© 1997 American Heart Association, Inc.
Articles |
High Prevalence of Hyperhomocysteinemia and Asymptomatic Vascular Disease in Siblings of Young Patients With Vascular Disease and Hyperhomocysteinemia
From the Institute for Cardiovascular Research, Vrije Universiteit, Amsterdam, The Netherlands (S.C. de J., C.D.A.S., A.J.C.M., M. van den B., F.C.V., J.A.R.) and the Departments of Surgery, (S.C. de J., A.J.C.M., M. van den B., E.J.B., J.A.R.), Internal Medicine, (C.D.A.S.), Cardiology, (F.C.V., J.B.), Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands
Correspondence to Dr S.C. de Jong, Department of Surgery, Academisch Ziekenhuis Vrije Universiteit, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
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Abstract |
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Abstract Hyperhomocysteinemia (HHC) is associated with an increased risk of atherosclerotic vascular disease and may be inherited. Fasting and postmethionine HHC are independent risk factors that overlap to a limited extent. To study the familial occurrence of HHC, we investigated the prevalence of HHC (both fasting and after methionine) among 450 siblings of 167 consecutive young patients with vascular disease and postmethionine HHC. Furthermore, all subjects with postmethionine HHC (n=125) were invited for noninvasive vascular testing; 101 (80.8%) agreed. Of those with a normal postmethionine plasma level (n=325), we randomly selected 73 subjects for further studies; 53 agreed (72.6%). Thus, a total of 154 siblings underwent ultrasonography of the carotid arteries, measurement of ankle-brachial pressure indices at rest and after a treadmill exercise test, and exercise electrocardiographic stress testing. We observed HHC after methionine, fasting, or both, in 27.8% (95% CI, 23.7 to 31.9), 11.1% (CI, 8.2 to 14.0) and 8.7% (CI, 6.1 to 11.3) of the siblings. Abnormal peripheral, coronary, or carotid artery tests were observed in 35.7% (CI, 28.1 to 43.3), 7.1% (CI, 3.0 to 11.2), and 7.1% (CI, 3.0 to 11.2). Univariate and multivariate analyses revealed weak evidence of a relationship with homocysteine levels.
In conclusion, we found a high prevalence of HHC and asymptomatic vascular disease in siblings of young patients with vascular (mainly peripheral arterial) disease and HHC. Our data raise the possibility that homocysteine does not play a major role in the early, asymptomatic phases of vascular disease, at least among siblings of young patients with vascular disease.
Key Words: hyperhomocysteinemia • vascular testing • vascular disease
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Introduction |
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Hyperhomocysteinemia, whether in the fasting state or after an oral methionine load, is associated with an increased risk of atherosclerotic vascular disease and thromboembolism.1,2 Fasting and postmethionine HHC are independent risk factors that overlap to a limited extent.3 In young patients with vascular disease, the prevalence of postmethionine HHC is high.1,4 In 1992, pooled data revealed a prevalence of postmethionine HHC of 32%, 24%, and 21% in young patients with peripheral, cerebral, and coronary arterial occlusive disease.5,6
Fasting plasma homocysteine levels are genetically influenced, as shown by studies in twins7 and in siblings of young patients with coronary arterial occlusive disease8-10 in whom the prevalence of fasting HHC is 12% to 14%.9,10 Information on the familial occurrence of postmethionine HHC, however, is limited.11,12 Recently, Franken et al have reported fasting and postmethionine HHC in 21% and 32% of 96 family members of 21 young patients with vascular disease and postmethionine HHC.12 In addition, no data exist on the prevalence of vascular disease among the clinically healthy members of these families.
In view of these considerations, we investigated the prevalence of HHC (both fasting and after methionine) among 450 siblings of 167 consecutive young patients with vascular disease and postmethionine HHC. Furthermore, using noninvasive tests13-18 that have prognostic value with regard to the development of vascular disease,19-21 we studied the prevalence of early signs of peripheral, carotid, and coronary arterial disease and analyzed whether this was related to the presence of HHC.
