© 1999 American Heart Association, Inc.
Thrombosis |
Inherited Prothrombotic Conditions and Premature Ischemic Stroke
Sex Difference in the Association With Factor V Leiden
From Unita' di Aterosclerosi e Trombosi, I.R.C.C.S. "Casa Sollievo della Sofferenza", S. Giovanni Rotondo, Divisione di Ematologia, Unità di Coagulazione, Ospedale "A. Cardarelli", Napoli, Istituto di Patologia Generale e Oncologia, Seconda Università di Napoli, Istituto di Patologia Medica e Divisione Neurologia, Università Cattolica Roma, and Istituto di Medicina Interna e Geriatria, Università di Palermo, Italy.
Correspondence to Maurizio Margaglione, MD, Unità di Aterosclerosi e Trombosi, IRCCS "Casa Sollievo della Sofferenza", viale Cappuccini, San Giovanni Rotondo (FG) 71013, Italy.
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Abstract |
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Abstract—At a young age, ischemic stroke is an uncommon event in which prothrombotic factors are likely to play an important role. In 202 referred cases, 105 men and 97 women, median age 39 years (range, 3 to 50), with a history of ischemic stroke and in 1036 age frequency-matched apparently healthy individuals from the same ethnic background, we have investigated whether inherited prothrombotic conditions increase the risk of ischemic stroke. Neither abnormal plasma levels of natural anticoagulants and fibrinogen nor significant increase of the prothrombin A20210 allele was found in stroke cases compared with controls. Hypertension (odds ratio [OR], 22.61), male sex (OR, 2.30), smoking (OR, 2.78) and alcohol habits (OR, 0.14), a personal history of venous thromboembolism (OR, 4.53), a family history of stroke (OR, 1.93), high circulating levels of fibrinogen (P=0.0190), and total cholesterol (P=0.101) were all independently associated with ischemic stroke. Compared with noncarriers, carriers of the factor V (FV) Leiden mutation (OR, 2.56), and to a lesser extent, of the methylenetetrahydrofolate reductase (MTHFR) TT genotype (OR, 1.60), had an independent higher estimated risk of having a history of ischemic stroke. The relationship with the FV Leiden mutation was greater in women (OR, 3.95). Thus, in addition to established determinants, FV Leiden mutation is independently associated with the occurrence of ischemic stroke in this setting. The greater association in women suggests the possibility of an interaction of this genotype with female hormones.
Key Words: stroke • genes • thrombosis • polymorphisms
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Introduction |
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Ischemic stroke is uncommon at a young age and, despite extensive investigations, a large proportion of ischemic strokes are of undetermined etiology.1 2 3 A series of studies have been carried out to elucidate the mechanisms of this ischemic event. The results of the studies are not entirely consistent, but the majority of the data support the concept that thrombosis, rather than atherosclerosis, is important for juvenile ischemic stroke. Abnormalities within the gene loci encoding for natural anticoagulants (antithrombin, protein C, and protein S)4 5 6 and for fibrinogen7 8 have been shown to be rather uncommonly associated with ischemic stroke. Other acquired (antiphospholipid antibodies, hyperhomocysteinemia, high plasma levels of fibrinogen, hypofibrinolysis) and inherited (resistance to activated protein C, prothrombin A20210 allele) prothrombotic conditions have also been studied. Hyperhomocysteinemia has been documented to be an independent risk factor for stroke.9 10 Hyperhomocysteinemia is often related to a thermolabile variant of the enzyme methylenetetrahydrofolate reductase (MTHFR),11 a C
T transition12 at
nucleotide position 677. Mutations of the coagulation
factor V (FV) Leiden and of the prothrombin (a GA transition at
nucleotide position 20210) genes account for a large number
of cases of venous thromboembolism.13 14 15 Some evidence
suggests a role for these gene variants in the risk of
arterial thrombosis leading to stroke.16 17 18 19 20 21
However, these claims have been challenged.22 23 24 25 26 27 28 29
We have investigated a relatively young (
50 years of age) population
with a history of ischemic stroke, in whom a genetic and
prothrombotic influence is conceivable to be most clearly evident, to
assess the relationship with carriership of hereditary prothrombotic
risk factors.
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Methods |
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Patients
Between May 1996 and June 1998, 202 individuals aged
50 years,
105 men and 97 women, with a history of ischemic stroke were
referred to one of the participating centers for a thrombophilic
workup. All of the subjects had survived an ischemic stroke
from 3 to 12 months before being enlisted. In each case, a nuclear
magnetic resonance and/or CT scan confirmed the clinical diagnosis and
served to define the type of stroke.30 The median age
at the time of the first thrombotic episode was 39.0 years (range, 3 to
50). Three patients suffered from autoimmune disease and 1 had a
T-cell lymphoma.
