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1.  Zinc Supplementation Inhibits Complement Activation in Age-Related Macular Degeneration 
PLoS ONE  2014;9(11):e112682.
Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. AMD is a multifactorial disorder but complement-mediated inflammation at the level of the retina plays a pivotal role. Oral zinc supplementation can reduce the progression of AMD but the precise mechanism of this protective effect is as yet unclear. We investigated whether zinc supplementation directly affects the degree of complement activation in AMD and whether there is a relation between serum complement catabolism during zinc administration and the complement factor H (CFH) gene or the Age-Related Maculopathy susceptibility 2 (ARMS2) genotype. In this open-label clinical study, 72 randomly selected AMD patients in various stages of AMD received a daily supplement of 50 mg zinc sulphate and 1 mg cupric sulphate for three months. Serum complement catabolism–defined as the C3d/C3 ratio–was measured at baseline, throughout the three months of supplementation and after discontinuation of zinc administration. Additionally, downstream inhibition of complement catabolism was evaluated by measurement of anaphylatoxin C5a. Furthermore, we investigated the effect of zinc on complement activation in vitro. AMD patients with high levels of complement catabolism at baseline exhibited a steeper decline in serum complement activation (p<0.001) during the three month zinc supplementation period compared to patients with low complement levels. There was no significant association of change in complement catabolism and CFH and ARMS2 genotype. In vitro zinc sulphate directly inhibits complement catabolism in hemolytic assays and membrane attack complex (MAC) deposition on RPE cells. This study provides evidence that daily administration of 50 mg zinc sulphate can inhibit complement catabolism in AMD patients with increased complement activation. This could explain part of the mechanism by which zinc slows AMD progression.
Trial Registration
The Netherlands National Trial Register NTR2605
doi:10.1371/journal.pone.0112682
PMCID: PMC4231060  PMID: 25393287
2.  A Circulating MicroRNA Profile Is Associated with Late-Stage Neovascular Age-Related Macular Degeneration 
PLoS ONE  2014;9(9):e107461.
Age-related macular degeneration (AMD) is the leading cause of severe vision impairment in Western populations over 55 years. A growing number of gene variants have been identified which are strongly associated with an altered risk to develop AMD. Nevertheless, gene-based biomarkers which could be dysregulated at defined stages of AMD may point toward key processes in disease mechanism and thus may support efforts to design novel treatment regimens for this blinding disorder. Circulating microRNAs (cmiRNAs) which are carried by nanosized exosomes or microvesicles in blood plasma or serum, have been recognized as valuable indicators for various age-related diseases. We therefore aimed to elucidate the role of cmiRNAs in AMD by genome-wide miRNA expression profiling and replication analyses in 147 controls and 129 neovascular AMD patients. We identified three microRNAs differentially secreted in neovascular (NV) AMD (hsa-mir-301-3p, pcorrected = 5.6*10−5, hsa-mir-361-5p, pcorrected = 8.0*10−4 and hsa-mir-424-5p, pcorrected = 9.6*10−3). A combined profile of the three miRNAs revealed an area under the curve (AUC) value of 0.727 and was highly associated with NV AMD (p = 1.2*10−8). To evaluate subtype-specificity, an additional 59 AMD cases with pure unilateral or bilateral geographic atrophy (GA) were analyzed for microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p. While we found no significant differences between GA AMD and controls neither individually nor for a combined microRNAs profile, hsa-mir-424-5p levels remained significantly higher in GA AMD when compared to NV (pcorrected<0.005). Pathway enrichment analysis on genes predicted to be regulated by microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p, suggests canonical TGFβ, mTOR and related pathways to be involved in NV AMD. In addition, knockdown of hsa-mir-361-5p resulted in increased neovascularization in an in vitro angiogenesis assay.
doi:10.1371/journal.pone.0107461
PMCID: PMC4159338  PMID: 25203061
3.  Nutritional Risk Factors for Age-Related Macular Degeneration 
BioMed Research International  2014;2014:413150.
