Polymorphism of Transforming Growth Factor TGFB and Its Receptors TGFBRI, TGFBRII, TGFBRIIII Genes in Western Siberia Patients with Open-Angle Glaucoma.

Polymorphism of genes of transforming growth factor TGFB and its receptors (TGFBRI, TGFBRII, and TGFBRIIII) in patients with primary open-angle glaucoma was analyzed. The frequency of the TGFBRII CC genotype in patients is increased relative to the control group (OR=6.10, p=0.0028). Heterozygosity in this polymorphic position is reduced (OR=0.18, p=0.0052). As the effects of TGF-ß is mediated through its receptors, we analyzed complex of polymorphic variants of the studied loci in the genome of patients. Two protective complexes consisting only of receptor genes were identified: TGFBRI TT:TGFBRII CG (OR=0.10, p=0.02) and TGFBRII CG:TGFBRIII CG (OR=0.09, p=0.01). The study showed an association of TGFBRII polymorphism with primary open-angle glaucoma and the need to study functionally related genes in the development of the disease, which should contribute to its early diagnosis and prevention.

SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma.

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients' surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Role of N6-methyladenosine-related lncRnas in pseudoexfoliation glaucoma.

To explore the role of lncRNA m6A methylation modification in aqueous humour (AH) of patients with pseudoexfoliation glaucoma (PXG). Patients with open-angle PXG under surgery from June 2021 to December 2021 were selected. Age- and gender-matched patients with age-related cataract (ARC) were chosen as control. Patients underwent detailed ophthalmic examinations. 0.05-0.1 ml AH were extracted during surgery for MeRIP-Seq and RNA-Seq. Joint analysis was used to screen lncRNAs with differential m6A methylation modification and expression. Online software tools were used to draw lncRNA-miRNA-mRNA network (ceRNA). Expression of lncRNAs and mRNAs was confirmed using quantitative real-time PCR. A total of 4151 lncRNAs and 4386 associated m6A methylation modified peaks were identified in the PXG group. Similarly, 2490 lncRNAs and 2595 associated m6A methylation modified peaks were detected in the control. Compared to the ARC group, the PXG group had 234 hypermethylated and 402 hypomethylated m6A peaks, with statistically significant differences (| Fold Change (FC) |≥2, p < 0.05). Bioinformatic analysis revealed that these differentially methylated lncRNA enriched in extracellular matrix formation, tight adhesion, TGF- ß signalling pathway, AMPK signalling pathway, and MAPK signalling pathway. Joint analysis identified 10 lncRNAs with differential m6A methylation and expression simultaneously. Among them, the expression of ENST000000485383 and ROCK1 were confirmed downregulated in the PXG group by RT-qPCR. m6A methylation modification may affect the expression of lncRNA and participate in the pathogenesis of PXG through the ceRNA network. ENST000000485383-hsa miR592-ROCK1 May be a potential target pathway for further investigation in PXG m6A methylation.

Identification of a circulating three-miRNA panel for the diagnosis of primary open angle glaucoma.

PURPOSE: Conventional diagnosis of primary open angle glaucoma (POAG) needs a combination of ophthalmic examinations. An efficient assay is urgently needed for a timely POAG diagnosis. We aim to explore differential expressions of circulating microRNAs (miRNA) and provide novel miRNA biomarkers for POAG diagnosis. METHODS: A total of 180 POAG patients and 210 age-related cataract (ARC) patients were enrolled. We collected aqueous humor (AH) and plasma samples from the recruited patients. The expressions of candidate miRNAs were measured using quantitative real time polymerase chain reaction. The diagnostic ability of candidate miRNAs was analyzed by receiver operating characteristic curve. RESULTS: The expressions of miR-21-5p and miR-29b-3p were downregulated significantly in AH and plasma of POAG and miR-24-3p expression was significantly increased in AH and plasma of POAG, comparing with those of ARC. A three-miRNA panel was constructed by a binary logistic regression. And the panel could differentiate between POAG and ARC with an area under the curve of 0.8867 (sensitivity = 78.0%, specificity = 83.3%) in aqueous humor and 0.7547 (sensitivity = 73.8%, specificity = 81.2%) in plasma. Next, we verified the three-miRNA panel working as a potential diagnostic biomarker stable and reliable. At last, we identified related function and regulation pathways in vitro. CONCLUSIONS: In conclusion, we built and identified a circulating three-miRNA panel as a potential diagnostic biomarker for POAG. It may be developed into an efficient assay and help improve the POAG diagnosis in the future.

