MTHFR 677C&gt;T (rs1801133) variant is associated with homocysteinemia but not with clinical severity in patients with peripheral arterial occlusive disease

doi:10.21203/rs.3.rs-1523928/v1

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Research Article

MTHFR 677C>T (rs1801133) variant is associated with homocysteinemia but not with clinical severity in patients with peripheral arterial occlusive disease

https://doi.org/10.21203/rs.3.rs-1523928/v1

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Objective: The aim was to evaluate the association between the MTHFR 677C>T (rs1801133) variant with susceptibility and severity of peripheral arterial occlusive disease (PAOD) and levels of homocysteine (Hcy).

Subject and Methods: A case-control study enrolled 157 patients with PAOD and unrelated 113 controls. The severity and anatomoradiological categories of the PAOD were assessed by Fontaine classification and Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC), respectively. The MTHFR 677C>T was genotyped using real-time polymerase chain reaction and Hcy serum levels were determined using chemiluminescence microparticles assay.

Results: The PAOD patients showed higher Hcy than controls but the values did not differ according to Fontaine and TASC categories. The MTHFR 677C>T variant was not associated with PAOD, the clinical stage, and the anatomoradiological categories. However, patients carrying the TT genotype (recessive model) or CT+ TT genotypes (dominant model) presented higher levels of Hcy than those carrying other genotypes. In conclusion, the T allele of MTHFR 677C>T variant was associated with hyperhomocysteinemia in PAOD patients, but not in controls.

Conclusion: Our data suggested a possible interaction between MTHFR 677C>T variant and the presence of other genetic, epigenetic or environment factors associated with PAOD on modulation of the Hcy metabolism.

Peripheral arterial occlusive disease

methylenetetrahydrofolate reductase

MTHFR 677C>T genetic variant

homocysteine

Fontaine classification.

Peripheral arterial occlusive disease (PAOD) is a global public health concern as result of a severe arterial stenosis and insufficient blood supply to the distal limb causing intermittent claudication and resting pain, decreases the quality of life of the patients in terms of mobility and level of independence and can lead to disability and even death [1]. It is characterized by atherosclerotic lesions involving non-cardiac and non-cerebral arteries and disturbances in the axial vessels and at the microcirculatory level, which may lead to critical ischemia of lower limbs. It is significantly PAOD is closely associated with an increased risk for incident cardiovascular diseases, cerebrovascular diseases, and mortality [2–7].

The underlying pathological mechanism of PAOD and the potential pathways and core genes had gained considerable attention in recent years with the aim of identifying potential therapeutic targets [8]. Over the last decade, much progress has been made in the capability of efficiently identifying genetic variants through whole genome sequencing efforts, genome-wide association studies (GWAS), and epigenetic profiling [9–11]. A better understanding of the genetics of PAOD may allow earlier identification of those at risk for PAOD and may open the door for precision medicine-based approaches to treatment.

Although familial aggregation and heritability estimates suggest a significant genetic contribution, little is known about the genetic susceptibility to PAOD in non-European populations [12–15]. GWAS of the ankle-brachial index (ABI) and PAOD (defined as an ABI < 0.90) showed that the variants associated with the ABI differ by Hispanic/Latino ethnic groups [16].

Hyperhomocysteinemia (HHcy) is involved in endothelial dysfunction, an important step in atherosclerosis development [17], and a relation between HHcy and PAOD has been showed [18]. The plasma concentration of Hcy is influenced by a complex interaction of environmental and genetic factors [19]. The 677C > T (rs1801133) variant of the MTHFR, which codes the methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in the remethylation of Hcy to methionine, is the most common genetic cause of HHcy [20, 21] This variant consists in a C (cytosine) to T (thymine) transition at the nucleotide 677 in the exon 4 [22]. HHcy and MTHFR 677 C > T genetic variant emerged as independent risk factor for subclinical atherosclerosis, implying genetic influences as potential contributors to the increased burden of atherosclerotic disease characterizing the PAOD. The association between PAOD and MTHFR variants has been studied in literature with controversial results; the Linz Peripheral Arterial Disease (LIPAD) study does not correlate MTHFR 677C > T and PAOD [23], while a meta-analysis of nine appropriate studies showed that the homozygosity for the T allele was associated with an increased risk of PAOD [24].

