The protective role of water intake in age-related eye diseases: insights from a Mendelian randomization study.

Age-related eye diseases (AREDs), including age-related cataracts (ARCs), age-related macular degeneration (AMD), diabetic retinopathy (DR), and glaucoma, are a leading cause of visual loss globally. This study aimed to explore the effects of dietary water intake on AREDs using Mendelian randomization. In the European population, genome-wide association study (GWAS) summary statistics of water intake and AREDs were obtained from the UK Biobank database and the FinnGen Consortium, respectively. The causal associations between water intake and ARED risks were explored by univariable and multivariable MR analyses, followed by sensitivity analyses to test the robustness of the results and detect potential pleiotropy bias. Water intake was associated with reduced risks of ARCs (odds ratio [OR]: 0.61; 95% confidence interval [CI]: 0.46-0.83; P = 1.44 × 10-3) and DR (OR: 0.52; 95% CI: 0.36-0.76; P = 5.47 × 10-4), and a suggestive reduced risk of AMD (OR: 0.42; 95% CI: 0.20-0.88; P = 2.18 × 10-2). Water intake had no effect on glaucoma (OR: 1.16; 95% CI: 0.72-1.88; P = 0.549). After adjusting confounders, the causal effects of water intake on ARCs and DR persisted. Our study provides evidence of the preventive role of water intake in ARCs and DR from a genetic perspective.

A bird's eye view on the use of whole exome sequencing in rare congenital ophthalmic diseases.

Phenotypic and genotypic heterogeneity in congenital ocular diseases, especially in anterior segment dysgenesis (ASD), have created challenges for proper diagnosis and classification of diseases. Over the last decade, genomic research has indeed boosted our understanding in the molecular basis of ASD and genes associated with both autosomal dominant and recessive patterns of inheritance have been described with a wide range of expressivity. Here we describe the molecular characterization of a cohort of 162 patients displaying isolated or syndromic congenital ocular dysgenesis. Samples were analyzed with diverse techniques, such as direct sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing (WES), over 20 years. Our data reiterate the notion that PAX6 alterations are primarily associated with ASD, mostly aniridia, since the majority of the cohort (66.7%) has a pathogenic or likely pathogenic variant in the PAX6 locus. Unexpectedly, a high fraction of positive samples (20.3%) displayed deletions involving the 11p13 locus, either partially/totally involving PAX6 coding region or abolishing its critical regulatory region, underlying its significance. Most importantly, the use of WES has allowed us to both assess variants in known ASD genes (i.e., CYP1B1, ITPR1, MAB21L1, PXDN, and PITX2) and to identify rarer phenotypes (i.e., MIDAS, oculogastrointestinal-neurodevelopmental syndrome and Jacobsen syndrome). Our data clearly suggest that WES allows expanding the analytical portfolio of ocular dysgenesis, both isolated and syndromic, and that is pivotal for the differential diagnosis of those conditions in which there may be phenotypic overlaps and in general in ASD.

Role of Serine Protease Inhibitors A1 and A3 in Ocular Pathologies.

Serine protease inhibitors A1 (SerpinA1) and A3 (SerpinA3) are important members of the serpin family, playing crucial roles in the regulation of serine proteases and influencing various physiological processes. SerpinA1, also known as α-1-antitrypsin, is a versatile glycoprotein predominantly synthesized in the liver, with additional production in inflammatory and epithelial cell types. It exhibits multifaceted functions, including immune modulation, complement activation regulation, and inhibition of endothelial cell apoptosis. SerpinA3, also known as α-1-antichymotrypsin, is expressed both extracellularly and intracellularly in various tissues, particularly in the retina, kidney, liver, and pancreas. It exerts anti-inflammatory, anti-angiogenic, antioxidant, and antifibrotic activities. Both SerpinA1 and SerpinA3 have been implicated in conditions such as keratitis, diabetic retinopathy, age-related macular degeneration, glaucoma, cataracts, dry eye disease, keratoconus, uveitis, and pterygium. Their role in influencing metalloproteinases and cytokines, as well as endothelial permeability, and their protective effects on Müller cells against oxidative stress further highlight their diverse and critical roles in ocular pathologies. This review provides a comprehensive overview of the etiology and functions of SerpinA1 and SerpinA3 in ocular diseases, emphasizing their multifaceted roles and the complexity of their interactions within the ocular microenvironment.

