Search

Background Mutations in the human crumbs homologue 1 (CRB1) gene are associated with a spectrum of inherited retinal diseases. However, functional studies demonstrating the impact of individual CRB1 mutations on gene expression are lacking for most variants. Here, we investigated the effect of two CRB1 variants on pre‐mRNA splicing using neural retinal organoids (NRO) derived from a patient with recessive rod‐cone dystrophy caused by compound heterozygous mutations in CRB1 (c.1892A>G and c.2548G>A). Methods The patient received ophthalmological examinations including multimodal imaging. NRO were differentiated from induced pluripotent stem cells (iPSCs) derived from the patient and a control subject. CRB1 transcripts were characterized by RT‐PCR and Sanger sequencing. Results The Patient displayed retinal thickening with disorganization of retinal layers and preservation of para‐arteriolar retinal pigment epithelium. Both patient and control iPSC produced NRO containing photoreceptor progenitor cells expressing CRB1 mRNA. Patient NRO expressed a novel CRB1 transcript displaying skipping of exon 6. CRB1 transcripts containing the c.2548G>A substitution in exon 7 were expressed in patient NRO. Conclusions Together, these results confirm the pathogenicity of the c.1892A>G and c.2548G>A CRB1 variants in a family with recessive adult‐onset rod‐cone dystrophy and further demonstrate the effects of these variants on pre‐mRNA splicing. This data provide important insights into the pathogenic mechanisms associated with these variants., Here, we demonstrate the pathogenicity of the c.1892A>G and c.2548G>A CRB1 variants in a family with recessive adult‐onset rod‐cone dystrophy and further demonstrate the effects of these variants on pre‐mRNA splicing. In patient‐derived retinal organoids, the c.1892A>G variant was shown to cause exon 6 skipping during CRB1 mRNA splicing, while the c.2548G>A variant results in expression of a CRB1 transcript bearing a missense mutation.

Aim To determine whether macrovascular disease assessed by carotid ultrasonography and arterial stiffness by pulse wave velocity are independently associated with diabetic retinopathy in type 2 diabetes. Methods A random subgroup of surviving participants with type 2 diabetes from the Fremantle Diabetes Study Phase II were invited to take part in this sub-study in 2018-2019. In addition to standardized questionnaires, a physical examination and fasting biochemical tests, each underwent dilated colour fundus photography, carotid arterial ultrasonography with measurement of the intima-media thickness (IMT) and quantification of the degree of stenosis, and pulse wave analysis calculation of the carotid-femoral pulse wave velocity (cfPWV). The cross-sectional association between arterial disease parameters and diabetic retinopathy was assessed using generalized estimating equation models which enabled both eyes to be included in the analysis. Results Some 270 participants [mean ± sd age 72 ± 9 years, 153 (57%) men and median (IQR) diabetes duration 15 [11-22) years] were included in analysis. Of 524 assessable eyes, 82 (16%) had diabetic retinopathy. In multivariable analysis, significant independent associates of diabetic retinopathy were age at diabetes diagnosis (inversely), HbA1c , insulin treatment and urinary albumin to creatinine ratio (all P ≤ 0.022), as well as cfPWV [odds ratio (OR) 1.13, 95% confidence interval (CI) 1.03, 1.23 per 1 m/s increase; P = 0.008] and common carotid artery (CCA) IMT ≥ 1 mm (OR 2.95, 95% CI 1.21, 7.23; P = 0.018). Conclusions The association between diabetic retinopathy and CCA IMT suggests that carotid disease may share cardiovascular risk factors with diabetic retinopathy. The association between diabetic retinopathy and cfPWV may reflect the consequences of altered intravascular haemodynamics.

Usher syndrome is a genetic disorder causing neurosensory hearing loss and blindness from retinitis pigmentosa (RP). Adaptive techniques such as braille, digital and optical magnifiers, mobility training, cochlear implants, or other assistive listening devices are indispensable for reducing disability. However, there is currently no treatment to reduce or arrest sensory cell degeneration. There are several classes of treatments for Usher syndrome being investigated. The present article reviews the progress this research has made towards delivering commercial options for patients with Usher syndrome.

Background Deletion–insertion (delins) variants in the retina‐specific ATP‐binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for, This work epitomizes the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and illustrates the use of patient‐derived fibroblasts to evaluate the effect on splicing of a novel ABCA4 indel variant, and as a method for pathogenicity assessment of other novel ABCA4 variants.

Purpose: Macular Integrity Assessment (MAIA) microperimetry is used widely in clinical trials and routine practice to assess paracentral scotoma. Current interpretation of MAIA is based on an assumed uniform 25 decibel (dB) cutoff for normal function irrespective of subject age and retinal location. We examined this convention by establishing an age- and loci-specific reference in healthy eyes and comparing this to the

IntroductionEye diseases and visual impairment more commonly affect elderly adults, thus, the majority of ophthalmic cohort studies have focused on older adults. Cohort studies on the ocular health of younger adults, on the other hand, have been few. The Raine Study is a longitudinal study that has been following a cohort since their birth in 1989–1991. As part of the 20-year follow-up of the Raine Study, participants underwent a comprehensive eye examination. As part of the 27- and 28-year follow-ups, eye assessments are being conducted and the data collected will be compared with those of the 20-year follow-up. This will provide an estimate of population incidence and updated prevalence of ocular conditions such as myopia and keratoconus, as well as longitudinal change in ocular parameters in young Australian adults. Additionally, the data will allow exploration of the environmental, health and genetic factors underlying inter-subject differential long-term ocular changes.Methods and analysisParticipants are being contacted via telephone, email and/or social media and invited to participate in the eye examination. At the 27-year follow-up, participants completed a follow-up eye screening, which assessed visual acuity, autorefraction, ocular biometry and ocular sun exposure. Currently, at the 28-year follow-up, a comprehensive eye examination is being conducted which, in addition to all the eye tests performed at the 27-year follow-up visit, includes tonometry, optical coherence tomography, funduscopy and anterior segment topography, among others. Outcome measures include the incidence of refractive error and pterygium, an updated prevalence of these conditions, and the 8-year change in ocular parameters.Ethics and disseminationThe Raine Study is registered in the Australian New Zealand Clinical Trials Registry. The Gen2 20-year, 27-year and 28-year follow-ups are approved by the Human Research Ethics Committee of the University of Western Australia. Findings resulting from the study will be published in health or medical journals and presented at conferences.Trial registration numberACTRN12617001599369; Active, not recruiting.

Herein, we review the safety, efficacy, regulatory standards and ethical implications of the use of stem cells in ocular disease. A literature review was conducted, registered clinical trials reviewed, and expert opinions sought. Guidelines and codes of conduct from international societies and professional bodies were also reviewed. Collated data is presented on current progress in the field of ocular regenerative medicine, future challenges, the clinical trial process and ethical considerations in stem cell therapy. A greater understanding of the function and location of ocular stem cells has led to rapid advances in possible therapeutic applications. However, in the context of significant technical challenges and potential long-term complications, it is imperative that stem cell practices operate within formal clinical trial frameworks. While there remains broad scope for innovation, ongoing evidence-based review of potential interventions and the development of standardized protocols are necessary to ensure patient safety and best practice in ophthalmic care.

