Age independent expression of myocilin in the human trabecular meshwork

Glaucoma is a leading cause of blindness. Primary open-angle glaucoma, the most common form of glaucoma, is known to increase in prevalence with age. One of its major risk factors is an elevated intraocular pressure believed to be related to the trabecular meshwork (TM), a specialized tissue located at the chamber angle of the eye. Myocilin, a gene linked to open-angle glaucomas (OAG), has been found to be expressed in the TM and a broad range of other ocular and non-ocular tissues. The purpose of this study was to examine the distribution of myocilin protein and mRNA in the TM of normal human eyes to determine whether age-related changes exist. Studies with glaucomatous eyes were carried out concurrently. Immunoperoxidase staining experiments demonstrated positive immunoreactivity for myocilin protein in both the cells and beams in all regions of the TM. The staining intensity and the myocilin level as determined by dot blot assays were not correlated with the ages of donors ranging from 8 weeks to 93 years. Neither did myocilin mRNA, detected by in situ hybridization in cells throughout the TM and quantified by relative quantitative RT-PCR, vary with age. Myocilin mRNA and protein expression in diseased tissues was either comparable to that of normal tissues or reduced. This study represents the first in-depth investigation of myocilin expression in relation to age in the TM of human eyes. The results indicate an age independence, argue thus against a direct role of myocilin and reiterate the involvement of additional factors in the pathogenesis of glaucoma.     

Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor

Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.     

A cellular assay distinguishes normal and mutant TIGR/myocilin protein.

Glaucoma is a blinding eye disease that affects approximately 70 000 000 people world-wide. Mutations in the gene TIGR / MYOC have been shown to cause the most common form of the disease, primary open angle glaucoma, in selected families. Amino acid sequence variants of the gene have been found in 2-4% of sporadic primary open angle glaucoma cases. Most variants are rare and it is often difficult to definitively distinguish between a deleterious mutation and a benign variant solely on the basis of relative frequencies in patient and control groups. The function of the TIGR/myocilin protein is unknown and an assay to functionally classify variants is lacking. We sought to develop a biochemical assay to distinguish different forms of TIGR/myocilin. We investigated the Triton X-100 detergent solubility characteristics of mutant and normal forms of the protein, expressed by transfection in cultured cells. We observed a clear difference in the behavior of the two types of TIGR/myocilin; all confirmed mutant proteins tested were substantially Triton insoluble, while normal protein and controls were completely soluble. We also tested seven ambiguous variant proteins and classified them as mutant or normal on the basis of their Triton solubility. The results in some cases validated, and in other cases contradicted, earlier classifications of these variants. To our knowledge, Triton solubility is the first example of a general difference in the properties of mutant and normal forms of TIGR/myocilin. The assay we have developed will be useful for discerning protein functional information from the location of mutations, will aid genetic counseling of individuals with TIGR/myocilin variants and may provide a clue to understanding a mechanism by which mutations in TIGR / MYOC cause glaucoma.     

Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma

Purpose: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective in lowering intraocular pressure by reducing the rate of aqueous humour secretion mediated by the CAs in the ciliary epithelium. In this work, we investigated the expression and tissue distribution of two recently discovered CA genes CA9 (CAIX) and CA12 (CAXII) in fetal, neonatal, and adult human eyes with and without glaucoma.

Methods: CAIX and CAXII expression in 16 normal and 10 glaucomatous eyes, and in cultured non-pigmented ciliary epithelial cells (NPE) from normal and glaucoma eye donors was assessed by immunostaining. In addition, northern blot hybridisation was performed to assess expression of CA4, CA9, and CA12 mRNA in cultured NPE cells from normal and glaucoma donors.

Results: CAXII was localised primarily to the NPE with its expression prominent during embryonic eye development but which decreased significantly in adults. CAIX expression in the NPE was very low. The epithelium of cornea and lens occasionally expressed both enzymes at low levels during development and in adult eye, and no expression was detected in the retina. The NPE from glaucoma eyes expressed higher levels of CAXII, but not CAIX, in comparison with normal eyes. This expression pattern was retained in cultured NPE cell lines. NPE cells from a glaucoma patient showed a five-fold increase in the CA12 mRNA level with no detectable expression of CA9 mRNA. Also, no expression of the CA4 gene encoding a GPI anchored plasma membrane protein was detected on these northern blots.

