In-Depth Retinal Sensitivity Assessment With the MP3 Type S Microperimeter: A Methods Study.

Purpose: Retinal sensitivity is frequently listed as an end point in clinical trials, often with long working practices. The purpose of this methods study was to provide a new workflow and reduced test time for in-depth characterization of retinal sensitivity. Methods: A workflow for the MP3-S microperimeter with detailed functional characterization of the retina under photopic, mesopic, and scotopic conditions was evaluated. Grids of 32 and 28 test positions for photopic/mesopic and scotopic, respectively, were tested in 12 healthy individuals and compared with an established 68-point grid for test time, mean sensitivity (MS), and bivariate contour ellipse area (BCEA). Results: The mean test time (range; ±SD) was 10.5 minutes (8.4-14.9; ±2.0) in the 68-point grid and 4.3 minutes (3.8-5.0; ±0.4) in the 32-point grid, which was significantly different (P < 0.0001). The mean of difference in test time (±SD; 95% confidence interval) was 6.1 minutes (±2.0; 4.6-7.6). MS and BCEA were significantly correlated between grids (r = 0.89 and 0.74; P = 0.0005 and 0.014, respectively). Mean test time of subjects who underwent the full protocol (n = 4) was 2.15 hours. Conclusions: The protocol suggested herein appears highly feasible with in-depth characterization of retinal function under different testing conditions and in a short test time. Translational Relevance: The protocol described herein allows for characterization of the retina under different testing conditions and in a short test time, which is relevant due to its potential for patient prognostication and follow-up in clinical settings and also given its increasing role as a clinical trial end point.

Structural and functional characterization of an individual with the M285R KCNV2 hypomorphic allele.

BACKGROUND: Disease-causing variants in the KCNV2 gene are associated with "cone dystrophy with supernormal rod responses," a rare autosomal recessive retinal dystrophy. There is no previous report of hypomorphic variants in the disease. MATERIAL AND METHODS: Medical history, genetic testing, ocular examination, high-resolution retinal imaging including adaptive optics scanning light ophthalmoscopy (AOSLO), and functional assessments. RESULTS: A 16-year-old male with mild cone-rod dystrophy presented with reduced central vision and photophobia. Genetic testing showed two variants in KCNV2, c.614_617dupAGCG (p.207AlafsTer166) and c.854T>G (p.Met285Arg), the latter which was previously considered benign. Electrophysiological assessment revealed the pathognomic electroretinogram waveforms associated with KCNV2-retinopathy. Optical coherence tomography showed discrete focal ellipsoid zone disruption, while fundus autofluorescence was normal. Non-waveguiding cones corresponding to areas of loss of photoreceptor integrity were visible on adaptive optics scanning light ophthalmoscopy. Retinal sensitivity and fixation were relatively preserved, with a demonstrable deterioration after 14 months of follow-up. CONCLUSIONS: We provide functional and structural evidence that the variant M285R is disease-causing if associated with a loss-of-function variant. To the best of our knowledge, this is the first hypomorphic allele reported in KCNV2.

Adaptive Optics Retinal Imaging in RDH12-Associated Early Onset Severe Retinal Dystrophy.

Purpose: RDH12 is among the most common genes found in individuals with early-onset severe retinal (EOSRD). Adaptive optics scanning light ophthalmoscopy (AOSLO) enables resolution of individual rod and cone photoreceptors in the retina. This study presents the first AOSLO imaging of individuals with RDH12-associated EOSRD. Methods: Case series of patients who attended Moorfields Eye Hospital (London, UK). Spectral-domain optical coherence tomography, near-infrared reflectance (NIR), and blue autofluorescence imaging were analyzed. En face image sequences of photoreceptors were recorded using either of two AOSLO modalities. Cross-sectional analysis was undertaken for seven patients and longitudinal analysis for one patient. Results: Nine eyes from eight patients are presented in this case series. The mean age at the time of the assessment was 11.2 ± 6.5 years of age (range 7-29). A subfoveal continuous ellipsoid zone (EZ) line was present in eight eyes. Posterior pole AOSLO revealed patches of cone mosaics. Average cone densities at regions of interest 0.5° to the fovea ranged from 12,620 to 23,660 cells/mm2, whereas intercell spacing ranged from 7.0 to 9.7 µm. Conclusions: This study demonstrates that AOSLO can provide useful high-quality images in patients with EOSRD, even during childhood, with nystagmus, and early macular atrophy. Cones at the posterior pole can appear as scattered islands or, possibly later in life, as a single subfoveal conglomerate. Detailed image analysis suggests that retinal pigment epithelial stress and dysfunction may be the initial step toward degeneration, with NIR being a useful tool to assess retinal well-being in RDH12-associated EOSRD.

