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NDT Advance Access originally published online on September 27, 2006
Nephrology Dialysis Transplantation 2007 22(1):154-162; doi:10.1093/ndt/gfl512
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Effect of MTHFR C677T genotype on survival in type 2 diabetes patients with end-stage diabetic nephropathy

Carsten A. Böger1, Mike Stubanus2, Thomas Haak3, Angela K. Götz1, Johanna Christ1, Ute Hoffmann1, Günter A. J. Riegger1, Bernhard K. Krämer1 for the GENDIAN Study Group

1Klinik und Poliklinik für Innere Medizin II, Klinikum der Universität Regensburg, 2Medizinische Klinik IV, Abteilung für Nephrologie und Allgemeinmedizin Universitätsklinikum Freiburg and 3Diabetes Zentrum Mergentheim, Germany

Correspondence and offprint requests to: Dr Carsten A. Böger, Klinik und Poliklinik für Innere Medizin II, Klinikum der Universität Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Germany. Email: carsten.boeger{at}klinik.uni-regensburg.de



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. The MTHFR C677T single nucleotide polymorphism TT genotype is associated with increased levels of plasma homocysteine and possibly an effect on cardiovascular mortality. We evaluated the effect of C677T genotype on mortality in a large end-stage renal disease (ESRD) cohort.

Methods. C677T genotype was determined in 439 Caucasians with end-stage diabetic nephropathy (DNP) (cases) recruited from 30 dialysis centres in Southern Germany. A total of 482 type 2 diabetes patients without DNP (no microalbuminuria) at inclusion served as a genotype control collective. Patients were prospectively followed for 4 years. Primary endpoint was all-cause mortality.

Results. In contrast to controls, the genotype distribution in cases was not in Hardy–Weinberg equilibrium (HWE, P = 0.003), due to a less than expected number of patients with the TT genotype. The requirements of HWE were met in cases with <2 years dialysis therapy prior to study inclusion (n = 219). TT genotype was associated with a decreased body mass index (P = 0.002) and long diabetes duration in dialysis patients (P = 0.03). However, TT genotype was not associated with an increased risk of all-cause or cardiac mortality in the total dialysis collective or the subgroup. Also, we observed no association of MTHFR genotype with cardiovascular morbidity in cases or controls (P > 0.05), or with an increased rate of progression to novel microalbuminuria.

Conclusion. MTHFR 677TT genotype was significantly underrepresented in patients with ESRD in our study, but was not associated with premature mortality in these patients. We found no evidence for survival bias due to C677T genotype in the ESRD cohort, or bias due to genetically determined accelerated progression to novel microalbuminuria in the controls. However, we cannot exclude that the TT genotype protects from progression from microalbuminuria to more advanced stages of DNP, or that TT genotype is associated with premature mortality before a patient progresses to ESRD.

Keywords: cardiovascular mortality; ESRD; genetics; MTHFR (C677T) polymorphism; progression of diabetic nephropathy; type 2 diabetes mellitus



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Hyperhomocysteinaemia is a major risk factor for thrombosis and atherosclerosis [1–3]. In dialysis-dependent end-stage renal disease (ESRD) patients, elevated fasting total plasma homocysteine (tHcy) levels may contribute independently to the excess incidence of fatal and non-fatal cardiovascular disease [4]. In ESRD patients, elevated tHcy may result from the deterioration in renal function or from genetic variation [5,6]. Genetic polymorphisms are thought to be a major contributor to elevated tHcy in ESRD patients by determining the function of 5,10-methylenetetrahydrofolate reductase (MTHFR), a folate-dependent enzyme crucial for the remethylation of homocysteine to methionine. The commonly occurring cytosine to thymidine substitution C->T at nucleotide 677 (C677T) of the coding region alters a highly conserved amino acid (alanine to valine) at position 222 of the amino acid sequence and is linked to elevated tHcy levels in homozygotes as compared with heterozygotes or wild-type (genotype CC) individuals [6]. This would suggest a recessive mode of inheritance for a disease trait due to the mutation. Accordingly, haemodialysis patients homozygous for 677T have increased homocysteine values [7,8]. Homozygosity for 677T has been shown to predispose to premature atherosclerosis [6,9], coronary artery disease (CAD) [10] and diabetic retinopathy [11], though results have been conflicting [3]. In an Italian dialysis cohort, there was no association of MTHFR C677T genotype with cardiovascular end points or mortality [12].

