MTHFR Gene Polymorphisms (rs1801133 and rs1801131) and Their Association with Type 2 Diabetes Mellitus and Diabetic Foot Ulcers: A Study from Karachi, Pakistan

By Saleem Ullah Atta Abro¹, Qamar Aziz Siddique¹, Iftikhar Ahmed Siddique², Zahid Miyan³, Asher Fawwad², Qurratulain Saleem⁴

Affiliations

1. Department of Physiology, Baqai Medical University, Karachi, Pakistan
2. Department of Biochemistry and Research, Baqai Medical University, Karachi, Pakistan
3. Department of Medicine, Baqai Institute of Diabetology and Endocrinology, Baqai Medical University, Karachi, Pakistan
4. Department of Community Medicine, Karachi Medical and Dental College, Karachi, Pakistan

doi: 10.29271/jcpsp.2025.08.975

ABSTRACT
Objective: To explore the methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms (rs1801133 and rs1801131) and their association with type 2 diabetes mellitus (T2D) and diabetic foot ulcers (DFU).
Study Design: A cross-sectional study.
Place and Duration of the Study: Department of Physiology, Baqai Medical University, Karachi, Pakistan, from June 2023 to June 2024.
Methodology: One hundred and thirty-eight individuals were enrolled and categorised into healthy (n = 46), type 2 diabetic (n = 46), and diabetic foot ulcer patients (n = 46). Patients having insulin-dependent diabetes mellitus were excluded. The blood samples of participants were collected, and MTHFR (rs1801133 and rs1801131) gene polymorphisms were determined using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Statistical analysis included the Pearson’s Chi-square test, with the risk assessment based on 95% confidence intervals (CI) and odds ratios (OR).
Results: MTHFR [rs1801133(C677T)] heterozygous CT allele was present in 91%, i.e. 42 out of 46 DFU patients (p = 0.002). In contrast, the MTHFR [rs1801131(A1298C)] homozygous CC allele was observed in 39% of healthy controls. Significant associations were found across the three groups for both MTHFR mutations (p <0.05). The CT genotype of MTHFR showed a risk associated with DFU (OR = 4.59, CI: 1.38-15.2), while the homozygous CC genotype was also significantly associated with DFU (OR = 2.22, CI: 0.80-6.11).
Conclusion: The findings suggest that the genetic polymorphism of the MTHFR genotype CT of rs1801133 is associated with DFU among the clinical population from Karachi, Pakistan.

Key Words: Diabetes mellitus, Diabetic foot ulcer, Gene, Genetic polymorphisms, MTHFR C677T” or “A1298C.

INTRODUCTION

Type 2 Diabetes mellitus (T2DM) is an extremely ubiquitous and devastating endocrine condition that impairs the metabolism of lipids, proteins, and carbohydrates in the human body. T2DM is characterised by hyperglycaemia, resulting from insulin resistance at the cellular level. This condition may cause several complications, one of which is diabetic foot ulcers (DFU).¹ Globally, 537 million people aged 20 to 79 currently have T2DM. This number is expected to soar to 643 million by 2030, translating to one  in  every  ten  adults  being  affected.

Nearly seventy-three million individuals worldwide may have Diabetes mellitus (DM) by 2045.² According to the International Diabetes Federation, the number of individuals diagnosed with non-insulin-dependent DM in Pakistan is expected to reach 6.9 million for those aged 20 to 79 years by 2030. This figure is expected to rise to 11.5 million by 2025, ranking Pakistan among the countries with the highest diabetes rates. Over the next decade, diabetes-related fatalities in Pakistan are anticipated to increase by 51%. Furthermore, an estimated 15% of more than 150 million individuals affected by DM globally may develop DFU.³

