Publications by authors named "Anneke I den Hollander"

241 Publications

Evaluating the Occurrence of Rare Variants in the Complement Factor H Gene in Patients With Early-Onset Drusen Maculopathy.

JAMA Ophthalmol 2021 Oct 14. Epub 2021 Oct 14.

Donders Institute for Brain, Cognition and Behaviour, Nijmegen, the Netherlands.

Importance: Early-onset drusen maculopathy (EODM) is a severe disease and can lead to advanced macular degeneration early in life; however, genetic and phenotypic characteristics of individuals with EODM are not well studied.

Objective: To identify genotypic and phenotypic characteristics of individuals with EODM.

Design, Setting, And Participants: This case-control study collected data from the European Genetic Database from September 2004 to October 2019. A total of 89 patients with EODM diagnosed at 55 years or younger and 91 patients with age-related macular degeneration (AMD) diagnosed at 65 years or older were included.

Exposures: Coding regions of CFH, CFI, C3, C9, CFB, ABCA4, PRPH2, TIMP3, and CTNNA1 genes were sequenced, genetic risk scores (GRS) were calculated based on 52 AMD-associated variants, and phenotypic characteristics on color fundus photographs were analyzed comparing patients with EODM and AMD.

Main Outcomes And Measures: GRS, frequency of rare genetic complement variants, and phenotypic characteristics.

Results: This case-control study included 89 patients with EODM (mean [SD] age, 51.8 [8.7] years; 58 [65.2%] were female) and 91 patients with AMD (mean [SD] age, 77.6 [6.1] years; 45 [49.5%] female). At a mean (SD) age of 56.4 (7.3) years, 40 of 89 patients with EODM (44.9%) were affected by geographic atrophy or choroidal neovascularization. A lower GRS was observed in patients with EODM compared with patients with AMD (1.03 vs 1.60; P = .002), and 27 of 89 patients with EODM (30.3%) carried rare variants in the CFH gene compared with 7 of 91 patients with AMD (7.7%). Carriership of a rare CFH variant was associated with EODM (odds ratio, 7.2; 95% CI, 2.7-19.6; P < .001). A large macular drusen area (more than 50% covered with drusen) was observed in patients with EODM (24 of 162 eyes [14.8%]) compared with patients with AMD (9 of 164 eyes [5.5%]) (odds ratio, 4.57; 95% CI, 1.5-14.1; P = .008).

Conclusions And Relevance: A large proportion of patients with EODM in this study carried rare CFH variants, with most of the identified CFH variants clustered in the first 7 complement control protein domains affecting factor H and factor H-like 1. Because EODM frequently leads to advanced macular degeneration at an early age and can result in many years of vision loss, this study supports targeting the complement system and sequencing the CFH gene in patients with EODM to improve genetic counseling and future treatments for AMD.
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http://dx.doi.org/10.1001/jamaophthalmol.2021.4102DOI Listing
October 2021

Systemic complement levels in patients with age-related macular degeneration carrying rare or low frequency variants in the CFH gene.

Hum Mol Genet 2021 Sep 11. Epub 2021 Sep 11.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. Genetic variants in the complement factor H (CFH) gene are associated with AMD, but the functional consequences of many of these variants are currently unknown. In this study we aimed to determine the effect of 64 rare and low frequency variants in the CFH gene on systemic levels of factor H (FH) and complement activation marker C3bBbP using plasma samples of 252 carriers and 159 non-carriers. Individuals carrying a heterozygous nonsense, frameshift or missense variant in CFH presented with significantly decreased FH levels, and significantly increased C3bBbP levels in plasma compared to non-carrier controls. FH and C3bBbP plasma levels were relatively stable over time in samples collected during follow-up visits. Decreased FH and increased C3bBbP concentrations were observed in carriers compared to non-carriers of CFH variants among different AMD stages, with the exception of C3bBbP levels in advanced AMD stages, which were equally high in carriers and non-carriers. In AMD families, FH levels were decreased in carriers compared to non-carriers, but C3bBbP levels did not differ. Rare variants in the CFH gene can lead to reduced FH levels or reduced FH function as measured by increased C3bBbP levels. The effects of individual variants in the CFH gene reported in this study will improve the interpretation of rare and low frequency variants observed in AMD patients in clinical practice.
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http://dx.doi.org/10.1093/hmg/ddab256DOI Listing
September 2021

PRPH2 mutation update: In silico assessment of 245 reported and 7 novel variants in patients with retinal disease.

Hum Mutat 2021 Aug 19. Epub 2021 Aug 19.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

Mutations in PRPH2, encoding peripherin-2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the total 252 PRPH2 variants, more than half (n = 137) were missense variants. All variants were uploaded into the Leiden Open source Variation and ClinVar databases. Our study underscores the need for experimental assays for variants of unknown significance to improve pathogenicity classification, which would allow us to better understand genotype-phenotype correlations, and in the long-term, hopefully also support the development of therapeutic strategies for patients with PRPH2-associated IRD.
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http://dx.doi.org/10.1002/humu.24275DOI Listing
August 2021

Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Am J Hum Genet 2021 08 13;108(8):1367-1384. Epub 2021 Jul 13.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, 6525EX, the Netherlands; Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, 6525GA, the Netherlands. Electronic address:

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10), FHR-2 (p = 1.47 × 10), FHR-3 (p = 1.05 × 10) and FHR-4A (p = 1.22 × 10) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10 and p = 2.81 × 10, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387287PMC
August 2021

Systemic complement activation levels in Stargardt disease.

PLoS One 2021 25;16(6):e0253716. Epub 2021 Jun 25.

Department of Ophthalmology, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: Preclinical research provides evidence for the complement system as a potential common pathway in Stargardt disease (STGD1) and age-related macular degeneration (AMD) leading to retinal pigment epithelium (RPE) loss. However, systemic complement activation has not yet been assessed in STGD1 patients. We conducted a cross-sectional case-control study to assess systemic complement activation in STGD1 patients and its association with disease severity.

