Trabecular Meshwork Inducible Glucocorticoid Response Research Articles

Prevalence of Glu323Lys Mutation of the TIGR/MYOC Gene and Risk Factors amongst Primary Open-angle Glaucoma Patients in Ouagadougou, Burkina Faso.

Glaucoma is a group of degenerative diseases of the optic nerve whose predisposing factors may be genetic. The objective of this study was to estimate the frequency of the Glu323Lys mutation as a genetic risk factor for glaucoma. A cross-sectional study over 6 months from October 2020 to March 2021 in Ouagadougou, Burkina Faso. A total of 89 samples of patients with primary open-angle glaucoma (POAG) were collected. The frequency of the Glu323Lys mutation of the myocilin, trabecular meshwork inducible glucocorticoid response (TIGR/MYOC) gene by polymerase chain reaction (PCR)-restriction fragment length polymorphism. In glaucoma patients, only homozygous nonmutated guanine-guanine (GG) and heterozygous mutated adenine-guanine (AG) genotypes were found in 96.63 and 3.37% of cases, respectively. Around 69.66% of patients had a family history of glaucoma, 28.09% had a history of hypertension, and 7.86% had a history of diabetes. The frequency of the Glu323Lys mutation of the TIGR/MYOC gene was 3.37% in the glaucoma population in Ouagadougou. A case-control study is necessary to know the contribution of the Glu323Lys mutation as a genetic risk factor for glaucoma in our study population. This study constituted the beginning of genetic investigations of glaucoma in our context and showed a low Glu323Lys mutation. Traoré L, Sanou J, Bakyono BS, et al. Prevalence of Glu323Lys Mutation of the TIGR/MYOC Gene and Risk Factors amongst Primary Open-angle Glaucoma Patients in Ouagadougou, Burkina Faso. J Curr Glaucoma Pract 2023;17(2):79-84.

Clinical Analysis of Steroids Induced Glaucoma: A Systematic Review

For more than 50 years, the elevation in intraocular pressure that can impede the administration of topical or systemic corticosteroids has been recognized. There has been increasing attention in this steroid-responsive phenomena since the identification of the myocilin gene (formerly known as the trabecular meshwork inducible glucocorticoid response or TIGR gene). In addition, the increasingly popular use of injectable intraocular steroids to treat clinically substantial subretinal fluid and macular oedema has increased the incidence. Animal studies, cell biology, molecular biology, and a better grasp of genetics have all contributed to a better understanding of the response's mechanics. The electronic MEDLINE, Embase, Cochrane, and PubMed databases were searched. Additionally, the bibliography of all relevant articles and textbooks were manually searched. To determine the link between plasma cortisol levels and steroid-induced glaucoma, plasma cortisol levels can be tested once every four hours. It can also be done on a bigger group of people. The steroid provocative test will aid in the identification of individuals who are high steroid responders in the general population. It can also be used in patients who require intravitreal triamcinolone injections. Genetic research could shed further light on the aetiology of steroid-induced glaucoma.

English

Myocilin (OMIM601652) MYOC gene, first revealed as a protein isolated from cultured human Trabecular Meshwork (TM) cells after prolong dexame thasone treatment. Hence given the name TIGR (Trabecular Meshwork Inducible Glucocorticoid Response Protein). The human myocilin gene, a concealed acidic glycoprotein,contains 504 amino acids, positioned at chromosome 1(1q24.3 ‐ 1q25.2), and filled with olfactomedin do main at its C terminus. Myocilin exists in various tissues throughout the eye and many other organs. M utant myocilin cause endoplasmic reticulum pressure in eye angle tissues, including the trabec ular meshwork, damaging the cells inside the meshwork, ultimately leading to structural changes in the outflow pathway, and high intraocular pressure.TM is vital in controlling pressure and mu tations in MYOC.It has been recognized as the cause of juvenile and adult onset of primary open a ngle glaucoma.

Myocilin Interacts with Syntrophins and Is Member of Dystrophin-associated Protein Complex

Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.