Phenotyping and genotyping inherited retinal diseases: Molecular genetics, clinical and imaging features, and therapeutics of macular dystrophies, cone and cone-rod dystrophies, rod-cone dystrophies, Leber congenital amaurosis, and cone dysfunction syndromes.

Inherited retinal diseases (IRD) are a leading cause of blindness in the working age population and in children. The scope of this review is to familiarise clinicians and scientists with the current landscape of molecular genetics, clinical phenotype, retinal imaging and therapeutic prospects/completed trials in IRD. Herein we present in a comprehensive and concise manner: (i) macular dystrophies (Stargardt disease (ABCA4), X-linked retinoschisis (RS1), Best disease (BEST1), PRPH2-associated pattern dystrophy, Sorsby fundus dystrophy (TIMP3), and autosomal dominant drusen (EFEMP1)), (ii) cone and cone-rod dystrophies (GUCA1A, PRPH2, ABCA4, KCNV2 and RPGR), (iii) predominant rod or rod-cone dystrophies (retinitis pigmentosa, enhanced S-Cone syndrome (NR2E3), Bietti crystalline corneoretinal dystrophy (CYP4V2)), (iv) Leber congenital amaurosis/early-onset severe retinal dystrophy (GUCY2D, CEP290, CRB1, RDH12, RPE65, TULP1, AIPL1 and NMNAT1), (v) cone dysfunction syndromes (achromatopsia (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, ATF6), X-linked cone dysfunction with myopia and dichromacy (Bornholm Eye disease; OPN1LW/OPN1MW array), oligocone trichromacy, and blue-cone monochromatism (OPN1LW/OPN1MW array)). Whilst we use the aforementioned classical phenotypic groupings, a key feature of IRD is that it is characterised by tremendous heterogeneity and variable expressivity, with several of the above genes associated with a range of phenotypes.

Deep phenotyping of PROM1-associated retinal degeneration.

BACKGROUND/AIMS: The purpose of this study was to investigate retinal structure in detail of subjects with autosomal-dominant (AD) and autosomal-recessive (AR) PROM1-associated retinal degeneration (PROM1-RD), study design: institutional, cross-sectional study. METHODS: Four eyes from four subjects (three with AD and one with AR) PROM1-RD were investigated by ophthalmic examination including best-corrected visual acuity (BCVA) and multimodal retinal imaging: fundus autofluorescence (FAF), spectral-domain optical coherence tomography (SD-OCT) and adaptive optics scanning light ophthalmoscopy. Quantitative assessment of atrophic lesions determined by FAF, thickness of individual retinal layers and cone photoreceptor quantification was performed. RESULTS: BCVA ranged from 20/16 to 20/200. Initial pathological changes included the presence of hyperautofluorescent spots on FAF imaging, while later stages demonstrated discrete areas of atrophy. In all patients, thinning of the outer retinal layers on SD-OCT with varying degrees of atrophy could be detected depending on disease-causing variants and age. Cone density was quantified both in central and/or at different eccentricities from the fovea. Longitudinal assessments were possible in two patients. CONCLUSIONS: PROM1-RD comprises a wide range of clinical phenotypes. Depending on the stage of disease, the cone mosaic in PROM1-RD is relatively preserved and can potentially be targeted by cone-directed interventions.

First-in-Human Gene Therapy Trial of AAV8-hCARp.hCNGB3 in Adults and Children With CNGB3-associated Achromatopsia.

