gene mutation and other predictive factors of cardiovascular disease (CVD) in patients on maintenance hemodialy- .... Modular Analytics E170 (Elecsys module).
Predictive Factors of Cardiovascular Disease in Patients on Maintenance Hemodialysis Ramazan Danis, MD; Sehmus Ozmen, MD; Davut Akin, MD; Fatma Celik, PhD; Orhan Yazanel, MD Drs. Danis, Ozmen, Akin, and Yazanel are with the Department of Nephrology and Dr. Celik is with the Nutrition and Dietetics Division of the Dicle University School of Medicine, Diyarbakir, Turkey.
OBJECTIVE: The purpose of this study was to identify the role of the methylenetetrahydrofolate reductase (MTHFR) gene mutation and other predictive factors of cardiovascular disease (CVD) in patients on maintenance hemodialysis. METHODS: A total of 144 patients with end-stage renal disease (ESRD) undergoing maintenance hemodialysis for at least 6 months were recruited. The mean age of patients was 44.84 16.23 years. Serum folate, vitamin B12, homocysteine (tHcy), albumin, lipids, MTHFR C677T mutation, and smoking habits were analyzed relative to the presence of CVD. RESULTS: Forty-two of the 144 patients (29.1%) had CVD. Mean age, duration of dialysis, body mass index, CRP, Kt/V, male sex, diabetes mellitus, and currently smoking were found to be significantly higher in patients with CVD than in those without CVD (p 0.05). The means of serum folate, vitamin B12, tHcy, albumin, and percentage of past smokers were not significant (p 0.05). MTHFR C677T mutation was not found to be associated with CVD risk factors. The mean differences in tHcy levels between the TT and CC and the CT and CC genotype allele groups were significant. CONCLUSION: We recommend that duration of ESRD and CRP predictors with traditional risk factors be taken into account when examining CVD. We suggest that if patients with ESRD have normal B12 and folate levels, abnormal tHcy level and tHcy metabolism are not risk factors for CVD in patients with ESRD. We do not recommend evaluation of the MTHFR C677T mutation and tHcy level in maintenance hemodialysis patients. The discriminative ability of risk predictors in CVD found in the study present new ideas for researchers studying this subject.
C
ardiovascular disease is a major cause of morbidity and mortality in patients with chronic renal failure, especially in those on maintenance hemodialysis.1 Traditional risk factors (diabetes mellitus, smoking, dyslipidemia, high blood pressure, sedentary lifestyle, and obesity) and cardiovascular risk factors related to uremia (hypervolemia, anemia, hyperdynamic status induced by vascular access, and secondary hyperparathyroidism) do not fully explain the high incidence of cardiovascular disease in these patients.2 More data continue to arise suggesting a role for other factors, known as “emergent,” “new,” or “non-classic”
factors.3 Among these “new” cardiovascular risk factors, homocysteine (tHcy) and inflammation markers (C-reactive protein) should be emphasized. Mild to moderate hyperhomocysteinemia is very common among patients undergoing hemodialysis. The prevalence of hyperhomocysteinemia in the general population is approximately 5%, in dialysis patients it reaches 80%–90%.4,5 Several recent studies showed that in dialysis patients, hyperhomocysteinemia was a risk factor for cardiovascular complications.6-8 In addition to nutritional deficiencies in B vitamins and genetic factors [C677T mutation of the methylenetetrahydrofolate
reductase (MTHFR) gene], renal failure is one of the most frequent clinical causes of hyperhomocysteinemia. Despite homocysteine’s role in vascular pathophysiology in vitro and in animal studies, clinical and epidemiologic evidence is mixed, and the lack of clinical trials leaves the question of causality open. Although several processes may explain the close correlation between kidney function and plasma tHcy concentration, the exact mechanism or mechanisms by which renal failure leads to hyperhomocysteinemia are not completely understood. A common variant of a key enzyme in tHcy metabolism, the C677T mutation February 2008 Dialysis & Transplantation 1
CVD in Hemodialysis
(also known as the thermolabile variant) of MTHFR, on chromosome 21, is emerging as a possible genetic contributor to hyperhomocysteinemia and cardiovascular disease (CVD). The C677T point mutation of MTHFR results in a valine substitution for alanine. With 2 copies of this variant (TT), serum activity of MTHFR is reduced to 30%, whereas serum activity of heterozygotes (CT) is 65% that of wild-type (CC) individuals.9 Substantial evidence has accrued that in the general population atherosclerosis underlies a systemic microinflammatory process and that serum C-reactive protein (CRP) is a reliable risk marker of atherosclerosis complications. In patients with end-stage renal disease (ESRD), serum CRP is a strong predictor of death and adverse cardiovascular events.10,11 In addition, it remains an open question what the influence is of nutritional status and chronic inflammation conditions, as expressed by elevated CRP, in chronic renal failure.12-15 The aim of this study was to identify the role of the MTHFR gene mutation and other predictive factors of cardiovascular disease in patients on maintenance hemodialysis.
