Purpose:Mucopolysaccharidosis type VI (MPS VI) is a rare autosomal recessive lysosomal storage disorder caused by pathogenic variants in ARSB, leading to deficiency of N-acetylgalactosamine 4-sulfatase and accumulation of glycosaminoglycans. Although enzyme replacement therapy (ERT) alleviates systemic symptoms, its efficacy for ocular complications is limited. Because ocular manifestations may require distinct therapeutic approaches, a precise understanding of the underlying retinal pathology is essential. This study aimed to characterize ocular and retinal involvement in MPS VI through clinical and experimental analyses. Methods:Comprehensive ophthalmic examinations were performed in siblings with MPS VI, and histological and electrophysiological assessments were conducted in an MPS VI rat model. Retinal morphology, retinal pigment epithelium (RPE) integrity, and electroretinographic responses were evaluated. Results:In patients, no apparent photoreceptor degeneration was detected, although subtle functional impairment could not be excluded. Consistently, MPS VI rats exhibited preserved photoreceptor structures but reduced electroretinogram amplitudes. Although RPE abnormalities were not evident in patients, rats showed pronounced RPE alterations, suggesting RPE involvement as a potential origin of retinal dysfunction. Conclusions:Our findings suggest that retinal dysfunction in MPS VI may primarily arise from RPE pathology rather than photoreceptor loss. Detailed retinal evaluations in aging patients are warranted, and therapeutic approaches targeting the RPE, such as localized ERT or RPE cell transplantation may provide future benefits.

Purpose:The purpose of this study was to investigate the association between entropy values derived from the retinal pigment epithelium (RPE) layer using polarization-sensitive optical coherence tomography (PS-OCT) and RPE cellular metrics obtained by adaptive optics transscleral flood illumination (AO-TFI), including cell density, spacing, regularity, and dispersion. We further aimed to determine which cellular structural alterations in retinitis pigmentosa (RP) are reflected by entropy. Methods:Healthy controls and patients with RP who underwent both PS-OCT and AO-TFI imaging were included. Spearman correlation coefficients were calculated to assess the relationships between polarimetric entropy in PS-OCT and each AO-TFI-derived parameter. To account for repeated measurements within the same eye and the effects of group and eccentricity, linear mixed-effects models were constructed to evaluate the independent associations between entropy and AO-TFI metrics after adjustment for these factors. Results:Polarimetric entropy showed the strongest positive correlation with RPE cell density among the AO-TFI parameters and was negatively correlated with spacing of RPE cells. No clear associations were observed with regularity or dispersion. In linear mixed-effects models, the association between entropy and RPE cell density differed between groups, with a significant association observed in the RP group but not in healthy controls. Conclusions:PS-OCT-derived polarimetric entropy was associated with RPE cell density measured by AO-TFI, with the association primarily observed in RP rather than in healthy controls. These findings suggest that correspondence between the two imaging modalities may contribute to quantitative assessment of outer retinal-RPE complex alterations and has potential utility as a biomarker in retinal degenerative diseases.

PURPOSE:To investigate genotype-phenotype correlations in a nationwide Japanese cohort of patients with RPGRIP1-associated retinal dystrophy. DESIGN:Retrospective, multicenter cohort study. METHODS:Japanese patients with biallelic pathogenic RPGRIP1 variants diagnosed with Leber congenital amaurosis (LCA) or achromatopsia (ACHM) were recruited from university hospitals throughout Japan. Genetic analyses included polymerase chain reaction for detection of the exon 18 deletion variant (exon 18-DEL), whole-exome sequencing, and whole-genome sequencing. Ophthalmic evaluations comprised best-corrected visual acuity (BCVA), visual field testing, full-field electroretinography, and multimodal retinal imaging. RESULTS:Thirty-four patients from 26 families (23 [18 families] with ACHM and 11 [8 families] with LCA) were included. Fourteen distinct RPGRIP1 variants were identified, with exon 18-DEL being the most prevalent (43 of 68 alleles, 63.2%). In ACHM, variants were predominantly clustered around exon 18, whereas LCA showed more diverse variant combinations. BCVA was significantly worse in LCA than in ACHM, although the rate of BCVA decline did not differ between phenotypes. Multimodal imaging demonstrated relatively preserved macular structure even in older patients with LCA, while ACHM showed age-dependent progressive outer retinal degeneration. Electroretinography revealed near-complete loss of rod and cone function in early infancy in LCA, whereas rod function was initially preserved in ACHM but gradually deteriorated. CONCLUSIONS:RPGRIP1 variants cause two distinct clinical phenotypes, LCA and ACHM, with clear genotype-phenotype correlations. The exon 18-DEL, observed in both phenotypes, likely represents a founder variant in the Japanese population. These findings expand the clinical spectrum of RPGRIP1-associated retinal dystrophy and have implications for molecular diagnosis, prognostic counseling, and future therapeutic development.

