Abstract
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a protein involved in LDL-cholesterol metabolism. The single-nucleotide polymorphism (SNP) rs11591147 has been associated with lower LDL-cholesterol and a lower risk of coronary heart disease. Because PCSK9 has high affinity to the LDL receptor, inhibiting PCSK9 is a testable therapeutic target for lipid-lowering therapy. Currently, several approaches to inhibit PCSK9 are under development, but it is unknown what the effects of those inhibitors will be on cognition or noncardiovascular clinical events. In this study, we assessed the association between rs11591147 and cognitive performance, activities of daily living (ADL), and noncardiovascular clinical events within 5,777 participants of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). Rs11591147 was associated with 10% to 16% lower LDL cholesterol levels (P = 3.62 × 10−12), but was not associated with cognitive performance, ADL, or noncardiovascular clinical events in the PROSPER study. Our findings suggest that lower cholesterol levels due to genetic variation in the PCSK9 gene are not associated with cognitive performance, functional status, or noncardiovascular clinical events.
Keywords: genetic variation, single-nucleotide polymorphism, lipid lowering, PROSPER
Elevated plasma concentration of LDL-cholesterol is a major causal risk factor for cardiovascular disease. Lowering LDL-cholesterol levels is one of the primary goals of therapy in its prevention and treatment (1). Present lipid-lowering therapy mainly depends on treatment with HMG-CoA reductase inhibitors (statins), but new lipid-lowering drugs are under development that can decrease circulating lipid levels even more (2). A promising new therapeutic target for lipid-lowering therapy is proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a protein involved in LDL-cholesterol metabolism (3–5). PCSK9 modulates plasma LDL-cholesterol levels by promoting the degradation of LDL receptors (LDLRs) (6).
Several “loss-of-function” and “gain-of-function” mutations have been described in the PCSK9 gene (4). Within the Atherosclerosis Risk in Communities study, “loss-of-function” mutations, for example rs11591147, were associated with lower LDL-cholesterol levels through increased LDLR levels, resulting in a lower prevalence of peripheral arterial disease and a reduced risk of coronary heart disease (7, 8). A recent meta-analysis showed 12% lower LDL-cholesterol levels and 28% lower ischemic heart disease (IHD) risk among carriers of the rs11591147 mutation in the PCSK9 gene (9).
On the other hand, PCSK9 may be involved in processes associated with neurodegenerative disorders like Alzheimer's disease. A Canadian study found a two-fold increased expression of the PCSK9 in the human frontal cortex of autopsy-confirmed Alzheimer's disease cases compared with age-matched controls (10). Furthermore, men carrying either the rs2483205-C, rs483462-A, or rs662145-T allele showed a significant increased risk for Alzheimer's disease. However, those single-nucleotide polymorphisms (SNPs) are not known to be associated with LDL-cholesterol levels or with cardiovascular disease risk.
Several approaches to inhibit PCSK9 are currently under development, but it is not known whether those inhibitors will have influence on cognitive performance or clinical events. In old age, cholesterol levels are not associated with cognitive performance, and it has previously been shown, in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER), that pravastatin therapy in old age did not affect cognitive decline over a median follow-up of 3.2 years (11, 12). However, to investigate whether lipid lowering via PCSK9 inhibition might have other clinical effects in addition to lipid lowering, we assessed the relation between lower LDL levels associated with genetic variation within the PCSK9 SNP rs11591147 and cognitive function, activities of daily living (ADL), and noncardiovascular clinical events in an elderly population where these events occur frequently.
METHODS
Study population
All data come from the PROSPER study. Detailed descriptions of PROSPER have been published elsewhere (13, 14). Briefly, PROSPER was an investigator-driven, prospective multinational randomized placebo-controlled trial to assess whether treatment with pravastatin reduces the risk of major vascular events in the elderly. Between December 1997 and May 1999, subjects in Scotland (Glasgow), Ireland (Cork), and The Netherlands (Leiden) were screened and enrolled. Men and women aged 70–82 years were recruited if they had preexisting vascular disease or were at increased risk for such disease because of smoking, diabetes, or hypertension. A total of 5,804 subjects were randomly assigned to pravastatin or placebo. The protocol of the PROSPER study was approved by the medical ethics committees of each participating institution. Written informed consent was obtained from all participating subjects.
