Identification of Novel FZD4 Mutations in Familial Exudative Vitreoretinopathy and Investigating the Pathogenic Mechanisms of FZD4 Mutations.

Purpose: The purpose of this study is to report five novel FZD4 mutations identified in familial exudative vitreoretinopathy (FEVR) and to analyze and summarize the pathogenic mechanisms of 34 of 96 reported missense mutations in FZD4. Methods: Five probands diagnosed with FEVR and their family members were enrolled in the study. Ocular examinations and targeted gene panel sequencing were conducted on all participants. Plasmids, each carrying 29 previously reported FZD4 missense mutations and five novel mutations, were constructed based on the selection of mutations from each domain of FZD4. These plasmids were used to investigate the effects of mutations on protein expression levels, Norrin/ß-catenin activation capacity, membrane localization, norrin binding ability, and DVL2 recruitment ability in HEK293T, HEK293STF, and HeLa cells. Results: All five novel mutations (S91F, V103E, C145S, E160K, C377F) responsible for FEVR were found to compromise Norrin/ß-catenin activation of FZD4 protein. After reviewing a total of 34 reported missense mutations, we categorized all mutations based on their functional changes: signal peptide mutations, cysteine mutations affecting disulfide bonds, extracellular domain mutations influencing norrin binding, transmembrane domain (TM) 1 and TM7 mutations impacting membrane localization, and intracellular domain mutations affecting DVL2 recruitment. Conclusions: We expanded the spectrum of FZD4 mutations relevant to FEVR and experimentally demonstrated that missense mutations in FZD4 can be classified into five categories based on different functional changes.

Investigating the Impact of Dimer Interface Mutations on Norrin's Secretion and Norrin/ß-Catenin Pathway Activation.

Purpose: This study aimed to investigate the impact of 21 NDP mutations located at the dimer interface, focusing on their potential effects on protein assembly, secretion efficiency, and activation of the Norrin/ß-catenin signaling pathway. Methods: The expression level, secretion efficiency, and protein assembly of mutations were analyzed using Western blot. The Norrin/ß-catenin signaling pathway activation ability after overexpression of mutants or supernatant incubation of mutant proteins was tested in HEK293STF cells. The mutant norrin and wild-type (WT) FZD4 were overexpressed in HeLa cells to observe their co-localization. Immunofluorescence staining was conducted in HeLa cells to analyze the subcellular localization of Norrin and the Retention Using Selective Hook (RUSH) assay was used to dynamically observe the secretion process of WT and mutant Norrin. Results: Four mutants (A63S, E66K, H68P, and L103Q) exhibited no significant differences from WT in all evaluations. The other 17 mutants presented abnormalities, including inadequate protein assembly, reduced secretion, inability to bind to FZD4 on the cell membrane, and decreased capacity to activate Norrin/ß-catenin signaling pathway. The RUSH assay revealed the delay in endoplasmic reticulum (ER) exit and impairment of Golgi transport. Conclusions: Mutations at the Norrin dimer interface may lead to abnormal protein assembly, inability to bind to FZD4, and decreased secretion, thus contributing to compromised Norrin/ß-catenin signaling. Our results shed light on the pathogenic mechanisms behind a significant proportion of NDP gene mutations in familial exudative vitreoretinopathy (FEVR) or Norrie disease.

Intra-Anterior Chamber Injection of Ranibizumab in Advanced Pediatric Vitreoretinal Diseases.