Polygenic risk score-based phenome-wide association for glaucoma and its impact on disease susceptibility in two large biobanks.

BACKGROUND: Glaucoma is a leading cause of worldwide irreversible blindness. Considerable uncertainty remains regarding the association between a variety of phenotypes and the genetic risk of glaucoma, as well as the impact they exert on the glaucoma development. METHODS: We investigated the associations of genetic liability for primary open angle glaucoma (POAG) with a wide range of potential risk factors and to assess its impact on the risk of incident glaucoma. The phenome-wide association study (PheWAS) approach was applied to determine the association of POAG polygenic risk score (PRS) with a wide range of phenotypes in 377, 852 participants from the UK Biobank study and 43,623 participants from the Penn Medicine Biobank study, all of European ancestry. Participants were stratified into four risk tiers: low, intermediate, high, and very high-risk. Cox proportional hazard models assessed the relationship of POAG PRS and ocular factors with new glaucoma events. RESULTS: In both discovery and replication set in the PheWAS, a higher genetic predisposition to POAG was specifically correlated with ocular disease phenotypes. The POAG PRS exhibited correlations with low corneal hysteresis, refractive error, and ocular hypertension, demonstrating a strong association with the onset of glaucoma. Individuals carrying a high genetic burden exhibited a 9.20-fold, 11.88-fold, and 28.85-fold increase in glaucoma incidence when associated with low corneal hysteresis, high myopia, and elevated intraocular pressure, respectively. CONCLUSION: Genetic susceptibility to POAG primarily influences ocular conditions, with limited systemic associations. Notably, the baseline polygenic risk for POAG robustly associates with new glaucoma events, revealing a large combined effect of genetic and ocular risk factors on glaucoma incidents.

Causal Associations of Glaucoma and Age-Related Macular Degeneration with Cataract: A Bidirectional Two-Sample Mendelian Randomisation Study.

Common age-related eye disorders include glaucoma, cataract, and age-related macular degeneration (AMD); however, little is known about their relationship with age. This study investigated the potential causal relationship between glaucoma and AMD with cataract using genetic data from multi-ethnic populations. Single-nucleotide polymorphisms (SNPs) associated with exposure to cataract were selected as instrumental variables (IVs) from genome-wide association studies using meta-analysis data from BioBank Japan and UK Biobank. A bidirectional two-sample Mendelian randomisation (MR) study was conducted to assess the causal estimates using inverse variance weighted, MR-Egger, and MR pleiotropy residual sum and outlier tests. SNPs with (p < 5.0 × 10-8) were selected as IVs for cataract, primary open-angle glaucoma, and AMD. We found no causal effects of cataract on glaucoma or AMD (all p > 0.05). Furthermore, there were no causal effects of AMD on cataract (odds ratio [OR] = 1.02, p = 0.400). However, glaucoma had a substantial causal effect on cataract (OR = 1.14, p = 0.020). Our study found no evidence for a causal relationship of cataract on glaucoma or AMD and a casual effect of AMD on cataract. Nonetheless, glaucoma demonstrates a causal link with cataract formation, indicating the need for future investigations of age-related eye diseases.

Exploring the role of apolipoprotein E gene promoter polymorphisms in susceptibility to normal-tension glaucoma in a Korean population.

Glaucoma, particularly primary open-angle glaucoma (POAG), poses a significant global health concern. Distinguished by intraocular pressure (IOP), POAG encompasses high-tension glaucoma (HTG) and normal-tension glaucoma (NTG). Apolipoprotein E (APOE) is a multifaceted protein with roles in lipid metabolism, neurobiology, and neurodegenerative diseases. However, controversies persist regarding the impact of APOE single-nucleotide polymorphisms (SNPs) on open-angle glaucoma and NTG. This study aimed to identify APOE-specific SNPs influencing NTG risk. A cohort of 178 patients with NTG recruited from Uijeongbu St. Mary's Hospital and 32,858 individuals from the Korean Genome and Epidemiology Study (KoGES) cohort were included in the analysis. Genotype and haplotype analyses were performed on three promoter SNPs (rs449647, rs769446, and rs405509) and two exonic SNPs (rs429358 and rs7412) located on chromosome 19. Among the five SNPs, rs769446 genotypes exhibited significant differences between cases and controls. The minor allele C of rs769446 emerged as a protective factor against NTG. Furthermore, haplotype analysis of the five SNPs revealed that the A-T-G-T-T haplotype was a statistically significant risk factor for NTG. This study indicated an association between APOE promoter SNPs and NTG in the Korean population. Further studies are required to understand how APOE promoter SNPs contribute to NTG pathogenesis.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Intraocular pressure across the lifespan of Tg-MYOCY437H mice.

