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Psaty BM, Smith NL, Lemaitre RN, et al. Hormone Replacement Therapy, Prothrombotic Mutations, and the Risk of Incident Nonfatal Myocardial Infarction in Postmenopausal Women. JAMA. 2001;285(7):906–913. doi:10.1001/jama.285.7.906
Author Affiliations: Departments of Medicine (Drs Psaty and Lemaitre), Epidemiology (Drs Psaty, Smith, Heckbert, LaCroix, and Rosendaal), and Health Services (Dr Psaty), Cardiovascular Health Research Unit, University of Washington, Seattle; Fred Hutchinson Cancer Research Center, Seattle, Wash (Dr LaCroix); Center for Health Studies, Group Health Cooperative, Seattle, Wash (Dr LaCroix); Department of Hematology (Drs Vos and Rosendaal), and Clinical Epidemiology (Dr Rosendaal), Leiden University Medical Center, Leiden, the Netherlands.
Context Estrogens are known to be prothrombotic, and findings from the Heart
and Estrogen/progestin Replacement Study suggest that in women with clinically
recognized heart disease, hormone replacement therapy (HRT) may be associated
with early harm and late benefit in terms of coronary events.
Objective To assess whether, as hypothesized, prothrombotic mutations modify the
association between HRT use and incidence of first myocardial infarction (MI).
Design and Setting Population-based, case-control study conducted in a Seattle-based health
Participants Cases were 232 postmenopausal women aged 30 to 79 years who had their
first nonfatal MI between 1995 and 1998. Controls were a stratified random
sample of 723 postmenopausal women without MI who were frequency-matched to
cases by age, calendar year, and hypertension status.
Main Outcome Measure Risk of first nonfatal MI based on current use of HRT and the presence
or absence of coagulation factor V Leiden and prothrombin 20210 G→A variants
among cases and controls, stratified by hypertension.
Results One hundred eight MI cases and 387 controls had hypertension and 124
MI cases and 336 controls did not. Among hypertensive women, the prothrombin
variant was a risk factor for MI (odds ratio [OR], 4.32; 95% confidence interval
[CI], 1.52-12.1) and, in this stratum, there was also a significant interaction
between use of HRT and presence of the prothrombin variant on risk of MI.
Compared with nonusers of HRT with wild-type genotype, women who were current
users and who had the prothrombin variant (n = 8) had a nearly 11-fold increase
in risk of a nonfatal MI (OR, 10.9; 95% CI, 2.15-55.2). The interaction with
the prothrombin variant was more pronounced in analyses assuming 100% compliance
than in those assuming 80% compliance with HRT. The interaction was absent
among nonhypertensive women and was less pronounced if hypertensive and nonhypertensive
women were combined into 1 group. No interaction was found for factor V Leiden
in either hypertensive or nonhypertensive women. Among hypertensive women,
the estimates were affected only in trivial ways by adjustment, and the interaction
with the prothrombin variant was specific to HRT.
Conclusions Our results suggest that among postmenopausal hypertensive women, the
association between HRT use and MI risk differed between those with and without
the prothrombin 20210 G→A variant. If these findings are confirmed in
other studies, screening for the prothrombin variant may permit a better assessment
of the risks and benefits associated with HRT in postmenopausal women.