Importance: Anti-vascular endothelial growth factor (VEGF) treatment through intravitreal or subretinal administrations has been proven effective for VEGF-driven pediatric vitreoretinal diseases but are not feasible for advanced cases, such as shallow traction retinal detachments or peripheral circumferential retinal detachments which adhere to the lens. Intra-anterior chamber injection (IAcI) of anti-VEGF may be a viable alternative in such cases but needs evaluation. Objective: To investigate the effects and safety of IAcI of anti-VEGF to treat VEGF-driven pediatric vitreoretinal diseases. Design, Setting, and Participants: This was a retrospective observational case series study conducted at Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine in China. The study included 14 eyes of 13 children diagnosed with vitreoretinal disease exhibiting elevated vascular activity between January and August 2023. Intervention: IAcI with ranibizumab. Main Outcomes and Measures: Retinal vascular abnormalities, vitreous hemorrhage resolution, and complications 1 month and 3 months after injection. Results: Of 13 patients included in this study, 12 were male. The mean age was 4.6 years (range, 1 month to 9 years). Six patients were diagnosed with familial exudative vitreoretinopathy, 4 with morning glory syndrome, 1 with retinopathy of prematurity, and 2 with chronic retinal detachments of unknown causes. At 1-month postoperative follow-up, vascular activity had decreased in 14 of 14 eyes. At 3-month follow-up, vascular activity had resolved in 7 of 14 eyes, persisted in 6 of 14 eyes, and reactivated in 1 of 14 eyes. On final observation, no complications were reported. Conclusions and Relevance: These findings support the possibility of treatment using IAcI with ranibizumab to decrease retinal vascular abnormalities in familial exudative vitreoretinopathy or retinopathy of prematurity or related conditions, but further studies are needed to understand more precise benefits and risks. This approach might be considered in cases where intravitreal or subretinal injection are not feasible, recognizing the limitations of these findings and that longer-term outcomes still need to be monitored.

Serum amyloid A aggravates endotoxin-induced ocular inflammation through the regulation of retinal microglial activation.

Serum amyloid A (SAA) are major acute-phase response proteins which actively participate in many inflammatory diseases. This study was designed to explore the function of SAA in acute ocular inflammation and the underlying mechanism. We found that SAA3 was upregulated in endotoxin-induced uveitis (EIU) mouse model, and it was primarily expressed in microglia. Recombinant SAA protein augmented intraocular inflammation in EIU, while the inhibition of Saa3 by siRNA effectively alleviated the inflammatory responses and rescued the retina from EIU-induced structural and functional damage. Further study showed that the recombinant SAA protein activated microglia, causing characteristic morphological changes and driving them further to pro-inflammatory status. The downregulation of Saa3 halted the amoeboid change of microglia, reduced the secretion of pro-inflammatory factors, and increased the expression of tissue-reparative genes. SAA3 also regulated the autophagic activity of microglial cells. Finally, we showed that the above effect of SAA on microglial cells was at least partially mediated through the expression and signaling of Toll-like receptor 4 (TLR4). Collectively, our study suggested that microglial cell-expressed SAA could be a potential target in treating acute ocular inflammation.

Description and surgical management of epiretinal membrane due to combined hamartoma of the retina and retinal pigment epithelium.

Purpose: To outline the characteristics of Combined Hamartoma of the Retina and Retinal Pigmentation Epithelium (CHRRPE) and provide a comprehensive overview of surgical management of epiretinal membrane (ERM) caused by CHRRPE. Main text: CHRRPE is a rare ocular tumor. It clinically mimics other diseases such as retinoblastoma and choroidal melanoma. The present study reviewed the multimodal imaging of CHRRPE, highlighted the multimodal imaging modalities which are useful for revealing the unique features of CHRRPE and hence allowing physicians to confirm the diagnosis.Although most of CHRRPEs are benign harmatoma, progressive visual loss may occur because of the traction of the tumor and other complications. It is treated through surgical removal of the ERM caused by CHRRPE to free retina from the traction. Currently, there is no consensus on the surgical management of CHRRPE. Therefore, the current review was designed to explore the surgical management of ERM caused by CHRRPE and hence provide updated data on this subject. Conclusions: Multimodal imaging technologies, especially optical coherence tomography (OCT), significantly contributes to the diagnosis of CHRRPE and visual prognosis. Surgical management of CHRRPE through removal of ERM is beneficial in patients with worsening VA which is secondary to ERM which is associated with CHRRPE. However, the strategy is limited to patients with long-standing poor vision. However, earlier surgical therapy and subsequent postoperative amblyopia therapy can be explored for children of amblyogenic age.