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Methods |
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Selection of Siblings
In April 1993, we started a HHC screening program among consecutive patients with clinically manifest peripheral, cerebral, and/or coronary arterial occlusive disease with onset before the age of 55 years or a history of obstetrical complications. Peripheral arterial occlusive disease was defined by the presence of intermittent claudication, confirmed by an ankle/arm index <0.9 and/or a decrease of >0.15 of the index after treadmill testing, or ischemic rest pain, gangrenous ulcers, or amputation for ischemia. Cerebral arterial occlusive disease was defined by the presence of symptomatic cerebral vascular disease (ischemic stroke or transient ischemic attack, WHO [World Health Organization] clinical definitions) and, in the case of stroke, confirmed by CT scan. Coronary arterial occlusive disease was defined as the presence of myocardial infarction (WHO clinical definition plus new Q waves on electrocardiography and/or diagnostic enzyme changes). Obstetric complications were defined as multiple spontaneous abortions, solutio placentae, and intrauterine fetal death.
From April 1993 to January 1995, we screened 737 patients, 167 of whom
had postmethionine HHC: 96, 34, and 35 with peripheral,
cerebral, and coronary arterial occlusive disease,
and 2 with a history of obstetric complications. The siblings (n=652;
[mean, 3.9; range, 1 to 13], 628 of whom were alive at the time of
our study) of these 167 patients were then invited for further studies;
475 (72.9%) agreed to participate. We excluded siblings who had a
history of venous thrombosis, myocardial infarction, stroke, or
peripheral arterial occlusive disease; those
<18 years or 
65 years of age; pregnant women or women who were
planning pregnancy; and those who had impaired renal (serum
creatinine >150 µmol/L) and/or liver
function (abnormal serum transaminase concentrations and/or presence of
physical signs). Thus, a total of 25 (5.6%) siblings were
excluded.
All other siblings (n=450; 167 families) underwent a methionine loading test. None used vitamin supplements. We subsequently invited all subjects with postmethionine HHC (n=125; 27.8%) for noninvasive vascular testing; 101 (80.8%) agreed. Of those with a normal postmethionine plasma homocysteine level (n=325; 72.2%), we randomly selected 73 subjects for further studies, 53 (72.6%) of whom agreed. Eventually, 154 siblings from 66 different families underwent noninvasive tests. All siblings gave informed consent for these studies, which were approved by the local ethics committee.
Methionine Loading Test and Other Clinical and Laboratory
Data
After an overnight fast, venous blood samples were taken between
9 and 10 AM. Subjects were asked to refrain from smoking and from using
alcohol from 10 PM. on the evening before blood sampling. A second
blood sample was obtained 6 hours after an oral methionine load (0.1
g/kg body weight). Plasma samples were stored at -30°C until
use. Plasma levels of homocysteine were determined within 1 week from
blood sampling and measured as total homocysteine by using high
pressure liquid chromatography with
fluorescence detection.22 Reference
values for fasting and postmethionine homocysteine in our laboratory
are <18 and <54 µmol/L in men, <15 and <51
µmol/L in premenopausal women, and <19 and <69
µmol/L in postmenopausal women.23
We recorded age, body weight, height, menopausal status (postmenopause was defined as absence of menstrual bleeds for >1 year), current and past smoking habits, the presence of diabetes mellitus (WHO criteria), and the presence of hypertension and hypercholesterolemia (both as diagnosed by a physician). Body mass index was calculated as weight divided by height2.
Just before the methionine loading test, venous blood samples were taken for measurement of serum lipids (total and high density lipoprotein cholesterol and triglycerides [enzymatically]), glucose (glucose oxidase method), vitamin B6 (by fluorescence high-pressure liquid chromatography), vitamin B12 (radioassay, Becton Dickinson, France), folic acid (radioassay, Becton Dickinson, France), and creatinine (modified Jaffé reaction). All blood samples were stored at -70°C until use.
We also assessed methylenetetrahydrofolate reductase (MTHFR) genotype. DNA was obtained from the buffy coat of EDTA blood. The mutation involves a C to T mutation at nucleotide 677, which converts an alaninine to a valine residue. The alteration creates a HinFI restriction site, which was used for mutation analysis. The PCR conditions and the sequence of the primers used in the amplification of the part of the gene containing the mutation were taken from Frosst et al.24 HinFI restriction enzyme analysis of the PCR products and subsequent electrophoresis in a 3% agarose gel were used to determine the mutation status of the subject. "+" is used to indicate the presence of the mutant allele. From 14 siblings, MTHFR genotype was not available.