For many years, recommendations about the use of hormone replacement
therapy (HRT) in postmenopausal women have been based largely on observational
studies, which suggest that HRT reduces the risk of coronary heart disease.1-3 The likely mechanisms
are numerous4 and include the beneficial effects
of estrogens on lipids.5 But estrogens are
also known to be prothrombotic.6 In men with
prostate cancer or cardiovascular disease7,8
and in women on oral contraceptives,9 high
doses or potent formulations of estrogens are associated with thrombotic complications,
including myocardial infarction (MI), stroke, and venous thrombosis. In postmenopausal
women, HRT is also a risk factor for venous thrombosis.10-13
The results of the Heart and Estrogen/progestin Replacement Study (HERS)
have renewed interest in the potential adverse effects of HRT.12,13
In this randomized clinical trial of secondary prevention, combined HRT was
no better than placebo at preventing coronary events in postmenopausal women
(relative risk [RR], 0.99; 95% confidence interval [CI], 0.81-1.22). In post-hoc
analyses, treatment was associated with early harm during the first year of
follow-up (RR, 1.52; 95% CI, 1.01-2.29) and a late benefit during follow-up
years 4 and 5 (RR, 0.75; 95% CI, 0.50-1.13). One broad hypothesis offered
to explain this pattern of risks was the possibility of "an immediate prothrombotic,
proarrhythmic, or proischemic effect of treatment that is gradually outweighed
by a beneficial effect on the underlying progression of atherosclerosis."12,13 According to this interpretation,
a subgroup—perhaps defined by a clinical characteristic, an environmental
exposure, or a genetic trait—is susceptible to an early adverse effect
of estrogens while the rest of the population benefits from estrogen therapy.
When a therapy produces effects that differ by more than chance variation
in 2 subgroups, an interaction is present.
Genetic variants are excellent candidates for such interactions. Vandenbroucke
et al14 have described an interaction between
oral contraceptives and factor V Leiden on the risk of deep venous thrombosis.
While factor V Leiden and the prothrombin 20210 G→A variant are clearly
associated with the risk of venous thrombosis,15
reports of an association between these prothrombotic variants and coronary
heart disease have been inconsistent, some finding an increased risk,16-19 while
others do not.20-24
Whether HRT places women with either of these 2 prothrombotic variants at
an especially high risk of MI remains unknown. Before the results of HERS
were published or known to us, we initiated a population-based, case-control
study to assess this interaction as an a priori hypothesis.
The setting for this project was Group Health Cooperative (GHC), a Seattle,
Wash–based health maintenance organization with an enrollment of more
than 400 000. The methods have been described previously25,26
and will be summarized only briefly here. The study was reviewed and approved
by human subjects committees at both GHC and the University of Washington,
Seattle. All subjects gave written informed consent before initiating the
study. During the period of this study, the preferred oral estrogens at the
GHC were esterified estrogens.
Cases were female GHC enrollees who survived an incident MI between
January 1995 and December 1998. Potential cases were identified from 2 sources:
(1) the computerized discharge abstracts for the 2 GHC hospitals; and (2)
the GHC claims databases, which include bills for all services provided by
non-GHC physicians and health care facilities. We have used and evaluated
similar methods in previous case-control studies.25-28
Due to different funding sources, cases were stratified on hypertension status
as assessed by the computerized pharmacy database. Controls were a stratified
random sample of postmenopausal female GHC enrollees sampled from the GHC
computerized enrollment files on the basis of person-time.29
Controls were frequency matched to the cases by age (within decade), calendar
year, and hypertension status at a ratio of approximately 3 to 1. Controls
met the same eligibility criteria as the cases, but they had not had an MI.
Grants from the National Institutes of Health and the American Heart Association
funded primary data collection on cases with and without hypertension, respectively.
All women had an index date. For the cases, the index date was the date
of admission for the first acute MI; and for the controls, the index date
was a computer-generated random date within the same calendar year for which
they had been chosen as controls. For all women, we only collected risk factor
data available before the index date. This approach ensured that cases and
controls met the same eligibility criteria. All women were aged 30 to 79 years
at their index dates. For all subjects, we excluded women who were members
of GHC for less than 1 year or who did not have at least 4 physician visits
prior to their index dates; women who had had a prior MI; and women who were
not postmenopausal. Additionally, we excluded cases whose index event was
a complication of a procedure or a surgery.
Data collection included a review of the GHC outpatient medical record,
a telephone interview, and a venous blood sample from women who consented
to participate. Based on the medical record, research assistants determined
eligibility and collected information about the following traditional risk
factors for coronary heart disease: blood pressure and pulse; height and weight;
cholesterol level, smoking status, family history, hysterectomy status, marital
status, and use of health services; medical conditions such as angina, hypertension,
diabetes, congestive heart failure, stroke, and peripheral vascular disease.