Multi-omics analysis in human retina uncovers ultraconserved cis-regulatory elements at rare eye disease loci.

Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases.

Diagnostic genome sequencing improves diagnostic yield: a prospective single-centre study in 1000 patients with inherited eye diseases.

PURPOSE: Genome sequencing (GS) is expected to reduce the diagnostic gap in rare disease genetics. We aimed to evaluate a scalable framework for genome-based analyses 'beyond the exome' in regular care of patients with inherited retinal degeneration (IRD) or inherited optic neuropathy (ION). METHODS: PCR-free short-read GS was performed on 1000 consecutive probands with IRD/ION in routine diagnostics. Complementary whole-blood RNA-sequencing (RNA-seq) was done in a subset of 74 patients. An open-source bioinformatics analysis pipeline was optimised for structural variant (SV) calling and combined RNA/DNA variation interpretation. RESULTS: A definite genetic diagnosis was established in 57.4% of cases. For another 16.7%, variants of uncertain significance were identified in known IRD/ION genes, while the underlying genetic cause remained unresolved in 25.9%. SVs or alterations in non-coding genomic regions made up for 12.7% of the observed variants. The RNA-seq studies supported the classification of two unclear variants. CONCLUSION: GS is feasible in clinical practice and reliably identifies causal variants in a substantial proportion of individuals. GS extends the diagnostic yield to rare non-coding variants and enables precise determination of SVs. The added diagnostic value of RNA-seq is limited by low expression levels of the major IRD disease genes in blood.

Artificial Intelligence-based database for prediction of protein structure and their alterations in ocular diseases.

The aim of the study is to establish an online database for predicting protein structures altered in ocular diseases by Alphafold2 and RoseTTAFold algorithms. Totally, 726 genes of multiple ocular diseases were collected for protein structure prediction. Both Alphafold2 and RoseTTAFold algorithms were built locally using the open-source codebases. A dataset with 48 protein structures from Protein Data Bank (PDB) was adopted for algorithm set-up validation. A website was built to match ocular genes with the corresponding predicted tertiary protein structures for each amino acid sequence. The predicted local distance difference test-Cα (pLDDT) and template modeling (TM) scores of the validation protein structure and the selected ocular genes were evaluated. Molecular dynamics and molecular docking simulations were performed to demonstrate the applications of the predicted structures. For the validation dataset, 70.8% of the predicted protein structures showed pLDDT greater than 90. Compared to the PDB structures, 100% of the AlphaFold2-predicted structures and 97.9% of the RoseTTAFold-predicted structure showed TM score greater than 0.5. Totally, 1329 amino acid sequences of 430 ocular disease-related genes have been predicted, of which 75.9% showed pLDDT greater than 70 for the wildtype sequences and 76.1% for the variant sequences. Small molecule docking and molecular dynamics simulations revealed that the predicted protein structures with higher confidence scores showed similar molecular characteristics with the structures from PDB. We have developed an ocular protein structure database (EyeProdb) for ocular disease, which is released for the public and will facilitate the biological investigations and structure-based drug development for ocular diseases. Database URL:  http://eyeprodb.jsiec.org.

Burden and clinical profile of genetic eye diseases in children in Nigeria: a descriptive cross-sectional study.

Introduction: ophthalmic genetics is rapidly evolving globally but is still nascent in much of sub-Saharan Africa, with gaps in knowledge about the burden in the region. This study evaluated the burden and manifestations of genetic eye diseases in children in Ibadan, Nigeria. Methods: this was a hospital-based cross-sectional study in which new and follow-up paediatric eye clinic patients were recruited consecutively at the University College Hospital, Ibadan. Children with genetic eye diseases had comprehensive ocular and systemic examinations, and their pedigrees were charted to determine the probable modes of inheritance. The main outcome variables were the proportion of study participants with genetic eye diseases, the probable modes of inheritance, and the clinical diagnoses. Summary statistics were performed using means and standard deviations for numerical variables and proportions for categorical variables. Results: fifty-two (12%) of 444 children had genetic eye diseases, and their mean (SD) age was 88.8 ± 50.4 months. Thirteen different phenotypic diagnoses were made following the evaluation of the 52 children, including primary congenital glaucoma (13, 25%) and familial non-syndromic cataracts (8, 15%). The probable modes of inheritance were derived from the pedigree charts, and 30 (58%) conditions were presumed to be sporadic. Conclusion: this study demonstrated a significant burden and a wide range of paediatric genetic eye diseases in this tertiary referral centre in Nigeria. This information provides invaluable evidence for planning ophthalmic genetic services.

