HUMAN MUTATION 28(9), 856^865, 2007

RESEARCH ARTICLE

Large-Scale Population-Based Metabolic

Phenotyping of Thirteen Genetic Polymorphisms
Related to One-Carbon Metabolism
Åse Fredriksen,1 Klaus Meyer,1 Per Magne Ueland,1 Stein Emil Vollset,1 Tom Grotmol,2
and Jørn Schneede1
1
LOCUS for Homocysteine and Related Vitamins, University of Bergen, Bergen, Norway; 2Cancer Registry of Norway, Oslo, Norway

Communicated by Klaus Meyer

Several polymorphisms of genes involved in one-carbon metabolism have been identified. The reported
metabolic phenotypes are often based on small studies providing inconsistent results. This large-scale study of
10,601 population-based samples was carried out to investigate the association between a panel of biochemical
parameters and genetics variants related to one-carbon metabolism. Concentrations of total homocysteine
(tHcy), folate, vitamin B12 (cobalamin), methylmalonic acid (MMA), vitamin B2 (riboflavin), vitamin B6
(PLP), choline, betaine, dimethylglycine (DMG), cystathionine, cysteine, methionine, and creatinine were
determined in serum/plasma. All subjects were genotyped for 13 common polymorphisms: methylenetetrahy-
drofolate reductase (MTHFR) c.665C4T (known as 677C4T; p.Ala222Val) and c.1286A4C (known as
1298A4C; p.Glu429Ala); methionine synthase (MTR) c.2756A4G (p.Asp919Gly); methionine synthase
reductase (MTRR) c.66A4G (p.Ile22Met); methylenetetrahydrofolate dehydrogenase (MTHFD1) c.1958G4A
(p.Arg653Gln); betaine homocysteine methyltransferase (BHMT) c.716G4A (known as 742G4A;
p.Arg239Gln); cystathionine b-synthase (CBS) c.844_845ins68 and c.699C4T (p.Tyr233Tyr); transcobalamin-II
(TCN2) c.67A4G (p.Ile23Val) and c.776C4G (p.Pro259Arg); reduced folate carrier-1 (SLC19A1) c.80G4A
(p.Arg27His); and paraoxonase-1 (PON1) c.163T4A (p.Leu55Met) and c.575A4G (p.Gln192Arg). The
metabolic profile in terms of the measured vitamins and metabolites were investigated for these 13 polymorphisms.
We confirmed the strong associations of MTHFR c.665C4T with tHcy and folate, but also observed significant
(Po0.01) changes in metabolite concentrations according to other gene polymorphisms. These include MTHFR
c.1286A4C (associations with tHcy, folate and betaine), MTR c.2756A4G (tHcy), BHMT c.716G4A
(DMG), CBS c.844_845ins68 (tHcy, betaine), CBS c.699C4T (tHcy, betaine, cystathionine) and TCN2
c.776C4G (MMA). No associations were observed for the other polymorphisms investigated. Hum Mutat
28(9), 856–865, 2007. r 2007 Wiley-Liss, Inc.

KEY WORDS: MTHFR; homocysteine; one-carbon metabolism; metabolic phenotyping; NORCCAP

INTRODUCTION consistently demonstrated that the MTHFR c.665T-allele is

associated with elevated tHcy under conditions of impaired folate
Homocysteine and folate are key components of one-carbon
status, and the MTHFR c.665C4T polymorphism has been linked
metabolism. Elevated concentrations of total homocysteine (tHcy)
to most pathologies associated with hyperhomocysteinemia
in plasma or inadequate folate status have been associated with
[Ueland et al., 2001]. The MTHFR c.665C4T polymorphism is
many pathological states, including cardiovascular disease, cogni-
in linkage disequilibrium with a second nearby SNP; i.e., MTHFR
tive dysfunction, adverse pregnancy outcomes, osteoporosis, and
c.1286A4C (p.Glu429Ala; known as 1298A4C). The effects of
cancer. Plasma tHcy serves as a marker of impaired folate and
cobalamin status [Fowler, 2005], but is also influenced by a variety
of lifestyle and nutritional factors. The numerous disease The Supplementary Material referred to in this article can be
associations have also stimulated the investigation of polymorphic accessed at http://www.interscience.wiley.com/jpages/1059 -7794/
variants of genes involved in one-carbon metabolism, their suppmat.
metabolic effects, and their relation to risk of chronic diseases Received 16 August 2006; accepted revised manuscript 14 February
[Gellekink et al., 2005; Molloy, 2004]. 2005.
Correspondence to: A
se Fredriksen, Section for Pharmacology,
The flavoenzyme, methylenetetrahydrofolate reductase
Institute of Medicine, University of Bergen, Armauer Hansens hus,
(MTHFR; MIM] 607093), has a major impact on folate 5021 Bergen, Norway. E-mail: ase.fredriksen@farm.uib.no
distribution by reducing 5,10-methylenetetrahydrofolate to 5- Grant sponsors: Norwegian Cancer Society; Foundation to Promote
methyltetrahydrofolate, which is the primary methyl donor for Research into Functional Vitamin B12-De¢ciency.
remethylation of homocysteine to methionine. A SNP of DOI 10.1002/humu.20522
MTHFR, c.665C4T (p.Ala222Val; known as 677C4T), was Published online 13 April 2007 in Wiley InterScience (www.inter
described by Frosst et al. [1995]. Numerous studies have since science.wiley.com).

