Rare genetic variation in VE-PTP is associated with central serous chorioretinopathy, venous dysfunction and glaucoma.

Central serous chorioretinopathy (CSC) is a fluid maculopathy whose etiology is not well understood. Abnormal choroidal veins in CSC patients have been shown to have similarities with varicose veins. To identify potential mechanisms, we analyzed genotype data from 1,477 CSC patients and 455,449 controls in FinnGen. We identified an association for a low-frequency (AF=0.5%) missense variant (rs113791087) in the gene encoding vascular endothelial protein tyrosine phosphatase (VE-PTP) (OR=2.85, P=4.5×10 -9 ). This was confirmed in a meta-analysis of 2,452 CSC patients and 865,767 controls from 4 studies (OR=3.06, P=7.4×10 -15 ). Rs113791087 was associated with a 56% higher prevalence of retinal abnormalities (35.3% vs 22.6%, P=8.0x10 -4 ) in 708 UK Biobank participants and, surprisingly, with varicose veins (OR=1.31, P=2.3x10 -11 ) and glaucoma (OR=0.82, P=6.9x10 -9 ). Predicted loss-of-function variants in VEPTP, though rare in number, were associated with CSC in All of Us (OR=17.10, P=0.018). These findings highlight the significance of VE-PTP in diverse ocular and systemic vascular diseases.

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

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

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Consensus Guidelines for Ocular Surveillance of von Hippel-Lindau Disease.

PURPOSE: To develop guidelines for ocular surveillance and early intervention for individuals with von Hippel-Lindau (VHL) disease. DESIGN: Systematic review of the literature. PARTICIPANTS: Expert panel of retina specialists and ocular oncologists. METHODS: A consortium of experts on clinical management of all-organ aspects of VHL disease was convened. Working groups with expertise in organ-specific features of VHL disease were tasked with development of evidence-based guidelines for each organ system. The ophthalmology subcommittee formulated questions for consideration and performed a systematic literature review. Evidence was graded for topic quality and relevance and the strength of each recommendation, and guideline recommendations were developed. RESULTS: The quality of evidence was limited, and no controlled clinical trial data were available. Consensus guidelines included: (1) individuals with known or suspected VHL disease should undergo periodic ocular screening (evidence type, III; evidence strength, C; degree of consensus, 2A); (2) patients at risk of VHL disease, including first-degree relatives of patients with known VHL disease, or any patient with single or multifocal retinal hemangioblastomas (RHs), should undergo genetic testing for pathologic VHL disease gene variants as part of an appropriate medical evaluation (III/C/2A); (3) ocular screening should begin within 12 months after birth and continue throughout life (III/C/2A); (4) ocular screening should occur approximately every 6 to 12 months until 30 years of age and then at least yearly thereafter (III/C-D/2A); (5) ocular screening should be performed before a planned pregnancy and every 6 to 12 months during pregnancy (IV/D/2A); (6) ultra-widefield color fundus photography may be helpful in certain circumstances to monitor RHs, and ultra-widefield fluorescein angiography may be helpful in certain circumstances to detect small RHs (IV/D/2A); (7) patients should be managed, whenever possible, by those with subspecialty training, with experience with VHL disease or RHs, or with both and ideally within the context of a multidisciplinary center capable of providing multiorgan surveillance and access to genetic testing (IV/D/2A); (8) extramacular or extrapapillary RHs should be treated promptly (III/C/2A). CONCLUSIONS: Based on available evidence from observational studies, broad agreement was reached for a strategy of lifelong surveillance and early treatment for ocular VHL disease. These guidelines were endorsed by the VHL Alliance and the International Society of Ocular Oncology and were approved by the American Academy of Ophthalmology Board of Trustees. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Measurement of the Inner Macular Layers for Monitoring of Glaucoma: Confounding Effects of Age-Related Macular Degeneration.