PURPOSE: To assess the safety and efficacy of AAV8-hCARp.hCNGB3 in participants with CNGB3-associated achromatopsia (ACHM). DESIGN: Prospective, phase 1/2 (NCT03001310), open-label, nonrandomized clinical trial. METHODS: The study enrolled 23 adults and children with CNGB3-associated ACHM. In the dose-escalation phase, adult participants were administered 1 of 3 AAV8-hCARp.hCNGB3 dose levels in the worse-seeing eye (up to 0.5 mL). After a maximum tolerated dose was established in adults, an expansion phase was conducted in children ≥3 years old. All participants received topical and oral corticosteroids. Safety and efficacy parameters, including treatment-related adverse events and visual acuity, retinal sensitivity, color vision, and light sensitivity, were assessed for 6 months. RESULTS: AAV8-hCARp.hCNGB3 (11 adults, 12 children) was safe and generally well tolerated. Intraocular inflammation occurred in 9 of 23 participants and was mainly mild or moderate in severity. Severe cases occurred primarily at the highest dose. Two events were considered serious and dose limiting. All intraocular inflammation resolved following topical and systemic steroids. There was no consistent pattern of change from baseline to week 24 for any efficacy assessment. However, favorable changes were observed for individual participants across several assessments, including color vision (n = 6/23), photoaversion (n = 11/20), and vision-related quality-of-life questionnaires (n = 21/23). CONCLUSIONS: AAV8-hCARp.hCNGB3 for CNGB3-associated ACHM demonstrated an acceptable safety and tolerability profile. Improvements in several efficacy parameters indicate that AAV8-hCARp.hCNGB3 gene therapy may provide benefit. These findings, with the development of additional sensitive and quantitative end points, support continued investigation.

Change in Cone Structure Over 24 Months in USH2A-Related Retinal Degeneration.

PURPOSE: To describe cone structure changes using adaptive optics scanning laser ophthalmoscopy (AOSLO) in the Rate of Progression of USH2A-related Retinal Degeneration (RUSH2A) study. DESIGN: Multicenter, longitudinal natural history study. METHODS: AOSLO images were acquired at 4 centers, twice at baseline and annually for 24 months in this natural history study. For each eye, at least 10 regions of interest (ROIs) with ≥50 contiguous cones were analyzed by masked, independent graders. Cone spacing Z-scores, standard deviations from the normal mean at the measured location, were compared between graders and tests at baseline. The association of cone spacing with clinical characteristics was assessed using linear mixed effects regression models weighted by image quality score. Annual rates of change were calculated based on differences between visits. RESULTS: Fourteen eyes of 14 participants were imaged, with 192 ROIs selected at baseline. There was variability among graders, which was greater in images with lower image quality score (P < .001). Cone spacing was significantly correlated with eccentricity, quality score, and disease duration (P < .02). On average, the cone spacing Z-score increased 0.14 annually (about 9%, P < .001). We observed no significant differences in rate of change between disease type (Usher syndrome or retinitis pigmentosa), imaging site, or grader. CONCLUSIONS: Using current methods, the analysis of quantitative measures of cone structure showed some challenges, yet showed promise that AOSLO images can be used to characterize progressive change over 24 months. Additional multicenter studies using AOSLO are needed to advance cone mosaic metrics as sensitive outcome measures for clinical trials. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Foveal Cone Structure in Patients With Blue Cone Monochromacy.

Purpose: Blue cone monochromacy (BCM) is a rare inherited cone disorder in which both long- (L-) and middle- (M-) wavelength sensitive cone classes are either impaired or nonfunctional. Assessing genotype-phenotype relationships in BCM can improve our understanding of retinal development in the absence of functional L- and M-cones. Here we examined foveal cone structure in patients with genetically-confirmed BCM, using adaptive optics scanning light ophthalmoscopy (AOSLO). Methods: Twenty-three male patients (aged 6-75 years) with genetically-confirmed BCM were recruited for high-resolution imaging. Eight patients had a deletion of the locus control region (LCR), and 15 had a missense mutation-Cys203Arg-affecting the first two genes in the opsin gene array. Foveal cone structure was assessed using confocal and non-confocal split-detection AOSLO across a 300 × 300 µm area, centered on the location of peak cell density. Results: Only one of eight patients with LCR deletions and 10 of 15 patients with Cys203Arg mutations had analyzable images. Mean total cone density for Cys203Arg patients was 16,664 ± 11,513 cones/mm2 (n = 10), which is, on average, around 40% of normal. Waveguiding cone density was 2073 ± 963 cones/mm2 (n = 9), which was consistent with published histological estimates of S-cone density in the normal eye. The one patient with an LCR deletion had a total cone density of 10,246 cones/mm2 and waveguiding density of 1535 cones/mm2. Conclusions: Our results show that BCM patients with LCR deletions and Cys203Arg mutations have a population of non-waveguiding photoreceptors, although the spectral identity and level of function remain unknown.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

