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.

PdmIRD: missense variants pathogenicity prediction for inherited retinal diseases in a disease-specific manner.

Accurate discrimination of pathogenic and nonpathogenic variation remains an enormous challenge in clinical genetic testing of inherited retinal diseases (IRDs) patients. Computational methods for predicting variant pathogenicity are the main solutions for this dilemma. The majority of the state-of-the-art variant pathogenicity prediction tools disregard the differences in characteristics among different genes and treat all types of mutations equally. Since missense variants are the most common type of variation in the coding region of the human genome, we developed a novel missense mutation pathogenicity prediction tool, named Prediction of Deleterious Missense Mutation for IRDs (PdmIRD) in this study. PdmIRD was tailored for IRDs-related genes and constructed with the conditional random forest model. Population frequencies and a newly available prediction tool were incorporated into PdmIRD to improve the performance of the model. The evaluation of PdmIRD demonstrated its superior performance over nonspecific tools (areas under the curves, 0.984 and 0.910) and an existing eye abnormalities-specific tool (areas under the curves, 0.975 and 0.891). We also demonstrated the submodel that used a smaller gene panel further slightly improved performance. Our study provides evidence that a disease-specific model can enhance the prediction of missense mutation pathogenicity, especially when new and important features are considered. Additionally, this study provides guidance for exploring the characteristics and functions of the mutated proteins in a greater number of Mendelian disorders.

Patient experiences and perceived value of genetic testing in inherited retinal diseases: a cross-sectional survey.

This study evaluated patient experiences with genetic testing for inherited retinal diseases (IRDs) and the association between underlying knowledge, testing outcomes, and the perceived value of the results. An online survey was distributed to adults with IRDs and parents/guardians of dependents with IRDs who had had genetic testing. Data included details of genetic testing, pre- and post- test perceptions, Decision Regret Scale, perceived value of results, and knowledge of gene therapy. Of 135 responses (85% from adults with IRDs), genetic testing was primarily conducted at no charge through public hospitals (49%) or in a research setting (30%). Key motivations for genetic testing were to confirm IRD diagnosis and to contribute towards research. Those who had received a genetic diagnosis (odds ratio: 6.71; p < 0.001) and those self-reported to have good knowledge of gene therapy (odds ratio: 2.69; p = 0.018) were more likely to have gained confidence in managing their clinical care. For over 80% of respondents, knowing the causative gene empowered them to learn more about their IRD and explore opportunities regarding clinical trials. Key genetic counselling information needs include resources for family communications, structured information provision, and ongoing genetic support, particularly in the context of emerging ocular therapies, to enhance consistency in information uptake.

Characterization of a novel heterozygous frameshift variant in NDP gene that causes familial exudative vitreoretinopathy in female patients.

Familial exudative vitreoretinopathy (FEVR) is a severe inherited disease characterized by defective retinal vascular development. With genetic and clinical heterogeneity, FEVR can be inherited in different patterns and characterized by phenotypes ranging from moderate visual defects to complete vision loss. This study was conducted to unravel the genetic and functional etiology of a 4-month-old female FEVR patient. Targeted gene panel and Sanger sequencing were utilized for genetic evaluation. Luciferase assays, western blot, quantitive real-time PCR, and immunocytochemistry were performed to verify the functional defects in the identified candidate variant. Here, we report a 4-month-old girl with bilateral retinal folds and peripheral avascularization, and identified a novel frameshift heterozygous variant c.37dup (p.Leu13ProfsTer13) in NDP. In vitro experiments revealed that the Leu13ProfsTer13 variant led to a prominent decrease in protein levels instead of mRNA levels, resulting in compromised Norrin/ß-catenin signaling activity. Human androgen receptor assay further revealed that a slight skewing of X chromosome inactivation could partially cause FEVR. Thus, the pathogenic mechanism by which heterozygous frameshift or nonsense variants in female carriers cause FEVR might largely result from a loss-of-function variant in one X chromosome allele and a slightly skewed X-inactivation. Further recruitment of more FEVR-affected females carrying NDP variants and genotype-phenotype correlation analysis can ultimately offer valuable information for the prognosis prediction of FEVR.

