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Eliot R Dow,1– 3 Karen M Chen,1 Cindy S Zhao,1 Austen N Knapp,1 Anuradha Phadke,4 Kirsti Weng,4 Diana V Do,1 Vinit B Mahajan,1 Prithvi Mruthyunjaya,1 Theodore Leng,1 David Myung1,2 1Department of Ophthalmology, Byers Eye Institute at Stanford, Palo Alto, CA, USA; 2Department of Ophthalmology, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA; 3Department of Ophthalmology, Duke Eye Center, Durham, NC, USA; 4Department of Internal Medicine, Stanford Health Care, Palo Alto, CA, USACorrespondence: David Myung, Byers Eye Institute at Stanford, Palo Alto, CA, 94303, USA, Tel +1 650 723-6995, Email djmyung@stanford.eduPurpose: We examine the rate of and reasons for follow-up in an Artificial Intelligence (AI)-based workflow for diabetic retinopathy (DR) screening relative to two human-based workflows.Patients and Methods: A DR screening program initiated September 2019 between one institution and its affiliated primary care and endocrinology clinics screened 2243 adult patients with type 1 or 2 diabetes without a diagnosis of DR in the previous year in the San Francisco Bay Area. For patients who screened positive for more-than-mild-DR (MTMDR), rates of follow-up were calculated under a store-and-forward human-based DR workflow (“Human Workflow”), an AI-based workflow involving IDx-DR (“AI Workflow”), and a two-step hybrid workflow (“AI–Human Hybrid Workflow”). The AI Workflow provided same-day results, whereas the other workflows took 1– 5 days. Patients were surveyed by phone about follow-up decisions.Results: Under the AI Workflow, 279 patients screened positive for MTMDR. Of these, 69.2% followed up with an ophthalmologist within 90 days. Altogether 70.5% (N=48) of patients who followed up chose their location based on primary care referral. Under the Human Workflow and AI–Human Hybrid Workflow, 12.0% (N=14/117) and 11.7% (N=12/103) of patients with a referrable screening result followed up compared to 35.5% of patients under the AI Workflow (N=99/279; χ2df=2 = 36.70, p < 0.00000001).Conclusion: Ophthalmology follow-up after a positive DR screening result is approximately three-fold higher under the AI Workflow than either the Human Workflow or AI–Human Hybrid Workflow. Improved follow-up behavior may be due to the decreased time to screening result.Keywords: telemedicine, teleophthalmology, fundus photography, deep learning, machine learning, referral

Abdourahman Cham,1,2 Mayank Bansal,3 Himanshu K Banda,4 Young Kwon,1 Paul S Tlucek,1 Alexander G Bassuk,5 Stephen H Tsang,6,7 Warren M Sobol,8 James C Folk,1 Steven Yeh,4 Vinit B Mahajan1,2 1Department of Ophthalmology and Visual Sciences, 2Omics Laboratory, University of Iowa, Iowa City, IA, USA; 3Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India; 4Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA, 5Department of Pediatrics, University of Iowa, Iowa City, IA, 6Barbara and Donald Jonas Laboratory of Stem Cells and Regenerative Medicine and Bernard and Shirlee Brown Glaucoma Laboratory, Department of Pathology and Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 7Edward S Harkness Eye Institute, New York-Presbyterian Hospital, New York, NY, 8Retina Physicians & Surgeons, Inc., Dayton, OH, USA Objective: The objective of this study was to review the treatment outcomes of patients with secondary glaucoma in cases of autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV), a hereditary autoimmune uveitis due to mutations in CAPN5. Patients and methods: A retrospective, observational case series was assembled from ADNIV patients with secondary glaucoma. The main outcome measures were intraocular pressure (IOP), visual acuity, use of antiglaucoma medications, ocular surgeries, and adverse outcomes. Perimetry and optic disk optical coherence tomography (OCT) were also analyzed. Results: Nine eyes of five ADNIV patients with secondary glaucoma were reviewed. Each received a fluocinolone acetonide (FA) implant for the management of posterior uveitis. Following implantation, no eyes developed neovascular glaucoma. Five eyes (in patients 1, 2, and 5) required Ahmed glaucoma valve surgery for the management of steroid-responsive glaucoma. Patient 2 also developed angle closure with iris bombe and underwent laser peripheral iridotomy. Patient 4 had both hypotony and elevated IOP that required periodic antiglaucoma medication in the FA-implanted eye. Patient 3 did not develop steroid-response glaucoma in either eye. Optic disk examinations were obscured by fibrosis and better assessed with OCT. Conclusion: ADNIV patients show combined mechanism secondary glaucoma best assessed by OCT of the optic disk. The FA implants have reduced uveitic and neovascular glaucoma. Nevertheless, IOP management remains complex due to steroid-response glaucoma, angle closure glaucoma, and hypotony. Keywords: ADNIV, CAPN5, calpain-5, fluocinolone acetonide, secondary glaucoma, uveitis

David RP Almeida,1 Eric K Chin,2,3 Shaival S Shah,3 Benjamin Bakall,3 Karen M Gehrs,3 H Culver Boldt,3 Stephen R Russell,3 James C Folk,3 Vinit B Mahajan3,41VitreoRetinal Surgery PA, Minneapolis, MN, 2Retina Consultants of Southern California, Riverside, CA, 3Vitreoretinal Service, Department of Ophthalmology and Visual Sciences, 4Omics Laboratory, University of Iowa, Iowa City, IA, USABackground: The role of pars plana vitrectomy (PPV) for endophthalmitis has evolved over recent decades but the literature is lacking on comparisons between small-gauge and 20-gauge vitrectomy.Objective: To evaluate evolving etiological and microbiological trends in patients undergoing vitrectomy for endophthalmitis and to compare culture-positive rates and visual outcomes between small-gauge (23- and 25-gauge) and 20-gauge instrumentation during vitrectomy for endophthalmitis.Methods: Ten-year retrospective comparative case series and prospective laboratory in vitro testing. Tertiary care academic referral center. Patients who underwent PPV for endophthalmitis between 2003 and 2013. Vitreous biopsies were obtained in all cases. The effect of vitrectomy gauge (20-, 23-, and 25-gauge) and vitreous cutting rate (1,500 and 5,000 cuts per minute) on the viability of bacterial culture was evaluated in an in vitro prospective laboratory investigation.Main outcome measures: Comparison of etiology, microbiology culture-positive rates, and visual outcomes between small-gauge and 20-gauge instrumentation in patients undergoing PPV for infectious endophthalmitis.Results: A total of 61 cases of vitrectomy for endophthalmitis were identified over a 10-year period; of these, 34 were treated with small-gauge (23- and 25-gauge) vitrectomy and 27 were treated with 20-gauge vitrectomy. In the small-gauge group, 12 cases (35.3%) yielded culture-positive results versus 20 cases (74.1%) with culture positivity in the 20-gauge cohort (P=0.002). The most common cause of endophthalmitis was cataract surgery and the most frequently identified organism was coagulase-negative Staphylococci in both groups. There was no significant difference in mean postoperative visual acuities between groups (P=0.33). Etiological trends indicate an increase in endophthalmitis due to intravitreal injection in the small-gauge group (n=9) compared to the 20-gauge group (n=3) (P=0.001). In vitro laboratory testing revealed no significant difference in rates of culture growth for different vitrectomy gauge sizes or vitreous cutting speeds.Conclusion and relevance: Small-gauge vitrectomy for endophthalmitis yields final visual outcomes comparable to 20-gauge instrumentation. A significant difference in culture-positive rates was observed between small-gauge and 20-gauge instrumentation for vitrectomy in endophthalmitis; however, laboratory testing indicates this is not related to either vitreous gauge size or cutter speed. Intravitreal injections are emerging as a common etiology of vitrectomy for endophthalmitis.Keywords: endophthalmitis, vitrectomy, vitreous biopsy