Material and Methods
Study Population A total of 144 ESRD patients (69 women, 75 men) were recruited, all patients who had been undergoing maintenance hemodialysis for at least 6 month at various dialysis centers in the southeastern region of Turkey. The mean age of these patients was 44.84 16.23 years. The mean duration of dialysis therapy was 43.65 30.7 months. The causes of renal failure in the 144 patients were hypertension, 24.6%; diabetic nephropathy, 18%; chronic glomerulonephritis, 9.8%; urologic disease, 15.6%; polycystic kidney disease, 1.6%; other, 5.7%; and unknown causes, 24.6%. Almost all patients were anuric or oliguric and were being treated 3 times a week for 4.5 hours with standard bicarbonate hemodialysis with a high-flux hemodialysis membrane. Baseline clinical variables were collected at the time of enrollment. These included diabetes status, smoking history, duration of renal replacement therapy, and 2 Dialysis & Transplantation February 2008
dry weight (the prescribed post-dialysis weight). In addition, the most recent monthly dialysis adequacy parameters were collected for all subjects. Adequacy parameters included Kt/V and urea reduction rate (URR).
Diagnosis of CVD CVD was defined as present if there was a medical history of coronary artery intervention, myocardial infarction, stroke, transient ischemic attack, or other clinical evidence of CVD, as documented in hospital discharge summaries or history and physical examination.
Biochemical Measurement Peripheral venous blood samples were collected after an overnight fast at the beginning of the dialytic treatment. Biochemical analysis of serum lipid profile (total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides) and albumin was performed with standard automated routines. Plasma folate and vitamin B12 concentrations were measured with the electrochemiluminescence immunoassay, intended for use on the Roche Elecsys 2010 and Modular Analytics E170 (Elecsys module) immunoassay analyzers. The normal range for serum folate was 2–9 ng/mL. The normal range for vitamin B12 was 240–900 pg/mL. tHcy concentration was determined with the Immulite 2500 analyzer (Diagnostic Products Corporation, Los Angeles, Calif.). The normal range for serum tHcy was 5–12 umd/L. CRP was determined using nelphelometry with a Beckman-Coulter Immage immunochemistry system (Fullerton, Calif.). The normal range for serum CRP was 0–8 mg/L. Kt/V was calculated in all patients.
Genetic Analysis DNA isolation. K3EDTA-anticoagulated venous blood samples (2 mL) were collected from all subjects for DNA analysis. DNA samples were isolated from whole blood with a MagNA Pure LC DNA Isolation Kit I using a MagNA Pure LC Automated DNA isolation instrument (Roche Molecular Biochemicals, Basel, Switzerland). DNA samples were stored
at 20ºC until the mutations were investigated. The MTHFR C677T mutation was detected with LightCycler FastStartDNA Master Hybridizations probes (Roche Molecular Biochemicals), amplifications primers (Metabion, Planegg, Germany), hybridizasyon probes (TIB MOLBIOL, LightMix for the detection of human MTHFR C677T). Mutationrelated gene regions were amplified in 20 L of PCR capillary tubes. After preparation of the master mixture, 15 L of the reaction mixture and 5 L of genomic DNA or control template were added to each LightCycler capillary tube. For the negative control, PCR-grade water was added instead of template. The capillary tubes were sealed and briefly centrifuged in a microcentrifuge and then placed in the LightCycler carousel. The PCR products were detected using 3-fluorescein (FLU)–labeled and 5-Red 640–labeled probes. When both probes hybridize in close proximity, fluorescence resonance energy transfer occurs, producing a specific fluorescence emission of LC-Red as a result of FLU excitation. The fluorescence intensity depends on the amount of specific PCR products. Amplification per cycle can be monitored with the LightCycler instrument. At the end of the amplification process, the LightCycler instrument increases the temperature, and the fluorescence obtained is plotted against the temperature. The mutations are then identified by their characteristic curves. Total assay time was approximately 40 minutes. The resulting melting peaks allowed discrimination between the homozygous (wild-type or mutant) and the heterozygous genotypes.
