The Journal of Nutrition
Methylenetetrahydrofolate Reductase Variants
Associated with Hypertension and
Cardiovascular Disease Interact with Dietary
Polyunsaturated Fatty Acids to Modulate
Plasma Homocysteine in Puerto Rican Adults1–3
Tao Huang,4,5,7Katherine L. Tucker,6Yu-Chi Lee,5Jimmy W. Crott,5Laurence D. Parnell,5Jian Shen,5
Caren E. Smith,5Jose M. Ordovas,5Duo Li,4,7* and Chao-Qiang Lai5*
4Department of Food Science and Nutrition, Zhejiang University, Hangzhou, 310029 China;5Jean Mayer USDA Human Nutrition
Research Center on Aging at Tufts University, Boston, MA 02111;6Department of Health Sciences, Northeastern University, Boston, MA
02115; and7APCNS Centre of Nutrition and Food Safety, Hangzhou, China
Although methylenetetrahydrofolate reductase (MTHFR) genetic variants are associated with plasma homocysteine (Hcy)
and cardiovascular disease (CVD), little is known whether dietary fatty acid intake modulates these associations. The goal
was to examine the interaction of MTHFR variants with dietary fatty acids influencing plasma Hcy in 995 Boston Puerto
Rican adults. We found that plasma Hcy concentration was negatively correlated with (n-3) PUFA intake (r = 20.117; P =
0.022), and the ratio of (n-3):(n-6)PUFA in the diet (r = 20.122; P = 0.009).Further, 2 functional MTHFR variants, 1298A.C
and 677C.T, which are not in linkage disequilibrium in this population, were significantly associated with hypertension
(OR = 1.72, P = 0.024, and OR = 1.60, P = 0.002, respectively). In addition, the 1298A.C variant was significantly
associated with CVD (OR = 3.32; P = 0.030). Importantly, this variant exhibited significant interactions with intakes of total
and (n-6) PUFA and the (n-3):(n-6) PUFA ratio of the diet. The plasma Hcy concentration of carriers of risk allele 1298C was
greater than that of noncarriers only when participants had consumed a high-PUFA diet (.7.8% energy) but was not
greater when they had low intake of PUFA (#7.8% energy). In addition, participants with combined genotypes of both
SNP (677 TT with 1298 AC or CC) who consumed high levels of (n-3) PUFA (.0.66% energy) had lower plasma Hcy
compared with those who had the same genotype and consumed low levels of (n-3) PUFA (#0.66% energy). Our study
suggests that dietary PUFA intake modulates the effect of 2 MTHFR variants on plasma Hcy in Boston Puerto Rican
adults. J. Nutr. 141: 654–659, 2011.
(HHcy)8has been suggested as an important risk factor for
cardiovascular diseases (CVD) (1–3). Moderately elevated
plasma Hcy concentration tends to be seen in patients with
thepast severaldecades, hyperhomocysteinemia
coronary and peripheral vascular diseases compared with the
general population (4–6). The major causes of HHcy include
impairment of renal function, deficiencies of plasma folate,
vitamin B-12, and vitamin B-6, and dietary and genetic factors
(7,8). Recently, we investigated the relationship between (n-3)
PUFA and plasma Hcy in Chinese and Australian populations and
provided evidence that increased (n-3) PUFA concentrations in
plasma phospholipids and platelet phospholipids were associated
with a protective effect on CVD and lower plasma Hcy concen-
trations (9,10). Human intervention studies also demonstrated
that dietary (n-3) PUFA can decrease plasma Hcy (11–13). To
understand how fatty acids regulate Hcy metabolism, we con-
ducted a feeding study in rats and found that tuna oil and salmon
oil rich in (n-3) PUFA regulate both gene expression and enzyme
activity of constituents of Hcy metabolism (14). However, the
question remains how dietary PUFA intake regulates Hcy metab-
olism inhumans.Methylenetetrahydrofolatereductase (MTHFR)
catalyzes the conversion of 5,10-methylenetetrahydrofolate to
1Supported by the China Scholarship Council, the NIH, National Institute on Aging
grant no. 5P01AG023394-02, NIH/National Heart, Lung and Blood Institute grant
nos. HL54776 and HL078885, contracts 53-K06–5-10 and 58–1950-9–001 from the
USDA Research Service and the National Natural Science Foundation of China (no.
2Author disclosures: T. Huang, K. L. Tucker, Y-C. Lee, J. Crott, L. D. Parnell,
J. Shen, J. M. Ordovas, D. Li, and C-Q. Lai, no conflicts of interest.
3Supplemental Table 1 and Figure 1 are available with the online posting of this
paper at jn.nutrition.org.
8Abbreviations used: BPRHS, Boston Puerto Rican Health Study; CVD, cardio-
vascular disease; Hcy, homocysteine; HHcy, hyperhomocysteinemia; LD, linkage
disequilibrium; PLP, pyridoxal phosphate; SNP, single nucleotide polymorphism.
* To whom correspondence should be addressed. E-mail: chaoqiang.lai@ars.
ã 2011 American Society for Nutrition.
