The Asp9 Asn Mutation in the Lipoprotein Lipase Gene Is Associated With Increased Progression of Coronary Atherosclerosis
Background Many patients suffering from premature coronary artery disease report a family history for such events. A mutation in a particular gene, which confers susceptibility for atherosclerosis, will be found more frequently in individuals suffering from coronary atherosclerosis than in the general population. We have recently reported the identification of an Asp9 Asn substitution in the lipoprotein lipase (LPL) enzyme. We analyzed the impact of this mutation on the progression of coronary atherosclerosis and the effect of pravastatin in both carriers and noncarriers.
Methods and Results All patients were enrolled in the quantitative coronary angiographic clinical trial REGRESS, which studied the impact of pravastatin therapy on coronary atherosclerosis. The Asp9 Asn mutation was identified in 38 of 819 (4.8%) patients. Carriers of the mutation more often had a positive family history of cardiovascular disease and lower HDL cholesterol levels than noncarriers. In the placebo group, carriers showed more progression of coronary atherosclerosis than noncarriers: mean reduction of the minimum obstruction diameter of −0.25 mm versus −0.12 mm (P=.029) and increase of percentage diameter stenosis of 6.4% versus 1.4% (P=.004). Moreover, the adjusted relative risk for a clinical event for carriers was calculated at 2.16 (95% CI, 1.09 to 4.29; P=.027). Although the lipid-lowering effect of pravastatin was attenuated in carriers, it appeared that these patients showed a response similar to noncarriers in terms of less progression of atherosclerosis and event-free survival.
Conclusions This study shows that heterozygosity for a mutation in the LPL gene, which causes only subtle changes in fasting plasma lipids, may promote the progression of coronary atherosclerosis and diminish clinical event–free survival.
Lipoprotein lipase is a multifunctional protein; it is anchored to the vascular endothelium where it constitutes the rate-limiting step in the breakdown of triglycerides in lipoproteins such as chylomicrons and VLDL.1 Recently, LPL has also been shown to serve as a ligand for the LDL receptor–related protein (LRP) and to affect secretion and uptake of VLDL and LDL cholesterol.2
Chylomicronemia was considered the clinical phenotype of LPL deficiency, a rare autosomal recessive disorder, and originally chosen for the elucidation of LPL gene mutations at the molecular level.1
However, more recently, it was established that heterozygosity for mutations in the LPL gene is associated with partial LPL activity and a milder clinical phenotype.3 4 In this disorder, lipoprotein abnormalities are more subtle and consist of increased triglycerides, marked reduced HDL2 cholesterol, decreased LpAI, and depletion of apo CIII in HDL as a marker of inefficient lipolysis, among others.5
These lipid and lipoprotein abnormalities would predict a relatively increased risk of premature CAD and increased progression of CAD, which is consistent with the findings in both the Cholesterol Lowering Atherosclerosis Study (CLAS) and the Monitored Atherosclerosis Regression Study (MARS), in which decreased apo CIII in HDL was a good predictor of the progression of coronary atherosclerosis.6 7
Identifying patients at increased risk for premature CAD is important, because these patients might benefit from early lipid-lowering treatment. Thus far, however, it has proven to be difficult to identify patients at increased risk for (progression of) premature CAD when lipoprotein disturbances were only moderate. We therefore initiated the study of the nature and frequency of LPL gene mutations in individuals with coronary artery disease and subtle combined hyperlipidemia. In individuals with combined hyperlipidemia, a particular mutation, ie, an aspartic acid to an asparagine residue at position 9 (Asp9 Asn) in the mature LPL protein, could be identified in a relatively high proportion (4% to 6%).8 This mutation proved to be accompanied by the high-triglyceride–low-HDL cholesterol phenotype. More recently, genetic material of individuals with clinically manifest coronary atherosclerosis documented by quantitative coronary angiography became available to us. These individuals were participants in a multicenter angiographic clinical trial, the Regression Growth Evaluation Statin Study (REGRESS) carried out in the Netherlands, which studied the impact of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor pravastatin on the evolution of atherosclerotic disease of the coronary arteries in patients with normal and moderately disturbed lipid and lipoprotein levels. This study has been published in detail elsewhere.9
We hypothesized that the presence of the Asp9 Asn LPL mutation in a given patient would confer increased susceptibility to atherosclerosis and therefore be associated with more progression of CAD. Moreover, since a considerable number of placebo-treated patients were available for follow-up, the natural history of this mutation could be determined. Furthermore, since carriers and noncarriers were evenly distributed over placebo- and pravastatin-treated groups, we had the opportunity to analyze the effect of pravastatin on all above-mentioned characteristics of these Asp9 Asn LPL mutation carriers. The above-mentioned analyses were also considered for other mutations in the LPL gene, ie, Gly188 Glu and Pro207 Leu, which are also associated with lipoprotein disturbances; however, the incidence of these mutations in the REGRESS population (considered to be representative for the majority of cardiology patients seen in general practice) was far too low to allow meaningful conclusions with regard to an increased risk for progression of CAD; therefore, these mutations are not discussed here.
