Apolipoprotein(a) genetic polymorphism and serum lipoprotein(a) concentration in patients with peripheral vascular disease

ATHEROSCLEROSIS Atherosclerosis

104 (1993) 87-94

Apolipoprotein(a) genetic polymorphism and serum lipoprotein(a) concentration in patients with peripheral vascular disease J. Pedro-Boteta, M. Senti”, T. Augueta, X. NoguCs”, J. Rub&Prat*“, F. Vidal-Barraquerb

C. Aub6”,

‘Department of Medicine, Hospital del Mar, Institute Municipal de Invesrigacibn Mkdica, Universidad Autdnoma de Barcelona, Barcelona, Spain hDepartment of Vascular Surgery, Hospital de la Esperanza, Barcelona, Spain

(Received

18 March

1993; revision

received

13 July 1993; accepted

2 August

1993)

Abstract

Serum lipoprotein(a) (Lp(a)) levels were measured in 89 men with peripheral vascular disease (PVD) and 129 (100 male and 29 woman) healthy controls. Apolipoprotein(a) genetic polymorphism was determined by immunoblotting in all subjects. Patients with PVD had significantly higher serum Lp(a) levels than controls. Apolipoprotein(a) phenotype frequencies in patients with PVD did not differ from those of the control group. Both patients and controls with phenotype S2 had higher serum Lp(a) levels than those with phenotype S4. It should be emphasized that serum Lp(a) levels were significantly higher in PVD patients than controls for those with phenotype S2, S3/S4 and S4. Raised serum Lp(a) levels together with other lipoprotein abnormalities in patients with PVD imply a high cardiovascular risk. Genetic polymorphism clearly influences serum Lp(a) levels both in patients and controls. In patients with PVD, environmental and/or other genetic factors must play a role in raising Lp(a) levels. Key words:

Lipoprotein(a);

Phenotype; Peripheral vascular disease

1. Introduction

Lipoprotein(a) (Lp(a)) described by Berg in 1963 [l] constitutes a newly recognized cardiovascular risk factor with implications in atherogenic and thrombogenic processes. Since in 1971 DahlCn et al. [2] described the association between angina pectoris and the presence of a pre-beta lipoprotein * Corresponding

author,

Department

of Medicine,

002l-9150/93/$06.00 0 1993 Elsevier Scientific SSDI 002 I-9150(93)05130-W

Hospital

Publishers

band, several studies have identified Lp(a) as a marker for coronary heart disease [3-71 and cerebrovascular disease [S-l 11. Lp(a) represents a low-density lipoprotein (LDL)-like particle with apolipoprotein (apo) B-100 linked to ape(a). The latter is a glycoprotein coded by a single gene locus on the long arm of chromosome 6, and is strongly similar to plasminogen. On the other hand, ape(a)

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size polymorphism is related to plasma levels and density distribution of Lp(a) [ 12- 161. Recently, Seed et al. (171 analyzed the relationship between serum Lp(a) concentration and ape(a) phenotype and coronary heart disease in patients with familial hypercholesterolaemia. These authors demonstrated that high Lp(a) levels are associated with a small isoform which moves rapidly in electrophoresis of serum, and that a low concentration is related to larger isoforms; thus, the Lp(a) concentration is determined at least in part by the genetic polymorphism. However, the reason for this association is unknown, and the hypothesis of Utermann et al. [ 181 that the LDL-receptor gene affects plasma Lp(a) levels is not widely accepted. The high incidence of peripheral vascular disease (PVD) in smokers, patients with diabetes mellitus and hypertensive subjects is well known [ 191. On the other hand, in a previous study we analyzed the role of lipoprotein disturbances in PVD [20]. However, to our knowledge none of the few studies of Lp(a) on PVD [21-231 analyzed the role of ape(a) phenotypes. Therefore, the aim of the present study was to determine whether patients with PVD have a particular ape(a) phenotype and assess its influence on serum Lp(a) levels in non-diabetic male patients with atherosclerosis of the lower limbs. 2. Patients and methods 2.1. Subjects Eighty-nine male patients with PVP (age-range 44-87 years, mean 65.5 years) were included in the study. The diagnosis of PVD was established by clinical history, physical examination and Doppler pressure at rest and’after exercise with stress testing [24]. Twenty-one patients had aortoiliac involvement, according to the femoral wave-form [25], and the remaining 68 patients with distal involvement were classified according to the ankle/ brachial index (ABI) as moderate (ABI 0.6-0.9) and severe (ABI 0.5 or less) ischaemia [24]. The results of Doppler examination of the lower limbs had remained constant throughout the 6 months prior to the study. Patients with coronary heart disease (including asymptomatic patients with electrocardiographic evidence of previous myocar-

