Effects of the C677T and A1298C polymorphisms of the MTHFR gene on the genetic predisposition for diabetic nephropathy

Dariusz Moczulski, Hanna Fojcik, Ewa Zukowska-Szczechowska, Ilona Szydlowska and Wladyslaw Grzeszczak

Department of Internal Medicine, Diabetology and Nephrology, Silesian School of Medicine, Zabrze, Poland

Correspondence and offprint request to: Hanna Fojcik, Department of Internal Medicine, Diabetology and Nephrology, Silesian School of Medicine, Zabrze, Poland. Email: hfojcik{at}poczta.onet.pl



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Methylenetetrahydrofolate reductase (MTHFR) is a regulatory enzyme of homocysteine metabolism. The C677T polymorphism of the MTHFR gene has been reported to be associated with elevated plasma homocysteine in patients with low folic acid intake. A recently reported second common polymorphism, A1298C, may increase homocysteine, but only in individuals carrying the T677 allele. This study aimed to investigate the influence of the C677T and A1298C polymorphisms of the MTHFR gene on the development of diabetic nephropathy in Caucasian patients with type 2 diabetes.

Methods. We genotyped 429 type 2 diabetic patients for the C677T and A1298C polymorphisms using standard PCR-based protocols, and divided them into three groups based on renal status: 159 patients with normoalbuminuria, 149 with microalbuminuria, and 121 with persistent proteinuria and chronic renal failure (CRF). The C677T and A1298C genotype frequencies were compared among the three groups.

Results. Although the frequencies of the CT and TT genotypes of the C677T polymorphism tended to increase with each stage of diabetic nephropathy (53, 56 and 63% in normoalbuminuria, microalbuminuria and proteinuria/CRF, respectively), these differences were not significant. When male and female patients were analysed separately, the effect was seen only in males. The CT + TT genotype was present in 46% of male patients with normoalbuminuria, in 57% with microalbuminuria and in 68% with proteinuria/CRF (OR = 2.46; 95% CI 1.13–5.38). There were no differ-ences in the A1298C polymorphism among the three groups.

Conclusions. These findings indicate that the C677T polymorphism is a risk factor for diabetic nephrop-athy in male patients with type 2 diabetes.

Keywords: diabetic nephropathy; genetic susceptibility; homocysteine; MTHFR; type 2 diabetes mellitus



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The methylenetetrahydrofolate reductase (MTHFR) gene is located on chromosome 1 (1p36.3). Human MTHFR catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-metyltetrahydrofolate, a co-substrate for homocysteine remethylation to metionine. Severe MTHFR deficiency is associated with hyperhomocysteinaemia. Homocysteine occupies a pivotal position in the metabolism of the essential amino acid, methionine. It is at the junction point of the transsulfuration pathway, the formation of cysteine, and the excretion of sulphur, and is pivotal for the remethylation of homocysteine to methionine with conservation of the carbon skeleton [1].

Elevated homocysteine levels have been identified as a risk factor for diabetic nephropathy in type 2 diabetes [2,3]. In addition, increased plasma homocysteine is an independent risk factor for several vasculopathies including arteriosclerosis, acute myocardial infraction, cerebrovascular diseases, arterial and venous thrombosis [47]. Hyperhomocysteinaemia has also been observed in families with neural tube defects [8].

The best-characterized polymorphism of the MTHFR gene is the C677T transition. This mutation causes an alanine to valine amino acid substitution. The result is a thermo labile MTHFR variant that has reduced catalytic activity and which can be stabilized by folate. Homozygosity for this MTHFR variant predisposes individuals for the development of hyperhomocysteinaemia, especially during periods of folate insufficiency [9]. Noiri et al. [3] reported an increased frequency of the CT and TT genotypes in male haemodialysed patients with type 2 diabetes as well as a correlation between the presence of the C677T allele and the progression of renal failure [3].

An additional polymorphism of the MTHFR gene, A1298C, causes a substitution of glutamate for an alanine residue. The A1298C polymorphism has been associated with an increased risk for neural tube defects [8], and has been shown to have reduced enzymatic activity in individuals carrying the T677 allele.

