The role of hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) C677T mutation in patients with retinal artery occlusion

The Role of Hyperhomocysteinemia and Methylenetetrahydrofolate Reductase (MTHFR) C677T Mutation in Patients With Retinal Artery Occlusion MARTIN WEGER, MD, OLAF STANGER, MD, HANNES DEUTSCHMANN, MD, FRANZ JOSEF LEITNER, MD, WILFRIED RENNER, PHD, OTTO SCHMUT, PHD, ¨ RGEN SEMMELROCK, PHD, AND ANTON HAAS, MD JU

● PURPOSE:

Hyperhomocysteinemia has been established as an important risk factor for cardiovascular diseases. The aim of the present study was to investigate whether hyperhomocysteinemia and/or homozygosity for the methylenetetrahydrofolate reductase (MTHFR) C677T mutation are associated with an increased risk for retinal artery occlusion (RAO). ● DESIGN: Retrospective case-control study. ● METHODS: We studied 105 consecutive patients with retinal artery occlusion and 105 age and sex-matched control subjects. Fasting plasma homocysteine levels were determined by high-performance liquid chromatography, while genotypes of the MTHFR C677T mutation were determined by polymerase chain reaction. ● RESULTS: Mean plasma homocysteine levels were significantly higher in patients with RAO compared with control subjects (12.2 ⴞ 4.8 ␮mol/l vs 10.3 ⴞ 3.4 ␮mol/l; P ⴝ .003). Hyperhomocysteinemia was defined by the 95th percentile of control plasma homocysteine levels as 15.8 ␮mol/l. Twenty (19.1%) patients with RAO exceeded this level and were therefore classified as hyperhomocysteinemic compared with 5 (4.8%) control subjects (P ⴝ .003). The odds ratio for these patients was calculated at 4.7 (95% confidence interval [CI], 1.5–15.1). Mean plasma folate levels were significantly lower in patients than in the control group (5.6 ⴞ 2.3 ng/ml vs. 6.3 ⴞ 2.5 ng/ml; P ⴝ .04). The prevalence of the homozygous genotype of methylenetetrahydrofolate Accepted for publication March 3, 2002. From the Department of Ophthalmology (M.W., F.J.L., O.S., A.H.), Department of Cardiac Surgery, Atherothrombosis Research Group (O.S.), Division of Angiology, Department of Internal Medicine (W.R.), and the Department of Laboratory Medicine (J.S.), Karl-Franzens University, Graz, Austria. Reprint requests to Martin Weger, MD, Department of Ophthalmology, Auenbruggerplatz 4, A-8036 Graz, Austria; fax: (⫹43) 316-3853261; e-mail: [email protected] 0002-9394/02/$22.00 PII S0002-9394(02)01471-X

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reductase C677T mutation did not significantly differ between patients and controls. ● CONCLUSIONS: Our results suggest that hyperhomocysteinemia, but not homozygosity, for the MTHFR C677T mutation is associated with RAO. (Am J Ophthalmol 2002;134:57– 61. © 2002 by Elsevier Science Inc. All rights reserved.)

R

ETINAL ARTERY OCCLUSION (RAO) IS A COMMON

vision-threatening disease affecting primarily patients older than 60 years. Occlusion of the central retinal artery or one of its branches leads to ischemic infarction of the affected retinal tissue. Embolism, hemorrhage under an atheroslerotic plaque, and intraluminal thrombosis, have been suggested to play a major role in the pathomechanism of RAO.1 Arterial hypertension, diabetes mellitus, cardiac valvular disease, carotid atherosclerosis, and elevated plasma lipoprotein(a) levels have been identified, among others, as risk factors.2,3 Nevertheless, not all cases can be fully explained by the known risk factors alone. Therefore, identification of other risk factors seems to be essential. Mildly elevated plasma levels of homocysteine were found to be associated with atherosclerosis, venous thrombosis, myocardial infarction, carotid artery stenosis, and stroke, suggesting a role for homocysteine in the pathogenesis of atherothrombotic disease.4 –7 Homocysteine is a sulfur-containing amino acid derived from methionine metabolism. Elevated plasma concentrations are caused by a variety of factors, including renal insufficiency, nutritional deficiencies (such as folate, vitamin B6, and vitamin B12), medications, and genetic polymorphisms of the enzymes involved in homocysteine metabolism.8,9 Intracellularly, homocysteine is either reconverted to methionine through the remethylation pathway or degraded to cysteine and cystathionine through the trans-

