Lack of evidence for GWAS signals of exfoliation glaucoma working via monogenic loss-of-function mutation in the nearest gene.

PURPOSE: Exfoliation syndrome (XFS) is a systemic disease of elastin-rich tissues involving a deposition of fibrillar exfoliative material (XFM) in the anterior chamber of the eye, which can promote glaucoma. The purpose of this study was to create mice with CRISPR/Cas9-induced variations in candidate genes identified from human genome-wide association studies (GWAS) and screen them for indices of XFS. METHODS: Variants predicted to be deleterious were sought in the Agpat1, Cacna1a, Loxl1, Pomp, Rbms3, Sema6a, and Tlcd5 genes of C57BL/6J mice using CRISPR/Cas9-based gene editing. Strains were phenotyped by slit-lamp, SD-OCT imaging, and fundus exams at 1-5 mos of age. Smaller cohorts of 12-mos-old mice were also studied. RESULTS: Deleterious variants were identified in six targets; Pomp was recalcitrant to targeting. Multiple alleles of some targets were isolated, yielding 12 strains. Across all genotypes and ages, 277 mice were assessed by 902 slit-lamp exams, 928 SD-OCT exams, and 358 fundus exams. Homozygosity for Agpat1 or Cacna1a mutations led to early lethality; homozygosity for Loxl1 mutations led to pelvic organ prolapse, preventing aging. Loxl1 homozygotes exhibited a conjunctival phenotype of potential relevance to XFS. Multiple other genotype-specific phenotypes were variously identified. XFM was not observed in any mice. CONCLUSIONS: This study did not detect XFM in any of the strains. This may have been due to species-specific differences, background dependence, or insufficient aging. Alternatively, it is possible that the current candidates, selected based on proximity to GWAS signals, are not effectors acting via monogenic loss-of-function mechanisms.

Unsuccessful transscleral cyclophotocoagulation in oculocutaneous albinism.

Purpose: To report a case of unsuccessful transscleral cyclophotocoagulation in a patient with OCA1A tyrosinase-negative oculocutaneous albinism. Observations: A 35-year-old Asian female with molecularly diagnosed OCA1A (tyrosinase-negative) oculocutaneous albinism and unilateral severe mixed mechanism glaucoma underwent transscleral cyclophotocoagulation on two separate occasions to treat elevated intraocular pressure. The intraocular pressure remained markedly elevated approximately 1 month following two separate treatments of transscleral cyclophotocoagulation while using high energy settings. The poor efficacy of both cyclophotocoagulation treatments was most likely due to a lack of melanin in the setting of oculocutaneous albinism. Conclusions and importance: Cyclophotocoagulation in patients with oculocutaneous albinism is less likely to yield a desired lowering of intraocular pressure due to the absence of melanin.

GJA3 Genetic Variation and Autosomal Dominant Congenital Cataracts and Glaucoma Following Cataract Surgery.

Importance: The p.Asp67Tyr genetic variant in the GJA3 gene is responsible for congenital cataracts in a family with a high incidence of glaucoma following cataract surgery. Objective: To describe the clinical features of a family with a strong association between congenital cataracts and glaucoma following cataract surgery secondary to a genetic variant in the GJA3 gene (NM_021954.4:c.199G>T, p.Asp67Tyr). Design, Setting, and Participants: This was a retrospective, observational, case series, genetic association study from the University of Iowa spanning 61 years. Examined were the ophthalmic records from 1961 through 2022 of the family members of a 4-generation pedigree with autosomal dominant congenital cataracts. Main Outcomes and Measures: Frequency of glaucoma following cataract surgery and postoperative complications among family members with congenital cataract due to the p.Asp67Tyr GJA3 genetic variant. Results: Medical records were available from 11 of 12 family members (7 male [63.6%]) with congenital cataract with a mean (SD) follow-up of 30 (21.7) years (range, 0.2-61 years). Eight of 9 patients with congenital cataracts developed glaucoma, and 8 of 8 patients who had cataract surgery at age 2 years or younger developed glaucoma following cataract surgery. The only family member with congenital cataracts who did not develop glaucoma had delayed cataract surgery until 12 and 21 years of age. Five of 11 family members (45.5%) had retinal detachments after cataract extraction and vitrectomy. No patients developed retinal detachments after prophylactic 360-degree endolaser. Conclusions and Relevance: The GJA3 genetic variant, p.Asp67Tyr, was identified in a 4-generation congenital cataract pedigree from Iowa. This report suggests that patients with congenital cataract due to some GJA3 genetic variants may be at especially high risk for glaucoma following cataract surgery. Retinal detachments after cataract extraction in the first 2 years of life were also common in this family, and prophylactic retinal endolaser may be indicated at the time of surgery.

