Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:129-136

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carmena, R.
Right arrow Articles by Davignon, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carmena, R.
Right arrow Articles by Davignon, J.
Right arrowPubmed/NCBI databases
*Genetics Home Reference
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:129-136.)
© 1996 American Heart Association, Inc.

Articles

Lp(a) Levels and Atherosclerotic Vascular Disease in a Sample of Patients With Familial Hypercholesterolemia Sharing the Same Gene Defect

Rafael Carmena; Suzanne Lussier-Cacan; Madeleine Roy; Anne Minnich; Arno Lingenhel; Florian Kronenberg; Jean Davignon

From the Endocrine Service, Hospital Clinico Universitario, Valencia, Spain (R.C.); the Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Quebec, Canada (S.L.-C., M.R., A.M., J.D.); and the Institute for Medical Biology and Genetics, University of Innsbruck, Innsbruck, Austria (A.L., F.K.).

Correspondence to Dr S. Lussier-Cacan, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, Canada H2W 1R7. E-mail cacans@ircm.umontreal.ca.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Abstract There is considerable variation in the severity of cardiovascular disease among patients with familial hypercholesterolemia (FH). Some reports have suggested that plasma lipoprotein(a) [Lp(a)] levels may explain such variation and that FH subjects deficient in LDL receptors, especially those with coronary heart disease, tend to have elevated Lp(a) levels. We have investigated the possible role of the LDL receptor in determining plasma Lp(a) levels in a genetically homogeneous FH population and the contribution of Lp(a) to cardiovascular risk. A total of 98 FH subjects and 66 healthy first- and second-degree relatives from 30 families with FH due to the French-Canadian >10-kilobase deletion of the LDL receptor gene were studied. A reference group of 392 normolipidemic French-Canadian participants in a Heart Health Survey was used for comparison. FH subjects were subdivided into subsets of 63 individuals free from atherosclerotic vascular disease (AVD) and 35 individuals with AVD. A complete cardiovascular evaluation was performed, and plasma lipid, lipoprotein, and Lp(a) levels were measured in all subjects in the absence of medication. Apolipoprotein (a) [apo(a)] phenotype was determined in 112 of FH and non-FH subjects. The log-transformed values for plasma Lp(a) were not significantly different among the three groups: 0.98±0.54 (mean±SD) in FH subjects with AVD, 0.89±0.51 in FH subjects without AVD, and 0.82±0.64 in their relatives. The distribution of the apo(a) phenotypes did not differ between the FH and non-FH groups. Comparison of two age- and sex-matched subgroups of FH subjects, with and without AVD, failed to show any differences in Lp(a) level. However, mean Lp(a) log values in the reference group (n=392) were significantly lower than values obtained for the total FH group (0.79±0.57 versus 0.92±0.52, respectively; P<.05) but were not different from those of the unaffected family members. Thus, in our sample, the LDL receptor appears not to influence plasma Lp(a) levels; rather, these levels reflect shared apo(a) genes. The cardiovascular risk in this group of subjects with FH was related to age, male sex, total and LDL cholesterol, and higher apoB but not Lp(a) levels.


Key Words: familial hypercholesterolemia {bullet} lipoprotein(a) • atherosclerotic complications


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Lp(a) is a macromolecular complex in human plasma that combines structural elements from the lipoprotein and blood clotting systems. High plasma levels are associated with premature CHD,1 2 3 4 CVD,5 restenosis after coronary artery bypass surgery,6 accelerated CHD in cardiac transplant recipients,7 PVD,8 9 and cardiovascular disease in hemodialysis patients.10 The two protein components of Lp(a) [apo(a) and apoB-100] are covalently linked.11 Apo(a) shares considerable homology with plasminogen, and the apo(a) gene is located on chromosome 6, adjacent to that of plasminogen.12 13

Plasma levels of Lp(a) and the molecular weight of apo(a) vary widely among populations, independently of race, age, and sex, and are largely genetically determined.13 14 15 Lp(a) concentrations are relatively resistant to most dietary and drug interventions.16 17 Because it is an apoB-100–containing particle, Lp(a) binds to the LDL receptor.18 However, its site of degradation remains uncertain, and whether Lp(a) is catabolized via receptor-mediated uptake is still controversial. Overexpression of the LDL receptor leads to accelerated catabolism of Lp(a) in transgenic mice,19 but in humans, drug treatments known to enhance LDL receptor–mediated catabolism of apoB-containing particles have no effect on plasma Lp(a) concentrations.16 20 21 Plasma Lp(a) levels correlate strongly with Lp(a) production rate but not with fractional catabolic rate.22

Considerable variation in the clinical severity of cardiovascular disease among FH heterozygotes has long been recognized. It has been suggested that plasma concentrations of Lp(a) in FH patients may explain such variation.21 23 24 Previous reports concerning plasma Lp(a) levels in FH subjects deficient in LDL receptors have provided contradictory results. In some studies,21 23 25 the plasma levels of Lp(a) were higher in FH patients than normolipidemic subjects. In the study by Mbewu et al,25 plasma Lp(a) concentrations were higher in FH heterozygotes with or without CHD than in unaffected relatives or in subjects with other types of primary hypercholesterolemia with similarly elevated LDL cholesterol levels. The authors concluded that an elevated plasma Lp(a) concentration should be regarded as a component of the clinical syndrome of FH.25 In another study,23 plasma Lp(a) levels in heterozygous FH patients were three times those of normal individuals with the same apo(a) phenotype. These results suggest that the inherited LDL receptor defect in FH could be associated with relatively high plasma Lp(a) concentrations.

Other studies24 26 showed that plasma Lp(a) concentrations were higher in FH than in non-FH subjects, but only in those who were afflicted with CHD. Moreover, it has also been reported27 28 that plasma Lp(a) concentrations in FH patients free from CHD are not significantly different from those in their first-degree, CHD-free, normolipidemic relatives. With the exception of one study,27 the FH populations examined have been heterogeneous with respect to the LDL receptor gene defect.

