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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 describe ocular adverse events and retinal changes during fibroblast growth factor receptor (FGFR) inhibitor (AZD4547) anticancer therapy., Methods: This is a sub-study examining ocular adverse effects from AZD4547 therapy (single-centre, open-label, single arm phase II clinical trial). Comprehensive ocular examinations were performed 3 weekly in 24 patients. Macular optical coherence tomography (OCT) scan (30 0  × 25 0 ) was obtained at each visit and OCT parameters [central 1 mm retinal thickness (CRT) and total macular volume in central 6 mm] extracted. OCT scans were subdivided into outer (ELM to RPE) and inner (ELM to ILM) layers to compare outer and inner retinal changes., Results: In 24 patients, AZD4547 was associated with eyelash elongation (n = 5, 21%) and punctate corneal erosion (n = 2, 8%). One patient developed clinically significant posterior capsular opacification during the study. OCT data were available in 23 patients, retinal changes ranged from an asymptomatic increased visibility of the interdigitation zone (IDZ) (n = 10, 43%) to multilobular subretinal fluid pockets (n = 5, 22%), which was associated with mild visual acuity loss. In a subset of patients (n = 9) with pre-AZD4547 dosing OCT baseline, CRT increased by mean (SD) of 9 (4) μm in those with IDZ change only compared with 64 (38) μm in those with other retinal changes. Retinal changes tended to be bilateral, self-limiting and improved over time without medical intervention., Conclusions: The ocular signs and symptoms did not result in dose cessation. Posteriorly, FGFR inhibition leads to outer retinal changes ranging from increased visibility of IDZ to distinct, multiple fluid pockets., (© 2021 Royal Australian and New Zealand College of Ophthalmologists.)

Background : Mutations in the RCC1 and BTB domain-containing protein 1 ( RCBTB1 ) gene have been implicated in a rare form of retinal dystrophy. Herein, we report the clinical features of a 45-year-old Singaporean-Chinese female and her presymptomatic sibling, who each possesses compound heterozygous mutations in RCBTB1 . Expression of RCBTB1 in patient-derived cells was evaluated. Materials and Methods : The natural history was documented by a series of ophthalmic examinations including electroretinography, fundus autofluorescence imaging, spectral-domain optical coherence tomography, visual field, microperimetry, and adaptive optics retinal imaging. Patient DNA was genetically analysed using a 537-gene Next Generation Sequencing panel and targeted Sanger sequencing. Expression of RCBTB1 in lymphocytes, fibroblasts, and induced pluripotent stem cells (iPSC) derived from the proband and healthy controls was characterized by quantitative PCR, Sanger sequencing, and western blotting. Results : The proband presented with left visual distortion at age 40 due to extrafoveal chorioretinal atrophy. Atrophy expanded at 1.3 (OD) and 1.0 (OS) mm 2 /year. Total macular volume declined by 0.09 (OD) and 0.13 (OS) mm 3 /year. Microperimetry demonstrated enlarging scotoma in both eyes. Generalised cone dysfunction was demonstrated by electroretinography. A retinal dystrophy panel testing revealed biallelic frameshifting mutations, c.170delG (p.Gly57Glufs*12) and c.707delA (p.Asn236Thrfs*11) in RCBTB1 . The level of RCBTB1 mRNA expression was reduced in patient-derived lymphocytes compared to controls. RCBTB1 protein was detected in control fibroblasts and iPSC but was absent in patient-derived cells. Conclusions : Atrophy expansion rate and macular volume change are feasible endpoints for monitoring RCBTB1 -associated retinopathy. We provide further functional evidence of pathogenicity for two disease-causing variants using patient-derived iPSCs.

Optical Coherence Tomography (OCT) enables non-invasive imaging of the retina and is used to diagnose and manage ophthalmic diseases including glaucoma. We present the first large-scale genome-wide association study of inner retinal morphology using phenotypes derived from OCT images of 31,434 UK Biobank participants. We identify 46 loci associated with thickness of the retinal nerve fibre layer or ganglion cell inner plexiform layer. Only one of these loci has been associated with glaucoma, and despite its clear role as a biomarker for the disease, Mendelian randomisation does not support inner retinal thickness being on the same genetic causal pathway as glaucoma. We extracted overall retinal thickness at the fovea, representative of foveal hypoplasia, with which three of the 46 SNPs were associated. We additionally associate these three loci with visual acuity. In contrast to the Mendelian causes of severe foveal hypoplasia, our results suggest a spectrum of foveal hypoplasia, in part genetically determined, with consequences on visual function., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: APK is a paid consultant to Aerie, Allergan, Google Health, Novartis, Reichert, Santen and Thea. LRP is consultant for Verily, Eyenovia, Bausch+Lomb, and Nicox. JW is consultant for Allergan, Editas, Maze, Regenxbio and has a sponsored research grant from Aerpio.