We evaluated the effect of the MTHFR single nucleotide polymorphism (SNP) genotype on morbidity and mortality in a large cohort of type 2 diabetic patients with ESRD due to diabetic nephropathy (DNP). A total of 482 type 2 diabetes patients without DNP (no microalbuminuria) served as genotype controls.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Subjects
Subjects studied are from the GENDIAN case control study (‘Genetic and clinical predictors of morbidity, mortality and diabetic nephropathy with end-stage renal disease in diabetes mellitus type 2’). This study and its phenotyping and biosampling procedures have been previously described [13–15]. Briefly, cases were 477 prevalent Caucasian patients with type 2 diabetes mellitus and ESRD recruited from 30 dialysis centres in Southern Germany from August 1999 to January 2000, and followed prospectively. Last follow-up was performed on 4 December 2003. Thirteen patients were excluded due to overt clinical infection, thus leaving 464 patients for further study.

To assess potential deviations of expected MTHFR C677T allele frequency, and differences of anthropometric variables and comorbidity by MTHFR C677T genotype inherent to an ESRD population, we included GENDIAN's nested case control component in the analysis of this study.

A total of 482 Caucasian type 2 diabetes mellitus patients without renal insufficiency or microalbuminuria in two morning urine samples collected on two consecutive days were recruited from a large diabetes clinic in Southern Germany from September 2000 to September 2001 and defined as controls. At inclusion, none of the controls had signs of renal disease in history, laboratory, and physical examination and urine analysis. At a follow-up examination performed from September 2004 to June 2005, the following parameters were determined: microalbuminuria as determined by semiquantitative urinary albumin and urinary creatinine determination (MicroalbustixTM, Bayer, Germany), serum creatinine as determined by the treating physician, date of follow-up examination, new cardiovascular morbidity, cause of death if any, arterial blood pressure, body weight and current medication. At the follow-up, n = 148 of the controls remained normalbuminuric and represented the ‘genotype controls’ used for comparison with ESRD cases, n = 98 had microalbuminuria and were defined as microalbuminuric cases of the nested case control study component, and in n = 43, the dipstick test was inconclusive. Twenty-five patients died before follow-up, and 168 patients did not complete the follow-up examination.

Clinical phenotyping was performed by a standardized questionnaire and reviewing the patients’ charts as previously described. Briefly, cardiovascular risk factor profile (diabetes mellitus, arterial hypertension, smoking history, dyslipidamia), date of onset of each risk factor, cardiovascular morbidity profile and comedication including folic acid supplementation was determined. In addition, we determined the date of birth, and in ESRD patients date of diagnosis of DNP and of the start of dialysis therapy.

The study was approved by the Ethics Committee of the Medical Faculty of the University of Regensburg (Study Nr: 97/38). The study is in accordance with the Declaration of Helsinki. All patients gave informed consent to participation in the study.

Serum parameters
Whole blood samples of 10 ml were drawn prior to haemodialysis sessions, and centrifuged within 6 h. Serum was frozen at –80°C until analyses were performed. Serum samples were not available for study in 15 cases. C-reactive protein (CRP) was determined with the C-Reactive Protein Assay (BIOMED, Oberschleissheim, Germany, Prod. No.: 123440) as previously described [13].

Genotyping
We used genomic DNA extracted by standard methods for determining MTHFR C677T genotype by PCR. The following primers were used: forward primer: 5' – CAA AGG CCA CCC CGA AGC – 3'; reverse primer: 5' – AGG ACG GTG CGG TGA GAG TG – 3'. PCR reactions were carried out under the following conditions: 5 min 95°C (one cycle); 15 s 94°C, 45 s 58°C, 40 s 72°C (35 cycles); 5 min 72°C (one cycle). The resulting amplification product (246 bp) was digested with Hinf I restriction endonuclease. Following digestion, restriction fragments (175 and 71 bp) were size fractioned on a 2% agarose gel. Genotype analysis was carried out by two independent investigators who were unaware of the clinical data. Wherever there was any ambiguity, the PCR reaction, Hinf I digestion and scoring were repeated. MTHFR C677T genotyping was not unambiguous in 10 cases and 6 controls.

