Modulator Effects of the Methylenetetrahydrofolate Reductase C677T Polymorphism on Response to Vitamin B12 Therapy and Homocysteine Metabolism

Abstract

In this study, our aim was to investigate the association of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on the vitamin B12 therapy response in 95 patients with vitamin B12 deficiency and 92 healthy control subjects using vitamin B12, plasma total homocysteine (tHcy), and folate as the main measure of outcome. MTHFR C677T genotypes were determined by polymerase chain reaction–restriction fragment length polymorphism techniques. There were no differences in the distribution of MTHFR genotypes in the cases versus the controls. Mean concentrations of plasma tHcy and B12 vitamin were 18.84 μM and 142.47 pg/mL in patients with TT (10.5%) genotypes. Furthermore, mean concentrations of B12 vitamin after cobalamin therapy were 697.62, 656.64, and 488.76 pg/mL in patients with the CC, CT, and TT genotypes, respectively. The MTHFR 677 TT genotype has decreasing effect in B12 vitamin and increasing effect in tHcy. In comparison with the patients having CC and CT genotypes, patients with the TT genotype had a lower response to vitamin B12 therapy.

Introduction

V ıtamın B12 defıcıency is common among the elderly in developed countries and in developing populations (Allen, 2009). Vitamin B12 (cobalamin) has a major role in DNA synthesis, hematologic, and neuropsychiatric diseases (Oh and Brown, 2003). A plasma vitamin B12 concentration of <148 pM (200 pg/mL) is defined as vitamin B12 deficiency (Allen, 2009). Vitamin B12 deficiency is mainly caused by inadequate dietary intake or malabsorbtion of vitamins in foods (Allen, 2009). Deficiency of vitamin B12 should be suspected in patients who have unexplained neurophyschiatric symptoms, and patients with gastrointestinal diseases such as sore tongue, anorexia, and diarrhea (Hvas and Nexo, 2006). Once diagnosed, efficient treatment can be ensured either by injections every 2–3 months or by a daily dose of 1 mg vitamin B12 (Hvas and Nexo, 2006). The prevalence of vitamin B12 deficiency in the general population varies with age affecting ≤3% of those aged 20–39 years, ≈4% of those aged 40–59 years, and ≈6% of people aged ≥70 years old (Allen, 2009). Vitamin B12–related enzymatic reactions in humans are conversion of methylmalonic acid (MMA) to succinyl-CoA using vitamin B12 as the cofactor and conversion of homocysteine to methionine using vitamin B12 and folic acid as the cofactors. Genetic abnormalities (MTHFR polymorphism) and vitamin B deficiencies are important factors responsible for elevated total homocysteine (tHcy) concentrations (Ganji and Kafai, 1999).

Homocysteine is a sulfur amino acid formed as a result of metabolism of sulfurous methionine to cysteine, or remethylation by folate to methionine supplied from dietary proteins (Taylor et al., 2000; Yates and Lucock, 2003). Several conditions including cardiovascular disease, end-stage renal disease, neurodegenerative disease, osteoporotic fractures, and neural tube defects have been linked to moderately elevated tHcy levels (Taylor et al., 2000; Tsai et al., 2009). Genetic background also affects tHcy level. The methylation of homocysteine to methionine is catalyzed by the 5,10-methylenetetrahydrofolate reductase (MTHFR) (MIM 236250) enzyme by converting 5,10-methylenetetrahydrofolate to predominant circulating form of folate irreversibly, which the enzyme plays a central role in folate metabolism (Ozbek et al., 2008).