Leber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

Axial Length Distributions in Patients With Genetically Confirmed Inherited Retinal Diseases.

Purpose: We investigated axial length (AL) distributions in inherited retinal diseases (IRDs), comparing them with reference cohorts. Methods: AL measurements from IRD natural history study participants were included and compared with reference cohorts (TwinsUK, Raine Study Gen2-20, and published studies). Comparing with the Raine Study cohort, formal odds ratios (ORs) for AL ≥ 26 mm or AL ≤ 22 mm were derived for each IRD (Firth's logistic regression model, adjusted for age and sex). Results: Measurements were available for 435 patients (median age, 19.5 years). Of 19 diseases, 10 had >10 participants: ABCA4 retinopathy; CNGB3- and CNGA3-associated achromatopsia; RPGR-associated disease; RPE65-associated disease; blue cone monochromacy (BCM); Bornholm eye disease (BED); TYR- and OCA2-associated oculocutaneous albinism; and GPR143-associated ocular albinism. Compared with the TwinsUK cohort (n = 322; median age, 65.1 years) and Raine Study cohort (n = 1335; median age, 19.9 years), AL distributions were wider in the IRD groups. Increased odds for longer ALs were observed for BCM, BED, RPGR, RPE65, OCA2, and TYR; increased odds for short AL were observed for RPE65, TYR, and GPR143. In subanalysis of RPGR-associated disease, longer average ALs occurred in cone-rod dystrophy (n = 5) than rod-cone dystrophy (P = 0.002). Conclusions: Several diseases showed increased odds for longer AL (highest OR with BCM); some showed increased odds for shorter AL (highest OR with GPR143). Patients with RPE65- and TYR-associated disease showed increased odds for longer and for shorter eyes. Albinism genes were associated with different effects on AL. These findings add to the phenotype of IRDs and may yield insights into mechanisms of refractive error development.

Novel disease-causing variant in RDH12 presenting with autosomal dominant retinitis pigmentosa.

AIM: To describe the clinical and molecular features of a novel, autosomal dominant RDH12-retinopathy. METHODS: Retrospective cross-sectional study. Twelve individuals from a four-generation British pedigree underwent ophthalmic examination, genotyping using next generation sequencing, including whole genome sequencing and multimodal retinal imaging including fundus photography, optical coherence tomography (OCT), autofluorescence imaging and adaptive optics (AO) scanning light ophthalmoscopy were performed. Visual electrophysiology was performed in a subset. RESULTS: Eight family members were confirmed as affected by genotyping heterozygous for RDH12 c.763delG. Visual acuity ranged from -0.1 to 0.2 logMAR. Affected individuals had constricted visual fields. A parafoveal and peripapillary ring of hyper-autofluorescence was seen initially, and with progression the area of perifoveal hypo-autofluorescence increased to involve the parafoveal area. Mild retinal thinning was identified on OCT imaging with reduction in both foveal total retinal and outer nuclear layer thickness. Cone densities along the temporal meridian were reduced in affected individuals compared with normative values at all temporal eccentricities studied. One individual with incomplete penetrance, was identified as clinically affected primarily on the basis of AO imaging. Full-field electroretinography demonstrated a rod-cone pattern of dysfunction and large-field pattern electroretinography identified peripheral macular dysfunction. CONCLUSIONS: This novel heterozygous variant RDH12 c.763delG is associated with a rod-cone dystrophy with variable expression. Determination of the degree of penetrance may depend on the modality employed to phenotypically characterise an individual. This rare and specific heterozygous (dominant) variant is predicted to result in a gain of function, that causes disease in a gene typically associated with biallelic (recessive) variants.

