Inherited retinal dystrophies (IRDs) are characterized by their high clinical and genetic heterogeneity. Despite significant advances in the identification of genes associated with IRDs, many individuals and families still have not received a definite molecular diagnosis. Here, we performed clinical examinations and conducted genetic testing in five families with IRD. Whole exome sequencing in the five index cases revealed a heterozygous missense variant, c.209G > A, p.(Gly70Glu) in the ARL3 gene (NM_004311.4). A de novo occurrence was demonstrated in one affected individual and autosomal dominant inheritance in nine affected individuals from four families. Their phenotypes displayed variable expressivity, and ranged from rod-cone to cone-rod dystrophy with photophobia. Human induced pluripotent stem cells (hiPSCs) were generated from dermal fibroblasts from the individual with the de novo ARL3 variant and were differentiated to retinal pigment epithelium cells (RPE) and retinal organoids. Immunofluorescence analyses in these models showed decreased INPP5E localization within the cilia of RPE and connecting cilia of retinal organoids, as well as reduced PDE6⍺ in the organoid outer segments, suggesting that the p.(Gly70Glu) variant causes IRD by defective lipidated protein transport in photoreceptors and/or RPE. This is the first study of ARL3 dysfunction in human retinal cells, highlighting its importance for retinal homeostasis, as well as a variability in the clinical presentation of ARL3-associated IRD.

Rare eye diseases such as inherited retinal diseases (IRDs) are challenging to diagnose genetically. IRDs are typically monogenic disorders and represent a leading cause of blindness in children and working-age adults worldwide. A growing number are now being targeted in clinical trials, with approved treatments increasingly available. However, access requires a genetic diagnosis to be established sufficiently early. Critically, the timely identification of a genetic cause remains challenging. We demonstrate that a deep learning algorithm, Eye2Gene, trained on a large multimodal imaging dataset of individuals with IRDs (n = 2,451) and externally validated on data provided by five different clinical centres, provides better-than-expert-level top-five accuracy of 83.9% for supporting genetic diagnosis for the 63 most common genetic causes. We demonstrate that Eye2Gene's next-generation phenotyping can increase diagnostic yield by improving screening for IRDs, phenotype-driven variant prioritization and automatic similarity matching in phenotypic space to identify new genes. Eye2Gene is accessible online (app.eye2gene.com) for research purposes.

Variants in six pre-mRNA processing factors cause autosomal dominant Retinitis Pigmentosa (adRP). The RP9 gene encodes a seventh splicing factor, and in 2002, we published RP9 variants c.410A>T; p.(H137L) and c.509A>G; p.(D170G) as likely causes of adRP in a large multigenerational RP9-linked family and a single case, respectively. It has since been suggested these variants might be artefacts due to simultaneous amplification of the RP9P pseudogene, and no further pathogenic variants have been reported. We therefore rescreened two members of the RP9-linked family by genome sequencing. Examination of the 2 Mb locus defined by crossovers in the original family revealed no other plausible causative variants. Alignment of both short and long-read sequences confirmed that p.(H137L) is in the RP9 gene, not the pseudogene. Screening for p.(H137L) in 1961 RP/Rod-cone dystrophy (RCD) cases from the Leeds patient cohort and UK 100,000 Genomes Project (100kGP) database revealed four further carriers. Including the original family, this variant was therefore present in 5/1962 RP/RCD probands, and is absent from gnomAD, constituting statistically significant enrichment in RP cases. Long-read sequencing of p.(H137L) in available carriers showed this is a UK founder allele. The RP9 p.(D170G) allele was also confirmed as gene, not pseudogene, derived, but is present in 22 individuals in the 100kGP cohort, none with RP, as well as >200 individuals in gnomAD and Biobank, suggesting it is non-pathogenic. In conclusion, RP9 p.(H137L) is strongly associated with RP and remains the only plausible variant accounting for the condition in a large multi-generation adRP family.