Administration Method and Potential Efficacy of Hyaluronidase for Hyaluronic Acid Filler-Related Vision Loss: A Systematic Review.

BACKGROUND: With the global increase in the use of injectable fillers, more cases with serious adverse events such vision loss are being reported. This article aims to review the cases of hyaluronic acid (HA) filler-related vision loss and to discuss the potential efficacy of hyaluronidase (HYASE) treatment via different given methods. METHODS: A total of 29 articles presenting 144 cases of HA filler-related vision loss were included in this study. RESULTS: Most cases of HA filler-related vision impairment were reported from China, followed by Korea. The majority of cases were seen in women. The nose, forehead and glabella were the most commonly injection sites. All cases had vision impairment and nearly all cases were unilateral with immediate onset of visual signs and symptoms. Ophthalmic artery occlusion (OAO) and central retinal artery occlusion (CRAO) were the two most commonly involved arterial obstruction patterns featured with a very poor prognosis followed by branch retinal artery occlusion (BRAO), the most favorable involved arterial pattern for a better prognosis. HYASE given subcutaneously and intra-arterially helped with visual recovery to different degrees, while retrobulbar HYASE seemed to be less helpful. CONCLUSION: Complications after HA-based filler injection are extremely rare but can cause disastrous visual impairment. HYASE given subcutaneously and intra-arterially helped with visual recovery to different extents, and the efficacy might be reinforced when performed together, while retrobulbar HYASE seemed to be less helpful. However, to accurately access the efficacy of HYASE via different administration methods, further randomized controlled trials are needed. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Two-step widefield fundus fluorescein angiography-assisted laser photocoagulation in pediatric retinal vasculopathy: A pilot study.

Purpose: To introduce the procedures of two-step fundus fluorescein angiography (FFA) and evaluate its utility in the management of pediatric retinal vasculopathy. Materials and methods: In this retrospective study, medical records of 12 patients who received two-step FFA were studied. The two-step FFA consisted of step 1 [low-dose (LD)] FFA at an intravenous dose of 1.5 mg/kg fluorescein, followed by step 2 [reduced dose (RD)] FFA at a dose of 6.2 mg/kg fluorescein. Demographic data, including age, gender, diagnosis, weight, gestational age, birth weight, and weight on the examination day were taken, were collected. The results of two-step FFA and treatment were recorded. Results: A total of 20 eyes were studied. The top 5 common FFA changes in RD-FFA included peripheral avascular zone (15 eyes), fluorescein leakage (10 eyes), supernumerous vascular branching (10 eyes), neovascularization (NV) (8 eyes), and absence of the foveal avascular zone (6 eyes). LD-FFA was efficient to show all the NV without severe vitreous dye in 8/8 (100.0%) eyes with NV, partial peripheral avascular zone in 11/15 (73.3%) eyes, while RD-FFA always offered more information in all the eyes. Thirteen eyes had laser photocoagulation under the guidance of LD-FFA. In 4 (30.8%) eyes, RD-FFA revealed more lesions and an immediate relaser was performed. Laser photocoagulation was successfully performed in all the 13 eyes in one session without being rearranged. After a median follow-up of 28.1 months, all the eyes were in a stable status. Conclusion: Step-one LD-FFA acted as a pre-FFA to show the NV, and step-two RD-FFA acted as a double-check. The modified strategy may be a helpful clinical adjuvant in the laser photocoagulation of pediatric retinal disorders, especially for young ophthalmologists.

Integrated analysis of multiple bioinformatics studies to identify microRNA-target gene-transcription factor regulatory networks in retinoblastoma.