We investigated the influence of the LDL receptor on plasma Lp(a) levels in a genetically homogeneous population, namely, FH subjects from 30 families carrying the French-Canadian >10-kb deletion29 of the LDL receptor gene. Plasma lipoprotein lipid, Lp(a), and apoB levels were measured in affected and unaffected members. The first goal of the study was to compare plasma Lp(a) concentrations in heterozygous FH patients and their unaffected relatives to assess the influence of the LDL receptor status on plasma Lp(a) concentrations. Our second goal was to investigate the contribution of Lp(a) to cardiovascular risk by comparing its plasma level in FH heterozygotes with and without atherosclerosis. The results show that within families carrying the same molecular defect of the LDL receptor gene, Lp(a) levels do not differ between FH and non-FH subjects. In addition, we found that our FH patients with atherosclerotic complications did not have higher Lp(a) levels than those free from AVD.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Patients
During screening for the French-Canadian >10-kb deletion of the LDL receptor gene29 in the families of 30 hypercholesterolemic probands, a total of 226 subjects were studied at the Lipid Clinic of the Clinical Research Institute of Montreal. In all subjects, a full medical, familial, and dietary history was obtained and a complete physical examination was performed. Venous blood was taken in the morning after a 12-hour overnight fast. During the preceding 8 weeks, FH subjects had followed their regular diet and none had been treated with lipid-lowering drugs or medication known to affect lipid metabolism. In the probands, the diagnosis of FH was based on clinical criteria, and subjects found to be carriers of the >10-kb deletion were selected. In relatives, the diagnosis of FH was based on the presence of a total cholesterol level >6.2 mmol/L or LDL cholesterol >5.2 mmol/L in the presence of the >10-kb deletion of the LDL receptor gene. Xanthomata were detected in 74.5% of FH subjects.

The initial study group comprised 30 families. For the purpose of the present study, spouses were excluded (n=25); 6 of the probands were also excluded because of incomplete data or on the basis of homozygosity for FH. Thus, the study group comprised 24 probands, all positive for the >10-kb deletion, and 171 first- and second-degree relatives. Among them, 78 (children, siblings, or parents of the probands) carried the >10-kb deletion, whereas the remaining 93 were negative. From the latter group, 24 subjects were excluded because of hyperlipidemia, arteriosclerotic complications, or failure to return for a complete physical examination once the blood tests had been reported to be normal. In this report, 4 FH and 3 unaffected children were excluded from the comparative analyses. The final study group of 164 subjects included 98 FH patients who carried the same >10-kb deletion of the LDL receptor gene and their 66 unaffected, normolipidemic relatives who were free from atherosclerotic complications. The FH patients were aged 18 to 73 years and the unaffected relatives 18 to 65 years. All were white and lived in or near Montreal. This study was approved by the institutional ethics committee.

A resting ECG and a flat x-ray film of the abdomen for detection of possible aortic calcifications were performed in all subjects. In addition, if the medical history or clinical examination so warranted, an exercise ECG, 201Tl myocardial scintigram, coronary artery angiogram, or Doppler echocardiography of the carotid arteries and the lower-limb arteries was performed. CHD was diagnosed if there was a documented history of previous myocardial infarction, coronary artery bypass surgery, or angina pectoris with positive exercise ECG and thallium test or abnormal coronary angiogram (stenosis of >70 percent in a major vessel). A total of 29 subjects had CHD (mean age of onset, 40 years; range, 24 to 61 years); of these, 11 had had coronary bypass surgery. CVD was diagnosed by previous medical history or physical exam (presence of carotid bruits) and positive (>25% stenosis) Doppler echocardiography of the carotid arteries. Thirteen subjects suffered from CVD (mean age for onset of symptoms, 49 years; range, 28 to 61 years). Atherosclerosis of the lower-limb arteries (PVD) was diagnosed in 7 subjects (mean age for onset of symptoms, 58 years; range, 29 to 65 years) by medical history of intermittent claudication, Doppler echocardiography, and arteriography if indicated. Seventeen subjects were diagnosed with CHD only, 6 with CHD and CVD, and 4 with CHD and PVD; the association of these three conditions was established in 2 patients. Five patients presented with CVD only, whereas PVD alone was diagnosed in only 1 case. Thus, 29 (83%) of the 35 patients with atherosclerosis had CHD. Calcifications of the abdominal aorta and/or iliac arteries were detected in 19 patients with proven atherosclerosis. In 1 asymptomatic 56-year-old FH woman, minimal linear aortic calcifications without dilations were detected; complete cardiovascular evaluation, including coronary angiogram, was normal, and her plasma Lp(a) value was 3.0 mg/dL. Aortic calcification alone, as found in this case, was not considered to be a sufficient criterion for inclusion in the atherosclerosis group. In this report, CHD, CVD, and PVD are designated AVD.

Thereafter, the FH patients were classified according to the presence or absence of AVD. The 63 FH heterozygotes free from AVD constituted group A of the study (26 men and 37 women), the 35 FH patients with AVD constituted group B (19 men and 16 women), and the 66 unaffected relatives constituted group C (33 men and 33 women). One patient in group B had non–insulin-dependent diabetes well controlled with diet. Hypertension was diagnosed if blood pressure levels were above 140/90 mm Hg on more than two occasions or in subjects taking antihypertensive medication. Smoking history was obtained in all cases, and subjects were classified as present or former smokers or as nonsmokers.