J. Pedro-Botet et al. /Atherosclerosis

104 (1993) 87-94

dial infarction), cerebrovascular disease, diabetes mellitus, liver, renal or other endocrine diseases were excluded. Thirty-nine patients (43.8%) had never been smokers or were ex-smokers, and the remaining 50 patients (56.1%) were current smokers at the time of the study. Mean f SD. body mass index of patients was 23 f 2. One hundred and twenty-nine healthy subjects (100 men, 29 women; age range 23-94 years; mean 60 years) served as controls and were recruited from hospital and university staff, non-retributed blood donors, and subjects who attended the ophthalmology outpatient clinic for visual acuity examination. This control group was judged free of any illness by history, clinical examination and routine laboratory data. Sixty-live (50.4%) had never been smokers or were ex-smokers. Body mass index of controls was 25 f 3. None of the subjects were receiving therapy known to cause changes in lipid and lipoprotein profiles. 2.2. Lipoprotein analysis Aliquots for serum Lp(a) measurement and for serum delipidation prior to ape(a) phenotyping were stored at -20°C for no more than 4 months. Serum concentration of Lp(a) was quantified by enzymoimmunoanalysis [26] in microplates TintElize Lp(a) (Biopool, Umel, Sweden) containing polyclonal anti-Lp(a) antibodies. The lower limit of Lp(a) sensitivity was 1 mg/dl, and the intra- and interassay variation coefficients 5% and 7%, respectively. Ape(a) phenotyping was performed by SDWpolyacrylamide gel electrophoresis followed by immunoblotting. Serum (10 ~1) was delipidated according to Utermann et al. [18] by extraction with 2.5 ml of ethanol/ether, 4:l (vol/vol) for 16 h at -20°C. Proteins were collected by centrifugation at 3000 rev./min for 10 min and washed once with ether for 2 h at -20°C. The resultant protein pellet was dissolved by addition of 200 ~1 of a solution containing 5% (wt/vol) SDS/O.02 M ethylmorpholine, pH 8.6, 5 ~1 of 2mercaptoethanol, plus 10 ~1 of 1.5% (wt/vol) bromophenol blue in glycerol and boiled for 5 min at 100°C. The mixture (30 ~1) was applied to 4-12% polyacrylamide gradient gels and run for 10 h at 30 V in a mini-cell EI 6001 vertical electrophoresis unit (Linus, San Francisco, CA). After

J. Pedro-Botet et al. / Afherosclerosis 104 (1993) 87-94

89

electrophoresis, gels were placed on nitrocellulose membranes and electroblotted for 12 h at 30 V in an electrophoresis unit (Hoeffer Scientific Inst, San Francisco, CA). The nitrocellulose membranes were blocked by a skim milk powder solution and the ape(a) bands visualized by a double-antibody procedure. The first antibody was a sheep anti-human Lp(a) (Immuno, Vienna, Austria), and a goat anti-sheep peroxidase conjugate (Sigma, St Louis, MO) was used as the second antibody at a 1:300 dilution. Blots were washed repeatedly with a Tris/NaCl solution, pH 10.2 and developed in 3,3’-diaminobenzidine peroxide substrate. The respective phenotypes were designated according to Utermann et al. [ 13,161, and accurate phenotyping was achieved by placing a standard serum (Immuno) with Sl/S2/S3/F bands in every gel. Isoforms B and S4 were identified according to their electrophoretic mobility halfway between F and Sl for the former and the latter with respect to S3. Since immunoblotting methods have not yet been adequately standardized, ape(a) isoforms which do not migrate exactly with any of the standard isoforms were included as the closest standard respective isoforms.

