Molecular Vision最新文献

Purpose: Biallelic variants in the retinal pigment epithelium-specific 65-kDa protein (RPE65) gene are linked to several inherited retinal diseases (IRDs), including Leber congenital amaurosis (LCA), early-onset severe retinal dystrophy (EOSRD), and retinitis pigmentosa (RP). This study screened patients from a tertiary center in Brazil with IRDs for RPE65 variants to characterize the associated phenotypes.

Methods: LCA, EOSRD, and RP diagnoses were based on predefined clinical criteria. Patients underwent comprehensive clinical evaluations and retinal imaging. Genomic DNA was analyzed using a next-generation sequencing panel for IRDs, covering 238 genes.

Results: RPE65 variants were identified in seven of the 68 patients screened. Of these, three were homozygous, and four were compound heterozygous for the identified mutant alleles. A total of six variants were detected, of which one was novel. The p.Leu341Ser (c.1022T>C) mutation was the most prevalent, being found in four of seven patients. Visual loss onset ranged from birth to the third decade of life. A consistent clinical feature observed in all patients was some degree of pigmentary change upon peripheral retinal examination.

Conclusions: RPE65 variants were found in 10.3% of cases in this series, associated with LCA, EOSRD, and RP. These variants were consistently linked with pigmentary changes in the peripheral retina and exhibited variable manifestations regarding arteriolar attenuation, disc pallor, and macular appearance. In this series, the prevalence of the p.Leu341Ser (c.1022T>C) mutation was 57%.

Objective: Retinopathy of prematurity (ROP) is a pathological condition characterized by abnormal proliferation of retinal vessels and it represents the primary cause of visual impairment in preterm infants. There is increasing backing for the involvement of genetic factors in the onset of ROP.

Methods: A prospective cohort study assessed the allele frequency and genotype distribution of gene polymorphisms in angiogenesis, inflammation and oxygen-sensing pathways in preterm infants with severe ROP. The role of genetic polymorphism in ROP development was investigated using next-generation sequencing (NGS) combined with candidate genes and data mining methods.

Results: A total of 47 confirmed severe ROP cases and gestational age, birthweight and days of oxygen therapy plus 35 similar control infants were enrolled in this study. In the initial hypothesis-generating survey, we selected a p value of 0.01 to minimize false positives while retaining true positives. Using this criterion, we identified 19 single-nucleotide polymorphisms across 11 genes that were associated with the occurrence of ROP (ZNF717, IHH, SEC22B, IGSF3, HYDIN), GGT1, FRG1, CDC27, LRRC37A3, CTAGE4 and ADAMTS7; all p<0.001). Compared with the control group, 62 single-nucleotide polymorphisms in 19 candidate genes (VEGF, EPO, EPAS-1, HIF1A, RUNX1, ESR1, CFH, PDGFB, JAK, STAT, IGF-1, IGFBP2, GPX4, TLR4, ROS1, CYP, TP53BP1, NOS3, TNF) representing angiogenic, inflammation, oxygen-sensing pathways and proliferative retinopathic diseases were found to be associated with the development of severe ROP (all p<0.01).

Conclusions: Using NGS gene analysis suggests that genetic risk factors may play an important role in susceptibility to the development of ROP.

Purpose: To explore the genetic variants of 14 keratoconus trios containing subclinical parents.

Methods: Trio-based whole-exome sequencing was performed in 14 keratoconus trios containing subclinical parents. The variants identified in candidate genes of keratoconus were analyzed by multiple bioinformatics tools.

Results: We identified 12 variants in 10 candidate genes of keratoconus (COL5A1, TGFBI, CAST, MPDZ, WNT10A, MYOF, ERMP1, MAP3K19, COL1A1, and WNT16). All variants were novel, not previously reported, and defined as uncertain significance according to the American College of Medical Genetics and Genomics guidelines. All variants were heterozygous and autosomal dominant cosegregated in keratoconus families.

Conclusions: We found that the candidate variants identified in clinically diagnosed patients and their subclinical parents may cause keratoconus through an autosomal dominant inheritance pattern, with different variable expressivity. This study indicates that genetic testing may play an important role in identifying patients with latent keratoconus and high-risk individuals for corneal ectasia after refractive surgery.

