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Subspecialties Basic & Translational Research, Retina

Age-related Methylation Degeneration

Patients with neovascular AMD rely on intravitreal injections of anti-VEGF – an onerous treatment which slows, but does not prevent, vision loss. Those with the non-neovascular form are similarly poorly served: they have no therapeutic options beyond vitamin supplements, and in the UK national health service, they are not even regularly monitored. Indeed, patients with non-neovascular AMD – despite representing the largest AMD patient group – have seen no significant change in their prognosis for decades. Yet interruption of the disease in this group of patients – as well as being desirable in itself – would avoid the undesirable treatments and unavoidable consequences associated with progression to neovascular AMD. Surely the early/intermediate AMD stages deserve more attention?

That is the rationale behind Louise Porter’s work on epigenetic changes – alterations in DNA methylation – in the retinal pigment epithelium (RPE) of eyes with AMD. The focus on RPE makes sense; not only is RPE is a primary site of AMD pathogenesis, but also RPE changes are thought to precede photoreceptor dysfunction and death. In particular, the decreased chromatin accessibility in AMD tissue (1) suggests that early AMD may be driven by epigenetic dysfunction in RPE. Furthermore, by focusing on the RPE of early-intermediate AMD, Porter increased the probability of identifying epigenetic changes that actually drive disease, as opposed to the global, but etiologically irrelevant, epigenetic changes that occur once the disease is established.

Challenges faced by the researchers included the difficulty in collecting a large enough panel of good quality ocular tissue. This was exacerbated by the existence of gender-related methylation differences, which dictated the use of larger sample sizes (identification of AMD versus control methylation differences at sexually dimorphic loci requires a per gender analysis, which in turn requires sufficient material for statistical analysis in each gender). Nevertheless, Porter’s strategy paid off. Firstly, the study identified genes that were differentially methylated in AMD versus control RPE (2); secondly, the RNA analysis indicated that three of these genes – SKI, GTF2H4 and TNXB – were differentially expressed in diseased and control tissue; thirdly – and perhaps most excitingly of all – it turned out that SKI and GTF2H4 had not previously been directly implicated in AMD, and therefore constitute novel targets.

The findings make perfect sense, in that all three genes tie into disease processes and anatomical structures relevant to AMD. Thus, the SKI proto-oncogene is a negative regulator of TGF-beta signalling, has a role in RPE cell migration and senescence, and modulates the complement over-activation found in early AMD. Methylation of SKI therefore could increase TGF-beta signalling and complement dysfunction. By contrast, GTF2H4 has a role intranscription-dependant DNA repair mechanisms; methylation-associated reduced GTF2H4 activity therefore could impede the repair of DNA lesions induced by oxidative stress and ultimately favour apoptosis. Finally, TNXB, which has a role in ECM maturation and collagen fibrillogenesis, has been shown to localise in Bruch’s membrane and choroid complex, suggesting a role in RPE architecture.

The finding that early AMD may be driven by methylation changes in a few genes is hugely important, as such aberrations can be corrected by therapeutic intervention. Porter’s study therefore opens up new avenues of functional study and drug development in the early-intermediate AMD field. At present, Porter is investigating the effect of methylation-targeting drugs on transcriptional activity at the loci identified in her study, and at the same time attempting to replicate the study findings in larger populations. The aim is to generate data that will support investment in the development of drugs that target differentially methylated loci and that ultimately will benefit AMD patients, who are in desperate need of new therapies.

The Mechanics of Methylation Analysis

  • Collected donated and consented-for-research eyes from Manchester Eye Bank
  • Dissected and and examined eyes to identify signs of AMD (drusen, pigmentary changes, geographic atrophy)
  • Eyes categorised as AMD (n=25) or normal controls (n=19)
  • RPE cells were mechanically removed; DNA extracted and subjected to genome-wide DNA methylation profiling (used the Illumina 450k array – characterizes methylation at >460,000 sites within the human genome)
  • Stratified bioinformatic analysis (correcting for gender, disease-state, and batch effect) identified genomic sites with different methylation levels in AMD versus control eyes
  • Methylation data were validated and replicated in an independent set of samples (30 AMD and 25 normal controls) using a different technique (bisulfite pyrosequencing: permits quantitative analysis of genomic methylation with single nucleotide resolution)
  • Global methylation changes in RPE tissues were assessed with long interspersed nucleotide element-1 (LINE-1) analysis
  • Independent RPE samples were subjected to RT-qPCR analysis to assess gene expression changes

Results: AMD-related differential gene methylation found in genes including SKI proto-oncogene (p=1.18x10E-9), general transcription factor GTF2H4 (p=7.03x10E-7) and tenascin X TNXB (p=6.30x10E-6); these were also differentially expressed in AMD of independent donors.

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  1. J Wang et al., “ATAC-Seq analysis reveals a widespread decrease of chromatin accessibility in age-related macular degeneration”, Nat Commun, 9, 1364. doi: 10.1038/s41467-018-03856-y (2018). PMID: 29636475.
  2. LF Porter et al., “Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals different methylation of the SKI, GTF2H4 and TNXB genes”, Clinical Epigenetics, (2019). PMID: 30642396.
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