Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

Common variants at 9p21 and 8q22 are associated with increased susceptibility to optic nerve degeneration in glaucoma.

Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normal-pressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], OR = 0.69 [95%CI 0.63-0.75], p = 1.86×10⁻¹8), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], OR = 1.32 [95%CI 1.21-1.43], p = 3.87×10⁻¹¹). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], OR = 0.58 [95% CI 0.50-0.67], p = 1.17×10⁻¹²) and a probable regulatory region on 8q22 (rs284489 [G], OR = 0.62 [95% CI 0.53-0.72], p = 8.88×10⁻¹°). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], OR = 0.59 [95% CI 0.41-0.87], p = 0.004 and rs284489 [G], OR = 0.76 [95% CI 0.54-1.06], p = 0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted p = 0.009). These results suggest that neuro-protective therapies targeting TGF-beta signaling could be effective for multiple forms of glaucoma.

Genome-wide association study and meta-analysis of intraocular pressure.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma and is influenced by genetic and environmental factors. Recent genome-wide association studies (GWAS) reported associations with IOP at TMCO1 and GAS7, and with primary open-angle glaucoma (POAG) at CDKN2B-AS1, CAV1/CAV2, and SIX1/SIX6. To identify novel genetic variants and replicate the published findings, we performed GWAS and meta-analysis of IOP in >6,000 subjects of European ancestry collected in three datasets: the NEI Glaucoma Human genetics collaBORation, GLAUcoma Genes and ENvironment study, and a subset of the Age-related Macular Degeneration-Michigan, Mayo, AREDS and Pennsylvania study. While no signal achieved genome-wide significance in individual datasets, a meta-analysis identified significant associations with IOP at TMCO1 (rs7518099-G, p = 8.0 × 10(-8)). Focused analyses of five loci previously reported for IOP and/or POAG, i.e., TMCO1, CDKN2B-AS1, GAS7, CAV1/CAV2, and SIX1/SIX6, revealed associations with IOP that were largely consistent across our three datasets, and replicated the previously reported associations in both effect size and direction. These results confirm the involvement of common variants in multiple genomic regions in regulating IOP and/or glaucoma risk.

Shear behavior of bovine scleral tissue.

Ocular tissue properties have been widely studied in tension and compression for humans and a variety of animals. However, direct shear testing of the tissues of the sclera appear to be absent from the literature even though modeling, analyses, and anatomical studies have indicated that shear may play a role in the etiology of primary open angle glaucoma (POAG). In this work, the mechanical behavior of bovine scleral tissue in shear has been studied in both out-of-plane and in-plane modes of deformation. Stress-strain and relaxation tests were conducted on tissue specimens at controlled temperature and hydration focusing on trends related to specimen location and orientation. There was generally found to be no significant effect of specimen orientation and angular location in the globe on shear stiffness in both modes. The in-plane response, which is the primary load carrying mode, was found to be substantially stiffer than the out-of-plane mode. Also, within the in-plane studies, tissue further from the optic nerve was stiffer than the near tissue. The viscosity coefficient of the tissue varied insignificantly with distance from the optic nerve, but overall was much higher in-plane than out-of-plane.

Constitutive behavior of ocular tissues over a range of strain rates.

The constitutive behavior of bovine scleral and corneal tissues is measured in tension and compression, at quasi-static and moderate strain rates. Experiments are conducted at strain rates up to about 50 strain per second by a pneumatic testing system developed to overcome noise and measurement difficulties associated with the time dependent test of low impedance materials. Results for the tissues at room and the natural bovine body temperatures are similar and indicate that ocular tissue exhibits nonlinear stiffening for increasing strain rates, a phenomena termed rate hardening. For example, at a tensile strain rate of 29/s, corneal tissue is found to develop 10 times the stress that it does quasi-statically at the same strain. Thus, conventional constitutive models will grossly underpredict stresses occurring in the corneo-scleral shell due to moderate dynamic events. This has implication to the accuracy of ocular injury models, the study of the stress field in the corneo-scleral shell for glaucoma research and tonometry measurements. The measured data at various strain rates is represented using the general framework of a constitutive model that has been used to represent biological tissue mechanical data. Here it is extended to represent the measured data of the ocular tissues over the range of tested strain rates. Its form allows for straightforward incorporation in various numerical codes. The experimental and analytical methods developed here are felt to be applicable to the test of human ocular tissue.

Effects of timolol on MYOC, OPTN, and WDR36 RNA levels.

OBJECTIVES: To evaluate if timolol affects expression of 3 open-angle glaucoma genes and to study its ability to modulate dexamethasone-induced up-regulation of MYOC. METHODS: We used quantitative polymerase chain reaction assay of glaucoma gene transcript levels from human trabecular meshwork (HTM) cultures exposed to 3 different doses of timolol. Three HTM cell cultures were grown with or without 1 of 3 timolol doses in the presence or absence of dexamethasone. RESULTS: All 3 concentrations of timolol reduced MYOC RNA levels in 1 HTM culture compared with an untreated control and showed negligible effects in the other 2 cultures. Timolol had no effect on dexamethasone-induced MYOC transcript levels in any of the 3 cultures. Timolol, dexamethasone, and dexamethasone plus timolol had a negligible effect on OPTN and WDR36 RNA levels. CONCLUSIONS: Timolol can reduce MYOC RNA levels in HTM cultures from some individuals. Timolol does not alter OPTN or WDR36 levels or ameliorate MYOC induction by dexamethasone in vitro. CLINICAL RELEVANCE: It remains to be determined whether timolol could reduce production of misfolded myocilin protein by HTM cells in individuals with MYOC missense mutations. A broader survey of interindividual variation in response to timolol as well as mechanistic studies are still needed before potential therapeutic implications can be explored.

