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A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase

TL;DR: This work has identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes and may represent an important genetic risk factor in vascular disease.
Abstract: Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular and coronary heart disease. Elevated levels of plasma homocysteine can result from genetic or nutrient-related disturbances in the trans-sulphuration or re-methylation pathways for homocysteine metabolism. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the re-methylation of homocysteine to methionine. Reduced MTHFR activity with a thermolabile enzyme has been reported in patients with coronary and peripheral artery disease. We have identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes. The mutation in the heterozygous or homozygous state correlates with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of a mutagenized cDNA containing the mutation confirms its effect on thermolability of MTHFR. Finally, individuals homozygous for the mutation have significantly elevated plasma homocysteine levels. This mutation in MTHFR may represent an important genetic risk factor in vascular disease.

Summary (1 min read)

A common mutation in methylenetetrahydrofolate reductase

  • The three genotypes were all significantly different (P < .01) with respect to enzyme thermolability.
  • Treatment of extracts at 46°C for five minutes revealed that the enzyme containing the substitution was significantly more thermolabile than the wild -type enzyme .
  • This hypothesis is compatible with the well-documented influence of folate on homocysteine levels7,14 and with the reported correction of mild hyperhomocysteinaemia by folic acid in individuals with premature vascular disease14 and thermolabile MTHFR.15 Enzyme activity and plasma homocysteine were determined as previously reported.
  • Each value represents mean ± standard error.

Methodology

  • Primers were designed from the cDNA sequence to generate 250-300 bp fragments which overlapped 50-75 bp at each end.
  • The individuals, all French Canadian, were not examined clinically or biochemically.
  • Of the 13 cardiovascular patients, eight had cerebrovascular arteriosclerosis, and five had peripheral arteriosclerosis.
  • The entire replacement fragment and the cloning sites were sequenced to verify that no additional changes were introduced by PCR.
  • Western analysis was performed using the Amersham ECL kit with antiserum generated against purified porcine liver MTHFR.12 Enzymatic assays were performed by established procedures.

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University of Nebraska - Lincoln University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln
College of Law, Faculty Publications Law, College of
5-1995
A candidate genetic risk factor for vascular disease: A common A candidate genetic risk factor for vascular disease: A common
mutation in methylenetetrahydrofolate reductase mutation in methylenetetrahydrofolate reductase
P. Frosst
McGill University
H. J. Blom
University Hospital Nijmegen, Netherlands
R. Milos
McGill University
P. Goyette
McGill University
Christal A. Sheppard
University of Nebraska-Lincoln
, christalsheppard@unl.edu
See next page for additional authors
Follow this and additional works at: https://digitalcommons.unl.edu/lawfacpub
Part of the Legal Studies Commons
Frosst, P.; Blom, H. J.; Milos, R.; Goyette, P.; Sheppard, Christal A.; Matthews, R. G.; Boers, G. J.H.; den
Heijer, M.; Kluijtmans, L. A.J.; van den Heuvel, L. P.; and Rozen, Rima, "A candidate genetic risk factor for
vascular disease: A common mutation in methylenetetrahydrofolate reductase" (1995).
College of Law,
Faculty Publications
. 124.
https://digitalcommons.unl.edu/lawfacpub/124
This Article is brought to you for free and open access by the Law, College of at DigitalCommons@University of
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Authors Authors
P. Frosst, H. J. Blom, R. Milos, P. Goyette, Christal A. Sheppard, R. G. Matthews, G. J.H. Boers, M. den
Heijer, L. A.J. Kluijtmans, L. P. van den Heuvel, and Rima Rozen
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
lawfacpub/124