Methods: Systemic concentrations of complement component C3 and its degradation product C3d were compared between 80 STGD1 patients and 80 controls that were frequency matched for age and sex. The C3d/C3 ratio was used as parameter of systemic complement activation. Within the STGD1 cohort, we additionally examined the association between the C3d/C3 ratio, demographic and behavioural factors (age, sex, smoking and BMI), and measures of disease severity (age at onset, visual acuity, and area of atrophy).

Results: The C3d/C3 ratio did not significantly differ between patients (mean C3d/C3 ratio 3.5±1.4) and controls (mean C3d/C3 ratio 3.6±1.0), mean difference -0.156 (p = 0.804, independent samples t-test). The overall effect size was 8% (95% confidence interval, 3-15%). Elevated C3d/C3 ratios (>8.1) were found in three patients who all had a concomitant inflammatory condition at the time of blood draw. Within the patient cohort, C3 levels were associated with sex (mean difference -134, p = 0.001, independent samples t-test) and BMI (correlation coefficient 0.463, p<0.001, Spearman's Correlation).

Conclusions: Systemic complement levels were not elevated in STGD1 patients compared to age and sex matched controls and was not associated with STGD1 severity. Considering the continued absent proof of a systemic contribution of the complement system to RPE loss in STGD1 patients, we hypothesize that complement activation in STGD1 is more likely a local process. In light of upcoming complement-targeted therapies, further studies are needed that measure complement levels in the eye of STGD1 patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0253716PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8232401PMC
June 2021

Increased pro-MMP9 plasma levels are associated with neovascular age-related macular degeneration and with the risk allele of rs142450006 near .

Mol Vis 2021 2;27:142-150. Epub 2021 Apr 2.

Department of Ophthalmology, Radboud University Medical Center, Donders Institute for Brain, Cognition and Behaviour, Nijmegen, The Netherlands.

Purpose: To evaluate the plasma levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitors of metalloproteinase 3 (TIMP3) in neovascular age-related macular degeneration (nAMD) patients compared to controls, and to explore the potential effect of AMD-associated genetic variants on MMP9 and TIMP3 protein levels.

Methods: nAMD and control patients were selected from the European Genetic Database (EUGENDA) based on different genotypes of rs142450006 near and rs5754227 near Plasma total MMP9, active MMP9 and TIMP3 levels were measured using the enzyme linked immunosorbent assay (ELISA) and compared between nAMD patients and controls, as well as between different genotype groups.

Results: nAMD patients had significantly higher total MMP9 levels compared to controls (median 46.58 versus 26.90 ng/ml; p = 0.0004). In addition, the median MMP9 level in the homozygous genotype group for the AMD-risk allele (44.23 ng/ml) was significantly higher than the median for the heterozygous genotype group (26.90 ng/ml; p = 0.0082) and the median for the homozygous group for the non-risk allele (28.55 ng/ml; p = 0.0355). No differences were detected for the active MMP9. TIMP3 levels did not significantly differ between the AMD and control groups, nor between the different genotype groups for rs5754227.

Conclusions: The results of our MMP9 analyses indicate that nAMD patients have on average higher systemic MMP9 levels than control individuals, and that this is partly driven by the rs142450006 variant near . This finding might be an interesting starting point for further exploration of MMP9 as a therapeutic target in nAMD, particularly among individuals carrying the risk-conferring allele rs142450006.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056465PMC
April 2021

ERAP2 Increases the Abundance of a Peptide Submotif Highly Selective for the Birdshot Uveitis-Associated HLA-A29.

Front Immunol 2021 25;12:634441. Epub 2021 Feb 25.

Department of Ophthalmology, University Medical Center Utrecht, University of Utrecht, Utrecht, Netherlands.

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU.
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http://dx.doi.org/10.3389/fimmu.2021.634441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7950316PMC
September 2021

Implications of genetic variation in the complement system in age-related macular degeneration.

Prog Retin Eye Res 2021 Sep 19;84:100952. Epub 2021 Feb 19.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525, GA, Nijmegen, the Netherlands; Department of Human Genetics, Radboud University Medical Center, 6525, GA, Nijmegen, the Netherlands. Electronic address:

Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in the Western world. While AMD is a multifactorial disease, the complement system was identified as one of the main pathways contributing to disease risk. The strong link between the complement system and AMD was demonstrated by genetic associations, and by elevated complement activation in local eye tissue and in the systemic circulation of AMD patients. Several complement inhibitors have been and are being explored in clinical trials, but thus far with limited success, leaving the majority of AMD patients without treatment options to date. This indicates that there is still a gap of knowledge regarding the functional implications of the complement system in AMD pathogenesis and how to bring these towards clinical translation. Many different experimental set-ups and disease models have been used to study complement activation in vivo and in vitro, and recently emerging patient-derived induced pluripotent stem cells and genome-editing techniques open new opportunities to study AMD disease mechanisms and test new therapeutic strategies in the future. In this review we provide an extensive overview of methods employed to understand the molecular processes of complement activation in AMD pathogenesis. We discuss the findings, advantages and challenges of each approach and conclude with an outlook on how recent, exciting developments can fill in current knowledge gaps and can aid in the development of effective complement-targeting therapeutic strategies in AMD.
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http://dx.doi.org/10.1016/j.preteyeres.2021.100952DOI Listing
September 2021

Microfluidic organ-on-a-chip model of the outer blood-retinal barrier with clinically relevant read-outs for tissue permeability and vascular structure.

Lab Chip 2021 01 21;21(2):272-283. Epub 2020 Dec 21.

Applied Stem Cell Technologies, Technical Medical Centre, University of Twente, PO Box 217, 7500 AE, Enschede, The Netherlands.

