Lens Regeneration: The Application of iSyTE and In Silico Approaches to Evaluate Gene Expression in Lens Organoids.

Several protocols have been established for the generation of lens organoids from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other cells with regenerative potential in humans or various animal models. It is important to examine how well the regenerated lens organoids reflect lens biology, in terms of its development, homeostasis, and aging. Toward this goal, the iSyTE database (integrated Systems Tool for Eye gene discovery; https://research.bioinformatics.udel.edu/iSyTE/ ), a bioinformatics resource tool that contains meta-analyzed gene expression data in wild-type lens across different embryonic, postnatal, and adult stages, can serve as a resource for comparative analysis. This article outlines the approaches toward effective use of iSyTE to gain insights into normal gene expression in the mouse lens, enriched expression in the lens, and differential gene expression in select mouse gene-perturbation cataract/lens defects models, which in turn can be used to evaluate expression of key lens-relevant genes in lens organoids by transcriptomics (e.g., RNA-sequencing (RNA-seq), microarrays, etc.) or other downstream methods (e.g., RT-qPCR, etc.).

PHARC syndrome: an overview.

PHARC, polyneuropathy, hearing loss, cerebellar ataxia, retinitis pigmentosa and cataracts, or PHARC is a very rare progressive neurodegenerative autosomal recessive disease caused by biallelic mutations in the ABHD12 (a/b-hydrolase domain containing 12) gene, which encodes a lyso-phosphatidylserine (lyso-PS) lipase. The Orpha number for PHARC is ORPHA171848. The clinical picture of PHARC syndrome is very heterogeneous with a wide range of age at onset for each symptom, making a clinical diagnosis very challenging. Differential diagnoses of the disease include Refsum disease, Charcot-Marie-Tooth disease, and Usher syndrome. Many aspects of the disease, such as the biochemistry and pathophysiology, are still not fully understood. We generated a clinical overview of all PHARC patients, including their mutations, described in literature so far. Furthermore, we give an outline of the most recent developments in research on the pathophysiology of PHARC syndrome in an attempt to gain more insight into and increase awareness of the heterogeneity of the disease. We included 58 patients with PHARC from 37 different families with 27 known ABHD12 mutations. The age at onset (from early childhood to late thirties) and the severity of each feature of PHARC varied widely among patients. Demyelinating polyneuropathy was reported in 91% of the patients. In 86% of patients, hearing loss was present and 74% had cerebellar ataxia, the most variable symptom of PHARC. Retinitis pigmentosa and cataracts occurred in 82% and 86% of patients, respectively. Due to the rareness of the disease and the variable clinical phenotype, a diagnosis of PHARC is often delayed and mostly only made after an extensive genetic work-up. Therefore, we recommend adding the ABHD12 gene to diagnostic gene panels for polyneuropathy, cerebellar ataxia, hearing loss, retinal dystrophy, and cataracts. In addition, a full clinical work-up, neurological (with EMG and neuroimaging of the brain) and ophthalmological (with ERG) examination and audiological tests are indispensable to obtain a comprehensive overview of the clinical phenotype as some symptoms in PHARC may be very subtle and easily overlooked if not tested for. In conclusion, we strongly recommend that patients with (suspected) PHARC should be evaluated in a multidisciplinary setting involving ophthalmologists, audiologists, neurologists, and geneticists to ensure the best possible care. Furthermore, we discuss whether PHARC is a spectrum with various incomplete phenotypes even later in life, or whether it is a syndrome in which the clinical symptoms are variable in severity and age of onset.

Influence of Cataract Causing Mutations on αA-Crystallin: A Computational Approach.

