- Email: [email protected]
Serum apolipoprotein E is associated with long-term risk of Alzheimer’s disease: The Rotterdam Study
Accepted Manuscript Title: Serum apolipoprotein E is associated with long-term risk of Alzheimer’s disease: the Rotterdam Study Author: Frank J. Wolters Peter J. Koudstaal Albert Hofman Cornelia M.van Duijn M.Arfan Ikram PII: DOI: Reference:
S0304-3940(16)30085-4 http://dx.doi.org/doi:10.1016/j.neulet.2016.02.018 NSL 31846
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
3-11-2015 8-1-2016 9-2-2016
Please cite this article as: Frank J.Wolters, Peter J.Koudstaal, Albert Hofman, Cornelia M.van Duijn, M.Arfan Ikram, Serum apolipoprotein E is associated with long-term risk of Alzheimer’s disease: the Rotterdam Study, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.02.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Serum apolipoprotein E is associated with long-term risk of Alzheimer’s disease: the Rotterdam Study. Frank J. Wolters1,2, MD Peter J. Koudstaal2, MD PhD Albert Hofman1, MD PhD Cornelia M. van Duijn1, PhD M. Arfan Ikram*1,2,3, MD PhD
1 Department of Epidemiology, Erasmus Medical Centre, Rotterdam, the Netherlands 2 Department of Neurology, Erasmus Medical Centre, Rotterdam, the Netherlands 3 Department of Radiology, Erasmus Medical Centre, Rotterdam, the Netherlands
* Corresponding author contact details: Tel 0031107043488 Fax 0031107044657 Email: [email protected]
Manuscript type: brief communication Number of tables: 1 Number of figures: 1 Number of tables for online supplement: 1 Word count abstract: 242 Word count text body, including references: 1448
Highlights
Serum apoE is highly correlated to APOE genotype;
ApoE is associated with long-term risk of AD, independent of APOE genotype;
Risk prediction for AD may be slightly improved by determining serum apoE.
Abstract Background: Serum levels of apolipoprotein E (apoE) have been suggested as potential biomarker for dementia, but the long-term association between apoE and risk of dementia is uncertain.
Methods: Between 1990 and 1993, we measured serum apoE by immunoassay in 1042 nondemented individuals (mean±SD age 68.4±7.3 years; 59.3% women) from the population-based Rotterdam Study. Follow-up for dementia was complete until 2014. We used Cox models to determine the risk of dementia and Alzheimer’s disease in relation to apoE, adjusting for age, sex, educational level, cardiovascular risk factors, and additionally APOE genotype. Results: Serum apoE was strongly associated to APOE genotype (p-trend=1.0E-51, r2=0.21). In men, apoE tended to be lower with age, whereas in women the opposite was observed (p-trend=0.07 and 0.08, respectively). During a median follow-up of 15.7 years (IQR 9.7-21.7), 220 participants developed dementia, of whom 180 had Alzheimer’s disease. Lower serum apoE was associated with an increased risk of dementia (HR, 95%CI, per SD decrease: 1.25, 1.05-1.48) and in particular Alzheimer’s disease (1.51, 1.23-1.86), which remained statistically significant for Alzheimer’s disease after additionally adjusting for APOE genotype (1.28, 1.01-1.62). When stratifying analyses in 5-year time frames, risk estimates were similar throughout the study period. Serum apoE tended to marginally improve 20-year prediction of Alzheimer’s disease (IDI 0.008, 95%CI -0.001–0.026, p=0.086), but not all dementia. Conclusion: Serum apoE is associated with long-term risk of Alzheimer’s disease in the general population, independent of APOE genotype. Additional predictive value of serum apoE was limited.
Keywords: APOE, serum apolipoprotein, dementia, Alzheimer
1. Introduction Worldwide, approximately 45 million people are living with dementia, and this number is projected to nearly double every 20 years till 2050.[1,2] Although symptoms of dementia typically arise late in life, subclinical pathological changes in the brain occur up to decades before onset of symptoms.[3] Early identification of individuals at high risk of dementia is therefore essential to prevent manifestation of the disease. A reliable biomarker could aid in timely application of preventive strategies, selecting participants for neuroprotective trials, and disease monitoring. Various cerebrospinal fluid biomarkers have been assessed for these purposes in clinical populations, but plasma biomarkers that would allow long-term risk stratification in the general population are lacking.[3]
Apolipoprotein E genotype (APOE) is the major genetic risk factor for Alzheimer’s disease (AD), increasing lifetime risk for ε4 carriers 3 to 12-fold.[4] Various cross-sectional studies have shown that plasma levels of apolipoprotein E (apoE) are lower in patients with AD,[5] and a recent Danish population-study found that lower levels of apoE are associated with increased risk of dementia and AD.[6] However, median follow-up duration in the latter study was only 4 years, precluding any conclusion about long-term associations, which are most relevant for risk prediction. We aimed to determine the long-term association and predictive value of serum apoE for dementia and AD in a population-based study.
