Choroidal endothelial and macrophage gene expression in atrophic and neovascular macular degeneration.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.

Transcriptomic and Chromatin Accessibility Analysis of the Human Macular and Peripheral Retinal Pigment Epithelium at the Single-Cell Level.

Some human retinal diseases are characterized by pathology that is restricted to specific cell types and to specific regions of the eye. Several disease entities either selectively affect or spare the macula, the retina region at the center of the posterior pole. Photoreceptor cells in the macula are involved in high-acuity vision and require metabolic support from non-neuronal cell types. Some macular diseases involve the retinal pigment epithelium (RPE), an epithelial cell layer with several metabolic-support functions essential for the overlying photoreceptors. In the current study, the ways in which RPE confers region-specific disease susceptibility were determined by examining heterogeneity within RPE tissue from human donors. RPE nuclei from the macular and peripheral retina were profiled using joint single-nucleus RNA and ATAC sequencing. The expression of several genes differed between macular and peripheral RPE. Region-specific ATAC peaks were found, suggesting regulatory elements used exclusively by macular or peripheral RPE. Across anatomic regions, subpopulations of RPE were identified that appeared to have differential levels of expression of visual cycle genes. Finally, loci associated with age-related macular degeneration were examined for a better understanding of RPE-specific disease phenotypes. These findings showed variations in the regulation of gene expression in the human RPE by region and subpopulation, and provide a source for a better understanding of the molecular basis of macular disease.

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.

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.

Choriocapillaris Degeneration in Geographic Atrophy.

Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculae from controls (n = 99), early AMD (n = 35), or clinically diagnosed with geographic atrophy (GA; n = 9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross-sectional area of capillary lumens divided by length) and of vascular lumen/stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni-corrected P = 0.024) with greater loss in GA (Bonferroni-corrected P < 10-9), even in areas of intact retinal pigment epithelium. In contrast, changes in lumen/stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P > 0.05), suggesting choriocapillaris changes are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration, predominantly in the microvasculature, contributes to dry AMD progression. Addressing capillary loss in AMD remains an important translational target.

Nanophthalmos patient with a THR518MET mutation in MYRF, a case report.

BACKGROUND: Nanophthalmos has a significant genetic background and disease-causing mutations have been recently been reported in the myelin regulatory factor (MYRF) gene. We report clinical features in a patient with nanophthalmos and a Thr518Met MYRF mutation. CASE PRESENTATION: A three-year-old male was discovered to have nanophthalmos after first presenting to the emergency department for a frontal headache, eye pain, emesis, and lethargy. Imaging studies (CT and MRI) were negative except for increased posterior fossa cerebrospinal fluid. Subsequent examinations revealed nanophthalmos (short axial eye lengths 18.1 mm OD and 18.3 mm OS), microcornea, and a large crystalline lens. Peripheral chorioretinal pigment abnormalities were also observed. He experienced episodes of marked ocular hypertension (53 mmHg OD and 60 mmHg) likely due to intermittent angle closure precipitated by nanophthalmos. The ocular hypertension was responsive to topical medicines. Genetic analysis of known nanophthalmos genes MFRP and TMEM98 were negative, while a novel mutation, Thr518Met was detected in MYRF. The Thr518Met mutation was absent from 362 matched normal controls and was extremely rare in a large population database, allele frequency of 0.000024. The Thr518Met mutation altered a highly conserved amino acid in the MYRF protein and three of four algorithms suggested that this mutation is likely pathogenic. Finally, molecular modeling showed that the Thr518Met mutation is damaging to MYRF structure. Together these data suggest that the Thr518Met mutation causes nanophthalmos. CONCLUSIONS: Nanophthalmos may present at an early age with features of angle closure glaucoma and a Thr518Met mutation in MYRF was detected in a patient with nanophthalmos. Prevalence data, homology data, mutation analysis data, and protein modeling data suggest that this variant is pathogenic and may expand the phenotypic range of syndromic nanophthalmos caused by MYRF mutations to include central nervous system abnormalities (increased posterior fossa cerebrospinal fluid).

