|Molecular Vision 2007;
Received 5 June 2007 | Accepted 10 September 2007 | Published 10 September 2007
Identification of mutations in the myocilin (MYOC) gene in Taiwanese patients with juvenile-onset open-angle glaucoma
1Institute of Medicine and 2Genetics Laboratory, Department of BioMedical Sciences, Chung Shan Medical University, Taichung, Taiwan; 3Department of Ophthalmology, Chi-Mei Medical Center Liou-Ying Campus, Tainan, Taiwan
Correspondence to: Dr. S-Y Li, Genetics Laboratory, Department of BioMedical Sciences, Chung Shan Medical University, Taichung, Taiwan, Republic of China; Phone: 886-4-24730022, ext. 11800; FAX: 886-4-24757412; email: firstname.lastname@example.org
Purpose: To investigate mutations in the promoter and coding regions of the myocilin (MYOC) gene in Taiwanese patients suffering from juvenile-onset open-angle glaucoma (JOAG).
Methods: MYOC was analyzed for mutations in 48 unrelated Taiwanese probands with JOAG and in 100 healthy control subjects. Genomic DNA was extracted from peripheral blood leukocytes and then subjected to PCR to amplify exons, flanking introns and promoter regions of the MYOC gene. The amplified products were screened for base mutations by autosequence. Data from the two groups were then compared using the χ2 test. Finally, the levels of MYOC transcripts were predicted by a neural network prediction system to study whether the intron mutations have any effect on the level of mRNA expression.
Results: The analysis revealed four MYOC mutations and six polymorphisms. The prevalence of MYOC gene mutations in this study was 12.5% (6/48). The mutations included one nonsense mutation (Arg46Stop; 3/6), one missense mutation (Val56Ala; 1/6), one intron mutation (c.604+228A>T; 1/6) as well as one mutation in the 3'-untranslated region (c.1515+73G>C; 1/6). In addition, although c.604+228A>T is an intron mutation and does not alter the content of the amino acid residue, the neural network prediction system revealed that it can potentially create a novel accept splice site during transcription. This mutation might affect the protein structure and consequently the normal function of myocilin.
Conclusions: Our results indicate that the c.136C>T (Arg46Stop), c.158T>C (Val56Ala), c.604+228A>T, and c.1515+73G>C mutations of MYOC may be associated with JOAG. In addition, we suggest that the c.136C>T (Arg46Stop) mutation of MYOC is a hot spot in Taiwanese patients with JOAG.
Glaucoma, a progressive optic neuropathy characterized by high intraocular pressure and retinal nerve fiber-layer damage, typically results in visual-field defects. The disease can also lead to blindness if the condition is not appropriately treated in a timely fashion . Glaucoma is a heterogeneous and multifarious disease reportedly caused by the interaction of several environmental factors. One form of glaucoma, namely primary open-angle glaucoma (POAG), is, to the best of our knowledge, a complex disease of unknown cause. POAG, which leads to progressive loss of the visual field, has been subdivided into two groups according to age at onset, namely: chronic open-angle glaucoma (COAG), a condition typically diagnosed after the age of 40 years, and juvenile open-angle glaucoma (JOAG), a disease most-often diagnosed in patients that are younger than 35 years . The definition of juvenile glaucoma is not consistent in the literature. The condition is variously described as a subset of infantile glaucoma and as an early onset form of chronic open-angle glaucoma. Whether this disease actually represents a distinct clinical entity, or not, has also been questioned by a number of researchers .
To date, at least seven gene loci (GLC1A-G) have been linked to POAG. Amongst these loci, three genes, MYOC encoding myocilin, OPTN encoding optineurin, and the WDR36 gene encoding a protein of unknown function have been considered to harbor the mutations that result in POAG [4-6]. MYOC is the gene primarily mutated in sufferers of juvenile-onset open-angle glaucoma , whereas OPTN appears to be the principal site of mutation in individuals with low-pressure POAG . Recently, WDR36 has been reported to represent a novel causative gene for adult-onset POAG .
