp analysis revealed no effect of retinoschisin-deficiency on Kv channel mediated potassium ion currents in vitro. Together, our data suggest that Kv2.1 and Kv8.2 together with retinoschisin and the retinal Na/K-ATPase are integral parts of a macromolecular complex at the photoreceptor inner segments. Defective compartmentalization of this complex due to retinoschisin-deficiency may be a crucial step in initial XLRS pathogenesis.

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.

Genetic Association Analysis of Anti-VEGF Treatment Response in Neovascular Age-Related Macular Degeneration.

Anti-VEGF treatment for neovascular age-related macular degeneration (nAMD) has been FDA-approved in 2004, and since then has helped tens of thousands of patients worldwide to preserve vision. Still, treatment responses vary widely, emphasizing the need for genetic biomarkers to robustly separate responders from non-responders. Here, we report the findings of an observational study compromising 179 treatment-naïve nAMD patients and their reaction to treatment after three monthly doses of anti-VEGF antibodies. We show that established criteria of treatment response such as visual acuity and central retinal thickness successfully divides our cohort into 128 responders and 51 non-responders. Nevertheless, retinal thickness around the fovea revealed significant reaction to treatment even in the formally categorized non-responders. To elucidate genetic effects underlying our criteria, we conducted an undirected genome-wide association study followed by a directed replication study of 30 previously reported genetic variants. Remarkably, both approaches failed to result in significant findings, suggesting study-specific effects were confounding the present and previous discovery studies. Of note, all studies so far are greatly underpowered, hampering interpretation of genetic findings. In consequence, we highlight the need for an extensive phenotyping study with sample sizes exceeding at least 15,000 to reliably assess anti-VEGF treatment responses in nAMD.

Incidence, progression and risk factors of age-related macular degeneration in 35-95-year-old individuals from three jointly designed German cohort studies.

OBJECTIVE: To estimate age-related macular degeneration (AMD) incidence/progression across a wide age range. METHODS AND ANALYSIS: AMD at baseline and follow-up (colour fundus imaging, Three Continent AMD Consortium Severity Scale, 3CACSS, clinical classification, CC) was assessed for 1513 individuals aged 35-95 years at baseline from three jointly designed population-based cohorts in Germany: Kooperative Gesundheitsforschung in der Region Augsburg (KORA-Fit, KORA-FF4) and Altersbezogene Untersuchungen zur Gesundheit der Universität Regensburg (AugUR) with 18-year, 14-year or 3-year follow-up, respectively. Baseline assessment included lifestyle, metabolic and genetic markers. We derived cumulative estimates, rates and risk factor association for: (1) incident early AMD, (2) incident late AMD among no AMD at baseline (definition 1), (3) incident late AMD among no/early AMD at baseline (definition 2), (4) progression from early to late AMD. RESULTS: Incidence/progression increased by age, except progression in 70+-year old. We observed 35-55-year-old with 3CACSS-based early AMD who progressed to late AMD. Predominant risk factor for incident late AMD definition 2 was early AMD followed by genetics and smoking. When separating incident late AMD definition 1 from progression (instead of combined as incident late AMD definition 2), estimates help judge an individual's risk based on age and (3CACSS) early AMD status: for example, for a 65-year old, 3-year late AMD risk with no or early AMD is 0.5% or 7%, 3-year early AMD risk is 3%; for an 85-year old, these numbers are 0.5%, 21%, 12%, respectively. For CC-based 'early/intermediate' AMD, incidence was higher, but progression was lower. CONCLUSION: We provide a practical guide for AMD risk for ophthalmology practice and healthcare management and document a late AMD risk for individuals aged <55 years.

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

MicroRNAs (miRNAs) are small post-transcriptional regulators that offer promising targets for treating complex diseases. To this end, hsa-miR-4513 is an excellent candidate as this gene harbors within its conserved heptametrical seed sequence a frequent polymorphism (rs2168518), which has previously been associated with several complex phenotypes. So far, little is known about the biological mechanism(s) underlying these associations. In an initial step, we now aimed to identify allele-specific target genes of hsa-miR-4513. We performed RNA sequencing in a miRNA overexpression model in human umbilical vein endothelial cells transfected with separated hsa-miR-4513 alleles at rs2168518, namely hsa-miR-4513-G and hsa-miR-4513-A. Genes specifically regulated by the rs2168518 alleles were independently verified by quantitative reverse transcription PCR (qRT-PCR), western blot analysis and allele-specific miRNA binding via a luciferase reporter assay. By a text-based search publicly available databases such as Online Mendelian Inheritance in Man and Mouse Genome Informatics were utilized to link target genes of hsa-miR-4513 to previously described phenotypes. Overall, we identified 23 allele-specific hsa-miR-4513 target genes and replicated 19 of those independently via qRT-PCR. Western blot analysis and luciferase reporter assays conducted for an exemplary subsample further confirmed the allele-specific regulation of these genes by hsa-miR-4513. Remarkably, multiple allele-specific target genes identified are linked via text retrieval to several phenotypes previously reported to be associated with hsa-miR-4513. These genes offer promising candidates for ongoing research on the functional pathobiological impact of hsa-miR-4513 and its seed polymorphism rs2168518. This could give rise to therapeutic applications targeting this miRNA.

