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Vascular calcification (VC) is a common manifestation of end-stage renal disease (ESRD) (1, 2). The presence of VC is associated with aortic stiffness and is predictive of subsequent cardiovascular disease (CVD) and increased mortality (2). The exact pathophysiology of VC in ESRD patients is unclear. Recent studies have reported that VC is an active process regulated by various genes and proteins (2). The 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with decreasing enzyme activity and increasing homocysteine (Hcy) levels (3). Elevated plasma Hcy is one of the suggested risk factors for atherosclerosis due to endothelial dysfunction and oxidative stress (4). There is evidence for an association between increased plasma Hcy and the MTHFR polymorphism with an increased risk for developing CVD (5, 6). However, few studies have reported on the relationship between the MTHFR C677T polymorphism and VC in patients on chronic hemodialysis. Therefore, the goal of this study was to evaluate the degree of VC (7) and analyze the association with the MTHFR C677T polymorphism.
After obtaining approval from the Institutional Review Board of Bundang CHA General Hospital, we recruited 152 patients. Inclusion criteria were ages 20-90 yr and the diagnosis of ESRD in patients that received chronic hemodialysis treatments for more than 3 months. Patients with acute infectious diseases or unstable vital signs were excluded. All patients provided written informed consent.
VC was evaluated by examining plain radiography of the pelvis and hands (7). The total final scores ranged from 0 to 8. Arterial stiffness was assessed using a commercially available device (VP-2000, Colin Corporation, Komaki, Japan) that measures the pulse wave velocity (PWV). Pulse wave forms were obtained from the carotid and femoral artery sites.
Genomic DNA was extracted from peripheral blood leukocytes using Puregene DNA extraction kits (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocols. The MTHFR C677T genotypes were identified as previously described (8).
A routine clinical workup of all patients within 1 month of enrollment included fasting blood samples before the mid-week dialysis. Hcy levels were measured by chemiluminescence immunoassay. The level of total intact parathyroid hormone (iPTH) was evaluated by electrochemiluminescence immunoassay.
Statistical comparison between the groups was performed using the independent samples t-test, ANOVA, and chi-square tests. Predictive factors for VC were examined using logistic regression analysis. P values less than 0.05 were considered statistically significant.
In terms of patients' genotypes, 28.9% had CC, 47.4% had CT, and 23.7% had TT (Table 1). The patients with the TT genotype had higher VC scores than patients with the CC or CT genotype (P = 0.002). The TT genotype tended to be associated with a higher PWV, pulse pressure (PP), and prevalence of CVD; however, the differences were not statistically significant. Only the prevalence of peripheral vascular disease (PVD) was significantly increased in patients with a T allele (P = 0.006). The study group was divided into two subgroups by 60 yr of age, based on a median age of 56.5 yr; the mean ages of the subgroups were 47.38 ± 9.4 and 69.83 ± 6.2 yr. In young patients (≤ 60 yr), MTHFR C677T mutations were associated with higher VC scores (P = 0.004). In young patients, MTHFR C677T mutations were a significant predictive factor for cerebrovascular accident (CVA) (P = 0.031) and PVD (P = 0.006), except ischemic heart disease. The MTHFR C677T polymorphism was not associated with plasma Hcy levels.
A total of 57 (37.5%) patients presented with VC. Extensive VC scores (> 3) were observed in 25 (16.4%) of all patients. Table 2 provides data indicating that the existence of VC was associated with a higher frequency of CVD and diabetes (P = 0.001). Patients with VC had significantly higher systolic blood pressures, PWVs, and PPs (P < 0.05). The mean albumin levels were significantly lower in patients with VC. The frequencies of the MTHFR C677T mutation significantly differed between patients with or without VC (P = 0.003). However, the plasma Hcy levels did not significantly differ between the subgroups.
To identify predictors of VC, multiple logistic regression analysis was performed using the existence of VC as the dependent variable and 10 selected variables, including age and gender, as the independent variables. Age, diabetes, and MTHFR C677T mutations were determined to be independent predictors of the existence of VC (Table 3). The adjusted odds ratio (OR) for the TT genotype for the risk of extensive VC score (> 3) was 1.66 (95% confidence interval [CI]; 1.41-19.47, P = 0.013) for all patients, and 2.41 (95% CI; 1.70-73.29, P = 0.012) for patients younger than 60 years of age (data not shown).