Statistical analysis
Ten cases were excluded from further analysis in the study due to missing genotype data, 15 due to missing CRP data and 13 due to overt clinical infection, leaving 439 cases in the final analysis.

Twenty-five controls (one of which with missing genotype) were excluded since they had died before follow-up. Three controls (all three had microalbuminuria at follow-up) were excluded due to missing genotype data, 43 due to an inconclusive urine dipstick test at follow-up and 168 (two of which with missing genotype) due to incomplete follow-up data, thus leaving 95 controls with new microalbuminuria (cases in the nested case control study) and 148 controls to be classified as genotype controls in the nested case control study.

Survival analysis of ESRD cases was performed by the Kaplan–Meier method, comparing groups using the log-rank test. Censoring was performed for renal transplantation (n = 11), loss to follow-up (n = 2) and if the patient was alive at the last examination (n = 126). Duration of dialysis therapy from study inclusion onwards was the time variable. The primary endpoint was all-cause mortality (ACM, n = 300). Cause of death was assessed where possible. Death due to myocardial infarction (n = 35), malignant arrhythmia (n = 27) or acute cardiac failure (n = 32) was defined as the combined secondary endpoint ‘cardiac’, and death due to pneumonia (n = 10) or septicaemia of any cause (n = 45) as ‘infectious’. Cause of death was unknown in n = 112, and other causes of death were cerebral ischaemia (n = 9), intracranial haemorrhage (n = 10), gastrointestinal bleeding (n = 4), cancer (n = 8), high grade aortic stenosis (n = 3) and cachexia (n = 5). To correct for covariates, a Cox proportional hazard ratio (HR) model was applied: in addition to the univariate model with MTHFR genotype as independent variable (additive, dominant and recessive model for 677T), we estimated two multivariate models, one containing a priori covariates only, and one with stepwise forward selection of further covariates in addition to a priori covariates. Model 1 covariates (inclusion of a priori covariates only) were: MTHFR C677T genotype (reference for additive and dominant models: 677CC; reference for recessive model: 677CC and CT combined), age at the begin of dialysis therapy (years), duration of dialysis at study inclusion (years), gender (reference: female gender) and presence of peripheral arterial disease (PAD) Fontaine Stage IV (reference no. 13; reference: no PAD IV). Model 2 covariates were: inclusion of a priori covariates as detailed in model 1, and stepwise forward selection of the following covariates (threshold: P = 0.05): smoking history (reference: never smoked), duration of diabetes at dialysis intitiation (years), body mass index (BMI; kg/m2), systolic blood pressure (mmHg), history of CAD, myocardial infarction or cerebral ischaemia (reference: no such history), medication with angiotensin converting enzyme (ACE) or angiotension-II (AT-II) receptor 1 antagonists, hydroxymethylglutaryl-coenzyme A reductase (HMG-COA-reductase) inhibitors, platelet inhibitors (acetylsalicylic acid, ticlopidin or clopidogrel), calcium channel antagonists and ß-blockers (reference: no therapy). In model 2, the Wald method was used for stepwise forward selection of covariates.

In a separate analysis, the dialysis patients were stratified for folic acid supplementation and the above survival analysis was performed for each stratum to test for a possible confounding role of folic acid supplementation on the effect of MTHFR genotype on the endpoints. In a further separate analysis, folic acid supplementation (reference: no supplementation) was entered as an a priori covariate in models 1 and 2 of the multivariate analysis detailed earlier.

Power calculations for survival analysis were performed with the ‘PS Power and Sample Size Calculations’ software package, Version 2.1.30 [16]. For the recessive mode of inheritance (MTHFR C677T reference: 677CC and CT combined), the study was powered with 0.82 to detect a HR of 2.0, with a 0.05 type I error probability, given an accrual period of 6 months and a follow-up period of 48 months, a ratio of ‘controls’ (MTHFR 677CC and CT combined) to ‘experimental’ group (MTHFR 677TT) of 12.7 and a median survival of the ‘control’ group of 2.3 years. For the dominant mode of inheritance (MTHFR C677T reference: 677CC), the study was powered with 0.82 to detect an HR of 1.32, with a 0.05 type I error probability, given an accrual period of 6 months and a follow-up period of 48 months, a ratio of ‘controls’ (MTHFR 677CC) to ‘experimental’ group (MTHFR 677CT and TT combined) of 0.75 and a median survival of the ‘control’ group of 2.2 years.

