Effects of fluorescent protein tdTomato on mouse retina.

Fluorescent proteins (FPs) have been widely used to investigate cellular and molecular interactions and trace biological events in many applications. Some of the FPs have been demonstrated to cause undesirable cellular damage by light-induced ROS production in vivo or in vitro. However, it remains unknown if one of the most popular FPs, tdTomato, has similar effects in neuronal cells. In this study, we discovered that tdTomato expression led to unexpected retinal dysfunction and ultrastructural defects in the transgenic mouse retina. The retinal dysfunction mainly manifested in the reduced photopic electroretinogram (ERG) responses and decreased contrast sensitivity in visual acuity, caused by mitochondrial damages characterized with cellular redistribution, morphological modifications and molecular profiling alterations. Taken together, our findings for the first time demonstrated the retinal dysfunction and ultrastructural defects in the retinas of tdTomato-transgenic mice, calling for a more careful design and interpretation of experiments involved in FPs.

Daily Low-Level Red Light for Spherical Equivalent Error and Axial Length in Children With Myopia: A Randomized Clinical Trial.

Importance: Treatments are needed to slow progression of or reduce incidence of myopia. Objective: To evaluate the efficacy and safety of daily 650-nm low-level red light (LLRL) for myopia treatment. Design, Setting, and Participants: Single-masked, randomized clinical trial at 1 site in China. Baseline measurements were completed from August to September 2021. Participants were children aged 6 to 12 years with spherical equivalent error (SER) of -6 diopters (D) to 3 D. Data were analyzed from March to July 2023. Interventions: Irradiation daily with 650-nm LLRL for 3 minutes twice daily 4 or more hours apart or no intervention. Main Outcomes and Measures: Primary outcomes were changes in cycloplegia SER and axial length (AL) at 6- and 12-month follow-up visits. Safety was assessed on masked fundus photograph evaluations. Results: A total of 336 children were randomly allocated into the LLRL group or control group in a 1:1 ratio. The control group contained 86 female patients (51.2%), and the treatment group contained 90 female patients (53.6%). The mean (SD) age, SER, and AL were 9.0 (1.9) years, -1.3 (1.5) D, and 23.8 (1.0) mm for all patients. A total of 161 (95.8%) in the LLRL group and 159 (94.6%) in the control group returned for the 6-month follow-up. A total of 157 (93.5%) in the LLRL group and 152 (90.5%) in the control group returned for the 12-month follow-up. Mean (SD) changes in SER were 0.15 (0.16) D and -0.26 (0.21) D for the LLRL group and the control group, respectively (difference, -0.41 D; 95% CI, -0.48 to -0.34 D; P < .001), at 6 months and 0.24 (0.27) D and -0.65 (0.33) D for the LLRL group and the control group, respectively (difference, -0.89 D; 95% CI, -0.95 to -0.83 D; P < .001), at 12 months. Mean (SD) changes in AL were -0.06 (0.08) mm and 0.13 (0.12) mm for the LLRL group and control group, respectively (difference, 0.19 mm; 95% CI, 0.16 to 0.22 mm; P < .001), at 6 months and -0.11 (0.10) mm and 0.26 (0.16) mm for the LLRL group and control group, respectively (difference, 0.37 mm; 95% CI, 0.34 to 0.40 mm; P < .001). Masked fundus photograph review did not identify retinal changes in either group. Conclusions and relevance: These findings suggest daily use of 650-nm LLRL for 1 year can slow progression of SER and AL without safety concerns identified. Confirmation of these findings at independent sites seems warranted, as well as determining whether these effects can be sustained with or without continued treatment and whether LLRL has any effect on pathological myopia. Trial Registration: ChiCTR2200058963.

A Novel Grading System for Diffuse Chorioretinal Atrophy in Pathologic Myopia.

