The Influence of MTHFR C677T Variants on Neuropathy Risk Among T2dm Patients Receiving Monotherapy Metformin

ABSTRACT


INTRODUCTION
In 2019, the International Diabetes Federation revealed its staggering global statistics that 463 million people between the ages of 20-79 years old diagnosed with diabetes.Notably, Indonesia was ranked 7 th marking the only country in Southeast Asia among 10 countries with the highest diabetics populations in 2019, tallying 10.7 million cases.Furthermore, a national health survey conducted in 2018 reported that the Special Region of Yogyakarta (DIY) ranked as the third province that has the highest prevalence of diabetes in Indonesia (International Diabetes Foundation, 2019;Kementerian Kesehatan Republik Indonesia, 2018).Intriguingly, neuropathy stands as one of the prevalent microvascular complications in T2DM patients (Amelia et al., 2019), and the risk of neuropathy escalates with metformin consumption as its side effects (Miyan & Waris, 2020).
Metformin is well known as the first-line therapy for type 2 diabetes mellitus (T2DM), recommended by the American Diabetes Association and The European Association for The Study of Diabetes (American Diabetes Association, 2020; Cosentino et al., 2020).However, metformin consumption is associated with the reduction of vitamin B12 levels and an increase in homocysteine and methylmalonic acid levels in the blood.Furthermore, metformin is posited to stimulate the expression of angiogenic factors in peripheral nerves.Consequently, patients who take metformin often reported neuropathy as a side effect of metformin (Hashem et al., 2021;Kim et al., 2019;Ni et al., 2017).
Previous studies reported neuropathy associated with methylenetetrahydrofolate reductase (Jiménez-Ramírez et al., 2017).The MTHFR enzyme plays a pivotal role in intracellular folate metabolism and homeostasis.
Volume 35 Issue 1 (2024) MTHFR catalyzes the irreversible conversion of 5,10-, methylenetetrahydrofolate to 5-methyltetrahydrofolate which serves as the primary methyl donor for the re-methylation of homocysteine to methionine.Notably, metformin consumption disrupts folate-like activity.Thus, MTHFR might take a role in those mechanisms (Owen et al., 2021).Furthermore, pharmacogenomic as personalized medicine is now considered to prevent side effects and optimize therapeutic effectiveness (Schwartz et al., 2016).The MTHFR gene is one of the genetic targets observed related to neuropathy (Dell'Edera et al., 2018;Wu et al., 2017).
The MTHFR gene encodes MTHFR, and variation in the C677T could reduce MTHFR activity by as much as 50%.Numerous studies have reported mutations in the MTHFR gene as a contributing factor in diabetic neuropathy (Kakavand Hamidi et al., 2017;Mottaghi et al., 2019;Russo et al., 2016;Wu et al., 2017).Sadewa et al. (2002) conducted a profiling genetic variation of MTHFR C677T in the Javanese population.Furthermore, Suryandari et al. (2012) also conducted genetic variations of MTHFR C677T among men in Indonesia, though their study primarily focused on its implication for fertility.
However, no prior study has specifically investigated the relationship between the genetic variation of MTHFR C677T in T2DM patients consuming metformin on neuropathy risk.The two aforementioned previous studies (Russo et al., 2016;Yigit et al., 2013) have not revealed the molecular impact when the mutation occurs.Therefore, there is critical need to evaluate the genetic variation of MTHFR C677T in relation to the risk of diabetic neuropathy among T2DM patients consuming metformin as a monotherapy in DI.Yogyakarta, especially in the Sleman District.This research could help fill an existing knowledge gap and provide valuable insights into the potential implications of this genetic variation on neuropathy in this specific patient population.

Research design and sampling
This cross-sectional study was conducted with the necessary approval granted by the Medical and Health Research Ethics Committee (MHREC), Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada -Dr.Sardjito General Hospital in Yogyakarta, Indonesia (for patients recruitment) and Research Ethics Committee of Faculty of Health Science at Universitas Respati Yogyakarta (for SNP C677T detection).Prior to data collection, written informed consent was obtained from all participating patients.All procedures and data collection obeyed the Declaration of Helsinki.
We conducted this study at ten primary healthcare facilities in Sleman, Province of DI Yogyakarta, Indonesia.Patients were included if they had a type 2 diabetes mellitus diagnosis and had been on metformin treatment for a minimum of three consecutive months.Patients were individually diagnosed by licensed medical practitioners based on medical judgments and in accordance with the criteria stipulated by the American Diabetes Association (ADA).We used the standard Neuropathy Symptom Score (NSS) and Neuropathy Disability Score (NDS) criteria to diagnose diabetic neuropathy.
Patients were excluded if they had uncontrolled hypertension, severe complications related to poor glucose control (such as amputations, foot ulcers, dialysis, and blindness due to severe diabetic retinopathy), renal disease, impaired hepatic function, or pregnancy.