Transgenic C57BL/6 mice expressing human myocilinY437 (Tg-MYOCY437H) are a well-established model for primary open-angle glaucoma (POAG). While the reduced trabecular meshwork (TM) cellularity due to severe endoplasmic reticulum (ER) stress has been characterized as the etiology of this model, there is a limited understanding of how glaucomatous phenotypes evolve over the lifespan of Tg-MyocY437H mice. In this study, we compiled the model's intraocular pressure (IOP) data recorded in our laboratory from 2017 to 2023 and selected representative eyes to measure the outflow facility (Cr), a critical parameter indicating the condition of the conventional TM pathway. We found that Tg-MYOCY437H mice aged 4-12 months exhibited significantly higher IOPs than age-matched C57BL/6 mice. Notably, a decline in IOP was observed in Tg-MYOCY437H mice at 17-24 months of age, a phenomenon not attributable to the gene dosage of mutant myocilin. Measurements of the Cr of Tg-MYOCY437H mice indicated that the age-related IOP reduction was not a result of ongoing TM damage. Instead, Hematoxylin and Eosin staining, immunohistochemistry analysis, and transmission electron microscopic examination revealed that this reduction might be induced by degenerations of the non-pigmented epithelium in the ciliary body of aged Tg-MYOCY437H mice. Overall, our findings provide a comprehensive profile of mutant myocilin-induced ocular changes over the Tg-MYOCY437H mouse lifespan and suggest a specific temporal window of elevated IOP that may be ideal for experimental purposes.

Targeting mechanics-induced trabecular meshwork dysfunction through YAP-TGFß Ameliorates high myopia-induced ocular hypertension.

High myopia is a risk factor for primary open angle glaucoma (POAG). The pathological mechanism of high myopia induced POAG occurrence is not fully understood. In this study, we successfully established the guinea pig model of ocular hypertension with high myopia, and demonstrated the susceptibility of high myopia for the occurrence of microbead-induced glaucoma compared with non-myopia group and the effect of YAP/TGF-ß signaling pathway in TM pathogenesis induced by high myopia. Moreover, we performed stretching treatment on primary trabecular meshwork (TM) cells to simulate the mechanical environment of high myopia. It was found that stretching treatment disrupted the cytoskeleton, decreased phagocytic function, enhanced ECM remodeling, and promoted cell apoptosis. The experiments of mechanics-induced human TM cell lines appeared the similar trend. Mechanically, the differential expressed genes of TM cells caused by stretch treatment enriched YAP/TGF-ß signaling pathway. To inhibit YAP/TGF-ß signaling pathway effectively reversed mechanics-induced TM damage. Together, this study enriches mechanistic insights of high myopia induced POAG susceptibility and provides a potential target for the prevention of POAG with high myopia.

Influence of the calcium voltage-gated channel auxiliary subunit (CACNA2D1) absence on intraocular pressure in mice.

The etiology of elevated intraocular pressure (IOP), a major risk factor for glaucoma (optic nerve atrophy), is poorly understood despite continued efforts. Although the gene variant of CACNA2D1 (encoding α2δ1), a calcium voltage-gated channel auxiliary subunit, has been reported to be associated with primary open-angle glaucoma, and the pharmacological mitigation of α2δ1 activity by pregabalin lowers IOP, the cellular basis for α2δ1 role in the modulation of IOP remains unclear. Our recent findings reveled readily detectable levels of α2δ1 and its ligand thrombospondin in the cytoskeletome fraction of human trabecular meshwork (TM) cells. To understand the direct role of α2δ1 in the modulation of IOP, we evaluated α2δ1 null mice for changes in IOP and found a moderate (∼10%) but significant decrease in IOP compared to littermate wild type control mice. Additionally, to gain cellular insights into α2δ1 antagonist (pregabalin) induced IOP changes, we assessed pregabalin's effects on human TM cell actin cytoskeletal organization and cell adhesive interactions in comparison with a Rho kinase inhibitor (Y27632), a known ocular hypotensive agent. Unlike Y27632, pregabalin did not have overt effects on cell morphology, actin cytoskeletal organization, or cell adhesion in human TM cells. These results reveal a modest but significant decrease in IOP in α2δ1 deficient mice, and this response appears to be not associated with the contractile and cell adhesive characteristics of TM cells based on the findings of pregabalin effects on isolated TM cells. Therefore, the mechanism by which pregabalin lowers IOP remains elusive.