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 gene.

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.

Performance of Breast Cancer Polygenic Risk Scores in 760 Female CHEK2 Germline Mutation Carriers.

BACKGROUND: Genome-wide association studies suggest that the combined effects of breast cancer (BC)-associated single nucleotide polymorphisms (SNPs) can improve BC risk stratification using polygenic risk scores (PRSs). The performance of PRSs in genome-wide association studies-independent clinical cohorts is poorly studied in individuals carrying mutations in moderately penetrant BC predisposition genes such as CHEK2. METHODS: A total of 760 female CHEK2 mutation carriers were included; 561 women were affected with BC, of whom 74 developed metachronous contralateral BC (mCBC). For PRS calculations, 2 SNP sets covering 77 (SNP set 1, developed for BC risk stratification in women unselected for their BRCA1/2 germline mutation status) and 88 (SNP set 2, developed for BC risk stratification in female BRCA1/2 mutation carriers) BC-associated SNPs were used. All statistical tests were 2-sided. RESULTS: Both SNP sets provided concordant PRS results at the individual level (r = 0.91, P < 2.20 × 10-16). Weighted cohort Cox regression analyses revealed statistically significant associations of PRSs with the risk for first BC. For SNP set 1, a hazard ratio of 1.71 per SD of the PRS was observed (95% confidence interval = 1.36 to 2.15, P = 3.87 × 10-6). PRSs identify a subgroup of CHEK2 mutation carriers with a predicted lifetime risk for first BC that exceeds the surveillance thresholds defined by international guidelines. Association of PRS with mCBC was examined via Cox regression analysis (SNP set 1 hazard ratio = 1.23, 95% confidence interval = 0.86 to 1.78, P = .26). CONCLUSIONS: PRSs may be used to personalize risk-adapted preventive measures for women with CHEK2 mutations. Larger studies are required to assess the role of PRSs in mCBC predisposition.

Altered Protein Function Caused by AMD-associated Variant rs704 Links Vitronectin to Disease Pathology.

Purpose: Vitronectin, a cell adhesion and spreading factor, is suspected to play a role in the pathogenesis of age-related macular degeneration (AMD), as it is a major component of AMD-specific extracellular deposits (e.g., soft drusen, subretinal drusenoid deposits). The present study addressed the impact of AMD-associated non-synonymous variant rs704 in the vitronectin-encoding gene VTN on vitronectin functionality. Methods: Effects of rs704 on vitronectin expression and processing were analyzed by semi-quantitative sequencing of VTN transcripts from retinal pigment epithelium (RPE) cells generated from human induced pluripotent stem cells (hiPSCs) and from human neural retina, as well as by western blot analyses on heterologously expressed vitronectin isoforms. Binding of vitronectin isoforms to retinal and endothelial cells was analyzed by western blot. Immunofluorescence staining followed extracellular matrix (ECM) deposition in cultured RPE cells heterologously expressing the vitronectin isoforms. Adhesion of fluorescently labeled RPE or endothelial cells in dependence of recombinant vitronectin or vitronectin-containing ECM was investigated fluorometrically or microscopically. Tube formation and migration assays addressed effects of vitronectin on angiogenesis-related processes. Results: Variant rs704 affected expression, secretion, and processing but not oligomerization of vitronectin. Cell binding and influence on RPE-mediated ECM deposition differed between AMD-risk-associated and non-AMD-risk-associated protein isoforms. Finally, vitronectin affected adhesion and endothelial tube formation. Conclusions: The AMD-risk-associated vitronectin isoform exhibits increased expression and altered functionality in cellular processes related to the sub-RPE aspects of AMD pathology. Although further research is required to address the subretinal disease aspects, this initial study supports an involvement of vitronectin in AMD pathogenesis.