Although some studies have assessed the involvement of the MTHFR 677C > T variant with HHcy and the development of PAOD, data are still scarce and contradictory [23–25]. Moreover, there are no studies that have evaluated the involvement of the MTHFR 667C > T genetic variant with the severity of the PAOD in the Brazilian population. Since, in studies carried out with other populations, the results were quite diverse, it is necessary a better understanding of the genetic characteristics and behavior of PAOD in our population. In this context, the aim of the present study was to evaluate the association between the MTHFR 677C > T variant with the susceptibility and severity of PAOD, as well as with serum levels of Hcy in the Brazilian population.

This is a case-control study, in which 270 unrelated participants were consecutively enrolled, of both sexes and aged between 37 and 76 years old. Of them, 113 were healthy individuals (controls without PAOD) attended at the Londrina Regional Blood Center, and 157 were patients diagnosed with PAOD who were attended at the Hemodynamics Sector of the University Hospital of Londrina, both institutions located at Londrina, Paraná State, South Brazil. Exclusion criteria (for PAOD patients and controls) were the presence of cancer, chronic inflammatory, infectious, and autoimmune diseases, and the use of supplements such as folic acid and vitamin B12. The ABI < 0.9 determined by continuous wave doppler were the criteria used for the diagnosis of PAOD. Continuous wave doppler was performed with DF-7001 VN Portable Vascular Doppler (Medpej, Ribeirão Preto, São Paulo, Brazil). ABI measurement technique was performed according to ACC/AHA guidelines for the management of patients with PAOD [26]. The ABI values were calculated by dividing the higher of the two ankle systolic pressures in one leg with the greater brachial artery value of systolic pressure. ABI values were calculated up to two decimal places [27].

Subsequently, the patients with PAOD were categorized according to their severity using the Fontaine's clinical classification into five groups: I- Asymptomatic, IIa- Mild claudication, IIb- Severe claudication, III- Pain at Rest, and IV- Trophic Injury [27]. The PAOD group was also evaluated regarding the severity and the different anatomical distributions using the anatomoradiological categories according to the Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II), including the aortoiliac segment and infra-inguinal (in this study called as femoropopliteal and it included the popliteotibial territory) segment [28]. TASC category was defined by a vascular surgeon after analyzing an arteriography (Siemens, Berlin and Munich, Germany) that was performed in hemodynamic sector of the University Hospital of Londrina.

The study was approved by the Institutional Research Ethics Committees of State University of Londrina, Paraná, Brazil (CAAE: 2.400.074) and all of the individuals invited were informed in detail about the research and gave written Informed Consent. All procedures were conducted in accordance with the principles outlined in the Declaration of Helsinki.

Demographic, clinical and laboratory data

Prior to blood collection, anthropometric measurements (weight, height and waist circumference) were obtained and the body mass index (BMI) was calculated. The measurement of systemic blood pressure was also performed, followed by filling out a questionnaire with data on age, ethnicity (self-reported as Caucasian and non-Caucasian), comorbidities, smoking and use of medications. The data related to the clinical and anatomical classification of the disease, in addition to the patient's surgical history, were provided by a vascular surgeon or accessed in the medical records. The systemic arterial hypertension (SAH), dyslipidemia, and type 2 diabetes mellitus (T2DM) diagnosis agreed with the guidelines of European Society of Hypertension/European Society of Cardiology [29], with the Third Report of the National Cholesterol Education program [30] and with the American Diabetes Association [31], respectively.

Venous blood collection (10 mL) was performed after 8 hours of fasting with no anticoagulant and with EDTA anticoagulant using Vacutainer System tubes (Becton-Dickinson, New Jersey, U.S). The plasma, serum and buffy coat were stored in a -80°C until the analyzes were performed. The serum levels of Hcy, vitamin B12 and folate were determined by chemiluminescence microparticles assay (Architect i2000SR, Abbott Laboratory, Abbott Park, IL, USA).

