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.

Quantification and image-derived phenotyping of retinal ganglion cell nuclei in the nee mouse model of congenital glaucoma.

The nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4 to 15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A- nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3000 nuclei/mm2 apparent spreading was observed, in which BRN3A- nuclei-regardless of genotype-became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.

Association of Genetic Variants With Primary Open-Angle Glaucoma Among Individuals With African Ancestry.

Importance: Primary open-angle glaucoma presents with increased prevalence and a higher degree of clinical severity in populations of African ancestry compared with European or Asian ancestry. Despite this, individuals of African ancestry remain understudied in genomic research for blinding disorders. Objectives: To perform a genome-wide association study (GWAS) of African ancestry populations and evaluate potential mechanisms of pathogenesis for loci associated with primary open-angle glaucoma. Design, Settings, and Participants: A 2-stage GWAS with a discovery data set of 2320 individuals with primary open-angle glaucoma and 2121 control individuals without primary open-angle glaucoma. The validation stage included an additional 6937 affected individuals and 14 917 unaffected individuals using multicenter clinic- and population-based participant recruitment approaches. Study participants were recruited from Ghana, Nigeria, South Africa, the United States, Tanzania, Britain, Cameroon, Saudi Arabia, Brazil, the Democratic Republic of the Congo, Morocco, Peru, and Mali from 2003 to 2018. Individuals with primary open-angle glaucoma had open iridocorneal angles and displayed glaucomatous optic neuropathy with visual field defects. Elevated intraocular pressure was not included in the case definition. Control individuals had no elevated intraocular pressure and no signs of glaucoma. Exposures: Genetic variants associated with primary open-angle glaucoma. Main Outcomes and Measures: Presence of primary open-angle glaucoma. Genome-wide significance was defined as P < 5 × 10-8 in the discovery stage and in the meta-analysis of combined discovery and validation data. Results: A total of 2320 individuals with primary open-angle glaucoma (mean [interquartile range] age, 64.6 [56-74] years; 1055 [45.5%] women) and 2121 individuals without primary open-angle glaucoma (mean [interquartile range] age, 63.4 [55-71] years; 1025 [48.3%] women) were included in the discovery GWAS. The GWAS discovery meta-analysis demonstrated association of variants at amyloid-ß A4 precursor protein-binding family B member 2 (APBB2; chromosome 4, rs59892895T>C) with primary open-angle glaucoma (odds ratio [OR], 1.32 [95% CI, 1.20-1.46]; P = 2 × 10-8). The association was validated in an analysis of an additional 6937 affected individuals and 14 917 unaffected individuals (OR, 1.15 [95% CI, 1.09-1.21]; P < .001). Each copy of the rs59892895*C risk allele was associated with increased risk of primary open-angle glaucoma when all data were included in a meta-analysis (OR, 1.19 [95% CI, 1.14-1.25]; P = 4 × 10-13). The rs59892895*C risk allele was present at appreciable frequency only in African ancestry populations. In contrast, the rs59892895*C risk allele had a frequency of less than 0.1% in individuals of European or Asian ancestry. Conclusions and Relevance: In this genome-wide association study, variants at the APBB2 locus demonstrated differential association with primary open-angle glaucoma by ancestry. If validated in additional populations this finding may have implications for risk assessment and therapeutic strategies.

Biological Correlations and Confounders for Quantification of Retinal Ganglion Cells by Optical Coherence Tomography Based on Studies of Outbred Mice.

Purpose: Despite popularity of optical coherence tomography (OCT) in glaucoma studies, it's unclear how well OCT-derived metrics compare to traditional measures of retinal ganglion cell (RGC) abundance. Here, Diversity Outbred (J:DO) mice are used to directly compare ganglion cell complex (GCC) thickness measured by OCT to metrics of retinal anatomy measured ex vivo with retinal wholemounts and optic nerve histology. Methods: J:DO mice (n = 48) underwent fundoscopic and OCT examinations, with automated segmentation of GCC thickness. RGC axons were quantified from para-phenylenediamine-stained optic nerve cross-sections and somas from BRN3A-immunolabeled retinal wholemounts, with total inner retinal cellularity assessed by TO-PRO and subsequent hematoxylin staining. Results: J:DO tissues lacked overt disease. GCC thickness, RGC abundance, and total cell abundance varied broadly across individuals. GCC thickness correlated significantly to RGC somal density (r = 0.58) and axon number (r = 0.44), but not total cell density. Retinal area and nerve cross-sectional area varied widely. No metrics were significantly influenced by sex. In bilateral comparisons, GCC thickness (r = 0.95), axon (r = 0.72), and total cell density (r = 0.47) correlated significantly within individuals. Conclusions: Amongst outbred mice, OCT-derived measurements of GCC thickness correlate significantly to RGC somal and axon abundance. Factors limiting correlation are likely both biological and methodological, including differences in retinal area that distort sampling-based estimates of RGC abundance. Translational Relevance: There are significant-but imperfect-correlations between GCC thickness and RGC abundance across genetic contexts in mice, highlighting valid uses and ongoing challenges for meaningful use of OCT-derived metrics.

