Open Access
Glaucoma  |   May 2017
A Histologic Categorization of Aqueous Outflow Routes in Familial Open-Angle Glaucoma and Associations With Mutations in the MYOC Gene in Japanese Patients
Author Affiliations & Notes
  • Teruhiko Hamanaka
    Department of Ophthalmology, Japanese Red Cross Medical Center, Shibuyaku, Tokyo, Japan
  • Masae Kimura
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Tetsuro Sakurai
    Center of General Education, Tokyo University of Science, Suwa, Chino-shi, Nagano, Japan
  • Nobuo Ishida
    Ishida Eye Clinic, Joetsu-shi, Niigata, Japan
  • Jun Yasuda
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Masao Nagasaki
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Naoki Nariai
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Atsushi Endo
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Kei Homma
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Fumiki Katsuoka
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Yoichi Matsubara
    Research Institute, National Center for Child Health and Development, Tokyo, Japan
  • Masayuki Yamamoto
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Nobuo Fuse
    Department of Integrative Genomics, Tohoku Medical Megabank Organization, Aoba-ku, Sendai, Miyagi, Japan
  • Correspondence: Nobuo Fuse, Department of Integrative Genomics, Tohoku Medical Megabank Organization, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8573, Japan; fusen@megabank.tohoku.ac.jp
Investigative Ophthalmology & Visual Science May 2017, Vol.58, 2818-2831. doi:10.1167/iovs.16-20646
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      Teruhiko Hamanaka, Masae Kimura, Tetsuro Sakurai, Nobuo Ishida, Jun Yasuda, Masao Nagasaki, Naoki Nariai, Atsushi Endo, Kei Homma, Fumiki Katsuoka, Yoichi Matsubara, Masayuki Yamamoto, Nobuo Fuse; A Histologic Categorization of Aqueous Outflow Routes in Familial Open-Angle Glaucoma and Associations With Mutations in the MYOC Gene in Japanese Patients. Invest. Ophthalmol. Vis. Sci. 2017;58(5):2818-2831. doi: 10.1167/iovs.16-20646.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: This study evaluated specific relationships between pathogenic mechanisms and genetic polymorphisms in primary open-angle glaucoma (POAG). We analyzed the morphologies of trabeculectomy specimens obtained from patients with familial POAG.

Methods: We used light microscopy and transmission electron microscopy to examine specimens obtained from 17 eyes of 14 patients with familial POAG. We also conducted exome analyses of two families and used targeted Sanger sequencing to analyze samples obtained from the remaining patients.

Results: The POAG cases examined in this study were divided into two groups based on morphologic characteristics. Group A eyes (7 eyes from 5 patients) had an abnormally thick trabecular meshwork (TM), whereas group B eyes (10 eyes from 9 patients) had a TM of normal thickness. The characteristics of the outflow routes in group A eyes were remarkable and included apoptotic TM cells, abnormally thickened TM basement membranes, fused TM beams, and occluded Schlemm's canals. All group A patients harbored mutations (F369L, P370L, T377M, and T448P) in the myocilin (MYOC) gene that were not found in group B patients.

Conclusions: Although age matching of morphologic changes in the outflow routes was impossible due to the small sample size, this study suggests that abnormal TM cells may cause sequential damage in abnormally thickened TM basement membranes, TM cell apoptosis, TM beam fusion, and the occlusion of Schlemm's canals. The four detected MYOC mutations appeared to be associated with morphologic changes in the TM and the underlying pathogenesis of a subtype of familial POAG.

Several risk factors for glaucoma progression have been identified, but the precise mechanisms that underlie progressive neurodegenerative damage in the axons of the optic nerve have not yet been fully defined. It is expected that identifying associations between genetic variations and glaucoma susceptibility will contribute to establishing personalized health care options. Indeed, there has been such progress in a related field. Rare mutations in the BRCA gene are used to more accurately predict breast cancer risk in some patients,1 demonstrating that investigations of relationships between morphologic aspects and genetic data provide valuable information that can be used in diagnostic and therapeutic strategies aimed at personalizing medicine and health care. 
Primary open-angle glaucoma (POAG) is a prevalent eye disorder. Approximately 0.3% of the adult population have this disorder,2 and POAG is the most frequent cause of acquired blindness in Japan.3 Clinically, POAG is characterized by higher IOP (21 mm Hg or higher), normal anterior chamber angles (open angle), and progressive defects in the visual field. Reducing IOP remains the only proven strategy for delaying the progression of the disease.46 Medications and surgical procedures aimed at reducing IOP are therefore the major treatments for POAG. 
POAG seems to be a heterogeneous disorder attributable to interactions among multiple genes and environmental factors. Morphologic studies have revealed that there are specific abnormalities in the trabecular meshwork (TM)710 and Schlemm's canal (SC) in POAG eyes.11 Additionally, it has been noted that a subset of POAG patients show a familial predisposition.12 In fact, the Human Genome Organisation lists 17 chromosomal loci as responsible for the development of POAG (http://www.genenames.org/index.html, in the public domain). Among these 17 loci, myocilin (MYOC, GLC1A),13 optineurin (GLC1E),14 and TBK1 (GLC1P)15 have been identified as loci with germline mutations in POAG patients. However, whether there are correlations between specific genotypes and histopathologies in the outflow pathways of POAG patients is not currently known. To the best of our knowledge, only one collaborative study has been performed using a genetic and clinicopathologic approach to characterize outflow routes,16 and it showed that specific CYP1B1 mutations may be linked to phenotypes associated with severe or moderate angle abnormalities. 
We divided the cohort into familial and nonfamilial POAG patients (including juvenile open-angle glaucoma [JOAG]) and investigated morphologic abnormalities in outflow routes by analyzing trabeculectomy specimens that were obtained at the Japanese Red Cross Medical Center (Shibuyaku, Tokyo, Japan). We found that there were characteristic differences between these two groups.17 Specifically, we discovered that in some familial POAG (F-POAG) patients, the TM cells disappeared and the TM beams became abnormally thick. It is strongly speculated that these abnormalities may constitute a special type of POAG and may have a common gene mutation. Therefore, in this study, we extended our methods to investigate the histopathology of the TM in F-POAG patients in conjunction with gene mutation analysis. To explore the mechanism underlying the increased IOP observed in this specific type of POAG patient, we also used light and transmission electron microscopy to perform detailed histopathological investigations of the TM and SC. Based on the results of these experiments, we classified the F-POAG subjects into group A patients, with abnormally thick TM beams, and group B patients, with TM beams with a normal thickness. We also used exome analysis to search for common genetic variations in a subset of group A patients, and we identified mutations in the MYOC gene that seemed to be tightly associated with the morphologic changes we observed in the TM and the pathogenesis underlying this subtype of F-POAG. 
Materials and Methods
Patients and Clinical Parameters
This study was approved by the Institutional Review Boards of Tohoku University and the Japanese Red Cross Medical Center. Routine ophthalmologic examinations were performed in all patients. Clinical parameters and spherical equivalence, cup-to-disc (C:D) ratio and visual field examinations were performed and evaluated by Aulhorn-Greve classification. Maximum preoperative IOP and records related to previous surgeries were obtained from patient records (Tables 1, 2). Gonioscopic examinations were performed in all subjects. All patients were provided with sufficient explanation of the purpose of this study, including genome analysis, and informed consent was obtained. POAG was defined as a patient with glaucoma indicated by an open, normal appearing anterior chamber angle with cupping of the optic nerve and elevated IOP of more than 21 mm Hg without other underlying disease. We restricted our analysis to select the subset of POAG with a maximum IOP greater than 21 mm Hg. Patients with glaucoma at a primary angle closure or secondary glaucoma (e.g., exfoliation, trauma, uveitis, or steroid use) were excluded from the study. None of the patients was treated with laser trabeculoplasty. As summarized in Figure 1, the patients in group A (7 eyes of 5 patients), which included those with abnormally thick TM beams, and group B (10 eyes of 9 patients), which had normal TM beams, were selected from the Japanese Red Cross Medical Center trabeculectomy library and serially asked to undergo an examination for genetic screening as part of the routine postoperation follow-up. The 5 group A patients were from 4 families, with 2 patients from the same family, because there were few patients who typically exhibited abnormally thick TM beams. Then, we used light and transmission electron microscopy to examine the specimens obtained from the 17 eyes of the 14 F-POAG patients who belonged to 13 families. Definition of F-POAG was a pedigree in which POAG was diagnosed in at least one pair of first-degree relatives (i.e., sibling-sibling or parent-child). 
Table 1
 
Clinical Data of Trabeculectomy Patients With or Without Abnormally Thick TM as Assessed by Microscopy Inspection
Table 1
 
Clinical Data of Trabeculectomy Patients With or Without Abnormally Thick TM as Assessed by Microscopy Inspection
Table 2
 
Characteristics of POAG Cases in DNA Analyses
Table 2
 
Characteristics of POAG Cases in DNA Analyses
Figure 1
 
Experimental design. Trabeculectomy specimens were obtained from 17 eyes of 14 POAG patients selected from the Japanese Red Cross Medical Center trabeculectomy library. Samples were divided into two groups based on morphologic characteristics, and molecular genetic analyses were performed. Then, detailed histopathological examinations were carried out, and the relationships between genotype and phenotype were analyzed.
Figure 1
 
