Investigating the impact of asymmetric macular sensitivity on visual acuity chart reading in choroideraemia.

INTRODUCTION: Degeneration in choroideraemia, unlike typical centripetal photoreceptor degenerations, is centred temporal to the fovea. Once the fovea is affected, the nasal visual field (temporal retina) is relatively spared, and the preferred retinal locus shifts temporally. Therefore, when reading left to right, only the right eye reads into a scotoma. We investigate how this unique property affects the ability to read an eye chart. METHODS: Standard- and low-luminance visual acuity (VA) for right and left eyes were measured with the Early Treatment of Diabetic Retinopathy Study (ETDRS) chart. Letters in each line were labelled by column position. The numbers of letter errors for each position across the whole chart were summed to produce total column error scores for each participant. Macular sensitivity was assessed using microperimetry. Central sensitivity asymmetry was determined by the temporal-versus-nasal central macular difference and subsequently correlated to a weighted ETDRS column error score. Healthy volunteers and participants with X-linked retinitis pigmentosa GTPase regulator associated retinitis pigmentosa (RPGR-RP) were used as controls. RESULTS: Thirty-nine choroideraemia participants (median age 44.9 years [IQR 35.7-53.5]), 23 RPGR-RP participants (median age 30.8 years [IQR 26.5-40.5]) and 35 healthy controls (median age 23.8 years [IQR 20.3-29.0]) were examined. In choroideraemia, standard VA in the right eye showed significantly greater ETDRS column errors on the temporal side compared with the nasal side (p = 0.002). This significantly correlated with greater asymmetry in temporal-versus-nasal central macular sensitivity (p = 0.04). No significant patterns in ETDRS column errors or central macular sensitivity were seen in the choroideraemia left eyes, nor in RPGR-RP and control eyes. CONCLUSION: Difficulty in tracking across lines during ETDRS VA testing may cause excess errors independent of true VA. VA assessment with single-letter optotype systems may be more suitable, particularly for patients with choroideraemia, and potentially other retinal diseases with asymmetric central macular sensitivity or large central scotomas including geographic atrophy.

Reduced Retinal Pigment Epithelial Autophagy Due to Loss of Rab12 Prenylation in a Human iPSC-RPE Model of Choroideremia.

Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in CHM, encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of CHM, the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited CHM-/- iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. CHM-/- iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in CHM-/- cells by transduction with gene replacement (ShH10-CMV-CHM) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.

A cohort study of 19 patients with gyrate atrophy of the choroid and retina (GACR).

PURPOSE: Gyrate atrophy of the choroid and retina (GACR) is an autosomal recessive inherited metabolic disorder (IMD) characterised by progressive retinal degeneration, leading to severe visual impairment. The rapid developments in ophthalmic genetic therapies warrant knowledge on clinical phenotype of eligible diseases such as GACR to define future therapeutic parameters in clinical trials. METHODS: Retrospective chart analysis was performed in nineteen patients. Data were analysed using IBM SPSS Statistics version 28.0.1.1. RESULTS: Nineteen patients were included with a mean age of 32.6 years (range 8-58). Mean age at onset of ophthalmic symptoms was 7.9 years (range 3-16). Median logMAR of visual acuity at inclusion was 0.26 (range -0.18-3.00). Mean age at cataract surgery was 28.8 years (n = 11 patients). Mean spherical equivalent of the refractive error was -8.96 (range -20.87 to -2.25). Cystoid maculopathy was present in 68% of patients, with a loss of integrity of the foveal ellipsoid zone (EZ) in 24/38 eyes. Of the 14 patients treated with dietary protein restriction, the four patients who started the diet before age 10 showed most benefit. CONCLUSION: This study demonstrates the severe ophthalmic disease course associated with GACR, as well as possible benefit of early dietary treatment. In addition to visual loss, patients experience severe myopia, early-onset cataract, and CME. There is a loss of foveal EZ integrity at a young age, emphasising the need for early diagnosis enabling current and future therapeutic interventions.

Comparison of The Results of Sponsored Genetic Testing Panels for Inherited Retinal Diseases.