Purpose: Serum pro-brain natriuretic peptide (BNP) is a 108-amino-acid prohormone that inhibits vascular endothelial growth factor (VEGF) secretion, protecting pericytes from cell death and decreasing retinal vascularization. The purpose of this study was to investigate the correlation of serum pro-BNP with optical coherence tomography (OCT) indices in diabetic retinopathy.

Methods: This cross-sectional study investigated 96 consecutive subjects aged between 40 and 65 years: controls n = 24, no diabetic retinopathy (NoDR) n = 24, non-proliferative diabetic retinopathy (NPDR) n = 24, and proliferative diabetic retinopathy (PDR) n = 24. Same-day analysis of blood samples for serum pro-BNP levels was performed and spectral-domain OCT (SD-OCT) was used to measure the following OCT indices: OCT angiography (OCTA) superficial vessel density (SVD), deep vessel density (DVD), and foveal avascular zone (FAZ); OCT retinal nerve fiber layer (RNFL); and OCT ganglion cell analysis (GCA).

Results: The mean serum pro-BNP levels for the control, NoDR, NPDR, and PDR groups were 14.07 ± 11.51, 27.35 ± 11.81, 280.44 ± 106.13, and 122.33 ± 43.66 pg/ml, respectively. The mean values of the various OCT parameters correlated with serum pro-BNP were OCTA SVD (r = $-$ 0.360), OCTA DVD (r = 0.408), OCTA FAZ (r = 0.475), OCT RNFL (r = $-$ 0.215) and OCT GCA (r = $-$ 0.285; p<0.001).

Conclusions: The serum pro-BNP levels were higher in the NPDR group than in the NoDR group and much lower in the PDR group than in the NPDR group, reflecting a lowering of the protective barrier. These results correlated with the changes in various OCT indices.

Purpose: Intravitreal injection of adeno-associated virus (AAV) vectors is a good approach for transducing retinal ganglion cells (RGCs) in mice. It allows for high transduction efficiency and is relatively specific to RGCs. To deliver vectors, most studies use a transscleral approach that can have potentially negative effects, causing damage to the lens or retina. We optimized the intravitreal injection method using a transpupillary approach to minimize ocular damage and efficiently transfect RGCs.

Methods: C57BL/6J mice were anesthetized, and their irises were dilated. The eyeball was held with forceps while a small, full-thickness incision was made halfway between the center and periphery of the cornea. Using a bent 35-gauge blunt needle, the tip was navigated through the incision across the anterior chamber to reach the distal aspect of the pupil. The needle was inserted through the pupil, swept around the lens, and entered the vitreous, delivering expression vectors containing cytomegalovirus (CMV) promoter-driving green fluorescent protein (AAV-CMV-GFP) into the vitreous chamber. Fourteen days after injection, live fluorescent fundus images were taken, followed by immunostaining for GFP.

Results: With the improved injection technique, the lens remained clear and undamaged. Fundus imaging and GFP staining showed that over 90% of the mouse retinas sustained no visible damage. Retinas injected via the transpupillary approach also exhibited GFP transduction throughout the ganglion cell layer.

Conclusions: Transpupillary intravitreal injection reduces the potential risk compared to the transscleral approach, offering a promising and efficient method for delivering reporter genes to RGCs and ensuring high levels of gene expression without damage to the lens or retina.

Purpose: To date, the assessment of lens epithelial cell viability has been proposed only in cell cultures or isolated capsule models. This study aimed to develop a method for quantifying the viability of epithelial cells on whole ex vivo crystalline lenses by triple labeling Hoechst 33342, ethidium homodimer, and calcein-acetoxymethyl (HEC).

Methods: Two models of induced cell death were used to study the performance and potential applications of the technique. First, ten fresh pairs of six-month-old porcine lenses were retrieved. On one lens of each pair, an easily identifiable localized lesion was induced by a calibrated cryo-application, while the other remained intact. Both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Ten other pairs of lenses were used in the second experiment. On one lens of each pair, a diffuse epithelial lesion was induced by incubation in staurosporine (STS) solution (0.5 µM in CorneaMax) for 24 h at 37 °C. The other lens of each pair was incubated in CorneaMax solution without STS for 24 h at 37 °C. The day after, both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Images were acquired with a macroscope (macro zoom) and analyzed with ImageJ. Calcein-AM and ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed us to count cell nuclei per unit area. Viable epithelial cell density (vECD) was defined as the number of viable cells per unit area. Different strategies were developed to reduce background noise.