Endpoints
Mean follow-up duration was 3.2 years (range 2.8–4.0), and participants were reviewed every 3 months. During those visits, all endpoints were adjudicated by a study Endpoint Committee. The primary outcome in PROSPER was the combined endpoint of definite or suspected death from coronary heart disease, nonfatal myocardial infarction, and fatal or nonfatal stroke. Furthermore several noncardiovascular clinical events were recorded. Cancer incidence was a tertiary study endpoint. Infections and diabetes were reported as serious adverse effects. Because the association between PCSK9 and cardiovascular events has been investigated previously in PROSPER, the focus in the present paper was on noncardiovascular events, functional status, and cognitive performance.
Cognitive performance and ADL
The mini-mental state examination (MMSE) was used to measure global cognitive performance. The MMSE scores range from 0 (very severe cognitive impairment) to 30 points (optimal cognitive function). Participants with poor cognitive performance (MMSE <24) were not eligible for inclusion in the PROSPER study. Four neuropsychological performance tests were used to measure various cognitive domains. The Stroop color-word test for attention and the letter-digit coding test (LDT) for processing speed were used to measure executive function. The outcome parameter for the Stroop test was the total number of seconds to complete the third Stroop card containing 40 items. The outcome variable for the LDT was the total number of correct entries in 60 s. Memory was assessed with the 15-picture learning test testing immediate and delayed recall. The main outcome parameters were the accumulated number of recalled pictures over the three learning trials and the number of pictures recalled after 20 min. To assess change of functional status, the 20-point Barthel and instrumental activities of daily living (IADL) questionnaires, using a modified version of the OARS questionnaire, were performed. The Barthel index is a measure of basic ADL, and the IADL measures extended ADL. The reliability and sensitivity of these tests in an elderly population have been published elsewhere (15).
Cognitive performance and ADL were tested at six different time-points during the study: before randomization; at baseline; after 9, 18, and 30 months; and at the end of the study. The time-point of this last measurement was between 36 and 48 months and was different for the participants; therefore, we performed the analyses with their individually varying time-points but report the results for the mean of these time-points (42 months). The prerandomized measurement was discarded in the analysis to preclude possible learning effects.
Genotyping
Rs11591147 of the PCSK9 gene was genotyped previously in PROSPER using Taq Man® SNP genotyping assay (Applied Biosystems; Foster City, CA). A detailed description of the genotyping has been published elsewhere (16). Rs11591147 genotyping succeeded in 5,777 of the 5,804 PROSPER participants. ApoE phenotype was determined on plasma samples by Western blotting, using the method of Havekes et al. (17).
To assess the association between other PCSK9 SNPs and cholesterol levels, cognitive performance, and clinical events, we used data of the PHArmacogenetic study of Statins in the Elderly at risk (PHASE), a genome-wide association study (GWAS) in the PROSPER participants (18). The GWAS was conducted using the Illumina 660-Quad beadchips following the manufacturer's instructions. After a stringent quality control, 557,192 SNPs were available for analysis. To maximize the availability of genetic data and coverage of the genome, imputation up to 2.5 million autosomal CEPH HapMap SNPs was performed with MACH imputation software based on the Hapmap build II release 23. Detailed descriptions of the PHASE project have been published elsewhere (18). PCSK9 SNPs were selected from the GWAS using PLINK software version 1.07 (http://pngu.mgh.harvard.edu/purcell/plink/) (19).
Statistical analysis
Cross-sectional associations between rs11591147 and cognitive function were assessed using a general linear model adjusted for age, sex, country, education, apoE phenotype, and, where appropriate, version of test used. Repeated cross-sectional associations with cognitive function were assessed with a linear mixed model for repeated measurements and were additionally adjusted for follow-up time. Cross-sectional associations between rs11591147 and history of clinical events were assessed with logistic regression analysis, which was adjusted for age, sex, and country. Longitudinal associations were assessed with Cox regression and were additionally adjusted for pravastatin use. Power calculation was performed using Quanto version 1.2.4, May 2009. The study had 99% power to detect a 2% increased incidence of diabetes by the rs11591147 variant, assuming a 5% incidence rate in the rs11591147 wild-type carriers. PASW statistics software (version 17.0.2; SPSS, Inc., Chicago, IL) was used for all statistical analyses. P values <0.05 were considered statistically significant.
RESULTS
Table 1 shows the baseline characteristics of study subjects, stratified by country of origin. The mean age of all subjects was 75.3 years and about 50% of the participants were female. The association between the PCSK9 SNP rs11591147 and lipoprotein levels and vascular events has previously been described (16). To summarize these findings, carriers of one rs11591147 variant had 10% lower LDL-cholesterol levels (P = 3.62 × 10−12) (Table 2) and a nonsignificant 9% reduced risk of vascular disease (16).
TABLE 1.