Frequencies 70

2.3. Statistical analysis Contingency tables of ape(a) phenotypes and allele frequencies in patients and controls were compared by Pearson’s x2 statistic and a likelihood-ratio test, The equality of Lp(a) levels among phenotypes was tested using the Kruskal-Wallis analysis of variance. Comparison between two groups was made using the Mann Whitney nonparametric test. Estimated frequencies of ape(a) alleles were calculated according to the method of Wiener et al. [27,28]. Significance levels were set at 0.05 in all cases. 3.

Results

Serum Lp(a) concentration was significantly raised in PVD patients compared with controls (21 f 2.2 mg/dl and 13 f 1.3 mg/dl, mean f Serum Lp(a) S.E.M., respectively, P < 0.005). levels showed no significant differences between patients with moderate distal ischaemia and those with severe distal ischaemia (21 f 2.8 mgidl and 19 f 4.3, respectively). The frequency distribution of serum Lp(a) concentration in patients with PVD and controls was skewed to the left, especially among controls (Fig. 1). Furthermore, 33 pa-

(2)

1

60-

50 -

40 -

m

Control8

blZ9)

m

Pstlents

w39)

302010 -

O-

o-s

10-19

20-29

30-39

40-49

50-59

60-69

70-79

Lp(ah mg/dl Fig. I. Frequency

distribution

of Lp(a) levels in controls

and in patients

with peripheral

vascular

disease.

90

J. Pedro-Borer

et al. /Atherosclerosis

104 (1993)

87-94

Table I Frequency of ape(a) phenotypes and Lp(a) concentrations in controls and patients with peripheral vascular disease Phenotype

Controls (n = 129)

Patients (n = 89)

Phenotype frequency No. of subjects

F F/S2 F/S3 F/S4 B B/S2 B/S4 Sl SllS2 SllS3 SI/S4 s2 S2lS3 S2lS4 s3 S3IS4 s4

Lp(a) (mean (S.E.M.)) mg/dl

%,

Lp(a) (mean (S.E.M.)) mg/dl

No. of patients

2 4 4 I I I I I

1.6 3.1 3.1 0.8 0.8 0.8 0.8 0.8

5 I I3 5 I2 31 I1 28

3.9 0.8 IO.1 3.9 9.3 24.0 8.5 21.7

22 (9.6)

8

6.2

2 3

2.2 3.4

I

I.1

I 3

1.1 3.4

IO (2.5) I8 (10.0) I7 (4.9) I5 (2.3) I7 (5.9) 7 (1.4)

I I4 8 5 21 I 20

1.1 15.7 8.9 5.6 23.6 7.9 22.5

4 (0.5)

3

3.4

42 (12.5) 25 (18.8)

-

‘Null’

Phenotype frequency

25”

-

44”

22 (4.5)* 22 (7.4) II (2.7) 22 (4.6) 43 (9.8)* II (l.9)* 4”

Pearson’s x2 test, P:NS. “At least three observations were made to determine the mean Lp(a) concentration and at least four to determine the S.E.M. *P < 0.05 with respect

to controls.

Table 2 Estimated frequencies of ape(a) alleles in controls and patients with peripheral vascular disease Allele

Frequency of allele Controls (n = 129)

All patients

Distal ischaemia

(n = 89)

Moderate (n = 46)

Severe (n = 22)

F B SI s2 s3 s4

0.028 0.014 0.014 0.150 0.296 0.275

0.050 0.025 0.025 0.217 0.289 0.278

0.037 0.000 0.037 0.225 0.285 0.203

0.117 0.117 0.000 0.169 0.274 0.349

‘Null’

0.232

0.153

0.245

0.000

Pearson’s x2 test, P:NS.