The MTHFR gene is located on chromosome 1p36.3. Single-nucleotide polymorphisms (SNPs) of folate-dependent enzymes that influence Hcy focused on a common missense mutation C677T in exon 4 of the MTHFR gene that causes an alanine to valine (Ala222Val) amino acid substitution in the folate-binding site of the enzyme rendering MTHFR enzyme thermolabile and reduce MTHFR activity (Frosst et al., 1995). Mutation in MTHFR, especially the C677T polymorphism, is an important genetic determinant of elevated tHcy levels as a consequence of the remethylation of homocysteine to methionine (Moriyama et al., 2002; Hustad et al., 2007). Those bearing the TT homozygote mutant genotype have higher circulating tHcy concentrations, which are between 1.5 and 2.6 μM (∼15–25% higher) on average, than the subjects carrying the C allele (Brattstrom et al., 1998; Klerk et al., 2002). MTHFR C677T is among several common polymorphic mutations that are tested for genetic-nutrient interaction due to its involvement in hyperhomocysteinemia and folate metabolism (Tsai et al., 2009). Since MTHFR genetic polymorphisms alter enzymes involved in tHcy metabolism, and vitamin deficiency may result in variation in tHcy, we investigated the effect of polymorphic variant of C677T of MTHFR on the homocysteine, folate, and vitamin B12 levels in cases with vitamin B12 deficiency before and after vitamin B12 treatment by comparing with age- and gender-matched healthy controls.

Materials and Methods

Subjects and study design

We studied the MTHFR C677T polymorphism in 90 control subjects and 94 subjects with vitamin B12 deficiency by using plasma tHcy and folate as the main measure. The control and vitamin B12–deficient subjects had a mean age of 38.7±13.47 years and 34.2±14.96 years, respectively. Data were collected via questionnaires about medical and family history. The study was conducted according to the Helsinki declaration and was approved by Ethics Committee of the Afyon Kocatepe University. All cases and controls were enrolled in this study under informed consent.

Analysis of MTHFR C677T mutation

Genomic DNA obtained from both case and control groups was isolated from peripheral blood by using a commercially available DNA extraction kit (Omega; Omega Bio Tek) according to the manufacturer's instructions. The following primer sequences were used for the amplification of the SNP of C677T located in exon 4 of the MTHFR gene producing a polymerase chain reaction (PCR) product of 198 base pairs (bp): forward 5′ TGAAGGAGAAGGTGTCTG CGGGA 3′ and reverse 5′ GGACGGTGCGGTGAGAGTG 3′. The PCR conditions were as previously described by Yilmaz et al. (2004). The C677T (rs1801133) missense mutation was determined by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) using HinfI restriction enzyme. The amplified and digested PCR products were analyzed by ethidium bromide staining in 2% agarose gel. The C→T substitution initially gives a product of 198 bp and digestion with HinfI enzyme creates 175 and 23 bp fragments. A subset of the patients was additionally evaluated for the presence of MTHFR C677T mutation using strip assay reverse-hybridization multiplex polymerase chain reaction tests for target genes (FV-PT-MTHFR strip assay^®; Viennalab Labordiagnostika GmgH) to confirm the RFLP results.

Biochemical analysis

Venous blood that was drawn into serum tubes without anticoagulant was centrifuged at 5000 rpm for 5 min and the sera were separated and immediately stored at −20°C until analysis. tHcy, serum folate, and vitamin B12 levels were determined in all of the subjects. After medical treatment, tHcy, folate, and vitamin B12 levels were evaluated in the patient group again.

Serum vitamin B12 and folate levels were quantified with the Cobas e601 Immunoassay System using commercial kits supplied by Roche Diagnostics GmbH. The Cobas e601 Immunoassay System is an electrochemiluminescent immunoassay for the quantitative determination of vitamin B12 and folate levels in human serum. Vitamin B12 assay employs a competitive test principle using an intrinsic factor specific for vitamin B12. Vitamin B12 in the sample competes with the added vitamin B12 labeled with biotin for the binding sites on the ruthenium-labeled intrinsic factor complex. Reference range was between 197 and 886 pg/mL. The folate assay also employs a competitive test principle using natural folate binding protein (FBP) specific for folate. Folate in the sample competes with the added folate labeled with biotin for the binding sites on the ruthenium-labeled FBP. Reference range of folate was between 4.6 and 18.7 ng/mL. The tHcy levels were determined by using high-pressure liquid chromatography with fluorescence detection with instrument and kits supplied by Chromsystems Diagnostics GmbH. Reference range of tHcy was 0–12 μM. Triacylglycerol (TG), total cholesterol (TChol), high-density lipoprotein-cholesterol (HDL-Chol), low-density lipoprotein-cholesterol (LDL-Chol), and very low density lipoprotein-cholesterol (VLDL-Chol) levels were determined in a Cobas c501 automated analyzer using commercial kits supplied by Roche Diagnostics GmbH. Reference ranges of TG, TChol, HDL-Chol, LDL-Chol, and VLDL-Chol are 0–200, 0–200, 40–60, 0–130, and 0–100 mg/dL, respectively.

