Molecular Analysis of the Myocilin Gene in Chinese Subjects with Chronic Primary-Angle Closure Glaucoma

Abstract

purpose. Mutations in the myocilin (MYOC) gene have been implicated in juvenile as well as late-onset primary open-angle glaucoma (POAG). Overall, MYOC mutations account for 3% to 5% of cases of POAG worldwide, making it the most significant gene identified so far in glaucoma. Although there are some similarities in the phenotype of POAG and in particular chronic primary angle-closure glaucoma (PACG), little is known about the role of MYOC in the causation of PACG. To address this, the MYOC gene was screened in a cohort of 106 patients with chronic PACG.

methods. Genomic DNA was extracted from leukocytes of the peripheral blood and exons 1 to 3 of the MYOC gene were PCR amplified and subjected to bidirectional sequencing and analysis.

results. One hundred six patients with chronic PACG of Chinese ethnicity were studied. Sequencing of the MYOC gene in these patients revealed eight sequence variants. Of these, one was a nonsense change, three were missense changes, two were synonymous codon changes, and two were changes in noncoding sequences. These included the Arg46Stop and Thr353Ile mutations, which have been reported in individuals with POAG. However, all the sequence alterations identified have been found in normal Chinese subjects.

conclusions. The results of this study do not support a role for MYOC mutations in the pathogenesis of chronic PACG in the Chinese.

Glaucoma, a group of heterogeneous optic neuropathies characterized by progressive visual field loss, is the leading cause of irreversible blindness worldwide. ^{1 2} Categorized according to the anatomy of the anterior chamber angle; there are two main forms of glaucoma: primary open-angle (POAG) and primary angle-closure (PACG) glaucoma. PACG is a major form of glaucoma in Asians, ^{3 4} compared with POAG, which is the predominant disease among whites and Africans. ^{5 6} The disease is responsible for most bilateral glaucoma-caused blindness in Singapore, China, and India, and it is estimated that PACG blinds more people than POAG worldwide. ^{9 10 11 12}  

Glaucoma has a major genetic basis, estimated to account for at least a third of all cases. ^{13 14 15 16} Genetic heterogeneity is illustrated by the >15 loci and seven glaucoma-causing genes identified to date. ¹⁷ Among them, two genes, MYOC encoding myocilin and OPTN encoding optineurin, have been identified as harboring mutations causing POAG. ^{18 19 20} Myocilin (MYOC, MIM 601652) at GLC1A on chromosome 1 at q21-q31 was the first gene to be identified for POAG. ^{19 20} The MYOC gene comprises three exons, and mutations have primarily been identified in the third exon of MYOC, which encodes the olfactomedin domain. MYOC mutations have been identified in populations of white, Asian, and African origin and overall, such mutations account for 3% to 5% of cases of adult POAG worldwide, ^{21 22 23 24 25} making it the most significant gene identified so far in glaucoma. 

The mechanism of action of myocilin in the causation of POAG is still unknown. MYOC is preferentially expressed in the anterior segment of the eye, where high amounts of myocilin mRNA have been detected in the trabecular meshwork (TM), sclera, ciliary body, and iris. ^{26 27 28 29} The finding that mutant MYOC proteins form aggregates that are not secreted suggests that mutant MYOC accumulates in TM cells and disturbs normal cellular function, resulting in impaired outflow of aqueous humor, elevated intraocular pressure (IOP), and glaucoma. ³⁰ However, as MYOC is expressed in the retina, it is also possible that MYOC causes glaucoma at the retinal ganglion cell level. 

The role of the myocilin gene in PACG remains to be established. Worldwide, the most common form of PACG is the chronic asymptomatic type, in which affected individuals have painless progressive visual loss associated with increased IOP and optic disc cupping. The clinical phenotype has some similarities to POAG, the main differences being the configuration of the angle and a stronger association between IOP and severity of optic neuropathy. ³¹ Ultrastructural analysis of the TM of patients with chronic PACG has shown changes in the TM similar to those seen in POAG, such as loss of endothelial cells and reactive repair processes. ³² Two recent studies in Canadian glaucoma probands reported the presence of MYOC mutations in a few individuals with PACG. ^{33 34} Of 17 subjects with PACG studied, Faucher et al. ³³ found two with MYOC mutations—one each with Pro481Leu and Gln368STOP. ³³ Vincent et al. ³⁴ also reported a patient with mixed POAG-PACG patient affected by the Gly399Val mutation. These reports provided preliminary evidence that PACG subjects may carry MYOC mutations, but were limited by a small sample size. The role of MYOC in PACG is worthy of investigation, as it may provide an insight into the pathogenesis of the glaucomatous disease process. The presence of MYOC mutations in subjects with PACG would imply a common mechanism of glaucomatous damage for both POAG and PACG. We therefore investigated the role of myocilin in PACG by screening the gene in a panel of 106 Chinese subjects with chronic PACG. 