r 2007 WILEY-LISS, INC.

 HUMAN MUTATION 28(9), 856^865, 2007 857

this SNP on tHcy and folate in blood are weaker [Kim, 2005], and
could not always be detected.
Numerous other polymorphisms of genes involved in one-carbon
metabolism have been identified. These include: methionine synthase
(MTR; MIM] 156570) c.2756A4G (p.Asp919Gly) [Leclerc et al.,
1996]; methionine synthase reductase (MTRR; MIM] 602568)
c.66A4G (p.Ile22Met) [Wilson et al., 1999]; methylenetetrahydro-
folate dehydrogenase (MTHFD1; MIM] 172460) c.1958G4A
(p.Arg653Gln) [Hol et al., 1998]; betaine homocysteine methyl-
transferase (BHMT; MIM] 602888) c.716A4G (p.Arg239Gln,
known as 742G4A) [Heil et al., 2000]; cystathionine b-synthase
(CBS; MIM] 236200) c.844_845ins68 [Sebastio et al., 1995] and
c.699C4T (p.Tyr233Tyr) [Kraus et al., 1998]; transcobalamin-II
(TCN2; MIM] 275350) c.67A4G (p.Ile23Val) [Li et al., 1995] and
c.776C4G (p.Pro259Arg) [Li et al., 1993]; reduced folate carrier-1
(SLC19A1; MIM] 600424) c.80G4A (p.Arg27His) [Chango et al.,
2000]; and paraoxonase-1 (PON1; MIM] 168820) 163T4A
(p.Leu55Met) [Adkins et al., 1993] and c.575A4G (p.Gln192Arg)
[Humbert et al., 1993]. The enzymatic reactions involved and their
role in one-carbon metabolism are summarized in Fig. 1. The
metabolic effects of these polymorphisms, often in terms of
concentrations of folate and cobalamin, and their metabolic markers,
tHcy and methylmalonic acid (MMA), have been assessed, and
variable and inconsistent results have been obtained. This is probably
explained by insufficient statistical power to detect moderate FIGURE 1. One-carbon metabolism and related gene polymorph-
metabolic effects in studies involving a small number of subjects isms. AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosyl-
methionine; BHMT, betaine homocysteine methyltransferase;
(often less than 500 subjects). The fact that the frequencies of the Cysta, cystathionine; Cys, cysteine; CBS, cystathionine
least common variants are often less than 10% adds to these b-synthase; CH2 THF, methylenetetrahydrofolate; CH 3THF,
methodological difficulties. methyltetrahydrofolate; DMG, dimethylglycine; Hcy, homocys-
The aim of the present work was to carry out metabolic profiling teine; Hcy-tl, homocysteine thiolactone; Met, methionine;
of genetic polymorphisms related to one-carbon metabolism in a MTHFD1, trifunctional enzyme methylenetetrahydrofolate dehy-
drogenase/methenyltetrahydrofolate cyclohydrolase/formylte-
large-scale population-based study to obtain sufficient statistical trahydrofolate synthetase; MTHFR, methylenetetrahydrofolate
power to detect moderate associations in small subgroups. We reductase; MTR, methionine synthase; MTRR, methionine
measured a panel of 13 metabolites and 13 polymorphisms of nine synthase reductase; PON1, paraoxonase-1; SLC19A1, reduced
genes in a large population of 10,601 subjects. folate carrier-1;TCN2, transcobalamin-II;THF, tetrahydrofolate.