Genetic changes and testing associated with childhood glaucoma: A systematic review.

Many forms of childhood glaucoma have been associated with underlying genetic changes, and variants in many genes have been described. Currently, testing is variable as there are no widely accepted guidelines for testing. This systematic review aimed to summarize the literature describing genetic changes and testing practices in childhood glaucoma. This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic review and Meta-Analyses (PRISMA) 2020 guidelines and registered with Prospero (ID CRD42023400467). A comprehensive review of Pubmed, Embase, and Cochrane databases was performed from inception through March 2, 2023 using the search terms: (glaucoma) AND (pediatric OR childhood OR congenital OR child OR infant OR infantile) AND (gene OR genetic OR genotype OR locus OR genomic OR mutation OR variant OR test OR screen OR panel). Information was extracted regarding genetic variants including genotype-phenotype correlation. Risk of bias was assessed using the Newcastle-Ottawa Scale. Of 1,916 records screened, 196 studies met inclusion criteria and 53 genes were discussed. Among study populations, mean age±SD at glaucoma diagnosis was 8.94±9.54 years and 50.4% were male. The most common gene discussed was CYP1B1, evaluated in 109 (55.6%) studies. CYP1B1 variants were associated with region and population-specific prevalence ranging from 5% to 86% among those with primary congenital glaucoma. MYOC variants were discussed in 31 (15.8%) studies with prevalence up to 36% among patients with juvenile open angle glaucoma. FOXC1 variants were discussed in 25 (12.8%) studies, which demonstrated phenotypic severity dependent on degree of gene expression and type of mutation. Overall risk of bias was low; the most common domains of bias were selection and comparability. Numerous genes and genetic changes have been associated with childhood glaucoma. Understanding the most common genes as well as potential genotype-phenotype correlation has the potential to improve diagnostic and prognostic outcomes for children with glaucoma.

Novel ancestry-specific primary open-angle glaucoma loci and shared biology with vascular mechanisms and cell proliferation.

Primary open-angle glaucoma (POAG), a leading cause of irreversible blindness globally, shows disparity in prevalence and manifestations across ancestries. We perform meta-analysis across 15 biobanks (of the Global Biobank Meta-analysis Initiative) (n = 1,487,441: cases = 26,848) and merge with previous multi-ancestry studies, with the combined dataset representing the largest and most diverse POAG study to date (n = 1,478,037: cases = 46,325) and identify 17 novel significant loci, 5 of which were ancestry specific. Gene-enrichment and transcriptome-wide association analyses implicate vascular and cancer genes, a fifth of which are primary ciliary related. We perform an extensive statistical analysis of SIX6 and CDKN2B-AS1 loci in human GTEx data and across large electronic health records showing interaction between SIX6 gene and causal variants in the chr9p21.3 locus, with expression effect on CDKN2A/B. Our results suggest that some POAG risk variants may be ancestry specific, sex specific, or both, and support the contribution of genes involved in programmed cell death in POAG pathogenesis.

Lack of Association between LOXL1 Variants and Pigment Dispersion Syndrome/Pigmentary Glaucoma: A Meta-Analysis.

The phenotypic similarities between exfoliation syndrome (XFS)/exfoliation glaucoma (XFG) and pigment dispersion syndrome (PDS)/pigmentary glaucoma (PG), particularly their association with material deposition in the eye's anterior segment, have prompted investigations into genetic commonalities. This study focuses on the LOXL1 gene, conducting a comprehensive meta-analysis of three candidate gene association studies. We analyzed three single nucleotide polymorphisms (SNPs) of LOXL1: rs1048661, rs3825942, and rs2165241. Our results reveal nominal significance for the exonic SNPs rs1048661 and rs3825942 (p ≤ 0.01), but show no significant association for the intronic SNP rs2165241 (p = 0.83) with PDS/PG. There was homogeneity across study cohorts (I2 = 0), and sensitivity analyses and funnel plots confirmed a lower likelihood of bias in our findings. The lack of a statistically significant association between LOXL1 variants and PDS/PG at p < 0.05 was attributable to the insufficient statistical power of the pooled data, which ranged from 5% to 37% for the three SNPs. This study suggests no association between LOXL1 variants and PDS/PG. Further validation and exploration of XFS/XFG-associated genes in larger and more diverse cohorts would be helpful to determine the genetic correlation or distinctiveness between these conditions.