A control group of 392 subjects was also studied. Four subjects for every FH patient (180 men and 212 women) were selected on the basis of age and sex from among the 1096 normocholesterolemic, AVD-free, French-Canadian adults (aged >=18 years; 514 men and 582 women) who participated in the Quebec Heart Health Survey.30

Laboratory Methods
All blood samples were obtained after a 12- to 14-hour fast and drawn into evacuated tubes containing EDTA (1.5 mg/dL). Plasma was stored at 4°C until analysis within 3 to 4 days. Plasma lipoproteins were separated31 and cholesterol and triglyceride levels were measured by enzymatic techniques as previously described.32 HDL cholesterol was measured after precipitation of apoB-containing lipoproteins with heparin-manganese; LDL cholesterol was calculated by use of Friedewald's formula in a few samples when ultracentrifugation was not performed. Total plasma apoB was measured by electroimmunoassay.30 Lp(a) levels were determined by a commercially available ELISA (Terumo Medical Corp). This assay uses a monoclonal antibody against apo(a) that does not cross-react against plasminogen and a second polyclonal antibody directed against the apo(a) portion of Lp(a).4 The coefficient of variation between assays is 10.8% at 35 mg/dL. The presence of the >10-kb deletion of the LDL receptor gene29 was tested by Southern blot analysis.33

Phenotyping for apo(a) was performed in Dr G. Utermann's laboratory in Innsbruck, Austria,23 on plasma samples from 112 subjects (64 FH subjects and 48 unaffected relatives).

Statistical Analysis
Data were analyzed with the Statistical Analysis System (SAS Institute Inc).34 One-way and two-way ANOVAs were used for comparison of mean values between sexes within the three groups. The Student's t test procedure was used whenever comparisons were limited to two groups. Median values for Lp(a) were obtained by the univariate procedure and evaluated by a nonparametric test (median test or Brown-Mood test). The relations between variables were evaluated by linear regression, and the {chi}2 statistic was used for evaluation of frequency distributions after elimination of subgroups with n<5.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
Table 1Down shows clinical features of the FH patients in the present study. There were no differences in sex distribution between the two groups. Patients with AVD (group B) were significantly older than those without (group A). Cigarette consumption (present or former smokers) was higher in group B than in group A subjects (69% versus 51%, respectively), but this difference was not statistically significant. The prevalence of hypertension was not significantly different between the two groups.


View this table:
[in this window]
[in a new window]
  		Table 1. Clinical Characteristics of FH Patients With and Without AVD

As expected, subjects in group C (men and women) had significantly (P<.001) lower concentrations of mean plasma total cholesterol, LDL cholesterol, and apoB and higher HDL cholesterol values than did those of the same sex in groups A and B (Table 2Down). No differences in mean plasma triglyceride levels were observed, although the log-transformed triglyceride levels were significantly lower in group C than the other groups (men and women together).


View this table:
[in this window]
[in a new window]
  		Table 2. Mean Plasma Lipid, Lipoprotein, Lp(a), and Apo B Concentrations in FH Patients and Normal Relatives

Statistical comparisons conducted between groups A and B independently of sex showed significant differences for age (P<.001), total and LDL cholesterol levels (P<.02), and apoB levels (P<.01) (data not shown). Mean plasma total and LDL cholesterol and apoB concentrations were significantly higher in FH men with AVD (group B) than in FH men without AVD (group A) (Table 2Up). This was not observed in women. Other differences in lipoprotein lipid levels (in the expected direction) did not reach statistical significance. Men in group B had significantly lower HDL cholesterol and higher apoB levels than did women in that group (P<.05 for both). Plasma Lp(a) levels were indistinguishable between FH heterozygotes (A and B) and their normolipidemic relatives (group C) or between the FH heterozygotes with (group B) and without (group A) AVD (Table 2Up). As reported in most populations, the distribution of Lp(a) was highly skewed in our sample. Analyses were therefore conducted on log-transformed as well as nontransformed data. No significant differences in Lp(a) levels were detected among the three groups, whether the analysis was conducted on absolute or log-transformed values (Table 2Up). No differences in frequency distributions or cumulative frequency curves for Lp(a) were apparent among the three groups (Fig 1Down). No significant differences in median scores were found among the two subgroups of FH patients and their normal relatives (not shown). In addition, a threshold value of 20 mg/dL was used to separate subjects in groups A (without AVD) and B (with AVD). There was no significant difference in the prevalence of AVD in FH patients with values of <=20 mg/dL compared with those with values >20 mg/dL (data not shown; {chi}2=1.00). No significant difference was found between the FH and non-FH groups in the distribution of the most frequent apo(a) phenotypes (Table 3Down) nor between the FH subjects with and without AVD (data not shown; {chi}2=10.60, P=.16). In most cases, the phenotypes with the S3 and S4 isoforms were associated with low Lp(a) levels, whereas S2 predominated in subjects with Lp(a) >30 mg/dL (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Graphs show percent frequency distributions (A) and cumulative frequency curves (B) of Lp(a) levels in FH subjects without atherosclerotic complications (group A), with atherosclerotic complications (group B), and their normolipidemic first- and second-degree relatives (group C).


View this table:
[in this window]
[in a new window]
  		Table 3. Apo(a) Phenotype Distribution in FH Subjects and in Normolipidemic Relatives

Because plasma Lp(a) concentrations were similar in groups A and B, the mean values of FH subjects from the six families in which data were available for 8 or more members were compared with those of their normolipidemic relatives (Table 4Down). Although FH subjects had significantly higher plasma total and LDL cholesterol and apoB concentrations, in each pedigree, mean plasma Lp(a) concentrations in FH subjects were statistically indistinguishable from those in their normolipidemic relatives. Thirteen men and 14 women from groups A and B were matched for age and sex. Subjects in group B tended to have higher total and LDL cholesterol and apoB values than subjects in group A, but no differences in mean, median, or log-transformed Lp(a) plasma concentrations were found (Table 5Down).