Multivariate logistic regression analysis was performed to analyse the effect of MTHFR C677T genotype on differences in comorbidities between ESRD cases and normalbuminuric controls: case/control status (reference: control), MTHFR C677T genotype (additive model, df = 1; reference: CC), age at the start of dialysis (ESRD cases, years) or follow-up (normalbuminuric controls, years) and gender (reference: female) were entered into the equation. History of the comorbidities ‘coronary artery disease’, ‘myocardial infarction’, ‘cerebral ischaemia’ and ‘peripheral arterial disease Fontaine Stage IV’ were set as the dependent variables in four separate analyses.

Results are expressed as mean (±1 SD), unless stated otherwise. Comparisons of continuous variables between groups were performed by Student's t-test, analysis of variance (ANOVA), Wilcoxon-W or by Kruskal–Wallis tests and of categorical variables by Pearson's {chi}2 test where applicable. Statistical significance in all the tests was accepted at P < 0.05. Statistical analysis was performed with the SPSS® Version 12.0 software package (Chicago, USA).



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Baseline characteristics
Baseline characteristics of cases and genotype controls are given in Table 1. Male and female ESRD patients differed significantly with respect to age, BMI, smoking history, inflammation, PAD status and history of coronary intervention. The differences in other comorbidities were not significant. Similar differences were observed between male and female controls. In the comparison of ESRD and control patients, there were significant differences in the comorbidity profiles, and thus comedication, in both genders.


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  		Table 1. Demographic and clinical characteristics of type 2 diabetes patients with ESRD due to diabetic nephropathy and without microalbuminuria after 4-year follow-up. Except where indicated, data (±1 SD) are given as determined at study inclusion

 
MTHFR genotype distribution
Patients with ESRD due to DNP showed a distribution of MTHFR genotype that deviated significantly from a distribution expected in a population with genotype distribution in Hardy–Weinberg equilibrium (HWE) [17,18] (P = 0.003, Table 2). Cases with TT genotype were significantly underrepresented. In contrast, requirements for HWE were met in cases with dialysis duration <2 years before study inclusion, and in controls (total collective, subgroups of patients with and without novel microalbuminuria after a 4-year follow-up, Table 2).


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  		Table 2. Genotypic and allelic frequencies of the MTHFR (C677T) polymorphism in type 2 diabetic patients with progressive stages of diabetic nephropathy

 
Association of MTHFR genotype with anthropometric variables
In cases with ESRD due to DNP the frequency of the TT genotype was, formally significant, higher in patients with a history of CAD than in patients without such a history (9.4 vs 4.3%, P = 0.044). However, there were no differences in the presence of TT genotype between cases with and without a history of myocardial infarction (9.0 vs 6.6%, P = 0.39), PAD Stage IV (7.9 vs 6.8%, P = 0.65) or cerebral ischaemia (7.5 vs 7.2%, P = 0.9). In controls with (n = 95) or without (n = 148) novel microalbuminuria, comorbidity was evenly distributed in patients with or without TT genotype (data not shown).

In cases with the TT genotype, mean duration of diabetes at initiation of dialysis therapy was significantly higher than in patients with CT or CC genotype. In spite of a shorter duration of dialysis therapy, cases with TT genotype had a lower BMI, higher CRP and lower low-density liboprotein (LDL), though these differences were significant only for BMI. Similar results were obtained in the separate analysis of males and females. In genotype controls, there were no significant differences between genotypes (Table 3).


