Genetics in ophthalmology: molecular blueprints of retinoblastoma.

This review presents current knowledge on the molecular biology of retinoblastoma (RB). Retinoblastoma is an intraocular tumor with hereditary and sporadic forms. 8,000 new cases of this ocular malignancy of the developing retina are diagnosed each year worldwide. The major gene responsible for retinoblastoma is RB1, and it harbors a large spectrum of pathogenic variants. Tumorigenesis begins with mutations that cause RB1 biallelic inactivation preventing the production of functional pRB proteins. Depending on the type of mutation the penetrance of RB is different. However, in small percent of tumors additional genes may be required, such as MYCN, BCOR and CREBBP. Additionally, epigenetic changes contribute to the progression of retinoblastoma as well. Besides its role in the cell cycle, pRB plays many additional roles, it regulates the nucleosome structure, participates in apoptosis, DNA replication, cellular senescence, differentiation, DNA repair and angiogenesis. Notably, pRB has an important role as a modulator of chromatin remodeling. In recent years high-throughput techniques are becoming essential for credible biomarker identification and patient management improvement. In spite of remarkable advances in retinoblastoma therapy, primarily in high-income countries, our understanding of retinoblastoma and its specific genetics still needs further clarification in order to predict the course of this disease and improve therapy. One such approach is the tumor free DNA that can be obtained from the anterior segment of the eye and be useful in diagnostics and prognostics.

Modification of the Suprachoroidal Buckling Technique for the Treatment of Rhegmatogenous Retinal Detachment.

PURPOSE: To describe modification of the suprachoroidal buckling technique for the treatment of rhegmatogenous retinal detachment (RRD), which may improve the safety profile. METHODS: A single-surgeon foot-pedal-controlled automated suprachoroidal injection (SCI) of sodium hyaluronate 1%, namely ProVisc (Alcon Laboratories, Fort Worth, TX) was used for the treatment of RRD. MicroDose Injection Kit (MedOne Surgical, Sarasota, FL) including a connector and a 1-mL syringe, designed for subretinal injection, was used to adapt Constellation Vision System (Alcon Laboratories) console for SCI of ProVisc from the 1-mL syringe. RESULTS: This approach enables better surgeon control during SCI. Three highly myopic eyes of three patients with primary macula-on RRD and single superior peripheral retinal break were treated. Complete retinal reattachment was achieved in all eyes without complications. CONCLUSION: Injecting ProVisc under foot-pedal control provides a more precise and potentially safer suprachoroidal buckling technique compared with the manual technique with more variable injection speed and pressure.

Novel Variant IMPDH1 c.134A>G, p.(Tyr45Cys): Phenotype-Genotype Correlation Revealed Likely Benign Clinical Significance.

Pathogenic variants in IMPDH1 are associated with autosomal dominant retinitis pigmentosa 10 (RP10), and Leber congenital amaurosis 11. This case report of a 13-year-old girl with Down's syndrome and keratoglobus is aimed at linking the novel variant IMPDH1 c.134A>G, p.(Tyr45Cys), a variant of uncertain significance, to a clinical phenotype and to provide grounds for the objective assignment of its benign features. RP10 is characterized by the early onset and rapid progression of ocular symptoms, beginning with nyctalopia in childhood, accompanied by typical RP fundus changes. As evidenced via thorough clinical examination and testing, none of the RP10 characteristics were present in our patient. On the contrary, our patient who was heterozygous for IMPDH1 c.134A>G, p.(Tyr45Cys) showed no signs of peripheral retinal dystrophy, and did not manifest any disease characteristics typical of the IMPDH1 gene mutation. Consequently, we conclude that the variant did not contribute to the phenotype. According to standards and guidelines for the interpretation of sequence variants, IMPDH1 c.134A>G, p.(Tyr45Cys) revealed likely benign features.