Controls
While patients were being recruited, we interviewed 1272
apparently healthy employees of the Casa Sollievo della Sofferenza
Hospital, San Giovanni Rotondo, Southern Italy. The 1084 subjects, who
were aged 50 years or less, were invited to participate in the study.
Of them, 33 refused and untypability was observed the blood specimens
of another 12 subjects; thus, 1039 subjects were enrolled. Three male
subjects had documented evidence of coronary heart disease
after their enrollment and were excluded from the analysis. All
subjects were white, and all of their parents and grandparents had been
born in the same region. The male to female ratio was 0.74 (males=440,
42.5%; females=596, 57.5%).
A complete clinical summary, with emphasis on personal and family history for stroke, angina pectoris, myocardial infarction, peripheral arterial disease, venous thromboembolism, and vascular risk factors (high blood pressure, hyperlipidemia, diabetes mellitus, cigarette smoking, alcohol consumption), was obtained from all subjects by a specially trained staff according to a previously described questionnaire.31 In addition to detailed and specific questions about symptoms of ischemic heart disease, peripheral vascular disease, and previous vascular surgery (as defined according to the World Health Organization questionnaire for cardiovascular disease), the questionnaire contained specific questions concerning stroke and the habitual use of drugs. Hypertension was defined as a longstanding use of antihypertensive drugs or as a systolic blood pressure >140 mm Hg and/or a diastolic blood pressure >90 mm Hg in the sitting position on at least 3 different occasions at the time of admission. Subjects with fasting blood glucose levels >7.8 mmol/L or on treatment with diets and drugs lowering plasma glucose levels were classified as diabetics. Alcohol consumers were divided into current drinkers and past consumers or subjects who never drank. Smokers were divided into subjects who currently smoke and those who never did. A family history for arterial and venous thrombosis was defined as the occurrence of stroke, myocardial infarction, or venous thromboembolism in parents and first-degree siblings. After approval of the local Ethics Committees, the study was carried out according to the Principles of the Declaration of Helsinki; informed consent was obtained from all the subjects.
Materials
Deoxynucleotide triphosphatase (dNTP), KCl,
MgCl2, gelatin, and mineral oil were from Perkin
Elmer-Cetus; proteinase K was from USB, Corp; and HEPES, Tris-HCl,
EDTA, ethidium bromide, and SDS, were from Sigma Chemical Co.
Restriction enzymes, HinfI, MnlI, and
HindIII were from New England Biolabs Inc. The
concentrations of total cholesterol were detected
enzymatically with commercially available reagents (Roche). Both the
reagent and the apparatus (CoA Data 2000) for the
measurement of the fibrinogen were from Boehringer Mannheim.
Blood Collection and Coagulation Studies
Blood samples were collected into vacuum plastic tubes
containing 0.129 mol/L trisodium citrate and centrifuged at
2000g for 15 minutes to obtain platelet-poor plasma. The
latter was frozen and stored in small aliquots at -70°C until
assayed. Fibrinogen, antithrombin, protein C, amidolytic and
immunological (Behring), and total and free protein S antigen (ELISA,
Diagnostica Stago) were determined in all patients at the
Center of recruitment, as reported elsewhere.32 33
Pooled normal plasma from 65 normal donors served as control plasma.
Clotting assays were performed on a KC4 Amelung coagulometer. Normal
ranges were 80% to 120% (antithrombin), 70% to 140% (protein C),
70% to 140% (protein S, total), 60% to 130% (protein S, free), 3.3
to 6.8 mmol/L (total cholesterol), and 2.0 to 4.5 g/L
(fibrinogen). Interassay and intraassay coefficients of all the
variables never exceed 8.0% and 5.0%, respectively.
DNA Extraction and Analysis
DNA was extracted from peripheral blood leukocytes
according to standard protocols.31 Amplification was
carried out on 50-µL volume samples in a Perkin Elmer-Cetus thermal
cycler. A 220-bp DNA fragment of the FV gene that included the
nucleotide 1691 was amplified and digested with
MnlI as previously described,34 with some
modifications.35 To identify the GA mutation of the
prothrombin gene, a 345-bp fragment was obtained and digested using the
HindIII endonuclease.15 Screening for the
MTHFR CT677 substitution was performed by amplification of a 198-bp
DNA fragment followed by HinfI digestion as
described,12 with modifications.36
Statistical Analysis
All of the analyses were performed according to the
Statistical Package for Social Science (SPSS 6.1 for Macintosh). The
significance of differences in means was evaluated by
nonparametric tests, whereas the 
2
statistic or the Fisher's exact test, as appropriate, tested the
significance of differences in proportions. The allele frequencies
were estimated by gene counting, and genotypes were scored. The
observed numbers of each FV Leiden, prothrombin, or MTHFR
genotypes were compared with those expected for a population in
Hardy-Weinberg equilibrium using the 2 test.