MATERIALS AND METHODS

Subjects 1100 g for 10 minutes. Whole blood, the plasma and serum
fraction were stored at
201C for 6 to 12 months, and then
A total of 13,823 subjects were drawn randomly from the transferred to the research laboratory, where they were stored at
population registries in Oslo and Telemark County between 1999
801C until analysis.
and 2001 for participation in the Norwegian Colorectal Cancer
Prevention (NORCCAP) study [Bretthauer et al., 2002]. Vitamin and Metabolite Determination
Participants were of both genders, homogeneous with respect to
ethnicity, and their age ranged from 50 to 64 years. Exclusion Folate and vitamin B12 (cobalamin) were determined in serum,
criteria were previous open colorectal surgery, need of long-lasting and tHcy, MMA, vitamin B2 (riboflavin), vitamin B6 (pyridoxal 50 -
attention and nursing services, on-going cytotoxic or radiotherapy phosphate, PLP), choline, betaine, dimethylglycine (DMG),
for malignant disease, severe chronic cardiopulmonary disease, cystathionine, cysteine, methionine, and creatinine were determined
lifelong anticoagulant therapy, a coronary episode requiring in plasma, using published methods [Holm et al., 2003; Midttun
hospital admission during the last 3 months, cerebrovascular et al., 2005; Molloy and Scott, 1997; Windelberg et al., 2005].
accident during the last 3 months, and resident abroad or postal
return of unopened mail. Using these criteria, 535 individuals were DNA Extraction and Genotyping
excluded and for 10,601 eligible subjects, we obtained blood DNA was extracted from the 25 mL cell pellet obtained after
samples where metabolites and genotypes were determined for the removal of the plasma fraction, using a Geno M-96 workstation
present work. The study was approved by the Regional Ethics (Geno Vision, Qiagen, Oslo, Norway). MTHFR c.665C4T
Committee and The Data Inspectorate. Written informed consent genotyping was performed by real-time PCR with 50 exonuclease
was obtained from all participants. (Taqmans, Applied Biosystems, Foster City, CA) probes [Ulvik
and Ueland, 2001]. The other polymorphisms were determined by
Collection of Blood
a matrix-assisted laser-desorption/ionization-time-of-flight (MAL-
Blood samples were collected from nonfasting subjects into DI-TOF) mass spectrometry (MS) assay as described [Meyer et al.,
EDTA Vacutainer tubes and tubes without additive. EDTA 2004]. The amount of DNA was variable and small in this sample
samples were immediately put on ice, whereas serum was allowed collection, and in order to increase accuracy of the genotyping,
to clot for 1 hr at room temperature. Samples were centrifuged at samples were analyzed twice by the MALDI-TOF MS assay and

Human Mutation DOI 10.1002/humu

858 HUMAN MUTATION 28(9), 856^865, 2007

TABLE 1. Characteristics of the Study Population

Totala Mena Womena Pb

Age (years) 55.00 (51.00^63.00) 55.00 (51.00^63.00) 55.00 (51.00^63.00) 0.189

tHcy (mM) 10.23 (6.78^16.43) 10.87 (7.51^17.08) 9.57 (6.40^15.70) o0.001
Folate (nM) 13.70 (6.59^39.44) 12.84 (6.39^34.86) 14.75 (6.83^43.34) o0.001
Cobalamin (pM) 307.20 (171.95^535.90) 304.60 (171.70^518.40) 309.70 (172.70^555.62) 0.001
MMA (mM) 0.169 (0.110^0.288) 0.169 (0.109^0.289) 0.170 (0.111^0.287) 0.331
Ribo£avin (nM) 10.50 (4.11^55.92) 9.92 (4.00^46.31) 11.00 (4.23^65.81) o0.001
PLP (nM) 48.00 (18.70^152.37) 48.70 (19.62^134.00) 47.20 (17.60^170.05) 0.082
Choline (mM) 8.62 (5.79^12.90) 9.04 (6.12^13.45) 8.26 (5.56^12.10) o0.001
Betaine (mM) 35.40 (19.12^58.30) 39.66 (25.44^63.04) 31.00 (16.80^52.00) o0.001
DMG (mM) 3.73 (2.44^5.93) 3.95 (2.70^6.27) 3.50 (2.30^5.59) o0.001
Cystathionine (mM) 0.190 (0.091^0.525) 0.202 (0.099^0.579) 0.177 (0.086^0.471) o0.001
Cysteine (mM) 283.70 (237.13^338.17) 285.79 (240.08^338.63) 281.49 (234.47^337.58) o0.001
Methionine (mM) 21.41 (15.26^33.31) 22.37 (16.03^34.34) 20.36 (14.82^32.33) o0.001
Creatinine (mM) 68.94 (50.60^92.17) 75.55 (58.26^96.78) 62.60 (48.00^81.30) o0.001
a
Values are median (¢fth^95th percentiles) of individuals.
b
By Mann-Whitney U test.