Purpose: To describe the thickness profiles of the full retinal and outer retinal layers (ORL) at the macula in healthy young adults, and associations with best-corrected visual acuity (BCVA)., Methods: In total, 1604 participants (19-30 years) underwent an eye examination that included measurements of their BCVA, axial length, and autorefraction. The retinal thickness at the foveal pit and at the nine Early Treatment of Diabetic Retinopathy Study macular regions (0.5-mm radius around the fovea, and superior, inferior, temporal, and nasal quadrants of the inner and outer rings of the macula) were obtained using spectral-domain optical coherence tomography imaging. A custom program was used to correct for transverse magnification effects because of different axial lengths., Results: The median full retinal and ORL thicknesses at the central macula were 285 µm and 92 µm. The full retina was thinnest centrally and thickest at the inner macula ring, whereas the ORL was thickest centrally and gradually decreased in thickness with increasing eccentricity. There was no association between axial length and the full retinal or ORL thickness. Increased thicknesses of the full retina at the central macula was associated with better BCVA; however, the effect size was small and not clinically significant., Conclusions: This article mapped the full retinal and ORL thickness profile in a population-based sample of young healthy adults., Translational Relevance: Thickness values presented in this article could be used as a normative reference for future studies on young adults and in clinical practice.

Background: Mutations in the splicing factor pre-messenger RNA processing factor 31 ( PRPF31 ) gene cause autosomal dominant retinitis pigmentosa 11 (RP11) through a haplo-insufficiency mechanism. We describe the phenotype and progression of microperimetry and autofluorescence endpoints in an Indigenous Australian RP11 family., Patients and Methods: Ophthalmic examination, optical coherence tomography, fundus autofluorescence and microperimetry were performed at baseline and every 6-12 months. Baseline and annual change in best-corrected visual acuity (BCVA), microperimetry mean sensitivity (MS) and number of scotoma loci, residual ellipsoid zone (EZ) span and hyperautofluorescent ring (HAR) area were reported. Next-generation and Sanger sequencing were performed in available members., Results: 12 affected members from three generations were examined. Mean (SD, range) age at onset of symptoms was 11 (4.5, 4-19) years. MS declined steadily from the third decade and EZ span and HAR area declined rapidly during the second decade. Serial microperimetry showed negligible change in MS over 2-3 years. However, mean EZ span, near-infrared and short-wavelength HAR area reduction was 203 (6.4%) µm/year, 1.8 (8.7%) mm 2 /year and 1.1 (8.6%) mm 2 /year, respectively. Genetic testing was performed on 11 affected and 10 asymptomatic members and PRPF31 c.1205 C > A (p.Ser402Ter) mutation was detected in all affected and two asymptomatic members (non-penetrant carriers)., Conclusions: Our findings suggest that in the studied cohort, the optimal window for therapeutic intervention is the second decade of life and residual EZ span and HAR area can be considered as efficacy outcome measures. Further studies on larger samples with different PRPF31  mutations and longer follow-up duration are recommended.

Background: Heterozygous c.440 G > T mutation in the S-antigen visual arrestin (SAG) gene has been described as a cause of autosomal dominant retinitis pigmentosa (adRP) in a series of patients of Hispanic origin. This study presents the early and late clinical features and disease progression rates in an Australian family with SAG adRP., Materials and Methods: An observational case series of four family members with adRP. They were examined clinically, with multi-modal retinal imaging and electroretinography (ERG) to ascertain phenotype. Disease progression rate was measured using optical coherence tomography (OCT) and fundus autofluorescence (FAF). A retinal dystrophy panel was used for the proband and cascade testing with targeted Sanger sequencing was conducted in other available family members., Results: The proband presented at 36 years of age with profoundly reduced full-field ERG responses despite a sector RP phenotype. This progressed to a classic RP pattern over several decades leaving a small residual island of central visual field. The horizontal span of the residual outer nuclear layer and the area of hyperautofluorescent ring contracted at a rate of 8-11% and 9-14% per year, respectively. DNA sequencing confirmed the segregation of SAG c.440 G > T mutation with disease., Conclusion: SAG adRP presents with a reduced full-field ERG response consistent with a rod-cone dystrophy in mid-life despite a sector RP phenotype. Centripetal progression of the disease into the macula can be tracked by OCT and FAF imaging.