Purpose To present the visual and safety outcomes of surgery for primary idiopathic macular holes including predictors of visual acuity and the impact of combined phacovitrectomy surgery. Design Registry-style, prospective, nonrandomized, observational cohort study. Participants Patients with idiopathic macular holes undergoing primary surgery. Methods Surgeons were invited to submit clinical details of all macular hole cases receiving surgery. Baseline demographic and clinical information, and details of surgical intervention were collected. Primary follow-up data were collected at 3 months postoperatively or before revision surgery, and surgeons were also asked to submit data at 12 and 24 months postoperatively. Main Outcome Measures Visual acuity improvement ≥15 letters and ≥0 letters, change in mean visual acuity, visual acuity ≥70 letters (20/40), retinal detachment, and endophthalmitis. Results A total of 2455 eyes of 2366 patients were included in the study, and hole closure was achieved in 95.6% of eyes with a single procedure. Mean baseline vision was 48.3 letters. The proportion of successful eyes improving ≥15 letters at 3, 12, and 24 months was 59.1%, 69.4%, and 68.2%, respectively. The mean improvement in acuity at 3, 12, and 24 months was 16.0, 19.2, and 23.6 letters, and 92.4%, 93.4%, and 95.8% improved ≥0 letters at 3, 12, and 24 months, respectively. Eyes receiving SF6 gas had better visual acuities at all time points postoperatively (adjusted effect 3.4, 3.1, and 4.6 letters better at 3, 12, and 24 months vs. longer-acting gas, respectively). Combined phacovitrectomy in phakic eyes was associated with better corrected visual acuity postoperatively (vs. vitrectomy surgery alone), a difference that vanished when eyes went on to have subsequent cataract surgery. The rate of retinal detachment postoperatively was 1.3%, and the odds of detachment were greater in eyes receiving longer-acting gases versus SF6 gas (adjusted odds ratio, 2.2; 95% confidence interval, 1.04–4.77; P = 0.039). There were no reported cases of endophthalmitis. Conclusions Macular hole surgery substantially improved acuity in approximately two thirds of patients and was seldom associated with loss of vision. SF6 gas was associated with better visual outcomes, an effect that warrants further study.

The human iPSC line LEIi006-A was generated from dermal fibroblasts from a patient with retinitis pigmentosa using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for p53. The iPSC cells carry compound heterozygous mutations (c.1892A > G and c.2548G > A) in the CRB1 gene. LEIi006-A expressed pluripotent stem cell markers, had a normal karyotype and could be differentiated into endoderm, mesoderm and ectodermal lineages, as well as retinal organoids.

We report the generation of the human iPSC line LEIi004-A from a patient with late-onset non-syndromic retinitis pigmentosa caused by compound heterozygous mutations in the CLN3 gene. Reprogramming of primary dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA. To create a coisogenic control line, one CLN3 variant was corrected in the patient-iPSC using CRISPR/Cas9 gene editing to generate the iPSC line LEIi004-A-1.

Purpose To define the range and life cycles of cuticular drusen phenotypes using multimodal imaging and to review the histologic characteristics of cuticular drusen. Design Retrospective, observational cohort study and experimental laboratory study. Participants Two hundred forty eyes of 120 clinic patients with a cuticular drusen phenotype and 4 human donor eyes with cuticular drusen (n = 2), soft drusen (n = 1), and hard drusen (n = 1). Methods We performed a retrospective review of clinical and multimodal imaging data of patients with a cuticular drusen phenotype. Patients had undergone imaging with various combinations of color photography, fluorescein angiography, indocyanine green angiography, near-infrared reflectance, fundus autofluorescence, high-resolution OCT, and ultrawide-field imaging. Human donor eyes underwent processing for high-resolution light and electron microscopy. Main Outcome Measures Appearance of cuticular drusen in multimodal imaging and the topography of a cuticular drusen distribution; age-dependent variations in cuticular drusen phenotypes, including the occurrence of retinal pigment epithelium (RPE) abnormalities, choroidal neovascularization, acquired vitelliform lesions (AVLs), and geographic atrophy (GA); and ultrastructural and staining characteristics of druse subtypes. Results The mean age of patients at the first visit was 57.9±13.4 years. Drusen and RPE changes were seen in the peripheral retina, anterior to the vortex veins, in 21.8% of eyes. Of eyes with more than 5 years of follow-up, cuticular drusen disappeared from view in 58.3% of eyes, drusen coalescence was seen in 70.8% of eyes, and new RPE pigmentary changes developed in 56.2% of eyes. Retinal pigment epithelium abnormalities, AVLs, neovascularization, and GA occurred at a frequency of 47.5%, 24.2%, 12.5%, and 25%, respectively, and were significantly more common in patients older than 60 years of age (all P P Conclusions Although the ultrastructural characteristics of cuticular drusen appear more similar to those of hard drusen, their lifecycle and macular complications are more comparable with those of soft drusen. Cuticular drusen phenotype may confer a unique risk for the development of GA and neovascularization.

Purpose To characterize the ultrastructural and functional correlates of hydroxychloroquine (HCQ)-induced subclinical bull’s eye lesion seen on near-infrared reflectance (NIR) imaging. Methods An asymptomatic 54-year-old male taking HCQ presented with paracentral ring-like scotoma, abnormal multifocal electroretinography (mfERG) and preserved ellipsoid zone on optical coherence tomography (OCT). Dense raster OCT was performed to create en face reflectivity maps of the interdigitation zone. Macular Integrity Assessment (MAIA) microperimetry and mfERG findings were compared with NIR imaging, en face OCT, retinal thickness profiles and wave-guiding cone density maps derived from flood-illumination adaptive optics (AO) retinal photography. Results The bull’s eye lesion is an oval annular zone of increased reflectivity on NIR with an outer diameter of 1450 µm. This region corresponds exactly to an area of preserved interdigitation zone reflectivity in en face OCT images and of normal cone density on AO imaging. Immediately surrounding the bull’s eye lesion is an annular zone (3°–12° eccentricity) of depressed retinal sensitivity on MAIA and reduced amplitude density on mfERG. Wave-guiding cone density at 2° temporal was 25,400 per mm2. This declined rapidly to 12,900 and 1200 per mm2 at 3° and 4°. Conclusion Multimodal imaging illustrated pathology in the area surrounding the NIR bull’s eye, characterized by reduced reflectance, wave-guiding cone density and retinal function. Further studies are required to investigate whether the bull’s eye on NIR imaging and en face OCT is prominent or consistent enough for diagnostic use.

Background Leber congenital amaurosis (LCA) is a severe visual impairment responsible for infantile blindness, representing ~5% of all inherited retinal dystrophies. LCA encompasses a group of heterogeneous disorders, with 24 genes currently implicated in pathogenesis. Such clinical and genetic heterogeneity poses great challenges for treatment, with personalized therapies anticipated to be the best treatment candidates. Unraveling the individual genetic etiology of disease is a prerequisite for personalized therapies, and could identify potential treatment candidates, inform patient management, and discriminate syndromic forms of disease. Methods We have genetically analyzed 45 affected and 82 unaffected individuals from 34 unrelated LCA pedigrees using predominantly next-generation sequencing and Array CGH technology. Results We present the molecular findings for an Australian LCA cohort, sourced from the Australian Inherited Retinal Disease Registry & DNA Bank. CEP290 and GUCY2D mutations, each represent 19% of unrelated LCA cases, followed by NMNAT1 (12%). Genetic subtypes were consistent with other reports, and were resolved in 90% of this cohort. Conclusion The high resolution rate achieved, equivalent to recent findings using whole exome/genome sequencing, reflects the progression from hypothesis (LCA Panel) to non-hypothesis (RD Panel) testing and, coupled with Array CGH analysis, is a highly effective first-tier test for LCA.

In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures., Highlights • Macromolecular crowding promoted deposition of extracellular matrix by ARPE19 cells. • ARPE19 cells deposited matrix proteins found in the inner layers of Bruch's membrane. • ARPE19-ECM displayed similar microstructure to Bruch's membrane. • ARPE19-ECM promoted pigmentation in human retinal pigment epithelial cell cultures. • ARPE19-ECM promoted RPE differentiation from pluripotent stem cells.

