Bietti crystalline dystrophy (BCD), an autosomal recessive inherited retinal disorder caused by mutations in the CYP4V2 gene, has long remained therapeutically challenging. Recent advances in adeno-associated virus–based gene therapy have emerged as promising therapeutic strategies for patients with BCD. This review synthesizes current knowledge regarding the molecular genetic mechanisms underlying BCD pathogenesis and examines recent developments in diagnostic approaches and gene therapeutic interventions. We specifically analyze the clinical outcomes of three investigational gene therapy products—ZVS101e, NGGT001, and VGR-R01—focusing on their preliminary efficacy, safety profiles, and tolerability. Key parameters evaluated include dosing strategies, routes of administration, adverse event profiles, and improvements in best-corrected visual acuity. The collective evidence suggests these therapeutic candidates show potential for decelerating disease progression and enhancing visual function. Future optimization of these approaches should carefully consider administration sites and modalities, injection volumes, and disease severity at intervention. With gene replacement therapy for BCD advancing through late-stage clinical development, regulatory approval and clinical implementation may be anticipated in the near future.

PurposeCysteinyl leukotriene receptor 1 (CysLTR1), originally described as a proinflammatory G protein-coupled receptor, has been shown to possess diverse nonimmunological properties. One of these functions is to modulate glucose-stimulated insulin secretion in β cells. Furthermore, the inhibition of CysLTR1 increases retinal cell survival in early diabetic retinopathy. Nevertheless, the potential of CysLTR1 to modulate glucose levels in retinal vascular cells, such as endothelial cells (ECs) and pericytes (PCs), is unknown. Therefore, we determined the intracellular glucose levels in retinal cells in vitro after the inhibition of CysLTR1 under standard and high-glucose culture conditions.MethodsPrimary human ECs, PCs, and the ARPE-19 cell line were cultured under standard (5.5 mmol/l glucose + 27.5 mmol/l mannitol) and high-glucose (33.0 mmol/l) conditions in the absence and presence of the specific CysLTR1 antagonists montelukast and zafirlukast for 1, 3, and 7 days. CysLTR1 expression was determined by immunofluorescence microscopy. CysLT secretion was measured by enzyme-linked immunosorbent assay. The effects of high glucose and CysLTR1 inhibition on cell viability and intracellular glucose levels were analyzed by luminescence-based assays. Furthermore, the transendothelial and transepithelial electrical resistance of the ECs and ARPE-19 monolayers was measured.ResultsCysLTR1 inhibition under standard glucose culture conditions increased the cellular glucose levels in retinal ECs, PCs, and ARPE-19 cells after 1 and 3 days of treatment. Under high-glucose culture conditions, CysLTR1 inhibition for 1 day reduced the intracellular glucose level in ARPE-19 cells. However, CysLTR1 inhibition for 3 days increased the level of intracellular glucose in ARPE-19 cells under high-glucose culture conditions. Furthermore, CysLTR1 inhibition reduced the tightness of the EC and ARPE-19 monolayers under standard culture conditions but increased the tightness of the ARPE-19 monolayers under high-glucose conditions.ConclusionsCysLTR1 is considered a potential target for the treatment of type 2 diabetes and early diabetic retinopathy. Our data revealed that CysLTR1 activity directly regulates cellular glucose levels in retinal cells, supporting these hypotheses. Interestingly, the effect of CysLTR1 activity on glucose levels was reversed under acute metabolic stress. Thus, the activity of CysLTR1 appears to be more complex in terms of glucose metabolism and needs to be studied in more detail.

