Kunitoshi Ohara

Mutations in the TIGR gene in familial primary open-angle glaucoma in Japan.

To the Editor:As described in an editorial by Raymond (1997)xMolecular genetics of the glaucomas: mapping of the first five “GLC” loci. Raymond, V. Am J Hum Genet. 1997; 60: 272–277PubMedSee all ReferencesRaymond (1997), glaucoma is characterized by progressive excavation of the optic disk, with both loss of retinal nerve fiber and visual field defects. This disease is one of the most common causes of bilateral blindness, and it is estimated that by the year 2000 ∼66.8 million people worldwide will be affected by it (Quigley 1996xNumber of people with glaucoma worldwide. Quigley, HA. Br J Ophthalmol. 1996; 80: 389–393Crossref | PubMedSee all ReferencesQuigley 1996). Recently, the glaucoma geneGLC1A was shown to be identical to the trabecular meshwork–inducible glucocorticoid response (TIGR) gene (TIGR) (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). The TIGR gene was cloned by Polansky and colleagues (Nguyen et al. 1993xGlucocorticoid (GC) effects on HTM cells: molecular biology approaches. Nguyen, TD, Huang, W, Bloom, E, and Polansky, JR. : 331–343See all ReferencesNguyen et al. 1993; Polansky et al. 1997xCellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Polansky, JR, Fauss, DJ, Chen, P, Chen, H, Lutjen-Drecoll, E, Johnson, D, Kurtz, RM et al. Ophthalmologica. 1997; 211: 126–139Crossref | PubMedSee all ReferencesPolansky et al. 1997) and, also, was called “myocilin” (gene MYOC) when it was cloned by Kubota et al. (1997)xA novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping. Kubota, R, Noda, S, Wang, Y, Minoshima, S, Asakawa, S, Kudoh, J, Mashima, Y et al. Genomics. 1997; 41: 360–369Crossref | PubMed | Scopus (250)See all ReferencesKubota et al. (1997). Three different mutations in the gene were shown to be responsible for the development of primary open-angle glaucoma (POAG), the most common form of glaucoma (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). The prevalence of these mutations was reported to be 4.4% in familial POAG patients and 2.9% in unselected POAG patients (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). We investigated whether Japanese patients with familial POAG carry identical or other mutants on the same gene. As a result, two new mutations in the TIGR gene were found. The prevalence of mutations in the TIGR gene in Japanese patients with familial POAG was also investigated.Peripheral blood samples were collected, with informed consent, from 52 POAG patients of 50 pedigrees with a family history. The patients were diagnosed with POAG, by ocular and systemic examinations. The family history was obtained by direct interview with the patients. Subjects having at least one relative with POAG within the third degree of relationship were defined as belonging to a pedigree having familial POAG. They all had an elevated intraocular pressure (⩾22 mmHg), open-angle (Shaffer grade III or IV), visual-field loss characteristic of glaucoma, and glaucomatous optic-disk damage. Blood samples from five normal healthy volunteer were also obtained, as controls, with informed consent. Genomic DNA was purified from these blood samples by use of a QIAGEN QIAamp Blood Kit. A DNA fragment encoding a portion of the TIGR protein (amino acid residues 317–476, exon 3; GenBank accession number U85257-AF001620) was amplified, with samples of the purified genomic DNA used as templates, by PCR. The nucleotide sequences of primers used are 5′-ATACTGCCTAGGCCACTGGA (sense strand) and 5′-CATGCTGCTGTACTTATAGCGG (antisense strand). A 150-ng template was mixed with 10 μl of 10 × buffer, 8 μl of the deoxynucleotides mixture, 10 pmol of each primer, and 0.5 μl of Taq polymerase (AmpliTaq Gold; Perkin Elmer), to produce a 100−μl PCR mixture. The nucleotide sequences of both strands of the PCR products were directly determined with the terminator cycle–sequencing method, by use of fluorescent dideoxynucleotides and an automatic DNA sequencer (Applied Biosystems). Mutation was recognized by the approximately