The Ins and Outs of Clusterin: Its Role in Cancer, Eye Diseases and Wound Healing.

Clusterin (CLU) is a glycoprotein originally discovered in 1983 in ram testis fluid. Rapidly observed in other tissues, it was initially given various names based on its function in different tissues. In 1992, it was finally named CLU by consensus. Nearly omnipresent in human tissues, CLU is strongly expressed at fluid-tissue interfaces, including in the eye and in particular the cornea. Recent research has identified different forms of CLU, with the most prominent being a 75-80 kDa heterodimeric protein that is secreted. Another truncated version of CLU (55 kDa) is localized to the nucleus and exerts pro-apoptotic activities. CLU has been reported to be involved in various physiological processes such as sperm maturation, lipid transportation, complement inhibition and chaperone activity. CLU was also reported to exert important functions in tissue remodeling, cell-cell adhesion, cell-substratum interaction, cytoprotection, apoptotic cell death, cell proliferation and migration. Hence, this protein is sparking interest in tissue wound healing. Moreover, CLU gene expression is finely regulated by cytokines, growth factors and stress-inducing agents, leading to abnormally elevated levels of CLU in many states of cellular disturbance, including cancer and neurodegenerative conditions. In the eye, CLU expression has been reported as being severely increased in several pathologies, such as age-related macular degeneration and Fuch's corneal dystrophy, while it is depleted in others, such as pathologic keratinization. Nevertheless, the precise role of CLU in the development of ocular pathologies has yet to be deciphered. The question of whether CLU expression is influenced by these disorders or contributes to them remains open. In this article, we review the actual knowledge about CLU at both the protein and gene expression level in wound healing, and explore the possibility that CLU is a key factor in cancer and eye diseases. Understanding the expression and regulation of CLU could lead to the development of novel therapeutics for promoting wound healing.

Unleashing the potential of CRISPR multiplexing: Harnessing Cas12 and Cas13 for precise gene modulation in eye diseases.

Gene therapy is a flourishing field with the potential to revolutionize the treatment of genetic diseases. The emergence of CRISPR-Cas9 has significantly advanced targeted and efficient genome editing. Although CRISPR-Cas9 has demonstrated promising potential applications in various genetic disorders, it faces limitations in simultaneously targeting multiple genes. Novel CRISPR systems, such as Cas12 and Cas13, have been developed to overcome these challenges, enabling multiplexing and providing unique advantages. Cas13, in particular, targets mRNA instead of genomic DNA, permitting precise gene expression control and mitigating off-target effects. This review investigates the potential of Cas12 and Cas13 in ocular gene therapy applications, such as suppression of inflammation and cell death. In addition, the capabilities of Cas12 and Cas13 are explored in addressing potential targets related with disease mechanisms such as aberrant isoforms, mitochondrial genes, cis-regulatory sequences, modifier genes, and long non-coding RNAs. Anatomical accessibility and relative immune privilege of the eye provide an ideal organ system for evaluating these novel techniques' efficacy and safety. By targeting multiple genes concurrently, CRISPR-Cas12 and Cas13 systems hold promise for treating a range of ocular disorders, including glaucoma, retinal dystrophies, and age-related macular degeneration. Nonetheless, additional refinement is required to ascertain the safety and efficacy of these approaches in ocular disease treatments. Thus, the development of Cas12 and Cas13 systems marks a significant advancement in gene therapy, offering the potential to devise effective treatments for ocular disorders.

339ª Reunião do Conselho Nacional de Saúde - 15 feb. 2023

eyeVarP: A computational framework for the identification of pathogenic variants specific to eye disease.