Results: There was no interfering lens autofluorescence for the exposure times used. The vECD median was 2,840 cells/mm² [10th-90th percentiles = 2,479-3,494] for cryo-injured lenses versus 3,364 cells/mm² [2,919-3,739] for healthy lenses (p = 0.002). The vECD median was 3,804 cells/mm² [10th-90th percentiles = 2,922-4,862] for lenses treated with STS versus 3,896 cells/mm² [3,169-4,980] for healthy lenses (p = 0.002).

Conclusions: Thanks to simple sample preparation, triple HEC staining allows fluorescence imaging of a large series of a whole lens to respect the architecture of the epithelium. It will be particularly useful for cytotoxicity studies of new therapies targeting the lens.

Purpose: This study identified the genetic causes of Axenfeld-Rieger syndrome (ARS) in a Chinese family and evaluated their clinical phenotype and clinical treatment.

Methods: We recruited a Chinese family with ARS. The proband presented with bilateral ectopic pupils, periumbilical redundancy, craniofacial abnormalities, and dental abnormalities after birth and was diagnosed with ARS. The symptoms were the same for her younger brother. Blood samples were collected from four family members: the proband, her brother, and her parents. Whole-genome sequencing (WGS) was performed to identify probable genetic variants in the proband. To confirm the identified variants, samples from the other family members were subjected to quantitative polymerase chain reaction (qPCR) and Sanger sequencing.

Results: Based on the results of WGS, we suspected a deletion region and an inversion region around the PITX2 gene. Through qPCR and Sanger sequencing, we identified a complex rearrangement involving a 6.15 Mb deletion on Chromosome 4, including the PITX2 coding region (Hg38; chr4:110617776-116769011), a 45.71 Mb inversion (Hg38; chr4:116769011-162481408), and a 14-bp deletion (Hg38; chr4:162481409-162481422). Interestingly, the father's copy number was normal, but Sanger sequencing revealed the same breakpoints. This indicated that the father is a balanced rearrangement carrier, and the children are unbalanced rearrangement carriers. While similar deletions and many breakpoints in this region have been reported, this specific rearrangement is novel.

Conclusions: Using WGS, qPCR, and Sanger, we found a complex genomic rearrangement with the deletion of PITX2 in a Chinese family with ARS. The clinical characteristics of the affected individuals were reported. The current findings broaden our understanding of the phenotype and variant spectrum associated with ARS caused by PITX2 deletion.

Purpose: This study aimed to investigate the association between proton pump inhibitors and dry eye disease (DED) among hospitalized SARS-CoV-2 patients.

Methods: We conducted a retrospective cohort study using electronic medical records from patients hospitalized for SARS-CoV-2 between April 2020 and December 2023. Eligible participants were aged over 18 years and hospitalized for SARS-CoV-2 without preexisting DED. Exclusions included ICU admissions, malignancies, recent ocular interventions, or chronic medications known to induce dry eye disease. Logistic regression adjusted for age, gender, and vaccination status evaluated associations between gastrointestinal (GI) medications and the subsequent development of dry eye disease within 6 months following hospital discharge.

Results: The age and gender distributions within the cohort were representative of the general SARS-CoV-2 infected population. Among 1165 patients, 167 (14.3%) developed dry eye disease post-hospitalization. Laxative use (lactulose and polyethylene glycol) correlated positively with dry eye disease (OR 1.939, p = 0.016; OR 2.094, p = 0.015, respectively). Metoclopramide treatment showed the strongest association (OR 13.413, p < 0.001), with over 50% incidence in affected patients. Conversely, omeprazole showed an inverse correlation with dry eye disease (OR 0.332, p < 0.001). Polypharmacy increased the odds of DED (odds ration [OR] 1.629, p = 0.015), while age, gender, and vaccination status did not significantly influence the outcomes.

Conclusions: Our findings emphasize significant correlations between GI medications and dry eye disease in SARS-CoV-2 survivors. Proton pump inhibitors may mitigate the risk of dry eye disease, contrasting with adverse effects linked to laxatives and metoclopramide. Vasoactive intestinal peptide (VIP), which links gut and lacrimal gland functions, is a strong candidate for the basis of the underlying pathophysiological mechanisms.