The REGRESS study and design have been described elsewhere.9 In summary, REGRESS is a randomized, placebo-controlled multicenter study to assess the effect of 2 years of treatment with pravastatin 40 mg on progression and regression of angiographically documented coronary atherosclerosis in 885 male patients with a normal to moderately raised serum cholesterol, ie, between 4 and 8 mmol/L (155 to 310 mg/dL) and triglycerides <4.0 mmol/L (354 mg/dL), as determined after an overnight fast. After certification, the patients were divided into one of three cohorts according to the type of primary management elected at the participating center: a percutaneous transluminal coronary angioplasty (PTCA) cohort, a coronary artery bypass grafting (CABG) cohort, and a medical management cohort. In each cohort, patients were then randomized to receive pravastatin 40 mg once daily or matching placebo. Patients and physicians were blinded to the randomization throughout the study.
A number of substudies were performed in addition to the main angiographic study, including specialized lipid and lipoprotein and genetic studies.
Quantitative Coronary Arteriography
Coronary angiograms were analyzed quantitatively by the Cardiovascular Measurement System (CMS, MEDIS Medical Imaging Systems, Nuenen, the Netherlands). The quantitative coronary arteriographic procedures are described in detail elsewhere.9 To standardize coronary vasomotor tone, 5 to 10 minutes before coronary angiography, 5 to 10 mg isosorbide dinitrate was administered sublingually. Primary end points were (a) the change in average MOD per patient and (b) the change in average MSD per patient. Change in MOD mainly reflects focal progression-regression of atherosclerosis, and change in MSD mainly reflects diffuse progression-regression of atherosclerosis. Although we prefer to use parameters that provide absolute measurements (MOD and MSD), percentage diameter stenosis (% D-stenosis) was included as a secondary end point because of its widespread use and clinical relevance to describe angiographic changes when judged by expert readers. If a segment or lesion was adequately visualized in two (preferably orthogonal) projections and free of significant foreshortening in both views, the average values of the parameters in both projections were calculated. To calculate average MOD, MSD, and % D-stenosis per patient, the MOD, MSD, and % D-stenosis of all qualifying segments or obstructions were added and divided by the number of contributing segments or obstructions.
The following clinical events (according to prespecified criteria) were analyzed during the study and identified before unblinding: myocardial infarction (fatal or nonfatal); coronary heart disease death (other than known fatal myocardial infarction); nonscheduled PTCA or CABG; stroke and transient ischemic attack; and death (all other causes).
Lipids and Lipoproteins
All lipid laboratory tests were carried out at the Lipid Reference Laboratory, as published previously.9 Serum cholesterol, HDL cholesterol, and triglycerides were measured on fasting blood samples by standard techniques at all visits. Total cholesterol was measured with an enzymatic kit (Boehringer Mannheim) and calibrated with a human serum calibrator. HDL cholesterol was measured after precipitation of apolipoprotein B–containing lipoproteins with a 4% tungstate solution and centrifugation, and the triglycerides were analyzed enzymatically (Bayer/Technicon) by a technique that included free glycerol. LDL cholesterol was calculated according to the Friedewald formula.
