Centre for Eye Research Australia (CERA) - Research Publications

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    A semi-automated pipeline for quantifying drusen-like deposits in human induced pluripotent stem cell-derived retinal pigment epithelium cells.
    Hall, J ; Daniszewski, M ; Cheung, S ; Shobhana, K ; Kumar, H ; Liang, HH ; Beetham, H ; Cho, E ; Abbott, C ; Hewitt, AW ; Simpson, KJ ; Guymer, RH ; Paull, D ; Pébay, A ; Lidgerwood, GE (Elsevier BV, 2023-08-30)
    Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.
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    Reticular Pseudodrusen on the Risk of Progression in Intermediate Age-Related Macular Degeneration
    Wu, Z ; Kumar, H ; Hodgson, LAB ; Guymer, RH (ELSEVIER SCIENCE INC, 2022-07)
    PURPOSE: To examine the association between reticular pseudodrusen (RPD) and progression to late age-related macular degeneration (AMD) in individuals with intermediate AMD. DESIGN: Prospective cohort study. METHODS: Two hundred eighty eyes from 140 participants with bilateral large drusen underwent multimodal imaging (MMI), including optical coherence tomography (OCT), near-infrared reflectance (NIR), fundus autofluorescence, and color fundus photography (CFP), at 6-monthly intervals up over a 36-month follow-up period. The presence of RPD per eye was determined based on either a combined MMI criterion, or each individual imaging modality, and their extent measured on combined OCT and NIR imaging. The association between the presence of RPD on different imaging modalities, and their extent, with the development of late AMD (including OCT-defined atrophy) was evaluated. RESULTS: The presence of RPD on MMI, or any of its individual modalities, at baseline was not significantly associated with an increased rate of developing late AMD, with or without adjusting for risk factors for AMD progression (age, drusen volume on OCT, and pigmentary abnormalities on CFP; all P ≥ 0.205). The extent of RPD present was also not significantly associated with an increased rate of developing late AMD, with or without adjustment for risk factors for AMD progression (both P ≥ 0.522). CONCLUSIONS: In this cohort with bilateral large drusen, the presence of RPD was not significantly associated with an increased risk of developing late AMD. Additional longitudinal studies in all stages of AMD are needed to understand the implications of RPD on vision loss in this condition.
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    P2X7-mediated alteration of membrane fluidity is associated with the late stages of age-related macular degeneration
    Drysdale, C ; Park, K ; Vessey, KA ; Huang, X ; Caruso, E ; Li, Y ; Wong, J ; Wiley, JS ; Fletcher, E ; Guymer, RH ; Gu, BJ (SPRINGER, 2022-12)
    We have shown deficits in monocyte phagocytosis from patients with age-related macular degeneration (AMD). Cell membrane fluidity is known to affect phagocytic capacity and leucocyte functionality more generally. Therefore, we examined membrane fluidity of peripheral blood leucocytes in human patients with AMD and in the P2X7 null mouse model of AMD using flow cytometry with a fluorescent probe for fluidity, TMA-DPH. The results showed that membrane fluidity was decreased in all leucocyte types of late AMD relative to healthy controls (HC) including monocytes, neutrophils and lymphocytes but this was not apparent in earlier stages of AMD. Further analysis of factors contributing to membrane fluidity indicated that pre-treatment of monocytes and lymphocytes with ATP greatly increased membrane fluidity in humans and mice. Evidence from P2X7 null mice and P2X7 antagonists confirmed that these ATP-driven increases in membrane fluidity were mediated by P2X7 but were not associated with the classic P2X7 functions of pore formation or phagocytosis. Analysis of P2X7 expression indicated that receptor levels were elevated in classic monocytes of late AMD patients, further suggesting the P2X7 may contribute to altered plasma membrane properties. Our findings identified a novel biological function of P2X7 in modulating membrane fluidity of leucocytes and demonstrated reduced membrane fluidity in cellular changes associated with the late stage of AMD.
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    Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration
    Senabouth, A ; Daniszewski, M ; Lidgerwood, GE ; Liang, HH ; Hernandez, D ; Mirzaei, M ; Keenan, SN ; Zhang, R ; Han, X ; Neavin, D ; Rooney, L ; Sanchez, MIGL ; Gulluyan, L ; Paulo, JA ; Clarke, L ; Kearns, LS ; Gnanasambandapillai, V ; Chan, C-L ; Nguyen, U ; Steinmann, AM ; McCloy, RA ; Farbehi, N ; Gupta, VK ; Mackey, DA ; Bylsma, G ; Verma, N ; MacGregor, S ; Watt, MJ ; Guymer, RH ; Powell, JE ; Hewitt, AW ; Pebay, A (NATURE PORTFOLIO, 2022-07-26)
    There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.