Baseline characteristics of PROSPER participants stratified by country
| Scotland, N = 2,516 | Ireland, N = 2,166 | The Netherlands, N = 1,095 | |
| Continuous variables (mean, SD) | |||
| Age (years) | 75.3 (3.4) | 75.5 (3.3) | 75.1 (3.3) |
| Body mass index (kg/m2) | 26.7 (4.2) | 27.0 (4.4) | 26.7 (3.8) |
| Total cholesterol (mmol/l) | 5.7 (0.9) | 5.6 (0.9) | 5.8 (0.9) |
| LDL cholesterol (mmol/l) | 3.8 (0.8) | 3.7 (0.8) | 3.9 (0.8) |
| HDL cholesterol (mmol/l) | 1.3 (0.4) | 1.3 (0.4) | 1.2 (0.3) |
| Categorical variables (n, %) | |||
| Males | 1,237 (49.2) | 980 (45.2) | 577 (52.7) |
| Current smoker | 707 (28.1) | 580 (26.8) | 267 (24.4) |
| History of hypertension | 1,443 (57.4) | 1,430 (66.0) | 703 (64.2) |
| History of diabetes | 213 (8.5) | 220 (10.2) | 184 (16.8) |
| History of angina | 811 (32.2) | 518 (23.9) | 222 (20.3) |
| History of claudication | 229 (9.1) | 113 (5.2) | 46 (4.2) |
| History of myocardial infarction | 379 (15.1) | 255 (11.8) | 139 (12.7) |
| History of stroke or TIA | 265 (10.5) | 222 (10.2) | 161 (14.7) |
| History of vascular diseasea | 1,239 (49.2) | 842 (38.9) | 473 (43.2) |
| Genotype, MAF (%) | |||
| Rs11591147 (GT) | 2.1 | 1.8 | 1.2 |
TIA, transient ischemic attack; MAF, minor allele frequency.
Any of stable angina, intermittent claudication, stroke, transient ischemic attack (TIA), myocardial infarction, peripheral artery disease surgery, or amputation for vascular disease more than 6 months before study entry.
TABLE 2.
Rs11591147, cholesterol levels, cognitive performance, and functional status at baseline and during follow-up
| Rs11591147 | ||||
| Wt/Wt | Wt/Var | Var/Var | ||
| N = 5,337 | N = 193 | N = 2 | P-trend | |
| Baseline | ||||
| Cholesterol levels | ||||
| Total cholesterol (mmol/l) | 5.64 (0.03) | 5.30 (0.07) | 5.04 (0.59) | 1.18 × 10−8 |
| LDL-cholesterol (mmol/l) | 3.66 (0.03) | 3.29 (0.06) | 3.08 (0.52) | 3.62 × 10−12 |
| Functional status | ||||
| Barthel score | 19.77 (0.03) | 19.70 (0.06) | 19.57 (0.51) | 0.21 |
| Instrumental activities of daily living score | 13.61 (0.04) | 13.63 (0.08) | 12.98 (0.70) | 0.90 |
| Cognitive performance | ||||
| Mini mental state examination score | 28.00(0.05) | 28.05 (0.12) | 28.14 (1.04) | 0.67 |
| Stroop-Color-Word test | 65.73 (0.90) | 63.87 (1.97) | 65.72 (16.63) | 0.31 |
| Picture Learning test immediate | 9.41 (0.07) | 9.26 (0.15) | 10.03 (1.22) | 0.32 |
| Picture Learning test delayed | 10.27 (0.09) | 10.13 (0.21) | 10.41 (1.74) | 0.48 |
| Letter-digit Coding test | 23.44 (0.25) | 23.24 (0.54) | 21.39 (4.57) | 0.63 |
| Repeated cross-sectional | ||||
| Functional status | ||||
| Barthel score | 19.61 (0.03) | 19.55 (0.08) | 19.40 (0.66) | 0.37 |
| Instrumental activities of daily living score | 13.29 (0.05) | 13.32 (0.10) | 11.24 (0.88) | 0.90 |
| Cognitive performance | ||||
| Mini mental state examination score | 28.04 (0.05) | 28.12 (0.12) | 28.37 (1.02) | 0.40 |
| Stroop-Color-Word test | 66.48 (0.86) | 63.96 (1.89) | 66.91 (17.19) | 0.16 |
| Picture Learning test immediate | 9.37 (0.06) | 9.29 (0.13) | 9.20 (1.16) | 0.51 |
| Picture Learning test delayed | 10.01 (0.08) | 9.96 (0.18) | 10.94 (1.63) | 0.86 |
| Letter-digit Coding test | 22.85 (0.23) | 23.12 (0.49) | 22.45 (4.44) | 0.57 |
Estimates and P values at baseline were assessed with general linear model adjusted for sex, age, and country. Analyses on cognition were additionally adjusted for education and apoE genotype, and where appropriate, version of test used. Repeated cross-sectional estimates and P values were assessed with linear mixed model and additionally adjusted for follow-up time. Values are means (SE).