results were compared. Conflicting genotypes were inspected years. The median (5th–95th percentiles) concentrations of tHcy,
manually, corrected or defined as undetermined. The MTHFD1 folate, cobalamin, MMA, riboflavin, PLP, choline, betaine, DMG,
c.1958G4A polymorphism was included in the assay using the sense cystathionine, cysteine, creatinine, and methionine are listed for
primer, 50 -CTGGTTTCCACAGGGCACTC-30 , and antisense pri- the total study group and separately for men and women in
mer, 50 -ACAAACCCTTCTGGGCCAAC-30 for PCR, and the Table 1. The concentrations of tHcy, betaine, choline, DMG,
primer 50 -TCCTCCATCATTGCAGACC-30 as extension probe. cystathionine, cysteine, methionine and creatinine were higher
The polymorphism numbering is based on the cDNA sequences and folate, cobalamin, and riboflavin lower in men than in women.
with GenBank accession numbers NM_005957.3 for MTHFR, MMA and PLP were not significantly different according to
NM_000254.1 for MTR, NM_002454.1 for MTRR, NM_005956.2 gender.
for MTHFD1, NM_001713.1 for BHMT, NM_000071.1 for CBS,
M60396.1 for TCN2, U19720.1 for SLC19A1, and NM_000446.3 Genotype Distributions
for PON1, with11 corresponding to the A of the ATG translation
DNA samples from all subjects were genotyped for 13
initiation codon in the reference sequence.
polymorphisms related to one-carbon metabolism. Their success
Statistical Methods rates (ratio of successfully genotyped samples to total number of
samples), genotypes, and allele frequencies are summarized in
Descriptive measures include medians with fifth and 95th Table 2. The success rates vary between 1.00 for the MTHFR
percentiles, and means. Adjusted mean values were corrected for c.665C4T and 0.85 for the SLC19A1 c.80G4A, respectively.
age, sex, creatinine, and center and were computed by analysis of Allele frequencies are compared with those in the literature
covariance. Differences between women and men were assessed [Brophy et al., 2001; Janosikova et al., 2005; Pepe et al., 1999] and
with the Mann-Whitney U test. Because of skewed distributions, are in accordance with published data.
logarithmic transformation was applied to all analyte concentra- The observed genotype frequencies are shown in Fig. 2. The
tions. Three samples with extreme MMA values (42 mM) were distributions were in HWE (0.0001oDo0.0021) for the genotypes
excluded from the analysis. A test for linear trend across genotypes of MTHFR c.665C4T, MTR c.2756A4G, and MTHFD1
(wild types, heterozygotes, homozygotes; coded 0, 1, 2) was c.1958A4G. Deviations from HWE were observed for the other
estimated by linear regression analysis with adjustment for age, polymorphisms ranging from the lowest D 5 0.0041 for TCN2
sex, creatinine, and center. P-valueso0.01 were considered c.67A4G to the highest D 5 0.0675 for SLC19A1 c.80G4A.
statistically significant. Hardy-Weinberg equilibrium (HWE) was Linkage disequilibrium was detected for all polymorphisms
measured by the chi-squared test and equilibrium was defined as located in the same gene (Table 3), and the following coefficients
P40.05. In addition, the disequilibrium coefficient D was D0 (95% CI) were calculated:
0.95 (
0.93,
0.97) for MTHFR
determined [Weir, 1996]. The linkage disequilibrium between c.665C4T and c.1286A4C,
0.89 (
0.85,
0.93) for CBS
two loci was described by the normalized disequilibrium coefficient c.844_845ins68 and c.699C4T,
0.41 (
0.35,
0.46) for TCN2
D0 [Lewontin, 1964]. The sign of the D0 coefficient indicates c.67A4G and c.776C4G, and
0.84 (
0.81,
0.87) for PON1
whether the rare alleles are associated (positive) or whether the c.163T4A and c.575A4G.
rare allele at one locus is associated with the most common allele
at the other locus (negative). SPSS 12 for Windows (SPSS, Associations Between Genotypes
Chicago, IL) was used for calculation of descriptive statistics and and Biochemical Variables
for the regression analysis. Haploview (www.broad.mit.edu/mpg/
haploview/index.php) was used for determination of the linkage The concentrations of 12 biochemical parameters according to
coefficient D0 with 95% confidence interval (CI). 13 polymorphisms were calculated as mean values for each
genotype, and the changes according to the number of the rare
allele for each polymorphism were assessed by analysis for trend
RESULTS
(Supplementary Table S1; available online at http://www.inters-
Characteristics of the Study Population
cience.wiley.com/jpages/1059–7794/suppmat). Due to linkage
The study population consisted of 10,601 subjects, 49.2% disequilibrium between the polymorphisms located in the same
males. The median (5th–95th percentiles) age was 55 (51–63) genes, the variants of MTHFR c.665C4T and c.1286A4C, CBS