Importance:Understanding the pathogenic mechanisms of Fuchs endothelial corneal dystrophy (FECD) could contribute to developing gene-targeted therapies. Objective:To investigate associations between demographic data and age at first keratoplasty in a genetically refined FECD cohort. Design, Setting, and Participants:This retrospective cohort study recruited 894 individuals with FECD at Moorfields Eye Hospital (London) and General University Hospital (Prague) from September 2009 to July 2023. Ancestry was inferred from genome-wide single nucleotide polymorphism array data. CTG18.1 status was determined by short tandem repeat and/or triplet-primed polymerase chain reaction. One or more expanded alleles (≥50 repeats) were classified as expansion-positive (Exp+). Expansion-negative (Exp-) cases were exome sequenced. Main Outcomes and Measures:Association between variants in FECD-associated genes, demographic data, and age at first keratoplasty. Results:Within the total cohort (n = 894), 77.3% of patients were Exp+. Most European (668 of 829 [80.6%]) and South Asian (14 of 22 [63.6%]) patients were Exp+. The percentage of female patients was higher (151 [74.4%]) in the Exp- cohort compared to the Exp+ cohort (395 [57.2%]; difference, 17.2%; 95% CI, 10.1%-24.3%; P < .001). The median (IQR) age at first keratoplasty of the Exp + patients (68.2 years [63.2-73.6]) was older than the Exp- patients (61.3 years [52.6-70.4]; difference, 6.5 years; 95% CI, 3.4-9.7; P < .001). The CTG18.1 repeat length of the largest expanded allele within the Exp+ group was inversely correlated with the age at first keratoplasty (β, -0.087; 95% CI, -0.162 to -0.012; P = .02). The ratio of biallelic to monoallelic expanded alleles was higher in the FECD cohort (1:14) compared to an unaffected control group (1:94; P < .001), indicating that 2 Exp+ alleles were associated with increased disease penetrance compared with 1 expansion. Potentially pathogenic variants (minor allele frequency, <0.01; combined annotation dependent depletion, >15) were only identified in FECD-associated genes in 13 Exp- individuals (10.1%). Conclusions and Relevance:In this multicenter cohort study among individuals with FECD, CTG18.1 expansions were present in most European and South Asian patients, while CTG18.1 repeat length and zygosity status were associated with modifications in disease severity and penetrance. Known disease-associated genes accounted for only a minority of Exp- cases, with unknown risk factors associated with disease in the rest of this subgroup. These data may have implications for future FECD gene-targeted therapy development.

The U4 small nuclear RNA (snRNA) forms a duplex with the U6 snRNA and, together with U5 and ~30 proteins, is part of the U4/U6.U5 tri-snRNP complex, located at the core of the major spliceosome. Recently, recurrent de novo variants in the U4 RNA, transcribed from the RNU4-2 gene, and in at least two other RNU genes were discovered to cause neurodevelopmental disorder. We detected inherited and de novo heterozygous variants in RNU4-2 (n.18_19insA and n.56T>C) and in four out of the five RNU6 paralogues (n.55_56insG and n.56_57insG) in 135 individuals from 62 families with non-syndromic retinitis pigmentosa (RP), a rare form of hereditary blindness. We show that these variants are recurrent among RP families and invariably cluster in close proximity within the three-way junction (between stem-I, the 5' stem-loop and stem-II) of the U4/U6 duplex, affecting its natural conformation. Interestingly, this region binds to numerous splicing factors of the tri-snRNP complex including PRPF3, PRPF8 and PRPF31, previously associated with RP as well. The U4 and U6 variants identified seem to affect snRNP biogenesis, namely the U4/U6 di-snRNP, which is an assembly intermediate of the tri-snRNP. Based on the number of positive cases observed, deleterious variants in RNU4-2 and in RNU6 paralogues could be a significant cause of isolated or dominant RP, accounting for up to 1.2% of all undiagnosed RP cases. This study highlights the role of non-coding genes in rare Mendelian disorders and uncovers pleiotropy in RNU4-2, where different variants underlie neurodevelopmental disorder and RP.