2. Materials & methods 2.1. Study population: The current study was embedded within the population-based Rotterdam Study, details of which have been described previously.[7] In brief, the initial study population consisted of 7983 individuals in the Ommoord district in Rotterdam, the Netherlands. Baseline examinations took place from 1990-1993. Of 7152 participants who visited the research centre, we determined serum apoE in a random subset of 1042 non-demented individuals. During the second follow-up visit from 1997-1999, measurements were repeated in a random subset of 338 of these individuals. The Rotterdam Study has been approved by the medical ethics committee according to the Population Study Act Rotterdam Study, executed by the Ministry of Health, Welfare and Sports of the Netherlands. Written informed consent was obtained from all participants.
2.2. Measurements of apoE and covariates: Blood samples were drawn by venipuncture from nonfasting subjects at baseline and from fasting subjects at follow-up, and samples were stored at -80°C. Serum apoE levels were measured by enzyme-linked immunosorbent assay (ELISA) at baseline, and plasma levels via multiplex immunoassay on human multianalyte profiles (Myriad RBM Inc., Austin TX, USA; http://rbm.myriad.com) during follow-up. APOE genotype was determined using polymerase chain reaction on coded DNA samples. Assessment of educational level and cardiovascular risk factors was done as described previously.[8]
2.3. Diagnosis of dementia: Methods of dementia diagnosis have been outlined elsewhere.[8] Follow-up for dementia was near complete until 1st January 2014.
2.4. Statistical analysis: Analyses included all non-demented participants in whom serum apoE was determined. Missing covariate data (maximum 11.4%) were imputed using 5-fold multiple imputation. We first determined the correlation of apoE with age, sex, and APOE genotype. We then
determined the risk of dementia and Alzheimer’s disease in relation to serum apoE levels, using Cox regression models. We tested for interaction on the multiplicative scale of apoE with age or sex. We determined the predictive value of serum apoE over that of age, sex, educational level, and APOE genotype, expressed as changes in the area under the receiver operating characteristic curve (AUC) and integrated discrimination improvement (IDI). In the subset of non-demented participants for whom we had a second consecutive apoE measurement, we determined the additive predictive value of this measurement for incident dementia, regarding the time in between first and second measurement as immortal person time. Analyses for prediction were done using R version 3.2.2 (packages ‘risksetROC’ and ‘survIDINRI’). All other analyses were done using SPSS Statistics version 21.0 (IBM Corp, Armonk, NY, USA). Alpha (type 1 error) was set at 0.05.
3. Results Serum apoE was measured in 1042 eligible individuals (mean±SD age 68.4±7.3, 59.3% women). Baseline characteristics of the study population are presented in supplemental table 1. During a median follow-up of 15.7 years (IQR 9.7-21.7) 220 individuals developed dementia, of whom 180 (81.8%) Alzheimer’s disease. Serum apoE tended to be slightly lower with age in men, whereas in women the opposite was observed (p-trend=0.07 and 0.08, respectively; Figure 1A). Serum apoE was highest for the ε2/ε2, and lowest for the ε4/ε4 genotype (p-trend=1.0E-53; r2=0.21; Figure 1B). Of all initial study participants, 328 without dementia had apoE measured again after a mean follow-up of 6.7 (SD 0.3) years. Correlation between the two subsequent measurements was high (Pearson’s r=0.62; Figure 1C). Lower serum apoE at baseline was associated with an increased risk of dementia, and in particular Alzheimer’s disease (Table 1). These associations were attenuated, but still statistically significant for Alzheimer’s disease, after additional adjustment for APOE genotype (Table 1). There was no evidence of effect modification by age or sex (pinteraction>0.20). When stratifying analyses in 5-year time frames, risk estimates were similar throughout the study period (data not shown). Compared to a model with age, sex, educational level and APOE genotype, adding serum apoE tended to marginally improve 20-year prediction of Alzheimer’s disease (AUC 0.727 vs. 0.732; IDI 0.008, 95% CI -0.001 to 0.026, p=0.086), but not all dementia (AUC 0.717 vs. 0.718; IDI 0.003, 95% CI -0.002 to 0.019, p=0.326). Incorporation of repeated apoE measurements after 6.7 years did not improve prediction for either Alzheimer’s disease or any type of dementia (data not shown).
4. Discussion In this population-based study, serum levels of apoE were associated with the risk of Alzheimer’s disease, independent of APOE genotype. Importantly, these associations were sustained up till 20 years of follow-up. Nevertheless, the added prognostic value of serum apoE over age, sex, education and APOE genotype in our study was only marginal.