Cardiovascular disease was defined as a history of angina, stroke, claudication,
or vascular procedures, including coronary bypass, angioplasty, carotid endarterectomy,
or peripheral vascular bypass. Research assistants were not blinded to case-control
The GHC computerized pharmacy database was used to assess current HRT
use at the index date. Since 1976, the GHC pharmacy database has included
a record for all prescriptions dispensed to GHC enrollees. Each pharmacy record
includes a patient identifier, the drug type and dose, the date, the quantity
dispensed, and dosing instructions. For determining current use, we searched
the pharmacy data for the hormone prescription immediately preceding the reference
date. When a woman (who was at least 80% compliant) received enough pills
to last until her index date, she was counted as a current user; otherwise,
she was counted as a nonuser. For 80% compliance, a woman who received 100
pills with instructions to take 1 pill per day was counted as a current user
for 125 days (from 100/0.8) after the prescription dispensing date. Current
progestin use was defined in the same way. In preplanned sensitivity analyses,
we reanalyzed data that defined current HRT use assuming 100% rather than
80% compliance. For 100% compliance, a woman who received 100 pills with instructions
to take 1 pill per day was counted as a current user for 100 days after the
prescription dispensing date. Only users of oral estrogens with or without
progestins were classified as users.
A blood specimen was drawn from the antecubital vein into tubes containing
edetic acid and kept at 4°C until the blood was initially processed. Buffy
coats were prepared, washed in saline, and stored at −70°C. Specimens
were shipped on dry ice to the laboratory in Leiden, the Netherlands. The
DNA was extracted from the white blood cells using standard salting-out procedures.30 The status of the prothrombin variant (20210 G→A)
was assessed by the presence of a HindIII restriction
site in the polymerase chain reaction fragment.31
The presence of factor V Leiden (1691 G→A) was assessed by the loss of
an MnlI restriction site as originally described
by Bertina et al.32 Laboratory personnel were
blinded to case-control status and to HRT status.
In comparing case and control characteristics, we used the t test for continuous variables, the χ2 test for categorical
variables, and the Fisher exact test. Because of the stratified sampling,
the analysis was stratified on hypertension status. Odds ratios (ORs) and
CIs were estimated in the standard way,33,34
and logistic regression was used for multivariable analysis. The ORs estimating
the association between HRT and MI were also calculated separately in the
2 strata defined by genotype: (1) women with a prothrombotic variant (susceptible
women); and (2) women with the wild-type (normal) genotype (nonsusceptible
women). All statistical tests were 2-tailed.
For the primary analysis, we classified women as current or not current
users of HRT at their index dates by the 80% compliance method. The current
users were compared with the noncurrent users. Women with a prothrombotic
variant were compared with women with the wild-type genotype. Formal tests
for interaction were carried out with both case-control and case-only methods,35,36 which are more efficient and powerful
than case-control methods.37 Both methods estimate
the synergy index (SI), which is a ratio of the OR in the susceptible women
to the OR in the nonsusceptible women. An SI of 1 means that the ORs in the
2 subgroups were the same and that there was no interaction on the multiplicative
scale; a SI of greater than 1 means that the joint effect of gene and the
drug were supramultiplicative compared with their expected effect, which is
the product of their individual effects.
Of the 955 women, 108 cases and 387 controls had hypertension, and 124
cases and 336 controls were in the stratum without hypertension. The control-to-case
matching ratio was higher in the hypertensive (3.6:1) than in the nonhypertensive
(2.7:1) women. Genotype assays were available for 950 women for factor V Leiden
and for 953 women for the prothrombin variant. Factor V Leiden was present
in 23 hypertensive and 25 nonhypertensive women, and no homozygotes were present.