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  		Table 3. MTHFR genotype and anthropometric variables

 
Comorbidity differed significantly between ESRD cases and normalbuminuric controls (Table 1). In a multivariate logistic regression model, this association was due to case/control status (for all comorbidities), age (for CAD and myocardial infarction only) and gender (for CAD and myocardial infarction only), but not due to MTHFR genotype [multivariate odds ratio (OR) of MTHFR genotype corrected for case/control status, age and gender = 1.19, 0.99, 0.93, 1.02 for history of CAD, myocardial infarction, PAD Stage IV and cerebral ischaemia, respectively, P ≥ 0.2].

Folic acid supplementation
Folic acid supplementation was provided to 52.0 and 55.1% of females and males, respectively (Table 1). There was no statistically significant difference in the frequency of folic acid supplementation between genotypes or in mean daily dose of folic acid supplemented (Table 4).


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  		Table 4. Folic acid supplementation in cases with ESRD due to DNP (n = 439). Folic acid dose means are calculated only for patients receiving such supplementation

 
Survival analysis
In the Kaplan–Meier analysis, MTHFR C677T genotype had no effect on the risk for ACM (additive model: see Figure 1; for the dominant and recessive models: P = 0.44, P = 0.45, respectively) or any of the secondary endpoints (cardiac: P = 0.94, P = 0.84 and P = 0.74 for the additive, dominant and recessive models, respectively; infectious: P = 0.21, P = 0.16 and P = 0.47 for the additive, dominant and recessive models, respectively). In the Cox regression analysis, there was no significant impact of MTHFR genotype on ACM (recessive model: see Table 5; for the additive and dominant models of model 2 multivariate analysis: HR = 0.94, P = 0.53 and HR = 0.9, P = 0.39, respectively) or secondary endpoints (additive mode of inheritance, multivariate model 2: HR for cardiac endpoints = 1.01, P = 0.98; HR for infectious endpoints = 0.74, P = 0.2—dominant mode of inheritance, multivariate model 2: HR for cardiac endpoints = 1.0, P = 1.0; HR for infectious endpoints = 0.64, P = 0.11—recessive mode of inheritance, multivariate model 2: HR for cardiac endpoints = 1.03, P = 0.94; HR for infectious endpoints = 1.0, P = 1.0).


Figure 1
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  		Fig. 1. Kaplan–Meier analysis of the effect of MTHFR C677T genotype on survival. Endpoint is—ACM P = 0.49 (log-rank test).

 

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  		Table 5. Effect of MTHFR C677T genotype on ACM in the complete collective (n = 439). HRs are given for MTHFR TT patients (i.e. recessive model: reference: [CC+CT])

 
Similarly, in patients with <2 years of dialysis therapy at study inclusion, there was no significant influence of C677T genotype on survival (multivariate model 2: HR for additive model for 677T = 0.93, P = 0.62; HR for recessive model for 677T = 0.99, P = 0.98) or secondary endpoints. Also, when the effect of MTHFR C677T genotype on ACM or secondary endpoints was calculated only in those patients with known cause of death and in those not dead at follow-up, similar results were obtained (data not shown).

Further, there was no effect of MTHFR C677T genotype (additive, dominant or recessive model) on the primary or secondary endpoints in univariate or multivariate analysis when the patients were stratified for folic acid supplementation (model 2 multivariate analysis: patients with folic acid supplementation—HR of 677TT patients for cardiac mortality = 1.18, P = 0.75; patients without folic acid supplementation—HR of 677TT patients for cardiac mortality = 0.68, P = 0.54; other data not shown) or when folic acid supplementation was an a priori covariate in the multivariate analyses (model 2 multivariate analysis: HR of 677TT patients for cardiac mortality = 1.02, P = 0.96). Similar data were obtained in the analysis of patients with <2 years dialysis therapy at study initiation (data not shown).



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Our study of the effect of MTHFR genotype on mortality in a cohort with ESRD exclusively due to DNP confirms previous findings in a general Italian ESRD cohort, in which there was no significant effect of MTHFR C677T genotype on survival [12]. Though our ESRD patients with TT genotype had had diabetes longer, and the syndrome of chronic inflammation more frequently (including lower BMI and LDL), these patients were not at a higher risk for premature mortality in comparison with patients with CC or CT genotype.