Abstract
Hyperhomocysteinaemia has been identied as a risk factor for
cerebrovascular, peripheral vascular, and coronary heart dis-
ease.
1-4
Elevated levels of plasma homocysteine can result from
genetic or nutrient-related disturbances in the trans-sulphuration
or re-methylation pathways for homocysteine metabolism.
1,5–7
5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes
the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetra-
hydrofolate, the predominant circulatory form of folate and carbon
donor for the re-methylation of homocysteine to methionine. Re-
duced MTHFR activity with a thermolabile enzyme has been re-
ported in patients with coronary and peripheral artery diseases.
6
We have identied a common mutation in MTHFR which alters
a highly-conserved amino acid; the substitution occurs at a fre-
quency of approximately 38% of unselected chromosomes. The
mutation in the heterozygous or homozygous state correlates with
reduced enzyme activity and increased thermolability in lympho-
cyte extracts; in vitro expression of a mutagenized cDNA contain-
ing the mutation conrms its effect on thermolability of MTHFR. Fi-
nally, individuals homozygous for the mutation have signicantly
elevated plasma homocysteine levels. This mutation in MTHFR
may represent an important genetic risk factor in vascular disease.
Severe MTHFR deciency, the most common inborn error
of folate metabolism, results in hyperhomocysteinaemia, ho-
mocystinuria, and hypomethioninaemia. Patients with severe
MTHFR deciency (0-20% residual activity in cultured bro-
blasts) present in infancy or adolescence with developmental
delay, motor and gait dysfunction, seizures, psychiatric distur-
bances, and other neurological abnormalities; they are also at
risk for vascular complications.
8
Individuals with 50% resid-
ual activity, due to a thermolabile form of the reductase, were
rst reported in approximately 17% of 212 North American
patients with coronary artery disease.
5
A recent study of the
Netherlands population identied the thermolabile variant in
different forms of premature vascular disease,
6
and estimated
its incidence to be 7% of vascular patients. The presence of a
thermolabile MTHFR is predictive of coronary artery stenosis,
independent of other risk factors, such as age, smoking, hyper-
cholesterolaemia, and hypertension.
9
Our recent isolation of a cDNA for human MTHFR
10
has
enabled us to identify nine mutations in this gene, in the se-
verely-decient group of patients, by SSCP analysis and direct
sequencing of PCR fragments.
10,11
Using the same procedures,
we identied a C to T substitution at nucleotide (nt) 677, which
converts an alanine to a valine residue (Figure 1a). This altera-
tion creates a HinfI site (Figure 1b), which was used to screen
114 unselected French Canadian chromosomes; the allele fre-
quency of the substitution was 0.38. The frequency of the three
genotypes were as follows: –/–, 37%; +/–, 51%; and +/+, 12%
(+ indicates the presence of the HinfI site and a valine residue).
As these individuals were not examined clinically or biochem-
ically, they cannot be considered as a control group.
We next performed genotypic analysis and measured en-
zyme activity and thermolability in a total of 40 lymphocyte
pellets from patients with premature vascular disease and
controls (Table I). We selected 13 vascular patients from our
previous study, among whom ve were considered to have
thermolabile MTHFR.
6
From a large reference group of 89
controls, we studied all seven individuals who had thermo-
labile MTHFR, and selected at random an additional 20 con-
trols with normal MTHFR from the same reference group.
The mean MTHFR activity for individuals homozygous for
the Ala to Val substitution (+/+) was approximately 30% of
the mean activity for (–/–) individuals. Heterozygotes had a
mean MTHFR activity of 65% compared to (–/–) individuals,
intermediate between values for (–/–) and (+/+) individuals.
The ranges of activities showed some overlap for the hetero-
zygous and (–/–) genotypes, but homozygous (+/+) individ-
uals showed virtually no overlap with the other two groups.
A one-way analysis of variance yielded a P value <.0001;
Published in Nature Genetics 10 (May 1995), pp. 111–113.
Copyright © 1995 Nature Publishing Group. Used by permission.
Submitted December 7, 1994; accepted March 1995.
A candidate genetic risk factor for vascular disease:
A common mutation in methylenetetrahydrofolate reductase
P. Frosst,
1
H.J. Blom,
2
R. Milos,
1
P. Goyette,
1
C. A. Sheppard,
3
R. G. Matthews,
3
G.J.H. Boers,
4
M. den Heijer,
2
L.A.J. Kluijtmans,
2
L. P. van den Heuvel,
2
and R. Rozen
1
1. Departments of Human Genetics, Pediatrics and Biology, McGill University, Montreal Children’s Hospital, Montreal, Canada H3H 1P3
2. Department of Pediatrics, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands
3. Biophysics Research Division and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA
4. Department of Medicine, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands
5. Department of Hematology, Municipal Hospital Leyenburg, 2545 CH The Hague, The Netherlands
Corresponding author — R. Rozen
111
Figure 1. Sequence change and restriction enzyme analysis for the al-
anine to valine substitution. a) Sequence of two individuals, a homo-
zygote for the alanine residue and a homozygote for the valine resi-
due. The antisense strands are depicted. The primers for analysis of
the A→V change are: 5’-TGAAGGAGAA GGTGTCTGCG GGA-3’ (ex-
onic) and 5’-AGGACGGTGC GGTGAGAGTG-3’ (intronic); these prim-
ers generate a fragment of 198 bp. b) The substitution creates a HinfI
recognition sequence which digests the 198 bp fragment into 175 and
23 bp fragments; the latter fragment has been run off the gel. All three
possible genotypes are shown.