The prothrombin variant was present in 15 hypertensive and 15 nonhypertensive
women. One additional nonhypertensive control was homozygous for the variant
Within each stratum, frequency matching produced a control group with
a mean age close to that of the cases (Table 1). In both the hypertensive and the nonhypertensive strata,
diabetes, smoking, physical activity, cholesterol, high-density lipoprotein
cholesterol, glucose, family history, and a history of angina differed between
cases and controls in the expected manner. Among nonhypertensive women, weight
and systolic and diastolic blood pressure also differed significantly between
cases and controls. Among the hypertensive women, weight, systolic blood pressure,
and mean time enrolled in GHC were similar in the cases and controls.
Table 2 summarizes the main
effects of HRT, factor V Leiden, and the prothrombin variant on MI risk. Among
hypertensive women, current HRT use and factor V Leiden were only weakly associated
with MI risk. On the other hand, the prothrombin variant was associated with
an increased risk of MI in hypertensive women (OR, 4.32; 95% CI, 1.52-12.1);
after adjustment for covariates, the OR increased to 7.02 (95% CI, 2.27-21.8).
Among nonhypertensive women, there was little association of HRT or either
prothrombotic variant with case-control status, before or after adjustment
Table 3 summarizes the stratified
analyses for the prothrombin variant. Hypertensive women who had the wild-type
genotype for prothrombin and who were not current users of HRT served as the
reference group. Among women with the wild-type genotype, HRT use was associated
with a small reduction in the risk of nonfatal MI (OR, 0.89; 95% CI, 0.56-1.42).
Compared with the reference group, women who had the prothrombin variant and
who were not current users of HRT had only a small increase in the OR to 1.45
(95% CI, 0.28-7.66). In the absence of an interaction (on a multiplicative
scale), the expected joint effects of the prothrombin variant and current
HRT use would be 1.29. Compared with the reference group, the 8 women who
had the prothrombin variant and who were current users of HRT had an almost
11-fold increase in the risk of MI (OR, 10.9; 95% CI, 2.15-55.2; P = .002).
The analyses stratified on susceptibility also appear in Table 3. Among the 478 nonsusceptible, hypertensive women (with
prothrombin wild type), HRT use was associated with a slight decrease in the
risk of MI (OR, 0.89). Among the 15 susceptible, hypertensive women (with
prothrombin variant), HRT use was associated with an increased risk of MI
(OR, 7.50; 95% CI, 0.76-74.2; P = .10). The case-control
estimate of the SI was 8.38 (95% CI, 0.81-86.8; P
= .06). The case-only estimate of the SI was similar at 5.57 (95% CI, 1.07-29.1; P = .03) and indicated a significant interaction between
HRT and the prothrombin variant on the risk of nonfatal MI among hypertensive
women. When the analysis was restricted to whites only (data not shown), the
case-control SI was 8.62 (95% CI, 0.83-89.4); the corresponding case-only
SI was 5.38 (95% CI, 1.03-28.15). When we assumed 100% rather than 80% compliance,
the evidence of an interaction became more pronounced with a case-control
SI of 23.9 (95% CI, 1.61-354; P = .008).
Among women without hypertension, no cases had the prothrombin variant
and used HRT. There was no evidence of an interaction. Combining estimates
from these 2 strata, defined by hypertension status, did not appear to be
appropriate. If hypertensive and nonhypertensive women were combined into
a single group, the interaction would have been less pronounced (SI, 2.0;
95% CI, 0.45-8.95).
Table 4 summarizes the data
for factor V Leiden in the same format used for the prothrombin variant. Again,
women who were not current users by the 80% compliance method and who did
not have factor V Leiden served as the reference group. Individually or jointly,
there was little association between HRT use or factor V Leiden and MI risk.
None of the CIs for these associations excluded the null hypothesis. The SIs
estimated by both the case-control and the case-only methods were small and
did not differ from the null hypothesis of no interaction.
