Comparative analysis of in-silico tools in identifying pathogenic variants in dominant inherited retinal diseases.

Inherited retinal diseases (IRDs) are a group of rare genetic eye conditions that cause blindness. Despite progress in identifying genes associated with IRDs, improvements are necessary for classifying rare autosomal dominant (AD) disorders. AD diseases are highly heterogenous, with causal variants being restricted to specific amino acid changes within certain protein domains, making AD conditions difficult to classify. Here, we aim to determine the top-performing in-silico tools for predicting the pathogenicity of AD IRD variants. We annotated variants from ClinVar and benchmarked 39 variant classifier tools on IRD genes, split by inheritance pattern. Using area-under-the-curve (AUC) analysis, we determined the top-performing tools and defined thresholds for variant pathogenicity. Top-performing tools were assessed using genome sequencing on a cohort of participants with IRDs of unknown etiology. MutScore achieved the highest accuracy within AD genes, yielding an AUC of 0.969. When filtering for AD gain-of-function and dominant negative variants, BayesDel had the highest accuracy with an AUC of 0.997. Five participants with variants in NR2E3, RHO, GUCA1A, and GUCY2D were confirmed to have dominantly inherited disease based on pedigree, phenotype, and segregation analysis. We identified two uncharacterized variants in GUCA1A (c.428T>A, p.Ile143Thr) and RHO (c.631C>G, p.His211Asp) in three participants. Our findings support using a multi-classifier approach comprised of new missense classifier tools to identify pathogenic variants in participants with AD IRDs. Our results provide a foundation for improved genetic diagnosis for people with IRDs.

Loss-of-function variants in UBAP1L cause autosomal recessive retinal degeneration.

PURPOSE: Inherited retinal diseases (IRDs) are a group of monogenic conditions that can lead to progressive blindness. Their missing heritability is still considerable, due in part to the presence of disease genes that await molecular identification. The purpose of this work was to identify novel genetic associations with IRDs. METHODS: Patients underwent a comprehensive ophthalmological evaluation using standard-of-care tests, such as detailed retinal imaging (macular optical coherence tomography and short-wavelength fundus autofluorescence) and electrophysiological testing. Exome and genome sequencing, as well as computer-assisted data analysis were used for genotyping and detection of DNA variants. A minigene-driven splicing assay was performed to validate the deleterious effects of 1 of such variants. RESULTS: We identified 8 unrelated families from Hungary, the United States, Israel, and The Netherlands with members presenting with a form of autosomal recessive and nonsyndromic retinal degeneration, predominantly described as rod-cone dystrophy but also including cases of cone/cone-rod dystrophy. Age of disease onset was very variable, with some patients experiencing first symptoms during their fourth decade of life or later. Myopia greater than 5 diopters was present in 5 of 7 cases with available refractive data, and retinal detachment was reported in 2 cases. All ascertained patients carried biallelic loss-of-function variants in UBAP1L (HGNC: 40028), a gene with unknown function and with homologies to UBAP1, encoding a protein involved in ubiquitin metabolism. One of these pathogenic variants, the intronic NM_001163692.2:c.910-7G>A substitution, was identified in 5 unrelated families. Minigene-driven splicing assays in HEK293T cells confirmed that this DNA change is responsible for the creation of a new acceptor splice site, resulting in aberrant splicing. CONCLUSION: We identified UBAP1L as a novel IRD gene. Although its function is currently unknown, UBAP1L is almost exclusively expressed in photoreceptors and the retinal pigment epithelium, hence possibly explaining the link between pathogenic variants in this gene and an ocular phenotype.

Successful Treatment of Clinically Diagnosed Cancer-Associated Retinopathy with Intravitreal Dexamethasone Implant Followed by 0.18 mg Fluocinolone Implant without Systemic Immunosuppression.

PURPOSE: To report a case of clinically diagnosed cancer-associated retinopathy (CAR) successfully treated with intravitreal corticosteroid implants without systemic immunosuppression. METHODS: Case report with multimodal imaging. RESULTS: An 80-year-old man without known systemic malignancy presented with debilitating shimmering, hemeralopia and rapidly progressive bilateral vision loss following uncomplicated cataract surgery. Mild vitritis, extensive photoreceptor loss, mottling of retinal pigmentary epithelium (RPE), and mild vascular attenuation were found in both eyes. Full field electroretinogram (ffERG) showed severe bilateral rod-cone dysfunction. Infectious etiologies and vitreoretinal lymphoma were ruled out. During cancer workup, intravitreal corticosteroid treatment was offered. Significant anatomical improvement with reconstitution of ellipsoid zone, improved RPE irregularities and functional improvement, were observed 3 weeks after bilateral intravitreal dexamethasone implants (Ozurdex). 2 months later, patient received bilateral intravitreal 0.18mg fluocinolone acetonide implants (YUTIQ). Later, a colonic adenocarcinoma was found (pathologic stage pT3 pN0). Patient recovered well from surgery and no chemotherapy was needed. 9 months since bilateral intravitreal fluocinolone acetonide implants (11 months since bilateral intravitreal dexamethasone implants), best corrected vision maintained at 20/25-2 OD, 20/20 OS without ongoing treatments. Bilateral reconstitution of ellipsoid zones and nearly resolution of RPE irregularities remained stable. Repeat ffERG demonstrated improved cone response OS and stable diminished rod response OU. Patient reports resolution of ocular symptoms. CONCLUSION: The sustained improvements with intravitreal corticosteroid monotherapy suggest potential advantages using local therapy over systemic treatment. Long term follow-up is warranted. Further research is needed to evaluate the efficacy of using 0.18mg fluocinolone implant (YUTIQ) to treat CAR.