Noninvasive Vascular Testing
The vascular laboratory studies were performed by two trained
technicians. Doppler-assisted systolic blood pressure
measurements were taken from the brachial, the posterior tibial, the
dorsalis pedis, and the peroneal artery on both sides, using 12-cm
cuffs which were automatically inflated and deflated (Medasonics
Vaculab). Recordings of the pressures started after a 15-minute
resting period at a room temperature of 23°C. For each leg, an ABPI
was calculated by dividing the highest systolic pressure in the
ankle artery by the highest systolic pressure in the upper arm.
The subjects then walked on a treadmill (4 km/h, 10% slope) for 1
minute, after which the ankle pressure was
measured.14,18 An ABPI was defined as abnormal if
<0.90 in one or both legs at rest.15 A treadmill
exercise test was defined as abnormal if the pressure dropped by
30 mm Hg or more.14,18
Ultrasonography of the carotid arteries was performed using a color Duplex scanner (Acuson 128) with a 7.5 MHz linear transducer in combination with a 5 MHz pulsed Doppler. Subjects were examined supine with the head rotated away from the side under investigation. Recordings were taken from the proximal and distal common carotid, the external carotid, and from the proximal and distal internal carotid arteries on both sides. Hemodynamically significant stenosis was assessed by flow velocity criteria according to the Seattle group,16,25 with one modification: in young subjects (as in this study) an elevated peak velocity (>1.25 m/s) in the common carotid artery may be a consequence of turbulent flow in a normal vessel and was thus considered normal if there were no signs of stenosis on real-time B-mode ultrasonography.26 Each carotid branch was classified as normal or abnormal with systolic peak flow velocity >1.25 m/s as cutoff (which is equivalent to a stenosis of >50%).27,28
Exercise electrocardiographic stress testing was performed on a Quinton
5000 treadmill according to the Bruce protocol.29
All subjects stopped because of exhaustion. The exercise test was
reviewed by two cardiologists. The interobserver agreement was 80% and
after further review 100%. An abnormal test result was defined as a
horizontal ST-segment depression of 1 mm at 60 m/s beyond the
J-point, a downsloping ST-segment depression of 1 mm at the J-point,
and/or an upsloping ST-segment depression of 2 mm at 80 ms beyond the
J-point on any of the 12-lead ECGs obtained at 1-minute intervals
during exercise and for the first 5 minutes of the recovery period,
versus the baseline recording.29
All tests were interpreted while "blinded" to other clinical and laboratory data.
Statistical Analysis
Data were analyzed using the statistical package SPSS
for Windows 6.1. Descriptive data are given as mean (SD). Skewed data
were logarithmically transformed. Continuous variables were tested
by Student's t-test (for means). Percentages were compared
by 
2 tests.
We assessed the prevalences of HHC (fasting, postmethionine, or both) and of abnormal vascular tests among the siblings. We then used univariate analyses to study the relation between results on noninvasive vascular testing and possible determinants thereof: fasting and postmethionine homocysteine levels, presence of fasting or postmethionine HHC, delta homocysteine levels (defined as the difference between postmethionine and fasting homocysteine levels), MTHFR genotype, serum vitamin levels, and other cardiovascular risk factors (age, sex, smoking habits, body mass index, blood pressure [as a continuous variable or as presence versus absence of hypertension], serum lipids, presence or absence of hypercholesterolemia, serum glucose, and type of vascular disease in the index patient). Variables that had a probability-value of <.2 in the univariate analyses were entered (forward and backward) into a multivariate logistic model to examine whether the relationship between outcomes on vascular testing and homocysteine and other cardiovascular risk factors were modulated. In the multivariate models, homocysteine, blood pressure, and cholesterol, when used as variables, were entered either as a continuous variable or as presence versus absence of the variable. Furthermore, MTHFR genotype, when used as variable, was never entered into the same multivariate model as homocysteine, vitamin B6, vitamin B12, or folic acid. We also investigated whether interactions between homocysteine and other cardiovascular risk factors modulated the relations between homocysteine and outcomes on noninvasive vascular testings. All odds ratios (ORs) are given with their 95% confidence interval (CI) in parentheses, and contrast the odds in those with versus those without the risk factor of interest (eg, hypertension) or are expressed per unit change of the risk factor (eg, per 1 year of age and per 1 µmol/L of plasma homocysteine). All testing was two-tailed with 0.05 as the level of significance.