Manuscript received October 27, 2010. Initial review completed November 23, 2010. Revision accepted December 09, 2010.
First published online January 26, 2011; doi:10.3945/jn.110.134353.
at ZheJiang University on May 31, 2011
Supplemental Material can be found at:
5-methyltetrahydrofolate, an important enzymatic process in
folate metabolism and in remethylation of Hcy into methionine.
In humans, 2 putative functional variants at MTHFR, 677C.
T and 1298A.C, are known to be associated with HHcy
(15,16). The homozygous MTHFR 677TT genotype results in a
thermolabile enzyme with reduced activity and consequentially
decreased concentration of plasma folate and increased plasma
Hcy concentration (17,18). Similarly, 1298C also yields de-
creased MTHFR activity (19). MTHFR 1298CC genotypes are
associated with increased risk of hypertension and higher Hcy in
essential hypertensive participants (15). Although the determi-
nants of Hcy and the relationship between MTHFR variants and
plasma Hcy have been most extensively evaluated with B
vitamins and folate, the interaction of fatty acids with MTHFR
polymorphisms on plasma Hcy concentration remains inadequately
described. Considering the relationship between (n-3) PUFA and the
criticalenzymes involved in Hcy metabolism,thegoalof the present
study was to test the hypothesis that fatty acid intake modulates the
effects of MTHFR variants on Hcy metabolism.
The population of Puerto Rican adults living in the Boston,
Massachusetts metropolitan area has a disproportionate health
burden, including high prevalence of hypertension and CVD (20).
Thus, we examined the association between MTHFR variants and
hypertension and CVD and assessed the interaction between dietary
fatty acids and MTHFR variants on plasma Hcy in this population.
Study design and participants. The current study was conducted in a
nested fashion within the ongoing Boston Puerto Rican Health Study
(BPRHS), described in detail elsewhere (21). Briefly, areas of high Puerto
Rican density in the Boston metropolitan area were identified from the
year 2000 census and 1 Puerto Rican adult from households with at least
1 Puerto Rican between 45 and 75 y of age was randomly selected for
participation. Interviews were conducted in the home and, in addition to a
host of health-related and anthropometric data, detailed data were
collected on dietary intake using a questionnaire previously adapted from
the NCI/Block FFQ and validated for this population (22). Fasting blood
samples were collected in the volunteer’s home the morning following the
health interviews. Approval for the Boston PuertoRican Health Studywas
obtained from the Institutional Review Board of the New England
Medical Center and Tufts University Health Sciences and the current
de-identified samples and data (NIH exemption category 4).
Genetic analysis. DNAwas isolated from blood samples using QIAamp
DNA Blood Mini kits according to the manufacturer’s instructions
(QIAGEN). MTHFR 1298A.C (rs1801131) and MTHFR 677C.T
(rs1801133) were genotyped by using TaqMan SNP genotyping kits with
Measurement of anthropometric and plasma biochemical param-
eters. Anthropometric variables, including height and weight, were
measured by standard techniques. BMI was calculated as weight (kg)/
height (m)2. Blood samples were collected by venipuncture from all
participants while they were fasting. Plasma total Hcy was measured
using HPLC with fluorescence detection as previously described (24).
Plasma pyridoxal phosphate (PLP) was determined using the radio-
enzymatic method of Camp et al. (25). Plasma folate and vitamin B-12
were measured using Immulite Chemiluminescent kits according to the
manufacturer’s instructions (Diagnostic Products /Siemens). Hyperten-
sion was identified as 1 of the following: 1) a positive response to the
question “Have you ever been told by a physician that you had high blood
pressure/hypertension?”; 2) reported use of blood pressure medication; or
3) high systolic ($140 mmHg) or diastolic ($90 mmHg) blood pressure.
CVD was defined as a positive response to the question “Have you ever
beentoldbya physician thatyouhad heart disease”ortosimilar questions
on heart attack or stroke or reported use of CVD medication. Smokers
or drinkers were defined as a positive response to the question “Do
you currently smoke/drink?” Thus, past smokers or drinkers were not
considered as a smoker or drinker. Using the American Diabetes Asso-
ciation criteria (26), participants were classified as having type 2 diabetes
when the fasting plasma glucose concentration was $126 mg/dL
($7.0 mmol/L) or use of insulin or diabetes medication was reported.
Physical activity was estimated as a physical activity score based on the
Paffenbarger questionnaire (27).
Dietary assessment. Dietary intake was assessed using a FFQ that was
designed for and tested in this population (17). Dietary data were linked
to the Minnesota Nutrient Data system (1999, version 25) for nutrient
analysis. Fatty acid intakes were expressed as percentages of total energy
intake and were included in analyses as both continuous and categorical
variables. To construct categorical variables, intakes were classified into
2 groups according to the median intake of the population.
