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Introduction: Autosomal 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., Methods: Genetically 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., Results: Using 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., Discussion: In 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 de Bruijn, Panneman, Weisschuh, Cadena, Boonen, Holtes, Astuti, Cremers, Leijsten, Corominas, Gilissen, Skowronska, Woodley, Beggs, Toulis, Chen, Cheetham, Hardcastle, McLaren, Lamey, Thompson, Chen, de Roach, Urwin, Sullivan, Roosing.)

Background: Fuchs endothelial corneal dystrophy (FECD) is the most common repeat-mediated disease in humans. It exclusively affects corneal endothelial cells (CECs), with ≤81% of cases associated with an intronic TCF4 triplet repeat (CTG18.1). Here, we utilise optical genome mapping (OGM) to investigate CTG18.1 tissue-specific instability to gain mechanistic insights., Methods: We applied OGM to a diverse range of genomic DNAs (gDNAs) from patients with FECD and controls (n = 43); CECs, leukocytes and fibroblasts. A bioinformatics pipeline was developed to robustly interrogate CTG18.1-spanning DNA molecules. All results were compared with conventional polymerase chain reaction-based fragment analysis., Findings: Analysis of bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin. However, clusters of CTG18.1 molecules, encompassing ∼1800-11,900 repeats, were exclusively detected in diseased CECs from expansion-positive cases. Additionally, both progenitor allele size and age were found to influence the level of leukocyte-specific CTG18.1 instability., Interpretation: OGM is a powerful tool for analysing somatic instability of repeat loci and reveals here the extreme levels of CTG18.1 instability occurring within diseased CECs underpinning FECD pathophysiology, opening up new therapeutic avenues for FECD. Furthermore, these findings highlight the broader translational utility of FECD as a model for developing therapeutic strategies for rarer diseases similarly attributed to somatically unstable repeats., Funding: UK Research and Innovation, Moorfields Eye Charity, Fight for Sight, Medical Research Council, NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Grantová Agentura České Republiky, Univerzita Karlova v Praze, the National Brain Appeal's Innovation Fund and Rosetrees Trust., Competing Interests: Declaration of interests The authors declare no competing interests. AED has previously acted as a paid consultant for Triplet Therapeutics Ltd, LoQus23 Therapeutics Ltd, Design Therapeutics Ltd and had a research collaboration with ProQR Therapeutics. AED has an ongoing research collaboration with Prime Medicine., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of interests All the authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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&#95;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., (© 2024. The Author(s).)