Experimental design. Trabeculectomy specimens were obtained from 17 eyes of 14 POAG patients selected from the Japanese Red Cross Medical Center trabeculectomy library. Samples were divided into two groups based on morphologic characteristics, and molecular genetic analyses were performed. Then, detailed histopathological examinations were carried out, and the relationships between genotype and phenotype were analyzed.
Histologic Parameters
Using the above-described criteria, 507 trabeculectomy specimens from POAG patients were selected from the Japanese Red Cross Medical Center trabeculectomy library. The specimens were obtained from September 1994 to January 2015 for this study. During the trabeculectomy surgery, a square-shaped flap (5 × 5 mm) was made parallel to the limbus. After the flap was raised, a rectangle cut of the deep sclera (1.5 × 3.5 mm), which was adjusted to include the gray zone at the center and to be parallel to the limbus, was made using a razor blade. The succeeding standardized methods of the trabeculectomy procedure were conducted according to previously described methods.18 The trabeculectomy samples in the library were fixed according to standard procedures, using a mixture of 2.5% or 5% formalin and 1% glutaraldehyde at the time the trabeculectomy was performed. The fixed specimens were divided into 4 to 5 pieces. During the dissection, the trabeculectomy specimens were cut by placing the uveal side up to avoid mechanical compression to the specimens and then embedded in paraffin for routine hematoxylin-eosin (H&E) staining and light microscopy. In cases for which further investigation was warranted, transmission electron microscopy was performed using specimens that had been embedded in Epon (Hexion, Edmonton, Alberta, Canada). To investigate the function of endothelia in the SC and TM, we performed immunohistochemical staining for thrombomodulin, CD34, and D240 (podoplanin) in all eyes of groups A and B according to previously described methods.17,19 Data for SC length (anterior-to-posterior distance), percentage of thrombomodulin-negative area (PTNA), the SC endothelial cell density (SC-ECD) and the number of Sondermann's canals were determined from light microscopy images of thrombomodulin-stained tissues (Table 3). The nuclei of the corneoscleral and uveal meshwork were counted in all eyes of groups A and B, using H&E-stained images. Trabecular meshwork beam thickness was also measured by dividing the uveal meshwork and corneoscleral meshwork near the center of SC or supposed area of SC by using the H&E-stained images. These parameters were independently measured by two ophthalmologists (TH, NF) who were blinded to clinical data when assessing TM thickness, and the values from 2 or 3 blocks from the same individuals were averaged. Repeatability was evaluated by interobserver comparison, and the difference was within 10%. 
Table 3
 
Histologic Data and Parameters of Trabeculectomy Specimens
Table 3
 
Histologic Data and Parameters of Trabeculectomy Specimens
Molecular Genetic Analysis
Genomic DNA was extracted from peripheral blood leukocytes and purified using a QIAamp blood kit (Qiagen, Hilden, Germany). For families A and C, we performed whole- exome sequencing to identify mutations that cosegregated with POAG or associated phenotypes. Genomic DNA libraries were prepared, and exon capture was performed using SureSelect human All Exon kit version 5 (Agilent Technologies, Santa Clara, CA, USA). The exon libraries were sequenced using a HiSeq2500 platform according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Paired 101-base pair reads were aligned to the reference human genome (hGRC37, hg19) using alignment software (version 0.7.5a-r405; BWA-MEM, http://bio-bwa.sourceforge.net, in the public domain). Reads that were potential duplicates based on polymerase chain reaction (PCR) amplifications were removed by using Picard software (http://picard.sourceforge.net/, in the public domain). Single-nucleotide variants (SNVs) and insertions-deletions (indels) were identified using software (version 2.5-2; Genome Analysis Toolkit with UnifiedGenotyper, http://www.broadinstitute.org/gatk, in the public domain). Single-nucleotide variants and indels were filtered against the variants in the Whole-Genome Japanese Reference Panel 1KJPN (distributed by Tohoku Medical Megabank Organization; https://ijgvd.megabank.tohoku.ac.jp/download/, in the public domain).20 Variants were manually viewed on an integrative genomics viewer to eliminate strand bias and reduce false-positive calls. 
Screening for MYOC gene mutations was performed by comparing DNA sequences that were amplified by PCR from regions of the MYOC gene in both affected and unaffected individuals. The PCR primers used to amplify the DNA fragments consisting of amino acid residues in MYOC were designed based on exon 3 sequences and are available upon request. PCR was performed using an amplification mixture (50 μL) containing 200 ng of template genome DNA, 0.5 μM of primers and ExTaq polymerase (Takara, Shiga, Japan). Amplifications were performed using the following program: an initial denaturation step at 95°C for 5 minutes; and then 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Purified fragments were sequenced directly using a BigDye Terminator Cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA) using an automated DNA sequencer (model 3700 Genetic Analyzer; PerkinElmer, Foster City, CA, USA). 
Results
Clinical Findings
Of the 507 trabeculectomy specimens obtained from the POAG patients who were selected from the Japanese Red Cross Medical Center trabeculectomy library (samples were collected from September 1994 to January 2015), 7 that were obtained from 5 Japanese F-POAG patients were characterized as having abnormally thick TM and are referred to as group A. In the Japanese Red Cross Medical Center trabeculectomy library, the percentage of POAG cases with an abnormally thick TM was approximately 1%; thus, cases with this phenotype are rare. During the initial stage of this study, we selected these seven cases by inspecting the sections using light microscopy. Nine F-POAG patients with normal TM consented to a blood examination during the postoperative follow-up. These cases were used as controls and are referred to as group B. The mean ± SD average ages at diagnosis of patients in group A and group B were 36.4 ± 16.9 and 44.6 ± 21.9 years (P = 0.454), respectively, and the average ages at trabeculectomy of patients in group A and group B were 51.7 ± 17.1 and 57.2 ± 15.05 years (P = 0.507), respectively. Some of the clinical parameters are shown in Table 1. There were no significant differences between group A and group B parameters. 
Molecular Genetic Analysis by Exome Analysis
We then conducted molecular genetic analyses of the group A cases with a special focus on determining the causal gene associated with the abnormally thick TM. We first conducted an exome analysis of two families in group A. The proband of the first family that we examined in this study (family A) was a 53-year-old female (Fig. 2, II: 2, no. 1; Table 2, age at DNA examination) who had a diagnosis of POAG and subsequently underwent trabeculectomy of her right eye. Her maximum IOP was 26 mm Hg, and her C:D ratio was 0.95. Her visual field score was 4 (Table 1). Her father also had POAG, and his maximum IOP was 33 mm Hg. His optic discs showed total cupping. 
Figure 2
 
The pedigree structures are shown of two families with POAG and the mutations in the MYOC gene that were identified in the exome analysis. (A) Family A had a T377M mutation, and family C had an F369L mutation. Individual 2 in family A had ocular hypertension. Individual 8 in family C had homozygous the mutations P.F369L/P.F369L. Roman numerals for generation and Arabic numerals for individuals within the generation. Arrows indicate probands; asterisks indicate the individuals for whom trabecular specimens were available. (B) Sanger sequence analyses validated two of the MYOC mutations that were detected in patients with familial POAG.
Figure 2
 
The pedigree structures are shown of two families with POAG and the mutations in the MYOC gene that were identified in the exome analysis. (A) Family A had a T377M mutation, and family C had an F369L mutation. Individual 2 in family A had ocular hypertension. Individual 8 in family C had homozygous the mutations P.F369L/P.F369L. Roman numerals for generation and Arabic numerals for individuals within the generation. Arrows indicate probands; asterisks indicate the individuals for whom trabecular specimens were available. (B) Sanger sequence analyses validated two of the MYOC mutations that were detected in patients with familial POAG.
In the exome analysis, we found that the proband harbored a C-to-T mutation in exon 3 of the MYOC gene that resulted in a threonine 377-to-methionine substitution (II:2, no. 1 MYOC POAG in Fig. 2; Table 2). We also found that her father harbored a T377M mutation (I:1, no. 9 MYOC POAG), as did her son (III:1, no. 2 MYOC POAG), who also displayed ocular hypertension. 
The proband of the second family (family C) was a 41-year-old female who underwent trabeculectomy in both eyes (Fig. 2, II:2, no. 6; Table 2). Her maximum IOP was 30 mm Hg in the right eye and 45 mm Hg in the left eye. Her C:D ratios were 0.6 in the right eye and 0.7 in the left eye (Table 1). Her mother was 77 years old (I:2) and also had a diagnosis of POAG, not JOAG. In the exome sequence analysis, the proband was found to have a heterozygous F369L (phenylalanine 369-to-leucine) mutation (II:2, no. 6 MYOC POAG in Fig. 2; Table 2), and her mother harbored the homozygous F369L mutation (I:2, no. 8 MYOC POAG). This mutation has been reported to be dominant.21 The proband's daughter (III:1, no. 10) was found to have normal IOP with no glaucoma, and her son (III:2, no. 7) was also found to have no abnormalities in either eye. Neither of these subjects harbored a MYOC mutation (Fig. 2). 
During exome sequencing of families A and C, an average of 108 and 95 million reads, respectively, were generated, and 99.8% and 99.9% of the reads, respectively, were mapped to the reference genome. The average coverage of each exome was 137-fold and 136-fold, respectively. We also conducted Sanger sequencing using samples obtained from these individuals, and the findings regarding the T377M mutation in family A and F369L mutation in family C were reproducible. None of the family members harbored any of the other glaucoma causal mutations that have been established in recent POAG gene analyses, including OPTN, WDR36, and ASB10, or in a recent congenital glaucoma gene analyses including CYP1B1, FOXC1, and PITX2
Molecular Genetic Analysis by Sanger Sequencing
We extended our molecular genetic analyses of POAG cases using Sanger sequencing. The proband of the third family (family H, II:1) was a 65-year-old male who underwent trabeculectomy in his right eye. His maximum IOP was 30 mm Hg, and his C:D ratio was 0.9 (Table 1). In the Sanger sequence analysis, he was found to have a P370L (proline 370-to-leucine) mutation in the MYOC gene (II:1, no. 17 MYOC POAG in Fig. 3; Table 2). His mother also had POAG, but we were unable to obtain a sample from her. 
Figure 3
 