Purpose. To evaluate the role of nutritional factors, serum lipids, and lipoproteins in late age-related macular degeneration (late AMD). Methods. Intake of red meat, fruit, fish, vegetables, and alcohol, smoking status, and body mass index (BMI) were ascertained questionnaire-based in 1147 late AMD cases and 1773 controls from the European Genetic Database. Serum levels of lipids and lipoproteins were determined. The relationship between nutritional factors and late AMD was assessed using logistic regression. Based on multivariate analysis, area-under-the-curve (AUC) was calculated by receiver-operating-characteristics (ROC). Results. In a multivariate analysis, besides age and smoking, obesity (odds ratio (OR): 1.44, P = 0.014) and red meat intake (daily: OR: 2.34, P = 8.22 × 10−6; 2–6x/week: OR: 1.67, P = 7.98 × 10−5) were identified as risk factors for developing late AMD. Fruit intake showed a protective effect (daily: OR: 0.52, P = 0.005; 2–6x/week: OR: 0.58, P = 0.035). Serum lipid and lipoprotein levels showed no significant association with late AMD. ROC for nutritional factors, smoking, age, and BMI revealed an AUC of 0.781. Conclusion. Red meat intake and obesity were independently associated with increased risk for late AMD, whereas fruit intake was protective. A better understanding of nutritional risk factors is necessary for the prevention of AMD.
doi:10.1155/2014/413150
PMCID: PMC4101976  PMID: 25101280
4.  Analysis of Rare Variants in the C3 Gene in Patients with Age-Related Macular Degeneration 
PLoS ONE  2014;9(4):e94165.
Age-related macular degeneration (AMD) is a progressive retinal disorder affecting over 33 million people worldwide. Genome-wide association studies (GWASs) for AMD identified common variants at 19 loci accounting for 15–65% of the heritability and it has been hypothesized that the missing heritability may be attributed to rare variants with large effect sizes. Common variants in the complement component 3 (C3) gene have been associated with AMD and recently a rare C3 variant (Lys155Gln) was identified which exerts a large effect on AMD susceptibility independent of the common variants. To explore whether additional rare variants in the C3 gene are associated with AMD, we sequenced all coding exons in 84 unrelated AMD cases. Subsequently, we genotyped all identified variants in 1474 AMD cases and 2258 controls. Additionally, because of the known genetic overlap between AMD and atypical hemolytic uremic syndrome (aHUS), we genotyped two recurrent aHUS-associated C3 mutations in the entire cohort. Overall, we identified three rare variants (Lys65Gln (P = 0.04), Arg735Trp (OR = 17.4, 95% CI = 2.2–136; P = 0.0003), and Ser1619Arg (OR = 5.2, 95% CI = 1.0–25; P = 0.05) at the C3 locus that are associated with AMD in our EUGENDA cohort. However, the Arg735Trp and Ser1619Arg variants were not found to be associated with AMD in the Rotterdam Study. The Lys65Gln variant was only identified in patients from Nijmegen, the Netherlands, and thus may represent a region-specific AMD risk variant.
doi:10.1371/journal.pone.0094165
PMCID: PMC3988049  PMID: 24736606
5.  Impact of the Common Genetic Associations of Age-Related Macular Degeneration upon Systemic Complement Component C3d Levels 
PLoS ONE  2014;9(3):e93459.
Age-related macular degeneration (AMD) is a common condition that leads to severe vision loss and dysregulation of the complement system is thought to be associated with the disease. To investigate associations of polymorphisms in AMD susceptibility genes with systemic complement activation, 2655 individuals were genotyped for 32 single nucleotide polymorphisms (SNPs) in or near 23 AMD associated risk genes. Component 3 (C3) and its catabolic fragment C3d were measured in serum and AMD staging was performed using multimodal imaging. The C3d/C3 ratio was calculated and associations with environmental factors, SNPs and various haplotypes of complement factor H (CFH) genes and complement factor B (CFB) genes were analyzed. Linear models were built to measure the influence of genetic variants on the C3d/C3 ratio. The study cohort included 1387 patients with AMD and 1268 controls. Higher C3d/C3 ratios were found for current smoker (p = 0.002), higher age (p = 1.56×10−7), AMD phenotype (p = 1.15×10−11) and the two SNPs in the C3 gene rs6795735 (p = 0.04) and rs2230199 (p = 0.04). Lower C3d/C3 ratios were found for diabetes (p = 2.87×10−6), higher body mass index (p = 1.00×10−13), the SNPs rs1410996 (p = 0.0001), rs800292 (p = 0.003), rs12144939 (p = 4.60×10−6) in CFH, rs4151667 (p = 1.01×10−5) in CFB and individual haplotypes in CFH and CFB. The linear model revealed a corrected R-square of 0.063 including age, smoking status, gender, and genetic polymorphisms explaining 6.3% of the C3d/C3 ratio. After adding the AMD status the corrected R-square was 0.067. In conclusion, none of the evaluated genetic polymorphisms showed an association with increased systemic complement activation apart from two SNPs in the C3 gene. Major genetic and non-genetic factors for AMD were not associated with systemic complement activation.