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., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Stargardt disease is one of the most common forms of inherited retinal disease and leads to permanent vision loss. A diagnostic feature of the disease is retinal flecks, which appear hyperautofluorescent in fundus autofluorescence (FAF) imaging. The size and number of these flecks increase with disease progression. Manual segmentation of flecks allows monitoring of disease, but is time-consuming. Herein, we have developed and validated a deep learning approach for segmenting these Stargardt flecks (1750 training and 100 validation FAF patches from 37 eyes with Stargardt disease). Testing was done in 10 separate Stargardt FAF images and we observed a good overall agreement between manual and deep learning in both fleck count and fleck area. Longitudinal data were available in both eyes from 6 patients (average total follow-up time 4.2 years), with both manual and deep learning segmentation performed on all (n = 82) images. Both methods detected a similar upward trend in fleck number and area over time. In conclusion, we demonstrated the feasibility of utilizing deep learning to segment and quantify FAF lesions, laying the foundation for future studies using fleck parameters as a trial endpoint.

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., Competing Interests: Disclosure: J. Charng, None; T.M. Lamey, None; J.A. Thompson, None; T.L. Mclaren, None; M.S. Attia, None; I.L. McAllister, None; I.J. Constable, None; D.A. Mackey, None; J.N. De Roach, None; F.K. Chen, None, (Copyright 2020 The Authors.)

A common inherited retinal disease is caused by mutations in RHO expressed in rod photoreceptors that provide vision in dim ambient light. Approximately half of all RHO mutations result in a Class B phenotype where mutant rods are retained in some retinal regions but show severe degeneration in other regions. We determined the natural history of dysfunction and degeneration of retained rods by serially evaluating patients. Even when followed for more than 20 years, rod function and structure at some retinal locations could remain unchanged. Other locations showed loss of both vision and photoreceptors but the rate of rod vision loss was greater than the rate of photoreceptor degeneration. This unexpected divergence in rates with disease progression implied the development of a rod function deficit beyond loss of cells. The divergence of progression rates was also detectable over a short interval of 2 years near the health-disease transition in the superior retina. A model of structure-function relationship supported the existence of a large rod function deficit which was also most prominent near regions of health-disease transition. Our studies support the realistic therapeutic goal of improved night vision for retinal regions specifically preselected for rod function deficit in patients.

SIX1/SIX6 polymorphism has been shown to be associated with glaucoma. Studies have also found that, in older adults, retinal nerve fibre layer (RNFL) thickness is significantly thinned with each copy of the risk allele in SIX1/SIX6. However, it is not known whether these genetic variants exert their effects in younger individuals. Comparing a healthy young adult with an older adult cohort (mean age 20 vs 63 years), both of Northern European descent, we found that there was no significant RNFL thinning in each copy of the risk alleles in SIX1/SIX6 in the eyes of younger individuals. The older cohort showed an unexpectedly thicker RNFL in the nasal sector with each copy of the risk allele for both the SIX1 (rs10483727) and SIX6 (rs33912345) variants. In the temporal sector, thinner RNFL was found with each copy of the risk allele in rs33912345 with a decrease trend observed in rs10483727. Our results suggest that SIX1/SIX6 gene variants exert their influence later in adult life.

Importance: Atropine eyedrops are a promising treatment for slowing myopia progression in East Asian children. However, its effects on children in Australia, including those of non-Asian background, have not been well-studied., Background: The Western Australia Atropine for the Treatment of Myopia (WA-ATOM) study aims to determine the efficacy and long-term effects of low-dose atropine eyedrops in myopia control. This paper describes the study rationale, methodology and participant baseline characteristics., Design: Single-centre, double-masked, randomized controlled trial., Participants: Children (6-16 years) with spherical equivalent ≤-1.50 D in each eye, astigmatism ≤1.50 D and myopia progression by ≥0.50 D/year., Methods: Enrolled children were randomly assigned 2:1 to receive 0.01% atropine or placebo eyedrops. Participants are examined every 6 months during first 3 years of the study (2-year treatment phase followed by a 1-year washout phase), and then at a 5-year follow-up (2 years after the end of the washout phase)., Main Outcome Measures: Annual progression rate of myopia and axial length, tolerability to eyedrops and incidence and severity of unwanted effects., Results: Out of 311 children who were referred, 242 were suitable for study participation, and 153 were subsequently enrolled. The baseline characteristics of enrolled participants are presented., Conclusions and Relevance: Outcomes of the WA-ATOM study will inform on the efficacy, tolerability, safety and long-term effects of low-dose atropine eyedrops in myopia control in Australian children. The impact of ocular sun exposure, iris colour and parental myopia on the efficacy of low-dose atropine will also be assessed., (© 2020 Royal Australian and New Zealand College of Ophthalmologists.)