Purpose: During ocular trauma, excessive proliferation and transdifferentiation of corneal stromal fibroblasts cause haze/fibrosis in the cornea. Transforming growth factor β (TGFβ) plays a key role in corneal fibrosis through the Smad signaling pathway. The aberrant activity of TGFβ signaling during ocular trauma (viz. mechanical, infectious, chemical, or surgically altered TGFβ/Smad signaling) leads to regulating the predominant expression of myogenic proteins and the extracellular matrix (ECM). We sought to investigate the functional role of Smad3 in corneal wound repair and stromal ECM assembly using Smad3^+/+ wild-type and Smad3^-/- deficient mice.

Methods: Corneal injury was introduced with the topical application of an alkali-soaked 2-mm filter disc on the central cornea in the Smad3^+/+ (C57BL/6J) and Smad3^-/- (129-Smad3^tm1Par/J) mouse strains. Slit-lamp and stereo microscopy were used for clinical assessment and corneal haze grading in live animals. Hematoxylin and eosin and Masson's trichrome staining were used to study comparative morphology and collagen level alterations between the groups. Real-time qRT-PCR, western blot, and immunohistochemistry were used to measure changes in profibrotic genes at the mRNA and protein levels.

Results: Slit-lamp clinical exams and stereo microscopy detected notably less opaque cornea in the eyes of Smad3^-/- compared with Smad3^+/+ mice at 3 weeks (p<0.01) in live animals. Corneal tissue sections of Smad3^-/- mice showed significantly fewer α-smooth muscle actin-positive cells compared with those of the Smad3^+/+ animals (p<0.05). The corneas of the Smad3^-/- mice showed significantly lower mRNA levels of pro-fibrotic genes, α-smooth muscle actin, fibronectin, and collagen I (p<0.05, p<0.01, and p<0.001). In addition, the matrix metalloproteinase and tissue inhibitors of metalloproteinase levels were significantly increased (p<0.001) in the corneal tissue during alkali injury in both Smad3^+/+ wild-type and Smad3^-/- deficient mice.

Conclusions: The significant changes in profibrotic genes and stromal ECM proteins revealed a direct role of Smad3 in stromal ECM proteins and TGFβ/Smad-driven wound healing. Smad3 appears to be an attractive molecular target for limiting abnormal stroma wound healing to treat corneal fibrosis in vivo.

Background: Age-related macular degeneration (AMD) is a complex condition involving multiple factors. The condition is associated with numerous inflammatory indicators, including cytokines. Single-nucleotide polymorphisms in cytokine genes can also modify gene expression, perhaps contributing to the development of the disease. The objective of the present study was to examine the correlation among IL-6 SNPs (rs1800795, rs1800796, and rs1800797) and the serum levels of IL-6 in AMD patients treated at the Regional Institute of Ophthalmology of Pt. B.D. Sharma PGIMS, Rohtak (Haryana), India.

Methods: This case-control study included 131 patients diagnosed with AMD using precise ophthalmic examinations, such as slit lamp examination, fundoscopy, and ocular coherence tomography. To provide a basis for comparison, we also enlisted 100 healthy individuals as controls. Serum IL-6 protein levels were measured in both patients and controls using an enzyme-linked immunosorbent assay kit (ELISA). Genotyping IL-6 SNPs was performed using the PCR and DNA Sanger sequencing technique.

Results: IL-6 serum levels were considerably elevated in individuals with AMD compared to the control group (p < 0.05). Statistically significant differences were seen in the genotype frequencies of rs1800795 (p = 0.027) and rs1800797 (p = 0.0011) among the AMD patients and the healthy controls. Furthermore, strong correlations were observed between rs1800795 and the likelihood of developing AMD based on the heterozygous (OR = 2.04; p = 0.025), dominant (OR = 1.80; p = 0.035), and over-dominant models (OR = 2.10; p = 0.0094). Additionally, there were notable associations between rs1800797 and vulnerability to AMD through heterozygous (OR = 3.21; p = 0.009), dominant (OR = 2.74; p = 0.004), and over-dominant (OR = 3.11; p = 0.002) models. The rs1800795, rs1800796, and rs1800797 haplotypes C-G-A and G-G-A were linked to an elevated risk of AMD (p = 0.005, p = 0.024. respectively).

Conclusions: Our findings indicated a significant elevation in IL-6 serum levels among the AMD patient group compared to the control group. The interleukin-6 gene polymorphisms rs1800795 and rs1800797 were linked to an elevated risk of AMD in our study population.