doi:10.1371/journal.pone.0093459
PMCID: PMC3968152  PMID: 24675670
6.  A Novel Large In-Frame Deletion within the CACNA1F Gene Associates with a Cone-Rod Dystrophy 3-Like Phenotype 
PLoS ONE  2013;8(10):e76414.
Cone-rod dystrophies (CORDs) represent a heterogeneous group of monogenic diseases leading to early impairment of vision. The majority of CORD entities show autosomal modes of inheritance and X-linked traits are comparably rare. So far, three X-chromosomal entities were reported (CORDX1, -X2 and -X3). In this study, we analysed a large family of German origin with solely affected males over three generations showing a CORDX-like phenotype. Due to the heterogeneity of cone-rod dystrophies, we performed a combined linkage and X-exome sequencing approach and identified a novel large intragenic in-frame deletion encompassing exons 18 to 26 within the CACNA1F gene. CACNA1F is described causative for CORDX3 in a single family originating from Finland and alterations in this gene have not yet been reported in other CORDX pedigrees. Our data independently confirm CACNA1F as the causative gene for CORDX3-like phenotypes and detailed clinical characterization of the family expands the knowledge about the phenotypic spectrum of deleterious CACNA1F alterations.
doi:10.1371/journal.pone.0076414
PMCID: PMC3790679  PMID: 24124559
7.  Rare Primary Mitochondrial DNA Mutations and Probable Synergistic Variants in Leber’s Hereditary Optic Neuropathy 
PLoS ONE  2012;7(8):e42242.
Background
Leber’s hereditary optic neuropathy (LHON) is a maternally inherited blinding disorder, which in over 90% of cases is due to one of three primary mitochondrial DNA (mtDNA) point mutations (m.11778G>A, m.3460G>A and m.14484T>C, respectively in MT-ND4, MT-ND1 and MT-ND6 genes). However, the spectrum of mtDNA mutations causing the remaining 10% of cases is only partially and often poorly defined.
Methodology/Principal Findings
In order to improve such a list of pathological variants, we completely sequenced the mitochondrial genomes of suspected LHON patients from Italy, France and Germany, lacking the three primary common mutations. Phylogenetic and conservation analyses were performed. Sixteen mitochondrial genomes were found to harbor at least one of the following nine rare LHON pathogenic mutations in genes MT-ND1 (m.3700G>A/p.A132T, m.3733G>A-C/p.E143K-Q, m.4171C>A/p.L289M), MT-ND4L (m.10663T>C/p.V65A) and MT-ND6 (m.14459G>A/p.A72V, m.14495A>G/p.M64I, m.14482C>A/p.L60S, and m.14568C>T/p.G36S). Phylogenetic analyses revealed that these substitutions were due to independent events on different haplogroups, whereas interspecies comparisons showed that they affected conserved amino acid residues or domains in the ND subunit genes of complex I.