OBJECTIVE: To investigate the confounding effect of nonexudative age-related macular degeneration (AMD), specifically drusen and outer retinal atrophy, on the architecture and automated segmentation of the inner retinal layers as measured with OCT. DESIGN: Observational cross-sectional study. SUBJECTS: Two hundred sixty-three consecutive eyes with nonexudative AMD were identified through a retrospective chart review. Exclusion criteria were a diagnosis of glaucoma or glaucoma suspect, other retinal pathology affecting the macula, axial length > 26.5 mm or spherical equivalent less than -6 diopters, any other optic nerve or neurologic disorders, or poor image quality. METHODS: Drusen were automatically segmented on macular OCT B-scans with a publicly available and validated deep learning approach. Automated segmentation of the inner plexiform layer (IPL)/inner nuclear layer (INL) boundary was carried out with the device's proprietary software. MAIN OUTCOME MEASURES: Quality of segmentation of the IPL/INL boundary as a function of drusen size and presence of inner retinal layer displacement in the area of macular pathology (drusen or atrophy). RESULTS: One hundred twenty-five eyes (65 patients) met the inclusion criteria. Drusen size varied between 16 and 272 µm (mean, 118 µm). Automated segmentation had a 22% chance of failure if the drusen height was between 145 and 185 µm and was most likely to fail with drusen heights above 185 µm. When drusen height was normalized by total retinal thickness, segmentation failed 36% of the time when the drusen to total retinal thickness ratio was 0.45 or above. Images were likely to show displacement of inner retinal layers with drusen heights above 176 µm and a normalized drusen height ratio of 0.5 or higher. Eighty-seven percent of images with outer retinal atrophy displayed incorrect segmentation. CONCLUSIONS: Outer retinal diseases can alter the retinal topography and affect the segmentation accuracy of the inner retinal layers. Large drusen may cause segmentation error and compression of the inner macular layers. Geographic atrophy confounds automated segmentation in a high proportion of eyes. Clinicians should be cognizant of the effects of outer retinal disease on the inner retinal layer measurements when interpreting the results of macular OCT imaging in patients with glaucoma.

Membrane Attack Complex Mediates Retinal Pigment Epithelium Cell Death in Stargardt Macular Degeneration.

Recessive Stargardt disease (STGD1) is an inherited retinopathy caused by mutations in the ABCA4 gene. The ABCA4 protein is a phospholipid-retinoid flippase in the outer segments of photoreceptors and the internal membranes of retinal pigment epithelial (RPE) cells. Here, we show that RPE cells derived via induced pluripotent stem-cell from a molecularly and clinically diagnosed STGD1 patient exhibited reduced ABCA4 protein and diminished activity compared to a normal subject. Consequently, STGD1 RPE cells accumulated intracellular autofluorescence-lipofuscin and displayed increased complement C3 activity. The level of C3 inversely correlated with the level of CD46, an early negative regulator of the complement cascade. Persistent complement dysregulation led to deposition of the membrane attack complex on the surface of RPE cells, decrease in transepithelial resistance, and subsequent cell death. These findings are strong evidence of complement-mediated RPE cell damage in STGD1, in the absence of photoreceptors, caused by reduced CD46 regulatory protein.

Systems genomics in age-related macular degeneration.

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Assessing Variant Causality and Severity Using Retinal Pigment Epithelial Cells Derived from Stargardt Disease Patients.

Purpose: Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease. Methods: Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system. Results: Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease. Conclusions: Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue. Translational Relevance: This "Disease in a Dish" approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.

Gene-Based Association Testing of Dichotomous Traits With Generalized Functional Linear Mixed Models Using Extended Pedigrees: Applications to Age-Related Macular Degeneration.

Genetics plays a role in age-related macular degeneration (AMD), a common cause of blindness in the elderly. There is a need for powerful methods for carrying out region-based association tests between a dichotomous trait like AMD and genetic variants on family data. Here, we apply our new generalized functional linear mixed models (GFLMM) developed to test for gene-based association in a set of AMD families. Using common and rare variants, we observe significant association with two known AMD genes: CFH and ARMS2. Using rare variants, we find suggestive signals in four genes: ASAH1, CLEC6A, TMEM63C, and SGSM1. Intriguingly, ASAH1 is down-regulated in AMD aqueous humor, and ASAH1 deficiency leads to retinal inflammation and increased vulnerability to oxidative stress. These findings were made possible by our GFLMM which model the effect of a major gene as a fixed mean, the polygenic contributions as a random variation, and the correlation of pedigree members by kinship coefficients. Simulations indicate that the GFLMM likelihood ratio tests (LRTs) accurately control the Type I error rates. The LRTs have similar or higher power than existing retrospective kernel and burden statistics. Our GFLMM-based statistics provide a new tool for conducting family-based genetic studies of complex diseases. Supplementary materials for this article, including a standardized description of the materials available for reproducing the work, are available as an online supplement.