The significance of the difference of observed alleles and
genotypes between the groups were tested using the
2 analysis after grouping homozygous
and heterozygous carriers of the FV Leiden mutation, homozygous and
heterozygous carriers of the A20210 prothrombin
allele, and homozygous and heterozygous carriers of the C677 MTHFR
allele. Prevalence odds ratios [ORs], considered as prevalence of
existing disease, and 95% confidence intervals [CIs] were calculated
employing the Normal approximation. Logistic-regression models
calculated adjusted ORs and 95% CIs. Statistical significance was
taken as P<0.05.
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Results |
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Clinical characteristics of the study sample as a whole, and after stratification according to sex, are shown in Table 1
. Cases were older
(P<0.0001), more often current smokers (OR, 2.75; 95% CI,
2.01 to 3.74), and less often alcohol consumers (OR, 0.23; 95% CI,
0.16 to 0.33) than controls. Among subjects with ischemic
stroke, there was a higher proportion of males (OR, 1.47; 95% CI, 1.08
to 1.98), hypertensives (OR, 27.58; 95% CI, 15.47 to 49.16), diabetics
(OR, 8.12; 95% CI, 3.27 to 20.12), a history of myocardial infarction
(P<0.0001, Fisher's exact test) and venous thromboembolism
(OR, 5.86; 95% CI, 2.78 to 12.33). In addition, cases more frequently
had a family history of stroke (OR, 2.77; 95% CI, 1.92 to 4.00) and
venous thromboembolism (OR, 1.93; 95% CI, 1.07 to 3.51) than controls.
The 2 groups also differed with respect to total
cholesterol (P<0.0001) and plasma fibrinogen
levels (P<0.0001). Similar findings were observed when the
entire sample was stratified according to sex (Table 1).
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No case or control carried inherited abnormalities of antithrombin, protein C, and protein S, or abnormally low fibrinogen plasma levels. In patients, mean±SD levels of antithrombin were 105.3±8.2% and those of protein C 112.0±11.6%. Total and free plasma levels of protein S were 103.2±6.5% and 99.7±5.8%, respectively.
Among cases, 50 individuals (24.8%; 95% CI, 18.8 to 30.8) were
homozygous for the T allele of the MTHFR gene, the frequency of the
T allele being 49.3% (95% CI, 44.4 to 54.2). One hundred
ninety-six healthy subjects (18.9%; 95% CI, 16.5 to 21.3;
P=0.05733) were T677 MTHFR homozygotes. Their T allele
frequency was 43.7% (95% CI, 41.6 to 45.8; P=not
significant [n.s.]). The median age at the time of the
ischemic stroke was 36.1 years (range, 3 to 50) in T677 MTHFR
homozygotes and 40.0 years (range, 11 to 50) in noncarriers
(Mann-Whitney U test, P=0.05). In this setting,
carriers of the FV Leiden mutation were 30 (14.9%; 95% CI, 10.0 to
19.8), 29 heterozygotes and 1 homozygote, among cases, and 43
heterozygotes (4.2%; 95% CI, 3.0 to 5.4; 2,
34.884; P<0.0001) among controls in this setting. The OR
associated with FV Leiden was 4.03 (95% CI, 2.46 to 6.60). The median
age at the time of the ischemic stroke was 44.5 years (range,
17 to 49) in carriers of the FV Leiden mutation and 39.0 years (range,
3 to 50) in noncarriers (Mann-Whitney U test,
P=0.08). The prothrombin A20210
mutation was detected in 10 cases (5.0%; 95% CI, 2.0 to 8.0), 9
heterozygotes and 1 homozygote, and in 43 controls, all heterozygotes
(4.2%; 95% CI, 3.0 to 5.4; P=n.s.). The median age at the
time of the ischemic stroke was 46.0 years (range, 32 to 49) in
carriers of the prothrombin A20210 mutation and
39.0 years (range, 3 to 50) in noncarriers (Mann-Whitney U
test, P=0.08). Frequencies of all the mutations were similar
in men and in women (Table 1
). The observed distribution of
genotypes showed no significant difference compared with that
predicted from the Hardy-Weinberg equilibrium
(2 test).