View this table:
[in this window]
[in a new window]
  		Table 4. Plasma Lipid, Lipoprotein, Lp(a), and Apo B Concentrations in Normolipidemic and Hypercholesterolemic Subjects From Six Kindreds With FH


View this table:
[in this window]
[in a new window]
  		Table 5. FH Subjects With (Group B) and Without (Group A) AVD Matched for Age and Sex

Lp(a) levels appeared higher in the families we studied than in most populations. To see if this was true of the French-Canadian population in general, we compared FH subjects (combined groups A and B) and their healthy relatives (group C) with an unrelated control group of normocholesterolemic, AVD-free, French-Canadian subjects (reference group), who were matched for age and sex (Table 6Down). Significant differences were observed for total, LDL, and HDL cholesterol and apoB levels and for Lp(a) log-transformed values. The tendency for mean Lp(a) levels to be higher in the unaffected relatives of FH subjects than in members of the control group was removed by log transformation (Table 6Down). Median scores were not significantly different among the three groups. To preclude the possibility that shared genetics within the FH group could bias our conclusions, the comparison was repeated on a sample of 30 unrelated FH patients versus a reference group of 120 subjects. The same differences were noted for all traits with the exception of log-transformed Lp(a) values, which were no longer statistically different (P=.17; data not shown).


View this table:
[in this window]
[in a new window]
  		Table 6. Plasma Lipid, Lipoprotein, and Apo B Levels in FH Subjects, Their Normal Relatives, and a Reference Group of Healthy Individuals

Fig 2Down shows the frequency distributions and cumulative frequencies of Lp(a) levels in FH subjects (combined A plus B subsets), in their non-FH relatives (group C), and in the control group. Fewer elevated Lp(a) values were observed in the last group. The 90th percentile was 35 and 54 mg/dL in the control subjects and FH subjects, respectively. No significant correlations were found between Lp(a) plasma concentrations or their log-transformed values and the plasma lipid and lipoprotein concentrations in any of the study groups (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Graphs show percent frequency distributions (A) and cumulative frequency curves (B) of Lp(a) levels in the total group of FH subjects, in their non-FH first- and second-degree relatives, and in a control group of unrelated individuals.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
The first principal finding of the present study was that plasma Lp(a) levels were not different between 98 FH patients (all carriers of the >10-kb deletion of the LDL receptor gene) and their normocholesterolemic, arteriosclerosis-free relatives. Second, no significant differences were found between Lp(a) levels in FH subjects with (group B) and without (group A) atherosclerotic complications (AVD). Cohen et al35 observed large variations in Lp(a) levels between unrelated subjects with identical apo(a) isoforms. These variations were ascribed to sequence variation in the Lp(a) gene among families. Thus, our within-family comparison of Lp(a) levels between FH and non-FH subjects in a genetically homogeneous sample eliminated a potentially important source of genetic variability inherent in previous studies.

The question of whether the LDL receptor contributes to the regulation of Lp(a) concentration is of considerable interest and controversy. In support of a role for the LDL receptor, several reports demonstrated twofold to threefold higher plasma Lp(a) levels in FH patients than in healthy control subjects.21 23 25 A study36 on the effect of apo E polymorphism on plasma levels of Lp(a) in normolipidemic individuals concluded that Lp(a) levels, like LDL cholesterol levels, are influenced by apo E polymorphism. Studies in transgenic mice also indicated that the LDL receptor may be important for the clearance of Lp(a) from plasma.19 However, several lines of evidence do not support this hypothesis. Turnover studies in humans suggested that plasma Lp(a) levels are primarily regulated by the rate of synthesis of the apo(a) protein.22 In vitro, the affinity of Lp(a) for the LDL receptor is less than that of LDL.37 A study38 of two families affected with familial defective apoB-100 reported that the catabolism of Lp(a) particles containing the mutant apoB was not retarded by their inability to bind to the LDL receptor, suggesting that Lp(a) is not catabolized significantly by this pathway. In addition, hypolipidemic drugs known to enhance LDL receptor–mediated catabolism of apoB–containing particles do not reduce plasma Lp(a) concentrations.16 20 21 Finally, a study conducted in homozygous and heterozygous FH patients recently showed that the LDL receptor is not required for normal catabolism of Lp(a) in humans.39 In fact, a receptor activity apparently unique for Lp(a) has been characterized in human macrophages.40

The present study showed that plasma Lp(a) levels and their log-transformed values were similar between FH subjects and their normolipidemic first- and second-degree relatives. Our results contrast with those of Mbewu et al,25 who found higher Lp(a) levels in FH heterozygotes (regardless of the presence or absence of CHD) than in their unaffected first- and second-degree relatives. In contrast to other previous studies,21 23 24 35 our data do not support a major role for the LDL receptor in Lp(a) metabolism. Our conclusion agrees with that of Ghiselli et al,28 who found no significant differences between plasma Lp(a) concentrations in 28 FH patients without coronary artery disease and their unaffected relatives.

Lp(a) levels are largely determined by genetic variation in the apo(a) gene.13 14 15 Sandholzer et al41 demonstrated that alleles at the apo(a) locus determine the risk for CHD through their effects on Lp(a) concentrations across multiple populations with large differences in CHD frequency and risk factor profiles. The small apo(a) isoforms that are associated with higher Lp(a) levels are more frequent in groups of patients with CHD.42 In the present study, Lp(a) levels tended to aggregate within families, as did the distribution of apo(a) phenotypes. The fact that the Lp(a) plasma values of our FH cases and their healthy relatives showed no significant difference is therefore expected on the basis of shared genes if the LDL receptor were not important for Lp(a) catabolism. As further support for the shared-gene hypothesis, when unaffected relatives of our FH subjects (group C) were compared with an unrelated control group of French Canadians, an insignificant tendency for higher plasma Lp(a) levels was observed in group C, but the distribution was widely scattered and thus log values were similar. However, the log Lp(a) plasma concentrations of our FH subjects were significantly higher than those of the unrelated group, raising the interesting possibility that the LDL receptor defect may have an impact on Lp(a) production.

Leitersdorf et al43 studied several families with four defined FH mutations. The differences in Lp(a) levels between affected and unaffected individuals varied in magnitude among the mutations but did not parallel those of LDL levels, suggesting that higher Lp(a) levels in FH subjects could not be accounted for by retarded catabolism via the LDL receptor pathway. In another study of 58 FH subjects, Hegele et al44 reported a lack of association of Lp(a) levels with a polymorphism in the LDL receptor gene. Furthermore, in a study of 153 pairs of twins, Berg45 reported a lack of association between plasma Lp(a) concentration and variability at the LDL receptor locus associated with hypercholesterolemia.