Population admixture. Population admixture was calculated using
STRUCTURE 2.2 based on 100 single nucleotide polymorphisms (SNP)
selected as ancestry informative markers specifically for Puerto Rican
populations (20,28). Using the estimated admixture of each participant,
Statistical analyses. Data analyses were performed using SPSS version
12 (SPSS) or SAS 9.1. All continuous dependent variables that were not
normally distributed were Box-Cox transformed (29) prior to statistical
analysis. Gender differences in demographic, anthropometric, and
biochemical characteristics were examined using a t test. Correlations
between dietary fatty acid compositions and plasma Hcy were estimated
as a Pearson correlation coefficient after adjustment for potential
confounding factors and exclusion of outliers for (n-3) PUFA (.1.35%
energy) and (n-6) PUFA intake (.13.5% energy) using the simplest
statistical outlier detection techniques (informal box plots), as described
by Kentala et al. (30). Men and women were first examined separately
for any gender effect. To ensure adequate statistical power, men and
women were analyzed together when there was no gender-specific
influence on phenotypes. Chi-square tests were conducted to examine
whether genotype frequencies of the selected SNP were in Hardy-
Weinberg equilibrium. The relationships among MTHFR genotypes,
dietary intakes, and anthropometric measures were assessed using linear
regression models. The interactions between dietary fatty acid intakes
and genotypes were tested in a multivariate interaction model while
controlling for potential confounders, including age, sex, population
admixture, diabetes status, tobacco and alcohol use, dietary energy, and
plasma folate, vitamin B-12, and PLP concentrations. The population
medians for total SFA, MUFA, PUFA, and (n-3) and (n-6) PUFA intakes
were used as cutoffs to dichotomize these variables. Differences between
groups were considered significant at P # 0.05.
Clinical characteristics of populations and genetic variants
at MTHFR. Information about demographic, biochemical, die-
tary intake, and genotypic data are provided in Table 1. Men had
significantly higher plasma Hcy and lower plasma folate than
women. No gender differences were observed in dietary fatty
acids or plasma PLP or vitamin B-12. Genotype frequencies did
not deviate from Hardy-Weinberg equilibrium expectation. Allele
frequenciesof the minor alleles of MTHFR
(rs1801131) and MTHFR 677C.T (rs1801133) were 0.358
i.e. not in linkage disequilibrium (r2= 0.002; P = 0.95).
Correlations between dietary fatty acid compositions and
plasma Hcy. Plasma Hcy concentration was negatively corre-
lated with (n-3) PUFA expressed as total energy intake (r =
20.12; P = 0.022), and with the ratio of (n-3):(n-6) PUFA
Genotype-fatty acid interactions on homocysteine 655
at ZheJiang University on May 31, 2011
(r = 20.12; P = 0.009), after adjustment for potential con-
founding factors (Supplemental Table 1). However, plasma Hcy
was not correlated with the intakes of other fatty acids.
Association between MTHFR genotype and hypertension
and CVD. MTHFR 677C.T showed a significant association
with hypertension (OR = 1.60 for TT vs. CC, P = 0.009; OR =
1.60 for TT+CT vs. CC, P = 0.002, respectively) (Table 2).
Participants homozygous for the minor allele (TT) or carriers for
T (TT+CT) had a 60% higher likelihood of hypertension than
did homozygotes (CC), but this variant was not associated with
CVD. The second variant, 1298A.C, was also associated with
hypertension (OR = 1.72 for CC vs. AA; P = 0.024). In addition,
1298A.C variants displayed a significant association with CVD
(OR = 3.32 for CC vs AA; P = 0.030). Minor allele (1298C)
homozygotes were more than 3-fold as likely to have CVD than
homozygotes for the major allele (AA).
Association between 2 MTHFR variants and plasma Hcy.
MTHFR 1298A.C was significantly associated with plasma
Hcy (Table 3). Plasma Hcy concentrations were higher in
participants homozygous for the 1298C risk allele compared
with carriers of the 1298A allele (P = 0.011). In contrast,
participants homozygous for 1298C had significantly lower
plasma folate compared with carriers of 1298A. MTHFR
677C.T was marginally associated with plasma Hcy under a
recessive model (i.e. CC+CT vs. TT). Homozygotes for the risk
allele of 677T had higher plasma Hcy compared with carriers of
677C (P = 0.050; Table 3). In addition, we evaluated the
combined effect of the 2 MTHFR 677C.T and 1298A.C
polymorphisms on Hcy concentration. Participants homozygous
for 677T, when carrying 1298C, had higher plasma Hcy
concentrations than all other genotypes (Fig. 1).
Interaction of MTHFR variants with dietary fat intakes on
plasma Hcy. We observed an interaction between total PUFA
consumption and MTHFR 1298A.C on plasma Hcy (P =
0.003) (Table4). PlasmaHcy concentrations in participants who
carried the 1298C allele were higher than those of noncarriers
(P = 0.039) when they had high daily PUFA intakes (.7.8%
energy) but were not significantly (P = 0.27) different when they
had low daily PUFA intakes (#7.8% energy).