Background/Objectives: Gene therapy's emergence has made molecular diagnosis for inherited retinal diseases clinically significant. Free genetic testing panels have improved testing access in clinical practice, yet the interpretation of results, especially variants of unknown significance (VUS), remains challenging and requires expertise. This study shares our experience in utilizing sponsored IRD panel tests by Invitae and Blueprint Genetics (BG), reporting their positivity rates, and comparing their reclassification of variants through amendments. Methods: This retrospective study analyzed genetic test reports from patients who underwent testing via Invitae or BG panels. A positive test was determined if there was a pathogenic mutation in an autosomal dominant gene, two pathogenic mutations in an autosomal recessive gene, or a pathogenic mutation in an X-linked gene in a male patient. Results: The testing positivity rates were 34.9% for Invitae (n = 109) and 42.1% for BG (n = 107). Invitae had more pathogenic variants per report (0.87 vs. 0.58 variants, p = 0.0038) and issued more amendments than BG (0.54 vs. 0.03 amendments; p < 0.01). Of the Invitae variant classification changes, 66.2% switched a VUS to benign. In the BG group, 75% of variant reclassifications changed a VUS to pathogenic. As a result of the Invitae amendments, 88% did not change the overall report result. Conclusions: While free-of-charge genetic testing panels offer valuable insights for diagnosing IRD, limitations such as low diagnostic yield and variant classification discrepancies persist between Invitae and BG. VUS should not be considered pathogenic in the clinical decision-making process. Careful interpretation of genetic testing is required.

Differential gene expression between central and peripheral retinal regions in dogs and comparison with humans.

The dog retina contains a central macula-like region, and there are reports of central retinal disorders in dogs with shared genetic etiologies with humans. Defining central/peripheral gene expression profiles may provide insight into the suitability of dogs as models for human disorders. We determined central/peripheral posterior eye gene expression profiles in dogs and interrogated inherited retinal and macular disease-associated genes for differential expression between central and peripheral regions. Bulk tissue RNA sequencing was performed on 8 mm samples of the dog central and superior peripheral regions, sampling retina and retinal pigmented epithelium/choroid separately. Reads were mapped to CanFam3.1, read counts were analyzed to determine significantly differentially expressed genes (DEGs). A similar analytic pipeline was used with a published bulk-tissue RNA sequencing human dataset. Pathways and processes involved in significantly DEGs were identified (Database for Annotation, Visualization and Integrated Discovery). Dogs and humans shared the extent and direction of central retinal differential gene expression, with multiple shared biological pathways implicated in differential expression. Many genes implicated in heritable retinal disorders in dogs and humans were differentially expressed between central and periphery. Approximately half of genes associated with human age-related macular degeneration were differentially expressed in human and dog tissues. We have identified similarities and differences in central/peripheral gene expression profiles between dogs and humans which can be applied to further define the relevance of dogs as models for human retinal disorders.

Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci.

BACKGROUND: Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. RESULTS: To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4. CONCLUSIONS: Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.

Neovascular Glaucoma in MELAS syndrome.

Purpose: To describe examination and findings in a case of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) with particular focus on the ocular sequelae from diabetes. Observations: Neovascular glaucoma is not a common manifestation of MELAS. Conclusions and Importance: We present a rare case of neovascular glaucoma in a patient with MELAS with a history of diabetes, hearing loss, and macular dystrophy. MELAS should be suspected in patients with this constellation of symptoms.

Autosomal Dominant Retinitis Pigmentosa Secondary to TOPORS Mutations: A Report of a Novel Mutation and Clinical Findings.