tients (37%) and 25 controls (19%) showed serum Lp(a) concentrations above 20 mg/dl (P c 0.005). Using 30 mg/dl as a cut-off, the distribution was 24 (27%) and 17 (13%), respectively (P < 0.05). Results of ape(a) phenotyping of the patients are shown in Table 1, and the estimated frequencies of ape(a) alleles in Table 2. Ape(a) phenotype frequencies were not significantly different between patients and controls. Specifically, the most prevalent S3 and S4 ape(a) phenotypes in the control group were also the most common in PVD patients. Similarly, no ape(a) phenotype was overrepresented in the patient group with respect to controls. The most revealing finding was that Lp(a) levels were significantly increased in patients with S4, S3lS4 and S2 phenotypes compared with controls with the same phenotype (Table 1). In controls, the non-parametric Kruskal-Wallis test showed a highly significant difference (P = 0.001)

J. Pedro-Borer et al. /Atherosclerosis Table 3 Homozygote

and heterozygote

ape(a)

91

104 (1993) 87-94

phenotypes

and serum Lp(a) levels in controls

No.

Lp(a) (mgidl

*P < 0.05 with respect to homozygote controls.

Phenotype

Moderate No. of patients

F FlS2 B Sl SllS2 s2 WS3 S2lS4 s3 S3IS4 s4 ‘Null’ Pearson’s

I 2 I 2 8 5 2 II 4 7 3 x2 test, P:NS.

in patients

(n = 46) o/u

2.2 4.3 0.0 2.2 4.3 17.4 10.9 4.3 23.9 a.7 15.2 6.5

Lp(a) (mgidl * S.E.M.)

59 21

17.2 f 2.27 29.5 f 4.8”

status, **P < 0.005 with respect to homozygote

among the Lp(a) concentrations of the different ape(a) phenotypes. Serum Lp(a) levels were also significantly different among ape(a) phenotypes in patients with PVD (P < 0.05). When the cut-off of 20 mg/dl of serum Lp(a) was used, differences in the distribution of ape(a) isoforms were found, with S2 phenotype being more prevalent in patients with serum Lp(a) levels above 20 mg/dl (P < 0.05), but not in controls. No significant differences were found in the frequencies of homozygote and heterozygote phenotypes between patients and controls. However, in both groups, serum Lp(a) levels were significantly higher among heterozygote phenotypes than homozygote phenotypes (Table 3).

Table 4 Ape(a) phenotype frequencies severe distal ischaemia

No ?? S.E.M.)

10.6 f 1.2 20.2 f 2.9**

16 45

Homozygote Heterozygote

with peripheral

vascular

disease

PVD patients

Controls

Phenotype

and patients

with moderate

and

Severe (n = 22) No. of patients

I I 2 2 2 5 I 8

“/u

0.0 4.5 4.5 0.0 0.0 9.1 9.1 9.1 22.1 4.5 36.4 0.0

status, tP < 0.01 with respect to homozygote

Results of ape(a) phenotyping of the 46 patients with moderate distal ischaemia and the 22 patients with severe distal ischaemia according to ABI measured by Doppler, are shown in Table 4, and estimated frequencies of ape(a) alleles in the two groups are shown in Table 2. Comparison of the ape(a) phenotypes failed to show signiticant differences between the moderate and severe distal ischaemia groups. 4. Discussion It is known that individuals who seek medical attention for intermittent claudication represent only part of those with PVD, whereas a larger group is asymptomatic or shows only limited or atypical findings. All the patients included in this study were symptomatic. In fact, several reports indicate that the prevalence rate of intermittent claudication in populations with an average age similar to that of our study group is about l-4% [29-311. On the other hand, the actual prevalence of PVD has been variously assessed with noninvasive tests, and seems to be approximately 11-150/o for subjects over 60 years of age [31]. It has become increasingly clear that genetic predisposition plays a role in the pathogenesis of atherosclerosis, particularly when disease occurs at a relatively young age. There is general agreement that nutritional and lifestyle factors preferentially cause disease in subjects with a genetic predisposition. Although the genetic determination of Lp(a) is unquestionable, it seems that ape(a) genetic polymorphism could account for 40% of the variability of serum Lp(a) concentration in different populations [32]. However, more

