PJZ_58_3_1121-1128

Association of MTHFR C677T with Obesity in Human Female Population

Rakhshanda Rani* and Muhammad Irfan

Department of Zoology, Wildlife and Fisheries, Faculty of Science, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan

ABSTRACT

The hypothalamo-pituitary-thyroid axis is responsible for the maintenance of the metabolic processes in the human body. The hypothalamus releases thyroid releasing hormone (TRH) which stimulates the pituitary gland to release thyroid stimulating hormone (TSH) which in turn leads to stimulation of the thyroid gland and release of thyroxine (T4) and its active derivative triiodothyroxine (T3). Malfunctioning of any component at any level of the hierarchy of the axis may lead to disorders of metabolism including obesity. The thyrotropin releasing hormone (TRH) is a hypothalamic peptide hormone that possesses a broad spectrum of effects mainly determined by its stimulatory effects on energy metabolism together with iodine containing hormones of the thyroid. The objective of this study was to determine association of a specific methylene tetrahydrofolate reductase (MTHFR) single nucleotide polymorphism (SNP) with obesity (BMI) in human females at early age. This research included the obese and normal females visiting the hospitals of Islamabad and Rawalpindi. Blood samples were used for isolation of DNA. MTHFR gene was amplified. A particular restriction enzyme was used identify polymorphisms. The DNA fragments of the wild-type and mutant varieties were obtained on the gel. The allele frequency of the C to T polymorphism was determined by counting alleles through electrophoresis gel analysis. Chi-square analysis was used to determine the Hardy-Weinberg equilibrium of the alleles in the population. By adjusting the effects of confounding factors such age and socioeconomic characteristics, logistic regression analysis was used to establish the correlation between the polymorphism and BMI. Statistical significance was set at a p value of <0.05. MTHFR C677T was not linked to obesity in women in this study.

Article Information

Received 30 August 2023

Revised 05 May 2024

Accepted 12 May 2024

Available online 09 July 2025

(early access)

Published 25 March 2026

Authors’ Contribution

RR: Collected samples, performed the experiments and analyzed the data and wrote the original draft of manuscript. MI: Conceived idea and planned the experiments, provided resources and supervised the study. All authors read and approved the manuscript.

Key words

Obesity, SNP, MTHFR gene, Allelic frequency, Homocysteine, Body mass index

DOI: https://dx.doi.org/10.17582/journal.pjz/20230830151236

* Corresponding author: [email protected]

0030-9923/2026/0003-1121 $ 9.00/0

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Methylene tetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate. This reduction reaction creates methyl donor that plays a key role for the conversion of homocysteine (Hcy) to methionine (Yamada et al., 2001). Methionine synthase catalyze this conversion along with vitamin B12 as a cofactor and this catalyst always coexist with vitamin B12 in all mammalian tissues. Methionine is incorporated in dietary protein, and act as a predecessor of S-adenosylmethionine. When methionine is converted to Hcy, S-adenosylmethionine acts as a methyl donor. Similarly, in transsulfuration process the Hcy can also be converted to cysteine through vitamin B6 dependent pathway (Selhub, 1999).

The chromosome 1 at 1p36.3 possesses the gene that codes for 5,10-methylenetetrahydrofolate reductase (MTHFR). This gene consists of 2.2 kb long complementary DNA sequence and 11 exons (Goyette et al., 1998). 70-77 kDa subunits of dimeric proteins are encoded by this cDNA. The cDNA of human has the most catalytic activity and binding sites as compared to catalytic activity of porcine and bacterial enzymes (Daubner and Matthews, 1982; Goyette et al., 1994).

Goyette et al. (1994, 1996, 1998) have reported 15 mutations of MTHFR gene that are associated with enzymatic deficiency, out of which 14 are infrequent and cause severe enzymatic deficiency and one is common that causes slight enzymatic shortage. A point mutation at C677T in the MTHFR gene replaces alanine to valine in the enzyme (Rosenberg et al., 2002). Kang et al. (1988) and Rozen (1997) reported that thermostability of MTHFR enzyme was reduced due to this mutation and the enzyme showed decreased activity at 37 ºC or higher temperature. As compared to normal subjects the activity of MTHFR enzyme in homozygous subjects is reduced to 50-60% at 37 ºC and 65% at 46 ºC. The plasma Hcy level rise in the homozygous mutated subjects due to incapability of the MTHFR enzyme to convert 5, 10-methylenetetrahydrofolate into 5-methyltetrahydrofolate. The Hcy level is higher in homozygous mutated subjects and slightly elevated in heterozygous mutated subjects as compared to the normal subjects (Rozen, 1997).