Somatic Mutations within Myocilin due to Aging May Be a Potential Risk Factor for Glaucoma.

Glaucoma is a chronic optic neuropathy that leads to irreversible vision loss. Aging and family history are the two most important risk factors of glaucoma. One of the most studied genes involved in the onset of open-angle glaucoma is myocilin (MYOC). About 105 germline mutations within MYOC are known to be associated with glaucoma and result in endoplasmic reticulum (ER) stress, which leads to trabecular meshwork (TM) cell death and subsequent intraocular pressure (IOP) elevation. However, only about 4% of the population carry these mutations. An analysis of MYOC somatic cancer-associated mutations revealed a notable overlap with pathogenic glaucoma variants. Because TM cells have the potential to accumulate somatic mutations at a rapid rate due to ultraviolet (UV) light exposure, we propose that an accumulation of somatic mutations within MYOC is an important contributor to the onset of glaucoma.

Assessment of Causality Between Diet-Derived Antioxidants and Primary Open-Angle Glaucoma: A Mendelian Randomization Study.

Purpose: This study aimed to investigate the genetic causal relationships among diet-derived circulating antioxidants, primary open-angle glaucoma (POAG), and glaucoma-related traits using two-sample Mendelian randomization (MR). Methods: Genetic variants associated with diet-derived circulating antioxidants (retinol, ascorbate, ß-carotene, lycopene, α-tocopherol, and γ-tocopherol) were assessed as absolute and metabolic instrumental variables. POAG and glaucoma-related traits data were derived from a large, recently published genome-wide association study database; these traits included intraocular pressure (IOP), macular retinal nerve fiber layer (mRNFL) thickness, macular ganglion cell-inner plexiform layer (mGCIPL) thickness, and vertical cup-to-disc ratio (vCDR). MR analyses were performed per outcome for each exposure. Results: We found no causal association between six diet-derived antioxidants and POAG using the International Glaucoma Genetics Consortium data. For absolute antioxidants, the odds ratios (ORs) ranged from 1.011 (95% confidence interval [CI], 0.854-1.199; P = 0.895) per natural log-transformed ß-carotene to 1.052 (95% CI, 0.911-1.215; P = 0.490) for 1 µmol/L of ascorbate. For antioxidant metabolites, the OR ranged from 0.998 (95% CI, 0.801-1.244; P = 0.989) for ascorbate to 1.210 (95% CI, 0.870-1.682; P = 0.257) for γ-tocopherol, using log-transformed levels. A similar result was obtained with the FinnGen Biobank. Furthermore, our results showed no significant genetic association between six diet-derived antioxidants and glaucoma-related traits. Conclusions: Our study did not support a causal association among six diet-derived circulating antioxidants, POAG, and glaucoma-related traits. This suggests that the intake of antioxidants may not have a preventive effect on POAG and offers no protection to retinal nerve cells. Translational Relevance: This study provides valid evidence regarding the use of diet-derived antioxidants for glaucoma patients.

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma.

INTRODUCTION: Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterized by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. We aimed to identify genetic and morphological variation that can be attributed to trabecular meshwork cell (TMC) dysfunction and raised IOP in POAG. METHODS: 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescence coupled with CellProfiler image analysis allowed for single-cell morphological profiling. RESULTS: Many gene knockouts invoked DEGs relating to matrix metalloproteinases and interferon-induced proteins. We have prioritized genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified, suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, while knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSION: High-throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

Integrating genetic regulation and single-cell expression with GWAS prioritizes causal genes and cell types for glaucoma.

