Investigating the pharmacogenetic basis of Age-related Macular Degeneration (AMD) with anti-VEGF treatments
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Riaz, MoeenDate
2017Affiliation
Ophthalmology (Eye & Ear Hospital)Metadata
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PhD thesisAccess Status
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© 2018 Dr. Moeen Riaz
Abstract
Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss and blindness in elderly people in the developed world. The two main vision threating subtypes are geographic atrophy (GA) and neovascular AMD (nAMD). The latter is responsible for 90% of vision loss attributed to the disease. Currently, the treatment for nAMD is through the use of intravitreal injections of anti-Vascular endothelial growth factor (anti-VEGF) agents. Randomized controlled clinical trials and retrospective studies conducted for anti-VEGF treatment in nAMD patients have shown good improvements in visual outcomes in the majority of patients but due to unknown reasons, a broad range of outcome responses have also been reported. Approximately 5-10% of patients typically show no improvement in the readout of visual acuity (VA) (loss of > 15 Early Treatment Diabetic Retinopathy Study (ETDRS) letters) and exhibit a continuous decline in VA over the course of long term anti-VEGF therapy.
A number of studies have indicated factors that might influence the response to treatment, which includes demographic, clinical and genetics factors. Among these, genetic factors are considered as one important determinant which likely result in a pharmacogenetic anti-VEGF treatment response. Genetic factors have been investigated for their association with change in VA following anti-VEGF treatment but these have been based on VEGF pathway genes as well as known AMD risk genes including CFH, ARMS2/HTRA1, VEGFR2, EPAS1 and APOE. Currently, findings from these genetic studies appear inconsistent with regard to their associations with anti-VEGF treatment outcomes (detailed in Chapter 2). This indicates that it is possible that other genetic factors are involved in determining response to anti-VEGF treatment in nAMD patients. Identifying these potential genetic variants across the human genome requires the use of sophisticated genetic techniques such as genome wide association studies (GWAS) and whole exome sequencing (WES).
The objective of my PhD is to undertake pharmacogenetic studies to identify genetic variants which could influence anti-VEGF treatment outcome in nAMD patients. To accomplish this, I have used strategies which include candidate gene analysis and the next generation genetic techniques: GWAS and WES.
Firstly, in Chapter 4, I assessed the genetic association of several previously described single nucleotide polymorphisms (SNPs) rs4576072 (VEGFR2), rs6828477 (VEGFR2), rs2070296 (NRP1) and rs9679290 (EPAS1) with regard to treatment outcome in nAMD after anti-VEGF treatment. A total of 207 nAMD patients were genotyped for these 4 SNPs with readout outcome measures of change in visual acuity (VA), retinal fluid clearance and central macular thickness (CMT) as measured by optical coherence tomography (OCT) following up-to 1 year of anti-VEGF treatment. Statistical analysis revealed no significant association between the genotypes of these four variants with change in VA at 3, 6 and 12 months of anti-VEGF treatment (P > 0.05 for each). Furthermore, there was no evidence of an association between each of the four variants with retinal fluid clearance or CMT change at 3 months of treatment (P >0.05 for each). A systematic search of Pubmed, Web of Science and Embase in August 2016 revealed two previous studies that had reported on the association of SNPs rs4576072 and rs682847 in the VEGFR2 gene with treatment outcomes (reported using only change in VA) following anti-VEGF therapy in nAMD. A meta-analysis of my own study with the two previously published studies showed no evidence of association between the genotypes of rs4576072 or rs682847 with mean change in VA at 3 or 12 months of treatment (P >0.05 each). In conclusion, there was insufficient evidence to ascribe a genetic association between the variants rs4576072, rs682847, rs2070296 and rs9679290 and anti-VEGF treatment outcome in nAMD patients.