The αA-crystallin protein plays a vital role in maintaining the refractive index and transparency of the eye lens. Significant clinical studies have emerged as the αA-crystallin is prone to aggregation, resulting in the formation of cataracts with varied etiologies due to mutations. This work aims to comprehend the structural and functional role of cataract-causing mutations in αA-crystallin, particularly at N-Terminal and α-Crystallin Domains, using in-silico approaches including molecular dynamics simulation. About 19 mutants of αA-crystallin along with native structure were simulated for 100 ns and the post-simulations analyses reveal pronounced dynamics of αA-crystallin due to the enhanced structure flexibility as its native compactness was lost and is witnessed mainly by the mutants R12L, R21L, R21Q, R54L, R65Q, R116C and R116H. It is observed that αA-crystallin discloses the NTD motions as the dominant one and the same was endorsed by the linear variation between Rg and the center-of-mass of αA-crystallin. Interestingly, such enhanced dynamics of αA-crystallin mutants associated with the structure flexibility is internally modulated by the dynamic exchange of secondary structure elements ß-sheets and coils (R2 = 0.619) during simulation. Besides, the observed pronounced dynamics of dimer interface region (ß3-L6-ß4 segment) of ACD along with CTD dynamics also gains importance. Particularly, the highly dynamic mutants are also characterized by enhanced non-covalent and hydrophobic interactions which renders detrimental effects towards its stability, and favours possible protein unfolding mechanisms. Overall, this study highlights the mutation-mediated structural distortions in αA-crystallin and demands the need for further potential development of inhibitors against cataract formation.

Incidental finding of a BRCA2 variant following whole genome sequencing to molecularly diagnose bilateral congenital cataracts.

A male patient in his 20s with a history of bilateral congenital cataracts and nystagmus presented to the genetic eye disease clinic at Moorfields Eye Hospital to enquire about genetic testing for family decision-making and access to preimplantation genetic testing. He had a history of lensectomy with best-corrected visual acuities of logMAR 0.60 and 1.00 in the right and left eye. Whole genome sequencing (WGS) was conducted, which included targeted analysis of a panel of 115 lens-related genes and incidental findings, for which patients are unable to opt-out. Genetic testing identified the causative variant c.134T>C (p.Leu45Pro) in the CRYGC gene. A pathogenic variant in BRCA2 was also identified as a secondary finding. This was unexpected given the absence of a strong family history of breast or ovarian cancer. This case illustrates the importance of genetic counselling and informing patients about the implications of incidental findings that arise from WGS.

Measurement of absolute abundance of crystallins in human and αA N101D transgenic mouse lenses using 15N-labeled crystallin standards.

Stable isotope labeled standards of all major human lens crystallins were created to measure the abundance of lens endogenous crystallins from birth to adulthood. All major human crystallins (αA, αB, ßA2, ßA3/A1, ßA4, ßB1, ßB2, ßB3, γA, γB, γC, γD, γS) were cloned with N-terminal 6 x His tagged SUMO for ease of purification and the ability to generate natural N-termini by SUMO protease cleavage when producing crystallins for structure/function studies. They were then expressed in 15N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as an artificial lens standard. The absolute quantification method was tested using soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses spiked with the 15N artificial lens standard. Proteins were trypsinized, relative ratios of light and heavy labeled peptides determined using high-resolution precursor and data independent MS2 scans, and data analysis performed using Skyline software. Crystallin abundances were measured in both human donor lenses and in transgenic mouse αA N101D cataract lenses. Technical replicates of human crystallin abundance measurements were performed with average coefficients of variation of approximately 2% across all 13 crystallins. αA crystallin comprised 27% of the soluble protein of 5-day-old lens and decreased to 16% by 18-years of age. Over this time period αB increased from 6% to 9% and the αA/αB ratio decreased from 4.5/1 to 2/1. γS-crystallin also increased nearly 2-fold from 7% to 12%, becoming the 3rd most abundant protein in adult lens, while ßB1 increased from 14% to 20%, becoming the most abundant crystallin of adult lens. Minor crystallins ßA2, ßB3, and γA comprised only about 1% each of the newborn lens soluble protein, and their abundance dropped precipitously by adulthood. While 9 of the SUMO tagged crystallins were useful for purification of crystallins for structural studies, γA, γB, γC, and γD were resistant to cleavage by SUMO protease. The abundance of WT and N101D human αA in transgenic mouse lenses was approximately 40-fold lower than endogenous mouse αA, but the deamidation mimic human αA N101D was less soluble than human WT αA. The high content of αA and the transient abundance of ßA2, ßB3, and γA in young lens suggest these crystallins play a role in early lens development and growth. ßB1 becoming the most abundant crystallin may result from its role in promoting higher order ß-crystallin oligomerization in mature lens. The full set of human crystallin expression vectors in the Addgene repository should be a useful resource for future crystallin studies. 15N labeling of these crystallins will be useful to accurately quantify crystallins in lens anatomic regions, as well as measure the composition of insoluble light scattering crystallin aggregates. The standards will also be useful to measure the abundance of crystallins expressed in transgenic animal models.