Purpose: Mutations in Topoisomerase I-binding RS protein (TOPORS) have been previously documented and have been described to result in pathological autosomal dominant retinitis pigmentosa (adRP). In our study, we describe the various genotypes and clinical/phenotypic manifestations of TOPORS-related mutations of our unique patient population in Rural Appalachia. Methods: The medical records of 416 patients with inherited retinal disease at the West Virginia University Eye Institute who had undergone genetic testing between the years of 2015-2022 were reviewed. Patients found to have pathologic RP and mutations related to TOPORS were then analyzed. Results: In total, 7 patients (ages 12-70) were identified amongst three unique families. All patients were female in our study. The average follow-up period was 7.7 years. A mother (70 yr) and daughter (51 yr) had a novel heterozygous nonsense point mutation in TOPORS c.2431C > T, p.Gln811X (Exon 3) that led to premature termination of the desired protein resulting in early onset vision loss, cataract formation, and visual field restriction. The mother developed a full-thickness macular hole which was successfully repaired. Five other patients were found to have previously described TOPORS mutations. Visual field loss was progressive with age in both cohorts. Conclusions: Seven patients at our institution were identified to have mutations in TOPORS resulting in autosomal dominant retinitis pigmentosa. Two patients were found to have novel truncating mutations in the TOPORS gene resulting in profound night blindness and visual field loss, recurrent macular edema, and in one individual, epiretinal membrane formation leading to a macular hole which was able to be successfully repaired.

Beyond the phenotype: Exploring inherited retinal diseases with targeted next-generation sequencing in a Turkish cohort.

This research aims to compile recent clinical and genetic data from Turkish patients with inherited retinal disorders and evaluate the effectiveness of targeted Next-generation sequencing panels. The study included Turkish individuals with hereditary retinal diseases who visited the Medical Genetic Department of Erciyes University between 2019 and 2022. One proband per family was selected based on eligibility. We used Hereditary Disorder Solution (HDS) by Sophia Genetics and performed next-generation sequencing (NGS) with Illumina NextSeq-500. Bioinformatics analysis using Sophia DDM® SaaS algorithms and ACMG guidelines classified genomic changes. The study involved 354 probands. Disease-causing variants were found in 58.1% of patients, with ABCA4, USH2A, RDH12, and EYS being the most frequently implicated genes. Forty-eight novel variants were detected. This study enhances the knowledge of clinical diagnoses, symptom onset, inheritance patterns, and genetic details for Turkish individuals with hereditary retinal disease. It contributes to broader health strategies by enabling comparisons with other studies.

Aberrant homeodomain-DNA cooperative dimerization underlies distinct developmental defects in two dominant CRX retinopathy models.

Paired-class homeodomain transcription factors (HD TFs) play essential roles in vertebrate development, and their mutations are linked to human diseases. One unique feature of paired-class HD is cooperative dimerization on specific palindrome DNA sequences. Yet, the functional significance of HD cooperative dimerization in animal development and its dysregulation in diseases remain elusive. Using the retinal TF Cone-rod Homeobox (CRX) as a model, we have studied how blindness-causing mutations in the paired HD, p.E80A and p.K88N, alter CRX's cooperative dimerization, lead to gene misexpression and photoreceptor developmental deficits in dominant manners. CRXE80A maintains binding at monomeric WT CRX motifs but is deficient in cooperative binding at dimeric motifs. CRXE80A's cooperativity defect impacts the exponential increase of photoreceptor gene expression in terminal differentiation and produces immature, non-functional photoreceptors in the CrxE80A retinas. CRXK88N is highly cooperative and localizes to ectopic genomic sites with strong enrichment of dimeric HD motifs. CRXK88N's altered biochemical properties disrupt CRX's ability to direct dynamic chromatin remodeling during development to activate photoreceptor differentiation programs and silence progenitor programs. Our study here provides in vitro and in vivo molecular evidence that paired-class HD cooperative dimerization regulates neuronal development and dysregulation of cooperative binding contributes to severe dominant blinding retinopathies.

PRPS1-associated retinopathy: a diagnostic odyssey.

PURPOSE: This study describes how the diagnosis of Usher syndrome was revised to PRPS1-associated retinopathy and Charcot-Marie-Tooth disease type 5. CASE REPORT: A 38-year-old female with bilaterally subnormal vision and non-congenital hearing loss was initially diagnosed with Usher syndrome, based on finding variants in three genes (MYO7A, USH2A, and PCDH15), was re-evaluated at the inherited retinal disorders clinic. She had asymmetric retinopathy and right macular pseudocoloboma. She was also found to have myopathic facies, poor grip strength and atrophy of the calf muscles. Whole exome sequencing including variants in PRPS1 showed a variant (NM_002764.4:c.287 G > A; p.Arg96Gln), which was not detected by targeted Sanger sequencing of the DNA from her mother and sister. CONCLUSION: The constellation of asymmetric retinopathy and non-congenital hearing impairment should prompt the clinician to search for other diagnoses that may not be covered by an Usher syndrome next generation sequencing panel. Interpretation of genetic testing results should be correlated with a detailed clinical phenotype.