The outer blood-retinal barrier (oBRB) tightly controls the transport processes between the neural tissue of the retina and the underlying blood vessel network. The barrier is formed by the retinal pigment epithelium (RPE), its basal membrane and the underlying choroidal capillary bed. Realistic three-dimensional cell culture based models of the oBRB are needed to study mechanisms and potential treatments of visual disorders such as age-related macular degeneration that result from dysfunction of the barrier tissue. Ideally, such models should also include clinically relevant read-outs to enable translation of experimental findings in the context of pathophysiology. Here, we report a microfluidic organ-on-a-chip model of the oBRB that contains a monolayer of human immortalized RPE and a microvessel of human endothelial cells, separated by a semi-permeable membrane. Confluent monolayers of both cell types were confirmed by fluorescence microscopy. The three-dimensional vascular structures within the chip were imaged by optical coherence tomography: a medical imaging technique, which is routinely applied in ophthalmology. Differences in diameters and vessel density could be readily detected. Upon inducing oxidative stress by treating with hydrogen peroxide (HO), a dose dependent increase in barrier permeability was observed by using a dynamic assay for fluorescence tracing, analogous to the clinically used fluorescence angiography. This organ-on-a-chip of the oBRB will allow future studies of complex disease mechanisms and treatments for visual disorders using clinically relevant endpoints in vitro.
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http://dx.doi.org/10.1039/d0lc00639dDOI Listing
January 2021

The Effect of Genetic Variants Associated With Age-Related Macular Degeneration Varies With Age.

Invest Ophthalmol Vis Sci 2020 12;61(14):17

Department of Ophthalmology, University Hospital of Cologne, Cologne, Germany.

Purpose: The prevalence of age-related macular degeneration (AMD) increases dramatically with age. This large collaborative study investigates the effects of 51 late-AMD-associated genetic variants in different ages, focusing on individuals above the age of 90 years.

Methods: The study included 27,996 individuals of the International AMD Genomics Consortium; 14,539 showed late AMD (51.9%) and 13,457 were controls (48.1%). Four age groups were compiled: 60 to 69 years, n = 6514, AMD = 2210 (33.9%); 70 to 79 years, n = 12228, AMD = 6217 (51.7%); 80 to 89 years, n = 8285, AMD = 5326 (64.3%); and ≥90 years, n = 969, AMD = 686 (70.8%). The effect sizes of 51 AMD-associated genetic variants were calculated for all age groups and were compared among the age groups.

Results: Six variants were associated with late AMD in individuals ≥ 90 years of age (P ≤ 0.0006). For rs10922109 and rs570618 (both in CFH), the minor allele (MA) was protective, and minor allele frequency (MAF) increased with age in cases and controls. For rs116503776 in C2/CFB/SKIV2L, the MA was protective, and MAF increased in cases. For rs3750846 in ARMS2/HTRA1, the MA increased risk, and MAF was lower in cases with increasing age. For rs6565597 in NPLOC4/TSPAN10, the MA increased risk. For rs5754227 in SYN3/TIMP3, the MA was protective, and there was no consistent variation in MAF with age. Variants in CFH and ARMS2 showed lower effect sizes at greater age. Interaction analysis showed strong age-related effects for rs570618 (P = 2.24 × 10-7) and rs3750846 (P = 0.001). Total genetic risk was lower in individuals ≥ 90 years old (area under the curve [AUC], 0.795) than in those 70 to 79 years old (AUC, 0.831; P = 0.03).

Conclusions: Effect sizes and MAF of genetic risk factors for late AMD differed among the age groups. These results could guide future work on AMD risk assessment in older individuals.
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http://dx.doi.org/10.1167/iovs.61.14.17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745630PMC
December 2020

Quantitative multiplex profiling of the complement system to diagnose complement-mediated diseases.

Clin Transl Immunology 2020 9;9(12):e1225. Epub 2020 Dec 9.

Laboratory of Medical Immunology Department of Laboratory Medicine Radboud Institute for Molecular Life Sciences Radboud University Medical Center Nijmegen The Netherlands.

Objectives: Complement deficiencies are difficult to diagnose because of the variability of symptoms and the complexity of the diagnostic process. Here, we applied a novel 'complementomics' approach to study the impact of various complement deficiencies on circulating complement levels.

Methods: Using a quantitative multiplex mass spectrometry assay, we analysed 44 peptides to profile 34 complement proteins simultaneously in 40 healthy controls and 83 individuals with a diagnosed deficiency or a potential pathogenic variant in 14 different complement proteins.

Results: Apart from confirming near or total absence of the respective protein in plasma of complement-deficient patients, this mass spectrometry-based profiling method led to the identification of additional deficiencies. In many cases, partial depletion of the pathway up- and/or downstream of the absent protein was measured. This was especially found in patients deficient for complement inhibitors, such as angioedema patients with a C1-inhibitor deficiency. The added value of complementomics was shown in three patients with poorly defined complement deficiencies.

Conclusion: Our study shows the potential clinical utility of profiling circulating complement proteins as a comprehensive read-out of various complement deficiencies. Particularly, our approach provides insight into the intricate interplay between complement proteins due to functional coupling, which contributes to the better understanding of the various disease phenotypes and improvement of care for patients with complement-mediated diseases.
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http://dx.doi.org/10.1002/cti2.1225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724921PMC
December 2020

Genetic Risk, Lifestyle, and Age-Related Macular Degeneration in Europe: The EYE-RISK Consortium.

Ophthalmology 2021 07 28;128(7):1039-1049. Epub 2020 Nov 28.

Department of Ophthalmology, Erasmus University Medical Center, Rotterdam, The Netherlands; Department of Epidemiology, Erasmus University Medical Center, Rotterdam, The Netherlands; Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands; Institute of Molecular and Clinical Ophthalmology, Basel, Switzerland. Electronic address:

Purpose: Age-related macular degeneration (AMD) is a common multifactorial disease in the elderly with a prominent genetic basis. Many risk variants have been identified, but the interpretation remains challenging. We investigated the genetic distribution of AMD-associated risk variants in a large European consortium, calculated attributable and pathway-specific genetic risks, and assessed the influence of lifestyle on genetic outcomes.

Design: Pooled analysis of cross-sectional data from the European Eye Epidemiology Consortium.

Participants: Seventeen thousand one hundred seventy-four individuals 45 years of age or older participating in 6 population-based cohort studies, 2 clinic-based studies, and 1 case-control study.

