Quantification of Fundus Autofluorescence Features in a Molecularly Characterized Cohort of More Than 3500 Inherited Retinal Disease Patients from the United Kingdom
William WoofThales A. C. de GuimarãesSaoud Al‐KhuzaeiMalena Daich VarelaSagnik SenPallavi BaggaBernardo MendesMital ShahPaula BurkeDavid ParrySiying LinGunjan NaikBiraja GhoshalBart LiefersDun Jack FuMichalis GeorgiouQuang NguyenAlan Sousa da SilvaYichen LiuYu Fujinami‐YokokawaDayyanah SumodheePraveen J. PatelJennifer FurmanIsmail MoghulMariya MoosajeeJuliana Maria Ferraz SallumSamantha De SilvaBirgit LorenzFrank G. HolzKaoru FujinamiAndrew R. WebsterOmar A. MahrooSusan M. DownesSavita MadhusudhanKonstantinos BalaskasMichel MichaelidesNikolas Pontikos
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Abstract Purpose To quantify relevant fundus autofluorescence (FAF) image features cross-sectionally and longitudinally in a large cohort of inherited retinal diseases (IRDs) patients. Design Retrospective study of imaging data (55-degree blue-FAF on Heidelberg Spectralis) from patients. Participants Patients with a clinical and molecularly confirmed diagnosis of IRD who have undergone FAF 55-degree imaging at Moorfields Eye Hospital (MEH) and the Royal Liverpool Hospital (RLH) between 2004 and 2019. Methods Five FAF features of interest were defined: vessels, optic disc, perimacular ring of increased signal (ring), relative hypo-autofluorescence (hypo-AF) and hyper-autofluorescence (hyper-AF). Features were manually annotated by six graders in a subset of patients based on a defined grading protocol to produce segmentation masks to train an AI model, AIRDetect, which was then applied to the entire MEH imaging dataset. Main Outcome Measures Quantitative FAF imaging features including area in mm 2 and vessel metrics, were analysed cross-sectionally by gene and age, and longitudinally to determine rate of progression. AIRDetect feature segmentation and detection were validated with Dice score and precision/recall, respectively. Results A total of 45,749 FAF images from 3,606 IRD patients from MEH covering 170 genes were automatically segmented using AIRDetect. Model-grader Dice scores for disc, hypo-AF, hyper-AF, ring and vessels were respectively 0.86, 0.72, 0.69, 0.68 and 0.65. The five genes with the largest hypo-AF areas were CHM , ABCC6 , ABCA4 , RDH12 , and RPE65 , with mean per-patient areas of 41.5, 30.0, 21.9, 21.4, and 15.1 mm 2 . The five genes with the largest hyper-AF areas were BEST1 , CDH23 , RDH12 , MYO7A , and NR2E3 , with mean areas of 0.49, 0.45, 0.44, 0.39, and 0.34 mm 2 respectively. The five genes with largest ring areas were CDH23 , NR2E3 , CRX , EYS and MYO7A, with mean areas of 3.63, 3.32, 2.84, 2.39, and 2.16 mm 2 . Vessel density was found to be highest in EFEMP1 , BEST1 , TIMP3 , RS1 , and PRPH2 (10.6%, 10.3%, 9.8%, 9.7%, 8.9%) and was lower in Retinitis Pigmentosa (RP) and Leber Congenital Amaurosis genes. Longitudinal analysis of decreasing ring area in four RP genes ( RPGR, USH2A, RHO, EYS ) found EYS to be the fastest progressor at -0.18 mm 2 /year. Conclusions We have conducted the first large-scale cross-sectional and longitudinal quantitative analysis of FAF features across a diverse range of IRDs using a novel AI approach.Keywords:
Stargardt disease
Autofluorescence
Fundus (uterus)
ABCA4
Stargardt disease is a juvenile macular degeneration most often inherited in an autosomal recessive pattern, characterized by decreased vision in the first 2 decades of life. This report presents a clinical case of Stargardt disease: a 10-year-old female patient complained of blurry vision, and in a 4-year period, her visual acuity was reduced from OD=0.3 and OS=0.3 to OD=0.08 and OS=0.1, respectively. A genetic analysis revealed a rare combination of 2 homozygous recessive mutations in the ABCA4 gene, which caused Stargardt disease. The presence of different genetic mechanisms leading to a severe disease phenotype can challenge molecular geneticists, ophthalmologists, and genetic counselors.