T2DM is influenced by nutritional deficiencies, genetic factors, epigenetic influences, and environmental conditions.⁴ Methy-lenetetrahydrofolate reductase (MTHFR) is an essential enzyme that changes 5, 10-methylenetetrahydrofolate into 5-methyltetrahydrofolate. The metabolism of homocysteine (Hcy) and folate in the human body is crucial for the remethylation Hcy to methionine, helping to maintain Hcy levels within the normal range. The short arm of human chromosome 1 (1p36.3) contains the Methylenetetrahydrofolate reductase gene.⁵ The two primary single-nucleotide polymorphisms (SNPs) are rs1801133 (Ala222Val) and rs1801131 (Glu429Ala). These genetic variations decrease the effectiveness of MTHFR enzymatic activity, impairing the breakdown of Hcy and leading to elevated circulating Hcy levels. High Hcy levels are associated with oxidative stress (OS), which disrupts the methylation process and impairs nitric oxide production. Individuals with these two variations of the MTHFR gene may experience a significant reduction in methyl folate synthesis, which is approximately 17% for MTHFR [rs1801131(A1298C)] and up to 70% for MTHFR [rs1801133 (C677T)]. This decrease in folate availability threatens DNA stability and interferes with methylation pathways or DNA methylation. The interaction between MTHFR SNPs, Hcy levels, and folate deficiency contributes to various health complications, including DM, diabetic foot ulcers (DFU), gestational diabetes, thrombosis, coronary artery disease, nephropathy, retinopathy, microangiopathy, and neuropathy.⁶ MTHFR polymorphism shows a stronger association with T2DM in Asians than in European and African populations.⁷ The [rs1801133 (C677T)] and [rs1801131 (A1298C)] polymorphisms of the MTHFR gene have been extensively studied for their risk regarding T2DM, though conflicting  results  have  emerged.⁸

The genetic variation between healthy individuals, T2DM, and patients with foot ulcers can vary significantly across different populations. No research has been conducted on the association between the MTHFR gene polymorphisms and healthy individuals and those with diabetes, both with and without foot ulcers, in Karachi, Pakistan. This study was conducted to explore the potential links between the MTHFR SNPs (rs1801133 and rs1801131) and the risk of developing T2DM, as well as the likelihood of foot ulcer development among diabetic individuals  in  Karachi,  Pakistan.

METHODOLOGY

The research was conducted at the Department of Physiology, Baqai Medical University, Karachi, Pakistan, from June 2023 to June 2024. The Ethics Committee approved the study, and the Board of Advanced Studies and Research at the Baqai Medical University, Karachi, Pakistan, provided approval on July 12, 2023 (Ref: BMU-EC/01-2023). A targeted sampling method was used to select individuals eligible for the research during this period. The OpenEpi online tool was used to determine the sample size.⁹ The population prevalence of MTHFR was used to get a total sample size of 138.¹⁰ The confidence level was set at 95%, with a power of 1% significance level. The inclusion criteria included Pakistani nationals, healthy adults (based on HbA1c and FBG levels), type 3 diabetes patients who had been diagnosed for more than six months, and patients with DFU (ischaemic or neuropathic). The exclusion criteria included patients with type 1 diabetes, complications of diabetes such as ischaemic heart disease, retinopathy, nephropathy, and stroke (except for cases involving foot ulcers), pregnancy-induced diabetes or gestational DM, and those who diagnosed with deep vein  thrombosis  (DVT)  or  ulcers.

All eligible participants were informed about the objectives and nature of the study. Consent was obtained from all the participants prior to taking the blood samples. Individuals with insulin-dependent DM were excluded from the study. Participants’ data, including demographic information, anthropometric measurements such as body mass index (BMI), and details regarding family history, including diabetes, cardiovascular disease, thrombosis, recurrent pregnancy loss, neural tube defects, and male infertility, as well as genetic polymorphisms related to the MTHFR enzyme. Blood samples were collected in EDTA CP bottles, and the results of the genetic tests were communicated  directly  to  participants.

A total of 138 participants were chosen and screened for two mutations (rs1801133 and rs1801131). As directed by the GJC DNA purification kit, blood samples were collected to isolate genomic DNA. The results showed the average DNA concentration was between 80 and 100 ng/µL. The genotype of the MTHFR gene variant (rs1801131) was determined using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique.¹¹ This method utilised four primers to amplify three DNA fragments with sizes of 414 base pair (bp), 220 bp, and 244 bp. One primer set generated an amplicon for the C allele (244 bp band), while the other set produced an amplicon for the A allele (220 bp band) in order to produce allele-specific amplicons.

rs1801131-in-F 5'-GGGGAGGAGCTGACCAGTGAGGA-3.
rs1801131-in-R 5'-AAAGAACGAAGACTTCAAAGACACCTG-3.
rs1801131-ou-F 5'-GAGTCAGGGGCAGAATTTACAGGAATG-3.
rs1801131-ou-R 5'-TTCTCCCTTTGCCATGTCCACAG-3.