Human Mutation DOI 10.1002/humu

 HUMAN MUTATION 28(9), 856^865, 2007 859

Reference sequences: MTHFR (GenBank: NM_005957.3), MTR (GenBank: NM_000254.1), MTRR (GenBank: NM_002454.1), MTHFD1 (GenBank: NM_005956.2), BHMT (GenBank: NM_001713.1), CBS (Gen-
Bank: NM_000071.1),TCN2 (GenBank: M60396.1), SLC19A1 (GenBank: U19720.1), and PON1 (GenBank: NM_000446.3) with 11 corresponding to the A of the ATG translation initiation codon in the reference
c.844_845ins68 and c.699C4T, TCN2 c.67A4G and

Janosikova et al. [2005]

Janosikova et al. [2005]
Janosikova et al. [2005]
Janosikova et al. [2005]
Janosikova et al. [2005]
Janosikova et al. [2005]

Janosikova et al. [2005]

Janosikova et al. [2005]
Janosikova et al. [2005]
Janosikova et al. [2005]
c.776C4G, and PON1 c.163T4A and c.575A4G polymorph-

Brophy et al. [2001]

Brophy et al. [2001]
isms were evaluated in the stratum of the wild-type genotype of
References

Pepe et al. [1999]

the linked polymorphism.
The strongest associations were observed for the MTHFR
c.665C4T SNP. tHcy increased (Po0.001), whereas folate
Literature

(Po0.001), betaine (Po0.001), DMG (Po0.001), and PLP

(Po0.001) decreased according to the number of T-alleles. Also
MTHFR c.1286A4C affected tHcy, folate and betaine as tHcy
increased (Po0.001), while folate (Po0.001) and betaine
of rare allele
Frequency

0.06^0.40
0.29^0.42

0.27^0.60
0.44^0.45
0.30^0.40

0.35^0.59

0.23^0.32
0.05^0.40

0.45^0.47
(Po0.008) decreased with the number of MTHFR c.1286C alleles.
0.19^0.22

0.13^0.47
0.33

0.13
The MTR c.2756A4G transition was inversely associated with
tHcy (Po0.001).
BHMTc.716G4A had a significant effect on the product of the
BHMT reaction, DMG, which decreased according to the number
of BHMT c.716A alleles (P 5 0.001).
of rare allele
Frequency

The CBS c.844_845ins68 was negatively associated with tHcy

0.60
0.28
0.34

0.28
0.09

0.44

0.28
0.33

0.33
0.45

0.42
0.12
0.19

(Po0.001) and positively associated with betaine (Po0.001).