The pedigree structures of two families with POAG and mutations in the MYOC gene that were validated using Sanger sequencing are shown. (A) Family H had a P370L mutation, and family N had a T448P mutation. No information was obtained from the parents in family N. Arrows indicate probands; asterisks indicate the individuals for whom trabeculectomy specimens were available. (B) Sanger sequencing analyses show four of the MYOC mutations that were detected in the patients with familial POAG.
Figure 3
 
The pedigree structures of two families with POAG and mutations in the MYOC gene that were validated using Sanger sequencing are shown. (A) Family H had a P370L mutation, and family N had a T448P mutation. No information was obtained from the parents in family N. Arrows indicate probands; asterisks indicate the individuals for whom trabeculectomy specimens were available. (B) Sanger sequencing analyses show four of the MYOC mutations that were detected in the patients with familial POAG.
The proband of the fourth family (family N, II:1) was a 39-year-old female in whom POAG was diagnosed and who subsequently underwent trabeculectomy of her left eye. Her maximum IOP was 22 mm Hg, and her C:D ratio was 0.9 (Table 1). She was found to have a T448P (threonine 448-to-proline) mutation in the MYOC gene (II:1, no. 25 MYOC POAG in Fig. 3; Table 2). None of the 9 patients in group B harbored any of the MYOC mutations. 
Gonioscopic Observation and Detailed Histopathological Examinations of POAG Tissues
Based on the results of the clinical and molecular genetic analyses, we hypothesized that TM thickness was tightly linked to gain-of-function mutations in the MYOC gene. To test this hypothesis, we performed detailed histopathological analyses of patient eyes. 
We first evaluated angle appearance data. The appearance of all angles was observed using a slit lamp. In MYOC POAG (group A) patients, we observed that the angles were wide open and without abnormalities (Figs. 4A, 4C, inset), but sometimes there was pigmentation in the TM in the older cases (Fig. 4A, inset). The most conspicuous changes in the TM in MYOC patients were the appearance of very thick TM beams and the disappearance of TM cells (Fig. 4A), which were not observed in non-MYOC patients (Fig. 4B). The thickened TM beams were fused to each other (Fig. 4A). Trabecular meshwork cells were completely absent not only in the uveal and corneoscleral meshwork but also in juxtacanalicular tissues (JCT) in older patients (Fig. 4A). In contrast, in the eyes of non-MYOC patients, TM thickness appeared to be normal, and many TM cells remained in the JCT or what appeared to be the region of the SC (Fig. 4B, solid stars). Fused beams were also observed in younger patients (Fig. 4C), who displayed a loss of TM cells (Fig. 4D). A marginated nucleus or karyolysis (Fig. 4D) was often observed in TM cells in MYOC patients. Thickened TM beams were mainly the result of a thickened basement membrane (BM), which is composed of granular materials and periodic structures (Fig. 4D, inset). 
Figure 4
 
Light microscopy and transmission electron microscopy images show the SC and TM in the eyes of MYOC and non-MYOC patients. (A) Light microscopy images of the SC and TM in the eye of MYOC (T377M) patient 9 (family A), who was an 82-year-old male (age at trabeculectomy). Inset shows a gonioscopic image taken in the same patient of the supposed region in which the trabeculectomy was performed. The angle appeared normal, with moderate pigmentation in the TM (inset). Note the very thick TM beams, the lack of TM cells, and the nearly occluded SC. Fused TM beams were observed in JCT (star). (B) Light microscopy images of the eye after the second trabeculectomy in non-MYOC patient 11 (family D), an a 58-year-old male. Inset Light microscopy image of H&E staining in tissues obtained after the first trabeculectomy in the same patient. Note that the spaces in the TM within the corneoscleral meshwork and JCT were very scarce and that the length of the SC was very short. Stars indicate the assumed SC region. (C, D) The eye of MYOC (carrying mutation T448P) patient 25 (family N) is shown. ([C] inset) Gonioscopic image of the supposed region of trabeculectomy taken in the same patient. Note that the TM beams are thick and fused to each other within the corneoscleral meshwork and JCT ([C] open star). The anterior and posterior tips of the SC split are shown during tissue preparation ([C] solid stars). (D) Transmission electron microscopy images. Note that the beams in the corneoscleral meshwork are fused to each other, the loss of TM cells is evident (open star in D), and the remaining TM cells are swollen and show evidence of karyolysis or marginated nuclei. (N) The transmission electron microscopy image in the inset shows a large magnification of the boxed area. A very thick BM containing granular materials ([D] star in the inset) and periodic structures ([D] arrows) were observed. CC, collector channel; SCE, endothelium of Schlemm's canal.
Figure 4
 
Light microscopy and transmission electron microscopy images show the SC and TM in the eyes of MYOC and non-MYOC patients. (A) Light microscopy images of the SC and TM in the eye of MYOC (T377M) patient 9 (family A), who was an 82-year-old male (age at trabeculectomy). Inset shows a gonioscopic image taken in the same patient of the supposed region in which the trabeculectomy was performed. The angle appeared normal, with moderate pigmentation in the TM (inset). Note the very thick TM beams, the lack of TM cells, and the nearly occluded SC. Fused TM beams were observed in JCT (star). (B) Light microscopy images of the eye after the second trabeculectomy in non-MYOC patient 11 (family D), an a 58-year-old male. Inset Light microscopy image of H&E staining in tissues obtained after the first trabeculectomy in the same patient. Note that the spaces in the TM within the corneoscleral meshwork and JCT were very scarce and that the length of the SC was very short. Stars indicate the assumed SC region. (C, D) The eye of MYOC (carrying mutation T448P) patient 25 (family N) is shown. ([C] inset) Gonioscopic image of the supposed region of trabeculectomy taken in the same patient. Note that the TM beams are thick and fused to each other within the corneoscleral meshwork and JCT ([C] open star). The anterior and posterior tips of the SC split are shown during tissue preparation ([C] solid stars). (D) Transmission electron microscopy images. Note that the beams in the corneoscleral meshwork are fused to each other, the loss of TM cells is evident (open star in D), and the remaining TM cells are swollen and show evidence of karyolysis or marginated nuclei. (N) The transmission electron microscopy image in the inset shows a large magnification of the boxed area. A very thick BM containing granular materials ([D] star in the inset) and periodic structures ([D] arrows) were observed. CC, collector channel; SCE, endothelium of Schlemm's canal.
Immunohistochemical Staining for D240 and Transmission Electron Microscopy to Analyze TM Cells in Mutated MYOC POAG Patients
Immunohistochemical staining for D240 clearly demonstrated that TM cells were largely absent in MYOC patients (Fig. 5A). The morphologic abnormalities reflected by the absence of TM cells and TM spaces clearly supported the observations made following H&E staining (Figs. 4A, 4C). The pattern of D240 immunohistochemical staining observed in non-MYOC patients showed that D240 had a clearly different expression profile, with strong positive staining in the TM despite the presence of local unstained areas (Fig. 5B). 
Figure 5
 
Light microscopy images show the SC and TM in the eye of MYOC (carrying mutation T377M) patient 1 (family A) and in the eyes of non-MYOC patient 19 (family J). (A) Immunohistochemical staining for D240 (podoplanin) is shown in the right eye of MYOC patient 1. Inset shows a gonioscopic image of the supposed region in which the trabeculectomy was performed. The image was taken in the same patient. Note that most of the TM cells in the JCT have disappeared and that the spaces in the TM have also disappeared as a result of the fusion of the TM beams. (B) Immunohistochemical staining for D240 is shown in the eyes of non-MYOC patient 19. Inset shows H&E-stained tissues. Note that denser staining was observed there, despite the presence of locally unstained areas (stars), than was observed in the MYOC patient (A).
Figure 5
 