Purpose:The purpose of this study was to simultaneously visualize cone photoreceptors (cone) and retinal pigment epithelium (RPE) cells at the cellular level in Stargardt disease (STGD) using adaptive optics (AO) imaging. In addition, the feasibility of quantitative analysis was explored. Design:Observational case series. Subjects:Patients with STGD. Methods:Cone and RPE cells were simultaneously imaged at the cellular level using AO flood-illumination ophthalmoscopy (AO-FIO) and AO transscleral flood illumination (AO-TFI). Quantitative metrics were derived and normalized as z-scores to provide an exploratory assessment of the feasibility of cellular-level quantification. Main Outcome Measures:Cellular-level cone and RPE integrity and abnormalities assessed by simultaneous AO-FIO and AO-TFI imaging. Results:Using AO-FIO and AO-TFI, cone and RPE cells could be simultaneously visualized in eyes with STGD. In a subset of cases, cellular-level quantitative analysis was feasible. Imaging findings varied across patients and retinal regions, showing heterogeneous degrees of structural alteration in cone and RPE cells. In the quantitatively analyzed cases, cone density z-scores showed marked reductions, with the lowest values of -3.28 in case 1 and -2.90 in case 2, whereas RPE cell density z-scores showed only mild deviations and remained within the reference range in the analyzed regions. Conclusions:Adaptive optics flood-illumination ophthalmoscopy and AO-TFI enable simultaneous cellular-level visualization of cone and RPE cells in STGD. This study characterized cellular-level structural features in STGD and demonstrated the feasibility of quantitative analysis in a subset of cases. These findings could contribute to a deeper understanding of STGD pathology and support future investigations aimed at treatment optimization. Financial Disclosures:Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Purpose: The purpose of this study was to validate a rule-based automated method for detecting retinal pigment epithelium (RPE) cells in adaptive optics (AOs) transscleral flood illumination (AO-TFI) images by comparison with manual annotation and inter-grader agreement. Methods: This cross-sectional study analyzed AO-TFI images from six eyes with retinitis pigmentosa (RP) and five healthy eyes. Regions of interest (ROIs) meeting predefined image quality criteria were selected. After background correction and contrast inversion, RPE cells were detected using a rule-based approach based on local maxima detection and distance filtering. Detection parameters were optimized using 30 ROIs and applied without modification to an independent validation set of 39 ROIs. Automated detections were compared with manual annotations (ground truth [GT]) using nearest-neighbor matching, and Precision, Recall, and F1-score were calculated. Inter-grader agreement between two graders (GT1 versus GT2) was assessed. Results: Automated detection showed stable performance across ROIs. F1-scores ranged from 0.55 to 0.89 in healthy eyes and from 0.70 to 0.92 in RP eyes, with a micro-averaged F1-score of 0.80. In the linear mixed-effects model (LMM), the F1-score of automated detection was significantly lower than inter-grader agreement, and this difference was consistently observed regardless of disease status. F1-scores remained stable across the analyzed signal-to-noise ratio (SNR) range. Conclusions: The rule-based automated detection method applied to AO-TFI images showed lower performance than inter-grader agreement in both healthy and RP eyes, while demonstrating stable detection performance regardless of disease status. This approach represents a proof-of-concept for the feasibility of quantitative analysis of RPE cells using AO-TFI. Further validation using larger cohorts and independent datasets is warranted. Translational Relevance: The automated RPE cell detection method based on AO-TFI evaluated in this study represents an initial step toward standardized quantitative assessment of RPE. With further validation, it may contribute to the development of imaging biomarkers and their application in clinical research.