The Lipid Reference Laboratory is an international member of the USA National Cholesterol Reference Method Laboratory Network chaired by the Centers for Disease Control and Prevention (Atlanta, Ga).
Genomic DNA was isolated from white cells as described previously,4 dissolved in 10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0, and stored at 4°C.
Polymerase chain reaction of exon 2 of patients and control subjects was performed as described earlier by Mailly et al.8 After amplification, the polymerase chain reaction product was digested with Taq-1 (2 hours, 65°C) and subsequently subjected to electrophoresis through a 6% agarose gel (Agarose MP, Boehringer Mannheim). In case of the guanosine to adenosine substitution at position 9 of the LPL gene a Taq-1 restriction site is lost.
Patients with and without the Asp9 Asn mutation in exon 2 of the LPL gene were compared with each other with respect to relevant baseline characteristics, change of lipid values, and the change of angiographic parameters. Differences with respect to baseline parameters were analyzed with the Student's t test or the Pearson χ2 test where appropriate. We report changes in mean values for MOD (as for MSD) instead of changes in median values as calculated in the original REGRESS publication9 because for the present analysis, we were interested in the interaction between the presence of the Asp9 Asn mutation and pravastatin medication, and this interaction cannot be estimated using median values. Results for angiographic changes were in essence the same with the use of mean or median values for change in MOD. The change of the lipid values and the angiographic parameters were analyzed with two-way ANCOVA with randomized therapy (placebo or pravastatin) and the presence of the Asp9 Asn LPL mutation as fixed factors and baseline values as covariates. Time to first clinical event was analyzed with the Cox model. The differential effect of pravastatin treatment for patients with and without the mutation was assessed with the test for interaction between treatment and presence of the mutations. A value of P≤.05 was considered significant.
Eight hundred nineteen patients were available for analysis of the LPL gene; 419 of these patients received pravastatin and 401 placebo. Of these patients, a total of 60 patients (35 pravastatin, 25 placebo) did not have a pair of matching angiograms (baseline angiogram lost or no follow-up angiogram available [7 deaths]), and of 123 patients (68 pravastatin, 55 placebo), the follow-up angiogram was considered not informative because all analyzable segments were considered influenced by CABG or PTCA and were therefore excluded from quantitative angiographic analysis.9 This “drop-out” rate did not differ significantly between the two treatment groups (P=.26). Thus, the effect of pravastatin treatment on angiographic progression could be evaluated in 637 patients (78%).
In total, 38 of 819 patients (4.8%; 95% CI, 3.3% to 6.3%) carried the Asp9 Asn mutation in exon 2 of the LPL gene: 23 in the placebo group (5.7%) and 15 in the pravastatin group (3.6%) (P=.14). Of these patients, a total of 14 patients (6 pravastatin, 8 placebo) had no (informative) pair of matching angiograms (no follow-up angiogram available or the follow-up angiogram was considered not informative because all analyzable segments were considered influenced by CABG or PTCA and were therefore excluded from quantitative angiographic analysis).9 Thus, the effect of the Asp9 Asn mutation on angiographic progression could be evaluated in 24 of the 38 patients (64%). Some baseline characteristics of patients with and without this mutation are reported in Table⇓s 1 and 2. Patients carrying the Asp9 Asn mutation had significantly more often a positive family history for cardiovascular disease (defined as myocardial infarction, sudden cardiac death, CABG, or PTCA <60 years of age in a first-degree relative) (P=.03) and had a lower HDL cholesterol level at baseline (P=.01) than patients without the mutation. Unadjusted levels of total cholesterol, LDL cholesterol, and triglycerides showed a trend toward elevation in Asp9 Asn LPL mutation carriers that did not reach statistical significance (P=.11, .14, and .13, respectively).
Lipids and Lipoproteins
Mean changes of the lipid and lipoprotein parameters are reported in Table 3⇓. Within the group of patients who received pravastatin, the reduction of total and LDL cholesterol appeared to be less in patients with the mutation compared with patients without the mutation. (P=.021 for total cholesterol and P=.008 for LDL cholesterol). Within the placebo groups, no such differences were observed. However, despite their extent, the differences between patients with and patients without the mutation with regard to the change of lipid levels did not differ significantly between the placebo and the pravastatin groups (interaction tests: P=.18 for total cholesterol, P=.08 for LDL cholesterol, P=.63 for HDL cholesterol, and P=.80 for triglycerides).
