Kazuhide Kawase

A spectrum of FOXC1 mutations suggests gene dosage as a mechanism for developmental defects of the anterior chamber of the eye

Mutations in the forkhead transcription-factor gene (FOXC1), have been shown to cause defects of the anterior chamber of the eye that are associated with developmental forms of glaucoma. Discovery of these mutations was greatly facilitated by the cloning and characterization of the 6p25 breakpoint in a patient with both congenital glaucoma and a balanced-translocation event involving chromosomes 6 and 13. Here we describe the identification of novel mutations in the FOXC1 gene in patients with anterior-chamber defects of the eye. We have detected nine new mutations (eight of which are novel) in the FOXC1 gene in patients with anterior-chamber eye defects. Of these mutations, five frameshift mutations predict loss of the forkhead domain, as a result of premature termination of translation. Of particular interest is the fact that two families have a duplication of 6p25, involving the FOXC1 gene. These data suggest that both FOXC1 haploinsufficiency and increased gene dosage can cause anterior-chamber defects of the eye.

Evaluation of optineurin sequence variations in 1,048 patients with open-angle glaucoma

PURPOSE To investigate the association of sequence variations in the optineurin (OPTN) gene in patients with open-angle glaucoma. DESIGN Prospective case control study. METHODS The OPTN gene was screened for sequence variations using a combination of single-strand conformational polymorphism analysis and automated DNA sequencing. A total of 1,299 subjects (1048 glaucoma patients and 251 controls) were screened for variations in the four portions of the gene that had been previously associated with glaucoma. A subset of these subjects (376 patients and 176 controls) was screened for variations in the entire coding sequence. Twenty-four percent of the patients and 35% of the controls were Japanese, whereas the remainder were predominantly Caucasian. Allele frequencies were compared with the Fisher exact test. RESULTS The OPTN sequence variations were not significantly associated with any form of high-tension open-angle glaucoma. One proband with familial normal-tension glaucoma was found to harbor the previously reported Glu50Lys variation. Another previously reported change, Met98Lys, was associated with normal-tension glaucoma in Japanese but not in Caucasian patients. CONCLUSIONS This study provides some additional evidence for the association of the Glu50Lys OPTN sequence variation with familial normal tension glaucoma. However, because familial normal-tension glaucoma is so rare, this change seems to be responsible for less than 0.1% of all open-angle glaucoma. The Arg545Gln variation is likely to be a nondisease-causing polymorphism. The Met98Lys change may be associated with a fraction of normal-tension glaucoma in patients of Japanese ethnicity.

Low-dose and High-dose Mitomycin Trabeculectomy as an Initial Surgery in Primary Open-angle Glaucoma

PURPOSE The purpose of the study is to determine the optimum regimen of intraoperative administration of mitomycin as an adjunct to trabeculectomy. METHODS Of 11 patients with primary open-angle glaucoma, 22 eyes that had not undergone any surgical intervention were included. In each patient, one eye was randomly allocated to a mitomycin 0.2-mg group and the fellow eye to a mitomycin 0.02-mg group. Mitomycin was applied for 5 minutes only once during trabeculectomy. The follow-up period was 6 to 17 months. RESULTS Eleven (100%) eyes in the 0.2-mg group and 7 (63.6%) in the 0.02-mg group achieved successful control of intraocular pressure with or without topical antiglaucoma medication. Transient hypotony maculopathy (18%) and cataract progression (18%) were noted in the 0.2-mg group exclusively. The incidence of other complications was similar between the two groups. CONCLUSION These data suggest that the most appropriate dose of mitomycin for primary surgery seems to be in between the two doses tested in the current study.

Overexpression of Optineurin E50K Disrupts Rab8 Interaction and Leads to a Progressive Retinal Degeneration in Mice

Glaucoma is one of the leading causes of bilateral blindness affecting nearly 8 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) and is often associated with elevated intraocular pressure (IOP). However, patients with normal tension glaucoma (NTG), a subtype of primary open-angle glaucoma (POAG), develop the disease without IOP elevation. The molecular pathways leading to the pathology of NTG and POAG are still unclear. Here, we describe the phenotypic characteristics of transgenic mice overexpressing wild-type (Wt) or mutated optineurin (Optn). Mutations E50K, H486R and Optn with a deletion of the first (amino acids 153–174) or second (amino acids 426–461) leucine zipper were used for overexpression. After 16 months, histological abnormalities were exclusively observed in the retina of E50K mutant mice with loss of RGCs and connecting synapses in the peripheral retina leading to a thinning of the nerve fiber layer at the optic nerve head at normal IOP. E50K mice also showed massive apoptosis and degeneration of entire retina, leading to approximately a 28% reduction of the retina thickness. At the molecular level, introduction of the E50K mutation disrupts the interaction between Optn and Rab8 GTPase, a protein involved in the regulation of vesicle transport from Golgi to plasma membrane. Wt Optn and an active GTP-bound form of Rab8 complex were localized at the Golgi complex. These data suggest that alternation of the Optn sequence can initiate significant retinal degeneration in mice.

