Association analysis of MTHFR (rs1801133 and rs1801131) gene polymorphism towards the development of type 2 diabetes mellitus in Dali area population from Yunnan Province, China

Introduction

Type 2 diabetes mellitus (T2DM) is one of the most prevalent metabolic diseases and is characterized by high glucose levels and insulin resistance. In China, approximately 140 million adults suffer from diabetes mellitus, and this number will reach 170 million by 2045 (Magliano, Boyko & IDF Diabetes Atlas 10th edition scientific committee, 2021). It has been verified that the progression of T2DM is remarkably affected by multiple genetic factors. Genetics can determine the susceptibility of an individual to developing T2DM (Tinajero & Malik, 2021). Therefore, the identification of inheritable susceptibility genes for T2DM can provide valuable information and a reference for the clinical prevention of T2DM.

Evidence has indicated that individuals with insufficient intake of folic acid, also known as folate, vitamin B9, and vitamin B11, are more likely to suffer from T2DM (Hayden & Tyagi, 2021). 5,10-methylenetetrahydrofolate reductase (MTHFR) is a key enzyme involved in folate metabolism. MTHFR, located at human chromosome 1p36.3, participates in the catalysis of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (Goyette et al., 1994). The decrease of this enzyme activity leads to the increase of homocysteine (Hcy) level, which induces vascular endothelial injury and dysfunction and contributes to cardiovascular diseases, diabetes, tumors, and other diseases (Ponce-Ruiz et al., 2020; Lapik, Ranjit & Galchenko, 2021; Gupta et al., 2018; Wang et al., 2022; Fan et al., 2020; Nasri et al., 2019). The MTHFR gene has numerous single nucleotide polymorphisms (SNPs) while two of them, rs1801133 (MTHFR C677T) and rs1801131 (MTHFR A1298C) were extensively investigated concerning the risk of T2DM, although the results were conflicting (Pathak et al., 2022; Shorikov et al., 2022; El Alami et al., 2024). MTHFR C677T polymorphism showed a more significant association with T2DM in the Asian population than in the European and African populations (Meng et al., 2019). Such differences might be caused by different genetics and environment. Therefore, the association between these variations and T2DM needs to be verified in different areas and ethnic groups.

The excessive oxidative stress produced by abnormalities in glucose metabolism usually found in T2DM patients (Darenskaya, Kolesnikova & Kolesnikov, 2021). It was reported that single nucleotide polymorphisms (SNPs) of genes encoding the antioxidant enzymes, such asglutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), and paraoxonase (PON), affect the oxsidative stress (Oh et al., 2007; Min et al., 2006; Senoner & Dichtl, 2019; Abd El Azeem et al., 2021; Wang et al., 2014). Previous studies have demonstrated thatfolate was involved in metabolic reactions for combating oxidative stress (Asbaghi et al., 2021; Jakovljevic Uzelac et al., 2020; Zhou et al., 2024). However, none of these studies investigated the effect of MTHFR gene polymorphism on oxidative stress in patients with T2DM.

The Dali Bai Autonomous Prefecture is located in the northwest of Yunnan Province, China. So far, few studies have investigated the genetic susceptibility to developing T2DM in the population of the Dali area. Here, we conducted a case-control study to reveal the potential association of MTHFR C677T (rs1801133) and A1298C (rs1801131) loci polymorphisms with T2DM in the population of the Dali area of Yunnan Province, China.

Materials and Methods

Results

Association of MTHFR C677T and A1298C polymorphisms with the risk of developing T2DM

The competitive allele-specific PCR (KASP) method was used to evaluate the MTHFR C677T and A1298C polymorphisms (Fig. 1). The Hardy-Weinberg equilibrium was used to assess the control group, and no significant deviation was identified (Table S1). Furthermore, the power of the sample size in this study was greater than 0.9 in both the T2DM and control groups. As shown in Table 2, both MTHFR C677T and A1298C polymorphisms were significantly associated with the development of T2DM. The frequencies of the TT genotype (P = 0.030) of MTHFR C677T and the CC genotype (P = 0.032) of MTHFR A1298C were significantly higher in patients with T2DM than in the control group. In addition, patients with T2DM presented a significantly higher frequency of the T allele (P = 0.047) of MTHFR C677T. The risk assessments also found that homozygous mutants of MTHFR C677T and A1298C had a 1.750 and 3.132 increased odds of developing T2DM, respectively. Meanwhile, both MTHFR C677T and A1298C polymorphisms were found to be related to T2DM in the recessive model, which conferred 1.608- and 2.988-fold odds, respectively. In addition, logistic regression model showed that MTHFR C677T locus TT genotype and A1298C locus CC genotype could increase the odds of T2DM (Table S2). These results indicated that homozygous mutants of MTHFR C677T (TT genotype) and MTHFR A1298C (CC genotype) appeared to be potential risk factors for developing T2DM in the Dali area of Yunnan Province.