Our findings are also in agreement with recent studies on mortality risk in kidney transplant recipients [19], on CAD risk in patients with heterozygous familial hypercholesterolaemia [20] and on CAD in the general population [21], which all failed to show an association of MTHFR 677TT genotype with cardiovascular endpoints, but in contrast to a meta-analysis with evidence for a weak association of MTHFR 677TT genotype with coronary heart disease [22].

However, in contrast to the Italian ESRD cohort [12], we observed a significant underrepresentation of patients with MTHFR 677TT genotype. In contrast, MTHFR genotype did not deviate from that expected from a population in HWE in our cohort of type 2 diabetes mellitus genotype controls without DNP and in the subgroup of ESRD patients with dialysis duration <2 years.

Whenever, within a population, each person's two gene alleles are drawn independently and at random from the gene pool, then a population is said to be in HWE. Deviations from this equilibrium arise, e.g. when there is non-random mating (e.g. inbreeding), when certain genotypes provide the carrier with a survival advantage or disadvantage or due to the recruiting strategy.

Our ESRD cohort is drawn from 30 dialysis centres in Southern Germany and the control cohort from a diabetes centre that derives its patients from across Germany, but predominantly from Southern Germany. We are not aware of any consanguinity within the cohorts. Accordingly, the observed deviation from HWE in the ESRD cohort is unlikely to be due to non-random mating. The deviation from HWE could be due to the recruiting strategy in spite of the fact that other polymorphisms do not deviate from HWE in our ESRD cohort [14]: we recruited all prevalent ESRD patients with DNP in the participating dialysis centres. Thus, in light of the previously published association of MTHFR 677TT genotype with elevated homocysteine levels, its association with cardiovascular morbidity [9,10] and the adherence to HWE in our subgroup with ESRD <2 years but not in the total ESRD cohort, a survival bias in our ESRD cohort seems possible. Hereby, a subgroup of ESRD patients with 677TT genotype would not survive long enough on dialysis therapy or remain on dialysis without transplantation to be included in our cohort of prevalent ESRD patients. This assumption is supported by our observation that ESRD patients with 677TT genotype have a lower duration of dialysis therapy at study inclusion.

However, by this hypothesis, one would expect a survival difference by genotype in the subgroup of patients in adherence with HWE (i.e. patients with dialysis duration <2 years), as we have previously described for variants in the RANTES gene [23]. We did not observe higher mortality in the total collective (Figure 1, Table 5) or the subgroup, nor did we observe a higher rate of renal transplantation in patients with MTHFR 677TT genotype (renal transplantation was performed in five, four and two ESRD patients with CC, CT and TT MTHFR C677T genotype, respectively). Though this may have been due to the fact that the study was underpowered to detect small-to-medium gene effects in the recessive model, the Kaplan–Meier graphic suggests that any MTHFR gene effect detected in larger collectives would be negligible.

A further, speculative reason for deviation from HWE in the ESRD cohort could be a ‘protection’ of 677TT patients from ESRD. This could be due to either increased mortality before patients reach ESRD, or protection from progression to higher degrees of DNP. Mortality in the genotype control group was too low to adequately address the first issue (in the initial control cohort of 476 successfully genotyped patients, 11, nine and four patients with CC, CT and TT MTHFR C677T genotype died, respectively). We observed no association of MTHFR C677T genotype with progression to novel microalbuminuria in the initial control cohort of 476 patients (Table 2). Thus, the reasons for the observed deviation from HWE in the ESRD cohort remain obscure. It is possible that population stratification, which we did not examine in this study, plays a role, or that the C677T locus is associated with another locus, within the MTHFR gene or in another gene, with an as yet undetermined effect on our cohort's composition. A further polymorphism, the A1298C variant, within the MTHFR gene has inconsistently been shown to affect plasma homocysteine levels [24–27]. It appears that the 677CT/1298AC compound heterozygote genotype is associated with similarly elevated homocysteine levels as 677TT homozygotes in healthy individuals [24] but not in dialysis [24] or renal transplant recipient patients [25]. In a smaller study on healthy individuals, no effect of MTHFR C677T/A1298C compound genotype was observed on plasma homocysteine levels [26]. In 530 CAD patients, the A1298C MTHFR genotype had no effect on plasma homocysteine levels, whilst patients homozygous for 677T had significantly higher values than CC or CT patients [27]. In light of this evidence and of data indicating near complete linkage disequilibrium between the two polymorphisms [28], it is unlikely that knowledge of the A1298C genotype of our collective will clarify the issue of deviation from HWE. Also, any significant association of A1298C genotype with increased or decreased risk of primary or secondary endpoints, though unlikely due to near complete linkage disequilibrium with the C677T locus, would thus have to be viewed with suspicion.