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., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: AED has previously acted as a paid consultant for Triplet Therapeutics Ltd, LoQus23 Therapeutics Ltd, Design Therapeutics Ltd and had a research collaboration with ProQR Therapeutics. AED’s has an ongoing research collaboration with Prime Medicine., (Copyright: © 2024 Bhattacharyya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Design: Retrospective case series., 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., Results: Forty pedigrees were investigated. Twenty-nine 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., 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., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: To characterise the phenotype and genotype of concurrent keratoconus and Fuchs endothelial corneal dystrophy (KC + FECD)., Methods: We recruited 20 patients with concurrent KC + FECD for a retrospective observational case series from the United Kingdom and the Czech Republic. We compared eight parameters of corneal shape (Pentacam, Oculus) with two groups of age-matched controls who had either isolated keratoconus (KC) or isolated FECD. We genotyped probands for an intronic triplet TCF4 repeat expansion (CTG18.1) and the ZEB1 variant c.1920G >T p.(Gln640His)., Results: The median age at diagnosis of patients with KC + FECD was 54 (interquartile range 46 to 66) years, with no evidence of KC progression (median follow-up 84 months, range 12 to 120 months). The mean (standard deviation (SD)) of the minimum corneal thickness, 493 (62.7) μm, was greater than eyes with KC, 458 (51.1) μm, but less than eyes with FECD, 590 (55.6) μm. Seven other parameters of corneal shape were more like KC than FECD. Seven (35%) probands with KC + FECD had a TCF4 repeat expansion of ≥50 compared to five controls with isolated FECD. The average of the largest TCF4 expansion in cases with KC + FECD (46 repeats, SD 36 repeats) was similar to the age-matched controls with isolated FECD (36 repeats, SD 28 repeats; p = 0.299). No patient with KC + FECD harboured the ZEB1 variant., Conclusions: The KC + FECD phenotype is consistent with KC but with superimposed stromal swelling from endothelial disease. The proportion of cases with a TCF4 expansion is similar in concurrent KC + FECD and age-matched controls with isolated FECD., (© 2023 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Purpose: To review and describe in detail the clinical course, functional and anatomic characteristics of RP2-associated retinal degeneration., Design: Retrospective case series., Participants: Male participants with disease-causing variants in the RP2 gene., Methods: Review of all case notes and results of molecular genetic testing, retinal imaging (fundus autofluorescence [FAF] imaging, OCT), and electrophysiology assessment., Main Outcome Measures: Molecular genetic testing, clinical findings including best-corrected visual acuity (BCVA), qualitative and quantitative retinal imaging analysis, and electrophysiology parameters., Results: Fifty-four molecularly confirmed patients were identified from 38 pedigrees. Twenty-eight disease-causing variants were identified, with 20 not previously clinically characterized. Fifty-three patients (98.1%) presented with retinitis pigmentosa. The mean age of onset (range ± standard deviation [SD]) was 9.6 years (1-57 ± 9.2 years). Forty-four patients (91.7%) had childhood-onset disease, with mean age of onset of 7.6 years. The most common first symptom was night blindness (68.8%). Mean BCVA (range ± SD) was 0.91 logarithm of the minimum angle of resolution (logMAR) (0-2.7 ± 0.80) and 0.94 logMAR (0-2.7 ± 0.78) for right and left eyes, respectively. On the basis of the World Health Organization visual impairment criteria, 18 patients (34%) had low vision. The majority (17/22) showed electroretinogram (ERG) evidence of a rod-cone dystrophy. Pattern ERG P50 was undetectable in all but 2 patients. A range of FAF findings was observed, from normal to advanced atrophy. There were no statistically significant differences between right and left eyes for ellipsoid zone width (EZW) and outer nuclear layer (ONL) thickness. The mean annual rate of EZW loss was 219 μm/year, and the mean annual decrease in ONL thickness was 4.93 μm/year. No patient with childhood-onset disease had an identifiable ellipsoid zone (EZ) after the age of 26 years at baseline or follow-up. Four patients had adulthood-onset disease and a less severe phenotype., Conclusions: This study details the clinical phenotype of RP2 retinopathy in a large cohort. The majority presented with early-onset severe retinal degeneration, with early macular involvement and complete loss of the foveal photoreceptor layer by the third decade of life. Full-field ERGs revealed rod-cone dystrophy in the vast majority, but with generalized (peripheral) cone system involvement of widely varying severity in the first 2 decades of life., Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references., (Copyright © 2022 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To characterize the phenotype observed in a case series with macular disease and determine the cause., Design: Multicenter case series., Participants: Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration., Methods: Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing., Main Outcome Measures: Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients., Results: All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor., Conclusions: Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease., (Copyright © 2022 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: Blue cone monochromacy (BCM) is a rare inherited cone disorder in which both long- (L-) and middle- (M-) wavelength sensitive cone classes are either impaired or nonfunctional. Assessing genotype-phenotype relationships in BCM can improve our understanding of retinal development in the absence of functional L- and M-cones. Here we examined foveal cone structure in patients with genetically-confirmed BCM, using adaptive optics scanning light ophthalmoscopy (AOSLO)., Methods: Twenty-three male patients (aged 6-75 years) with genetically-confirmed BCM were recruited for high-resolution imaging. Eight patients had a deletion of the locus control region (LCR), and 15 had a missense mutation-Cys203Arg-affecting the first two genes in the opsin gene array. Foveal cone structure was assessed using confocal and non-confocal split-detection AOSLO across a 300 × 300 µm area, centered on the location of peak cell density., Results: Only one of eight patients with LCR deletions and 10 of 15 patients with Cys203Arg mutations had analyzable images. Mean total cone density for Cys203Arg patients was 16,664 ± 11,513 cones/mm2 (n = 10), which is, on average, around 40% of normal. Waveguiding cone density was 2073 ± 963 cones/mm2 (n = 9), which was consistent with published histological estimates of S-cone density in the normal eye. The one patient with an LCR deletion had a total cone density of 10,246 cones/mm2 and waveguiding density of 1535 cones/mm2., Conclusions: Our results show that BCM patients with LCR deletions and Cys203Arg mutations have a population of non-waveguiding photoreceptors, although the spectral identity and level of function remain unknown.

Purpose: To generate a prognostic model to predict keratoconus progression to corneal crosslinking (CXL)., Design: Retrospective cohort study., Methods: We recruited 5025 patients (9341 eyes) with early keratoconus between January 2011 and November 2020. Genetic data from 926 patients were available. We investigated both keratometry or CXL as end points for progression and used the Royston-Parmar method on the proportional hazards scale to generate a prognostic model. We calculated hazard ratios (HRs) for each significant covariate, with explained variation and discrimination, and performed internal-external cross validation by geographic regions., Results: After exclusions, model fitting comprised 8701 eyes, of which 3232 underwent CXL. For early keratoconus, CXL provided a more robust prognostic model than keratometric progression. The final model explained 33% of the variation in time to event: age HR (95% CI) 0.9 (0.90-0.91), maximum anterior keratometry 1.08 (1.07-1.09), and minimum corneal thickness 0.95 (0.93-0.96) as significant covariates. Single-nucleotide polymorphisms (SNPs) associated with keratoconus (n=28) did not significantly contribute to the model. The predicted time-to-event curves closely followed the observed curves during internal-external validation. Differences in discrimination between geographic regions was low, suggesting the model maintained its predictive ability., Conclusions: A prognostic model to predict keratoconus progression could aid patient empowerment, triage, and service provision. Age at presentation is the most significant predictor of progression risk. Candidate SNPs associated with keratoconus do not contribute to progression risk., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third ( n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Purpose: The aim of the study was to describe the phenotype and molecular genetic causes of X-linked megalocornea (MGC1). We recruited four British, one New Zealand, one Vietnamese and four Czech families., Methods: All probands and three female carriers underwent ocular examination and Sanger sequencing of the CHRDL1 gene. Two of the probands also had magnetic resonance imaging (MRI) of the brain., Results: We identified nine pathogenic or likely pathogenic and one variant of uncertain significance in CHRDL1, of which eight are novel. Three probands had ocular findings that have not previously been associated with MGC1, namely pigmentary glaucoma, unilateral posterior corneal vesicles, unilateral keratoconus and unilateral Fuchs heterochromic iridocyclitis. The corneal diameters of the three heterozygous carriers were normal, but two had abnormally thin corneas, and one of these was also diagnosed with unilateral keratoconus. Brain MRI identified arachnoid cysts in both probands, one also had a neuroepithelial cyst, while the second had a midsagittal neurodevelopmental abnormality (cavum septum pellucidum et vergae)., Conclusion: The study expands the spectrum of pathogenic variants and the ocular and brain abnormalities that have been identified in individuals with MGC1. Reduced corneal thickness may represent a mild phenotypic feature in some heterozygous female carriers of CHRDL1 pathogenic variants., (© 2021 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: To compare foveal hypoplasia and the appearance of the ellipsoid zone (EZ) at the fovea in patients with genetically confirmed achromatopsia (ACHM) and blue cone monochromacy (BCM)., Design: Retrospective, multi-center observational study., Subjects: Molecularly confirmed patients with ACHM (n = 89) and BCM (n = 33)., Methods: We analyzed high-resolution spectral domain optical coherence tomography (SD-OCT) images of the macula from aforementioned patients with BCM. Three observers independently graded SD-OCT images for foveal hypoplasia (i.e. retention of one or more inner retinal layers at the fovea) and four observers judged the integrity of the EZ at the fovea, based on an established grading scheme. These measures were compared with previously published data from the ACHM patients., Main Outcome Measures: Presence of foveal hypoplasia and EZ grade., Results: Foveal hypoplasia was significantly more prevalent in ACHM than in BCM (p<0.001). In addition, we observed a significant difference in the distribution of EZ grades between ACHM and BCM, with grade II EZ being by far the most common phenotype in BCM (61% of patients). In contrast, ACHM patients had a relatively equal prevalence of EZ grades I, II, and IV. Interestingly, grade IV EZ was 2.6 times more prevalent in ACHM compared to BCM, while grade V EZ (macular atrophy) was present in 3% of both the ACHM and BCM cohorts., Conclusions: The higher incidence of foveal hypoplasia in ACHM than BCM supports a role for cone activity in foveal development. Although there are differences in EZ grades between these conditions, the degree of overlap suggests EZ grade is not sufficient for definitive diagnosis, in contrast to previous reports. Analysis of additional OCT features in similar cohorts may reveal differences with greater diagnostic value. Finally, the extent to which foveal hypoplasia or EZ grade is prognostic for therapeutic potential in either group remains to be seen, but motivates further study.