David RP Almeida,1 Eric K Chin,1 Ryan M Tarantola,1 Elizabeth O Tegins,1 Christopher A Lopez,1 Herbert Culver Boldt,1 Karen M Gehrs,1 Elliott H Sohn,1 Stephen R Russell,1 James C Folk,1 Vinit B Mahajan1,2 1Department of Ophthalmology and Visual Sciences, 2Omics Laboratory, University of Iowa Hospitals and Clinics, Iowa City, IA, USA Purpose: To determine the outcomes in patients with rhegmatogenous retinal detachment (RRD) secondary to viral retinitis. Patients and methods: This was a retrospective, consecutive, noncomparative, interventional case series of 12 eyes in ten patients with RRD secondary to viral retinitis. Results of vitreous or aqueous biopsy, effect of antiviral therapeutics, time to retinal detachment, course of visual acuity, and anatomic and surgical outcomes were investigated. Results: There were 1,259 cases of RRD during the study period, with 12 cases of RRD secondary to viral retinitis (prevalence of 0.95%). Follow-up was available for a mean period of 4.4 years. Varicella zoster virus was detected in six eyes, herpes simplex virus in two eyes, and cytomegalovirus in two eyes. Eight patients were treated with oral valacyclovir and two patients with intravenous acyclovir. Lack of optic nerve involvement correlated with improved final visual acuity of 20/100 or greater. Pars plana vitrectomy (n=12), silicone-oil tamponade (n=11), and scleral buckling (n=10) provided successful anatomic retinal reattachment in all cases, with no recurrent retinal detachment and no cases of hypotony during the follow-up period. Conclusion: Varicella zoster virus was the most frequent cause of viral retinitis, and lack of optic nerve involvement was predictive of a favorable visual acuity prognosis. Vitrectomy with silicone-oil tamponade and scleral buckle placement provided stable anatomical outcomes. Keywords: viral retinitis, acute retinal necrosis, herpetic retinitis, vitrectomy, retinal detachment

John J Chen,1,2,* Lucas J Wendel,1,3,* Emily S Birkholz,1 John G Vallone,4 Anne L Coleman,5,6 Fei Yu,7 Vinit B Mahajan1,3,8 1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA; 2Mayo Clinic, Rochester, MN, USA; 3Vitreoretinal Service, University of Iowa, Iowa City, IA, USA; 4Department of Pathology, University of Southern California, 5Department of Ophthalmology, 6Department of Epidemiology, School of Public Health, 7Department of Biostatistics, University of California, Los Angeles, CA, USA; 8Omics Laboratory, University of Iowa, Iowa City, IA, USA *These authors contributed equally to this work Background: While metabolic syndrome has been strongly implicated as a risk factor for macrovascular diseases, such as stroke and cardiovascular disease, its relationship with microvascular diseases, including diabetic retinopathy, has been less defined. The purpose of this pilot study was to investigate the association between metabolic syndrome and the presence and severity of diabetic retinopathy.Methods: A retrospective case–control chart review at the University of Iowa ophthalmology and primary care clinics included 100 patients with proliferative diabetic retinopathy (PDR), 100 patients with nonproliferative diabetic retinopathy (NPDR), 100 diabetic patients without diabetic retinopathy, and 100 nondiabetic patients who were randomly selected. Using the International Diabetes Foundation definition, the prevalence of metabolic syndrome and the number of components of metabolic syndrome were compared among these groups.Results: The prevalence of metabolic syndrome in patients with diabetes was 69.3%, which was significantly higher than that in patients without diabetes (27%; P

David RP Almeida,1 Eric K Chin,1 H Culver Boldt,1 Vinit B Mahajan1,2 1Department of Ophthalmology and Visual Sciences; 2Omics Laboratory, University of Iowa Hospitals and Clinics, Iowa City, IA, USA Abstract: We present a case of a 41-year-old man who was referred for evaluation of a ­choroidal tumor with a remote history of scleral buckle placement for traumatic retinal detachment. Ocular imaging, echography, and magnetic resonance imaging could not rule out a neoplastic process so the patient was taken for surgical exploration where a hemorrhagic cyst was discovered. This is the first case in the literature of a silicone scleral buckle–associated hemorrhagic cyst presenting as orbital mass. Keywords: choroidal mass, choroidal lesion, choroidal melanoma, echography, optical coherence tomography, magnetic resonance imaging

Vinit B Mahajan,1,2 Jonathan H Lin31Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA; 2Omics Laboratory, University of Iowa Hospitals and Clinics, Iowa City, IA, USA; 3Departments of Pathology and Ophthalmology, University of California San Diego, La Jolla, CA, USAPurpose: To describe immunohistopathological findings in autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV).Methods: An enucleated eye specimen from a patient with Stage V ADNIV was examined using standard histopathological methods and lymphocyte markers.Results: A c.731T>C CAPN5 mutation resulted in a p.Leu244Pro substitution in calpain-5. The eye showed exudative retinal detachment and neovascularization, intraocular fibrosis, and features of phthisis bulbi. Chronic inflammatory CD3-positive cell infiltrates were identified throughout the uvea, vitreous and retina, consistent with chronic uveitis.Conclusion: Mutations in CAPN5 trigger autoimmune uveitis characterized by inflammatory T-cells and severe neovascularization.Keywords: autosomal dominant neovascular inflammatory vitreoretinopathy, ADNIV, CAPN5, uveitis, CD3