112 P. Fr o ss t et a l . i n Nat u r e G e N e t i c s 10 (1995)
a pairwise Bonferroni t test showed that all three genotypes
were signicantly different with P < 0.01 for the three possi-
ble combinations.
The three genotypes were all signicantly different (P < .01)
with respect to enzyme thermolability. The mean residual ac-
tivity after heat inactivation (5 min at 46°C) was 67% (–/–),
56% (+/–) and 22% (+/+). While the degree of thermolability
overlaps somewhat for (–/–) individuals and heterozygotes,
individuals with two mutant alleles had a distinctly lower
range. Every individual with the (+/+) genotype had residual
activity <35% after heating, and specic activity <50% of that
of the (–/–) genotype.
Fasting homocysteine levels in (+/+) individuals were al-
most twice the value for (+/–) and (–/–) individuals. The dif-
ferences among genotypes for plasma homocysteine were
maintained when homocysteine was measured following six
hours of methionine loading. A one-way anova yielded a P <
.01 for the fasting and post-methionine homocysteine levels.
A pairwise Bonferroni t test showed that homozygous mutant
individuals had signicantly elevated homocysteine levels (P
< .05),compared to either (+/–) or (–/–) individuals.
We have used the original MTHFR cDNA (1.3 kb) to isolate
a 2.2 kb cDNA, which contained an additional 900 bp at the 3’
end; the latter contained a termination codon, 100 bp of 3’ UTR
and a poly A tail (GenBank# UO9806). The open reading frame
of 1980 bp predicts a protein of 74.6 kDa. The puried porcine
liver enzyme has been shown to have subunits of 77 kDa.
12
Western analysis (Figure 2a) of several human tissues and of
porcine liver reveals a polypeptide of 77 kDa, as well as an ad-
ditional polypeptide of approximately 70 kDa in human fetal
liver and in porcine liver, suggesting the presence of isozymes.
The wild-type cDNA and a mutagenized cDNA, containing
the Ala to Val substitution, were expressed in E. coli to yield
a protein of approximately 70 kDa (Figure 2a), which co-mi-
grates with the smaller polypeptide mentioned above. Treat-
ment of extracts at 46°C for ve minutes revealed that the
enzyme containing the substitution was signicantly more
thermolabile than the wild -type enzyme (P < .001; Figure 2b).
The expression experiments were not designed to measure
differences in specic activity before heating, since variation
in efciencies of expression could contribute to difculties in
interpretation. Curiously though, the specic activity for the
mutant construct was higher in both experiments. It is possi-
ble that the mutant protein has increased stability in E. coli, or
that inclusion bodies in our extracts contributed to differences
in recovery of properly-assembled enzyme.
The alanine residue is conserved in porcine MTHFR and
in the corresponding bacterial metF genes.
10
We have also ob-
served a region of homology in the human dihydrofolate re-
ductase (DHFR) gene,
11
although the alanine residue itself is
not conserved; this region of amino acids 130-149 of DHFR
contains Thr136, which has been implicated in folate binding
of human DHFR.
13
This region in MTHFR might also be in-
volved in folate binding, and the enzyme may be stabilized in
the presence of folate. This hypothesis is compatible with the
well-documented inuence of folate on homocysteine levels
7,14
and with the reported correction of mild hyperhomocysteinae-
mia by folic acid in individuals with premature vascular dis-
ease
14
and thermolabile MTHFR.
15
Table 1. Correlation between MTHFR genotype and enzyme activity,
thermolability, and plasma homocysteine level
Genotype
–/– +/– +/+
n=19 n=9 n=12
Specic activity
a,b
22.9±1.7 15.0±0.8 6.9±0.6
(nmol CH
2
O/mg protein/hr) (11.8–33.8) (10.2–18.8) (2.6–10.2)
Residual activity 66.8±1.5 56.2±2.8 21.8±2.8
after heatings
a,b
(%) (55–76) (41–67) (10–35)
Plasma homocysteine
a,c
12.6±1.1 13.8±1.0 22.4±2.9
(μM)(after fasting) (7–21) (9.6–20) (9.6–42)
Plasma homocysteine
a,c
41.3±5.0
d
41±2.8 72.6±11.7
e
(μM)(post-methionine load) (20.9-110) (29.1-54) (24.4-159)
Enzyme activity and plasma homocysteine were determined as pre-
viously reported.
6
Each value represents mean ± standard error. The
range is given in parentheses below the mean. a. one-way anova P <
.01. b. paired t test for all combinations P < .01. c. paired t test P < .05
for +/+ group versus +/– group or –/– group; P > .05 for +/– versus –/–
group. d. n = 18. e. n = 11.
Figure 2. Expression analysis of MTHFR in E. coli. a) Western blot of bacterial extracts and tissues. Two μg of bacterial extract protein was used
for lanes 1-3. The tissues (lanes 4-6) were prepared by homogenization in 0.25 M sucrose with aprotinin and leupeptin, followed by sonication
on ice. The extracts were spun for 15 min in a microcentrifuge at 14,000g and 100 μg of supernatant protein was used for western analysis. h,
human; p, porcine. b) Thermolability assay of bacterial extracts. Two separate experiments (with 3-4 replicates construct experiment) were per-
formed to measure thermostable activity of the wild-type and mutagenized MTHFR cDNAs. The values shown represent mean ± standard error for
each experiment, as % of residual activity after heating. The means of the specic activities before heating (expressed as nmol formaldehyde/hr/
mg protein) were as follows: 3.8 and 5.3 (Exp. 1) and 6.2 and 7.5 (Exp. 2) for MTHFR and MTHFR A→V, respectively.