Statistical Analysis Continuous variables are presented as means SD, with frequency expressed as a percentage. The significance of differences in means between 2 groups were calculated with Student’s t-test. Comparisons among multiple groups was performed by one-way analysis of variance (ANOVA) with the Bonferroni post hoc test for continuous variables. Logistic regression analysis was used to test possible risk factors for
CVD in Hemodialysis
Results In this study analysis, 42 of the 144 dialysis patients (29.1 %) had CVD. Means SD,
95% confidence intervals (95% CIs), and characteristics of the hemodialysis patients with and without cardiovascular disease (as percentages) are summarized in Table I. Mean age, duration of dialysis, BMI, CRP, Kt/V, percentage of men, diabetes status, and current smoking were found to be significantly higher in patients with CVD than in those without CVD ( p 0.05). However, serum folate, vitamin B12, tHcy, albumin, and percentage of past smokers were not significantly different (p 0.05) between the groups. The C677T mutation of MTHFR was not found to be associated with CVD risk factors in our patients. The results of clinical characteristics by C677T mutation of MTHFR genotype are summarized in Table II. There were 12 patients (8.3%) with 2 copies (TT) of the allele, 50 patients (34.7%) with 1 copy (CT), and 80 patients (55.5%) with the wild-type allele (CC). ANOVA followed by the Bonferroni post
hoc test showed that the differences in mean tHcy level between the TT and CC and the CT and CC genotype allele groups were significant. There was no significant difference in other related parameters among the TT, CT, and CC genotype allele groups (Table II). The logistic regression model were based on with and without cardiovascular diseases. The outcomes of age, BMI, diabetes status, dialysis duration (DD), smoking status, and CRP, which were significant in logistic regression model were presented by Table III. By examining the results of Table III, the odds ratios (95% CI) of age, BMI, diabetes status, DD, smoking status, and CRP were found to be: 1.126 (1.022–1.240), 1.056 (1.020–1.094), 2.879 (1.017–8.149), 1.039 (1.005–1.074), 12.055 (1.172–123.98), and 2.789 (1.76– 4.41), respectively. Receiver operating characteristic (ROC) curve analyses were applied
œ
development of cardiovascular disease in patients on maintenance hemodialysis. Age (years), sex, diabetes status, smoking status, duration of dialysis (months), body mass index (BMI), serum folate, vitamin B12, tHcy, albumin, triglyceride, total cholesterol, LDL cholesterol, TT genotype C677T variant, Hb, CRP, CT, genotype, C677T variant, Kt/V, and URR were included in the model. The forward Wald selection procedure was performed in the execution of the logistic regression analysis. All statistical tests were two-tailed, with the level of significance established at p .05. All analyses were conducted using SPSS software (version 15.0, SPSS Inc., Chicago, Ill.).
TABLE I. Characteristics of the study patients (n = 144). Variable
No cardiovascular disease (n ⴝ 102)
Mean SD Age (year)
(95% CI)
42.1 16.2
37.6–46.6
Cardiovascular disease (n ⴝ 42)
Mean SD
p
(95% CI)
55.6 8.99
49.9–61.4
0.001
Male (%)
48.0
66.6
0.05
Diabetic (%)
14.7
40.5
0.001
Past smokers (%)
22.5
14.2
0.05
Current smokers (%)
27.5
59.5
0.001
Duration of dialysis (months)
40.1 24.2
35.4–44.8
49.7 26.3
41.7–57.6
0.043
BMI (kg/m2)
20.1 4.61
19.2–21.0
21.9 4.27
20.6–23.2
0.015
Serum folate (ng/mL)
9.54 6.39
7.77–11.3
10.5 6.20
6.53–14.4
0.740
Vitamin B12 (pg/mL)
574.7 423.6
456.7–692.6
671.3 552.8
20.1 9.28
17.5–22.7
18.6 5.80
Homocysteine (umd/L)
320.1–1002.6
0.394
14.9–22.3
0.800
3.67 0.59
3.51–3.83
3.59 0.51
3.27–3.91
0.567
Triglyceride (mg/dL)
201.0 139.7
162.1–239.9
200.5 115.9
126.8–274.1
0.459
Total cholesterol (mg/dL)
171.0 37.3
160.6–181.4
169.9 32.4
149.3–190.5
0.378
LDL cholesterol (mg/dL)
101.9 25.9
94.0–109.7
95.7 15.3
Albumin (g/dL)
TT genotype C677T variant (%)
7.84
83.9–107.5
0.491 0.05
11.9
Hemoglobin (g/dL)
10.9 1.74
10.5–11.2
10.9 1.57
10.0–11.4
0.794
CRP (mg/L)
5.50 10.7
3.22–7.77
10.5 23.1
0.61–21.6
0.042
CT genotype C677T variant (%)
34.3
0.05
40.5
Kt/V
1.21 0.25
1.14–1.28
1.11 0.22
0.96–1.25
0.044
URR (%)
69.0 6.33
67.2–70.7
67.3 5.00
64.1–70.4
0.133
February 2008 Dialysis & Transplantation 3
CVD in Hemodialysis
TABLE II. Clinical characteristics by C677T variant of MTHFR. Variable
TT (n 12)
CT (n 50)
CC (n 80)
p
Mean SD
Mean SD
Age (year)
43.9 16.3
45.6 15.0
Mean SD 45.8 16.9
0.940
Duration of dialysis (months)
47.5 38.3
40.0 29.8
42.2 29.5
0.774
Male
58.3
52.0
61.3
0.05
Diabetic (%)
16.7
18.0
25.0
0.05
Cardiovascular disease (%)
30.0
25.6
19.1
0.05
Serum folate
11.6 6.29
10.6 6.56
9.72 5.85
0.591
Vitamin B12
525.2 224.3
673.9 516.4
672.6 470.2
0.642
Homocysteine*