MTHFR C677T genotyping

The genomic DNA was extracted with the Biopur DNA extraction kit (Biometrix Diagnóstica, Curitiba, Paraná, Brazil) according to the manufacturer's instructions, with some modifications, such as the volume of buffy coat (200 µL) and the temperature of the elution buffer (70° C). DNA samples were quantified using a NanoDrop 2000c™ spectrophotometer (Thermo Scientific, Waltman, MA, USA) at 260 nm and purity was assessed by measuring the 260/280 nm ratio. The MTHFR 677C > T (rs1801133) variant was determined using real time polymerase chain reaction (qPCR) through the TaqMan® (Thermo Fisher Scientific, Waltham, Massachusetts, EUA) method. It was used a validated assay (C_32060205_10, Life Technologies Corporation, Carlsbad, CA, USA) with specific primers and fluorescent probes for the genotype determination [VIC/FAM] GGC AAA CAA TAA ATG TAATAG TAG G[C/T]A AAT TTG TGC TAT GTT AGA GGT CTT). The level of fluorescence of the qPCR products were evaluated using the Quantum Studio VI (Applied Biosystems, Foster City, CA, USA).

Statistical analysis

Analysis of contingency tables (χ2 or Fisher’s exact test) were employed to check the associations between categorical variables and diagnostic groups. Categorical variables were expressed as absolute number (n) and percentage (%). We assessed the differences in continuous variables between groups using the Mann-Whitney test and were expressed as median and interquartile range 25.0%-75.0% (IQR). Genotype and allele frequencies were compared by chi-squared test. Odds ratios (OR) were calculated with 95% confidence interval (95% CI). Binary logistic regression analysis was performed to assess the effect of the genetic variants in the study groups and OR and 95% CI were determined. For all statistical tests p < 0.05 was considered for significance level. The number of samples allowed to reach 80.0% of power. Statistical analyses were performed using IBM SPSS windows version 24 (SPSS, Inc., Chicago, IL, USA).

The study included 60 (38.2%) female and 97 (61.8%) male with PAOD with median age of 69 years (IQR: 62–67). When compared to control group, the PAOD patients were older (p < 0.001) and had higher levels of Hcy (p < 0.020). However, they did not differ about sex (p = 0.116), ethnicity (p = 0.119), BMI (p = 0.355), folate (p = 0.242), and vitamin B12 (p = 0.885) levels (Table 1).

Table 1

Sociodemographic and clinical data of peripheral arterial occlusive disease (PAOD) patients and controls from Brazilian population
	Control (n = 113)	PAOD (n = 157)	p value*
Age (year)	43 (37–48)	69 (62–76)	< 0.001
Sex (Female/Male)	54 (47.8)/59 (52.2)	60 (38.2)/97 (61.8)	0.116
Ethnicity (C/NC)	88 (77.9)/25 (22.1)	109 (69.4)/48 (30.6)	0.123
BMI (kg/m²)	25.81 (22.95–28.80)	25.31 (21.64–29.73)	0.355
Hcy (µmol/L)	9.91 (8.47–11.30)	13.66 (10.61–16.74)	< 0.020
Folate (ng/mL)	5.4 (3.9–7.5)	7.3 (5.1–10.6)	0.242
Vitamin B12 (pg/mL)	320 (230–450)	316 (236–440)	0.885
All results of Mann Whitney test. x²: results of analyses of contingency tables. Continuous variables were expressed as median and interquartile range (25%-75%) and categorical variables were expressed as absolute number (n) and percentage (%). C: Caucasian; NC: Non-Caucasian; BMI: Body mass index. Hcy: Homocysteine. *Adjusted by age, sex, ethnicity, and body mass index.

Other characteristics of the PAOD patients were recorded, such as 37 (24.2%) were smokers, 68 (44.4%) were ex-smokers, 98 (71.5%) were diagnosed with T2DM, 73 (47.4%) were diagnosed with dyslipidemia, 114 (74%) were diagnosed with SAH, and 58 (39.2%) showed HHcy (Hcy ≥ 15 µmol/L). Moreover, 31 (19.9%) patients underwent endovascular revascularization, 66 (42.3%) had conventional surgery, and 59 (37.8%) adopted conservative therapy.

