Centre for Eye Research Australia (CERA) - Research Publications

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    Remote Control in Formation of 3D Multicellular Assemblies Using Magnetic Forces
    Jafari, J ; Han, X-L ; Palmer, J ; Tran, PA ; O'Connor, AJ (AMER CHEMICAL SOC, 2019-05)
    Cell constructs have been utilized as building blocks in tissue engineering to closely mimic the natural tissue and also overcome some of the limitations caused by two-dimensional cultures or using scaffolds. External forces can be used to enhance the cells' adhesion and interaction and thus provide better control over production of these structures compared to methods like cell seeding and migration. In this paper, we demonstrate an efficient method to generate uniform, three-dimensional cell constructs using magnetic forces. This method produced spheroids with higher densities and more symmetrical structures than the commonly used centrifugation method for production of cell spheroids. It was also shown that shape of the cell constructs could be changed readily by using different patterns of magnetic field. The application of magnetic fields to impart forces on the cells enhanced the fusion of these spheroids, which could be used to produce larger and more complicated structures for future tissue engineering applications.
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    A village in a dish model system for population-scale hiPSC studies
    Neavin, DR ; Steinmann, AM ; Farbehi, N ; Chiu, HS ; Daniszewski, MS ; Arora, H ; Bermudez, Y ; Moutinho, C ; Chan, C-L ; Bax, M ; Tyebally, M ; Gnanasambandapillai, V ; Lam, CE ; Nguyen, U ; Hernandez, D ; Lidgerwood, GE ; Graham, RM ; Hewitt, AW ; Pebay, A ; Palpant, NJ ; Powell, JE (NATURE PORTFOLIO, 2023-06-09)
    The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and different conditions. Human induced pluripotent stem cells are uniquely suited to study these context-dependent effects but cell lines from hundreds or thousands of individuals are required. Village cultures, where multiple induced pluripotent stem lines are cultured and differentiated in a single dish, provide an elegant solution for scaling induced pluripotent stem experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned to an induced pluripotent stem line using single-cell sequencing and illustrating that the genetic, epigenetic or induced pluripotent stem line-specific effects explain a large percentage of gene expression variation for many genes. We demonstrate that village methods can effectively detect induced pluripotent stem line-specific effects, including sensitive dynamics of cell states.
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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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    Genome-Wide Meta-analysis Identifies Risk Loci and Improves Disease Prediction of Age-Related Macular Degeneration
    He, W ; Han, X ; Ong, J-S ; Wu, Y ; Hewitt, AW ; Mackey, DA ; Gharahkhani, P ; Macgregor, S (ELSEVIER SCIENCE INC, 2024-01)
    PURPOSE: To identify age-related macular degeneration (AMD) risk loci and to establish a polygenic prediction model. DESIGN: Genome-wide association study (GWAS) and polygenic risk score (PRS) construction. PARTICIPANTS: We included 64 885 European patients with AMD and 568 740 control participants (with overlapped samples) in the UK Biobank, Genetic Epidemiology Research on Aging (GERA), International AMD Consortium, FinnGen, and published early AMD GWASs in meta-analyses, as well as 733 European patients with AMD and 20 487 control participants from the Canadian Longitudinal Study on Aging (CLSA) and non-Europeans from the UK Biobank and GERA for polygenic risk score validation. METHODS: A multitrait meta-analysis of GWASs comprised 64 885 patients with AMD and 568 740 control participants; the multitrait approach accounted for sample overlap. We constructed a PRS for AMD based on both previously reported as well as unreported AMD loci. We applied the PRS to nonoverlapping data from the CLSA. MAIN OUTCOME MEASURES: We identified several single nucleotide polymorphisms associated with AMD and established a PRS for AMD risk prediction. RESULTS: We identified 63 AMD risk loci alongside the well-established AMD loci CFH and ARMS2, including 9 loci that were not reported in previous GWASs, some of which previously were linked to other eye diseases such as glaucoma (e.g., HIC1). We applied our PRS to nonoverlapping data from the CLSA. A new PRS was constructed using the PRS method, PRS-CS, and significantly improved the prediction accuracy of AMD risk compared with PRSs from previously published datasets. We further showed that even people who carry all the well-known AMD risk alleles at CFH and ARMS2 vary considerably in their AMD risk (ranging from close to 0 in individuals with low PRS to > 50% in individuals with high PRS). Although our PRS was derived in individuals of European ancestry, the PRS shows potential for predicting risk in people of East Asian, South Asian, and Latino ancestry. CONCLUSIONS: Our findings improve the knowledge of the genetic architecture of AMD and help achieve better accuracy in AMD prediction. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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    RNA-targeting strategies as a platform for ocular gene therapy
    Kumar, S ; Fry, LE ; Wang, J-H ; Martin, KR ; Hewitt, AW ; Chen, FK ; Liu, G-S (Elsevier, 2023-01)
    Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.