Purpose The Laser Intervention in Early Stages of Age-Related Macular Degeneration (LEAD) study is an investigation of the safety and efficacy of subthreshold nanosecond laser treatment to slow the progression of intermediate age-related macular degeneration (AMD). This report presents the novel study design and baseline characteristics. Design Multicenter, double-masked, randomized controlled, medical device feasibility clinical trial. Participants Persons with bilateral drusen >125 μm within 1500 μm of the fovea, monocular best-corrected visual acuity (BCVA) ≥20/40, and microperimetric retinal sensitivity of Methods Participants were randomized to nanosecond or sham laser treatment. Twelve laser or sham spots are applied to the macular region of the study eye. Participants are reviewed in visits every 6 months with functional testing and MMI for 36 months and are re-treated at each visit (until 30 months) if an end point is not reached in the study eye. Main Outcome Measures Progression to late AMD or MMI-defined anatomic end points in the study eye. Results A total of 292 participants across 6 centers were enrolled, with 145 participants randomized to arm 1 and 147 participants randomized to arm 2. Population characteristics at baseline were as follows: median age 70 years, 73% female, 90% Anglo-Saxon, and 3% current smokers. Baseline ocular characteristics of the study eyes were BCVA of 83 letters (20/25); low luminance visual acuity (LLVA) of 68 letters (20/50); hyperpigmentation, 33%; reticular pseudodrusen, 23%; square root drusen area (SD-OCT), 0.77 mm; square root drusen area (color photographs), 0.92 mm; cube root drusen volume (SD-OCT), 0.26 mm; average retinal sensitivity, 26 dB; and worst point retinal sensitivity, 20 dB. Only lutein supplement use was significantly different between treatment arms. Conclusions The LEAD study uses novel inclusion/exclusion criteria and end points in an attempt to optimize our study design. Risk characteristics for progression to study end points are equally distributed between treatment arms.

Background The culture of retinal progenitors from an accessible adult stem cell source such as the limbus could provide a useful autologous source of retinal cell therapies. The human corneoscleral limbus contains multipotent stem cells that can be cultured as floating neurospheres. Previous work in rodents has demonstrated neuronal and photoreceptor differentiation from limbal neurosphere cultures. Here, this study has examined undifferentiated cultured adult human limbal neurospheres as donor cells for retinal cell therapies by transplantation into a rat model of retinal degeneration. Methods Gene expression in limbal neurospheres was examined by immunostaining and western blot. Human limbal neurospheres were transplanted into the subretinal space of Royal College of Surgeon's rats. Rats were monitored by optical coherence tomography for 6 weeks then processed for retinal histology. Results Human limbal neurospheres expressed the neural lineage markers, Nestin, sex determining region box-2 and N-cadherin, and the retinal transcription factors microphthalmia-associated transcription factor, sex determining region box-2 and orthodentical homeobox-2. Human limbal neurospheres could be cultured to express NeuN, neurofilament and rhodopsin. Rats receiving saline or no injection underwent complete degeneration of the retinal outer nuclear layer after 3 weeks. In contrast, rats injected with human limbal neurospheres or retinal pigment epithelial cells maintained the outer nuclear layer for up to 6 weeks. Gene expression in transplanted limbal neurospheres was inconsistent with the production of mature retinal pigment epithelial or photoreceptor cells. Conclusions Human limbal neurospheres represent an accessible source of autologous donor cells for the treatment of retinal diseases.

Evan N Wong,1,2 William H Morgan,1,3 Fred K Chen1,3 1Centre for Ophthalmology and Visual Science (Incorporating Lions Eye Institute), The University of Western Australia, 2Department of Ophthalmology, Sir Charles Gairdner Hospital, 3Department of Ophthalmology, Royal Perth Hospital, Perth, WA, Australia Purpose: To determine the intersession test–retest variability (TRV) of CenterVue Macular Integrity Assessment (MAIA) microperimeter in glaucoma patients with fixation-threatening field defects.Methods: This is a prospective case–control study of 27 participants consisting of 13 patients with stable primary open-angle glaucoma and 14 control subjects including 5 healthy individuals and 9 retinal patients (5 with non-neovascular age-related macular degeneration and 4 with inherited retinal disease). Each participant underwent three microperimetry tests in one eye at 1-month intervals. Each test used an identical test strategy of 10-2 Cartesian grid and 4-2 staircase algorithm. We investigated TRV by calculating the coefficient of repeatability (CR) for mean sensitivity (MS) and point-wise sensitivity (PWS) for glaucomatous subjects and retinal and normal subjects. 95% confidence intervals (CIs) for CRs were calculated.Results: There was no significant change in MS, and the median durations of microperimetry sessions were 9´26Ë®, 8´52Ë®, and 8´46Ë® across the three study visits. The intersession CRs for MS were 1.1, 2.5, and 1.8 dB, and the average CRs for PWS were 3.5, 7.4, and 8.6 dB for healthy controls and retinal and glaucoma patients, respectively. For test loci with 25–34 dB at baseline, CRs for PWS were 8.2 (95% CI: 7.5–8.9) and 4.3 (95% CI: 4.0–4.6) dB for glaucoma and control subjects, respectively.Conclusion: We found differences in TRV of test loci depending on the baseline sensitivity value. Glaucoma patients had significantly worse TRV for loci that had sensitivity values within the normal range at baseline. The estimated CR has implications for sample size calculation in future glaucoma treatment trials using microperimetry as a clinical endpoint. Keywords: microperimetry, MAIA perimeter, glaucoma, retinal dystrophy, optic neuropathy

OBJECTIVE To use optical coherence tomography angiography (OCTA) to determine whether retinal microvascular parameters are associated with carotid arterial disease in people with type 2 diabetes. RESEARCH DESIGN AND METHODS Participants (community-based) underwent detailed assessments including carotid ultrasonography and OCTA. Ultrasound images were assessed for mean intima-media thickness (IMT) and the presence of stenosis. OCTA image analysis provided measures of vessel density, foveal avascular zone (FAZ) area, blood flow areas, and retinal thickness. For each OCTA variable, the most parsimonious model was generated using generalized estimating equations, then ipsilateral and contralateral carotid disease–related variables were added to determine their significance. RESULTS A total of 474 eyes from 261 participants (mean ± SD age 72.0 ± 9.3 years, 57.1% males, median diabetes duration 15.4 years [interquartile range 11.1–22.4]) were analyzed. When carotid variables were added to the most parsimonious models, the ipsilateral natural logarithm of common carotid artery IMT (coefficient −2.56 [95% CI −4.76, −0.35], P = 0.023) and presence of any ipsilateral stenosis (−0.82 [−1.48, −0.17], P = 0.014) were statistically significantly associated with a lower parafoveal density in the deep capillary plexus. A mean bifurcation IMT ≥1 mm was associated with a decreased vessel density in the 300-μm ring surrounding the FAZ (coefficient −0.79 [−1.50, −0.08], P = 0.030)). Contralateral carotid disease–related variables were also significantly associated with retinal microvascular parameters. CONCLUSIONS This is the first study to show that carotid disease is an independent associate of retinal microvascular disease assessed by OCTA in type 2 diabetes. Appropriately intensive management of carotid disease may improve the retinal microcirculation.