Purpose: Over a third of patients with Acanthamoeba keratitis (AK) experience severe inflammatory complications (SICs). This study aimed to determine if some contact lens (CL) wearers with AK were predisposed to SICs due to variations in key immune genes., Methods: CL wearers with AK who attended Moorfields Eye Hospital were recruited prospectively between April 2013 and October 2014. SICs were defined as scleritis and/or stromal ring infiltrate. Genomic DNA was processed with an Illumina Low Input Custom Amplicon assay of 58 single nucleotide polymorphism (SNP) targets across 18 genes and tested for association in PLINK., Results: Genomic DNA was obtained and analyzed for 105 cases of AK, 40 (38%) of whom experienced SICs. SNPs in the CXCL8 gene encoding IL-8 was significantly associated with protection from SICs (chr4: rs1126647, odds ratio [OR] = 0.3, P = 0.005, rs2227543, OR = 0.4, P = 0.007, and rs2227307, OR = 0.4, P = 0.02) after adjusting for age, sex, steroids prediagnosis, and herpes simplex keratitis (HSK) misdiagnosis. Two TLR-4 SNPs were associated with increased risk of SICs (chr9: rs4986791 and rs4986790, both OR = 6.9, P = 0.01). Th-17 associated SNPs (chr1: IL-23R rs11209026, chr2: IL-1β rs16944, and chr12: IL-22 rs1179251) were also associated with SICs., Conclusions: The current study identifies biologically relevant genetic variants in patients with AK with SICs; IL-8 is associated with a strong neutrophil response in the cornea in AK, TLR-4 is important in early AK disease, and Th-17 genes are associated with adaptive immune responses to AK in animal models. Genetic screening of patients with AK to predict severity is viable and this would be expected to assist disease management.

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease.

The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

RP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2-associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene-edited isogenic RP2 knockout (RP2 KO) induced pluripotent stem cells (iPSCs) and RP2 patient-derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient-derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. Adeno-associated virus-mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Central corneal thickness (CCT) is one of the most heritable human traits, with broad-sense heritability estimates ranging between 0.68 to 0.95. Despite the high heritability and numerous previous association studies, only 8.5% of CCT variance is currently explained. Here, we report the results of a multiethnic meta-analysis of available genome-wide association studies in which we find association between CCT and 98 genomic loci, of which 41 are novel. Among these loci, 20 were significantly associated with keratoconus, and one (RAPSN rs3740685) was significantly associated with glaucoma after Bonferroni correction. Two-sample Mendelian randomization analysis suggests that thinner CCT does not causally increase the risk of primary open-angle glaucoma. This large CCT study explains up to 14.2% of CCT variance and increases substantially our understanding of the etiology of CCT variation. This may open new avenues of investigation into human ocular traits and their relationship to the risk of vision disorders.