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Results |
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Among the 450 siblings (Table 1
),
136 (30.2%; CI, 26.0 to 34.4) had HHC (postmethionine and/or fasting);
125 (27.8%; CI, 23.7 to 31.9) had postmethionine HHC, 50 (11.1%; CI,
8.2 to 14.0) had fasting HHC, and 39 (8.7%; CI, 6.1 to 11.3) had both
(P<.05 for the comparison of postmethionine versus fasting
or both fasting and postmethionine HHC). Fasting HHC without
postmethionine HHC was found in 11 siblings (2.4%; CI, 1.0 to 3.8) and
postmethionine HHC without fasting HHC was found in 86 siblings
(18.9%; CI, 15.3 to 22.5) (Fig 1).
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We performed noninvasive vascular tests in 154 siblings (Table 2). The siblings with postmethionine HHC
who underwent vascular testing were older than those who did not (44.5
versus 37.3 years; P<.001), but did not differ importantly
with respect to other cardiovascular risk factors (data
not shown). The siblings with normal postmethionine homocysteine levels
who underwent vascular testing, compared with those who did not, were
older (47.9 versus 42.1 years; P<.001) and had higher
fasting (11.6 versus 10.0 µmol/L; P=.002) and
postmethionine homocysteine levels (40.9 versus 37.5
µmol/L; P=.031) but were similar with respect to
other cardiovascular risk factors (data not shown).
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The prevalence of 1 abnormal result on the noninvasive vascular
testing was 43.5% (CI, 35.7 to 51.3). Abnormal results on
peripheral, carotid and coronary artery testing
were observed in 35.7% (CI, 28.1 to 43.3), 7.1% (CI, 3.0 to 11.2) and
7.1% (CI, 3.0 to 11.2) (Fig 2).
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Table 3 shows that the siblings who had
one or more abnormal results on vascular testing had lower fasting
homocysteine levels, a higher prevalence of hypertension, higher
diastolic blood pressures, and higher serum
triglyceride levels. Postmethionine homocysteine levels did
not differ between the groups (Table 3).
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Univariate and multivariate
analyses showed that abnormal results on
peripheral, carotid, and coronary artery testing
were significantly associated with, respectively, smoking and blood
pressure, blood pressure, and serum cholesterol (Table 4). Multivariate
analyses indicated that the relationships with postmethionine
and fasting homocysteine levels were not significant.
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Univariate and multivariate analyses with abnormal results on vascular testing as dependent variable and vitamin levels or MTHFR genotype as independent variable did not reveal any significant relationships (data not shown).
Our study included only two siblings of index patients with an
obstetric history (Table 3
). Omitting these siblings from the
analyses did not materially affect the results (data not
shown).
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Discussion |
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This large study shows that, among the clinically healthy siblings of young patients with atherosclerotic, mainly peripheral vascular disease and elevated levels of homocysteine after methionine loading, (1) the prevalence of postmethionine and fasting HHC is high (28 and 11%), as is (2) the prevalence of asymptomatic vascular disease, with 36%, 7% and 7% having an abnormal result on noninvasive testing of the peripheral, carotid, and coronary arteries, respectively, and 44% having at least one abnormal test result. Remarkably, there was no clear association between the presence of HHC and that of asymptomatic vascular disease.