INTRODUCTION: This study aims to quantitatively assess diffuse chorioretinal atrophy (DCA) in pathologic myopia and establish a standardized classification system utilizing artificial intelligence. METHODS: A total of 202 patients underwent comprehensive examinations, and 338 eyes were included in the study. The methodology involved image preprocessing, sample labeling, employing deep learning segmentation models, measuring and calculating the area and density of DCA lesions. Lesion severity of DCA was graded using statistical methods, and grades were assigned to describe the morphology of corresponding fundus photographs. Hierarchical clustering was employed to categorize diffuse atrophy fundus into three groups based on the area and density of diffuse atrophy (G1, G2, G3), while high myopic fundus without diffuse atrophy was designated as G0. One-way analysis of variance (ANOVA) and nonparametric tests were conducted to assess the statistical association with different grades of DCA. RESULTS: On the basis of the area and density of DCA, the condition was classified into four grades: G0, G1 (0 < density ≤ 0.093), G2 (0.093 < density ≤ 0.245), and G3 (0.245 < density ≤ 0.712). Fundus photographs depicted a progressive enlargement of atrophic lesions, evolving from punctate-shaped to patchy with indistinct boundaries. DCA atrophy lesions exhibited a gradual shift in color from brown-yellow to yellow-white, originating from the temporal side of the optic disc and extending towards the macula, with severe cases exhibiting widespread distribution throughout the posterior pole. Patients with DCA were significantly older [34.00 (27.00, 48.00) vs 29.00 (26.00, 34.00) years], possessed a longer axial length (28.85 ± 1.57 vs 27.11 ± 1.01 mm), and exhibited a more myopic spherical equivalent [- 13.00 (- 16.00, - 10.50) vs - 9.09 ± 2.41 D] compared to those without DCA (G0) (all P < 0.001). In eyes with DCA, a trend emerged as grades increased from G1 to G3, showing associations with older age, longer axial length, deeper myopic spherical equivalent, larger area of parapapillary atrophy, and increased fundus tessellated density (all P < 0.001). CONCLUSIONS: The novel grading system for DCA, based on assessments of area and density, serves as a reliable measure for evaluating the severity of this condition, making it suitable for widespread application in the screening of pathologic myopia.

Modeling autosomal dominant retinitis pigmentosa by using patient-specific retinal organoids with a class-3 RHO mutation.

Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) causes progressive vision loss and is potentially incurable, accounting for 25% of adRP cases. Studies on RHO-adRP mechanism were at large based on the biochemical and cellular properties, especially class-3. Nonetheless, the absence of an appropriate model for class-3 RHO-adRP has impeded comprehensive exploration. Here, induced pluripotent stem cells (iPSCs) were generated from a healthy control and two sibling RP patients with the same point mutation, c.403C>T (p.R135W). The first three-dimensional (3D) retinal organoid model of a class-3 RHO point mutation from patient-derived iPSCs was generated. Significant defects were observed in rod photoreceptors in terms of localization, morphology, transcriptional profiling and single cell resolution, to better understand the human disease resulting from RHO mutations from a developmental perspective. This first human model of class-3 RHO-adRP provides a representation of patient's retina in vitro and displays features of RHO-adRP retinal organoids relevant for therapeutic development.

Genetic control of DNA methylation is largely shared across European and East Asian populations.

DNA methylation is an ideal trait to study the extent of the shared genetic control across ancestries, effectively providing hundreds of thousands of model molecular traits with large QTL effect sizes. We investigate cis DNAm QTLs in three European (n = 3701) and two East Asian (n = 2099) cohorts to quantify the similarities and differences in the genetic architecture across populations. We observe 80,394 associated mQTLs (62.2% of DNAm probes with significant mQTL) to be significant in both ancestries, while 28,925 mQTLs (22.4%) are identified in only a single ancestry. mQTL effect sizes are highly conserved across populations, with differences in mQTL discovery likely due to differences in allele frequency of associated variants and differing linkage disequilibrium between causal variants and assayed SNPs. This study highlights the overall similarity of genetic control across ancestries and the value of ancestral diversity in increasing the power to detect associations and enhancing fine mapping resolution.

Rationale and protocol paper for the Asia Pacific Network for inherited eye diseases.