Conclusions/Significance
Our findings indicate that these nine substitutions are all primary LHON mutations. Therefore, despite their relative low frequency, they should be routinely tested for in all LHON patients lacking the three common mutations. Moreover, our sequence analysis confirms the major role of haplogroups J1c and J2b (over 35% in our probands versus 6% in the general population of Western Europe) and other putative synergistic mtDNA variants in LHON expression.
doi:10.1371/journal.pone.0042242
PMCID: PMC3411744  PMID: 22879922
8.  Complement Factor D in Age-Related Macular Degeneration 
Complement factor D catalyzes a critical step in the alternative complement activation pathway. The authors report a significant elevation in plasma CFD concentrations in age-related macular degeneration (AMD) patients compared with controls and a weak genetic association between CFD gene variants and AMD.
Purpose.
To examine the role of complement factor D (CFD) in age-related macular degeneration (AMD) by analysis of genetic association, copy number variation, and plasma CFD concentrations.
Methods.
Single nucleotide polymorphisms (SNPs) in the CFD gene were genotyped and the results analyzed by binary logistic regression. CFD gene copy number was analyzed by gene copy number assay. Plasma CFD was measured by an enzyme-linked immunosorbent assay.
Results.
Genetic association was found between CFD gene SNP rs3826945 and AMD (odds ratio 1.44; P = 0.028) in a small discovery case-control series (462 cases and 325 controls) and replicated in a combined cohorts meta-analysis of 4765 cases and 2693 controls, with an odds ratio of 1.11 (P = 0.032), with the association almost confined to females. Copy number variation in the CFD gene was identified in 13 out of 640 samples examined but there was no difference in frequency between AMD cases (1.3%) and controls (2.7%). Plasma CFD concentration was measured in 751 AMD cases and 474 controls and found to be elevated in AMD cases (P = 0.00025). The odds ratio for those in the highest versus lowest quartile for plasma CFD was 1.81. The difference in plasma CFD was again almost confined to females.
Conclusions.
CFD regulates activation of the alternative complement pathway, which is implicated in AMD pathogenesis. The authors found evidence for genetic association between a CFD gene SNP and AMD and a significant increase in plasma CFD concentration in AMD cases compared with controls, consistent with a role for CFD in AMD pathogenesis.
doi:10.1167/iovs.11-7933
PMCID: PMC3230905  PMID: 22003108
9.  Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components 
PLoS ONE  2011;6(8):e22959.
Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3.
doi:10.1371/journal.pone.0022959
PMCID: PMC3149070  PMID: 21829675
10.  In vitro emulsification assessment of new silicone oils 
Aim
To investigate whether the emulsification of conventional silicone oils can be reduced by adding small amounts of silicone molecules of a very long chain length.
Methods
Siluron 1000, Siluron 2000, Siluron 5000, Acri.Sil-Ol 5000, Oxane 5700, Densiron 68 LV, Densiron 68 and Densiron 68 HV (0.5 ml) were each tested along with either plasma or serum (0.5 ml) in a glass cuvette. Emulsification was induced by sonication and documented by photography. The total area of emulsified oil was assessed using the ImageJ software.
Results
The addition of small amounts of very-long-chain silicone molecules significantly reduced the emulsification for 1000 cSt silicone oil (Siluron 2000) and for 1000 cSt silicone oil with an admixture of F6H8 (Densiron 68 HV).
Conclusion
New low-viscosity silicone oils show a reduced emulsification similar to that of 5000 cSt oils. In future, it may be possible to avoid using 5000 cSt oils. The findings may foster silicone oil surgery in general, and in particular the application of small-incision techniques.
doi:10.1136/bjo.2009.170852
PMCID: PMC2976467  PMID: 19955199
Silicone oil; polydimethylsiloxane; emulsification; vitreous; treatment surgery
11.  Fundus autofluorescence and spectral-domain optical coherence tomography findings suggesting tissue remodelling in retinal pigment epithelium tear 
The British Journal of Ophthalmology  2012;96(9):1211-1216.
Aim
To study tissue remodelling and wound healing after retinal pigment epithelium (RPE) tears due to age-related macular degeneration.
Methods
Retrospective longitudinal study of 36 eyes (33 patients) with RPE tears. Imaging was performed using fundus autofluorescence (FAF) (λ=488 nm) and spectral-domain optical coherence tomography (SD-OCT). Presence of intraretinal hyper-reflective dots in SD-OCT, which correlated with hyperfluorescent dots in FAF, indicating RPE migration was studied. Morphology of subretinal mass and RPE layer integrity in the RPE denuded area over time were examined.
