Department of Clinical and Experimental Medicine,
University of Naples Federico II
Department of Neurological Sciences,
University of Naples Federico II,
Naples, Italy
Coagulation Service,
IRCCS H.S. Raffaele,
Milan, Italy
To the Editor:
Case-control1 2 and prospective3 4 studies
have suggested an association between moderate hyperhomocysteinemia and
risk of ischemic stroke. Homozygosity for the C-to-T
substitution at nucleotide 677 of the gene of
5,10-methylenetetrahydrofolate
reductase (MTHFR) is associated with a 50% reduction of the activity
of this enzyme5 and is the most common inherited cause of
moderate hyperhomocysteinemia. In 1996 Klujitmans et al6
reported a threefold increase in the risk of early-onset
cardiovascular disease in homozygotes for the C677T
MTHFR mutation. However, the association of this genetic marker with
arterial vascular events has been disputed by a nested
case-control study.7 Markus et al8 recently
failed to show an association between cerebrovascular disease and the
MTHFR genotype, a comparable prevalence of homozygosity for the
C677T MTHFR mutation being detectable in a population of 345 patients
with ischemic stroke or transient ischemic attacks
(TIA) and in 161 control subjects (10.7% versus 13.7%, respectively).
Nor were nonfasting log homocysteine plasma levels able to identify
subjects with a stroke history in that setting, as judged by the
analysis of a subgroup of patients (n=160) and control subjects
(n=75) in whom this amino acid was measured. However, as expected, the
authors found a significant relationship between MTHFR genotype
and homocysteine levels, the latter being also independently related to
log serum folate.
In the frame of a larger study on juvenile thrombotic events, we
have evaluated a population of 60 consecutive patients with a history
of early-onset ischemic stroke (29 females and 31 males, aged 5
to 64 years [mean age, 38; mean age at time of diagnosis, 34; range 4
to 49 years]) documented within 72 to 96 hours from the event by CT
and/or MRI scans. Subjects who had suffered from TIA or who exhibited
abnormalities of carotid and/or vertebral arteries were excluded from
the study. As many as 182 subjects matched for sex and age, without a
history of thrombosis, served as controls. Total fasting plasma
homocysteine (tHcy), ie, the sum of free and protein-bound forms plus
cysteine-homocysteine mixed disulfide, was measured by isolation of the
amino acid by high-performance liquid
chromatography and fluorescence
detection.9 Hyperhomocysteinemia was defined as tHcy
values above the 95th percentile of the distribution within the general
population (>19.5 µM for males and >15 µM for females in our
population).9 The C677T MTHFR mutation was studied by
endonuclease digestion with HinfI of the polymerase chain
reaction–amplified products.10 As summarized in the
table
, among patients the prevalence of homozygous C677T
MTHFR mutation (+/+) was 36.7% (22 of 60); among control subjects, it
was 21.4% (39 of 182 individuals; P<.05,
Several concepts should be taken into account in understanding
differences between the present findings and those of Markus et al.
In our setting, only individuals with juvenile stroke were enrolled.
Patient mean age in the study of Markus et al was 65.7 years. An
unknown proportion of patients with TIA was evaluated in that report;
patients with TIA were excluded from our study. As a whole, in our
study fasting hyperhomocysteinemia and the MTHFR mutation allowed
identification of almost 40% of subjects with a history of stroke. It
is known that nonfasting plasma homocysteine levels help to identify
higher numbers of thrombophilic subjects with this
metabolic abnormality.9 However, factors other
than the C677T MTHFR mutation play a role in regulating nonfasting
homocysteine levels. The strong linkage between C677T homozygosity and
fasting hyperhomocysteinemia in this setting further supports this
association. The high frequency of the mutation in our setting may be
the result of an inappropriate sample selection and may have greatly
contributed to the significant and independent association between the
genetic marker and ischemic stroke reported here. However,
figures on the frequency of the C677T homozygous state in Italian
populations are comparable with those reported by our and other
groups,10 11 and the combined data are consistent
with the possibility that genotype-based observations can be
applied only to ethnic groups of similar genetic background.
