GWAS study using DNA pooling strategy identifies association of variant rs4910623 in OR52B4 gene with anti-VEGF treatment response in age-related macular degeneration

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Pooled DNA based GWAS to determine genetic association of SNPs with visual acuity (VA) outcome in anti-vascular endothelial growth factor (anti-VEGF) treated neovascular age-related macular degeneration (nAMD) patients. We performed pooled DNA based GWAS on 285 anti-VEGF treated nAMD patients using high density Illumina 4.3 M array. Primary outcome was change in VA in Early Treatment Diabetic Retinopathy Study (ETDRS) letters after 6 months of anti-VEGF treatment (patients who lost ≥5 ETDRS letters classified as non-responders and all remaining classified as responders). GWAS analysis identified 44 SNPs of interest: 37 with strong evidence of association (p < 9 × 10−8), 2 in drug resistance genes (p < 5 × 10−6) and 5 nonsynonymous changes (p < 1 × 10−4). In the validation phase, individual genotyping of 44 variants showed three SNPs (rs4910623 p = 5.6 × 10−5, rs323085 p = 6.5 × 10−4 and rs10198937 p = 1.30 × 10−3) remained associated with VA response at 6 months. SNP rs4910623 also associated with treatment response at 3 months (p = 1.5 × 10−3). Replication of these three SNPs in 376 patients revealed association of rs4910623 with poor VA response after 3 and 6 months of treatment (p = 2.4 × 10−3 and p = 3.5 × 10−2, respectively). Meta-analysis of both cohorts (673 samples) confirmed association of rs4910623 with poor VA response after 3 months (p = 1.2 × 10−5) and 6 months (p = 9.3 × 10−6) of treatment in nAMD patients

Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

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

Clinical correlation to differences in ranibizumab and aflibercept vascular endothelial growth factor suppression times

Aim To determine clinical correlations to intraocular vascular endothelial growth factor A (VEGF-A) suppression times (VSTs) on the treatment of neovascular age-related macular degeneration (nAMD) with ranibizumab (Lucentis) or aflibercept (Eylea). Methods Seven of 89 treatment-naive nAMD eyes showed persistent choroidal neovascular membrane (CNV) activity throughout a spectral domain optical coherence tomography (SD-OCT)-driven pro re nata (PRN) regimen of intravitreal ranibizumab injections over 284months. The treatment was switched to PRN aflibercept injections and patients were followed for another 152months. A total of 160 aqueous humour specimens were collected before the intravitreal injections, and their VEGF-A concentrations were assayed by Luminex multiplex bead analysis (Luminex, Austin, Texas, USA). Intraocular VEGF-A concentrations were correlated to CNV activity shown by SD-OCT. Results The mean duration of suppression of VEGF-A concentrations in aqueous humour below the lower limit of quantification of our assay was 34 +/- 5 (26-69) days for ranibizumab and 67 +/- 14 (49-89) days for aflibercept (p<0.001). The percentual reduction of central retinal volume (CRV) 6weeks after injection was higher for aflibercept compared with ranibizumab (p=0.009). The time point of clinical re-activity occurred about 50% earlier than the respective VST for each ranibizumab and aflibercept. Conclusions The VST under aflibercept treatment exceeded that under ranibizumab treatment by a factor of 2. This difference correlated with differential clinical CRV reduction 6weeks after the respective injection. For both medications, clinical activity was found at a time point as early as 50% of the individual VST. Trial registration number NCT01213667, post-result

Suppression of Intraocular Vascular Endothelial Growth Factor During Aflibercept Treatment of Age-Related Macular Degeneration

