Association of homocysteine with type 2 diabetes: a meta-analysis implementing Mendelian randomization approach

The pathophysiology of type 2 diabetes mellitus (T2DM) characterized by a high incidence of vascular complications is complex and multifactorial [1]. The major cause of T2DM and its complications has not yet been clarified. In recent years, plasma homocysteine (Hcy) level, a sulphur-containing non-protein amino acid in the metabolism of methionine, has been reported to be associated with the vascular complications of diabetes [2]. In patients with diabetes, elevated Hcy levels were associated with insulin resistance [3] and nephropathy [4]. Of note, several studies have demonstrated that elevated Hcy levels predict the risk of death or coronary events in patients with T2DM [5, 6]. However, conflicting results regarding the Hcy level in patients with diabetes have been reported. Some studies found that plasma Hcy levels were increased [7, 8], unchanged [9–11], or decreased [12, 13] in patients with T2DM.

The candidate gene approach is widely used for identifying genes involved in complex human diseases [14]. 5,10 methylene-tetrahydrofolate reductase (MTHFR) is the key rate-limiting enzyme required for the conversion of dietary folate to 5-methyltetrahydrofolate, the methyl group donor required for the remethylation of Hcy to methionine in vivo [15]. Hyperhomocysteinemia (HHcy) may be due to the presence of a thermolabile isoform of this key enzyme. A single base pair (677C > T) substitution in the human MTHFR gene predicts phenotypic expression of a heat-sensitive variant with reduced enzymatic activity [16]. Elevated Hcy level caused by MTHFR genetic variants has been demonstrated to be associated with insulin resistance [17–19]. Hcy exerts detrimental effects on a number of cell lineages including endothelial cells and neuronal cells through production of reactive oxygen species (ROS) [20]. Both acute and prolonged exposure to Hcy had detrimental effects on beta cell glucose metabolism, insulin secretory responsiveness and cell viability [20, 21]. Hcy generates ROS in a redox-cycling reaction that explains the decline in viability of insulin-secreting cells, leading to reduced glucokinase phosphorylating ability, diminished insulin secretory responsiveness and cell death [22]. Based on its biological functions, this variant has been considered an ideal candidate for genetic polymorphism for predisposition to diabetes [23], as it is common in many populations studied to date and the genotype correlates highly with the plasma Hcy level in a dose-dependent fashion [24].

In recent years, there have been a number of case–control studies investigating the association between the 677 C > T polymorphism in the MTHFR gene and T2DM or diabetes-related complications [12, 16, 25–40]. However, these studies have reported conflicting results, which has prevented a definitive conclusion to date. The associations of Hcy, MTHFR polymorphism and T2DM are still inconclusive.

To provide an answer to these contradictory results and elucidate the possible mechanism, a meta-analysis of the published literature regarding the risk for T2DM associated with an elevated Hcy level and the MTHFR 677C > T was conducted. In this meta-analysis, the estimate of the genetic association of each individual study and a pooled estimate of this association were obtained. Furthermore, to test the hypothesis that elevated Hcy is causally related to increased risk of T2DM, Mendelian randomization analysis, which is an epidemiological approach based on the fact that individuals inherit genetic variants randomly from their parents, was conducted.

Overall, we found that MTHFR 677 T was significantly associated with elevated plasma Hcy concentration. The pooled mean Hcy concentration was greater in subjects with T2DM than in those without T2DM. Our findings from Mendelian randomization approach supported the hypothesis that elevated Hcy is causally related to increased risk of T2DM.

Previous studies have given conflicting results regarding the circulating Hcy levels in patients with diabetes. An association between Hcy level and diabetic complications was seen in type I diabetes, but not among T2DM [7]. Very recently, a study on 105 patients with T2DM and normal renal function also found that in patients with T2DM, basal level of Hcy was 35% lower compared with healthy subjects [12]. The reduced Hcy level may be explained by an influence of hyperglycemia on acceleration in the hepatic transsulfuration pathway due to insulin disorder [12], subsequently, elevated glucocorticoids decreased Hcy level [55]. Cell culture and animal studies also showed that the diabetic status may reduce rather than increase the circulating Hcy level due to enhanced Hcy catabolism [55, 56]. However, it should be emphasized that this data was derived from animal models of experimental diabetes and cannot provide solid evidence that a similar relationship between diabetic status and Hcy level exists in patients with diabetes. Moreover, many other studies found that plasma Hcy levels were significantly higher in diabetic patients than in subjects without diabetes. In the present meta-analysis, the main analysis for weighted mean difference in Hcy between subjects with and without T2DM in all included studies revealed significant heterogeneity between the studies. Therefore, we used random model to estimate the pooled mean Hcy. Similarly, the absolute pooled mean Hcy concentration in individuals with T2DM was also significantly higher than that in control subjects in random model. We speculated that a large part of the discrepancy resulted from the heterogeneity of patients included in these studies with regard to the stage of disease, status of renal function or presence of macroangiopathy [4, 9]. Furthermore, the opposing influences on circulating Hcy levels exercised by the diabetic status per se and by a number of risk factors that relate to or are accentuated by diabetes may also help explain the controversy [25].