Methods: Age-related macular degeneration was diagnosed and graded based on fundus photographs. Data on genetics, lifestyle, and diet were harmonized. Minor allele frequencies and population-attributable fraction (PAF) were calculated. A total genetic risk score (GRS) and pathway-specific risk scores (complement, lipid, extra-cellular matrix, other) were constructed based on the dosage of SNPs and conditional β values; a lifestyle score was constructed based on smoking and diet.

Main Outcome Measures: Intermediate and late AMD.

Results: The risk variants with the largest difference between late AMD patients and control participants and the highest PAFs were located in ARMS2 (rs3750846) and CHF (rs570618 and rs10922109). Combining all genetic variants, the total genetic risk score ranged from -3.50 to 4.63 and increased with AMD severity. Of the late AMD patients, 1581 of 1777 (89%) showed a positive total GRS. The complement pathway and ARMS2 were by far the most prominent genetic pathways contributing to late AMD (positive GRS, 90% of patients with late disease), but risk in 3 pathways was most frequent (35% of patients with late disease). Lifestyle was a strong determinant of the outcome in each genetic risk category; unfavorable lifestyle increased the risk of late AMD at least 2-fold.

Conclusions: Genetic risk variants contribute to late AMD in most patients. However, lifestyle factors have a strong influence on the outcome of genetic risk and should be a strong focus in patient management. Genetic risks in ARMS2 and the complement pathway are present in most late AMD patients but are mostly combined with risks in other pathways.
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http://dx.doi.org/10.1016/j.ophtha.2020.11.024DOI Listing
July 2021

Association of plasma trace element levels with neovascular age-related macular degeneration.

Exp Eye Res 2020 12 21;201:108324. Epub 2020 Oct 21.

Wellcome Trust Centre for Cell-Matrix Research, Division of Cell-Matrix Biology & Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester, M13 9PT, UK; Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester, M13 9PT, UK. Electronic address:

Although the triggers causing angiogenesis in the context of neovascular age-related macular degeneration (nAMD) are not fully understood, oxidative stress is likely involved. Oxidative stress in the eye can occur through exposure of macular tissues to sunlight and local or systemic exposure to oxidative stressors associated with environmental or lifestyle factors. Because trace elements have been implicated as regulators of oxidative stress and cellular antioxidant defense mechanisms, we hypothesized that they may play a role as a risk factor, modifying the progression toward nAMD. Herein, we determined whether levels of human plasma trace elements are different in 236 individuals with nAMD compared to 236 age-matched controls without AMD. Plasma levels of 16 trace elements including arsenic, barium, calcium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, molybdenum, lead, antimony, selenium, vanadium and zinc were measured using inductively coupled plasma mass spectrometry. Associations of trace elements with demographic, environmental and lifestyle factors and AMD-associated genetic variants were assessed. Elevated levels of barium and cadmium and reduced levels of chromium were observed in nAMD patients compared to controls. Mean plasma concentrations of barium were 1.35 μg/L (standard deviation [SD] 0.71) in nAMD and 1.15 μg/L (SD 0.63) in controls (P = 0.001). Mean levels of chromium were 0.37 μg/L (SD 0.22) in nAMD and 0.46 μg/L (SD 0.34) in controls (P = 0.001). Median levels for cadmium, which were not normally distributed, were 0.016 μg/L (interquartile range [IQR] 0.001-0.026) in nAMD and 0.012 μg/L (IQR 0.001-0.022) in controls (P = 0.002). Comparison of the Spearman's correlation coefficients between nAMD patients and controls identified a difference in correlations for 8 trace elements. Cadmium levels were associated with the smoking status (P < 0.001), while barium levels showed a trend of association with the usage of antihypertensive drugs. None of the AMD-associated genetic variants were associated with any trace element levels. In conclusion, in this case-control study we detected elevated plasma levels of barium and cadmium and reduced plasma levels of chromium in nAMD patients. An imbalance in plasma trace elements, which is most likely driven by environmental and lifestyle factors, might have a role in the pathogenesis of AMD. These trace elements may be incorporated as biomarkers into models for prediction of disease risk and progression. Additionally, population-based preventive strategies to decrease Cd exposure, especially by the cessation of smoking, could potentially reduce the burden of nAMD. Future studies are warranted to investigate whether supplementation of Cr would have a beneficial effect on nAMD.
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http://dx.doi.org/10.1016/j.exer.2020.108324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773981PMC
December 2020

Genetic and environmental risk factors for extramacular drusen.

Mol Vis 2020 4;26:661-669. Epub 2020 Oct 4.

Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, Germany.

Purpose: To analyze risk factors for extramacular drusen (EMD) in patients with age-related macular degeneration (AMD) and healthy control individuals.

Methods: This case-control study included 1,520 patients from the prospective multicenter European Genetic Database (EUGENDA). Color fundus photographs and optical coherence tomography scans were evaluated for the presence of AMD and EMD. EMD was considered present if ten or fewer drusen including at least one intermediate-sized drusen were detected outside the macula. Association of EMD was evaluated with various genetic and non-genetic risk factors (31 single nucleotide polymorphisms, systemic complement activation, smoking, cardiovascular factors, and sunlight exposure) using logistic regression models adjusted for age, gender, and AMD.

Results: EMD was found in 608 subjects (40%) and AMD in 763 (50%) of 1,520 participants. EMD was strongly associated with AMD (p = 2.83 × 10-63, odds ratio [OR] 7.63). After adjustment for AMD, age (p = 0.06, OR 1.02), female gender (p = 3.34 × 10-24, OR 4.44), history of sunlight exposure ≥ 8 h /day (p = 0.0004, OR 1.99), serum complement activation (p = 0.004, OR 1.61), and polymorphisms in ARMS2 (p = 0.00016, OR 1.43) and CFI (p = 0.043, OR 1.20) were identified as risk factors for EMD. The final prediction model including these variants showed an area under the curve of 0.820.