Chapter 5 describes a cost effective pooled DNA based GWAS on 285 anti-VEGF treated nAMD patients. The primary outcome measure was change in VA in ETDRS letters after 6 months of anti-VEGF treatment: patients who lost ≥5 (ETDRS) letters were classified as non-responders while all remaining patients were classified as responders. GWAS analysis identified 44 SNPs associated with change in VA at 6 months of anti-VEGF treatment. In a technical validation phase, individual genotyping of these 44 variants showed three SNPs (rs4910623 p = 5.6 × 10-5, rs323085 p = 6.5 × 10-4 and rs10198937 p = 1.30 × 10-3) that remained suggestively associated with VA response at 6 months of anti-VEGF treatment. Replication of these three SNPs in an independent cohort of 376 anti-VEGF nAMD patients of European origin followed by meta-analysis of both cohorts (673 samples) confirmed association of the SNP rs4910623 in the Olfactory Receptor Family 52 Subfamily B Member 4 (OR52B4) gene with poor VA response after 3 months (p = 1.2 × 10-5) and 6 months (p = 9.3 × 10-6) of anti-VEGF treatment in nAMD patients.
In Chapter 6, I have undertaken a multi-centre GWAS on anti-VEGF treated nAMD cohorts identified via the International AMD Gene Consortium (IAMDGC) custom exome chip. In the discovery GWAS phase, a total of 678 anti-VEGF treated nAMD patients from 5 different clinical sites were included and the main outcome measure was change in VA at 3 months of anti-VEGF treatment. Linear regression analysis was undertaken on imputed genetic variants for each cohort with change in VA, followed by meta-analysis. Six SNPs rs241692, rs12138564, rs13002976, rs242939, rs2237435 and rs927203 revealed suggestive association with change in VA (p<1x10-5) at three months of treatment. Replication of these six variants was undertaken in an additional 6 anti-VEGF treated cohorts from outside the IAMDGC and subsequent overall meta-analysis of both the discovery + replication cohorts (n=2058) was undertaken. A variant, rs12138564 in the Chaperonin Containing TCP1 Subunit 3 (CCT3) gene remained associated with change in VA (p = 1.3x10-5) after three months of anti-VEGF treatment. The gene based burden test for non-synonymous rare variants (MAF<0.01) showed significant association of three genes Chromosome 10 Open Reading Frame 88 (C10orf88), Unc-93 homolog B1 (UNC93B1) and Cytoskeleton associated protein 2 like (CKAP2L) with change in VA associated (corrected p = 4.2 x10-7, p=6.0x10-7 and p = 1.3x10-6 respectively) at three months of anti-VEGF treatment. In addition pathway analysis revealed aldosterone signalling and G protein pathway as the top biological processes associated with change in VA (p<1x10-4) following treatment.
Chapter 7 presents a Whole Exome Sequencing (WSE) study on 54 anti-VEGF treated AMD patients to identify genetic variants that influence the qualitative measure of retinal fluid presence (non-responders) relative to the retinal fluid dryness (responders) measured on OCT after 3 monthly doses of anti-VEGF injections. After downstream quality control and statistical analysis of the WES data, five SNPs; rs8786, rs36078271, rs2275123, rs1753430 and rs3801790 showed evidence of association with fluid presence (p ≤ 1x10-4) after 3 months of anti-VEGF treatment. Replication of these top variants in an independent anti-VEGF treated cohort (n=161) followed by meta-analysis of both cohorts (n=215) revealed association of rs1753430 and rs3801790 in the olfactory receptor family 6 subfamily J member 1 (OR6J1) and dedicator of cytokinesis 4 (DOCK4) genes with presence of retinal fluid after three months of anti-VEGF treatment (p = 0.002, and p = 0.01, respectively). Furthermore, the gene enrichment pathway analysis revealed the G-protein coupled receptor (GPCR) pathway as the top functional process (corrected p=8x10-15), suggesting involvement of cell signalling molecules in anti-VEGF treatment response in nAMD patients.
In conclusion, I was able to undertake candidate gene, GWAS and WES in anti-VEGF treated nAMD patients, identifying variants in four novel genes; OR52B4, CCT3, OR6J1 and DOCK4, associated with varying treatment outcomes in nAMD. Findings from this thesis indicate multiple genetic factors are likely involved in varied response to anti-VEGF therapy and by investigating the biological role of these four novel genes in abnormal angiogenesis. It will aid in the future identification of new drug targets as potential alternatives or adjunctive treatments to existing anti-VEGF therapy. Additionally, it is the first step towards optimizing and personalizing treatment based on a pharmacogenetic approach for patients with nAMD.
Keywords
pharmacogenetics; neovascular AMD; GWAS; exome sequencing; rare variants; anti-VEGF treatmentExport Reference in RIS Format
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