Paul S Tlucek,1 James C Folk,1 Warren M Sobol,2 Vinit B Mahajan1,3 1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA; 2Retina Physicians and Surgeons, Dayton, OH, USA; 3Omics Laboratory, University of Iowa, Iowa City, IA, USA Objective: To review fibrosis of fluocinolone acetonide (FA) implants in subjects with CAPN5 autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). Methods: A retrospective case series was assembled from ADNIV patients in which there was fibrotic encapsulation of a fluocinolone acetonide implant. CAPN5 genotypes and surgical repair techniques were reviewed. Results: Two eyes of two ADNIV patients developed a fibrotic capsule over the fluocinolone acetonide implant. Both patients had Stage IV disease. Patient A had a c.731T > C mutation in the CAPN5 gene and patient B had a c.728G > T mutation. The fibrotic membrane was surgically excised and the implant function was restored. Conclusion: The exuberant fibrotic response in later stages of ADNIV may be resistant to local immunosuppression with steroids. Surgical excision of fibrotic membranes over FA implants can reestablish local steroid delivery in cases of severe proliferative vitreoretinopathy. Keywords: autosomal dominant neovascular inflammatory vitreoretinopathy, ADNIV, CAPN5, calpain-5, Retisert, fluocinolone acetonide, fibrotic encapsulation

Hannah A Rowell,1,2 Alexander G Bassuk,3,4 Vinit B Mahajan1,21Omics Laboratory, 2Department of Ophthalmology and Visual Sciences, 3Department of Pediatrics, 4Department of Neurology, University of Iowa, Iowa City, IA, USABackground: The purpose of this study was to describe the clinical findings in a set of monozygotic twins with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) over a 23-year period.Methods: A pair of female twins were examined between 26 and 49 years of age. The concordance and discordance of their clinical features were determined. The CAPN5 gene was sequenced using genomic DNA.Results: Both twins of an affected father demonstrated Stage I ADNIV with mild vitreous cells and a negative b-wave on electroretinography. Genetic analysis confirmed a guanine to thymine nucleotide (c.728G>T, pArg243Leu) mutation in the CAPN5 gene. Over the course of 23 years, each twin progressed to stage III disease, showing posterior uveitis, cystoid macular edema, intraocular fibrosis, early retinal neovascularization, retinal degeneration, and cataract. Disease progression varied moderately between each twin and was asymmetrical between eyes. Twin A had 20/70 and 20/125 in the right and left eye, respectively, and underwent vitrectomy surgery and intravitreal injections with bevacizumab for recurrent cystoid macular edema. Twin B maintained 20/20 and 20/40 in the right and left eye, respectively without intervention.Conclusion: There was asymmetry between the eyes and some discordance in the rate of disease progression in these monozygotic twins with ADNIV. The overall high disease concordance suggests genetic factors play a major role in clinical manifestations in CAPN5 vitreoretinopathy.Keywords: autosomal dominant neovascular inflammatory vitreoretinopathy, ADNIV, CAPN5, calpain-5, monozygotic twins

In this study, the authors sought to determine whether single nucleotide polymorphisms in the estrogen receptor alpha (ESR1) and matrix metalloproteinase 2 (MMP2) genes are associated with age-related maculopathy (ARM) in older women. Subjects comprised a random sample of Caucasian women aged > or =74 years participating in the Study of Osteoporotic Fractures year 10 follow-up (n = 906) in 1997-1998. Fundus photographs were graded for ARM using a modification of the Wisconsin Age-Related Maculopathy Grading System. The prevalences of early ARM and late ARM were 46% and 4%, respectively. The MMP2 rs2287074 single nucleotide polymorphism (G-->A) was associated with ARM. The A allele was present in 47%, 43%, and 30% of subjects with no, early, and late ARM, respectively (p = 0.01), and was associated with lower odds of any ARM (for AG vs. GG, odds ratio = 0.80, 95% confidence interval: 0.65, 0.99; for AA vs. GG, odds ratio = 0.64, 95% confidence interval: 0.42, 0.98). An interaction with use of postmenopausal hormone therapy was significant (p = 0.02). The MMP2 rs2287074 A allele may be associated with a lower likelihood of ARM in older Caucasian women, particularly those who have never used hormone therapy. The role of MMP2 rs2287074 in ARM should be further elucidated. [ABSTRACT FROM AUTHOR]

Purpose: We aim to present a case of disseminated fusariosis that occurred in the setting of immunosuppression and presented with bilateral endogenous endophthalmitis, along with a literature review of Fusarium endophthalmitis, highlighting management strategies., Observation: A 70-year-old male with acute myeloid leukemia who had recently undergone a bone marrow transplant noted bilateral floaters and decreased vision. He was found to have bilateral Fusarium endophthalmitis, with subsequent evidence of fungemia and fusariosis in his skin and joints. Despite aggressive local and systemic treatment, he succumbed to the disease. Endophthalmitis was initially stabilized with pars plana vitrectomy and intravitreal amphotericin and voriconazole until the patient transitioned to comfort measures. A review of 31 cases demonstrates that outcomes are poor and that the disease must be treated aggressively, often both systemically and surgically., Conclusion: This case highlights the recalcitrance of Fusarium bacteremia and Fusarium endophthalmitis.

Traumatic Brain Injury (TBI) induces neuroinflammatory response that can initiate epileptogenesis, which develops into epilepsy. Recently, we identified anti-convulsive effects of naltrexone, a mu-opioid receptor (MOR) antagonist, used to treat drug addiction. While blocking opioid receptors can reduce inflammation, it is unclear if post-TBI seizures can be prevented by blocking MORs. Here, we tested if naltrexone prevents neuroinflammation and/or seizures post-TBI. TBI was induced by a modified Marmarou Weight-Drop (WD) method on 4-week-old C57BL/6J male mice. Mice were placed in two groups: non-telemetry assessing the acute effects or in telemetry monitoring for interictal events and spontaneous seizures both following TBI and naltrexone. Molecular, histological and neuroimaging techniques were used to evaluate neuroinflammation, neurodegeneration and fiber track integrity at 8 days and 3 months post-TBI. Peripheral immune responses were assessed through serum chemokine/cytokine measurements. Our results show an increase in MOR expression, nitro-oxidative stress, mRNA expression of inflammatory cytokines, microgliosis, neurodegeneration, and white matter damage in the neocortex of TBI mice. Video-EEG revealed increased interictal events in TBI mice, with 71% mice developing post-traumatic seizures (PTS). Naltrexone treatment ameliorated neuroinflammation, neurodegeneration, reduced interictal events and prevented seizures in all TBI mice, which makes naltrexone a promising candidate against PTS, TBI-associated neuroinflammation and epileptogenesis in a WD model of TBI., (© 2024. The Author(s).)