Conclusions: The comprehensive analysis of various risk factors revealed a common genetic and pathological pathway of EMD with AMD. Future longitudinal studies are needed to evaluate the role of EMD in otherwise healthy subjects as an expanded phenotype of AMD.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553721PMC
July 2021

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium.

Nutrients 2020 Oct 6;12(10). Epub 2020 Oct 6.

Wellcome Wolfson Institute for Experimental Medicine, Queen's University of Belfast, Belfast BT97BL, Northern Ireland, UK.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.
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http://dx.doi.org/10.3390/nu12103051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601425PMC
October 2020

Development of a Genotype Assay for Age-Related Macular Degeneration: The EYE-RISK Consortium.

Ophthalmology 2020 Jul 25. Epub 2020 Jul 25.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

Purpose: To develop a genotype assay to assess associations with common and rare age-related macular degeneration (AMD) risk variants, to calculate an overall genetic risk score (GRS), and to identify potential misdiagnoses with inherited macular dystrophies that mimic AMD.

Design: Case-control study.

Participants: Individuals (n = 4740) from 5 European cohorts.

Methods: We designed single-molecule molecular inversion probes for target selection and used next generation sequencing to sequence 87 single nucleotide polymorphisms (SNPs), coding and splice-site regions of 10 AMD-(related) genes (ARMS2, C3, C9, CD46, CFB, CFH, CFI, HTRA1, TIMP3, and SLC16A8), and 3 genes that cause inherited macular dystrophies (ABCA4, CTNNA1, and PRPH2). Genetic risk scores for common AMD risk variants were calculated based on effect size and genotype of 52 AMD-associated variants. Frequency of rare variants was compared between late AMD patients and control individuals with logistic regression analysis.

Main Outcome Measures: Genetic risk score, association of genetic variants with AMD, and genotype-phenotype correlations.

Results: We observed high concordance rates between our platform and other genotyping platforms for the 69 successfully genotyped SNPs (>96%) and for the rare variants (>99%). We observed a higher GRS for patients with late AMD compared with patients with early/intermediate AMD (P < 0.001) and individuals without AMD (P < 0.001). A higher proportion of pathogenic variants in the CFH (odds ratio [OR] = 2.88; P = 0.006), CFI (OR = 4.45; P = 0.005), and C3 (OR = 6.56; P = 0.0003) genes was observed in late AMD patients compared with control individuals. In 9 patients, we identified pathogenic variants in the PRPH2, ABCA4, and CTNNA1 genes, which allowed reclassification of these patients as having inherited macular dystrophy.

Conclusions: This study reports a genotype assay for common and rare AMD genetic variants, which can identify individuals at intermediate to high genetic risk of late AMD and enables differential diagnosis of AMD-mimicking dystrophies. Our study supports sequencing of CFH, CFI, and C3 genes because they harbor rare high-risk variants. Carriers of these variants could be amendable for new treatments for AMD that currently are under development.
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http://dx.doi.org/10.1016/j.ophtha.2020.07.037DOI Listing
July 2020

Integrating Metabolomics, Genomics, and Disease Pathways in Age-Related Macular Degeneration: The EYE-RISK Consortium.

Ophthalmology 2020 12 14;127(12):1693-1709. Epub 2020 Jun 14.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

Purpose: The current study aimed to identify metabolites associated with age-related macular degeneration (AMD) by performing the largest metabolome association analysis in AMD to date, as well as aiming to determine the effect of AMD-associated genetic variants on metabolite levels and investigate associations between the identified metabolites and activity of the complement system, one of the main AMD-associated disease pathways.

Design: Case-control association analysis of metabolomics data.

Participants: Five European cohorts consisting of 2267 AMD patients and 4266 control participants.

Methods: Metabolomics was performed using a high-throughput proton nuclear magnetic resonance metabolomics platform, which allows quantification of 146 metabolite measurements and 79 derivative values. Metabolome-AMD associations were studied using univariate logistic regression analyses. The effect of 52 AMD-associated genetic variants on the identified metabolites was investigated using linear regression. In addition, associations between the identified metabolites and activity of the complement pathway (defined by the C3d-to-C3 ratio) were investigated using linear regression.

Main Outcome Measures: Metabolites associated with AMD.

Results: We identified 60 metabolites that were associated significantly with AMD, including increased levels of large and extra-large high-density lipoprotein (HDL) subclasses and decreased levels of very low-density lipoprotein (VLDL), amino acids, and citrate. Of 52 AMD-associated genetic variants, 7 variants were associated significantly with 34 of the identified metabolites. The strongest associations were identified for genetic variants located in or near genes involved in lipid metabolism (ABCA1, CETP, APOE, and LIPC) with metabolites belonging to the large and extra-large HDL subclasses. Also, 57 of 60 metabolites were associated significantly with complement activation levels, independent of AMD status. Increased large and extra-large HDL levels and decreased VLDL and amino acid levels were associated with increased complement activation.

Conclusions: Lipoprotein levels were associated with AMD-associated genetic variants, whereas decreased essential amino acids may point to nutritional deficiencies in AMD. We observed strong associations between the vast majority of the AMD-associated metabolites and systemic complement activation levels, independent of AMD status. This may indicate biological interactions between the main AMD disease pathways and suggests that multiple pathways may need to be targeted simultaneously for successful treatment of AMD.
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http://dx.doi.org/10.1016/j.ophtha.2020.06.020DOI Listing
December 2020

Effect of rare coding variants in the CFI gene on Factor I expression levels.

Hum Mol Genet 2020 08;29(14):2313-2324

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.
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http://dx.doi.org/10.1093/hmg/ddaa114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424754PMC
August 2020

Genes Involved in Energy Metabolism Are Differentially Expressed During the Day-Night Cycle in Murine Retinal Pigment Epithelium.

Invest Ophthalmol Vis Sci 2020 05;61(5):49

,.

Purpose: The functional interaction between photoreceptors and retinal pigment epithelium (RPE) cells is essential for vision. Phagocytosis of photoreceptor outer segments (POSs) by the RPE follows a circadian pattern; however, it remains unknown whether other RPE processes follow a daily rhythm. Therefore, our aim was to identify RPE processes following a daily rhythm.