Purpose To investigate the relationship between patient-reported outcome (PRO) questionnaire responses and time to late age-related macular degeneration (AMD; neovascular AMD [nAMD] or multimodal imaging [MMI]-defined atrophy) among individuals with bilateral large drusen, and the prognostic value of baseline PROs for 36-month AMD status. Design Exploratory analyses from a multicenter randomized controlled trial of an AMD intervention (Australian New Zealand Clinical Trials Registry identifier, ACTRN12612000704897). Participants Sham treatment group of the Laser Intervention in Early Stages of Age-Related Macular Degeneration (LEAD) Study (n = 141; age, 50–88 years; 77% female). Methods The 28-item Impact of Vision Impairment (IVI-28) and 10-item Night Vision Questionnaire (NVQ-10) were administered at the baseline visit. The PRO scores were derived using rating scale models. Multivariate Cox regression adjusting for demographics and clinical measures of vision (low-luminance visual acuity, low-luminance deficit, and microperimetric sensitivity) from the poorer-performing eye was used to investigate the association between PRO scores and time to late AMD in either eye. Multivariate competing-risk regression was used to estimate cause-specific subhazard ratios for nAMD and atrophy in either eye. Cross-validated logistic lasso models were used to estimate the predicted probability of AMD at 36 months. The area under the receiver operating characteristic curve was assessed to compare prognostic accuracy between models with and without PROs. Main Outcome Measure Time until nAMD or atrophy in either eye. Results The PRO scores were skewed toward higher functional vision. Higher IVI-28 scores were associated with a lower risk of progression to MMI-defined atrophy (20 events: adjusted hazard ratio, 0.65/logit increase; P = 0.002) but not nAMD (10 events; P = 0.562). Insufficient evidence was found of an association between NVQ-10 score and rate of progression to late AMD (P ≥0.149). Baseline IVI-28 scores were found to contribute to the prognosis of atrophy at the 36-month visit (P = 0.010). Conclusions On average, PROs were associated with an increased risk of progression from intermediate AMD to MMI-defined atrophy. Continuing development of instruments to record PROs in the early stages of AMD have the potential to produce inexpensive and efficient tools to assist in the assessment of disease severity and risk of AMD progression.

Purpose: To determine the impact of an AngioVue software upgrade on total retinal thickness (RT) and inner retinal vessel density (VD) measurements derived from optical coherence tomography angiography (OCTA). Methods:OptovueOCTA images (3×3mm) from126 individuals (105healthy eyes and 72 eyes with retinal disease)were acquired before an upgrade of the AngioVue software, which resulted in an inward shift of the outer boundary of the inner retinal vessels and improved Bruch’s membrane segmentation. Total RT and inner retinal VD values were extracted before and after the software upgrade for comparison. Bias and limits of agreement (LA) were calculated. Results: The mean (SD) age of participants was 46 (17) years. Mean (LA) foveal RT increased by 3.0 (–11 to +17) and 3.7 (–11 to +18) μm (P < 0.001) and parafoveal RT increased by 9.7 (–3.8 to +23) and 6.4 (–2.5 to +15) μm (P < 0.001) in healthy and diseased retina, respectively.Mean (LA) foveal inner retinal VD decreased by 6.6 (2.5–11) and 7.7 (0.4–15) percentage units (P < 0.001) and parafoveal inner retinal VD decreased by 4.1 (1.2–7.0) and 4.7 (0.5–8.9) percentage units (P < 0.001) in healthy and diseased retina, respectively. Conclusions: The AngioVue software upgrade resulted in an unexpected increase in total RT and an expected reduction in inner retinal VD measurements in all regions due to altered segmentation. Translational Relevance: RT and VD measures derived from the newer AngioVue software version are not directly comparable to the reported normative data derived from the older software.

Background Congenital mydriasis and retinal arteriolar tortuosity are associated with the life-threatening multisystemic smooth muscle dysfunction syndrome (MSMDS) due to mutations in the gene, ACTA2, which encodes alpha-smooth muscle actin (α-SMA). Previous reports attributed MSMDS-related congenital mydriasis to the absence of iris sphincter muscle. Similarly, it has been hypothesized that abnormal proliferation of the vascular smooth muscle cells causes the marked tortuosity of retinal arterioles in MSMDS. In this report, high-resolution ocular imaging reveals unexpected findings that reject previous hypotheses. Case presentation The proband is a 37-year-old female with a history of neonatal patent ductus arteriosus (PDA) ligation, left-sided choreiform movements at the age of 11 and a transient aphasia with right-sided weakness at the age of 30. Her older sister also had PDA ligation and congenital mydriasis but no neurological deficit up to age 41. Magnetic resonance angiogram demonstrated cerebrovascular lesions resembling but distinct from Moyamoya disease, characterised by internal carotid artery dilatation, terminal segment stenosis and absent basal collaterals. Their mother had poorly reactive pupils with asymptomatic cerebral arteriopathy resembling her daughters. All three had prominent retinal arteriolar tortuosity. The daughters were heterozygous and the mother was a somatic mosaic for a novel c.351C > G (p.Asn117Lys) transversion in ACTA2. Iris optical coherence tomography (OCT) showed a hyporeflective band anterior to the pigment epithelium indicating the presence of dysfunctional sphincter muscle. Adaptive optics retinal imaging showed no thickening of the arteriolar vessel wall whilst OCT angiography showed extreme corkscrew course of arterioles suggesting vessel elongation. Conclusions In addition to the known association between Met46, Arg179 and Arg258 substitutions and ACTA2-related arteriopathy, this case illustrates the possibility that Asn117 also plays an important role in α-SMA function within the cerebrovascular smooth muscle cell. MSMDS-related congenital mydriasis is due to reduced iris sphincter contractility rather than its absence. Retinal arteriolar tortuosity might be due to longitudinal proliferation of arteriolar smooth muscle cells. The described cerebrovascular and ocular signs are consistent with predicted effects of the novel Asn117Lys substitution in ACTA2.

Purpose To examine the added predictive value of microperimetric sensitivity and low luminance deficit (LLD; difference between photopic and low luminance visual acuity (VA)) to information from colour fundus photography (CFP) for progression to late age-related macular degeneration (AMD) in individuals with bilateral large drusen. Methods 140 participants with bilateral large drusen underwent baseline microperimetry testing, VA measurements and CFP. They were then reviewed at 6-monthly intervals to 36 months, to determine late AMD progression. Microperimetry pointwise sensitivity SD (PSD), LLD and the presence of pigmentary abnormalities on CFPs were determined. Predictive models based on these parameters were developed and examined. Results Baseline microperimetry PSD and presence of pigmentary abnormalities were both significantly associated with time to develop late AMD (p≤0.004), but LLD was not (p=0.471). The area under the receiver operating characteristic curve (AUC) for discriminating between eyes that progressed to late AMD based on models using microperimetry PSD (AUC=0.68) and LLD (AUC=0.58) alone was significantly lower than that based on CFP grading for the presence of pigmentary abnormalities (AUC=0.80; both p Conclusions While microperimetry, but not LLD, was significantly and independently associated with AMD progression at the population level, this study observed that both measures were suboptimal at predicting progression at the individual level when compared to conventional CFP grading and their addition to the latter did not improve predictive performance.

Clinical evaluation of macular disease symptoms has evolved beyond visual acuity assessment alone. Despite common usage of the Amsler grid by eye care professionals for documenting visual distortion and scotoma, more reliable and precise clinical tools have been developed to quantify these symptoms. The M-CHARTS have emerged as a commonly used tool in monitoring the natural history of tractional maculopathies and surgical outcomes of epiretinal membrane peel and repair of macular holes and rhegmatogenous retinal detachment. Structure–function correlation studies using the M-CHARTS have confirmed the directional relationship between retinal displacement and visual distortion. Fundus-tracking perimetry is becoming a commonly useful clinical tool in quantifying the extent and depth of central and paracentral scotoma. It has been used for detecting diffuse retinal dysfunction in the perifoveal regions affected by macular pucker and possible iatrogenic side effects of internal limiting membrane staining and peeling in macular surgery. Retinal sensitivity is also used as clinical endpoints in novel retinal therapies including cell transplantation and gene replacement. The clinician assessing macular function needs to understand the basic principles, basis and limitation of these tests and choose the optimal combination of testing strategies based on clinical history, retinal imaging and current literature.