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    Disparities in access to anti-vascular endothelial growth factor treatment for neovascular age-related macular degeneration
    Finger, RP ; Xie, J ; Fotis, K ; Parikh, S ; Cummins, R ; Mitchell, P ; Guymer, RH (WILEY, 2017-03)
    BACKGROUND: Late neovascular age-related macular degeneration (nvAMD) is very common and causes irreversible severe visual loss unless treated swiftly with vascular endothelial growth factor (VEGF) inhibitors. Although publicly subsidized access to treatment may be inequitable, which is why we assessed treatment provision across Australia. DESIGN: Secondary analysis of Australian data. PARTICIPANTS: All Pharmaceutical Benefits Scheme (including Repatriation PBS) beneficiaries. METHODS: Treatment and incidence data were obtained from Medicare Australia, the Royal Australian and New Zealand College of Ophthalmologists, Optometry Australia, the Blue Mountains Eye Study and the Australian Bureau of Statistics. Data were mapped using geographical information software, and factors associated with treatment provision were assessed using multiple linear regression models. MAIN OUTCOME MEASURE: Unmet need (%) for anti-VEGF treatment for nvAMD. RESULTS: On average, we estimated 7316 incident cases of nvAMD not to be treated per year from 2010 to 2014 (50.1% of total). Number of ophthalmologists and optometrists (per 1000, β = -0.024; 95% confidence interval [CI] -0.041, -0.007) and being located in remote regions (β = 0.186; 95% CI 0.110, 0.262) were associated with percentage of untreated cases. A higher proportion of the population speaking a language other than English at home was associated in univariate analyses only (β = 0.0015; 95% CI -0.0004, 0.0027; P = 0.007). CONCLUSION: A large proportion of incident nvAMD is not treated with anti-VEGF. Not receiving treatment is more likely in regional or remote areas and areas with fewer service providers. Not speaking English at home may further limit access. Service delivery models for more equitable service provision are needed.
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    Ellipsoid zone on optical coherence tomography: a review
    Tao, LW ; Wu, Z ; Guymer, RH ; Luu, CD (WILEY, 2016-07)
    Emergence of the high-resolution optical coherence tomography has allowed better delineation of retinal layers, and many of the anatomical correlations of these layers have now been agreed upon. However, some anatomical correlates still remain contentious, such as the second hyper-reflective band, which is now termed ellipsoid zone. Despite the lack of consensus of the actual origin of the ellipsoid zone, there has been much interest in evaluating its integrity and intensity in different disease processes. This review paper aims to provide an overview of the ellipsoid zone and its clinical and research applications.
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    Relationship between reticular pseudodrusen and choroidal thickness in intermediate age-related macular degeneration: response
    Ho, CYD ; Lek, JJ ; Aung, KZ ; McGuinness, MB ; Luu, CD ; Guymer, RH (WILEY, 2018-11)
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    Relationship between reticular pseudodrusen and choroidal thickness in intermediate age-related macular degeneration
    Ho, CYD ; Lek, JJ ; Aung, KZ ; McGuinness, MB ; Luu, CD ; Guymer, RH (WILEY, 2018-07)
    IMPORTANCE: Reticular pseudodrusen (RPD) is strongly associated with late age-related macular degeneration (AMD) but their aetiology remains unknown. RPD have been associated with reduced choroidal thickness (ChT) but most studies are limited by small sample size and varying severity of AMD. BACKGROUND: To investigate the relationship between choroidal thickness and RPD in eyes with intermediate AMD (iAMD), controlling for variables known to influence ChT. DESIGN: Retrospective cohort study. PARTICIPANTS: Participants were recruited from Centre for Eye Research Australia. METHODS: Colour fundus photographs, fundus auto fluorescence, near-infrared and spectral-domain ocular coherence tomography (OCT) were graded for RPD. ChT was measured from enhanced-depth imaging OCT scans at the centre of fovea, 1500 and 3000 μm nasal, temporal, superior and inferior from centre of fovea. MAIN OUTCOME MEASURES: ChT between RPD and non-RPD group. RESULTS: A total of 297 eyes from 152 subjects were included. A total of 84 (28%) had RPD and were older than non-RPD group (75.1 ± 5.4 years and 68.7 ± 6.9 years, respectively; P < 0.001). In unadjusted analysis, the RPD group was significantly associated with thinner choroids across all measured locations (P ≤ 0.022). After adjustment for variables, the presence of RPD was no longer associated with ChT (P ≥ 0.132 for all locations) but age (P < 0.001) and refractive error (P = 0.002) remained significantly associated with ChT. CONCLUSIONS AND RELEVANCE: Age and refractive error, rather than RPD, was significantly associated with reduced ChT in eyes with iAMD. Choroidal insufficiency may be a less important variable in RPD aetiology than previously considered.
