|Molecular Vision 2006;
Received 19 April 2006 | Accepted 2 July 2006 | Published 7 July 2006
Methylenetetrahydrofolate reductase gene polymorphisms c.677C/T and c.1298A/C are not associated with open angle glaucoma
Departments of 1Ophthalmology and 2Health Sciences, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan
Correspondence to: Fumihiko Mabuchi, MD, PhD, Department of Ophthalmology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo-shi, Yamanashi, 409-3898, Japan; Phone: 81-55-273-9657; FAX: 81-55-273-6757; email: email@example.com
Purpose: To assess whether or not the c.677C/T and c.1298A/C genetic polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with open angle glaucoma (OAG).
Methods: Genomic DNA was examined in a cohort of 131 Japanese patients with normal tension glaucoma (NTG), 133 patients with primary open angle glaucoma (POAG), and 106 control subjects. The mean age at the time of blood sampling was 62.8±13.3 years (mean±SD) in the patients with NTG, 61.8±15.4 years in the patients with POAG, and 65.0±10.5 years in the control subjects. MTHFR c.677C/T and c.1298A/C genotype and allele frequencies were determined using pyrosequencing analysis, and the findings were compared between the OAG patients and control subjects. The frequencies of compound MTHFR c.677C/T and c.1298A/C genotypes were also compared between OAG patients and control subjects.
Results: No significant differences were observed (p>0.05, χ2 test or Fisher's exact test) regarding the MTHFR c.677C/T genotype (TT: 14.5%, CT: 44.3%, CC: 41.2% for patients with NTG; TT: 20.3%, CT: 41.4%, CC: 38.3% for patients with POAG; TT: 17.9%, CT: 36.8%, CC: 45.3% for control subjects) and c.1298A/C (CC: 0%, AC: 38.9%, AA: 61.1% for patients with NTG; CC: 2.3%, AC: 32.3%, AA: 65.4% for patients with POAG; CC: 0.9%, AC: 41.5%, AA: 57.6% for control subjects). There were no allele frequencies between the NTG or POAG patients and the control subjects. In addition, no significant differences (p>0.05, χ2 test) were found in the frequencies of the compound MTHFR c.677C/T and c.1298A/C genotypes between the NTG or POAG patients and the control subjects.
Conclusions: The MTHFR c.677C/T and c.1298A/C polymorphisms were not found to be associated with NTG and POAG. Further studies in the different ethnic populations should be performed to elucidate the relationship between MTHFR and OAG.
Glaucoma is a major cause of blindness, affecting 70 million individuals worldwide . Open angle glaucoma (OAG) is the most common form of glaucoma, and it is clinically characterized by progressive optic neuropathy and visual field changes corresponding to the excavation of the optic disc and an open anterior chamber angle. As the pathogenesis of glaucomatous optic neuropathy, elevated intraocular pressure (IOP) is considered to be a major risk factor . In addition, the risk factors for OAG include age, ethnicity, refractive error, a family history of glaucoma, and some aspects of vascular function . Although OAG is caused by mutations in myocilin , optineurin , or WDR36  in a small proportion of families with autosomal dominant inheritance, these mutations are rare in patients with sporadic OAG. It is generally acknowledged that OAG is a complex trait and multiple genes are thus considered to contribute to the phenotype and increase the individual susceptibility to glaucomatous optic neuropathy .
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate, which is required for the remethylation of homocysteine to methionine. Polymorphisms in the MTHFR gene, such as c.677C/T and c.1298A/C, are associated with a reduced enzyme activity and mild hyperhomocysteinemia . Recently, hyperhomocysteinemia has been shown to be associated with primary open angle glaucoma (POAG) and capsular glaucoma . Junemann et al.  reported higher prevalence of the C677T variant in POAG, thus suggesting that this variant in MTHFR is a genetic risk factor for POAG. However, as of yet, no studies regarding the effects of MTHFR genotype on normal tension glaucoma (NTG) have been performed. In addition, there have also been no studies that assessed the association between the c.1298A/C genotype and OAG. Previous studies on MTHFR and POAG was exclusively performed on white populations, and there have so far been no studies on other populations.