Table 2:

Genotype and allele distribution of MTHFR C677T and A1298C polymorphisms in the study population and risk analyses of developing T2DM.

Genotypes/Alleles	Control (n = 272) n (%)	T2DM (n = 445) n (%)	OR (95% CI)	P-value
MTHFR C677T
CC	110 (40.44)	156 (35.06)	1.000 (reference)	–
CT	135 (49.63)	222 (49.89)	1.160 (0.838–1.604)	0.371
TT	27 (9.93)	67 (15.06)	1.750 (1.052–2.911)	0.030*
C	355 (65.26)	534 (60.00)	1.000 (reference)	–
T	189 (34.74)	356 (40.00)	1.252 (1.003–1.563)	0.047*
Dominant model	–	–	1.258 (0.922–1.716)	0.148
Recessive model	–	–	1.608 (1.000–2.586)	0.048*
Overdominant model	–	–	0.990 (0.732–1.338)	0.947
MTHFR A1298C
AA	180 (66.18)	273 (61.35)	1.000 (reference)	–
AC	88 (32.35)	153 (34.38)	1.146 (0.830–1.583)	0.407
CC	4 (1.47)	19 (4.27)	3.132 (1.048–9.357)	0.032*
A	448 (82.35)	699 (78.54)	1.000 (reference)	–
C	96 (17.65)	191 (21.46)	1.275 (0.971–1.674)	0.080
Dominant model	–	–	1.233 (0.899–1.690)	0.193
Recessive model	–	–	2.988 (1.006–8.879)	0.039*
Overdominant model	–	–	0.913 (0.662–1.258)	0.577

DOI: 10.7717/peerj.18334/table-2

Notes:

OR

adds ratios

95% CI

95% confidence interval

*P < 0.05

Linkage disequilibrium and haplotype analysis of the MTHFR C677T and A1298C polymorphisms

As shown in Fig. 2, there existed linkage disequilibrium between the MTHFR C677T and A1298C polymorphisms in the study population. Haplotype analysis was performed for the MTHFR gene polymorphisms, and four different haplotypes, C-A, T-A, C-C, and T-C, could be formed between the MTHFR C677T and A1298C polymorphisms. The C-A, T-A, and C-C haplotypes were used for analysis because the frequency of the T-C haplotype was less than 0.03. Compared with the control group, the distribution frequency of the T-A haplotype was significantly higher in the T2DM group and conferred a 1.305-fold increased odds of developing T2DM (P = 0.030) (Table 4).

Discussion

Numerous studies have been conducted considering the association of genetic polymorphisms with the development of T2DM in various populations, which have revealed controversial results. To the best of our knowledge, this is the first study to evaluate the association of the MTHFR C677T (rs1801133) and A1298C (rs1801131) polymorphisms with the risk of developing T2DM in the population of the Dali area of Yunnan Province, China. We identified that the MTHFR C677T and A1298C polymorphisms significantly increase the risk of developing T2DM among the population in the Dali area.

There are various studies describing the association between MTHFR C677T and T2DM susceptibility. Sarkar, Chatterjee & Bandyopadhyay (2021) collected samples from 104 patients with T2DM and 176 healthy controls and found that the CT genotype of MTHFR C677T exhibited a significantly higher risk of developing T2DM compared to the CC genotype in the Bengalee Hindu caste population of West Bengal, India. A meta-analysis on 6,783 subjects published by Zhu et al. (2014) showed that the MTHFR C677T locus T allele may be a genetic risk factor of T2DM in the Chinese population.There was the same association between MTHFR C677T polymorphism and T2DM susceptibility in the Middle East and North Africa region population through a meta-analysis (El Alami et al., 2024). However, El Hajj Chehadeh et al. (2016) collected 169 T2DM patients and 209 healthy controls and their findings demonstrate that the MTHFR gene polymorphisms are not related to T2DM in the Emirati population. In addition, no association was also observed between MTHFR polymorphism and T2DM in 118 Taiwanese or 359 Bahraini (Al-Harbi et al., 2015; Chang et al., 2011). Polymorphisms of the MTHFR gene vary widely by area and ethnicity, thus suggesting that any association between SNPs and T2DM risk may likewise depend on the area and population ethnicity. This case-control study revealed an obvious association of the MTHFR C677T and A1298C polymorphisms with T2DM susceptibility in the population of the Dali area, which was similar to the findings of Zhu et al. (2014). To investigate the interactive effects of the derived allele combinations on T2DM risk, haplotype analysis was performed for the MTHFR C677T and A1298C loci. We found that the T-A haplotype conferred an increased risk of developing T2DM in the population of the Dali area. Our results provide strong evidence that MTHFR C677T and A1298C are significantly associated with the development of T2DM in the population of the Dali area. Thus, it is necessary to clarify the roles of these SNPs in different areas.