Mu tat i on in MtHFr a s c an d i d at e g e n et i c r i s k Fa c to r F o r va s cu l ar d i se a se 113
Our data have identied a common genetic change in
MTHFR which results in thermolability; our experiments do
not directly address the relationship between this change and
vascular disease. Nonetheless, this mutation represents a di-
agnostic test for evaluation of MTHFR thermolability in hy-
perhomocysteinaemia. Large case-control studies are required
to evaluate the frequency of this genetic change in various
forms of occlusive arterial disease and to examine the interac-
tion between this genetic marker and dietary factors, such as
folate intake. Well-dened populations need to be examined,
as the limited data set thus far suggests that population-spe-
cic allele frequencies may exist. More importantly, however,
the identication of a candidate genetic risk factor for vascu-
lar disease, which may be inuenced by nutrient intake, rep-
resents a critical step in the design of appropriate therapies for
the homocysteinaemic form of arteriosclerosis.
Methodology
Mutation identication. Primers were designed from the cDNA se-
quence to generate 250-300 bp fragments which overlapped 50-75 bp
at each end. When PCR amplication of human genomic DNA yielded
larger fragments than expected for the coding region alone, these frag-
ments were presumed to contain introns and were sequenced directly
(Cycle Sequencing kit, GIBCO). Intronic primer sequences were ob-
tained with this strategy. PCR products were analyzed by a non-ra-
dioactive SSCP protocol as described.
10
Fragments showing a shift
on SSCP gels were subcloned into Bluescript and sequenced (Seque-
nase kit, USB). To conrm the sequence changes, a new PCR was per-
formed with genomic DNA; the PCR product was digested with HinfI
and analyzed by polycrylamide gel electrophoresis.
Clinical material. To determine the frequency of the A→V mutation,
DNA from 57 individuals from Quebec was analyzed by PCR and re-
striction digestion. The individuals, all French Canadian, were not ex-
amined clinically or biochemically. The 40 individuals analyzed in Ta-
ble 1 have been described.
6
Of the 13 cardiovascular patients, eight
had cerebrovascular arteriosclerosis, and ve had peripheral arterio-
sclerosis. Five had thermolabile MTHFR, while eight had thermosta-
ble MTHFR (>33% residual activity after heating). Controls and patients
were all Dutch-Caucasian, between 20–60 years of age. None of these in-
dividuals used vitamins that could alter homocysteine levels. Enzyme
assays and homocysteine determinations have also been reported.
6
Constructs for expression analysis. A human colon carcinoma cDNA
library (gift of Nicole Beauchemin, McGill University) was screened by
plaque hybridization with the original 1.3 kb cDNA
10
to obtain addi-
tional coding sequences. A cDNA of 2.2 kb was isolated, which contained
900 additional bp at the 3’ end (Genbank accession number UO9806). Se-
quencing was performed on both strands for the entire cDNA. Addi-
tional 5’ sequences (800 bp) were obtained from a human kidney cDNA
library (Clontech), but these sequences did not contain additional coding
sequences and were therefore used for the PCR-based mutagenesis only
(see below) and not for the expression analysis. The two cDNAs (2.2 kb
and 800 bp) were ligated using the EcoRI site at nt 199 and inserted into
Bluescript (Stratagene). The 2.2 kb cDNA was subcloned into the expres-
sion vector pTrc99A (Pharmacia) using the NcoI site at nt 11 and the XbaI
site in the polylinker region of both vectors. Sequencing was performed
across the cloning sites to verify the wild-type construct.
PCR-based mutagenesis, using the cDNA-containing Bluescript
vector as template, was used to create the A to V mutation identied.
16
Vent polymerase (NEB) was used to reduce PCR errors. The following
primers were used: primer 1, bp -200 to -178, sense; primer 2, bp 667
to 687, antisense, containing a mismatch, A, at nt 677; primer 3, 667 to
687, sense, containing a mismatch, T, at nt 677; primer 4, bp 1092 to
1114, antisense. PCR was performed using primers 1 and 2 to generate
a product of 887 bp, and using primers 3 and 4 to generate a product
of 447 bp. The two PCR fragments were isolated from a 1.2% agarose
gel by Geneclean (BIO 101). A nal PCR reaction, using primers 1 and
4 and the rst two PCR fragments as template, was performed to gen-
erate a 1.3 kb band containing the mutation. The 1.3 kb fragment was
digested with NcoI and MscI, and inserted into the wild-type cDNA-