Purpose: To describe the natural history of Leber congenital amaurosis (LCA) associated with GUCY2D variants (GUCY2D-LCA) in a cohort of children and adults, in preparation for trials of novel therapies., Design: Retrospective case series., Methods: Participants: Patients with GUCY2D-LCA at a single referral center., Procedures: Review of clinical notes, retinal imaging including fundus autofluorescence (FAF) and optical coherence tomography (OCT), electroretinography (ERG), and molecular genetic testing., Main Outcome Measures: Demographic data, symptoms at presentation, visual acuity, evidence of progression, OCT and FAF findings, ERG assessment, and molecular genetics., Results: Twenty-one subjects with GUCY2D-LCA were included, with a mean follow-up ± standard deviation (SD) of 10 ± 11.85 years. Marked reduction in visual acuity (VA) and nystagmus was documented in all patients within the first 3 years of life. Fifty-seven percent (n = 12) exhibited photophobia and 38% (n = 8) had nyctalopia. VA was worse than hand motion in 71% of the patients (n = 15). Longitudinal assessment of VA showed stability in all patients, except 1 patient who experienced deterioration over a follow-up of 44 years. Hyperopia was reported in 13 of the 17 subjects (71%) with available refraction data. Eighteen subjects had either normal fundus appearance (n = 14) or a blond fundus (n = 3), while only 4 of the eldest subjects had mild retinal pigment epithelium (RPE) atrophy (mean, 49 years; range 40-54 years). OCT data were available for 11 subjects and 4 different grades of ellipsoid zone (EZ) integrity were identified: (1) continuous/intact EZ (n = 6), (2) focally disrupted EZ (n = 2), (3) focally disrupted with RPE changes (n = 2), and (4) diffuse EZ disruption with RPE changes (n = 1). All examined subjects had stable OCT findings over the long follow-up period. Full-field ERGs showed evidence of a severe cone-rod dystrophy in 5 of 6 patients and undetectable ERGs in 1 subject. Novel genotype-phenotype correlations are also reported., Conclusion: GUCY2D-LCA is a severe early-onset retinal dystrophy associated with very poor VA from birth. Despite the severely affected photoreceptor function, the relatively preserved photoreceptor structure based on EZ integrity until late in the disease in the majority of subjects suggests a wide therapeutic window for gene therapy trials., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Name of the disease (synonyms) CUGC for posterior polymorphous corneal dystrophy (PPCD).OMIM# of the disease 122000; 609141; 618031.Name of the analysed genes or DNA/chromosome segments OVOL2 (PPCD1); ZEB1 (PPCD3); GRHL2 (PPCD4).OMIM# of the gene(s) 616441; 189909; 608576. Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for variants in theOVOL2, ZEB1andGRHL2gene(s) in a diagnostic setting, predictive and parental settings and for risk assesment in relatives.

Purpose: To characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients., Design: Multicenter case series, in vitro experimentation, and retrospective analysis of previously reported variants., Participants: Seven families with apparently autosomal dominant EOMD., Methods: Patients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed., Main Outcome Measures: Detailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD., Results: All affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1G→T het; c.619+1G→A het, c.380G→A, p.(Arg127His) het; c.694C→T p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291T→A, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch's membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1 R127H , FHL-1 A415f/s , and FHL-1 C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1., Conclusions: Early-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities., (Copyright © 2019 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Importance: Keratoconus is an important cause of visual loss in young adults, but little is known about its genetic causes. Understanding the genetic determinants of corneal biomechanical factors may in turn teach us about keratoconus etiology., Objectives: To identify genetic associations with corneal biomechanical properties and to examine whether these genetic variants are associated with keratoconus., Design, Setting, and Participants: A stage 1 discovery and replication genome-wide association study (GWAS) of corneal biomechanical properties was performed in 2 cross-sectional populations (6645 participants from the European Prospective Investigation into Cancer and Nutrition [EPIC]-Norfolk Eye Study and 2384 participants from the TwinsUK study). In stage 2, the association of genetic determinants identified in stage 1 with keratoconus was examined in a case-control study. A total of 752 patients with keratoconus were compared with 974 TwinsUK participants (undergoing direct sequencing) or 13 828 EPIC-Norfolk participants (undergoing genotyping and imputation) who were not part of the stage 1 analysis. Data were collected from March 1, 1993, through March 13, 2017, and analyzed from November 1, 2015, through February 1, 2018., Exposures: In stage 1, allele dosage at genome-wide single-nucleotide polymorphisms (SNPs); in stage 2, allele dosage at SNPs with genome-wide significance (P < 5 × 10-8) in stage 1 and not previously reported as associated with corneal disease., Main Outcomes and Measures: In stage 1, corneal hysteresis (CH) and corneal resistance factor (CRF), measured with the Ocular Response Analyzer (ORA); in stage 2, association with keratoconus compared with controls., Results: Among 6645 participants in the discovery cohort (3635 women (54.7%); mean age, 69 years [range, 48-92 years]), 7 genome-wide significant loci associated with CH or CRF were identified that were independently replicated. Two further suggestive loci were identified after meta-analysis. To date, 5 of the identified loci, at ANAPC1, ADAMTS8, ADAMTS17, ABCA6, and COL6A1, have not previously been reported as associated with corneal disease. The ABCA6 locus (rs77542162) was associated with keratoconus using the TwinsUK (odds ratio [OR], 0.50; 95% CI, 0.27-0.92; P = .03) and EPIC-Norfolk controls (OR, 0.39; 95% CI, 0.22-0.70; P = .002). The other loci were associated with keratoconus using TwinsUK (OR per effect allele for ADAMTS8, 0.51 [95% CI, 0.37-0.71; P = 7.9 × 10-5]; for COL6A1, 1.65 [95% CI, 1.05-2.59; P = .03]) or EPIC-Norfolk (OR per effect allele for ANAPC1, 0.78 [95% CI, 0.68-0.89; P = 3.7 × 10-4]; for ADAMTS17, 0.82 [95% CI, 0.68-0.99; P = .04]) controls., Conclusions and Relevance: Five loci that are associated with corneal biomechanical properties and that have suggestive associations with keratoconus were reported. These findings suggest the role of type VI collagen, extracellular matrix, and connective-tissue development for corneal biomechanics and keratoconus and the role of CH and CRF as biomarkers for keratoconus.