Our findings also differ from those of other studies21 24 26 which indicated that FH patients with CHD had higher Lp(a) levels than those without. In the present study, no significant difference in plasma Lp(a) concentrations was found between FH subjects with and without AVD, even after subjects were matched for age and sex. When FH subjects and their relatives were compared, there were no differences in the apo(a) phenotype distribution, nor were significant differences apparent in the prevalence of apo(a) isoforms between FH subjects with and without AVD.

Elevated Lp(a) has been proposed as a risk factor for different forms and manifestations of atherosclerosis, such as CHD,1 2 3 4 CVD,5 and PVD.9 10 High plasma Lp(a) concentration has been proposed as a strong and independent risk factor for CHD in FH patients.21 26 In the FH sample we studied, which carried the same molecular defect of the LDL receptor gene, such an elevation is apparently not a major factor in the development of premature atherosclerosis. In a study of the association between CHD and common risk factors in a sample of 263 FH patients, including 89 with coronary disease, who carried the same >10-kb deletion of the LDL receptor gene, we recently reported that Lp(a) levels were not a significant predictor of CHD either in univariate or multivariate analyses.46 In the present study, cardiovascular risk factors other than high Lp(a) levels, such as age, male sex, and higher apoB and LDL cholesterol levels, did correlate with the presence of CHD and other atherosclerotic complications in our FH patients. Among subjects with AVD, FH women were on average 8 years older than men. Furthermore, we found that AVD arose in our FH patients in the absence of elevated Lp(a) values. In fact, 57% of the FH patients with AVD (group B) had an Lp(a) plasma level <11 mg/dL (data not shown). In FH patients from an ethnically heterogeneous population, Bowden et al42 found no difference in the plasma Lp(a) levels of FH patients with and without CHD. Our present results extend this observation to a homogeneous sample of FH patients.

Interpretation of the apparent disagreement of our current results with some previous reports should take into account several considerations. The diagnosis of FH in our case subjects was confirmed by demonstration of a molecular defect of the LDL receptor gene, the so-called French-Canadian >10-kb deletion.29 Our FH sample differed from others21 24 25 26 in that all subjects carried the same LDL receptor genetic defect and, unlike those in the study of Seed et al,26 they were not receiving hypolipidemic drugs at the time of the study.

Most but not all epidemiological studies support an association between high plasma Lp(a) levels and atherosclerosis. In a prospective study, Jauhiainen et al47 showed a similar mean, median, and distribution of plasma Lp(a) concentrations in Helsinki Heart Study participants with and without coronary events. Another study48 also failed to show significant differences in the median Lp(a) levels of elderly men and women with and without CHD. Furthermore, a recent prospective study in middle-aged American men (the Physicians' Health Study) found no evidence of an association between Lp(a) levels and myocardial infarction risk.49 On the other hand, two recent prospective studies50 51 have reported that elevated plasma Lp(a) values are an independent risk factor for CHD in white men. In a German population sample, Lp(a) levels correlated positively with severity of coronary lesions assessed by angiography in men with suspected CHD.52 One possible factor contributing to the apparently contradictory effects of Lp(a) levels in CHD risk is the suggestion of Scanu and Edelstein53 that certain apo(a) mutations can impart a lower atherothrombogenic potential to Lp(a).

In addition, methodological variables, such as storage and lack of standardization of analytical techniques, may contribute to disagreements in these epidemiological studies. A recent report54 that compared two methods for determination of Lp(a) concluded that Lp(a) levels in subjects bearing low molecular weight apo(a) isoforms were underestimated by the method developed by Terumo. The use of this technique in the present study is unlikely to explain the lack of differences among the various subgroups of our sample, since the distribution of apo(a) isoforms was similar in the three study groups. Our laboratory recently reported that oxidized Lp(a) showed increased immunoreactivity to anti-Lp(a) monoclonal antibodies.55 Thus, it is possible that in FH patients, in whom the turnover of cholesterol-rich lipoproteins is considerably reduced, a proportion of circulating Lp(a) is present as oxidized Lp(a) and that some techniques sensitive to this fraction overestimate plasma Lp(a) levels.

Our criteria for classifying patients with CHD were similar to those used by others.21 25 26 Also, the average age of CHD onset in our patients was 40 years, similar to that reported by Seed et al.26 In the present study, all of the FH subjects carried the same deletion of the LDL receptor gene, and their first- and second-degree relatives were included for comparison. It is likely that the relative genetic homogeneity of the presently described population is partially responsible for the differences between our results and those in some previously reported studies. In our population, Lp(a) levels appear to be independent of the LDL receptor status and unrelated to the presence of atherosclerotic complications in FH subjects. Thus, the present study of FH kindreds with the same LDL receptor gene deletion, in which potential sources of genetic variability are minimized, supports neither the involvement of the LDL receptor in clearance of Lp(a) from plasma nor the atherogenicity of Lp(a).


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
AVD = atherosclerotic vascular disease
CHD = coronary heart disease
CVD = cerebrovascular disease
FH = familial hypercholesterolemia
kb = kilobase
Lp(a) = lipoprotein(a)
PVD = peripheral vascular disease


*    Acknowledgments
 
This work was supported by grants from the Medical Research Council of Canada/Ciba-Geigy Canada Ltd University-Industry Program (UI-11407), the Heart and Stroke Foundation of Quebec, La Succession J.A. DeSève, and Fondo de Investigaciones Sanitarias de la Seguridad Social from the Spanish Ministry of Health. We are indebted to Dr Gerd Utermann of Innsbruck, Austria, for the determination of the apo(a) phenotype in our subjects and to Dr Daniel Bouthillier and the clinical and technical staff of the Clinical Research Institute of Montreal for their help with this study. We also thank Johanne Duhaime, Louis-Jacques Fortin, Lisa Tassoni, and Judith Lauzon for data processing.

