Due to the growing size and aging of the world’s population, the global incidence of stroke is alarmingly high, with approximately 16.9 million people experiencing a stroke each year [1]. Numerous conventional risk factors contribute to the onset and progression of stroke, such as diabetes mellitus, hypertension, atrial fibrillation, hyperlipidemia, and so on. These factors have been extensively studied, and their underlying mechanisms are well-established [2]. As a result, many primary prevention strategies have been developed and implemented to reduce stroke incidence, particularly in developed countries [3]. Additional effective stroke prevention strategies are still required to be developed to further decrease the global burden of stroke.

Nevertheless, there are several other potential risk factors that have not been thoroughly investigated, and homocysteine (Hcy) is one such non-traditional risk factor for stroke [2, 4]. Moreover, the predictive accuracy of the Framingham Stroke Risk Score could be improved by incorporating four biomarkers (including Hcy), based on the findings of Framingham’s offspring cohort [5]. Prior study has shown that elevated levels of Hcy was independently linked to the incidence of stroke in young individuals [6]. Hyperhomocysteinemia (HHcy) is currently recognized as a risk factor for cardiovascular and cerebrovascular diseases. However, its underlying mechanism is complex and not yet fully understood [7]. HHcy has been found in approximately 60.6% of stroke patients, and this is associated with low serum B12 level [8]. There is indeed a causal relationship between high plasma Hcy levels and ischemic stroke [4, 9]. Intracerebral hemorrhage constitutes 10–15% of all stokes cases and is associated with high morbidity and mortality rates. Elevated plasma Hcy levels can damage endothelial function, disrupt methylation reactions, increase oxidative stress, and alter the structure of proteins. These effects can indirectly contribute to the occurrence and development of atherosclerosis [10]. The elevated plasma Hcy levels could be influenced by various factors, including nutrition, diet, disease, medication, and heredity [11].

Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme that regulates the conversion of N5, N10-methylene tetrahydrofolate to 5-methyltetrahydrofolate, a key step in folic acid metabolism. This process is essential for DNA methylation and repair, which are important for maintaining the integrity and stability of genetic material [12]. Recently, there has been increased interest in the TT genotypes of the MTHFR C677T polymorphism as a research topic [13]. Prior studies have shown that individuals with the C677T genotype of MTHFR gene have approximately twice the plasma Hcy levels compared to those with the normal genotype [13]. The impact of the MTHFR C677T mutation on the relationship between plasma Hcy levels and stroke is well-documented [14]. Nevertheless, it is still ambiguous whether MTHFR C677T mutation could influence the response to Hcy lowering treatment in stroke patients with HHcy.

Herein, the aim of our study was to explore the potential link between MTHFR C677T gene polymorphism and the risk of stroke, as well as to evaluate the effectiveness of Hcy lowering treatment in stroke patients with HHcy. Our findings could potentially help alleviate the social and economic burden associated with stroke by informing the development of effective prevention and treatment strategies for at-risk populations.

Stroke can result in long-term disability, making it crucial to prioritize early detection and active management of potential risk factors. This can effectively prevent the occurrence of stroke [3]. The role of genetic factors in the pathogenesis of stroke has been confirmed through associations between specific gene variants and the risk of stroke occurrence. However, conflicting results [19, 20] have made it difficult to determine the effects of these polymorphisms on the risk of stroke development. This ambiguity may be due to the heterogeneity of cerebral infarction [20, 21].

HHcy has been identified as an independent risk factor for cerebrovascular and cardiovascular atherosclerotic occlusive diseases [22]. It is a relatively new risk factor for ischemic stroke, with approximately 60% of stroke patients exhibiting HHcy, which may be associated with lower levels of serum B12 [23]. Additionally, there is a positive correlation between plasma Hcy levels and ischemic stroke [24, 25]. The National Health and Nutrition Survey conducted a survey in 1999–2004, analyzing the effects of high Hcy ($\ge$10 µmol/L) on stroke using 12,683 samples. The results showed that high Hcy levels were independently associated with stroke (OR = 1.52; 95% CI, 1.01–2.29; p = 0.045) after adjusting for 17 covariates. This correlation was found to decrease with age, which was consistent with previous studies [6, 26].

A Meta-analysis revealed that the TT genotype of MTHFR C677T gene polymorphism had a greater influence on plasma Hcy levels in Asian countries than in non-Asian countries [27]. Our study revealed that the frequency of the TT genotype in stroke patients was as high as 60.5%, which was higher than that reported in the Caucasian population [28]. Our study did not find a significant difference in the frequency of the TT genotype between patients with ischemic stroke and those with hemorrhagic stroke. However, the proportion of patients with insufficient treatment effects was significantly higher in those with the TT genotype (83.3%) compared to those with the CC and CT genotype (46.9%). This finding was consistent with the results of a prospective study conducted in a similar ethnic background and disease context [24]. Our study also emphasized the significance of early detection of plasma Hcy levels in the Chinese population with the TT genotype of the MTHFR gene polymorphism. Early detection can aid in identifying individuals who are at a higher risk of developing HHcy and experiencing suboptimal treatment outcomes.

