Relation Between Folate Status, a Common
Mutation in Methylenetetrahydrofolate
Reductase, and Plasma Homocysteine
Concentrations
1. Paul F. Jacques, ScD;
2. Andrew G. Bostom, MD, MS;
3. Roger R. Williams, MD;
4. R. Curtis Ellison
, MD, MS;
5. John H. Eckfeldt
, MD, PhD;
6. Irwin H. Rosenberg, MD;
7. Jacob Selhub, PhD;
8. Rima Rozen, PhD
+ Author Affiliations
1. From the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts
University, Boston, Mass (P.F.J., A.G.B., I.H.R., J.S.); the NHLBI Family Heart Study,
University of Utah Cardiovascular Genetics Research Clinic, Salt Lake City (R.R.W.);
the NHLBI Family Heart Study, Framingham, Mass, and Boston (Mass) University
School of Medicine (R.C.E.); the NHLBI Family Heart Study Central Laboratory,
Department of Laboratory Medicine and Pathology, University of Minnesota,
Minneapolis (J.H.E.); and the Departments of Human Genetics, Pediatrics, and Biology,
McGill University, Montreal (Quebec) Children’s Hospital (R.R.).
1. Correspondence to Rima Rozen, Montreal Children’s Hospital, 2300 Tupper St,
Montreal, Quebec, Canada H3H 1P3.
Next Section
Abstract
Background Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5-
methyltetrahydrofolate, the major carbon donor in remethylation of homocysteine to methionine.
A common MTHFR mutation, an alanine-to-valine substitution, renders the enzyme thermolabile
and may cause elevated plasma levels of the amino acid homocysteine.
Methods and Results To assess the potential interaction between this mutation and vitamin
coenzymes in homocysteine metabolism, we screened 365 individuals from the NHLBI Family
Heart Study. Among individuals with lower plasma folate concentrations (<15.4 nmol/L), those
with the homozygous mutant genotype had total fasting homocysteine levels that were 24%
greater (P<.05) than individuals with the normal genotype. A difference between genotypes was
not seen among individuals with folate levels ≥15.4 nmol/L.
Conclusions Individuals with thermolabile MTHFR may have a higher folate requirement for
regulation of plasma homocysteine concentrations; folate supplementation may be necessary to
prevent fasting hyperhomocysteinemia in such persons.
Key Words:
enzymes
homocysteine
amino acids
metabolism
genetics
Homocysteine is a sulfur amino acid whose metabolism is at the intersection of two metabolic
pathways: remethylation and transsulfuration.
1
In remethylation, the primary methyl donor for
the vitamin B
12
-dependent conversion of homocysteine to methionine is 5-
methyltetrahydrofolate, the principal circulating form of folate. 5-Methyltetrahydrofolate is
synthesized from 5,10-methylenetetrahydrofolate by the enzyme MTHFR. In the transsulfuration
pathway, homocysteine condenses with serine to form cystathionine in an irreversible reaction
catalyzed by the PLP-containing enzyme CBS. These pathways can be disrupted by genetic
defects in the two enzymes CBS and MTHFR or by deficiencies of folate, vitamin B
12
, and
vitamin B
6
.
Because of the existence of a cellular homocysteine export mechanism, plasma normally
contains a small amount of homocysteine, averaging 10 μmol/L.
2
This export mechanism
complements the catabolism of homocysteine to help maintain low intracellular concentrations
of this potentially cytotoxic sulfur amino acid. In hyperhomocysteinemia, plasma homocysteine
concentrations are elevated, indicating that homocysteine metabolism has in some way been
disrupted. The more severe cases of hyperhomocysteinemia are primarily due to defects in the
genes encoding CBS
3
and MTHFR.
4
A potential consequence of even moderate elevations of plasma homocysteine may be an
increased risk of occlusive vascular disease.
5
Accordingly, investigation of the determinants of
moderate hyperhomocysteinemia has intensified. Inadequate status of nutritional coenzymes in
homocysteine metabolism, at least in the elderly, appears to be a major determinant of moderate
hyperhomocysteinemia.
5
6
However, recent evidence suggests that common enzyme mutations
may also be important determinants of hyperhomocysteinemia. In 1988, Kang et al
7
reported a
variant of MTHFR that was distinguishable from the normal enzyme by its lower specific
activity and its heat sensitivity and suggested that this thermolabile variant was an inherited
autosomal recessive trait that is present in ≈5% of the general population and 17% of patients
with coronary disease.
8
Subsequently, one of us (R.R.) and coworkers isolated the cDNA for
human MTHFR
4
and demonstrated that thermolabile MTHFR is caused by an alanine-to-valine
(Ala-to-Val) missense mutation.
9
Twelve percent of French Canadians were shown to have the
homozygous mutant genotype for this polymorphic variant.
9
The impact of thermolabile MTHFR on hyperhomocysteinemia remains equivocal. Kang et al
8
demonstrated that even though plasma homocysteine levels were higher among individuals with
thermolabile MTHFR than among those with normal enzyme activity, many of those with the
thermolabile enzyme did not have hyperhomocysteinemia. Furthermore, the
hyperhomocysteinemia seen in the original study of Kang et al
7
was associated with low plasma
folate concentrations, and folate supplementation normalized the plasma homocysteine
concentrations. These data suggested that folate status might play a crucial role in the
development of hyperhomocysteinemia in individuals with the thermolabile defect.
To test the hypothesis that homocysteine concentrations in individuals with thermolabile
MTHFR are dependent on folate status, we examined the influence of plasma folate
concentration on the relation between the MTHFR thermolabile polymorphism and plasma
homocysteine concentrations, using data from the NHLBI FHS.