Significance of G-X-W motif in the myocilin olfactomedin domain

Myocilin, trabecular meshwork inducible glucocorticoid response, also known as MYOC, is encoded by the MYOC gene in humans. The myocilin protein is a secreted 55–57-kDa glycoprotein whose known structural features include a myosin-like domain, a leucine zipper region, and an olfactomedin domain [1]. Most of the diseases causing mutations lie on the olfactomedin domain, which is highly conserved among species. This suggests that the domain may play an important role in myocilin function. The olfactomedin domain was first identified in extracellular matrix protein of the olfactory neuroepithelium [2]. A number of metazoans are known to have this extracellular protein domain. Some of the olfactomedin containing metazoan proteins are latrophilins, myocilins, optimedins, and noelins [3]. The mouse and human genomes encode at least 12 olfactomedin-related gene products. These proteins have a variable N-terminal domain and a conserved olfactomedin C-terminal domain [4]. The initial member of the olfactomedin family was identified as a secreted glycoprotein in the bullfrog olfactory neuroepithelium [2]. Olfactomedin family members are expressed in many tissues and exhibit tissue-specific expression patterns [5]. Several olfactomedin proteins are expressed in the nervous system. They include olfactomedin-1 (noelin-1) in brain and retina [6], olfactomedin–noelin–tiarin protein 1 (ONT1) in midbrain and hindbrain roof plate and axial and paraxial mesoderm [7], tiarin in non-neural ectoderm [8], olfactomedin-2 in brain and retina, optimedin (olfactomedin-3) in brain, and retina [9], myocilin in the eye [10], and gliomedin in Schwann cells [11]. Retinoic acid (RA), a metabolite of vitamin A, profoundly affects cell proliferation, cell differentiation, and morphogenesis in several vertebrate embryonic tissues including the neural crest [12–15]. RA receptors and cellular RA binding proteins both are expressed in neural crest derivatives [16, 17]. RA treatment alters the expression pattern of homeobox (Hox)-containing genes, which are being implicated in segmental specification in various embryonic tissues including the neural crest [18]. Some of the olfactomedins are shown to have key roles in the development of nervous system. Noelins are needed for development of the neural crest and regulation of neuronal fate [19, 20]. Tiarin affects dorsalization in the developing spinal cord [7, 8]. ONT1, another member of the olfactomedin family, is responsible for development of neural crest cells [7]. Optimedin is a downstream target of Pax6 [21], a transcription factor that is critical for central nervous system development. Gliomedin, a member of the olfactomedin protein family, is expressed by Schwann cells and is required for molecular assembly of developing nodes of Ranvier in the peripheral nervous system [11]. Olfactomedin domain-containing proteins play important roles in neurogenesis and dorsal ventral patterning. Lipocalins transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. The members of the family show high affinity and selectivity for hydrophobic molecules. The presence of six- or eight-stranded β-barrel and highly conserved motifs/short conserved region in their amino acid sequences is a common feature of lipocalin [22]. They are structurally and functionally diverse and include the subfamilies of kernel and outlier lipocalins. The sequences of most members of the core or kernel lipocalins are characterized by the three short conserved stretches of residues, while the outlier lipocalin group share only one or two of these conserved stretches.

Keeping an Eye on Myocilin: A Complex Molecule Associated with Primary Open-Angle Glaucoma Susceptibility

MYOC encodes a secretary glycoprotein of 504 amino acids named myocilin. MYOC is the first gene to be linked to juvenile open-angle glaucoma (JOAG) and some forms of adult-onset primary open-angle glaucoma (POAG). The gene was identified as an up-regulated molecule in cultured trabecular meshwork (TM) cells after treatment with dexamethasone and was originally referred to as trabecular meshwork-inducible glucocorticoid response (TIGR). Elevated intraocular pressure (IOP), due to decreased aqueous outflow, is the strongest known risk factor for POAG. Increasing evidence showed that the modulation of the wild-type (wt) myocilin protein expression is not causative of glaucoma while some misfolded and self-assembly aggregates of mutated myocilin may be associated with POAG in related or unrelated populations. The etiology of the disease remains unclear. Consequently, a better understanding of the molecular mechanisms underlyingPOAG is required to obtain early diagnosis, avoid potential disease progression, and develop new therapeutic strategies. In the present study, we review and discuss the most relevant studies regarding structural characterizations, expressions, molecular interactions, putative functions of MYOC gene and/or its corresponding protein in POAG etiology.