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Inherited retinal dystrophies, including Stargardt disease, are a group of genetic eye diseases that currently lack effective treatment options. The CRISPR-Cas9 gene editing system and human induced pluripotent stem cells (hiPSCs) offer a promising avenue for treating Stargardt disease, a devastating genetic eye disorder. In a recent study published in Molecular Therapy–Nucleic Acids, Siles and colleagues demonstrated the accurate correction of two pathogenic variants in the ABCA4 gene, which triggers Stargardt disease, in hiPSCs without any adverse effects.1Siles L. Ruiz-Nogales S. Navinés-Ferrer A. Méndez-Vendrell P. Pomares E. Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients.Mol. Ther. Nucleic Acids. 2023; 32: 64-79Google Scholar This finding paves the way for personalized medicine and emerging gene and cell therapies for inherited retinal dystrophies. Stargardt disease (STGD1) is a genetic disorder inherited in an autosomal recessive manner that affects the retina and causes vision loss. It is the second most prevalent pathology in this group and results in progressive retinal degeneration and vision loss in both children and adults.2Koenekoop R.K. The gene for Stargardt disease, ABCA4, is a major retinal gene: a mini-review.Ophthalmic Genet. 2003; 24: 75-80Google Scholar STGD1 is caused by mutations in the ATP-binding cassette (ABC) transporter subfamily A4 gene (ABCA4), which encodes a protein involved in the transport of various molecules across cell membranes. These mutations lead to the accumulation of toxic substances in the retina, which leads to the death of photoreceptors. Stargardt disease is the most common form of inherited macular dystrophy, affecting 1 in 8,000 to 10,000 people worldwide. There are more than 1,500 known pathogenic variants of the ABCA4 gene, most of which are missense or nonsense mutations. Mutations in non-coding regions are also being studied due to their effects on transcriptomic and proteomic complexity. The inherited retinal disease STGD1 leads to macular degeneration and visual loss, for which there is currently no cure. Developing therapeutic approaches to prevent disease progression is therefore crucial. Several therapeutic approaches have been developed to modulate Stargardt disease, including gene therapy, cell replacement therapy, and gene editing. Recent developments in gene editing technology, such as CRISPR-Cas9, have enabled the possibility of permanent gene correction.3Wang J.Y. Doudna J.A. A decade of genome editing is only the beginning.Science. 2023; 379eadd8643Google Scholar Notably, CRISPR-Cas9 technology has made significant progress in the biotechnology and biomedicine sectors. CRISPR-Cas9-mediated gene editing has been used to study, model, and potentially treat inherited eye disorders, but there are concerns about the potential risks associated with this technology. In this study, the authors aimed to correct two pathogenic variants from two STGD1 patients, who are unrelated, carrying compound heterozygous mutations using CRISPR-Cas9 technology. One of the variants related to STGD1 corresponds to a single-base substitution in an intronic region between exons 28 and 29 (c.4253+4C>T), which is predicted to cause a splicing defect. The other variant corresponds to an insertion of a GT in exon 22 of the ABCA4 gene (c.3211_3212insGT), which is expected to result in a frameshift.4Ozgül R.K. Durukan H. Turan A. Oner C. Ogüs A. Farber D.B. Molecular analysis of the ABCA4 gene in Turkish patients with Stargardt disease and retinitis pigmentosa.Hum. Mutat. 2004; 23: 523Google Scholar,5Allikmets R. Singh N. Sun H. Shroyer N.F. Hutchinson A. Chidambaram A. Gerrard B. Baird L. Stauffer D. Peiffer A. et al.A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.Nat. Genet. 1997; 17: 122-246Google Scholar The authors effectively edited both pathogenic variants using single-stranded oligodeoxynucleotide and CRISPR-Cas9-mediated repair without causing genomic alterations in the predicted off-targets, as confirmed by whole-genome and Sanger sequencing.6Simkin D. Papakis V. Bustos B.I. Ambrosi C.M. Ryan S.J. Baru V. Williams L.A. Dempsey G.T. McManus O.B. Landers J.E. et al.Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.Stem Cell Rep. 2022; 17: 993-1008Google Scholar Additionally, they found that gene editing did not compromise the expression of pluripotency markers in corrected clones compared with parental ones, indicating that the edited cells remained pluripotent. These findings encourage the investigation of CRISPR-Cas9 gene editing to revert pathogenic variants as a promising tool for STGD1 research and a potential therapeutic strategy for this inherited retinal dystrophy. Importantly, deep-intronic mutations account for only a small proportion of ABCA4 described variants, and the approach used in this study shows precise single-nucleotide gene editing in the ABCA4 sequence without detected off-target genomic alterations. As a result, the CRISPR-Cas9 investigation used in this work provides a promising approach for possible therapy of the STGD1 disease. Furthermore, this suggests that CRISPR-Cas9 gene editing can be a promising tool for future research and treatment of inherited retinal dystrophies. The results of the study are promising, as the researchers were able to correct the mutation in the ABCA4 gene with high efficiency in the patient-derived hiPSCs. The use of hiPSCs, which are generated from the patient's own cells, provides an exciting opportunity for personalized medicine as it eliminates the risk of immune rejection and allows for the development of patient-specific therapies. This research holds great potential for developing gene therapies for patients with Stargardt disease and other inherited retinal diseases. However, there are some important caveats and issues that warrant further analysis before this technology can be used in clinical settings. The efficiency of CRISPR-Cas9 gene editing may vary depending on the location and type of the mutation, and off-target affects can lead to unintended mutations and potentially harmful consequences.7Javaid D. Ganie S.Y. Hajam Y.A. Reshi M.S. CRISPR/Cas9 system: a reliable and facile genome editing tool in modern biology.Mol. Biol. Rep. 2022; 49: 12133-12150Google Scholar,8Zeballos C.M.A. Gaj T. Next-generation CRISPR technologies and their applications in gene and cell therapy.Trends Biotechnol. 2021; 39: 692-705Google Scholar Additionally, the study was conducted in vitro, and further testing in animal models and clinical trials is necessary to assess the safety and efficacy of the gene-edited cells. Moreover, the study only corrected the mutation in the hiPSCs, and it remains to be seen whether these corrected cells can be successfully differentiated into the desired cell types, such as retinal pigment epithelium cells or photoreceptor cells. Additionally, the long-term stability and safety of the corrected cells need to be evaluated, including the potential for off-target affects and immunogenicity. Ongoing clinical trials with human embryonic stem cell-derived retinal pigment epithelium cells for treating Stargardt disease are aimed at evaluating the safety of subretinal transplantation of these differentiated cells.9Huang D. Heath Jeffery R.C. Aung-Htut M.T. McLenachan S. Fletcher S. Wilton S.D. Chen F.K. Stargardt disease and progress in therapeutic strategies.Ophthalmic Genet. 2022; 43: 1-26Google Scholar In conclusion, the use of CRISPR-Cas9 to correct mutations in hiPSCs derived from Stargardt disease patients is a significant advancement in gene editing and personalized medicine. However, further analysis and testing are necessary to fully evaluate the safety, efficacy, and long-term stability of this technology before it can be applied in clinical settings. S.K. and P.R.C. conceived and wrote this commentary. The authors have no conflict of interest to declare.
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Stargardt disease, a progressive retinal disorder, is associated with bi-allelic variants in ABCA4, a protein that is expressed in the retina. Induced pluripotent stem cell lines (RMCGENi005-A, SCTCi018-A, SCTCi017-A) were generated by lentivirus reprogramming of fibroblasts derived from Stargardt patients carrying different bi-allelic ABCA4 variants. All the generated lines showed pluripotent characteristics and no chromosomal aberrations. The availability of these lines will allow us to generate patient-derived photoreceptor precursor cells and retinal organoids to further study ABCA4 and thereby, Stargardt disease in relevant model systems carrying the patient's genetic background.