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sulfuration pathway.8 Remethylation to methionine depends on the availability of 5-methyltetrahydrofolate, which is formed by the riboflavin-dependent enzyme, 5-10 methylenetetrahydrofolate reductase (MTHFR). Kang and associates identified a common qualitative variant of the MTHFR enzyme resulting from a single amino acid substitution (alanin to valin, caused by a 677C 3 T nucleotide exchange).10 This point mutation is characterized by its in vitro heat sensitivity and an approximately 50% lower enzyme activity compared with normal.11 Prevalence for homozygosity of the MTHFR C677T mutation was determined at 5% to 15% in subjects of Caucasian descent.12,13 However, previous reports on an association between the C677T MTHFR variant and cardiovascular diseases have yielded conflicting results and the role of this common genetic polymorphism thus remains unclear.14,15 In recent studies hyperhomocysteinemia was suggested to be a risk factor for RAO.16,17 However, the number of patients enrolled in these studies was small and only one study group reported the distribution of MTHFR C677T genotypes.18 The aim of our study was therefore to investigate whether both hyperhomocysteinemia and MTHFR C677T mutation are associated with RAO in a larger cohort of patients.

intake of vitamin B12/B6, folate, and medications known to influence plasma homocysteine concentrations such as estrogens, carbamazepine, phenytoin, antifolates (methotrexate, trimethoprim), and tricyclic antidepressants were excluded from the study.9,19 According to the exclusion criteria, another 46 patients diagnosed with RAO were not enrolled in this study (22 for taking one or more of the above-cited medications, eight for malignancy, eight for impaired renal function, four for refusal to take part in the study, and four suffered from arteritis temporalis). Arterial hypertension was defined by a history of arterial hypertension, systolic blood pressure ⱖ 160 mm Hg and/or diastolic blood pressure ⱖ 95 mm Hg and/or the intake of antihypertensive drugs. Subjects were classified as diabetics when being treated for insulin or noninsulin-dependent diabetes mellitus. Myocardial infarction was defined by a history of myocardial infarction confirmed by electrocardiographic findings. Subjects were classified as having suffered from stroke by a history of stroke with neurologic deficits lasting longer than 24 hours. According to their smoking status subjects were defined as non, ex-, and current smokers (ex-smokers had to have stopped smoking for at least 1 year). Blood samples were drawn from the antecubital vein between 7:00 AM and 8:00 AM after an overnight fast of at least 8 hours. Samples for homocysteine determination were processed immediately, centrifuged at 4 C (3000 g for 10 minutes), and stored at ⫺70 C until analysis. Measurements of plasma homocysteine in ethylene diamine tetraacetic acid plasma were performed using high performance liquid chromatography and fluorescence detection according to the method of Araki with modifications by Ubbink and Vester.20 –22 This procedure involved a reducing step, thus the method did not distinguish between homocysteine and its oxidized analogues. Vitamin B12 and folate were determined with an Abbott AxSym-analyzer using a Microparticle Enzyme Immunoassay (vitamin B12) or an “ion capture” technology (folate). Genomic DNA was extracted from peripheral blood lymphocytes by standard techniques, and the MTHFR mutation analysis was performed by polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP) according to Frosst and associates12 by a technician unaware of the status of the DNA sample. Descriptive statistics were used to calculate frequencies and percentages of discrete variables. Continuous data are given as mean ⫾ standard deviation (SD). We performed the Kolmogorov–Smirnov test to assess normal distribution and the Levine test to assess homogeneity of variances. Means were compared using the Mann–Whitney rank-sum test and proportions were compared using ␹2 test statistics. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression analysis. A P value ⬍ .05 was considered to be significant. Statistical

DESIGN THIS STUDY WAS DESIGNED AS A RETROSPECTIVE CASE-

control study.