Purpose: Knowing the surgical safety of anterior chamber liquid biopsies will support the increased use of proteomics and other molecular analyses to better understand disease mechanisms and therapeutic responses in patients and clinical trials. Manual review of operative notes from different surgeons and procedures in electronic health records (EHRs) is cumbersome, but free-text software tools could facilitate efficient searches., Design: Retrospective case series., Participants: A total of 1418 aqueous humor liquid biopsies from patients undergoing intraocular surgery., Methods: Free-text EHR searches were performed using the Stanford Research Repository cohort discovery tool to identify complications associated with anterior chamber paracentesis and subsequent endophthalmitis. Complications of the surgery unrelated to the biopsy were not reviewed., Main Outcome Measures: Biopsy-associated intraoperative complications and endophthalmitis., Results: A total of 1418 aqueous humor liquid biopsies were performed by 17 experienced surgeons. EHR free-text searches were 100% error-free for surgical complications, >99% for endophthalmitis (<1% false positive), and >93.6% for anesthesia type, requiring manual review for only a limited number of cases. More than 85% of cases were performed under local anesthesia without ocular muscle akinesia. Although the most common indication was cataract (50.1%), other diagnoses included glaucoma, diabetic retinopathy, uveitis, age-related macular degeneration, endophthalmitis, retinitis pigmentosa, and uveal melanoma. A 50- to 100-μL sample was collected in all cases using either a 30-gauge needle or a blunt cannula via a paracentesis. The median follow-up was >7 months. There was only one minor complication (0.07%) identified: a case of a small tear in Descemet membrane without long-term sequelae. No other complications occurred, including other corneal injuries, lens or iris trauma, hyphema, or suprachoroidal hemorrhage. There was no case of postoperative endophthalmitis., Conclusions: Anterior chamber liquid biopsy during intraocular surgery is a safe procedure and may be considered for large-scale collection of aqueous humor samples for molecular analyses. Free-text EHR searches are an efficient approach to reviewing intraoperative procedures., Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (© 2024 by the American Academy of Ophthalmology.)

Purpose: The purpose of this study was to profile protein expression liquid vitreous biopsies from patients with uveal melanoma (UM) using mass spectrometry to identify prognostic biomarkers, signaling pathways, and therapeutic targets., Methods: Vitreous biopsies were collected from two cohorts in a pilot study: comparative control eyes with epiretinal membranes (ERM; n = 3) and test eyes with UM (n = 8). Samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identified proteins were compared to data from a targeted multiplex ELISA proteomics platform., Results: A total of 69 significantly elevated proteins were detected in the UM vitreous, including LYVE-1. LC-MS/MS identified 62 significantly upregulated proteins in UM vitreous that were not previously identified by ELISA. Analysis of differential protein expression by tumor molecular classification (gene expression profiling [GEP] and preferentially expressed antigen in melanoma [PRAME]) further identified proteins that correlated with these classifications. Patients with high-risk GEP tumors displayed elevated vitreous expression of HGFR (fold-change [FC] = 2.66E + 03, P value = 0.003) and PYGL (FC = 1.02E + 04, P = 1.72E-08). Patients with PRAME positive tumors displayed elevated vitreous expression of ENPP-2 (FC = 3.21, P = 0.04), NEO1 (FC = 2.65E + 03, P = 0.002), and LRP1 (FC = 5.59E + 02, P value = 0.01). IGF regulatory effectors were highly represented (P value = 1.74E-16). Cross-platform analysis validated seven proteins identified by ELISA and LC-MS/MS., Conclusions: Proteomic analysis of liquid biopsies may provide prognostic information supporting gene expression of tumor biopsies. The use of multiple protein detection platforms in the same patient samples increases the sensitivity of candidate biomarker detection and allows for precise characterization of the vitreous proteome.

Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objective: Detection of diabetic retinopathy (DR) outside of specialized eye care settings is an important means of access to vision-preserving health maintenance. Remote interpretation of fundus photographs acquired in a primary care or other nonophthalmic setting in a store-and-forward manner is a predominant paradigm of teleophthalmology screening programs. Artificial intelligence (AI)-based image interpretation offers an alternative means of DR detection. IDx-DR (Digital Diagnostics Inc) is a Food and Drug Administration-authorized autonomous testing device for DR. We evaluated the diagnostic performance of IDx-DR compared with human-based teleophthalmology over 2 and a half years. Additionally, we evaluated an AI-human hybrid workflow that combines AI-system evaluation with human expert-based assessment for referable cases., Design: Prospective cohort study and retrospective analysis., Participants: Diabetic patients ≥ 18 years old without a prior DR diagnosis or DR examination in the past year presenting for routine DR screening in a primary care clinic., Methods: Macula-centered and optic nerve-centered fundus photographs were evaluated by an AI algorithm followed by consensus-based overreading by retina specialists at the Stanford Ophthalmic Reading Center. Detection of more-than-mild diabetic retinopathy (MTMDR) was compared with in-person examination by a retina specialist., Main Outcome Measures: Sensitivity, specificity, accuracy, positive predictive value, and gradability achieved by the AI algorithm and retina specialists., Results: The AI algorithm had higher sensitivity (95.5% sensitivity; 95% confidence interval [CI], 86.7%-100%) but lower specificity (60.3% specificity; 95% CI, 47.7%-72.9%) for detection of MTMDR compared with remote image interpretation by retina specialists (69.5% sensitivity; 95% CI, 50.7%-88.3%; 96.9% specificity; 95% CI, 93.5%-100%). Gradability of encounters was also lower for the AI algorithm (62.5%) compared with retina specialists (93.1%). A 2-step AI-human hybrid workflow in which the AI algorithm initially rendered an assessment followed by overread by a retina specialist of MTMDR-positive encounters resulted in a sensitivity of 95.5% (95% CI, 86.7%-100%) and a specificity of 98.2% (95% CI, 94.6%-100%). Similarly, a 2-step overread by retina specialists of AI-ungradable encounters improved gradability from 63.5% to 95.6% of encounters., Conclusions: Implementation of an AI-human hybrid teleophthalmology workflow may both decrease reliance on human specialist effort and improve diagnostic accuracy., Financial Disclosures: Proprietary or commercial disclosure may be found after the references., (© 2023 Published by Elsevier Inc. on behalf of the American Academy of Ophthalmology.)