Primary open-angle glaucoma (POAG), characterized by retinal ganglion cell death, is a leading cause of irreversible blindness worldwide. However, its molecular and cellular causes are not well understood. Elevated intraocular pressure (IOP) is a major risk factor, but many patients have normal IOP. Colocalization and Mendelian randomization analysis of >240 POAG and IOP genome-wide association study (GWAS) loci and overlapping expression and splicing quantitative trait loci (e/sQTLs) in 49 GTEx tissues and retina prioritizes causal genes for 60% of loci. These genes are enriched in pathways implicated in extracellular matrix organization, cell adhesion, and vascular development. Analysis of single-nucleus RNA-seq of glaucoma-relevant eye tissues reveals that the POAG and IOP colocalizing genes and genome-wide associations are enriched in specific cell types in the aqueous outflow pathways, retina, optic nerve head, peripapillary sclera, and choroid. This study nominates IOP-dependent and independent regulatory mechanisms, genes, and cell types that may contribute to POAG pathogenesis.

Causal relationships between Gut microbiota and primary open-angle Glaucoma: A Mendelian randomization and mediation analysis of Glaucoma endophenotypes.

Primary open-angle glaucoma (POAG) is a widespread condition responsible for irreversible blindness, and its prevalence is expected to increase substantially in the coming decades. Despite its significance, the exact cause of POAG remains elusive, necessitating a comprehensive exploration of its pathogenesis. Emerging research suggests a potential link between alterations in gut microbiota composition and POAG. However, establishing causality in these associations remains a challenge. In this study, we employed Mendelian randomization (MR) analysis to investigate the potential causal relationships between gut microbiota (GM) and POAG. Significant bacteria taxa were further analyzed with POAG endophenotypes. We utilized data from genome-wide association studies (GWAS) for GM and POAG, as well as for glaucoma endophenotypes, including intraocular pressure (IOP), retinal nerve fiber layer (RNFL) thickness, vertical cup-to-disc ratio (VCDR), and central corneal thickness (CCT). Univariable, multivariable MR and mediation effect analysis were conducted. Our analysis revealed that certain taxa, including phylum Euryarchaeota, genus Odoribacter, Rumnicoccaceae UCG009, Ruminiclostridium9, unknown genus id.2071, and Eubacterium rectale group, were associated with an increased risk of POAG. On the other hand, family Victivallaceae, Lacchnospiraceae, genus Lachnoclostridium, Oscillospira, Ruminococcaceae UCG011, Alloprevotella, and Faecalibacterium were found to be associated with a decreased risk of POAG. Furthermore, some of these taxa were found to be connected to glaucoma endophenotypes. Through further multivariable MR analysis, it was determined that IOP, VCDR, and CCT might played mediating roles between GM and POAG. In conclusion, this study utilizes MR analysis to elucidate potential causal associations between GM and POAG, providing insights into specific GM taxa that influence POAG risk and related endophenotypes. These findings emphasize the potential role of the gut microbiota in the pathogenesis of POAG and pave the way for future research and therapeutic interventions.

Genistein stimulates the viability and prevents myofibroblastic transformation in human trabecular meshwork cells stimulated by TGF-ß.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma leading to blindness. The search for ways to prevent/treat this entity is one of the main challenges of today's ophthalmology. One of such solution seems to be biologically active substances of natural origin, such as genistein (GEN), which can affect the function of isolated trabecular meshwork by the inhibition of protein tyrosine kinase. However, the role of GEN in viability as well as myofibroblastic transformation in human trabecular meshwork cells stimulated by TGF-ß is unknown. Using human trabecular meshwork cells (HTMCs) we investigated the effect of genistein on cell viability and myofibroblastic transformation stimulated by TGF-ß1 and TGF-ß2. Using Real-Time PCR, western blot and immunofluorescence we determined the effect on the expression changes of αSMA, TIMP1, collagen 1 and 3 at mRNA and protein level. We found that genistein increases the viability of HTMCs (1, 2, 3 µg/ml; P < 0.05 and 4, 5, 10, 15, 20 µg/ml; P < 0.01). Moreover, we found that addition of 10, 15 and 20 µg/ml is able to prevent myofibroblastic transformation of HTMCs by decreasing αSMA, TIMP1, collagen 1 and 3 mRNA and protein expression (P < 0.01). Based on the obtained results, we can conclude that genistein is a potential factor that can prevent the myofibroblastic transformation of HTMCs accompanying glaucoma. Describing GEN influence on myofibroblastic transformation processes in HTMC allows us to conclude that it can be considered a potential therapeutic agent or a substance supporting treatment in patients with glaucoma.