Expanding the clinical phenotype in patients with disease causing variants associated with atypical Usher syndrome.

Atypical Usher syndrome (USH) is poorly defined with a broad clinical spectrum. Here, we characterize the clinical phenotype of disease caused by variants in CEP78, CEP250, ARSG, and ABHD12.Chart review evaluating demographic, clinical, imaging, and genetic findings of 19 patients from 18 families with a clinical diagnosis of retinal disease and confirmed disease-causing variants in CEP78, CEP250, ARSG, or ABHD12.CEP78-related disease included sensorineural hearing loss (SNHL) in 6/7 patients and demonstrated a broad phenotypic spectrum including: vascular attenuation, pallor of the optic disc, intraretinal pigment, retinal pigment epithelium mottling, areas of mid-peripheral hypo-autofluorescence, outer retinal atrophy, mild pigmentary changes in the macula, foveal hypo-autofluorescence, and granularity of the ellipsoid zone. Nonsense and frameshift variants in CEP250 showed mild retinal disease with progressive, non-congenital SNHL. ARSG variants resulted in a characteristic pericentral pattern of hypo-autofluorescence with one patient reporting non-congenital SNHL. ABHD12-related disease showed rod-cone dystrophy with macular involvement, early and severe decreased best corrected visual acuity, and non-congenital SNHL ranging from unreported to severe.This study serves to expand the clinical phenotypes of atypical USH. Given the variable findings, atypical USH should be considered in patients with peripheral and macular retinal disease even without the typical RP phenotype especially when SNHL is noted. Additionally, genetic screening may be useful in patients who have clinical symptoms and retinal findings even in the absence of known SNHL given the variability of atypical USH.

Characterization of the Spectrum of Ophthalmic Changes in Patients With Alagille Syndrome.

Purpose: The purpose of this study was to characterize the phenotypic spectrum of ophthalmic findings in patients with Alagille syndrome. Methods: We conducted a retrospective, observational, multicenter, study on 46 eyes of 23 subjects with Alagille syndrome. We reviewed systemic and ophthalmologic data extracted from medical records, color fundus photography, fundus autofluorescence, optical coherence tomography, visual fields, electrophysiological assessments, and molecular genetic findings. Results: Cardiovascular abnormalities were found in 83% of all cases (of those, 74% had cardiac murmur), whereas 61% had a positive history of hepatobiliary issues, and musculoskeletal anomalies were present in 61% of all patients. Dysmorphic facies were present in 16 patients, with a broad forehead being the most frequent feature. Ocular symptoms were found in 91%, with peripheral vision loss being the most frequent complaint. Median (range) Snellen visual acuity of all eyes was 20/25 (20/20 to hand motion [HM]). Anterior segment abnormalities were present in 74% of the patients; of those, posterior embryotoxon was the most frequent finding. Abnormalities of the optic disc were found in 52%, and peripheral retinal abnormalities were the most frequent ocular finding in this series, found in 96% of all patients. Fifteen JAG1 mutations were identified in 16 individuals; of those, 6 were novel. Conclusions: This study reports a cohort of patients with Alagille syndrome in which peripheral chorioretinal changes were more frequent than posterior embryotoxon, the most frequent ocular finding according to a number of previous studies. We propose that these peripheral chorioretinal changes are a new hallmark to help diagnose this syndrome.

Assessing the Clinical Utility of Expanded Macular OCTs Using Machine Learning.

Purpose: Optical coherence tomography (OCT) is widely used in the management of retinal pathologies, including age-related macular degeneration (AMD), diabetic macular edema (DME), and primary open-angle glaucoma (POAG). We used machine learning techniques to understand diagnostic performance gains from expanding macular OCT B-scans compared with foveal-only OCT B-scans for these conditions. Methods: Electronic medical records were extracted to obtain 61 B-scans per eye from patients with AMD, diabetic retinopathy, or POAG. We constructed deep neural networks and random forest ensembles and generated area under the receiver operating characteristic (AUROC) and area under the precision recall (AUPR) curves. Results: After extracting 630,000 OCT images, we achieved improved AUROC and AUPR curves when comparing the central image (one B-scan) to all images (61 B-scans). The AUROC and AUPR points of diminishing return for diagnostic accuracy for macular OCT coverage were found to be within 2.75 to 4.00 mm (14-19 B-scans), 4.25 to 4.50 mm (20-21 B-scans), and 4.50 to 6.25 mm (21-28 B-scans) for AMD, DME, and POAG, respectively. All models with >0.25 mm of coverage had statistically significantly improved AUROC/AUPR curves for all diseases (P < 0.05). Conclusions: Systematically expanded macular coverage models demonstrated significant differences in total macular coverage required for improved diagnostic accuracy, with the largest macular area being relevant in POAG followed by DME and then AMD. These findings support our hypothesis that the extent of macular coverage by OCT imaging in the clinical setting, for any of the three major disorders, has a measurable impact on the functionality of artificial intelligence decision support. Translational Relevance: We used machine learning techniques to improve OCT imaging standards for common retinal disease diagnoses.