This protocol describes an ex vivo model to quantify enzymatic effects on vitreous liquefaction using porcine eyes in a transwell-plate system via induced syneresis. It provides a standardized dissection process and performs critical steps for gel-liquid separation with high precision, minimal tissue loss, and scalability. The protocol can be applied to other studies investigating vitreous liquefaction or gelatinous tissue analysis and can also serve to study vitreous liquefaction in vivo as it may occur during aging or disease progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Purpose: To determine the prevalence of SARS-CoV-2 in tear samples and to investigate whether it correlates with ocular findings and patients’ prognosis in Brazil. Methods: Tears were collected using Schirmer strips (SS) and conjunctival swabs (CS) from patients hospitalized with laboratory-confirmed SARS-CoV-2 infection. Samples were analyzed using qRT-PCR. Demographic and clinical data, ocular symptoms, and Schirmer tests (ST) were collected from patients. Charlson Comorbidity Index (CCI) was used to rate comorbidities, and patients were monitored until hospital discharge or death. Results: There were 61 hospitalized patients, 33 of which were diagnosed with COVID-19. Within the confirmed COVID-19 patients, SARS-CoV-2 was detected in 18.2% (n = 6) of CS and 12.1% (n = 4) of SS samples. Subjective and objective parameters for dry eye syndrome (e.g., ST COVID-19: 8.3 ± 6.4mm, non-COVID-19: 8.9 ± 6.6mm, p > 0.05) were comparable between COVID-19 (n = 33) and non-COVID-19 patients (n = 28). Among the 16 COVID-19 patients exhibiting ocular symptoms, only tearing was reported significantly more frequently when tear samples were positive for SARS-CoV-2 (p < 0.05). Strikingly, patients whose tears tested positive for SARS-CoV-2 had significantly inferior CCI (pos.: 34.0 ± 31.8%, neg.: 67.6 ± 36.4%, p < 0.05) and higher mortality rates (pos.: 50.0%, neg.: 7.4%, p < 0.01). Conclusions: SARS-CoV-2 was detected with a prevalence of 18.2% on the ocular surface. Decreased CCI and increased mortality rate in the positive tear group suggests that viral detection may relate to prognosis and highlight the need of personal protective measures for healthcare professionals. Most of the patients, regardless of COVID-19 diagnosis, had low tear production and eye discomfort, possibly pointing to the need for artificial tear use during hospitalization.

Introduction: Leber congenital amaurosis (LCA) type 2, due to disease-causing variants in RPE65, is characterized by severe visual loss in early infancy. Current treatments include voretigene neparvovec-rzyl (VN) for RPE65-associated LCA. Herein, we present the long-term follow-up of a patient treated with VN using quantitative autofluorescence (488 nm excitation)., Case Report: A 9-year-old girl with a diagnosis of LCA with biallelic variants in RPE65 presented for evaluation. The patient underwent VN treatment at the age of 11. The patient returned to clinic at age of 19 at which time imaging revealed evidence of chorioretinal atrophy. Quantitative autofluorescence performed prior to gene therapy and at 6- and 8-year follow-up revealed a central area of fundus autofluorescence., Discussion: This case report demonstrates acquisition of fundus autofluorescence at 6- and 8-year follow-up despite the development of chorioretinal atrophy., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

While color fundus photos are used in routine clinical practice to diagnose ophthalmic conditions, evidence suggests that ocular imaging contains valuable information regarding the systemic health features of patients. These features can be identified through computer vision techniques including deep learning (DL) artificial intelligence (AI) models. We aim to construct a DL model that can predict systemic features from fundus images and to determine the optimal method of model construction for this task. Data were collected from a cohort of patients undergoing diabetic retinopathy screening between March 2020 and March 2021. Two models were created for each of 12 systemic health features based on the DenseNet201 architecture: one utilizing transfer learning with images from ImageNet and another from 35,126 fundus images. Here, 1277 fundus images were used to train the AI models. Area under the receiver operating characteristics curve (AUROC) scores were used to compare the model performance. Models utilizing the ImageNet transfer learning data were superior to those using retinal images for transfer learning (mean AUROC 0.78 vs. 0.65, p-value < 0.001). Models using ImageNet pretraining were able to predict systemic features including ethnicity (AUROC 0.93), age > 70 (AUROC 0.90), gender (AUROC 0.85), ACE inhibitor (AUROC 0.82), and ARB medication use (AUROC 0.78). We conclude that fundus images contain valuable information about the systemic characteristics of a patient. To optimize DL model performance, we recommend that even domain specific models consider using transfer learning from more generalized image sets to improve accuracy.

Purpose: To identify vitreous molecular biomarkers associated with autoimmune retinopathy (AIR)., Design: Case-control study., Participants: We analyzed six eyes from four patients diagnosed with AIR and eight comparative controls diagnosed with idiopathic macular holes and epiretinal membranes., Methods: Vitreous biopsies were collected from the participants and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) or multiplex ELISA., Outcome Measures: Protein expression changes were evaluated by 1-way ANOVA (significant p-value <0.05), hierarchical clustering, and pathway analysis to identify candidate protein biomarkers., Results: There were 16 significantly upregulated and 17 significantly downregulated proteins in the vitreous of three AIR patients compared to controls. The most significantly upregulated proteins included lysozyme C (LYSC), zinc-alpha-2-glycoprotein (ZA2G), complement factor D (CFAD), transforming growth factor-beta induced protein (BGH3), beta-crystallin B2, and alpha-crystallin A chain. The most significantly downregulated proteins included disco-interacting protein 2 homolog (DIP2C), retbindin (RTBDN), and amyloid beta precursor like protein 2 (APLP2). Pathway analysis revealed that vascular endothelial growth factor (VEGF) signaling was a top represented pathway in the vitreous of AIR patients compared to controls. In comparison to a different cohort of three AIR patients analyzed by multiplex ELISA, a commonly differentially expressed protein was neuronal cell adhesion molecule (NrCAM) with p-values of 0.027 in the LC-MS/MS dataset and 0.035 in the ELISA dataset., Conclusion: Protein biomarkers such as NrCAM in the vitreous may eventually help diagnose AIR., Competing Interests: Conflict of Interest: No conflicting relationship exists for any author.