PurposeTo characterize the aggregation behavior of the γD-crystallin protein in an acidic environment with a focus on the formation of intermediate species. The research employs fluorescence correlation spectroscopy to unravel the intricate molecular events leading to aggregation, contributing to a comprehensive understanding of cataract formation.MethodsThe kinetics of γD-crystallin protein aggregation were studied with a reversed-phase high-performance liquid chromatography sedimentation assay, a ThT binding assay, and light scattering. We used fluorescence correlation spectroscopy (FCS) to recognize intermediate aggregate species and characterized them with Fourier transform infrared spectroscopy (FTIR). Further, the morphologic characterization of aggregates was done by transmission electron microscopy (TEM), and their hydrophobic characteristics were analyzed using the 8-anilino-1-naphthalenesulfonic acid binding assay.ResultsA negligible lag phase was observed in the aggregation kinetic experiments of the γD-crystallin protein. Pentamer, 25-mer, and higher oligomer intermediates were formed on the aggregation pathway. Conformation studies by FCS and FTIR have shown that oligomers are rich in cross-β sheet and random coil structure; however, they constitute more α-helix and less cross-β sheet structure than fibrils. TEM analysis revealed the approximate size of oligomers (diameter ~10 nm), protofibrils (~15 nm), and fibrils (~15 to ~35 nm).ConclusionsIn this study, we reported the presence of various intermediate aggregate species formed on the aggregation pathway of γD-crystallin protein at low pH. This will open new areas of research in understanding the detailed aggregation mechanism and aggregation hotspot within unfolded γD-crystallin monomers. The insights gained will also pave the way for future research in the realm of amyloid formation in cataract.

PurposeHair anomalies represent a common associated symptom of congenital cataracts. Early diagnosis is crucial for treatment and predicting prognosis. However, the insidious and nonspecific nature of the symptoms in young children makes diagnosis challenging, often necessitating tools such as whole-exome sequencing (WES) for accurate assessment. This study aims to propose a simple and expedient approach to guide clinical management by analyzing phenotype-genotype correlations.MethodsA prospective cohort study was conducted among participants who underwent clinical examinations and WES between 2021 and 2023. Bioinformatic analysis was performed. In total, 170 unrelated congenital cataract probands were tested. The suspected pathogenic variants were validated through Sanger sequencing in both the probands and available family members. Correlation analyses were then performed, integrating clinical characteristics, cataract phenotype, and genotype data.ResultsNine probands presented with both cataracts and hair anomalies. Potential pathogenic variants were detected in all patients with hair anomalies, including a novel variant in LSS. Phenotype-genotype analysis supports the classification of patients into two groups: hypotrichosis 14 and ichthyosis follicularis with atrichia and photophobia syndrome 2, based on the cataract phenotype, severity of the hair anomalies, and the presence of corneal pannus. These patients should be monitored closely for the development and progression of glaucoma and corneal lesions.ConclusionsWe identified nine probands with hair anomalies in our large cohort of congenital cataract probands. Using WES and comprehensive clinical examinations, we established definitive diagnoses, broadened the phenotype and genotype, and proposed phenotype-genotype correlations.

PurposeCyclic nucleotide-gated (CNG) channels are ligand-gated ion channels that transduce light signals into electrical signals in the retinal photoreceptors. Pathogenic variants in CNG channel genes are reported to cause inherited retinal dystrophies (IRDs). The current study used targeted panel sequencing to describe the mutational spectrum of CNG channel genes in familial cases of IRDs from eight consanguineous Pakistani families.MethodsThe current study included consanguineous Pakistani families with at least two affected members. DNA was extracted from whole blood samples by the phenol-chloroform method. Two affected members from each family were initially analyzed using targeted panel sequencing of 344 known IRD genes. The pathogenicity of candidate variants was assessed using the American College of Medical Genetics and Genomics guidelines. Segregation testing was performed by Sanger sequencing.ResultsResults of eight IRD families revealed a total of four reported variants in CNGA3 (c.827A>G, c.955T>C, c.1641C>A, c.1810C>T) and three novel variants, including c.1633A>T, c.800G>T, and c.1153T>C in CNGA1, CNGA3, and CNGB3 genes, respectively, segregating in each respective family. Among disease-causing variants identified in our study cohort, 87.5% were missense. Furthermore, one of the reported missense variants (i.e., c.1641C>A in CNGA3) was segregating in two unrelated families. All identified variants were homozygous and segregated in an autosomal recessive form.ConclusionsCNGA3 was the most frequently mutated gene in our study cohort. Only the c.1641C>A variant of CNGA3 was repeated in two families, showing genetic diversity. The identification of three novel pathogenic variants in CNG channel genes in the present study reaffirms the allelic and genetic heterogeneity of IRDs in the Pakistani population.