PURPOSE: Disease-specific pathogenic variant prediction tools that differentiate pathogenic variants from benign have been improved through disease specificity recently. However, they have not been evaluated on disease-specific pathogenic variants compared with other diseases, which would help to prioritize disease-specific variants from several genes or novel genes. Thus, we hypothesize that features of pathogenic variants alone would provide a better model. METHODS: We developed an eye disease-specific variant prioritization tool (eyeVarP), which applied the random forest algorithm to the data set of pathogenic variants of eye diseases and other diseases. We also developed the VarP tool and generalized pipeline to filter missense and insertion-deletion variants and predict their pathogenicity from exome or genome sequencing data, thus we provide a complete computational procedure. RESULTS: eyeVarP outperformed pan disease-specific tools in identifying eye disease-specific pathogenic variants under the top 10. VarP outperformed 12 pathogenicity prediction tools with an accuracy of 95% in correctly identifying the pathogenicity of missense and insertion-deletion variants. The complete pipeline would help to develop disease-specific tools for other genetic disorders. CONCLUSION: eyeVarP performs better in identifying eye disease-specific pathogenic variants using pathogenic variant features and gene features. Implementing such complete computational procedure would significantly improve the clinical variant interpretation for specific diseases.

Roles of non-coding RNAs in eye development and diseases.

The prevalence of ocular disorders is dramatically increasing worldwide, especially those that cause visual impairment and permanent loss of vision, including cataract, glaucoma, age-related macular degeneration, and diabetic retinopathy. Extensive evidence has shown that ncRNAs are key regulators in various biogenesis and biological functions, controlling gene expression related to histogenesis and cell differentiation in ocular tissues. Aberrant expression and function of ncRNA can lead to dysfunction of visual system and mediate progression of eye disorders. Here, we mainly offer an overview of the role of precise modulation of ncRNAs in eye development and function in patients with eye diseases. We also highlight the challenges and future perspectives in conducting ncRNA studies, focusing specifically on the role of ncRNAs that may hold expanded promise for their diagnostic and therapeutic applications in various eye diseases. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders.

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

Evaluation of somatic and/or germline mosaicism in congenital malformation of the eye.

Microphthalmia, Anophthalmia and Coloboma (MAC) form a spectrum of congenital eye malformations responsible for severe visual impairment. Despite the exploration of hundreds of genes by High-Throughput Sequencing (HTS), most of the patients remain without genetic diagnosis. One explanation could be the not yet demonstrated involvement of somatic mosaicism (undetected by conventional analysis pipelines) in those patients. Furthermore, the proportion of parental germline mosaicism in presumed de novo variations is still unknown in ocular malformations. Thus, using dedicated bioinformatics pipeline designed to detect mosaic variants, we reanalysed the sequencing data obtained from a 119 ocular development genes panel performed on blood samples of 78 probands with sporadic MAC without genetic diagnosis. Using the same HTS strategy, we sequenced 80 asymptomatic parents of 41 probands carrying a disease-causing variant in an ocular development gene considered de novo after Sanger sequencing of both parents. Reanalysis of the previously sequencing data did not find any mosaic variant in probands without genetic diagnosis. However, HTS of parents revealed undetected SOX2 and PAX6 mosaic variants in two parents. Finally, this work, performed on two large cohorts of patients with MAC spectrum, provides for the first time an overview of the interest of looking for mosaicism in ocular development disorders. Somatic mosaicism does not appear to be frequent in MAC spectrum and might explain only few diagnoses. Thus, other approaches such as whole genome sequencing should be considered in those patients. Parental mosaicism is however not that rare (around 5%) and challenging for genetic counselling.

RNA-targeting strategies as a platform for ocular gene therapy.

Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.

Molecular Mechanisms of Alu and LINE-1 Interspersed Repetitive Sequences Reveal Diseases of Visual System Dysfunction.