Table 2 shows the association between rs11591147 and cognitive performance and functional status. There were no associations between rs11591147 and cognitive performance and functional status, either at baseline or during follow-up (all P > 0.1).
Next, we assessed the association between rs11591147 and history of diabetes and hypertension at baseline. Carrying a variant of rs11591147 was not associated with a history of diabetes or hypertension (both P > 0.4) (Table 3). Also during follow-up, there was no association between rs11591147 and the incidence of cancer, diabetes, and infections (Table 3).
TABLE 3.
Rs11591147 and history or incidence of noncardiovascular clinical events
| Rs11591147 | |||||
| Wt/Wt | Wt/Var | Var/Var | |||
| N = 5,571 | N = 204 | N = 2a | |||
| N events | OR (95% CI) | OR (95% CI) | OR (95% CI) | P | |
| History of diabetes | 617 | 1 (ref) | 0.82 (0.49–1.36) | NA | 0.44 |
| History of hypertension | 3,575 | 1 (ref) | 1.08 (0.80–1.45) | NA | 0.63 |
| HR (95% CI) | HR (95% CI) | HR (95% CI) | |||
| Serious cancer | 441 | 1 (ref) | 0.86 (0.49–1.49) | NA | 0.59 |
| Incident diabetes | 291 | 1 (ref) | 1.39 (0.81–2.39) | NA | 0.23 |
| Incident infection | 313 | 1 (ref) | 0.90 (0.48–1.68) | NA | 0.73 |
Odds ratios (ORs) and corresponding P values were assessed with logistic regression analysis adjusted for age, sex, and country. Hazard ratios (HRs) and P values were assessed with Cox proportional hazard models adjusted for age, sex, country, and pravastatin use.
Not possible to perform logistic or Cox regression with n = 2.
To cover the entire PCSK9 gene, we also assessed the association between other PCSK9 tag SNPs (rs2094470, rs2479415, rs7525649, rs17111495, rs10888896, rs4927193, rs2479411, rs499718, rs10888897, rs529787, rs562556, rs615563, rs10465832, rs505151, rs662145, rs9326034) and cholesterol levels, cognitive performance, functional status, and clinical events, using data of the PHASE study (18). Three SNPs, rs2479415, rs2182833, and rs11206514, were associated with LDL-cholesterol levels (all P < 0.01). However, none of these SNPs were also associated with cognitive performance, functional status, or noncardiovascular clinical events (see supplementary Table I II). Also, when we combined the PCSK9 SNPs into haplotypes, there were no associations between the haplotypes, cholesterol levels, cognitive performance, functional status, or clinical events (data not shown).
DISCUSSION
In this study, we assessed the association between the PCSK9 SNP rs11591147, cognitive performance, functional status and noncardiovascular clinical events in the PROSPER study. No association between rs11591147 and cognitive performance, functional status, or nonvascular clinical events was observed. Because the SNP was significantly associated with lower cholesterol and with cardiovascular disease protection (7, 9, 16), these data do not lend arguments that drugs inhibiting PCSK9 will have other (adverse) effects.
The association between the PCSK9 SNP rs11591147 and lipid levels and vascular disease risk has been investigated previously in PROSPER (16). In this analysis, carriers of the rare variant of rs11591147 had significantly 10% lower LDL-cholesterol levels (P < 0.001). These lower LDL-cholesterol levels are well in line with the findings of a meta-analysis including seven general population studies with a total of 1,639 carriers of the rs11591147 minor allele and 59,298 noncarriers (9). In this meta-analysis, rs11591147 was associated with 12% lower LDL-cholesterol levels in carriers. The same article also reported a meta-analysis combining studies that investigated the risk of IHD, including 11,339 cases and 55,359 control subjects. The rs11591147 SNP was associated with a reduction in risk of IHD of 28% (fixed effect OR: 0.72, 95% CI: 0.62–0.84). This effect is higher than the previously reported results in PROSPER where rs11591147 was associated with a nonsignificant reduced risk of vascular disease of 9%.