Statistical analysis

Statistical analyses were performed using SPSS 13.0 (SPSS). Data were expressed as mean±standard deviation for continuous variables. Shapiro–Wilks test was used for defining whether the data were normally distributed. For normally distributed data, the Student's t-test was used to compare means between two groups. However, analysis of variance or test for linear trends was used to compare means of three or more groups. When the groups were not normally distributed, comparisons between two groups were performed using Mann-Whitney U tests. Kruskal–Wallis test was used to compare three or more groups. Chi-square statistics were used for categorical variables. Distributions of allele and genotypes were compared by chi-square test and correlations were expressed as median values and 95% confidence intervals. Hardy–Weinberg equilibrium was tested by chi-square analysis with one degree of freedom. A value of p<0.05 was considered statistically significant. The statistical power was calculated using the QUANTO 1.2 program. For the less-frequent allele frequency of 34% for MTHFR C677T with p=0.05, the study had a power >80 (odds ratio=2.0, mode of inheritance: log-additive, population risk: 10%) (Gauderman and Morrison, 2006).

Results

General characteristics

The clinical characteristics of the cases and controls are shown in Table 1. Overall, 95 patients and 92 controls were enrolled in the study. Mean ages of the patients with vitamin B12 deficiency and control subjects were 34.21 and 38.72 years, respectively. Women accounted for 69.4% of patients and 78.2% of controls. The controls on average had a higher HDL-Chol, VLDL-Chol, folate, and vitamin B12 levels than the patients, but the patients' triglyceride levels were higher than the controls. Male patients had higher tHcy than control men (15.26 vs. 12 μM). After the first month of medical therapy, the evaluations, and the measurements of tHcy, folate, and vitamin B12 levels were carried out again for only 58 of the patients (Table 2). Some patients did not apply for control after medical therapy, their vitamin B12, folate, and tHcy levels cannot be measured and this caused the difference in the number of patients in the genetic test and biochemical analysis.

Table 1.

Clınıcal Data of the Study and Control Groups

	Patient (n=95)	Control (n=92)
Gender (female/male) (n)	66/29 (95)	72/20 (92)
Age (years)	34.21±14.96 (95)	38.72±13.47 (92)
Body mass index (kg/m²)	29.2±6.5 (95)	29.0±8.3 (92)
Systolic pressure (mmHg)	120.73±20.11 (95)	125.22±23.8 (92)
Diastolic pressure (mmHg)	86.00±64.82 (95)	82.55±13.83 (92)
Total-cholesterol (mg/dL)	181.60±37.36 (86)	188.47±43.99 (88)
HDL-cholesterol^a (mg/dL)	45.36±10.86 (90)	49.97±12.10 (89)
LDL-cholesterol (mg/dL)	108.43±31.07 (83)	114.02±33.09 (87)
VLDL-cholesterol^a (mg/dL)	29.57±20.70 (89)	31.35±60.41 (90)
Triglyceride^b (mg/dL)	144.22±85.84 (87)	122.30±106.33 (88)
Homocysteine (μM)	12.30±6.63 (95)	11.56±6.42 (91)
Folate^c (ng/mL)	7.54±3.03 (84)	9.02±3.10 (86)
Vitamin B12^c (pg/mL)	156.17±29.69 (95)	318.01±91.93 (92)

n, number of individuals. The results are shown as mean±SD.

p<0.05.

p<0.01.

p<0.001.

HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low density lipoprotein; SD, standard deviation.

Table 2.

Homocysteıne, Folate, and Vıtamın B12 Levels of Patıents Before and After Fırst Month of Vıtamın B12 Treatment

Before therapy			After therapy
Homocysteine (μM)	Folate (ng/mL)	Vitamin B12 (pg/mL)	Homocysteine (μM)	Folate (ng/mL)	Vitamin B12 (pg/mL)
11.98±5.56 (51)	7.63±2.90 (51)	154.87±27.062 (58)	13.60±6.08 (51)	8.36±2.98 (51)	660.46±439.71 (58)

Prevalence of the MTHFR mutation

The MTHFR genotype was determined in 184 subjects. The distribution of the MTHFR genotypes is summarized in Table 3. Distribution of genotypes in controls and patients did not show a significant departure from Hardy–Weinberg equilibrium, p=0.88 and p=0.68, respectively. Allele frequencies for the C and T alleles were 0.66 and 0.34 in patients and 0.65 and 0.34 in controls, respectively (p=0.93). There was no significant distribution of MTHFR genotypes between patients and control subjects (p=0.92). Overall, 10.6% of the cases and 12.2% of the controls carried the 677 TT genotype (p=0.80). The heterozygous status was 44.4% and 46.8% for control and patient groups, respectively (p=0.82).

Table 3.

Genotype and Allele Dıstrıbutıon of C677T (rs1801133) Genotypes

Genotype	Control (n=90)	Patient (n=94)	p-Value	OR (%95 CI)	p-Value ^b
C677T			0.921
CC	39 (43.3%)	40 (42.6%)		1^a
CT	40 (44.4%)	44 (46.8%)		1.07 (0.58–1.98)	0.823
TT	11 (12.2%)	10 (10.6%)		0.89 (0.34–2.32)	0.806
CT+TT^b	51 (56.6%)	54 (57.4%)		1.03 (0.58–1.85)	0.915
Alelles			0.935
C	118 (65.6%)	124 (66%)		1^a
T	62 (34.4%)	64 (34%)		0.982 (0.64–1.51)	0.935
	H-W p=0.88	H-W p=0.68

Reference genotype/allele.

Calculations CC versus CT, TT, and CT+TT.

OR, odds ratio; CI, confidence intervals.

Relation between homocysteine, folate, vitamin B12, and MTHFR mutation

MTHFR genotypes and clinical parameters including homocysteine, folate, and vitamin B12 levels are shown in Table 4. Mean homocysteine concentration was 12.3 μM in patients. The tHcy levels of the patients with vitamin B12 deficiency were higher in TT (18.84±9.73 μM) than in heterozygous CT (10.48±4.95 μM) and homozygous wild type CC individuals (12.61±6.39 μM; p<0.05). There was no difference in control subjects. Vitamin B12 level was higher in heterozygous CT (164.45±27.47 pg/mL) than wild type CC (150.94±27.87 pg/mL) patients, and was much lower in homozygous TT (142.47±38.52 pg/mL) mutants. In control subjects, vitamin B12 level was higher in homozygous TT (354.46±104.96.52 pg/mL) mutants than in CC (314.35±95.78 pg/mL) and CT (310.74±83.43 pg/mL) genotypes. In addition, we also investigated whether MTHFR CC, CT, and TT genotypes were correlated with the altered tHcy, folate, and vitamin B12 by considering vitamin B12 therapy as the main outcome. In this respect, vitamin B12, folate, and tHcy were studied in subjects with the CC, CT, and TT genotypes before and after vitamin B12 therapy. In CC and CT subjects, vitamin B12 levels dramatically increased after therapy, but in TT individuals the response to therapy was not as effective as in other genotypes (Table 5). Furthermore, there was a significant increase in plasma tHcy and folate levels after vitamin B12 therapy in heterozygote CT patients as p=0.008 and p<0.001, respectively (Table 5).

