The genetic mechanisms of primary angle closure glaucoma.

Primary Angle Closure Glaucoma (PACG) is one of the most common types of glaucoma affecting over 15 million individuals worldwide. Family history and ethnicity are strongly associated with the development of the disease, suggesting that one or more genetic factors contribute to PACG. Although strictly heritable disease-causing mutations have not been identified, a number of recent association studies have pointed out genetic factors that appear to contribute to an individual's risk to develop PACG. In addition, genetic factors have been identified that modify PACG endophenotypes for example, axial length. Herein we review the current literature on this important topic.

Lipofuscin in human glaucomatous optic nerves.

Lipofuscin accumulation has been observed in a number of neurodegenerative diseases. We recently found that autofluorescent particles also occur in the aged human optic nerve. In this study we sought to determine the nature of these particles and their correlation with aging, age-related macular degeneration (AMD) and primary open angle glaucoma (POAG). Groups of eight optic nerves from patients diagnosed with primary open angle glaucoma, age-related macular degeneration, age-matched controls and four optic nerves derived from controls younger than 42 years were used for the study. All samples were fixed in paraformaldehyde and frozen frontal sections were prepared. Sections were analyzed with fluorescence microscopy, bright field microscopy, Sudan black staining and spectrofluorometry using a confocal laser scanning microscope. Sections were photographed and analyzed to establish the distribution, quantity, and size of the autofluorescent particles. Additionally, transmission electron microscopy was used to determine the ultrastructural location of the granules. On unstained sections under light microscopy granules are detectable as pale brown inclusions and are easily stained with oil-soluble dyes, such as Sudan black. Granules fluoresce when excited at all tested wavelengths but lose their fluorescence after staining with Sudan black. These particles are distributed throughout the axonal columns, but not in the septa, and appear to be located within the glia ensheathing optic nerve axons. The histologic properties of the granules seen in the optic nerve sections correspond to lipofuscin aggregates, a result of incomplete degradation of oxidized proteins. Our morphometric analyses indicate that overall the optic nerves from control, glaucoma, and AMD donors contain similar amounts of lipofuscin. However, optic nerves derived from donors with glaucoma contain lipofuscin particles that are larger than those observed in the age-matched control and AMD groups. Furthermore optic nerves from glaucoma donors display a smaller diameter than those from age-matched controls resulting in a higher concentration of lipofuscin in glaucomatous optic nerves.

Novel drug delivery systems for glaucoma.

Reduction of intraocular pressure (IOP) by pharmaceutical or surgical means has long been the standard treatment for glaucoma. A number of excellent drugs are available that are effective in reducing IOP. These drugs are typically applied as eye drops. However, patient adherence can be poor, thus reducing the clinical efficacy of the drugs. Several novel delivery systems designed to address the issue of adherence and to ensure consistent reduction of IOP are currently under development. These delivery systems include contact lenses-releasing glaucoma medications, injectables such as biodegradable micro- and nanoparticles, and surgically implanted systems. These new technologies are aimed at increasing clinical efficacy by offering multiple delivery options and are capable of managing IOP for several months. There is also a desire to have complementary neuroprotective approaches for those who continue to show progression, despite IOP reduction. Many potential neuroprotective agents are not suitable for traditional oral or drop formulations. Their potential is dependent on developing suitable delivery systems that can provide the drugs in a sustained, local manner to the retina and optic nerve. Drug delivery systems have the potential to improve patient adherence, reduce side effects, increase efficacy, and ultimately, preserve sight for glaucoma patients. In this review, we discuss benefits and limitations of the current systems of delivery and application, as well as those on the horizon.

Intraocular pressure measurement in mice: a comparison between Goldmann and rebound tonometry.

PURPOSE: The development of mouse models of glaucoma requires methods to accurately measure the intraocular pressure (IOP) in this species. The aim of this study was to compare the accuracy of IOP measurements in mice between modified Goldmann and rebound tonometers. METHODS: IOP was measured either with a modified Goldmann or a rebound tonometer while simultaneously measuring the IOP using invasive manometry in enucleated eyes and in vivo. The level of IOP was controlled hydrostatically. The agreement and correlation between the IOP determined by invasive manometry and by either noninvasive method was evaluated. In addition, the IOP was determined by both noninvasive methods in a cohort of mice with laser-induced ocular hypertension (OHT), and the agreement and correlation between the two tonometry methods were evaluated. RESULTS: Measured IOP by either noninvasive tonometer correlated well with those recorded simultaneously by invasive manometry (r=0.98 for rebound and r=0.94 for Goldmann). In mice with OHT, the IOP correlation between rebound and modified Goldmann was moderate (r=0.71); the IOP measured by modified Goldmann tonometry was consistently higher than that by rebound by approximately 5 mmHg. However, the relative per cent increases in IOP were similar between the two methods. CONCLUSION: Both noninvasive methods of IOP measurements in mice are suitable to detect changes in IOP although rebound tonometry correlated better with the invasive manometry readings. The results suggest that the relative, rather than absolute, IOP offers a more reliable means of correlating findings from studies using different tonometers.