Thrombospondin Mutations and Patients With Primary Congenital Glaucoma in a United States Population.

Mutations in the thrombospondin 1 ( THBS1 ) gene have been previously reported in primary congenital glaucoma (PCG) pedigrees that exhibit autosomal dominant inheritance with low penetrance. We sought to determine the role of THBS1 mutations in a cohort of 20 patients with PCG and 362 normal controls from Iowa using a combination of Sanger sequencing and whole exome sequencing. We detected 16 different THBS1 variants, including 4 rare, nonsynonymous variants (p.Thr611Met, p.Asn708Lys, p.Gln1089His, and p.Glu1166Lys). However, none of these variants were judged to be disease-causing mutations based on: 1) prevalence in cases and controls from Iowa, 2) prevalence in the public database gnomAD, 3) mutation analysis algorithms, and 4) THBS1 DNA sequence conservation. These results indicate THBS1 mutations are not a common cause of PCG in patients from Iowa and may be a rare cause of PCG overall.

Familial normal tension glaucoma genetics.

Glaucoma is defined by characteristic optic nerve damage and corresponding visual field defects and is the leading cause of irreversible blindness in the world. Elevated intraocular pressure (IOP) is a strong risk factor for developing glaucoma. However, glaucoma can occur at any IOP. Normal tension glaucoma (NTG) arises with IOPs that are within what has been defined as a normal range, i.e., 21 mm Hg or less, which may present challenges in its diagnosis and management. Identifying inheritance patterns and genetic mutations in families with NTG has helped elucidate mechanisms of NTG, however the pathophysiology is complex and not fully understood. Approximately 2% of NTG cases are caused primarily by mutations in single genes, optineurin (OPTN), TANK binding kinase 1 (TKB1), or myocilin (MYOC). Herein, we review pedigree studies of NTG and autosomal dominant NTG caused by OPTN, TBK1, and MYOC mutations. We review identified mutations and resulting clinical features of OPTN-associated and TBK1-associated NTG, including long-term follow up of these patients with NTG. In addition, we report a new four-generation pedigree of NTG caused by a Glu50Lys OPTN mutation, including six family members with a mean follow up of 17 years. Common features of OPTN -associated NTG due to Glu50Lys mutation included early onset of disease with an IOP <21 mm Hg, marked optic disc cupping, and progressive visual field loss which appeared to stabilize once an IOP of less than 10 mm Hg was achieved. Lastly, we review risk factor genes which have been identified to contribute to the complex inheritance of NTG.

Alpha-gal syndrome (AGS) in a glaucoma suspect with narrow iridocorneal angles.