METHODS THE PRESENT RETROSPECTIVE CASE-CONTROL STUDY IN-

cluded 105 consecutive patients with RAO and 105 controls matched for age and sex. Patients diagnosed with RAO between January 1996 and December 2001 were eligible for inclusion in the study. All participants were examined at the Department of Ophthalmology and gave written informed consent before enrollment. The study was approved by the Ethics Committee of the KarlFranzens University, Graz, Austria. The diagnosis of RAO was made by ophthalmoscopic fundus examination revealing superficial retinal whitening in the distribution of the involved retinal artery. Accordingly, 49 patients were classified as suffering from branch retinal artery occlusion (BRAO) and 56 from central retinal artery occlusion (CRAO). The control group included 105 sex and age- (age of controls, ⫾ 2 years) matched consecutive subjects, who were referred to our department for other reasons than retinal vascular occlusion, anterior ischemic optic neuropathy, and vasculitis. Patients and control subjects were matched one to one. Subjects with renal and liver dysfunction, malignancy, 58

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TABLE 1. Baseline Characteristics of Patients With RAO and Controls

Female Male Mean age (years ⫾ SD) Range (years) Risk factors Arterial hypertension Diabetes mellitus Stroke Myocardial infarction Smoking status Past Current Never

RAO (n ⫽ 105)

Controls (n ⫽ 105)

46 (43.8) 59 (56.2) 69.1 ⫾ 10.6 40–89

46 (43.8) 59 (56.2) 68.9 ⫾ 10.6 39–89

70 (66.5) 15 (14.3) 17 (16.1) 12 (11.4)

44 (41.8) 19 (18.1) 3 (2.9) 8 (7.6)

37 (35.2) 21 (20.0) 47 (44.8)

25 (23.8) 23 (21.9) 57 (54.3)

TABLE 2. Mean Plasma Levels of Homocysteine, Folate, and Vitamin B12 in Patients and Controls RAO (n ⫽ 105)

P Value

Controls (n ⫽ 105)

Homocysteine (␮mol/l) 12.2 ⫾ 4.8 10.3 ⫾ 3.4 Plasma folate (ng/ml) 5.6 ⫾ 2.3 6.3 ⫾ 2.5 490.9 ⫾ 300.1 457.1 ⫾ 248.1 Vitamin B12 (pg/ml)

.001 .387 .001 .310 .866

Significance P Value

.003 .040 .548

RAO ⫽ retinal artery occlusion. Results are given as mean ⫾ SD. Normal range for plasma folate, 5.3– 11.7 ng/ml; for vitamin B12, 159 –1057 pg/ml. Hyperhomocysteinemia was defined as a plasma homocysteine concentration of ⱖ15.8 ␮mol/l.

RAO ⫽ retinal artery occlusion; SD ⫽ standard deviation. Numbers are given as n (%).

analysis was performed with the SPSS statistical package (SPSS, version 9.0.1998, Chicago, Illinois, USA).

RESULTS THE STUDY GROUP CONSISTED OF 105 PATIENTS (46 FE-

males and 59 males). The mean age of patients was 69.1 ⫾ 10.6 years (range, 40 – 89 years) and 68.9 ⫾ 10.6 years (range, 39 – 89 years) in controls, respectively. The mean time interval between occurrence of RAO and blood sampling for plasma homocysteine was 14.2 months (range, 1– 46 months). Baseline parameters and clinical characteristics of both groups are shown in Table 1. Arterial hypertension and stroke were significantly more frequent in the RAO group than in the control group (Table 1). Mean plasma homocysteine levels were significantly higher in patients than in controls (Table 2). Distribution of plasma homocysteine concentrations of both patients and controls is shown in Figure 1. Hyperhomocysteinemia defined by the 95th percentile of plasma homocysteine levels in the control group was determined as ⱖ 15.8 ␮mol/l. Twenty (19.1%) patients were therefore classified as hyperhomocysteinemic compared with 5 (4.8%) controls (P ⫽ .003). The odds ratio for these patients was 4.7 (95% CI, 1.5–15.1). An increase of 1 ␮mol/l of plasma homocysteine was associated with an odds ratio of 1.12 (95% CI, 1.04 –1.21). Adjustment for arterial hypertension and stroke did not significantly alter this calculated odds ratio (OR, 1.08; 95% CI, 1.01–1.17). Mean plasma folate levels were significantly lower in patients compared with controls (Table 2). Subnormal VOL. 134, NO. 1