In this study, we investigated whether these two polymorphisms in MTHFR were associated with POAG and NTG in the Japanese population.
Japanese patients with OAG were recruited from ophthalmology clinics from the University of Yamanashi Hospital, Enzan City Hospital, Uenohara Town Hospital, and Oizumi Clinic in Yamanashi or Nagano prefectures, Japan, using criteria previously described . The diagnosis of OAG required open angles on a gonioscopic examination, typical glaucomatous cupping of the optic disc (diffuse or focal thinning of the disc rims), and visual field defects characteristic of glaucoma by automated static perimetry (Humphrey Visual Field Analyzer 30-2, Humphrey Instruments, San Leandro, CA). In addition, patients with POAG showed evidence of at least one previous measurement of IOP that was more than 21 mmHg with a Goldman applanation tomometer. Patients with NTG showed an IOP of less than 21 mmHg each time they were tested (at least three IOP measurements before treatment), and no signs of intracranial disease that would cause optic nerve atrophy in either the ray computerized tomography or magnetic resonance imaging findings. Patients were excluded if they had any other ocular diseases that would have caused an increase in the IOP, such as a period of steroid administration, trauma or uveitis, or if they had a history of eye surgery before the diagnosis of glaucoma.
As control subjects, Japanese patients with eye problems unrelated to OAG or optic nerve diseases, such as refractive error or cataract, were recruited from the participant institutions. The control subjects were over 40 years old, had an IOP of below 21 mmHg (at least one IOP measurement), had no glaucomatous cupping of the optic disc (no thinning of the disc rim, and cup-to-disc ratio less than 0.4), and had no family or personal history of glaucoma. All participants received a comprehensive ophthalmologic examination and had peripheral blood drawn. The study protocol was approved by the Ethics Committee of the University of Yamanashi, and informed consent was obtained from all study participants. The study was conducted in accordance with the Declaration of Helsinki.
Genomic DNA genotyping
Genomic DNA was purified with a FlexiGene® DNA Kit (Qiagen, Valencia, CA). The MTHFR c.677C/T and c.1298A/C genotypes were determined using pyrosequencing analysis. The following primers were used for amplification and sequencing (5'-end biotinylation for the PCR forward primer was performed): c.677C>T (Ala222Val; rs1801133); PCR forward primer-biotin labeled 5'-Bio-AGT CAT GAG CCC AGC CAC TC-3'; PCR reverse primer 5'-GTA AGC AAC GCT GTG CAA GTT C-3'; sequencing reverse primer 5'-CGT GAT GAT GAA ATC G-3'; c.1298A>C (Glu429Ala; rs1801131); PCR forward primer biotin labeled 5'-Bio-CTT TGG GGA GCT GAA GGA C-3'; PCR reverse primer 5'-CAT TCC GGT TTG GTT CTC C-3'; sequencing reverse primer 5'-AAC AAA GAC TTC AAA GAC AC-3'.