22.7 14.0
20.5 9.54
16.8 6.65
0.045*
Albumin
3.81 0.40
3.59 0.61
3.65 0.55
0.597
C-reactive protein Hemoglobin
5.92 13.2
6.58 12.5
6.54 15
0.990
11.06 2.08
10.48 1.53
11.05 1.77
0.234
*Homocysteine variable compared by ANOVA test (p 0.045). Mean differences between TT and CT were not found to be significant (p 0.05). However, the mean differences between TT and CC and between CT and CC were significant using the Bonferroni post hoc test.
to examine the performance of age, BMI, smoker, DM status, and dialysis duration as diagnostic tests for CVD Area under the curve values of age, BMI, smoking, DM status, and dialysis duration for CVD were 0.750, 0.722, 0.729, 0.633, and 0.659, respectively.
Discussion The mortality rate of patients with ESRD is high and increasing. CVD is the most common cause of death among HD patients, followed by cerebrovascular disease, infection, and malignancy. This finding on CVD might be considered a result of the close relationship between cardiovascular and renal pathologies. One- and 5-year survival rates of our patients with ESRD were found to be 88% and 90.4%, respectively.16 CVD was the principle cause of that in our patients with ESRD. Our current cross-sectional study investigated the association of tHcy level, traditional risk factors, and CVD in a cohort of hemodialysis patients. We found that mean age, duration of ESRD, BMI, CRP, Kt/V, percentage of men, diabetes mellitus, and current smoking were significantly higher in patients with CVD than in those without CVD. But only age, BMI, diabetes status, ESRD duration, smoking status, and CRP were significant in the 4 Dialysis & Transplantation February 2008
logistic regression model. The finding that diabetes, smoking, and age are traditional risk factors associated with cardiovascular diseases is consistent with prior studies in the ESRD population.17 The predictive role of duration of ESRD in our patients may be explained by the uremic milieu, and perhaps hemodialysis procedure-related risk factors are likely to contribute to CVD events. Hemodynamic and metabolic risk factors, peculiar to chronic uremia, enhance the cardiac risk of dialysis patients. The death of myocytes in chronic uremia may be exacerbated by diminished perfusion, hyperparathyroidism, and perhaps, inadequate dialysis.18 The significantly low Kt/V value in the CVD group is a marker of inadequate dialysis in subjects with CVD. But a low Kt/V value was not a predictive factor in our analysis. The predictive role of duration of ESRD in CVD is a new finding not previously reported in investigating CVD factors in dialysis population.19-24 Our results are similar to these previous reports in mean age, ESRD etiology, duration of ESRD, and number of patients enrolled.19-24 However, our result for duration of ESRD (49.7 months in the CVD group) was found to be longer than that in the previous studies. This discrepancy may be a result of high coronary artery calcification in those with long-duration HD.25
Hyperhomocysteinemia was suspected as a cardiovascular risk factor in chronic renal failure and was examined in several cross-sectional and mechanistic studies.5,7,8,26-33 Mild to moderate hyperhomocysteinemia is very common among patients undergoing hemodialysis. In the present study homocysteinemia levels were found to be moderately elevated. This result could not be attributed to nutritional deficiencies lost in dialysis because our patients had normal serum folate and B12 vitamin levels. Elevation of the tHcy level could be explained by the presence of renal failure or the MTHFR gene genotype mutation. Serum tHcy level was significantly higher in subjects with the MTHFR gene mutation (homozygote or heterozygote) when compared with those who had the wild type. The association between tHcy and risk for CVD is not a consistent finding in ESRD. Since the early enthusiasm over the proposal of hyperhomocysteinemia as a risk factor, minimal confirmation has been sustained. Some cross-sectional studies reported higher levels of tHcy in ESRD patients with CVD7,8,26,34-36; others reported no difference in tHcy level22,36 or even, paradoxically, lower tHcy levels in CVD patients.20,33,3840 Likewise, prospective studies revealed either a graded increase in relative risk with the rise of tHcy22,41-44 or a worse outcome in patients with lower tHcy,33,39,45,46
TABLE III. Outcomes of logistic regression and odds ratios. Variable
Age DD

SE
Wald
OR (95% CI)
p
.119
.049
5.770
1.126 (1.022–1.240)
0.016 0.024
.038
.017
5.068
1.039 (1.005–1.074)
Smoking
2.489
1.189
4.383
12.055 (1.172–123.98)
0.036
BMI
0.055
0.018
9.551
1.056 (1.020–1.094)
0.002
DM
1.057
.531
3.967
2.879 (1.017–8.149)
0.046
CRP
1.026
.234
4.384
2.789 (1.76–4.41)
0.044
10.275
3.513
Constant
8.553
0.003
Correct classification: 94.45%.