Table 2 summarizes the clinical and anatomical characteristics of the PAOD patients. Regarding the clinical characteristics, there were no patients in Fontaine's classification I included in the study. There were 35 (22.4%) and 33 (21.2%) patients with Fontaine's classes IIa and IIb patients, respectively. The group of patients with pain at rest, corresponding to Fontaine's class III, presented 11 (7.1%) individuals and patients with trophic injury, the most severe stage of the disease, allocated to Fontaine's class IV, presented 77 (49.4%) patients. Fontaine's classes IIa and IIb were grouped, since the prognosis and treatment of choice are similar, adding up to a total of 68 (43.6%) patients. Similarly, classes III and IV were also joined, resulting in a total of 88 (56.4%) individuals. Regarding the different anatomical distributions of PAOD, 40 (25.8%) patients had disease in the aortoiliac segment; of them, 7 (4.5%) were TASC A, 5 (3.2%) were TASC B, 12 (7.7%) were TASC C and 16 were (10.3%) TASC D. Other 115 (74.2%) patients were free of disease in that territory. In relation to the infra-inguinal segment, in the study it was called femoropopliteal, 134 (86.5%) of the patients had disease in this territory, while 21 (13.5%) had no significant disease in this topography. As in the aortoiliac segment, the patients with femoropopliteal territory, the classes A and B showed less frequency, with 9 (5.8%) TASC A and 17 (11.0%) TASC B, while the majority had more advanced disease, with 36 (23.2%) TASC C and 71 (45.8%) TASC D. In addition, we obtained 19 (12.3%) patients with concomitant disease in the aortoiliac and femoropopliteal territories. When we grouped the cases in which the treatment of choice is similar, without distinction of the arterial territory, we obtained 23 (14.8%) of the patients in the classes of TASC A + B and 132 (85.2%) in the classes C + D. However, Hcy levels did not differ according to the clinical stage of the PAOD evaluated by Fontaine classification (p = 0.257) (Fig. 1A) and the anatomoradiological categories evaluated by TASC II classification (p = 0.678) (Fig. 1B). All data were adjusted by age, ethnicity, BMI, folate, and vitamin B12.

Table 2

Clinical characteristics of the peripheral arterial occlusive disease, according to Fontaine classification and anatomical localization from Brazilian population
Characteristics	Patients (n = 157)
Fontaine classification*
I - asymptomatic	0 (0.0)
IIa - mild claudication	35 (22.4)
IIb - severe claudication	33 (21.2)
III - pain at rest	11 (7.1)
IV - trophic injury	77 (49.4)
Intermittent claudication (IIa + IIb)	68 (43.6)
Critical limb ischemia (III + IV)	88 (56.4)
Aorto-Iliac (No/Yes)**	115 (74.2)/40 (25.8)
TASC A	7 (4.5)
TASC B	5 (3.2)
TASC C	12 (7.7)
TASC D	16 (10.3)
Femoropopliteal (No/Yes)*	21 (13.5)/134 (86.5)
TASC A	9 (5.8)
TASC B	17 (11.0)
TASC C	36 (23.2)
TASC D	71 (45.8)
Aorto-Iliac/Femoropopliteal (No/Yes)**	136 (87.7)/19 (12.3)
All patients without distinction of the arterial territory: TASC A + B/C + D **	23 (14.8)/132 (85.2)
The categorical variables were expressed as absolute number (n) and percentage (%). In one patient, this variable was not recorded. * In two patients, this variable was not recorded. TASC: Inter-Society Consensus for the Management of Peripheral Arterial Disease.