The mean levels of apoE in our study ranged from 2.9 mg/dL measured in serum of non-fasting subjects at baseline, to 4.6 mg/dL in plasma taken after fasting at follow-up nearly 7 years later. Correlation between measurements was high (r=0.62), but the levels at follow-up were thus higher on an absolute scale. Although this may be related to physiological processes, apoE levels are generally found somewhat higher in studies that measured plasma levels,[5,6] which may be explained by interactions between analytes and clotting factors or other additives.[5] Taking this into account, measured levels in our study were comparable to those obtained in other European and Asian studies,[5,6] albeit higher values have been reported for North-American populations.[5] To understand these differences and determine a reference standard for serum apoE levels, clear reporting of circumstances of blood withdrawal and methods of analyses in future studies is essential.
Within the central nervous system, apoE is produced mainly by astrocytes and plays an essential role in cholesterol transport and β-amyloid clearance.[9] In peripheral tissue, apoE is produced primarily by the liver and macrophages, and mediates lipoprotein metabolism.[9] ApoE in serum and CSF are thought to act independently, as animal work suggests only very limited transport of apoE (and other lipoproteins) across the blood-brain barrier in physiological conditions,[10,11] and phenotypes of APOE may differ between CSF and plasma.[12] Similarly, levels of apoE in CSF, but less so in serum, have been found to correlate with CSF levels of amyloid-β42.[12,13] The association between serum apoE and Alzheimer’s disease, however, does suggest that peripheral apoE levels do relate to pathology in the central nervous system. This is supported by similar correlations between APOE genotype and apoE levels in cerebrospinal fluid (CSF) and plasma in a large study,[13] albeit only with plasma levels in a smaller sample.[12] Upon direct comparison, correlation between serum and CSF apoE is low,[12-14] but may be much higher in patients with Alzheimer’s disease than in healthy controls.[14] This might point to increased blood brain barrier permeability in patients with dementia,[15] allowing circulating serum apoE to cross the blood-brain barrier into the central nervous system, and vice versa.
Risk estimates in our study were virtually unaffected by adjustment for cardiovascular risk factors, suggesting any effect of peripheral apoE on dementia occurs independent of cardiovascular risk factors. Although a few studies have reported modification of the effect of cardiovascular risk factors on dementia by APOE genotype,[16-18] no such studies are done with serum apoE, and our study was insufficiently powered to address this question. The independent associations for apoE and APOE genotype with dementia we found, suggest that both may contribute in clinical diagnostics or risk stratification. Nevertheless, prediction of Alzheimer’s disease improved only marginally by taking into account apoE levels in our study. Furthermore, although serum apoE seems to vary with age,[6] a second measurement of apoE nearly 7 years apart did not contribute to risk prediction in a subsample of our study. As correlations between repeated measures were high, this may indicate that any (pathophysiological) changes in serum apoE levels occur early in life and subsequently change proportionally in the absence of disease modifying intervention. Future studies assessing the predictive value of serum apoE in combination with other serum biomarkers are warranted for more precise prediction of dementia. The main strength of our study is the long follow-up time. Studying pre-clinical changes of dementia as early as possible is important, not only to understand the earliest pathological changes, but also to aid long-term risk prediction. In turn, the ability to improve risk stratification and risk prediction over a long time horizon is essential to develop health care policies and properly design clinical trials. Although we believe our findings are valid, there are certain limitations to take into account. First, our sample size was relatively limited, which may explain a lack of statistical significance for the association between apoE and all dementia, especially as risk estimates were similar to those reported in the Danish population.[6] Second, as participants of the Rotterdam Study are predominantly Caucasian, our findings may not be applicable to other ethnicities. Third, the sensitivity of immunoassays for measuring different isoforms of apoE has been debated, as a previous mass spectrometry analysis did not show a correlation between serum apoE and Alzheimer’s disease.[19] However, mass spectrometry reported correlations between apoE and genotype, as well as between apoE and sex, are in agreement with our findings.[19] Moreover, the correlation between different types of immunoassays was high, and the measurement error due to insensitivity of immunoassays would only be expected to dilute effect estimates. In conclusion, serum apoE is independently associated with long-term risk of Alzheimer’s disease, and may hold potential as an easily accessible biomarker for early detection of individuals at high risk of developing Alzheimer’s disease. Nevertheless, excess predictive power in our study was limited, highlighting the need for development and concurrent use of additional serum biomarkers.
Author contributions: All authors have made a substantial intellectual contribution to design of the study (AH, MAI, CMD), acquisition of data (FJW, PJK), analysis and interpretation of data (FJW, MAI), drafting the manuscript (FJW), or revising it critically for important intellectual content (AH, PJK, CMD, MAI). All authors approved the final version of the manuscript for publication. MAI had full access to the data and takes responsibility for data integrity and accuracy of data analysis.