[Analysis of cytogenetic indexes and occupational characteristics of radiation workers with lens opacity].

Objective: To analyse the characteristics of micronuclus and chromosomal aberrations and the lens opacity in different working years, ages and work type of radiation workers with lens opacity. Methods: In January 2022, we selected 400 radiation workers with lens opacity who had undergone occupational health examination in Hangzhou Hospital for the Prevention and Treatment of Occupational Disease from March 2014 to December 2021 as research objects. To analyze the differences of micronucleus, chromosome aberration and the lens opacification rates of different length of service, type of work, age radiologic workers. Results: The results showed that radiation the micronucleus abnormality (0.59‰) and chromosome aberration (2.67%) in peripheral blood in workers who served more than 20 years were significantly higher than that of micronucleus abnormality (0.18‰) and chromosome aberration (0.31%) in workers served less than 20 years, the differences were statistically significant (P<0.05). The micronucleus abnormality (0.32‰) and chromosome aberration (0.57%) in the interventionist were higher than oral radiation workers (0.12‰ and 0.19%) and diagnostic radiologist (0.05‰ and 0.12%), the differences were statistically signcficant (χ(2)=23.98, 6.72, P<0.05) . Conclusion: The rates of micronucleus and chromosome aberration of the interventionaist are higher than oral radiation workers and the traditional radiologist. Workers engaged in radiation should improve personal protection and undertake regular occupational health examinations.

Causal relationship between hyperthyroidism and cataracts: a two-sample bi-directional mendelian randomization study.

Clinical studies and meta-analyses have suggested a link between thyroid dysfunction and lens opacification. The objective of this study is to investigate the causal relationship between hyperthyroidism and the development of cataracts using the Mendelian randomization approach, with the aim of filling the gap in knowledge about the systemic effects of hyperthyroidism on ocular health. Leveraging genetic variants as instrumental variables, the analysis used extensive datasets from the UK Biobank and the FinnGen Database and employed three common approaches for causal inference (the Inverse Variance Weighted method, a regression based method the Weighted Median estimator, and MR-Egger regression) and accompanying sensitivity analyses to ensure robustness. The results demonstrate that there is a strong likely causal relationship, with hyperthyroidism increasing the risk of developing senile cataracts (OR = 361.09, 95%CI 5.024 to 2.60 × 104, P = 0.007). A sensitivity analysis provided no evidence of horizontal pleiotropy and no significant outliers, suggesting the results are robust. In conclusion, our study established a significant causal relationship between hyperthyroidism and increased risk of cataract development, underscoring the importance of considering the systemic effects of hyperthyroidism in clinical and public health interventions and policies. Future research should focus on elucidating the molecular mechanisms underlying this association, exploring the potential benefits of early intervention in hyperthyroidism to prevent cataract development, and investigating whether these findings translate across different ethnic populations. Additionally, further GWAS studies aimed at identifying genetic variants associated with both hyperthyroidism and cataracts are warranted to confirm and expand upon our results.

Ferulic Acid Protects Human Lens Epithelial Cells Against UVA-Induced Oxidative Damage by Downregulating the DNA Demethylation of the Keap1 Promoter.

Ultraviolet (UV) radiation-triggered production of reactive oxygen species (ROS) is a primary contributor to apoptosis in human lens epithelial cells (HLECs), which can ultimately result in cataract formation. The nuclear factor erythroid-2-related factor 2 (Nrf2)-Kelch ECH associating protein 1 (Keap1) pathway, a fundamental oxidative stress regulation mechanism, plays a crucial role in the development of cataracts. Ferulic acid (FA), recognized for its potent antioxidant properties can activate the Nrf2 signaling pathway to mitigate oxidative damage and cell apoptosis. In this study, we have demonstrated the protective effects of FA in reducing UVA-induced oxidative damage and apoptosis in HLECs through the modulation of the Keap1/Nrf2 pathway, as evidenced by both cellular and animal experiments. HLECs and Lens were exposed to 10 J/cm2 UVA radiation with or without prior treatment with FA. We found that UVA radiation increased oxidative damage and cell apoptosis in HLECs, ultimately leading to opacification of rat lenses, while FA was able to attenuate both oxidative damage and cell apoptosis in HLECs and reduce the degree of lens opacification. FA upregulated the expression of antioxidant response factors of the Keap1/Nrf2 pathway and downregulated the expression of apoptosis-related genes in HLECs, as demonstrated by Western blot and RT-qPCR analyses. We also found that UVA radiation increased the degree of demethylation of the Keap1 promoter in HLECs, whereas FA reduced the level of Keap1 promoter demethylation as determined by DNA sequencing. Additionally, UVA upregulated the expression of DNA active demethylase of the Keap1 promoter in HLECs, Dnmt1, Dnmt3a, and Dnmt3b, as shown by immunofluorescence, Western blot, and RT-qPCR, however, FA attenuated the activity of the passive demethylase TET1 in addition to the active demethylases. These results demonstrated that UVA radiation can cause oxidative damage, cell apoptosis, and rat lens opacification by increasing the demethylation of the Keap1 promoter in lens epithelial cells. Conversely, FA was shown to reduce oxidative damage, inhibit cell apoptosis, and decrease rat lens opacification by increasing the methylation of the Keap1 promoter. These findings suggest that FA could be therapeutically beneficial in preventing and mitigating cataracts induced by UVA radiation.