PCR reactions were performed in a total volume of 25 μl containing 10 ng genomic DNA, 5 pmol of each primer, 2.5 mM of each dNTP, 20 mM of MgCl2, and 1.25 U of Taq polymerase (TaKaRa Ex TaqTM, Takara Bio Inc., Otsu, Shiga, Japan). Amplification was carried out with the initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. A final extension at 72 °C for 7 min completed the reactions. The preparation of single-stranded DNA is necessary prior to pyrosequencing. A solution of 25 μl of biotinylated PCR product combined with 40 μl of 2X binding buffer (10 mM Tris-HCl, 2 M NaCl, 1 mM EDTA, 0.1% Tween® 20 [pH 7.6]) was immobilized onto streptavidin-coated sepharose beads (Amersham Biosciences, Amersham Place, Buckinghamshire, UK) by incubation at room temperature for 10 min with constant agitation (1,400 rpm) to keep the beads dispersed. With a Vacuum Prep Tool (Biotage AB, Uppsala, Sweden), a vacuum was applied and the beads with immobilized PCR products were moved to a separate trough, where 70% ethanol was aspirated through the filter probes. The prep tool was then placed in a trough of 0.2 M NaOH to denature and release the single-stranded DNA while 5'-biotinylated strands remained immobilized on the beads. Next, the beads were washed (10 mM Tris-acetate buffer [pH 7.6]) and transferred to a 96 well PSQ96 plate (Biotage AB) containing 40 μl of 1X annealing buffer (20 mM Tris-acetate, 2 mM Mg-acetate [pH 7.6]) and 10 pmol of sequencing primer per sample. With the vacuum pressure switched off, a gentle shake of the prep tool released the beads into the PSQ96 plate, which was heated (90 °C, 2 min) and then left to cool at room temperature to allow the annealing of the sequencing primer. Pyrosequencing was performed at 28 °C using an automated pyrosequencer (PSQ96MA; Biotage AB). The PSQ96 plate was placed into the process chamber of a PSQ96MA instrument and enzymes, substrates, and nucleotides from the PyroGold Reagent (Biotage AB) were dispensed. A charge-coupled device (CCD) camera registered the light emitted from each incorporated nucleotide. An analysis of the programs was performed with PSQ96MA SNP (version 2.1) software (Biotage AB).
Data were analyzed using SAS statistical software (version 9.1, SAS Institute Inc., Cary, NC). χ2 analysis of the Hardy-Weinberg equilibrium for MTHFR genotypes were performed for patients and controls. Genotypic and allelic frequency differences were estimated by a χ2 test and a Fisher's exact test, respectively. Statistical analyses of the compound MTHFR c.677C/T and c.1298A/C genotypes were conducted using the χ2 test. A value of p<0.05 was considered to be statistically significant.
This study enrolled 131 Japanese patients with NTG, 133 patients with POAG, and 106 control subjects. The mean age at the time of blood sampling was 62.8±13.3 years (mean±SD) in the patients with NTG, 61.8±15.4 years in the patients with POAG, and 65.0±10.5 years in the control subjects. The mean age at the time of diagnosis was 56.8±13.7 and 53.7±16.0 years in patients with NTG and POAG, respectively. The mean and standard deviation of the maximum known IOP was 18.5±1.9 and 29.2±9.7 mmHg in patients with NTG and POAG patients, respectively.
The MTHFR genotype and allele frequencies were in Hardy-Weinberg equilibrium in both the patients with NTG and POAG and the control subjects. The genotype and allele frequencies of MTHFR c.677C/T and c.1298A/C polymorphisms in the OAG patients and the control subjects are shown in Table 1. There were no significant differences in the MTHFR genotype and allele frequencies between the NTG or POAG patients and control subjects. The frequencies of the compound MTHFR c.677C/T and c.1298A/C genotypes in the OAG patients and the control subjects are shown in Table 2. There were no significant differences in the compound MTHFR genotype frequencies between the NTG or POAG patients and control subjects.