Purpose: Alpha-gal syndrome (AGS) is an allergy to non-primate mammalian carbohydrate (galactose-alpha-1,3-galactose) which may cause anaphylaxis. Allergic patients must avoid ophthalmic drugs containing animal-derived ingredients. Observations: We report a 59-year-old non-Hispanic white woman who was referred for a glaucoma evaluation. She had been diagnosed with AGS after a tick bite in 2017. Ophthalmic exam revealed potentially occludable, narrow iridocorneal angles and laser iridotomy was recommended. Prior to performing the iridotomy, we investigated the ophthalmic medications required for the procedure to identify options that are free of animal-derived products and safe to use. Laser iridotomy was performed without complications or allergy to medications. Conclusions: Ophthalmologists need to be aware of both the presence of AGS as well as the identity of ophthalmic medications that are safe to use in patients with this condition to avoid potentially lethal allergic responses.

Specifications of the ACMG/AMP variant curation guidelines for myocilin: Recommendations from the clingen glaucoma expert panel.

The standardization of variant curation criteria is essential for accurate interpretation of genetic results and clinical care of patients. The variant curation guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 are widely used but are not gene specific. To address this issue, the Clinical Genome Resource (ClinGen) Variant Curation Expert Panels (VCEP) have been tasked with developing gene-specific variant curation guidelines. The Glaucoma VCEP was created to develop rule specifications for genes associated with primary glaucoma, including myocilin (MYOC), the most common cause of Mendelian glaucoma. Of the 28 ACMG/AMP criteria, the Glaucoma VCEP adapted 15 rules to MYOC and determined 13 rules not applicable. Key specifications included determining minor allele frequency thresholds, developing an approach to counting probands and segregations, and reviewing functional assays. The rules were piloted on 81 variants and led to a change in classification in 40% of those that were classified in ClinVar, with functional evidence influencing the classification of 18 variants. The standardized variant curation guidelines for MYOC provide a framework for the consistent application of the rules between laboratories, to improve MYOC genetic testing in the management of glaucoma.

Prevalence of Open-angle Glaucoma in the Faroese Population.

PURPOSE: The Faroe Islands are home to 50,000 genetically isolated people in the North Atlantic. The prevalence of open-angle glaucoma (OAG) in the Faroese population is unknown. Consequently, we conducted a survey to determine the prevalence of OAG in the Faroese population. We also investigated the role of known glaucoma-causing genes in Faroese OAG. MATERIALS AND METHODS: We conducted a prospective survey of known and newly diagnosed glaucoma patients at the Faroese National Hospital, Landssjukrahusid, Tórshavn between October 1, 2015 to December 31, 2017. In addition we reviewed the only eye care provider in the Faroese Islands by scrutinizing electronic medical records between 2009 and June 15, 2014, October 1, 2015 and the partly overlapping prescriptions for ocular hypotensive medications in 2016 to identify patients with either a diagnosis of glaucoma, a diagnosis of ocular hypertension or a prescription for ocular hypotensive medications. Next, we prospectively confirmed diagnoses with complete eye examinations. Patient DNA samples were tested for variations in known glaucoma-causing genes [myocilin (MYOC), optineurin (OPTN), and TANK binding kinase 1 (TBK1)]. RESULTS: We determined the age-related prevalence of OAG January 1, 2017 in individuals 40 years or older to be 10.7/1000 (1.07%) and highly age-related. A diagnosis of OAG was present in 264 patients, of whom 211 (79.9%) had primary OAG (including normal tension glaucoma), 49 (18.6%) had pseudoexfoliation glaucoma, and 4 (1.5%) had pigmentary glaucoma. Among patients receiving medications for glaucoma, nearly 50% had primary OAG, while the majority of the rest had ocular hypertension or secondary glaucoma. No disease-causing variants were detected in MYOC, OPTN, or TBK1. CONCLUSIONS: The calculated prevalence of OAG in the Faroe Islands was 1.07%. The absence of MYOC, OPTN, or TBK1 disease-causing variants in Faroese primary OAG patients suggests that a different, potentially unique set of genes may be contributing to the pathogenesis of glaucoma in this population.

Chimeric Helper-Dependent Adenoviruses Transduce Retinal Ganglion Cells and Müller Cells in Human Retinal Explants.