BACKGROUND: Short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINE-1s) are the abundant and well-characterized repetitive elements in the human genome. METHODS: For this review, all relevant original research studies were assessed by searching electronic databases, including PubMed, Google Scholar, and Web of Science, by using relevant keywords. Accumulating evidence indicates that the disorder of gene expression regulated by these repetitive sequences is one of the causes of the diseases of visual system dysfunction, including retinal degenerations, glaucoma, retinitis punctata albescens, retinitis pigmentosa, geographic atrophy, and age-related macular degeneration, suggesting that SINEs and LINE-1s may have great potential implications in ophthalmology. RESULTS: Alu elements belonging to the SINEs are present in more than one million copies, comprising 10% of the human genome. CONCLUSION: This study offers recent advances in Alu and LINE-1 mechanisms in the development of eye diseases. The current study could advance our knowledge of the roles of SINEs and LINE-1s in the developing process of eye diseases, suggesting new diagnostic biomarkers, therapeutic strategies, and significant points for future studies.

This study reveals the Alu and LINE-1 interspersed repetitive sequences involved in the diseases of visual system dysfunction.This study shows the disorder of gene expression regulated by SINEs and LINE-1s sequences is one of the causes of the diseases of visual system dysfunction.This study suggests recent advances in Alu and LINE-1 mechanisms are involved in eye diseases.

Epigenetics in Eye Development and Ocular Disorders: A Brief Review.

Epigenetics is a powerful regulator of gene expression. With advanced discoveries in underlying molecular mechanisms that can alter chromatin response to internal and external signals, epigenetic alterations have been implicated in various developmental pathways and human disorders. The extent to which this epigenetic effect contributes to eye development and progression of ocular disorders is currently less defined. However, emerging evidence suggests that epigenetic changes are relevant in the development of eye and ocular disorders like pterygium, age-related macular degeneration, glaucoma and more. This brief review will discuss the relevance of epigenetic mechanisms like DNA methylation, histone modifications, polycomb proteins and noncoding RNAs in the context of eye development and selected ocular disorders.

Mendelian randomization analyses in ocular disease: a powerful approach to causal inference with human genetic data.

Ophthalmic epidemiology is concerned with the prevalence, distribution and other factors relating to human eye disease. While observational studies cannot avoid confounding factors from interventions, human eye composition and structure are unique, thus, eye disease pathogenesis, which greatly impairs quality of life and visual health, remains to be fully explored. Notwithstanding, inheritance has had a vital role in ophthalmic disease. Mendelian randomization (MR) is an emerging method that uses genetic variations as instrumental variables (IVs) to avoid confounders and reverse causality issues; it reveals causal relationships between exposure and a range of eyes disorders. Thus far, many MR studies have identified potentially causal associations between lifestyles or biological exposures and eye diseases, thus providing opportunities for further mechanistic research, and interventional development. However, MR results/data must be interpreted based on comprehensive evidence, whereas MR applications in ophthalmic epidemiology have some limitations worth exploring. Here, we review key principles, assumptions and MR methods, summarise contemporary evidence from MR studies on eye disease and provide new ideas uncovering aetiology in ophthalmology.

Effectiveness of Whole-Exome Sequencing for the Identification of Causal Mutations in Patients with Suspected Inherited Ocular Diseases.

Background: Genetic eye disorders, affecting around one in 1000 people, encompass a diverse group of diseases causing severe visual deficiency. The recent adoption of next-generation sequencing techniques, including whole-exome sequencing (WES), in medicine has greatly enhanced diagnostic rates of genetically heterogeneous diseases. Objectives: The objectives of the study were to assess the diagnostic yield of WES in a cohort of Mexican individuals with suspected genetic eye disorders and to evaluate the improvement of diagnostic rates by reanalysis of WES data in patients without an initial molecular diagnosis. Methods: A total of 90 probands with ocular anomalies of suspected genetic origin were ascertained. Patients underwent WES in leukocytic DNA. Bioinformatics analysis and Sanger sequencing were used to confirm the disease-causing variants. Only variants identified as pathogenic or likely pathogenic were considered as causal. Results: Initial analysis revealed causal mutations in 46 cases (51%). Reanalysis of WES data 12 months after first analysis resulted in the identification of additional causal variants in 6 patients (7%), increasing the molecular diagnostic yield to 58%. The highest diagnostic rates by disease categories corresponded to hereditary retinal dystrophies (77%) and to anomalies of the anterior segment of the eye (47%). Conclusions: Our study demonstrates that WES is an effective approach for genetic diagnosis of genetic ocular diseases and that reanalysis of WES data can improve the diagnostic yield.