The small number of hypertensive women with the prothrombin variant
(n = 15) limited the ability to adjust for potential confounding factors. Table 5 summarizes the effect of adjustment
for a number of covariates one at a time. The unadjusted OR estimates are
the same as in Table 3: 0.89 for
HRT use, 1.45 for the prothrombin variant, and 10.9 for the combination of
HRT use and the prothrombin variant. Adjustments for age, calendar year, race,
smoking, diabetes, cardiovascular disease, systolic blood pressure, or cholesterol
each had little effect on the point estimates. The lowest adjusted OR for
the combination of HRT use and the prothrombin variant was 10.4, and the lower
limit of the 95% CIs never went below 1.97.
Table 6 summarizes the findings
of a series of analyses that attempted to identify other potential interactions
with the prothrombin variant on the risk of MI, primarily among women with
hypertension. The SIs for HRT use at 80% and 100% compliance were 8.38 (95%
CI, 0.81-86.8) and 23.9 (95% CI, 1.61-354), respectively. No other characteristic
was associated with such an elevated SI. For most of the characteristics,
the ORs for MI risk were similar among those with and without the prothrombin
variant. For the association between cardiovascular disease (such as angina)
and MI risk, the OR was higher among those with the prothrombin variant (OR,
6.00; 95% CI, 0.48-75.4) than among those without the prothrombin variant
(OR, 2.92; 95% CI, 1.79-4.78). The SI for cardiovascular disease was 2.05
(95% CI, 0.16-27.0). Zero entries precluded the ability to examine an interaction
for smoking and for diabetes in the hypertensive women, but there was no evidence
of an interaction in the population as a whole.
Among the controls, 85.7% of HRT users were taking esterified estrogens,
and most of the others were taking conjugated estrogens. Estrogen patches
were rarely used at GHC (2 cases and 2 controls). Among the 8 hypertensive
women currently taking HRT and having the prothrombin variant, the 6 cases
had a slightly smaller mean number of lifetime prescriptions for estrogens
than the 2 controls (31 vs 39; P = .71). One of the
6 cases had started HRT 7 months prior to her MI while the other 5 had been
regular users for a least 1 year before their MI event. Among the 6 hypertensive
cases who had the prothrombin variant and who were current users of HRT, all
6 were using esterified estrogens; 5 of the 6 were taking estrogen alone rather
than in combination with a progestin; and 5 were taking 0.625 mg/d and 1 was
taking 0.3 mg/d. The antihypertensive medications used by these 6 women were
similar to the agents typically used at GHC, and 3 of the 6 were taking atenolol.
In this population-based, case-control study, the prothrombin 20210
G→A variant was a risk factor for MI among hypertensive women. There
was also a significant interaction between the use of HRT and the prothrombin
variant on the risk of MI among women with hypertension. Compared with the
reference group, the 8 women who were current HRT users and who had the prothrombin
variant had a nearly 11-fold increase in the risk of a nonfatal MI (OR, 10.9;
95% CI, 2.15-55.2). The 95% CI of the SI as assessed by the more powerful
case-only method, a formal test for a multiplicative interaction, excluded
the null hypothesis (Table 3).
The interaction was more pronounced for the 100% compliance method than for
the 80% compliance method of defining current HRT use (Table 3). No such interaction was found for nonhypertensive women
(Table 3) or for factor V Leiden
in either the hypertensive or the nonhypertensive women (Table 4). Among the hypertensive women, the estimates were affected
only in trivial ways by adjustment for potential confounding factors (Table 5). The interaction with the prothrombin
variant was specific to HRT use and not to other characteristics (Table 6).
The prothrombin variant has been associated with an increased incidence
of venous thrombosis and with elevated levels of prothrombin in plasma.31 In other observational studies, the prothrombin variant
has been associated with the incidence of MI in some studies17-19
but not in others.20,22-24
While previous studies have identified potential interactions of oral contraceptive
use with factor V Leiden14 and of HRT use with
activated protein C resistance38 on the incidence
of venous thrombosis, there are no previous epidemiologic reports of an interaction
between HRT use and prothrombotic variants on the risk of MI. The only cross-sectional
analysis, conducted in women with hyperlipidemia, assessed HRT use at the
time of the clinic visit rather than at the time of the cardiovascular event.39
This study had a number of limitations. Due to the low prevalence of
the prothrombin variant, it was not possible to adjust simultaneously for
multiple potential confounding factors in a single model.40
While the healthy user effect is often invoked to explain the association
between HRT use and a decreased risk of cardiovascular events,41,42
such biases are less likely to be important in studies of genetic variants
as risk factors. In addition to the possibility of confounding, potential
alternative explanations for the findings of genetic association studies include
linkage disequilibrium and population admixture. The results were also based
on a small number of women with the genetic variant, so there remained considerable
statistical uncertainty around the risk estimates reported in this study.