Pathogenic variants in the CYP21A2 gene cause isolated autosomal dominant congenital posterior polar cataracts.

BACKGROUND: Congenital cataracts are the most common cause of visual impairment worldwide. Inherited cataract is a clinically and genetically heterogeneous disease. Here we report disease-causing variants in a novel gene, CYP21A2, causing autosomal dominant posterior polar cataract. Variants in this gene are known to cause autosomal recessive congenital adrenal hyperplasia (CAH). METHODS: Using whole-exome sequencing (WES), we have identified disease-causing sequence variants in two families of British and Irish origin, and in two isolated cases of Asian-Indian and British origin. Bioinformatics analysis confirmed these variants as rare with damaging pathogenicity scores. Segregation was tested within the families using direct Sanger sequencing. RESULTS: A nonsense variant NM_000500.9 c.955 C > T; p.Q319* was identified in CYP21A2 in two families with posterior polar cataract and in an isolated case with unspecified congenital cataract phenotype. This is the same variant previously linked to CAH and identified as Q318* in the literature. We have also identified a rare missense variant NM_000500.9 c.770 T > C; p.M257T in an isolated case with unspecified congenital cataract phenotype. CONCLUSION: This is the first report of separate sequence variants in CYP21A2 associated with congenital cataract. Our findings extend the genetic basis for congenital cataract and add to the phenotypic spectrum of CYP21A2 variants and particularly the CAH associated Q318* variant. CYP21A2 has a significant role in mineralo- and gluco-corticoid biosynthesis. These findings suggest that CYP21A2 may be important for extra-adrenal biosynthesis of aldosterone and cortisol in the eye lens.

Agreement Between Spectral-Domain and Swept-Source Optical Coherence Tomography Retinal Thickness Measurements in Macular and Retinal Disease.

INTRODUCTION: To assess inter-device agreement in optical coherence tomography-derived retinal thickness measurements in patients with known macular conditions between spectral-domain and swept-source optical coherence tomography (OCT). METHODS: Two hundred seventy-two subjects were included in the study. They consisted of 91 male (33.5%) and 181 female (66.5%) subjects, and 132 left (48.5%) and 140 right (51.5%) eyes. Each subject underwent spectral-domain OCT (SD-OCT, Spectralis, Heidelberg Engineering; RTVue XR Avanti XR HD, Optovue) and swept-source OCT (SS-OCT; DRI-OCT-1, Atlantis, Topcon) in a single imaging session performed by the same clinical trial-certified technician. The comparison of retinal thickness reproducibility between devices was performed using Bland-Altman analyses and across the entire data set using the intraclass correlation coefficient (ICC). RESULTS: The ICC of the retinal thickness measurements (95% confidence interval) made using all three OCT instruments was 0.81 (0.77-0.84). The mean difference in mean retinal thickness between Spectralis SD-OCT and Topcon SS-OCT was 59.1 µm (95% limit of agreement [LoA] -21.7 to 139.8 µm). The mean difference in mean retinal thickness between Optovue SD-OCT and Topcon SS-OCT was 21.8 µm (95% LoA -34.7  to 78.3 µm). CONCLUSIONS: Retinal layer thickness measurements vary between SS-OCT and SD-OCT devices. We describe inter-device agreement in retinal thickness between SS-OCT and SD-OCT in patients with macular conditions. Clinicians should be aware of the differences in retinal thickness values when imaging patients using different OCT devices and should consider using the same OCT device model in order to monitor clinical change. TRIAL REGISTRATION: ClinicalTrials.gov Identifier (NCT02828215).

Comparing Retinal Structure in Patients with Achromatopsia and Blue Cone Monochromacy Using OCT.