equal peak intensity of two fluorescent dyes at the mutation site. When a mutation was detected, the whole procedure of PCR and sequencing was repeated, and the existence of the mutation was confirmed.Of the 52 patients from the 50 pedigrees, 2 patients of one family (a father and a daughter [who was the proband]) carried a heterozygous C→T mutation at the second nucleotide position in the codon corresponding to the 370th amino acid residue of the TIGR protein, resulting in an amino acid change from proline to leucine (Pro370Leu) (figs. 1A1A and 22). The father was diagnosed with POAG at age 26 years old, the daughter at age 16 years. One other patient, from a different pedigree, had a heterozygous G→A mutation at the first nucleotide position in the codon corresponding to the 367th amino acid residue, resulting in an amino acid change from glycine to arginine (Gly367Arg) (fig. 1Bfig. 1B). She was diagnosed with POAG at age 45 years. Both mutations were different from those reported elsewhere (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). In the pedigree with the Pro370Leu mutation, the mother and a sister of the proband were examined and proved to have neither symptoms of glaucoma nor mutations in the TIGR gene portion examined (fig. 2fig. 2). Therefore, the mutation was inherited in an autosomal dominant manner. The patient with the Gly367Arg mutation had a family history, in that at least her two aunts and five cousins had POAG; but we could not obtain blood samples from her relatives. The prevalence of the mutations in the TIGR gene was 4.0% (2/50 families), which was comparable to that reported in a previous study (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997).Figure 1Chromatograms of nucleotide sequences from patients with mutations (A and B) and from a normal control (C). The double peak of cytosine (blue line) and thymine (red line) (A, red arrow) represents a heterozygous mutation in the codon corresponding to the 370th amino acid residue of the TIGR protein (Pro370Leu). The double peak of guanine (black line) and adenine (green line) (B, green arrow) represents a heterozygous mutation at the codon of 367th amino acid residue (Gly367Arg).View Large Image | View Hi-Res Image | Download PowerPoint SlideFigure 2Pedigree of patients with the Pro370Leu mutation. The proband is indicated by an arrow.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe present study has revealed that mutations in the TIGR gene are also responsible for familial POAG in Japan. The mutations in the third exon of the TIGR gene were found to be associated with ∼4% of Japanese familial POAG patients. The prevalence of the mutations in the gene was comparable in Japanese patients and in the population previously studied. The mutated sites, however, were different from those reported in the previous study, and no common mutations were found. Therefore, the distribution of the mutated sites in the TIGR gene in Japanese POAG may be different from those in other races. Known mutated sites in the TIGR-gene disease alleles are at the 364th, 367th, 368th, 370th, and 437th amino acid residues. The apparent focus of the reported mutations—around the 367th amino acid residue—indicates that the amino acid change around this position may play a critical role in the pathogenesis of POAG. The TIGR-protein product is overexpressed with glucocorticoid stimulation and is thought to contribute to steroid-responsive intraocular-pressure increase, by the obstruction of aqueous outflow (Nguyen et al. 1993xGlucocorticoid (GC) effects on HTM cells: molecular biology approaches. Nguyen, TD, Huang, W, Bloom, E, and Polansky, JR. : 331–343See all ReferencesNguyen et al. 1993; Polansky et al. 1997xCellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Polansky, JR, Fauss, DJ, Chen, P, Chen, H, Lutjen-Drecoll, E, Johnson, D, Kurtz, RM et al. Ophthalmologica. 1997; 211: 126–139Crossref | PubMedSee all ReferencesPolansky et al. 1997). Further investigations of the TIGR gene will reveal more information about the pathogenesis of POAG.

Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model

Ischemic retinal diseases, such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, are a major cause of blindness worldwide. Angiostatin is an internal peptide fragment of plasminogen that inhibits endothelial proliferation in vitro and tumor growth in vivo. We now demonstrate that HIV vector encoding angiostatin (HIV-angiostatin) can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Intravitreal injections of HIV-angiostatin led to stable expression of the angiostatin gene in retinal tissue. Retinal neovascularization was histologically quantitated by a masked protocol. Retinal neovascularization in the eye injected with HIV-angiostatin was reduced in 90% (9/10; P=0.025) of animals, compared with the eye injected with phosphate-buffered saline. Reduction of histologically evident neovascular nuclei per 6-μm section averaged 68%, with maximal inhibitory effects of 87%. Neovascularization was not reduced in the eyes injected with HIV vector encoding enhanced green fluorescent protein. This is the first report that HIV-angiostatin can reduce neovascular cell nuclei in a murine proliferative retinopathy model. These data suggest that the anti-angiogenic activity of angiostatin has therapeutic potential for the treatment of retinal neovascularization.

Membranous Outgrowth Suggesting Lens Epithelial Cell Proliferation in Pseudophakic Eyes

PURPOSE We sought to determine the incidence and structure of membranous outgrowth, which extends from the anterior capsular opening onto the intraocular lens surface in pseudophakic eyes. METHODS Thirty-four eyes of 31 patients with age-related cataract were prospectively studied. No patient had any abnormality other than cataract. Each patient underwent continuous circular capsulorhexis, phacoemulsification, and implantation within the capsule of a three-piece posterior chamber lens. A slit lamp and specular microscope were used to observe and photograph the intraocular lens surface and anterior capsular opening every day for the first postoperative week, and at days 14, 21, and 28. We counted the number of eyes with the membranous outgrowth and graded the outgrowth according to its shape and length at each postoperative period. RESULTS In total, 27 of 34 (79%) eyes had the membranous outgrowth from the anterior capsular opening onto the intraocular lens surface. The membrane was first observed on day 3. Three of 34 eyes had the dendritic or fan-shaped structure, which extended less than 0.5 mm from the capsular edge. The membranes were most frequently found on day 7. Twenty-five of 34 eyes had the outgrowth in various grades. After four weeks, no membranes were observed. CONCLUSIONS The time course and structure of the membranous outgrowth we observed were comparable to those of the outgrowth of lens epithelial cells under tissue culture conditions. The membranous outgrowth may be the result of a transient but active proliferation of human lens epithelial cells onto the intraocular lens surface.

Branch Retinal Vein Occlusion in a Child With Ocular Sarcoidosis

PURPOSE/METHODS A 13-year-old girl had unilateral iridocyclitis and periphlebitis. The periphlebitis exacerbated, and macular edema as well as branch retinal vein occlusion developed. The patient was treated with systemic corticosteroids. RESULTS/CONCLUSIONS The lesions responded well to systemic corticosteroids. Histologic diagnosis of sarcoidosis was obtained by transbronchial lung biopsy. Ocular lesions in this child were similar to those seen in adult sarcoidosis. Branch retinal vein occlusion may occur as a rare vascular complication of sarcoidosis.

Inhibitory Effects of Arg-Gly-Asp (RGD) Peptide on Cell Attachment and Migration in a Human Lens Epithelial Cell Line

Posterior capsule opacification (PCO) after cataract surgery is caused by growth of residual human lens epithelial (HLE) cells on the posterior capsule. We have shown that extracellular matrix (ECM) is an essential factor for HLE cell attachment and migration. The purpose of this study was to examine the inhibitory effects of Arg-Gly-Asp (RGD) peptide on cell attachment and migration in an HLE cell line. HLE cell line cells (SRA 01/04) that were obtained by transfection of large T antigen of SV40 were cultured in the absence of serum. The culture dishes were coated with type IV collagen, laminin or fibronectin, and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) RGD peptide (0.1, 0.3, 1.0, 2.0 mg/ml) was added to the medium. The number of attached cells was counted after 90 min of incubation, and the inhibitory effects of GRGDSP RGD peptide on cell attachment were calculated. Cell attachment on the fibronectin-coated dishes was inhibited by GRGDSP RGD peptide at concentrations higher than 0.3 mg/ml; the inhibitory rate was 80% at a concentration of 2.0 mg/ml. The inhibition of cell attachment by GRGDSP RGD peptide on laminin-coated dishes appeared only at a concentration of 2.0 mg/ml, whereas no effects were observed on the type IV collagen-coated dishes. The inhibitory effects of GRGDSP RGD peptide on cell migration were measured in medium containing 2.0 mg/ml of GRGDSP RGD peptide after 1, 3, 5 and 7 days of culture. Cell migration was inhibited by GRGDSP RGD peptide from 1 day of culture on the fibronectin-coated dishes and from 5 days of culture on the laminin-coated dishes, whereas no effects were observed on the type IV collagen-coated dishes. GRGDSP RGD peptide inhibited cell attachment and migration on laminin and fibronectin that have RGD sequences. These data suggested that RGD peptide may have the potential to prevent PCO.