EYS is one of the major causative genes of autosomal recessive retinitis pigmentosa, particularly in Asian, and is expected to be a key target for future therapies. However, reliable biomarkers have not established. This KEYS study aimed to evaluate whether structural and functional parameters could serve as potential biomarkers of disease progression in EYS-associated retinitis pigmentosa (EYS-RP). We prospectively observed 49 patients with EYS-RP at Kobe Eye Hospital over a 2-year period. Visual field testing was performed using the Humphrey Field Analyzer 10- 2 (HFA 10-2). The mean total deviation (TD) was divided into central and surrounding regions for analysis, and the mean deviation (MD) was also evaluated. Horizontal and vertical EZ widths were measured by OCT. Over 2 years, our study showed that visual field progressed in surrounding and central and horizontal EZ width decreased. In subgroup analysis, a decline in horizontal EZ width and correlations between visual field parameters and EZ width were observed only in the higher MD based on the mean baseline HFA-MD value. HFA and horizontal EZ width parameters may serve as useful biomarkers of disease progression in EYS-RP, although their effectiveness varies by disease severity.

This study was conducted to evaluate the longitudinal morphological changes in cone density (CD) in patients with the eyes shut homolog (EYS)-associated retinitis pigmentosa (RP) using adaptive optics (AO) fundus imaging and to assess its potential as a biomarker for disease progression. As a sub-analysis of the KEYS study, this prospective observational study was conducted at the Kobe Eye Center, involving 50 patients with EYS-RP; 27 eyes from 27 patients who were eligible for adaptive optics fundus imaging were included in this analysis. Ellipsoid zone (EZ) length was assessed using optical coherence tomography, and mean deviation (MD) values were obtained from Humphrey visual field testing. CD showed a significant reduction in all regions of interest as early as 6 months from baseline. In contrast, a significant decrease in EZ length was observed only at 24 months, while MD values did not exhibit significant changes throughout the observation period. AO fundus imaging demonstrated high sensitivity in detecting early structural changes in EYS-RP. These findings contribute to a deeper understanding of the natural history of the disease, and suggest that CD measurement may serve as a supplementary indicator for assessing disease progression.

Purpose: To explore the safety and efficacy of the allogeneic induced pluripotent stem cell (iPSC)–derived retinal pigment epithelium (RPE) strip transplantation for patients with RPE degeneration. Design: Single-arm, open-label, interventional study. Participants: Three eyes from 3 patients clinically diagnosed with RPE impairment disease; 1 patient had dry age-related macular degeneration (AMD), and remaining 2 patients had MERTK-associated retinitis pigmentosa. Intervention: Allogeneic iPSC-derived RPE strip transplantation was performed by a 25-gauge pars plana vitrectomy. The RPE strips were prepared by incubating iPSC-derived RPE cells in 2-mm-wide grooves in the mold. Artificial retinal detachment was generated using a 38-gauge subretinal cannula, and the RPE strips were injected into the retinal bleb using a 31-gauge cannula with the maximum graft dose limited to 2 strips. Main Outcome Measures: The reduction of RPE abnormal area by the engraftment of transplanted allogeneic iPSC-derived RPE cells, which was measured by analyzing fluorescein angiography with an automated evaluation program at pretransplantation and up to 52 weeks posttransplantation. Results: The primary endpoint of reducing abnormal areas of RPE through the survival of the transplanted graft cells was achieved in all patients at 52 weeks posttransplantation. Visual function assessments confirmed significant vision-related quality of life improvement and potential retinal sensitivity restoration in 1 patient with dry AMD. The successful subretinal delivery of the iPSC-derived RPE strips was confirmed during and immediately after surgery. The engraftment of RPE cells migrated out from the strips was observed using polarization-sensitive OCT specifically and visualized as characteristic hexagonal cells via adaptive optics imaging in all patients. While no serious adverse events occurred, suspected immune reactions to graft cells and epiretinal membrane formation were observed in 1 patient each. Conclusions: A decrease in the RPE abnormal area by reliable delivery of allogeneic iPSC-derived RPE strips was achieved in all 3 cases with no serious adverse events. Further long-term studies and larger cohorts with better preoperative vision are warranted to evaluate the safety and efficacy of RPE strip transplantation. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Purpose:To evaluate the safety and therapeutic effects of induced pluripotent stem (iPS) cell-derived retinal pigment epithelium (RPE) transplantation for RPE65-associated Leber congenital amaurosis (RPE65-LCA). Observations:A 46-year-old male patient with RPE65-LCA underwent allogeneic iPS cell-derived RPE transplantation. The patient's best-corrected visual acuity (VA) prior to treatment was 2.0 (logMAR). A cell suspension of iPS cell-derived RPE was transplanted into the subretinal space. On day 15 post-transplantation, intraocular pressure (IOP) increased to 46 mmHg due to local steroid treatment, resulting in a decrease in VA to light perception (LP). Retinal imaging on day 71 revealed that most transplanted cells had migrated and formed an epiretinal membrane (ERM). The ERM was surgically removed on day 112. Two years post-transplantation, the patient reported improved vision, with VA improving to 1.4 (logMAR) from LP. Full-field stimulus testing (FST) and microperimetry demonstrated increased retinal sensitivity. These improvements have been maintained for up to 4 years post-treatment. Conclusions and importance:Although this case raised safety concerns regarding the use of cell suspension for RPE transplantation, RPE transplantation may still serve as a potential therapeutic option for patients with RPE65-associated retinopathy, particularly those who are not eligible or older age for RPE65 gene therapy.