We next examined the interaction between PUFA and
MTHFR genotype by splitting total PUFA into (n-3) and (n-6)
PUFA. (n-6) PUFA normalized to total energy intake was
dichotomized into high (.7.1% energy) and low (#7.1%
energy) daily (n-6) PUFA intakes based on the median intake of
the population. After adjusting for potential confounders
(including age, sex, tobacco smoking, alcohol drinking, popu-
lation admixture, diabetes, dietary energy, plasma folate, plasma
vitamin B-12, and plasma PLP), we observed that MTHFR
1298A.C displayed a significant interaction with dietary (n-6)
PUFA, influencing the plasma Hcy concentration (P = 0.039)
(Fig. 2). When daily intake of (n-6) PUFA was high (.7.1%
energy), carriers of the 1298C allele had higher plasma Hcy than
1298AA homozygotes (P = 0.032), whereas under a low
(#7.1% energy) daily intake of (n-6) PUFA, the 1298C carriers
did not differ in plasma Hcy concentrations from the AA
homozygotes (P = 0.21) (Table 4).
Using the same statistical models, we also found an interac-
tion between the dietary (n-3):(n-6) PUFA ratio and the MTHFR
677C.T SNP (P = 0.027). Plasma Hcy concentrations in
participants who harbored the 677T allele were higher than
Demographic, anthropometric, biochemical, and
genotype data in BPRHS participants1
Smoker, n (%)
Drinker, n (%)
Total fat, % of energy
Total SFA, % of energy
Total MUFA, % of energy
Total PUFA, % of energy
(n-3) PUFA, % of energy
(n-6) PUFA, % of energy
Plasma vitamin B-12,4pg/mL
Plasma PLP, nmol/L
Plasma Hcy, mmol/L
Hypertension, n (%)
CVD, n (%)
MTHFR 1298 A.C, n (%)
MTHFR 677 C.T, n (%)
57.6 6 7.6
29.7 6 5.1
9.2 6 3.4
2696 6 1321
31.9 6 5.4
9.7 6 2.5
11.6 6 2.1
7.9 6 1.7
0.68 6 0.17
6.9 6 1.7
17.7 6 8.7
526.6 6 276.1
61.4 6 60.3
10.7 6 6.2
57.8 6 7.2
33.1 6 6.9*
1.5 6 0.5*
2175 6 1115*
30.7 6 5.2*
9.3 6 2.2
11.2 6 2.1
7.6 6 1.8
0.67 6 0.16
7.2 6 1.6
20.1 6 9.4*
549.6 6 284.3
59.2 6 63.3
8.8 6 4.2*
1Values are mean 6 SD, or n (%).*Different from men, P,0.01.
21 kcal = 4.184 kJ.
31 ng/mL = 2.3 nmol/L.
41 pg/mL = 0.74 pmol/L.
Association between MTHFR variants and hypertension and CVD in BPRHS participants
677 C.TTT vs CC
TT vs CT
TT+CT vs CC
CC vs AA
CC vs AC
137 vs 486
137 vs 502
639 vs 486
66 vs 598
66 vs 439
0.030 1298 A .C
1n = sample size.
2P were calculated by logistic regression models and adjusted for sex, smoking, drinking, BMI, age, diabetes, population admixture, plasma
folate, vitamin B-12, and PLP.
656 Huang et al.
at ZheJiang University on May 31, 2011
those of noncarriers (P = 0.001) when the diet had a high (n-3):
(n-6) ratio (.0.09% energy), whereas carriers and noncarriers
did not differ when the (n-3):(n-6) consumption ratio was low
(#0.09% energy) (P = 0.20). We did not observe any significant
interactions on Hcy between these 2 SNP and total MUFA, total
SFA, and total fat intake (Table 4).
Analysis of combining the 2 MTHFR SNP identified a
significant interaction with dietary (n-3) PUFA intake on plasma
Hcy (P = 0.024) (Supplemental Fig. 1). Participants who carried
2 risk alleles (677TT + 1298AC or CC) and consumed high (n-3)
PUFA (.0.66% energy) had significantly lower Hcy than those
consuming low (n-3) PUFA (#0.66% energy). No other signif-
icant interaction between individual SNP and (n-3) PUFA was
HHcy has been associated with CVD and is thus considered an
important risk factor for this disease (1,2,31). The mechanism
underlying this correlation, however, is not clearly understood.
Although in the past 2 decades many studies have demonstrated
the protective effects of fatty acids on CVD (32–34), correlations
between plasma/platelet phospholipid fatty acids and Hcy in
humans (12) and rats (14) have been reported. These studies
suggested that fatty acids are central to Hcy metabolism (12,13).
Increased consumption of dietary (n-3) PUFA increases the
concentration of (n-3) PUFA in plasma phospholipids. This is
associated with a protective effect on CVD and lowers plasma
Hcy concentrations (9). An increased ratio of (n-3):(n-6) PUFA
in platelet phospholipids was also associated with decreased
thrombotic risks, such as plasma Hcy, in middle-aged and
geriatric hyperlipemia patients in Hangzhou, China (10). In the
present study, we confirmed that plasma Hcy concentrations
were negatively correlated with dietary (n-3) PUFA and with the
ratio of (n-3):(n-6) PUFA. These correlations, however, remain
weak even after removal of the outliers, a result perhaps of the
confounding factors and the low (n-3) PUFA intake (,0.7%
energy) in this population.