Table 4.

Homocysteıne, Folate, and Vıtamın B12 Levels by Methylenetetrahydrofolate Reductase C677T (MTHFR) Genetıc Polymorphısm ın the Cases wıth Vıtamın B12 Defıcıency and the Controls

	Patient			Control
Genotype
Determinant	CC	CT	TT	CC	CT	TT
Homocysteine	12.61±6.39 (42)^a	10.48±4.95 (43)	18.84±9.73 (10)^b	11.02±6.64 (40)	12.01±6.54 (40)	11.86±5.46 (11)
Folate	7.94±3.49 (36)	7.37±2.71 (40)	6.56±2.14 (8)	9.49±3.20 (37)	9.05±3.07 (37)	7.48±2.60 (12)
Vitamin B12	150.94±27.87 (42)	164.45±27.47 (43)	142.47±38.52 (10)^c	314.35±95.78 (40)	310.74±83.43 (40)	354.46±104.96 (12)

n, number of individuals.

p<0.05 for CC versus TT with patient.

p<0.01 for CT versus TT with patients.

p<0.05 for CC versus CT with patient.

Table 5.

Homocysteıne, Folate, and Vıtamın B12 Levels by Methylenetetrahydrofolate Reductase (MTHFR) Genetıc Polymorphısm ın the Cases wıth Vıtamın B12 Defıcıency

Genotypes	Determinant	Before therapy \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$({\overline x} \pm SS)$$ \end{document}	After therapy \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$({\overline x} \pm SS)$$ \end{document}	p-Value
CC	Homocysteine (n=28)	12.46±5.92	13.85±7.63	0.539
	Folate (n=26)	7.89±3.61	7.99±3.04	0.416
	Vitamin B12 (n=30)	147.83±24.80	697.62±494.85	<0.001^a
CT	Homocysteine (n=18)	9.92±4.18	13.52±3.68	0.008^a
	Folate (n=20)	7.54±2.02	9.37±2.81	<0.001^a
	Vitamin B12 (n=22)	165.21±24.69	656.64±409.26	<0.001^a
TT	Homocysteine (n=5)	16.74±5.20	12.58±3.36	0.14
	Folate (n=5)	6.71±1.71	6.30±2.19	0.50
	Vitamin B12 (n=6)	152.18±37.98	488.76±191.66	0.03^a

n, number of individuals. The results are shown as mean±SD.

Statistically significant.