A CASE OF INTRARETINAL PERIPAPILLARY NEOVASCULARIZATION IN ABCA4-RELATED RETINOPATHY.

PURPOSE: To report a case of ABCA4-related retinopathy and potential complications. METHODS: The authors describe a case report of intraretinal neovascularization in a patient with ABCA4-related retinopathy and describe the multimodal retinal imaging findings. RESULTS: A 49-year-old woman presents with cystoid macular edema and diffuse intraretinal and perivascular hyperpigmentation in both eyes. Genetic testing confirmed ABCA4-related retinopathy. Fluorescein angiography revealed peripapillary intraretinal neovascularization in the absence of any other identifiable retinal pathology. CONCLUSION: To the authors' knowledge, this is the first reported case of intraretinal neovascularization-associated ABCA4-related retinopathy. Ancillary testing of ABCA4-related retinopathy with fluorescein angiography or optical coherence tomography angiography may be helpful in identifying this rare complication.

Novel mutation in SLC4A7 gene causing autosomal recessive progressive rod-cone dystrophy.

BACKGROUND: Recent advances in genetic sequencing techniques have improved the overall diagnostic yield for finding genetic causes for inherited retinal dystrophies (IRD). Rod-cone dystrophy is the most common IRD and is characterized by the primary involvement of the rod photoreceptors. Over 80 causal genes have been identified so far giving clinicians insight into the pathogenesis. SLC4A7 encodes a sodium bicarbonate cotransporter responsible acid disposal which, within the retina, is prevalent in the photoreceptor synaptic terminals. Thus far, there have been no published reports of variants in this gene known to cause rod-cone dystrophy. MATERIAL AND METHODS: Case report of a rod-cone dystrophy patient with a novel mutation in SLC4A7, whole exome sequencing with CLIA-certified NGS and Sanger confirmation, and, review of a SLC4A7 knockout mouse model phenotype. RESULTS: A 66-year-old male presented with slowly progressing night blindness, constricted visual field and relatively stable visual acuity. Fundus examination showed diffuse intraretinal pigment in the mid- and peripheral retina, diffuse retinal pigment epithelial atrophy, and intact macula in both eyes. There has been mild macular edema in both eyes which remained stable with the use of topical dorzolamide eyedrops. Whole exome sequencing found, and a subsequent vision panel confirmed, the pathogenic variant to be a homozygous frameshift mutation in SLC4A7 which results in termination of the protein. CONCLUSIONS: We report a case of progressive rod-cone dystrophy caused by a novel mutation in SLC4A7, a gene coding the sodium bicarbonate cotransporter NBC3, underscoring the importance of ion homeostasis for photoreceptor function and maintenance.

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

Predictive genetics for AMD: Hype and hopes for genetics-based strategies for treatment and prevention.

Age-related macular degeneration (AMD) is a complex disease with multiple genetic and environmental risk factors. In the age of molecular genetics, many investigators have established a link between genes and development or progression of the disease. This later evolved to determine whether phenotypic features of AMD have distinct genetic profiles. Molecular genetics have subsequently been introduced as factors in risk assessment models, increasing the predictive value of these tools. Models seek to predict either development or progression of disease, and different AMD-related genes aid our understanding of these respective features. Several investigators have attempted to link molecular genetics with treatment response, but results and their clinical significance vary. Ocular and systemic biomarkers may interact with established genes, promising future routes of ongoing clinical assessment. Our understanding of AMD molecular genetics is not yet sufficient to recommend routine testing, despite its utility in the research setting. Clinicians must be wary of misusing population-based risk models from genetic and biomarker associations, as they are not necessarily relevant for individual counseling. This review addresses the known uses of predictive genetics, and suggests future directions.