Congenital Stationary Night Blindness Structure, Function and Genotype-Phenotype Correlations in a Cohort of 122 Patients.

OBJECTIVE: To examine the molecular causes of Schubert-Bornschein (S-B) congenital stationary night blindness (CSNB), clinically characterize in detail, and assess genotype-phenotype correlations for retinal function and structure. DESIGN: Retrospective, longitudinal, single-center case series. PARTICIPANTS: One hundred twenty-two patients with S-B CSNB attending Moorfields Eye Hospital, United Kingdom. METHODS: All case notes, results of molecular genetic testing, and OCT were reviewed. MAIN OUTCOME MEASURES: Molecular genetics, presenting complaints, rates of nystagmus, nyctalopia, photophobia, strabismus, color vision defects and spherical equivalent refraction (SER). Retinal thickness, outer nuclear layer (ONL) thickness, and ganglion cell layer + inner plexiform layer (GCL+IPL) thickness from OCT imaging. RESULTS: X-linked (CACNA1F and NYX) and autosomal recessive (TRPM1, GRM6, GPR179 and CABP4) genotypes were identified. The mean (± standard deviation) reported age of onset was 4.94 ± 8.99 years. Over the follow-up period, 95.9% of patients reported reduced visual acuity (VA), half had nystagmus, and 64.7% reported nyctalopia. Incomplete CSNB (iCSNB) patients more frequently had nystagmus and photophobia. Nyctalopia was similar for iCSNB and complete CSNB (cCSNB). Color vision data were limited but more defects were found in iCSNB. None of these clinical differences met statistical significance. There was no significant difference between groups in VA, with a mean of 0.46 logarithm of the minimum angle of resolution, and VA remained stable over the course of follow-up. Complete congenital stationary night blindness patients, specifically those with NYX and TRPM1 variants, were more myopic. CACNA1F patients showed the largest refractive variability, and the CABP4 patient was hyperopic. No significant differences were found in OCT structural analysis during the follow-up period. CONCLUSIONS: Retinal structure in CSNB is stationary and no specific genotype-structure correlates were identified. Visual acuity seems to be relatively stable, with rare instances of progression. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

GNB1-Related Rod-Cone Dystrophy: A Case Report.

Introduction: The GNB1 (guanine nucleotide-binding protein, ß1) gene encodes for the ubiquitous ß1 subunit of heterotrimeric G proteins, which are associated with G-protein-coupled receptors (GPCRs). GNB1 mutations cause a neurodevelopmental disorder characterized by a broad clinical spectrum. A novel variant has recently been confirmed in a case of rod-cone dystrophy. Case Presentation: We describe the second confirmed case of a classical rod-cone dystrophy associated with a mutation located in exon 6 of GNB1 [NM_002074.5:c.217G>C, p.(Ala73Pro)] in a 56-year-old patient also presenting mild intellectual disability, attention deficit/hyperactivity disorder, and truncal obesity. Conclusion: This paper confirms the role of GNB1 in the pathogenesis of a classic rod-cone dystrophy and highlights the importance of including this gene in the genetic analysis panel for inherited retinal diseases.

Cell-cell interaction in the pathogenesis of inherited retinal diseases.