Discussion

We analyzed MTHFR C677T polymorphism in 94 cases with vitamin B12 deficiency and 90 healthy adults, and also quantified the tHcy, folate, and vitamin B12 levels in the patient group before and after vitamin B12 therapy. Gender- and age-related characteristics were previously demonstrated as the factors that contribute to tHcy, folate, and vitamin B12 levels (Gielchinsky et al., 2001; Papandreou et al., 2006; Papoutsakis et al., 2006). Ganji and Kafai (1999) and Jacques et al. (2001) reported in their cohort studies that the tHcy level was significantly higher in men than women as our study found. These results suggest metabolic causes for the differences in tHcy between men and women. Thus, the gender and age distribution in our study may have impacted the results. There was no difference in the distribution of MTHFR genotypes between patients and control subjects (p=0.92). In our study, the relative frequency of the 677 T allele was 34%, and it was 42%, 43.8%, 38.2%, and 33.3% in Spanish, Italian, French, and Portuguese populations, respectively (Castro et al., 2003). In the current study, the prevalence of the mutant homozygous 677 TT genotypes, which were lower than Spanish, Italian, and French population, was 12.2% and 10.5% in control and patient groups, respectively (15.8%, 18%, and 18.2% for Spanish, Italian, and French population, respectively) (Castro et al., 2003). In healthy subjects compatible with our results, the prevalence of the 677 TT genotype in the Turkish population has been previously reported to be 9.1%, 10.5%, 9.3%, and 6.5%, respectively (Yilmaz et al., 2004, 2006; Ozbek et al., 2008; Ozarda et al., 2009). Moderately increased tHcy levels were associated with the 677 TT mutation, particularly in the presence of low plasma folate levels confirming our results (van der Put et al., 1995; Brattstrom et al., 1998; Botto and Yang, 2000). In a study by de Bree et al. (2003), they reported that TT subjects had lower plasma folate concentrations than CT and CC subjects in any folate intake level and this was compatible with our results both in patient and control groups. Zittan et al. (2007) reported that the frequency of vitamin B12 deficiency among the homozygous TT group (29.8%) was higher than the heterozygous or subjects without mutation. And also, in the same study, homocysteine level was elevated in homozygote TT when compared with the subjects of CT and CC genotypes. We also report that the levels of vitamin B12 are low in homozygote mutant patients. In our study, tHcy levels were significantly higher in the cases with vitamin B12 deficiency than healthy subjects. After vitamin B12 therapy, tHcy levels decreased in 677 TT homozygotes as compatible with the literature (De Bree et al., 2003; von Castel-Dunwoody et al., 2005). However, after vitamin B12 therapy there was an increase in 677 CT and 677 CC subjects' homocysteine level but this was not statistically significant and also not seen in other studies (De Bree et al., 2003; von Castel-Dunwoody et al., 2005). Vitamin B12 and folate deficiencies elevate plasma homocysteine levels (Kang et al., 1987; Ubbink et al., 1993). Furthermore, when we compared folate levels in patient and control groups, there was a significant difference revealing a decrease in folate level in the patients with vitamin B12 deficiency (p<0.001). In the presence of decreased MTHFR activity because of the C677T transition, decrease in plasma folate levels would be expected. Our results are compatible with this expectation in both patient and control groups, and also vitamin B12 therapy affected only 677 CT patients by increasing folate levels (7.54–9.37 ng/mL). Ma et al. (1996) and Herrmann et al. (1999) reported that patients with the 677 TT mutation responded well to the folate treatment, which lowered the plasma homocysteine levels. But there was no report on the vitamin B12 treatment response in patients with the 677 TT mutation. In the current study, patients with 677 TT had less response to vitamin B12 treatment than the other genotypes. To confirm the response of MTHFR C677T mutant patients to vitamin B12 therapy, a study including a larger number of individuals should be carried out.

Footnotes

Acknowledgment

This work was supported by grants from Afyon Kocatepe University Research Fund (09.TIP.19).

Disclosure Statement

No competing financial interests exist.

References

Allen

L.H.

2009. How common is vitamin B-12 deficiency? Am J Clin Nutr, 89:693S–696S.

Botto

L.D.

, Yang

2000. 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review. Am J Epidemiol, 151:862–877.

Brattstrom

, Wilcken

D.E.

, Ohrvik

, Brudin

1998. Common methylenetetrahydrofolate reductase gene mutation leads to hyperhomocysteinemia but not to vascular disease: the results of a meta-analysis. Circulation, 98:2520–2526.

Castro

, Rivera

, Ravasco

, Jakobs

, Blom

H.J.

, Camilo

M.E.

, De Almeida

I.T.

2003. 5,10-Methylenetetrahydrofolate reductase 677C→T and 1298A→C mutations are Genetic determinants of elevated homocysteine. Q J Med, 96:297–303.

De Bree

, Verschuren

W.M.M.

, Bjorke-Monsen

A-L.

, van der Put

N.M.

, Heil

S.G.

, Trijbels

F.J.

, Blom

H.J.

2003. Effect of the methylenetetrahydrofolate reductase 677C→T mutation on the relations among folate intake and plasma folate and homocysteine concentrations in a general population sample. Am J Clin Nutr, 77:687–693.

Frosst

, Blom

H.J.

, Milos

, Goyette

, Sheppard

C.A.