PURPOSE: There are major gaps in our knowledge of hereditary ocular conditions in the Asia-Pacific population, which comprises approximately 60% of the world's population. Therefore, a concerted regional effort is urgently needed to close this critical knowledge gap and apply precision medicine technology to improve the quality of lives of these patients in the Asia-Pacific region. DESIGN: Multi-national, multi-center collaborative network. METHODS: The Research Standing Committee of the Asia-Pacific Academy of Ophthalmology and the Asia-Pacific Society of Eye Genetics fostered this research collaboration, which brings together renowned institutions and experts for inherited eye diseases in the Asia-Pacific region. The immediate priority of the network will be inherited retinal diseases (IRDs), where there is a lack of detailed characterization of these conditions and in the number of established registries. RESULTS: The network comprises 55 members from 35 centers, spanning 12 countries and regions, including Australia, China, India, Indonesia, Japan, South Korea, Malaysia, Nepal, Philippines, Singapore, Taiwan, and Thailand. The steering committee comprises ophthalmologists with experience in consortia for eye diseases in the Asia-Pacific region, leading ophthalmologists and vision scientists in the field of IRDs internationally, and ophthalmic geneticists. CONCLUSIONS: The Asia Pacific Inherited Eye Disease (APIED) network aims to (1) improve genotyping capabilities and expertise to increase early and accurate genetic diagnosis of IRDs, (2) harmonise deep phenotyping practices and utilization of ontological terms, and (3) establish high-quality, multi-user, federated disease registries that will facilitate patient care, genetic counseling, and research of IRDs regionally and internationally.

Foveal Microstructure and Visual Outcomes after Pars Plana Vitrectomy in Patients with Different Types of Epiretinal Membrane Foveoschisis.

INTRODUCTION: The aim of this study was to evaluate the clinical characteristics and surgical outcomes of the epiretinal membrane foveoschisis (ERM-FS) with different morphological types. METHODS: This retrospective observational study reviewed 44 consecutive ERM-FS patients who underwent ERM surgery. According to the optical coherence tomography images, ERM-FS was classified into three groups: group A, FS crossed the fovea with the foveola elevated; group B, FS located at the foveal edges with a near-normal central foveal point thickness; and group C, FS with undermined foveal edges with a near-normal central foveal point thickness. RESULTS: There were 10 eyes in group A, 20 eyes in group B, and 14 eyes in group C. Preoperatively, eyes in group A had the best best-corrected visual acuity (BCVA), the thickest central foveal point thickness, and the highest ellipsoid zone (EZ) intact rate among the three groups. After surgery, a resolution of foveoschisis was observed in 40.0%, 45.0%, and 50.0% of the eyes in group A, group B, and group C (p = 0.928), respectively. BCVA was significantly improved postoperatively. Although there was no significant difference in BCVA among the three groups at 1 month postoperatively, BCVA of group A was the best at 4 and 10 months. Correlation analysis indicated that the type of ERM-FS, baseline BCVA, central foveal point thickness, and postoperative EZ continuity (all p < 0.05) were important factors for the final BCVA. CONCLUSIONS: The damage to the retinal structure and visual function was milder in group A ERM-FS. Our study emphasized the necessity of OCT-based subtyping in patients with ERM-FS.

Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSCs.

RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing. RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation, RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.

RNA fusion in human retinal development.

Chimeric RNAs have been found in both cancerous and healthy human cells. They have regulatory effects on human stem/progenitor cell differentiation, stemness maintenance, and central nervous system development. However, whether they are present in human retinal cells and their physiological functions in the retinal development remain unknown. Based on the human embryonic stem cell-derived retinal organoids (ROs) spanning from days 0 to 120, we present the expression atlas of chimeric RNAs throughout the developing ROs. We confirmed the existence of some common chimeric RNAs and also discovered many novel chimeric RNAs during retinal development. We focused on CTNNBIP1-CLSTN1 (CTCL) whose downregulation caused precocious neuronal differentiation and a marked reduction of neural progenitors in human cerebral organoids. CTCL is universally present in human retinas, ROs, and retinal cell lines, and its loss-of-function biases the progenitor cells toward retinal pigment epithelial cell fate at the expense of retinal cells. Together, this work provides a landscape of chimeric RNAs and reveals evidence for their critical role in human retinal development.