Inherited retinal diseases (IRDs) are genetically and phenotypically diverse, and they cause significant morbidity worldwide. Importantly, IRDs may be amenable to precision medicine strategies, and thus the molecular characterisation of causative variants is becoming increasingly important with the promise of personalised therapies on the horizon. ABCA4, involved in the translocation of visual cycle derivatives, is a well-established, frequent cause of IRDs worldwide, with pathogenic variants implicated in phenotypically diverse diseases. Identification of causative ABCA4 variants in some individuals, however, has been enigmatic, and resolution of this issue is currently a hotbed of research. Recent evidence has indicated that hypomorphic alleles, which cause disease under certain conditions, may account for some of the missing causal variants. It has been postulated that the ABCA4 c.5603AT (p.Asn1868Ile) variant, previously considered benign, be reclassified as hypomorphic when in cis configuration with c.2588GC (p.Gly863Ala/Gly863del), a variant previously considered to be pathogenic in its own right. We are exploring this relationship within an Australian cohort to test this theory.

The segmentation of tissue layers in optical coherence tomography (OCT) images of the internal lining of the eye (the retina and choroid) is commonly performed for clinical and research purposes. However, manual segmentation of the numerous scans is time consuming, tedious and error-prone. Fortunately, machine learning-based automated approaches for image segmentation tasks are becoming more common. However, poor performance of these methods can result from a lack of quantity or diversity in the data used to train the models. Recently, generative adversarial networks (GANs) have demonstrated the ability to generate synthetic images, which may be useful for data augmentation purposes. Here, we propose the application of GANs to construct chorio-retinal patches from OCT images which may be used to augment data for a patch-based approach to boundary segmentation. Given the complexity of GAN training, a range of experiments are performed to optimize performance. We show that it is feasible to generate 32×32 versions of such patches that are visually indistinguishable from their real variants. In the best case, the segmentation performance utilizing solely synthetic data to train the model is nearly comparable to real data on all three layer boundaries of interest. The difference in mean absolute error for the inner boundary of the inner limiting membrane (ILM) [0.50 vs. 0.48 pixels], outer boundary of the retinal pigment epithelium (RPE) [0.48 vs. 0.44 pixels] and choroid-scleral interface (CSI) [4.42 vs. 4.00 pixels] shows the performance using synthetic data to be only marginally inferior. These findings highlight the potential use of GANs for data augmentation in future work with chorio-retinal OCT images.

Importance Individual differences in axial length affect the lateral magnification of in vivo retinal images and as a result can affect the accuracy of quantitative measurements made from these images. As measurements from optical coherence tomography angiography (OCTA) images are becoming increasingly used in the diagnosis and monitoring of a wide range of diseases, evaluating which studies use correctly scaled images is crucial to their interpretation. Objective To perform a systematic literature review to assess the percentage of articles that report correcting the scale of their OCTA images for individual differences in retinal magnification. Evidence Review A PubMed (MEDLINE) search was conducted for articles on OCTA retinal imaging published between June 1, 2015, and June 1, 2018. Initial results included 7552 articles. Initial exclusion criteria removed studies of animal models, as well as reviews, letters, replies, comments, and image-based or photographic essays. Articles not written in English and those that required purchase from non–English language websites were excluded. Articles that did not use OCTA for imaging the retina were also excluded. Remaining articles were reviewed in detail to assess whether the OCTA measurements required correct lateral scaling, and if so, whether axial length was reported or used to scale the images. We also determined the number of articles that mentioned the lack of correct lateral scaling as a limitation of the study. Findings A total of 989 articles were included in the detailed review. Of these, 509 were determined to require correct image scaling for their analyses, but only 41 (8.0%) report measuring and using axial length to correct the lateral scale of their OCTA images. Furthermore, of the 468 articles that did not correctly scale their images, only 18 (3.8%) mentioned this as a limitation to their study. Conclusions and Relevance These findings suggest that most peer-reviewed articles in PubMed that use quantitative OCTA measurements use incorrectly scaled images. This could call into question the conclusions of such studies and warrants consideration by OCTA manufacturers, physicians, authors, journal reviewers, and journal editors.

Importance All automated image quality indicators for en face optical coherence tomography angiography (OCTA) images require gold standard validation for determining optimum thresholds. Background A manual grading system (gold standard) for OCTA images was validated and compared to two automated image quality indicators: signal strength index (SSI) and scan quality index (SQI) generated by different software versions of the Optovue OCTA device. Design Retrospective cross‐sectional study. Participants A total of 52 eyes of 52 healthy individual and 77 eyes of 51 patients with retinal vascular diseases. Methods A total of 129 OCTA images of the superficial vascular plexus were graded manually by three independent examiners. Each image was assigned grades 1 to 4 (1‐2, unacceptable; 3‐4, acceptable) masked to the software‐generated quality indicators. Main Outcome Measures Inter‐grader agreement and comparison of the utility of SSI and SQI in discriminating between acceptable and unacceptable OCTA images. Results There was a substantial agreement between the three graders (κ = 0.63). Mean SSI and SQI was significantly different between acceptable and unacceptable images (P ˂ .001). SQI outperformed SSI in separating acceptable from unacceptable images (areas under the receiver operating characteristic curve: 0.87 vs 0.80) and the optimum cut‐off was ≥7 for SQI and ≥70 for SSI for acceptable images. Up to 30% of images with quality indicators reaching the optimum SQI and SSI cut‐off thresholds still had unacceptable quality on manual grading. Unacceptable images were found in 33% and 66% of healthy and diseased eyes, respectively. Conclusions and Relevance SQI is closely related to manual grading but we caution reliance on the optimized threshold to determine image quality. SQI is superior to SSI in discriminating between acceptable and unacceptable images.

Purpose Nascent geographic atrophy (nGA) describes features on OCT imaging previously observed to precede the development of atrophy. This study sought to prospectively evaluate the predictive ability of nGA for the conventional clinical endpoint of geographic atrophy (GA) as defined on color fundus photography (CFP). Design Prospective, longitudinal, observational study. Participants A total of 284 eyes from 142 participants with bilateral large drusen and without nGA nor late age-related macular degeneration (AMD) at baseline were included. Methods OCT volume scans and CFP images were obtained from all participants at baseline and then at 6-month intervals for up to 36 months. OCT and CFP images were graded independently for the presence of nGA and GA, respectively. Eyes that developed neovascular AMD were censored at the day of its detection. Main Outcome Measures Time to development of GA. Results A total 12 eyes from 10 participants progressed to GA over 36 months of follow-up, and nGA was detected in 10 of these eyes (83%) at a preceding visit (median, 13 months prior; interquartile range, 6–25 months). A total of 40 eyes from 28 participants developed nGA or GA over 36 months of follow-up, and the probability of progression to nGA and GA after 36 months was 20% (95% confidence interval [CI], 14%–28%) and 9% (95% CI, 6%–13%), respectively. After the detection of nGA, the probability of progression to GA was 38% (95% CI, 15%–55%) after 24 months. The development of nGA was associated with a markedly increased risk of progression to GA compared with when it did not develop (adjusted hazard ratio, 78.1; 95% CI, 13.6–448.0; P Conclusions This study prospectively demonstrated that nGA was a strong predictor for the development of GA, providing supportive evidence of the potential value of nGA as a surrogate endpoint in future intervention trials for the early stages of AMD to improve their feasibility substantially.

Purpose: To develop a fully automatic method, based on deep learning algorithms, for determining the locations of cone photoreceptors within adaptive optics scanning laser ophthalmoscope images and evaluate its performance against a dataset of manually segmented images. Methods:A fully convolutional network (FCN) based on U-Net architecture was used to generate prediction probability maps and then used a localization algorithm to reduce the prediction map to a collection of points. The proposed method was trained and tested on two publicly available datasets of different imaging modalities, with Dice overlap, false discovery rate, and true positive reported to assess performance. Results:The proposed method achieves a Dice coefficient of 0.989, true positive rate of 0.987, and false discovery rate of 0.009 on the first confocal dataset; and a Dice coefficient of 0.926, true positive rate of 0.909, and false discovery rate of 0.051 on the second split detector dataset. Results compare favorably with a previously proposed method, but this method provides quicker (25 times faster) evaluation performance. Conclusions:The proposed FCN-based method demonstrates that deep learning algorithms can achieve accurate cone localizations, almost comparable to a human expert, while labeling the images. Translational Relevance:Manual cone photoreceptor identification is a timeconsuming task due to the large number of cones present within a single image; using the proposed FCN-based method could support the image analysis task, drastically reducing the need for manual assessment of the photoreceptor mosaic.