For the MTHFR gene's [rs1801133 (C677T)], the temperature at which rs1801133 was integrated was 56°C. Rotational temperatures of 50°C for rs1805087 and 57°C for rs662 were employed for rs1801133. For these samples, there were no touchdowns in the results. After a 45-second annealing period, the temperature was maintained at 72°C for one minute. Additionally, 41 to 45 cycles were performed at 95°C for 45 seconds each, following a seven-minute relaxation period. Using specific primers, the outer region of the MTHFR gene was amplified, producing a 224 bp fragment that helps to regulate DNA integrity. While the primers F0 and RI amplified the T allele, producing a 177 bp fragment, the primers FI and Ro generated a 101  bp  fragment  corresponding  to  the  C  allele.

rs1801133-in-F 5'-AGGAGAAGGTGTCTGCGGGCGT-3.
rs1801133-in-R5-AAGAAAAGCTGCGTGATGATGAAATAGG-3.
rs1801133-ou-F5'-AAGCATATCAGTCATGAGCCCAGCC-3.
rs1801133-ou-R 5'-GGGAAGAACTCAGCGAACTCAGCAC-3.

PCR reaction was performed using an Applied Biosystems Veriti thermal cycler. The reaction mixture had a final reaction volume of 25 microlitres and included a DNA template with a concentration of 100 ng to 300 ng, 10 µM of each primer, and 12.5 µl of GoTaq Green Master Mix (Promega Inc.). The cycling conditions comprised 30 cycles of denaturation for 30 seconds after an initial denaturation step of five minutes at 95°C. For the first cycle, the annealing temperature was set at 67°C. For the subsequent cycles, which lasted 30 seconds each, the temperature was lowered to 58°C (optimum annealing temperature 58-60°C). After 30 seconds of elongation at 72°C, a last expansion was performed for seven minutes at the same temperature.

Data were compiled and analysed by using 23.0 version of SPSS IBM. The outcomes for Group 1 (control and healthy), Group 2 (T2DM), and Group 3 (DFU) were exhibited as counts and proportions derived from MTHFR [rs1801133 (C677T)] and MTHFR rs1801131. The central age (in years) and BMI (in kg/m²) were computed as means with standard deviations for each category of the study. These averages were interpreted using a one-way ANOVA. The association between MTHFR genes was assessed by applying the Pearson’s Chi-square test. Risk was estimated using 95% confidence intervals (CI) and odds ratios (OR). Statistical relevance was validated with a value of p <0.05.

RESULTS

Table I illustrates the comparison of age and BMI between the study participants. The results indicated that the mean age was 37.2 ± 9.9 years, and their mean BMI was 28.6 ± 5.7 kg/m² in the healthy group. In contrast, the T2DM patients had an average age of 51.5 ± 12.5 years, and a mean BMI was 28.8 ± 5.7 kg/m². For patients with DFU, the average age was 53.7 ± 7.8 years, and BMI was 26.0 ± 3.9 kg/m². Using a one-way ANOVA, the three groups' mean age (in years) and BMI (in kg/m²) showed significant differences (p <0.05).

Table II represents the association between MTHFR rs1801131 and rs1801133 and the study subjects and displays the findings across different categories. In the healthy group, the frequency of MTHFR rs1801133 was noted as 30.4% homozygous CC and 69.6% CT heterogeneous alleles. For MTHFR rs1801131, AA homozygous was noted as 41.3%, CC homozygous as 39.1%, and CA heterogeneous alleles as 19.6%. 

In the T2DM group, MTHFR rs1801133 was found as 41.3% CC homozygous and 58.7% CT heterogeneous alleles. For MTHFR rs1801131, AA homozygous was noted as 54.3%, CC homozygous as 26.1%, and CA heterogeneous alleles as 19.6%.

In the DFU group, MTHFR rs1801133 was found to have 8.7% CC homozygous and 91.3% CT heterogeneous alleles. For MTHFR rs1801131, AA homozygous was noted as 41.3%, homozygous CC as 15.2%, and CA heterogeneous alleles as 43.5% (Figure 1). MTHFR rs1801133 and MTHFR rs1801131 were significantly (p <0.05) associated with all three study groups by applying the Pearson’s Chi-square test.

Table III presents an association between MTHFR SNP [rs180 1131 (A1298C) and rs1801133 (C677T)] and DM, both with and without foot ulcer complications.