Study on MYOC/TIGR gene mutations in primary open-angle glaucoma

To study the mutations in the trabecular meshwork induced-glucocorticoid response protein gene (TIGR/MYOC) in Chinese POAG patients. It was a case-control study. One hundred and eighteen Chinese patients with POAG and 150 control subjects without POAG were screened for coding sequence of the MYOC gene by polymerase chain reaction, single-strand conformation polymorphism (SSCP) and DNA sequence. Restriction endonuclease analysis was used to detect the SSCP in 150 gender- and aged-matched controls. Statistical significance of the difference in frequencies of MYOC mutations between POAG patients and the controls was determined by the chi-squared test. Three coding sequence variants that lead to amino acid changes were identified in MYOC gene. One mutation of MYOC gene was novel, I288M, which was present in one patient with POAG and 3 control subjects. G12R and Y353I have been reported previously. I288M and Y353I were found in both the subjects with and without POAG, the difference of the frequency of these two mutations between POAG and the controls was χ(2) = 0.07, P = 0.791 and χ(2) = 0.56, P = 0.453, respectively. Coding sequence mutations of MYOC were found in 4.23% POAG patients. Frequency of G12R mutation in POAG patients was significantly greater than that in the controls (χ(2) = 4.37, P = 0.037). One novel variant locus in the MYOC/TIGR (I288M) was found in the present study, but this mutation is not associated with POAG. G12R is the most common mutation in MYOC locus in Chinese subjects with POAG. Approximately 4.23% sporadic POAG patients have mutation in the exon 1 in MYOC/TIGR.

Corticosteroid-induced glaucoma: a review of the literature

The intraocular pressure rise that can complicate the use of topical or systemic corticosteroid has been recognised for 50 years. More recently, following isolation of the myocilin gene (previously known as the trabecular meshwork inducible glucocorticoid response or TIGR gene), there has been renewed interest in this steroid-responsive phenomenon. Furthermore, the currently fashionable use of injectable intraocular steroids in the management of clinically significant subretinal fluid and macular oedema has resulted in an increased incidence. Animal studies, cell biology, molecular biology, and an improved knowledge of genetics have provided a better understanding of the mechanisms behind the response. The purpose of this review is to describe the risk factors for developing corticosteroid-induced glaucoma, to discuss the underlying mechanisms and genetics of the condition and to present management options.

Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants.

To investigate mechanism(s) by which mutations in the olfactomedin domain of myocilin (MYOC), also known as the trabecular meshwork-induced glucocorticoid response (TIGR) gene, cause autosomal dominant open-angle glaucoma, the structure and properties of wild-type (WT) MYOC protein were examined, when expressed alone or simultaneously with the Q368X or K423E disease-associated polypeptides. Myocilin was analyzed in human aqueous humor and human trabecular meshwork (HTM) tissues. COS-7 and immortalized human trabecular meshwork (iHTM) cell lines were transfected with expression vectors encoding WT MYOC, mutated, and/or epitope-tagged cDNAs. MYOC proteins were characterized by double-epitope tagging procedures and/or Western blot analysis. MYOC polypeptides formed highly similar oligomers in aqueous humor, HTM tissues, transfected COS-7, and iHTM cell lines. These complexes ranged in size from 116 kDa to more than 200 kDa. The smallest complex, approximately 116 kDa, resulted from dimerization between two MYOC monomers. Expression of a 150-kDa complex was strongest in aqueous humor. Cotransfections of the WT construct with either the Q368X or K423E cDNA produced MYOC(WT)/MYOC(mutant) heterodimers and higher molecular weight hetero-oligomeric complexes. WT homo-oligomeric complexes were secreted in the extracellular media of both cell lines whereas the Q368X and K423E mutant/mutant homomultimers and heteromeric WT/mutant oligomers remained sequestered intracellularly. Formation of heteromeric WT/mutant complexes may provide a critical mechanism by which mutant myocilin polypeptides produce autosomal dominant open-angle glaucoma. The intracellular sequestration of abnormal WT/mutant complexes could lead to the malfunction of MYOC-expressing cells and to POAG potentially involving a dominant negative effect.