Case Report: Longitudinal Evaluation and Treatment of a Melanoma-Associated Retinopathy Patient.

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous metastatic melanoma in which patients develop vision deficits that include reduced night vision, poor contrast sensitivity, and photopsia. MAR is caused by autoantibodies targeting TRPM1, an ion channel found in melanocytes and retinal ON-bipolar cells (ON-BCs). The visual symptoms arise when TRPM1 autoantibodies enter ON-BCs and block the function of TRPM1, thus detection of TRPM1 autoantibodies in patient serum is a key criterion in diagnosing MAR. Electroretinograms are used to measure the impact of TRPM1 autoantibodies on ON-BC function and represent another important diagnostic tool for MAR. To date, MAR case reports have included one or both diagnostic components, but only for a single time point in the course of a patient's disease. Here, we report a case of MAR supported by longitudinal analysis of serum autoantibody detection, visual function, ocular inflammation, vascular integrity, and response to slow-release intraocular corticosteroids. Integrating these data with the patient's oncological and ophthalmological records reveals novel insights regarding MAR pathogenesis, progression, and treatment, which may inform new research and expand our collective understanding of the disease. In brief, we find TRPM1 autoantibodies can disrupt vision even when serum levels are barely detectable by western blot and immunohistochemistry; intraocular dexamethasone treatment alleviates MAR visual symptoms despite high levels of circulating TRPM1 autoantibodies, implicating antibody access to the retina as a key factor in MAR pathogenesis. Elevated inflammatory cytokine levels in the patient's eyes may be responsible for the observed damage to the blood-retinal barrier and subsequent entry of autoantibodies into the retina.

Using multi-modal imaging to refine the phenotype of PRPH2-associated retinal degeneration.

OBJECTIVE: To refine retinal PRPH2-associated retinal degeneration (PARD) phenotypes using multimodal imaging. DESIGN: Retrospective review of clinical records and multimodal imaging. SUBJECTS: Patients who visited the inherited retinal degeneration (IRD) clinic at two tertiary referral eye centers with molecularly confirmed IRD due to PRPH2 variants. METHODS: Retinal imaging was reviewed using ultra-widefield (UWF) pseudocolor, UWF fundus autofluorescence (FAF), and spectral-domain optical coherence tomography (SD-OCT). Phenotypes were identified in the macular or peripheral region. A combined phenotype was considered if any phenotypes were present in both macular and peripheral regions. Mixed phenotypes in the macula or peripheral retina were considered if there were two distinct phenotypes identified in the same eye. The presence or absence of atrophy in the macular or peripheral area was also noted. MAIN OUTCOME MEASURE: Grading of multimodal imaging by phenotype and atrophy. RESULTS: A total of 144 eyes of 72 patients were included in this study. The majority of the eyes had combined macular and peripheral phenotypes (89/14, 61.8%), while 44 (30.6%) eyes had isolated macular findings, and 11 (7.6%) had isolated peripheral findings. Twenty-five eyes were classified with mixed macular phenotypes while fundus flavimaculatus dystrophy type was the most common combined macular and peripheral phenotype (54/144, 37.5%): n = 10 with macular dystrophy and macular flavimaculatus dystrophy, and n = 15 with butterfly pattern dystrophy and macular flavimaculatus dystrophy. Nearly half of the eyes (71/144, 49.3%) were identified to have concomitant outer retinal atrophy. Fundus flavimaculatus type dystrophy was also associated with the highest proportion of concomitant atrophy (57/71, 80.3%). CONCLUSION: PARD demonstrates a wide array of phenotypes using multimodal imaging. We report that combinations of classically described phenotypes were often seen. Additionally, macular and peripheral atrophy were often associated with PARD phenotypes. Refinement of PARD phenotypes using newer multimodal imaging techniques will likely assist diagnosis and future clinical trials.

A proposal for an updated staging system for LCHADD retinopathy.