References
1.
Coull BM, Malinow MR, Beamer N, Sexton
G, Nordt F, deGarmo P. Elevated plasma homocyst(e)ine
concentration as a possible independent risk factor for stroke.
Stroke. 1990;21:572–576.
2.
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;324:1149–1155.[Abstract]
3.
Verhoef P, Hennekens CH, Malinow MR, Kok FJ, Willett
WC, Stampfer MJ. A prospective study of plasma homocyst(e)ine
and risk of ischemic stroke. Stroke. 1994;25:1924–1930.[Abstract]
4.
Perry IJ, Refsum H, Morris RW, Ebrahim SB, Ueland PM,
Shaper AG. Prospective study of serum total homocysteine
concentration and risk of stroke in middle-aged British men.
Lancet. 1995;346:1395–1398.[Medline]
[Order article via Infotrieve]
5.
Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA,
Matthews RG, Boers GHJ, den Heijer M, Kluijtmans LAJ, van den Heuvel
LP, Rozen R. A candidate genetic risk factor for vascular
disease: a common mutation in
methylenetetrahydrofolate
reductase. Nat Genet. 1995;10:111–113.[Medline]
[Order article via Infotrieve]
6.
Klujitmans LAJ, van den Heuvel LPWJ, Boers GHJ, Frosst
P, Stevens EMB, van Oost BA, den Heijer M, Trijbles FJM, Rozen R, Blom
HJ. Molecular genetic analysis in mild
hyperhomocysteinemia: a common mutation in the
methylene-tetrahydrofolate reductase gene is a genetic risk factor for
cardiovascular disease. Am J Hum
Genet. 1996;58:35–41.[Medline]
[Order article via Infotrieve]
7.
Ma J, Stampfer MJ, Hennekens CH, Frosst P, Sehlub J,
Horsford J, Malinow MR, Willett WC, Rozen R.
Methylene-tetrahydrofolate reductase polymorphism, plasma
folate, homocysteine, and risk of myocardial infarction in US
physicians. Circulation. 1996;94:2410–2416.
8.
Markus HS, Nadira A, Swaminathan R, Sankaralingam A,
Molloy J, Powell J. A common polymorphism in the
methylenetetrahydrofolate reductase
gene, homocysteine, and ischemic cerebrovascular
disease. Stroke. 1997;28:1739–1743.
9.
Fermo I, Vigano' D'Angelo S, Paroni R, Mazzola G,
Calori G, D'Angelo A. Prevalence of moderate
hyperhomocysteinemia in patients with early-onset venous and
arterial thrombosis. Ann Intern Med. 1995;123:747–753.
10.
de Franchis R, Mancini FP, D'Angelo A, Sebastio G,
Fermo I, De Stefano V, Margaglione M, Mazzola G, Di Minno G, Andria G.
Elevated total plasma homocysteine and 677C
11.
Sacchi E, Tagliabue L, Duca F, Mannucci PM. High
frequency of the C677T mutation in the
methylenetetrahydrofolate reductase
(MTHFR) gene in Northern Italy. Thromb Haemost. 1997;78:963–964.[Medline]
[Order article via Infotrieve]
Department of Clinical Neurosciences,
Institute of Psychiatry,
London, UK
We were interested to see the results of this study in an
Italian population which suggests that the C677T MTHFR gene
polymorphism may be a risk factor for early-onset stroke.