TIGR is upregulated in the chronic glial scar in response to central nervous system injury and inhibits neurite outgrowth

Reactive astrocytes respond to central nervous system (CNS) injury and disease by elaborating a glial scar that is inhibitory to axonal regeneration. To identify genes that may be involved in the astrocytic response to injury, we used differential display polymerase chain reaction and an in vivo model of the CNS glial scar. Expression of the trabecular meshwork inducible glucocorticoid response (TIGR) gene was increased in gliotic tissue compared with the uninjured cerebral cortex. Increased TIGR expression by reactive astrocytes was confirmed by in situ hybridization, quantitative reverse transcriptase–polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Although mutations of the TIGR gene have been implicated in glaucoma, a function for TIGR has not been reported. Since TIGR is secreted, we assessed a possible role in inhibition of neuronal regeneration with an in vitro bioassay and found that this protein is a potent inhibitor of neurite outgrowth. Thus, TIGR is a newly identified component of the CNS glial scar that is likely to contribute to neuronal regenerative failure characteristic of the mammalian CNS.

Transcription profiling in Drosophila eyes that overexpress the human glaucoma-associated trabecular meshwork-inducible glucocorticoid response protein/myocilin (TIGR/MYOC).

The availability of the human genome sequence together with sequenced genomes of several model organisms provides an unprecedented opportunity to utilize comparative genomic approaches for the discovery of genes that contribute to human disease. We have used transgenic flies to establish an experimental paradigm for the discovery of genes that might be involved in the development of glaucoma, a prevalent disease affecting a large segment of the population. Inherited mutations in the trabecular meshwork-inducible glucocorticoid response protein/myocilin (TIGR/MYOC) are associated with juvenile glaucoma and some cases of adult primary open angle glaucoma. The interrelationships between TIGR/MYOC and the development of glaucoma, however, are not understood. We show that overexpression of human TIGR/MYOC in the eyes of Drosophila melanogaster results in distortion of ommatidia accompanied by fluid discharge. High-density oligonucleotide microarrays identified altered expression of 50 transcripts in response to TIGR/MYOC overexpression, including homologs of aquaporin-4 and cytochrome-P450, previously associated with glaucoma, and several proteins of unknown function. We found that expression of Swiss Cheese, a neurodegenerative protein, increased 34-fold and that its human ortholog, neuropathy target esterase, is also upregulated in response to adenovirus-mediated overexpression of TIGR/MYOC in perfused postmortem human eyes. Our observations establish the Drosophila eye as an advantageous system for the discovery of genes that are associated with glaucoma.

A Multidomain TIGR/Olfactomedin Protein Family with Conserved Structural Similarity in the N-terminal Region and Conserved Motifs in the C-terminal Region

Based on the similarity between the TIGR (trabecular-meshwork inducible glucocorticoid response) (also known as myocilin) and olfactomedin protein families identified throughout the length of the TIGR protein, we have identified more distantly related proteins to determine the elements essential to the function/structure of the TIGR and olfactomedin proteins. Using a sequence walk method and the Shotgun program, we have identified a family including 31 olfactomedin domain-containing sequences. Multiple sequence alignments and secondary structure analyses were used to identify conserved sequence elements. Pairwise identity in the olfactomedin domain ranges from 8 to 64%, with an average pairwise identity of 24%. The N-terminal regions of the proteins fall into two subgroups, one including the TIGR and olfactomedin families and another group of apparently unrelated domains. The TIGR and olfactomedin sequences display conserved motifs including a residual leucine zipper region and maintain a similar secondary structure throughout the N-terminal region. The correlation between conserved elements and disease-associated mutations and apparent polymorphisms in human TIGR was also examined to evaluate the apparent importance of conserved residues to the function/structure of TIGR. Several residues have been identified as essential to the function and/or structure of the human TIGR protein based on their degree of conservation across the family and their implication in the pathogenesis of primary open-angle glaucoma. Additionally, we have identified a group of chitinase sequences containing several of the highly conserved motifs present in the C-terminal region of the olfactomedin domain-containing sequences.