Vitamin B, specifically mecobalamin, has been proven to effectively lower plasma Hcy levels. The standard clinical practice is to supplement with a combination of Vitamin B9 (folic acid), Vitamin B12, and Vitamin B6 [24]. This treatment regimen has been found to reduce the risk of stroke occurrence, and is widely recommended as a preventive measure against stroke [3]. Prior study indicated that the dosage of 0.8 mg folic acid per day exerted sufficient effect on lowering plasma Hcy level [29].

However, there was still insufficient evidence to support the notion that long-term intake and higher doses of folic acid can enhance the treatment effects. Additionally, the safety of such practices still requires careful consideration. Triple combinations of folic acid tablets, Vitamin B6, and mecobalamin have been utilized in the treatment of patients with HHcy. Chinese guidelines for the secondary prevention of ischemic stroke and transient ischemic attack recommend the supplementation of folic acid, Vitamin B6, and Vitamin B12 in recent ischemic stroke or transient ischemic attack patients, or those with elevated plasma Hcy level [15]. Furthermore, the guidelines also suggested that the supplementation of the three compound vitamins can effectively lower Hcy levels and significantly reduce the risk of stroke occurrence [3].

In our study, the majority of patients with CT and CC genotypes experienced a satisfactory treatment outcome following standard combination therapy. After one month of vitamin combination supplements, there was limited effect in lowering Hcy levels in patients with the TT genotype. That is to say, patients who were more likely to be influenced by hereditary factors did not show an obvious response to treatment, which may require prolonged treatment cycles. A previous study also reported that even though folic acid and Vitamin B12 levels were normal in HHcy patients with the MTHFR C677T TT genotype, they could still benefit from folic acid supplementation [30]. However, there is still a lack of studies on how to modify treatment in a few patients with increased folic acid and Vitamin B12 levels. Additionally, co-morbidities associated with oxidative stress may act synergistically with genetic polymorphisms that limit the anti-oxidative stress capacity of MTHFR (and cystathionine $\beta$-synthase) [31]. For instance, conditions such as diabetes, hypertension, dyslipidemia and hyperuricemia are all correlated with oxidative stress, and these co-existing conditions may exacerbate the impact of genetic polymorphisms on oxidative stress. As a result, the importance of considering the influence of co-morbidities and genetic variations when preventing and treating diseases that are related to oxidative stress should be highlighted.

An increased plasma Hcy level can be influenced by various factors, including diet, heredity, and drugs [32]. Our study evaluated the treatment effects of stroke patients with HHcy and demonstrated that the TT genotype of the MTHFR C677T gene contributes to the genetic susceptibility of stroke. This genotype is more common in the Chinese population, which may further increase the risk of stroke occurrence. Therefore, primary hospitals could conduct genotype detection to assist in stratifying risk factors. It is recommended to administrate standardized folic acid in combination with Vitamin B to patients with HHcy, particular those with MTHFR C677T TT genotype. Plasma Hcy level should be screened as age increases, and targeted treatment to lower Hcy levels should be initiated as early as possible.

The present study has some potential limitations that should be acknowledged. Firstly, it is a retrospective study conducted at a single center with a limited sample size. Therefore, our findings need to be validated in larger multicenter prospective trials. Secondly, we only investigated the MTHFR gene’s role in regulating Hcy levels, and other genes that may affect Hcy levels were not examined. Additionally, long-term follow-up beyond one month is necessary to explore the treatment duration’s impact. Thirdly, future studies should consider how to manage abnormal Hcy elevation in HHcy patients with the TT genotype, including but not limited to dosage modification, alternative agents, or various administration methods.

Our findings indicated that the TT genotype and T allele of the MTHFR C677T gene polymorphism were independently associated with insufficient Hcy lowering treatment effects in stroke patients with HHcy.

MTHFR, methylenetetrahydrofolate reductase; Hcy, homocysteine; HHcy, hyperhomocysteinemia; PCR-RFLP, polymerase chain reaction–restriction fragment length polymorphism analysis; SD, standard deviation; OR, odd rations.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

ZL and ZZ prepared the conception and design of the study, participated in the acquisition and analysis of data. ZL and ZZ wrote the original draft of the manuscript and critically revised the manuscript. MH, QY, CL, BH and JW participated in the acquisition and analysis of data, critically revised and edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All data collection, storage and processing were done in compliance with the Helsinki Declaration. All patients provided signed, informed consent and the study number (2017-022) was approved by the ethics committee of Quanzhou First Hospital Affiliated to Fujian Medical University.

We thank all patients and their families for participating in this study.

This research received no external funding.

The authors declare no conflict of interest.