Apolipoprotein ɛ4 is Associated with Dementia and Cognitive Impairment Predominantly Due to Alzheimer’s Disease and Not with Vascular Cognitive Impairment: A Singapore-Based Cohort

Abstract

Background and Objective:

While the association for apolipoprotein ɛ4 allele (APOE4) with Alzheimer’s disease (AD) has been consistently confirmed, the association with vascular cognitive impairment (VCI) is unclear. We therefore explored the relationship of APOE with both AD and cerebrovascular disease (CeVD) by examining the prevalence of APOE4 in AD, AD with CeVD and vascular dementia (VaD), as well as in cognitive impairment no dementia (CIND) with and without CeVD.

Methods:

We performed a case-control study with subjects recruited from memory clinics and the community. All subjects underwent standardized brain neuroimaging, clinical and neuropsychological assessments, following which they were classified using research criteria.

Results:

A total of 411 subjects; 92 controls with no cognitive impairment (NCI), 77 CIND without CeVD, 87 CIND with CeVD, 55 AD without CeVD, 68 AD with CeVD, and 32 VaD patients were recruited. Compared to NCI (16.3%), the prevalence of APOE4 carriers was significantly higher only in CIND (37.7%) and AD in the absence of CeVD (45.5%), but not in the three subgroups of VCI, namely CIND with CeVD (20.7%), AD with CeVD (27.9%) and VaD (25.0%). Logistic regression analyses also showed that APOE4 carriers were more likely to have CIND without CeVD (Odds Ratio [OR]: 3.34; 95% Confidence Interval [CI]: 1.59–7.03) and AD without CeVD (OR: 7.21; 95% CI: 2.74–18.98), but no such association was observed in the VCI subgroups.

Conclusion:

APOE4 is significantly associated with dementia and CIND due to AD pathology, but not with VCI.

Keywords

Alzheimer’s disease apolipoprotein ɛ4 cerebrovascular disease cognitive impairment no dementia vascular cognitive impairment vascular dementia

INTRODUCTION

Apolipoprotein E (APOE) is a lipid-binding protein involved in cholesterol transportation, neuronal repair, and regeneration in the central nervous system [1]. It is encoded by three polymorphic alleles on chromosome 19, namely ɛ2, ɛ3, and ɛ4, of which APOE ɛ4 allele (APOE4) has been recognized as the most important genetic risk factor for sporadic late-onset Alzheimer’s disease (AD). The prevalence of APOE4 among AD patients is as high as 48.7% globally [2], and APOE4 carriers, both homo- and heterozygotes, have an increased risk of developing AD [3]. In addition to being a well-established risk factor for dementia due to AD, various studies have also suggested associations between APOE4 and mild cognitive impairment (MCI) [4, 5], a pre-clinical stage of dementia, as well as conversion from MCI to AD [6, 7].

In contrast, the role of APOE polymorphisms in vascular cognitive impairment (VCI) is less clear. In view of the increasingly recognized role of vascular pathology in cognitive impairment, VCI describes a continuum of cognitive disorders attributable to cerebrovascular disease (CeVD) either alone, for instance vascular dementia (VaD) and vascular cognitive impairment no dementia (i.e., CIND with CeVD), or in conjunction with other pathologies such as AD (i.e., AD with CeVD) [8]. It has been reported that the presence of APOE4 could influence the association between CeVD and cognitive decline, with a likely synergistic effect between APOE4 and CeVD [9]. Moreover, APOE4 has also been linked to several vascular risk factors of VCI such as atherosclerosis and stroke [10, 11]. While recent meta-analyses showed statistically significant but weaker association between APOE4 and VaD compared to AD [12, 13], inconsistent results have been reported on the associations for APOE4 with CIND with CeVD [14 –17] and AD with CeVD [16 –28].

In the present study, we thus examined the prevalence of APOE alleles in a well phenotyped group of patients with cognitive impairment and dementia due to AD and/or CeVD. We hypothesized that the association of APOE with dementia and cognitive impairment would be not be significant in patients with VCI.

MATERIALS AND METHOD

Study population

The present study adopts a case-control design. Cases (CIND and dementia) were recruited from two study sites in Singapore (memory clinics in the National University Hospital and Saint Luke’s Hospital). Cognitively normal controls (No Cognitive Impairment, NCI) were recruited from both memory clinics and the community in the same catchment area as cases [29]. Ethics approval for this study was obtained from National Healthcare Group Domain-Specific Review Board (DSRB) (DSRB reference: 2010/00017; study protocol number: DEM4233). The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained for all participants in their preferred language prior to study recruitment.