Background: Deletion-insertion (delins) variants in the retina-specific ATP-binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data., Methods: Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RNA extracted from patient-derived fibroblasts was analyzed by RT-PCR to evaluate splicing behavior of the ABCA4 variants., Results: Affected members carrying the novel c.6031&#95;6044delinsAGTATTTAACCAATATTT variant in exon 44 presented with contrasting phenotypes; from early-onset cone-rod dystrophy to late-onset macular dystrophy. This variant resulted in a 56-nucleotide deletion in the mutant allele by activation of a cryptic splice acceptor site which disrupts the reading frame and results in a premature termination codon (p.Ile2003LeufsTer41). If translated, the crucial functional domains near the C-terminus would be truncated from the ABCA4 protein., Conclusion: This work demonstrates the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and the use of patient-derived fibroblasts to evaluate the effect of a novel ABCA4 delins variant on splicing to complement in silico pathogenicity assessment., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

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 <25 dB cutoff., Methods: Retrospective MAIA results from healthy eyes were analyzed for prevalence of loci with <25 dB. At each locus, a new reference cutoff was derived from quantile regression of sensitivity against age at the 2.5th percentile. Two clinical cases of serial MAIA testing were analyzed using the new approach and compared to the <25 dB cutoff., Results: Fifty-four and 56 age-matched (range: 16-75 years) healthy eyes underwent small (37 loci) and large (68 loci) grid testing, respectively. Retinal sensitivity <25 dB was found in 5% of the small grid (1998 data points) and 10% of the large grid (3808 data points). These were found predominantly in older subjects and at the central point or in the perifoveal region. Quantile regression at each individual locus showed age-related decline with a median gradient of 0.6 dB/decade., Conclusions: We caution against using <25 dB cutoff in MAIA interpretation and advocate an age- and loci-specific cutoff criterion., Translational Relevance: Our study suggests that MAIA interpretation is influenced by the criterion used for defining abnormal pointwise measurement., Competing Interests: Disclosure: J. Charng, None; P.G. Sanfilippo, None; M.S. Attia, None; M. Dolliver, None; S. Arunachalam, None; A.L. Chew, None; E.N. Wong, None; D.A. Mackey, None; F.K. Chen, None, (Copyright 2020 The Authors.)

Introduction: Eye 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 Analysis: Participants 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 Dissemination: The 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 Number: ACTRN12617001599369; Active, not recruiting., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose : To consider the effect of including past sun exposure in estimating heritability and familial correlation of myopia-related traits. Methods : We calculate familial correlation and heritability of anterior chamber depth (ACD), axial length (AL), corneal curvature (CC), and spherical equivalent (SphE), with or without past sun exposure as a covariate, in a large number of unrelated nuclear families from the Raine Study (parents: Gen1, offspring: Gen2) residing in Perth, Australia, a city with a high amount of daily sunlight. Past sun exposure was objectively measured using conjunctival ultraviolet autofluorescence (CUVAF) photography. Results : When sun exposure was not included in the analysis, both familial correlation (correlation±SE; ACD: 0.308 ± 0.065, AL: 0.374 ± 0.061, CC: 0.436 ± 0.063, SphE: 0.281 ± 0.070) and heritability (ACD: 0.606 ± 0.104, AL: 0.623 ± 0.098, CC: 0.793 ± 0.079, SphE: 0.591 ± 0.106) were significant for all traits (all P < .001). However, there was no significant change in both familial correlation and heritability estimates when sun exposure was included as an additional covariate. Conclusions : Past sun exposure does not affect the estimation of the additive genetic component in myopia-related traits.

Gene therapy for adRP due to RHO mutations was recently shown to prevent photoreceptor death in a canine model of Class B disease. Among translational steps to be taken, one is to determine a method to detect efficacy in a human clinical trial. The relatively slow progression of adRP becomes a difficulty for clinical trials requiring an answer to whether there is slowed progression of degeneration in response to therapy. We performed a single-center, retrospective observational study of cross-sectional and longitudinal data. The study was prompted by our identification of a pericentral disease distribution in Class B RHO -adRP. Ultrawide optical coherence tomography (OCT) scans were used. Inferior retinal pericentral defects was an early disease feature. Degeneration further inferior in the retina merged with the pericentral defect, which extended into superior retina. In about 70% of patients, there was an asymmetric island of structure with significantly greater superior than inferior ellipsoid zone (EZ) extent. Serial measures of photoreceptor structure by OCT indicated constriction in superior retinal extent within a two-year interval. We conclude that these results should allow early-phase trials of therapy in RHO -adRP to move forward by inclusion of patients with an asymmetric extent of photoreceptor structure and by monitoring therapeutic effects over two years in the superior retina, a reasonable target for subretinal injection.