Background: Inherited retinal dystrophies describe a heterogeneous group of retinal diseases that lead to the irreversible degeneration of rod and cone photoreceptors and eventual blindness. Recessive loss-of-function mutations in Tubulin Tyrosine Ligase Like 5 (TTLL5) represent a recently described cause of inherited cone-rod and cone dystrophy. This study describes the unusual phenotypes of three patients with autosomal recessive mutations in TTLL5. Examination of these patients included funduscopic evaluation, spectral-domain optical coherence tomography, short-wavelength autofluorescence, and full-field electroretinography (ffERG). Genetic diagnoses were confirmed using whole exome capture. Protein modeling of the identified variants was performed to explore potential genotype-phenotype correlations., Results: Genetic testing revealed five novel variants in TTLL5 in three unrelated patients with retinal dystrophy. Clinical imaging demonstrated features of sectoral cone-rod dystrophy and cone dystrophy, with phenotypic variability seen across all three patients. One patient also developed high-frequency hearing loss during a similar time period as the onset of retinal disease, potentially suggestive of a syndromic disorder. Retinal structure findings were corroborated with functional measures including ffERG findings that supported these diagnoses. Modeling of the five variants suggest that they cause different effects on protein function, providing a potential reason for genotype-phenotype correlation in these patients., Conclusions: The authors report retinal phenotypic findings in three unrelated patients with novel mutations causing autosomal recessive TTLL5-mediated retinal dystrophy. These findings broaden the understanding of the phenotypes associated with TTLL5-mediated retinal disease and suggest that mutations in TTLL5 should be considered as a potential cause of sectoral retinal dystrophy in addition to cone-rod and cone dystrophies., (© 2022. The Author(s).)

Purpose: To report that variants in the gene for a large lamina basal component protein, COL6A6 (collagen type VI alpha 6 chain, Col6α6), linked to chromosome 3p22.1 causes retinitis pigmentosa (RP) in patients with autosomal dominant transmission (adRP)., Methods: A positional-cloning approach, whole exome sequencing, and modeling were used. The proband and several affected family members have been phenotyped and followed for over 12 years., Results: A heterozygous missense variant, c.509C>G (p. Ser170Cys) in exon 2 of COL6A6 (comprised of 36 exons and 2236 amino acids), was observed in a four- generation family and is likely to cause the adRP phenotype. It was identified in 10 affected members. All affected family members had a distinct phenotype: late-onset rod cone dystrophy, with good retained visual acuity, until their late 70s. Immunohistochemistry of human retina showed a dot-like signal at the base of the inner segments of photoreceptors and outer plexiform layer (OPL). The structural modeling of the N7 domain of Col6α6 suggests that the mutant might result in the abnormal cellular localization of collagen VI or malformation of collagen fibers resulting in the loss of its unique filament structure., Conclusions: COL6A6 is widely expressed in human tissues and evolutionary conserved. It is thought to interact with a range of extracellular matrix components. Our findings suggest that this form of RP has long-term useful central visual acuity and a mild progression, which are important considerations for patient counseling.

Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR-Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR-Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically., (© 2022. The Author(s).)

Ocular drug implants (ODIs) are beneficial for treating ocular diseases. However, the lack of a robust injection approach for small-eyed model organisms has been a major technical limitation in developing ODIs. Here, we present a cost-effective, minimally invasive protocol to deliver ODIs into the mouse vitreous called Mouse Implant Intravitreal Injection (MI3). MI3 provides two alternative surgical approaches (air-pressure or plunger) to deliver micro-scaled ODIs into milli-scaled eyes, and expands the preclinical platforms to determine ODIs' efficacy, toxicity, and pharmacokinetics. For complete details on the use and execution of this protocol, please refer to Sun et al. (2021)., Competing Interests: The authors declare no competing interests.

Using small molecule drugs to treat eye diseases carries benefits of specificity, scalability, and transportability, but their efficacy is significantly limited by a fast intraocular clearance rate. Ocular drug implants (ODIs) present a compelling means for the slow and sustained release of small molecule drugs inside the eye. However, methods are needed to inject small molecule ODIs into animals with small eyes, such as mice, which are the primary genetic models for most human ocular diseases. Consequently, it has not been possible to fully investigate efficacy and ocular pharmacokinetics of ODIs. Here, we present a robust, cost-effective, and minimally invasive method called "mouse implant intravitreal injection" (MI3) to deliver ODIs into mouse eyes. This method will expand ODI research to cover the breadth of human eye diseases modeled in mice., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Translational Relevance: Mitochondria are viable therapeutic targets for a broad spectrum of ocular diseases.

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS coronavirus 2 (SARS-CoV-2) viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, avoralstat, PCI-27483, antipain, and soybean trypsin inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, avoralstat significantly reduced lung tissue titers and mitigated weight loss when administered prophylactically to mice susceptible to SARS-CoV-2, indicating its potential to be repositioned for coronavirus disease 2019 (COVID-19) prophylaxis in humans.

Ocular disorders originating in the retina can result in a partial or total loss of vision, making drug delivery to the retina of vital importance. However, effectively delivering drugs to the retina remains a challenge for ophthalmologists due to various anatomical and physicochemical barriers in the eye. This review introduces diverse administration routes and the accordant pharmacokinetic profiles of ocular drugs to aid in the development of safe and efficient drug delivery systems to the retina with a focus on peptidomimetics as a growing class of retinal drugs, which have great therapeutic potential and a high degree of specificity. We also discuss the pharmacokinetic profiles of small molecule drugs due to their structural similarity to small peptidomimetics. Lastly, various formulation strategies are suggested to overcome pharmacokinetic hurdles such as solubility, retention time, enzymatic degradation, tissue targeting, and membrane permeability. This knowledge can be used to help design ocular delivery platforms for peptidomimetics, not only for the treatment of various retinal diseases, but also for the selection of potential peptidomimetic drug targets.

Rare diseases are an emerging global health priority. Although individually rare, the prevalence of rare "orphan" diseases is high, affecting approximately 300 million people worldwide. Treatments for these conditions are often inadequate, leaving the disease to progress unabated. Here, we review the clinical features and pathophysiology of neovascular inflammatory vitreoretinopathy (NIV), a rare inflammatory retinal disease caused by mutations in the CAPN5 gene. Although the prevalence of NIV is low (1 in 1,000,000 people), the disease mimics more common causes of blindness (e.g. uveitis, retinitis pigmentosa, proliferative diabetic retinopathy, and proliferative vitreoretinopathy) at distinct clinical stages. There is no cure for NIV to date. We highlight how personalized proteomics helped identify potential stage-specific biomarkers and drug targets in liquid vitreous biopsies. The NIV vitreous proteome revealed enrichment of molecular pathways associated with common retinal pathologies and implicated superior targets for therapeutic drug repositioning. In addition, we review our pipeline for collecting, storing, and analyzing ophthalmic surgical samples. This approach can be adapted to treat a variety of rare genetic diseases., Competing Interests: Conflict of Interest Disclosures: None reported. Role of the Sponsor: The funding organizations had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Purpose: In a mouse model of blast-mediated traumatic brain injury (bTBI), interleukin-1 (IL-1)-pathway components were tested as potential therapeutic targets for bTBI-mediated retinal ganglion cell (RGC) dysfunction. Sex was also evaluated as a variable for RGC outcomes post-bTBI., Methods: Male and female mice with null mutations in genes encoding IL-1α, IL-1β, or IL-1RI were compared to C57BL/6J wild-type (WT) mice after exposure to three 20-psi blast waves given at an interblast interval of 1 hour or to mice receiving sham injury. To determine if genetic blockade of IL-1α, IL-1β, or IL-1RI could prevent damage to RGCs, the function and structure of these cells were evaluated by pattern electroretinogram and optical coherence tomography, respectively, 5 weeks following blast or sham exposure. RGC survival was also quantitatively assessed via immunohistochemical staining of BRN3A at the completion of the study., Results: Our results showed that male and female WT mice had a similar response to blast-induced retinal injury. Generally, constitutive deletion of IL-1α, IL-1β, or IL-1RI did not provide full protection from the effects of bTBI on visual outcomes; however, injured WT mice had significantly worse visual outcomes compared to the injured genetic knockout mice., Conclusions: Sex does not affect RGC outcomes after bTBI. The genetic studies suggest that deletion of these IL-1 pathway components confers some protection, but global deletion from birth did not result in a complete rescue.