Subjects carrying the T-allele of the silent CBS c.699C4T
polymorphism had decreased tHcy (P 5 0.002) and increased
betaine (P 5 0.003) and slightly increased cystathionine
homozygotes (%)

(P 5 0.001). These small effects from the CBS c.699C4T

3765 (38.2)
2065 (20.4)

2020 (20.0)
1292 (12.8)

1282 (12.9)
1332 (13.5)

2187 (24.1)
Number of

polymorphism motivated us to investigate the relation between

871 (8.6)

858 (8.5)
847 (8.0)

381 (3.8)

179 (1.8)
152 (1.5)
Characteristics of the Study Population

the substrate (homocysteine) and product (cystathionine) of the

CBS reaction according to CBS c.699C4T genotype. Fig. 3
demonstrates that the tHcy/cystathionine ratio declined according
to the number of CBS c.699T alleles (Po0.001).
Both TCN2 c.776C4G (Po0.001) and c.67A4G (P 5 0.011)
Current study

were positively associated with MMA (Supplementary Table S1).

heterozygotes (%)

3891 (38.5)
4299 (40.6)

3922 (39.8)
4336 (42.8)

4218 (42.8)

4009 (40.3)
3992 (39.5)
4994 (49.4)

1575 (15.6)

2000 (19.9)
3059 (30.1)

3183 (35.1)
4783 (47.4)
Number of

NULL EFFECTS
MTRR c.66A4G, MTHFD1 c.1958G4A, SLC19A1 c.80G4A,
and PON1 c.163T4A and c.575A4G were not significantly
associated with changes in any of the biochemical parameters
investigated. However MTHFD1 c.1958G4A and SLC19A1
wild-types (%)

4647 (46.8)
4506 (44.5)

4596 (46.7)
3060 (30.2)

8364 (82.9)

5356 (53.0)
3278 (32.5)
3693 (40.7)
7885 (78.3)
6707 (66.1)

5236 (51.8)
5452 (51.5)
Number of

1883 (19.1)

c.80G4A had borderline significant effects on folate. Cobalamin,

TABLE 2.

riboflavin, choline, cysteine, and methionine were not significantly

influenced by any of the investigated polymorphisms.

E¡ect Size
Success

For all significant associations, the relative changes of the mean

0.96
0.96

0.85
1.00

0.94
0.93
0.95
0.95
0.95
0.93
0.95
0.95

0.95
rate

concentrations between homozygous rare and wild types were

illustrated in Fig. 4. The strongest effects were found for the
MTHFR c.665C4T polymorphism, with 32% difference in tHcy,
Protein change

29% in folate, 11% in DMG, 10% in betaine, and 8% in PLP. For

p.Arg653Gln
p.Arg239Gln

p.Pro259Arg
p.Asp919Gly
p.Glu429Ala

p.Gln192Arg
p.Ala222Val

p.Leu55Met
p.Tyr233Tyr

p.Arg27His
p.Ile22Met

p.Ile23Val

the MTHFR c.1286A4C polymorphism, the differences were 5%

in tHcy, 9% in folate, and 3% in betaine. The effects of BHMT
^

c.716G4A, CBS c.844_845ins68, CBS c.699C4T, and TCN2

c.776C4G were in the range of 2 to 7%.
c.844_845ins68

DISCUSSION
DNA change

c.1958G4A
c.2756A4G
c.1286A4C
c.665C4T

c.699C4T

c.776C4G

c.575A4G
c.716G4A

c.163T4A
c.80G4A
c.66A4G

c.67A4G

This study demonstrates that the polymorphisms MTHFR

c.665C4T and c.1286A4C, MTR c.2756A4G, BHMT
c.716G4A, CBS c.844_845ins68 and c.699C4T, and TCN2
c.776C4G were associated with the concentrations of biochem-
ical parameters related to one-carbon metabolism in a population
of 10,601 healthy adults. The strongest associations were observed
SLC19A1
MTHFD1

sequence.
MTHFR
MTHFR

for the MTHFR c.665C4T polymorphism, which had profound

BHMT
MTRR

PON1
PON1
TCN2
TCN2
Gene

MTR

effects on homocysteine and folate status, whereas MTRR

CBS
CBS

c.66A4G, MTHFD1 c.1958G4A, SLC19A1 c.80G4A, and

Human Mutation DOI 10.1002/humu

860 HUMAN MUTATION 28(9), 856^865, 2007

FIGURE 2. Genotype frequencies.The observed genotype distributions are given as colored bars and the departure from HWE is given
as disequilibrium coe⁄cient D.