The retina is part of the central nervous system specialized for vision. Inherited retinal diseases (IRD) are a group of clinically and genetically heterogenous disorders that lead to progressive vision impairment or blindness. Although each disorder is rare, IRD accumulatively cause blindness in up to 5.5 million individuals worldwide. Currently, the pathophysiological mechanisms of IRD are not fully understood and there are limited treatment options available. Most IRD are caused by degeneration of light-sensitive photoreceptors. Genetic mutations that abrogate the structure and/or function of photoreceptors lead to visual impairment followed by blindness caused by loss of photoreceptors. In healthy retina, photoreceptors structurally and functionally interact with retinal pigment epithelium (RPE) and Müller glia (MG) to maintain retinal homeostasis. Multiple IRD with photoreceptor degeneration as a major phenotype are caused by mutations of RPE- and/or MG-associated genes. Recent studies also reveal compromised MG and RPE caused by mutations in ubiquitously expressed ciliary genes. Therefore, photoreceptor degeneration could be a direct consequence of gene mutations and/or could be secondary to the dysfunction of their interaction partners in the retina. This review summarizes the mechanisms of photoreceptor-RPE/MG interaction in supporting retinal functions and discusses how the disruption of these processes could lead to photoreceptor degeneration, with an aim to provide a unique perspective of IRD pathogenesis and treatment paradigm. We will first describe the biology of retina and IRD and then discuss the interaction between photoreceptors and MG/RPE as well as their implications in disease pathogenesis. Finally, we will summarize the recent advances in IRD therapeutics targeting MG and/or RPE.

Cost-of-illness studies of inherited retinal diseases: a systematic review.

BACKGROUND: While health care and societal costs are routinely modelled for most diseases, there is a paucity of comprehensive data and cost-of-illness (COI) studies for inherited retinal diseases (IRDs). This lack of data can lead to underfunding or misallocation of resources. A comprehensive understanding of the COI of IRDs would assist governmental and healthcare leaders in determining optimal resource allocation, prioritizing funding for research, treatment, and support services for these patients. METHODS: Following PRISMA guidelines, a literature search was conducted using Medline, EMBASE and Cochrane databases, from database inception up to 30 Jun 2023, to identify COI studies related to IRD. Original studies in English, primarily including patients with IRDs, and whose main study objective was the estimation of the costs of IRDs and had sufficiently detailed methodology to assess study quality were eligible for inclusion. To enable comparison across countries and studies, all annual costs were standardized to US dollars, adjusted for inflation to reflect their current value and recalculated on a "per patient" basis wherever possible. The review protocol was registered in PROSPERO (registration number CRD42023452986). RESULTS: A total of nine studies were included in the final stage of systematic review and they consistently demonstrated a significant disease burden associated with IRDs. In Singapore, the mean total cost per patient was roughly US$6926/year. In Japan, the mean total cost per patient was US$20,833/year. In the UK, the mean total cost per patient with IRD ranged from US$21,658 to US$36,549/year. In contrast, in the US, the mean total per-patient costs for IRDs ranged from about US$33,017 to US$186,051 per year. In Canada, these mean total per-patient costs varied between US$16,470 and US$275,045/year. Non-health costs constituted the overwhelming majority of costs as compared to healthcare costs; 87-98% of the total costs were due to non-health costs, which could be attributed to diminished quality of life, poverty, and increased informal caregiving needs for affected individuals. CONCLUSION: IRDs impose a disproportionate societal burden outside health systems. It is vital for continued funding into IRD research, and governments should incorporate societal costs in the evaluation of cost-effectiveness for forthcoming IRD interventions, including genomic testing and targeted therapies.

Novel ATF6 homozygous variant in a Chinese patient with achromatopsia.

BACKGROUND: ATF6-associated Achromatopsia (ACHM) is a rare autosomal recessive disorder characterized by reduction of visual acuity, photophobia, nystagmus, and poor color vision. METHODS: Detailed ophthalmological examinations were performed in a Chinese patient with ACHM. Whole exome sequencing and Sanger sequencing were performed to detect the disease-causing gene in the patient. RESULTS: A 6-year-old girl presented photophobia, low vision and reduced color discrimination. Small yellow lesion in the macula of both eyes was observed. FAF demonstrated hypofluorescence in the macular fovea. OCT images revealed interruption of ellipsoid and interdigitation zone in the foveal area and a loss of the foveal pit. ERG showed relatively normal rod responses and unrecordable cone responses. Sequencing result identified a novel splicing variant c.354 + 6T>C in the ATF6 gene (NM_007348.4). CONCLUSIONS: We reported detailed clinical features and genetic analysis of a new Chinese ATF6-associated patient with ACHM.