containing expression vector by replacing the sequences between the
NcoI site at bp 11 and the MscI site at bp 943. The entire replacement
fragment and the cloning sites were sequenced to verify that no addi-
tional changes were introduced by PCR.
Expression analysis. Overnight cultures of JM 105 containing vector,
wild-type, or mutagenized MTHFR cDNA were grown at 37 °C in 2
× YT media with .05 mg ml
–1
ampicillin. Fresh 10 ml cultures of each
were inoculated with approximately 50 μl of overnight cultures and
grown at 37 °C to an O.D. of 1 at 420 nM. Cultures were then induced
for 2 h with 1 mM IPTG and pelleted. The cells were resuspended in
TE buffer with 2 μg ml
–1
aprotinin and leupeptin (3.5 × wet weight
of cells). Cell suspensions were sonicated on ice for 3 × 15 s and cen-
trifuged for 30 min at 4 °C to pellet cell debris and unlysed cells. The
supernatant was removed and assayed for protein concentration with
the Bio-Rad protein assay. Western analysis was performed using the
Amersham ECL kit with antiserum generated against puried porcine
liver MTHFR.
12
Enzymatic assays were performed by established pro-
cedures.
17
Thermolability was assessed by pre-treating the extracts at
46 °C for 5 min before determining activity. Specic activities (nmol
formaldehyde/h/mg protein) were calculated for the 2 cDNA-con-
taining constructs after subtraction of the values obtained with vector
alone (to subtract background E. coli MTHFR activity).
Acknowledgments We thank E. Stevens, H. van Lith-Zanders,
C Mandel, and N. Beauchemin for their contribution to this work.
This work was supported by the Medical Research Council of Can-
ada (R.R.), the Canadian Heart and Stroke Foundation (R.R.), in part
by the Netherlands Heart Foundation (H.B.), and by NIH Grant R37
GM24908 (CA.S. and R.G.M.). R.R. is a Principal Investigator of the
MRC Group in Medical Genetics.
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Citations
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Journal ArticleDOI
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Journal ArticleDOI
04 Oct 1995-JAMA
TL;DR: Higher folic acid intake by reducing tHcy levels promises to prevent arteriosclerotic vascular disease and under different assumptions, 13,500 to 50,000 CAD deaths annually could be avoided.
Abstract: Objective. —To determine the risk of elevated total homocysteine (tHcy) levels for arteriosclerotic vascular disease, estimate the reduction of tHcy by folic acid, and calculate the potential reduction of coronary artery disease (CAD) mortality by increasing folic acid intake. Data Sources. —MEDLINE search for meta-analysis of 27 studies relating homocysteine to arteriosclerotic vascular disease and 11 studies of folic acid effects on tHcy levels. Study Selection and Data Extraction. —Studies dealing with CAD, cerebrovascular disease, and peripheral arterial vascular disease were selected. Three prospective and six population-based case-control studies were considered of high quality. Five cross-sectional and 13 other case-control studies were also included. Causality of tHcy's role in the pathogenesis of vascular disease was inferred because of consistency across studies by different investigators using different methods in different populations. Data Synthesis. —Elevations in tHcy were considered an independent graded risk factor for arteriosclerotic vascular diseases. The odds ratio (OR) for CAD of a 5-μmol/L tHcy increment is 1.6(95% confidence interval [Cl], 1.4 to 1.7) for men and 1.8 (95% Cl, 1.3 to 1.9) for women. A total of 10% of the population's CAD risk appears attributable to tHcy. The OR for cerebrovascular disease (5-μmol/L tHcy increment) is 1.5 (95% Cl, 1.3 to 1.9). Peripheral arterial disease also showed a strong association. Increased folic acid intake (approximately 200 μg/d) reduces tHcy levels by approximately 4 μmol/L. Assuming that lower tHcy levels decrease CAD mortality, we calculated the effect of (1) increased dietary folate, (2) supplementation by tablets, and (3) grain fortification. Under different assumptions, 13 500 to 50 000 CAD deaths annually could be avoided; fortification of food had the largest impact. Conclusions. —A 5-μmol/L tHcy increment elevates CAD risk by as much as cholesterol increases of 0.5 mmol/L (20 mg/dL). Higher folic acid intake by reducing tHcy levels promises to prevent arteriosclerotic vascular disease. Clinical trials are urgently needed. Concerns about masking cobalamin deficiency by folic acid could be lessened by adding 1 mg of cobalamin to folic acid supplements. ( JAMA . 1995;274:1049-1057)