RPE65 c.353G>A, p.(Arg118Lys): A Novel Point Mutation Associated with Retinitis Pigmentosa and Macular Atrophy

Precise genetic diagnosis in RPE65-mediated retinitis pigmentosa (RP) is necessary to establish eligibility for genetic treatment with voretigene neparvovec: a recombinant adeno-associated viral vector providing a functional RPE65 gene. This case report aims to report a novel RP-related point mutation RPE65 c.353G>A, p.(Arg118Lys), a variant of uncertain significance associated with a severe clinical presentation and the striking phenotypic feature of complete macular atrophy. We report the case of a 40-year-old male with inherited retinal dystrophy, all features typical for the RPE65-associated RP, and marked macular atrophy. Genetic testing identified that the patient was a compound heterozygote in trans form with two heterozygous variants: RPE65 c.499G>T, p.(Asp167Tyr) and RPE65 c.353G>A, p.(Arg118Lys). Furthermore, short-wavelength and near-infrared autofluorescence patterns exhibited deficiencies specific to mutations in the visual cycle genes. To the best of our knowledge, RPE65 c.353G>A, p.(Arg118Lys) is the first described point mutation on this locus, among all other reported insertional mutations, currently classified as likely benign and of uncertain significance. We concluded that this variant contributed to the pathological phenotype, demonstrating its significance clearly to be reclassified as likely pathogenic. This being the case, patients with this specific variant in homozygous or compound heterozygous form would be likely candidates for genetic treatment with voretigene neparvovec.

Macular perfusion normative data acquired with optical coherence tomography angiography in healthy four-year-old Caucasian children.

BACKGROUND: The purpose of this cross-sectional study involving healthy emmetropic four-year-old Caucasian children was to provide a macular perfusion normative database acquired with optical coherence tomography angiography (OCTA). One eye of each examinee underwent OCTA imaging. The following parameters were analyzed using AngioTool Image J software: vessels area (VA), vessels density (VD), total number of junctions (TNJ), junctions density (JD), total vessel length (TVL), average vessel length (AVL), total number of endpoints (TNEP), lacunarity (L), vessel diameter index (VDI), tortuosity (T) and foveal avascular zone (FAZ). Average central macular thickness (CMT) and average central macular volume (CMV) were measured. RESULT: Sixty-two eyes of 62 children of average age 50.4 ± 3.8 months were examined. VA, VD, and T increased from the inner towards the outer layers of the retina. The intermediate capillary plexus had the highest JD and TNEP and narrowest FAZ. Retinal sexual differentiation was supported with higher values of the retinal VA, VDI and TNEP, and chorioretinal VA, VDI and L in males. The choriocapillaris presented with the highest VD, AVL, and T and the lowest L and TNEP. CONCLUSION: The study provides the first detailed normative database of the macular vascular network in the youngest uniform cohort of emmetropic four-year-old children.

The CroMyop study: myopia progression in Croatian children and adolescents-a 15-year retrospective analysis.

Purpose: Myopia is a major global health issue, especially among children and adolescents. Understanding its traits and progression is vital for proper management and prevention. This study aimed to fill a gap in research by analyzing demographic and refractive data concerning myopia among children and adolescents in Croatia, with the goal of providing insights into myopia prevalence, progression rates, and associated risk factors within the Croatian population. Design: This retrospective study utilized a comprehensive dataset from pediatric ophthalmology clinics at the University Eye Department, University Hospital "Sveti Duh," Zagreb, Croatia. The dataset included electronic medical records spanning from January 2008 to July 2023, encompassing demographic and refractive data. Methods: Data analysis focused on individuals aged 4 to 18 years who were diagnosed with primary myopia and/or compound myopic astigmatism. Ophthalmic examinations, including visual acuity tests, cycloplegic refraction, and assessments for eye comorbidities, were conducted by experienced pediatric ophthalmologists. Statistical analysis, including t-tests, survival analysis, and logistic regression, was performed to assess myopia prevalence, progression rates, and associated factors. These analyses were adjusted for covariates such as age, parental myopia, and gender. Results: The study included 895 individuals, 51 premyopes, 813 low myopes, and 31 high myopes. The average age of diagnosis was 11.37 ± 3.59 years for premyopes, 11.18 ± 3.53 years for low myopes, and 11.44 ± 4.35 years for high myopes. The fastest progression occurred in 2021 and 2022, -0.5 ± 0.12 D/y for premyopes and - 0.45 ± 0.1 D/y for low myopes. Premyopic progression to low myopia was associated with age 7-9 years (HR 2.42, 1.53 to 3.21) and both parents being myopic (HR 920.27. 850.16 to 950.53). Low myopic individuals with both myopic parents displayed the fastest 11-24 months after first visit progression rates, -0.69 (-0.52 to -0.87) D/y, while the 7-9 age group demonstrated -0.36 (-0.24 to -0.45) D/y. Low myopes aged 7-9 years with baseline SE between -6 D and -4 D were more strongly associated with ≤ - 0.5 D progression (OR = 2.0, 95% CI -1.00 to 2.39). Conclusion: This study highlights the importance of environmental factors, genetics, and age in addressing myopia progression among Croatian youth, urging further research for effective local intervention strategies.