Patients and Methods

Subjects with chronic PACG were recruited from the glaucoma service of the Singapore National Eye Centre and National University Hospital, Singapore. Written informed consent was obtained from all subjects, and the study had the approval of the Ethics Committees of the two hospitals and was performed according to the tenets of the Declaration of Helsinki. Standardized inclusion criteria for chronic PACG were used:

1.  
   The presence of glaucomatous optic neuropathy, which was defined as disc excavation with loss of neuroretinal rim tissue with a cup-to-disc ratio of 0.7 or greater, when examined with a 78-D biomicroscopic lens.
2.  
   Visual field loss detected with static automated white-on-white threshold perimetry (program 24-2 SITA, model 750; Humphrey Field Analyzer; Carl Zeiss Meditec, Dublin, CA) that is consistent with glaucomatous optic nerve damage. This was defined as Glaucoma Hemifield Test results outside normal limits and/or an abnormal pattern SD with P < 5% occurrence in the normal population.
3.  
   A closed angle on indentation gonioscopy. A closed angle was defined as the presence of at least a 180° angle in which the posterior pigmented TM was not visible on gonioscopy, and with evidence of peripheral anterior synechiae in any part of the angle.

Subjects with a history of acute symptomatic angle closure as well as cases of secondary angle closure such as neovascularization of the iris, uveitis, trauma, lens intumescence, or subluxation were excluded. 

Genomic DNA was extracted from leukocytes of the peripheral blood and exons 1 to 3 of the myocilin gene were amplified by polymerase chain reaction (PCR) with a thermocycler (DNA Theromocycler 9700; Applied Biosystems, Inc. [ABI], Foster City, CA). Primers were obtained according to previously published sequences. ³⁵ PCR reactions were performed in 50 μL reaction volumes containing 10 mM Tris-HCl, (pH 8.9), 50 mM KCl, 1.5 mM MgCl₂, 25 picomoles of each primer, 200 μM each dNTP, 50 to 100 ng of patient genomic DNA, and 0.7 U Taq thermostable DNA polymerase (Promega, Madison, WI). Cycling parameters were 3 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at the melting temperature (T_m) of the primers (52–62°C), and 30 seconds at 72°C, with a final 5-minute extension at 72°C. PCR products were purified using PCR clean-up columns (GFX; Amersham, Arlington Heights, IL). Sequence variations were identified by automated bidirectional sequencing with dye terminator chemistry (BigDye Terminator, ver. 3.1; ABI). An automated DNA sequencer (Prism 3100; ABI) was used. Primers for sequence reactions were the same as those for the PCR reaction. 

Results

A total of 106 subjects with chronic PACG were studied. All subjects were of Chinese ethnicity (Table 1) . There were 69 (65%) women, and the mean age was 73.5 ± 8.1 years (range, 54–94). 

Eight MYOC sequence alterations were identified in our study subjects (Table 2) . Of these, one was a nonsense change, three were missense changes, two were synonymous codon changes, and two were changes in noncoding sequences. Only one sequence change, an intronic variant (604+16G>T), was novel. All other sequence alterations have been reported earlier in Chinese samples. ^{35 36 37} The promoter polymorphism 1-83 G→A always occurred with the Arg76Lys polymorphism. This haplotype may be specific to the Asian population, since it has only been observed in Chinese ^{35 37} and Japanese ³⁸ populations. 

The Arg46Stop change was found in one patient with chronic PACG. The patient concerned was an 82-year-old lady with chronic PACG first diagnosed at age 75. At presentation, the vertical cup-to-disc ratio was 0.8 in both eyes, and gonioscopy revealed 360° of closed angles with extensive peripheral anterior synechiae. The axial length was 21.05 mm and anterior chamber depth 2.09 mm in the right eye. 

Discussion

In 1999, Fingert et al. ²⁵ defined a disease-causing mutation in MYOC as one that alters the amino acid sequence of MYOC, is present in one or more subjects with glaucoma but in <1% of the general population, and is absent in normal control subjects. According to these criteria, the results of this study indicate that MYOC mutations do not play a major role in the causation of chronic PACG, as none of the sequence variations found are thought to be disease causing. This is in contrast to POAG, where MYOC mutations are thought to account for 3% to 5% of all cases. ^{21 22 23 24 25} It is likely that POAG and PACG, though both leading to glaucomatous optic neuropathy, have different underlying mechanisms. In PACG, the main predisposing factor appears to be pretrabecular obstruction of the outflow channels. Glaucoma probably develops secondary to the high IOP induced by this obstruction. In contrast, pretrabecular obstruction is absent in POAG, and other factors are likely to be involved in the pathophysiology of the glaucomatous process, such as increased outflow resistance at the trabecular level ³⁹ or increased susceptibility of the optic nerve to damage from raised IOP. ⁴⁰  