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    Multi-focal electro-retinogram response following sub-threshold nano-second laser intervention in age-related macular degeneration
    Luu, CD ; Makeyeva, G ; Caruso, E ; Baglin, E ; Sivarajah, P ; Wu, Z ; Guymer, RH (WILEY, 2020-09)
    IMPORTANCE: The effect of sub-threshold nano-second laser (SNL) treatment on retinal function remains unknown. BACKGROUND: SNL treatment has been studied as a potential intervention in intermediate age-related macular degeneration (iAMD). This study investigated the longitudinal effect of SNL treatment on retinal function. DESIGN: This was a sub-study of the LEAD trial; a 36-month, multi-centre, randomized and sham-controlled trial. PARTICIPANTS: Subjects with iAMD. METHODS: Eligible participants were assigned randomly to receive SNL or sham treatment to the study eye at 6-monthly visits. Multi-focal electro-retinography (mfERG) was performed at each study visit from a study site. The mfERG responses were grouped into three regions (central, middle and outer rings) and compared between the SNL and sham group. MAIN OUTCOME MEASURES: mfERG P1 response amplitude and implicit time. RESULTS: Data were collected from 50 subjects (26 in the SNL group, 24 in the sham group). At baseline, the P1 amplitudes of both the study eyes and the fellow eyes were similar between the groups at all rings. In the sham group, the P1 amplitude gradually decreased over time (P < .05). In the SNL group, there was an improvement in P1 amplitude which became statistically significant at the 36-month visit, detected in both the treated and fellow eyes at the central (P = .005) and middle ring (P = .007) but not at the outer ring (P = .070). No difference in P1 implicit time detected between the groups (P > .05). CONCLUSIONS AND RELEVANCE: SNL treatment improved electro-physiological function. mfERG could be useful for monitoring AMD progression and evaluating the efficacy of SNL treatment.
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    A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants
    Fritsche, LG ; Igl, W ; Bailey, JNC ; Grassmann, F ; Sengupta, S ; Bragg-Gresham, JL ; Burdon, KP ; Hebbring, SJ ; Wen, C ; Gorski, M ; Kim, IK ; Cho, D ; Zack, D ; Souied, E ; Scholl, HPN ; Bala, E ; Lee, KE ; Hunter, DJ ; Sardell, RJ ; Mitchell, P ; Merriam, JE ; Cipriani, V ; Hoffman, JD ; Schick, T ; Lechanteur, YTE ; Guymer, RH ; Johnson, MP ; Jiang, Y ; Stanton, CM ; Buitendijk, GHS ; Zhan, X ; Kwong, AM ; Boleda, A ; Brooks, M ; Gieser, L ; Ratnapriya, R ; Branham, KE ; Foerster, JR ; Heckenlively, JR ; Othman, MI ; Vote, BJ ; Liang, HH ; Souzeau, E ; McAllister, IL ; Isaacs, T ; Hall, J ; Lake, S ; Mackey, DA ; Constable, IJ ; Craig, JE ; Kitchner, TE ; Yang, Z ; Su, Z ; Luo, H ; Chen, D ; Hong, O ; Flagg, K ; Lin, D ; Mao, G ; Ferreyra, H ; Starke, K ; von Strachwitz, CN ; Wolf, A ; Brandl, C ; Rudolph, G ; Olden, M ; Morrison, MA ; Morgan, DJ ; Schu, M ; Ahn, J ; Silvestri, G ; Tsironi, EE ; Park, KH ; Farrer, LA ; Orlin, A ; Brucker, A ; Li, M ; Curcio, CA ; Mohand-Said, S ; Sahel, J-M ; Audo, I ; Benchaboune, M ; Cree, AJ ; Rennie, CA ; Goverdhan, SV ; Grunin, M ; Hagbi-Levi, S ; Campochiaro, P ; Katsanis, N ; Holz, FG ; Blond, F ; Blanche, H ; Deleuze, J-F ; Igo, RP ; Truitt, B ; Peachey, NS ; Meuer, SM ; Myers, CE ; Moore, EL ; Klein, R ; Hauser, MA ; Postel, EA ; Courtenay, MD ; Schwartz, SG ; Kovach, JL ; Scott, WK ; Liew, G ; Tan, AG ; Gopinath, B ; Merriam, JC ; Smith, RT ; Khan, JC ; Shahid, H ; Moore, AT ; McGrath, JA ; Laux, R ; Brantley, MA ; Agarwal, A ; Ersoy, L ; Caramoy, A ; Langmann, T ; Saksens, NTM ; de Jong, EK ; Hoyng, CB ; Cain, MS ; Richardson, AJ ; Martin, TM ; Blangero, J ; Weeks, DE ; Dhillon, B ; van Duijn, CM ; Doheny, KF ; Romm, J ; Klaver, CCW ; Hayward, C ; Gorin, MB ; Klein, ML ; Baird, PN ; den Hollander, AI ; Fauser, S ; Yates, JRW ; Allikmets, R ; Wang, JJ ; Schaumberg, DA ; Klein, BEK ; Hagstrom, SA ; Chowers, I ; Lotery, AJ ; Leveillard, T ; Zhang, K ; Brilliant, MH ; Hewitt, AW ; Swaroop, A ; Chew, EY ; Pericak-Vance, MA ; DeAngelis, M ; Stambolian, D ; Haines, JL ; Iyengar, SK ; Weber, BHF ; Abecasis, GR ; Heid, IM (NATURE PUBLISHING GROUP, 2016-02)
    Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.