Genetic variant examination
DNA isolation from blood samples was performed using the Tissue Genomic DNA Extraction Mini Kit 100 Prep (Proteinase K) following the manufacturer's (Favorgen) procedure.Subsequently, the quality of DNA was checked by electrophoresis and detected on a UV transilluminator.The MTHFR C677T mutation was analysed with PCR-based restriction fragment length polymorphism (RFLP) assay using restriction enzyme NlaIV.
The MTHFR gene of 351 bp was amplified by using primers: 5'-GCGAACTCAGCACTCCACC-3' (forward) and 5'-TCATCCCTCGCCTTGAACAG-3' (reverse).Each PCR mixture (25 µL) contained DNA isolate, Go Taq Green Master Mix, primers (12.5 pmoles of each).The mixture was heated at 95°C for 5 min and underwent 35 cycles of amplification: denaturation at 95°C for 20 s, annealing at 60°C for 30 s, extension at 72°C for 30 s.The final extension took 5 min at 72°C.The PCR reaction was performed in the Bio-Rad T100 Thermal Cycler.After amplification, the 351 bp PCR product was digested with NlaIV in a 30 µL reaction solution containing 10 µL of PCR product, 3 µL of 10X NE buffer, 16 µL nuclease-free water, and 2 units of NlaIV at 37°C for 2 h.The enzyme deactivated at 65°C for 20 min.
The PCR product was analysed on 2% agarose gels and was photographed on an ultraviolet transilluminator.There were two variations of interpretation.First, wild-type (CC) individuals were identified by only a 214 and 137 bp fragment; heterozygotes (CT) by the 214, 137 bp, and 351 bp; and homozygote variants (TT) by the 351 bp.Second, CC was identified by 137 bp, 125 bp, and 189 bp; CT by 263 bp, 127 bp, 125 bp, and 89 bp; and TT by 263 bp and 89 bp.The author designed the primer and restriction enzyme to detect 2 SNPs.Therefore, there were variations in interpretation.However, it has already been confirmed and validated by the repetition of RFLP on multiple samples (Figure 1).

Statistical Analysis
The results are presented as the mean ± standard deviation (SD) and frequency (percentage).To assess the distribution of genotypes among the patients and controls, the Hardy-Weinberg equilibrium was evaluated using the chi-square (χ2) test.The relationships between the C677T mutation and diabetic neuropathy of the patients were analyzed using the χ2 test.The χ2 test was used to compare categorical variables appropriately, and odds ratios and 95% confidence intervals were used to assess the risk factors.Regression logistic was performed to evaluate the adjusted odd ratio (AOR) after adjusting for age, BMI, blood pressure, HbA1c level, smoking status, and T2DM family history.All p values were twotailed, and p values less than 0.05 were considered statistically significant.

RESULTS AND DISCUSSIONS
Our study recruited 103 patients who had received a first-time diagnosis with T2DM at primary health care and were prescribed metformin as monotherapy (Table I).The average age, HbA1c level, and fasting blood glucose (FBG) of the participants were 52.99 years, 10.47%, and 192.91 mg/dL, respectively.It was clear that our patients indicated an elderly group based on the mean of their age, but the mean of HbA1c and FBG were unexpectedly high for newly diagnosed T2DM patients.The majority of our respondents were female and tended to be obese; and blood pressure was controlled relatively.45.6% of patients were diagnosed with neuropathy, but we did not definitively confirm whether it was caused by T2DM or metformin.Notably, our respondents were naïve metformin and newly diagnosed with T2DM.Therefore, we assumed that the neuropathy was induced by metformin consumption over the course of three months.
Only one patient exhibited the mutant TT genotype, while more than 50% of patients presented the heterozygote CT genotype (Table II).Assuredly, our study's SNP of C677T was inconsistent with Hardy-Weinberg Equilibrium (p<0.05).Consequently, this present study only compared clinical characteristics among CC and CT genotypes.The clinical characteristics of the C677T genotype demonstrated no significant difference between wild-type homozygote and heterozygote (p>0.05).
The crude odd ratio (COR), adjusted odd ratio (AOR), 95%CI, and p-value related to the association between C677T and neuropathy risk in the genotype, allele, and dominant models (Table III).We performed association statistics in the genotype model between CT and CC to neuropathy risk because only one patient has TT genotype.The odds ratio in the genotype model was different between COR and AOR.The CT had lower odds of neuropathy than CC in COR (COR = 0.98; 95%CI = 0.44 -2.17), whereas its odds were higher in AOR (AOR =1,15; 95%CI = 0.46 -2.86), although it was not statistically significant in both (p=0.97 and p=0.80 in COR and AOR, respectively).We revealed that the T allele as a mutant had COR = 0.93 (95%CI = 0.51 -1.61, p-value = 0.82) and AOR = 0.87 (95%CI = 0.46 -1.63, p-value -0.66) to neuropathy risk in the allele model.The recessive model could not be calculated due to the limited presence of the TT genotype.Moreover, the CT and TT genotype had COR = 0.95 (95%CI = 0.43 -2.11, p-value = 0.91) and AOR = 0.83 (95%CI = 0.35 -1.96, p-value = 0.67).Overall, the findings in the allele and dominant model were similar, indicating that the mutant had a lower risk of neuropathy than the wild type, either in COR or AOR.