Importantly, the heterogeneity may also result from the genetic background of the patients. A common variant (MTHFR 677 C > T) of the enzyme MTHFR involved in the Hcy metabolic pathway may affect Hcy concentration [57]. Among the diabetic group, patients carrying the T allele showed a increased Hcy level in comparison to those having the native genotype CC [23]. This contradicted a previous report which suggested that double heterozygosity was associated with reduced enzyme activity and high Hcy level [58]. Interestingly, the MTHFR 677 C > T appears to have a sex-specific effect on Hcy, Hcy level was slightly higher in men than in women [59]. Men carrying TT genotype appear to be at higher risk of HHcy than women with this genotype [60]. Plasma estrogen was found to decrease in MTHFR CC, but not in TT homozygous postmenopausal women [61]. The precise mechanism by which estrogen could affect Hcy metabolism is not known. But the sex-specific effect may contribute to the previous contradicted results. Our meta-analysis gave strong evidence that the MTHFR was associated with plasma Hcy in patients with T2DM. MTHFR 677TT have significantly higher plasma Hcy than other genotypes, which are consistent with previous results in diabetic subjects [25].

Because elevated plasma Hcy level in patients with T2DM has been reported, it may be readily postulated that the 677 C > T MTHFR gene polymorphism might be involved in the development of T2DM [23]. However, no literature data directly associates the MTHFR-linked Hcy metabolism with T2DM. To date, the relationship between the MTHFR 677 C > T polymorphism and T2DM is less clear. Previous studies found an association between mutant homozygous genotype for MTHFR 677 C > T and diabetic retinopathy in individuals with T2DM [62]. Similarly, another study has also found that the MTHFR 677 C > T predisposes T2DM patients to the development of diabetic retinopathy [63]. One study suggested the MTHFR mutant genotype as a possible risk factor for the development of left ventricular hypertrophy in T2DM [28]. The TT genotype was more frequent in T2DM patients than in healthy controls (13.9% vs. 10.3%). Of interest is that the Brazilian population holds the lowest frequency ever reported for the TT genotype (9%) [53], and the highest (19.1%) is in Chinese population [34]. In fact, the association between the MTHFR 677 C > T and diabetes was not found in some studies including Brazil [53], China [34], Germany [25]. The reasons why there are conflicting results in studies concerning MTHFR677 C > T and T2DM are still not understood, however, an important reason for conflicting results may relate to the different genetic background of patients included in the studies. The frequency of MTHFR 677TT varies in different ethnic groups, for example, the 677TT genotype frequency is the lowest in Moroccan patients and controls (8.87% and 9.92%, respectively) [39]. Similarly, low frequency of 677TT (9%) was also reported in a Brazilian population [53]. In contrast, a high frequency (19.1%) of the 677TT genotype was reported in a Chinese population [34]. Furthermore, small sample size, study design flaws or other biases may be more common reasons than true racial heterogeneity for the observed discrepancies between studies addressing genetic risks [64, 65]. The meta-analysis presented here included data from case–control association studies that investigated the relationship between MTHFR polymorphism and T2DM. The strength of the present analysis, however, is based on the aggregation of published case–control studies. The overall results indicated an association of MTHFR 677 C > T with T2DM and MTHFR TT genotype increases the risk of T2DM.

Our study is the first meta-analysis to date which has pooled all the data available to investigate the associations of MTHFR-linked Hcy and T2DM. Therefore we have provided the most extensive data on this issue. As with all meta-analyses, some limitations are also presented here. First, the lack of clinical homogeneity between the subjects in the included studies, although our inclusion criteria ensured that the selected studies were broadly similar, it was not possible to eliminate all sources of heterogeneity. Second, we pooled the data from different ethnicities together, genetic heterogeneity among diverse ethnic populations leads to some unavoidable bias. Third, many studies did not include information regarding vitamin status at the time of Hcy measurement, which prevented examination of the interaction between nutritional status, Hcy, genotypes, and diabetic risk. Differences in sampling protocols and methods of Hcy measurement may have contributed to variation between studies. Hcy measurement using different high-performance liquid chromatography methods has been reported to vary by 6 to 7% among different laboratories [66]. Fourthly, exclusion of studies which didn’t provide adequate information might contribute to the publication bias. We also cannot exclude the possibility of bias related to the exclusion of non-English language publications, although Begg’s and Egger’s tests did not show evidence for the presence of substantial publication bias with respect to the small effect sizes. Fifthly, although we observed elevated Hcy is causally related to increased risk of T2DM, recent clinical trial showed that lowering Hcy levels by daily supplementation with folic acid and vitamins B₆ and B₁₂ did not reduce the risk of developing T2D among women at high risk for CVD [67]. We specialated that the associations of Hcy with T2D may be modified by environmental factors such as dietary folate, as well as the potential gene-environment interactions involved in the development of T2D. Finally, the association of the MTHFR variant with T2D was not observed in large-scale GWAS studies with huge sample sizes. The possible reason is that this association is likely to be due to the combined effect of genes, environmental factors, and their interactions. However, most investigators conducting GWAS do not consider gene-environment interactions in their search for new genes due to a current lack of efficient statistical methods for detecting interactions in high-volume genetic data.

In summary, our results provided evidence that the TT genotype of MTHFR C677T contributes to susceptibility to T2DM, and supported the hypothesis that elevated Hcy is causally related to increased risk of T2DM. The existence of gene-environment interactions may explain the discrepancy of results obtained in individual genetic association studies. Therefore, case–control studies that investigate gene-environment interactions might help further elucidate the genetics of T2DM.

Written informed consent was obtained from the patient for the publication of this report and any accompanying images.