Organization of the human IMPG2 gene and its evaluation as a candidate gene in age-related macular degeneration and other retinal degenerative disorders.

PURPOSE: To characterize the genomic organization of human IMPG2, the gene encoding the retinal interphotoreceptor matrix (IPM) proteoglycan IPM 200, to evaluate its relationship to IPM 150, and to evaluate its involvement in inherited retinopathies, such as age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis. METHODS: After isolation of human genomic clones, the structure of IMPG2 was determined by sequence analysis. Mutational analyses were conducted on genomic DNA isolated from 316 probands using single-strand conformation polymorphism analysis. RESULTS: The IMPG2 gene is organized into 19 exons, and the structure of the gene is highly similar to that of the IMPG1 gene, which encodes another retinal proteoglycan, IPM 150. Mutational analyses indicate that the observed sequence changes are present at approximately equal rates in donors with and without retinal disease. Additional data derived from RT-PCR and Northern blot analysis show that IMPG2 is processed in the human retina into multiple alternatively sized transcripts that may represent splicing isoforms. CONCLUSIONS: Analysis of the overall relationship of human IMPG2 (located on chromosome 3q12.2-12.3) to human IMPG1 (located on chromosome 6q14) suggests that these genes have evolved from a common ancestral gene. Although this is an excellent candidate gene for hereditary retinopathies, single-strand conformation polymorphism analyses provided no evidence that variations in IMPG2 coding region are responsible for the inherited retinopathies examined.

Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F.

We have determined the molecular basis for Usher syndrome type 1F (USH1F) in two families segregating for this type of syndromic deafness. By fluorescence in situ hybridization, we placed the human homolog of the mouse protocadherin Pcdh15 in the linkage interval defined by the USH1F locus. We determined the genomic structure of this novel protocadherin, and found a single-base deletion in exon 10 in one USH1F family and a nonsense mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, we demonstrated expression of PCDH15 in the retina and cochlea by RT-PCR and immunohistochemistry. This report shows that protocadherins are essential for maintenance of normal retinal and cochlear function.

Genomic organization and mutation analysis of the gene encoding lecithin retinol acyltransferase in human retinal pigment epithelium.

PURPOSE: To determine the structure of the human lecithin retinol acyltransferase (LRAT) gene, map its chromosomal localization, and screen for mutations in humans with various hereditary retinal degenerations. METHODS: Using DNA probes specific for LRAT, a bacterial artificial chromosome (BAC) clone containing the LRAT gene was isolated, subcloned into DNA fragments and relevant subclones characterized by sequencing. Exon-intron junctions were determined by comparison with the cDNA sequence previously published. Southern blot analysis was performed on human genomic DNA samples digested with several restriction enzymes. Fluorescence in situ hybridization (FISH) analysis of normal metaphase chromosomes derived from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes and radiation hybrid mapping were used for localization of the LRAT gene. Single-strand conformation polymorphism analysis (SSCP) was used to screen for potential mutations in patients with age-related macular degeneration, Leber congenital amaurosis, retinitis pigmentosa, and cone-rod dystrophy. RESULTS: The human LRAT gene is organized into three exons of 219, 541, and 2058 bp and two introns of 103 and 4117 bp. Southern blot analysis of digested genomic DNA revealed a single band, suggesting a single copy of the LRAT gene. The human LRAT gene was localized to chromosome 4q31.2, a locus having no previous association with human eye disease. Additionally, the bovine LRAT homologue sequence was deduced and a general LRAT protein topology is suggested. No polymorphisms that segregated with retinal disease phenotypes were identified in 374 unrelated probands. CONCLUSIONS: The organization of the LRAT gene, based on cDNA clones derived from the retinal pigment epithelium (RPE) has been determined. Its structure is less complex than other acyltransferases such as lecithin cholesterol acyltransferase (LCAT) and acyl CoA acyltransferase (ACAT). The absence of polymorphisms in the probands examined suggests a very low mutation level in the LRAT gene from the diseases analyzed.