A possible mechanism underlying the correlation between
PUFA and plasma Hcy concentration is taken from the obser-
vation that fatty acids modulate expression of a gene encoding
an enzyme(s) involved in the metabolism of plasma Hcy (9). In
animal studies, expression of Mthfr, encoding an enzyme central
to Hcy metabolism, was modulated by (n-3) PUFA (14). In the
present study and others (14,17), MTHFR variants are associ-
ated with plasma Hcy concentrations. Furthermore, in this
population, we also observed that carriers of MTHFR 677T or
1289C had an increased risk of hypertension and that 1289C
was associated with an elevated risk of CVD. Importantly, we
found that dietary PUFA consumption significantly interacted
with MTHFR variants (677C.T and 1298A.C) on plasma
Hcy. Persons carrying risk allele 1298C had higher plasma Hcy
than the noncarriers (AA) only when consuming a high-PUFA
diet (.7.8% energy) (Table 4) but did not when consuming low
concentrations of PUFA (#7.8% energy). Additionally, partic-
ipants with combined genotypes of both SNP (677TTwith 1298
AC or CC) who consumed higher (n-3) PUFA tended to exhibit
low Hcy. Thus, combined with our previous results (14), this
finding strengthens support for a regulatory role by PUFA on
Hcy metabolism acting through MTHFR. Although (n-3) PUFA
regulates expression of MTHFR in cell culture (data not shown),
we observed only weak interactions between the 2 MTHFR
variants and (n-3) PUFA intake. This may be the consequence of
low (n-3) PUFA intake (0.7% energy) in this population. In the
typical Western diet, consumption of (n-6) PUFA is ~20- to 25-
fold greater than that of (n-3) PUFA (35). Persons consuming
a vegetarian diet only, with little saturated fat, have a low (n-3):
(n-6) PUFA ratio in plasma and can still develop CVD as elderly
persons (36). The predominance of (n-6) PUFA in the typical diet
results from the abundance of linoleic acid [18:2(n-6)], which is
Association between the MTHFR variants and plasma homocysteine and B vitamin group in BPRHS participants1
Hcy and B
MTHFR 1298 A.CP-value MTHFR 677 C.TP-value
AA, n = 498 AC, n = 369 CC, n = 53P2
CC, n = 404 CT, n = 424 TT, n = 117P2
9.4 6 5.5
19.6 6 0.4
527.4 6 12.2
59.7 6 3.1
9.2 6 4.2
19.3 6 0.5
557.5 6 16.8
59.0 6 2.9
10.9 6 4.8
17.3 6 1.3
539.2 6 39.9
69.0 6 11.6
9.1 6 4.0
19.7 6 9.3
537.3 6 272.5
57.6 6 56.1
9.2 6 3.7
19.4 6 9.0
532.6 6 275.5
61.8 6 71.4
9.7 6 4.1
19.1 6 10.4
605.6 6 335.4
60.5 6 51.5
1Values are mean 6 SD. Plasma Hcy data were transformed before analysis.
2P for additive model.
3P for recessive model (MTHFR 1298A.C: AA+AC vs. CC; MTHFR 677 C.T: CC+CT vs. TT).
4P for dominant model (MTHFR 1298A.C: AA vs. AC+CC; MTHFR 677 C.T: CC vs. CT+TT).
51 ng/mL = 2.3 nmol/L.
61 pg/mL = 0.74 pmol/L.
variants on plasma homocysteine. Mean plasma Hcy was estimated
and plotted based on the combined genotypes of 2 SNP, MTHFR
677C.T and 1298A.C, after adjustment for potential confounders
(age, sex, smoking, drinking, BMI, diabetes, population admixture,
plasma folate, plasma vitamin B-12,plasma PLP, and total energy).
The sample size of each genotype is given inside each bar. *Different
from all other groups, P , 0.05. Values are adjusted means 6 SEM.
The combined effect of MTHFR 677C.T and 1298A.C
Genotype-fatty acid interactions on homocysteine 657
at ZheJiang University on May 31, 2011
high in soy, corn, safflower, and sunflower oils. In contrast, there
is lower intake of the (n-3) homolog of linoleic acid, a-linolenic
acid [18:3(n-3)], which is present in leafy green vegetables and in
flaxseed and canola oils (37). The indiscriminate recommenda-
tion to substitute (n-6) PUFA for saturated fats to lower serum
cholesterol concentrations could also contribute to excessive
intake of (n-6) PUFA in the current Western diet (38). High (n-6)
PUFA, or a low ratio of (n-3):(n-6) PUFA intake, may increase
plasma Hcy concentrations in carriers of the 1298C or 677T
allele, and, importantly, this may contribute to increased risk for
hypertension and CVD in these participants.
MTHFR 677C.T has been shown to be functional in that
the 677T allele shows reduced MTHFR enzyme activity (39).