Purpose: Despite numerous recent advances in retinal gene therapy using adeno-associated viruses (AAVs) as delivery vectors, there remains a crucial need to identify viral vectors with the ability to transduce specific retinal cell types and that have a larger carrying capacity than AAV. In this study, we evaluate the retinal tropism of 2 chimeric helper-dependent adenoviruses (HDAds), helper-dependent adenovirus serotype 5 (HDAd5)/3 and HDAd5/35, both ex vivo using human retinal explants and in vivo using rats. Methods: We transduced cultured human retinal explants with HDAd5/3 and HDAd5/35 carrying an eGFP vector and evaluated tropism and transduction efficiency using immunohistochemistry. To assess in vivo transduction efficiency, subretinal injections were performed in wild-type Sprague-Dawley rats. For both explants and subretinal injections, we delivered 10 µL (1 × 106 vector genomes/mL) and assessed tropism at 7- and 14-days post-transduction, respectively. Results: HDAd5/3 and HDAd5/35 both transduced human retinal ganglion cells (RGCs) and Müller cells, but not photoreceptors, in human retinal explants. However, subretinal injections in albino rats resulted in transduction of the retinal pigmented epithelium only, highlighting species-specific differences in retinal tropism and the value of a human explant model when testing vectors for eventual human gene therapy. Conclusions: Chimeric HDAds are promising candidates for the delivery of large genes, multiple genes, or neuroprotective factors to Müller cells and RGCs. These vectors may have utility for targeted therapy of neurodegenerative diseases primarily involving retinal ganglion or Müller cell types, such as glaucoma or macular telangiectasia type 2.

Diffusion Tensor Imaging of Visual Pathway Abnormalities in Five Glaucoma Animal Models.

Purpose: To characterize the visual pathway integrity of five glaucoma animal models using diffusion tensor imaging (DTI). Methods: Two experimentally induced and three genetically determined models of glaucoma were evaluated. For inducible models, chronic IOP elevation was achieved via intracameral injection of microbeads or laser photocoagulation of the trabecular meshwork in adult rodent eyes. For genetic models, the DBA/2J mouse model of pigmentary glaucoma, the LTBP2 mutant feline model of congenital glaucoma, and the transgenic TBK1 mouse model of normotensive glaucoma were compared with their respective genetically matched healthy controls. DTI parameters, including fractional anisotropy, axial diffusivity, and radial diffusivity, were evaluated along the optic nerve and optic tract. Results: Significantly elevated IOP relative to controls was observed in each animal model except for the transgenic TBK1 mice. Significantly lower fractional anisotropy and higher radial diffusivity were observed along the visual pathways of the microbead- and laser-induced rodent models, the DBA/2J mice, and the LTBP2-mutant cats compared with their respective healthy controls. The DBA/2J mice also exhibited lower axial diffusivity, which was not observed in the other models examined. No apparent DTI change was observed in the transgenic TBK1 mice compared with controls. Conclusions: Chronic IOP elevation was accompanied by decreased fractional anisotropy and increased radial diffusivity along the optic nerve or optic tract, suggestive of disrupted microstructural integrity in both inducible and genetic glaucoma animal models. The effects on axial diffusivity differed between models, indicating that this DTI metric may represent different aspects of pathological changes over time and with severity.

Automated segmentation of choroidal layers from 3-dimensional macular optical coherence tomography scans.