Introduction:This study aimed to classify the phenotypes of eyes shut homolog (EYS)-associated retinopathy based on visual impairment patterns and investigate their characteristics. Methods:This retrospective, single-center, cross-sectional study was conducted in 154 patients diagnosed with EYS-related retinopathy who underwent genetic testing between December 2017 and July 2023. Phenotyping was performed only in patients who underwent Goldmann perimetry (GP) and Humphrey visual field (HVF) 10-2 testing. Phenotypes were categorized as early, pericentral, typical, and advanced based on peripheral visual field preservation (GP: V-4e isopter extending beyond a 30-degree radius in ≥2 quadrants), central visual field impairment (HVF10-2: ≤20 points with 26 dB sensitivity), and macular impairment (logMAR ≥ 0.2). Genetic and ophthalmological characteristics were compared between the pericentral and typical types. Results:A total of 39 eyes from 39 patients with EYS-associated retinopathy (average age: 48.2 ± 11.9 years, 21 women) were analyzed. Ten pathogenic variants were identified, with the three major variants (p.G843E, p.S1653fs, and p.Y2935X) accounting for a combined allele frequency of 83.3%. The phenotypes were classified as early (n=3), pericentral (n=18), typical (n=9), and advanced (n=9). No significant differences were observed between the pericentral and typical types in terms of the presence of major variants or biallelic null variants. Age and age at onset also did not differ significantly. However, macular impairment was significantly more frequent in the pericentral type (61.8%) than in the typical type (11.1%) (P = 0.014). Discussion:In EYS-associated retinopathy, the pericentral type is considered a common phenotype, although its correlation with the genotype remains unclear. Despite preserved peripheral vision, careful monitoring is warranted due to the risk of macular impairment.

Purpose: The aim of this study was to explore the potential benefits of retinal pigment epithelium replacement therapy in patients with Bietti crystalline dystrophy (BCD) by assessing the disease pathology with the distinctive relationship between fundus autofluorescence (FAF) abnormality and visual field defect. Methods: Sixteen eyes from 16 patients with BCD and 16 eyes from 16 patients with RHO-associated retinitis pigmentosa were included. Fundus autofluorescence, optical coherence tomography, and Goldmann perimetry results were retrospectively reviewed and assessed using image analyses. Results: In patients with BCD, the FAF abnormality area was not correlated with the overall visual field defect area and median overall visual field defect area (57.5%) was smaller than FAF abnormality area (98.5%). By contrast, the ellipsoid zone width was significantly correlated with the central visual field area (r = 0.806, P < 0.001). In patients with RHO-associated retinitis pigmentosa, the FAF abnormality area and ellipsoid zone width were significantly correlated with the overall visual field defect area (r = 0.833, P < 0.001) and central visual field area (r = 0.887, P < 0.001), respectively. Conclusion: The FAF abnormality shown in patients with BCD involves retinal pigment epithelium degeneration without complete loss of photoreceptors or visual function. These results suggest that patients with BCD are good candidates for retinal pigment epithelium replacement therapy for preservation of residual visual function.

Inherited retinal dystrophies comprise a clinically complex and heterogenous group of diseases characterized by visual impairment due to pathogenic variants of over 300 different genes. Accurately identifying the causative gene and associated variant is crucial for the definitive diagnosis and subsequent selection of precise treatments. Consequently, well-validated genetic tests are required in the clinical practice. Here, we report the analytical and clinical validation of a next-generation sequencing targeted gene panel, the PrismGuide IRD Panel System. This system enables comprehensive genome profiling of 82 genes related to inherited retinal dystrophies. The PrismGuide IRD Panel System demonstrated 100% (n = 43) concordance with Sanger sequencing in detecting single-nucleotide variants, small insertions, and small deletions in the target genes and also in assessing their zygosity. It also identified copy-number loss in four out of five cases. When assessing precision, we evaluated the reproducibility of variant detection with 2,160 variants in 144 replicates and found 100% agreement in terms of single-nucleotide variants (n = 1,584) and small insertions and deletions (n = 576). Furthermore, the PrismGuide IRD Panel System generated sufficient read depth for variant calls across the purine-rich and highly repetitive open-reading frame 15 region of RPGR and detected all five variants tested. These results show that the PrismGuide IRD Panel System can accurately and consistently detect single-nucleotide variants and small insertions and deletions. Thus, the PrismGuide IRD Panel System could serve as useful tool that is applicable in clinical practice for identifying the causative genes based on the detection and interpretation of variants in patients with inherited retinal dystrophies and can contribute to a precise molecular diagnosis and targeted treatments.