Table 3 shows the frequency of the alleles and genotypes of the MTHFR 677C > T variant in the PAOD patients and controls. The groups did not differ regarding about the allele and genotypes distributions (p > 0.05), after adjusted by sex, ethnicity, age, and BMI. Table 4 shows the data related to the dominant and recessive models of statistical analysis of the MTHFR 677C > T obtained in the PAOD subgroup. The subgroups differed on ethnicity in both models. In addition, Hcy serum levels were higher in patients carrying the CT + TT genotypes than those carrying the CC genotype (p = 0.008, dominant model) while patients carrying the TT genotype had higher levels of Hcy when compared with those carrying the CC + CT genotypes (p = 0.019, recessive model), independently of sex, ethnicity, age, BMI, folic acid, vitamin B12, and dyslipidemia. There was no association between genotypes (dominant and recessive models) and Hcy serum levels in the control group (data not shown). Regarding the Fontaine and TASC classification, the frequency of the genotypes did not differ among the subgroups of PAOD patients.

Table 3

Distribution of genotypes and allele frequencies of *MTHFR* 677C > T variant in different genetic models between patients with peripheral arterial occlusive disease (PAOD) and controls from Brazilian population
Genetic model		Controls (n = 113)	PAOD (n = 157)	OR (95% CI)	p-value*
Allelic	C	156 (69.0)	209 (66.6)	Reference	-
	T	70 (31.0)	105 (33.4)	1.120 (0.778–1.628)	0.576
Codominant	CC	54 (47.8)	76 (48.4)	Reference	-
	CT	48 (42.5)	57 (36.3)	1.230 (0.423–3.579)	0.704
	TT	11 (9.7)	24 (15.3)	3.415 (0.655–17.79)	0.145
Dominant	CC	54 (47.8)	76 (48.4)	Reference	-
	CT + TT	59 (52.2)	81 (51.6)	1.484 (0.536–4.105)	0.447
Recessive	CC + CT	102 (90.3)	133 (84.7)	Reference	-
	TT	11 (9.7)	24 (15.3)	3.029 (0.660–13.91)	0.154
Overdominant	CC + TT	65 (57.5)	100 (63.7)	Reference	-
	CT	48 (42.5)	57 (36.3)	0.921 (0.346– 2.454)	0.870
χ2: results of analyses of contingency tables. Data were expressed as absolute number (n) and percentage (%). Bold values represent statistically significant values. *Adjusted by age, sex, ethnicity, and body mass index (BMI); OR: odds ratio; CI: confidence interval; C: major allele; T: minor allele.

Table 4

Results of the dominant and recessive model analysis of the association between the *MTHFR* 677C > T variant in patients with peripheral arterial occlusive disease (PAOD) and exploratory variables
Variable	CC (n = 76)	CT + TT (n = 81)	p value*	CC + CT (n = 133)	TT (n = 24)	p value*
Age (year)	71 (61–78)	68 (62–74)	0.321	69 (61–76)	71 (63–76)	0.963
Sex (Female/Male)	26 (34.2)/50 (65.8)	34 (42.0)/47 (58.0)	0.317	50 (37.6)/83 (62.4)	10 (41.7)/14 (58.3)	0.705
Ethnicity (C/NC)	44 (57.9)/ 32 (42.1)	65 (80.2)/16 (19.8)	0.002	88 (66.2)/45 (33.8)	21 (87.5)/3 (12.5)	0.037
BMI (kg/m²)	24.57 (21.26–27.92)	26.17 (22.43–30.61)	0.157	25.35 (21.67–29.73)	24.98 (20.31–29.37)	0.489
Homocysteine (µmol/L)	12.94 (10.02–15.98)	14.60 (10.86–17.83)	0.008	13.22 (10.20–15.97)	16.40 (13.65–23.77)	0.019
Folate (ng/mL)	7.1 (5.2–10.6)	7.5 (4.8–10.8)	0.945	7.5 (5.3–11.1)	6.3 (3.7–9.6)	0.126
Vitamin B12 (pg/mL)	341 (241–459)	299 (217–418)	0.202	318 (244–445)	276 (185–395)	0.596
Fontaine IIa + IIb/II + IV	32 (42.1)/44 (57.9)	36 (45.0)/44 (55.0)	0.716	56 (42.4)/76 (57.6)	12 (50.0)/12 (50.0)	0.548
TASC A + B/C + D^**	10 (13.3)/65 (86.7)	13 (16.2)/67 (83.8)	0.610	20 (15.2)/112 (84.8)	3 (13.0)/20 (87.0)	0.544
Mann Whitney test. Data were expressed by median and interquartile range (25%-75%). χ2: results of analyses of contingency tables. Data were expressed as absolute number (n) and percentage (%). Adjusted by age, sex, ethnicity, BMI, folate, vitamin B12, and dyslipidemia. C: Caucasian; NC: Non-Caucasian; BMI: Body Mass Index; TASC: Inter-Society Consensus for the Management of Peripheral Arterial Disease. *In two patients, this variable was not recorded.