Conclusions: Transmembrane CAIX and CAXII enzymes are expressed in the ciliary cells and, thus, may be involved in aqueous humour production. CA12 may be a targeted gene in glaucoma.

     

Expression patterns of focal adhesion associated proteins in the developing retina.

Adhesive interactions between integrin receptors and the extracellular matrix (ECM) are intimately involved in regulating development of a variety of tissues within the organism. In the present study, we have investigated the relationships between beta(1) integrin receptors and focal adhesion associated proteins during eye development. We used specific antibodies to examine the distribution of beta(1) integrin ECM receptors and the cytoplasmic focal adhesion associated proteins, talin, vinculin, and paxillin in the developing Xenopus retina. Immunoblot analysis confirmed antibody specificity and indicated that beta(1) integrins, talin, vinculin, and paxillin were expressed in developing retina and in the retinal-derived Xenopus XR1 glial cell line. Triple-labeling immunocytochemistry revealed that talin, vinculin, paxillin, and phosphotyrosine proteins colocalized with beta(1) integrins at focal adhesions located at the termini of F-actin filaments in XR1 cells. In the retina, these focal adhesion proteins exhibited developmentally regulated expression patterns during eye morphogenesis. In the embryonic retina, immunoreactivities for focal adhesion proteins were expressed in neuroepithelial cells, and immunoreactivity was especially strong at the interface between the optic vesicle and overlying ectoderm. At later stages, these proteins were expressed throughout all retinal layers with higher levels of expression observed in the plexiform layers, optic fiber layer, and in the region of the inner and outer limiting membrane. Strong immunoreactivities for beta(1) integrin, paxillin, and phosphotyrosine were expressed in the radially oriented Müller glial cells at later stages of development. These results suggest that focal adhesion-associated proteins are involved in integrin-mediated adhesion and signaling and are likely to be essential in regulating retinal morphogenesis.     

Identification of the region in the N-terminal domain responsible for the cytoplasmic localization of Myoc/Tigr and its association with microtubules.

Mutations in the MYOC/TIGR gene are associated with juvenile open-angle glaucoma and in some cases may be involved in the formation of sporadic primary open-angle glaucoma in humans. To better understand the functions of the MYOC/TIGR protein, its intracellular distribution was investigated using green fluorescent protein (GFP) as a marker. The results indicated that the recombinant mouse and human Myoc/Tigr-GFP proteins are located in the cytoplasm of the transfected cells in which they colocalize with microtubules. Deletion analysis demonstrated that the N-terminal region (positions 1-124 and 15-138 in the mouse and human proteins, respectively) encoded by exon 1 is critical for the cytoplasmic localization of Myoc/Tigr. Most of the known mutations in the human MYOC/TIGR gene implicated in juvenile and sporadic primary open-angle glaucoma formation are located outside the region responsible for the cytoplasmic localization of the protein. However, some of these mutations may alter the tertiary structure of the protein and subsequently modify its interaction with microtubules.     

Aggregated myocilin induces russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary open-angle glaucoma