OBJECTIVE: To develop an updated staging system for long-chain 3-hydroxyacyl coenzyme A dehydrogenase deficiency (LCHADD) chorioretinopathy based on contemporary multimodal imaging and electrophysiology. METHODS: We evaluated forty cases of patients with genetically confirmed LCHADD or trifunctional protein deficiency (TFPD) enrolled in a prospective natural history study. Wide-field fundus photographs, fundus autofluorescence (FAF), optical coherence tomography (OCT), and full-field electroretinogram (ffERG) were reviewed and graded for severity. RESULTS: Two independent experts first graded fundus photos and electrophysiology to classify the stage of chorioretinopathy based upon an existing published system. With newer imaging modalities and improved electrophysiology, many patients did not fit cleanly into a single traditional staging group. Therefore, we developed a novel staging system that better delineated the progression of LCHADD retinopathy. We maintained the four previous delineated stages but created substages A and B in stages 2 to 3 to achieve better differentiation. DISCUSSION: Previous staging systems of LCHADD chorioretinopathy relied on only on the assessment of standard 30 to 45-degree fundus photographs, visual acuity, fluorescein angiography (FA), and ffERG. Advances in recordings of ffERG and multimodal imaging with wider fields of view, allow better assessment of retinal changes. Following these advanced assessments, seven patients did not fit neatly into the original classification system and were therefore recategorized under the new proposed system. CONCLUSION: The new proposed staging system improves the classification of LCHADD chorioretinopathy, with the potential to lead to a deeper understanding of the disease's progression and serve as a more reliable reference point for future therapeutic research.

Perimacular Atrophy Following Voretigene Neparvovec-Rzyl Treatment in the Setting of Previous Contralateral Eye Treatment With a Different Viral Vector.

Purpose: To report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector. Design: Single-center, retrospective chart review. Methods: In this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated. Results: Three out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector. Conclusions: We observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes. Translational Relevance: Caution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.

Gene therapy in bestrophinopathies: Insights from preclinical studies in preparation for clinical trials.

The BEST1 gene encodes bestrophin-1, a homopentameric ion channel expressed in the retinal pigment epithelium (RPE), where it localizes to the basolateral plasma membrane. Pathogenic variants in this gene can cause different autosomal dominant and recessive inherited retinal diseases (IRDs), collectively named "bestrophinopathies." These disorders share a number of clinical and molecular features that make them an appealing target for gene therapy. Clinically, bestrophinopathies are often slowly progressive with a wide window of opportunity, and the presence of subretinal material (vitelliform deposits and/or fluid) as a hallmark of these conditions provides an easily quantifiable endpoint in view of future clinical trials. From a molecular standpoint, most BEST1 pathogenic variants have been shown to cause either loss of function (LOF) of the protein or a dominant-negative (DN) effect, with a smaller subset causing a toxic gain of function (GOF). Both LOF and DN mutations may be amenable to gene augmentation alone. On the other hand, individuals harboring GOF variants would require a combination of gene silencing and gene augmentation, which has been shown to be effective in RPE cells derived from patients with Best disease. In this article, we review the current knowledge of BEST1-related IRDs and we discuss how their molecular and clinical features are being used to design novel and promising therapeutic strategies.

Retinitis pigmentosa GTPase regulator-related retinopathy and gene therapy.

Retinitis pigmentosa GTPase regulator (RPGR)-related retinopathy is a retinal dystrophy inherited in a X-linked recessive manner that typically causes progressive visual loss starting in childhood with severe visual impairment by the fourth decade of life. It manifests as an early onset and severe form of retinitis pigmentosa. There are currently no effective treatments for RPGR-related retinopathy; however, there are multiple clinical trials in progress exploring gene augmentation therapy aimed at slowing down or halting the progression of disease and possibly restoring visual function. This review focuses on the molecular biology, clinical manifestations, and the recent progress of gene therapy clinical trials.

Optical coherence tomography angiography of choroidal neovascularization in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD).

Purpose: To report the clinical utility of optical coherence tomography angiography (OCTA) for demonstrating choroidal neovascularization (CNV) associated with Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency (LCHADD) retinopathy. Methods: Thirty-three participants with LCHADD (age 7-36 years; median 17) were imaged with OCTA and the Center for Ophthalmic Optics & Lasers Angiography Reading Toolkit (COOL-ART) software was implemented to process OCTA scans. Results: Seven participants (21 %; age 17-36 years; median 25) with LCHADD retinopathy demonstrated evidence of CNV by retinal examination or presence of CNV within outer retinal tissue on OCTA scans covering 3 × 3 and/or 6 × 6-mm. These sub-clinical CNVs are adjacent to hyperpigmented areas in the posterior pole. CNV presented at stage 2 or later of LCHADD retinopathy prior to the disappearance of RPE pigment in the macula. Conclusion: OCTA can be applied as a non-invasive method to evaluate the retinal and choroidal microvasculature. OCTA can reveal CNV in LCHADD even when the clinical exam is inconclusive. These data suggest that the incidence of CNV is greater than expected and can occur even in the early stages of LCHADD retinopathy.