We found fasting HHC in 11% of the siblings, which is similar to previous reports of families with premature coronary arterial occlusive disease (12% to 14%).9,10 Only one other study has reported the prevalence of HHC in families of patients with vascular disease and postmethionine HHC, and found HHC postmethionine, fasting, and both in 32%, 21% and 19%.12 The estimates in our study were similar for postmethionine HHC (28%), but lower for fasting and both postmethionine and fasting HHC (11% and 9%). These differences may be related to differences in study design, because Franken et al,12 who studied patients with arterial and venous disease, did not include consecutive patients and did not attempt to screen the entire family of the index patient as we did but performed methionine loading tests at the individual requests of family members. Even if we assume that all siblings who either died from, or had, vascular disease and were excluded from our study (n=34) had fasting HHC, the prevalence of fasting HHC (17%) would still be considerably lower than that in Franken's study. Thus, our results are likely to have been less affected by selection bias and to be more representative of families of young patients with arterial disease and postmethionine HHC.
We observed an abnormal result on peripheral artery testing
in 36% of the siblings, who had a mean age of 46 years. The Edinburgh
Artery Study found abnormal results of noninvasive vascular testing
(ie, an ABPI <0.9 and/or a reactive hyperemia-induced pressure
reduction of >20%) in 8% of the general population aged 55 to 74
years.17 Criqui et al found the prevalences of
large and small-vessel peripheral arterial
disease to be 3 and 10% of 158 subjects aged 38 to 59
years.30 A cross-sectional study performed among
a 50- to 74-year-old Caucasian population showed that 18.1% of the
subjects had signs of any peripheral arterial
disease (ankle-brachial pressure index <0.9, at least one monophasic
or absent Doppler flow curve or vascular
surgery).31 In a study of vascular disease in
relation to cystathionine-ß-synthase deficiency, 13 heterozygote
subjects (mean age, 46 years) had evidence of peripheral
arterial occlusive disease, ie, an ABPI <0.97 or
Doppler evidence of stenosis, in 8% and
54%.26 Our data indicate that the prevalence of
abnormal results on peripheral arterial testing
among siblings of young hyperhomocysteinemic patients with vascular
disease, two-thirds of whom had peripheral artery disease,
is much higher than that in the normal population, but, from the
limited data available,26 appears comparable to
that in subjects with heterozygote cystathionine-ß-synthase
deficiency. An abnormal result on carotid artery testing, equivalent to
a stenosis >50%, was observed in 7% of the siblings. A
population study in Finnish men (age range, 42 to 60) found
extracranial carotid stenosis of 20% or more by
ultrasonography in 3%.32 In 348
asymptomatic volunteers attending a health screening
program (age range, 24 to 91), 4 and 1% had extracranial carotid
artery stenoses greater than 50% and 80%,
respectively.33 A cross-sectional study performed
among a 50- to 74-year-old Caucasian population showed severe (50%)
stenosis by duplex scanning in 2.8%.34
Among 13 subjects with heterozygote cystathionine-ß-synthase
deficiency, carotid stenoses of <50% were frequently observed
(in 45%), but stenoses of 50% were never
found.26 These studies indicate that the
prevalence of carotid stenosis observed in our study is higher
than would be expected in the general population. We found an abnormal
result on electrocardiographic stress testing in 7% of the siblings.
This estimate does not appear to be higher than that expected in the
general population of comparable age, in whom prevalences varying from
5% to 13% have been reported in several large
studies.13,35,36
Therefore, it is likely that the prevalences of abnormal results on carotid and peripheral artery testing are considerably higher than would be expected in asymptomatic young persons. To our knowledge, no prior study has performed noninvasive tests in relatives of young vascular patients with postmethionine HHC. Nevertheless, such high prevalences are not unexpected in view of evidence indicating that, among healthy middle-aged subjects, a positive family history of vascular disease is independently predictive of death from all causes and from cardiovascular and ischemic heart disease.37 The prognostic value of noninvasive tests with regard to the development of vascular disease has been established.19-21,38 The results of our study thus suggest that siblings of young patients with vascular disease and postmethionine HHC have an increased risk of development of symptomatic vascular disease.
In general, classic risk factors showed the expected relationships with abnormal test results. Thus, abnormal results on peripheral, carotid and coronary artery testing were associated with, respectively, smoking and blood pressure, blood pressure, and serum cholesterol. Remarkably, there was at most weak evidence of a relationship with homocysteine levels. Univariate and multivariate analyses showed a weak, nonsignificant relation between abnormal coronary artery testing and postmethionine HHC and no relation between abnormal peripheral or carotid artery testing and HHC (by any measure).