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    Use of antihypertensive medications and the risk of glaucoma onset: Findings from the 45 and Up Study
    Yuan, Y ; Wang, W ; Shang, X ; Xiong, R ; Ha, J ; Zhang, L ; Zhu, Z ; He, M (WILEY, 2022-08)
    BACKGROUND: Abnormal blood pressure is a potential risk factor for glaucoma. However, the role of antihypertensive medications on glaucoma pathogenesis is controversial. This study aims to investigate the association between the use of antihypertensive medications and glaucoma onset. METHODS: This nested case-control study, based on a large-scale longitudinal cohort in Australia, retrieved participants' claims records on drugs and Medicare services from national health databases. Participants with three or more claim records of anti-glaucoma medications from 2009 to 2016 were classified as glaucoma patients; those with none were classified as controls. Claim records of antihypertensive medications were identified within the 5 years before glaucoma onset and contemporary periods in matched controls without glaucoma. The association between the use of antihypertensive medications and glaucoma onset was assessed by multivariable logistic regression models. RESULTS: A total of 6748 cases and 13 496 controls were analysed. Compared with controls, the proportion of users of antihypertensive medications was slightly higher in glaucoma patients (46.9% vs. 46.0%, p > 0.05). After adjustments for demographics, health-related factors and medical history, the association between the use of antihypertensive medications and glaucoma onset was nonsignificant (OR 0.95, 95% CI = 0.89-1.02). As for specific subtypes, only beta-blocking agents (BBA) (OR 0.82, 95% CI = 0.75-0.90) and diuretics (OR 0.85, 95% CI = 0.77-0.95) were significantly associated with reduced risks of glaucoma onset. CONCLUSIONS: This study indicated that the use of antihypertensive medications was not associated with glaucoma onset. Decreased risks of glaucoma onset in users of BBA and diuretics require further validation.
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    Diagnostic yield of candidate genes in an Australian corneal dystrophy cohort
    Souzeau, E ; Siggs, OM ; Mullany, S ; Schmidt, JM ; Hassall, MM ; Dubowsky, A ; Chappell, A ; Breen, J ; Bae, H ; Nicholl, J ; Hadler, J ; Kearns, LS ; Staffieri, SE ; Hewitt, AW ; Mackey, DA ; Gupta, A ; Burdon, KP ; Klebe, S ; Craig, JE ; Mills, RA (WILEY, 2022-10)
    Corneal dystrophies describe a clinically and genetically heterogeneous group of inherited disorders. The International Classification of Corneal Dystrophies (IC3D) lists 22 types of corneal dystrophy, 17 of which have been demonstrated to result from pathogenic variants in 19 identified genes. In this study, we investigated the diagnostic yield of genetic testing in a well-characterised cohort of 58 individuals from 44 families with different types of corneal dystrophy. Individuals diagnosed solely with Fuchs endothelial corneal dystrophy were excluded. Clinical details were obtained from the treating ophthalmologist. Participants and their family members were tested using a gene candidate and exome sequencing approach. We identified a likely molecular diagnosis in 70.5% families (31/44). The detection rate was significantly higher among probands with a family history of corneal dystrophy (15/16, 93.8%) than those without (16/28, 57.1%, p = .015), and among those who had undergone corneal graft surgery (9/9, 100.0%) compared to those who had not (22/35, 62.9%, p = .041). We identified eight novel variants in five genes and identified five families with syndromes associated with corneal dystrophies. Our findings highlight the genetic heterogeneity of corneal dystrophies and the clinical utility of genetic testing in reaching an accurate clinical diagnosis.
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    Sustained and rebound effect of repeated low-level red-light therapy on myopia control: A 2-year post-trial follow-up study
    Xiong, R ; Zhu, Z ; Jiang, Y ; Kong, X ; Zhang, J ; Wang, W ; Kiburg, K ; Yuan, Y ; Chen, Y ; Zhang, S ; Xuan, M ; Zeng, J ; Morgan, IG ; He, M (WILEY, 2022-12)
    BACKGROUND: To evaluate the long-term efficacy and safety of continued repeated low-level red-light (RLRL) therapy on myopia control over 2 years, and the potential rebound effect after treatment cessation. METHODS: The Chinese myopic children who originally completed the one-year randomised controlled trial were enrolled. Children continued RLRL-therapy were defined as RLRL-RLRL group, while those who stopped and switched to single-vision spectacle (SVS) in the second year were RLRL-SVS group. Likewise, those who continued to merely wear SVS or received additional RLRL-therapy were SVS-SVS and SVS-RLRL groups, respectively. RLRL-therapy was provided by an at-home desktop light device emitting red-light of 650 nm and was administered for 3 min at a time, twice a day and 5 days per week. Changes in axial length (AL) and cycloplegic spherical equivalence refraction (SER) were measured. RESULTS: Among the 199 children who were eligible, 138 (69.3%) children attended the examination and 114 (57.3%) were analysed (SVS-SVS: n = 41; SVS-RLRL: n = 10; RLRL-SVS: n = 52; RLRL-RLRL: n = 11). The baseline characteristics were balanced among four groups. In the second year, the mean changes in AL were 0.28 ± 0.14 mm, 0.05 ± 0.24 mm, 0.42 ± 0.20 mm and 0.12 ± 0.16 mm in SVS-SVS, SVS-RLRL, RLRL-SVS and RLRL-RLRL group, respectively (p < 0.001). The respective mean SER changes were -0.54 ± 0.39D, -0.09 ± 0.55D, -0.91 ± 0.48D, and -0.20 ± 0.56D (p < 0.001). Over the 2-year period, axial elongation and SER progression were smallest in RLRL-RLRL group (AL: 0.16 ± 0.37 mm; SER: -0.31 ± 0.79D), followed by SVS-RLRL (AL: 0.44 ± 0.37 mm; SER: -0.96 ± 0.70D), RLRL-SVS (AL: 0.50 ± 0.28 mm; SER: -1.07 ± 0.69D) and SVS-SVS group (AL: 0.64 ± 0.29 mm; SER: -1.24 ± 0.63D). No self-reported adverse events, functional or structural damages were noted. CONCLUSIONS: Continued RLRL therapy sustained promising efficacy and safety in slowing myopia progression over 2 years. A modest rebound effect was noted after treatment cessation.