When stratified according to types of stroke, 82 cases (40.6%) were
atherothrombotic, 14 (6.9%) cardioembolic, 46 (22.8%) occlusions of
small artery, 4 (2.0%) vasculitis, and 56 (27.7%) were of
undetermined etiology or a result of more than one cause. No
significant differences were observed when cases were analyzed
according to such stratification (P always >0.05). In the
whole sample, a personal history of venous thromboembolism was
associated with the FV Leiden mutation (Fisher exact test, 0.00549),
the prothrombin GA20210 mutation (Fisher exact
test, 0.03219), and the MTHFR TT genotype
(2, 3.98204; P=0.04599). A family
history of venous thromboembolism was associated with the presence of
FV Leiden mutation (Fisher exact test, 0.00405), but not the
prothrombin GA20210 mutation (Fisher exact
test, 0. 12620) and MTHFR TT genotype
(2, 0.26143; P=n.s.).
The data relative to FV Leiden were further analyzed.
Stratification according to smoking habit or a family history of stroke
showed an additive effect of the gene variant (Table 2). In contrast, the combined presence of
FV Leiden mutation and 1 or more vascular risk factors, ie,
hypertension, diabetes mellitus, myocardial infarction, and
hypercholesterolemia (Table 2), led to
risk estimates (OR, 10.72; 95% CI, 5.46 to 21.04) that exceeded the
separate effects of FV Leiden and the vascular risk factors,
respectively (OR, 1.93; 95% CI, 0.82 to 4.57; and OR, 1.94; 95% CI,
1.40 to 2.70).
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The findings from the univariate analysis were
further investigated in a logistic model after adjustment for age (in
years), sex, alcohol and smoking habits, hypertension, diabetes
mellitus, a history of myocardial infarction and of thromboembolic
episodes, a family history of stroke and of venous thromboembolism, and
plasma levels of fibrinogen and total cholesterol. (Table 3). Under these circumstances, FV Leiden
mutation was still independently and significantly associated with the
occurrence of ischemic stroke, whereas the MTHFR TT
genotype played a marginal, although significant, role. A
separate analysis for sex was carried out. An independent and
significant association between the occurrence of the cerebrovascular
event and the FV Leiden mutation (OR, 3.95; 95% CI, 1.55 to 10.05), a
personal history of venous thromboembolism (OR, 4.58; 95% CI, 1.55 to
13.50), and a family history of stroke (OR, 2.34; 95% CI, 1.23 to
4.49) was found in women. In men, significant associations were found
with smoking consumption (OR, 4.48; 95% CI, 2.38 to 8.40) and plasma
fibrinogen levels (OR, 1.65; 95% CI, 1.15 to 2.35, for an increase of
1 g/L). Hypertension (women, OR, 16.86; 95% CI, 6.50 to 43.86; men,
OR, 25.15; 95% CI, 8.77 to 72.11), total cholesterol
(women, OR, 1.36; 95% CI, 1.04 to 1.79; men, OR, 1.32; 95% CI, 1.01
to 1.73, for an increase of 1 mmol/L), and alcohol consumption
(women, OR, 0.12; 95% CI, 0.05 to 0.29; men, OR, 0.12; 95% CI, 0.06
to 0.23) were significantly related with the event in both sexes. In a
multiple logistic regression model, evidence for an interaction was
found for smoking habit and sex (P=0.0249), but not for FV
Leiden mutation and smoking habit, sex, family history of stroke, and
vascular risk factors (P always >0.1).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Atherosclerosis is a lifelong degenerative event whose progression leads to major ischemic complications such as myocardial infarction and ischemic stroke. In the young, because of the limited lag of time for atherosclerosis to progress, ischemic stroke is likely to be a well-suited clinical setting to investigate the role of prothrombotic conditions, in particular inherited ones, in the risk of ischemic stroke.