Vitreous hyper-reflective dots and the macular thickness after cataract surgery.

PURPOSE: To assess the association between vitreous hyper-reflective dots (VHD) and the macular thickness changes following uneventful phacoemulsification. METHODS: In this prospective cohort study optical coherence tomography (OCT) examinations were performed preoperatively and 1 week, 1 month and 3 months postoperatively in patients undergoing cataract surgery. OCT images were analyzed for retinal central subfield thickness (CST) and preretinal VHDs. Surgeries were recorded for the assessment of lens fragments in the space of Berger. RESULTS: 111 eyes of 97 patient were enrolled of whom 69 (62.2%) were female. VHDs were seen in 25 eyes (22.5%) at week 1; in 21 eyes (18.9%) at month 1 and in 3 eyes (2.7%) at month 3. In all eyes with VHDs retro-capsular lens fragments were visible immediately after phacoemulsification. The number of VHDs significantly decreased over the postoperative period. There was a moderate correlation between the number of VHDs and CST at 1 month (r = 0.426, p<0.001). In eyes with VHD the CST averaged 238.8±17.6 µm (214-266) at 1 week; 276.1±63.5 µm (231-481) at 1 month and 285.1±122.3 µm (227-785) at 3 months. In eyes with no detectable VHDs CST averaged 235.9±23.3 µm (192-311) at 1 week; 240.1±21.6 µm (200-288) at 1 month and 242.2±21.3 µm (205-289) at 3 months. Although the differences among the assessment points were relatively low, there was a significant difference in general (p<0.001, Friedman test). CONCLUSION: In conclusion, VHDs seem to cause macular thickening throughout the postoperative course. The origin of VHDs is still unknown; however, they presumably represent lens fragments that provoke subclinical inflammation.

Pathogenicity reclassification of the RPE65 c.1580A>G (p.His527Arg) - a case report.

BACKGROUND: It is of utmost importance to define the molecular diagnosis of patients with retinitis pigmentosa (RP) due to existing targeted therapeutic option: voretigene neparvovec.We provide clinical evidence for pathogenicity reclassification of variants of uncertain significance (VUSs) RPE65 c.1580A>G (p.His527Arg). MATERIALS AND METHODS: A case report of a 10-year-old boy with progressive vision loss. The patient manifested disease highly suggestive of RPE65 retinal dystrophy: nyctalopia, fairly good central vision, severely depressed full-field electroretinography responses and complete loss of peripheral fundus aut ofluorescence. RESULTS: Invitae Inherited Retinal Disorders Panel identified likely pathogenic mutation RPE65 c.499G>T (p.Asp167Tyr) and RPE65 c.1580A>G (p.His527Arg), variant of uncertain significance. Segregation analysis confirmed that these variants are in trans. CONCLUSIONS: We conclude that the variant RPE65 c.1580A>G (p.His527Arg) has contributed to the pathologic phenotype, demonstrating its significance clearly in the case presented, and should be reclassified according to the criteria of evidence as pathogenic. Therefore, patients with this specific variant in homozygous or compound heterozygous form would likely benefit from genetic treatment based on recombinant adeno-associated virus vector, providing a working RPE65 gene to act in place of a mutated RPE65 gene.

RPE65 c.393T>A, p.(Asn131Lys): Novel Sequence Variant Detected.

Background: Leber congenital amaurosis (LCA) is a monogenic, but genetically heterogenous disease, and at least 27 genes are implicated. This case report is aimed at providing evidence to link the novel variant RPE65 c.393T>A, p.(Asn131Lys), variant of uncertain significance (VUS), to clinical phenotype and to set the ground for objective assignment of pathogenicity confidence. Case Presentation. A case report of a female patient with LCA who manifested with nystagmus, night blindness, profound visual deficiency, and peripheral involvement of the retina consistent with RPE65 dystrophy. A thorough clinical examination, diagnostic evaluation, and genetic testing were performed. The patient was a compound heterozygote in trans form: RPE65 c.304G>T, p.(Glu102∗) pathogenic, and RPE65 c.393T>A, p.(Asn131Lys), VUS. The latter variant is absent in healthy controls and is considered harmful on in silico prediction. Conclusions: We conclude that RPE65 c.393T>A, p.(Asn131Lys) contributed to the pathologic phenotype, demonstrating its significance clearly in the case presented, and should be reclassified according to the criteria of evidence as likely pathogenic. This being the case, patients with this specific variant are likely candidates for genetic treatment.