3,722 citations

Journal ArticleDOI
TL;DR: In this article, an elevated level of total homocysteine (tHcy) in blood, denoted hyperhomocysteinemia, is emerging as a prevalent and strong risk factor for atherosclerotic vascular disease in the coronary, cerebral, and peripheral vessels, and for arterial and venous thromboembolism.
Abstract: An elevated level of total homocysteine (tHcy) in blood, denoted hyperhomocysteinemia, is emerging as a prevalent and strong risk factor for atherosclerotic vascular disease in the coronary, cerebral, and peripheral vessels, and for arterial and venous thromboembolism. The basis for these conclusions is data from about 80 clinical and epidemiological studies including more than 10,000 patients. Elevated tHcy confers a graded risk with no threshold, is independent of but may enhance the effect of the conventional risk factors, and seems to be a particularly strong predictor of cardiovascular mortality. Hyperhomocysteinemia is attributed to commonly occurring genetic and acquired factors including deficiencies of folate and vitamin B12. Supplementation with B-vitamins, in particular with folic acid, is an efficient, safe, and inexpensive means to reduce an elevated tHcy level. Studies are now in progress to establish whether such therapy will reduce cardiovascular risk.

2,099 citations


Cites background from "A candidate genetic risk factor for..."

  • ...mia under conditions of impaired folate status (29, 73, 74)....

    [...]

  • ...In 1995, Frosst et al suggested that the C677T polymorphism in the MTHFR gene is a candidate risk factor for vascular disease (29)....

    [...]

  • ...was later ascribed to a C677T mutation in MTHFR gene (29)....

    [...]

  • ...The same year, the C677T mutation in the MTHFR gene was identified and proposed as a candidate risk factor for vascular disease (29)....

    [...]

  • ...predictor of hyperhomocysteinemia in the general population (29, 73, 74), is...

    [...]

Journal ArticleDOI
TL;DR: In 1969, McCully reported autopsy evidence of extensive arterial thrombosis and atherosclerosis in two children with elevated plasma homocyst(e)ine concentrations and homocysteine thiolactone, and it has recently become clear that hyperhomocyst (e)inemia is an independent risk factor.
Abstract: In 1969, McCully made the clinical observation linking elevated plasma homocyst(e)ine concentrations with vascular disease.1 He reported autopsy evidence of extensive arterial thrombosis and atherosclerosis in two children with elevated plasma homocyst(e)ine concentrations and homocystinuria. On the basis of this observation, he proposed that elevated plasma homocyst(e)ine (hyperhomocyst(e)inemia) can cause atherosclerotic vascular disease. The term “homocyst(e)ine” is used to define the combined pool of homocysteine, homocystine, mixed disulfides involving homocysteine, and homocysteine thiolactone found in the plasma of patients with hyperhomocyst(e)inemia. Subsequent investigations have confirmed McCully's hypothesis, and it has recently become clear that hyperhomocyst(e)inemia is an independent risk factor . . .

1,981 citations

Journal ArticleDOI
23 Oct 2002-JAMA
TL;DR: This meta-analysis of observational studies suggests that elevated homocysteine is at most a modest independent predictor of IHD and stroke risk in healthy populations.
Abstract: CONTEXT: It has been suggested that total blood homocysteine concentrations are associated with the risk of ischemic heart disease (IHD) and stroke. OBJECTIVE: To assess the relationship of homocysteine concentrations with vascular disease risk. DATA SOURCES: MEDLINE was searched for articles published from January 1966 to January 1999. Relevant studies were identified by systematic searches of the literature for all reported observational studies of associations between IHD or stroke risk and homocysteine concentrations. Additional studies were identified by a hand search of references of original articles or review articles and by personal communication with relevant investigators. STUDY SELECTION: Studies were included if they had data available by January 1999 on total blood homocysteine concentrations, sex, and age at event. Studies were excluded if they measured only blood concentrations of free homocysteine or of homocysteine after a methionine-loading test or if relevant clinical data were unavailable or incomplete. DATA EXTRACTION: Data from 30 prospective or retrospective studies involving a total of 5073 IHD events and 1113 stroke events were included in a meta-analysis of individual participant data, with allowance made for differences between studies, for confounding by known cardiovascular risk factors, and for regression dilution bias. Combined odds ratios (ORs) for the association of IHD and stroke with blood homocysteine concentrations were obtained by using conditional logistic regression. DATA SYNTHESIS: Stronger associations were observed in retrospective studies of homocysteine measured in blood collected after the onset of disease than in prospective studies among individuals who had no history of cardiovascular disease when blood was collected. After adjustment for known cardiovascular risk factors and regression dilution bias in the prospective studies, a 25% lower usual (corrected for regression dilution bias) homocysteine level (about 3 micromol/L [0.41 mg/L]) was associated with an 11% (OR, 0.89; 95% confidence interval [CI], 0.83-0.96) lower IHD risk and 19% (OR, 0.81; 95% CI, 0.69-0.95) lower stroke risk. CONCLUSIONS: This meta-analysis of observational studies suggests that elevated homocysteine is at most a modest independent predictor of IHD and stroke risk in healthy populations. Studies of the impact on disease risk of genetic variants that affect blood homocysteine concentrations will help determine whether homocysteine is causally related to vascular disease, as may large randomized trials of the effects on IHD and stroke of vitamin supplementation to lower blood homocysteine concentrations.

1,953 citations


Cites background from "A candidate genetic risk factor for..."

  • ...may well help to assess the nature of this association.(69,70) Individuals who have a...

    [...]

  • ...gene that encodes the methylenetetrahydrofolate reductase enzyme have reduced enzyme activity and, as a consequence, have homocysteine levels that are about 25% higher than those with the CC genotype.(70,71) An accompanying article in THE JOURNAL (2002;288: 2023-2031) describes a meta-analysis of 40 studies of this genetic polymorphism in which individuals with the TT polymorphism have a 16% (95% CI, 5%28%) higher risk of IHD than those with the CC polymorphism....