Received July 6, 1995; accepted October 20, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

  1. Dahlén GH, Guyton JR, Attar M, Farmer JA, Kautz JA, Gotto AM Jr. Association of levels of lipoprotein Lp(a), plasma lipids and other lipoproteins with coronary artery disease documented by angiography. Circulation. 1986;74:758-765. [Abstract/Free Full Text]
  2. Rhoads GG, Dahlen G, Berg K, Morton NE, Dannenbury AL. Lp(a) lipoprotein as a risk factor for myocardial infarction. JAMA. 1986;256:2540-2544. [Abstract]
  3. Hoefler G, Harnoncourt F, Paschke E, Mitrl W, Pfeiffer KH, Kostner GM. Lipoprotein Lp(a): a risk factor for myocardial infarction. Arteriosclerosis. 1988;8:398-401. [Abstract/Free Full Text]
  4. Genest J Jr, Jenner JL, McNamara JR, Ordovas JM, Silberman SR, Wilson PWF, Schaefer EJ. Prevalence of lipoprotein (a) [Lp(a)] excess in coronary artery disease. Am J Cardiol. 1991;67:1039-1045. [Medline] [Order article via Infotrieve]
  5. Zenker G, Koltringer P, Bone G, Nieder-Korn K, Pfeiffer K. Lipoprotein (a) as a strong indicator for cerebrovascular disease. Stroke. 1986;17:942-948. [Abstract/Free Full Text]
  6. Cushing GL, Gaubatz JW, Nava ML, Burdick BJ, Bocan TM, Guyton JR, Weilbaecher D, DeBakey ME, Lawrie GM, Morrisett JD. Quantitation and localization of apolipoprotein(a) and B in coronary artery bypass vein grafts resected at reoperation. Arteriosclerosis. 1989;9:593-603. [Abstract/Free Full Text]
  7. Barbir M, Kushwaha S, Hunt B, Macken A, Thompson GR, Mitchell A, Robinson D, Yacoub M. Lipoprotein(a) and accelerated coronary artery disease in cardiac transplant recipients. Lancet. 1992;340:1500-1502. [Medline] [Order article via Infotrieve]
  8. Tyrrell J, Cooke T, Reilly M, Colgan M, Moore D, Shanik DG, Bergin C, Feely J. Lipoprotein [Lp(a)] and peripheral vascular disease. J Intern Med. 1992;232:349-352. [Medline] [Order article via Infotrieve]
  9. Pedro-Botet J, Sentí M, Auguet T, Nogués X, Rubiés-Prat J, Aubó C, Vidal-Barraquer F. Apolipoprotein(a) genetic polymorphism and serum lipoprotein(a) concentration in patients with peripheral vascular disease. Atherosclerosis. 1993;104:87-94. [Medline] [Order article via Infotrieve]
  10. Cressman MD, Heyka RJ, Paganini EP, O'Neil J, Skibinski CI, Hoff HF. Lipoprotein(a) is an independent risk factor for cardiovascular disease in hemodialysis patients. Circulation. 1992;86:475-482. [Abstract/Free Full Text]
  11. Utermann G, Weber W. Protein composition of Lp(a) lipoprotein from human plasma. FEBS Lett. 1983;154:357-361. [Medline] [Order article via Infotrieve]
  12. McLean JW, Tomlinson JE, Kuang W-J, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn RM. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987;330:132-137. [Medline] [Order article via Infotrieve]
  13. Utermann G. The mysteries of lipoprotein(a). Science. 1989;246:904-910. [Abstract/Free Full Text]
  14. Scanu AM, Fless GM. Lipoprotein(a): heterogeneity and biological relevance. J Clin Invest. 1990;85:1709-1715.
  15. Boerwinkle EC, Leffert CC, Lin J, Lackner C, Chiesa G, Hobbs HH. Apolipoprotein(a) gene accounts for greater than 90% of the variation in plasma lipoprotein(a) concentrations. J Clin Invest. 1992;90:52-60.
  16. Vessby B, Kostner G, Lithell H, Thomis J. Diverging effects of cholestyramine on apolipoprotein B and lipoprotein Lp(a). Atherosclerosis. 1982;44:61-71. [Medline] [Order article via Infotrieve]
  17. Brewer HB Jr. Effectiveness of diet and drugs in the treatment of patients with elevated Lp(a) levels. In: Scanu AM, ed. Lipoprotein(a). New York, NY: Academic Press Inc; 1990:211-220.
  18. Armstrong VW, Walli AK, Seidel D. Isolation, characterization, and uptake in human fibroblasts of an apo(a)-free lipoprotein obtained on reduction of lipoprotein(a). J Lipid Res. 1985;26:1314-1323. [Abstract]
  19. Hofmann SL, Eaton DL, Brown MS, McConathy WJ, Goldstein JL, Hammer RE. Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice. J Clin Invest. 1990;85:1542-1547.
  20. Thiery J, Armstrong VW, Schleef J, Creutzfeldt C, Creutzfeldt W, Seidel D. Serum lipoprotein Lp(a) concentrations are not influenced by an HMG CoA reductase inhibitor. Klin Wochenschr. 1988;66:462-463. [Medline] [Order article via Infotrieve]
  21. Wiklund O, Angelin B, Olofsson S-O, Eriksson M, Fager G, Berglund L, Bondjers G. Apolipoprotein (a) and ischaemic heart disease in familial hypercholesterolaemia. Lancet. 1990;335:1360-1363. [Medline] [Order article via Infotrieve]
  22. Krempler F, Kostner GM, Roscher A, Haslauer F, Bolzano K, Sandhofer F. Studies on the role of specific cell surface receptors in the removal of lipoprotein(a) in man. J Clin Invest. 1983;71:1431-1441.
  23. Utermann G, Hoppichler F, Dieplinger H, Seed M, Thompson G, Boerwinkle E. Defects in the low density lipoprotein receptor gene affect lipoprotein (a) levels: multiplicative interaction of two gene loci associated with premature atherosclerosis. Proc Natl Acad Sci U S A. 1989;86:4171-4174. [Abstract/Free Full Text]
  24. Houlston R, Quiney J, Mount J, Watts GF, Lewis B. Lipoprotein(a) and coronary heart disease in familial hypercholesterolaemia. Lancet. 1988;2:405. Letter.
  25. Mbewu AD, Bhatnagar D, Durrington PN, Hunt L, Ishola M, Arrol S, Mackness M, Lockley P, Miller JP. Serum lipoprotein(a) in patients heterozygous for familial hypercholesterolemia, their relatives, and unrelated control populations. Arterioscler Thromb. 1991;11:940-946. [Abstract/Free Full Text]
  26. Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson GR, Boerwinkle E, Utermann G. Relation of serum lipoprotein(a) concentration and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. N Engl J Med. 1990;322:1494-1499. [Abstract]
  27. Soutar AK, McCarthy SN, Seed M, Knight BL. Relationship between apolipoprotein(a) phenotype, lipoprotein(a) concentration in plasma, and low density lipoprotein receptor function in a large kindred with familial hypercholesterolemia due to the Pro664->Leu mutation in the LDL receptor gene. J Clin Invest. 1991;88:483-492.
  28. Ghiselli G, Gaddi A, Barozzi G, Ciarrocchi A, Descovich G. Plasma lipoprotein(a) concentration in familial hypercholesterolemic patients without coronary artery disease. Metabolism. 1992;41:833-838. [Medline] [Order article via Infotrieve]