Variants in RCBTB1 have been implicated in inherited retinal disease (IRD). Here, we generated induced pluripotent stem cells (iPSCs) from a 45-year-old female IRD patient harbouring compound heterozygous mutations in the RCBTB1 gene. Episomal plasmids containing OCT4, SOX2, KLF4, MYCL, LIN28, shRNA for TP53 and mir302/367 microRNA were employed to conduct the reprogramming of primary dermal fibroblasts. These iPSC lines provide a useful model for further investigations on the pathophysiological role of mutations in the RCBTB1 gene in IRD.

Analysis of specific somatic copy number alterations (SCNAs) using multiplex ligation-dependent probe amplification (MLPA) is used routinely as a prognostic test for uveal melanoma (UM). This technique requires relatively large amounts of input DNA, unattainable from many small fine-needle aspirate biopsy specimens. Herein, we compared the use of MLPA with whole-genome amplification (WGA) combined with low-pass whole-genome sequencing (LP-WGS) for detection of SCNA profiles in UM biopsy specimens. DNA was extracted from 21 formalin-fixed, paraffin-embedded UM samples and SCNAs were assessed using MLPA and WGA followed by LP-WGS. Cohen's κ was used to assess the concordance of copy number calls of each individual chromosome arm for each patient. MLPA and WGA/LP-WGS detection of SCNAs in chromosomes 1p, 3, 6, and 8 were compared and found to be highly concordant with a Cohen's κ of 0.856 (bias-corrected and accelerated 95% CI, 0.770–0.934). Only 13 of 147 (8.8%) chromosomal arms investigated resulted in discordant calls, predominantly SCNAs detected by WGA/LP-WGS but not MLPA. These results indicate that LP-WGS might be a suitable alternative or adjunct to MLPA for the detection of SCNAs associated with prognosis of UM, for cases with limiting tissue or DNA yields.

We report the generation of the iPSC line LEIi005-B from a patient with retinitis pigmentosa caused by a dominant nonsense mutation in the RP1 gene (c.2098G>T p.E700X). Reprogramming of dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for p53 to establish the clonal iPSC line LEIi005-B. LEIi005-B expressed pluripotent stem cell markers, had a normal karyotype and differentiated into endoderm, mesoderm and ectoderm.

Purpose To review the clinical features, microbiology spectrum, management, and outcomes of patients with endogenous endophthalmitis in Western Australia over a 16-year period. Methods This is a retrospective chart review of all patients with endogenous endophthalmitis who presented to all tertiary ophthalmology departments between 2000 and 2015 in Western Australia. Results Sixty-six eyes of 57 patients with endogenous endophthalmitis were identified, and follow-up data were available for a mean of 554 days. The average frequency was 1.6 per 1,000,000 population per year. Diabetes mellitus (33%) and intravenous drug use (30%) were the most common risk factors. Concurrent systemic infections included urinary tract infection (28%), pneumonia (23%), and endocarditis (21%). Among culture-positive cases (93%), 57% were bacterial and 43% were fungal. Visual acuity improved in 33 (50%) and declined in 15 eyes (22.7%). Baseline visual acuity and the presence of Gram-negative or filamentous fungi were the only predictors of final visual acuity (P = 0.023 and P = 0.001). Conclusion The population frequency of endogenous endophthalmitis has not changed over 16 years despite the changing profile of pathogen and risk factors. Similar to previous studies in Asian and Western countries, visual and anatomical prognosis depends on initial visual acuity and isolated pathogen. Gram-negative and filamentous fungi culture predicted a worse visual outcome.

Purpose: To report the distribution of peripapillary retinal nerve fiber layer (RNFL) thickness in healthy young adults, investigate factors associated with RNFL thickness, and report the percentage of outside normal limits (ONL) and borderline (BL) RNFL thickness classifications based on the optical coherence tomography (OCT) manufacturer reference database. Methods: Participants of the Raine Study Generation 2 cohort (aged 18–22 years) underwent spectral domain OCT imaging with an RNFL circle scan. Eyes with inadequate scans or optic nerve pathology were excluded. Linear mixed models were used to analyze associations. Results: Data were available for 1288 participants (mean age, 20.0 years). Mean RNFL thicknesses in right and left eyes, respectively, were global = 100.5 µm, 100.3 µm (P = 0.03); temporal = 73.1 µm, 68.9 µm (P < 0.001); superotemporal = 140.6 µm, 136.3 µm (P < 0.001); superonasal = 104.9 µm, 115.1 µm (P < 0.001); nasal = 79.7 µm, 79.1 µm (P = 0.09); inferonasal = 109.8 µm, 111.5 µm (P < 0.001); and inferotemporal = 143.2 µm, 143.6 µm (P = 0.51). Longer axial length was associated with thinner RNFL globally, nasally, inferotemporally, superotemporally, superonasally, and inferonasally, as well as thicker RNFL temporally. The prevalence of ONL and BL classifications was generally higher than the expected rates of 1% and 4%, respectively, in temporal sectors and lower than expected in nasal sectors. The prevalence of global BL classifications was lower than expected (right eye, 2.3%; left eye, 2.6%). Conclusions: Measured RNFL thickness differs with axial length and between right and left eyes. More reference data are needed to better define the normal limits of RNFL variation in different populations. Translational Relevance: This study provides an improved understanding of normal variation in RNFL thickness in young adults.

Background To determine the impact of retinal pigment epithelium (RPE) pathology on intersession repeatability of retinal thickness and volume metrics derived from Spectralis spectral-domain optical coherence tomography (SD-OCT). Design Prospective cross-sectional single centre study. Participants 56 eyes of 56 subjects were divided into 3 groups: (A) normal RPE band (25 eyes), (B) RPE elevation: macular soft drusen (13 eyes) and (C) RPE attenuation: geographic atrophy or inherited retinal diseases (18 eyes). Methods Each subject underwent three consecutive follow-up macular raster scans (61 B-scans at 119 micron separation) at 1-month intervals. Main outcome measures Retinal thicknesses and volumes for each zone of the macular subfields before and after manual correction of segmentation error. Coefficients of repeatability (CR) were calculated. Results Mean (range) age was 57 (21-88) years. Mean central subfield thickness (CST) and total macular volume (TMV) were 264 and 258 µm (p = 0.62), and 8.0 and 7.8 mm3 (p = 0.31), before and after manual correction. Intersession CR (95% confidence interval) for CST and TMV were reduced from 40 (38-41) to 8.3 (8.1-8.5) and 0.62 to 0.16 mm3 after manual correction of segmentation lines. CR for CST were 7.4, 23.5 and 66.7 µm before and 7.0, 10.9 and 7.6 µm after manual correction in groups A, B and C. Conclusions Segmentation error in eyes with RPE disease has a significant impact on intersession repeatability of Spectralis SD-OCT macular thickness and volume metrics. Careful examination of each B-scan and manual adjustment can enhance the utility of quantitative measurement. Improved automated segmentation algorithms are needed.

Mutations in the USH2A gene are the most common cause of Usher syndrome and autosomal recessive non-syndromic retinitis pigmentosa. Here, we describe the generation of three induced pluripotent stem cell lines from dermal fibroblasts derived from a patient carrying biallelic c.949C > A and c.1256G > T variants in the USH2A gene, using episomal reprogramming plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28, mir302/367 and shRNA targeting TP53. All three lines expressed pluripotency markers, displayed unaltered karyotypes as well as trilineage differentiation potential, and were negative for reprogramming episomes and mycoplasma.