This is also the most common genetic variant associated with
HHcy (40,41). In this population, however, we observed only a
weak association between 677C.T and Hcy concentration and
an association with CVD that did not reach significance (P =
0.86). This may be due to the high plasma folate concentrations
in this population, which consumes a diet high in rice fortified
with folic acid. The MTHFR 1298C allele decreased MTHFR
activity and increased plasma Hcy (15,39,42). We have con-
firmed this association in CVD patients, observing that MTHFR
1298C increased the risk of hypertension and CVD. Inconsistent
association between MTHFR variants and CVD, however, was
observed in other populations (43). The discrepancy of such
observations could result from differences in LD patterns at the
MTHFR locus and dissimilar dietary structure in diverse
populations. Such factors may affect the interaction between
MTHFR genotype and dietary PUFA intake on Hcy. The LD
between 677C.T and 1298A.C is strong (r2= 0.19; D’=0.91)
in the HapMap European population (http://hapmap.ncbi.nlm.
nih.gov/). In marked contrast, this Puerto Rican population
shows that these 2 variants are genetically independent. In
addition, substantial evidence establishes that this population is
genetically different from European populations. For example,
using 100 ancestry informative markers, we have estimated the
ancestry of this population on average to be 57.2% European,
27.4% African, and 15.4% Native American (16). This could
be 1 important factor that contributes to the inconsistencies
between populations. Other population characteristics, such as
dietary or nondietary environmental factors, and a small sample
size are all likely to also contribute. Indeed, the observation of a
strong interaction between the 2 variants analyzed here and
PUFA intake could be another important factor contributing to
the discrepancy between different study populations.
In summary, dietary fatty acid intake modulates the effect of
MTHFR genotypes on plasma Hcy in Boston Puerto Ricans.
MTHFR 677T increases the risk of hypertension and MTHFR
1298C increases the risk of both hypertension and CVD in
Boston Puerto Rican adults.
C-Q.L., D.L., and T.H. designed research; T.H., Y-C.L., and J.
W.C. conducted research; T.H. and C-Q.L. analyzed data; T.H.,
C.E.S., L.D.P., D.L., and C-Q.L. wrote the paper; and T.H. and
C-Q.L. had primary responsibility for final content. All authors
read and approved the final manuscript.
Interaction between MTHFR variants and dietary fatty acids on plasma Hcy in BPRHS participants
MTHFR 1298 A.C
MTHFR 677 C.T
P-trendP-interactionAA (n) AC+CC (n) CC (n) CT+TT (n)
9.5 6 0.322(26)
9.0 6 0.32 (27)
9.5 6 0.32 (25)
8.8 6 0.32 (24)
9.6 6 0.32 (26)
8.9 6 0.32 (25)
9.3 6 0.22 (24)
9.1 6 0.32 (27)
9.5 6 0.32 (22)
9.4 6 0.32 (29)
9.2 6 0.32 (26)
9.2 6 0.32 (25)
9.2 6 0.312(92)
9.6 6 0.31 (95)
9.2 6 0.21 (94)
9.2 6 0.21 (90)
9.5 6 0.21 (96)
9.0 6 0.31 (94)
9.1 6 0.31 (95)
9.6 6 0.21 (92)
9.4 6 0.32 (09)
9.3 6 0.31 (83)
9.8 6 0.21 (84)
8.8 6 0.32 (03)
9.1 6 0.312(92)
9.1 6 0.31 (77)
9.0 6 0.31 (87)
9.4 6 0.31 (80)
9.3 6 0.21 (87)
8.9 6 0.31 (82)
8.9 6 0.31 (86)
9.3 6 0.31 (80)
9.3 6 0.31 (95)
8.8 6 0.21 (74)
9.6 6 0.31 (86)
8.5 6 0.31 (83)
9.4 6 0.222(36)
9.2 6 0.32 (53)
9.5 6 0.32 (38)
9.1 6 0.32 (50)
9.5 6 0.32 (39)
9.2 6 0.32 (49)
9.4 6 0.32 (39)
9.4 6 0.32 (50)
9.3 6 0.32 (44)
9.3 6 0.32 (44)
9.4 6 0.32 (30)
9.4 6 0.32 (58)
Total MUFA 0.380.72
Total SFA0.71 0.68
(n-6) PUFA 0.0050.23
1Dichotomized values for fatty acids were adjusted for the total energy.
2Data in this column are mean 6 SEM. Plasma Hcy data were transformed before analysis.
3Data in this column are adjusted for age, sex, BMI, smoking, drinking, population admixture, diabetes, dietary energy, plasma folate, plasma vitamin B-12, and plasma PLP.
(n-6) PUFA intake on plasma homocysteine. MTHFR: Predicted values
were calculated from regression models containing (n-6) PUFA intake,
MTHFR 1298A.C genotype, their interaction terms, and potential
confounders (age, sex, smoking, drinking, BMI, diabetes, population
admixture, plasma folate, plasma vitamin B-12, plasma PLP, and total
energy). The sample sizes of each genotype of MTHFR 1298A.C are
as follows: n = 98 (AA), n = 22 (AC+CC). The P-interaction between
MTHFR 1298A.C genotype and (n-6) PUFA intake (percent energy) as
a continuous variable is 0.039. Values are adjusted means 6 SEM.