Photoreceptor ciliopathies constitute the most common molecular mechanism of the childhood blindness Leber congenital amaurosis. Ten patients with Leber congenital amaurosis carrying the c.2991+1655A>G allele in the ciliopathy gene centrosomal protein 290 (CEP290) were treated (ClinicalTrials.gov no. NCT03140969 ) with intravitreal injections of an antisense oligonucleotide to restore correct splicing. There were no serious adverse events, and vision improved at 3 months. The visual acuity of one exceptional responder improved from light perception to 20/400.

Purpose: To compare the phenotype of blue cone monochromacy (BCM) caused by large deletion mutations with those having the C203R missense mutation., Methods: BCM patients with large deletion mutations (n = 21; age range, 5-60 years), and with the C203R missense mutation (n = 13; age range, 5-70 years), were studied with optical coherence tomography, visual acuity, and perimetric sensitivity in a retrospective observational case series. Perceptual estimates of spatial resolution driven by rods, S-cones, and L/M-cones were obtained by the choice of chromatic gratings presented on varied adapting conditions with a modified microperimeter., Results: Both genotypes had abnormal foveal photoreceptor structure early in life. Patients with the C203R mutation, however, had decades-longer persistence of foveal photoreceptor outer nuclear layer thickness and a slower rate of development of inner segment/outer segment defects than did patients with large deletion mutations. At late ages, both genotypes had comparably severe losses of central structure. At the rod-rich hot spot, there was no difference in structure between cohorts with age. Grating acuities in all BCM patients were driven by S-cones and rods; the foveal structural differences were not reflected in a difference between cohorts in visual sensitivity and spatial resolution., Conclusions: A difference in structural phenotype due to the C203R mutation versus large deletion mutations in BCM was detected as a more prolonged persistence of foveal photoreceptor structure in patients with the missense mutation. This should be taken into account in planning natural history studies, selecting outcomes for clinical trials, and defining the time window for possible therapies.

Purpose: To determine the progression rate and the variability of rod and cone sensitivities in patients with X-linked retinitis pigmentosa (XLRP) caused by mutations in ORF15-RPGR., Methods: ORF15-RPGR-XLRP patients (n = 15) were studied prospectively over 2 years with static perimetry sampling the visual field under dark-adapted and light-adapted conditions on a 12° square grid covering 168° width and 84° height. Natural history of rod and cone sensitivity loss and test-retest variability were estimated. Data were analyzed pointwise as well as averaged across small regions of neighboring loci of approximately 80 mm2 (900 deg2) in size representing the likely extent of localized gene therapy injections., Results: Retinal loci with mild to moderate loss of sensitivity tended to be in the mid- to far-peripheral retina in most patients. When averaged across small regions, dark-adapted rod vision progressed at an average of 2 dB per year with a coefficient of repeatability (CR) of 6.3 dB, and light-adapted cone vision with white stimulus progressed at an average of 0.9 dB per year with a CR of 3.8 dB. For an average patient enrolled in an early-phase clinical trial, significant (α = 0.05) progression would be predicted to occur with 80% power in 4.5 years for rod vision and 6.1 years for cone vision. Localization of regions in the temporal hemifield and grouping of results from multiple patients would permit trial designs of shorter duration., Conclusions: Measurement of rod sensitivity under dark-adapted conditions averaged across a small region showed the greatest potential for detectability of progression in the shortest period.