In the current study, no significant differences were found between the genotype and allele frequencies of the MTHFR c.677C/T and c.1298A/C polymorphisms among POAG patients and control subjects. In addition, the frequencies of the compound MTHFR c.677C/T and c.1298A/C genotypes were similar among these diagnostic groups. The statistical power was above 89% to detect any significant difference of the allele frequencies among the diagnostic groups at α=0.05. These results contrast with those of Junemann et al. , who found a significantly higher prevalence of the c.677C/T polymorphism in 76 Caucasian patients with POAG in comparison to 71 control subjects. There are several possible explanations for these discrepancies. The finding of Junemann et al.  may have possibly been false positive because of their small cohort size, although our cohort size is also a limitation in the current study. MTHFR might have a more obvious effect in populations exposed to different environmental factors or with a different genetic background. Ethnic differences in the MTHFR allele frequencies have been reported [12,13]. The reason for dissimilar findings may reflect the ethnic difference in MTHFR allele frequencies. These findings indicate that the association between MTHFR and POAG may thus vary among different ethnic groups. An alternative explanation may be a linkage disequilibrium with the effect of a nearby polymorphism and a different case definition of the patients in each study. Homozygous or heterozygous MTHFR deficiencies are the most common etiological factors known to cause moderate hyperhomocysteinemia . If the activity of wild-type cDNA were designated to be 100%, then polymorphic enzymes containing the c.1298A/C and c.677C/T polymorphisms in the MTHFR gene separately would be considered to have 68% and 45% of the control activity, respectively, while the enzyme containing both polymorphisms would have 41% of the control activity . Bleich et al.  described increased levels of plasma homocysteine in patients with POAG. However, no significant difference was observed in the plasma homocysteine levels among the POAG patients and control subjects [15,16], and the association between homocysteine and POAG remains controversial. We performed a meta-analysis by adding the data of the current study to those of Junemann et al. , and the positive association was still found between the genotype and allele frequencies of the MTHFR c.677C/T polymorphisms among POAG patients and the control subjects. Larger-scale prospective studies in other ethnic populations should thus be performed to resolve any conflicting results regarding the association between the plasma homocysteine levels, MTHFR polymorphisms, and POAG.
Hyperhomocysteinemia has been reported to be associated with an increased incidence and progression of vascular diseases such as cardiovascular disease , arterial occlusive disease , atherosclerosis , and retinal vein occlusion . There is evidence that vascular deficits may contribute to the initiation and progression of OAG , and vascular deficits by hyperhomocysteinemia may also be associated with glaucomatous optic neuropathy, especially NTG. Moore et al.  showed that homocysteine was toxic to retinal ganglion cells (RGCs), while also inducing apoptotic cell death in RGCs by the overstimulation of N-methyl-D-aspartate (NMDA) receptors and caspase-3 activation. They also reported that this excitotoxic damage to RGCs was potentiated by the simultaneous elevation of homocysteine and glutamate . These findings suggest that hyperhomocysteinemia may contribute to RGC death by inducing apoptosis or excitotoxicity in patients with NTG. These reports have led to the hypothesis that MTHFR could be related to optic nerve damage in patients with NTG, and, in addition to POAG, we also investigated the association between the MTHFR polymorphisms and NTG. However, no significant differences were found between the genotype and allele frequencies of the MTHFR c.677C/T and c.1298A/C polymorphisms among the NTG patients and control subjects. The homocysteine concentrations used in many of the previous experiments were in the pharmacologic range and therefore may not be relevant to the levels encountered in clinical practice. To investigate the plasma homocysteine level in patients with NTG, it will therefore be necessary to elucidate the relationship between homocysteine and NTG.
We would like to thank Asaho Adachi, Ryoko Matsuda, and Chiho Yoshida from the SC BioSciences Corporation for technical support in determining the genotypes. This work was supported in part by Grant-in-Aid (Number 12670348) from the Ministry of Education, Science, Sports, and Culture, Japan.
1. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol 1996; 80:389-93.
2. Anderson DR. Glaucoma: the damage caused by pressure. XLVI Edward Jackson memorial lecture. Am J Ophthalmol 1989; 108:485-95.
3. Sommer A, Tielsch JM, Katz J, Quigley HA, Gottsch JD, Javitt J, Singh K. Relationship between intraocular pressure and primary open angle glaucoma among white and black Americans. The Baltimore Eye Survey. Arch Ophthalmol 1991; 109:1090-5.
4. Stone EM, Fingert JH, Alward WL, Nguyen TD, Polansky JR, Sunden SL, Nishimura D, Clark AF, Nystuen A, Nichols BE, Mackey DA, Ritch R, Kalenak JW, Craven ER, Sheffield VC. Identification of a gene that causes primary open angle glaucoma. Science 1997; 275:668-70.