, Matthews

R.G.

, Boers

G.H.J.

, Den Heijer

, Kluitmans

L.A.J.

, Van den Heuvel

L.P.W.J.

, Rosen

1995. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet, 1:111–113.

Ganji

, Kafai

M.R.

2009. Demographic, lifestyle, and health characteristics and serum B vitamin status are determinants of plasma total homocysteine concentration in the post-folic acid fortification period, 1999–2004. J Nutr, 139:345–352.

Gauderman

W.J.

, Morrison

J.M.

2006. QUANTO 1.1: a computer program for power and sample size calculations for Genetic-epidemiology studies. http://hydra.usc.edu/gxe. 2011 September 20.

Gielchinsky

, Elstein

, Green

, Miller

J.W.

, Elstein

, Algur

, Lahad

, Shinar

, Abrahamov

, Zimran

. 2001. High prevalence of low serum vitamin B12 in a multi-ethnic Israeli population. Br J Haematol, 115:707–709.

10.

Herrmann

, Quast

, Ullrich

, Schultze

, Bodis

, Geisel

1999. Hyperhomocysteinemia in high-aged subjects: relation of B-vitamins, folic acid, renal function and the methylenetetrahydrofolate reductase mutation. Atherosclerosis., 144:91–101.

11.

Hustad

, Midttun

Ø.

, Schneede

, Vollset

S.E.

, Grotmol

, Ueland

P.M.

2007. The methylenetetrahydrofolate reductase 677C→T polymorphism as a modulator of a B vitamin network with major effects on homocysteine metabolism. Am J Hum Genet, 80:846–855.

12.

Hvas

A.M.

, Nexo

2006. Diagnosis and treatment of vitamin B12 deficiency. An update. Haematologica, 91:1506–1512.

13.

Jacques

P.F.

, Bostom

A.G.

, Wilson

P.W.F.

et al. 2001. Determinants of plasma total homocysteine concentration in the Framingham Offspring cohort. Am J Clin Nutr, 73:613–621.

14.

Kang

S.S.

, Wong

P.W.

, Norusis

1987. Homocysteinemia due to folate deficiency. Metabolism, 36:458–462.

15.

Klerk

, Verhoef

, Clarke

, Blom

H.J.

, Kok

F.J.

, Schouten

E.G.

2002. MTHFR 677C→T polymorphism and risk of coronary heart disease: a meta-analysis. J Am Med Assoc, 288:2023–2031.

16.

, Stampfer

M.J.

, Hennekens

C.H.

, Frosst

, Selhub

, Horsfold

, Malinow

M.R.

, Willett

W.C.

, Rozen

1996. Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US physicians. Circulation, 94:2410–2416.

17.

Miller

J.W.

, Ribaya-Mercado

J.D.

, Russell

R.M.

, Shepard

D.C.

, Morrow

F.D.

, Cochary

E.F.

, Sadowsky

J.A.

, Gershoff

S.N.

, Selhub

1992. Effect of vitamin B-6 deficiency on fasting plasma homocysteine concentrations. Am J Clin Nutr, 55:1154–1160.

18.

Moriyama

, Okamura

, Kajinami

, Iso

, Inazu

, Kawashiri

, Mizuno

, Takeda

, Sakamoto

, Kimura

, Suzuki

, Mabuchi

2002. Effects of serum B vitamins on elevated plasma homocysteine levels associated with the mutation of methylenetetrahydrofolate reductase gene in Japanese. Atherosclerosis, 164:321–328.

19.

R.C.

, Brown

D.L.

2003. Vitamin B12 deficiency. Am Fam Physician, 67:979–986993–994.

20.

Ozarda

, Sucu

D.K.

, Hizli

, Aslan

2009. Rate of T alleles and TT genotype at MTHFR 677C→T locus or C alleles and CC genotype at MTHFR 1298A→C locus among healthy subjects in Turkey: impact on homocysteine and folic acid status and reference intervals. Cell Biochem Funct, 27:568–577.