Purpose: To compare foveal hypoplasia and the appearance of the ellipsoid zone (EZ) at the fovea in patients with genetically confirmed achromatopsia (ACHM) and blue cone monochromacy (BCM). Design: Retrospective, multi-center observational study. Subjects: Molecularly confirmed patients with ACHM (n = 89) and BCM (n = 33). Methods: We analyzed high-resolution spectral domain optical coherence tomography (SD-OCT) images of the macula from aforementioned patients with BCM. Three observers independently graded SD-OCT images for foveal hypoplasia (i.e. retention of one or more inner retinal layers at the fovea) and four observers judged the integrity of the EZ at the fovea, based on an established grading scheme. These measures were compared with previously published data from the ACHM patients. Main Outcome Measures: Presence of foveal hypoplasia and EZ grade. Results: Foveal hypoplasia was significantly more prevalent in ACHM than in BCM (p<0.001). In addition, we observed a significant difference in the distribution of EZ grades between ACHM and BCM, with grade II EZ being by far the most common phenotype in BCM (61% of patients). In contrast, ACHM patients had a relatively equal prevalence of EZ grades I, II, and IV. Interestingly, grade IV EZ was 2.6 times more prevalent in ACHM compared to BCM, while grade V EZ (macular atrophy) was present in 3% of both the ACHM and BCM cohorts. Conclusions: The higher incidence of foveal hypoplasia in ACHM than BCM supports a role for cone activity in foveal development. Although there are differences in EZ grades between these conditions, the degree of overlap suggests EZ grade is not sufficient for definitive diagnosis, in contrast to previous reports. Analysis of additional OCT features in similar cohorts may reveal differences with greater diagnostic value. Finally, the extent to which foveal hypoplasia or EZ grade is prognostic for therapeutic potential in either group remains to be seen, but motivates further study.

Long-Term Investigation of Retinal Function in Patients with Achromatopsia.

Purpose: To investigate the long-term natural history of retinal function of achromatopsia (ACHM). Methods: Subjects with molecularly confirmed ACHM were recruited in a prospective cohort study of mesopic microperimetry. Coefficient of repeatability and intraclass correlation coefficient (ICC) of mean sensitivity (MS) were calculated. Best-corrected visual acuity (BCVA), bivariate contour ellipse area (BCEA), contrast sensitivity (CS), MS, total volume (VTOT), and central field volume (V5°) from volumetric and topographic analyses were acquired. Correlation of functional parameters with structural findings from optical coherence tomography (OCT) was performed. Results: Eighteen subjects were recruited. Mean follow-up was 7.2 years. The MS test-retest repeatability coefficient was 1.65 decibels (dB), and the ICC was 0.973 (95% confidence interval, 0.837-0.98). Mean MS was similar for right and left eyes (16.97dB and 17.14dB, respectively). A negative significant correlation between logMAR BCVA and the retinal sensitivity indices (MS, VTOT, V5°) was found. A significant negative correlation between logCS and MS, VTOT, and V5° was also observed. BCVA and BCEA improved during follow-up. Mean CS, MS, VTOT, and V5° at final follow-up were similar to baseline. MS was similar between CNGA3- and CNGB3-ACHM. Patients with and without the presence of a foveal ellipsoid zone on OCT had similar MS (16.64 dB and 17.17 dB, respectively). Conclusions: We demonstrate a highly reproducible assessment of MS. Retinal function including MS, volumetric indices, and CS are stable in ACHM. Improvement of fixation stability and small changes of BCVA over time may be part of the natural history of the disease.

Intraobserver Repeatability and Interobserver Reproducibility of Foveal Cone Density Measurements in CNGA3- and CNGB3-Associated Achromatopsia.

Purpose: To examine repeatability and reproducibility of foveal cone density measurements in patients with CNGA3- and CNGB3-associated achromatopsia (ACHM) using split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Methods: Thirty foveae from molecularly confirmed subjects with ACHM, half of whom harbored disease-causing variants in CNGA3 and half in CNGB3, underwent nonconfocal split-detection AOSLO imaging. Cone photoreceptors within the manually delineated rod-free zone were manually identified twice by two independent observers. The coordinates of the marked cones were used for quantifying foveal cone density. Cone density and difference maps were generated to compare cone topography between trials. Results: We observed excellent intraobserver repeatability in foveal cone density estimates, with intraclass correlation coefficients (ICCs) ranging from 0.963 to 0.991 for CNGA3 and CNGB3 subjects. Interobserver reproducibility was also excellent for both CNGA3 (ICC = 0.952; 95% confidence interval [CI], 0.903-1.0) and CNGB3 (ICC = 0.968; 95% CI, 0.935-1.0). However, Bland-Altman analysis revealed bias between observers. Conclusions: Foveal cone density can be measured using the described method with good repeatability and reproducibility both for CNGA3- and CNGB3-associated ACHM. Any degree of bias observed among the observers is of uncertain clinical significance but should be evaluated on a study-specific basis. Translational Relevance: This approach could be used to explore disease natural history, as well as to facilitate stratification of patients and monitor efficacy of interventions for ongoing and upcoming ACHM gene therapy trials.