The aim of this study was to examine the influence of the C677T and A1298C polymorphisms of the MTHFR gene on the susceptibility for diabetic nephropathy in Caucasian patients with type 2 diabetes.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Selection of study patients
Between January 1, 1998 and December 31, 1998, we screened volunteer patients with type 2 diabetes (diagnosed according to the National Diabetes Data Group criteria) from the Outpatient Clinic for Diabetic Patients in Zabrze for microalbuminuria by using random urine samples tested with a semi-quantitative dipstick MICRAL II assay (Boehringer Mannheim, Mannheim, Germany). Patients (n = 1270) were screened for microalbuminuria and all dipstick-positive (>=20 mg albumin/dl) as well as dipstick-negative subjects with a known diabetes duration of at least 10 years were evaluated for renal status from repeated measurements of the albumin/creatinine ratio (ACR) in random urine samples, from serum creatinine concentrations and from careful reviews of medical files. Albumin concentration in urine was measured using an immunoturbidometric assay on a TurbiTimer with Albumin-Urine kits (Dade Behring Leiderbach, Germany).

Serum and urine creatinine concentrations were measured by spectrophotometry on a Kodak automated system. Normoalbuminuria was defined as an ACR (mg/mmol) of <1.9 for men and <2.8 for women [10]. Overt proteinuria was defined as an ACR >=28.2 for men and >=40.2 for women. Microalbuminuria was defined as ACRs between normoalbuminuria and overt proteinuria from the consensus of two out of three determinations (the interval between measurements was at least 2 weeks) combined with the absence of non-diabetic kidney disease (defined as acute or chronic urinary tract infection, asymmetrical kidney size, a rise in serum creatinine levels after ACE inhibition, a rapid decline in renal function or systemic symptoms of vasculitis).

Altogether, 429 patients met these criteria and were genotyped for the C677T and A1298C polymorphisms. In 21 additional subjects, genotypes were not assessed because of technical problems. Of the 429 genotyped subjects, 121 patients had overt proteinuria, with 46 of these having chronic renal failure (CRF; defined as persistent rise in serum creatinine >=1.5 mg/dl; 16 patients were dialysed), 149 patients had ACR in the microalbuminuria range and 159 control subjects had normoalbuminuria with a known diabetes duration >=10 years.

All patients also underwent fundus camera examination. Retinopathy status was graded by two independent observers as nihil, simplex, pre-proliferative or proliferative using fluorescing angiography and a third evaluation performed in case of discrepancies. From the total 450 genotyped and non-genotyped patients retinal status was available for 391 patients (59 patients did not attend eye examination or retinal status was difficult to obtain because bilateral cataract or other problems).

Patients were considered hypertensive if they were on antihypertensive medication or if systolic or diastolic blood pressure were >=140 or 90 mmHg (blood pressure was measured twice with a mercury sphygmomanometer, in supine position, after at least 10 min of rest, and results were averaged) at time of microalbuminuria screening and during the subsequent visit in the clinic. Of the 243 patients taking antihypertensive treatment, 162 were taking angiotensin-converting enzyme inhibitors.

Glycaemic control was assessed before examination by measuring glycated HbA1c blood levels on an HPLC Variant System (Bio-Rad, Hercules, CA, USA).

Total and protein-bound homocysteine was measured by automated high-performance liquid chromatography using a Hewlett-Packard 1100 Series system, according to the method of Bald et al. [11].

All enrolled patients gave written informed consent for participation in the study. The Ethics Committee of the Silesian School of Medicine approved the study protocol.

Genetic markers and genotyping
To detect the C677T and A1298C polymorphisms, PCR-based protocols and RLFP analysis were performed according to published techniques [8,9]. The C677T mutation introduces a HinfI restriction site that results in digestion of a 198-bp PCR amplicon into 175- and 23-bp fragments. The A1298C mutation abolishes a MobII restriction site, resulting in a 163-bp amplicon that is digested into four fragments of 84, 31, 30 and 18 bp. The wild-type 1298AA yields five fragments, and the 84-bp fragment is cut into 56- and 28-bp fragments producing base pair lengths of 56, 31, 30, 28 and 18. After digestion, the PCR product was resolved electrophoretically on the agarose gel.

We used the EH program (http://linkage.rockefeller.edu/ott/eh.htm) to test and estimate linkage disequilibrium between these two markers.

Statistical analysis
All statistical analyses were made with STATISTICA for Windows version 5.0. Quantitative variables were tested by analysis of variance, and frequencies were tested by {chi}2 test. The odds ratio (OR) was calculated and a 95% confidence interval (95% CI) was provided.