Purpose: To determine the change in vision and retinal structure in patients with the common c.2299delG mutation in the USH2A gene in anticipation of clinical trials of therapy., Design: Retrospective observational case series., Methods: Eighteen patients, homozygotes or compound heterozygotes with the c.2299delG mutation in USH2A, were studied with regard to visual acuity, kinetic perimetry, dark- and light-adapted static perimetry, optical coherence tomography (OCT), and autofluorescence (AF) imaging. Serial data were available for at least half of the patients, depending on the parameter analyzed., Results: The kinetics of disease progression in this specific molecular form of USH2A differed between the measured parameters. Visual acuity could remain normal for decades. Kinetic and light-adapted static perimetry across the entire visual field had similar rates of decline that were slower than those of rod-based perimetry. Horizontal OCT scans through the macula showed that inner segment/outer segment line width had a similar rate of constriction as co-localized AF imaging and cone-based light-adapted sensitivity extent. The rate of constriction of rod-based sensitivity extent across this same region was twice as rapid as that of cones., Conclusions: In patients with the c.2299delG mutation in USH2A, rod photoreceptors are the cells that express disease early and more aggressively than cones. Rod-based vision measurements in central or extracentral-peripheral retinal regions warrant monitoring in order to complete a clinical trial in a timely manner., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: To evaluate whether a practical method of imaging lenticular autofluorescence (AF) can provide an individualized measure correlated with age-related lens yellowing in older subjects undergoing tests involving shorter wavelength lights., Methods: Lenticular AF was imaged with 488-nm excitation using a confocal scanning laser ophthalmoscope (cSLO) routinely used for retinal AF imaging. There were 75 older subjects (ages 47-87) at two sites; a small cohort of younger subjects served as controls. At one site, the cSLO was equipped with an internal reference to allow quantitative AF measurements; at the other site, reduced-illuminance AF imaging (RAFI) was used. In a subset of subjects, lens density index was independently estimated from dark-adapted spectral sensitivities performed psychophysically., Results: Lenticular AF intensity was significantly higher in the older eyes than the younger cohort when measured with the internal reference (59.2 ± 15.4 vs. 134.4 ± 31.7 gray levels; P < 0.05) as well as when recorded with RAFI without the internal reference (10.9 ± 1.5 vs. 26.1 ± 5.7 gray levels; P < 0.05). Lenticular AF was positively correlated with age; however, there could also be large differences between individuals of similar age. Lenticular AF intensity correlated well with lens density indices estimated from psychophysical measures., Conclusions: Lenticular AF measured with a retinal cSLO can provide a practical and individualized measure of lens yellowing, and may be a good candidate to distinguish between preretinal and retinal deficits involving short-wavelength lights in older eyes.

The retina is an easily accessible out-pouching of the central nervous system (CNS) and thus lends itself to being a biomarker of the brain. More specifically, the presence of neuronal, vascular and blood-neural barrier parallels in the eye and brain coupled with fast and inexpensive methods to quantify retinal changes make ocular biomarkers an attractive option. This includes its utility as a biomarker for a number of cerebrovascular diseases as well as a drug pharmacology and safety biomarker for the CNS. It is a rapidly emerging field, with some areas well established, such as stroke risk and multiple sclerosis, whereas others are still in development (Alzheimer's, Parkinson's, psychological disease and cortical diabetic dysfunction). The current applications and future potential of retinal biomarkers, including potential ways to improve their sensitivity and specificity are discussed. This review summarises the existing literature and provides a perspective on the strength of current retinal biomarkers and their future potential., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Pupillary light reflex (PLR) is driven by outer retinal photoreceptors and by melanopsin-expressing intrinsically photosensitive retinal ganglion cells of the inner retina. To isolate the melanopic component, we studied patients with severe vision loss due to Leber congenital amaurosis (LCA) caused by gene mutations acting on the outer retina., Methods: Direct PLR was recorded in LCA patients (n = 21) with known molecular causation and severe vision loss. Standard stimuli (2.5 log scot-cd.m-2; ∼13 log quanta.cm-2.s-1; achromatic full-field) with 0.1- or 5-second duration were used in all patients. Additional recordings were performed with higher luminance (3.9 log scot-cd.m-2) in a subset of patients., Results: The LCA patients showed no detectable PLR to the standard stimulus with short duration. With longer-duration stimuli, a PLR was detectable in the majority (18/21) of patients. The latency of the PLR was 2.8 ± 1.3 seconds, whereas normal latency was 0.19 ± 0.02 seconds. Peak contraction amplitude in patients was 1.1 ± 0.9 mm at 6.2 ± 2.3 seconds, considerably different from normal amplitude of 4.2 ± 0.4 mm at 3.0 ± 0.4 seconds. Recordings with higher luminance demonstrated that PLRs in severe LCA could also be evoked with short-duration stimuli., Conclusions: The PLR in severe LCA patients likely represents the activation of the melanopic circuit in isolation from rod and cone input. Knowledge of the properties of the human melanopic PLR allows not only comparison to those in animal models but also serves to define the fidelity of postretinal transmission in clinical trials targeting patients with no outer retinal function.