Allele-specific antisense oligonucleotides for the treatment of BEST1-related dominantly inherited retinal diseases: An in vitro model.

Retinal dystrophies are a common health problem worldwide that are currently incurable due to the inability of retinal cells to regenerate. Inherited retinal diseases (IRDs) are a diverse group of disorders characterized by progressive vision loss caused by photoreceptor cell dysfunction. The eye has always been an attractive organ for the development of novel therapies due to its independent access to the systemic pathway. Moreover, anti-sense oligonucleotides (ASOs), which facilitate manipulation of unwanted mRNAs via degradation or splicing, are undergoing rapid development and have been clinically deployed for the treatment of several diseases. The primary aim of this study was to establish a reliable in vitro model utilizing induced photoreceptor-like cells (PRCs) for assessing the efficacy and safety of ASOs targeting the BEST1 gene. Despite advances in gene therapy, effective treatments for a broad range of IRDs remain limited. An additional aim was to develop an in vitro model for evaluating RNA-based therapeutics, specifically ASOs, for the treatment in IRDs. Firstly, a cell culture model was established by induction of PRCs from dermal fibroblasts via direct programming. The induced PRCs were characterized at both the transcriptomic and protein level. Then, a common single nucleotide polymorphism (SNP) was identified in the BEST1 gene (rs1800007) for targeting with ASOs. ASOs were designed using the GapmeR strategy to target multiple alleles of this SNP, which is potentially suitable for a large proportion of the population. The efficacy and possible off-target effects of these ASOs were also analyzed in the induced PRC model. The findings show that the selected ASOs achieved allele-specific mRNA degradation with virtually no off-target effects on the global transcriptome profile, indicating their potential as safe and effective therapeutic agents. The presented in vitro model is a valuable platform for testing personalized IRD treatments and should inspire further research on RNA-based therapeutics. To the best of our knowledge this study is the first to test RNA-based therapeutics involving the use of ASOs in an induced PRC model. Based on the present findings, it will be possible to establish an ex vivo disease model using dermal fibroblast samples from affected individuals. In other words, the disease model and the ASOs that were successfully designed in this study can serve as a useful platform for the testing of personalized treatments for IRDs.

Optical coherence tomography in children with inherited retinal disease.

Recent advances have led to therapeutic options becoming available for people with inherited retinal disease. In particular, gene therapy has been shown to hold great promise for slowing vision loss from inherited retinal disease. Recent studies suggest that gene therapy is likely to be most effective when implemented early in the disease process, making consideration of paediatric populations important. It is therefore necessary to have a comprehensive understanding of retinal imaging in children with inherited retinal diseases, in order to monitor disease progression and to determine which early retinal biomarkers may be used as outcome measures in future clinical trials. In addition, as many optometrists will review children with an inherited retinal disease, an understanding of the expected imaging outcomes can improve clinical care. This review focuses on the most common imaging modality used in research assessment of paediatric inherited retinal diseases: optical coherence tomography. Optical coherence tomography findings can be used in both the clinical and research setting. In particular, the review discusses current knowledge of optical coherence tomography findings in eight paediatric inherited retinal diseases - Stargardt disease, Bests disease, Leber's congenital amaurosis, choroideremia, RPGR related retinitis pigmentosa, Usher syndrome, X-linked retinoschisis and, Batten disease.

Long-term follow-up of torpedo maculopathy: a case series and mini-review.

BACKGROUND: Torpedo maculopathy (TM) is a rare, congenital condition characterized by an oval-shaped, chorioretinal lesion in the temporal macula of unknown etiology. To our knowledge, the longest reported follow-up of TM is 5 years. Herein we report 10 years of follow-up on two patients with TM to further characterize the long-term natural history of the condition. CASE REPORTS: Two patients with torpedo maculopathy were examined at baseline and then again at 5 years and 10 years from baseline. Eyes were evaluated using color fundus photography, automated perimetry, fundus autofluorescence and spectral domain optical coherence tomography. Visual function of both patients remained stable throughout the observation period. In case 1, there was no evidence of change in lesion morphology over the 10 year observation period. Case 2 showed progression of cystic degeneration of the neurosensory retina within the torpedo lesion. Case 1 reported a history of supernumerary teeth and underwent gene sequence with deletion/duplication analyses of the APC gene but no clinically significant variants were detected. CONCLUSIONS: Our findings support the position that TM is a nonprogressive condition with long-term stability of visual function. Genetic analysis of case 1 failed to detect any association with Gardner syndrome.