Methionine is only obtained by using the 5-MTHF as donor group. The use of Hcy in the biochemical cycle is maintained by 5-MTHF, because rise in plasma Hcy level is associated with vascular injury that can start or speed up atherogenic and thrombotic reactions. Loscalzo (1996) reported that hydrogen peroxide and superoxide free radicals are formed quickly by oxidation of free Hcy in plasma which causes injury to the biological cellular membrane by oxidation, or initiates the per-oxidation reactions of lipoprotein particles in plasma.

In obese individuals the levels of circular oxidative stress markers are high because of processes involving pro-oxidant reactions for example inflammatory adipokine synthesis similar to leptin, TNF- α (tumor necrosis-factor-alpha) and IL-β (interleukine-1β) by macrophages and adipocytes. Obese individuals have enhanced respiratory action and less utilization of anti-oxidant molecules and vitamins (Vincent and Taylor, 2006). It appears that in obese individuals little utilization of vitamins B12 and B6 and folic acid is linked with the existence of a greater prevalence of C677T of MTHFR that can be a factor responsible for low availability of cofactors and substrate necessary for the production of 5-MTHF.

Mojtabai (2008) established the relationship of low levels of folate with elevated body mass index. It is also assumed that obesity is influenced by folate levels through epigenetic control of the genes which regulate the body fat storage (Smith et al., 2008). The methylation of DNA cytosine and histone amino acid residues is done with methyl groups provided by folate that may change the epigenetic gene expression (Fuks, 2005). The methylation of dinucleotides of gene involved in food intake, fat storage and cellular physiology or body weight will be affected by any defect in the genes that are involved in methyl group metabolism. Therefore, in the present study we hypothesized that the genetic polymorphisms C677T of MTHFR gene may compromise the potency of MTHFR enzyme resulting in decreased levels of folate which is associated with obesity. The objectives of the present study was to determine association of MTHFR C677T polymorphism with obesity in humans.

Materials and Methods

Subjects

The present study included human normal and obese female subjects visiting various hospitals in Rawalpindi and Islamabad. All the subjects were from the same socioeconomic status and eating habits. The overweight subjects with minor illness like temperature, flu, and cough without any concurrent diseases and having obesity were recruited in the study after a written consent. The controls were normal weight female subjects taken from the same population.

Data of socio-demographic, physical health and lifestyle aspects was collected in the form of a predefined questionnaire.

Measurement of body mass index (BMI)

For determination of BMI, Harpender Standiometer was used to measure the standing height close to 0.1 cm. Digital weight scale was used to determine the weight with a precision of 0.1 Kg. Following formula was used to calculate the body mass index.

The subjects with BMI of 18-25 normal weight and above 30 were considered obese.

PCR amplification of MTHERC677T

Standard phenol-chloroform method was used to extract DNA from blood samples. HinfI restriction endonuclease was used to digest the PCR amplified products to determine polymorphisms (Irfan et al., 2016). The genotype and allelic frequency of the genetic polymorphism was obtained by directly counting bands on 3% agarose gel.

Biotechnology Information sequences database (http://www.ncbi.nlm.nih.gov) was used to construct primers and Primer 3 programme was used to design primers 5’-ACC CAC AGA AAA TAC CCA G-3’ (forward)

and 5’-TGC CCC ATT ATT TA-3’ (reverse) (Irfan et al., 2016) (http://www.patch.com.ac.uk/cgi.bin/primer3.cgi). The specificity, dimmer and multiple priming sites were determined by using PCR simulation programme amplify 1.2.

Statistical analysis

The Mean±S.E. of the quantitative variables was calculated. Chi-square analysis was used to determine the Hardy-Weinberg equilibrium of the alleles in the population. The association of the polymorphism with overweight condition and BMI was determined by logistic regression analysis adjusting the effects of confounding factors i.e., age, socioeconomic factors and lifestyle. A p-value < 0.05 was considered statistically significant.

Results and Discussion

Association of lifestyle and medical factors with obesity

The mean age of obese (37.18±1.232) were significantly (p<0.05) higher as compared to normal (31.50±1.011) subjects. However, the multiple logistic regression analysis shows no significant (p<0.05) association between age and obesity (Table I). Our data show that the female consuming extra meal (4th meal) are at a higher risk of obesity. Obsessive eating also increases the odds of obesity. A routine daily walk and increasing sleep time have protective role against obesity (Table I).

We found an association between obesity and liver disease. Digestive disease significantly (p<0.0) increases the odds ratio of obesity. There is also a significant (p<0.05) association of diabetes with obesity (Table I).

Though, previous studies reported an association between age and obesity (Cynthia et al., 2012; Amira et al., 2012) we did not find a significant association, which may be due to our age matched subjects. However, our results of association of obesity with an extra meal (4th meal) consumption and obsessive eating (a meal after every hour) and lack of physical activity are consistent with previous reports (Swinburn et al., 2004). It is well established previously that the obsessive and frequent eating of carbohydrates and fat rich foods may increase the fats deposition and central obesity especially in elderly as anabolic lipolytic hormones such as growth hormone (GH) and sex steroids decline gradually with age.