ObjectiveCystoid macular edema (CME) in retinitis pigmentosa (RP) is an important complication causing visual dysfunction. We investigated the effect of CME on photoreceptors in RP patients with previous or current CME, using an adaptive optics (AO) fundus camera.MethodsWe retrospectively observed the CME and ellipsoid zone (EZ) length (average of horizontal and vertical sections) by optical coherence tomography. The density and regularity of the arrangement of photoreceptor cells (Voronoi analysis) were examined at four points around 1.5° from superior to inferior and temporal to nasal. We also performed a multivariate analysis using CME duration, central macular thickness and transversal length of CME.ResultsWe evaluated 18 patients with previous or current CME (18 eyes; age, 48.7 ± 15.6 years) and 24 patients without previous or current CME (24 eyes; age, 46.0 ± 14.5 years). There were no significant differences in age, logMAR visual acuity, or EZ length. In groups with and without CME, cell density was 11967 ± 3148 and 16239 ± 2935 cells/mm2, and sequence regularity was 85.5 ± 3.4% and 88.5 ± 2.8%, respectively; both parameters were significantly different. The correlation between photoreceptor density and age was more negative in group with CME. The CME group tended toward greater reductions in duration of CME.ConclusionComplications of CME in RP patients may lead to a decrease in photoreceptor density and regularity. Additionally, a longer duration of CME may result in a greater reduction in photoreceptor density.

Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy and a major cause of blindness. RP is caused by several variants of multiple genes, and genetic diagnosis by identifying these variants is important for optimizing treatment and estimating patient prognosis. Next-generation sequencing (NGS), which is currently widely used for diagnosis, is considered useful but is known to have limitations in detecting copy number variations (CNVs). In this study, we re-evaluated CNVs in EYS, the main causative gene of RP, identified via NGS using multiplex ligation-dependent probe amplification (MLPA). CNVs were identified in NGS samples of eight patients. To identify potential CNVs, MLPA was also performed on samples from 42 patients who were undiagnosed by NGS but carried one of the five major pathogenic variants reported in Japanese EYS-RP cases. All suspected CNVs based on NGS data in the eight patients were confirmed via MLPA. CNVs were found in 2 of the 42 NGS-undiagnosed RP cases. Furthermore, results showed that 121 of the 661 patients with RP had EYS as the causative gene, and 8.3% (10/121 patients with EYS-RP) had CNVs. Although NGS using the CNV calling criteria utilized in this study failed to identify CNVs in two cases, no false-positive results were detected. Collectively, these findings suggest that NGS is useful for CNV detection during clinical diagnosis of RP.

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

This study aimed to investigate how the extent and central/peripheral location of the residual visual field (VF) in patients with late-stage inherited retinal diseases (IRDs) are related to retinal sensitivity detected using full-field stimulus testing (FST). We reviewed the results of Goldmann perimetry and FST from the medical records of patients with IRDs whose VF represents central (within 10°) and/or peripheral islands, or undetectable. In total, 19 patients (19 eyes) were analyzed in this study. The median value of residual VF area was 1.38%. The median values of rod and cone sensitivities were − 14.9 dB and 7.4 dB, respectively. Patients with only the peripheral island (− 33.9 dB) had better median rod sensitivity than other groups (only central, − 18.9 dB; both, − 3.6 dB). VF area significantly correlated with rod sensitivity (r = − 0.943, p = 0.005) in patients with only peripheral island, but not with cone sensitivity. Peripheral VF islands were significant contributors to FST results, especially rod sensitivity. With reduced or loss of central vision, the extent of residual peripheral VF significantly affected rod sensitivity, suggesting that FST can be useful in quantitatively estimating the overall remaining vision in patients with late-stage IRD.