Closure Grading and Visual Outcome in Patients with Large Idiopathic Macular Holes: A Spectral-Domain Optical Coherence Tomography Observation.

INTRODUCTION: So far, there has been no closure grade system synthesizing morphological and microstructural features for large idiopathic macular holes (IMHs) treated by vitrectomy and internal limiting membrane (ILM) peeling. This study aimed to propose a concise one and explore its relevance with visual acuity and the related preoperative factors. METHODS: Consecutive patients with large IMHs (minimum diameter >400 µm), undergoing vitrectomy and ILM peeling, obtaining primary closure and regularly followed-up were enrolled. Preoperative clinical charts and spectral-domain optical coherence tomography (SD-OCT) parameters were reviewed. SD-OCT images and best corrected visual acuity (BCVA) were assessed at 1, 4, and 10 months postoperatively. SD-OCT features at last visit were categorized by BCVA significance, and preoperative risk factors were analyzed. RESULTS: Sixty-eight eyes from 64 patients were enrolled. The 10-month postoperative SD-OCT images were categorized into closure grade 1, 2, and 3 with successively decreased BCVA (p < 0.001). During early follow-up, part of grades 2 and 3 could evolve into the upper grade, respectively, but grade 3 could never evolve into grade 1 and exhibited the least satisfactory long-term BCVA. Binary logistic regression showed that large minimum linear diameter (MLD) was a risk factor for grade 3 occurrence (p < 0.001), with a cutoff value of 625.5 µm from the receiver operating characteristic curve for MLD predicting grade 3 occurrence (p = 0.001). CONCLUSION: Long-term closure status of large IMHs could be categorized into three grades with BCVA significance. Large horizontal MLD is a risk factor for occurrence of grade 3 closure with unsatisfactory visual recovery.

Acute onset of decreased vision after violently vomiting: A case report.

PURPOSE: Reported cases of central retinal vein occlusion (CRVO) associated with violent vomiting are scarce all over the world. In this case, we reported a male patient with an acute decreased vision right after violently vomiting. METHODS: A 55-year-old male patient found himself with a sudden, painless decline in visual acuity in his left eye (LE) after violently vomiting due to alcohol consumption for 1 day. His best corrected visual acuity (BCVA) was 20/2000 in the LE. He has a history of moderate myopia in both eyes and well-controlled hypertension. After evaluating multimodal images, non-ischemic CRVO retinopathy was diagnosed. RESULTS: During the follow-up period, the patient's BCVA in the LE improved to 20/40 and the ocular examination data confirmed that the previous abnormal performances were nearly back to normal without any treatment. CONCLUSION: Non-ischemic CRVO retinopathy following violent vomiting suggests that vomiting-related mechanisms like Valsalva maneuvers and dehydration may contribute to CRVO through effects on ocular blood flow and thrombosis.

Fundus Tessellated Density of Pathologic Myopia.

PURPOSE: The study aimed to quantitatively evaluate the fundus tessellated density (FTD) in different categories of pathologic myopia (PM) using fundus photographs with the application of artificial intelligence. METHODS: A retrospective review of 407 PM (META-PM, Category 2-Category 4) eyes was conducted, employing a biomimetic mechanism of human vision and integrated image processing technologies for FTD extraction and calculation. Different regions of interest were analyzed, including circle O4.5 (optic disc centered, diameter of 4.5 mm) and circle M1.0, M3.0, M6.0 (macular centered, diameter of 1.0, 3.0, and 6.0 mm), using 2 partitioning methods ("X" and "+"). The density of patchy (Category 3) or macular atrophy (Category 4) areas was quantified. Univariate and multivariate linear regression analyses were performed to assess the association with FTD. RESULTS: The mean FTD of total PM eyes was 0.283, ranging from 0.002 to 0.500, and demonstrating a negative correlation with the PM category. In multivariate analysis, age was found to be significantly associated with FTD ( P <0.05), while axial length did not show a significant association. Fundus tessellation of circle O4.5 and circle M6.0 displayed associations with the FTD across different PM categories. The "X" partitioning method better fit the circle M6.0 region, while both methods were suitable for the circle O4.5 region. After excluding the patchy and macular atrophic areas, the mean FTD values were 0.346 in Category 2, 0.261 in Category 3, and 0.186 in Category 4. CONCLUSIONS: The study revealed a decreasing trend in FTD values across different categories of PM, regardless of the presence or absence of patchy or macular atrophic areas. Quantifying FTD in PM could be a valuable tool for improving the existing PM classification system and gaining insights into the origin of posterior staphyloma and visual field defects in high myopia.