  29. Hobbs HH, Brown MS, Russell DW, Davignon J, Goldstein JL. Deletion in the gene for low-density-lipoprotein receptor in a majority of French-Canadians with familial hypercholesterolemia. N Engl J Med. 1987;317:734-737. [Abstract]
  30. Santé Québec. Et votre coeur, ça va? Rapport de l'Enquête québécoise sur la santé cardiovasculaire 1990. Montréal, Canada: Ministère de la Santé et des Services Sociaux, Gouvernement du Québec; 1994:159.
  31. Lipid Research Clinics Program. Manual of Laboratory Operations: Lipid and Lipoprotein Analysis. Washington, DC: US Dept of Health, Education, and Welfare; 1974;1:1-81. US Dept of Health, Education, and Welfare publication NIH 75-628.
  32. Lussier-Cacan S, Bouthillier D, Davignon J. Apo E allele frequency in primary endogenous hypertriglyceridemia (type IV) with and without hyperapobetalipoproteinemia. Arteriosclerosis. 1985;5:639-643. [Abstract/Free Full Text]
  33. Ma Y, Bétard C, Roy M, Davignon J, Kessling A. Identification of a second `French Canadian' LDL receptor gene deletion and development of a rapid method to detect both deletions. Clin Genet. 1989;36:219-228. [Medline] [Order article via Infotrieve]
  34. SAS Institute Inc. SAS/STAT User's Guide, Version 6. 4th ed. Cary, NC: SAS Institute Inc; 1989.
  35. Cohen JC, Chiesa G, Hobbs HH. Sequence polymorphisms in the apolipoprotein(a) gene: evidence for dissociation between apolipoprotein(a) size and plasma lipoprotein(a) levels. J Clin Invest. 1993;91:1630-1636.
  36. de Knijff P, Kaptein A, Boomsma D, Princen HMG, Frants RR, Havekes LM. Apolipoprotein E polymorphism affects plasma levels of lipoprotein(a). Atherosclerosis. 1991;90:169-174. [Medline] [Order article via Infotrieve]
  37. Steyrer E, Kostner GM. Interaction of lipoprotein Lp(a) with the B/E receptor: a study using isolated bovine adrenal cortex and human fibroblast receptors. J Lipid Res. 1990;31:1247-1253. [Abstract]
  38. Perombelon YFN, Gallagher JJ, Myant NB, Soutar AK, Knight BL. Lipoprotein(a) in subjects with familial defective apolipoprotein B100. Atherosclerosis. 1992;92:203-212. [Medline] [Order article via Infotrieve]
  39. Rader DJ, Mann WA, Cain W, Kraft H-G, Usher D, Zech LA, Hoeg JM, Davignon J, Lupien P, Grossman M, Wilson JM, Brewer HB Jr. The low density lipoprotein receptor is not required for normal catabolism of Lp(a) in humans. J Clin Invest. 1995;95:1403-1408.
  40. Bottalico LA, Keeler GA, Fless GM, Tabas I. Cholesterol loading of macrophages leads to marked enhancement of native lipoprotein(a) and apoprotein(a) internalization and degradation. J Biol Chem. 1993;268:8569-8573. [Abstract/Free Full Text]
  41. Sandholzer C, Saha N, Kark JD, Rees A, Jaross W, Dieplinger H, Hoppichler F, Boerwinkle E, Utermann G. Apo(a) isoforms predict risk for coronary heart disease: a study of six populations. Arterioscler Thromb. 1992;12:1214-1226. [Abstract]
  42. Bowden JF, Pritchard PH, Hill JS, Frohlich JJ. Lp(a) concentration and apo(a) isoform size: relation to the presence of coronary artery disease in familial hypercholesterolemia. Arterioscler Thromb. 1994;14:1561-1568. [Abstract/Free Full Text]
  43. Leitersdorf E, Friedlander Y, Bard JM, Fruchart JC, Eisenberg S, Stein Y. Diverse effect of ethnicity on plasma lipoprotein(a) levels in heterozygote patients with familial hypercholesterolemia. J Lipid Res. 1991;32:1513-1519. [Abstract]
  44. Hegele RA, Sutherland S, Robertson M, Wu L, Emi M, Hopkins PN, Williams RR, Lalouel JM. The effect of genetic determinants of low density lipoprotein levels on lipoprotein(a). Clin Invest Med. 1991;14:146-151. [Medline] [Order article via Infotrieve]
  45. Berg K. Genetics and clinical importance of Lp(a) lipoprotein. Atherosclerosis Rev. 1991;23:63-75.
  46. Ferrières J, Lambert J, Lussier-Cacan S, Davignon J. Coronary artery disease in heterozygous familial hypercholesterolemia patients with the same LDL receptor gene mutation. Circulation. 1995;92:290-295. [Abstract/Free Full Text]
  47. Jauhiainen M, Koskinen P, Ehnholm C, Frick MH, Mänttäri M, Manninen M, Huttunen JK. Lipoprotein(a) and coronary heart disease risk: a nested case-control study of the Helsinki Heart Study participants. Atherosclerosis. 1991;89:59-67. [Medline] [Order article via Infotrieve]
  48. Simons L, Friedlander Y, Simons J, McCallum J. Lipoprotein(a) is not associated with coronary heart disease in the elderly: cross-sectional data from the Dubbo study. Atherosclerosis. 1993;99:87-95. [Medline] [Order article via Infotrieve]
  49. Ridker PM, Hennekens CH, Stampfer MJ. A prospective study of lipoprotein(a) and the risk of myocardial infarction. JAMA. 1993;270:2195-2199. [Abstract]
  50. Schaefer EJ, Lamon-Fava S, Jenner JL, McNamara JR, Ordovas JM, Davis CE, Abolafia JM, Lippel K, Levy RI. Lipoprotein(a) levels and risk of coronary heart disease in men: the Lipid Research Clinics Coronary Primary Prevention Trial. JAMA. 1994;271:999-1003. [Abstract]
  51. Cremer P, Nagel D, Labrot B, Mann H, Muche R, Elster H, Seidel D. Lipoprotein Lp(a) as predictor of myocardial infarction in comparison to fibrinogen, LDL cholesterol and other risk factors: results from the prospective Göttingen Risk Incidence and Prevalence Study (GRIPS). Eur J Clin Invest. 1994;24:444-453. [Medline] [Order article via Infotrieve]
  52. Budde T, Fechtrup C, Bösenberg E, Vielhauer C, Enbergs A, Schulte H, Assmann G, Breithardt G. Plasma Lp(a) levels correlate with number, severity, and length-extension of coronary lesions in male patients undergoing coronary arteriography for clinically suspected coronary atherosclerosis. Arterioscler Thromb. 1994;14:1730-1736. [Abstract/Free Full Text]
  53. Scanu AM, Edelstein C. Apolipoprotein(a): structural and functional consequences of mutations in kringle type 10 (or kringle 4-37). Clin Genet. 1994;46:42-45. [Medline] [Order article via Infotrieve]
  54. Taddei-Peters WC, Butman BT, Jones GR, Venetta TM, Macomber PF, Ranson JH. Quantification of lipoprotein(a) particles containing various apolipoprotein(a) isoforms by a monoclonal anti-apo(a) capture antibody and a polyclonal anti-apolipoprotein B detection antibody sandwich enzyme immunoassay. Clin Chem. 1993;39:1382-1389. [Abstract]
  55. Naruszewicz M, Giroux LM, Davignon J. Oxidative modification of lipoprotein(a) causes changes in the structure and biological properties of apo(a). Chem Phys Lipids. 1994;67/68:167-174.