Homocysteine levels were measured using a methodology similar to that
in our study but these were fasting levels in contrast to the levels we
measured, which were nonfasting and performed only in a subgroup of
patients. Consistent with their data and with previous studies,
we found that homocysteine levels were higher in subjects than in
controls, but this result did not reach significance (mean [SD] log
homocysteine level in cases, 1.32 [0.19] mmol/L; in controls,
1.27 [0.19] mmol/L; P=.09). This lack of significance
is likely to result from small sample numbers (homocysteine was
measured only in a subgroup) and from a weakening of any association by
the use of nonfasting levels. Nevertheless, this result is
consistent with the existing literature and the results of
Soriente et al on an association between raised homocysteine levels and
stroke risk. In contrast, there was not even a trend toward an increase
in the prevalence of homozygocity (TT) for the C677T MTHFR
polymorphism in our population (TT genotype
frequency in cases was 10.7% versus 13.7% in controls;
P=.34) or in T allele frequency (0.68 versus
0.67; P=.67). This suggests that in our population, which
was an unselected group of consecutive white patients presenting
with ischemic stroke and TIA, the MTHFR genotype is not
a major risk factor for stroke. As mentioned in our article, we also
performed subgroup analysis to determine whether the
polymorphism was a risk factor for patients with stroke compared
with patients with TIA and no CT infarct, but there was no difference
in TT frequency between the two groups (11.1% versus 9.5%;
P=.69). In addition, there was no association between
genotype and cerebrovascular disease when only the 157 case
subjects and 79 control subjects aged <65 years, were considered
(TT frequency of 13.5% in case subjects and 15.2% in
control subjects; P=.70). Unfortunately, because of an error
at the editing stage, this line was printed as
Therefore, the results of Soriente et al, which show a strikingly
increased TT genotype prevalence in stroke patients,
are at variance with ours. However, the association, once other risk
factors had been accounted for in a logistic regression model, was only
just significant, with a lower confidence interval of 1.1. These
results may reflect a real difference between the two populations. The
patients they studied were young, with a mean age of 34 and a youngest
age of 4, which is significantly lower than both the mean age of our
patient population and the mean age of our subgroup of patients aged
© 1998 American Heart Association, Inc.
Letters to the Editor
Homozygous C677T Mutation of the 5,10 Methylenetetrahydrofolate Reductase Gene and Hyperhomocysteinemia in Italian Patients With a History of Early-Onset Ischemic Stroke
2
test). Elevated fasting tHcy was detected in 13 of 60 patients (21.6%)
and in 18 of 182 control subjects (9.8%) (P<0.05,
2 test). Mean tHcy was 15.8±14.6 µM in patients and
12.5±7.8 µM in controls (P<.005, t-test). By
analyzing the distribution of hyperhomocysteinemic patients among the
different MTHFR genotypes, a significantly higher prevalence of
elevated tHcy was found among +/+ homozygotes compared with other
genotypes. Nine of the 22 +/+ patients (40.9%) showed fasting
plasma homocysteine levels above the 95th percentile of the
distribution. Only 4 of the 38 (10.5%) nonhomozygotes (+/- and -/-)
behaved similarly (P<.005 by the 2 test,
homozygotes versus nonhomozygotes). Accordingly, higher mean fasting
tHcy was detected in +/+ homozygous patients than in nonhomozygotes
(23.3±22.2 µM versus 11.8+4.9; P<.005. Similar to the
findings of Markus et al,8 plasma tHcy was inversely
related to folate levels (r=-.26; P<.001) in
this setting. Folate plasma levels did not differ among patients and
controls. In a logistic regression model, the homozygous C677T MTHFR
mutation was significantly associated with the event (odds ratio, 2.1;
95% confidence interval, 1.1 to 4.0; P=.02). By excluding
the C677T MTHFR mutation from the model, the association with tHcy
became significant (odds ratio, 1.03; 95% confidence interval, 1.001
to 1.06; P=.04). Accordingly, a multiple regression
analysis showed that C677T MTHFR mutation was a major
determinant of tHcy (ß coefficient=8.67; P<.001).