Glucose Transporter 1 Expression in Corneal Wound Repair under High Serum Glucose Level

PURPOSE To determine glucose transporter (GLUT) 1 mRNA and protein expression during corneal epithelial wound healing in diabetic rat. METHODS Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. At 10 days after injection, unilateral 3-mm epithelial debridement was carried out in the central cornea. At 2, 4, 6, and 24 hours after wounding, whole corneal epithelium was collected and GLUT1 protein and mRNA levels were determined by Western blotting and reverse transcription-polymerase chain reaction, respectively. Sugar content in collected samples was measured by the Anthrone reaction. Normal rats were used as controls. RESULTS Glucose transporter 1 protein and mRNA levels in unwounded cornea were similarly low in the diabetic and control groups. Healing of corneal wounds was slower in diabetic rats than in controls. After wounding, GLUT1 mRNA and protein expression in both groups were similarly enhanced compared to unwounded epithelium. Sugar content at all time points did not show significant alteration in any group, although in diabetic rats it was significantly higher than in controls throughout the time course. CONCLUSION Glucose transporter 1 expression in diabetic rat cornea showed little difference from that in normal rat cornea, suggesting minimal influence of GLUT1 on the delayed healing of diabetic corneal wounds.

Anterior chamber irrigation with an ozonated solution as prophylaxis against infectious endophthalmitis

Purpose: To assess the validity of anterior chamber irrigation with an ozonated solution as prophylaxis against endophthalmitis. Setting: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan. Methods: Viability of human corneal endothelium in culture was assessed by the WST‐8 assay, lactate dehydrogenase (LDH) release assay, and trypan blue exclusion assay after exposure to a 4 to 40 parts per million (ppm) solution for 10 to 60 seconds. The in vivo effect was observed 1 week after irrigation of a 4 ppm solution in the rabbit anterior chamber by trypan blue exclusion assay. Bactericidal efficacy of the anterior chamber irrigation with the 4 ppm solution was examined by bacterial colony count of the aqueous humor following methicillin‐resistant Staphylococcus aureus (MRSA) contaminated intraocular lens implantation in the porcine eye. Results: The WST‐8 assay revealed no significant reduction of viability with 10‐second exposure to a 4 ppm solution. Lactate dehydrogenase release and trypan blue exclusion assays similarly demonstrated little damage after 60‐second exposure to a 4 ppm solution. In the rabbit cornea 1 week after treatment, damage caused by 30‐second exposure to a 4 ppm solution was not significant. The MRSA colony count documented almost complete bactericidal action with 5‐second exposure to the 4 ppm solution when no ophthalmic viscosurgical device existed in the anterior chamber. Conclusion: Limited damage to the corneal endothelium after 10‐second exposure and potent bactericidal action with 5‐second exposure suggests the validity of anterior chamber irrigation with a 4 ppm ozonated solution as prophylaxis against endophthalmitis.

Age-related changes of human lens epithelial cells in vivo.

We examined the density and morphology of lens epithelial cells (LECs) in vivo in a group of normal volunteers and cataract patients by using a newly developed noncontact specular microscope. There was a statistically significant decrease in the cell density of LECs in a group of cataract patients over the age of 80 years. The coefficient of variation of the cell area and the number of large black spots that were observed in the enhanced specular images were not related to aging or cataract formation. Our data indicate that the cell density of LECs decreases after reaching the age of 80, but cataract formation does not affect the cell density or the coefficient of variation of the cell area until the age of 80.

Role of positioning holes in intraocular lens glare

ABSTRACT We performed laboratory studies to determine the role of positioning holes in posterior chamber intraocular lens glare. Several areas of the lens optic were illuminated independently, and the scattering light off the lens was photographed. A conventional lathe‐cut lens with four positioning holes on the optic produced the most intense light scattering. Scattering off nonilluminated areas was apparent, and it was suggested that the presence of positioning holes on the optic might contribute to the scattering. Optically complex areas including a hole, optic edge, a loop insertion, and optic‐haptic junction produced the scattering in all lenses of various designs. An internal reflex inside the optic may occur, and complex or irregular structures in the optic may be a source of light scattering and some intraocular lens glare.

Finishing of modern posterior chamber intraocular lenses

ABSTRACT We performed specular microscopy to study the surface finishes of modern posterior chamber intraocular lenses from 12 manufacturers. Specular microscopy detected surface contamination, severe polishing marks, subtle irregularities of the lens surface, roughly finished optic edges, and irregular edges of positioning holes in many lenses. The results indicated that the finishes on modern intraocular lenses from several manufacturers were not satisfactory, and surgeons should reconsider the quality of intraocular lenses.