Sampling and APOE genotyping

Non-fasting blood was collected in EDTA tubes and centrifuged at 2000 g, 4°C for 10 min. The upper plasma layer was removed, while the remaining buffy coat and erythrocyte layers were mixed and stored at –80°C until genomic DNA was extracted from 200μl to 300μl of the blood mixture using the Promega Wizard^® SV Genomic Purification system and diluted in 75μl of nuclease-free water. The quantity and quality of DNA extracted was assessed using the NanoDrop Spectrophotometer (ND-1000).

APOE genotyping was performed on the extracted genomic DNA via polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP), a well-validated method used in previous studies [30 –32]. A 244bp DNA segment encompassing the APOE polymorphisms is amplified using PCR in a 50μl reaction mixture containing 5μl each of the F4 forward primer (5^′-ACA GAA TTC GCC CCG GCC TGG TAC AC-3^′) and F6 reverse primer (5^′-TAA GCT TGG CAC GGC TGT CCA AGG A-3^′) (Integrated DNA Technologies), 10% Dimethyl Sulfoxide (DMSO), 25μl of GoTaq Green Master Mix, 2X (Promega, M7123), and 10μl of template DNA from DNA extraction. The PCR condition consists of an initial denaturation at 95°C for 5 min, and a 30 cycle of 95°C for 1 min, 60°C for 1 min and 70°C for 2 min. The PCR product was restriction digested with HhaI (New England Biolabs, R0139L) for 1 h at 37°C. The digested DNA products and the PCR product previously stored at 4°C were then separated by size using a 4% agarose gel (Low Range Ultra Agarose, Bio-rad, #161-3107) in gel electrophoresis and visualized with ethidium bromide under ultraviolet light.

Neuropsychological assessment

Cognitive tests, which included the Mini-Mental State Examination (MMSE), the Montreal Cognitive Assessment (MoCA), and a locally validated, detailed neuropsychological test battery were administered to all subjects by trained research psychologists. The formal test battery assessed seven cognitive domains – executive function, attention, language, visuomotor speed, visuoconstruction, verbal memory, and visual memory, as described previously [29].

Diagnosis of cognitive impairment and dementia

The diagnosis of CIND was determined by clinical judgment and was anchored in the following guidelines, as previously published [33], namely, self and/or informant report of problems with cognition and impairment in at least one domain of the neuropsychological test battery using education-adjusted cutoffs of 1.5 standard deviations below established normal means on individual tests. Failure in at least half of the tests in a domain constituted failure in that domain. CIND subjects who showed significant CeVD on neuroimaging scans (see the section on Neuroimaging below for details) were classified as CIND with CeVD.

The diagnosis of AD was based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria [34], whereas VaD was diagnosed using the National Institute of Neurological Disorders and Stroke and Association Internationale pour la Recherché et l’ Enseignement en Neurosciences (NINDS-AIREN) criteria [35]. Subjects who fulfilled the NINCDS-ADRDA criteria but also showed significant CeVD on neuroimaging scans (see the section on Neuroimaging below for details) were classified as AD with CeVD.

Neuroimaging

Magnetic resonance imaging (MRI) scans were performed on a 3T Siemens Magnetom Trio Tim scanner, using a 32-channel head coil, at the Clinical Imaging Research Centre, NUS. Several sequences were utilized including T1-weighted Magnetization Prepared Rapid Gradient Recalled Echo (MPRAGE), Fluid Attentuated Inversion Recovery (FLAIR), T2-weighted and Susceptibility Weighted Imaging. MRI markers for CeVD were graded using the following criteria, as described previously [29]:

Cortical infarcts were defined as focal lesions with involvement of cortical gray matter, signal following CSF intensity, hyperintense rim on FLAIR images, and tissue loss of variable magnitude, with prominent adjacent sulci and ipsilateral ventricular enlargement [36].

Lacunes were defined as lesions, 3–15 mm in diameter, with low signal on T1 weighted image and FLAIR; a high signal on T2 weighted image, and a hyperintense rim with center following the cerebrospinal fluid intensity [36]. They were distinguished from the perivascular spaces by the absence of hyperintense rim and fluid filled lesions that follow the typical course of the vessel [37].

White matter hyperintensities (WMH) were graded using Age Related White Matter Changes scale (ARWMC) [38].

Significant CeVD was defined as the presence of cortical infarcts and/or ≥2 lacunes and/or confluent WMH in two regions of the brain (ARWMC score ≥8).