Interaction between MTHFR 1298A.C genotype and
658Huang et al.
at ZheJiang University on May 31, 2011
1. McCully KS. Vascular pathology of homocysteinemia: implications for
the pathogenesis of arteriosclerosis. Am J Pathol. 1969;56:111–28.
Huang T, Yuan G, Zhang Z, Zou Z, Li D. Cardiovascular pathogenesis
in hyperhomocysteinemia. Asia Pac J Clin Nutr. 2008;17:8–16.
Refsum H, Nurk E, Smith AD, Ueland PM, Gjesdal CG, Bjelland I,
Tverdal A, Tell GS, Nygard O, et al. The Hordaland Homocysteine
Study: a community-based study of homocysteine, its determinants, and
associations with disease. J Nutr. 2006;136:S1731–40.
Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B,
Graham I. Hyperhomocysteinemia: an independent risk factor for
vascular disease. N Engl J Med. 1991;324:1149–55.
Harker LA, Slichter SJ, Scott CR, Ross R. Homocystinemia. Vascular
injury and arterial thrombosis. N Engl J Med. 1974;291:537–43.
Brude IR, Finstad HS, Seljeflot I, Drevon CA, Solvoll K, Sandstad B,
Hjermann I, Arnesen H, Nenseter MS. Plasma homocysteine concen-
tration related to diet, endothelial function and mononuclear cell gene
expression among male hyperlipidaemic smokers. Eur J Clin Invest.
Yeh YY, Yeh SM. Homocysteine-lowering action is another potential
cardiovascular protective factor of aged garlic extract. J Nutr.
McMahon JA, Skeaff CM, Williams SM, Green TJ. Lowering homo-
cysteine with B vitamins has no effect on blood pressure in older adults.
J Nutr. 2007;137:1183–7.
Li D, Mann NJ, Sinclair AJ. A significant inverse relationship between
concentrations of plasma homocysteine and phospholipid docosahex-
aenoic acid in healthy male subjects. Lipids. 2006;41:85–9.
10. Li D, Yu XM, Xie HB, Zhang YH, Wang Q, Zhou XQ, Yu P, Wang LJ.
Platelet phospholipid n-3 PUFA negatively associated with plasma
homocysteine in middle-aged and geriatric hyperlipaemia patients.
Prostaglandins Leukot Essent Fatty Acids. 2007;76:293–7.
11. Piolot A, Blache D, Boulet L, Fortin LJ, Dubreuil D, Marcoux C, Davignon
J, Lussier-Cacan S. Effect of fish oil on LDL oxidation and plasma
homocysteine concentrations in health. J Lab Clin Med. 2003;141:41–9.
12. Zeman M, Zak A, Vecka M, Tvrzicka E, Pisarikova A, Stankova B. N-3
fatty acid supplementation decreases plasma homocysteine in diabetic
dyslipidemia treated with statin-fibrate combination. J Nutr Biochem.
13. Pooya S, Jalali MD, Jazayery AD, Saedisomeolia A, Eshraghian MR,
Toorang F. The efficacy of omega-3 fatty acid supplementation on
plasma homocysteine and malondialdehyde levels of type 2 diabetic
patients. Nutr Metab Cardiovasc Dis. 2010;20:326–31.
14. Huang T, Wahlqvist ML, Li D. Docosahexaenoic acid decreases plasma
homocysteine via regulating enzyme activity and mRNA expression
involved in methionine metabolism. Nutrition. 2010;26:112–9.
15. Markan S, Sachdeva M, Sehrawat BS, Kumari S, Jain S, Khullar M.
MTHFR 677 CT/MTHFR 1298 CC genotypes are associated with
increased risk of hypertension in Indians. Mol Cell Biochem. 2007;
16. Lim U, Peng K, Shane B, Stover PJ, Litonjua AA, Weiss ST, Gaziano JM,
Strawderman RL, Raiszadeh F, et al. Polymorphisms in cytoplasmic serine
hydroxymethyltransferase and methylenetetrahydrofolate reductase affect
the risk of cardiovascular disease in men. J Nutr. 2005;135:1989–94.
17. Zetterberg H, Zafiropoulos A, Spandidos DA, Rymo L, Blennow K.
Gene-gene interaction between fetal MTHFR 677C.T and trans-
cobalamin 776C.G polymorphisms in human spontaneous abortion.
Hum Reprod. 2003;18:1948–50.
18. Fodinger M, Buchmayer H, Heinz G, Papagiannopoulos M, Kletzmayr
J, Perschl A, Vychytil A, Horl WH, Sunder-Plassmann G. Association of
two MTHFR polymorphisms with total homocysteine plasma levels in
dialysis patients. Am J Kidney Dis. 2001;38:77–84.
19. van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocke MC, Zielhuis GA,
Goorhuis-Brouwer SM, van der Biezen JJ, Kuijpers-Jagtman AM,
Steegers-Theunissen RP. Does the interaction between maternal folate
intake and the methylenetetrahydrofolate reductase polymorphisms
affect the risk of cleft lip with or without cleft palate? Am J Epidemiol.