Whole-genome DNA methylation (methylome) analysis is of broad interest to biomedical research due to its central role in human development and disease. However, generating high-quality methylomes at scale remains challenging due to inherent technical limitations. While imputation has the potential to help overcome this problem, no existing approach adequately addresses the scaling issue. Here, we present GIMMEcpg (Global Imputation of Mean cpg MEthylation), a novel imputation tool that scales efficiently from single samples to large cohort studies. GIMMEcpg uses a custom feature dataset built from known CpG sites within the same dataset to impute missing values by calculating the distance-weighted mean of the methylation value of the two immediately neighbouring CpG sites. We benchmarked GIMMEcpg for speed and accuracy against multiple imputation methods using downsampled datasets produced from high-quality (~100x) Whole Genome Bisulfite Sequencing (WGBS) data. With a 10x downsampled dataset, GIMMEcpg was able to process the dataset and impute 9.14 Million CpG sites within 7 seconds (R: 0.78, MAE: +5.6%, RMSE: +10.9%). Our results demonstrate that GIMMEcpg is 39-2,562 times faster than three existing methylation imputation tools (BoostMe, DeepCpG, and MethImpute) while maintaining comparable accuracy. To quantify GIMMEcpg's scalability, we applied it to the most extensive single collection of WGBS data (N=645 at variable coverage) from the EpiATLAS generated by the International Human Epigenome Consortium (IHEC). Using a single, standard CPU server, GIMMEcpg processed and imputed an additional 2.4 billion CpG methylation values across the 645 datasets in less than a day, enriching the EpiATLAS methylome resource by 20%. This demonstrates that GIMMEcpg scales to large cohort studies with only a subtle impact on accuracy, as illustrated by our benchmark. We also developed a machine learning variant, GIMMEcpg.ml, which delivers a higher accuracy compared to existing methodologies. Using the same 10x downsampled benchmarking dataset, GIMMEcpg.ml achieved a Person Correlation of 0.87 compared to the ground truth, representing an improvement of 0.11 over the best performing alternative method. Additionally, GIMMEcpg.ml has a Mean Absolute Error (MAE) of 8.67%, which is 2.63% lower than the most accurate performing alternative. While this enhanced accuracy comes at the cost of increased computation requirements, GIMMEcpg.ml is a useful tool where higher accuracy is preferred over scalability. For increased accessibility, GIMMEcpg is freely available under an MIT license as R and Python packages at https://github.com/ucl-medical-genomics/gimmecpg-r and https://github.com/ucl-medical-genomics/gimmecpg-python, respectively. ### Competing Interest Statement The authors have declared no competing interest.

Purpose:Fuchs endothelial corneal dystrophy (FECD) is a common, age-related cause of visual impairment. This systematic review synthesizes evidence from the literature on artificial intelligence (AI) models developed for the diagnosis and management of FECD. Methods:We conducted a systematic literature search in MEDLINE, PubMed, Web of Science, and Scopus from January 1, 2000, to June 31, 2024. Full-text studies utilizing AI for various clinical contexts of FECD management were included. Data extraction covered model development, predicted outcomes, validation, and model performance metrics. We graded the included studies using the Quality Assessment of Diagnostic Accuracies Studies 2 tool. This review adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations. Results:Nineteen studies were analyzed. Primary AI algorithms applied in FECD diagnosis and management included neural network architectures specialized for computer vision, utilized on confocal or specular microscopy images, or anterior segment optical coherence tomography images. AI was employed in diverse clinical contexts, such as assessing corneal endothelium and edema and predicting post-corneal transplantation graft detachment and survival. Despite many studies reporting promising model performance, a notable limitation was that only three studies performed external validation. Bias introduced by patient selection processes and experimental designs was evident in the included studies. Conclusions:Despite the potential of AI algorithms to enhance FECD diagnosis and prognostication, further work is required to evaluate their real-world applicability and clinical utility. Translational Relevance:This review offers critical insights for researchers, clinicians, and policymakers, aiding their understanding of existing AI research in FECD management and guiding future health service strategies.