Light microscopy images show the SC and TM in the eye of MYOC (carrying mutation T377M) patient 1 (family A) and in the eyes of non-MYOC patient 19 (family J). (A) Immunohistochemical staining for D240 (podoplanin) is shown in the right eye of MYOC patient 1. Inset shows a gonioscopic image of the supposed region in which the trabeculectomy was performed. The image was taken in the same patient. Note that most of the TM cells in the JCT have disappeared and that the spaces in the TM have also disappeared as a result of the fusion of the TM beams. (B) Immunohistochemical staining for D240 is shown in the eyes of non-MYOC patient 19. Inset shows H&E-stained tissues. Note that denser staining was observed there, despite the presence of locally unstained areas (stars), than was observed in the MYOC patient (A).
Transmission electron microscopy observation was performed, using all eyes of group A and group B to study the detailed changes in the TM cells and TM beams. In the transmission electron microscopy observations of MYOC patients, the TM cells became swollen, shrunken, (Fig. 6A) or more damaged during karyolysis (Fig. 4D). Even in TM cells with a normal nucleus, many small vesicles (Fig. 6B) contained a ground substance that appeared to be secreted toward a local thickening of the BM. The rough endoplasmic reticulum (RER) was dilated (Figs. 6B, 6C, DR) and contained membrane-bound inclusion-like bodies composed of granular materials, ground substances, or more dense materials (Fig. 6C). Inclusion bodies were not observed in any eyes of non-MYOC patients (Fig. 6D). 
Figure 6
 
(AC) Transmission electron microscopy images of the SC and TM in the right eye of 40-year-old MYOC patient 6 (family C; mutation F369L) and the eye of 38-year-old non-MYOC patient 21 (D). (A) The inner-most uveal meshwork contained swollen (black open stars), slightly swollen (black solid stars), and shrunken (white open star) TM cells. TM beams with extremely thick BM were surrounded by swollen TM cells (black open star) or had become naked. (B, C) Large magnifications of the boxed area shown in A. Many small vesicles ([B] arrows) containing a ground substance appeared to have been secreted toward the BM, where there was a local accumulation of BM (B). The RER had become dilated ([B, C] DR) and contained membrane-bound inclusion-like bodies that were composed of granular materials ([C] small, open arrowhead), ground substances ([C] small black solid arrowhead) or ground substances containing more dense materials (arrow heads). Part of the dilated RER was tangentially cut (star), and both of its edges appeared invaginated (solid and open arrows). (B) Large open arrows indicate a centriole. AC, anterior chamber. (D) TM cells in the UM of non-MYOC patients contained normal mitochondria (M), RER and endoplasmic reticulum (ER). No inclusion bodies were observed in the RER. (D) Large magnification of the boxed area in inset. EF, elastic fiber.
Figure 6
 
(AC) Transmission electron microscopy images of the SC and TM in the right eye of 40-year-old MYOC patient 6 (family C; mutation F369L) and the eye of 38-year-old non-MYOC patient 21 (D). (A) The inner-most uveal meshwork contained swollen (black open stars), slightly swollen (black solid stars), and shrunken (white open star) TM cells. TM beams with extremely thick BM were surrounded by swollen TM cells (black open star) or had become naked. (B, C) Large magnifications of the boxed area shown in A. Many small vesicles ([B] arrows) containing a ground substance appeared to have been secreted toward the BM, where there was a local accumulation of BM (B). The RER had become dilated ([B, C] DR) and contained membrane-bound inclusion-like bodies that were composed of granular materials ([C] small, open arrowhead), ground substances ([C] small black solid arrowhead) or ground substances containing more dense materials (arrow heads). Part of the dilated RER was tangentially cut (star), and both of its edges appeared invaginated (solid and open arrows). (B) Large open arrows indicate a centriole. AC, anterior chamber. (D) TM cells in the UM of non-MYOC patients contained normal mitochondria (M), RER and endoplasmic reticulum (ER). No inclusion bodies were observed in the RER. (D) Large magnification of the boxed area in inset. EF, elastic fiber.
Thrombomodulin Immunohistochemical Staining and Transmission Electron Microscopy Analyses of SC Canals in Mutated MYOC POAG Patients
Immunohistochemical staining for thrombomodulin enabled us to characterize the appearance of SC canals. We observed that the SC endothelium was sealed off in the thrombomodulin-negative area (Fig. 7A) and in regions lacking thrombomodulin staining (Fig. 7B). Different degrees of SC damage were observed even within a single trabeculectomy sample that was divided into three blocks, as seen in the images showing dropped-out SC endothelium (Fig. 7B), narrowed SC (Fig. 7C), and collapsed SC (Fig. 7D). In transmission electron microscopy observations, the SC endothelium had largely disappeared, even when the SC had a normal length, as shown in a light microscopy photograph (Fig. 8A, inset). 
Figure 7
 
Light microscopy images of the SC and TM in the eyes of MYOC (mutation F369L) patient 6 (family C) and MYOC (mutation T377M) patient 1 (family A). Immunohistochemical staining for thrombomodulin enabled us to observe the changes that had occurred in the SC endothelium (A–D). The SC endothelium had sealed off (small arrow) the negative thrombomodulin area (large arrow) in the right eye of patient 6 (A). There appeared to be no differences in the TM and SC after the first and second trabeculectomy ([A] inset, H&E stain). ([A] inset, arrow] Slit made during preparation. (B–D) Immunohistochemical staining for thrombomodulin in the left eye of MYOC (mutation T377M) patient 1 (family A). Note the different degree of SC damage that was observed in the three blocks that were obtained from the same trabeculectomy sample. The SC endothelium ([B] star) had dropped, and the SC had narrowed ([C] arrowheads) and collapsed ([D] arrowheads). These changes were clearer in the tissues stained for thrombomodulin than in those stained with H&E ([B–D] inset). Note the thickened and fused TM beams in A, C, and D but not in B. SC, Schlemm's canal; CC, collector channel.
Figure 7
 
Light microscopy images of the SC and TM in the eyes of MYOC (mutation F369L) patient 6 (family C) and MYOC (mutation T377M) patient 1 (family A). Immunohistochemical staining for thrombomodulin enabled us to observe the changes that had occurred in the SC endothelium (A–D). The SC endothelium had sealed off (small arrow) the negative thrombomodulin area (large arrow) in the right eye of patient 6 (A). There appeared to be no differences in the TM and SC after the first and second trabeculectomy ([A] inset, H&E stain). ([A] inset, arrow] Slit made during preparation. (B–D) Immunohistochemical staining for thrombomodulin in the left eye of MYOC (mutation T377M) patient 1 (family A). Note the different degree of SC damage that was observed in the three blocks that were obtained from the same trabeculectomy sample. The SC endothelium ([B] star) had dropped, and the SC had narrowed ([C] arrowheads) and collapsed ([D] arrowheads). These changes were clearer in the tissues stained for thrombomodulin than in those stained with H&E ([B–D] inset). Note the thickened and fused TM beams in A, C, and D but not in B. SC, Schlemm's canal; CC, collector channel.
Figure 8
 
Transmission electron microscopy and light microscopy images of the SC and TM in the eye of MYOC (mutation P370L) patient 17 ([A] family H) and the eye of non-MYOC patient 18 (B). The transmission electron microscopy images represent the region contained in the boxed areas of light microscopy ([A, B] inset). Inset Toluidine blue stain. The TM cells in the eye of the MYOC patient became very thin or disappeared in the JCT in the regions in which the TM spaces had become very small because there was an abundant accumulation of BM-like materials (stars). Only single SC endothelia (arrow) remained in the inner wall of the SC. Although the SC had split from the sclera (inset, arrow) at its posterior end during tissue preparation, the length of the SC appeared normal. In the eye of the non-MYOC patient, the SC endothelium appeared normal, and extracellular materials in the JCT appeared less compact than those in the eye of the MYOC patient.
Figure 8
 
Transmission electron microscopy and light microscopy images of the SC and TM in the eye of MYOC (mutation P370L) patient 17 ([A] family H) and the eye of non-MYOC patient 18 (B). The transmission electron microscopy images represent the region contained in the boxed areas of light microscopy ([A, B] inset). Inset Toluidine blue stain. The TM cells in the eye of the MYOC patient became very thin or disappeared in the JCT in the regions in which the TM spaces had become very small because there was an abundant accumulation of BM-like materials (stars). Only single SC endothelia (arrow) remained in the inner wall of the SC. Although the SC had split from the sclera (inset, arrow) at its posterior end during tissue preparation, the length of the SC appeared normal. In the eye of the non-MYOC patient, the SC endothelium appeared normal, and extracellular materials in the JCT appeared less compact than those in the eye of the MYOC patient.
Histologic Data and Parameters of Trabeculectomy Specimens
It should be noted that we observed different degrees of changes in SC length in MYOC POAG cases, as summarized in Table 3. Briefly, the SC appeared to be normal in younger MYOC patients (Figs. 4C, 7), but the SC endothelium had dropped out, as shown by thrombomodulin staining (Figs. 7A, 7B), and the naked portion of the SC endothelium was sealed off (Fig. 7A). Even in specimens in which the SC looked normal using light microscopy, the SC endothelium had extensively dropped out (Figs. 7B, 8). Severe occlusion or collapse of the SC was observed in the eyes of the members of family A (Figs. 4A, 5A, 7C–7D). The PTNA, SC-ECD, and Sondermann's canal data showed that there were no significant differences between groups A and B, but there was more severe damage in the SC within the PTNA in group A than in group B. 
In the non-MYOC POAG eyes (group B), the length of the SC varied from 0 to 235 μm (Table 3). Among histologic parameters, it is noteworthy that SC length was significantly longer in group A than in group B. However, there were no significant differences in PTNA (P = 0.077) or SC-ECD (P = 0.181) between group A and B. The SC tended to show more endothelial damage in MYOC POAG patients. There were 4 eyes that underwent trabeculectomy twice because of a failure to control IOP after the first trabeculectomy (Table 2). In these cases, the first trabeculectomy did not appear to alter the outflow routes (family C, II:2, MYOC POAG patient no. 6 and non-MYOC POAG patient no. 11, Figs. 4B, 7A). 
Nuclear Count in the Corneoscleral Meshwork (CSM) and Uveal Meshwork (UM)
Nuclei of the TM cells in the CSM and UM were counted. As shown in Table 3, the mean ± SD nuclei of the TM cells in the CSM and UM ranged from 16 to 26 (20.29 ± 3.73) and 26 to 38 (32.80 ± 3.16) in group A and B, respectively. The number of nuclei of the TM cells in the CSM and UM from the eyes of group A was significantly different from that of group B (Table 3; P = 0.00001). The numbers of nuclei in the TM cells in the CSM and UM were obviously decreased in group A patients compared to those in group B at a similar age (Fig. 9). Although statistical evaluation using age matching was impossible because of the small sample size, it was clear that the numbers of nuclei of the TM cells in the CSM and UM in group A were decreased at all ages (Fig. 10). 
Figure 9
 