Combining a prioritization strategy and functional studies nominates 5'UTR variants underlying inherited retinal disease.

BACKGROUND: 5' untranslated regions (5'UTRs) are essential modulators of protein translation. Predicting the impact of 5'UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5'UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). METHODS: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5'UTRs by different whole exome sequencing (WES) kits. The selected 5'UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5'UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. RESULTS: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5'UTR compared to that of their canonical isoform. Depending on the probe design, 3-20% of IRD genes have 5'UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5'UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. CONCLUSIONS: This study demonstrates the importance of 5'UTR variants implicated in IRDs and provides a systematic approach for 5'UTR annotation and validation that is applicable to other inherited diseases.

Non-vasogenic cystoid maculopathy in autosomal recessive bestrophinopathy: novel insights from NIR-FAF and OCTA imaging.

BACKGROUND: Autosomal Recessive Bestrophinopathy (ARB) is an inherited retinal disease caused by biallelic mutations in the BEST1 gene. Herein, we report the multimodal imaging findings of ARB presenting with cystoid maculopathy and investigate the short-term response to combined systemic and topical carbonic anhydrase inhibitors (CAIs). MATERIAL AND METHODS: An observational, prospective, case series on two siblings affected by ARB is presented. Patients underwent genetic testing and optical coherence tomography (OCT), blue-light fundus autofluorescence (BL-FAF), near-infrared fundus autofluorescence (NIR-FAF), fluorescein angiography (FA), MultiColor imaging, and OCT angiography (OCTA). RESULTS: Two male siblings, aged 22 and 16, affected by ARB resulting from c.598C>T, p.(Arg200*) and c.728C>A, p.(Ala243Glu) BEST1 compound heterozygous variants, presented with bilateral multifocal yellowish pigment deposits scattered through the posterior pole that corresponded to hyperautofluorescent deposits on BL-FAF. Vice versa, NIR-FAF mainly disclosed wide hypoautofluorescent areas in the macula. A cystoid maculopathy and shallow subretinal fluid were evident on structural OCT, albeit without evidence of dye leakage or pooling on FA. OCTA demonstrated disruption of the choriocapillaris throughout the posterior pole and sparing of intraretinal capillary plexuses. Six months of combined therapy with oral acetazolamide and topical brinzolamide resulted in limited clinical benefit. CONCLUSIONS: We reported two siblings affected by ARB, presenting as non-vasogenic cystoid maculopathy. Prominent alteration of NIR-FAF signal and concomitant choriocapillaris rarefaction on OCTA were noted in the macula. The limited short-term response to combined systemic and topical CAIs might be explained by the impairment of the RPE-CC complex.

Perspectives of carriers of X-linked retinal diseases on genetic testing and gene therapy: A global survey.

Female carriers of X-linked inherited retinal diseases (IRDs) are burdened with potentially passing their disease-causing variant to future generations, as well as exhibiting signs of retinal disease themselves. This study aimed to investigate carriers' experiences of genetic testing, emotions relating to having affected children, and their knowledge regarding genetic testing and gene therapy. An online survey was advertised to self-identified carriers worldwide. Two hundred and twenty-eight carriers completed the survey with mean age of 51 years (SD ± 15.0). A majority of respondents resided in the United States of America (51%), Australia (19%), and the United Kingdom (14%). Most carriers identified with feelings of guilt (70%), concern (91%), and anxiety (88%) for their child. Female carriers who had given birth to children had significantly greater gene therapy knowledge compared to carriers who had not (p < 0.05). Respondents agreed that their eyecare provider and general practitioner helped them understand their condition (63%), however, few carriers reported receiving psychological counselling (9%) or family planning advice (5%). Most respondents (78%) agreed that gene therapy should be available to carriers. This study emphasises the importance of providing appropriate counselling to female carriers and illustrates the motivation of many to participate in emerging treatment options, such as gene therapy.