Molecular characterization of the murine orthologue of the human retinal proteoglycan IPM 150.

PURPOSE: We recently identified a family of novel human proteoglycans/glycoproteins that are major constituents of the human interphotoreceptor matrix. Two members of this family, designated IPM 150 and IPM 200, have been extensively characterized. Although the IPM is thought to mediate crucial roles in retinal physiology, including retinal adhesion and photoreceptor cell viability, little is known about the roles of specific IPM constituents in these processes. In order to characterize the mouse IPM 150 orthologue, to initiate functional in vivo studies, and as a prerequisite towards future genetic manipulation, we cloned the murine orthologue of human IPM 150 and determined its chromosomal location. METHODS: A mouse retinal cDNA library was screened using an IMAGE clone with sequence similarity to human IPM 150. The genomic location of the mouse IPM 150 gene was determined by radiation hybrid analyses. RESULTS: We describe here the molecular structure of the murine orthologue of human IPM 150 and place the location of its gene on mouse chromosome 9. Among the tissues examined, expression of IPM 150 appeared to be restricted to the retina. CONCLUSIONS: Comparison of the human and murine IPM 150 core proteins revealed that the molecules are generally well conserved, although several potentially significant differences do exist. In addition, two highly conserved domains within the core proteins were identified. The data presented here represent a first step towards the development of experimental murine models, which may eventually be used to elucidate the mechanisms underlying retinal adhesion and photoreceptor survival.

Expression and characterization of the IPM 150 gene (IMPG1) product, a novel human photoreceptor cell-associated chondroitin-sulfate proteoglycan.

The interphotoreceptor matrix (IPM) occupies the extracellular space between the apical surface of the retinal pigmented epithelium and the external limiting membrane of the neural retina. This space contains two chondroitin sulfate proteoglycans, designated IPM 150 and IPM 200, which are likely to effect retinal adhesion and photoreceptor survival. In an effort to characterize human IPM 150, several cDNA clones encoding its core protein have been isolated from a human retinal cDNA library. Translation of overlapping cDNA sequences yields a novel core protein with a predicted molecular mass of 89.3 kDa. Northern and dot-blot analyses as well as the isolation of expressed sequence tags demonstrate that IPM 150 mRNA is expressed not only in the neural retina but also in several other non-ocular tissues. In situ hybridization analyses indicate that, in the eye, IPM 150 mRNA is expressed specifically by cone and rod photoreceptor cells. Characterization of IPM 150 proteoglycan core protein and identification of its site of synthesis are important steps towards understanding the architecture and biology of the IPM.

The genetics of age-related macular degeneration.

Age-related macular degeneration (AMD) is increasingly recognized as a complex genetic disorder in which one or more genes contribute to an individual's susceptibility for developing the condition. Twin and family studies as well as population-based genetic epidemiologic methods have convincingly demonstrated the importance of genetics in AMD, though the extent of heritability, the number of genes involved, and the phenotypic and genetic heterogeneity of the condition remain unresolved. The extent to which other hereditary macular dystrophies such as Stargardts disease, familial radial drusen (malattia leventinese), Best's disease, and peripherin/RDS-related dystrophy are related to AMD remains unclear. Alzheimer's disease, another late onset, heterogeneous degenerative disorder of the central nervous system, offers a valuable model for identifying the issues that confront AMD genetics.

Molecular characterization and genomic mapping of human IPM 200, a second member of a novel family of proteoglycans.

We report herein the characterization of the cDNA for a novel human chondroitin sulfate proteoglycan, designated IPM 200, and the chromosomal location of its gene, designated IMPG2. IPM 200 was isolated from the retinal interphotoreceptor matrix, a unique extracellular matrix that occupies the subretinal space between the apices of the retinal pigment epithelium and the neural retina. The cDNA contains an open reading frame of 3,726 bp that codes for a core protein with a deduced molecular weight of 138.5 kDa. The deduced IPM 200 core protein contains a putative transmembrane domain, two EGF-like repeats, numerous N- and O-linked glycosylation consensus sequences and one consensus sequence for glycosaminoglycan attachment. IMPG2 maps to human chromosome 3q12.2-12.3. Based on homologies within their amino acid sequences we propose that IPM 200 and a previously described human proteoglycan, IPM 150, form a new family of extracellular matrix glycoconjugates.