We have observed an inverse association between obesity and sleep time that is also reported previously. The values of BMI, body fat, waist and hip circumferences and fat mass index were higher for subjects with short sleeping time, especially for females (Garaulet et al., 2011; Taheri, 2006; Sun et al., 2009; Shaikh et al., 2009). The association between reduced sleep duration and obesity can be explained in different ways such as shorter sleep time leads to an excess of energy intake and lower energy expenditure as the awaking person tends to consume more and frequent food. On the other hand, insufficient sleep is associated with low anabolic lipolytic hormones (GH and sex steroids) and an anorexic (eating and fat deposition inhibiting) hormone, i.e., leptin. Appetite and hunger might be affected by this association, which leads to overeating and obesity. Hence in this way, the subjects with short sleep duration become obese because they have much time to eat and decreased energy expenditure due to fatigue and changed thermoregulation (Cauter and Knutson, 2008).

As in the present study, the association between obesity and digestive disease was also established similar to previous study (Giovannucci and Michaud, 2007). The poor digestion, reflux and digestive ulcers may lead to central obesity. The liver diseases result in disorders of fat metabolism and lead to deposition of fats. The previous studies also reported the association between obesity and thyroid or goiter (Zheng et al., 2015; Tamer et al., 2011). The hypothyroidism based goiter is associated with an increase in body weight and obesity (Scheen and Luyckx, 2002). It is also observed that obesity and insulin has a relation, but the status of cause and effect is dubious (Hussain et al., 2010).

Table I. Association of non-genetic factors with obesity.

Factors	Control n (%)	Obese n (%)	OR (95% CI)	p Value	AOR (95% CI)	p Value
Age	-	-	1.047 (1.019-1.075)	0.001	1.025 (0.962-1.092)	0.449
Diabetes	3 (3.0)	25 (25.5)	11.073 (3.219-38.091)	0.000	20.944 (3.063-143.217)	0.002
Extra meal (4th meal)	4 (4.0)	31 (31.6)	11.104 (3.745-32.930)	0.000	25.061 (4.330-145.062)	0.000
Digestive disease	1 (1.0)	59 (60.2)	149.769 (20.049-1.119E3)	0.000	305.196 (30.864-3.018E3	0.000
Goiter	3 (3.0)	9 (9.2)	3.270 (0.858-12.461)	0.083	NC	-
Frequent fast food	23 (23.0)	30 (30.6)	1.477 (0.784-2.783)	0.228	NC	-
Liver disease	3 (3.0)	39 (39.8)	21.373 (6.322-72.254)	0.000	18.483 (2.893-118.087)	0.002
Obsessive eating	2 (2.0)	23 (23.5)	15.027 (3.435-65.739)	0.000	18.341 (1.640-205.137	0.018
Daily walk	87 (87.0)	62 (63.3)	0.257 (0.126-0.525)	0.000	0.064 (0.016-0.262)	0.000
Sleeping time	-	-	0.652 (0.496-0.857)	0.002	0.874 (0.472-1.617)	0.667
Family history of thyroid disorders	3 (3.0)	6 (6.1)	2.109 (0.512-8.680)	0.301	NC	-

Logistic regressions; significant if p≤0.05; NC, not calculated. OR, odds ratio; AOR, adjusted odds ratio; CI, confidence interval

Association of MTHFR C677T with obesity

Genotype distribution of the MTHFR 677C>T polymorphism of the 198 subjects was analyzed: 117 (59. 09%) subjects were homozygous for the C allele (CC), 77 (38.89%) subjects were heterozygous (CT), and 4 (2.02%) subjects were homozygous for the T allele (TT). The minor allele (T) frequency of the MTHFR 677C>T polymorphism was 0.205, and the genotype distributions did not deviate from the Hardy-Weinberg equilibrium (p >0.05) (Table II).

The alleles and genotype distribution were compared between obese (98) and control (100) subjects. Out of 98 obese subjects 57 (58.2%) were homozygous for C allele (CC), 38 (38.8%) were heterozygous (CT) and 3 (3.1%) were homozygous for T allele (TT). Out of 100 control subjects 60 (60.0%) were homozygous for C allele (CC), 39 (39.9%) were heterozygous (CT) and 1 (1.0%) was homozygous for T allele (TT).

Furthermore, C allele was present in 152 (77.6%) obese and 159 (79.5%) control subjects. The frequency of T allele in obese and control was 44 (22.4%) and 41 (20.5%), respectively.

Allelic frequencies

According to the Table II the allelic frequencies were statistically similar (P>0.05) in the both groups of subjects suggesting lack of association between minor allele (T) and obesity.