Genome analysis of individuals affected by retinitis pigmentosa (RP) identified two rare nucleotide substitutions at the same genomic location on chromosome 11 (g.61392563 [GRCh38]), 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 (c.-69G>A, c.-69G>T [GenBank: NM_001173991.3]), in individuals of South Asian and African ancestry, respectively. Genotypes included 71 homozygotes and 3 mixed heterozygotes in trans with a predicted loss-of-function allele. Haplotype analysis showed single-nucleotide variants (SNVs) common across families, suggesting ancestral alleles within the two distinct ethnic populations. Clinical phenotype analysis of 62 available individuals from 49 families indicated a similar clinical presentation with night blindness in the first decade and progressive peripheral field loss thereafter. No evident systemic ciliopathy features were noted. Functional characterization of these variants by luciferase reporter gene assay showed reduced promotor activity. Nanopore sequencing confirmed the lower transcription of the TMEM216 c.-69G>T allele in blood-derived RNA from a heterozygous carrier, and reduced expression was further recapitulated by qPCR, using both leukocytes-derived RNA of c.-69G>T homozygotes and total RNA from genome-edited hTERT-RPE1 cells carrying homozygous TMEM216 c.-69G>A. In conclusion, these variants explain a significant proportion of unsolved cases, specifically in individuals of African ancestry, suggesting that reduced TMEM216 expression might lead to abnormal ciliogenesis and photoreceptor degeneration.

Corneal dystrophies are phenotypically and genetically heterogeneous, often resulting in visual impairment caused by corneal opacification. We investigated the genetic cause of an autosomal dominant corneal stromal dystrophy in a pedigree with eight affected individuals in three generations. Affected individuals had diffuse central stromal opacity, with reduced visual acuity in older family members. Histopathology of affected cornea tissue removed during surgery revealed mild stromal textural alterations with alcianophilic deposits. Whole genome sequence data were generated for four affected individuals. No rare variants (MAF < 0.001) were identified in established corneal dystrophy genes. However, a novel heterozygous missense variant in exon 4 of SPARCL1, NM_004684: c.334G > A; p.(Glu112Lys), which is predicted to be damaging, segregated with disease. SPARC-like protein 1 (SPARCL1) is a secreted matricellular protein involved in cell migration, cell adhesion, tissue repair, and remodelling. Interestingly, SPARCL1 has been shown to regulate decorin. Heterozygous variants in DCN, encoding decorin, cause autosomal dominant congenital stromal corneal dystrophy, suggesting a common pathogenic pathway. Therefore, we performed immunohistochemistry to compare SPARCL1 and decorin localisation in corneal tissue from an affected family member and an unaffected control. Strikingly, the level of decorin was significantly decreased in the corneal stroma of the affected tissue, and SPARCL1 appeared to be retained in the epithelium. In summary, we describe a novel autosomal dominant corneal stromal dystrophy associated with a missense variant in SPARCL1, extending the phenotypic and genetic heterogeneity of inherited corneal disease.

IntroductionAutosomal dominant retinitis pigmentosa type 17 (adRP, type RP17) is caused by complex structural variants (SVs) affecting a locus on chromosome 17 (chr17q22). The SVs disrupt the 3D regulatory landscape by altering the topologically associating domain (TAD) structure of the locus, creating novel TAD structures (neo-TADs) and ectopic enhancer-gene contacts. Currently, screening for RP17-associated SVs is not included in routine diagnostics given the complexity of the variants and a lack of cost-effective detection methods. The aim of this study was to accurately detect novel RP17-SVs by establishing a systematic and efficient workflow.MethodsGenetically unexplained probands diagnosed with adRP (n = 509) from an international cohort were screened using a smMIPs or genomic qPCR-based approach tailored for the RP17 locus. Suspected copy number changes were validated using high-density SNP-array genotyping, and SV breakpoint characterization was performed by mutation-specific breakpoint PCR, genome sequencing and, if required, optical genome mapping. In silico modeling of novel SVs was performed to predict the formation of neo-TADs and whether ectopic contacts between the retinal enhancers and the GDPD1-promoter could be formed.ResultsUsing this workflow, potential RP17-SVs were detected in eight probands of which seven were confirmed. Two novel SVs were identified that are predicted to cause TAD rearrangement and retinal enhancer-GDPD1 contact, one from Germany (DE-SV9) and three with the same SV from the United States (US-SV10). Previously reported RP17-SVs were also identified in three Australian probands, one with UK-SV2 and two with SA-SV3.DiscussionIn summary, we describe a validated multi-step pipeline for reliable and efficient RP17-SV discovery and expand the range of disease-associated SVs. Based on these data, RP17-SVs can be considered a frequent cause of adRP which warrants the inclusion of RP17-screening as a standard diagnostic test for this disease.