The results of this study suggest that there is a strong relationship between the incidence of MTHFR C677T mutations and VC in patients with ESRD on chronic hemodialysis. Compared to patients with the CC genotype, patients with CT and TT genotypes had adjusted ORs for VC of 1.39 and 1.58, respectively (P = 0.042 and 0.032). In the subgroup analysis, the correlation of the MTHFR C677T mutation and VC persisted in young patients (≤ 60 yr) (Table 1). A similar trend of increased VC scores, although not significant, was observed for the older patient group (> 60 yr). These results suggest that the harmful effect of having a mutant T allele at nucleotide position 677 may be diluted with age, which is another strong risk factor for VC.
The possible association between a genetic polymorphism and the development of VC has been of recent interest to investigators (8). A better understanding of the pathogenesis contributing to the increased VC in ESRD patients can provide a better perspective on the high cardiovascular mortality, which is partially explained by the traditional risk factors. Several recent reports have suggested that the MTHFR C677T polymorphism (9) is associated with the development of CVD in patients with ESRD. This is the first study to statistically evaluate the relationship between the extent of VC and MTHFR C677T polymorphism.
The mediator of vascular injury has been presumed to be plasma Hcy levels (10), which are increased in patients with mutant T allele (5). However, in this study, plasma Hcy levels were not associated with the MTHFR C677T polymorphism and VC. There are several possible explanations for these findings. Several randomized trials focused on reducing Hcy levels in the general population (11) or in patients with ESRD (12) failed to improve outcome. Perhaps, lifetime exposure to increased Hcy levels cannot be remedied by a few years of partial restoration of Hcy levels to normal. Alternatively, high Hcy levels may simply mark rather than mediate the injury caused by impaired MTHFR activity. Recently, the levels of tissue 5-methyl-tetrahydrofolate (5-MTHF), rather than plasma or tissue levels of Hcy, have been suggested to play a role in regulating endothelial function (13).
VC was present in 37.5% of the study participants, which is inconsistent with the rate of 74.8% reported in a study evaluating chronic hemodialysis patients using the same VC scoring method (7, 14). In this study, mean patient age was 56.83 ± 13.8 yr, which is lower than the mean patient age reported in the previous study (14). Unfortunately, there are limited data available on VC in Asian patients assessed using a simple radiogdraphy calcification score.
There was no significant relationship between MTHFR C677T polymorphism and CVD in this study. In the ESRD population, evidence for an association of the MTHFR C677T mutation with CVD is also inconsistent (9). Since ESRD patients carry a heavy cardiovascular disease burden, these conflicting results may reflect confounding by various risk factors. Thus, in our study, the mean age of the patients with the CC genotype was greater than those of the patients with CT or TT. In addition, methodological limitations constrain the interpretation of the findings from our small-sized cross-sectional study. The prevalence of PVD increased with the incidence of MTHFR C677T mutation for all patients, and the CVA also significantly increased for the young patients (≤ 60 yr). Further studies using a larger population are required to confirm these findings.
The MTHFR 677TT genotype was not associated with the PWV in this study. It is possible that the association of the MTHFR genotype with the PWV was attenuated by several therapeutic interventions such as correction of uremia (15), hypertension (16), hyperhomocysteinemia (17), or the use of rennin-angiotensin-aldosterone system antagonists (18), statins (19) and/or beta blockers (20).
This study has several limitations. It was a small-sized cross-sectional study. No variability was determined by repeated measures of the Hcy or PWV parameters. However, individual investigators, who were blind to the clinical data, independently reported the VC and PWV measurements. Dietary calcium intake was not quantified, and the 25-hydroxy vitamin D levels were not determined, which would have been useful for assessing VC in the ESRD population.
In conclusion, the present study indicates that the MTHFR C677T mutation is an important factor influencing VC in chronic hemodialysis patients. However, further large-scale studies are required to fully characterize the relationship between the extent of VC and MTHFR C677T mutational status.
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