Longitudinal Assessment of Remnant Foveal Cone Structure in a Case Series of Early Macular Telangiectasia Type 2.

Purpose: To determine the extent of remnant cone structure within early foveal ellipsoid zone (EZ) lesions in macular telangiectasia type 2 longitudinally using both confocal and split detector adaptive optics scanning light ophthalmoscopy (AOSLO). Methods: Spectral domain optical coherence tomography (SDOCT), confocal and split detector AOSLO were acquired from seven patients (10 eyes) with small (early) EZ lesions on SDOCT secondary to macular telangiectasia type 2 at baseline, 6 months, and 12 months. The presence of cone structure on AOSLO in areas of EZ loss as well as cones at 1° eccentricity, and their change over time were quantified. Results: By split detector AOSLO, remnant cone structure was identified within and on the borders of all foveal EZ lesions. Within the extent of these lesions, cone spacing ranged from 4.97 to 9.95 µm at baseline, 5.30 to 6.10 µm at 6 months, and 4.99 to 7.12 µm at 12 months. Four eyes with significantly smaller EZ lesions showed evidence of recovery of EZ reflectivity on SDOCT B-scans. Remnant cone structure was identified in some areas where EZ reflectivity recovered at the following time point. Eyes that showed recovery of EZ reflectivity had a continuous external limiting membrane. Conclusions: Remnant cone structure can persist within small SDOCT-defined EZ lesions, which can wax and wane in appearance over time. AOSLO can help to inform the interpretation of SDOCT imaging. Translational Relevance: The absence of EZ in early macular telangiectasia type 2 and other retinal conditions needs careful interpretation because it does not always indicate an absence of underlying cone structure. The integrity of the external limiting membrane may better predict the presence of remnant cone structure and recovery of EZ reflectivity.

Quantifying the Separation Between the Retinal Pigment Epithelium and Bruch's Membrane using Optical Coherence Tomography in Patients with Inherited Macular Degeneration.

Purpose: To describe and quantify Bruch's membrane (BM) and retinal pigment epithelium (RPE) separation using spectral-domain (SD) optical coherence tomography (OCT) in patients affected by inherited macular degenerations associated with BM thickening. Methods: Patients with molecularly confirmed Sorsby fundus dystrophy (SFD), dominant drusen (DD), and late-onset retinal degeneration (L-ORD) were included in this retrospective study. Each disease was classed as early stage if subjects were asymptomatic, intermediate stage if they had nyctalopia alone, and late stage if they described loss of central vision. The main outcome was measurement of BM-RPE separation on SD-OCT. The BM-RPE separation measurements were compared against those in normal age-matched controls. Results: Seventeen patients with SFD, 22 with DD, and eight with L-ORD were included. BM-RPE separation on SD-OCT demonstrated a high test-retest and interobserver reproducibility (intraclass correlation coefficients >0.9). BM-RPE separation was not identified in normal subjects. In SFD, there was greater BM-RPE separation in late-stage disease compared with intermediate-stage patients both at subfoveal (P < 0.05) and juxtafoveal (P < 0.01) locations. In DD, there was increased BM-RPE separation in late-stage disease compared with early stage at subfoveal (P < 0.001) and juxtafoveal (P < 0.05) topographies. There was no significant difference in BM-RPE separation between disease stages in L-ORD. Conclusions: BM-RPE separation is a novel, quantifiable phenotype in the three monogenic macular dystrophies studied, and may be an optical correlate of the histopathological thickening in BM that is known to occur. BM-RPE separation, as measured by OCT, varies with stage of disease in SFD and DD, but not in L-ORD. Translational Relevance: SFD, DD, and L-ORD are associated with BM thickening. In this group of patients, OCT assessment of macular structure identifies a separation of the usually single, hyperreflective line thought to represent BM and the overlying RPE. This separation is a novel and quantifiable feature of disease staging in SFD and DD.