whereas other studies did not find a difference between low and high tHcy levels.47,48 In the present study, tHcy level was not significantly different between those with and without CVD. It could be expressed that our results for tHcy level were not sufficiently high to yield significant difference in the presence of CVD, and this mild difference in tHcy level may be a result of the normal folate level in our population. Although Morimoto et al., Wrone et al., and Haviv et al. reported an association between MTHFR genotype and CVD, events in patients with ESRD.19,20,49 Kimura et al. and Aucella et al. found no association between MTHFR genotype and CVD events in such patients.50,51 In addition to these findings, recently, Marie et al. did not find any association between MTHFR genotype and ischemic cerebrovascular disease in patients without ESRD.52 In our findings, there was no significant difference between subjects with and without CVD in MTHFR mutant allele frequency. Our results were similar to those of the largest multicentric study, which reported that MTHFR C677T polymorphisms were not associated with cardiovascular disease or mortality.51 The lack of an association between total cholesterol level and the presence of CVD in our patients is compatible with reports by Cheung et al., Lowrie et al., and Kalantar-Zadeh et al., who concluded there was no association between total cholesterol and CVD events.17,53,54 The predictive value of CRP in CVD risk and mortality in HD patients has been shown in numerous studies, and CRP has been a new addition to CVD risk factor profiles.3,55 Epidemiological and clinical evidence suggest that atherosclerosis represents a low-grade inflammatory disease
of vascular endothelium, and certain markers such as CRP, an acute phage reactant, are elevated in the serum of such patients. These results were in accordance with the literature linking CRP and CVD in HD patients.14 Significantly, high CRP in subjects with CVD and its independent predictive role in logistic regression analysis in our study support the predictive value of CRP in CV risk. The discriminative ability of the predictors was found to be significant in the logistic regression model using ROC curves. A ROC curve, which is defined as a plot with test sensitivity as the vertical axis versus its 1-specificity, or false-positive rate, as the horizontal axis, is an effective method of evaluating the performance of predictors. The area under the receiver operating characteristic curve (AUC) is frequently used as a measure for the effectiveness of predictors. The accuracy of the test depends on how well the test separates the group being tested into those with and without the CVD in question. Accuracy is measured by the area under the ROC curve.56 The AUC values of all predictors were found to be significantly high. The AUC values were important criteria for determining the discriminative levels of the predictors for CVD—age, BMI, smoking, DM, DD, and CRP—which were .750, .722, .729, .633, .659, and .717, respectively.
Conclusion The findings of the current study recommended that duration of ESRD and CRP predictors with traditional risk factors be taken into account when examining
cardiovascular diseases. The predictive role of duration of ESRD in CVD may be a warning for screening patients with longduration ESRD for CVD. We suggest that if patients with ESRD have normal B12 and folate levels, an abnormal tHcy level or tHcy metabolism is not a risk factor for atherosclerotic disease in patients with ESRD. Therefore, we are not recommending evaluation of the MTHFR C677T mutation and tHcy level in maintenance hemodialysis patients, as the role of tHcy in promoting vascular injury in ESRD patients is still unclear and was not supported by the results of our study. However, it could be argued that the treatment still could be proposed for these patients because folic acid supplementation has no apparent side effects and is inexpensive. The discriminative ability of risk predictors in CVD found in the study presents new opinions for research in this subject.
Acknowledgement We thank Prof. Yusuf Celik for assisting with the statistical evaluation. D&T
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