Purpose: To use a deep learning model to develop a fully automated method (fully semantic network and graph search [FS-GS]) of retinal segmentation for optical coherence tomography (OCT) images from patients with Stargardt disease. Methods: Eighty-seven manually segmented (ground truth) OCT volume scan sets (5171 B-scans) from 22 patients with Stargardt disease were used for training, validation and testing of a novel retinal boundary detection approach (FS-GS) that combines a fully semantic deep learning segmentation method, which generates a per-pixel class prediction map with a graph-search method to extract retinal boundary positions. The performance was evaluated using the mean absolute boundary error and the differences in two clinical metrics (retinal thickness and volume) compared with the ground truth. The performance of a separate deep learning method and two publicly available software algorithms were also evaluated against the ground truth. Results: FS-GS showed an excellent agreement with the ground truth, with a boundary mean absolute error of 0.23 and 1.12 pixels for the internal limiting membrane and the base of retinal pigment epithelium or Bruch's membrane, respectively. The mean difference in thickness and volume across the central 6 mm zone were 2.10 µm and 0.059 mm3. The performance of the proposed method was more accurate and consistent than the publicly available OCTExplorer and AURA tools. Conclusions: The FS-GS method delivers good performance in segmentation of OCT images of pathologic retina in Stargardt disease. Translational Relevance: Deep learning models can provide a robust method for retinal segmentation and support a high-throughput analysis pipeline for measuring retinal thickness and volume in Stargardt disease.

Purpose Microperimetry is commonly used to assess retinal function. We perform cross-sectional and longitudinal analysis on microperimetry parameters in USH2A retinopathy and explore end points suitable for future clinical trials. Methods Microperimetry was performed using two grids, Grid 1 (18° diameter) and Grid 2 (6° diameter). In Grid 1, four parameters (number of nonscotomatous loci, mean sensitivity [MS], responding point sensitivity [RPS], and edge of scotoma sensitivity [ESS]) were analyzed. In Grid 2, number of nonscotomatous loci and MS were examined. Interocular symmetry was also examined. Longitudinal analysis was conducted in a subset of eyes. Results Microperimetry could be performed in 16 of 21 patients. In Grid 1 (n = 15; average age, 35.6 years), average number of nonscotomatous loci, MS, RPS, and ESS were 46.6 loci, 10.0 dB, 14.7 and 9.6 dB, respectively. In Grid 2 (n = 13; average age, 37.4 years), 12 eyes had measurable sensitivity across the entire grid. Average MS was 23.8 dB. Interocular analysis revealed large 95% confidence intervals for all parameters. Longitudinally, Grid 1 (n = 12, average follow-up 2.6 years) ESS showed the fastest rate of decline (-1.84 dB/y) compared with MS (-0.34 dB/y) and RPS (-0.90 dB/y). Conclusions Our data suggest that ESS may be more useful than MS and RPS in test grids that cover a large extent of the macula. We caution the use of contralateral eye as an internal control. Translational relevance ESS may decrease the duration or sample size of treatment trials in USH2A retinopathy.

Inherited retinal diseases (IRDs) are genetically and phenotypically diverse, and they cause significant morbidity worldwide. Importantly, IRDs may be amenable to precision medicine strategies, and thus the molecular characterisation of causative variants is becoming increasingly important with the promise of personalised therapies on the horizon. ABCA4, involved in the translocation of visual cycle derivatives, is a well-established, frequent cause of IRDs worldwide, with pathogenic variants implicated in phenotypically diverse diseases. Identification of causative ABCA4 variants in some individuals, however, has been enigmatic, and resolution of this issue is currently a hotbed of research. Recent evidence has indicated that hypomorphic alleles, which cause disease under certain conditions, may account for some of the missing causal variants. It has been postulated that the ABCA4 c.5603A>T (p.Asn1868Ile) variant, previously considered benign, be reclassified as hypomorphic when in cis configuration with c.2588G>C (p.Gly863Ala/Gly863del), a variant previously considered to be pathogenic in its own right. We are exploring this relationship within an Australian cohort to test this theory.

The process of vision begins in the retina, yet the role of biomechanical forces in the retina is relatively unknown and only recently being explored. This contribution describes a computational framework involving 3D fluid–structure interaction simulations derived from fundus images that work towards creating unique data on retinal biomechanics. We developed methods to convert 2D fundus photographs into 3D geometries that follow the curvature of the retina. Retina arterioles are embedded into a six-layer representation of the retinal tissue with varying material properties throughout the retinal tissue. Using three different human retinas (healthy, glaucoma, diabetic retinopathy) and by varying our simulation approaches, we report the effects of transient versus steady flow, viscosity assumptions (Newtonian, non-Newtonian and Fahraeus–Lindqvist effect) and rigid versus compliant retinal tissue, on resulting wall shear stress (WSS) and von Mises stress. In the retinal arterioles, the choice of viscosity model is important and WSS obtained from models with the Fahraeus–Lindqvist effect is markedly different from Newtonian and non-Newtonian models. We found little difference in WSS between steady-state and pulsatile simulations (

We report the generation of the human iPSC line LEIi007-A from a patient with autosomal recessive Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene (c.[5461-10 T > C];[4139C > T]). Reprogramming of patient dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA to establish the clonal iPSC line LEIl007-A. LEIl007-A displayed normal pluripotent stem cell colony morphology, expressed pluripotent stem cell markers, displayed a normal karyotype and differentiated into ectodermal, mesodermal and endodermal germ layer lineages.

We report the generation of the human iPSC line LEIi008-A from a patient with retinitis pigmentosa-11 caused by a dominant nonsense mutation in the PRPF31 gene (NM_015629.3:c.1205C > A p.(Ser402Ter)). A second line, LEIi009-A, was generated from a related non-penetrant carrier of the same mutation with no retinal disease. Reprogramming of patient dermal fibroblasts using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA generated cell lines displaying pluripotent stem cell marker expression, a normal karyotype and the capability to differentiate into the three germ layer lineages. Resource table Unique stem cell lines identifier LEIi008-A LEIi009-A Alternative names of stem cell lines 1093ips4 (LEIi008-A) 1374ips1 (LEIi009-A) Institution Lions Eye Institute, Nedlands, Western Australia, Australia Contact information of distributor Samuel McLenachan: smclenachan@lei.org.au Fred Chen: fredchen@lei.org.au Type of cell lines iPSC Origin Human Cell Source Dermal fibroblasts Clonality Clonal Method of reprogramming Episomal Multiline rationale Unaffected mother and affected son carrying same dominant PRPF31 mutation Gene modification Yes Type of modification Hereditary Associated disease Retinitis Pigmentosa 11 Gene/locus PRPF31/19q13.42 Method of modification N/A Name of transgene or resistance N/A Inducible/constitutive system N/A Date archived/stock date LEIi008-A: 18/12/17; LEIi009-A: 17/11/17 Cell line repository/bank https://hpscreg.eu/cell-line/LEIi008-A https://hpscreg.eu/cell-line/LEIi009-A Ethical approval Human Research Ethics Office, University of Western Australia (RA/4/1/7916)

Mutation of the CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, has recently been associated with late-onset, non-syndromic retinal dystrophy. Herein we describe the multimodal imaging, immunological and systemic features of an adult with compound heterozygous CLN3 mutations. A 50-year-old female with non-syndromic retinal dystrophy from the age of 36 years underwent multimodal retinal imaging, electroretinography, neuroimaging, immunological studies and genetic testing. CLN3 transcripts were amplified from patient leukocytes by reverse transcriptase polymerase chain reaction and characterized by Sanger sequencing. Visual acuity declined to 6/12 and 6/76 due to asymmetrical central scotoma. ERG responses became electronegative and patient’s serum contained anti-retinal antibodies. Final visual acuity stabilized at 6/60 bilaterally 3 years after peri-ocular steroid and rituximab infusion. Genetic testing revealed compound heterozygous CLN3 mutations: the 1.02 kb deletion and a novel missense mutation (c.175G>A). In silico, analyses predicted the c.175G>A mutation disrupted an exonic splice enhancer site in exon 3. In patient leukocytes, CLN3 expression was reduced and novel CLN3 transcripts lacking exon 3 were detected. Our case study shows that (1) non-syndromic CLN3 disease leads to rod and delayed primary cone degeneration resulting in constricting peripheral field and enlarging central scotoma and, (2) the c.175G>A CLN3 mutation, altered splicing of the CLN3 gene. Overall, we provide comprehensive clinical characterization of a patient with non-syndromic CLN3 disease.