Prospective studies have shown high fasting (or random) homocysteine levels to be associated with an increased risk of myocardial infarction and stroke;39,40 case-control studies suggest that both high fasting (or random) and high postmethionine homocysteine levels increase the risk of cerebrovascular, coronary, and peripheral artery disease.41-44 These studies have mainly been performed in middle-aged and elderly subjects, in whom atherothrombotic disease is likely to have a multifactorial etiology. Therefore, such studies cannot show whether hyperhomocysteinemia initiates atherosclerosis or accelerates its progression. Our finding of, at most, a weak relationhip between hyperhomocysteinemia and the presence of vascular disease raises the possibility that hyperhomocysteinemia accelerates the progression of atherothrombotic disease rather than initiating it. This hypothesis is compatible with our previous finding of a significant relation between homocysteine levels and the severity of atherosclerosis in young patients with clinical vascular disease,23 ie, those who had a more advanced stage of atherosclerosis than the subjects in the present study. The high prevalence of asymptomatic vascular disease in the siblings would then conceivably be related to classic risk factors and an additional unidentified familial factor, which may be weakly related or not related to HHC.
Other explanations can be adduced to explain our findings. First, the
noninvasive tests, although widely used, may yield an estimate of
vascular disease that is too imprecise to find a relation with
homocysteine levels. This is unlikely, because we did find the
expected, statistically significant relations between vascular
pathology and classic cardiovascular risk factors, such
as smoking, hypertension, and
hypercholesterolemia. Second, we cannot exclude
the possibility that measurement of carotid artery intima-media
thickness and brachial artery endothelium-dependent
vasodilatation,45 which are considered sensitive
tests of early atherosclerotic disease, might have yielded higher
prevalences of abnormal test results than the 7% each for carotid and
coronary artery testing that we found and thus might have
increased the statistical power to find a relation with
hyperhomocysteinemia. Nevertheless, lack of statistical power appears
an unlikely explanation for the absence of a relation between
peripheral artery test results and hyperhomocysteinemia, in
view of both the high prevalence of abnormal test results (36%) and
the narrow 95% confidence intervals of the risk estimates (Table 4).
Third, both the normohomocysteinemic and the hyperhomocysteinemic
siblings who underwent vascular testing were older and had fasting HHC
more often than did their family members who did not undergo vascular
testing. Although this may have led to a slight overestimation of the
prevalence of abnormal results on vascular testing, it should not have
biased the relation with homocysteine levels if such a relation
existed. Finally, the normohomocysteinemic siblings who underwent
vascular testing had higher fasting and postmethionine homocysteine
levels than those who did not. This may have reduced our chances of
finding differences between the normohomocysteinemic and
hyperhomocysteinemic siblings, because it narrowed the difference in
homocysteine levels between these groups. However, because the
homocysteine levels actually observed varied over a wide range, we were
able to investigate the association between vascular outcomes and
homocysteine level as a continuous variable. This analysis
led to a similar conclusion.
Our findings suggest that asymptomatic siblings of young patients with vascular disease and postmethionine HHC have a high prevalence of HHC and are at high risk of developing symptomatic vascular disease. Our study clearly shows that HHC is familial, but additional studies are required to investigate whether this is genetically46 or environmentally47 (eg, from similarities in food consumption) determined, or both. Our data further raise the possibility that homocysteine does not play a major role in the early, asymptomatic phases of vascular disease, at least in subjects with a family history of vascular (mainly peripheral artery) disease occurring at a young age. An essential test of the significance of hyperhomocysteinemia in such subjects is to study the effect on progression of asymptomatic lesions of homocysteine-lowering treatment in a randomized, placebo-controlled trial. Such a study is now in progress.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr de Jong is supported by the Praeventiefonds (Prevention Fund Grant No. 282272). Dr Stehouwer is the recipient of a fellowship from the Netherlands Organization for Scientific Research (NWO). The authors would like to thank Dr C Jakobs for measuring homocysteine levels and M. Bischoff and J. Scholma for their technical assistance.
Received June 20, 1997; accepted July 28, 1997.
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