Primary open-angle glaucoma with elevated intraocular pressure is a leading cause of blindness worldwide. Mutations of myocilin are known to play a critical role in the manifestation of the disease. Misfolded mutant myocilin forms secretion-incompetent intracellular aggregates. The block of myocilin secretion was proposed to alter the extracellular matrix environment of the trabecular meshwork, with subsequent impediment of aqueous humor outflow leading to elevated intraocular pressure. However, the molecular pathogenesis of myocilin-caused glaucoma is poorly defined. In this study, we show that heteromeric complexes composed of wild-type and mutant myocilin were retained in the rough endoplasmic reticulum, aggregating to form inclusion bodies typical of Russell bodies. The presence of myocilin aggregates induced the unfolded protein response proteins BiP and phosphorylated endoplasmic reticulum-localized eukaryotic initiation factor-2alpha kinase (PERK) with the subsequent activation of caspases 12 and 3 and expression of C/EBP homologous protein (CHOP)/GADD153, leading to apoptosis. Our findings identify endoplasmic reticulum stress-induced apoptosis as a pathway to explain the reduction of trabecular meshwork cells in patients with myocilin-caused glaucoma. As a consequence, the phagocytotic capacity of the remaining trabecular meshwork cell population would be insufficient for effective cleaning of aqueous humor, constituting a major pathogenetic factor for the development of increased intraocular pressure in primary open-angle glaucoma.     

Kir2.1通道在牛视网膜胶质细胞中的分布和表达-mRNA和蛋白水平的研究

本研究目的为在 m RNA和蛋白质水平探讨 Kir2 .1通道在牛视网膜胶质细胞中的分布和表达。从神经视网膜和视网膜色素上皮分离 m RNA和蛋白质 ,以 RT-PCR,Northern blot,Western blot和免疫荧光组织化学法检测 Kir2 .1m RNA和 Kir2 .1蛋白的表达。结果表明 ,RT-PCR扩增的 Kir2 .1c DNA产物在神经视网膜中强表达 ,在视网膜色素上皮中弱表达 ;Northern斑点杂交的 Kir2 .1c DNA仅在神经视网膜表达 ;Western斑点杂交检测 Kir2 .1蛋白单带在神经视网膜中表达 ;免疫荧光组织化学显示 Kir2 .1蛋白在 Muller细胞中分布 ,并且主要分布在 Muller细胞与神经元相接触的细胞膜区域 ,与 Muller细胞的特异性标记蛋白质抗谷氨酸合成酶抗体双重标记显示其分布特性重叠。在视网膜色素上皮未检测到 Kir2 .1蛋白的免疫活性。上述结果表明 ,Kir2 .1分布在视网膜胶质细胞的 Muller细胞 ,这种分布特性可能与完成 Muller细胞功能 -保持细胞外 K+ 的平衡、促进 K+的内流有重要关系     

Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma

Glaucoma is a progressive blinding disease characterized by gradual loss of vision due to optic neuropathy and retinal ganglion cell death. Increased intraocular pressure is a common feature of glaucoma that is thought to arise from an increased resistance to outflow of aqueous humor through the trabecular meshwork. Mutations of the myocilin gene are one cause of autosomal dominant juvenile- and adult-onset primary open angle glaucoma, but the mechanism by which mutant myocilins cause disease is poorly understood. We have found that disease-causing myocilin mutants are misfolded, are highly aggregation-prone and accumulate in large aggregates in the endoplasmic reticulum (ER) of human embryonic kidney cells and differentiated primary human trabecular meshwork (HTM) cells. In HTM cells, Pro370Leu mutant myocilin is not secreted under normal culture conditions and prolonged expression results in abnormal cell morphology and cell killing. Culturing HTM cells at 30 degrees C, a condition known to facilitate protein folding, promotes secretion of mutant myocilin, normalizes cell morphology and reverses cell lethality. Our results indicate that myocilin-associated glaucoma is an ER storage disease and suggest a progression of events in which chronic expression of misfolded, non-secreted myocilin leads to HTM cell death, trabecular meshwork dysfunction and, ultimately, a dominant glaucoma phenotype. The beneficial effects of facilitating folding and secretion of mutant myocilin suggest a new type of treatment for this form of glaucoma.     