T2DM is a complex, heterogeneous, and polygenic metabolic disease that is characterized by hyperglycemia and is accompanied by disorders of fat and protein metabolism (Abbas et al., 2013). Studies have found that dyslipidemia increases the risk of developing T2DM (Fang et al., 2022; Lee et al., 2022; Gheflati et al., 2019). Accordingly, significant changes were observed in the levels of FBG, TG, HDL-C, and APOA1 between the T2DM and control groups. It has also been reported that MTHFR C677T could affect LDL-C levels (Yilmaz et al., 2003; Pitsavos et al., 2006), and our findings indicate an association of MTHFR C677T and A1298C polymorphisms with FBG levels in patients with T2DM in the Dali area, Yunnan Province. However, no significant differences were found between MTHFR gene polymorphisms and the lipid metabolism parameters.

Individuals with T2DM often have a persistent low-grade inflammatory response and oxidative stress. Previous research has suggested that proinflammatory cytokines, such as TNF-α and interleukins (ILs), could impair insulin signaling and cause insulin resistance (Hotamisligil, Shargill & Spiegelman, 1993; Krogh-Madsen et al., 2006; Fain, 2006). The reactive oxygen species produced by oxidative stress, such as hydroxyl radicals, superoxide anions, and peroxides, have been linked to the development of insulin resistance and β-cell dysfunction (Babel & Dandekar, 2021; Rains & Jain, 2011). These inflammatory mediators and free radicals could also cause direct damage to vascular endothelial cells and accelerate the development of vascular diseases. In this study, we identified that the MTHFR C677T polymorphism was significantly associated with elevated TNF-α levels in patients with T2DM. Although no significant association was observed in the variables of oxidative stress, the increased level of MDA implied the potential role of MTHFR C677T in oxidative stress.

CVD is a common complication of diabetes and is a major cause of death and disability among individuals with diabetes (Napoli et al., 2020; Einarson et al., 2018). Studies have shown that the risk of CVD in patients with T2DM was 2–4 times higher than that in individuals without T2DM (Tuttle et al., 2021). Thus, a major goal in the prevention and control of T2DM is to reduce the risk of cardiovascular events. Early identification or screening of populations at high risk of developing T2DM and T2DM-related cardiovascular complications might provide a strategy for the early prevention of diabetes. Settin et al. (2015) found that the TT genotype of the MTHFR C677T locus could increase the risk of developing T2DM, and the CT and TT genotypes could increase the risk of developing diabetes complications such as peripheral neuropathy and ischemic heart disease. Furthermore, Hcy is a widely used biomarker for predicting cardiovascular events in patients with T2DM (Guieu, Ruf & Mottola, 2022; Raghubeer & Matsha, 2021). We found that patients with T2DM, who also had the high diabetic risk genotypes of MTHFR C677T and A1298C, had significantly higher Hcy levels, which suggests a potential increased risk of cardiovascular events. A sustained high level of Hcy could induce vascular endothelial injury, promote oxidative stress, and form atherosclerotic plaques, which may eventually result in cardiovascular events (Gospodarczyk et al., 2022). Furthermore, we analyzed the association of MTHFR gene polymorphisms with the risk of CVD in patients with T2DM. The results revealed that the frequencies of the CT and TT genotypes and the T allele of MTHFR C677T were higher in the patients with T2DM and CVD group compared to the patients with T2DM group, but no significant difference was found in these two groups. However, a further large-sample prospective study is required to verify these results.

In conclusion, we demonstrated that the MTHFR C677T and A1298C polymorphisms are important determinants for the incidence of T2DM in the population of the Dali area of Yunnan Province, China. Our findings provide additional information for the prevention and treatment of T2DM through folate supplementation in the local region. The sample size of this study was small, which may have a certain impact on the statistical results. In the future, we will continue to expand the sample size to explore the relationship between MTHFR C677T and A1298C polymorphism and the development of T2DM in Dali area of Yunnan Province, which has certain referencevalue for the early screening of high-risk groups in this area, and provides a new ideafor the prevention of T2DM patients.

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