Potential conflicts of interest: The authors declare no conflict of interest.
Acknowledgements: The Rotterdam Study is sponsored by the Erasmus Medical Centre and Erasmus University Rotterdam, The Netherlands Organization for Scientific Research (NWO), The Netherlands Organization for Health Research and Development (ZonMW), the Research Institute for Diseases in the Elderly (RIDE), The Netherlands Genomics Initiative, the Ministry of Education, Culture and Science, the Ministry of Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. Further support was obtained from the Netherlands Cardiovascular Research Initiative (CVON2012-06). None of the funding organisations or sponsors were involved in study design, in collection, analysis, and interpretation of data, in writing of the report, or in the decision to submit the article for publication.
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International AD. Policy Brief for G8 Heads of Government. The Global Impact of Dementia 2013-2050 [Internet]. www.alz.co.uk. [cited 2015 Mar 25]. Available from: http://www.alz.co.uk/research/G8policy-brief
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Prince M, Bryce R, Albanese E, Wimo A, Ribeiro W, Ferri CP. The global prevalence of dementia: a systematic review and metaanalysis. Alzheimers Dement. 2013;9(1):63–75.e2.
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Jack CR, Knopman DS, Jagust WJ, Petersen RC, Weiner MW, Aisen PS, et al. Tracking pathophysiological processes in Alzheimer's disease: an updated hypothetical model of dynamic biomarkers. Lancet Neurol. 2013;12(2):207–16.
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Verghese PB, Castellano JM, Holtzman DM. Apolipoprotein E in Alzheimer's disease and other neurological disorders. Lancet Neurol. 2011;10(3):241–52.
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Wang C, Yu JT, Wang HF, Jiang T, Tan CC, Meng XF, et al. Meta-analysis of peripheral blood apolipoprotein E levels in Alzheimer's disease. PLoS One. 2014;9(2).
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Rasmussen KL, Tybjærg-Hansen A, Nordestgaard BG, Frikke-Schmidt R. Plasma levels of apolipoprotein E and risk of dementia in the general population. Ann. Neurol. 2015;77(2):301–11.
[7]
Hofman A, Brusselle GGO, Murad SD, van Duijn CM, Franco OH, Goedegebure A, et al. The Rotterdam Study: 2016 objectives and design update. Eur J Epidemiol. 2015;30(8):661–708.
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Schrijvers EMC, Verhaaren BFJ, Koudstaal PJ, Hofman A, Ikram MA, Breteler MMB. Is dementia
incidence declining?: Trends in dementia incidence since 1990 in the Rotterdam Study. Neurology. 2012;78(19):1456–63. [9]
Liu C-C, Liu C-C, Kanekiyo T, Xu H, Bu G. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol. 2013;9(2):106–18.
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Martel CL, Mackic JB, Matsubara E, Governale S, Miguel C, Miao W, et al. Isoform-specific effects of apolipoproteins E2, E3, and E4 on cerebral capillary sequestration and blood-brain barrier transport of circulating Alzheimer's amyloid beta. J. Neurochem. 1997;69(5):1995–2004.
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Wang H, Eckel RH. What are lipoproteins doing in the brian? Trends Endocrinol Metab. 2014;25(1):814.
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Martinez-Morillo E, Hansson O, Atagi Y, Bu G, Minthon L, Diamandis EP, Nielsen HM. Total apolipoprotein E levels and specific isoform composition in cerebrospinal fluid and plasam from Alzheimer’s disease patients and controls. Acta Neuropathol. 2014;127:633-643.
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Cruchaga C, Kauwe JSK, Nowotny P, Bales K, Pickering EH, Mayo K, et al. Cerebrospinal fluid APOE levels: an endophenotype for genetic studies for Alzheimer's disease. Hum. Mol. Genet. 2012;21(20):4558–71.[14] Richens JL, Vere K-A, Light RA, Soria D, Garibaldi J, Smith AD, et al. Practical detection of a definitive biomarker panel for Alzheimer's disease; comparisons between matched plasma and cerebrospinal fluid. Int J Mol Epidemiol Genet. 2014;5(2):53–70.
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Zlokovic BV. Cerebrovascular effects of apolipoprotein E: implications for Alzheimer disease. JAMA Neurol. 2013;70(4):440–4.
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Kivipelto M, Rovio S, Ngandu T, Kåreholt I, Eskelinen M, Winblad B, et al. Apolipoprotein E epsilon4 magnifies lifestyle risks for dementia: a population-based study. J. Cell. Mol. Med. 2008;12(6B):2762– 71.
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Haan MN, Shemanski L, Jagust WJ, Manolio TA, Kuller L. The role of APOE epsilon4 in modulating effects of other risk factors for cognitive decline in elderly persons. JAMA. 1999;282(1):40–6.