Deviation from HWE may also be explained by the finding that the 677TT genotype may accelerate the progression from microalbuminuria to the proteinuric stage of DNP or from the proteinuric stage to ESRD [29]. Our study is not designed to detect this since we did not include patients in the different stages of DNP from microalbuminuria to proteinuria. Our control collective has a low risk of progressing to advanced stages of DNP or ESRD, given the long duration of diabetes mellitus. Importantly, it has been proposed that there are distinct genetic bases to the development of DNP with microalbuminuria and of advanced DNP with proteinuria with or without renal insufficiency [30].

Our study has drawbacks. First, it was underpowered to detect a minor gene effect. However, as noted previously by others [3], the risk implied by the observed HR of MTHFR C677T genotype appears small and negligible in light of other risk factors. Second, we did not measure homocysteine levels to support the notion that MTHFR genotype affects homocysteine levels. However, other groups have repeatedly shown an association of 677TT genotype with elevated levels [1,3,6] and this evidence would have been important only if we had found a significant association of MTHFR genotype with mortality.

Plasma homocysteine levels may have been lowered in patients receiving folic acid supplementation [31], and this may have reduced any effect of MTHFR C677T genotype on endpoints in patients receiving folic acid supplementation. However, in a separate analysis of patients without supplementation, an effect of MTHFR genotype could not be demonstrated. Our finding is thus in agreement with two large, recent studies showing no significant effect of folic acid supplementation on cardiovascular outcome in spite of significant reductions of plasma homocysteine levels achieved by supplementation [32,33].

In conclusion, our study fails to show an effect of MTHFR C677T genotype on mortality in type 2 diabetes mellitus patients with ESRD or on progression to novel microalbuminuria in type 2 diabetes controls.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This study was supported in part by grants from the ReForM-B and -C-Program of the Medical Faculty of the University of Regensburg, by Ortho-Biotech (Janssen Cilag) and by the 2005 MSD Diabetes Stipend to CAB. The support of the patients, the physicians and the staff at the Diabetes Zentrum Mergentheim and at the dialysis centres KfH Amberg, KfH Bayreuth, KfH Deggendorf, KfH Donauwörth, KfH Freising, KfH Freyung, KfH Fürth, KfH Hof, KfH Ingolstadt, KfH Kelheim, KfH München Elsenheimerstraße, KfH München-Schwabing, KfH Neumarkt, KfH Neusäß, KfH Oberschleißheim, KfH Passau, KfH Plauen, KfH Regensburg Günzstraße, KfH Regensburg Caritas-Krankenhaus, KfH Straubing, KfH Sulzbach-Rosenberg, KfH Weiden, Dialysezentrum Augsburg Dr. Kirschner, Dialysezentrum Bad Alexandersbad, KfH Bamberg, Dialysezentrum Emmering, Dialysezentrum Klinikum Landshut, Dialysezentrum Landshut, Dialysezentrum Pfarrkirchen and Dialysezentrum Schwandorf for participating in the study is gratefully acknowledged. We wish to thank Claudia Strohmeier and Gabriele Spatar for their expert technical assistance.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Harmon DL, Doyle RM, Meleady R, et al. (1999) Genetic analysis of the thermolabile variant of 5,10-methylenetetrahydrofolate reductase as a risk factor for ischemic stroke. Arterioscler Thromb Vasc Biol 19:208–211.[Abstract/Free Full Text]
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  3. Anderson JL, Muhlestein JB, Horne BD, et al. (2000) Plasma homocysteine predicts mortality independently of traditional risk factors and C-reactive protein in patients with angiographically defined coronary artery disease. Circulation 102:1227–32.
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Received for publication: 24. 4.06
Accepted in revised form: 1. 8.06


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