Anecdotal observations have suggested a link between congenital
deficiencies of natural anticoagulants (antithrombin, protein C, and
protein S) or fibrinogen and cerebral infarction.4 5 6 7 8 No
inherited abnormalities of natural anticoagulants nor of fibrinogen
were found in this setting. No association between the mutant
A20210 allele of the prothrombin gene and a
history of cerebral ischemia was observed in an Italian
case-control study.29 In the present report, in which
allele and genotype frequencies closely resembled those
reported in other Italian series,28 29 35 36 37 38 the data are
consistent with such a formulation, and imply that
abnormalities of fibrinogen, natural anticoagulants, and the mutant
A20210 allele of the prothrombin gene are a
rather uncommon cause of ischemic stroke in young adults. In
addition to established determinants (eg, hypertension and smoking
habit), FV Leiden mutation exhibited a significant and independent
relationship with the occurrence of ischemic stroke in this
setting. The association was still significant when all the
variables in which cases and controls differed were taken into
account in a multivariate logistic regression model. In
carriers of the FV Leiden mutation, the estimated risk of
ischemic stroke was at least additive to that observed in
current smokers, in subjects with a family history of stroke, and in
those with vascular risk factors. A borderline significant association
between the MTHFR TT genotype and the occurrence of
ischemic stroke was found. The CT substitution at
nucleotide 677 within the MTHFR gene is a relatively
frequent missense mutation.12 36 37 39 40 41 Especially in
settings carrying low plasma folate levels, the presence of MTHFR TT
homozygosity has been associated with moderate
hyperhomocysteinemia.37 39 40 41 The latter is a risk factor
for stroke.9 10 Our findings somewhat differ from those of
Markus et al,41 in which no association between MTHFR TT
homozygosity and cerebrovascular disease was found. The interaction
with plasma folate and the low informativeness of the marker used may
also account for inconsistencies. Plasma samples collected at the time
of the ischemic event do not allow for measurements of the
levels of homocysteine and folate in the present setting. Thus the
present data cannot rule out the possibility that differences in
the intake of folate account, at least in part, for the association of
the MTHFR TT genotype with the occurrence of ischemic
stroke.
Inconclusive results are present in the literature on the association between FV Leiden mutation and stroke.16 17 18 19 21 22 23 24 25 26 27 Although the inconsistency may reflect the play of chance, alternative explanations have to be considered. Whereas a relationship has been documented in younger adults or specific settings,16 17 18 19 21 the presence of the FV Leiden mutation was not associated with stroke in studies that enlisted older adults or elderly patients.22 23 24 25 26 27 For example, Catto et al24 studied 386 randomly selected elderly patients (median age, 74 years) with acute stroke. In the present study, median age was 39 years, 25.8% of women and 20% of men aged 30 years or less when the ischemic event took place. The US Physicians' Health Study23 included only male physicians. In the study by Press et al25 the percentage of men in the stroke group was 91%. In the present study, women were 48% of cases.
After stratification for sex, FV Leiden mutation was independently associated with an increased risk estimate of ischemic stroke in women (OR, 3.95). Recently, an increased risk of myocardial infarction has been reported in young women with FV Leiden mutation.42 Endogenous estrogens and oral contraceptives increase the resistance to activated protein C regardless of the presence of the FV Leiden mutation.43 In oral contraceptive users, FV Leiden mutation has been described to further increase the risk of venous thrombosis.44 In the present sample, the small number of oral contraceptive users (n=9) did not allow for a reliable analysis of the role of oral contraceptives alone and in combination with the FV Leiden mutation on the occurrence of ischemic stroke.
Epidemiological evidence suggests a U-shaped association between alcohol consumption and cardiovascular disease, with moderate intake protecting against stroke.45 46 In our study population, alcohol intake was associated with an independent lower risk of ischemic stroke (OR, 0.14). The beneficial effect of the alcohol consumption has been attributed to inhibition of the atherogenic potential of LDL cholesterol.46 We did not address this issue. However, the higher percentage of alcohol drinkers and the lower mean levels of total plasma cholesterol in the control group further suggest an important role for blood lipids in the pathogenesis of stroke.
Because we only enlisted subjects that survived their stroke event, one could argue that this could have led to biased results. Raised circulating levels of some parameters measured, such as fibrinogen, may have been caused by the disease process because fibrinogen is an acute-phase protein. However, established determinants of ischemic stroke (eg, hypertension, male sex, smoking and alcohol habits, family history of stroke, and high circulating levels of fibrinogen and total cholesterol) are all strongly and independently associated with the occurrence of ischemic stroke in the present setting. Case fatality in stroke largely depends on age, with 88% of all deaths from stroke occurring in people older than 65 years.2 Furthermore, it has not been proven whether subjects who died would have had an over- or under-representation of these gene variants. Thus, the data tend to exclude the possibility that the relationships that we have found are significantly affected by the selection of stroke survivors, and support the possibility that our case group is representative of a stroke population.
We conclude that FV Leiden mutation and, to a lesser extent MTHFR TT genotype, are independently associated with the occurrence of ischemic stroke in young adults aged 50 years or less. Similar to myocardial infarction, the relevance of FV Leiden mutation appears to be restricted to women in this setting, suggesting a role for endogenous and exogenous female hormones in such an association. The clinical implications of these data need to be addressed in prospective ad hoc studies.
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Acknowledgments |
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The financial support of Telethon-Italy (Grant no. E.C 804) is gratefully acknowledged.
Received October 9, 1998; accepted December 11, 1998.
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