21.

Ozbek

, Kucukali

C.I.

, Ozkok

, Orhan

, Aydin

2008. Effect of the methylenetetrahydrofolate reductase gene polymorphisms on homocysteine, folate and vitamin B12 in patients with bipolar disorder and relatives. Prog NeuroPsychopharmacol Biol Psychiatry, 32:1331–1337.

22.

Papandreou

, Mavromichalis

, Makedou

, Rousso

, Arvanitidou

2006. Reference range of total serum homocysteine level and dietary indexes in healthy Greek schoolchidren aged 6–15 years. Br J Nutr, 96:719–724.

23.

Papoutsakis

, Papoutsakis

, Yiannakouris

, Manios

, Papaconstantinou

, Magkos

, Schulpis

K.H.

, Zampelas

, Matalas

A.L.

2006. The effect of MTHFR (C677T) genotype on plasma homocysteine concentrations in healthy children is influenced by gender. Eur J Clin Nutr, 60:155–162.

24.

Selhub

, Jaques

P.F.

, Wilson

P.W.

, Rush

, Rosenberg

I.H.

1993. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. J Am Med Assoc, 270:2693–2698.

25.

Taylor

B.V.

, Qudit

G.Y.

, Evans

2000. Homocysteine, vitamins, and coronary artery disease. Can Fam Physician, 46:2236–2245.

26.

Tsai

M.Y.

, Loria

C.M.

, Cao

, Kim

, Siscovick

, Schreiner

P.J.

, Hanson

N.Q.

2009. Clinical utility of genotyping the 677C>T variant of methylenetetrahydrofolate reductase in humans is decreased in the post-folic acid fortification era. J Nutr, 139:33–37.

27.

Ubbink

J.B.

, Vermaak

W.J.

, van der Merwe

, Becker

P.J.

1993. Vitamin B-12, vitamin B-6, and folate nutritional status in men with hyperhomocysteinemia. Am J Clin Nutr, 57:47–53.

28.

van der Put

N.M.

, Steegers-Theunissen

R.P.

, Frosst

, Trijbels

F.J.

, Eskes

T.K.

, van den Heuvel

L.P.

, Mariman

E.C.

, den Heyer

, Rozen

, Blom

H.J.

1995. Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet, 346:1070–1071.

29.

von Castel-Dunwoody

K.M.

, Kauwell

G.P.A.

, Shelnutt

K.P.

, Vaughn

J. D

, Griffen

E.R.

, Maneval

D.R.

, Theriaque

D. W.

, Bailey

L.B.

2005. Transcobalamin 776C→G polymorphism negatively affects vitamin B-12 metabolism. Am J Clin Nutr, 81:1436–1441.

30.

Yates

, Lucock

2003. Interaction between common folate polymorphisms and B-vitamin nutritional status modulates homocysteine and risk for a thrombotic event. Mol Genet Metabol, 79:201–213.

31.

Yilmaz

, Agachan

, Ergen

, Karaalib

Z.E.

, Isbir

2004. Methylene Tetrahydrofolate reductase C677T mutation and left ventricular hypertrophy in Turkish patients with type II diabetes mellitus. J Biochem Mol Biol, 37:234–238.

32.

Yilmaz

, Isbir

, Agachan

, Ergen

, Farsak

, Isbir

2006. C677T Mutation of methylenetetrahydrofolate reductase gene and serum homocysteine levels in Turkish patients with coronary artery disease. Cell Biochem Funct, 24:87–90.

33.

Zittan

, Preis

, Asmir

, Cassel

, Lindenfelt

, Alroy

, Halon

D. A.

, Lewis

B.S

, Shiran

, Schliamser

J.E.

, Flugelman

M.Y.

2007. High frequency of vitamin B12 deficiency in asymptomatic individuals homozygous to MTHFR C677T mutation is associated with endothelial dysfunction and homocysteinemia. Am J Physiol Heart Circ Physiol, 293:H860–H865.