(A) Light microscopy images of the SC and TM in the left eye of 38-year-old MYOC (mutation F369L) patient 6 (family C), (C) 82-year-old (mutation T377M) patient 9 (family A), and (B) 46-year-old non-MYOC patient 33 and (D) 81-year-old patient 22, obtained at the second trabeculectomy. H&E staining. Nuclei of the TM cells in the CSM and UM were counted by making marks with squares in the figures. The nuclear count area of the TM cells is surrounded by the dotted line. The width of the nuclear count area was up to 300 μm (line with arrows) from the scleral spur (thick line). The number of nuclei in the eyes of the MYOC patients were less than those of the eyes of non-MYOC patients, even at younger or similar ages.
Figure 9
 
(A) Light microscopy images of the SC and TM in the left eye of 38-year-old MYOC (mutation F369L) patient 6 (family C), (C) 82-year-old (mutation T377M) patient 9 (family A), and (B) 46-year-old non-MYOC patient 33 and (D) 81-year-old patient 22, obtained at the second trabeculectomy. H&E staining. Nuclei of the TM cells in the CSM and UM were counted by making marks with squares in the figures. The nuclear count area of the TM cells is surrounded by the dotted line. The width of the nuclear count area was up to 300 μm (line with arrows) from the scleral spur (thick line). The number of nuclei in the eyes of the MYOC patients were less than those of the eyes of non-MYOC patients, even at younger or similar ages.
Figure 10
 
The association between the number of nuclei of the TM cells in the CSM and UM (vertical axis) and the age (horizontal axis) in group A and B. The solid circles and triangles indicate the number of nuclei of the TM cells in the eyes of the MYOC and non-MYOC patients, respectively. The open triangles and circle indicate the number of nuclei of the TM cells at the second trabeculectomy. It was obvious that the number of nuclei of the TM cells in the eyes of the MYOC patients was decreased than that of the non-MYOC patients at all ages.
Figure 10
 
The association between the number of nuclei of the TM cells in the CSM and UM (vertical axis) and the age (horizontal axis) in group A and B. The solid circles and triangles indicate the number of nuclei of the TM cells in the eyes of the MYOC and non-MYOC patients, respectively. The open triangles and circle indicate the number of nuclei of the TM cells at the second trabeculectomy. It was obvious that the number of nuclei of the TM cells in the eyes of the MYOC patients was decreased than that of the non-MYOC patients at all ages.
TM Thickness in CSM and UM
In transmission electron microscopy observation, the BM of the TM beams in MYOC POAG cases was 2.8 to 5.9 μm in thickness, several times thicker than that in non-MYOC POAG eyes. In light microscopy observation of H&E-stained images, the mean ± SD TM beam thickness in the UM ranged from 7.5 to 9.2 μm (8.30 ± 0.64 μm) and 3.2 to 5.3 μm (4.37 ± 0.90 μm) in groups A and B, respectively. The mean ± SD TM beam thickness in the CSM ranged from 8.4 to 10.5 μm (9.46 ± 0.70 μm) and 3.0 to 7.7 μm (5.38 ± 1.38 μm) in groups A and B, respectively (Table 3). The individual TM thickness in the CSM was difficult to measure in all eyes of MYOC patients and some eyes of non-MYOC patients because of the fusion of the TM beams (Fig. 11). There was a slight tendency toward increased TM beam thickness with age, except in the UM in MYOC patients (Fig. 12). The TM beam thickness in the UM (P = 2 × 10−8) and CSM (P = 1.4 × 10−6) was significantly greater in the eyes of MYOC patients than that in the eyes of non-MYOC patients (Table 3). 
Figure 11
 
(A) Light microscopy images of the UC and CSM beams in the right eye of 40-year-old MYOC (mutation F369L) patient 6 (family C); (C) 56-year-old (mutation T377M) patient 1 (family A); and 40-year-old non-MYOC patient 21 and (D) 58-year-old patient 11. H&E staining. All images were taken from the center of the SC, some of which were partly (B, D) or completely (C) collapsed. The UM (blue arrows) and CSM (black arrows) beams were much thicker in MYOC patients (A, C) than in the non-MYOC patients (B, D), even at the same ages. Fusion of the beams in the CSM (stars) was often observed in the eyes of MYOC patients.
Figure 11
 
(A) Light microscopy images of the UC and CSM beams in the right eye of 40-year-old MYOC (mutation F369L) patient 6 (family C); (C) 56-year-old (mutation T377M) patient 1 (family A); and 40-year-old non-MYOC patient 21 and (D) 58-year-old patient 11. H&E staining. All images were taken from the center of the SC, some of which were partly (B, D) or completely (C) collapsed. The UM (blue arrows) and CSM (black arrows) beams were much thicker in MYOC patients (A, C) than in the non-MYOC patients (B, D), even at the same ages. Fusion of the beams in the CSM (stars) was often observed in the eyes of MYOC patients.
Figure 12
 
Associations between the thickness of the TM beams in the UM (A) and CSM (B) (μm, vertical axis) and the subjects' ages (horizontal axis) in groups A and B are shown. Solid circles and triangles indicate the thickness of the UM or CSM beams in the eyes of the MYOC and non-MYOC patients, respectively. Open triangles and circle indicate the thickness of the UM or CSM at the second trabeculectomy. It was obvious that the UM and CSM beams in the eyes of MYOC patients were thicker than those of the non-MYOC patients at all ages.
Figure 12
 