Purpose: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1)., Methods: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis., Results: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically., Conclusion: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.

The aim of this study was to identify the molecular genetic cause of disease in posterior polymorphous corneal dystrophy (PPCD) probands of diverse origin and to assess the utility of massively parallel sequencing in the detection of ZEB1 mutations. We investigated a total of 12 families (five British, four Czech, one Slovak and two Swiss). Ten novel and two recurrent disease-causing mutations in ZEB1, were identified in probands by Sanger (n = 5), exome (n = 4) and genome (n = 3) sequencing. Sanger sequencing was used to confirm the mutations detected by massively parallel sequencing, and to perform segregation analysis. Genome sequencing revealed that one proband harboured a novel ∼0.34 Mb heterozygous de novo deletion spanning exons 1-7 and part of exon 8. Transcript analysis confirmed that the ZEB1 transcript is detectable in blood-derived RNA samples and that the disease-associated variant c.482-2A>G leads to aberrant pre-mRNA splicing. De novo mutations, which are a feature of PPCD3, were found in the current study with an incidence rate of at least 16.6%. In general, massively parallel sequencing is a time-efficient way to detect PPCD3-associated mutations and, importantly, genome sequencing enables the identification of full or partial heterozygous ZEB1 deletions that can evade detection by both Sanger and exome sequencing. These findings contribute to our understanding of PPCD3, for which currently, 49 pathogenic variants have been identified, all of which are predicted to be null alleles., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The purpose of this study was to identify the genetic cause and describe the clinical phenotype of Schnyder corneal dystrophy (SCD) in six unrelated probands., Methods: We identified two white Czech, two white British and two South Asian families with a clinical diagnosis of SCD. Ophthalmic assessment included spectral domain optical coherence tomography (SD-OCT) of one individual with advanced disease, and SD-OCT and confocal microscopy of a child with early stages of disease. UBIAD1 coding exons were amplified and Sanger sequenced in each proband. A fasting serum lipid profile was measured in three probands. Paternity testing was performed in one family., Results: A novel heterozygous c.527G>A; p.(Gly176Glu) mutation in UBIAD1 was identified in one Czech proband. In the second Czech proband, aged 6 years when first examined, a previously described de novo heterozygous c.289G>A; p.(Ala97Thr) mutation was found. Two probands of South Asian descent carried a known c.305G>A; p.(Asn102Ser) mutation in the heterozygous state. Previously reported heterozygous c.361C>T; p.(Leu121Phe) and c.308C>T; p.(Thr103Ile) mutations were found in two white British families. Although crystalline deposits were present in all probands the affected area was small in some individuals. Corneal arcus and stromal haze were the most prominent phenotypical feature in two probands. In the Czech probands, SD-OCT confirmed accumulation of reflective material in the anterior stroma. Crystalline deposits were visualized by confocal microscopy. Mild dyslipidemia was found in all three individuals tested., Conclusion: Although de novo occurrence of mutations in UBIAD1 is extremely rare, SCD should be considered in the differential diagnosis of bilateral corneal haze and/or crystal deposition, especially in children.

The following review summarizes the state of the art in representative aspects of gene therapy/translational medicine and evolves from a symposium held at the School of Veterinary Medicine, University of Pennsylvania on November 16, 2017 honoring Dr. Gustavo Aguirre, recipient of ARVO's 2017 Proctor Medal. Focusing on the retina, speakers highlighted current work on moving therapies for inherited retinal degenerative diseases from the laboratory bench to the clinic.

Purpose: Mutations in ARL2BP, encoding ADP-ribosylation factor-like 2 binding protein, have recently been implicated as a cause of autosomal recessive retinitis pigmentosa (arRP), with three homozygous variants identified to date. In this study, we performed next-generation sequencing to reveal additional arRP cases associated with ARL2BP variants., Methods: Whole-genome sequencing (WGS) or whole-exome sequencing (WES) was performed in 1,051 unrelated individuals recruited for the UK Inherited Retinal Disease Consortium and NIHR-BioResource Rare Diseases research studies. Sanger sequencing was used to validate the next-generation sequencing data, and reverse transcriptase (RT)-PCR analysis was performed on RNA extracted from blood from affected individuals to test for altered splicing of ARL2BP . Detailed phenotyping was performed, including clinical evaluation, electroretinography, fundus photography, fundus autofluorescence imaging, and spectral-domain optical coherence tomography., Results: Homozygous variants in ARL2BP (NM&#95;012106.3) were identified in two unrelated individuals with RP. The variants, c.207+1G>A and c.390+5G>A, at conserved splice donor sites for intron 3 and intron 5, respectively, were predicted to alter the pre-mRNA splicing of ARL2BP . RT-PCR spanning the affected introns revealed that both variants caused abnormal splicing of ARL2BP in samples from affected individuals., Conclusions: This study identified two homozygous variants in ARL2BP as a rare cause of arRP. Further studies are required to define the underlying disease mechanism causing retinal degeneration as a result of mutations in ARL2BP and any phenotype-genotype correlation associated with residual levels of the wild-type transcript.