Uncovering genetic loci and biological pathways associated with age-related cataracts through GWAS meta-analysis.

Age-related cataracts is a highly prevalent eye disorder that results in the clouding of the crystalline lens and is one of the leading causes of visual impairment and blindness. The disease is influenced by multiple factors including genetics, prolonged exposure to ultraviolet radiation, and a history of diabetes. However, the extent to which each of these factors contributes to the development of cataracts remains unclear. Our study identified 101 independent genome-wide significant loci, 57 of which are novel. We identified multiple genes and biological pathways associated with the cataracts, including four drug-gene interactions. Our results suggest a causal association between type 1 diabetes and cataracts. Also, we highlighted a surrogate measure of UV light exposure as a marker of cataract risk in adults.

Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

Disruption of Eph-ephrin bidirectional signaling leads to human congenital and age-related cataracts, but the mechanisms for these opacities in the eye lens remain unclear. Eph receptors bind to ephrin ligands on neighboring cells to induce canonical ligand-mediated signaling. The EphA2 receptor also signals non-canonically without ligand binding in cancerous cells, leading to epithelial-to-mesenchymal transition (EMT). We have previously shown that the receptor EphA2 and the ligand ephrin-A5 have diverse functions in maintaining lens transparency in mice. Loss of ephrin-A5 leads to anterior cataracts due to EMT. Surprisingly, both canonical and non-canonical EphA2 activation are present in normal wild-type lenses and in the ephrin-A5 knockout lenses. Canonical EphA2 signaling is localized exclusively to lens epithelial cells and does not change with age. Non-canonical EphA2 signaling is in both epithelial and fiber cells and increases significantly with age. We hypothesize that canonical ligand-dependent EphA2 signaling is required for the morphogenesis and organization of hexagonal equatorial epithelial cells while non-canonical ligand-independent EphA2 signaling is needed for complex membrane interdigitations that change during fiber cell differentiation and maturation. This is the first demonstration of non-canonical EphA2 activation in a non-cancerous tissue or cell and suggests a possible physiological function for ligand-independent EphA2 signaling.

Dental abnormalities observed in the oculo-facio-cardio-dental (OFCD) syndrome present in two Czech families bearing novel de novo BCOR pathogenic variants.

BACKGROUND: The oculo-facio-cardio-dental syndrome (OFCD) is an ultra-rare multiple congenital anomaly. This report describes clinical findings emphasising dental phenotype in five, molecularly confirmed, female cases from two Czech families. CASE PRESENTATION: Dental examinations were carried out. An orthopantomogram was taken in three patients, and all patients' intraoral cavities and teeth were photographed. Exome sequencing was performed in both probands. Results were validated by Sanger DNA sequencing which was also used to follow segregation of the variants in first-degree relatives. Dental abnormalities and congenital cataracts were present in all five cases, whilst other signs were variable and included facial dysmorphism, microphthalmia, and cardiac and skeletal abnormalities. Two individuals had cleft lip and/or cleft palate. Radiculomegaly occurred in three patients with permanent teeth and was diagnosed on orthopantomograms. Two patients had agenesis of permanent teeth. Malocclusion was also present in two patients due to crowding and a Class III malocclusion and mandibular overjet. De novo novel pathogenic variants in the BCOR gene were identified; c.2382del p.(Lys795Argfs*12) and c.3914dup p.(Gln1306Alafs*20) and co-segregated with the disease in each family. CONCLUSIONS: The OFCD syndrome has a unique dental phenotype and dentists should be aware of signs of this ultra-rare genetic disorder. All patients with congenital cataracts and dental abnormalities, including those without a family history, should be referred for genetic testing and indicated to specialised dental care.