Volume 35 Issue 1 (2024)
In brief, this present study could not find any significant association between MTHFR C677T and the risk of neuropathy, even after several confounding factors were added.
The genetic variation of C677T, recorded as rs1801133 in NCBI, is one of the prevalent MTHFR polymorphisms (Ni et al., 2017).The SNP is a point mutation that occurs in exon 4 of the MTHFR gene, converting amino acid at codon 222, valine, to alanine.The variation in the SNP C677T has been associated with a reduction in MTHFR activities (Liew & Gupta, 2015).The MTHFR enzyme has an important role in catalyzing homocysteine to methionine as a methyl donor.Therefore, activity induces hyper-homocysteine resulting in elevating peripheral neuropathy (Hsu et al., 2020).
MTHFR genetic variation combined with hyperhomocysteinemia was found to be associated  (Mottaghi et al., 2019;Hsu et al., 2020).Previous studies confirmed that metformin consumption leads to peripheral neuropathy through B12 deficiency and high homocysteine (Elhadd et al., 2018;Hashem et al., 2021).Neuropathy impacts the quality of life and contributes to high morbidity and mortality rates among T2DM patients (Dornas et al., 2021;Hicks et al., 2021).Therefore, this current study is the first in the Indonesian population to examine the variation of C677T in the MTHFR gene related to metformin and T2DM to neuropathy risk.
A key highlighted finding of our study is the demonstration of no significant association between the C677T genetic variant and neuropathy among T2DM patients consuming monotherapy metformin, even after adjusting for several confounding factors.Several studies have mentioned that C677T increases the risk of diabetic neuropathy.Moreover, C677T was superior to A1298C as a risk factor for neuropathy (Hamidi et al., 2017;Yigit et al., 2013).Still, another study involving a different racial population from our population has similar results to our findings.Russo et al. (2015) could not discover any significant association between C677T and the risk of neuropathy among metformin users.Additionally, we found one cohort study that found C677T had a significant association with neuropathy, but interestingly the variant did not correlate with homocysteine (Jiménez-Ramírez et al., 2017).
The discrepancy in these findings might be because of the unclear mechanism through which metformin impacts the MTHFR enzyme.
Theoretically, metformin has the potential to reduce vitamin B12 and interfere with the methylation cycle, thus leading to hyperhomocysteinemia (Owen et al., 2021).The collaboration of metformin and C677T in the MTHFR gene potentially induces neuropathy through hyperhomocysteinemia. Nevertheless, it was not clear whether the high homocysteine levels were induced by metformin or variant MTHFR.
While our study imposed certain restrictions by only recruiting patients with naive metformin who had received a first-time T2DM diagnosis, it is noteworthy that the mean HbA1c level was high.It is important to take into consideration that neuropathy is caused by uncontrolled diabetes (Amelia et al., 2019;Nguyen et al., 2019).Therefore, the high level of HbA1c could predispose our findings.Furthermore, other factors that might affect our findings were lifestyle, including diet and physical activities.Those could influence neuropathy through improvement in controlling blood sugar and insulin sensitivity (Zilliox & Russell, 2019).Indeed, the medication factor is an important and critical point that affects disease progression (Al-Temimi et al., 2021).Adherence to medication consumption might involve a confounding factor (da Silva et al., 2019).Therefore, pharmacists should escalate their communication skills to counsel the patient regarding medication adherence (Selvadurai et al., 2020).
This study does have several limitations.First, we did not control the baseline of HbA1c and blood glucose levels influencing neuropathy.Second, the homocysteine level was not measured in our study.If it was measured, it might explain the unclear path between metformin-MTHFRhomocysteine-neuropathy.
Therefore, we recommend that future studies establish more rigorous baseline criteria and examine homocysteine levels.

CONCLUSION
The current study revealed that genetic variant C677T in the MTHFR gene had no significant association with neuropathy risk among newly diagnosed T2DM patients consuming monotherapy metformin.Due to no proven association, more rigorous criteria and laboratory parameters related to neuropathy should be examined in future research endeavors.

Table I .
Characteristic of T2DM patients consuming monotherapy metformin.

Table II .
The profile of subject's characteristic according to genotype C677T *Compare between CC and CT genotype

Table III .
Association between MTHFR C677T genetic variation with neuropathy risk