The GLC1A (OMIM 137750) genetic locus has been mapped to chromosome 1q21-q31 by linkage analysis of the gene in probands and their family members with both juvenile-onset and adult-onset POAG . The MYOC gene (OMIM 601652) is associated with the GLC1A locus. Positional cloning strategies have revealed that most of the mutations in MYOC occur in three exons (606, 126, and 718 bp) and two major domains (MYOC Nh2-terminal and olfactomedin COOH-terminal) [8,9]. The gene codes for a 57 kDa protein named trabecular meshwork-induced glucocorticoid response protein (TIGR), but which is also referred to as myocilin (MYOC). It consists of a 5 kb promoter region containing several regulatory elements . The protein myocilin has not only been found to be distributed within the trabecular meshwork of eyes but it has also been found to be present in the ciliary body, the retina, and other ocular tissues, including the cornea, sclera, iris, and optic-nerve head . However, the exact mechanisms of action of MYOC and its role in the etiology of JOAG are largely unknown.
Mutations of MYOC associated with POAG and JOAG have been extensively investigated in different racial/ethnic populations [12,13]. To the best of our knowledge, however, no studies have reported on MYOC variants and their association with glaucoma in Taiwanese. Therefore, we investigated the promoter and coding regions of MYOC in 48 JOAG patients and 100 normal unrelated individuals in Taiwan, and tried to determine whether mutations in that gene are associated with the development of JOAG.
A total of 4,500 patients who presented to the department of Ophthalmology at the Kuo General Hospital were screened by chart review and 1,047 patients with symptoms of glaucoma were evaluated from 2003 to 2006. All study patients received more than two complete ocular examinations, each comprising slit-lamp testing, IOP measurement, fundus examination, and visual-field examination. Patients were defined as suffering from JOAG if they were younger than 35 years old and found to have an intraocular pressure (IOP) >22 mmHg, a cup/disc ratio >0.5 or one with an asymmetric appearance, a visual-field loss characteristic of glaucomatous change, and an open angle width ranging from Shaffer grade II to IV without any other apparent secondary cause (e.g. traumatically or surgically induced). Of the 1047 individuals screened, JOAG was diagnosed in 48 unrelated patients; these patients were included in the patient group for subsequent MYOC genetic analysis.
One hundred randomly selected normal individuals over 50 years of age were included as the control group. Individuals in the control group also received complete ocular examinations, as described above, in order to exclude the possibility of their suffering from glaucoma. The study protocol was approved by the Institutional Review Board of the Kuo General Hospital and was carried out in accordance with the World Medical Association's Declaration of Helsinki (2000). All patients provided signed informed consent to study participation subsequent to the details of the study having been explained in detail to them.
Detection of mutations in MYOC
DNA samples were collected from 10 ml of peripheral blood acquired from each of the 148 individuals and purified using a Gentra DNA Blood Kit (Gentra Systems, Inc., Minneapolis, MN) according to the manufacturer's directions. The quality and quantity of purified genomic DNA was determined by gel electrophoresis and spectrophotometry, respectively. Mutations in the promoter and coding regions of MYOC were screened by direct sequencing (GenBank NM_000261). Intragenic primers used for polymerase chain reaction (PCR) are listed in Table 1. In brief, PCR was carried out in a reaction volume of 25 μl containing 100 ng of genomic DNA, 200 μM dNTP, 0.25 units of proTaq DNA polymerase (Promega Corporation, Madison, WI), and 200 μM intragenic primers. The PCR products were purified using a PCR Purification Kit (Qiagen GmbH, Hilden, Germany) and then subjected to PCR-directed DNA sequencing using a DNA Sequencing Kit (Applied Biosystems Corporation, Foster City, CA). Sequencing was performed on an Applied Biosystems model 377 automated sequencer (Applied Biosystems Corporation). Sequence data were compared with the published sequence of MYOC (GenBank NM_000261).
Significant differences in allele frequencies between JOAG patients and controls were determined by the χ2 test. A p value <0.05 represented a statistically significant difference between JOAG patients and controls.
Splice site prediction by neural network
Normal and mutant DNA sequences of intron 1 and exon 3 in MYOC (GeneBank NT_004487 and AF049793) were analyzed by a neural network prediction system to predict acceptor and donor splice sites in these regions .
Mutations in 3 exons of MYOC (GenBank NM_000261), including the flanking intronic sequences, promoter region and coding region, were screened by PCR amplification and direct DNA sequence analysis. Six polymorphic sites of MYOC were determined in the 48 JOAG patients and 100 unrelated normal controls (Table 2). A p-value was calculated using the χ2 test, resulting more than 0.05 represented in all polymorphic sites between JOAG patients and controls. Further, we calculated the power of polymorphism variants (0.2>p>0.05) using PASS statistical software. We found that the power of these variants was low (Table 2). Therefore, we suggest that there were no significant differences in allelic frequencies of any of these polymorphic sites between the patients and controls.