Protein localization in the human eye and genetic screen of opticin

The opticin (OPTC) gene encodes a protein that is a member of the small leucine-rich repeat protein (SLRP) family. OPTC is located on chromosome 1q31-q32 within an age-related macular degeneration (AMD) susceptibility locus. We have developed an affinity-purified N-terminal anti-opticin antibody and used it to examine opticin expression in human eye tissues. The antibody was also used for opticin protein localization in human eye sections. Immunoblots of human eye tissues detected a predominant band of approximately 62 kDa in size in iris, trabecular meshwork/ciliary body, retina, vitreous, and optic nerve. Immunohistochemical experiments revealed that opticin is specifically localized in human cornea, iris, ciliary body, vitreous, choroid and retina. Due to opticin's protein profile in the eye, we have also screened OPTC for mutations in individuals with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG) or AMD. We identified four sequence variations, all of which were observed in normal controls except for the Arg229Cys change. Three amino acid substitutions (Ile182Thr, Arg229Cys and Arg325Trp) were in residues conserved in dog, mouse, pig and human. The Arg229Cys alteration was present in a homozygous state in one individual with neovascular AMD. Examination of the other AMD afflicted family members showed that the OPTC Arg229Cys variant did not segregate with the disorder within the family. The protein localization pattern of opticin and our preliminary screen of AMD patients suggest that a larger AMD patient screen may be warranted.     

Glaucoma-causing myocilin mutants require the Peroxisomal targeting signal-1 receptor (PTS1R) to elevate intraocular pressure

Glaucoma is a leading cause of worldwide irreversible visual impairment and blindness and is a clinically and genetically heterogenous group of optic neuropathies. Specific mutations in the myocilin (MYOC) gene cause primary open angle glaucoma (POAG) with varying age-of-onset and degree of severity. We show a mutation-dependent, gain-of-function association between human myocilin and the peroxisomal targeting signal type 1 receptor (PTS1R). There was correlation between the glaucoma phenotype and the specific MYOC mutations, with the more severe early-onset POAG mutations having a higher degree of association with PTS1R. Expression of human myocilin glaucomatous mutations in mouse eyes causes elevated intraocular pressure, which is a major phenotype of MYOC glaucoma. This is the first demonstration of a disease resulting from mutation-induced exposure of a cryptic signaling site that causes mislocalization of mutant protein to peroxisomes and the first disease-gene-based animal model of human POAG.     

Localization of retinoid binding proteins, retinoid receptors, and retinaldehyde dehydrogenase in the chick eye

Retinoids have many functions in the eye, including, perhaps, the visual guidance of ocular growth. Therefore, we identified where retinoid receptors, binding proteins, and biosynthetic enzymes are located in the ocular tissues of the chick as a step toward discovering where retinoids are generated and where they act. Using antibodies to interphotoreceptor retinoid binding protein (IRBP), cellular retinol binding protein (CRBP), cellular retinoic acid binding protein (CRABP), cellular retinaldehyde binding protein (CRALBP), retinaldehyde dehydrogenase (RALDH), and retinoic acid receptors (RAR and RXR), we localized these proteins to cells in the retina, retinal pigmented epithelium, choroid and sclera of the chick eye. IRBP was detected in the photoreceptor layer and pigmented epithelium; CRBP was in the pigmented epithelium; CRABP was in amacrine and bipolar cells in the retina; CRALBP was in Müller cells, pigmented epithelium, choroid, and fibrous sclera; RALDH was in retinal amacrine cells, pigmented epithelium, and choroid; RAR was in amacrine cells, choroid, and chondrocytes and fibroblasts in the sclera; and RXR was in amacrine and ganglion cells, bipolar cell nuclei, choroid, and chondrocytes. We also found that the growth-modulating toxins colchicine and quisqualate destroyed selectively different subsets of CRABP-containing amacrine cells. We conclude that the distribution of proteins involved in retinoid metabolism is consistent with a role of retinoids not only in phototransduction, but also in maintenance of cellular phenotype and visual guidance of ocular growth.     