The main results of this case-control study are that the T allele of the MTHFR 677C > T variant was associated with increased Hcy serum levels only PAOD patients but not in health controls, independently of extraneous variables but there are no associations between the genotypes and Fontaine or TASC classification. In this study, we demonstrated that patients with PAOD showed increased Hcy serum levels when compared with controls but the Hcy levels did not differ according to disease severity. To our knowledge, the present study is the first that evaluated the association between the MTHFR 677C > T variant, Hcy levels and the severity of PAOD in Brazilian patients.

Our data are in agreement with a previous study that found increased Hcy in PAOD patients compared with controls [32], and increased Hcy levels have been considered an important risk factor for cardiovascular diseases [33, 34]. Hcy may affect both atherogenesis and the resistance of the endothelium to thrombosis through different mechanisms including the direct toxic effect to endothelial cells, interference with the antithrombotic and vasodilator functions of nitric oxide [35], enhancement of leukocyte adhesion and extravasation, with the potency to contribute to vascular injury [36].

The MTHFR 677C > T (rs1801133) variant of MTHFR consists in a cytosine (C) to thymine (T) transition at the nucleotide 677 in the exon 4 and this nonsynonymous variant results in the alanine-to valine (Ala to Val) amino acid change leading to a thermolabile enzyme with reduced activity [34]. The reduced MTHFR enzyme activity and the increased thermolability lead to a decrease in 5-methyltetrahydrofolate and an increase in the accumulation of the substrate 5,10- methylenetetrahydrofolate and, consequently HHcy [19].

Our data showed that the allelic and genotypes frequency of the MTHFR 677C > T variant did not differ between the PAOD patients and control group in different genetic models that were evaluated, suggesting that this genetic factor, by itself, may not contribute to the development of PAOD in our population. These results are in agreement with previous studies reported in worldwide population [23} as well as in Brazilian population [25]. However, the observed frequency of the MTHRF 677C > T genotypes in the present study agrees with previous studies that showed the presence of the TT genotype among 5.0–15.0% of the general population and its role and impact on the Hcy levels may vary among different populations [37–40]. In addition, we demonstrated that patients with PAOD carrying the TT genotype (in recessive model) showed higher levels of Hcy than those carrying the CT + TT genotypes. Moreover, the patients with PAOD carrying the TT + CT genotypes (in dominant model) showed higher levels of Hcy than those carrying the CC genotype, suggesting that this variant may be related to HHcy in PAOD Brazilian patients. It is important to highlight that these associations were independently of age, sex, ethnicity, and BMI, folate, and vitamin B12.

Divergent results have been reported regarding the association between Hcy levels and MTHFR 677C > T genotypes. Khandanpour and coworkers [24] showed that PAOD was associated with elevated levels of Hcy and the TT genotype. However, other studies reported that the T allele could be a protector factor for PAOD [41], and that high levels of Hcy were associated with the T allele only in those individuals with decreased levels of folate, vitamin B12 or vitamin B6 [42]. These results can be justified by the fact that folate and vitamins B12 and B6 are, respectively, substrate and cofactors in the Hcy metabolic pathway [19]. Moreover, no significant association between the MTHFR 677C > T genotypes and Hcy levels was also reported [23, 43].

It is important to emphasize that our data showed that the presence of the T allele was associated with an increase in Hcy levels exclusively in the PAOD patients (but not in controls) with the CT + TT genotypes, regardless of the other risk factors. That association could be explained, in part, through the presence of other genetic and/or epigenetic factors associated with the disease that may modulate the metabolism of Hcy in these patients Several risk factors for PAOD could regulate gene expression through the epigenetic mechanisms. Studies reported the epigenetic effects of T2DM, SAH, Hcy levels, aging, smoking, and sedentary lifestyle in the DNA methylation and chromatin remodeling, histone acetylation, microRNA expression and regulation [44, 45].