A Retrospective Data Review Confirms That Topical Preservative-Free Hydrocortisone Improves Inflammation in Dry Eye Disease.

BACKGROUND: Inflammation is among the most important mechanisms in the pathogenesis of dry eye disease (DED), triggering the vicious circle of the disease. Reducing inflammation is an important target in dry eye disease treatment. Hydrocortisone is a low-potency corticosteroid with a low ocular penetration potential. AIM: To document the effect of topical preservative-free hydrocortisone 0.335% (PFH, Softacort®, Laboratoires Théa, France) on DED. METHODS: Retrospective data review of patients with mild to moderate DED, treated with PFH for 15 days. Clinical evaluations at Days 0 and 15 included the assessment of the central precorneal tear film thickness (CPTFT), fluorescein tear breakup time, Schirmer test, corneal grading staining (Oxford schema), ocular surface disease index (OSDI) spatial distribution of the precorneal tear film thickness, intraocular pressure (IOP) and local tolerance. RESULTS: Data from 13 women and 2 men were collected. Mean age±SD was 51±5 years for women and 53±4 years for men. Clinical signs and symptoms significantly (all p<0.05) improved after 15 days of treatment. A significant positive correlation between the percentage of change in left eye CPTFT and that in the contralateral eye CPTFT was observed (p=0.003) as well as for both eyes and the left eye FTBUT (p=0.03). For the percentage of change in OSDI, the only significant correlation was with the percentage of change in right eye and FTBUT (p=0.03). IOP remained unchanged. No adverse events were recorded. CONCLUSION: This retrospective data review confirms that topical PFH twice daily for 2 weeks significantly improves clinical signs and symptoms in patients with mild to moderate DED with no safety issues.

Expression patterns of Wnt signaling component, secreted frizzled­related protein 3 in astrocytoma and glioblastoma.

Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the cytoplasm an increased expression level of SFRP3 was identified in the high grade astrocytomas when compared with those of a low grade. This may suggest that SFRP3 acts as an agonist of Wnt signaling and promotes invasive behavior.

Wnt signaling transcription factors TCF-1 and LEF-1 are upregulated in malignant astrocytic brain tumors.

Since the discovery of the TCF/LEF family of transcription factors, their functions have been under intensive investigation in the area of cancer biology. The work presented in this paper focused on the changes in TCF-1 and LEF-1 expression levels in a set of astrocytic brain tumors. Protein expression was detected using immunohistochemistry and then evaluated by Ellipse software (ViDiTo, Slovakia). Statistical evaluations were performed with the SPSS statistical package, version 14.0 (SPSS Inc., Chicago, IL, USA). Strong TCF-1 and LEF-1 expression was observed in 51.6% and 71% of glioblastoma samples. Statistical analysis confirmed significant differences in protein expression levels associated to 3 important values, weak expression of TCF-1, weak expression of LEF-1 and strong expression of LEF-1. Analysis of variances performed on the total sample also indicated significant differences in the values of TCF-1 weak (F=2.804; p=0.045), LEF-1 weak (F=4.255; p=0.008) and LEF-1 strong (F=5.498; p=0.002) with regard to malignancy grade. Thus, glioblastomas were characterized by -in relative terms- the lowest values for weak expression of TCF-1 and LEF-1, combined with the highest values of LEF-1 strong expression. The F-ratios for two variables (LEF-1 strong and LEF-1 weak) indicated that differences between astrocytomas (II, III) and glioblastomas were statistically significant (p<0.02). Discriminant function analysis further showed that strong LEF-1 expression alone could discriminate between astrocytomas (II, III) and glioblastomas. Elevated TCF-1 and LEF-1 expression is characteristic of malignant gliomas. LEF-1, in particular, may serve as a potential marker for malignant transformation.