Purpose: The purpose of this paper was to discuss manifestations of primary mitochondrial dysfunctions and whether the retinal pigment epithelium or the photoreceptors are preferentially affected., Methods: A retrospective analysis was performed of patients with clinically and laboratory confirmed diagnoses of maternally inherited diabetes and deafness (MIDD) or Kearns-Sayre syndrome (KSS). Patients underwent full ophthalmic examination, full-field electroretinogram, and multimodal imaging studies, including short-wavelength autofluorescence, spectral domain-optical coherence tomography, and color fundus photography., Results: A total of five patients with MIDD and four patients with KSS were evaluated at two tertiary referral centers. Mean age at initial evaluation was 50.3 years old. Nascent outer retinal tubulations corresponding with faint foci of autofluorescence were observed in two patients with MIDD. Characteristic features of this cohort included a foveal sparing phenotype observed in 13 of 18 eyes (72%), global absence of intraretinal pigment migration, and preserved retinal function on full-field electroretinogram testing in 12 of 16 eyes (75%). One patient diagnosed with MIDD presented with an unusual pattern of atrophy surrounding the parapapillary region and one patient with KSS presented with an atypical choroideremia-like phenotype., Conclusions: MIDD and KSS are phenotypically heterogeneous disorders. Several features of disease suggest that primary mitochondrial dysfunction may first affect the retinal pigment epithelium followed by secondary photoreceptor loss. Similarities between primary mitochondrial degenerations and retinal disorders, such as age-related macular degeneration may suggest a primary role of mitochondria in the pathogenesis of these oligogenic disorders.

Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The purpose of this study was to characterize acute changes in inflammatory pathways in the mouse eye after blast-mediated traumatic brain injury (bTBI) and to determine whether modulation of these pathways could protect the structure and function of retinal ganglion cells (RGC). The bTBI was induced in C57BL/6J male mice by exposure to three 20 psi blast waves directed toward the head with the body shielded, with an inter-blast interval of one hour. Acute cytokine expression in retinal tissue was measured through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) four hours post-blast. Increased retinal expression of interleukin ( lL)-1β, IL-1α, IL-6, and tumor necrosis factor (TNF)α was observed in bTBI mice exposed to blast when compared with shams, which was associated with activation of microglia and macroglia reactivity, assessed via immunohistochemistry with ionized calcium binding adaptor molecule 1 and glial fibrillary acidic protein, respectively, one week post-blast. Blockade of the IL-1 pathway was accomplished using anakinra, an IL-1RI antagonist, administered intra-peritoneally for one week before injury and continuing for three weeks post-injury. Retinal function and RGC layer thickness were evaluated four weeks post-injury using pattern electroretinogram (PERG) and optical coherence tomography (OCT), respectively. After bTBI, anakinra treatment resulted in a preservation of RGC function and RGC structure when compared with saline treated bTBI mice. Optic nerve integrity analysis demonstrated a trend of decreased damage suggesting that IL-1 blockade also prevents axonal damage after blast. Blast exposure results in increased retinal inflammation including upregulation of pro-inflammatory cytokines and activation of resident microglia and macroglia. This may explain partially the RGC loss we observed in this model, as blockade of the acute inflammatory response after injury with the IL-1R1 antagonist anakinra resulted in preservation of RGC function and RGC layer thickness.

Background: Neurodegenerative diseases are incurable disorders caused by progressive neuronal cell death. Retinitis pigmentosa (RP) is a blinding neurodegenerative disease that results in photoreceptor death and progresses to the loss of the entire retinal network. We previously found that proteomic analysis of the adjacent vitreous served as way to indirectly biopsy the retina and identify changes in the retinal proteome., Methods: We analyzed protein expression in liquid vitreous biopsies from autosomal recessive (ar)RP patients with PDE6A mutations and arRP mice with Pde6ɑ mutations. Proteomic analysis of retina and vitreous samples identified molecular pathways affected at the onset of photoreceptor death. Based on affected molecular pathways, arRP mice were treated with a ketogenic diet or metabolites involved in fatty-acid synthesis, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle., Findings: Dietary supplementation of a single metabolite, ɑ-ketoglutarate, increased docosahexaeonic acid levels, provided neuroprotection, and enhanced visual function in arRP mice. A ketogenic diet delayed photoreceptor cell loss, while vitamin B supplementation had a limited effect. Finally, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on ɑ-ketoglutarate-treated mice revealed restoration of metabolites that correlated with our proteomic findings: uridine, dihydrouridine, and thymidine (pyrimidine and purine metabolism), glutamine and glutamate (glutamine/glutamate conversion), and succinic and aconitic acid (TCA cycle)., Interpretation: This study demonstrates that replenishing TCA cycle metabolites via oral supplementation prolongs retinal function and provides a neuroprotective effect on the photoreceptor cells and inner retinal network., Funding: NIH grants [R01EY026682, R01EY024665, R01EY025225, R01EY024698, R21AG050437, P30EY026877, 5P30EY019007, R01EY018213, F30EYE027986, T32GM007337, 5P30CA013696], NSF grant CHE-1734082., Competing Interests: Declaration of Competing Interest Dr. Wert has nothing to disclose. Mr. Velez has nothing to disclose. Dr. Kanchustambham has nothing to disclose. Mr. Shankar has nothing to disclose. Ms. Evans has nothing to disclose. Dr. Sengillo has nothing to disclose. Dr. Zare has nothing to disclose. Dr. Bassuk has nothing to disclose. Dr. Tsang has nothing to disclose. Dr. Mahajan has nothing to disclose., (Published by Elsevier B.V.)