BACKGROUND: Changes in choroidal thickness are associated with various ocular diseases, and the choroid can be imaged using spectral-domain optical coherence tomography (SD-OCT) and enhanced depth imaging OCT (EDI-OCT). NEW METHOD: Eighty macular SD-OCT volumes from 80 patients were obtained using the Zeiss Cirrus machine. Eleven additional control subjects had two Cirrus scans done in one visit along with enhanced depth imaging (EDI-OCT) using the Heidelberg Spectralis machine. To automatically segment choroidal layers from the OCT volumes, our graph-theoretic approach was utilized. The segmentation results were compared with reference standards from two independent graders, and the accuracy of automated segmentation was calculated using unsigned/signed border positioning/thickness errors and Dice similarity coefficient (DSC). The repeatability and reproducibility of our choroidal thicknesses were determined by intraclass correlation coefficient (ICC), coefficient of variation (CV), and repeatability coefficient (RC). RESULTS: The mean unsigned/signed border positioning errors for the choroidal inner and outer surfaces are 3.39 ± 1.26 µm (mean ± standard deviation)/- 1.52 ± 1.63 µm and 16.09 ± 6.21 µm/4.73 ± 9.53 µm, respectively. The mean unsigned/signed choroidal thickness errors are 16.54 ± 6.47 µm/6.25 ± 9.91 µm, and the mean DSC is 0.949 ± 0.025. The ICC (95% confidence interval), CV, RC values are 0.991 (0.977-0.997), 2.48%, 14.25 µm for the repeatability and 0.991 (0.977-0.997), 2.49%, 14.30 µm for the reproducibility studies, respectively. COMPARISON WITH EXISTING METHOD(S): The proposed method outperformed our previous method using choroidal vessel segmentation and inter-grader variability. CONCLUSIONS: This automated segmentation method can reliably measure choroidal thickness using different OCT platforms.

Exome-based investigation of the genetic basis of human pigmentary glaucoma.

BACKGROUND: Glaucoma is a leading cause of visual disability and blindness. Release of iris pigment within the eye, pigment dispersion syndrome (PDS), can lead to one type of glaucoma known as pigmentary glaucoma. PDS has a genetic component, however, the genes involved with this condition are largely unknown. We sought to discover genes that cause PDS by testing cohorts of patients and controls for mutations using a tiered analysis of exome data. RESULTS: Our primary analysis evaluated melanosome-related genes that cause dispersion of iris pigment in mice (TYRP1, GPNMB, LYST, DCT, and MITF). We identified rare mutations, but they were not statistically enriched in PDS patients. Our secondary analyses examined PMEL (previously linked with PDS), MRAP, and 19 other genes. Four MRAP mutations were identified in PDS cases but not in controls (p = 0.016). Immunohistochemical analysis of human donor eyes revealed abundant MRAP protein in the iris, the source of pigment in PDS. However, analysis of MRAP in additional cohorts (415 cases and 1645 controls) did not support an association with PDS. We also did not confirm a link between PMEL and PDS in our cohorts due to lack of reported mutations and similar frequency of the variants in PDS patients as in control subjects. CONCLUSIONS: We did not detect a statistical enrichment of mutations in melanosome-related genes in human PDS patients and we found conflicting data about the likely pathogenicity of MRAP mutations. PDS may have a complex genetic basis that is not easily unraveled with exome analyses.

Cell-Matrix Interactions in the Eye: From Cornea to Choroid.

The extracellular matrix (ECM) plays a crucial role in all parts of the eye, from maintaining clarity and hydration of the cornea and vitreous to regulating angiogenesis, intraocular pressure maintenance, and vascular signaling. This review focuses on the interactions of the ECM for homeostasis of normal physiologic functions of the cornea, vitreous, retina, retinal pigment epithelium, Bruch's membrane, and choroid as well as trabecular meshwork, optic nerve, conjunctiva and tenon's layer as it relates to glaucoma. A variety of pathways and key factors related to ECM in the eye are discussed, including but not limited to those related to transforming growth factor-ß, vascular endothelial growth factor, basic-fibroblastic growth factor, connective tissue growth factor, matrix metalloproteinases (including MMP-2 and MMP-9, and MMP-14), collagen IV, fibronectin, elastin, canonical signaling, integrins, and endothelial morphogenesis consistent of cellular activation-tubulogenesis and cellular differentiation-stabilization. Alterations contributing to disease states such as wound healing, diabetes-related complications, Fuchs endothelial corneal dystrophy, angiogenesis, fibrosis, age-related macular degeneration, retinal detachment, and posteriorly inserted vitreous base are also reviewed.