Methods: Murine RPE was isolated at Zeitgeber time (ZT) 0, 2, 4, 9, 14, and 19 (n = 5 per time point), after which RNA was isolated and sequenced. Genes with a significant difference in expression between time points (P < 0.05) were subjected to EnrichR pathway analysis to identify daily rhythmic processes.

Results: Pathway enrichment revealed 13 significantly enriched KEGG pathways (P < 0.01), including the metabolic pathway (P = 0.002821). Analysis of the metabolic pathway differentially expressed genes revealed that genes involved in adenosine triphosphate production, glycolysis, glycogenolysis, and glycerophospholipid were low at ZT0 (light onset) and high at ZT19 (night). Genes involved in fatty acid degradation and cholesterol synthesis were high at light onset and low at night.

Conclusions: Our transcriptome data suggest that the highest energy demand of RPE cells is at night, whereas POS phagocytosis and degradation take place in the morning. Furthermore, we identified genes involved in fatty acid and glycerophospholipid synthesis that are upregulated at night, possibly playing a role in generating building blocks for membrane synthesis.
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http://dx.doi.org/10.1167/iovs.61.5.49DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405837PMC
May 2020

Genotype- and Phenotype-Based Subgroups in Geographic Atrophy Secondary to Age-Related Macular Degeneration: The EYE-RISK Consortium.

Ophthalmol Retina 2020 12 1;4(12):1129-1137. Epub 2020 May 1.

Barcelona Macula Foundation, Barcelona, Spain; Institut de la Màcula, Hospital Quirón Teknon, Barcelona, Spain.

Purpose: Geographic atrophy (GA) secondary to age-related macular degeneration is considered a single entity. This study aimed to determine whether GA subgroups exist that can be defined by their genotype and phenotype.

Design: Retrospective analysis of cross-sectional data.

Participants: Individuals (196 eyes of 196 patients) 50 years of age or older with GA from the EYE-RISK database.

Methods: Participants were graded for the presence of each of the following fundus features on color fundus photography: large soft drusen, reticular pseudodrusen (RPD), refractile drusen, hyperpigmentation, location of atrophy (foveal vs. extrafoveal), and multifocal lesions. Genotypes of 33 single nucleotide polymorphisms previously assigned to the complement, lipid metabolism, or extracellular matrix (ECM) pathways and ARMS2 also were included, and genetic risk scores (GRSs) for each of those 3 pathways were calculated. Hierarchical cluster analysis was used to determine subgroups of participants defined by these features. The discriminative ability of genotype, phenotype, or both for each subgroup was determined with 10-fold cross-validated areas under the receiver operating characteristic curve (cvAUCs), and the agreement between predicted and actual subgroup membership was assessed with calibration plots.

Main Outcome Measures: Identification and characterization of GA subgroups based on their phenotype and genotype.

Results: Cluster analyses identified 3 subgroups of GA. Subgroup 1 was characterized by high complement GRS, frequently associated with large soft drusen and foveal atrophy; subgroup 2 generally showed low GRS, foveal atrophy, and few drusen (any type); and subgroup 3 showed a high ARMS2 and ECM GRS, RPD, and extrafoveal atrophy. A high discriminative ability existed between subgroups for the genotype (cvAUC, ≥0.94), and a modest discriminative ability existed for the phenotype (cvAUC, <0.65), with good calibration.

Conclusions: We identified 3 GA subgroups that differed mostly by their genotype. Atrophy location and drusen type were the most relevant phenotypic features.
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http://dx.doi.org/10.1016/j.oret.2020.04.014DOI Listing
December 2020

Family-based exome sequencing identifies rare coding variants in age-related macular degeneration.

Hum Mol Genet 2020 07;29(12):2022-2034

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen 6500, The Netherlands.

Genome-wide association studies (GWAS) have identified 52 independent variants at 34 genetic loci that are associated with age-related macular degeneration (AMD), the most common cause of incurable vision loss in the elderly worldwide. However, causal genes at the majority of these loci remain unknown. In this study, we performed whole exome sequencing of 264 individuals from 63 multiplex families with AMD and analyzed the data for rare protein-altering variants in candidate target genes at AMD-associated loci. Rare coding variants were identified in the CFH, PUS7, RXFP2, PHF12 and TACC2 genes in three or more families. In addition, we detected rare coding variants in the C9, SPEF2 and BCAR1 genes, which were previously suggested as likely causative genes at respective AMD susceptibility loci. Identification of rare variants in the CFH and C9 genes in our study validated previous reports of rare variants in complement pathway genes in AMD. We then extended our exome-wide analysis and identified rare protein-altering variants in 13 genes outside the AMD-GWAS loci in three or more families. Two of these genes, SCN10A and KIR2DL4, are of interest because variants in these genes also showed association with AMD in case-control cohorts, albeit not at the level of genome-wide significance. Our study presents the first large-scale, exome-wide analysis of rare variants in AMD. Further independent replications and molecular investigation of candidate target genes, reported here, would assist in gaining novel insights into mechanisms underlying AMD pathogenesis.
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http://dx.doi.org/10.1093/hmg/ddaa057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390936PMC
July 2020

Complement Activation Levels Are Related to Disease Stage in AMD.

Invest Ophthalmol Vis Sci 2020 03;61(3):18

,.

Purpose: To study the levels of complement activation in different disease stages of AMD and the influence of genetic polymorphisms in complement genes.

Methods: We included 797 patients with AMD and 945 controls from the European Genetic Database. Patients were grouped into five AMD stages: early AMD, intermediate AMD, central geographic atrophy, active choroidal neovascularization or inactive choroidal neovascularization. Differences in complement activation, as defined by the systemic C3d/C3 ratio, between AMD stages were evaluated using general linear modeling. In addition, we evaluated the influence of 18 genetic AMD polymorphisms in complement genes and their effect on complement activation. Differences in complement activation between stages were evaluated stratifying by complement associated haplotypes.