CRX haploinsufficiency compromises photoreceptor precursor translocation and differentiation in human retinal organoids.

BACKGROUND: The CRX-associated autosomal dominant retinopathies suggest a possible pathogenic mechanism of gene haploinsufficiency. However, based on reported human patient cases and studies with mouse models, it is hard to confirm the specific weight of haploinsufficiency in pathogenesis due to the interspecies gaps between gene expression and function. METHODS: We created monoallelic CRX by replacing one allele with tdTomato in human embryonic stem cells (hESCs) and subsequently dissect pathogenesis in hESCs-derived retinal organoids. We used transcriptome and immunofluorescence analyses to dissect phenotypic differences between CRX-monoallelic knockout and control wildtype organoids. For location analysis of CRX+ cells, a CRX-expression-tracing system was constructed in control hESCs. We implemented long-term live-cell imaging to describe the translocation of CRX+ cells between two groups in early organoid differentiation. The expression pattern of these dynamic differences was validated using RNA-seq and immunofluorescence assays. RESULTS: We identified delayed differentiation of outer nuclear layer (ONL) stratification along with thinner ONL, serious loss of photoreceptor outer segments, as well as downregulated expression of gene for phototransduction and inner/outer segment formation. By live-cell imaging and immunostaining, we observed the overtension of actomyosin network and the arrested translocation of monoallelic CRX+ cells in the early stage of retinal differentiation. CONCLUSIONS: We confirmed that gene haploinsufficiency is the mechanism for the dominant pathogenicity of CRX and discovered that CRX regulated postmitotic photoreceptor precursor translocation in addition to its specification of photoreceptor cell fates during human retinal development. These findings revealed a new underlying mechanism of CRX dominant pathogenesis and provided a new clue for the treatment of CRX-associated human retinopathies.

Timp1 Deletion Induces Anxiety-like Behavior in Mice.

The hippocampus is essential for learning and memory, but it also plays an important role in regulating emotional behavior, as hippocampal excitability and plasticity affect anxiety and fear. Brain synaptic plasticity may be regulated by tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a known protein inhibitor of extracellular matrix (ECM), and the expression of TIMP1 in the hippocampus can be induced by neuronal excitation and various stimuli. However, the involvement of Timp1 in fear learning, anxiety, and hippocampal synaptic function remains to be established. Our study of Timp1 function in vivo revealed that Timp1 knockout mice exhibit anxiety-like behavior but normal fear learning. Electrophysiological results suggested that Timp1 knockout mice showed hyperactivity in the ventral CA1 region, but the basic synaptic transmission and plasticity were normal in the Schaffer collateral pathway. Taken together, our results suggest that deletion of Timp1 in vivo leads to the occurrence of anxiety behaviors, but that Timp1 is not crucial for fear learning.

Initiation of China Alliance of Research in High Myopia (CHARM): protocol for an AI-based multimodal high myopia research biobank.