Genotype frequencies

The genotypic frequencies were not significantly (p>0.05) different in obese and normal females. Therefore, no significant (p>0.05) association between MTHFR C677T and obesity was observed. Both the heterozygous (CT) and mutated homozygous (TT) increase the odds of obesity statistically non-significantly (p>0.05) (Table II). However, the heterozygous and mutated homozygous (CT+TT) collectively showed a statistically significant (p<0.05) association (OR:1.776, CI: 0.287-2.954) with obesity, after adjusting odds ratios for age, diabetes, extra meal, digestive diseases, goiter, fast food frequent consumption, liver disease, obsessive eating, physical activity, sleeping time and family history of thyroid disorders.

The present study found no association between MTHFR C677T and obesity in human female obese population. However, it is observed that other factors such as lifestyle habits and medical condition are responsible to cause obesity.

A number of studies have been conducted to investigate the association of MTHFR C677T polymorphism with obesity in various populations (Tables III and IV). Although, the results of most of these studies are similar but they did not find significant relationship between C677T and obesity (Fan et al., 2015; Gara et al., 2011; Hernandez-Guerrero et al., 2013; Lewis et al., 2008; Settin et al., 2009; Thawnashom et al., 2005; Yin et al., 2012; Bazzaz et al., 2010). There are however two studies, i.e., Terruzzi et al. (2007) and Yang et al. (2014), based on a Caucasian and an Asian, respectively, that have shown a significant association between obesity and the polymorphism. Therefore, the differences in the results could be attributed to other factors such as variations in recruitment of subjects, sample size, ethnicity and geographic factors (Table IV). The results are also dependent on the general health, medical conditions (diabetes, liver diseases and digestive diseases, etc.) and lifestyle (frequency and type of food, sleeping and resting time and daily physical activity etc.), which were not addressed while selecting subjects in many of the previous studies.

Therefore, the present study was conducted to address the role of non-genetic factors along with MTHFR C677T polymorphism in obesity. The results reports no association between the polymorphism and obesity in spite of adjusting non-genetic factors significantly. However, there are few limitations in the present study that we could not measure folate and Hcy levels of the subjects. A mutation in the MTHFR such as C677T may reduce conversion of Hcy

Table II. Genotype and allelic frequencies of the MTHFR C677T mutation in the studied subjects.

Genotype	Obese n (%)	Control n (%)	OR (95% CI)	p Value	AOR (95 % CI)	p Value
CC	57 (58.2)	60 (60.0)	1		1
CT	38 (38.8)	39 (39.9)	1.026 (0.577-1.823)	0.931	0.739 (.217-2.518)	0.628
TT	3 (3.1)	1 (1.0)	3.158 (0.319-31.247)	0.325	20.002 (0.871-459.331)	0.061
CT+TT	41 (41.8)	40 (40.0)	1.079(0.612-1.902)	0.793	1.776 (0.287-2.954)	0.000
C	152 (77.6)	159 (79.5)	1		1
T	44 (22.4)	41 (20.5)	1.123(0.695-1.814)	0.637	1.182 (0.469-2.981)	0.723

Logistic regression adjusted for age, diabetes, extra meal, digestive diseases, goiter, fast food, liver disease, obsessive eating, daily walk, sleeping time and family history of thyroid disorders. Significant at p<0.05

into methionine may lead to hyper-homocysteinemia that is found to be responsible for various health conditions such as cardiovascular diseases, infertility and obesity. Similarly, lower levels of folate were also found to be responsible for the obesity (Rassoul et al., 2008; Klerk et al., 2002; Thawnashom et al., 2005; Vincent and Taylor, 2006).

Conclusion

We concluded that there is no association of MTHFR C677T with obesity in females. But, the extra meal (4th meal), obsessive eating, diabetes, digestive and liver diseases are among the major causes of obesity. However, a daily walk and increase in sleeping time have protective role. There are few limitations in the present study that we did not measure folate and homocysteine levels of the subjects. Therefore, further study is needed to measure the folate and Hcy levels in the obese patients along with MTHFR C677T polymorphism.

DeclarationS

Funding

This study was financially supported by Higher Education Commission of Pakistan via Access to scientific instrumentation (Grant number HEC/R and D/ASIP/2016/000063).

IRB approval

The study was approved by the Institutional Review Board at Shaheed Zulfiqar Ali Bhutto Medical University, PIMS Islamabad vide reference No. F. I-1/2015/ERB/SZABMU/ Dated: 26-1-2016.

Ethical statement

The research was approved by the ethical committee at Pakistan Institute of Medical Sciences (PIMS) Hospital, Islamabad and all experiments were performed in accordance with relevant guidelines and regulations. Informed consent of participants was taken in the form of a questionnaire.

Statement of conflict of interest

The authors have declared no conflict of interest.

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