PurposeAutophagy is involved in the pathological changes of traumatic optic neuropathy (TON), and the regulation of autophagy mediated by the AMPK-mTOR-ULK pathway is a potential therapeutic approach. Astragaloside IV (AS-IV) can regulate autophagy and play a therapeutic role in various diseases. This study aimed to observe the therapeutic effect of astragaloside on TON and the role of AMPK-MTOR-ULK pathway-mediated autophagy in this process.MethodsAfter the TON model was established, varying doses of AS-IV were administered as an intervention. Additionally, compound C (an AMPK inhibitor) or 3-methyladenine (an autophagy inhibitor) was administered intraperitoneally in conjunction with AS-IV. Samples were collected following a 7-day intervention period. Western blot analysis was conducted to measure the protein and phosphorylation levels of AMPK, mTOR, and ULK proteins. Moreover, western blot and quantitative reverse transcription PCR assays were used to quantify LC3 levels in retinal tissue. LC3 immunofluorescence was performed to examine autophagy levels in the ganglion cell layer (GCL), while transmission electron microscopy was employed to observe autophagosomes. Additionally, BRN3A immunofluorescence was used to label retinal ganglion cells (RGCs) in the GCL, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining was used to assess apoptosis within the GCL. Finally, optic nerve conduction function was evaluated using flash visual evoked potentials.ResultsAfter 7 days, the phosphorylation levels of AMPK, mTOR, and ULK proteins in retinal tissue exhibited significant changes following TON. AS-IV treatment enhanced LC3 messenger RNA and protein levels in TON model rats, and the autophagy-promoting effect of AS-IV was reversed by 3-methyladenine. Moreover, AS-IV elevated P-AMPK and P-ULK levels while decreasing P-mTOR levels. AS-IV also improved the survival rate of RGCs and reduced the P2 peak latency of flash visual evoked potentials. These effects were attenuated by the AMPK inhibitor compound C. Additionally, AS-IV increased the levels of AKT1 and P-AKT1 while decreasing P-S6RP levels in the retinal tissue of TON model rats.ConclusionsAS-IV can increase the survival rate of RGCs and improve visual function after TON, which may be related to the improvement of autophagy by regulating the AMPK-MTORC1-ULK pathway.

This study describes a distinct spectrum of latent transforming growth factor-β-binding protein 2 (LTBP2)-related ocular phenotypes in pediatric glaucoma with supporting genetic evidence and highlights our clinical experiences in its management. A total of 189 children with childhood glaucoma underwent whole-exome sequencing-based genetic testing. Of these, 24 children displayed LTBP2-related phenotypes, among whom 18 cases who tested positive for LTBP2 variants were included in the study. The identified variants were confirmed through Sanger sequencing whenever possible and analyzed using in silico tools. The clinical presentation, genetic variants, and management of these 18 cases were thoroughly reviewed and presented. All 36 eyes of the 18 children with biallelic LTBP2 variants exhibited megalocornea without Descemet break, iridodonesis, and ectopia lentis. Pupillary changes were noted in all eyes, with persistent pupillary membrane in 78% (28/36) and ectropion uveae in 19% (7/36) eyes. Secondary glaucoma was observed in 72% (26/36) eyes, requiring surgery in 13 of these. Retinal pathology was noted in 47% (17/36) eyes. Lensectomy was performed in 94% (34/36) eyes with a mean age of 4.09 ± 3.5 years. Logistic regression analysis suggested that older age at lensectomy increased the risk of secondary glaucoma (hazard ratio, 1.69; [95% Confidence Interval: 1.00, 2.86], p < 0.05). The identified LTBP2 variants included five stop-gain variations, six frameshift variations, and one substitution variation, with five being novel and seven classified as rare variants. The study expands the classic LTBP2-related phenotype spectrum in an Indian pediatric glaucoma cohort, highlighting additional features such as persistent pupillary membrane, ectropion uveae, and associated retinal pathology. These ocular manifestations were predominantly linked to nonsense LTBP2 variants. From a management standpoint, early lensectomy can help prevent secondary glaucoma, while timely identification and treatment of peripheral retinal pathology can reduce the risk of sight-threatening complications.