The Cochran–Armitage trend test was used to test for genotype frequency trends.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients with normoalbuminuria, microalbuminuria and proteinuria/CRF did not differ with regard to age at diagnosis of type 2 diabetes or body mass index (Table 1). Although patients with normoalbuminuria had longer known duration of type 2 diabetes than subjects with microalbuminuria and proteinuria/CRF, all subjects were recruited to have diabetes duration of at least 10 years to avoid misclassifications. As presented in Table 1, hypertension was more frequent in patients with microalbuminuria and proteinuria/CRF than in subjects with normoalbuminuria. The frequency of retinopathy, especially the pre-proliferative and proliferative types, was greater in patients with proteinuria/CRF than in subjects with normoalbuminuria and microalbumin-uria.


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Table 1. Clinical characteristics of type 2 diabetes patients stratified according to nephropathy status (means ± SD)

 
In non-dialysis patients there was a correlation between plasma homocysteine and serum creatinine in (r = 0.15, P-value = 0.0012).

The distribution of C677T genotypes in the three subgroups of is shown in Table 2.


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Table 2. Distribution of C677T genotypes in the three nephropathy status subgroups

 
The frequency of the CT + TT genotype, in both males and females combined, increased with each stage of diabetic nephropathy and ranged from 53, 56 to 63% in normoalbuminuria, microalbuminuria and proteinuria, respectively. However, when males and females were analysed separately, the effect was seen only in males. The CT + TT genotype was present in 46% of male patients having normoalbuminuria, in 57% having microalbuminuria and in 68% having proteinuria/CFR (OR = 2.46; 95% CI 1.13–5.38). The Cochran–Armitage trend test indicated a trend for increasing CT and TT genotype frequency with each stage of diabetic nephropathy. The P-value was 0.0049 for all patients and 0.011 for male patients.

Table 3 shows that there was no significant difference in the A1298C genotype distribution between the three subgroups.


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Table 3. Distribution of A1298C genotypes in the three nephropathy status subgroups

 
The frequency of 1298AC/677CT double heterozygotes is shown in Table 4. In male patients only, the double heterozygote genotype increased with each stage of diabetic nephropathy. The risk of diabetic nephropathy was higher for double heterozygotes (OR = 3.04; CI 95% 1.15–8.07) than for the C677T genotype (OR = 2.46; CI 95% 1.13–5.38).


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Table 4. Comparison of 1298AC/ 677CT double heterozygotes with other genotypes in the renal status subgroups

 
Patients with proteinuria/CFR had higher homocysteine levels than patients with normoalbuminuria and microalbuminuria. However, when patients with the CC genotype were compared with the CT and TT genotype, there was no correlation between the 677T allele and homocysteine levels.

Using the EH program, we estimated that there was complete linkage disequilibrium between markers 677 and 1298 in the MTHFR gene. There were four possible haplotypes of these two markers, but the CT haplotype was very rare. These haplotypes and their estimated frequencies, shown in Table 5, were not different among the renal status subgroups.


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Table 5. Four possible haplotypes and their estimated frequencies

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The present study showed that the C677T polymorphism is a risk factor for diabetic nephropathy in male patients with type 2 diabetes, and that the CT + TT genotype increases in frequency with each stage of diabetic nephropathy. These findings, which are in agreement with observations in a Japanese population [3], suggest that the MTHFR gene may be an aggravating factor for diabetic nephropathy in type 2 diabetes subjects, but only in male patients.

An association between the MTHFR gene polymorphism and diabetic nephropathy in type 2 diabetes was suggested previously by Neugebauer et al. [2]. In addition, Noiri et al. [3] were the first to examine the sex dependence of the MTHFR genotype distribution. An increased frequency of CT and TT genotypes has been reported previously in haemodialysed male patients with type 2 diabetes and the correlation between the presence of the 677T allele and the progression of renal failure has also been demonstrated.

The second polymorphism of the MTHFR gene, A1298C, was reported previously to be a risk factor for neural tube defects [8] and for cardiovascular disease [12]. The A1298C polymorphism by itself does not affect plasma homocysteine but may have a modest effect when present with the T677 variant [8].