Purpose: To assess residual cone structure in subjects with mutations in exon 2, 3, and 4 of the OPN1LW or OPN1MW opsin., Methods: Thirteen males had their OPN1LW/OPN1MW opsin genes characterized. The cone mosaic was imaged using both confocal and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO), and retinal thickness was evaluated using optical coherence tomography (OCT). Six subjects completed serial imaging over a maximum period of 18 months and cone density was measured across imaging sessions., Results: Ten subjects had an OPN1LW/OPN1MW "interchange" opsin mutation designated as LIAVA or LVAVA, which both introduce exon 3 splicing defects leading to a lack of functional photopigment in cones expressing LIAVA and greatly reduced functional photopigment in cones expressing LVAVA. Despite disrupted cone reflectivity and reduced numerosity, residual inner segments could be visualized. Similar patterns were observed in individuals with an exon 2 insertion, or an exon 4 splice defect, both of which are also expected to produce cones that are devoid of functional opsin protein. OCT revealed variably reduced retinal thickness. A significant inverse relationship was found between the proportion of waveguiding cones and axial length., Conclusions: Split-detection imaging revealed that the altered appearance of the cone mosaic in confocal images for subjects with exon 2, 3, and 4 mutations was generally due to disrupted waveguiding, rather than structural loss, making them possible candidates for gene therapy to restore cone function. The relative fraction of waveguiding cones was highly variable across subjects, which appears to influence emmetropization in these subjects.

Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Retinal dystrophies are a heterogeneous group of disorders of visual function leading to partial or complete blindness. We report the genetic basis of an unusual retinal dystrophy in five families with affected females and no affected males. Heterozygous missense variants were identified in the X-linked phosphoribosyl pyrophosphate synthetase 1 (PRPS1) gene: c.47C > T, p.(Ser16Phe); c.586C > T, p.(Arg196Trp); c.641G > C, p.(Arg214Pro); and c.640C > T, p.(Arg214Trp). Missense variants in PRPS1 are usually associated with disease in male patients, including Arts syndrome, Charcot-Marie-Tooth, and nonsyndromic sensorineural deafness. In our study families, affected females manifested a retinal dystrophy with interocular asymmetry. Three unrelated females from these families had hearing loss leading to a diagnosis of Usher syndrome. Other neurological manifestations were also observed in three individuals. Our data highlight the unexpected X-linked inheritance of retinal degeneration in females caused by variants in PRPS1 and suggest that tissue-specific skewed X-inactivation or variable levels of pyrophosphate synthetase-1 deficiency are the underlying mechanism(s). We speculate that the absence of affected males in the study families suggests that some variants may be male embryonic lethal when inherited in the hemizygous state. The unbiased nature of next-generation sequencing enables all possible modes of inheritance to be considered for association of gene variants with novel phenotypic presentation., (© 2017 Wiley Periodicals, Inc.)

Summary: Phenopolis is an open-source web server providing an intuitive interface to genetic and phenotypic databases. It integrates analysis tools such as variant filtering and gene prioritization based on phenotype. The Phenopolis platform will accelerate clinical diagnosis, gene discovery and encourage wider adoption of the Human Phenotype Ontology in the study of rare genetic diseases., Availability and Implementation: A demo of the website is available at https://phenopolis.github.io . If you wish to install a local copy, source code and installation instruction are available at https://github.com/phenopolis . The software is implemented using Python, MongoDB, HTML/Javascript and various bash shell scripts., Contact: n.pontikos@ucl.ac.uk., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Ciliary trafficking defects are the underlying cause of many ciliopathies, including Retinitis Pigmentosa (RP). Anterograde intraflagellar transport (IFT) is mediated by kinesin motor proteins; however, the function of the homodimeric Kif17 motor in cilia is poorly understood, whereas Kif7 is known to play an important role in stabilizing cilia tips. Here we identified the ciliary tip kinesins Kif7 and Kif17 as novel interaction partners of the small GTPase Arl3 and its regulatory GTPase activating protein (GAP) Retinitis Pigmentosa 2 (RP2). We show that Arl3 and RP2 mediate the localization of GFP-Kif17 to the cilia tip and competitive binding of RP2 and Arl3 with Kif17 complexes. RP2 and Arl3 also interact with another ciliary tip kinesin, Kif7, which is a conserved regulator of Hedgehog (Hh) signaling. siRNA-mediated loss of RP2 or Arl3 reduced the level of Kif7 at the cilia tip. This was further validated by reduced levels of Kif7 at cilia tips detected in fibroblasts and induced pluripotent stem cell (iPSC) 3D optic cups derived from a patient carrying an RP2 nonsense mutation c.519C > T (p.R120X), which lack detectable RP2 protein. Translational read-through inducing drugs (TRIDs), such as PTC124, were able to restore Kif7 levels at the ciliary tip of RP2 null cells. Collectively, our findings suggest that RP2 and Arl3 regulate the trafficking of specific kinesins to cilia tips and provide additional evidence that TRIDs could be clinically beneficial for patients with this retinal degeneration., (© The Author 2017. Published by Oxford University Press.)