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    Prevalence of diabetic retinopathy in women with pregestational diabetes during pregnancy and the postpartum
    Widyaputri, F ; Rogers, SL ; Khong, EWC ; Nankervis, AJ ; Conn, JJ ; Sasongko, MB ; Shub, A ; Fagan, XJ ; Guest, D ; Symons, RCA ; Lim, LL (WILEY, 2022-09)
    BACKGROUND: Diabetic retinopathy (DR) may be affected by pregnancy. The majority of prevalence data regarding DR in pregnancy predate the advent of contemporary guidelines for diabetes management during pregnancy. This study reports DR prevalence and associated risk factors in women with pregestational diabetes during pregnancy and the postpartum in Australia. METHODS: A total of 172 pregnant women with type 1 (T1DM) or type 2 diabetes diagnosed pre-pregnancy were prospectively recruited from two obstetrics hospitals in Melbourne (November 2017-March 2020). Eye examinations were scheduled in each trimester, at 3-, 6-, and 12-months postpartum. DR severity was graded from two-field fundus photographs by an independent grader utilising the Airlie House Classification. Sight-threatening DR (STDR) was defined as the presence of diabetic macular oedema or proliferative DR. RESULTS: Overall, 146 (84.9%) women had at least one eye examination during pregnancy. The mean age was 33.8 years (range 19-51), median diabetes duration was 7.0 years (IQR 3.0-17.0), 71 women (48.6%) had T1DM. DR and STDR prevalence during pregnancy per 100 eyes was 24.3 (95% CI 19.7-29.6) and 9.0 (95% CI 6.1-12.9); while prevalence in the postpartum was 22.2 (95% CI 16.5-29.3) and 10.0 (95% CI 5.4-17.9), respectively. T1DM, longer diabetes duration, higher HbA1c in early pregnancy, and pre-existing nephropathy were significant risk factors. CONCLUSIONS: The prevalence of DR in pregnant women was similar to the non-pregnant diabetic population in Australia. One in nine participants had STDR during pregnancy and the postpartum, highlighting the need to optimise DR management guidelines in pregnancy given the significant risk of vision loss.
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    Attitudes Toward Glaucoma Genetic Risk Assessment in Unaffected Individuals
    Hollitt, GL ; Siggs, OM ; Ridge, B ; Keane, MC ; Mackey, DA ; MacGregor, S ; Hewitt, AW ; Craig, JE ; Souzeau, E (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2022-10)
    PURPOSE: Integrating polygenic risk scores (PRS) into healthcare has the potential to stratify an individual's risk of glaucoma across a broad population. Glaucoma is the most common cause of irreversible blindness worldwide, therefore effective screening for glaucoma endorsed by the population is highly important. This study assessed the attitude of unaffected individuals toward PRS testing for glaucoma, and sought to identify factors associated with interest in testing. METHODS: We surveyed 418 unaffected individuals including 193 with a first-degree relative with glaucoma, 117 who had a recent eye examination, and 108 general members of the community. RESULTS: Overall, 71.3% of the individuals indicated an interest in taking a polygenic risk test for glaucoma. Interest was more likely in those who believed glaucoma to be a severe medical condition (odds ratio [OR] = 14.58, 95% confidence interval [CI] = 1.15-185.50, P = 0.039), those concerned about developing glaucoma (OR = 4.37, 95% CI = 2.32-8.25, P < 0.001), those with an intention to take appropriate measures regarding eye health (OR = 2.39, 95% CI = 1.16-4.95, P = 0.019), and those preferring to know if considered to be at-risk or not (OR = 4.52, 95% CI = 2.32-8.83, P < 0.001). CONCLUSIONS: Our results show strong interest in genetic risk assessment for glaucoma among unaffected individuals in Australia. TRANSLATIONAL RELEVANCE: These findings represent a valuable assessment of interest in glaucoma polygenic risk testing among potential target populations, which will be integral to the implementation and uptake of novel PRS-based tests into clinical practice.