20. Lai CQ, Tucker KL, Choudhry S, Parnell LD, Mattei J, Garcia-Bailo B,
Beckman K, Burchard EG, Ordovas JM. Population admixture associ-
ated with disease prevalence in the Boston Puerto Rican health study.
Hum Genet. 2009;125:199–209.
21. Tucker KL. Stress and nutrition in relation to excess development of
chronic disease in Puerto Rican adults living in the Northeastern USA.
J Med Invest. 2005;52 Suppl:252–8.
22. Tucker KL, Bianchi LA, Maras J, Bermudez OI. Adaptation of a food
frequency questionnaire to assess diets of Puerto Rican and non-
Hispanic adults. Am J Epidemiol. 1998;148:507–18.
23. Lai CQ, Tucker KL, Parnell LD, Adiconis X, Garcia-Bailo B, Griffith J,
Meydani M, Ordovas JM. PPARGC1A variation associated with DNA
damage, diabetes, and cardiovascular diseases: the Boston Puerto Rican
Health Study. Diabetes. 2008;57:809–16.
24. Araki A, Sako Y. Determination of free and total homocysteine in
human plasma by high-performance liquid chromatography with
fluorescence detection. J Chromatogr A. 1987;422:43–52.
25. Camp VM, Chipponi J, Faraj BA. Radioenzymatic assay for direct
measurement of plasma pyridoxal 5’- phosphate. Clin Chem. 1983;29:
26. American Diabetes Association. Standards of medical care in diabetes.
Diabetes Care. 2007;28 Suppl:4–36.
27. Lee IM, Paffenbarger RS Jr. Physical activity and stroke incidence: the
Harvard Alumni Health Study. Stroke. 1998;29:2049–54.
28. Falush D, Stephens M, Pritchard JK. Inference of population structure
using multilocus genotype data: linked loci and correlated allele
frequencies. Genetics. 2003;164:1567–87.
29. Box GEP, Cox DR. An analysis of transformations. J Roy Stat Soc.
1964; Series B:41.
30. Kentala E, Viikki K, Laurikkala J, Juhola M. Impact and management
of confounding values and outliers in a neurotologic expert system. Ann
N Y Acad Sci. 2001;942:472.
31. Dangour AD, Breeze E, Clarke R, Shetty PS, Uauy R, Fletcher AE.
Plasma homocysteine, but not folate or vitamin B-12, predicts mortality
in older people in the United Kingdom. J Nutr. 2008;138:1121–8.
32. Calder PC. n-3 Fatty acids and cardiovascular disease: evidence ex-
plained and mechanisms explored. Clin Sci (Lond). 2004;107:1–11.
33. Dewailly E, Blanchet C, Lemieux S, Sauve L, Gingras S, Ayotte P, Holub
BJ. n-3 Fatty acids and cardiovascular disease risk factors among the
Inuit of Nunavik. Am J Clin Nutr. 2001;74:464–73.
34. Kinsella JE, Lokesh B, Stone RA. Dietary n-3 polyunsaturated fatty
acids and amelioration of cardiovascular disease: possible mechanisms.
Am J Clin Nutr. 1990;52:1–28.
35. Simopoulos AP. Omega-3 fatty acids in health and disease and in
growth and development. Am J Clin Nutr. 1991;54:438–63.
36. Li D, Sinclair A, Wilson A, Nakkote S, Kelly F, Abedin L, Mann N,
Turner A. Effect of dietary alpha-linolenic acid on thrombotic risk
factors in vegetarian men. Am J Clin Nutr. 1999;69:872–82.
37. James MJ, Gibson RA, Cleland LG. Dietary polyunsaturated fatty
acids and inflammatory mediator production. Am J Clin Nutr. 2000;71:
38. Simopoulos AP. Essential fatty acids in health and chronic disease. Am J
Clin Nutr. 1999;70:S560–9.
39. van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes
TK, van den Heuvel LP, Blom HJ. A second common mutation in the
methylenetetrahydrofolate reductase gene: an additional risk factor for
neural-tube defects? Am J Hum Genet. 1998;62:1044–51.
40. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG,
Boers GJ, den Heijer M, Kluijtmans LA, et al. A candidate genetic risk
factor for vascular disease: a common mutation in methylenetetrahy-
drofolate reductase. Nat Genet. 1995;10:111–3.
41. Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic
polymorphism in methylenetetrahydrofolate reductase (MTHFR)
associated with decreased enzyme activity. Mol Genet Metab. 1998;
42. Kumar J, Das SK, Sharma P, Karthikeyan G, Ramakrishnan L, Sengupta
S. Homocysteine levels are associated with MTHFR A1298C polymor-
phism in Indian population. J Hum Genet. 2005;50:655–63.
43. Ye H, Yan JT, Shao JM, Zhang F, Hong ML, Wang DW. [A case-control
study on the relationship between stroke and plasma homocysteine level
and the mutation of MTHFR gene.] Zhonghua Liu Xing Bing Xue Za
Genotype-fatty acid interactions on homocysteine659
at ZheJiang University on May 31, 2011