Background: Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Employing it as a first-tier genetic testing can be favored due to the associated lower incremental cost per diagnosis compared to when using it later in the diagnostic pathway. However, there are technical limitations of WES that can lead to inaccurate negative variant callings. Our study presents these limitations through a re-evaluation of negative WES results using subsequent tests primarily driven by fundoscopic findings. These tests included targeted gene testing, inherited retinal gene panels, whole genome sequencing (WGS), and array comparative genomic hybridization., Results: Subsequent genetic testing guided by fundoscopy findings identified the following variant types causing retinitis pigmentosa that were not detected by WES: frameshift deletion and nonsense variants in the RPGR gene, 353-bp Alu repeat insertions in the MAK gene, and large exonic deletion variants in the EYS and PRPF31 genes. Deep intronic variants in the ABCA4 gene causing Stargardt disease and the GUCY2D gene causing Leber congenital amaurosis were also identified., Conclusions: Negative WES analyses inconsistent with the phenotype should raise clinical suspicion. Subsequent genetic testing may detect genetic variants missed by WES and can make patients eligible for gene replacement therapy and upcoming clinical trials. When phenotypic findings support a genetic etiology, negative WES results should be followed by targeted gene sequencing, array based approach or whole genome sequencing.

Increased calpain activity is linked to neuroinflammation including a heritable retinal disease caused by hyper-activating mutations in the calcium-activated calpain-5 (CAPN5) protease. Although structures for classical calpains are known, the structure of CAPN5, a non-classical calpain, remains undetermined. Here we report the 2.8 Å crystal structure of the human CAPN5 protease core (CAPN5-PC). Compared to classical calpains, CAPN5-PC requires high calcium concentrations for maximal activity. Structure-based phylogenetic analysis and multiple sequence alignment reveal that CAPN5-PC contains three elongated flexible loops compared to its classical counterparts. The presence of a disease-causing mutation (c.799G>A, p.Gly267Ser) on the unique PC2L2 loop reveals a function in this region for regulating enzymatic activity. This mechanism could be transferred to distant calpains, using synthetic calpain hybrids, suggesting an evolutionary mechanism for fine-tuning calpain function by modifying flexible loops. Further, the open (inactive) conformation of CAPN5-PC provides structural insight into CAPN5-specific residues that can guide inhibitor design., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Background: Mutations in the Kelch-like protein 7 (KLHL7) represent a recently described and, to date, poorly characterized etiology of inherited retinal dystrophy. Dominant mutations in KLHL7 are a cause of isolated, non-syndromic retinitis pigmentosa (RP). In contrast, recessive loss-of-function mutations are known to cause Crisponi or Bohring-Opitz like cold induced sweating syndrome-3 (BOS-3). In this study, the phenotype and progression of five unrelated patients with KLHL7 mediated autosomal dominant RP (adRP) are characterized. Clinical evaluation of these patients involved a complete ophthalmic exam, full-field electroretinography (ffERG), and imaging, including fundus photography, spectral domain optical coherence tomography (SD-OCT), short wavelength fundus autofluorescence (SW-AF), and near-infrared fundus autofluorescence (NIR-AF). Molecular diagnoses were performed using whole-exome sequencing or gene panel testing. Disease progression was monitored in three patients with available data for a mean follow up time of 4.5 ± 2.9 years. Protein modeling was performed for all variants found in this study in addition to those documented in the literature for recessive loss-of-function alleles causing Crisponi or Bohring-Opitz like cold-induced sweating syndrome., Results: Genetic testing in three patients identified two novel variants within the 3-box motif of the BACK domain: c.472 T > C:p.(Cys158Arg) and c.433A > T:p.(Asn145Tyr). Clinical imaging demonstrated hyperautofluorescent ring formation on both SW-AF and NIR-AF in three patients, with diffuse peripheral and peripapillary atrophy seen in all but one case. SD-OCT demonstrated a phenotypic spectrum, from parafoveal atrophy of the outer retina with foveal sparing to widespread retinal thinning and loss of photoreceptors. Incidence of cystoid macular edema was high with four of five patients affected. Protein modeling of dominant alleles versus recessive loss-of-function alleles showed dominant alleles localized to the BTB and BACK domains while recessive alleles were found in the Kelch domain., Conclusions: We report the phenotype in five patients with KLHL7 mediated adRP, two novel coding variants, and imaging biomarkers using SW-AF and NIR-AF. These findings may influence future gene-based therapies for adRP and pave the way for mechanistic studies that elucidate the pathogenesis of KLHL7-mediated RP.

The PROM1 ( prominin 1 ) gene encodes an 865-amino acid glycoprotein that is expressed in retinoblastoma cell lines and in the adult retina. The protein is localized to photoreceptor outer segment disc membranes, where it plays a structural role, and in the retinal pigment epithelium (RPE), where it acts as a cytosolic protein that mediates autophagy. Mutations in PROM1 are typically associated with cone-rod dystrophy 12 (OMIM#3612657), autosomal dominant retinal macular dystrophy 2 (OMIM#608051), autosomal recessive retinitis pigmentosa 41 (OMIM#612095), and Stargardt disease 4 (OMIM#603786). Here we describe the first case of PROM1 -associated Leber congenital amaurosis (LCA) in a 12-yr-old Asian male, caused by two not previously described deleterious frameshift variants in the compound heterozygous state. Clinical features include the presence of bull's eye maculopathy, pendular horizontal nystagmus, and photodysphoria consistent with the clinical diagnosis of LCA. The patient was evaluated using ophthalmic imaging, electroretinography, and whole-exome sequencing. Electroretinography revealed extinguished retinal activity., (© 2019 Ragi et al.; Published by Cold Spring Harbor Laboratory Press.)

Hypoxia associated with the high metabolic demand of rods has been implicated in the pathology of age-related macular degeneration (AMD), the most common cause of adult blindness in the developed world. The majority of AMD-associated severe vision loss cases are due to exudative AMD, characterized by neovascularization. To further investigate the causes and histopathology of exudative AMD, we conditionally induced hypoxia in a novel preclinical AMD model (Pde6gcreERT2/+;Vhl-/-) by targeting Vhl and used multimodal imaging and immunohistochemistry to track the development of hypoxia-induced neovascularization. In addition to developing a preclinical model that phenocopies exudative AMD, our studies revealed that the photoreceptor hypoxic response initiates and drives type 3 neovascularization, mainly in the outer retina. Activation of the VHL-HIF1a-VEGF-EPO pathway in the adult retina led to long-term neovascularization, retinal hemorrhages and compromised retinal layers. Our novel preclinical model would accelerate the testing of therapies that use metabolomic approaches to ameliorate AMD., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)