Statistical analysis

Statistical analysis was performed using SPSS v21 (Statistical Package for Social Science, SPSS Inc., USA). Intergroup differences between cases and controls were examined using the following statistical analyses, namely, analyses of variance (ANOVA) for mean age comparison, Mann-Whitney U test for non-parametric comparison on years of education, and Pearson’s chi-square test for comparison on the frequency distributions of gender, APOE genotype, and CeVD. Binary logistic regression analysis was used to evaluate the association of each APOE allele carrier in CIND and dementia, with reference to the NCI group. The model was adjusted for age, gender and education, and results were expressed as odds ratios (OR) and 95% confidence interval (CI). A p-value less than 0.05 is considered statistically significant.

RESULTS

A total of 411 subjects, 92 (22.4%) NCI, 77 (18.7%) CIND without CeVD, 87 (21.2%) CIND with CeVD, 55 (13.4%) AD without CeVD, 68 (16.5%) AD with CeVD, and 32 (7.8%) VaD, were recruited into this study and consented for blood collection from August 2010 to November 2014.

Tables 1 and 2 show the baseline characteristics and APOE genotype distribution in cases and NCI. Prior to classifying the cases into non-vascular and vascular subgroups, a significantly higher prevalence of APOE4 carrier was observed in both CIND (28.7%, p = 0.027) and AD (35.8%, p = 0.002) as compared to NCI (Table 1). However, upon further stratification, significant increases in APOE4 carrier frequencies were only observed in the absence of CeVD, i.e., in CIND without CeVD (37.7%, p = 0.002) and AD without CeVD (45.5%, p < 0.001), but not in the VCI subgroups, i.e., CIND with CeVD (20.7%, p = 0.450), AD with CeVD (27.9%, p = 0.075), and VaD (25.0%, p = 0.276) (Table 2).

Further logistic regression analyses showed that the presence of at least one APOE4 allele was significantly associated with CIND without CeVD (OR: 3.34; 95% CI: 1.59–7.03; p = 0.001) and AD without CeVD (OR: 7.21; 95% CI: 2.74–18.98; p < 0.001), but not with CIND with CeVD (OR: 1.60; 95% CI: 0.71–3.57; p = 0.255), AD with CeVD (OR: 1.94; 95% CI: 0.60–6.29; p = 0.272) and VaD (OR: 1.42; 95% CI: 0.48–4.19; p = 0.525) (Table 3).

To further examine the potential link between APOE genotype and CeVD, the prevalence of each APOE allele carrier among subjects with specific CeVD subtypes were assessed. Our results showed that APOE4 carrier frequencies did not differ significantly in subjects with WMH graded by ARWMC ≥8 (27.2%, p = 0.725), cortical infarct (18.0%, p = 0.068) as well as with the presence of ≥2 lacunes (29.3%, p = 0.751), when compared with subjects without these CeVD subtypes respectively.

Lastly, the effect of APOE genotype on the link between cognitive impairment and CeVD was also examined. While the majority of the demented patients had significant CeVD (64.5%), the trend remained unchanged by APOE genotype as 62.5% APOE2 carriers, 66.2% APOE3 carriers, and 51.9% APOE4 carriers had CeVD.

DISCUSSION

In this study, we have shown that the prevalence of APOE4 carriers was significantly higher among both CIND and AD subjects who did not have neuroimaging evidence of CeVD, but not in the three VCI subgroups, i.e., CIND with CeVD, AD with CeVD, and VaD. Moreover, subjects with at least one APOE4 allele were more likely to have CIND and AD without CeVD, whereas no association was found between APOE4 and VCI. Our hypothesis was further supported by the lack of association between APOE4 and specific CeVD subtypes defined on MRI, namely WMH, cortical infarct, and lacunes.

This study showed that 45.5% of the patients having AD without CeVD were APOE4 carriers, a frequency significantly higher than that of the NCI group. This is consistent with a recent meta-analysis which reported a relatively lower APOE4 carrier prevalence in Asian populations (41.9%) as compared to northern European populations (61.3%) [2]. Furthermore, the role of APOE4 in pre-dementia stages was also confirmed in the present study among subjects who were diagnosed as CIND without CeVD. Given that these subjects did not have clinical or neuroimaging evidence of CeVD and hence were likely to be in the early stages of AD, the association observed between APOE4 and CIND without CeVD is in keeping with our understanding of APOE function: APOE proteins bind amyloid-β (Aβ) and promote its clearance in an isoform-dependent pattern with E2 having the highest and E4 the lowest affinity to bind (E2>E3>E4), as demonstrated in animal models [39 –41] and autopsy studies [42 –44]. Thus, APOE genotyping may be a useful clinical tool for assessing the risk of having cognitive impairment and dementia due to AD.