Associations between the thickness of the TM beams in the UM (A) and CSM (B) (μm, vertical axis) and the subjects' ages (horizontal axis) in groups A and B are shown. Solid circles and triangles indicate the thickness of the UM or CSM beams in the eyes of the MYOC and non-MYOC patients, respectively. Open triangles and circle indicate the thickness of the UM or CSM at the second trabeculectomy. It was obvious that the UM and CSM beams in the eyes of MYOC patients were thicker than those of the non-MYOC patients at all ages.
In this study, we demonstrated that there was a lower number of TM cells in the MYOC patients than in the non-MYOC patients. We often found that in group A cases, the TM cells had degenerated and displayed karyolysis or marginated nuclei or had disappeared altogether from the TM beams. Several conspicuous changes were observed, including the disappearance of TM cells and the thickening and fusing of TM beams. Two related patients were in group A, and we could not exclude the possibility of selection bias; nonetheless, our results are the first to indicate that there is a correlation between these morphologic abnormalities and specific gene mutations in POAG. 
Discussion
In this study, we present data from a unique subgroup of F-POAG eyes that were classified according to morphologic features of the outflow route. The eyes in this group showed abnormally thick TM and apoptosis of TM cells. The probands had high intraocular pressure and poor responses to medical treatment. Molecular genetic analyses revealed that the patients in this group retained nonsynonymous mutations (F369L, P370L, T377M, and T448P) in the MYOC gene, whereas POAG cases with normal TM thickness did not harbor these mutations in the MYOC gene. In both groups, no causal mutations were found in other established POAG-associated genes, and no other plausible causal mutations were found in the MYOC gene. Based on these results, we propose that F-POAG patients can be divided into two groups: group A patients, who have an abnormally thick TM and harbor mutations in the MYOC gene, and group B patients, who have a TM of normal thickness and do not harbor MYOC gene mutations. Thus, our study is the first to provide evidence suggesting a correlation between morphologic abnormalities and specific gene mutations in POAG. 
More than 180 MYOC variants have so far been documented, and approximately 3% to 4% of POAG patients harbor a disease-causing MYOC mutation. The MYOC mutation database has been established and is available online (http://www.myocilin.com/variants.php, in the public domain). This database contains a list of all identified mutations and associated phenotypic data, including age at POAG onset, maximum recorded IOP, age-related penetrance, and whether surgical intervention was required. However, many histologic studies have been performed in POAG eyes, and some of these have focused on changes in the TM. However, no previous study has investigated the relationship between morphologic changes in the TM and MYOC mutations. 
In family C of this study, the proband and his mother had adult onset POAG that was associated with the F369L mutation.21 It is intriguing that the mother harbored a homozygous F369L mutation. It was reported in a very large French Canadian pedigree that subjects who inherited two copies of the affected haplotype did not show glaucoma.22 In contrast, the present case showed overt glaucoma even though she possessed two copies of the affected allele, suggesting that a MYOC POAG status may not be limited to heterozygotes. 
Previous reports of cases with the T377M,23 P370L,24 and T448P25 mutations have shown that phenotypes are generally associated with onset at a younger age, a higher peak IOP, and poor responses to medical treatment that potentially lead to surgical interventions. There have been many reports of the T377M and P370L mutations and JOAG. In this study, we found that a proband and his father in family A harbored the T377M mutation and had POAG. It is interesting that the patients in family A had diagnoses of POAG in later middle age (47 years old) or old age (61 years old), despite the presence of severe SC occlusion (Figs. 4A, 5A, 7C, 7D). Slow progression of the abnormalities in the TM and SC may be the reason for this delay in family A. The proband's 18-year-old son also harbored the T377M mutation and showed ocular hypertension in his most recent examination. We plan to follow his IOP and optic disc to implement early interventions. As for the F369L and T448P mutations, they are unique mutations found only in the Japanese population. The F369L mutation was reported by Ishikawa et al.21 and was associated with POAG. In our pedigree (family C), the proband and his mother had POAG. The T448P mutation was also reported in only two of the Japanese groups with very high IOP, and treatment of these patients with topical medications was insufficient; therefore, filtering surgeries were required. Our case with a P.T448P mutation (family N) indicated the presence of JOAG but had only mild IOP elevation. It is probable that we detected the early phase of JOAG. 
The lack of gonioscopic angle abnormalities in MYOC POAG patients suggested that MYOC POAG may not be caused by abnormalities associated with angle splitting during embryonic development. The characteristic morphologic changes that were observed in the eyes of group A patients included 1) marginated nuclei or karyolysis and swollen or shrunken cytoplasm that resulted in degeneration and disappearance of TM cells; 2) an abnormally thickened BM, resulting in thickened TM beams; 3) fused TM beams in CSM; and 4) occlusion of SC. 
Marginated nuclei or karyolysis and swollen or shrunken cytoplasm may indicate the presence of necrosis or apoptosis in TM cells.26 We counted the number of nuclei in TM cells in the CSM and UM in all eyes in groups A and B, including those from the second trabeculectomy. The number of TM cells in the JCT was excluded because it is difficult to differentiate TM cells from SC endothelium or fibroblasts that may have appeared after the occlusion of the SC.17 It was impossible to conduct an age-matched analysis in our study because of the small sample size; however, it was obvious that the number of nuclei in TM cells in the CSM and UM was decreased in group A at all ages (Figs. 9, 10). Dilated RER was observed during our transmission electron microscopy analyses. These structures contained membrane-bound, inclusion-like bodies and seemed to represent one of the most prominent changes in the cytoplasm of TM cells in MYOC POAG patients. Interestingly, it has been reported that mutated MYOC is retained in RER, where it aggregates to form inclusion bodies that are typical of Russell bodies.27 In our study, we observed that the inclusion-like bodies (Fig. 6C, small and large arrowheads) in the RER appeared to exist in the invaginated RER cisternae because they were enveloped by membranous structures.28 These changes have also been observed in regressing or dying normal hepatocytes in atrophic or starving livers.26 Thus, RER invagination may be a mechanism by which cytoplasmic atrophy occurs in TM cells in these patients. 
In MYOC POAG patients, BM-like materials contained granular materials and periodic structures (Fig. 4D). The granular materials (Fig. 4D) resembled the components of membrane-bound, inclusion-like bodies observed in dilated RER (Fig. 6C, open and solid arrow heads) in TM cells of MYOC POAG patients. These periodic structures and granular materials appeared to be components of the sheathing materials that surrounded the elastic fibers in the JCT in aged subjects. Ueda et al.29 reported that when these MYOC-associated structures were labeled with gold particles, they were heavily localized to the sheathing materials that surrounded elastic-like fibers. Interestingly, local accumulation of BM was observed, and many small vesicles containing ground substances appeared to be secreted toward these materials (Fig. 6B, solid arrows). Excessive production of BM-like materials by TM cells in MYOC POAG patients may be responsible for the thickened TM beams in UM and CSM. The UM (P = 2 × 10−8) and CSM beams (P = 1.4 × 10−6) were significantly thicker in MYOC patients than those in non-MYOC patients (Table 3) and tended to become slightly thicker with age (Fig. 12). Although age matching in both groups was impossible in this study, it was obvious that the UM and CSM were much thicker in the eyes of MYOC-POAG patients (Figs. 11, 12). 
Fusion of TM beams in CSM was often observed in the eyes of MYOC patients (Fig. 11). The fusion of TM beams in MYOC-POAG patients may have been caused by an abnormally thickened BM and the disappearance of TM cells, similar to what occurs during the aging process in TM in both normal and POAG patients.8 The fusion of the CSM may cause occlusion of SC by obstructing aqueous outflow, as discussed below. 
D240 (podoplanin) expression in TM cells has been previously reported.19,30 D240 is a good marker for detecting the disappearance of TM cells in PACG eyes.18 In this study, we also found that immunohistochemical staining for D240 provided excellent evidence that indicated that TM cells were absent and present in MYOC POAG and non-MYOC POAG patients, respectively. It may take a long time for an eye to develop an abnormally thickened BM that can elicit changes in the outflow routes, thereby leading to elevated IOP. It is therefore interesting that the widely reported MYOC mutation Q368X, which results in a truncation of the MYOC protein that reduces its length by 137 amino acids, has been observed in families with late onset POAG and moderate IOP.13,31,32 Myoc-deficient mice lack any discernible phenotype and have normal intraocular pressure.33 Because of these observations, we surmise that TM cells in MYOC POAG patients are likely to be normal at birth and to acquire abnormalities over the course of the patient's lifetime. Indeed, while an immature JCT is the main cause of juvenile glaucoma,9 the JCT appeared to be normally developed but became compact with an accumulation of BM-like materials after birth in all MYOC POAG patients. 
Different degrees of SC shortening were observed between the groups. We found that the length of the SC was significantly longer in MYOC POAG patients (223 ± 33.8 μm) than in non-MYOC POAG patients (116 ± 80.9 μm). It is important to discuss whether the changes in SC length were acquired or congenital. Developmental abnormalities in the SC have previously been reported in congenital glaucoma34 and POAG.17 Many TM cells remained in the JCT or SC region in non-MYOC patients (Fig. 4B) but not in MYOC patients, suggesting that the SC was developmentally immature in some of the non-MYOC patients.17 Although there were no significant differences in PTNA and SC-ECD between groups A and B, MYOC POAG patients tended to have more SC endothelial damage (Figs. 7A–7D, 8A). We propose that PTNA and SC-ECD are the best parameters for assessing SC endothelial damage.17 We determined that the SC endothelial abnormalities (Figs. 7A, 7B, 8A) and the shortened SC (Figs. 4A, 5A, 7C, 7D) that were observed in MYOC POAG patients may be secondary changes that result from impaired aqueous outflow. 
The weaknesses of this study are the lack of inclusion of unrelated subjects in the study cohort, which may result in biases arising from relatedness, the impossibility of age matching due to the rarity of the MYOC mutation and the small sample size. In addition, the enrollment of family members in group A introduced bias into emphases and judgments regarding the frequency of patients with MYOC mutations with the identified histologic features of an abnormally thick TM. Despite these weaknesses, this study suggests that abnormal TM cells cause a series of events, including the thickening of the BM, apoptosis in TM cells, the fusion of TM beams to CSM beams and, eventually, the occlusion of SC in MYOC POAG patients. To prevent blindness, it is important to detect and treat MYOC POAG early. Regenerative therapies aimed at blocking, reversing, or replacing the degeneration of TM cells may be candidate strategies for future treatments that can be implemented before irreversible changes occur in the TM and SC. This analysis shows that this approach could be potentially useful for clinically diagnosing and treating glaucoma, and our findings advance our ability to provide personalized medicine to affected patients. 
Acknowledgments
Supported by grants from the Ministry of Health, Labor and Welfare of Japan and from the Japan Agency for Medical Research and Development (AMED). 
Disclosure: T. Hamanaka, None; M. Kimura, None; T. Sakurai, None; N. Ishida, None; J. Yasuda, None; M. Nagasaki, None; N. Nariai, None; A. Endo, None; K. Homma, None; F. Katsuoka, None; Y. Matsubara, None; M. Yamamoto, None; N. Fuse, None 
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Figure 1
 
Experimental design. Trabeculectomy specimens were obtained from 17 eyes of 14 POAG patients selected from the Japanese Red Cross Medical Center trabeculectomy library. Samples were divided into two groups based on morphologic characteristics, and molecular genetic analyses were performed. Then, detailed histopathological examinations were carried out, and the relationships between genotype and phenotype were analyzed.
Figure 1
 
Experimental design. Trabeculectomy specimens were obtained from 17 eyes of 14 POAG patients selected from the Japanese Red Cross Medical Center trabeculectomy library. Samples were divided into two groups based on morphologic characteristics, and molecular genetic analyses were performed. Then, detailed histopathological examinations were carried out, and the relationships between genotype and phenotype were analyzed.
Figure 2
 