Mitomycin Concentration in Rabbit and Human Ocular Tissues after Topical Administration

The authors measured mitomycin C (MMC) concentrations in ocular tissues with high-performance liquid chromatography. Mitomycin C concentration after a single subconjunctival injection of the drug in rabbit eyes showed a rapid decrease with a half-life of 0.18 to 0.30 hours for the conjunctiva and 0.20 to 0.45 hours for the sclera at the injection site. Irrigating the ocular surface with 200 ml of saline after MMC application reduced the initial drug concentration to one fifth in the sclera and to one fifteenth in the conjunctiva but did not change the half-life. The MMC concentration in human trabeculectomy specimens obtained immediately after MMC application (0.2 mg/0.5 ml) and irrigation was 5.4 to 12.0 micrograms/g with a mean of 8.4 micrograms/g, a level similar to that in the rabbit sclera immediately after the irrigation after administration of the same MMC dose. These results indicate that MMC disappears rapidly from the ocular tissues and that irrigating the tissues significantly reduces the tissue concentration of MMC.

Ocular and systemic factors related to intraocular pressure in Japanese adults: the Tajimi study

Background: As intraocular pressure (IOP) and age are consistent risk factors of glaucoma, it is of special interest to know the association between IOP and possibly relating factors including age in Japan where a high prevalence of normal-tension glaucoma has been reported. The aim of this report was to evaluate the distribution of and factors related to applanation IOP in a population-based study in Japan. Methods: A randomly sampled group of 3021 residents (response rate 78.1%) of Tajimi City, aged 40 years or older, underwent screening examinations including measurements of IOP with Goldmann applanation tonometry and central corneal thickness. Results: Among right eyes without glaucoma, suspected glaucoma or other disorders which could affect correct IOP measurements, IOP averaged 14.6 (SD 2.7) and 14.5 (2.5) mm Hg in men and women, respectively, with no significant intergender difference (p = 0.342). Multiple regression analyses revealed that age was significantly negatively correlated with IOP (non-standardised beta (B) = −0.020/year, p = 0.0001). Higher body mass index (B = 0.14/BMI, p<0.0001), higher mean blood pressure (B = 0.022/mm Hg, p<0.0001), history of diabetes (p = 0.0019), thicker cornea (B = 0.014/μm, p<0.0001), higher myopia (B = 0.055/dioptres, p = 0.0043) and steeper corneal curvature (B = −0.72/mm, p = 0.0002) were also significantly correlated with higher IOP. Conclusions: In an adult Japanese population, applanation IOP averaged 14.5 mm Hg and was negatively correlated with age after adjusting for other related factors. A positive correlation between IOP and myopia was found.

Serum autoantibodies to heat shock proteins in glaucoma patients from Japan and the United States.

OBJECTIVE To study serum titers of antibodies against heat shock proteins in glaucoma patients from two different ethnic populations and to examine the relationship between serum antibody titers and glaucomatous damage. STUDY DESIGN Comparative, cross-sectional study. PARTICIPANTS Twenty-seven age-matched patients with primary open-angle glaucoma, 28 patients with normal pressure glaucoma, and a control group of 26 healthy subjects from Japan; and 21 age-matched patients with primary open-angle glaucoma, 40 patients with normal pressure glaucoma, and a control group of 20 healthy subjects from the United States. MAIN OUTCOME MEASURES Measurement of serum antibody titers and examination of optic disc and visual field parameters. METHODS Serum immunoreactivity against heat shock proteins, including hsp27, alphaB-crystallin, and human and bacterial hsp60, was studied by enzyme-linked immunosorbent assay (ELISA). The relationship of serum antibody titers to glaucoma parameters, including mean deviation, neural rim area-to-disc area ratio, and peripapillary atrophy area-to-disc area ratio was examined. RESULTS The serum ELISA titers of antibodies, including hsp27, alphaB-crystallin, human hsp60, and bacterial hsp60 antibodies, were higher in glaucoma patients compared with control subjects in both the Japanese and American groups (P: < 0.05). There was no association between the serum antibody titers and optic disc parameters in either group from Japan or the United States (P: > 0.05). CONCLUSIONS These findings suggest that increased titers of circulating antibodies against heat shock proteins occur in both Japanese and American patients with glaucoma. The lack of a relationship between the level of serum antibody titers and the degree of glaucomatous damage in either ethnic group suggests that increased antibodies to heat shock proteins do not occur as an epiphenomenon of the glaucomatous neurodegeneration process.