Results: Complement activation levels differed significantly between AMD disease stages. As compared with controls, the C3d/C3 ratio was higher in patients with intermediate AMD (P < 0.001) and central geographic atrophy (P = 0.001). Two polymorphisms in CFH (rs10922109 and rs570618) and one in CFB (rs116503776) were significantly associated with complement activation. The association between AMD disease stage and complement activation was more pronounced in patients with haplotypes associated with the highest complement activation.

Conclusions: In general, consecutive AMD disease stages showed increasing levels of complement activation, especially in individuals with a genetic burden in complement genes. These findings contribute to the discussion on the pathogenesis of AMD in relation to complement activation and might suggest refinement in patient selection and the optimum window of treatment with complement inhibitors. Prospective studies are needed to confirm these results.
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http://dx.doi.org/10.1167/iovs.61.3.18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401663PMC
March 2020

Major Predictive Factors for Progression of Early to Late Age-Related Macular Degeneration.

Ophthalmologica 2020 13;243(6):444-452. Epub 2020 Mar 13.

Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany.

Introduction: We present a prediction model for progression from early/intermediate to advanced age-related macular degeneration (AMD) within 5.9 years.

Objectives: To evaluate the combined role of genetic, nongenetic, and phenotypic risk factors for conversion from early to late AMD over ≥5 years.

Methods: Baseline phenotypic characteristics were evaluated based on color fundus photography, spectral-domain optical coherence tomography, and infrared images. Genotyping for 36 single-nucleotide polymorphisms as well as systemic lipid and complement measurements were performed. Multivariable backward logistic regression resulted in a final prediction model.

Results And Conclusions: During a mean of 5.9 years of follow-up, 22.4% (n = 52) of the patients (n = 232) showed progression to late AMD. The multivariable prediction model included age, CFH variant rs1061170, pigment abnormalities, drusenoid pigment epithelial detachment (DPED), and hyperreflective foci (HRF). The model showed an area under the curve of 0.969 (95% confidence interval 0.948-0.990) and adequate calibration (Hosmer-Lemeshow test, p = 0.797). In addition to advanced age and carrying a CFH variant, pigment abnormalities, DPED, and HRF are relevant imaging biomarkers for conversion to late AMD. In clinical routine, an intensified monitoring of patients with a high-risk phenotypic profile may be suitable for the early detection of conversion to late AMD.
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http://dx.doi.org/10.1159/000507196DOI Listing
August 2021

Risk factors for progression of age-related macular degeneration.

Ophthalmic Physiol Opt 2020 03 25;40(2):140-170. Epub 2020 Feb 25.

Departments of, Department of, Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: Age-related macular degeneration (AMD) is a degenerative disease of the macula, often leading to progressive vision loss. The rate of disease progression can vary among individuals and has been associated with multiple risk factors. In this review, we provide an overview of the current literature investigating phenotypic, demographic, environmental, genetic, and molecular risk factors, and propose the most consistently identified risk factors for disease progression in AMD based on these studies. Finally, we describe the potential use of these risk factors for personalised healthcare.

Recent Findings: While phenotypic risk factors such as drusen and pigment abnormalities become more important to predict disease progression during the course of the disease, demographic, environmental, genetic and molecular risk factors are more valuable at earlier disease stages. Demographic and environmental risk factors such as age and smoking are consistently reported to be related to disease progression, while other factors such as sex, body mass index (BMI) and education are less often associated. Of all known AMD variants, variants that are most consistently reported with disease progression are rs10922109 and rs570618 in CFH, rs116503776 in C2/CFB/SKIV2L, rs3750846 in ARMS2/HTRA1 and rs2230199 in C3. However, it seems likely that other AMD variants also contribute to disease progression but to a lesser extent. Rare variants have probably a large effect on disease progression in highly affected families. Furthermore, current prediction models do not include molecular risk factors, while these factors can be measured accurately in the blood. Possible promising molecular risk factors are High-Density Lipoprotein Cholesterol (HDL-C), Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), zeaxanthin and lutein.

Summary: Phenotypic, demographic, environmental, genetic and molecular risk factors can be combined in prediction models to predict disease progression, but the selection of the proper risk factors for personalised risk prediction will differ among individuals and is dependent on their current disease stage. Future prediction models should include a wider set of genetic variants to determine the genetic risk more accurately, and rare variants should be taken into account in highly affected families. In addition, adding molecular factors in prediction models may lead to preventive strategies and personalised advice.
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http://dx.doi.org/10.1111/opo.12675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155063PMC
March 2020

Differential day-night expression of tight junction components in murine retinal pigment epithelium.

Exp Eye Res 2020 04 21;193:107985. Epub 2020 Feb 21.

Dept of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands. Electronic address:

Strong communication and interaction between the retinal pigment epithelium (RPE) and the photoreceptor (PR) cells is essential for vision. RPE cells are essential for supporting and maintaining PR cells by transporting nutrients, waste products and ions, and phagocytosing photoreceptor outer segments (POS). POS phagocytosis follows a circadian pattern, taking place in the morning in human, mice and other organisms. However, it remains unknown whether other RPE processes follow a daily rhythm. To study the daily rhythm of RPE cells, we isolated murine RPE cells at six different time points during a 24 h period, after which RNA was isolated and sequenced. Murine RPE flatmounts were isolated at four different time points to study daily rhythm in protein abundance and localisation. EnrichR pathway analysis resulted in 13 significantly-enriched KEGG pathways (p < 0.01) of which seven showed a large number of overlapping genes. Several genes were involved in intracellular trafficking, possibly playing a role in nutrient transport, POS phagocytosis or membrane protein trafficking, with different expression patterns during the day-night cycle. Other genes were involved in actin cytoskeleton building, remodelling and crosslinking and showed a high expression in the morning, suggesting actin cytoskeleton remodelling at this time point. Finally, tight junction proteins Cldn2 and Cldn4 showed a difference in RNA and protein expression and tight junction localisation over time. Our study suggests that several important processes in the RPE follow a day-night rhythm, including intracellular trafficking, and processes involving the actin cytoskeleton and tight junctions. The differential protein localisation of Cldn2 in the RPE during the day-night cycle suggest that Cldn2 may facilitate paracellular water and sodium transport during the day.
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http://dx.doi.org/10.1016/j.exer.2020.107985DOI Listing
April 2020

Increased circulating levels of Factor H-Related Protein 4 are strongly associated with age-related macular degeneration.