Purpose : To determine the influence of choroidal boundary morphology on the accuracy of automated measurements of subfoveal choroidal thickness (SFCT) in swept source optical coherence tomography (SSOCT). Methods : A retrospective image analysis of foveal-centered horizontal line scans from normal and diseased eyes using the Topcon DRI OCT-1 Atlantis SSOCT was conducted. Subfoveal choroid-scleral junction (CSJ) and retina-choroidal junction (RCJ) morphologies were graded by two observers. Automated SFCT (A-SFCT) was compared with manual SFCT (M-SFCT) measurements from Bruch's membrane to the posterior limits of choroidal vessel, hyperreflective stroma, and hyporeflective lamina fusca. Agreement in boundary grading was assessed by Cohen's kappa. A-SFCT and M-SFCT were compared using Bland-Altman analysis and paired t-tests. Results : A total of 200 eyes of 100 patients with a mean (SD) age of 62 (18) years were included. The choroidal vessel, stromal, and lamina fusca boundaries were visible in 100%, 58%, and 38% of the eyes, respectively. Interobserver agreement in RCJ and CSJ grading was high (kappa = 0.974 and 0.851). Mean A-SFCT differed from M-SFCT by only 2 μm at posterior choroidal vessel boundary (P = 0.801). A-SFCT overestimated SFCT at the posterior vessel wall boundary by 17 μm (P = 0.026) and 23 μm (P = 0.001) in the presence of a visible posterior choroidal stroma and lamina fusca, respectively. Conclusions : Automated outer choroidal boundary segmentation tends to identify the posterior limit of the choroidal vessel. Agreement between A-SFCT and M-SFCT is reduced by the presence of posterior stromal layer and lamina fusca. A-SFCT should be interpreted with RCJ and CSJ boundary grading.

There is an urgent need for a more effective intervention to slow or prevent progression of age-related macular degeneration (AMD) from its early stages to vision-threatening late complications. Subthreshold nanosecond laser (SNL) treatment has shown promise in preclinical studies and a pilot study in intermediate AMD (iAMD) as a potential treatment. We aimed to evaluate the safety of SNL treatment in iAMD and its efficacy for slowing progression to late AMD.The Laser Intervention in Early Stages of Age-Related Macular Degeneration (LEAD) study is a 36-month, multicenter, randomized, sham-controlled trial.Two hundred ninety-two participants with bilateral large drusen and without OCT signs of atrophy.Participants were assigned randomly to receive Retinal Rejuvenation Therapy (2RTThe primary efficacy outcome was the time to development of late AMD defined by multimodal imaging (MMI). Safety was assessed by adverse events.Overall, progression to late AMD was not slowed significantly with SNL treatment compared with sham treatment (adjusted hazard ratio [HR], 0.61; 95% confidence interval [CI], 0.33-1.14; P = 0.122). However, a post hoc analysis showed evidence of effect modification based on the coexistence of reticular pseudodrusen (RPD; adjusted interaction P = 0.002), where progression was slowed for the 222 participants (76.0%) without coexistent RPD at baseline (adjusted HR, 0.23; 95% CI, 0.09-0.59; P = 0.002), whereas an increased progression rate (adjusted HR, 2.56; 95% CI, 0.80-8.18; P = 0.112) was observed for the 70 participants (24.0%) with RPD with SNL treatment. Differences between the groups in serious adverse events were not significant.In participants with iAMD without MMI-detected signs of late AMD, no significant difference in the overall progression rate to late AMD between those receiving SNL and sham treatment were observed. However, SNL treatment may have a role in slowing progression for those without coexistent RPD and may be inappropriate in those with RPD, warranting caution when considering treatment in clinical phenotypes with RPD. Our findings provide compelling evidence for further trials of the 2RT

Purpose To evaluate the feasibility of using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) for the management of uveal melanoma (UM). Patients and Methods Low-coverage whole-genome sequencing was used to determine somatic chromosomal copy number alterations (SCNAs) in primary UM tumors, ctDNA, and whole-genome amplified CTCs. CTCs were immunocaptured using an antimelanoma-associated chondroitin sulfate antibody conjugated to magnetic beads and immunostained for melanoma antigen recognised by T cells 1 (MART1)/glycoprotein 100 (gp100)/S100 calcium-binding protein β (S100β). ctDNA was quantified using droplet digital polymerase chain reaction assay for mutations in the GNAQ, GNA11, PLCβ4, and CYSLTR2 genes. Results SCNA analysis of CTCs and ctDNA isolated from a patient with metastatic UM showed good concordance with the enucleated primary tumor. In a cohort of 30 patients with primary UM, CTCs were detected in 58% of patients (one to 37 CTCs per 8 mL of blood), whereas only 26% of patients had detectable ctDNA (1.6 to 29 copies/mL). The presence of CTCs or ctDNA was not associated with tumor size or other prognostic markers. However, the frequent detection of CTCs in patients with early-stage UM supports a model in which CTCs can be used to derive tumor-specific SCNA relevant for prognosis. Monitoring of ctDNA after treatment of the primary tumor allowed detection of metastatic disease earlier than 18F-labeled fluorodeoxyglucose positron emission tomography in two patients. Conclusion The presence of CTCs in localized UM can be used to ascertain prognostic SCNA, whereas ctDNA can be used to monitor patients for early signs of metastatic disease. This study paves the way for the analysis of CTCs and ctDNA as a liquid biopsy that will assist with treatment decisions in patients with UM.

Robyn H. Guymer, Zhichao Wu, Fred K. Chen, Usha Chakravarthy, Jennifer J. Arnold ... Shane R.Durkin ... et al.

Background To evaluate and classify image artefacts in optical coherence tomography (OCT) angiography (OCTA) of the choroid in a group of patients with macular diseases. Design Retrospective observational study. Participants Five patients with age-related macular degeneration, three with central serous retinopathy, one with polypoidal choroidal vasculopathy and one with multiple evanescent white dot syndrome. Methods OCTA and OCT reflectivity (OCTR) maps were reviewed along with their fluorescein angiography and indocyanine green angiography. Sixty OCTA images (20 outer retina, 20 Sattler and 20 Haller layers) were graded for image artefacts by two examiners independently. Main Outcome Measures OCTA artefacts and their correlation with OCTR maps, angiography and OCT B-scans. Results Artefacts (frequency) were classified into (i) motion (70–100%), (ii) fringe washout (100%), (iii) decorrelation projection (0–20%), (iv) masking and unmasking (50–65%) and (v) stromal decorrelation signal (100%). Motion artefact in OCTA is characterized by horizontal dark lines or bands not apparent on OCTR map. Fringe washout creates signal void within choroidal vessels because of fast blood flow. Decorrelation projection from retinal vasculature and choroidal new vessels above the Bruch's membrane are seen within the choroidal OCTA image. Masking and unmasking artefacts occur in regions of pigment epithelial detachment and atrophy. Decorrelation signals can also be seen in the choroidal stroma. Conclusions Our classification system of artefact in choroidal OCTA establishes a common terminology for clinical interpretation. This is important in enhancing our understanding of the principles of OCTA acquisition, and it also serves as a bench mark for reading centres.