Enhanced optineurin E50K–TBK1 interaction evokes protein insolubility and initiates familial primary open-angle glaucoma

Glaucoma is the leading cause for blindness affecting 60 million people worldwide. The optineurin (OPTN) E50K mutation was first identified in familial primary open-angle glaucoma (POAG), the onset of which is not associated with intraocular pressure (IOP) elevation, and is classified as normal-tension glaucoma (NTG). Optineurin (OPTN) is a multifunctional protein and its mutations are associated with neurodegenerative diseases such as POAG and amyotrophic lateral sclerosis (ALS). We have previously described an E50K mutation-carrying transgenic (E50K-tg) mouse that exhibited glaucomatous phenotypes of decreased retinal ganglion cells (RGCs) and surrounding cell death at normal IOP. Further phenotypic analysis of these mice revealed persistent reactive gliosis and E50K mutant protein deposits in the outer plexiform layer (OPL). Over-expression of E50K in HEK293 cells indicated accumulation of insoluble OPTN in the endoplasmic reticulum (ER). This phenomenon was consistent with the results seen in neurons derived from induced pluripotent stem cells (iPSCs) from E50K mutation-carrying NTG patients. The E50K mutant strongly interacted with TANK-binding kinase 1 (TBK1), which prohibited the proper oligomerization and solubility of OPTN, both of which are important for OPTN intracellular transition. Treatment with a TBK1 inhibitor, BX795, abrogated the aberrant insolubility of the E50K mutant. Here, we delineated the intracellular dynamics of the endogenous E50K mutant protein for the first time and demonstrated how this mutation causes OPTN insolubility, in association with TBK1, to evoke POAG.

Expression of the glaucoma gene myocilin (MYOC) in the human optic nerve head

Glaucoma is a leading cause of blindness in the world, and several glaucoma genes have been recently identified. The glaucoma gene myocilin (MYOC) (also known as GLC1A and TIGR) is responsible for juvenile open‐angle glaucoma and a subset of adult‐onset primary open‐angle glaucoma. Previous studies have shown that MYOC is expressed in the trabecular meshwork, an ocular tissue involved in the development of ocular hypertension, which is often associated with the development of glaucoma. Because all forms of glaucoma involve pathogenic changes to the optic nerve head (ONH), including cupping, excavation, and collapse of the connective support tissue, we sought to determine whether MYOC mRNA and protein are expressed in cells of the human ONH. Reverse transcriptase PCR, in situ hybridization, immunofluorescent microscopy, and immunoblotting of polyacrylamide gels were used to demonstrate that myocilin is expressed in ONH tissue and cultured ONH cells. A secondary purpose was to determine whether MYOC sequence variations are associated with a clinically significant fraction of normal tension glaucoma (NTG), a form of glaucoma in which intraocular pressure is not appreciably elevated. Nonconservative MYOC sequence variations were observed in only 6 of 307 patients with NTG and 4 of 193 control individuals (P=1.0, Fishers exact test). Thus, although MYOC is expressed in a pattern that is consistent with its involvement in NTG, variations in the MYOC coding sequence are not commonly or significantly associated with this disease.