The outcomes indicated the following associations: Individuals carrying the MTHFR rs1801133CT genotype displayed an inverse association with DM, presenting an OR of 0.62 and a CI ranging from 0.26 to 1.46.
 

Figure  1:  Frequency  percentage  of  the  alleles  in  the  groups.

Table I: Association of mean age and mean BMI across study subjects.
 

Parameters	Healthy group (n = 46)	T2DM group (n = 46)	DFU group (n = 46)	p-values
	Mean ± SD	Mean ± SD	Mean ± SD
Age (years)	37.2 ± 9.9	51.5 ± 12.5	53.7 ± 7.8	<0.001
BMI (kg/m2)	28.6 ± 5.7	28.8 ± 5.7	26.0 ± 3.9	0.026*
*A p-value less than 0.05 was considered statistically significant using the one-way ANOVA.

Table II: Association of MTHFR SNP [rs1801131 and rs1801133] across study subjects.

Parameters	Healthy group (n = 46)	T2DM group (n = 46)	DFU group (n = 46)	p-values
MTHFR rs1801133 (C677T)
Wild type CC	14 (30.4%)	19 (41.3%)	4 (8.7%)	0.002*
Heterozygous mutation i-e CT	32 (69.6%)	27 (58.7%)	42 (91.3%)
Homozygous mutation i-e TT	00	00	00
MTHFR rs1801131 (A1298C)
Ref; AA	19 (41.3%)	25 (54.3%)	19 (41.3%)	0.014*
Homozygous mutation CC	18 (39.1%)	12 (26.10%)	7 (15.2%)
Heterozygous i-e mutation AC	9 (19.6%)	9 (19.6%)	20 (43.5%)
*A p-value less than 0.05 was considered statistically significant, * Pearson’s Chi-square test showed a value of p <0.05 significant.

Table III: Risk assessment for T2DM and foot ulcer in T2DM patients by using multinomial logistic regression with MTHFR (rs1801131 (A1298C) and rs1801133 (C677T).

Variables	OR (95% CI) T2DM	p-values	OR (95% CI) T2DM group	p-values
Homozygous i-e CC rs1801133 of MTHFR	Reference		Reference
Heterozygous i-e CT rs1801133 of MTHFR	0.62 (C.I. = 0.26-1.46)	0.27	4.59 (C.I. = 1.38-15.2)	0.013*
Homozygous i-e AA rs1801131 of MTHFR	Reference		Reference
Homozygous i-e CC rs1801131 of MTHFR	0.76 (C.I. = 0.25-2.28)	0.62	2.22 (C.I. = 0.80-6.11)	0.12
Heterozygous i-e CA rs1801131 of MTHFR	0.50 (C.I. = 0.19-1.30)	0.15	0.38 (C.I. = 0.13-1.14)	0.08
OR obtained using the multinomial logistic regression model (entered method), *p-value of less than 0.05 was considered statistically significant for OR.

Individuals possessing the MTHFR rs1801131CC genotype also reflected a negative association with DM, featuring an OR of 0.76 and a CI from 0.25 to 2.28. Individuals with the MTHFR rs1801131CA genotype illustrated an inverse association with DM, boasting an OR of 0.50, and a CI from 0.19 to 1.30.

Conversely, the relationships concerning DFU were as follows: Individuals with the MTHFR rs1801133 CT genotype, revealed a positive association with DFU, showing an OR of 4.59 and a CI from 1.38 to 15.2. Individuals bearing the MTHFR rs1801131 CC genotype, exhibited a positive connection with DFU, registering an OR of 2.22 and a CI from 0.80 to 6.11. Individuals with the MTHFR rs1801131 CA genotype, exhibited a negative association with DFU, having an OR of 0.38 and a CI from 1.13 to 1.14.

These results exhibit unique associations between the MTHFR SNP [rs1801131 (A1298C) and rs1801133 (C677T)] and the risk of DM and the incidence of foot ulcers in patients having T2DM.