Expression of Myocilin Mutants Sensitizes Cells to Oxidative Stress-Induced Apoptosis : Implication for Glaucoma Pathogenesis

Mutations in the myocilin gene are associated with juvenile and adult-onset primary open-angle glaucoma. However, the pathogenic mechanisms of myocilin-induced glaucoma are still largely unknown. To investigate these mechanisms, we developed stably transfected HEK293 cell lines expressing wild-type or mutant (Y437H and I477N) myocilins under an inducible promoter. Expression of two mutant myocilins led to different levels of endoplasmic reticulum stress and increased apoptosis after treatment of cells with hydrogen peroxide. The Y437H mutant myocilin cell line showed the highest sensitivity to the oxidant treatment. Several antioxidant genes were down-regulated in the Y437H mutant myocilin cell line, but not in other cell lines. The Y437H mutant myocilin cell line also produced more reactive oxygen species than other cell lines examined. Consistent with the data obtained in cultured cells, the endoplasmic reticulum stress marker, 78 kDa glucose-regulated protein, was up-regulated, whereas antioxidant proteins, paraoxonase 2 and glutathione peroxidase 3, were down-regulated in the eye angle tissue of 18-month-old transgenic mice expressing Y437H myocilin mutant. In addition, a pro-apoptotic factor, CCAAT/enhancer-binding protein-homologous protein, was up-regulated in the aged transgenic mouse angle tissue. Our results suggest that expression of mutated myocilins may have a sensitization effect, which can lead to a severe phenotype in combination with oxidative stress. Mutant myocilins may confer different sensitivity to oxidative stress depending on the mutation.Glaucoma is a group of chronic optic neuropathies characterized by the death of retinal ganglion cells, leading to a specific deformation of the optic nerve, known as glaucomatous cupping, and progressive loss of the visual field. Glaucoma is one of the leading causes of irreversible blindness in the world. It is estimated to affect more than 60 million and blind about 4.5 million people worldwide.¹ Elevated intraocular pressure (IOP) is the most important risk factor for the onset and progression of glaucoma. Different forms of glaucoma are classified by the appearance of the iridocorneal angle, which is important in regulating the drainage of aqueous humor from the eye. In primary open-angle glaucoma (POAG), the most common form of glaucoma in populations of European and African ancestry, there are no clear abnormalities in the geometry and appearance of the iridocorneal angle. Several theories have been suggested to explain possible deficiencies in the eye drainage structures that may lead to POAG.^2,3 Family history of POAG, old age, black race, myopia, and low diastolic perfusion pressure are well established risk factors for POAG.^2,4It is now well demonstrated that mutations in the MYOCILIN (MYOC) gene are associated with elevated IOP and with POAG.^5,6 They are found in more than 10% of cases of juvenile POAG occurring between the ages of 3 and 40 years⁴ and 3% to 4% of cases of adult onset POAG occurring after 40 years of age.^5,7,8 The MYOC gene is expressed in various ocular and nonocular tissues with the highest levels of expression in the trabecular meshwork (TM) and sclera.^6,9,10,11 It encodes a secreted protein that contains a leucine zipper in the N-terminus and the olfactomedin domain in the C-terminus.^12,13,14 The olfactomedin domain was originally identified in the protein isolated from frog olfactory neuroepithelim.¹⁵ This domain was subsequently found in several proteins in species ranging from Caenorhabditis elegans to Homo sapiens.¹⁶The functions of myocilin protein are still not well understood. Biochemical data indicate that myocilin may interact with several intracellular and extracellular matrix proteins.^{10,17,18,19,20,21} Our recent data demonstrated that myocilin interacts with several Wnt receptors in the Frizzled family as well as with Wnt antagonists in the secreted Frizzled-related protein family and Wnt inhibitory factor-1.²²The absence of open-angle glaucoma in an elderly woman homozygous for the Arg46Stop mutation²³ as well as the absence of glaucoma in people hemizygous for MYOC²⁴ suggests that the loss of functional myocilin is not sufficient for the development of glaucoma or critical for normal eye function. We suggested that the absence of a glaucoma phenotype with myocilin null mutations in the eye may be explained by the compensatory action of Wnt proteins.²²A glaucoma phenotype appears to be dependent on expression of mutated myocilin protein in eye tissues. More than 70 glaucoma-causing mutations have been identified in the MYOC gene and greater than 90% of them are located in the region encoding the olfactomedin domain.^7,25 Mutations that are associated with a severe glaucoma phenotype reduce the solubility of myocilin, lead to retention of the protein in the endoplasmic reticulum (ER), and prevent its secretion.^26,27,28,29 Moreover, secretion of wild-type myocilin is impeded in the presence of mutated myocilin protein, making these mutations dominant.^26,29,30,31 Accumulation of mutated myocilin in ER may be deleterious for cells and lead to cell death.^32,33 However, the expression of mutated myocilin allele (Tyr423His) specifically in the mouse iridocorneal angle did not lead to detectable TM cell death.^34,35Environment may contribute to the effects of mutated myocilin. In particular, it is well established that oxidative stress may be a factor in the progression of many diseases, including glaucoma.^36,37 Endothelial-like cells of the TM are in a direct contact with aqueous humor containing reactive oxygen species (ROS). Recent data demonstrate that the TM shows the highest sensitivity to the consequences of oxidative stress among the tissues of the eye anterior chamber.³⁸ Expression of some myocilin mutants may induce ER stress in vitro,^32,39 and ER stress in turn may cause oxidative stress.⁴⁰ We hypothesized that expression of mutated myocilin can make cells more sensitive to oxidative stress both in vitro and in vivo.In this article we demonstrate that expression of Y437H mutant myocilin in cultured cells and in transgenic mice in vivo induces the unfolded protein response (UPR) and suppresses expression of a number of proteins involved in the response to oxidative stress. Expression of myocilin mutants made cells more sensitive to oxidative stress, and different mutants provided different levels of sensitivity. Our data suggest that antioxidative agents should be assessed as therapeutic agents for myocilin-induced glaucoma.     