Results
7 of 36 eyes (19.4%) showed patchy or hazy hyperfluorescent areas in FAF, and the majority of eyes (83.3%) showed hyper-reflective dots, which possibly represent intraretinal RPE migration and hard exudates. Homogenous subretinal mass was encountered in about half of all cases. In one case (2.8%), the RPE layer proliferated and covered the defect.
Conclusions
SD-OCT and FAF showed a considerable amount of RPE proliferation, migration and repopulation. Intraretinal RPE migration did not form a functional RPE layer. A small defect might be repaired by cell proliferation. But this RPE proliferation is not sufficient to cover large defects.
doi:10.1136/bjophthalmol-2012-301750
PMCID: PMC3432490  PMID: 22826551
Retinal pigment epithelium tear; pigment migration; spectral-domain optical coherence tomography; fundus autofluorescence; macula; pharmacology; neovascularisation; drugs; clinical trial; treatment surgery
12.  Spectral-domain optical coherence tomography in subjects over 60 years of age, and its implications for designing clinical trials 
The British Journal of Ophthalmology  2012;96(10):1325-1330.
Aims
To study the variability of central retinal thickness (CRT), its concordance to the fellow eye, and the implications for designing future clinical trials using spectral-domain optical coherence tomography (SD-OCT).
Methods
Cross-sectional retrospective analysis of European Genetic Database. 632 eyes of 316 subjects over 60 years of age without macular pathology were examined using SD-OCT.
Results
Mean CRT was 280.22 µm and 281.02 µm for the right and left eyes, respectively. There was a strong concordance for all measured values between right and left eyes. Men had significantly thicker CRT than women. Variation up to 23 µm difference between both eyes was seen. To detect a change of at least 30 µm in CRT, a sample size of 90 or 176 per group is needed for a single-arm or double-arm study, respectively (α=0.05, power=0.80, no loss to follow up, assuming SD in future studies=100 µm).
Conclusions
Clinical trials using CRT as an endpoint are feasible in terms of sample size needed.
doi:10.1136/bjophthalmol-2012-301885
PMCID: PMC3595499  PMID: 22863948
Imaging; Macula; Retina
13.  Retinal layers measurements in healthy eyes and in eyes receiving silicone oil-based endotamponade 
Acta Ophthalmologica  2013;92(4):e292-e297.
Purpose
To characterize the concordance/symmetry of each retinal layers in individuals without macular pathology and to further characterize the localization of inner retinal thinning in eyes receiving silicone oil-based endotamponade.
Methods
Retinal layers of one hundred eyes of 50 individuals without macular pathology were imaged using spectral domain optical coherence tomography (SD-OCT) and manually segmented using ImageJ software (developed by Wayne Rasband, NIH, Bethesda, MD, USA). In the second part of the study, retrospective analysis of 3028 cases of pars plana vitrectomy in University Eye Hospital Cologne, Germany, was conducted, retrieving nine patients with silicone oil-based endotamponade with no macular condition interfering retinal layers measurements. These patients had retinal detachment not involving the macula due to various conditions. In these patients, retinal layer segmentation was performed and compared with the fellow eye.
Results
There is a moderate-to-high concordance for all retinal layers between the right and the left eye of the same individual. In eyes receiving silicone oil-based endotamponade, the inner retinal layers become subsequently thinner. Ganglion cell and inner plexiform layers contribute most to this thinning, that is, 0.537 ± 0.096 mm3 compared with 0.742 ± 0.117 mm3; p = 0.006. Outer retinal layers were not affected by silicone oil-based endotamponade (p = 0.439 for the differences of calculated outer retinal layers).
Conclusion
Ganglion cell and inner retinal layers become subsequently thinner after the use of silicone oil-based endotamponade. This study advocates the use of spectral domain optical coherence tomography for patient management with silicone oil endotamponade to early detect subsequent retinal thinning.
doi:10.1111/aos.12307
PMCID: PMC4153956  PMID: 24238324
retinal thickness; silicone oil; spectral domain optical coherence tomography

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