Toward low-cost gene therapy: mRNA-based therapeutics for treatment of inherited retinal diseases.

Inherited retinal diseases (IRDs) stem from genetic mutations that result in vision impairment. Gene therapy shows promising therapeutic potential, exemplified by the encouraging initial results with voretigene neparvovec. Nevertheless, the associated costs impede widespread access, particularly in low-to-middle income countries. The primary challenge remains: how can we make these therapies globally affordable? Leveraging advancements in mRNA therapies might offer a more economically viable alternative. Furthermore, transitioning to nonviral delivery systems could provide a dual benefit of reduced costs and increased scalability. Relevant stakeholders must collaboratively devise and implement a research agenda to realize the potential of mRNA strategies in equitable access to treatments to prevent vision loss.

Comparison of Worldwide Disease Prevalence and Genetic Prevalence of Inherited Retinal Diseases and Variant Interpretation Considerations.

One of the considerations in planning the development of novel therapeutic modalities is disease prevalence that is usually defined by studying large national/regional populations. Such studies are rare and might suffer from inaccuracies and challenging clinical characterization in heterogeneous diseases, such as inherited retinal diseases (IRDs). Here we collected reported disease prevalence information on various IRDs in different populations. The most common IRD, retinitis pigmentosa, has an average disease prevalence of ∼1:4500 individuals, Stargardt disease ∼1:17,000, Usher syndrome ∼1:25,000, Leber congenital amaurosis ∼1:42,000, and all IRDs ∼1:3450. We compared these values to genetic prevalence (GP) calculated based on allele frequency of autosomal-recessive IRD mutations. Although most values did correlate, some differences were observed that can be explained by discordant, presumably null mutations that are likely to be either nonpathogenic or hypomorphic. Our analysis highlights the importance of performing additional disease prevalence studies and to couple them with population-dependent allele frequency data.

GENETIC FACTORS AND CHARACTERISTICS ON SPECTRAL-DOMAIN OPTICAL COHERENCE TOMOGRAPHY ARE ASSOCIATED WITH CHOROIDAL THICKNESS IN ABCA4 -RELATED RETINOPATHY.

PURPOSE: To investigate the possible correlation factors of choroidal thickness in ABCA4 -related retinopathy. METHODS: A total of 66 patients were included in the cohort. It is a retrospective, cross-sectional laboratory investigation. The patients were tested using whole-exon sequencing and ophthalmic examinations, including slit-lamp examinations, best-corrected visual acuity, spectral-domain optical coherence tomography, fundus photograph, and fundus autofluorescence. RESULTS: Besides demographic characteristics (age, onset age, duration), we selected genetic factors and ocular characteristics on spectral-domain optical coherence tomography as the candidates related to choroidal thickness. Mutation type (inframe mutation or premature termination codon), epiretinal membrane, retinal pigment epithelium- Bruch membrane integrity, and macular curvature changes were identified as related factors to choroidal thickness in ABCA4 -related retinopathy after the adjustment of Logistic LASSO regression. CONCLUSION: Mutation type, epiretinal membrane, retinal pigment epithelium-Bruch membrane integrity, and macular curvature changes are related factors to choroidal thinning. These findings could provide us a further understanding for the pathological process and clinical features of ABCA4 mutation.

Whole exome sequencing of 491 individuals with inherited retinal diseases reveals a large spectrum of variants and identification of novel candidate genes.