View this table:
[in a new window]
Table 1. Prevalence of Homozygous C677T MTHFR Mutation and Elevated
tHcy Among Patients With a History of Early-Onset Ischemic
Stroke and Control Subjects 
T mutation of
5,10-methylenetetrahydrofolate
reductase gene in thrombotic vascular disease. Am J
Hum Genet. 1996;59:262–264.[Medline]
[Order article via Infotrieve]
Response
65 years rather than
the 
65 years in the original manuscript. The majority of studies to
date looking at the association between the MTHFR polymorphism and
cardiovascular risk, most of which have looked at
ischemic heart disease, have also failed to show a strong
association. 65 years. Another important factor could be the folate intake of the
population. Both our study and that of Soriente et al show a
significant interaction between serum folate, MTHFR genotype,
and homocysteine levels. Therefore, if the folic acid intake of their
population is lower than that of ours, the MTHFR genotype will
have a greater effect on determining serum homocysteine levels. An
alternative explanation is that this is a chance finding, which can
frequently happen with candidate gene association studies. Only 60
patients were studied. No information is given about how the control
population was recruited or whether the study individuals were
ethnically homogenous. In addition, a relatively high frequency of the
TT genotype was found both in cases and
controls.
This article has been cited by other articles:
 |
|
 |
|
  A Pezzini, M Grassi, E Del Zotto, D Assanelli, S Archetti, R Negrini, L Caimi, and A Padovani Interaction of homocysteine and conventional predisposing factors on risk of ischaemic stroke in young people: consistency in phenotype-disease analysis and genotype-disease analysis J. Neurol. Neurosurg. Psychiatry, October 1, 2006; 77(10): 1150 - 1156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Bosco, G. Anello, R. Ferri, R.-M. Gueant-Rodriguez, J.-L. Gueant, S. Cronin, and P. Kelly Heterogeneity of Association Between MTHFR and Stroke Among European Regions: Additional Population Studies Are Needed in Italy Stroke, March 1, 2006; 37(3): 761 - 762. [Full Text] [PDF]
|
|
|
|
|
|
S. Cronin, K. L. Furie, and P. J. Kelly Dose-Related Association of MTHFR 677T Allele With Risk of Ischemic Stroke: Evidence From a Cumulative Meta-Analysis Stroke, July 1, 2005; 36(7): 1581 - 1587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Casas, A. D. Hingorani, L. E. Bautista, and P. Sharma Meta-analysis of Genetic Studies in Ischemic Stroke: Thirty-two Genes Involving Approximately 18 000 Cases and 58 000 Controls Arch Neurol, November 1, 2004; 61(11): 1652 - 1661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Li, L. Sun, H. Zhang, Y. Liao, D. Wang, B. Zhao, Z. Zhu, J. Zhao, A. Ma, Y. Han, et al. Elevated Plasma Homocysteine Was Associated With Hemorrhagic and Ischemic Stroke, but Methylenetetrahydrofolate Reductase Gene C677T Polymorphism Was a Risk Factor for Thrombotic Stroke: A Multicenter Case-Control Study in China Stroke, September 1, 2003; 34(9): 2085 - 2090. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Kelly, J. Rosand, J. P. Kistler, V. E. Shih, S. Silveira, A. Plomaritoglou, and K. L. Furie Homocysteine, MTHFR 677C->T polymorphism, and risk of ischemic stroke: Results of a meta-analysis Neurology, August 27, 2002; 59(4): 529 - 536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.Y. Streifler, N. Rosenberg, A. Chetrit, R. Eskaraev, B.A. Sela, R. Dardik, A. Zivelin, B. Ravid, J. Davidson, U. Seligsohn, et al. Cerebrovascular Events in Patients With Significant Stenosis of the Carotid Artery Are Associated With Hyperhomocysteinemia and Platelet Antigen-1 (Leu33Pro) Polymorphism Stroke, December 1, 2001; 32(12): 2753 - 2758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Cardo, E. Monros, C. Colome, R. Artuch, J. Campistol, M. Pineda, and M. A. Vilaseca Children With Stroke: Polymorphism of the MTHFR Gene, Mild Hyperhomocysteinemia, and Vitamin Status J Child Neurol, May 1, 2000; 15(5): 295 - 298. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||