EQ-5D-5L health utility scores in Australian adults with inherited retinal diseases: A cross-sectional survey.

PURPOSE: Economic evaluations of interventions for ocular disease require utility scores that accurately represent quality of life in the target population. This study aimed to describe the distribution of EQ-5D-5L utility values among Australian adults with symptomatic inherited retinal diseases (IRDs) and to assess the relationship between these scores and vision-related quality of life. METHODS: A survey was administered predominantly online in 2021. Participants completed the EQ-5D-5L general health utility instrument, the EQ vertical visual analogue scale (EQ-VAS) and the National Eye Institute Visual Functioning Questionnaire (NEI-VFQ-25). Self-reported IRD diagnoses were classified as being associated with central or widespread retinal involvement. RESULTS: Responses from 647 participants aged 18-93 years were included, 50.1% were men and 77.6% had an IRD associated with widespread retinal involvement. The majority reported no problems with self-care and no pain/discomfort but did report anxiety/depression and problems with work, study, housework, or family/leisure activities. Most people with widespread involvement reported problems with mobility. Median EQ-5D-5L utility was 0.88 and 0.91 among people with widespread and central involvement, respectively (age and sex-adjusted p = 0.029); and median EQ-VAS was 75 and 80, respectively (adjusted p = 0.003). A moderate curvilinear correlation was observed between EQ-5D-5L and NEI-VFQ-25 composite score (Spearman's ρ 0.69), but not all people with poor vision-related quality of life had low EQ-5D-5L utility values. CONCLUSIONS: EQ-5D-5L health utility values are correlated with vision-related quality of life among adults with IRDs. However, the EQ-5D-5L may not be sensitive to the full impact of vision impairment on quality of life.

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.

Spectrum of Genetic Variants in the Most Common Genes Causing Inherited Retinal Disease in a Large Molecularly Characterized United Kingdom Cohort.

PURPOSE: Inherited retinal disease (IRD) is a leading cause of blindness. Recent advances in gene-directed therapies highlight the importance of understanding the genetic basis of these disorders. This study details the molecular spectrum in a large United Kingdom (UK) IRD patient cohort. DESIGN: Retrospective study of electronic patient records. PARTICIPANTS: Patients with IRD who attended the Genetics Service at Moorfields Eye Hospital between 2003 and July 2020, in whom a molecular diagnosis was identified. METHODS: Genetic testing was undertaken via a combination of single-gene testing, gene panel testing, whole exome sequencing, and more recently, whole genome sequencing. Likely disease-causing variants were identified from entries within the genetics module of the hospital electronic patient record (OpenEyes Electronic Medical Record). Analysis was restricted to only genes listed in the Genomics England PanelApp R32 Retinal Disorders panel (version 3.24), which includes 412 genes associated with IRD. Manual curation ensured consistent variant annotation and included only plausible disease-associated variants. MAIN OUTCOME MEASURES: Detailed analysis was performed for variants in the 5 most frequent genes (ABCA4, USH2A, RPGR, PRPH2, and BEST1), as well as for the most common variants encountered in the IRD study cohort. RESULTS: We identified 4415 individuals from 3953 families with molecularly diagnosed IRD (variants in 166 genes). Of the families, 42.7% had variants in 1 of the 5 most common IRD genes. Complex disease alleles contributed to disease in 16.9% of affected families with ABCA4-associated retinopathy. USH2A exon 13 variants were identified in 43% of affected individuals with USH2A-associated IRD. Of the RPGR variants, 71% were clustered in the ORF15 region. PRPH2 and BEST1 variants were associated with a range of dominant and recessive IRD phenotypes. Of the 20 most prevalent variants identified, 5 were not in the most common genes; these included founder variants in CNGB3, BBS1, TIMP3, EFEMP1, and RP1. CONCLUSIONS: We describe the most common pathogenic IRD alleles in a large single-center multiethnic UK cohort and the burden of disease, in terms of families affected, attributable to these variants. Our findings will inform IRD diagnoses in future patients and help delineate the cohort of patients eligible for gene-directed therapies under development. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.