For both the prothrombin variant and factor V Leiden, we had expected,
but did not find, similar interactions with HRT use. The cases in this study
all represent survivors of an MI, and it is possible that the interaction
may affect mortality rather than disease incidence. If, for instance, the
joint effects of factor V Leiden and HRT use are associated with a high case-fatality
rate, a case-control study of nonfatal MI would fail to detect an interaction.
On the other hand, survival bias is unlikely to induce the interaction seen
with the prothrombin variant and HRT use in this study.
For the prothrombin variant, the findings of an interaction among those
with hypertension were not confirmed by the findings among those without hypertension.
Several explanations, including chance, are possible. Three of the 6 hypertensive
cases who had the prothrombin variant and who were HRT users were also taking
the β-blocker atenolol, which may have improved their post-MI survival.
In hypertensive patients, moreover, the presence and the severity of target
organ disease is strongly associated with several prothrombotic abnormalities,
including elevated levels of D-dimer.43,44
As we have previously hypothesized,6 the presence
of subclinical or clinical cardiovascular disease, such as angina or hypertensive
disease, may be important in initiating the prothrombotic effects of estrogens,
including any potential interaction with the prothrombin variant. Rosendaal15 has described the risk of venous thrombosis in terms
of multiple interacting causes, and a similar model may be relevant for arterial
In this case-control study, which excluded women with a previous MI,
the interaction with the prothrombin variant was associated with current use
of HRT rather than with recent initiation of therapy as in HERS. If a prothrombotic
third factor such as coronary atherosclerosis or hypertensive target organ
disease is also required,43,44
the onset of the thrombotic event may be delayed until that third factor progresses
far enough or becomes severe enough, in the presence of the prothrombin variant
and HRT use, to precipitate an MI. If this third-factor hypothesis is true,
the findings of this study would be expected to differ from those of HERS
in terms of the time relationship between MI events and the initiation of
HRT. HERS was a secondary prevention trial that enrolled only women with clinical
coronary disease so that the hypothetical third factor was already present
from the outset when participants were randomized and began taking HRT.
Among hypertensive controls, the prothrombin variant was present in
only 1.8% of women. An uncommon susceptibility factor, such as the prothrombin
variant or another associated with a high risk of events among HRT users,
might account in part for the pattern of early harm and late benefit seen
in the HERS trials.12,13 Simulation
studies suggest that the putative unknown susceptibility factor has to exhibit
both a prevalence of 3% to 5% and a risk ratio of 13 to 25 in HRT users to
reproduce the pattern of early harm and late benefit seen in HERS. While the
prothrombin variant is a good candidate, other susceptibility factors are
also likely to be important.
The long-term goal of research in the area of pharmacogenetics is to
help clinicians individualize treatment for their patients and select drug
therapies that maximize either effectiveness, or safety, or both. If the HERS
findings are indeed the result of an interaction between HRT and a susceptibility
factor, there is an urgent public health need to identify the putative susceptibility
factor. Based on their biology and prevalence, the prothrombin variant and
factor V Leiden were both reasonable candidates. The findings of this study
suggest the possibility of an interaction between the prothrombin variant
and HRT use on the incidence of MI among women with hypertension, but they
need to be confirmed in other settings. If the findings are confirmed, or
if other drug-gene interactions are identified, clinicians may eventually
screen postmenopausal women for selected genetic variants that help characterize
a woman's expected risk or benefit from HRT for a variety of outcomes, including
MI, stroke, and venous thrombosis.