Mouse models and strain-dependency of Chédiak-Higashi syndrome-associated neurologic dysfunction.

Chédiak-Higashi syndrome (CHS) is a lethal disorder caused by mutations in the LYST gene that involves progressive neurologic dysfunction. Lyst-mutant mice exhibit neurologic phenotypes that are sensitive to genetic background. On the DBA/2J-, but not on the C57BL/6J-background, Lyst-mutant mice exhibit overt tremor phenotypes associated with loss of cerebellar Purkinje cells. Here, we tested whether assays for ataxia could measure this observed strain-dependency, and if so, establish parameters for empowering phenotype- and candidate-driven approaches to identify genetic modifier(s). A composite phenotypic scoring system distinguished phenotypes in Lyst-mutants and uncovered a previously unrecognized background difference between wild-type C57BL/6J and DBA/2J mice. Accelerating rotarod performance also distinguished phenotypes in Lyst-mutants, but at more advanced ages. These results establish that genetic background, Lyst genotype, and age significantly influence the severity of CHS-associated neurologic deficits. Purkinje cell quantifications likewise distinguished phenotypes of Lyst-mutant mice, as well as background differences between wild-type C57BL/6J and DBA/2J mice. To aid identification of potential genetic modifier genes causing these effects, we searched public datasets for cerebellar-expressed genes that are differentially expressed and/or contain potentially detrimental genetic variants. From these approaches, Nos1, Prdx2, Cbln3, Gnb1, Pttg1 were confirmed to be differentially expressed and leading candidates.

Myocilin Mutations in Patients With Normal-Tension Glaucoma.

Importance: Mutations in the myocilin (MYOC) gene are the most common molecularly defined cause of primary open-angle glaucoma that typically occurs in patients with high intraocular pressures (IOP). One MYOC mutation, p.Gln368Ter, has been associated with as many as 1.6% of primary open-angle glaucoma cases that had a mean maximum recorded IOP of 30 mm Hg. However, to our knowledge, the role of the p.Gln368Ter mutation in patients with normal-tension glaucoma (NTG) with an IOP of 21 mm Hg or lower has not been investigated. Objective: To evaluate the role of the p.Gln368Ter MYOC mutation in patients with NTG. Design, Setting, and Participants: In this case-control study of the prevalence of the p.Gln368Ter mutation in patients with NTG, cohort 1 was composed of 772 patients with NTG and 2152 controls from the United States (Iowa, Minnesota, and New York) and England and cohort 2 was composed of 561 patients with NTG and 2606 controls from the Massachusetts Eye and Ear Infirmary and the NEIGHBORHOOD consortium. Genotyping was conducted using real-time polymerase chain reaction that was confirmed with Sanger sequencing, the imputation of genome-wide association study data, or an analysis of whole-exome sequence data. Data analysis occurred between April 2007 and April 2018. Main Outcomes and Measures: Comparison of the frequency of the p.Gln368Ter MYOC mutation between NTG cases and controls with the Fisher exact test. Results: Of 6091 total participants, 3346 (54.9%) were women and 5799 (95.2%) were white. We detected the p.Gln368Ter mutation in 7 of 772 patients with NTG (0.91%) and 7 of 2152 controls (0.33%) in cohort 1 (P = .03). In cohort 2, we detected the p.Gln368Ter mutation in 4 of 561 patients with NTG (0.71%) and 10 of 2606 controls (0.38%; P = .15). When the cohorts were analyzed as a group, the p.Gln368Ter mutation was associated with NTG (odds ratio, 2.3; 95% CI, 0.98-5.3; P = .04). Conclusions and Relevance: In cohorts 1 and 2, the p.Gln368Ter mutation in MYOC was found in patients with IOPs that were 21 mm Hg or lower (NTG), although at a frequency that is lower than previously detected in patients with higher IOP. These data suggest that the p.Gln368Ter mutation may be associated with glaucoma in patients with normal IOPs as well as in patients with IOPs that are greater than 21 mm Hg.

Histochemical Analysis of Glaucoma Caused by a Myocilin Mutation in a Human Donor Eye.

Objective: Mutations in myocilin (MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation. Design: Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes. Subjects: An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51-66) with no known ocular disease were examined. Methods: Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork. Main Outcome Measure: Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects. Results: Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient's eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient's eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes. Conclusions: This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.