TABLE 3. Number of Genotype Combinations of Polymorphisms in the MTHFR, CBS,TCN2, and PON1 Genes

CC CT TT wt wt wt ins ins ins

MTHFR, c.665 CBS, c.844_845ins68

c.1286AA 1492 2217 783 c.699CC 3448 985 134
c.1286AC 2469 1848 16 c.699CT 3366 544 9
c.1286CC 1246 43 0 c.699TT 1298 24 0

CC CG GG AA AG GG

TCN2, c.776 PON1, c.575

c.67AA 2322 3745 1711 c.163TT 1728 2111 768
c.67AG 805 952 235 c.163TA 2326 1608 57
c.67GG 103 52 21 c.163AA 1150 107 6

PON1 c.163T4A and c.575A4G did not significantly affect the metabolite-genotypes relation may become substantial for the
investigated parameters. polymorphisms estimated with the highest error rate, in
particular SLC19A1 c.80G4A. Conceivably, decrease in folate
according to the number of A-alleles, which was of borderline
Study Design, Strength, and Weakness
significance, may actually reflect a true metabolic effect from this
The strength of this study is the large population, which allows polymorphism.
the detection of moderate associations, also in subgroups. Another
advantage is the number of parameters used for metabolite
Metabolic Phenotyping
profiling, which includes 13 markers and metabolites related to
one-carbon metabolism. This contrasts to most published studies This work confirmed the strong effect of MTHFR c.665C4T on
[Molloy, 2004], which measure tHcy and folate. Finally, the study homocysteine and folate status that has been consistently
population is homogeneous with respect to age and ethnicity, and demonstrated in numerous studies [Jacques et al., 1996; Ma
was recruited from an area with no mandatory folate fortification. et al., 1996; Rozen, 2000]. In addition, we observed a 10%
Since the age of the female subjects ranged from 50 to 64 years, reduction of betaine in subjects with c.665TT compared with the
the presented results are restricted to postmenopausal women. c.665CC genotype, which has not been previously reported. This
Among the 13 polymorphisms, three were in HWE, whereas 10 reduction may be linked to a decrease in serum folate, because
SNPs showed deviation from equilibrium. Deviation becomes betaine has previously been observed to be positively related to
significant at a small D of 0.0041 (TCN2 c.67A4G) due to the folate [Holm et al., 2005] and increases after supplementation
large size of the study population. The positive D [Leal, 2005] with folic acid [Melse-Boonstra et al., 2005].
demonstrates the underestimation of heterozygous genotypes. This We observed a simultaneous increase in tHcy and decrease
is explained by genotyping errors due to low amounts of DNA in in folate by the MTHFR c.1286AC and c.1286CC genotypes,
some blood samples, resulting in poor amplification of some PCR which has not been reported before. An increase of tHcy in
products and misinterpretation of some heterozygous genotypes as c.665CT/c.1286AC compared to c.665CT/c.1286AA has been
homozygous genotypes. Notably, the success rate was lowest for demonstrated in two studies [van der Put et al., 1998; Weisberg
polymorphisms, which suffered from the highest deviations from et al., 2001], while others reported an increase [Castro et al.,
HWE. Such reading errors may lead to underestimation of ‘‘true’’ 2003] or decrease [Friedman et al., 1999] of tHcy in c.665CC/
associations, especially for metabolites with highest assay impreci- c.1286CC compared to c.665CC/c.1286AA. Associations with
sion (high coefficient of variation). Such attenuation of the folate were often not reported [Castro et al., 2003; Dekou et al.,