Nat Commun 2020 02 7;11(1):778. Epub 2020 Feb 7.

Division of Evolution and Genomic Sciences, Faculty of Biology Medicine and Health, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value = 7.1 × 10), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value = 2.2 × 10), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD.
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http://dx.doi.org/10.1038/s41467-020-14499-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005798PMC
February 2020

Genome-wide association analyses identify two susceptibility loci for pachychoroid disease central serous chorioretinopathy.

Commun Biol 2019 12;2:468. Epub 2019 Dec 12.

Department of Ophthalmology, Donders Institute of Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands.

The recently emerged pachychoroid concept has changed the understanding of age-related macular degeneration (AMD), which is a major cause of blindness; recent studies attributed AMD in part to pachychoroid disease central serous chorioretinopathy (CSC), suggesting the importance of elucidating the CSC pathogenesis. Our large genome-wide association study followed by validation studies in three independent Japanese and European cohorts, consisting of 1546 CSC samples and 13,029 controls, identified two novel CSC susceptibility loci: and near (rs13278062, odds ratio = 1.35,  = 1.26 × 10; rs6061548, odds ratio = 1.63,  = 5.36 × 10). A T allele at rs13278062, a risk allele for CSC, is known to be a risk allele for AMD. This study not only identified new susceptibility genes for CSC, but also improves the understanding of the pathogenesis of AMD.
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http://dx.doi.org/10.1038/s42003-019-0712-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908630PMC
July 2020

GENETIC RISK FACTORS IN SEVERE, NONSEVERE AND ACUTE PHENOTYPES OF CENTRAL SEROUS CHORIORETINOPATHY.

Retina 2020 Sep;40(9):1734-1741

Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.

Purpose: To study genetic predispositions and differences between severe chronic central serous chorioretinopathy (cCSC), nonsevere cCSC, and acute central serous chorioretinopathy (aCSC).

Methods: One hundred seventy-three severe cCSC patients, 272 nonsevere cCSC patients, 135 aCSC patients, and 1,385 control individuals were included. Eight single-nucleotide polymorphisms were genotyped in the ARMS2 (rs10490924), CFH (rs800292, rs1061170, rs1065489, rs1329428, rs2284664, rs3753394), and NR3C2 (rs2070951). Additionally, C4B gene copy numbers were analyzed.

Results: A significant association in 5 single-nucleotide polymorphisms in the CFH gene could be reproduced among severe cCSC patients, including rs800292 (P = 0.0014; odds ratio [OR] = 1.93; 95% confidence interval [CI] = 1.51-2.47), rs1065489 (P = 2.22 × 10; OR = 0.49; 95% CI = 0.34-0.72), rs1329428 (P = 0.001; OR = 1.89; 95% CI = 1.49-2.40), rs2284664 (P = 1.21× 10; OR = 1.65; 95% CI = 1.28-2.13), and rs3753394 (P = 6.10× 10; OR = 0.61; 95% CI = 0.46-0.81). Carrying three C4B copies was protective for severe cCSC (P = 0.001; OR = 0.29; 95% CI = 0.14-0.61). No significant differences in allele frequencies could be found among the CSC phenotypes.

Conclusion: Acute CSC, nonsevere cCSC, and severe cCSC all showed a similar association with the CFH and C4B genes, and the three phenotypes could not be distinguished based on the genetics. This shows that despite the differences in clinical presentation and severity, there is an overlap in the genetic predisposition of different CSC phenotypes. Nongenetic factors may play a more important role in determining the clinical course of CSC.
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http://dx.doi.org/10.1097/IAE.0000000000002682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447124PMC
September 2020

CEP290 Mutation Spectrum and Delineation of the Associated Phenotype in a Large German Cohort: A Monocentric Study.

Am J Ophthalmol 2020 03 14;211:142-150. Epub 2019 Nov 14.

Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen, Germany. Electronic address:

Purpose: Gene therapy for Leber congenital amaurosis (LCA) is becoming available, and therefore it is crucial to identify eligible candidates. We report the spectrum and associated phenotype of CEP290 mutations in the largest German cohort observed by a single clinical site.

Design: Prospective cohort study.

Methods: Twenty-three patients with mutations in CEP290 were included. Genomic DNA was analyzed by Sanger sequencing or high-throughput sequencing for all retinitis pigmentosa-associated genes in patients, and segregation analysis was done in family members. Patients underwent functional and morphologic examinations, including fundus autofluorescence and spectral-domain optical coherence tomography.

Results: The most frequent mutation was c.2991+1655A>G, found in 87% of patients (20/23). Thirty percent of patients (7/23) carried the mutation in an apparent homozygous state and 57% (13/23) in a likely compound heterozygous state. The most common clinical diagnosis was LCA and/or early onset severe retinal dystrophy in 82% (19/23), followed by retinitis pigmentosa in 14% (3/23) and cone-rod dystrophy (4%, 1/23). Best-corrected visual acuity was severely reduced to residual light perception and hand motion vision, with the exception of 3 patients with best-corrected visual acuity of 0.8 (Snellen). The visual field was severely decreased and electroretinogram was undetectable in most cases; however, retinal layers at the fovea appeared to be relatively well preserved. Systemic disorders were not noticed.

Conclusions: c.2991+1655A>G is by far the most important CEP290 mutation, contributing to 87% of patients with the CEP290 mutation in Germany. In our cohort, a homozygous c.2991+1655A>G genotype presented with a more severe phenotype. National studies and further detailed phenotype analysis seem to be important to assess the need for and promise of specific gene therapies.
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http://dx.doi.org/10.1016/j.ajo.2019.11.012DOI Listing
March 2020
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