Analysis of myocilin gene mutations in Japanese patients with normal tension glaucoma and primary open-angle glaucoma

The myocilin gene was identified as a gene (MYOC) that caused primary open-angle glaucoma (POAG). Although a normal tension glaucoma (NTG) patient with the myocilin gene mutation was previously reported, no study using large numbers of patients with NTG has been reported. Single-strand conformation polymorphism analysis and subsequent sequence analysis were performed for genotyping the myocilin gene in 114 unrelated Japanese patients with NTG. One hundred and nineteen patients with POAG and 100 control subjects without glaucoma were studied as reference subjects. Five amino acid sequence changes of the myocilin were identified: Arg46Stop (one NTG), Arg76Lys (four NTG, 10 POAG, seven control), Arg158Gln (one NTG, one POAG, one control) found in only Japanese, Asp208Glu (four NTG, three POAG, one control), Pro481Ser (one control). Pro481Ser was novel. Arg76Lys always occurred with 1-83 from G to A in the promoter as it was reported in Chinese. Although some Japanese patients with NTG had sequence changes of the myocilin gene, there were no apparent specific mutations in patients with NTG.     

原发性开角型青光眼家系一新的myocilin基因突变

目的探讨一个中国人原发性开角型青光眼(primary open angle glaucoma,POAG)家系的my-ocilin基因缺陷。方法对一个5代POAG大家系进行全面的临床检查后,用聚合酶链反应扩增家系成员myocilin基因的所有外显子以及相邻部分内含子,对其产物直接测序。结果家系的遗传方式符合常染色体显性遗传。确诊年龄在26～59岁之间。在所有青光眼患者,可疑者以及4例尚未出现明显青光眼体征的家系成员发现携带myocilin基因T455K突变。无该突变的家系成员中无POAG患者及可疑者。突变位于myocilin蛋白C-末端非常保守的氨基酸残基。结论T455K突变是中国人所特有的新的致病性myocilin基因突变。突变的临床表型为混合发病年龄型POAG且具有很高的外显率。这个新基因突变的发现证实中国人的致病性myocilin基因突变类型与其他种族不同。     

Analysis of myocilin mutations in 1703 glaucoma patients from five different populations