    [...]

References
More filters
Book
01 Jan 1972
TL;DR: The metabolic basis of inherited disease, the metabolic basis for inherited disease as mentioned in this paper, The metabolic basis in inherited disease and inherited diseases, and inherited disease diagnosis and management, in the context of inherited diseases
Abstract: The metabolic basis of inherited disease , The metabolic basis of inherited disease , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

7,724 citations

Book
01 Jan 1989

5,306 citations

Journal ArticleDOI
TL;DR: Hyperhomocysteinemia is an independent risk factor for vascular disease, including coronary disease, and in most instances is probably due to cystathionine beta-synthase deficiency.
Abstract: Background. Hyperhomocysteinemia arising from impaired methionine metabolism, probably usually due to a deficiency of cystathionine β-synthase, is associated with premature cerebral, peripheral, and possibly coronary vascular disease. Both the strength of this association and its independence of other risk factors for cardiovascular disease are uncertain. We studied the extent to which the association could be explained by heterozygous cystathionine β-synthase deficiency. Methods. We first established a diagnostic criterion for hyperhomocysteinemia by comparing peak serum levels of homocysteine after a standard methionine-loading test in 25 obligate heterozygotes with respect to cystathionine β-synthase deficiency (whose children were known to be homozygous for homocystinuria due to this enzyme defect) with the levels in 27 unrelated age- and sex-matched normal subjects. A level of 24.0 μmol per liter or more was 92 percent sensitive and 100 percent specific in distinguishing the two groups. The ...

2,202 citations

Journal ArticleDOI
08 Dec 1993-JAMA
TL;DR: A strong association between homocysteine concentration and folate, vitamin B12, and vitamin B6 status, as well as age is indicated, suggesting that a substantial majority of the cases of high homocy steine in this older population can be attributed to vitamin status.
Abstract: Objective. —To describe the distribution of plasma homocysteine concentrations in an elderly population and to analyze the relationship between homocysteine level and intake of vitamins and serum levels of vitamins that serve as coenzymes in homocysteine metabolism. Design. —Cross-sectional analysis of homocysteine levels and vitamin blood levels and intake in elderly participants in the Framingham Study. Setting. —Population-based cohort in Framingham, Mass. Participants. —A total of 1160 adult survivors, aged 67 to 96 years, from the original Framingham Heart Study cohort. Main Outcome Measures. —Plasma homocysteine concentration correlated with plasma folate, vitamin B 12 , pyridoxal-5'-phosphate (PLP), and oral intakes of these vitamins, and the contribution of these vitamins to the prevalence of elevated homocysteine in the population. Results. —Homocysteine levels were positively correlated with age after controlling for vitamin concentrations. After controlling for age, sex, and levels of other vitamins, homocysteine exhibited a strong inverse association with plasma folate. When subjects were grouped by deciles of plasma folate, mean homocysteine was significantly higher in the lowest two folate deciles (15.6 and 13.7 μmol/L, respectively) than in the highest decile (11.0 μmol/L). Homocysteine demonstrated weaker, inverse associations with plasma vitamin B 12 and PLP. Similar inverse associations were demonstrated between homocysteine and intakes of folate and vitamin B 6 , but not vitamin B 12 . Prevalence of high homocysteine (>14 μmol/L) was 29.3% in this cohort, and was greatest among subjects with low folate status. Inadequate plasma concentrations of one or more B vitamins appear to contribute to 67% of the cases of high homocysteine. Conclusions. —These results indicate a strong association between homocysteine concentration and folate, vitamin B 12 , and vitamin B 6 status, as well as age. It is possible that a substantial majority of the cases of high homocysteine in this older population can be attributed to vitamin status. ( JAMA . 1993;270:2693-2698)

1,978 citations

Journal ArticleDOI
19 Aug 1992-JAMA
TL;DR: In this paper, a nested case-control study using prospectively collected blood samples was conducted to assess prospectively the risk of coronary heart disease associated with elevated plasma levels of homocyst(e)ine.
Abstract: Objective. —To assess prospectively the risk of coronary heart disease associated with elevated plasma levels of homocyst(e)ine. Design. —Nested case-control study using prospectively collected blood samples. Setting. —Participants in the Physicians' Health Study. Participants. —A total of 14916 male physicians, aged 40 to 84 years, with no prior myocardial infarction (Ml) or stroke provided plasma samples at baseline and were followed up for 5 years. Samples from 271 men who subsequently developed Ml were analyzed for homocyst(e)ine levels together with paired controls, matched by age and smoking. Main Outcome Measure. —Acute Ml or death due to coronary disease. Results. —Levels of homocyst(e)ine were higher in cases than in controls (11.1±4.0 [SD] vs 10.5±2.8 nmol/mL; P =.03). The difference was attributable to an excess of high values among men who later had MIs. The relative risk for the highest 5% vs the bottom 90% of homocyst(e)ine levels was 3.1 (95% confidence interval, 1.4 to 6.9; P =.005). After additional adjustment for diabetes, hypertension, aspirin assignment, Quetelet's Index, and total/high-density lipoprotein cholesterol, this relative risk was 3.4 (95% confidence interval, 1.3 to 8.8) ( P =.01). Thirteen controls and 31 cases (11%) had values above the 95th percentile of the controls. Conclusions. —Moderately high levels of plasma homocyst(e)ine are associated with subsequent risk of Ml independent of other coronary risk factors. Because high levels can often be easily treated with vitamin supplements, homocyst(e)ine may be an independent, modifiable risk factor. ( JAMA . 1992;268:877-881)