Differential expression profile prioritization of positional candidate glaucoma genes: the GLC1C locus.

OBJECTIVES: To develop and apply a model for prioritization of candidate glaucoma genes. METHODS: This Affymetrix GeneChip (Affymetrix, Santa Clara, Calif) study of gene expression in primary culture human trabecular meshwork cells uses a positional differential expression profile model for prioritization of candidate genes within the GLC1C genetic inclusion interval. RESULTS: Sixteen genes were expressed under all conditions within the GLC1C interval. TMEM22 was the only gene within the interval with differential expression in the same direction under both conditions tested. Two genes, ATP1B3 and COPB2, are of interest in the context of a protein-misfolding model for candidate selection. SLC25A36, PCCB, and FNDC6 are of lesser interest because of moderate expression and changes in expression. Transcription factor ZBTB38 emerges as an interesting candidate gene because of the overall expression level, differential expression, and function. CONCLUSIONS: Only 1 gene in the GLC1C interval fits our model for differential expression under multiple glaucoma risk conditions. The use of multiple prioritization models resulted in filtering 7 candidate genes of higher interest out of the 41 known genes in the region. CLINICAL RELEVANCE: This study identified a small subset of genes that are most likely to harbor mutations that cause glaucoma linked to GLC1C.

Gene expression profile of human trabecular meshwork cells in response to long-term dexamethasone exposure.

PURPOSE: Topical use of dexamethasone has long been associated with steroid induced-glaucoma, although the mechanism is unknown. We applied a strict filtering of comparative microarray data to more than 18,000 genes to evaluate global gene expression of cultured human trabecular meshwork cells in response to treatment with dexamethasone. METHODS: Three human trabecular meshwork cell primary cultures from nonglaucomatous donors were incubated with and without dexamethasone for 21 days. Relative gene expression was evaluated by analysis of U133A GeneChip and the results validated using quantitative polymerase chain reaction (PCR). RESULTS: Application of strict filtering to include only genes with statistically significant differences in gene expression across all three trabecular meshwork cell cultures produced a list of 1,260 genes. Significant changes in signal level were observed, including 23 upregulated and 18 downregulated genes that changed greater than three fold in each of three cell cultures. Using quantitative PCR we found changes greater than a thousand fold for two genes (SLP1 and SAA2) and changes greater than a hundred fold for another five genes (ANGPTL7, MYOC, SAA1, SERPINA3, and ZBTB16). CONCLUSIONS: Expression changes in trabecular meshwork cells in response to dexamethasone treatment indicate that a group of actins and actin-associated proteins are involved in the development of cross-linked actin networks that form in response to dexamethasone. A trend was identified toward decreased expression of protease genes accompanied by an increased expression of protease inhibitors. Such a trend in nonproteasomal proteolysis conceivably affects gene product levels above the level of transcription. Only two genes, MYOC and IGFBP2, showed significantly elevated expression after dexamethasone treatment in our study and the other three previously published reports of primary culture trabecular meshwork cell gene expression.

The NEIGHBOR consortium primary open-angle glaucoma genome-wide association study: rationale, study design, and clinical variables.

Primary open-angle glaucoma (POAG) is a common disease with complex inheritance. The identification of genes predisposing to POAG is an important step toward the development of novel gene-based methods of diagnosis and treatment. Genome-wide association studies (GWAS) have successfully identified genes contributing to complex traits such as POAG however, such studies frequently require very large sample sizes, and thus, collaborations and consortia have been of critical importance for the GWAS approach. In this report we describe the formation of the NEIGHBOR consortium, the harmonized case control definitions used for a POAG GWAS, the clinical features of the cases and controls, and the rationale for the GWAS study design.

Mutations in TCF8 cause posterior polymorphous corneal dystrophy and ectopic expression of COL4A3 by corneal endothelial cells.

Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV alpha 3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome.

A locus for posterior polymorphous corneal dystrophy (PPCD3) maps to chromosome 10.

Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder characterized by corneal endothelial abnormalities, which can lead to blindness due to loss of corneal transparency and sometimes glaucoma. We mapped a new locus responsible for PPCD in a family in which we excluded the previously reported PPCD locus on 20q11, and the region containing COL8A2 on chromosome 1. Results of a 317-marker genome scan provided significant evidence of linkage of PPCD to markers on chromosome 10, with single-point LOD scores of 2.63, 1.63, and 3.19 for markers D10S208 (at (circumflex)theta = 0.03), D10S1780 (at (circumflex)theta = 0.00), and D10S578 (at (circumflex)theta = 0.06). A maximum multi-point LOD score of 4.35 was found at marker D10S1780. Affected family members shared a haplotype in an 8.55 cM critical interval that was bounded by markers D10S213 and D10S578. Our finding of another PPCD locus, PPCD3, on chromosome 10 indicates that PPCD is genetically heterogeneous. Guttae, a common corneal finding sometimes observed along with PPCD, were found among both affected and unaffected members of the proband's sib ship, but were absent in the younger generations of the family. Evaluation of phenotypic differences between family members sharing the same affected haplotype raises questions about whether differences in disease severity, including differences in response to surgical interventions, could be due to genetic background or other factors independent of the PPCD3 locus.