No mutations of MYOC were found in any of the normal controls. However, mutations were identified in 6 of the 48 JOAG patients (Figure 1 and Table 3). The prevalence of MYOC mutations in this study was 12.5% (6/48). A heterozygous c.136C>T mutation was found in 3 of the 6 patients (3/48; 6.25%; Figure 1B); one patient had a heterozygous c.158T>C mutation (1/48; 2.08%; Figure 1D); one patient had a heterozygous c.604+228A>T mutation (1/48; 2.08%; Figure 1F); and one patient had a heterozygous c.1515+73G>C mutation (1/48; 2.08%; Figure 1H). Among the four heterozygous mutations present in the 6 JOAG patients, the c.136C>T and c.158T>C mutations located in the coding region led to amino acid changes within the MYOC protein. Heterozygous c.136C>T is a transversion mutation that leads to heterozygous arginine to stop codon substitution at codon 46 (Arg46Stop). Heterozygous c.158T>C is a transversion mutation that leads to heterozygous valine to alanine substitution at codon 53 (Val53Ala). The two heterozygous c.604+228A>T and c.1515+73G>C mutations were found in the intronic region and 3'-untranslation region of MYOC, respectively (Table 3).
To study whether the c.604+228A>T and c.1515+73G>C mutations had any effect on the level of mRNA expression, the levels of MYOC transcripts were predicted by a neural network prediction system. Using a cutoff of 0.4, a detection rate of 83.8% and a false-positive rate of 3.1%, we tested MYOC c.604+228A>T and c.1515+73G>C mutations. We found that the c.604+228A>T intron mutation created a novel accept splice site in the c.604+233-234AG of MYOC. When the mutation was introduced in MYOC, the neural network values of the novel accept splice site (c.604+233-234AG) at this position was 0.71 (Figure 2A). The result indicated that this novel splice site would increase MYOC mRNA by 234 nucleotides between 604 and 605, thereby disrupting the normal splicing of MYOC (Figure 2B). Such a MYOC transcript would lead to a frame shift mutation beginning at residue 201 and produce a premature termination stop at residue 215 during translation (Figure 2C). In contrast, the neural network prediction system found that a c.1515+73G>C mutation in the 3'-untranslated region would not affect the expression level of mRNA.
Juvenile-onset open angle glaucoma (JOAG) attacks relatively early in life when acquired via autosomal dominant inheritance, and is believed, by many clinicians, to be a different disease from common adult-onset glaucoma . The MYOC gene is primarily mutated in juvenile-onset subjects . In this study, six of the 48 (6/48, 12.5%) JOAG patients carried heterozygous mutations (c.136C>T, c.158T>C, c.604+228A>T, and c.1515+73G>C) in the MYOC gene. Although the frequency of mutations was low, the fact that those mutations were not detected in the 100 normal controls is significant. Similarly results have been reported that around 10% to 20% of JOAG patients carried mutation of MYOC gene and exhibited autosomal dominant inheritance .
The Mansergh et al.  study suggested that most Gln368Stop mutations descended from a common founder. Nevertheless, we did not observe the mutation in any of our study participants. Previous studies in Asia have reported that the Arg46Stop mutation was the most common familial mutation resulting in POAG and JOAG in Korea, Japan, and Hong Kong [17-20]. In agreement with these earlier findings, our study found that the Arg46Stop mutation in MYOC is a highest frequency factor (3/6) in Taiwanese patients with JOAG. Oppositely, there are reports indicated that the Arg46Stop mutation was found in the POAG patients and unrelated normal controls, and it was recognized as a polymorphic site in MYOC. The results from these reports are compared and summarized in Table 4 [17-27]. Despite the fact from the above reports, whether the Arg46Stop mutation is a truly disease-causing mutation or just a common polymorphism is still fuzzy. Since the Arg46Stop mutation was only found in 3 JOAG patients and not detected in the 100 unrelated normal control individuals in this study, we suggested that the Arg46Stop possibly is a disease-causing mutation. However, we cannot rule out the possibility that the Arg46Stop mutation may be in combination with other genetic or environmental factors.