A glaucoma locus, GLC1A, was identified previously on chromosome 1q. A gene within this locus (encoding the protein myocilin) subsequently was shown to harbor mutations in 2-4% of primary open angle glaucoma patients. A total of 1703 patients was screened from five different populations representing three racial groups. There were 1284 patients from primarily Caucasian populations in Iowa (727), Australia (390) and Canada (167). A group of 312 African American patients was from New York City and 107 Asian patients from Japan. Overall, 61 different myocilin sequence variations were identified. Of the 61 variations, 21 were judged to be probable disease-causing mutations. The number of probands found to harbor such mutations in each population was: Iowa 31/727 (4.3%), African Americans from New York City 8/312 (2.6%), Japan 3/107 (2.8%), Canada 5/167 (3.0%), Australia 11/390 (2.8%) and overall 58/1703 (3. 4%). Overall, 16 (76%) of 21 mutations were found in only one population. The most common mutation observed, Gln368Stop, was found in 27/1703 (1.6%) glaucoma probands and was found at least once in all groups except the Japanese. Studies of genetic markers flanking the myocilin gene suggest that most cases of the Gln368Stop mutations are descended from a common founder. Although the specific mutations found in each of the five populations were different, the overall frequency of myocilin mutations was similar ( approximately 2-4%) in all populations, suggesting that the increased rate of glaucoma in African Americans is not due to a higher prevalence of myocilin mutations.     

Expression of the Vax family homeobox genes suggests multiple roles in eye development

BACKGROUND: Homeobox gene products are thought to regulate target genes involved in the regional specification of tissues and organs. Several genes play roles in eye development. RESULTS: We isolated two new mouse homeobox genes, Vax1 and Vax2, expressed in retina primordium. The two genes share the same gene organization, consisting of three exons with predicted amino acid sequences identical in the homeodomain but diversified in other regions. At 9. 5 days post coitum, both Vax genes were expressed in optic vesicles. At 11.5-14.5 dpc, Vax1 became restricted to the optic stalk, while Vax2 was expressed in the ventral half of the neural retina. Mapping of Vax2 between D6Mit3 and D6Mit8 on chromosome 6 indicated a possible linkage with Emx1. CONCLUSIONS: The expression patterns of Vax genes suggest separate and specific involvement in eye development. Vax1 may contribute to differentiation of the neuroretina, pigmented epithelium and optic stalk, while Vax2 could function in the establishment of the dorso-ventral axis of the retina and the visual system. The chromosomal location of Vax2 suggests that Vax2-Emx1 and Vax1-Emx2 arose by chromosome duplication of the same ancestral gene.     

Immunolocalization of cellular retinol-, retinaldehyde- and retinoic acid-binding proteins in rat retina during pre- and postnatal development

Summary Cellular retinol-, retinaldehyde- and retinoic acid-binding proteins were localized in rat retina during pre- and postnatal development by indirect immunofluorescence. Cryostat tissue sections were prepared daily from embryonic day 11 until the day of birth (E11–22) and from postnatal days 1–32 (P1–32). Cellular retinaldehyde- and retinol-binding proteins were first detected in retinal pigment epithelium on E13 and E18, respectively, and in Müller cells at P1 and P15. Parallel studies showed that in adult retina cellular retinoic acid-binding protein is present in a subpopulation of GABAergic amacrine cells. During retinal differentiation, cellular retinoic acid-binding protein was first detected at E18 in cells sclerad to the developing inner plexiform layer, suggesting that this binding protein is expressed in amacrine cells very early during differentiation. During early ocular morphogenesis, cellular retinoic acid-binding protein was present in mesenchymal cells enveloping the eye (E12–15), in the neuroblastic layer of the retina (E13–15), in the nerve fibre layer (E14–15), and the developing optic nerve (E15). Our results suggest that retinoic acid, the natural ligand of cellular retinoic acid-binding protein, may be involved in neuronal differentiation in the inner retina. The studies further support a role for cellular retinoic acid-binding protein in mediating the effects of retinoic acid on developing neural crest cells and raise new questions about the role of cellular retinaldehyde-binding protein in the visual cycle and during development.