The major focus of our study was to investigate APOE4 prevalence in subjects with VCI, ranging from CIND with CeVD to VaD, as well as AD with CeVD. A significant association between APOE4 and VaD was not observed in the present study although the OR of 1.42 (95% CI: 0.48–4.19) falls within the range of pooled estimates (OR: 1.60; 95% CI: 1.34–1.92) reported for Asian populations by Sun et al.. There was also no significant increase in APOE4 prevalence among CIND with CeVD subjects, whose cognitive impairment was likely due to CeVD. Studies on APOE4 in CIND with CeVD are very limited, and they had shown contradictory results [14 –17]. While our study may not have sufficient power to detect statistical significance for these associations, it is clear that the associations for APOE4 with VaD (OR: 1.42; 95% CI: 0.48–4.19) and CIND with CeVD (OR: 1.60; 95% CI: 0.71–3.57) were much weaker than that with AD (OR: 7.21; 95% CI: 2.74–18.98) and CIND without CeVD (OR: 3.34; 95% CI 1.59–7.03), suggesting that APOE polymorphism might not play a crucial role in vascular pathology. This is supported by the lack of association between APOE4 and specific CeVD subtypes observed in the present study, as well as previous studies where APOE4 was not linked to CeVD, including brain atrophy and WMH, in dementia [45, 46].

Interestingly, we also did not observe significant association for APOE4 in AD with CeVD although AD was diagnosed clinically among this group of patients. Our study classified cases of AD with CeVD by using MRI to define CeVD, namely by WMH, cortical infarct and lacunes, which is not the case for many other studies, and this may explain the discrepancies between our current finding and others which reported significant association between APOE4 and AD with CeVD [16 –22]. While some of these studies defined CeVD as ischemic stroke, subcortical and/or cortical infarct [16 –18], others did not have the neuroimaging criteria specified [19 –22]. Nevertheless, our finding is consistent with others which also did not observe significant association, including one performed in Taiwan [23 –28]. It is important to note that there is a higher burden of CeVD and vascular risk factors in Asian populations [47, 48]. Hence a higher proportion of the AD with CeVD subjects in the present study as well as in the Taiwanese study may have their cognitive impairment driven by CeVD instead of AD pathology, resulting in a lack of association with APOE4. Further investigations into the neuropathological features, molecular biology and neuroimaging of the AD with CeVD cases may help to clarify the lack of association with APOE4.

Our study had some limitations. As the analyses were performed cross-sectionally, we were unable to establish the longitudinal relationship between APOE polymorphism and disease progression. Secondly, while the present study included all major ethnic groups from Singapore’s multi-racial population (Chinese, Indian, and Malay), it was not possible to examine the role of ethnicity in the association between APOE4 and cognitive impairment due to the small number within individual ethnic groups. Moreover, due to the small sample size, we might be underpowered to detect statistical significance for the weaker associations between APOE4 and the VCI groups. Lastly, while diagnostic criteria with high specificity were utilized together with MR brain imaging, AD and CeVD tend to be comorbid pathologies and most individuals’ brains in old age may have multiple pathologies. Hence, further studies with neuropathological verification are needed to confirm our present findings. However, a major strength of this study is the use of extensive and standardized clinical and neuropsychological assessment to diagnose the different types of cognitive impairment and dementia. The availability of 3T MRI neuroimaging also allowed the identification of significant CeVD, hence improving the diagnostic classification.

CONCLUSION

The present study has elucidated for the first time, the APOE genotype profile in demented, cognitive impaired and cognitively normal Singaporeans. We confirmed the risk conferred by APOE4 in both CIND and AD without CeVD, and provided novel evidence that APOE4 was not associated with VCI, i.e., CIND with CeVD, AD with CeVD, and VaD. This provides insights into the role of APOE4 in the pathophysiology of cognitive impairment and dementia, and may aid in differentiating between the subtypes of dementia. APOE-directed therapeutics, for instance by inducing APOE protein expression and subsequently promoting Aβ clearance [49], could be promising for AD and CIND without CeVD, but might not be as effective in cognitive impairment and dementia due to CeVD.

Footnotes

ACKNOWLEDGMENTS

We would like to thank the psychologists, laboratory personnel, and trial coordinators who assisted in data collection for this study.

This work was supported by the National Medical Research Council Centre Grant (NMRC/CG/013/2013) and the National Medical Research Council Senior Clinician Scientist Award (NMRC/CSA/032/2011) to Dr. C.L. Chen, and a seed grant for basic science from the National University Health System (NUHSRO/2011/005/STB/B2B-01) to Dr. B.S Wong. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors’ disclosures available online ().

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