Mendelian genes in primary open angle glaucoma.

Mutations in each of three genes, myocilin (MYOC), optineurin (OPTN), and TANK binding kinase 1 (TBK1), may cause primary open-angle glaucoma (POAG) that is inherited as a Mendelian trait. MYOC mutations cause 3-4% of POAG cases with IOP >21 mmHg, while mutations in OPTN, TBK1, and MYOC each cause ∼1% of POAG with IOP ≤21 mmHg, i.e. normal tension glaucoma. Identification of these disease-causing genes has provided insights into glaucoma pathogenesis. Mutations in MYOC cause a cascade of abnormalities in the trabecular meshwork including intracellular retention of MYOC protein, decreased aqueous outflow, higher intraocular pressure, and glaucoma. Investigation of MYOC mutations demonstrated that abnormal retention of intracellular MYOC and stimulation of endoplasmic reticular (ER) stress may be important steps in the development of MYOC-associated glaucoma. Mutations in OPTN and TBK1 cause a dysregulation of autophagy which may directly cause retinal ganglion cell damage and normal tension glaucoma. Discovery of these Mendelian causes of glaucoma has also provided a new set of potential therapeutic targets that may ultimately lead to novel, gene-directed glaucoma treatments.

Characterization of Chorioretinopathy Associated with Mitochondrial Trifunctional Protein Disorders: Long-Term Follow-up of 21 Cases.

PURPOSE: To assess long-term effects of genotype on chorioretinopathy severity in patients with mitochondrial trifunctional protein (MTP) disorders. DESIGN: Retrospective case series. PARTICIPANTS: Consecutive patients with MTP disorders evaluated at a single center from 1994 through 2015, including 18 patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and 3 patients with trifunctional protein deficiency (TFPD). METHODS: Local records from all visits were reviewed. Every participant underwent a complete ophthalmic examination and was evaluated by a metabolic physician and dietitian. Nine patients underwent ancillary funduscopic imaging including optical coherence tomography (OCT) and OCT angiography. MAIN OUTCOME MEASURES: The primary outcome measure was best-corrected visual acuity at the final visit. Secondary outcome measures included spherical equivalent refraction, visual fields, electroretinography B-wave amplitudes, and qualitative imaging findings. RESULTS: Participants were followed up for a median of 5.6 years (range 0.3-20.2 years). The median age of LCHADD participants at initial and final visits was 2.3 and 11.9 years, whereas that for TFPD participants at initial and final visits was 4.7 and 15.5 years, respectively. Four long-term survivors older than 16 years were included (3 with LCHADD and 1 with TFPD). The LCHADD participants demonstrated a steady decline in visual acuity from an average of 0.23 logarithm of the minimum angle of resolution (logMAR; Snellen equivalent, 20/34) at baseline to 0.42 logMAR (Snellen equivalent, 20/53) at the final visit, whereas TFPD patients maintained excellent acuity throughout follow-up. Participants with LCHADD, but not TFPD, showed an increasing myopia with a mean decrease in spherical equivalent refraction of 0.24 diopters per year. Visual fields showed sensitivity losses centrally associated with defects on OCT. Multimodal imaging demonstrated progressive atrophy of the outer retina in LCHADD, often preceded by the formation of outer retinal tubulations and choriocapillaris dropout. Electroretinography findings support the more severe clinical profile of LCHADD patients compared with TFPD patients; the function of both rods and cones are attenuated diffusely in LCHADD patients, but are within normal limits for TFPD patients. CONCLUSIONS: Despite improved survival with early diagnosis, medical management, and dietary treatment, participants with the LCHADD subtype of MTP disorder continue to demonstrate visually disabling chorioretinopathy. Multimodal imaging is most consistent with choriocapillaris loss exceeding photoreceptor loss.

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.

Glaucoma in a patient with Singleton-Merten syndrome.