INTRODUCTION: High myopia is a pressing public health concern due to its increasing prevalence, younger trend and the high risk of blindness, particularly in East Asian countries, including China. The China Alliance of Research in High Myopia (CHARM) is a newly established consortium that includes more than 100 hospitals and institutions participating across the nation, aiming to promote collaboration and data sharing in the field of high myopia screening, classification, diagnosis and therapeutic development. METHODS AND ANALYSIS: The CHARM project is an ongoing study, and its initiation is distinguished by its unprecedented scale, encompassing plans to involve over 100 000 Chinese patients. This initiative stands out not only for its extensive scope but also for its innovative application of artificial intelligence (AI) to assist in diagnosis and treatment decisions. The CHARM project has been carried out using a 'three-step' strategy. The first step involves the collection of basic information, refraction, axial length and fundus photographs from participants with high myopia. In the second step, we will collect multimodal imaging data to expand the scope of clinical information, for example, optical coherence tomography and ultra-widefield fundus images. In the final step, genetic testing will be conducted by incorporating patient family histories and blood samples. The majority of data collected by CHARM is in the form of images that will be used to detect and predict the progression of high myopia through the identification and quantification of biomarkers such as fundus tessellation, optic nerve head and vascular parameters. ETHICS AND DISSEMINATION: The study has received approval from the Ethics Committee of Beijing Tongren Hospital (TREC2022-KY045). The establishment of CHARM represents an opportunity to create a collaborative platform for myopia experts and facilitate the dissemination of research findings to the global community through peer-reviewed publications and conference presentations. These insights can inform clinical decision-making and contribute to the development of new treatment modalities that may benefit patients worldwide. TRIAL REGISTRATION NUMBER: ChiCTR2300071219.

Bietti's crystalline dystrophy: genotyping and deep qualitative and quantitative phenotyping in preparation for clinical trials.

PURPOSE: To qualitatively and quantitatively characterise the genotypes and phenotypes of Bietti's crystalline dystrophy (BCD) in a cohort of patients. DESIGN: Cross-sectional and observational study. METHODS: Clinically confirmed BCD patients were recruited for genotyping and phenotyping. Multiple retinal imaging modalities were employed. Atrophy in the fovea was adopted as major consideration for staging strategy, while percentage area of autofluorescence (AF) atrophy (PAFA) in the macula was determined for quantitation. RESULTS: In 74 clinically diagnosed BCD patients, c.802-8_810del17insGC was shown the predominant variant of the CYP4V2 gene (allele frequency 55.4%). Sixty-two cases (123 eyes) with full imaging data were classified according to a modified criterion into stages 1 (n=8, 6.50%), 2A (n=9, 7.32%), 2B (n=17, 13.82%), 3A (n=30, 24.39%) and 3B (n=59, 47.97%). The eyes of the stage 2B were particularly deemed 'high risk' due to atrophy near fovea, while in stage 3A, though with remarkable foveal atrophy, preserved retinal pigment epithelium/photoreceptor islands near the fovea were found in 14 eyes. A tendency of increase in PAFA with age was found (rs=0.31, p=0.014). Significant PAFA increase was shown through stages 1 to 3B, and best-corrected visual acuity (BCVA, Logarithm of the Minimum Angle of Resolution) was shown to moderately correlate with PAFA (rs=0.56, p<0.001). CONCLUSION: The PAFA might be an efficient biomarker for BCD severities correlating with BCVA. The highly heterogeneous chorioretinopathy and BCVA of BCD cases appear to be associated with disease stages, progression types and patients' ages. Foveal involvement should be of a major concern for consideration of potential therapeutic intervention.

The Evolution and Visual Prognosis of Glial Proliferation with Different Grades after Macular Hole Surgery: An Optical Coherence Tomography-Based Study.