Results with regard to angiographic parameters are shown in Table 4⇓. In the placebo group, the mean reduction of the MOD (progression of focal atherosclerosis) in patients carrying the Asp9 Asn LPL mutation was −0.25 mm (SD, 0.58), while in patients without the mutation the reduction was −0.12 mm (SD, 0.21) (P=.29). In the pravastatin group, the mean MOD in patients with the Asp9 Asn LPL mutation slightly increased +0.01 mm (SD, 0.17), and in patients without the mutation the mean MOD decreased −0.09 mm (SD, 0.34) (P=.40). The test for interaction between randomized therapy and the Asp9 Asn LPL mutation had a probability value of .06, suggesting a trend that the deleterious effect of the mutation on progression of coronary atherosclerosis could be reversed by pravastatin therapy.
With respect to the % D-stenosis during the trial, similar findings were observed. In the placebo group, the mean % D-stenosis increased +6.4% (SD, 16.4%) in patients with the mutation and +1.4% (SD, 5.6) in patients without the mutation (P=.004). In the pravastatin group, the mean % D-stenosis decreased −0.7% (SD, 4.2) in patients with the mutation, and in patients without the mutation the mean % D-stenosis increased +1.3% (SD, 9.1); this difference was not statistically significant (P=.52). The interaction test between randomized therapy and the Asp9 Asn mutation had a probability value of .038, indicating that the deleterious effect of the mutation on progression of coronary atherosclerosis could be overcome by pravastatin therapy.
With respect to the change of the MSD (change of diffuse atherosclerosis), no statistically significant differences were observed between patients with or without the Asp9 Asn LPL mutation either in the placebo or in the pravastatin group. Angiographic results for patients with or without the Asp9 Asn LPL mutation with regard to changes in lesions <50% diameter stenosis were essentially similar to the results for all lesions as shown in Table 4⇑. No influence of the Asp9 Asn mutation was observed with regard to changes in lesions ≥50% diameter stenosis either in the placebo group or in the pravastatin group.
Of the 38 patients carrying the Asp9 Asn LPL mutation, 9 experienced one or more clinical events during the trial (24%), while of the 782 patients without the mutation, a total of 110 experienced clinical events (14%). The relative risk of the presence of the Asp9 Asn LPL mutation for any clinical event within 2 years was therefore estimated as 1.85 (95% CI, 0.94 to 3.66; P=.08). Using a stepwise Cox regression model with randomized therapy (placebo/pravastatin), age, hypertension, positive family history, smoking, history of myocardial infarction, body mass index, systolic and diastolic blood pressures, left ventricular ejection fraction, extent of coronary artery disease, baseline nitrate, β-blocking and calcium channel–blocking therapy, and baseline lipids (total cholesterol, HDL and LDL cholesterol, and triglycerides), it appeared that randomized pravastatin therapy (P=.007) and calcium channel–blocking therapy (P=.038) were the only statistically independent prognostic factors for clinical event–free survival. Adjusted for these two prognostic variables, the relative risk for a clinical event of the Asp9 Asn LPL mutation was then estimated at 2.16 (95% CI, 1.09 to 4.29; P=.027). After 2 years of follow-up, 90% of the patients in the pravastatin group without the Asp9 Asn LPL mutation and 79% of patients carrying Asp9 Asn LPL mutation were event-free alive, while in the placebo group these numbers were 81% and 73%, respectively (Table 4⇑). The effect of pravastatin on the clinical event–free period was not significantly different for patients with and without the Asp9 Asn LPL mutation (interaction test: P=.52).
Identifying patients at increased risk for premature CAD and an increased progression of CAD is important because these patients might benefit from early lipid-lowering treatment. Thus far, however, it has proven to be difficult to identify patients at such an increased risk when lipoprotein disturbances were only moderate. We therefore initiated the study of the nature and frequency of LPL gene mutations in individuals with CAD and subtle combined hyperlipidemia.