Gene-by-environmental interactions are hypothesized to play an important role in the expression of a disease. Therefore, the effects of environmental factors in association with the risk factors for PAOD that were not evaluated in the present study may regulate the expression of genes involved in the Hcy metabolism and could explain why the high levels of Hcy were observed only in PAOD patients, independently of the risk factors controlled in this study. PAOD has complex phenotypes that depend on multiple other genetic and environmental factors, as well as epigenetic mechanisms [45] that could also explain the high levels of Hcy among the patients with PAOD carrying the T allele of MTHFR 677C > T variant. The reduced enzyme activity associated with the MTHFR 677C > T variant has been linked to decreased DNA methylation [46] and HHcy [47]. In turn, decreased DNA methylation may involve aberrant expression of structural and matrix proteins or reduced DNA integrity resulting in premature aging of the vascular tissue [48].

This study has some potential limitations. First, it is a case-control design, which does not allow inferences on causal relationship between the MTHFR 677C > T variant and the PAOD, as well as the Hcy levels and the severity of the disease. Second, we evaluated one MTHFR variant, but this gene presents other single nucleotide variants, and it might be interesting to evaluate whether genetic haplotypes play a role in subjects with PAOD. However, this study has some strengths, such as controlling confounding effects by the statistical analysis.

In conclusion, the T allele of MTHFR 677C > T variant was associated with HHcy in PAOD patients, but not in controls. In addition, this variant was not associated with the clinical stage and the anatomoradiological categories of PAOD. Our data suggested a possible interaction between MTHFR 677C > T variant and a presence of other genetic, epigenetic and environment factors associated with PAOD on modulation of metabolism of Hcy.

Ethics approval and consent to participate

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent for publication

The studied participants were informed about the present research, and a written consent form was taken from all of them before their enrollment. Moreover, all the authors and co-authors participated and contributed sufficiently in the research, and all of them concur with the submission. The manuscript has been approved by the responsible authorities where the work was carried out. The authors also concur that, if accepted, the manuscript shall not be published elsewhere in the same form in either the same or any other language, without the consent of the Editor-in Chief of Inflammation Research.

Human rights

All procedures performed in this study involving human participants were in accordance with the ethical standards of the Institution and/or National Research Committee and with the World Medical Association 1964 Helsinki Declaration.

Standards for reporting

The manuscript was prepared taken into account the recommendations of the guidelines hosted by the Strengthening the Reporting of Observational studies in Epidemiology (STROBE). STROBE is used for observational studies (cohort, case-control, or cross-sectional designs) according to the STROBE statement (www.strobe-statement.org)

Availability of data and materials

The dataset generated during and/or analyzed during the current study will be available from EMVR upon reasonable request and once the dataset has been fully exploited by the authors.

Competing interests

The authors declare that they have no competing interest. None of the authors are involved in the publication process or have a financial or other beneficial interest in the products or concepts mentioned in the submitted manuscript.

Funding

The study was supported by grants from Coordination for the Improvement of Higher Level of Education Personnel (CAPES) of Brazilian Ministry of Education: Finance Code 001; Institutional Program for Scientific Initiation Scholarship (PIBIC) of the National Council for Scientific and Technological Development (CNPq) of Brazil.

Authors' contributions

1) conception and design of the study: ANCS, RC, EMVR; 2) acquisition of data: GSS; IMC, TF; 3) laboratory analysis: IMC, TF, MABL; 4) statistical analysis: TF, ANCS; 5) analysis and interpretation of data: GSS, EMVR, ANCS; drafting of the manuscript, tables and figures: GSS, EMVR, ANCS; 6) manuscript review: GSS, EMVR, ANCS. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

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No competing interests reported.

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MTHFR 677C>T (rs1801133) variant is associated with homocysteinemia but not with clinical severity in patients with peripheral arterial occlusive disease

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Introduction

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Demographic, clinical and laboratory data

MTHFR C677T genotyping

Statistical analysis

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