The LDLR plays an important role in cholesterol metabolism by mediating the uptake of apoB-containing lipoprotein from the blood into liver cells (2). PCSK9 is involved in the cholesterol metabolism by promoting the degradation of LDLR (6). There are indications that cholesterol metabolism is associated with cognitive performance. High total serum cholesterol levels have been shown to be associated with lower cerebral spinal fluid levels of β-amyloid and larger amounts of β-amyloid deposition in brain autopsy studies (11). The involvement of PCSK9 in cholesterol metabolism raises the question of whether PCSK9 is also associated with cognitive function. A Canadian study investigated the relation between PCSK9 and Alzheimer's disease. In this study, variations in rs2483205, rs483462, and rs662145 were associated with an increased Alzheimer's disease risk (10). However, those SNPs are not known to be associated with cholesterol levels or in linkage disequilibrium with one of our SNPs associated with LDL-cholesterol. A study with Japanese Alzheimer's disease patients found no association between the PCSK9 SNPs rs11583680 and rs662145 and Alzheimer's disease (20). In our study, rs11591147 was not associated with cognitive performance.
Because PCSK9 has high affinity for the LDLR, inhibiting PCSK9 might be a promising therapeutic target for lipid-lowering therapy in addition to statin therapy. Several approaches to inhibit PCSK9 are currently under development, but it is unknown what the influences of those inhibitors will be on cognitive function or noncardiovascular events (2). In our study, rs11591147 associated with LDL-cholesterol levels did not affect cognitive performance, functional status, or noncardiovascular clinical events. On the basis of our results, one may expect that drugs inhibiting PCSK9 (21–23), to an extent comparable to the rs11591147 mutation, administered to a group aged 70–82 years with a history or increased risk of cardiovascular disease, are not likely to affect cognitive performance. This suggests that cholesterol lowering by PCSK9 inhibition may be a valid and safe way to reduce cardiovascular disease risk. However, because the inhibitors in development may cause larger LDL-cholesterol reductions compared with the rs11591147 mutation, definite conclusions about the adverse effects of cholesterol lowering due to PCSK9 inhibition cannot be assessed by this study.
One of the strengths of our study is our population size. We have prospective data for over 5,000 subjects on lipid parameters, cognitive function, and clinical endpoints. Also we have a follow-up of 42 months with very little lost to follow-up. Furthermore, our population is an appropriate study population to measure cognitive function, inasmuch as only subjects with a MMSE above 24 points were eligible for participation, which makes it a homogenous study group suitable for investigating cognitive function.
A possible limitation of our study is the limited power to detect subtle differences. Although the PROSPER study is one of the largest studies to investigate the association between genetic variations and lipid levels, cognitive function, and clinical events, we do not have infinite power to detect small effects. But we assume that it is unrealistic that we have missed clinically relevant effects. Another possible limitation is the ceiling effect of the ADL measurements. This may have restricted our ability to find significant associations between the PCSK9 mutation and ADL performance. However, previous analyses of the apoE genotype have proven that the PROSPER study is suitable for investigating genetic influences on cognition and ADL (24). A last possible limitation is the selection of the PROSPER participants. Participants were recruited when they had existing vascular disease or increased risk of such disease (13). Individuals particularly susceptible to the mutation leading not only to low LDL-cholesterol but also protective for cardiovascular disease may have been under-represented in our study.
In conclusion, it has previously been shown that the PCSK9 SNP rs11591147 is associated with lower LDL-cholesterol levels and lower cardiovascular risk. In our study, we failed to find associations between the variant and cognitive phenotypes. The implication of our finding is that it is unlikely that medication lowering LDL-cholesterol via inhibiting PCSK9 to an extent comparable to the effect of the mutation in our study population will affect cognitive performance, functional status, or noncardiovascular clinical events.
Acknowledgments
This work was performed as part of an ongoing collaboration of the PROSPER study group in the universities of Leiden, Glasgow, and Cork.
Footnotes
Abbreviations:
- ADL
- activities of daily living
- GWAS
- genome-wide association study
- IADL
- instrumental activities of daily living
- IHD
- ischemic heart disease
- LDLR
- LDL receptor
- LDT
- letter-digit coding test
- MMSE
- mini-mental state examination
- PCSK9
- proprotein convertase subtilisin-like/kexin type 9
- PHASE
- PHArmacogenetic study of Statins in the Elderly at risk
- PROSPER
- PROspective Study of Pravastatin in the Elderly at Risk
- SNP
- single-nucleotide polymorphism
The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement HEALTH-F2-2009-223004. A part of the genotyping was funded by The Netherlands Consortium for Healthy Ageing (NGI: 05060810). Prof. Dr. J. W. Jukema is an established clinical investigator of The Netherlands Heart Foundation (2001 D 032).
The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of three tables.
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