A 2-year-old girl presented for evaluation of glaucoma suspect status. Despite her lack of buphthalmia, her clear corneas, and absence of Haab's striae, she was found to have an intraocular pressure of 45 mm Hg in the right eye and 47 mm Hg in the left eye and significant optic nerve cupping. Both eyes were initially treated with goniotomies, trabeculotomies, and later Baerveldt glaucoma implants. She was diagnosed with Singleton-Merten syndrome from a DDX58 pathogenic variant, with congenital glaucoma, juvenile progressive high myopia, hypoplastic nails, and abnormal dentition.

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.

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.

Decreased Expression of Glial-Derived Neurotrophic Factor Receptors in Glaucomatous Human Retinas.

PURPOSE: The purpose of this study was to examine the expression of glial-derived neurotrophic factor (GDNF), the GDNF receptors GFRα1 and GFRα2, ciliary neurotrophic factor (CNTF), and the CNTF receptor CNTFRα in normal and glaucomatous human tissue. METHODS: Human retinas were collected from 8 donors that had been clinically diagnosed and treated for glaucoma, and also from 9 healthy control donors. Immunohistochemical analysis for each trophic factor and receptor was performed. The percent of each retinal section labeled with each antibody was quantified for the total retinal thickness, and separately for the retinal ganglion cell (RGC) complex + retinal nerve fiber layer (RNFL). The expression of each protein was correlated with measures of the subject's ocular histories. RESULTS: The percentage area immunopositive for GFRα2 was significantly decreased in the total retinal thickness containing all retinal layers and in the combined RGC complex + RNFL in glaucomatous eyes in both the peripapillary region and more peripheral retinal locations. We also observed a decrease in GFRα1 expression in the peripapillary RGC Complex + RNFL in glaucoma patients compared to healthy control patients. We also observed a relationship between GDNF and its receptors with several outcomes obtained from the medical record. No differences in CNTF or CNTFR labeling were observed. CONCLUSION: Decreases in GDNF receptor expression in glaucomatous tissue may limit the potential for neuroprotective therapy by supplementation with GDNF.

High Correlation between Glaucoma Treatment with Topical Prostaglandin Analogs and BDNF Immunoreactivity in Human Retina.

PURPOSE: To examine the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin-related kinase receptor-B (TrkB), in normal and glaucomatous human retinas. METHODS: Human retinas were collected from 8 donors who had been clinically diagnosed and treated for glaucoma, and from 9 control donors. Immunohistochemical analysis for BDNF and TrkB was performed. The percent of each retina expressing BDNF and TrkB was quantified for the total retinal thickness, and separately for the retinal ganglion cell (RGC) complex + retinal nerve fiber layer (RNFL). The expression of each protein was correlated with clinical outcomes obtained from the subject's ocular histories. RESULTS: There was no significant difference in BDNF or TrkB expression when comparing glaucomatous and control retinas. Correlation analysis revealed a significant relationship between BDNF expression and the use of prostaglandin analogs. TrkB expression was highly correlated with the last-measured intraocular pressure (IOP), the use of carbonic anhydrase inhibitors, the use of beta blockers, and the total number of drugs used for the treatment of glaucoma. CONCLUSION: Topical drugs used to treat glaucoma were associated with an increase in retinal BDNF and TrkB expression in human retina, independent of IOP, which may represent molecular evidence of neuroprotective pathway activation.

Slit-lamp Pars Plana Needle Hyaloidotomy-Zonulotomy-Iridotomy for the Treatment of Spontaneous Malignant Glaucoma.

An 88-year-old woman with a history of recent complicated pacemaker insertion presented with acute-onset malignant glaucoma recalcitrant to conservative medical therapy. Surgical intervention was discussed; however, given her complex cardiac history and recent postoperative state, the risk of anesthesia-related systemic adverse events was deemed unacceptably high. As such, a slit-lamp procedure was recommended to break the attack of malignant glaucoma. Here within, we report a novel technique of breaking an attack of malignant glaucoma by needling the anterior hyaloid face at the slit lamp. With this technique, a 25-G needle was entered through the pars plana and was advanced through the anterior hyaloid face, zonules, and peripheral iridotomy to create a unicameral eye and successfully break the malignant closure attack.