This article has been cited by other articles:


Home page
 Clin. Chem.Home page
D. T. Holmes, B. A. Schick, K. H. Humphries, and J. Frohlich
Lipoprotein(a) Is an Independent Risk Factor for Cardiovascular Disease in Heterozygous Familial Hypercholesterolemia
Clin. Chem., November 1, 2005; 51(11): 2067 - 2073.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
S. Gonbert, B. Saint-Jore, P. Giral, C. Doucet, J. Chapman, and J. Thillet
Molecular Analysis of Apo(a) Fragmentation in Polygenic Hypercholesterolemia: Characterization of a New Plasma Fragment Pattern
Arterioscler. Thromb. Vasc. Biol., August 1, 2001; 21(8): 1353 - 1358.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
J.T Real, F.J Chaves, I Martinez-Uso, A.B Garcia-Garcia, J.F Ascaso, and R Carmena
Importance of HDL cholesterol levels and the total/ HDL cholesterol ratio as a risk factor for coronary heart disease in molecularly defined heterozygous familial hypercholesterolaemia
Eur. Heart J., March 2, 2001; 22(6): 465 - 471.
[Abstract] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
H. G. Kraft, A. Lingenhel, F. J. Raal, M. Hohenegger, and G. Utermann
Lipoprotein(a) in Homozygous Familial Hypercholesterolemia
Arterioscler. Thromb. Vasc. Biol., February 1, 2000; 20(2): 522 - 528.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
M. E. Wittekoek, S. N. Pimstone, P. W. A. Reymer, L. Feuth, G.-J. Botma, J. C. Defesche, M. Prins, M. R. Hayden, and J. J. P. Kastelein
A Common Mutation in the Lipoprotein Lipase Gene (N291S) Alters the Lipoprotein Phenotype and Risk for Cardiovascular Disease in Patients With Familial Hypercholesterolemia
Circulation, March 3, 1998; 97(8): 729 - 735.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
A. F. Vuorio, H. Turtola, K.-M. Piilahti, P. Repo, T. Kanninen, and K. Kontula
Familial Hypercholesterolemia in the Finnish North Karelia : A Molecular, Clinical, and Genealogical Study
Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 3127 - 3138.
[Abstract] [Full Text]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
R. A. Hegele, W. C. Breckenridge, J. H. Brunt, and P. W. Connelly
Genetic Variation in Factor VII Associated with Variation in Plasma Lipoprotein(a) Concentration
Arterioscler. Thromb. Vasc. Biol., September 1, 1997; 17(9): 1701 - 1706.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carmena, R.
Right arrow Articles by Davignon, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carmena, R.
Right arrow Articles by Davignon, J.
Right arrowPubmed/NCBI database