AbstractHere, we demonstrate the utility of optical genome mapping (OGM) to interrogate the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (termed CTG18.1) and gain novel insights into the tissue-specific nature of the disease. Genomic DNA (gDNA) samples derived from peripheral blood leukocytes and primary corneal endothelial cells (CECs) were analysed by OGM. Concurrently, all samples were genotyped by standard PCR-based methods to classify their expansion status. Individuals with one or more CTG18.1-expanded alleles (≥50 CTG repeats) detected in their leukocyte-derived gDNA were classified as expansion-positive. A customised bioinformatics pipeline was developed to perform CTG18.1-targeted OGM analysis. All linearised gDNA molecules containing labels flanking CTG18.1 were extracted, corrected for the repeats on the reference human genome and sized. Analysis of paired bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin, but displayed significantly higher levels of instability within the diseased corneal endothelium. Clusters of CTG18.1 molecules of approximately 1,800-11,900 repeats, beyond the ranges observed in individual-matched leukocyte samples, were detected in all CEC gDNA samples from expansion-positive cases. In conclusion, OGM is a powerful method to analyse the somatically unstable CTG18.1 locus. More generally, this work exemplifies the broader utility of OGM in exploring somatically unstable short tandem repeat loci. Furthermore, this study has highlighted the extreme levels of tissue-specific CTG18.1 somatic instability occurring within the diseased corneal endothelium, which we hypothesise plays a pivotal role in driving downstream pathogenic mechanisms of CTG18.1-mediated FECD.

center dot PURPOSE: RP2 -associated retinopathy typically causes severe early onset retinitis pigmentosa (RP) in affected males. However, there is a scarcity of reports describing the clinical phenotype of female carriers. We tested the hypothesis that RP2 variants manifest in female carriers with a range of functional and anatomic characteristics. center dot DESIGN: Retrospective case series. center dot METHODS: Females with disease-causing variants in RP2 were identified from investigation of pedigrees affected by RP2 retinopathy. All case notes and results of molecular genetic testing, retinal imaging (fundus autofluorescence imaging, optical coherence tomography (OCT)), and electrophysiology were reviewed. center dot RESULTS: Forty pedigrees were investigated. Twentynine pedigrees had obligate carriers or molecularly confirmed female members with recorded relevant history and/or examination. For 8 pedigrees, data were available only from history, with patients reporting affected female relatives with RP in 4 cases and unaffected female relatives in the other 4 cases. Twenty-seven females from 21 pedigrees were examined by a retinal genetics specialist. Twenty-three patients (85%) reported no complaints and had normal vision and 4 patients had RP-associated complaints (15%). Eight patients had normal fundus examination (30%), 10 had a tapetal-like reflex (TLR; 37%), 5 had scattered peripheral pigmentation (19%), and the 4 symptomatic patients had fundus findings compatible with RP (15%). All asymptomatic patients with normal fundus, TLR, or asymptomatic pigmentary changes had a continuous ellipsoid zone on OCT when available. The electroretinograms revealed mild to severe photoreceptor dysfunction in 9 of 11 subjects, often asymmetrical, including 5 with pattern electroretinogram evidence of symmetrical (n = 4) or unilateral (n = 1 subject) macular dysfunction. center dot CONCLUSIONS: Most carriers were asymptomatic, exhibiting subclinical characteristics such as TLR and pigmentary changes. However, female carriers of RP2 variants can manifest RP. Family history of affected females with RP does not exclude X-linked disease. The phenotypic spectrum as described herein has prognostic and counselling implications for RP2 carriers and patients. (Am J Ophthalmol 2024;261: 112-120. (c) 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.