Here we report the identification of a common mutation, ie, an aspartic acid to an asparagine substitution at position 9 (Asp9 Asn) of the LPL protein. This Asp9 Asn mutation was identified in 4.8% of the patients who participated in the lipid-lowering angiographic clinical study REGRESS.9 Carriers of this Asp9 Asn LPL mutation had significantly more often a positive family history of cardiovascular disease and exhibited lower HDL cholesterol levels than noncarriers and tended toward elevated levels of LDL cholesterol and triglycerides. Indeed, it could be shown that these patients with only relatively subtle disturbances of lipoprotein metabolism due to the presence of this gene mutation exhibited accelerated progression of coronary atherosclerosis and a diminished clinical event–free survival.
Recently, it could be demonstrated that heterozygosity for a particular mutation in the LPL gene was associated with increased triglycerides, low HDL2 cholesterol, decreased LpAI, and reduced apo CIII in HDL as markers of inefficient lipolysis.5 This suggests that LPL activity in these mutation carriers may not be sufficient to keep plasma triglyceride levels within normal limits when adverse exogenous influences exert stress on the lipid transport system. In the angiographic regression trial CLAS, univariate on-trial predictors of progression in the placebo-treated group were total triglyceride levels, as well as levels of apo CIII in whole serum and in the VLDL fraction, accompanied by a reduction of apo CIII in HDL particles.6 The association of both triglyceride-rich lipoproteins and apo CIII levels with the progression of coronary atherosclerosis points to the pivotal role of the removal of these particles by LPL as a protective step against atherogenesis. Furthermore, in the MARS study, multivariate analysis indicated that higher on-trial apo CIII was the single risk factor that was independently associated with the progression of mild to moderate lesions (<50% diameter stenosis).7 In our patients we also observed that the most progression in the Asp9 Asn LPL mutation group was apparent for lesions with a <50% diameter stenosis.
In the presence of a partial defect in lipolysis, triglyceride-rich particles circulate for longer periods of time, increasing the probability of contact between these particles and the endothelial lining of the vascular tree. The association of triglyceride-rich lipoproteins with the progression of mild to moderate lesions may be of particular clinical importance, since recent studies have demonstrated that these may be the culprit lesions underlying myocardial infarction.10 11 In our study, the excess of angiographic progression seen in Asp9 Asn carriers was accompanied by a decreased 2-year event-free interval. As a substrate for free radical reactions, triglyceride-rich lipoproteins in general, and VLDL in particular, may facilitate lesion destabilization and rupturing of the central lipid core. Triglyceride-rich particles such as VLDL and IDL have been identified within atherosclerotic plaques acquired from human specimens.12 This mechanism may explain the clinical relevance of the presence of Asp9 LPL mutation as demonstrated in our study, with regard to not only coronary atherosclerosis but also to its clinical sequelae, in spite of only subtle lipid changes.
Relation of the Asp9 Asn LPL Mutation, Extent of Lipid Disturbances, and Progression of Atherosclerosis
When evaluating the effects of the Asp9 Asn LPL mutation, the question arose whether the relatively subtle changes in measured fasting plasma lipids could explain the increased progression of coronary atherosclerosis. Therefore, the patients carrying the mutation were matched with REGRESS patients who did not carry the mutation but had similar lipid disturbances.
Mutation carriers versus lipid-matched noncarriers (control patients)
Matching criteria for these control patients were for (1) HDL cholesterol: carrier HDL cholesterol ±0.05 mmol/L, for (2) triglycerides: carrier triglycerides ±0.10 mmol/L, and for (3) LDL cholesterol: carrier LDL cholesterol ±0.5 mmol/L. Thus defined, we identified 97 control patients. These control patients did not differ from the mutation carriers with regard to the baseline characteristics listed in Tables 1 and 2⇑⇑ (all P>.10), with the exception of familial heart disease, which again was more common in carriers of the mutation (P=.058). The extent of lipid lowering in the pravastatin group was not significantly different for mutation carriers versus their controls (all P>.10). The results with regard to change in angiographic parameters for mutation carriers versus control patients were essentially the same as shown in Table 4⇑ for mutation carriers versus (not matched) noncarriers in the placebo as well as in the pravastatin groups. Again, progression in the placebo group was significantly larger for carriers than for control patients (P=.016 and P=.035 for MOD and % D-stenosis, respectively), whereas in the pravastatin group no significant differences in progression between carriers and control patients were observed (all P>.15).