Confirmation of TBK1 duplication in normal tension glaucoma

Recently, we discovered chromosome 12q14 duplications in patients with normal tension glaucoma (NTG) (Fingert et al., 2011). Three different but overlapping chromosome 12q14 duplications that all spanned the TBK1, XPOT, and RASSF3 genes were identified. The duplication of TBK1 was judged to be the most likely cause of NTG in these patients because: 1) TBK1 associates with the product of another NTG gene, optineurin (Morton et al., 2008); 2) Duplication of TBK1 leads to increased transcription of TBK1 (Fingert et al., 2011); and 3) TBK1 is specifically expressed in cells affected by glaucoma pathophysiology (retinal ganglion cells and their axons) (Fingert et al., 2011). Finally, the population-based segment of our previous study identified chromosome 12q14 duplications that span TBK1 in 2 (1.3%) of 152 NTG patients from Iowa (Fingert et al., 2011) suggesting that copy number variations of TBK1 may be responsible for a fraction of all NTG cases. Here we report the first replication study to investigate the role of copy number variations (CNVs) of TBK1 in NTG pathogenesis. The study was approved by local Institutional Review Boards and informed consent was obtained from all study participants. We tested NTG patients and ethnically matched normal control subjects from Japan. NTG patients from New York and North Carolina were also studied. Subjects were tested for duplication of TBK1 using a quantitative PCR assay and microarray analysis of SNPs at chromosome 12q14. Patients were examined by fellowship-trained glaucoma specialists and received complete ophthalmic examinations including gonioscopy, standardized computerized Humphrey (Zeiss, San Leonardo, Ca.) SITA visual field testing, and stereoscopic optic nerve examination. Subjects were diagnosed with open-angle glaucoma when optic nerve damage and corresponding visual field defects were detected in at least one eye as previously described (Alward et al., 1998) and NTG was diagnosed when the maximum untreated IOP was ≤21 mmHg in both eyes. The study dataset consisted of 252 NTG patients and 202 controls from Japan, 29 NTG patients from North Carolina, and 28 NTG patients from New York. DNA samples from each subject were tested for duplication of the TBK1 gene using a quantitative PCR assay (TaqMan Copy Number Assay, Applied BioSystems, Carlsbad, CA) as previously described (Fingert et al., 2011). Briefly, a segment of the TBK1 gene was PCR amplified in triplicate for each DNA sample, as was a control amplicon from a different gene on a different chromosome. Experiments were conducted using a 7900HT PCR machine and data was analyzed to detect CNVs using CopyCaller software (Applied BioSystems, Carlsbad, CA) with default settings. Subjects that exhibited a CNV that spanned TBK1 were retested to confirm its presence. One of 252 (0.40%) unrelated Japanese NTG subjects (patient GGJ-414) was found to carry a TBK1 duplication using quantitative PCR. The duplication in patient GGJ-414 was confirmed using microarray analysis (Affymetrix microarrays, Santa, Clara, CA) as previously described (Fingert et al., 2011). No duplications were detected in the Japanese control subjects or in any of the patients from North Carolina or New York. Further analysis of the genetic data (Partek software package, St. Louis, MO) showed that patient GGJ-414 carried a chromosome 12q14 duplication that extended from 64,802,839 to 65,098,981 bps (human genome build hg19). This 300 kbp duplication encompassed the TBK1 gene as well as XPOT and RASSF3 and is similar in extent to the duplication previously reported in Caucasian NTG subject GGA-1159-1 (Fingert et al., 2011). Patient GGJ-414 (subject II-4 in Figure 1) is a Japanese woman who was diagnosed with NTG at age 42 and has a strong family of disease. Representative disc photos from family members are shown in Figure 2. Other members of this family were tested for the presence of the chromosome 12q14 duplication using the quantitative PCR assay described above. Patient GGJ-414 (Figure 1, subject II-4) has a sister with NTG (Figure 1, subject II-3) and two sons that were diagnosed as NTG suspects, based on large cup-to-disc ratios (Figure 1, subjects III-2 and III-3), also carried the TBK1 duplication. The maximum recorded untreated intraocular pressure for patient GGJ-414 was 18 mm Hg OD and 17 mm Hg OS and central corneal thickness was 521 microns OD and 528 microns OS. One family member (subject II-1, Figure 1) that carried the TBK1 duplication had not been diagnosed with glaucoma when he was last examined at 45 years of age. At this examination, subject II-1 had large cup to disc ratios (0.75 OD and 0.70 OS) and had abnormal glaucoma hemifield test OD. Subject II-1 has not been examined in nine years and lacks further information on glaucoma status. Figure 1 Japanese NTG pedigree Figure 2 Disc Photos Over the last 15 years, family-based linkage studies have identified several genes (i.e. myocilin and optineurin) that are capable of causing POAG with little influence from other genes or environmental factors. These single gene or Mendelian forms of glaucoma represent approximately 5% of POAG cases. The vast majority of glaucoma-causing mutations in myocilin and optineurin are missense and nonsense sequence changes. More recently, we reported that duplication of a segment of chromosome 12q14 that encompasses the TBK1 gene is associated with NTG. Our study first detected a chromosome 12q14 duplication in a large African American pedigree with NTG and later identified two different but overlapping chromosome 12q14 duplications in 2 (1.3%) of 152 unrelated Caucasian NTG patients (Fingert et al., 2011). This is the first confirmation that chromosome 12q14 duplications which encompass the TBK1 gene are associated with NTG. Although it remains possible that other neighboring genes in and around the chromosome 12q14 duplication have a role in the pathogenesis of NTG, there is strong data to suggest that it is duplication of TBK1 that leads to this form of NTG (Fingert et al., 2011). The discovery and confirmation that another gene, TBK1, leads to glaucoma potentially represents a significant advance in glaucoma genetics. TBK1 encodes a kinase that influences gene expression in the NF-κB signaling pathway. Two other NTG genes, optineurin (Rezaie et al., 2002) and toll-like receptor 4 (Zareparsi et al., 2005), also participate in NF-κB signaling. Together these data strongly suggest that this biological pathway has an important role in the pathogenesis of NTG in at least a subset of patients, perhaps via its influence on key cellular processes including apoptosis. Furthermore, duplication of TBK1 is associated with 0.40% to 1.3% of NTG in Caucasians, African Americans (Fingert et al., 2011), and Japanese, suggesting that this defect may have a role in NTG pathogenesis in many ethnicities.