PURPOSE: To determine the duration of suppression of aqueous humor concentrations of vascular endothelial growth factor (VEGF) in eyes with neovascular age-related macular degeneration (AMD) treated with aflibercept. DESIGN: Nonrandomized prospective clinical study. METHODS: Twenty-seven eyes of 27 neovascular AMD patients receiving intravitreal aflibercept injections on a pro re nata regimen driven by spectral-domain optical coherence tomography (SD OCT) were included in this study. A total of 132 aqueous humor specimens were collected before intravitreal aflibercept injections and their VEGF-A concentrations assayed by multiplex bead analysis. RESULTS: Mean aqueous humor VEGF concentrations before treatment initiation were 90.6 +/- 37.1 pg/mL (range 23.4-190.3 pg/mL). Intravitreal injection of aflibercept suppressed the aqueous VEGF concentrations to below the lower limit of quantification (71 +/- 18 days. The earliest time after injection at which the VEGF concentration recovered to above the lower limit of quantification was 55 days in 1 patient and >56 days, the recommended aflibercept treatment interval, in 20 patients. The aqueous VEGF recovery status of 6 patients was uncertain after 56 days. CONCLUSIONS: On average, VEGF concentrations in the aqueous humor were suppressed below the lower limit of quantification after intravitreal aflibercept injections for about 10 weeks. This aqueous suppression time suggests durable VEGF inhibition for most patients dosed with aflibercept every 8 weeks. (C) 2014 by Elsevier Inc. All rights reserved

A model of the ocular pharmacokinetics involved in the therapy of neovascular age-related macular degeneration with ranibizumab

Background To develop a model of the pharmacokinetics of vascular endothelial growth factor (VEGF-A) determined in samples of aqueous humour from patients with neovascular age-related macular degeneration (AMD) treated with ranibizumab (Lucentis). Methods Post hoc analysis of data from 31 eyes of 31 patients with AMD treated with ranibizumab gathered in a non-randomised, prospective clinical study. VEGF-A concentrations were measured in 440 aqueous humour samples by Luminex multiplex bead analysis (Luminex, Austin, Texas, USA). Results The kinetics of recovery of VEGF-A from suppression by ranibizumab were well described by a simple model: VEGF-A is produced at a constant individual rate; VEGF-A and ranibizumab disperse rapidly within the vitreous chamber and bind with a known affinity; both are eliminated at identical rates from the vitreous chamber in a constant but individual flow into the anterior chamber, and are finally cleared by draining into the peripheral circulation. Average rates of VEGF-A production were predicted to be 5.8 fmol/day (range: 2.7-10.1 fmol), and elimination half-times predicted to be 3.5 days (range: 2.3-5.5 days). The duration of complete VEGF-A suppression in the aqueous humour averaged 41 days (range: 28-67 days). Conclusions The ocular pharmacokinetics of VEGF-A and ranibizumab have been linked for the first time in a simple and plausible model which suggests that it might be possible to anticipate individual VEGF-A suppression times

Vascular endothelial growth factor suppression times in patients with diabetic macular oedema treated with ranibizumab

Background To measure vascular endothelial growth factor (VEGF) levels in aqueous humour from patients with diabetic macular oedema (DME) treated with ranibizumab and to determine how long VEGF was suppressed. Methods In this nonrandomised, prospective clinical study, 17 eyes of 17 patients were included in the study. A total of 110 aqueous humour samples were taken before an intravitreal ranibizumab injection in patients with DME. VEGF-A was measured by Luminex multiplex bead analysis (Luminex Inc, USA). Results VEGF was completely suppressed in all patients after ranibizumab injections for a mean of 33.7 days (SD +/- 5.1, range 27-42, median 34). VEGF suppression times were individually stable during the observation time of up to 16 months. There was no statistically significant difference of VEGF levels at baseline and before the beginning of a new injection series (123.6 pg/mL vs 125.1 pg/mL; p=1.0, Wilcoxon). Conclusions Monthly ranibizumab injections lead to a complete VEGF suppression in patients with DME. The long-term stability and the range of suppression times among individuals suggest that some patients could benefit from individual injection intervals

A randomized controlled clinical trial comparing 20 gauge and 23 gauge vitrectomy for patients with macular hole or macular pucker

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