Retinal Structure in RPE65-Associated Retinal Dystrophy.

Purpose: RPE65-associated retinal dystrophy (RPE65-RD) is an early onset, progressive, severe retinal dystrophy. We sought to characterize the natural history of retinal degeneration in affected individuals. Methods: We performed cross-sectional and longitudinal quantitative and qualitative assessments of retinal architecture in RPE65-RD using spectral domain optical coherence tomography (SD-OCT) and fundus autofluorescence (FAF) imaging. Twenty-six subjects (mean age, 14.8 years, range, 5-24 years) with RPE65-RD underwent SD-OCT and FAF imaging, of whom 14 subjects were followed up over time. Foveal thickness (FT), outer nuclear layer thickness (ONLT), ellipsoid zone width (EZW), and ellipsoid zone area (EZA) were calculated where possible. These were correlated with age, best corrected visual acuity (BCVA), and central 30° retinal sensitivity (V30). Intra-observer agreement, test-retest repeatability, and interocular symmetry were also investigated. Results: We identified structural interocular symmetry, the presence of autofluorescence in 46% (12/26) of subjects, and the presence of foveal hypoplasia (associated with significantly worse BCVA) in 50% of subjects. EZW and EZA were measurable in 67% (35/52) and 37% (19/52) of eyes, respectively, with both demonstrating good agreement on repeated measurement. The annual rate of progression using EZW was -300.63 µm/year, and -1.17 mm2/year in EZA. EZW was found to have a statistically significant correlation with BCVA and V30. Conclusions: We identified the presence of autofluorescence in half of our subjects, with foveal hypoplasia also noted in half of our cohort. EZW, and to a lesser extent EZA, were robust measures of retinal degeneration and represent valuable metrics to determine the impact of intervention. (ClinicalTrials.gov number NCT02714816.).

Photoreceptor Structure in GNAT2-Associated Achromatopsia.

Purpose: The purpose of this study was to report GNAT2-associated achromatopsia (GNAT2-ACHM) natural history, characterize photoreceptor mosaic, and determine a therapeutic window for potential intervention. Methods: Patients with GNAT2-ACHM were recruited from a single tertiary referral eye center (Moorfields Eye Hospital, London, UK). We performed longitudinal clinical evaluation and ophthalmic examination, and multimodal retinal imaging, including adaptive optics scanning light ophthalmoscopy, quantitative analysis of the cone mosaic, and outer nuclear layer (ONL) thickness, including cone densities evaluation in selected regions of interest and comparison with reported healthy controls. Results: All nine subjects (3 women) presented with nystagmus, decreased visual acuity (VA), light sensitivity, and highly variable color vision loss. One patient had normal color vision and better VA. Mean VA was 1.01 (±0.10) logarithms of the minimal angle of resolution (LogMAR) at baseline, and 1.04 (±0.10) LogMAR after a mean follow-up (range) of 7.6 years (1.7-12.8 years). Optical coherence tomography showed preservation of the foveal ellipsoid zone (EZ; n = 8; 88.9%), and EZ disruption (n = 1; 11.1%). Mean ONL thickness (range, ± SD) was 84.72 µm (28.57-113.33, ± 25.46 µm) and 86.47 µm (28.57-113.33, ± 24.65 µm) for right and left eyes, respectively. Mean cone densities (±SD) at 190 µm, 350 µm, and 500 µm from the foveal center, were 48.4 (±24.6), 37.8 (±14.7), and 30.7 (±9.9), ×103 cones/mm2, respectively. Mean cone densities were lower than these of unaffected individuals, but with an overlap. Conclusions: The cone mosaic in GNAT2-ACHM is relatively well preserved, potentially allowing for a wide therapeutic window for cone-directed interventions.