Myocilin is associated with mitochondria in human trabecular meshwork cells

The trabecular meshwork (TM) is a specialized tissue located at the chamber angle of the eye next to the cornea. This tissue is believed to be responsible for regulation of the aqueous humor outflow and control of the intraocular pressure (IOP). Alterations in functions of the TM may lead to IOP elevation and development of glaucoma, a major cause of blindness. The myocilin gene has recently been directly linked to open-angle glaucomas. The gene product was originally identified as a protein inducible in TM cells by treatment with glucocorticoids such as dexamethasone (DEX) and termed TIGR (TM inducible-glucocorticoid response). The exact nature and function of the myocilin protein so far still remain elusive. In this study, myocilin was localized to the perinuclear region of both DEX-treated and control TM cells. Its distribution overlapped considerably with that of mitochondria. Subcellular fractionation and Western blot analyses suggested a rather extensive association of myocilin with mitochondria. The DEX-treated TM cells were found to undergo apoptosis, when exposed to anti-Fas antibody, to a significantly higher degree than the untreated control cells. It appears that the TM cell integrity remains intact after DEX treatment. However, the induced myocilin or myocilin-mitochondria association seems to render the cells more susceptible to a second stress or challenge. This vulnerability may be the basis that ultimately leads to pathological consequences.

Targeted Disruption of the Myocilin Gene (Myoc) Suggests that Human Glaucoma-Causing Mutations Are Gain of Function.

Glaucoma is a heterogeneous eye disease and a major cause of blindness worldwide. Recently, primary open angle glaucoma (POAG)-associated mutations have been found in the trabecular meshwork inducible glucocorticoid response gene (TIGR), also known as the myocilin gene (MYOC), at the GLC1A locus on chromosome 1q21-q31. These mutations occurred in a subset of patients with juvenile- and adult-onset POAG and exhibited autosomal dominant inheritance. Ocular expression and its involvement in POAG suggest that TIGR/MYOC may have a role(s) in regulating intraocular pressure (IOP). Here, we report the generation and analysis of mice heterozygous and homozygous for a targeted null mutation in Myoc. Our study shows that Myoc mutant mice are both viable and fertile. Our in vivo findings further demonstrate that Myoc is not required for normal IOP or normal ocular morphology. The lack of a discernable phenotype in both Myoc-heterozygous and Myoc-null mice suggests that haploinsufficiency is not a critical mechanism for POAG in individuals with mutations in MYOC. Instead, disease-causing mutations in humans likely act by gain of function.

Association of a single nucleotide polymorphism in the TIGR/MYOCILIN gene promoter with the severity of primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is a highly prevalent optic neuropathy and a major cause of irreversible blindness, with elevation of intraocular pressure (IOP) being a primary risk factor. The trabecular meshwork-inducible glucocorticoid response (TIGR)/MYOCILIN (MYOC) gene coding region is mutated in 3-4% of POAG patients. Here, in a retrospective study of 142 POAG patients, we evaluated the influence on glaucoma phenotype of a novel biallelic polymorphism (-1000C/G) located in the upstream region of the MYOC gene. Allele frequencies were similar among patients and controls. However, the G allele (frequency 17.6%), also designated as MYOC.mt1, was associated with an increased IOP (+4.9 mmHg, p=0.0004) and a more damaged visual field (p=0.02). Both effects were predominant in females. Moreover, whereas IOP in MYOC.mt1 noncarriers decreased very markedly to the normal range between diagnosis and inclusion in the study (p=3 x 10(-5) in both males and females), reflecting successful therapy, it decreased less noticeably in MYOC.mt1+ male patients (p=0.005) and not at all in MYOC.mt1+ female patients. MYOC.mt1 appears therefore to be an indicator of poor IOP control and greater visual field damage in diagnosed POAG patients, potentially due to a lack of response to therapeutic intervention. Its typing might help in the selection of treatment paradigms for the management of POAG patients.

Ultrastructural localization of myocilin in human trabecular meshwork cells and tissues.

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.

Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma

PURPOSE: To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC). METHODS: Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites. RESULTS: In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%). CONCLUSIONS: Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.

Regulation of TIGR/MYOC gene expression in human trabecular meshwork cells.

Glucocorticoid (GC) treatment of human trabecular meshwork (HTM) cells produces delayed, progressive cellular and extracellular protein/glycoprotein inductions with characteristics matching those for intraocular pressure elevation with corticosteroid eyedrops. The cloning of the Trabecular Meshwork Inducible Glucocorticoid Response (TIGR) gene from this system has suggested possible environmental and genetic influences in relation to glaucoma mechanisms. As reported here, the major GC-induced increase of TIGR expression in HTM cells is reduced approximately 4-fold by basic fibroblast growth factor (bFGF, 100-1000 pM), with a somewhat smaller inhibition noted with the thyroid hormone triiodothyronine (T3, 100 nM). Such endogenous 'protective' factors could help balance stimulatory effects on TIGR gene expression from 'stress' and/or mechanical perturbations in the trabecular meshwork. TIGR coding region mutations affecting the gene's olfactomedin (OLF) homology domain may also perturb biosynthetic pathways and cellular homeostatic functions. Our recent studies have shown the OLF domain corresponds to a major translocational 'pause', an area where critical processes for normal TIGR biogenesis are expected to take place. Observations that Glu323Lys (and other mutations early in the OLF domain) altered the pattern of paused protein intermediates provide possible clues to previously unexplained pathogenetic mechanisms. HTM cell transfection studies using TIGR-green fluorescent protein (GFP) fusions showed increased and altered distribution of the expressed protein with constructs missing the OLF domain, an effect also found with the Pro370 Leu mutation for early-onset glaucoma. The data suggest an activation of stress/apoptotic pathways in HTM cells as a potential mechanism for environmental/genetic interactions in glaucoma pathogenesis.

The myocilin (MYOC) gene expression in the human trabecular meshwork

Purpose. We previously reported a novel cytoskeletal protein with a myosin-like domain which is localized in the ciliary rootlet and basal body of connecting cilium of photoreceptor and hence we named it ‘myocilin’. It was soon realized that myocilin is identical to a protein called TIGR (trabecular meshwork inducible glucocorticoid response protein) which was found to be responsible for the pathogenesis of juvenile open angle glaucoma. In this study, we employed in situ RNA hybridization to examine the myocilin (MYOC)/ TIGR gene expression in the trabecular meshworks of glaucomatous and nonglaucomatous eyes. Methods. The glaucomatous specimens were obtained by trabeculectomy from the patients with primary open angle glaucoma (POAG), chronic angle closure glaucoma (CACG) and steroid glaucoma, respectively, and the nonglaucomatous specimens were obtained from a victim of traffic accident at autopsy and from a patient with maxillary sinus carcinoma at enucleation for the operation. The in situ RNA hybridization was carried out with digoxigenin-labeled sense and antisense RNA probes. Results. In all cases, hybridization signals were detected primarily in the trabecular meshwork cells and secondarily in the fibroblast-like cells of corneoscleral wall. Conclusions. Myocilin gene is expressed clearly in the trabecular meshwork cells of both glaucomatous and nonglaucomatous eyes.

Detection of a New TIGR Gene Mutation in a Japanese Family With Primary Open Angle Glaucoma

Purpose: To describe a new mutation of the trabecular meshwork-inducible glucocorticoid response protein (TIGR) gene in a Japanese patient with familial primary open angle glaucoma (POAG). Methods: Standard ocular examinations were performed on the 44-year-old patient, his sister, and mother. DNA sequencing was used to identify the mutation. We also developed a DNA diagnostic method for detecting this missense mutation by polymerase chain reaction-induced mutation restriction analysis (PCR-IMRA). Results: The patient, father, and sister had been diagnosed as having POAG. The patient and his sister had a Thr448Pro mutation (C→A transition at the nucleotide number 1419) in exon 3. This mutation has not been reported before. Conclusions: Gene analysis is promising for an early diagnosis among the family members of familial POAG patients and will contribute to early therapy before an occurrence of irreversible visual impairment.