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Simon R, Girod M, Fonbonne C, Salvador A, Clément Y, Lantéri P, et al. Total ApoE and ApoE4 isoform assays in an Alzheimer's disease case-control study by targeted mass spectrometry (n=669): a pilot assay for methionine-containing proteotypic peptides. Mol. Cell Proteomics. 2012;11(11):1389–403.
Caption Figure 1: Serum levels of apoE by age (stratified for sex) (A), by APOE genotype (B), and the correlation between two different types of immunoassays 7 years apart (C). Values are depicted as group means with corresponding standard errors (A and B) and scatter plot of individual data points with regression line (C).
Table 1.
Serum levels of apoE and the risk of dementia
Serum apoE
Model I
Model II
Model III
Model IV
n/N
HR, 95% CI
HR, 95% CI
HR, 95% CI
HR, 95% CI
Highest tertile
44/342
REFERENCE
REFERENCE
REFERENCE
REFERENCE
Middle tertile
64/346
1.58, 1.08-2.33
1.37, 0.91-2.06
1.57, 1.06-2.33
1.37, 0.90-2.08
Lowest tertile
72/354
2.18, 1.49-3.20
1.62, 1.06-2.47
2.15, 1.43-3.22
1.53, 0.97-2.42
180/1042
1.51, 1.25-1.84
1.31, 1.05-1.62
1.51, 1.23-1.86
1.28, 1.01-1.62
Highest tertile
61/342
REFERENCE
REFERENCE
REFERENCE
REFERENCE
Middle tertile
77/346
1.37, 0.98-1.92
1.23, 0.86-1.77
1.36, 0.96-1.92
1.24, 0.86-1.79
Lowest tertile
82/354
1.76, 1.25-2.47
1.37, 0.94-1.99
1.74, 1.21-2.50
1.33, 0.89-2.00
220/1042
1.26, 1.07-1.47
1.10, 0.92-1.31
1.25, 1.05-1.48
1.07, 0.88-1.31
Alzheimer’s disease
Per SD decrease
All dementia
Per SD decrease
SD=standard deviation; HR=hazard ratio; CI=confidence interval. Model I: adjusted for age and sex. Model II: model I with additional adjustment for APOE genotype. Model III: adjusted for age, sex, level of education, systolic and diastolic blood pressure, antihypertensive medication, serum cholesterol and HDL, lipid lowering medication, body mass index, diabetes mellitus, and smoking. Model IV: model III with additional adjustment for APOE genotype.
S0304-3940(16)30085-4 http://dx.doi.org/doi:10.1016/j.neulet.2016.02.018 NSL 31846
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
3-11-2015 8-1-2016 9-2-2016
Please cite this article as: Frank J.Wolters, Peter J.Koudstaal, Albert Hofman, Cornelia M.van Duijn, M.Arfan Ikram, Serum apolipoprotein E is associated with long-term risk of Alzheimer’s disease: the Rotterdam Study, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.02.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Serum apolipoprotein E is associated with long-term risk of Alzheimer’s disease: the Rotterdam Study. Frank J. Wolters1,2, MD Peter J. Koudstaal2, MD PhD Albert Hofman1, MD PhD Cornelia M. van Duijn1, PhD M. Arfan Ikram*1,2,3, MD PhD
1 Department of Epidemiology, Erasmus Medical Centre, Rotterdam, the Netherlands 2 Department of Neurology, Erasmus Medical Centre, Rotterdam, the Netherlands 3 Department of Radiology, Erasmus Medical Centre, Rotterdam, the Netherlands
* Corresponding author contact details: Tel 0031107043488 Fax 0031107044657 Email: [email protected]
Manuscript type: brief communication Number of tables: 1 Number of figures: 1 Number of tables for online supplement: 1 Word count abstract: 242 Word count text body, including references: 1448
Highlights
Serum apoE is highly correlated to APOE genotype;
ApoE is associated with long-term risk of AD, independent of APOE genotype;
Risk prediction for AD may be slightly improved by determining serum apoE.
Abstract Background: Serum levels of apolipoprotein E (apoE) have been suggested as potential biomarker for dementia, but the long-term association between apoE and risk of dementia is uncertain.