[Clinical and genetic characterization of three families with Nance-Horan syndrome caused by NHS gene mutations].

Objective: To explore the clinical phenotypes and pathogenic gene variation characteristics of three Chinese Han ethnic families affected by Nance-Horan syndrome, a rare X-linked genetic disorder. Methods: A pedigree investigation study was conducted at the First Affiliated Hospital of Zhengzhou University, collecting clinical data from three Chinese Han families with Nance-Horan syndrome between February 2009 and September 2018. Detailed family histories, comprehensive ophthalmological and systemic examinations were documented. Pedigree charts were created, and genetic inheritance patterns were analyzed to preliminarily diagnose the probands and other affected individuals. Genomic DNA was extracted from peripheral blood samples of family members, and next-generation sequencing was used to screen for target gene variations, which were confirmed by Sanger sequencing. Pathogenicity of the genetic variants and their impact on three-dimensional protein structure were analyzed using MutationTaster and computer-aided protein modeling. Results: In Family 1, there are 5 patients, including 4 females (aged 42, 37, 9 and 7) and 1 males (aged 12). In Family 2, there are 5 patients, including 3 females (aged 54, 32 and 16) and 2 males (aged 26 and 9). In Family 3, there are 8 patients, including 5 females (aged 69, 42, 37, 35 and 14) and 3 males (aged 10, 7 and 4). All probands in the three families exhibited nuclear cataracts with typical congenital hereditary cataract features, but no noticeable abnormalities in facial appearance or teeth. Next-generation sequencing identified new variation sites in the NHS gene, specifically c.2519_2520del, exon3del, and c.3847C>T. These variations included nonsense mutation p.(Ser840*), exon deletion p.(?), and nonsense mutation p.(Gln1283*). Combined clinical and genetic sequencing results confirmed X-linked Nance-Horan syndrome in all three families. Bioinformatics analysis indicated these variation sites were pathogenic and resulted in abnormal three-dimensional protein structures, likely being the main cause of Nance-Horan syndrome. Conclusion: The majority of patients from the three Nance-Horan syndrome families studied were affected by congenital hereditary cataracts characterized by nuclear opacities.The NHS gene variations c.2519_2520del, exon3del, and c.3847C>T are newly identified pathogenic sites in Nance-Horan syndrome, reported for the first time across three different families.

METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence.

BACKGROUND: The increasing incidence of diabetes mellitus has established diabetic cataracts (DC) as a significant worldwide public health issue. The mechanisms underlying DC remain unknown, and effective prevention and treatment strategies are lacking. Accordingly, we aimed to explore the role and mechanism behind N6-methyladenosine (m6A) in DC progression. METHODS: Methyltransferase-like 3 (METTL3), p21, Beclin1, LC3, and p62 expression levels were measured in human tissues. This study assessed total m6A levels and common m6A-regulated biomarkers in both in vitro and in vivo DC models. Autophagy flux was detected in vitro through Ad-mCherry-GFP-LC3B and Monodansylcadaverine (MDC) staining. Cellular senescence was assessed utilizing the senescence-associated ß-galactosidase (SA-ß-Gal) assay. Furthermore, the effect of METTL3 on SIRT1 mRNA modification was demonstrated, and its mechanism was elucidated using RT-qPCR, western blot, RNA stability assays, and RIP analysis. RESULTS: METTL3, p21, and p62 expression levels were elevated in lens epithelial cells (LECs) from DC patients, while Beclin1 and LC3 levels were reduced. Silencing METTL3-mediated m6A modifications restored high-glucose-induced autophagy inhibition and prevented premature senescence in LECs. Notably, SIRT1720 and Metformin significantly enhanced autophagosome generation and delayed cellular senescence. The m6A-reading protein YTHDF2 bound to m6A modifications, and YTHDF2 silencing significantly reduced METTL3-mediated SIRT1 inactivation. CONCLUSIONS: METTL3 induces senescence in DC by destabilizing SIRT1 mRNA in an m6A-YTHDF2-dependent manner. The METTL3-YTHDF2-SIRT1 axis is a key target and potential pathogenic mechanism in DC.