Purpose: To determine outcome measures for a clinical trial of Leber congenital amaurosis (LCA) associated with mutations in the GUCY2D gene., Design: Retrospective observational case series., Methods: Twenty-eight patients with GUCY2D-LCA (aged 2-59 years) were studied clinically and with chromatic full-field sensitivity testing (FST), optical coherence tomography (OCT), pupillometry, and the NEI Visual Function Questionnaire (VFQ)., Results: FST permitted quantitation of cone and rod sensitivity in these patients with severe visual impairment. For most patients, the degree of rod and cone sensitivity losses showed a relationship, thereby providing an opportunity to divide patients into cohorts by severity of rod and cone dysfunction. OCT analyses indicated that retinal structure could be used not only as an objective safety measure but also as an exploratory efficacy outcome. A foveal bulge was not present in 67% of patients. The intensity of inner segment/outer segment (ellipsoid zone line) reflectivity was reduced significantly at the fovea and in the rod-dense superior retina. Based on OCT and FST parameters, most patients had dissociation of structure and function. Abnormal pupillometry sensitivity in the majority of GUCY2D-LCA patients provided another objective efficacy outcome. NEI VFQ scores showed a similar range of findings to those of other severe retinal diseases., Conclusion: Conventional outcome measures, such as visual acuity and the NEI VFQ, will need to be complemented by methods more specific to this GUCY2D-LCA population. Any therapeutic strategy should determine if there is an effect on rod as well as cone function and structure. FST provides a photoreceptor-based subjective outcome; and OCT in 2 retinal regions, fovea and superior retina, can assess photoreceptor structure. A change in the relationship of structure and function away from baseline becomes evidence of efficacy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: To determine efficacy outcome measures for clinical trials of Leber congenital amaurosis (LCA) associated with a common intronic mutation in the CEP290 gene., Methods: CEP290-LCA patients (ages 5-48) with the intronic mutation (c.2991+1655A>G) were studied as a retrospective observational case series using clinical methods and with full-field sensitivity testing (FST), optical coherence tomography (OCT), autofluorescence imaging (NIR-RAFI), transient pupillary light reflex (TPLR), oculomotor control and instability (OCI), a mobility course, and a questionnaire (NEI-VFQ). Patients were investigated cross-sectionally but a subset was able to be followed longitudinally., Results: With FST, there was no rod function; cone sensitivities had a wide range from not detectable to near normal. OCT analyses indicated retained central photoreceptors with abnormal distal laminae. Based on OCT and FST, most patients had dissociation of structure and function. TPLR was nondetectable in the majority of patients, with responders demonstrating severe losses in light sensitivity. OCI was abnormal in most patients. NEI-VFQ scores had a similar range to those of other severe retinopathies. Mobility scores were consistent with FST sensitivities. In patients examined with FST, OCT, and NIR-RAFI over long-term intervals (7-10 years), there was limited but detectable disease progression., Conclusions: Efficacy would be a quantitative change in foveal cone function and possibly distal laminar structure. FST provides a subjective photoreceptor-based outcome; OCT and NIR-RAFI can assess photoreceptor and RPE structure. TPLR and OCI can provide objective measures of postretinal transmission. Minimal change over a decade indicates that there is no practical value in natural history studies.

Mutations in the ORF15 exon of the RPGR gene cause a common form of X-linked retinitis pigmentosa, which often results in severe loss of vision. In dogs and mice, gene augmentation therapy has been shown to arrest the progressive degeneration of rod and cone photoreceptors. However, the distribution of potentially treatable photoreceptors across the human retinas and the rate of degeneration are not known. Here, we have defined structural and functional features of the disease in 70 individuals with ORF15 mutations. We also correlated the features observed in patients with those of three Rpgr-mutant (Rpgr-ko, Rd9, and Rpgr-cko) mice. In patients, there was pronounced macular disease. Across the retina, rod and cone dysfunction showed a range of patterns and a spectrum of severity between individuals, but a high symmetry was observed between eyes of each individual. Genotype was not related to disease expression. In the Rpgr-ko mice, there were intra-retinal differences in rhodopsin and cone opsin trafficking. In Rd9 and Rpgr-cko mice, retinal degeneration showed inter-ocular symmetry. Longitudinal results in patients revealed localized rod and cone dysfunction with progression rates of 0.8 to 1.3 log per decade in sensitivity loss. Relatively retained rod and cone photoreceptors in mid- and far-peripheral temporal-inferior and nasal-inferior visual field regions should be good targets for future localized gene therapies in patients., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

Purpose: To present stimuli with varied sizes, colors, and patterns over a large range of luminance., Methods: The filter bar used in scotopic MP1 was replaced with a custom slide-in tray that introduces light from an external projector driven by an additional computer. MP1 software was modified to provide retinal tracking information to the computer driving the projector. Retinal tracking performance was evaluated by imaging the system input and the output simultaneously with a high-speed video system. Spatial resolution was measured with achromatic and chromatic grating/background combinations over scotopic and photopic ranges., Results: The range of retinal illuminance achievable by the modification was up to 6.8 log photopic Trolands (phot-Td); however, in the current work, only a lower range over -4 to +3 log phot-Td was tested in human subjects. Optical magnification was optimized for low-vision testing with gratings from 4.5 to 0.2 cyc/deg. In normal subjects, spatial resolution driven by rods, short wavelength-sensitive (S-) cones, and long/middle wavelength-sensitive (L/M-) cones was obtained by the choice of adapting conditions and wavelengths of grating and background. Data from a patient with blue cone monochromacy was used to confirm mediation., Conclusions: The modified MP1 can be developed into an outcome measure for treatments in patients with severe retinal degeneration, very low vision, and abnormal eye movements such as those for whom treatment with optogenetics is planned, as well as for patients with cone disorders such as blue cone monochromacy for whom treatment with gene therapy is planned to improve L/M-cone function above a normal complement of rod and S-cone function.