The pedigree structures are shown of two families with POAG and the mutations in the MYOC gene that were identified in the exome analysis. (A) Family A had a T377M mutation, and family C had an F369L mutation. Individual 2 in family A had ocular hypertension. Individual 8 in family C had homozygous the mutations P.F369L/P.F369L. Roman numerals for generation and Arabic numerals for individuals within the generation. Arrows indicate probands; asterisks indicate the individuals for whom trabecular specimens were available. (B) Sanger sequence analyses validated two of the MYOC mutations that were detected in patients with familial POAG.
Figure 2
 
The pedigree structures are shown of two families with POAG and the mutations in the MYOC gene that were identified in the exome analysis. (A) Family A had a T377M mutation, and family C had an F369L mutation. Individual 2 in family A had ocular hypertension. Individual 8 in family C had homozygous the mutations P.F369L/P.F369L. Roman numerals for generation and Arabic numerals for individuals within the generation. Arrows indicate probands; asterisks indicate the individuals for whom trabecular specimens were available. (B) Sanger sequence analyses validated two of the MYOC mutations that were detected in patients with familial POAG.
Figure 3
 
The pedigree structures of two families with POAG and mutations in the MYOC gene that were validated using Sanger sequencing are shown. (A) Family H had a P370L mutation, and family N had a T448P mutation. No information was obtained from the parents in family N. Arrows indicate probands; asterisks indicate the individuals for whom trabeculectomy specimens were available. (B) Sanger sequencing analyses show four of the MYOC mutations that were detected in the patients with familial POAG.
Figure 3
 
The pedigree structures of two families with POAG and mutations in the MYOC gene that were validated using Sanger sequencing are shown. (A) Family H had a P370L mutation, and family N had a T448P mutation. No information was obtained from the parents in family N. Arrows indicate probands; asterisks indicate the individuals for whom trabeculectomy specimens were available. (B) Sanger sequencing analyses show four of the MYOC mutations that were detected in the patients with familial POAG.
Figure 4
 
Light microscopy and transmission electron microscopy images show the SC and TM in the eyes of MYOC and non-MYOC patients. (A) Light microscopy images of the SC and TM in the eye of MYOC (T377M) patient 9 (family A), who was an 82-year-old male (age at trabeculectomy). Inset shows a gonioscopic image taken in the same patient of the supposed region in which the trabeculectomy was performed. The angle appeared normal, with moderate pigmentation in the TM (inset). Note the very thick TM beams, the lack of TM cells, and the nearly occluded SC. Fused TM beams were observed in JCT (star). (B) Light microscopy images of the eye after the second trabeculectomy in non-MYOC patient 11 (family D), an a 58-year-old male. Inset Light microscopy image of H&E staining in tissues obtained after the first trabeculectomy in the same patient. Note that the spaces in the TM within the corneoscleral meshwork and JCT were very scarce and that the length of the SC was very short. Stars indicate the assumed SC region. (C, D) The eye of MYOC (carrying mutation T448P) patient 25 (family N) is shown. ([C] inset) Gonioscopic image of the supposed region of trabeculectomy taken in the same patient. Note that the TM beams are thick and fused to each other within the corneoscleral meshwork and JCT ([C] open star). The anterior and posterior tips of the SC split are shown during tissue preparation ([C] solid stars). (D) Transmission electron microscopy images. Note that the beams in the corneoscleral meshwork are fused to each other, the loss of TM cells is evident (open star in D), and the remaining TM cells are swollen and show evidence of karyolysis or marginated nuclei. (N) The transmission electron microscopy image in the inset shows a large magnification of the boxed area. A very thick BM containing granular materials ([D] star in the inset) and periodic structures ([D] arrows) were observed. CC, collector channel; SCE, endothelium of Schlemm's canal.
Figure 4
 
Light microscopy and transmission electron microscopy images show the SC and TM in the eyes of MYOC and non-MYOC patients. (A) Light microscopy images of the SC and TM in the eye of MYOC (T377M) patient 9 (family A), who was an 82-year-old male (age at trabeculectomy). Inset shows a gonioscopic image taken in the same patient of the supposed region in which the trabeculectomy was performed. The angle appeared normal, with moderate pigmentation in the TM (inset). Note the very thick TM beams, the lack of TM cells, and the nearly occluded SC. Fused TM beams were observed in JCT (star). (B) Light microscopy images of the eye after the second trabeculectomy in non-MYOC patient 11 (family D), an a 58-year-old male. Inset Light microscopy image of H&E staining in tissues obtained after the first trabeculectomy in the same patient. Note that the spaces in the TM within the corneoscleral meshwork and JCT were very scarce and that the length of the SC was very short. Stars indicate the assumed SC region. (C, D) The eye of MYOC (carrying mutation T448P) patient 25 (family N) is shown. ([C] inset) Gonioscopic image of the supposed region of trabeculectomy taken in the same patient. Note that the TM beams are thick and fused to each other within the corneoscleral meshwork and JCT ([C] open star). The anterior and posterior tips of the SC split are shown during tissue preparation ([C] solid stars). (D) Transmission electron microscopy images. Note that the beams in the corneoscleral meshwork are fused to each other, the loss of TM cells is evident (open star in D), and the remaining TM cells are swollen and show evidence of karyolysis or marginated nuclei. (N) The transmission electron microscopy image in the inset shows a large magnification of the boxed area. A very thick BM containing granular materials ([D] star in the inset) and periodic structures ([D] arrows) were observed. CC, collector channel; SCE, endothelium of Schlemm's canal.
Figure 5
 
Light microscopy images show the SC and TM in the eye of MYOC (carrying mutation T377M) patient 1 (family A) and in the eyes of non-MYOC patient 19 (family J). (A) Immunohistochemical staining for D240 (podoplanin) is shown in the right eye of MYOC patient 1. Inset shows a gonioscopic image of the supposed region in which the trabeculectomy was performed. The image was taken in the same patient. Note that most of the TM cells in the JCT have disappeared and that the spaces in the TM have also disappeared as a result of the fusion of the TM beams. (B) Immunohistochemical staining for D240 is shown in the eyes of non-MYOC patient 19. Inset shows H&E-stained tissues. Note that denser staining was observed there, despite the presence of locally unstained areas (stars), than was observed in the MYOC patient (A).
Figure 5
 
Light microscopy images show the SC and TM in the eye of MYOC (carrying mutation T377M) patient 1 (family A) and in the eyes of non-MYOC patient 19 (family J). (A) Immunohistochemical staining for D240 (podoplanin) is shown in the right eye of MYOC patient 1. Inset shows a gonioscopic image of the supposed region in which the trabeculectomy was performed. The image was taken in the same patient. Note that most of the TM cells in the JCT have disappeared and that the spaces in the TM have also disappeared as a result of the fusion of the TM beams. (B) Immunohistochemical staining for D240 is shown in the eyes of non-MYOC patient 19. Inset shows H&E-stained tissues. Note that denser staining was observed there, despite the presence of locally unstained areas (stars), than was observed in the MYOC patient (A).
Figure 6
 
(AC) Transmission electron microscopy images of the SC and TM in the right eye of 40-year-old MYOC patient 6 (family C; mutation F369L) and the eye of 38-year-old non-MYOC patient 21 (D). (A) The inner-most uveal meshwork contained swollen (black open stars), slightly swollen (black solid stars), and shrunken (white open star) TM cells. TM beams with extremely thick BM were surrounded by swollen TM cells (black open star) or had become naked. (B, C) Large magnifications of the boxed area shown in A. Many small vesicles ([B] arrows) containing a ground substance appeared to have been secreted toward the BM, where there was a local accumulation of BM (B). The RER had become dilated ([B, C] DR) and contained membrane-bound inclusion-like bodies that were composed of granular materials ([C] small, open arrowhead), ground substances ([C] small black solid arrowhead) or ground substances containing more dense materials (arrow heads). Part of the dilated RER was tangentially cut (star), and both of its edges appeared invaginated (solid and open arrows). (B) Large open arrows indicate a centriole. AC, anterior chamber. (D) TM cells in the UM of non-MYOC patients contained normal mitochondria (M), RER and endoplasmic reticulum (ER). No inclusion bodies were observed in the RER. (D) Large magnification of the boxed area in inset. EF, elastic fiber.
Figure 6
 
(AC) Transmission electron microscopy images of the SC and TM in the right eye of 40-year-old MYOC patient 6 (family C; mutation F369L) and the eye of 38-year-old non-MYOC patient 21 (D). (A) The inner-most uveal meshwork contained swollen (black open stars), slightly swollen (black solid stars), and shrunken (white open star) TM cells. TM beams with extremely thick BM were surrounded by swollen TM cells (black open star) or had become naked. (B, C) Large magnifications of the boxed area shown in A. Many small vesicles ([B] arrows) containing a ground substance appeared to have been secreted toward the BM, where there was a local accumulation of BM (B). The RER had become dilated ([B, C] DR) and contained membrane-bound inclusion-like bodies that were composed of granular materials ([C] small, open arrowhead), ground substances ([C] small black solid arrowhead) or ground substances containing more dense materials (arrow heads). Part of the dilated RER was tangentially cut (star), and both of its edges appeared invaginated (solid and open arrows). (B) Large open arrows indicate a centriole. AC, anterior chamber. (D) TM cells in the UM of non-MYOC patients contained normal mitochondria (M), RER and endoplasmic reticulum (ER). No inclusion bodies were observed in the RER. (D) Large magnification of the boxed area in inset. EF, elastic fiber.
Figure 7
 