Methods: Between 1990 and 1993, we measured serum apoE by immunoassay in 1042 nondemented individuals (mean±SD age 68.4±7.3 years; 59.3% women) from the population-based Rotterdam Study. Follow-up for dementia was complete until 2014. We used Cox models to determine the risk of dementia and Alzheimer’s disease in relation to apoE, adjusting for age, sex, educational level, cardiovascular risk factors, and additionally APOE genotype. Results: Serum apoE was strongly associated to APOE genotype (p-trend=1.0E-51, r2=0.21). In men, apoE tended to be lower with age, whereas in women the opposite was observed (p-trend=0.07 and 0.08, respectively). During a median follow-up of 15.7 years (IQR 9.7-21.7), 220 participants developed dementia, of whom 180 had Alzheimer’s disease. Lower serum apoE was associated with an increased risk of dementia (HR, 95%CI, per SD decrease: 1.25, 1.05-1.48) and in particular Alzheimer’s disease (1.51, 1.23-1.86), which remained statistically significant for Alzheimer’s disease after additionally adjusting for APOE genotype (1.28, 1.01-1.62). When stratifying analyses in 5-year time frames, risk estimates were similar throughout the study period. Serum apoE tended to marginally improve 20-year prediction of Alzheimer’s disease (IDI 0.008, 95%CI -0.001–0.026, p=0.086), but not all dementia. Conclusion: Serum apoE is associated with long-term risk of Alzheimer’s disease in the general population, independent of APOE genotype. Additional predictive value of serum apoE was limited.
Keywords: APOE, serum apolipoprotein, dementia, Alzheimer
1. Introduction Worldwide, approximately 45 million people are living with dementia, and this number is projected to nearly double every 20 years till 2050.[1,2] Although symptoms of dementia typically arise late in life, subclinical pathological changes in the brain occur up to decades before onset of symptoms.[3] Early identification of individuals at high risk of dementia is therefore essential to prevent manifestation of the disease. A reliable biomarker could aid in timely application of preventive strategies, selecting participants for neuroprotective trials, and disease monitoring. Various cerebrospinal fluid biomarkers have been assessed for these purposes in clinical populations, but plasma biomarkers that would allow long-term risk stratification in the general population are lacking.[3]
Apolipoprotein E genotype (APOE) is the major genetic risk factor for Alzheimer’s disease (AD), increasing lifetime risk for ε4 carriers 3 to 12-fold.[4] Various cross-sectional studies have shown that plasma levels of apolipoprotein E (apoE) are lower in patients with AD,[5] and a recent Danish population-study found that lower levels of apoE are associated with increased risk of dementia and AD.[6] However, median follow-up duration in the latter study was only 4 years, precluding any conclusion about long-term associations, which are most relevant for risk prediction. We aimed to determine the long-term association and predictive value of serum apoE for dementia and AD in a population-based study.
2. Materials & methods 2.1. Study population: The current study was embedded within the population-based Rotterdam Study, details of which have been described previously.[7] In brief, the initial study population consisted of 7983 individuals in the Ommoord district in Rotterdam, the Netherlands. Baseline examinations took place from 1990-1993. Of 7152 participants who visited the research centre, we determined serum apoE in a random subset of 1042 non-demented individuals. During the second follow-up visit from 1997-1999, measurements were repeated in a random subset of 338 of these individuals. The Rotterdam Study has been approved by the medical ethics committee according to the Population Study Act Rotterdam Study, executed by the Ministry of Health, Welfare and Sports of the Netherlands. Written informed consent was obtained from all participants.
2.2. Measurements of apoE and covariates: Blood samples were drawn by venipuncture from nonfasting subjects at baseline and from fasting subjects at follow-up, and samples were stored at -80°C. Serum apoE levels were measured by enzyme-linked immunosorbent assay (ELISA) at baseline, and plasma levels via multiplex immunoassay on human multianalyte profiles (Myriad RBM Inc., Austin TX, USA; http://rbm.myriad.com) during follow-up. APOE genotype was determined using polymerase chain reaction on coded DNA samples. Assessment of educational level and cardiovascular risk factors was done as described previously.[8]
2.3. Diagnosis of dementia: Methods of dementia diagnosis have been outlined elsewhere.[8] Follow-up for dementia was near complete until 1st January 2014.
2.4. Statistical analysis: Analyses included all non-demented participants in whom serum apoE was determined. Missing covariate data (maximum 11.4%) were imputed using 5-fold multiple imputation. We first determined the correlation of apoE with age, sex, and APOE genotype. We then
determined the risk of dementia and Alzheimer’s disease in relation to serum apoE levels, using Cox regression models. We tested for interaction on the multiplicative scale of apoE with age or sex. We determined the predictive value of serum apoE over that of age, sex, educational level, and APOE genotype, expressed as changes in the area under the receiver operating characteristic curve (AUC) and integrated discrimination improvement (IDI). In the subset of non-demented participants for whom we had a second consecutive apoE measurement, we determined the additive predictive value of this measurement for incident dementia, regarding the time in between first and second measurement as immortal person time. Analyses for prediction were done using R version 3.2.2 (packages ‘risksetROC’ and ‘survIDINRI’). All other analyses were done using SPSS Statistics version 21.0 (IBM Corp, Armonk, NY, USA). Alpha (type 1 error) was set at 0.05.