Several sequence variations in the MYOC gene were identified in this panel of PACG subjects. The Arg46Stop sequence variant, found in one patient with PACG, has already been identified in POAG, ^{35 36 37} normal-tension glaucoma, ³⁸ and normal individuals. ^{35 37} Currently, there is some debate as to whether this variant is a disease-causing mutation, a polymorphism, or a modifier, with respect to glaucoma disease phenotype. ⁴¹ Yoon et al. ³⁶ first identified this change in a homozygote with POAG, which suggests autosomal recessive inheritance. However, Pang et al. ³⁷ later identified this change in a 77-year-old homozygous individual who had no POAG and later still in heterozygote individuals with and without POAG. The Arg46Stop variation may thus reduce the expression of MYOC by half in heterozygotes and eliminate expression in homozygotes. However, as this change was prevalent in 2.2% of normal Chinese individuals, ³⁷ we believe that this change is a polymorphism found commonly within populations of Chinese and Mongoloid descent. The possibility however remains that Arg46Stop, possibly in combination with other genetic or environmental influences, may affect the disease phenotype. ⁴¹  

The Thr353Ile change was found in three patients with PACG in this study. This change occurs in a residue located in the olfactomedin homology region of the protein, which is conserved in all mammalian MYOC protein sequences tested (data not shown). However, the fact that this change has been identified in normal individuals suggests it is non–disease-causing, though again the possibility remains that Thr353Ile also affects risk of PACG. 

Although the amounts of myocilin mRNA observed in the retina and optic nerve are considerably lower than in the anterior segment of the eye, ^{29 42} it is possible that MYOC not only acts on the TM but also on the optic nerve. Studies have shown that myocilin is expressed in astrocytes of the optic nerve head at the lamina cribrosa ^{42 43 44 45} and is a component of the myelin sheath that surrounds postlaminar optic nerve axons. ⁴⁵ The fact that MYOC knock-out mice grow normally with normal IOP and ocular morphology ⁴⁶ and that a person missing one copy of the MYOC gene had normal IOP and no glaucoma, ⁴⁷ suggests that total absence of myocilin is harmless to optic nerve function. It is important to investigate further the influence of normal and “mutant” myocilin on the optic nerve, since some variants of MYOC may still increase susceptibility to retinal ganglion cell damage at high IOP. Such studies may also resolve and identify common pathogenic processes between PACG and POAG. 

Since Tornquist ⁴⁸ first suggested that PACG was transmitted by a single, dominant gene in 1953, there has been a paucity of research into the genetic basis of PACG. To date, a genetic locus for PACG has not been published, and there have been no other reports of candidate-gene-association studies related to PACG. This is probably related to the high prevalence of the condition in populations in which glaucoma research has not been a major focus. There may be underreporting of the family history, as in POAG, ¹⁶ leading to the impression that most affected patients are isolated cases. The late onset of the disease and the lack of accurate clinical information on previous generations are further obstacles to determining the genetics of the disorder. It is hoped that further research into the genetic basis of PACG will lead to improved understanding of the molecular basis of this major worldwide cause of blindness. 

 Supported by grants from the National University of Singapore and the Singapore Eye Research Institute.

 Submitted for publication September 30, 2004; revised November 7, 2004; accepted November 20, 2004.

 Disclosure: T. Aung, None; V.H.K. Yong, None; P.T.K. Chew, None; S.K.L. Seah, None; G. Gazzard, None; P.J. Foster, None; E.N. Vithana, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Demographic Features of Study Subjects

Demographic Feature	No. of Study Subjects (n = 106)
Gender
Male	37 (35%)
Female	69 (65%)
Race
Chinese	106 (100%)
Age (ys)
Mean ± SD	73.5± 8.1
Range	54–94
Median	74

Table 2.

 

MYOC Sequence Alterations Detected in Patients with Chronic PACG

Sequence Change	Location in Gene	Prevalence of Change			P
				PACG Patients (n = 106) (%) [95% CI]		Control Subjects* (n = ≥291) (%) [95% CI]
1–83 G→A	Promoter	16 (15.1%) [8.9%–23.4%]	70/388 (18.0%) [14.3%–22.2%]	0.48
Gly12Arg (34G→C)	Exon 1	1 (0.94%) [0.02%–5.1%]	8/388 (2.1%) [0.9%–4.0%]	0.69
Arg46Stop (136C→T)	Exon 1	1 (0.94%) [0.02%–5.1%]	9/402 (2.2%) [1.0%–4.2%]	0.70
Arg76Lys (227G→A)	Exon 1	16 (15.1%) [8.9%–23.4%]	70/388 (18.0%) [14.3%–22.2%]	0.48
Thr123Thr (369 C→T)	Exon 1	1 (0.94%) [0.02%–5.1%]	5/291 (1.7%) [0.6%–4.0%]	1.0
604+16G→T	Intron 1	1 (0.94%) [0.02%–5.1%]	—	—
Ile288Ile (864C→T)	Exon 3	1 (0.94%) [0.02%–5.1%]	4/291 (1.4%) [0.4%–3.5%]	1.0
Thr353Ile (1058C→T)	Exon 3	3 (2.8%) [0.6%–8.0%]	6/388 (1.6%) [0.6%–3.3%]	0.41

 Data were obtained from Pang et al. ³⁷ who investigated the frequency of the MYOC variants in ≥291 Chinese control subjects at least 50 years of age.