Recently, a study demonstrated a mutation-dependent, gain-of-function association between the human MYOC COOH-terminal sequence and the peroxisomal targeting signal type 1 receptor (PTS1R) . The interaction of MYOC with PTS1R varied depending on the particular MYOC mutation. They showed that the full-length wild type MYOC and Lys398Arg (a nondisease-causing polymorphism) did not appreciably interact with PTS1R. In contrast, mutant MYOC interacted with PTS1R either directly or indirectly. MYOC Tyr437His and Gly364Val mutants, which contain the peroxisomal targeting singnal type 1 (PTS1) site, showed the strongest levels of interaction with PTS1R and then led to the elevation of IOP. In addition, the Gln368Stop mutation, which lacks the PTS1, did not directly interact with PTS1R and elevate IOP. However, the Gln368Stop mutation can interact indirectly with PTS1R by oligomerization with wild type MYOC to elevate IOP . According to the above study, Arg46Stop and Gln368Stop mutations are similar to each other because both of them lack the PTS1 site. Therefore, we assume the Arg46Stop mutant probably contributes to the elevation of IOP leading to the development of juvenile-onset open angle glaucoma. Further study is needed to confirm that whether the Arg46Stop mutation is the cause for the MYOC functional change and IOP elevation.
The heterozygous c.158T>C mutation (Val53Ala) present in some of our JOAG patients, but which was not found in any of the individuals in the control group, has also been reported in a number of Hong Kong patients with angle-closure glaucoma (Table 4) . However, to the best of our knowledge, our study represents the first case where an observed alanine substitution at codon 53 (Val53Ala) was found to be associated with the presence of JOAG. As a result, we suggest that such a substitution is a probable risk factor for the development of juvenile-onset open angle glaucoma. Further investigation is warranted before any definite conclusions can be made about this specific substitution's role in the pathogenesis of JOAG.
This study is, to the best of our knowledge, the first to detect one heterozygous mutation, located at the c.604+228A>T locus, among JOAG patients. However, the pathogenic potential of c.604+228A>T mutation is unknown because its effect on the functional change of the gene and the protein has not been determined. The neural network prediction system revealed that the c.604+228A>T mutation in MYOC creates a novel accept splice site in c.604+233-234AG. Based on the results from similar studies, in which mutations occurring in intronic regions have been found to cause various levels of normal and aberrant transcripts , we presume that the c.604+228A>T mutation affects the splicing of MYOC, and leads to a dysfunctional MYOC protein. However, we cannot rule out the possibility that the interaction of MYOC mutations with mutations of other genes results in loss of sight. It is also possible that the nucleotide changes represent a rare or uncommon polymorphism not associated with the disease phenotype in the patient. Further investigation will be needed to understand how interference of the mutation contributes to JOAG.
The promoter region of MYOC comprises multiple steroid hormone-responsive elements and regulatory motifs . Mutations in the MYOC promoter areas have been reported to be associated with POAG . According to Colomb et al.  and Polansky et al. , the presence of c.-1000C>G (MYOC.mt1 variant) of MYOC suggests that a common polymorphism located in the promoter region of MYOC is related to an individual's rather poor response to glaucoma therapy and the markedly increased risk of progression of the disease for such an individual as compared to other individuals who do not feature this polymorphism [30,31]. The MYOC.mt1 variant appears to be an indicator of visual field damage for diagnosed POAG patients. In contrast, our study is not consistent with the above studies. We found that there were no significant differences in allelic and phenotypic frequencies of the MYOC.mt1 variant between patients and controls. Therefore, our suggest that this variant (MYOC.mt1) is not associated with the risk of developing JOAG in Taiwanese.
In conclusion, we have provided comprehensive genetic information on MYOC from study subjects with JOAG in Taiwan. On the basis of the above results, we suggest that c.136C>T (Arg46Stop), c.158T>C (Val56Ala), c.604+228A>T, and c.1515+73G>C mutations of MYOC may be associated with the development of JOAG in Taiwanese.
We thank all of the subjects who participated in the present project. We also thank Ju-Chin Yan for his technical assistance. Preparation of the manuscript is partially supported by Ms Me-Li Chen, Medical Research Department, Chi-Mei Medical Center.
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