1,714 citations

Frequently Asked Questions (13)
Q1. What have the authors contributed in "A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase" ?

8 Individuals with 50 % residual activity, due to a thermolabile form of the reductase, were first reported in approximately 17 % of 212 North American patients with coronary artery disease. A recent study of the Netherlands population identified the thermolabile variant in different forms of premature vascular disease,6 and estimated its incidence to be 7 % of vascular patients. The frequency of the three genotypes were as follows: –/–, 37 % ; +/–, 51 % ; and +/+, 12 % ( + indicates the presence of the HinfI site and a valine residue ). The authors next performed genotypic analysis and measured enzyme activity and thermolability in a total of 40 lymphocyte pellets from patients with premature vascular disease and controls ( Table I ). The authors selected 13 vascular patients from their previous study, among whom five were considered to have thermolabile MTHFR. From a large reference group of 89 controls, the authors studied all seven individuals who had thermolabile MTHFR, and selected at random an additional 20 controls with normal MTHFR from the same reference group. The Hague, The Netherlands Corresponding author — R. Rozen 111 Figure 1. The differences among genotypes for plasma homocysteine were maintained when homocysteine was measured following six hours of methionine loading. This hypothesis is compatible with the well-documented influence of folate on homocysteine levels7,14 and with the reported correction of mild hyperhomocysteinaemia by folic acid in individuals with premature vascular disease14 and thermolabile MTHFR. The purified porcine liver enzyme has been shown to have subunits of 77 kDa. 12 Western analysis ( Figure 2a ) of several human tissues and of porcine liver reveals a polypeptide of 77 kDa, as well as an additional polypeptide of approximately 70 kDa in human fetal liver and in porcine liver, suggesting the presence of isozymes. 

Severe MTHFR deficiency, the most common inborn error of folate metabolism, results in hyperhomocysteinaemia, homocystinuria, and hypomethioninaemia. 

Individuals with 50% residual activity, due to a thermolabile form of the reductase, were first reported in approximately 17% of 212 North American patients with coronary artery disease.5 

It is possible that the mutant protein has increased stability in E. coli, or that inclusion bodies in their extracts contributed to differences in recovery of properly-assembled enzyme. 

Patients with severe MTHFR deficiency (0-20% residual activity in cultured fibroblasts) present in infancy or adolescence with developmental delay, motor and gait dysfunction, seizures, psychiatric disturbances, and other neurological abnormalities; they are also at risk for vascular complications. 

The presence of a thermolabile MTHFR is predictive of coronary artery stenosis, independent of other risk factors, such as age, smoking, hypercholesterolaemia, and hypertension. 

Heterozygotes had a mean MTHFR activity of 65% compared to (–/–) individuals, intermediate between values for (–/–) and (+/+) individuals. 

The authors have also observed a region of homology in the human dihydrofolate reductase (DHFR) gene,11 although the alanine residue itself is not conserved; this region of amino acids 130-149 of DHFR contains Thr136, which has been implicated in folate binding of human DHFR.13 

The authors have used the original MTHFR cDNA (1.3 kb) to isolate a 2.2 kb cDNA, which contained an additional 900 bp at the 3’ end; the latter contained a termination codon, 100 bp of 3’ UTR and a poly A tail (GenBank# UO9806). 

Western analysis was performed using the Amersham ECL kit with antiserum generated against purified porcine liver MTHFR.12 Enzymatic assays were performed by established procedures. 

Their recent isolation of a cDNA for human MTHFR10 has enabled us to identify nine mutations in this gene, in the severely-deficient group of patients, by SSCP analysis and direct sequencing of PCR fragments. 

The purified porcine liver enzyme has been shown to have subunits of 77 kDa.12 Western analysis (Figure 2a) of several human tissues and of porcine liver reveals a polypeptide of 77 kDa, as well as an additional polypeptide of approximately 70 kDa in human fetal liver and in porcine liver, suggesting the presence of isozymes. 

This hypothesis is compatible with the well-documented influence of folate on homocysteine levels7,14 and with the reported correction of mild hyperhomocysteinaemia by folic acid in individuals with premature vascular disease14 and thermolabile MTHFR.15Enzyme activity and plasma homocysteine were determined as previously reported.