INTRODUCTION: The glial proliferation after macular hole (MH) surgery was divided into two types previously: those replacing the entire intraretinal layer and those involving only the inner foveal layers. The evolution and prognosis of the former type were elaborated on in previous studies, but the latter one has received limited attention. Therefore, this study aims to investigate the evolution of glial proliferation with varying grades after MH surgery and its effects on foveal microstructure and best-corrected visual acuity (BCVA). METHODS: In this retrospective research, we reviewed 202 eyes from 196 consecutive patients who underwent a successful idiopathic MHs repair. Based on optical coherence tomography images, glial proliferation was classified into three types: A-type, which replaced the entire intraretinal layer; B-type, located at the level of and above the external limiting membrane (ELM); and C-type, situated above the ELM. RESULTS: Of the 67 eyes that attended the 1-, 4-, and 10-month follow-up, A-type, B-type, C-type, and no glial proliferation were identified in 27 (40.3%), 17 (25.4%), 20 (29.8%), and 3 eyes (4.5%), respectively, at 1 month. Within 10 months, the prevalence of A-type glial proliferation significantly decreased (p < 0.001), but the changes in B-type (p = 0.261), C-type (p = 0.151), and no glial proliferation (p = 0.492) were not significant. In 32 of the 67 eyes, the grade of glial proliferation gradually improved, with A-type transforming into B- or C-type in 19 of 27 eyes (70.4%), B-type into C-type or no glial proliferation in 11 out of 17 eyes (64.7%), and C-type gradually disappearing in 2 out of 20 eyes (10.0%). Among the eyes that attended at least one follow-up (1 M, 202 eyes; 4 M, 161 eyes; 10 M, 97 eyes), those with A-type glial proliferation showed the most defective outer retinal layers, worst BCVA, and thinnest central fovea compared with the other two types at all follow-up time points (p < 0.001). Eyes with C-type glial proliferation exhibited significantly better photoreceptor layer status and BCVA compared with those with B-type glial proliferation. A-type glial proliferation at 1 month, which showed significant association with BCVA at 10 months, could be accurately predicted by the minimum linear diameter with a cut-off >547.5 µm (p < 0.001). CONCLUSION: Within 10 months, A-type glial proliferation substantially resolves but the prevalence of B- and C-type remains unchanged. B-type glial proliferation hinders the restoration of photoreceptors and impairs visual recovery despite being located within the inner retina.

Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3.

Inherited retinal dystrophies (IRDs) are a heterogeneous group of visual disorders caused by different pathogenic mutations in genes and regulatory sequences. The endoplasmic reticulum (ER) membrane protein complex (EMC) subunit 3 (EMC3) is the core unit of the EMC insertase that integrates the transmembrane peptides into lipid bilayers, and the function of its cytoplasmic carboxyl terminus remains to be elucidated. In this study, an insertional mutation c.768insT in the C-terminal coding region of EMC3 was identified and associated with dominant IRDs in a five-generation family. This mutation caused a frameshift in the coding sequence and a gain of an additional 16 amino acid residues (p.L256F-fs-ext21) to form a helix structure in the C-terminus of the EMC3 protein. The mutation is heterozygous with an incomplete penetrance, and cosegregates in all patients examined. This finding indicates that the C-terminus of EMC3 is essential for EMC functions and that EMC3 may be a novel candidate gene for retinal degenerative diseases.

Comprehensive Genetic Analysis Unraveled the Missing Heritability and a Founder Variant of BEST1 in a Chinese Cohort With Autosomal Recessive Bestrophinopathy.

Purpose: To describe the genetic landscape of BEST1 for a large Chinese cohort with autosomal recessive bestrophinopathy (ARB), identify the missing heritability, and report a common Chinese founder variant. Methods: We recruited 65 patients from 63 families with a clinical diagnosis of ARB. All patients underwent ophthalmic examinations and comprehensive genetic analyses, including Sanger DNA sequencing of BEST1 and whole genome sequencing (WGS). The effects of deep intronic variants (DIVs) on splicing were assessed using in vitro splicing assays in HEK293T cells and patient-derived peripheral blood mononuclear cells. Haplotype mapping was performed for 17 unrelated patients harboring variant c.867+97G>A. Results: We identified 54 distinct disease-causing variants of BEST1 in 63 pedigrees, 62 probands with biallelic variants, and one family with monoallelic variants. Sanger DNA sequencing of BEST1 initially detected 51 variants in 61 pedigrees, including 19 probands with one heterozygous variant. Subsequent WGS, combined with supplementary Sanger sequencing, revealed three missing DIVs (c.1101-491A>G, c.867+97G>A, and c.867+97G>T) in 20 families. The novel DIV c.1101-491A>G caused an abnormal splicing resulting in a 204-nt pseudoexon (PE) insertion, whereas c.867+97G>A/T relatively strengthened an alternative donor site, resulting in a 203-nt intron retention (IR). The PE and IR generated a premature termination codon downstream. Haplotype analysis identified c.867+97G>A as a common founder variant with an allele frequency of 16%. Conclusions: Our results expand the pathogenic variant spectrum of BEST1, and DIVs can explain almost all of the missing heritability. The c.867+97G>A DIV is a common founder variant for Chinese patients with ARB.