3. Results Serum apoE was measured in 1042 eligible individuals (mean±SD age 68.4±7.3, 59.3% women). Baseline characteristics of the study population are presented in supplemental table 1. During a median follow-up of 15.7 years (IQR 9.7-21.7) 220 individuals developed dementia, of whom 180 (81.8%) Alzheimer’s disease. Serum apoE tended to be slightly lower with age in men, whereas in women the opposite was observed (p-trend=0.07 and 0.08, respectively; Figure 1A). Serum apoE was highest for the ε2/ε2, and lowest for the ε4/ε4 genotype (p-trend=1.0E-53; r2=0.21; Figure 1B). Of all initial study participants, 328 without dementia had apoE measured again after a mean follow-up of 6.7 (SD 0.3) years. Correlation between the two subsequent measurements was high (Pearson’s r=0.62; Figure 1C). Lower serum apoE at baseline was associated with an increased risk of dementia, and in particular Alzheimer’s disease (Table 1). These associations were attenuated, but still statistically significant for Alzheimer’s disease, after additional adjustment for APOE genotype (Table 1). There was no evidence of effect modification by age or sex (pinteraction>0.20). When stratifying analyses in 5-year time frames, risk estimates were similar throughout the study period (data not shown). Compared to a model with age, sex, educational level and APOE genotype, adding serum apoE tended to marginally improve 20-year prediction of Alzheimer’s disease (AUC 0.727 vs. 0.732; IDI 0.008, 95% CI -0.001 to 0.026, p=0.086), but not all dementia (AUC 0.717 vs. 0.718; IDI 0.003, 95% CI -0.002 to 0.019, p=0.326). Incorporation of repeated apoE measurements after 6.7 years did not improve prediction for either Alzheimer’s disease or any type of dementia (data not shown).
4. Discussion In this population-based study, serum levels of apoE were associated with the risk of Alzheimer’s disease, independent of APOE genotype. Importantly, these associations were sustained up till 20 years of follow-up. Nevertheless, the added prognostic value of serum apoE over age, sex, education and APOE genotype in our study was only marginal.
The mean levels of apoE in our study ranged from 2.9 mg/dL measured in serum of non-fasting subjects at baseline, to 4.6 mg/dL in plasma taken after fasting at follow-up nearly 7 years later. Correlation between measurements was high (r=0.62), but the levels at follow-up were thus higher on an absolute scale. Although this may be related to physiological processes, apoE levels are generally found somewhat higher in studies that measured plasma levels,[5,6] which may be explained by interactions between analytes and clotting factors or other additives.[5] Taking this into account, measured levels in our study were comparable to those obtained in other European and Asian studies,[5,6] albeit higher values have been reported for North-American populations.[5] To understand these differences and determine a reference standard for serum apoE levels, clear reporting of circumstances of blood withdrawal and methods of analyses in future studies is essential.
Within the central nervous system, apoE is produced mainly by astrocytes and plays an essential role in cholesterol transport and β-amyloid clearance.[9] In peripheral tissue, apoE is produced primarily by the liver and macrophages, and mediates lipoprotein metabolism.[9] ApoE in serum and CSF are thought to act independently, as animal work suggests only very limited transport of apoE (and other lipoproteins) across the blood-brain barrier in physiological conditions,[10,11] and phenotypes of APOE may differ between CSF and plasma.[12] Similarly, levels of apoE in CSF, but less so in serum, have been found to correlate with CSF levels of amyloid-β42.[12,13] The association between serum apoE and Alzheimer’s disease, however, does suggest that peripheral apoE levels do relate to pathology in the central nervous system. This is supported by similar correlations between APOE genotype and apoE levels in cerebrospinal fluid (CSF) and plasma in a large study,[13] albeit only with plasma levels in a smaller sample.[12] Upon direct comparison, correlation between serum and CSF apoE is low,[12-14] but may be much higher in patients with Alzheimer’s disease than in healthy controls.[14] This might point to increased blood brain barrier permeability in patients with dementia,[15] allowing circulating serum apoE to cross the blood-brain barrier into the central nervous system, and vice versa.