Light microscopy images of the SC and TM in the eyes of MYOC (mutation F369L) patient 6 (family C) and MYOC (mutation T377M) patient 1 (family A). Immunohistochemical staining for thrombomodulin enabled us to observe the changes that had occurred in the SC endothelium (A–D). The SC endothelium had sealed off (small arrow) the negative thrombomodulin area (large arrow) in the right eye of patient 6 (A). There appeared to be no differences in the TM and SC after the first and second trabeculectomy ([A] inset, H&E stain). ([A] inset, arrow] Slit made during preparation. (B–D) Immunohistochemical staining for thrombomodulin in the left eye of MYOC (mutation T377M) patient 1 (family A). Note the different degree of SC damage that was observed in the three blocks that were obtained from the same trabeculectomy sample. The SC endothelium ([B] star) had dropped, and the SC had narrowed ([C] arrowheads) and collapsed ([D] arrowheads). These changes were clearer in the tissues stained for thrombomodulin than in those stained with H&E ([B–D] inset). Note the thickened and fused TM beams in A, C, and D but not in B. SC, Schlemm's canal; CC, collector channel.
Figure 7
 
Light microscopy images of the SC and TM in the eyes of MYOC (mutation F369L) patient 6 (family C) and MYOC (mutation T377M) patient 1 (family A). Immunohistochemical staining for thrombomodulin enabled us to observe the changes that had occurred in the SC endothelium (A–D). The SC endothelium had sealed off (small arrow) the negative thrombomodulin area (large arrow) in the right eye of patient 6 (A). There appeared to be no differences in the TM and SC after the first and second trabeculectomy ([A] inset, H&E stain). ([A] inset, arrow] Slit made during preparation. (B–D) Immunohistochemical staining for thrombomodulin in the left eye of MYOC (mutation T377M) patient 1 (family A). Note the different degree of SC damage that was observed in the three blocks that were obtained from the same trabeculectomy sample. The SC endothelium ([B] star) had dropped, and the SC had narrowed ([C] arrowheads) and collapsed ([D] arrowheads). These changes were clearer in the tissues stained for thrombomodulin than in those stained with H&E ([B–D] inset). Note the thickened and fused TM beams in A, C, and D but not in B. SC, Schlemm's canal; CC, collector channel.
Figure 8
 
Transmission electron microscopy and light microscopy images of the SC and TM in the eye of MYOC (mutation P370L) patient 17 ([A] family H) and the eye of non-MYOC patient 18 (B). The transmission electron microscopy images represent the region contained in the boxed areas of light microscopy ([A, B] inset). Inset Toluidine blue stain. The TM cells in the eye of the MYOC patient became very thin or disappeared in the JCT in the regions in which the TM spaces had become very small because there was an abundant accumulation of BM-like materials (stars). Only single SC endothelia (arrow) remained in the inner wall of the SC. Although the SC had split from the sclera (inset, arrow) at its posterior end during tissue preparation, the length of the SC appeared normal. In the eye of the non-MYOC patient, the SC endothelium appeared normal, and extracellular materials in the JCT appeared less compact than those in the eye of the MYOC patient.
Figure 8
 
Transmission electron microscopy and light microscopy images of the SC and TM in the eye of MYOC (mutation P370L) patient 17 ([A] family H) and the eye of non-MYOC patient 18 (B). The transmission electron microscopy images represent the region contained in the boxed areas of light microscopy ([A, B] inset). Inset Toluidine blue stain. The TM cells in the eye of the MYOC patient became very thin or disappeared in the JCT in the regions in which the TM spaces had become very small because there was an abundant accumulation of BM-like materials (stars). Only single SC endothelia (arrow) remained in the inner wall of the SC. Although the SC had split from the sclera (inset, arrow) at its posterior end during tissue preparation, the length of the SC appeared normal. In the eye of the non-MYOC patient, the SC endothelium appeared normal, and extracellular materials in the JCT appeared less compact than those in the eye of the MYOC patient.
Figure 9
 
(A) Light microscopy images of the SC and TM in the left eye of 38-year-old MYOC (mutation F369L) patient 6 (family C), (C) 82-year-old (mutation T377M) patient 9 (family A), and (B) 46-year-old non-MYOC patient 33 and (D) 81-year-old patient 22, obtained at the second trabeculectomy. H&E staining. Nuclei of the TM cells in the CSM and UM were counted by making marks with squares in the figures. The nuclear count area of the TM cells is surrounded by the dotted line. The width of the nuclear count area was up to 300 μm (line with arrows) from the scleral spur (thick line). The number of nuclei in the eyes of the MYOC patients were less than those of the eyes of non-MYOC patients, even at younger or similar ages.
Figure 9
 
(A) Light microscopy images of the SC and TM in the left eye of 38-year-old MYOC (mutation F369L) patient 6 (family C), (C) 82-year-old (mutation T377M) patient 9 (family A), and (B) 46-year-old non-MYOC patient 33 and (D) 81-year-old patient 22, obtained at the second trabeculectomy. H&E staining. Nuclei of the TM cells in the CSM and UM were counted by making marks with squares in the figures. The nuclear count area of the TM cells is surrounded by the dotted line. The width of the nuclear count area was up to 300 μm (line with arrows) from the scleral spur (thick line). The number of nuclei in the eyes of the MYOC patients were less than those of the eyes of non-MYOC patients, even at younger or similar ages.
Figure 10
 
The association between the number of nuclei of the TM cells in the CSM and UM (vertical axis) and the age (horizontal axis) in group A and B. The solid circles and triangles indicate the number of nuclei of the TM cells in the eyes of the MYOC and non-MYOC patients, respectively. The open triangles and circle indicate the number of nuclei of the TM cells at the second trabeculectomy. It was obvious that the number of nuclei of the TM cells in the eyes of the MYOC patients was decreased than that of the non-MYOC patients at all ages.
Figure 10
 
The association between the number of nuclei of the TM cells in the CSM and UM (vertical axis) and the age (horizontal axis) in group A and B. The solid circles and triangles indicate the number of nuclei of the TM cells in the eyes of the MYOC and non-MYOC patients, respectively. The open triangles and circle indicate the number of nuclei of the TM cells at the second trabeculectomy. It was obvious that the number of nuclei of the TM cells in the eyes of the MYOC patients was decreased than that of the non-MYOC patients at all ages.
Figure 11
 
(A) Light microscopy images of the UC and CSM beams in the right eye of 40-year-old MYOC (mutation F369L) patient 6 (family C); (C) 56-year-old (mutation T377M) patient 1 (family A); and 40-year-old non-MYOC patient 21 and (D) 58-year-old patient 11. H&E staining. All images were taken from the center of the SC, some of which were partly (B, D) or completely (C) collapsed. The UM (blue arrows) and CSM (black arrows) beams were much thicker in MYOC patients (A, C) than in the non-MYOC patients (B, D), even at the same ages. Fusion of the beams in the CSM (stars) was often observed in the eyes of MYOC patients.
Figure 11
 
(A) Light microscopy images of the UC and CSM beams in the right eye of 40-year-old MYOC (mutation F369L) patient 6 (family C); (C) 56-year-old (mutation T377M) patient 1 (family A); and 40-year-old non-MYOC patient 21 and (D) 58-year-old patient 11. H&E staining. All images were taken from the center of the SC, some of which were partly (B, D) or completely (C) collapsed. The UM (blue arrows) and CSM (black arrows) beams were much thicker in MYOC patients (A, C) than in the non-MYOC patients (B, D), even at the same ages. Fusion of the beams in the CSM (stars) was often observed in the eyes of MYOC patients.
Figure 12
 
Associations between the thickness of the TM beams in the UM (A) and CSM (B) (μm, vertical axis) and the subjects' ages (horizontal axis) in groups A and B are shown. Solid circles and triangles indicate the thickness of the UM or CSM beams in the eyes of the MYOC and non-MYOC patients, respectively. Open triangles and circle indicate the thickness of the UM or CSM at the second trabeculectomy. It was obvious that the UM and CSM beams in the eyes of MYOC patients were thicker than those of the non-MYOC patients at all ages.
Figure 12
 
Associations between the thickness of the TM beams in the UM (A) and CSM (B) (μm, vertical axis) and the subjects' ages (horizontal axis) in groups A and B are shown. Solid circles and triangles indicate the thickness of the UM or CSM beams in the eyes of the MYOC and non-MYOC patients, respectively. Open triangles and circle indicate the thickness of the UM or CSM at the second trabeculectomy. It was obvious that the UM and CSM beams in the eyes of MYOC patients were thicker than those of the non-MYOC patients at all ages.
Table 1
 
Clinical Data of Trabeculectomy Patients With or Without Abnormally Thick TM as Assessed by Microscopy Inspection
Table 1
 
Clinical Data of Trabeculectomy Patients With or Without Abnormally Thick TM as Assessed by Microscopy Inspection
Table 2
 
Characteristics of POAG Cases in DNA Analyses
Table 2
 
Characteristics of POAG Cases in DNA Analyses
Table 3
 
Histologic Data and Parameters of Trabeculectomy Specimens
Table 3
 
Histologic Data and Parameters of Trabeculectomy Specimens
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