Previous SectionNext Section
Methods
Subjects
Subjects were participants in the NHLBI FHS. The FHS was established to evaluate genetic and
nongenetic determinants of coronary heart disease in randomly sampled families and families
known to be at high risk for coronary heart disease. Subject examinations began in February
1994 and are expected to be complete by January 1996. Two of the four sites participating in the
FHS are involved in an ancillary homocysteine study, which is described in greater detail
elsewhere.
10
All persons at these sites undergoing the complete FHS phase II evaluation were
invited to participate in the ancillary project, with the following exclusion criteria: age <25 or
>69 years old, fasting for <10 hours, and lack of informed consent. This ancillary homocysteine
study protocol was approved by the FHS Steering Committee and Safety Monitoring Board and
the institutional review boards for Boston University School of Medicine, the University of Utah,
and Tufts–New England Medical Center. These preliminary analyses are based on the initial 365
subjects enrolled in the ancillary homocysteine study.
Fasting Blood Collection
Immediately upon arriving, before methionine loading, subjects underwent fasting (>10 hours)
phlebotomy. One 10-mL EDTA-containing vacuum tube was obtained, and the plasma was
promptly separated, divided into aliquots, and stored at −70°C. DNA was purified by a
commercially available salt precipitation method (Puragene from Gentra Systems, Inc).
Methionine Load Test
Methionine (100 mg/kg) was administered in 200 mL of fruit juice immediately after the fasting
phlebotomy. Four hours after the methionine load, a repeat plasma sample was obtained for
homocysteine determination.
Laboratory Determinations
As previously described,
6
total homocysteine in plasma was determined by high-performance
liquid chromatography with fluorometric detection, plasma folate by a 96-well plate microbial
(Lactobacillus casei) assay, plasma PLP by the tyrosine decarboxylase apoenzyme method, and
plasma vitamin B
12
by a radioassay.
MTHFR Genotype Determination
The polymerase chain reaction primers for amplification of the MTHFR mutation have been
described elsewhere.
9
The primers generate a 198-bp fragment. The MTHFR polymorphism, a
C-to-T substitution at bp 677, creates a HinfI recognition sequence. If the mutation is present,
HinfI digests the 198-bp fragment into a 175-bp and a 23-bp fragment. The fragments were
analyzed by polyacrylamide gel electrophoresis.
Statistical Methods
All plasma measures were positively skewed, and we used logarithmic transformations to
normalize their distributions. Thus, all means presented here are geometric means. To describe
the relationships between MTHFR thermolabile genotype and plasma homocysteine and vitamin
concentrations, we calculated the geometric mean levels of these factors in individuals with
normal (Ala/Ala), heterozygous (Val/Ala), and homozygous (Val/Val) mutant genotypes. We
used ANOVA to test for differences between genotypes and for interactions between genotype
and vitamin levels. Because age and sex adjustment had no influence on the observed results, we
present only the unadjusted data.
Previous SectionNext Section
Results
We measured total fasting and post–methionine load homocysteine concentrations. Mean total
fasting homocysteine concentrations were slightly greater for homozygotes of the MTHFR
thermolabile mutation, but there was a significant interaction between genotype and folate status
(Table֝
). Among those with plasma folate levels below the sample median (15.4 nmol/L), the
mean fasting homocysteine concentration was 24% greater (P<.05) for homozygotes with the
thermolabile mutation than for either the normal or heterozygous individuals. No association was
observed between genotype and fasting homocysteine concentrations in those with plasma folate
levels at or above the median. Neither PLP nor vitamin B
12
modified the association between
genotype and fasting homocysteine. Post–methionine load total homocysteine concentration was
unrelated to MTHFR genotype (Table֝
) irrespective of plasma vitamin concentrations. Mean
plasma vitamin concentrations were also unrelated to genotype.
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Table 1.
Total Plasma Homocysteine by MTHFR Genotype
Previous SectionNext Section
Discussion
Our data qualify the role of thermolabile MTHFR as a determinant of fasting homocysteine
levels, revealing an interaction between MTHFR thermolabile genotype and folate status.
Individuals who are homozygotes for the MTHFR thermolabile mutation have elevated fasting
homocysteine concentrations when plasma folate concentration is in the lower range but not
when folate is high. We further demonstrate that thermolabile MTHFR genotype is not
associated with the post–methionine load increase in plasma homocysteine, consistent with the
hypothesis that post–methionine load hyperhomocysteinemia is due primarily to defects in the
transsulfuration pathway.
11
In conclusion, these findings indicate that individuals with thermolabile MTHFR may have a
higher folate requirement for regulation of plasma homocysteine concentrations and, more
importantly, suggest a therapeutic strategy (ie, folate supplementation) to prevent fasting
hyperhomocysteinemia in such persons.
Previous SectionNext Section
Selected Abbreviations and Acronyms
CBS = cystathionine β-synthase
FHS = NHLBI Family Heart Study
MTHFR = methylenetetrahydrofolate reductase
PLP = pyridoxal-5′-phosphate
Previous SectionNext Section
Acknowledgments
This project was funded in part with federal funds from the US Department of Agriculture,
Agricultural Research Service under contract 53-3K06-01 and NHLBI contract N01-HC-25106
and by the Medical Research Council of Canada. The contents of this publication do not
necessarily reflect the views or policies of the US Department of Agriculture.
Received September 20, 1995.
Revision received October 23, 1995.
Accepted October 30, 1995.