DISCUSSION

The objective of this research was to assess the MTHFR allele polymorphisms (rs1801133 and rs1801131) and their association with T2DM and DFU among the inhabitants of Karachi, Pakistan. According to this study, significant differences were found in the mean values of age and BMI (healthy, T2DM, and DFU patients), with p <0.05, consistent with studies conducted in India and Albania.^12,13 Both studies concluded that age is the primary reason related to the onset and severity of foot ulcers in patients with T2DM, particularly in individuals over 50 years of age, while BMI is an independent predictor of diabetic foot in patients with T2DM. In contrast to this study’s findings, there were no significant associations among study participants.¹⁴

This observed MTHFR [rs1801133(C677T)] distribution in the present study’s participants is as follows: healthy controls had a higher prevalence of the CT heterozygous allele (69.6%), diabetic patients had a higher frequency of the CC genotype (41.3%), and DFU patients exhibited a markedly increased frequency of the CT heterozygous genotype (91.3%). This study found a significant association (p <0.05) of the MTHFR [rs1801133 (C677T)] genotype (wild type CC and heterozygous mutation CT). These findings align with earlier research conducted by previous researchers.^15-17 These differences in the frequencies of the MTHFR [rs1801133 (C677T)] gene alleles are related to the pathogenesis or progression of foot ulcers in T2DM patients, suggesting that this polymorphism may serve as a significant genetic marker.

The MTHFR [rs1801131 (A1298C)] distribution among participants of this study was observed as follows: healthy controls had an increased prevalence of the CC homozygous allele (39.1%), diabetic patients had a higher frequency of the AA homozygous allele (54.3%), and DFU patients had a markedly increased frequency of the CA heterozygous genotype (43.5%). A significant association (p <0.05) was observed for MTHFR rs1801131 (Ref; AA, homozygous mutation CC, and heterozygous mutation AC) in this study. These outcomes align with genotype frequencies noted by others and genome-wide associations attaining significance for diabetic complications.^18-21

The MTHFR [rs1801131(A1298C)] genotype has been observed as a new genetic susceptibility factor for diabetes complications, particularly cardiovascular diseases, neuropathy, retinal occlusive disease, and complications such as DFU.¹⁷ Genetic heterozygous mutations in the allele that decrease the methylenetetrahydrofolate reductase enzymatic activity can disrupt folate metabolism and elevate plasma Hcy levels, possibly resulting in increased OS and inflammatory reactions in diabetic patients.²²

MTHFR rs1801133 (C677T), particularly the heterozygous CT genotype, had a positive risk factor associated with DFU [OR = 4.59, C.I. (1.38-15.2)], while MTHFR rs1801131 gene, particularly the wild CC homozygous genotype, is a positive risk factor for DFU [OR = 2.22, CI (0.80-6.11)], which are often caused by nerve damage (neuropathy) and poor circulation, highlighting a significant association between MTHFR rs180 1133 (C677T i.e., CT genotype) and MTHFR rs1801131 (A1298C i.e., CC genotype) polymorphisms in this study.

This observation aligns with previous research indicating that the T allele is linked to elevated Hcy levels, specifically hyperhomocysteinaemia.²³ Hyperhomocysteinaemia may result in endothelial dysfunction, impaired microvascular circulation, and increased OS factors that lead to DFU.^24,25 While rs1801131 (A1298C) has a less severe effect on Hcy concentrations than rs1801133, studies suggest that individuals with the CC genotype may experience decreased MTHFR enzyme functionality, potentially resulting in vascular dysfunction and impaired wound healing.²⁶ These findings underscore the role of genetic predisposition in developing DFU, alongside traditional risk factors such as poor glycaemic control, peripheral arterial disease, and neuropathy. The significant association between MTHFR polymorphisms and DFU indicates that genetic screening could assist in identi-fying individuals at higher risk, enabling early preventive measures such as folate supplementation in diet, lifestyle modifications, and proactive wound care strategies, including timely therapeutic interventions. A small number of partici-pants were enrolled, as there was a limitation of resources.

CONCLUSION

The study suggests specific MTHFR gene polymorphisms may play a contributory role in the genetic susceptibility to DFU, highlighting their potential as biomarkers for early identification and risk stratification in diabetic populations.

ETHICAL  APPROVAL:
The study approval was obtained from the Ethics Committee of Baqai Medical University in accordance with the guidelines for human research and experimentation (Ref. No: BMU-EC/01-2023).

PATIENTS’  CONSENT:
Informed consent was obtained from all participants included in this study.

COMPETING  INTEREST:
The authors declared no conflict of interest.

AUTHORS’  CONTRIBUTION:
SUAA: Conception, writing, reviewing, editing, and principal investigation.
QAS: Conception and supervision.
IAS: Conception and co-supervision.
ZM: Data collection and curation.
AF: Conception and investigation.
QS: Critical reviewing, data analysis, and editing.
All authors approved the final version of the manuscript to be published.

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