Therefore, we conclude that the deleterious effects of the Asp9 Asn LPL mutation on the progression of coronary atherosclerosis could not (solely) be explained by the fasting plasma disturbances in lipids and lipoproteins.
The finding that the deleterious effects of the LPL mutation on progression of coronary atherosclerosis were not related to disturbances in fasting plasma lipids and lipoproteins may be explained from the lipoprotein metabolism viewpoint or from the genetic viewpoint.
Potential mechanisms related to lipoprotein metabolism
The following factors may play a role: (1) fasting lipid levels in heterozygotes for LPL deficiency actually underestimate the true extent of the disturbance in postprandial lipemia. LPL activity in these mutation carriers is probably not sufficient to keep levels of atherogenic triglyceride-rich lipoproteins within normal limits when adverse influences (food) exert stress on the lipid transport system. (2) There is a change in lipoprotein kinetics by which a higher production of remnant particles is partially offset by a higher turnover, which leads to an increased uptake of atherogenic particles into the vessel wall, resulting in enhanced progression of coronary atherosclerosis, and (3) dysfunction of LPL in the arterial wall.
Potential mechanisms related to genetic factors
There may be a specific linkage between the LPL gene and other genes related to atherogenesis. In a group of subjects with the Asp9 Asn mutation, some genes related to LPL rather than the LPL mutation itself may be responsible for the progression of atherosclerosis. We have previously shown by site-directed mutagenesis and expression studies that the mutant Asp9 Asn LPL protein exhibited 20% to 30% less hydrolytic activity and dimeric mass.8 This is consistent with similar experiments involving other naturally occurring LPL mutations and makes it unlikely that another molecular defect needs to be involved to explain the dyslipidemia and increased progression of coronary atherosclerosis as seen in our study. However, a specific linkage between the LPL gene and other genes related to atherogenesis cannot be ruled out.
Influence of Pravastatin on Parameters in Mutation Carriers
In our study, the effect of pravastatin on lipid and lipoprotein levels showed a trend toward attenuation in patients carrying the Asp9 Asn LPL mutation. However, it appeared that the deleterious effects of this mutation on progression of atherosclerosis could be (partially) reversed by pravastatin. This could not yet be demonstrated with regard to 2-year event-free survival. However, in view of the correlation between angiographic progression and subsequent clinical events as demonstrated by several groups,13 14 15 it is reasonable to assume that pravastatin therapy in Asp9 Asn LPL mutation carriers is also associated with improved clinical outcome, especially after longer follow-up. Properties other than lowering of LDL cholesterol of HMG-CoA reductase inhibitors, such as a direct effect on plaque structure and vascular tone, might contribute to this effect.10 11 16 17 It may be hypothesized, however, that other lipid-lowering drugs, such as fibric acid derivatives, are better suited to normalize the disturbed lipid and lipoprotein profiles seen in LPL heterozygotes. However, no results of randomized clinical trials are currently available to verify this supposition.
This is the first report to show that heterozygosity for a mutation in the LPL gene, which causes only subtle changes in fasting plasma lipids, can promote the progression of coronary atherosclerosis and can diminish clinical event–free survival. Although the lipid-lowering effect by pravastatin was attenuated in patients carrying the Asp9 Asn substitution, the deleterious effects of this mutant on the progression of atherosclerosis could be reversed by pravastatin.
Selected Abbreviations and Acronyms
|CAD||=||coronary artery disease|
|MOD||=||minimal obstruction diameter|
|MSD||=||mean segment diameter|
The REGRESS study was sponsored by Bristol-Myers Squibb Co, Princeton, NJ. We thank Valentin Fuster, MD, PhD, the Mount Sinai Medical Center, New York, for his careful review and helpful suggestions.
- Received January 24, 1996.
- Revision received April 25, 1996.
- Accepted May 1, 1996.
- Copyright © 1996 by American Heart Association
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