FIGURE 1. Total plasma homocysteine levels for cases and control patients.

plasma folate concentrations defined by a cut-off level of 5.3 ng/ml were found more frequently among patients with RAO than in control subjects (55 patients with RAO vs 42 control subjects; P ⫽ .09). In contrast, mean vitamin B12 levels did not significantly differ between patients and controls (Table 2). Genotype distribution of the MTHFR C677T mutation among patients with RAO and control subjects did not reveal any significant difference: Frequencies of CC, CT, and TT genotypes were 46 (43.8%), 46 (43.8%), and 13 (12.4%) among patients and 49 (46.7%), 46 (43.8%), and 10 (9.5%) among controls (P ⫽ .78).

DISCUSSION OUR STUDY DEMONSTRATES THAT HYPERHOMOCYSTEINE-

mia is associated with RAO. However, the prevalence of the homozygous genotype of MTHFR C677T mutation does not significantly differ between patients and controls. Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases, comparable to smoking and hyper-

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cholesterolemia.6,7 Various vitamin deficiencies such as low plasma folate and vitamin B12 levels are known to increase plasma homocysteine levels.23 In fact, plasma folate and homocysteine concentrations are inversely correlated and also age dependent. In the present study, mean plasma folate levels were significantly lower in patients with RAO compared with control subjects. Therefore, the higher plasma homocysteine levels may reflect relative folate deficiency in these patients. In contrast, mean plasma vitamin B12 levels did not differ significantly between both groups. An increase of 1 ␮mol/l of plasma homocysteine was associated with an odds ratio of 1.12. This effect was not substantially altered after adjustment for arterial hypertension and stroke, which were found significantly more often in patients than in controls. Homozygosity for the MTHFR 677T mutation has been suggested to cause hyperhomocysteinemia, especially in the presence of suboptimal folate intake.13–15,24 The prevalence of this mutation varies widely, ranging from 5% in Scandinavian countries to approximately 15% in Italy.4,24 We found a prevalence of 12.4% in our cohort of Austrians, further supporting the hypothesis of a geographic increase from North to South. In the present study, however, no significant difference in the distribution of MTHFR C677T genotypes could be observed between patients with RAO and control subjects. Thus, our results support the findings of Cahill and associates, who did not observe an increased prevalence of the mutation in Irish patients with RAO.18 We therefore assume that the MTHFR C677T mutation is not associated with RAO. The precise pathomechanism by which hyperhomocysteinemia leads to atherosclerosis is still unknown, but numerous studies have suggested that increased plasma homocysteine levels lead to endothelial dysfunction resulting in dysregulation of vascular tone, impaired generation and bioavailability of nitric oxide, increased proliferation of smooth muscle cells, and increased oxidative stress, which all play an important role in the development and progression of atherosclerosis.25–30 Reducing plasma homocysteine levels by 25% is easily achieved by a low dose of folic acid.9 Thus, treatment of hyperhomocysteinemia with folic acid can be considered to be effective, cheap, and safe. Moreover, folic acid supplementation has been shown to improve homocysteine-induced endothelial dysfunction.31,32 A potential limitation of our study may be its retrospective design. Patients were recruited some time after the occurrence of RAO, and measured plasma homocysteine may therefore not reflect plasma homocysteine concentrations at the time of occurrence of RAO. Thus, our study does not prove a causal role for hyperhomocysteinemia in the pathogenesis of RAO. The prevalence of stroke and arterial hypertension was significantly higher among patients with RAO than among controls. Nevertheless, in a logistic regression analysis including 60

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these factors, elevated plasma homocysteine levels remained an independent and significant risk factor for RAO. Prospective studies are warranted to further investigate the role of hyperhomocysteinemia and its contribution to the risk of RAO. ACKNOWLEDGMENTS

We thank Ms Gabriele Trummer, Ms Christa Wachswender, and Ms Anna Forjanics for their excellent technical assistance.

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