Novel Intragenic PAX6 Deletion in a Pedigree with Aniridia, Morbid Obesity, and Diabetes.

Purpose: Aniridia is a rare congenital eye disease, characterized by a constellation of symptoms including hypoplastic irides, foveal hypoplasia, early cataract, corneal stem cell deficiency, and glaucoma. Large chromosomal deletions spanning the PAX6 gene cause WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and intellectual disability [formerly called mental retardation]). We describe clinical and genetic studies of a three-generation pedigree with aniridia along with additional systemic conditions (morbid obesity, diabetes) suggesting the possibility of a contiguous-gene syndrome like WAGR.Methods: Clinical records were obtained and DNA was prepared from blood samples from three of the four patients and tested for mutations in the coding sequences of the PAX6 gene. The index patient also had cardiomyopathy and was tested for known cardiomyopathy genetic mutations using a next-generation DNA sequencing assay.Results: We discovered a novel intragenic PAX6 mutation, a 16 bp heterozygous deletion c.203delCCAGGGCAATCGGTGG, with Sanger sequencing that is the likely cause of autosomal dominant aniridia in this pedigree. This PAX6 deletion causes a frameshift in predicted protein translation and a subsequent premature termination, p.Pro68Leufs*6. The PAX6 deletion was detected in all three available family members with aniridia, the index patient, his mother, and his maternal aunt but was not observed in the Exome Aggregation Consortium (ExAC) database. Targeted sequencing of known cardiomyopathy genes in the index patient identified a second mutation, a 1.7 Mp deletion that spans the MYBPC3 gene.Conclusions: We report a pedigree with aniridia and other systemic abnormalities that were initially suspicious for a contiguous-gene syndrome like WAGR. However, genetic analysis of the pedigree revealed two independent genetic abnormalities on chromosome 11p: 1) a novel PAX6 mutation, and 2) a large chromosome deletion spanning MYBPC3, a known cardiomyopathy gene. It is unclear if morbid obesity and type II diabetes mellitus have a related genetic cause.

Gonioscopy-assisted Transluminal Trabeculotomy (GATT) is An Effective Procedure for Steroid-induced Glaucoma.

PRECIS: Gonioscopy-assisted transluminal trabeculotomy (GATT) is a novel technique that lowers intraocular pressure (IOP) by fracturing the trabecular meshwork. In this retrospective chart review, GATT was found to be effective at lowering the intraocular pressure for steroid-induced glaucoma (SIG). PURPOSE: GATT is a novel microinvasive glaucoma surgery that builds on traditional trabeculotomy techniques to decrease the proximal resistance of conventional outflow, and it is proposed to be an effective surgical treatment for SIG. The purpose of this study is to evaluate the efficacy of GATT for lowering the IOP in SIG. METHODS AND PATIENTS: A retrospective chart review was performed of all GATT procedures performed on patients with a predominant diagnosis of steroid-induced glaucoma between March 1, 2016 and March 30,2018 at the University of Michigan. Primary outcome measures include IOP, the number of IOP-lowering medications prescribed, and the topical steroid dosing over the duration of follow-up. RESULTS: A total of 13 patients with steroid-induced glaucoma underwent the GATT procedure. There was a significant reduction in the mean IOP at all postoperative visits. The average IOP decreased by 16.4 (55%) to 19.5 mm Hg (63%) between 3 and 24 months postoperatively. By 24 months, all patients had a reduction in IOP of >20%. The number of glaucoma medications also decreased significantly from an average of 3.1 medications preoperatively to an average of 0.8 medications at last follow-up. The majority of patients (>67%) required continued use of steroids at all postoperative visits. The most common postoperative complication was a transient hyphema (38%). No patients required a repeat glaucoma surgery or anterior chamber washout. CONCLUSIONS: This small case series suggests that GATT is an effective and safe surgical technique to decrease IOP and decrease medication burden in patients with predominantly steroid-induced glaucoma. To our knowledge, this is the first study looking specifically at the efficacy of GATT for predominantly SIG.