Identification of genes contributing to attenuation of rat model of galactose-induced cataract by pyruvate.

Cataracts are a disease that reduces vision due to opacity formation of the lens. Diabetic cataracts occur at young age and progress relatively quickly, so the development of effective treatment has been awaited. Several studies have shown that pyruvate inhibits oxidative stress and glycation of lens proteins, which contribute to onset of diabetic cataracts. However, detailed molecular mechanisms have not been revealed. In this study, we attempted to reduce galactose-induced opacity by pyruvate with rat ex vivo model. Rat lenses were extracted and cultured in galactose-containing medium to induce lens opacity. After opacity had developed, continued culturing with pyruvate in the medium resulted in a reduction of lens opacity. Subsequently, we conducted microarray analysis to investigate the genes that contribute to the therapeutic effect. We performed quantitative expression measurements using RT-qPCR for extracted genes that were upregulated in cataract-induced lenses and downregulated in pyruvate-treated lenses, resulting in the identification of 34 candidate genes. Functional analysis using the STRING database suggests that metallothionein-related factors (Mt1a, Mt1m, and Mt2A) and epithelial-mesenchymal transition-related factors (Acta2, Anxa1, Cd81, Mki67, Timp1, and Tyms) contribute to the therapeutic effect of cataracts.

Targeted Proteomics Profiling for Biomarker Discovery in Glaucoma Using the Olink Proteomics Platform.

Metabolic dysfunction plays a crucial role in the pathogenesis of glaucoma. In this study, we used Olink proteomics profiling to identify potential biomarkers for glaucoma. Aqueous humor samples were obtained from 44 cataract patients and 44 glaucoma patients. We identified 84 differentially expressed metabolic proteins between the glaucoma and the cataract group. Gene Ontology enrichment analysis highlighted the involvement of these proteins in ER-associated degradation pathway, regulation of interleukin-13 production, and DNA damage response pathway. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further revealed links to pathways, such as tyrosine and pyrimidine metabolism. Among these, ALDH1A1 emerged as a candidate with a significant diagnostic potential for glaucoma. ALDH1A1 also exhibited a prominent role in the protein-protein interaction network. Elevated levels of ALDH1A1 in the aqueous humor of glaucoma patients were confirmed both in clinical samples and in an ischemia/reperfusion model. Functional assays confirmed that elevated ALDH1A1 induced retinal ganglion cell (RGC) apoptosis in vitro and demonstrated its pro-apoptotic role in RGCs in vivo. Collectively, these findings not only underscore the significance of ALDH1A1 in glaucoma but also provide valuable insights into clinical decision-making and therapeutic strategies.

Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts.

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this "cataract-reversing" phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress-related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.

Altered Cell Clusters and Upregulated Aqp1 in Connexin 50 Knockout Lens Epithelium.

Purpose: To characterize the heterogeneity and cell clusters of postnatal lens epithelial cells (LECs) and to investigate the downstream targets of connexin 50 (Cx50) in the regulation of lens homeostasis and lens growth. To determine differentially expressed genes (DEGs) in the connexin 50 knockout (Cx50KO) lens epithelial cells that shed light on novel mechanism underlying the cataract and small size of the Cx50KO lenses. Methods: Single-cell RNA sequencing (scRNA-seq) of lens epithelial cells isolated from one-month-old Cx50KO and wild-type (WT) mice were performed. Differentially expressed genes were identified, and selected DEGs were further studied by quantitative real-time PCR (RT-qPCR) analysis and Western blot analysis. Results: The expression profiles of several thousand genes were identified by scRNA-seq data analysis. In comparison to the WT control, many DEGs were identified in the Cx50KO lens epithelial cells, including growth regulating transcriptional factors and genes encoding water channels. Significantly upregulated aquaporin 1 (Aqp1) gene expression was confirmed by RT-qPCR, and upregulated AQP1 protein expression was confirmed by Western blot analysis and immunostaining both in vivo and in vitro. Conclusions: Lens epithelial cells exhibit an intrinsic heterogeneity of different cell clusters in regulating lens homeostasis and lens growth. Upregulated Aqp1 in Cx50KO lens epithelial cells suggests that both connexin 50 and AQP1 likely play important roles in regulating water homeostasis in lens epithelial cells.