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To describe an automated method of quantification of specific fundus phenotypes and evaluate its performance in differentiating drusen, the hallmark lesions of age-related macular degeneration (AMD), from similar-looking bright lesions, the pisciform deposits or flecks typical of Stargardt disease (SD).Fundus macular images of 30 eyes of 30 subjects were studied. Fifteen subjects had a clinical diagnosis of AMD with at least 10 intermediate and/or 1 large drusen, and the other 15 had SD. As a test of bright-lesion separation, AMD and SD subjects were chosen from the heterogeneous phenotypes of each disorder, to be as visually similar as possible. Drusen and fleck properties were quantified from the color images by using an automated method, and a shape classifier was used to divide the images as characteristic of either AMD or SD. Image identification performance was quantified by using the area under the receiver operating characteristic curve (AUC).All SD subjects demonstrated at least one disease-associated variant of the ABCA4 gene. The method achieved an AUC of 0.936 for differentiating AMD from SD.Automated quantification of fundus phenotypes was achieved, and the results show that the method can differentiate AMD from SD, two distinctly different genetically associated disorders, by quantifying the properties of the bright lesions (drusen and flecks) in their fundus images, even when the images were visually selected to be similar. Quantification of fundus phenotypes may allow recognition of new phenotypes, correlation with new genotypes and may measure disease-specific biomarkers to improve management of patients with AMD or SD.
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Purpose To describe the clinical and molecular spectrum of Stargardt disease (STGD) in a cohort of Argentinean patients. Methods This retrospective study included 132 subjects comprising 95 probands clinically diagnosed with STGD and relatives from 16 of them. Targeted next-generation sequencing of the coding and splicing regions of ABCA4 and other phenocopying genes ( ELOVL4 , PROM1 , and CNGB3 ) was performed in 97 STGD patients. Results We found two or more disease-causing variants in the ABCA4 gene in 69/95 (73%) probands, a single ABC A4 variant in 9/95 (9.5%) probands, and no ABCA4 variants in 17/95 (18%) probands. The final analysis identified 173 variants in ABCA4 . Seventy-nine ABCA4 variants were unique, of which nine were novel. No significant findings were seen in the other evaluated genes. Conclusion This study describes the phenotypic and genetic features of STGD1 in an Argentinean cohort. The mutations p.(Gly1961Glu) and p.(Arg1129Leu) were the most frequent, representing almost 20% of the mutated alleles. We also expanded the ABCA4 mutational spectrum with nine novel disease-causing variants, of which eight might be associated with South American natives.
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Stargardt's disease (STGD1) is caused by mutations in the ABCA4 gene. Different lesions characterised by decreased autofluorescence levels are found in fundus autofluorescence (FAF) from STGD1 patients and could be used as outcome indicators for disease progression. We investigated the fate of foci with reduced autofluorescence (FRA) within the heterogeneous background of STGD1 patients using FAF imaging. Genetically confirmed STGD1 patients presenting heterogeneous background autofluorescence on high-quality FAF images at a minimum of two visits at least 12 months apart were chosen. A grid centred on the fovea was used to define five different zones. Within each zone, five FRA were randomly selected for each eye. The eccentricity of foci was determined at different time points for each patient. Analysis of 175 randomly chosen FRA showed consistent centrifugal displacement over time, most notably in eyes showing areas with definitely decreased autofluorescence. Interestingly, FRA did not leave an area of hypo-autofluorescence on FAF in locations where they were previously located. These findings may help to better understand STGD1 progression, improve FAF interpretation, and shed light on the nature of heterogeneous background.
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Abstract Mutations in ATP-binding cassette transporter type A4 ( ABCA4 ) have been linked to several forms of inherited retinal diseases (IRDs) besides the classically defined Stargardt disease (STGD), known as ABCA4 retinopathies. ABCA4 is a sizeable locus harboring 50 exons; thus, its analysis has revealed a rich area of genetic information comprising at least 1,200 disease-causing mutations of varied severity and types. Due to the clinical and genetic heterogeneity, diagnosing ABCA4 retinopathies is challenging. To date, no ABCA4 -retinopathy has been detected in Lebanon. Using next-generation sequencing, we sought to pinpoint the mutation spectrum in seven families with different forms of IRDs: STGD, rod-cone and cone-rod dystrophies (RCD and CRD, respectively). Eight ABCA4 mutations were found, including one novel; c.4330G>C; p.(Trp1408Cys). Three families were diagnosed with CRD, two with STGD, and two others with RCD. In conclusion, our study revealed a novel ABCA4 mutation and showed significant genotypic and phenotypic heterogeneity in Lebanon.
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