BACKGROUND: Inherited retinal diseases (IRDs) include a range of vision loss conditions caused by variants in different genes. The clinical and genetic heterogeneity make identification of the genetic cause challenging. Here, a cohort of 491 unsolved cases from our cohort of Israeli and Palestinian families with IRDs underwent whole exome sequencing (WES), including detection of CNVs as well as single nucleotide variants (SNVs). METHODS: All participants underwent clinical examinations. Following WES on DNA samples by 3 billion, initial SNV analysis was performed by 3 billion and SNV and CNV analysis by Franklin Genoox. The CNVs indicated by the programme were confirmed by PCR followed by gel electrophoresis. RESULTS: WES of 491 IRD cases revealed the genetic cause of disease in 51% of cases, of which 11% were due wholly or in part to CNVs. In two cases, we clarified previously incorrect or unclear clinical diagnoses. This analysis also identified ESRRB and DNM1 as potential novel genes. CONCLUSION: This analysis is the most extensive one to include CNVs to examine IRD causing genes in the Israeli and Palestinian populations. It has allowed us to identify the causative variant of many patients with IRDs including ones with unclear diagnoses and potential novel genes.

Analysis of the Outer Retinal Bands in ABCA4 and PRPH2-Associated Retinopathy using OCT.

PURPOSE: To evaluate the outer retinal bands using OCT in ABCA4- and PRPH2-associated retinopathy and develop a novel imaging biomarker to differentiate between these 2 genotypes. DESIGN: Multicenter case-control study. PARTICIPANTS: Patients with a clinical and genetic diagnosis of ABCA4- or PRPH2-associated retinopathy and an age-matched control group. METHODS: Macular OCT was used to measure the thickness of the outer retinal bands 2 and 4 by 2 independent examiners at 4 retinal loci. MAIN OUTCOME MEASURES: Outcome measures included the thicknesses of band 2, band 4, and the band 2/band 4 ratio. Linear mixed modeling was used to make comparisons across the 3 groups. Receiver operating characteristic (ROC) analysis determined the optimal cutoff for the band 2/band 4 ratio to distinguish PRPH2- from ABCA4-associated retinopathy. RESULTS: We included 45 patients with ABCA4 variants, 45 patients with PRPH2 variants, and 45 healthy controls. Band 2 was significantly thicker in patients with PRPH2 compared with ABCA4 (21.4 vs. 15.9 µm, P < 0.001) variants, whereas band 4 was thicker in patients with ABCA4 variants than those with PRPH2 variants (27.5 vs. 21.7 µm, P < 0.001). Similarly, the band 2/band 4 ratio was significantly different (1.0 vs. 0.6 for PRPH2 vs. ABCA4, P < 0.001). The area under the ROC curve was 0.87 for either band 2 (> 18.58 µm) or band 4 (< 26.17 µm) alone and 0.99 (95% confidence interval: 0.97-0.99) for the band 2/band 4 ratio with a cutoff threshold of 0.79, providing 100% specificity. CONCLUSIONS: We report an altered outer retinal band profile whereby the band 2/band 4 ratio was able to discriminate between PRPH2- and ABCA4-associated retinopathy. This may have future clinic utility in predicting the genotype and provide further insight into the anatomic correlate of band 2. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

RETINAL VASCULAR DISEASE IN LIMB-GIRDLE MUSCULAR DYSTROPHY.

PURPOSE: To report bilateral retinal vascular occlusive disease in limb-girdle muscular dystrophy (LGMD). METHODS: Case report. RESULTS: A 34-year-old Asian woman was referred for evaluation and management of central retinal vein occlusion. Ultra-wide-field fluorescein angiography showed resolving initial peripheral retinal vein occlusion in one eye and peripheral venular segmental staining in the fellow asymmetric eye. Genetic testing established the diagnosis of LGMD. CONCLUSION: Similar to other forms of muscular dystrophy, LGMD is caused by genetic abnormalities in sarcolemma proteins, a key structural component that connects the intracellular cytoskeleton of a myofiber to the extracellular matrix. Like other muscular dystrophies, LGMD may be associated with retinal vascular abnormalities noted. In this case, retinal vascular smooth muscle dysfunction was seen in LGMD, analogous to reported vascular abnormalities in other muscular dystrophies such as facioscapulohumeral dystrophy and Duchenne muscular dystrophy.