Human Mutation DOI 10.1002/humu

 HUMAN MUTATION 28(9), 856^865, 2007 861

2001; Gueant-Rodriguez et al., 2005; Weisberg et al., 2001]. A c.665/c.1286 genotype combinations, and the results have been
reduction of betaine was also observed for the MTHFR published in a separate work [Ulvik et al., 2007] (http://
c.1286A4C variant, but was only one-third of the decrease www.springerlink.com/content/051213l4t2254328/).
found for the c.665C4T. In addition, we have investigated in MTR and MTRR are both involved in homocysteine remethyla-
detail the changes in folate and tHcy levels according to MTHFR tion [Finkelstein, 1990]. The decrease in tHcy according to the
number of MTR c.2756G alleles observed by us, confirms the results
from many previous studies [Chen et al., 2001; Harmon et al., 1999;
Tsai et al., 2000; Yates and Lucock, 2002], but this is not a
consistent finding [Hyndman et al., 2000; Klerk et al., 2003; Ma
et al., 1999; Morita et al., 1999; Ulvik et al., 2004; van der Put
et al., 1997]. In contrast to published results [Chen et al., 2001;
Hyndman et al., 2000; Klerk et al., 2003], we and others [Harmon
et al., 1999; Ma et al., 1999] found no relation of MTR
c.2756A4G genotypes to the concentrations of folate or
cobalamin. Furthermore, there are reports on association of MTRR
c.66A4G polymorphism with tHcy [Gaughan et al., 2001; Gueant-
Rodriguez et al., 2005], which could not be confirmed by us or
others [Brilakis et al., 2003; Jacques et al., 2003].
The relation of the BHMT c.716G4A polymorphism to tHcy
has so far been investigated in three studies [Heil et al., 2000;
Morin et al., 2003b; Weisberg et al., 2003] and no associations
have been found. We did not find any significant effect of this SNP
on tHcy, but a significant decrease of DMG according to the
number of c.716A alleles. This is the first indication that BHMT
c.716G4A might have metabolic effects.
Cystathionine b-synthase is the rate limiting step in the
transsulfuration pathway that degrades superfluous homocysteine
[Finkelstein, 1990]. A few studies [Janosikova et al., 2003; Tsai
et al., 1999; Tsai et al., 2000] have reported lower levels of
FIGURE 3. The relation of the homocysteine/cystathionine ratio
postmethionine load (PML) tHcy in subjects with the CBS
to the CBS c.699C4Tgenotypes.The ratio between the substrate
(homocysteine) and product (cystathionine) is plotted according c.844_845ins68. In accordance with the role of cystathionine
to genotype as means with 95% CI after adjustment for age, sex, b-synthase in homocysteine homeostasis, they suggested that the
creatinine, and center. P-value for trend is given. 68-bp insertion may enhance transsulfuration efficiency by

FIGURE 4. E¡ect sizes for the associations of gene polymorphisms with concentrations of metabolites. The e¡ect sizes are given as
relative di¡erence in means between the two homozygous genotypes. Mean values of the wild types were used as reference.

Human Mutation DOI 10.1002/humu

862 HUMAN MUTATION 28(9), 856^865, 2007

increasing the CBS activity or amount of the CBS mRNA. tHcy in Implications and Conclusion
subjects not loaded with methionine or in the fasting state has
The MTHFR c.665C4T polymorphism has a well-established
shown no relation to the c.844_845ins68 [Bowron et al., 2005;
and marked effect on folate and homocysteine status [Ueland
Janosikova et al., 2003; Kluijtmans et al., 1997, 2003], which is in
et al., 2001]. The reports on metabolic phenotypes of most other
agreement with homocysteine remethylation rather than transsul-
genetic polymorphisms related to one-carbon metabolism have
furation as the main determinant of this tHcy modality [Refsum
been inconsistent. The present study has sufficient power to
et al., 2004]. However, we observed a minor but significant
demonstrate that MTHFR c.1286A4C, MTR c.2756A4G,
reduction of tHcy in subjects with the insertion. This probably
BHMT c.716G4A, CBS c.844_845ins68, CBS c.699C4T, and
reflects the power of the present study to detect small changes in
TCN2 c.776C4G have significant associations with concentra-
tHcy according to genotype. Notably, the 68-bp insertion was
tions of vitamins or metabolites in plasma/serum. However, one
associated with a significant increase in betaine, which adds to the
can not infer from the small effect size measured for some genetic
general observation [Holm et al., 2005] that conditions that lower
variants that their biological importance is minor, because small
tHcy increase betaine.
changes in extracellular markers may reflect substantial perturba-
Our observation that the 68-bp insertion is in strong linkage
tion of intracellular metabolism. Therefore, the metabolic profile
disequilibrium with the CBS c.699C4T (D0 5
0.89) is in
demonstrated for several polymorphisms in the present study
accordance with earlier reported data [De Stefano et al., 1998].
should stimulate further studies on their biological effects as well
Published results [Aras et al., 2000; Kruger et al., 2000]
are their relations to chronic diseases.
demonstrate a tHcy lowering effect of the CBS c.699T alleles in
subjects after PML or folate supplementation. Likewise, we found a
decrease in tHcy and increase in betaine with increasing number ACKNOWLEDGMENTS
of CBS c.699 T alleles. Thus, the metabolic phenotype of the CBS
c.699C4T polymorphism is similar to that of the 68-bp insertion, We thank the members of the Section for Pharmacology for
except for the decline of the tHcy/cystathionine ratio, which was sample handling and acquisition of the metabolic data.
confined to the CBS c.699C4T. Since the c.699C4T transition
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Human Mutation DOI 10.1002/humu