92

recently the same investigators [33] estimated that the ape(a) gene was responsible for greater than 90% of the variation in plasma Lp(a) concentrations, In addition to smoking and diabetes mellitus, we have previously emphasized the role of lipoprotein disturbances as a major risk factor for PVD in subjects with raised serum cholesterol and triglyceride levels and also in ‘normolipidaemic’ subjects [20]. In recent years, attention has been drawn to both atherogenic and thrombogenic properties of Lp(a), and raised serum levels have been found in patients with coronary heart disease [3-71 and cerebrovascular disease [8-l 11. The present study confirms that Lp(a) levels’ are significantly increased in PVD [21,23], to a magnitude similar to that seen in other atherosclerotic localizations. However, we failed to find a relationship between severity of the disease and serum Lp(a) levels. Using SDS-PAGE and immunoblotting, at least six different common isoforms designated F, B, Sl, S2, S3, and S4 have been described [13,16,18]. The relation of ape(a) electrophoretic migration to molecular weight is in the order of F > B > Sl > S2 > S3 > S4, where B represents migration similar to apo B, F is faster, and S is slower [ 13,16,18]. The originally designated ape(a) phenotypes B, Sl and S2, and possibly also F, seem to be associated with high serum Lp(a) concentrations and phenotypes S3 and S4 with low concentrations [ 13,16,18]. This association indicates the inverse relation between ape(a) size and Lp(a) plasma concentration. Although other authors have demonstrated additional isoforms [ 14,15,34-361, the identification of more isoforms does not change this relation [37]. In the present study, ape(a) isoform frequencies in the control group were similar to those previously described in other European [18] and also Japanese populations [7] but different from those of other ethnic groups [38]. However, the allele F both in controls and patients was more prevalent, which gave rise to F homozygote states or alternatively heterozygotes with ‘null’ allele and heterozygotes with alleles S2, S3 and S4. This may be related to the inclusion of a standard with an F isoform in the immunoblotting run which

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facilitates its identification. It is noteworthy that the distribution of phenotypes in patients did not differ from that of the control group. As described in the general population and patients with coronary heart disease [7,13,17,18], in the present study, both controls and PVD patients with S2 phenotype had higher serum Lp(a) levels than those with S4 phenotype. Similarly, it should be emphasized that serum Lp(a) levels were significantly increased in patients with S2, S3lS4 and S4 phenotypes compared with controls with the same phenotype. Furthermore, in both patients and controls, heterozygotes showed significantly higher Lp(a) levels than homozygotes. These results are in agreement with those reported by Abe et al. [7] and Gaubatz et al. [14] and suggest that heterozygote status has a greater risk for atherosclerosis than homozygote status. On the other hand, in the two populations reported here, the frequency of double-band phenotypes varied from 30.3% to 34.8% of all phenotypes. Despite the possible underestimation of heterozygote status, this ratio was higher than that recently reported [ 17,37-391, similar to that described by Gaubatz et al. [14], and slightly lower than that reported by Helmhold et al. [34]. Our results show that the severity of distal ischaemia does not appear to depend on the influence of ape(a) polymorphism on serum Lp(a) concentration. Nevertheless, the relatively small number of patients did not permit us to establish differences in ape(a) phenotype prevalence between those with moderate and severe distal ischaemia. In conclusion, men with PVD have higher serum Lp(a) levels than healthy subjects, as described in coronary heart disease and ischaemic cerebrovascular disease. Although ape(a) genetic polymorphism clearly influences serum Lp(a) levels in patients with PVD, as occurs in the general population, environmental and/or other genetic factors not related to ape(a) polymorphism must play a role in raised serum Lp(a) levels in these patients. 5. Acknowledgements Supported by grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social

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(0624/91). We thank Christine O’Hara for her help with the preparation of the manuscript.

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