Pre-mRNA splicing factors play a fundamental role in regulating transcript diversity both temporally and spatially. Genetic defects in several spliceosome components have been linked to a set of non-overlapping spliceosomopathy phenotypes in humans, among which skeletal developmental defects and non-syndromic retinitis pigmentosa (RP) are frequent findings. Here we report that defects in spliceosome-associated protein CWC27 are associated with a spectrum of disease phenotypes ranging from isolated RP to severe syndromic forms. By whole-exome sequencing, recessive protein-truncating mutations in CWC27 were found in seven unrelated families that show a range of clinical phenotypes, including retinal degeneration, brachydactyly, craniofacial abnormalities, short stature, and neurological defects. Remarkably, variable expressivity of the human phenotype can be recapitulated in Cwc27 mutant mouse models, with significant embryonic lethality and severe phenotypes in the complete knockout mice while mice with a partial loss-of-function allele mimic the isolated retinal degeneration phenotype. Our study describes a retinal dystrophy-related phenotype spectrum as well as its genetic etiology and highlights the complexity of the spliceosomal gene network., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Importance: Steroid 5α-reductase type 3 congenital disorder of glycosylation (SRD5A3-CDG) is a rare disorder of N-linked glycosylation. Its retinal phenotype is not well described but could be important for disease recognition because it appears to be a consistent primary presenting feature., Objective: To investigate a series of patients with the same mutation in the SRD5A3 gene and thereby characterize its retinal manifestations and other associated features., Design, Setting and Participants: Seven affected individuals from 4 unrelated families with early-onset retinal dystrophy as a primary manifestation underwent comprehensive ophthalmic assessment, including retinal imaging and electrodiagnostic testing. Developmental and systemic findings were also recorded. Molecular genetic approaches, including targeted next-generation sequencing, autozygosity mapping, and apex microarray, were tried to reach a diagnosis; all participants were mutation negative. Whole-exome sequencing or whole-genome sequencing was used to identify the causative variant. Biochemical profiling was conducted to confirm a CDG type I defect. Patient phenotype data were collected over the course of ophthalmic follow-up, spanning a period of 20 years, beginning March 20, 1997, through September 15, 2016., Main Outcomes and Measures: Detailed clinical phenotypes as well as genetic and biochemical results., Results: The cohort consisted of 7 participants (5 females and 2 males) whose mean (SD) age at the most recent examination was 17.1 (3.9) years and who were all of South Asian ethnicity. Whole-exome sequencing and whole-genome sequencing identified the same homozygous SRD5A3 c.57G>A, p.(Trp19Ter) variant as the underlying cause of early-onset retinal dystrophy in each family. Detailed ocular phenotyping identified early-onset (aged ≤3 years) visual loss (mean [SD] best-corrected visual acuity, +0.95 [0.34] logMAR [20/180 Snellen]), childhood-onset nyctalopia, myopia (mean [SD] refractive error, -6.71 [-4.22]), and nystagmus. Six of the 7 patients had learning difficulties and psychomotor delay. Fundus autofluorescence imaging and optical coherence tomographic scans were abnormal in all patients, and electrodiagnostic testing revealed rod and cone dysfunction in the 5 patients tested., Conclusions and Relevance: Mutations in the SRD5A3 gene may cause early-onset retinal dystrophy, a previously underdescribed feature of the SRD5A3-CDG disorder that is progressive and may lead to serious visual impairment. SRD5A3 and other glycosylation disorder genes should be considered as a cause of retinal dystrophy even when systemic features are mild. Further delineation of SRD5A3-associated eye phenotypes can help inform genetic counseling for prognostic estimation of visual loss and disease progression.

Mutations in GCNT2 have been associated with the rare adult i blood group phenotype with or without congenital cataract. We report a novel homozygous frameshift mutation c.1163&#95;1166delATCA, p.(Asn388Argfs*20) as the cause of congenital cataract in two affected siblings. Blood group typing confirmed that both affected males have the rare adult i phenotype, supporting the hypothesis that the partial association of I/i phenotype and congenital cataract is due to the differential expression of GCNT2 isoforms., Competing Interests: The authors declare no conflict of interest.

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs ∗ 63), c.1996C>T (p.Arg666 ∗ ), c.2632G>T (p.Glu878 ∗ ), and c.2738&#95;2761del (p.Arg913&#95;Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352&#95;2353insC (p.Arg785Glnfs ∗ 23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Purpose: The majority of anterior corneal dystrophies are caused by dominant mutations in TGFBI (transforming growth factor β-induced) collectively known as the epithelial-stromal TGFBI dystrophies. Most cases of epithelial basement membrane dystrophy (EBMD) are thought to result from a degenerative (nongenetic) process; however, a minority of cases are associated with specific TGFBI mutations. We evaluated the spectrum of TGFBI mutations and associated phenotypes in a United Kingdom cohort with typical epithelial-stromal TGFBI dystrophies and an EBMD cohort., Methods: We recruited 68 probands with a clinical diagnosis of epithelial-stromal TGFBI dystrophy and 23 probands with bilateral EBMD. DNA was extracted from peripheral leukocytes, and TGFBI was bi-directly Sanger sequenced., Results: Nine TGFBI mutations were identified. The most common occurred at the mutation hot-spot residues R124 and R555 in 61 probands; these individuals had a genotype-phenotype correlation consistent with prior reports. Four probands with lattice corneal dystrophy carried a mutation in exon 14: p.(A620D), p.(V625D), and p.(H626R). We identified a p.(G623D) mutation in five probands, including two probands from the EBMD cohort. These subjects typically had an onset of severe recurrent corneal epithelial erosion in the fourth decade with mild diffuse or geographic subepithelial corneal opacities and only small anterior stromal lattice structures in older individuals. Symptoms of painful epithelial erosion improved markedly following phototherapeutic keratectomy., Conclusions: There was a strong correlation between genotype and phenotype for the majority of TGFBI mutations. In this cohort, the p.(G623D) mutation caused a greater proportion of TGFBI-associated disease than anticipated, associated with variable phenotypes including individuals diagnosed with EBMD.