In our study, the frequency of the A1298C genotypes did not differ among patients with different stages of diabetic nephropathy. However, when the C677T and A1298C genotypes were combined, we found an increasing frequency of the C677T and A1298C double heterozygotes at each stage of diabetic nephropathy in male patients with type 2 diabetes. The risk of diabetic nephropathy was higher for double heterozygotes than for the C677T genotype.

These results suggest that two variants of the MTHFR gene should be assessed as genetic risk factors for diabetic nephropathy in male patients with type 2 diabetes. However, caution is advised in interpreting these results due to the small number of 1298AC/677CT double heterozygote patients and because weak, this association may disappear in a larger population.

The C677T polymorphism may have a greater association with diabetic nephropathy than A1298C because of the localization of these two variants. The C677T polymorphism is in the exon 4, which is within the N-terminal catalytic domain of the enzyme, whereas the A1298C polymorphism is in the exon 7, which is within the C-terminal regulatory domain. The more dynamic effect of C677T is due to its location within the catalytic region. The A1298C polymorphism may affect enzyme regulation, possibly by influencing S-adenosylmethionine, which is an allosteric inhibitor of MTHFR and which is known to bind in the C-terminal region [13]. Van der Put et al. [8] reported a similar effect while studying risk factors for neural tube defects. Double heterozygotes for both polymorphisms had reduced MTHFR-specific activity and had higher homocysteine levels whereas the A1298C mutation by itself had no effect [8]. In addition, Weisberg et al. [14] found that the A1298C mutation reduced MTHFR activity, but to a lesser extent than the C677T mutation. Consequently, effects on both MTHFR activity and homocysteine levels may become significant only when an individual carries both mutations or has poor nutrient status [14].

Although our data suggest that the C677T and A1298C polymorphisms of the MTHFR gene are responsible for diabetic nephropathy, these mutations may be in linkage disequilibrium with another marker involved in this pathology. To examine this possibility, we performed a haplotype analysis using the EH program. We failed to find a correlation among haplotypes and renal status indicating that it was the analysed polymorphisms rather than another marker involved in the genetic susceptibility to diabetic nephropathy.

Although our patients with persistent proteinuria and CRF had higher plasma homocystine levels, a stratification of patients by renal status, did not produce a correlation between the C677T genotype and homocysteine levels. This lack of correlation may have been secondary to single homocysteine determinations per individual. We measured homocysteine only once per patient and it should be measured several times at various intervals to obtain an accurate assessment of homocysteine status.

In our proteinuric patients, 25.6% were free of retinopathy. As reported by Christensen et al. [15], ~30% of these patients can have non-diabetic glomerulopathies [15]. We added these patients to this group because we did not see significant differences between patients with and without retinopathy.

Our finding that only men with MTHFR polymorphisms had a predisposition for the development of diabetic nephropathy was puzzling. Several scenarios may explain this association. First, this finding may be related to lifestyle patterns. For example, cigarette smoking, a lack of vitamin supplements and coffee consumption have all been related to hyperhomocystinaemia [16]. It is well known that there are more smokers among men and this may represent an aggravating factor for hyperhomocystinaemia and vascular complications. Secondly, homocysteine levels have been correlated with fat-free mass and testosterone but inversely with estradiol [17]. This gender difference in homocysteine levels may be secondary to differences in fat-free mass or in hormone levels. Hormone replacement therapy is known to affect homocysteine blood levels but the duration is inconsistent [18] and is weak in women having the C677T polymorphism. As a third scenario, there is a direct metabolic relationship between creatinine and homocysteine. Creatinine originates from metabolism in skeletal muscles, and the amount of released creatinine is therefore determined by muscle mass. The formation of creatine, the precursor of creatinine, depends on a methyl donation by S-adenosylmethionine, leading to the formation of homocysteine. Thus, the level of homocysteine would be expected to reflect both muscle mass and creatinine concentration. This may explain why men generally have higher concentrations of both homocysteine and creatinine than women [19].

Together, these causes lead to hyperhomocys-teinaemia, and men with the C677T polymorphism and the risk factors mentioned previously may be more susceptible for developing diabetic nephropathy and vascular diseases than women, who are more protected from hyperhomocysteinaemia.

In conclusion, the data suggest that the C677T polymorphism in the MTHFR gene may be a risk factor for diabetic nephropathy in male patients with type 2 diabetes.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 9. 4.02
Accepted in revised form: 22. 1.03





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