5. Rezaie T, Child A, Hitchings R, Brice G, Miller L, Coca-Prados M, Heon E, Krupin T, Ritch R, Kreutzer D, Crick RP, Sarfarazi M. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 2002; 295:1077-9.
6. Monemi S, Spaeth G, DaSilva A, Popinchalk S, Ilitchev E, Liebmann J, Ritch R, Heon E, Crick RP, Child A, Sarfarazi M. Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1. Hum Mol Genet 2005; 14:725-33.
7. Friedman JS, Walter MA. Glaucoma genetics, present and future. Clin Genet 1999; 55:71-9.
8. Weisberg IS, Jacques PF, Selhub J, Bostom AG, Chen Z, Curtis Ellison R, Eckfeldt JH, Rozen R. The 1298A-->C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis 2001; 156:409-15.
9. Bleich S, Junemann A, von Ahsen N, Lausen B, Ritter K, Beck G, Naumann GO, Kornhuber J. Homocysteine and risk of open-angle glaucoma. J Neural Transm 2002; 109:1499-504.
10. Junemann AG, von Ahsen N, Reulbach U, Roedl J, Bonsch D, Kornhuber J, Kruse FE, Bleich S. C677T variant in the methylentetrahydrofolate reductase gene is a genetic risk factor for primary open-angle glaucoma. Am J Ophthalmol 2005; 139:721-3.
11. Mabuchi F, Tang S, Ando D, Yamakita M, Wang J, Kashiwagi K, Yamagata Z, Iijima H, Tsukahara S. The apolipoprotein E gene polymorphism is associated with open angle glaucoma in the Japanese population. Mol Vis 2005; 11:609-12 <http://www.molvis.org/molvis/v11/a72/>.
12. Rady PL, Szucs S, Grady J, Hudnall SD, Kellner LH, Nitowsky H, Tyring SK, Matalon RK. Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) in ethnic populations in Texas; a report of a novel MTHFR polymorphic site, G1793A. Am J Med Genet 2002; 107:162-8.
13. Esfahani ST, Cogger EA, Caudill MA. Heterogeneity in the prevalence of methylenetetrahydrofolate reductase gene polymorphisms in women of different ethnic groups. J Am Diet Assoc 2003; 103:200-7.
14. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10:111-3.
15. Wang G, Medeiros FA, Barshop BA, Weinreb RN. Total plasma homocysteine and primary open-angle glaucoma. Am J Ophthalmol 2004; 137:401-6.
16. Altintas O, Maral H, Yuksel N, Karabas VL, Dillioglugil MO, Caglar Y. Homocysteine and nitric oxide levels in plasma of patients with pseudoexfoliation syndrome, pseudoexfoliation glaucoma, and primary open-angle glaucoma. Graefes Arch Clin Exp Ophthalmol 2005; 243:677-83.
17. Jacobsen DW. Homocysteine and vitamins in cardiovascular disease. Clin Chem 1998; 44:1833-43.
18. Aronow WS, Ahn C. Association between plasma homocysteine and peripheral arterial disease in older persons. Coron Artery Dis 1998; 9:49-50.
19. Temple ME, Luzier AB, Kazierad DJ. Homocysteine as a risk factor for atherosclerosis. Ann Pharmacother 2000; 34:57-65.
20. Chua B, Kifley A, Wong TY, Mitchell P. Homocysteine and retinal vein occlusion: a population-based study. Am J Ophthalmol 2005; 139:181-2.
21. Chung HS, Harris A, Evans DW, Kagemann L, Garzozi HJ, Martin B. Vascular aspects in the pathophysiology of glaucomatous optic neuropathy. Surv Ophthalmol 1999; 43:S43-50.
22. Moore P, El-sherbeny A, Roon P, Schoenlein PV, Ganapathy V, Smith SB. Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine. Exp Eye Res 2001; 73:45-57.