Objective. To examine whether retinal electrophysiology is a useful surrogate marker of drug penetrance into the central nervous system (CNS). Materials and Methods. Brain and retinal electrophysiology were assessed with full-field visually evoked potentials and electroretinograms in conscious and anaesthetised rats following systemic or local administrations of centrally penetrant (muscimol) or nonpenetrant (isoguvacine) compounds. Results. Local injections into the eye/brain bypassed the blood neural barriers and produced changes in retinal/brain responses for both drugs. In conscious animals, systemic administration of muscimol resulted in retinal and brain biopotential changes, whereas systemic delivery of isoguvacine did not. General anaesthesia confounded these outcomes. Conclusions. Retinal electrophysiology, when recorded in conscious animals, shows promise as a viable biomarker of drug penetration into the CNS. In contrast, when conducted under anaesthetised conditions confounds can be induced in both cortical and retinal electrophysiological recordings.

The electroretinogram (ERG, retina) and visual evoked potential (VEP, brain) are widely used in vivo tools assaying the integrity of the visual pathway. Current recordings in preclinical models are conducted under anesthesia, which alters neural physiology and contaminates responses. We describe a conscious wireless ERG and VEP recording platform in rats. Using a novel surgical technique to chronically implant electrodes subconjunctivally on the eye and epidurally over the visual cortex, we are able to record stable and repeatable conscious ERG and VEP signals over at least 1 month. We show that the use of anaesthetics, necessary for conventional ERG and VEP measurements, alters electrophysiology recordings. Conscious visual electrophysiology improves the viability of longitudinal studies by eliminating complications associated with repeated anaesthesia. It will also enable uncontaminated assessment of drug effects, allowing the eye to be used as an effective biomarker of the central nervous system.

Purpose: To determine whether there is an age-dependent susceptibility in retinal function in response to repeated anterior chamber cannulation with or without intraocular pressure (IOP) elevation., Methods: Baseline electroretinograms were measured in 3- and 18-month-old Sprague-Dawley rats (n = 16 each group). Following baseline assessment, eyes were randomly assigned to undergo a 60-min anterior chamber cannulation with IOP either left at baseline (sham, 15 mm Hg) or elevated to 60 mm Hg. This was repeated three additional times, with each episode separated by 1 week. At weeks 1 to 3, dark-adapted retinal function was assessed immediately before cannulation, with final functional assessment at week 4., Results: Both sham and IOP elevated eyes of older rats showed retinal dysfunction, which became more pronounced with the number of repeated insults. This effect was largest for responses arising from the inner retina. Repeated insult in younger eyes did not produce a change in amplitude but an increase in the sensitivity to light of photoreceptoral and bipolar cell components of the electroretinogram., Conclusions: Repeated trauma, not IOP, produces permanent retinal dysfunction in older eyes. Younger eyes appear to be able to withstand this type of injury by upregulating sensitivity of outer and middle retinal responses to maintain normal inner retinal function.

Purpose: To investigate the effect of old age (3 vs. 18 months) on the retinal function of albino (Sprague-Dawley [SD]) and pigmented (Long-Evans [LE]) rats., Methods: Electroretinograms (ERG) were recorded in both albino (SD; 3 months old n = 16, 18 months old n = 16) and pigmented (LE; 3 months n = 16, 18 months n = 5) rats. Data are analyzed for photoreceptor, ON-bipolar, and retinal ganglion cell (RCG) amplitudes as well as photoreceptor and ON-bipolar cell sensitivities., Results: In the pigmented strain, senescence results in decreased photoreceptor output, but ON-bipolar and retinal ganglion cell amplitudes were preserved, due to a relative increase in ON-bipolar cell sensitivity. In the albino rats, although ageing decreased both photoreceptor and ON-bipolar cell amplitudes, increased photoreceptor sensitivity produced a relative sparing of retinal ganglion cell amplitude., Conclusions: Both strains show evidence of retinal plasticity with senescence, albeit at different retinal levels. The exact mechanisms underlying sensitivity changes require further investigation. Nevertheless, given the findings of previous human studies, pigmented rats appear to be a more appropriate model for human ageing. Future work using animals to study the effect of ageing need careful consideration in strain selection.