Retinitis pigmentosa GTPase regulator (RPGR) gene sequence variants account for the vast majority of X linked retinitis pigmentosa (RP), which is one of the most severe forms of RP. Symptoms of nyctalopia typically begin in childhood, with increasing loss of peripheral visual field during teenage years, and progressive central visual loss during the second to fourth decade of life. There is however marked intrafamilial and interfamilial phenotypic heterogeneity in affected males and carrier females. There is now a far greater understanding of the range of phenotypes associated with variants in this gene; including rod-cone dystrophy, cone-rod dystrophy, cone dystrophy, macular dystrophy and non-ocular phenotypes. There are also increasingly established genotype-phenotype associations and structure-function correlations. RPGR is involved in ciliary function, with ciliary dysfunction now recognised as the mechanism underlying a large proportion of inherited retinal disease. There has been significant progress in identifying naturally occurring animal models and developing novel models to define the underlying disease mechanisms and to test gene replacement therapy, in addition to advances in human retinal imaging, culminating in completed and planned clinical trials. These significant developments will be discussed., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Purpose: To describe the clinical signs of gelatinous drop-like corneal dystrophy (GDLD) in a consanguineous Colombian family and determine the underlying genetic cause., Methods: We performed ocular examination of available family members and bidirectionally Sanger sequenced the GDLD-associated gene, TACSTD2. In one individual, the presence of subepithelial amyloid was confirmed with biopsy., Results: The parents were consanguineous and 5 of their 10 children had GDLD. Typical mulberry subepithelial deposits with subepithelial vascularization were present in 3 individuals; 2 individuals only had mild polymorphic anterior stromal opacity. We identified a homozygous TACSTD2 missense mutation, c.551A>G, p.(Tyr184Cys), in the affected family members. Both parents were heterozygous for the mutation, and unaffected siblings were either heterozygous or homozygous wild-type for this allele. In the Colombian population, this mutation has a minor allele frequency of 0.53%., Conclusion: The clinical presentation of GDLD in this family was variable and does not solely support an age-dependent progression of the phenotype, suggesting that environmental or other genetic factors can modify phenotypic expression. The relatively high prevalence of this mutation in the Colombian population suggests that other individuals may have undiagnosed subclinical disease.

Importance: Mutations in phototransduction and retinal signaling genes are implicated in many retinopathies. To our knowledge, GNB3 encoding the G-protein β subunit 3 (Gβ3) has not previously been implicated in human disease., Observations: In this brief report, whole-exome sequencing was conducted on a patient with distinct inherited retinal disease presenting in childhood, with a phenotype characterized by nystagmus, normal retinal examination, and mild disturbance of the central macula on detailed retinal imaging. This sequencing revealed a homozygous GNB3 nonsense mutation (c.124C>T; p.Arg42Ter). Whole-exome sequencing was conducted from April 2015 to July 2015., Conclusions and Relevance: Expressed in cone photoreceptors and ON-bipolar cells, Gβ3 is essential in phototransduction and ON-bipolar cell signaling. Knockout of Gnb3 in mice results in dysfunction of cone photoreceptors and ON-bipolar cells and a naturally occurring chicken mutation leads to retinal degeneration. Identification of further affected patients may allow description of the phenotypic and genotypic spectrum of disease associated with GNB3 retinopathy.

A substantial proportion of patients with posterior polymorphous corneal dystrophy (PPCD) lack a molecular diagnosis. We evaluated 14 unrelated probands who had a clinical diagnosis of PPCD who were previously determined to be negative for mutations in ZEB1 by direct sequencing. A combination of techniques was used including whole-exome sequencing (WES), single-nucleotide polymorphism (SNP) array copy number variation (CNV) analysis, quantitative real-time PCR, and long-range PCR. Segregation of potentially pathogenic changes with disease was confirmed, where possible, in family members. A putative run of homozygosity on chromosome 10 was identified by WES in a three-generation PPCD family, suggestive of a heterozygous deletion. SNP array genotyping followed by long-range PCR and direct sequencing to define the breakpoints confirmed the presence of a large deletion that encompassed multiple genes, including ZEB1. Identification of a heterozygous deletion spanning ZEB1 prompted us to further investigate potential CNVs at this locus in the remaining probands, leading to detection of two additional heterozygous ZEB1 gene deletions. This study demonstrates that ZEB1 mutations account for a larger proportion of PPCD than previously estimated, and supports the hypothesis that haploinsufficiency of ZEB1 is the underlying molecular mechanism of disease for PPCD3.

Purpose: Mutations in the coding sequence of the L and M opsin genes are often associated with X-linked cone dysfunction (such as Bornholm Eye Disease, BED), though the exact color vision phenotype associated with these disorders is variable. We examined individuals with L/M opsin gene mutations to clarify the link between color vision deficiency and cone dysfunction., Methods: We recruited 17 males for imaging. The thickness and integrity of the photoreceptor layers were evaluated using spectral-domain optical coherence tomography. Cone density was measured using high-resolution images of the cone mosaic obtained with adaptive optics scanning light ophthalmoscopy. The L/M opsin gene array was characterized in 16 subjects, including at least one subject from each family., Results: There were six subjects with the LVAVA haplotype encoded by exon 3, seven with LIAVA, two with the Cys203Arg mutation encoded by exon 4, and two with a novel insertion in exon 2. Foveal cone structure and retinal thickness was disrupted to a variable degree, even among related individuals with the same L/M array., Conclusions: Our findings provide a direct link between disruption of the cone mosaic and L/M opsin variants. We hypothesize that, in addition to large phenotypic differences between different L/M opsin variants, the ratio of expression of first versus downstream genes in the L/M array contributes to phenotypic diversity. While the L/M opsin mutations underlie the cone dysfunction in all of the subjects tested, the color vision defect can be caused either by the same mutation or a gene rearrangement at the same locus.

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)