Aqueous Misdirection After Trabeculectomy in a Down Syndrome Patient With Angle-closure Glaucoma.

Down syndrome is a genetic disease caused by trisomy of chromosome 21 that is characterized by numerous systemic abnormalities including intellectual disability, stereotypical facies, and congenital heart malformations. Ocular abnormalities are commonly seen with Down syndrome including corneal disease (keratoconus), refractive error, and atypical irides (Brushfield spots). We report the first case of aqueous misdirection in a patient with Down syndrome after trabeculectomy. Patients with Down syndrome often have small, hyperopic eyes with narrow iridocorneal angles and may be at increased risk for aqueous misdirection associated with surgical procedures. Awareness of this risk may aid surgical planning and postoperative management.

Recombinant adenovirus causes prolonged mobilization of macrophages in the anterior chambers of mice.

PURPOSE: Ocular tissues of mice have been studied in many ways using replication-deficient species C type 5 adenovirus (Ad5) as a tool for manipulating gene expression. Whereas refinements to injection protocols and tropism have led to several advances in targeting cells of interest, there remains a relative lack of information concerning how Ad5 may influence other ocular cell types capable of confounding experimental interpretation. Here, a slit lamp is used to thoroughly photodocument the sequelae of intraocular Ad5 injections over time in mice, with attention to potentially confounding indices of inflammation. METHODS: A cohort of C57BL/6J mice was randomly split into three groups (Virus, receiving unilateral intracameral injection with 5×107 plaque-forming units (pfu) of a cargo-less Ad5 construct; Saline, receiving unilateral balanced salt solution injection; and Naïve, receiving no injections). From this initial experiment, a total of 52 eyes from 26 mice were photodocumented via slit lamp at four time points (baseline and 1, 3, and 10 weeks following initiation of the experiment) by an observer masked to treatments and other parameters of the experimental design. Following the last in vivo exam, tissues were collected. Based on the slit-lamp data, tissues were studied via immunostaining with the macrophage marker F4/80. Subsequently, three iterations of the original experiment were performed with otherwise identical experimental parameters testing the effect of age, intravitreal injection, and A195 buffer, adding slit-lamp photodocumentation of an additional 32 eyes from 16 mice. RESULTS: The masked investigator could use the sequential images from each mouse in the initial experiment to assign each mouse to its correct treatment group with near perfect fidelity. Virus-injected eyes were characterized by corneal damage indicative of intraocular injection and a prolonged mobilization of clump cells on the surface of the iris. Saline-injected eyes had only transient corneal opacities indicative of intraocular injections, and Naïve eyes remained normal. Immunostaining with F4/80 was consistent with ascribing the clump cells visualized via slit-lamp imaging as a type of macrophage. Experimental iterations using Ad5 indicate that all virus-injected eyes had the distinguishing feature of a prolonged presence of clump cells on the surface of the iris regardless of injection site. Mice receiving an intraocular injection of Ad5 at an advanced age displayed a protracted course of corneal cloudiness that prevented detailed visualization of the iris at the last time point. CONCLUSIONS: Because the eye is often considered an "immune privileged site," we suspect that several studies have neglected to consider that the presence of Ad5 in the eye might evoke strong reactions from the innate immune system. Ad5 injection caused a sustained mobilization of clump cells-that is, macrophages. This change is likely a consequence of either direct macrophage transduction or a secondary response to cytokines produced locally by other transduced cells. Regardless of how these cells were altered, the important implication is that the adenovirus led to long-lasting changes in the environment of the anterior chamber. Thus, these findings describe a caveat of Ad5-mediated studies involving macrophage mobilization, which we encourage groups to use as a bioassay in their experiments and consider in interpretation of their ongoing experiments using adenoviruses.