Background: In children, retinoblastoma (RB) is one of the most common primary malignant ocular tumors and has a poor prognosis and high mortality. To understand the molecular mechanisms of RB, we identified microRNAs (miRNAs), key genes and transcription factors (TFs) using bioinformatics analysis to build potential miRNA-gene-TF networks. Methods: We collected three gene expression profiles and one miRNA expression profile from the Gene Expression Omnibus (GEO) database. We used the limma R package to identify overlapping differentially expressed genes (DEGs) and differentially expressed miRNAs in RB tissues compared to noncancer tissues. The robust rank aggregation (RRA) method was implemented to identify key genes among the DEGs. Then, miRNA-key gene-TF networks were built using the online tools TransmiR and miRTarBase. Next, we used RT-qPCR to confirm the results. Results: We identified 180 DEGs in RB tissues compared to nontumor tissues using integrative analysis, among which 109 genes were upregulated and 71 were downregulated. Gene ontology (GO) analysis revealed that these DEGs were primarily involved with chromosome segregation, condensed chromosome and DNA replication origin binding. The most highly enriched pathways obtained in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were cell cycle, DNA replication, homologous recombination, P53 signaling pathway and pyrimidine metabolism. Furthermore, two key differentially expressed miRNAs (DEMs) were also established: let-7a and let-7b. Finally, the potential regulatory networks of miRNA-target gene-TFs were examined. Conclusions: This study identified key genes and built miRNA-target gene-TF regulatory networks in RB, which will deepen our understanding of the molecular mechanisms involved in the development of RB. These key genes and miRNAs may be potential targets and biomarkers for RB diagnosis and therapy.

A Novel Subfoveal Perfluorocarbon Liquid Removal Technique Combining a 25-Gauge Retrobulbar Needle With a Built-in 30-Gauge Needle.

Introduction: To report a novel combining a 25-gauge retrobulbar needle with a built-in 30-gauge needle surgical technique for subfoveal perfluorocarbon liquid (PFCL) removal. Materials and Methods: Fourteen eyes of 14 patients who underwent subfoveal PFCL removal with a 25-gauge retrobulbar needle combined with a built-in 30-gauge needle were studied. The 30-gauge needle was inserted into the 25-gauge retrobulbar needle. The bent tip of the built-in 30-gauge needle was used to create a 30-gauge retinotomy at the farthest edge of the subfoveal PFCL droplet. Then, a flute cannula was used to aspirate the PFCL through the previously created retinotomy. The best-corrected visual acuity (BCVA) was determined, previous surgical history and post-operative complications were recorded. Results: Fourteen cases were analyzed. Most eyes (92.85%) showed an improvement in BCVA after surgery. The mean change in the BCVA was -0.7 ± 0.72 logarithm of the minimum angle of resolution (logMAR) units (p = 0.006). Post-operative complications included a self-healing macular hole in one eye and vitreous hemorrhage in one eye. Post-operative optical coherence tomography confirmed removal of the subfoveal PFCL with restoration of the macular fovea. Conclusion: Combining a 25-gauge retrobulbar needle with a built-in 30-gauge needle to remove subfoveal PFCL is easy to perform and carries little potential risk of subretinal impairment. This method also provides relatively good macular contour with functional improvement.

A novel frameshift variant in the TSPAN12 gene causes autosomal dominant FEVR.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is an inherited blinding eye disease with abnormal retinal vascular development. We aim to broaden the variant spectrum of FEVR and provide a basis for molecular diagnosis and genetic consultation. METHODS: We recruited five FEVR patients from one large Chinese family. Whole-exome sequencing (WES) and Sanger sequencing were applied to sequence, analyze, and verify variants on genomic DNA samples. Immunocytochemistry, western blot, qPCR, and luciferase assay were performed to test the influence of the variant on the protein expression and activity of the Norrin/ß-catenin pathway. RESULTS: We identified a novel heterozygous frameshift variant c.533dupC (p.D179Rfs*6) in Tetraspanin 12 (TSPAN12) gene that is related to FEVR. This variant caused degradation of the entire TSPAN12 protein, which failed to activate Norrin/ß-catenin signaling, possibly causing FEVR. CONCLUSION: Our study revealed a novel frameshift variant D179Rfs*6 in TSPAN12 that is inherited in an autosomal dominant manner. We found that D179Rfs*6 caused a failure to activate Norrin/ß-catenin signaling. This finding broadens the variant spectrum of TSPAN12 and provides invaluable information for the molecular diagnosis of FEVR.