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ORIGINAL Methylenetetrahydrofolate Reductase Gene Polymorphism C677T is Associated With Increased Risk of Coronary Heart Disease in Chinese Type 2 Diabetic Patients

Objective Chronic cardiovascular diseases induced by long-term poor blood glucose control are the main cause of death in patients with type 2 diabetes mellitus (T2DM). Previous research reports that methylenetetrahydrofolate reductase gene ( MTHFR ) polymorphisms might influence the occurrence of coronary heart disease (CHD) in T2DM patients. The purpose of this study was to evaluate whether MTHFR C677T and A1298C mutations are associated with the risk of CHD in T2DM patients. Methods A total of 197 subjects with T2DM were studied, of which 95 patients with CHD. The genotypes of MTHFR C677T and A1298C were analyzed by using the dideoxy chain-termination method, and compared between patients with CHD and those without CHD. Results We found that the frequency of the 677T allele was significantly higher in T2DM patients with CHD than those without CHD ( P =0.011). However, there was no have reported no association between CHD and the MTHFR 677T allele in patients with T2DM. [29-31] The contradictory results exist in various populations, suggesting that further studies including a larger sample size and different populations are required before confirming the role of the MTHFR 677T allele in CHD development. Our results showed that there was no significant difference in the haplotypes of MTHFR (C677T and A1298C) gene between T2DM patients with CHD and those without CHD. These findings suggested that the haplotypes of the MTHFR (C677T and A1298C) gene are not associated with susceptibility of CHD in patients with T2DM.

conclusions are not consistent whether the polymorphism of MTHFR could be a predictor for the CHD in T2DM patients. [6,[18][19][20][21][22][23] The main purpose of this study was to evaluate the frequency of C677T and A1298C mutations in the Chinese population with T2DM and to assess whether their polymorphisms are associated with the risk of CHD in T2DM.

Study subjects and data collection
A total of 197 subjects with T2DM were included in this study, of which 95 patients were combined with CHD. All of the study subjects were older than 30 years old.
Basic information including gender, age, height and weight was collected by questionnaire.
Diabetes was diagnosed according to the diabetes diagnostic criteria published by the World Health Organization (WHO). [24] CHD was diagnosed according to the patient's physical examination, supplementary examination, and medical history. All of the diagnoses of CHD were consistent with the diagnostic criteria for International Society of Cardiology and WHO. [25] Exclusion criteria: pregnancy and lactating women; patients with primary heart disease; severe kidney disease with serum creatinine (Scr) levels>132 μmol/L (serious kidney disease may be an important factor leading to atherosclerosis); patients taking folic acid or vitamin B 6 or B 12

Clinical characteristics of the study patients
The clinical characteristics of the participants are shown in Table 1. There were no significant differences in the variables including gender, age, FPG, lipid, blood pressure, or Scr among T2DM subjects with or without CHD (P>0.05). with the sequencing map shown in Figure 1. All 197 subjects with T2DM were genotyped at MTHFR C677T, and only 167 subjects (100 with CHD and 67 without CHD) were genotyped at A1298C.
The distributions of MTHFR C677T and A1298C genotypes in T2DM patients with or without CHD are shown in Table 2 and Table 3. The frequencies of MTHFR 677CT+TT in T2DM patients with and without CHD were 90.53% and 78.43%, respectively. The frequencies of the CT+TT genotype and T allele were more common in T2DM patients with CHD than in those without CHD (P=0.020, 0.011).
However, the frequencies of MTHFR 1298AC+CC genotype or alleles showed no significant difference between these two groups (P>0.05).
There were three haplotypes at the two loci of MTHFR (C677T and A1298C) when the haplotype frequency is >3%. The haplotypes of the MTHFR (C677T and A1298C) are showed in Table 4. No significant difference was found in any of the examined haplotypes between T2DM patients with CHD and those without CHD.

Interaction between MTHFR (C677T and A1298C) gene polymorphism and Hcy levels
The Hcy levels in T2DM patients with CHD were significantly higher than those without CHD (14.26±6.09 vs. 11.76±4.13 μmol/L; P=0.001). We divided the diabetic patients into two groups according to Hcy levels (lower levels: ≤15.0 μmol/L; higher levels: >15 μmol/L) and assessed the relationship between MTHFR polymorphism and CHD development. Data showed that the frequency of the MTHFR 677CT+TT genotype was significantly higher in patients with CHD than that in those without CHD in the lower Hcy levels group (P=0.006), while no significant difference was observed in the higher Hcy levels group (P>0.05). There was no significant difference in the distribution of A1298C genotypes (AA vs. AC+CC) between patients with and without CHD, neither in lower nor in higher Hcy levels groups (P>0.05) ( Table 5).  Hcy: homocysteine.

DISCUSSION
In the pathway of Hcy metabolism MTHFR is a key enzyme that is able to re-methylate Hcy into methionine. [7] Dysfunctional mutations of the MTHFR gene are related to a decrease in MTHFR activity, resulting in an increase in the plasma concentration of Hcy. [8,10,11] Several studies have confirmed the important role of Hhcy in vascular complications. [12,13] An elevated plasma level of Hcy is associated with endothelial dysfunction, prethrombotic state and coronary atherosclerosis in patients with T2DM. [6,[14][15][16][17] These effects of Hcy could be explanatory of a close correlation between the mutations of MTHFR gene that cause Hhcy and the presence of CHD in patients with T2DM.
In the present study, the data showed that the 677T allele was significantly associated with an increased risk of CHD in patients with T2DM, while no association was found between A1298C polymorphism and CHD development. These results are in accordance with previous research results conducted in including Czech, Polish and Chinese participants. [20,[26][27][28] Although MTHFR 677T has been verified to be a risk allele for CHD in the past. However, opposite conclusions still exist. Several studies On the other hand, studies have confirmed that an elevated plasma levels of Hcy has been identified as a potential risk factor for the development of CHD in patients with T2DM. [6,[32][33][34] Our findings also show that plasma Hcy values were significantly higher in T2DM patients with CHD than those without CHD. Clinically, Hcy could be a useful predictive biomarker for CHD risk in type 2 diabetic patients. However, for the patients with normal Hcy levels, it is necessary to find another reliable biomarker to predict CHD. In our study, MTHFR C677T mutation showed great value in predicting CHD in T2DM patients with lower Hcy levels. We have found that the MTHFR 667T allele could be used as a biomarker of CHD risk in T2DM patients with "normal" Hcy levels. However, for patients with higher Hcy levels, it seems that MTHFR gene polymorphism could not be a valuable marker to predict CHD progression.
The small sample size is one of the main limitations of this study. Our study was conducted with 197 T2DM patients. Therefore, there was insufficient statistical power to detect correlation. In particular, the sample size of the present study was limited when stratified by Hcy levels. The limitation of the sample size may cause deviations in the statistical results. Furthermore, this study was performed with a Chinese population, and the results need further investigation to confirm whether they could