Risk estimates in our study were virtually unaffected by adjustment for cardiovascular risk factors, suggesting any effect of peripheral apoE on dementia occurs independent of cardiovascular risk factors. Although a few studies have reported modification of the effect of cardiovascular risk factors on dementia by APOE genotype,[16-18] no such studies are done with serum apoE, and our study was insufficiently powered to address this question. The independent associations for apoE and APOE genotype with dementia we found, suggest that both may contribute in clinical diagnostics or risk stratification. Nevertheless, prediction of Alzheimer’s disease improved only marginally by taking into account apoE levels in our study. Furthermore, although serum apoE seems to vary with age,[6] a second measurement of apoE nearly 7 years apart did not contribute to risk prediction in a subsample of our study. As correlations between repeated measures were high, this may indicate that any (pathophysiological) changes in serum apoE levels occur early in life and subsequently change proportionally in the absence of disease modifying intervention. Future studies assessing the predictive value of serum apoE in combination with other serum biomarkers are warranted for more precise prediction of dementia. The main strength of our study is the long follow-up time. Studying pre-clinical changes of dementia as early as possible is important, not only to understand the earliest pathological changes, but also to aid long-term risk prediction. In turn, the ability to improve risk stratification and risk prediction over a long time horizon is essential to develop health care policies and properly design clinical trials. Although we believe our findings are valid, there are certain limitations to take into account. First, our sample size was relatively limited, which may explain a lack of statistical significance for the association between apoE and all dementia, especially as risk estimates were similar to those reported in the Danish population.[6] Second, as participants of the Rotterdam Study are predominantly Caucasian, our findings may not be applicable to other ethnicities. Third, the sensitivity of immunoassays for measuring different isoforms of apoE has been debated, as a previous mass spectrometry analysis did not show a correlation between serum apoE and Alzheimer’s disease.[19] However, mass spectrometry reported correlations between apoE and genotype, as well as between apoE and sex, are in agreement with our findings.[19] Moreover, the correlation between different types of immunoassays was high, and the measurement error due to insensitivity of immunoassays would only be expected to dilute effect estimates. In conclusion, serum apoE is independently associated with long-term risk of Alzheimer’s disease, and may hold potential as an easily accessible biomarker for early detection of individuals at high risk of developing Alzheimer’s disease. Nevertheless, excess predictive power in our study was limited, highlighting the need for development and concurrent use of additional serum biomarkers.
Author contributions: All authors have made a substantial intellectual contribution to design of the study (AH, MAI, CMD), acquisition of data (FJW, PJK), analysis and interpretation of data (FJW, MAI), drafting the manuscript (FJW), or revising it critically for important intellectual content (AH, PJK, CMD, MAI). All authors approved the final version of the manuscript for publication. MAI had full access to the data and takes responsibility for data integrity and accuracy of data analysis.
Potential conflicts of interest: The authors declare no conflict of interest.
Acknowledgements: The Rotterdam Study is sponsored by the Erasmus Medical Centre and Erasmus University Rotterdam, The Netherlands Organization for Scientific Research (NWO), The Netherlands Organization for Health Research and Development (ZonMW), the Research Institute for Diseases in the Elderly (RIDE), The Netherlands Genomics Initiative, the Ministry of Education, Culture and Science, the Ministry of Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. Further support was obtained from the Netherlands Cardiovascular Research Initiative (CVON2012-06). None of the funding organisations or sponsors were involved in study design, in collection, analysis, and interpretation of data, in writing of the report, or in the decision to submit the article for publication.
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Caption Figure 1: Serum levels of apoE by age (stratified for sex) (A), by APOE genotype (B), and the correlation between two different types of immunoassays 7 years apart (C). Values are depicted as group means with corresponding standard errors (A and B) and scatter plot of individual data points with regression line (C).
Table 1.
Serum levels of apoE and the risk of dementia
Serum apoE
Model I
Model II
Model III
Model IV
n/N
HR, 95% CI
HR, 95% CI
HR, 95% CI
HR, 95% CI
Highest tertile
44/342
REFERENCE
REFERENCE
REFERENCE
REFERENCE
Middle tertile
64/346
1.58, 1.08-2.33
1.37, 0.91-2.06
1.57, 1.06-2.33
1.37, 0.90-2.08
Lowest tertile
72/354
2.18, 1.49-3.20
1.62, 1.06-2.47
2.15, 1.43-3.22
1.53, 0.97-2.42
180/1042
1.51, 1.25-1.84
1.31, 1.05-1.62
1.51, 1.23-1.86
1.28, 1.01-1.62
Highest tertile
61/342
REFERENCE
REFERENCE
REFERENCE
REFERENCE
Middle tertile
77/346
1.37, 0.98-1.92
1.23, 0.86-1.77
1.36, 0.96-1.92
1.24, 0.86-1.79
Lowest tertile
82/354
1.76, 1.25-2.47
1.37, 0.94-1.99
1.74, 1.21-2.50
1.33, 0.89-2.00
220/1042
1.26, 1.07-1.47
1.10, 0.92-1.31
1.25, 1.05-1.48
1.07, 0.88-1.31
Alzheimer’s disease
Per SD decrease
All dementia
Per SD decrease
SD=standard deviation; HR=hazard ratio; CI=confidence interval. Model I: adjusted for age and sex. Model II: model I with additional adjustment for APOE genotype. Model III: adjusted for age, sex, level of education, systolic and diastolic blood pressure, antihypertensive medication, serum cholesterol and HDL, lipid lowering medication, body mass index, diabetes mellitus, and smoking. Model IV: model III with additional adjustment for APOE genotype.