Alterations in anterior lens capsule structure and LTBP-2 expression in primary angle-closure glaucoma.

OBJECTIVE: This study investigated the role of latent-transforming growth factor ß-binding protein 2 (LTBP-2) in primary angle-closure glaucoma (PACG) by analysing its expression and the ultrastructure of the anterior lens capsule in PACG patients with age-related cataract (ARC). METHODS: Tissue samples of the anterior lens capsule were collected from patients undergoing cataract phacoemulsification surgery. Patients in the experimental group were diagnosed with primary angle-closure (PAC) combined with ARC (PAC+ARC) and PACG combined with ARC (PACG+ARC). The control group consisted of patients with only ARC. The techniques used included scanning electron microscopy, real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), western blotting and immunofluorescence. RESULTS: Ultrastructural analysis revealed disordered connections in PAC+ARC, loose connections in PACG+ARC and well-ordered connections in ARC. RT-qPCR and western blotting showed significantly lower LTBP-2 mRNA and protein expression in PAC+ARC and PACG+ARC than in ARC, with PAC+ARC having the lowest levels. Immunofluorescence confirmed these findings, showing varying LTBP-2 fluorescence intensities across groups. CONCLUSION: The study identified ultrastructural changes in the anterior lens capsules in PACG accompanied by reduced LTBP-2 expression, especially in PAC+ARC patients. This suggests a potential role for LTBP-2 in PACG development, warranting further investigation.

Genotype-phenotype spectrum and correlation of PHARC Syndrome due to pathogenic ABHD12 variants.

BACKGROUND: A comprehensive understanding of the genetic basis of rare diseases and their regulatory mechanisms is essential for human molecular genetics. However, the genetic mutant spectrum of pathogenic genes within the Chinese population remains underrepresented. Here, we reported previously unreported functional ABHD12 variants in two Chinese families and explored the correlation between genetic polymorphisms and phenotypes linked to PHARC syndrome. METHODS: Participants with biallelic pathogenic ABHD12 variants were recruited from the Chinese Deafness Genetics Cohort. These participants underwent whole-genome sequencing. Subsequently, a comprehensive literature review was conducted. RESULTS: Two Han Chinese families were identified, one with a compound heterozygous variant and the other with a novel homozygous variant in ABHD12. Among 65 PHARC patients, including 62 from the literature and 3 from this study, approximately 90% (57 out of 63) exhibited hearing loss, 82% (50 out of 61) had cataracts, 82% (46 out of 56) presented with retinitis pigmentosa, 79% (42 out of 53) experienced polyneuropathy, and 63% (36 out of 57) displayed ataxia. Seventeen different patterns were observed in the five main phenotypes of PHARC syndrome. A total of 33 pathogenic variants were identified in the ABHD12. Compared with other genotypes, individuals with biallelic truncating variants showed a higher incidence of polyneuropathy (p = 0.006), but no statistically significant differences were observed in the incidence of hearing loss, ataxia, retinitis pigmentosa and cataracts. CONCLUSIONS: The diagnosis of PHARC syndrome is challenging because of its genetic heterogeneity. Therefore, exploring novel variants and establishing genotype-phenotype correlations can significantly enhance gene diagnosis and genetic counseling for this complex disease.

Clinicogenetic characterization of cerebrotendinous xanthomatosis in Brazil.

There are few cerebrotendineous xanthomatosis (CTX) case series and observational studies including a significant number of Latin American patients. We describe a multicenter Brazilian cohort of patients with CTX highlighting their clinical phenotype, recurrent variants and assessing possible genotype-phenotype correlations. We analyzed data from all patients with clinical and molecular or biochemical diagnosis of CTX regularly followed at six genetics reference centers in Brazil between March 2020 and August 2023. We evaluated 38 CTX patients from 26 families, originating from 4 different geographical regions in Brazil. Genetic analysis identified 13 variants in the CYP27A1 gene within our population, including 3 variants that had not been previously described. The most frequent initial symptom of CTX in Brazil was cataract (27%), followed by xanthomas (24%), chronic diarrhea (13.5%), and developmental delay (13.5%). We observed that the median age at loss of ambulation correlates with the age of onset of neurological symptoms, with an average interval of 10 years (interquartile range 6.9 to 11 years). This study represents the largest CTX case series ever reported in South America. We describe phenotypic characteristics and report three new pathogenic or likely pathogenic variants.