Evaluation of the Choroid in Eyes With Retinitis Pigmentosa and Cystoid Macular Edema.

Purpose: To study the anatomical choroidal features associated with the presence of cystoid macular edema (CME) in eyes with retinitis pigmentosa (RP). Methods: A total of 159 eyes (from 159 patients) with a diagnosis of RP were enrolled in this retrospective cross-sectional case-control study and divided into two groups based on the presence (67 eyes) or absence (92 eyes) of CME. Retinal and choroidal features were evaluated on spectral domain optical coherence tomography including central macular thickness (CMT) and subfoveal choroidal thickness (CT). Total choroidal area (TCA), choroidal luminal area (LA), and choroidal stromal area (SA) were measured and the choroidal vascularity index (CVI) was calculated in all study eyes. Results: Average age was 49.2 ± 14.9 and 47.1 ± 15.5 years (P = 0.40) and logMAR Snellen visual acuity (VA) was 0.4 ± 0.6 (median 0.3, 20/40) and 0.2 ± 0.4 (median 0.1, 20/25) in the RP groups with and without CME, respectively (P = 0.05). Mean CMT was 334.1 ± 93.5 and 252.6 ± 47.6 µm in the RP groups with and without CME, respectively (P < 0.001). The subfoveal CT was significantly increased in the RP group with versus without CME (294.2 ± 110.9 µm vs. 198.1 ± 75.5 µm, respectively, P < 0.001). In patients with CME, the CVI was lower (P < 0.001) and the TCA, LA, and SA were all significantly higher (P < 0.001). Conclusions: In patients with CME associated with RP, the choroid exhibited significantly greater subfoveal thickening and decreased CVI. The choroid may be an important factor to consider in the etiology of CME in patients with RP.

Utility of In Vitro Mutagenesis of RPE65 Protein for Verification of Mutational Pathogenicity Before Gene Therapy.

Importance: Next-generation sequencing can detect variants of uncertain significance (VUSs), for some of which gene therapy would not be advantageous. Therefore, the pathogenicity of compound heterozygous or homozygous variants should be confirmed before bilateral vitrectomy and administration of voretigene neparvovec-rzyl. Objective: To describe an in vitro mutagenesis assay for assessing the pathogenicity of variants in the RPE65 gene. Design, Setting, and Participants: This case series was conducted at 2 tertiary referral centers. Clinical history, imaging, and electrophysiologic testing results were reviewed from September 5, 2008, to December 31, 2019. Participants were 4 pediatric patients with Leber congenital amaurosis who were evaluated for or met the inclusion criteria for phase 1 to 3 clinical trials or were referred for voretigene neparvovec-rzyl treatment. Main Outcomes and Measures: A functional assay was used to confirm the pathogenicity of novel RPE65 VUSs in 4 patients with Leber congenital amaurosis. Results: Four patients with Leber congenital amaurosis had VUSs in RPE65. Patients 1 and 2 were siblings with the homozygous VUS c.311G>T p.(G104V). Patient 3 was a compound heterozygote with 1 known pathogenic allele, c.1202_1203insCTGG p.(Glu404AlafsTer4), and 1 VUS, c.311G>T p.(G104V), which segregated to separate alleles. Patient 4 was also a compound heterozygote with 1 pathogenic variant, c.11 + 5G>A, and 1 variant in trans, c.1399C>T p.(P467S). In vitro mutagenesis revealed that the G104V and P467S RPE65 proteins were catalytically inactive (0% isomerase activity). Patients 1 and 2 were excluded from participation in a phase 1 trial owing to high Adeno-associated virus 2 capsid-neutralizing antibodies. Patients 3 (G104V) and 4 (P467S) underwent successful surgical gene therapy with voretigene neparvovec-rzyl, and their response to lower white light intensity and visual field increased in fewer than 30 days after gene therapy intervention. Conclusions and Relevance: Findings from this study suggest that, in patients with missense mutations in RPE65, functional assays of protein function can be performed to assess the pathogenicity of variants in both compound heterozygous and homozygous cases. Given the potential risks of gene therapy operations, in vitro RPE65 activity testing should be considered to avoid the possibility of treating a false genotype.