Inherited retinal dystrophies (IRDs) represent a clinically and genetically heterogeneous group of genetic disorders that involve photoreceptors and/or retinal pigment epithelium degeneration. IRDs may occur as an isolated condition or may represent an ocular manifestation of a multisystemic disorder referred as syndromic IRD. To increase the understanding of the molecular determinants of syndromic IRD-related genes in the Pakistani population, we revealed the genetic profile of 13 consanguineous Pakistani families using capture panel sequencing. We performed comprehensive molecular testing on 72 IRD segregating Pakistani families using targeted capture panel sequencing of 344 known genes. The pathogenicity of candidate variants was assessed using American College of Medical Genetics and Genomics guidelines, followed by Sanger sequencing for segregation analysis. Causative variants in previously reported syndromic IRDs genes were detected in 13/72 (18%) IRD families, including 5/72 (6.94%), 4/72 (5.55%), 2/72 (2.8%), 1/72(1.38%) and 1/72 (1.38%) in Usher syndrome, Bardet-Biedl syndrome, Batten disease, retinitis pigmentosa with situs inversus and Stickler syndrome segregated families, respectively. Disease-causing variants included nine previously reported and six novel homozygous variants, i.e., c.1143G>C in USH2A, c.470G>A in MYO7A, c.877-2A>G in PCDH15, c.347C>T in ARL6, c.581C>T in CLN5 and c.100+1G>T in ARL2BP gene segregation with disease phenotype in eight families. Two heterozygous variants of the USH2A gene, i.e., c.12093C>A and c.9815C>T, were segregated in a compound heterozygous form in family RP243. Furthermore, RP151 showed segregation of a heterozygous variant c.247G>A in a Stickler syndrome gene, i.e., COL2A1, in an autosomal dominant manner. This study reaffirms the clinical and genetic heterogeneity of syndromic IRD-associated genes and confirms the usefulness of molecular methods in advancing our understanding of these conditions in consanguineous populations. The most commonly mutated Bardet-Biedl syndrome gene was ARL6 (75%) and the most commonly mutated Usher syndrome genes were USH2A (40%) and MYO7A (40%). Our data could serve as a reference for future studies and the development of treatment modalities for affected families of Pakistani origin.

Neonatal mice were divided into five groups: one normal group and four with oxygen-induced retinopathy (OIR), including OIR, phosphate-buffered saline, UA and Lucentis groups. On postnatal day 17 (P17), mice were euthanized and one eye was collected for retinal analysis using fluorescence microscopy. Protein and messenger ribonucleic acid (mRNA) levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-9 and cyclo-oxygenase-2 (COX-2) were detected. HRCECs cultured under high-glucose conditions were treated with UA to assess its effects on proliferation and molecular expression. UA significantly reduced RNV area in OIR mice and protected astrocytes from hypoxia-induced damage (p<0.01). VEGF, MMP-2, MMP-9 and COX-2 levels were lower in the UA group compared with the OIR and phosphate-buffered saline groups (p<0.05), but slightly higher than in normal controls (p<0.01). Lucentis reduced VEGF levels but did not significantly affect MMP-2, MMP-9 or COX-2. In HRCECs, UA inhibited high-glucose-induced proliferation and reduced VEGF, MMP-2, MMP-9 and COX-2 expression in a time- and dose-dependent manner. UA inhibits RNV by reducing VEGF, MMP-2, MMP-9 and COX-2 expression, protecting astrocytes and suppressing HRCEC proliferation under high-glucose conditions, highlighting its therapeutic potential for retinal neovascular diseases.