Genetic risk factor APOEε4 associates with plasma amyloid beta in amnestic mild cognitive impairment and alzheimer’s disease

Rocksy F.V. Situmeang,1,2,4 Eka J. Wahjoepramono,2,3 Cahyono Kaelan,5,6 Jan S. Purba,7 Budhianto Suhadi,3 Suryani As'ad,8 Amiruddin Aliah,5,8 Ilham J. Patellongi,8 Syarifuddin Wahid6,8

Background

APOEε4 is a strong genetic risk factor for Alzheimer’s disease (AD). AD itself has been associated with reduced Aβ clearance from the brain and plasma. Understanding the potential pathogenic link between APOEε4 and plasma Aβ might allow for earlier identification of people at risk of developing AD. The aim of this study is to find out the correlation between APOEε4 and plasma Aβ in amnestic mild cognitive impairment (aMCI) and AD patients.

 

Methods

This is a comparative cross-sectional study of patients attending a memory clinic in Siloam Hospital Lippo Karawaci, Tangerang, during the period of 2013-2014. Subjects were categorized into three categories: normal aging, aMCI, and AD. We performed blood test to examine APOEε4, plasma Aβ4o level, and plasma Aβ42 level. All data analyses were performed using correlation test and logistic regression.

 

Results

Sixty subjects (normal aging = 23, aMCI = 17, AD = 20) were included. There were 19 (31.7%) subjects with APOEε4 positive. Subjects carrying ε4 allele were more likely to have AD by 3.9-fold than subjects with APOE ε4 allele negative. There is a significant difference between the mean of plasma Aβ40 in aMCI group and AD group. We also found correlation between APOEε4 (+) and higher plasma Aβ42 (p<0.05).

 

Conclusion

There is a correlation between APOEε4 and plasma Aβ42 level, which supports the hypothesis that this genetic isoform accelerates the rate and progression of AD through Aβ-dependent pathways.

 

Keywords

alzheimer's disease, amnestic mild cognitive impairment, APOEε4, plasma amyloid beta

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. It is approximately occurred in 13% of people over the age of 65 to 45% people over the age of 85.1 One of the strongest pathomechanism of AD is amyloid cascade hypothesis that points to the imbalance between production and clearance of amyloid-β (Aβ) peptides in the brain which results in accumulation and aggregation of Aβ in the form of soluble Aβ oligomers, intraneuronal Aβ, and amyloid plaques injure synapses, and ultimately leads to neurodegeneration and dementia.2,3

The strongest risk factor for AD is the ε4 allele of the apolipoprotein E (APOE).4-6 There are three polymorphic alleles of APOE gene: ε2, ε3, and ε4, which APOEε3 allele is the most frequent in worldwide population (77.9%), followed by ε4 (13.7%), and ε2 (8.4%).7 Risk of AD was increased in individual with one copy of ε4 allele (heterozygote) and higher in two copies (homozygote).7 APOEε4 is associated with lower age of AD onset. Mean age at clinical AD onset in ε4 homozygote, ε4 heterozygote, and ε4 noncarriers are 68 years, 76 years, and 84 years, respectively.4,8

Mild cognitive impairment (MCI) is a transitional stage between normal cognitive function and dementia. Amnestic MCI (aMCI) is associated with increased risk of AD,9 is estimated to progress into clinically diagnosable AD in 10–15% of cases per year, compared to a rate of 1–2% per year among healthy elderly individuals.10 APOEε4 affects memory performance in people with MCI,11,12 both in middle age (40–59 years) and elderly (60–85 years) people.13,14 In addition, MCI patients with APOE ε4 experience more-rapid decline in several cognitive and functional assessments,12,15,16 which leads to increased risk of progression from MCI to AD-type dementia.17-19 Consistent with younger age of AD onset in individuals with APOEε4, it also applies in aMCI.20 These findings indicate that the APOEε4 genotype in patients with MCI can serve as a predictive factor for determination of clinical outcome and the risk of conversion to AD.21

APOE provides instruction to make a protein called Apo lipoprotein E- lipoproteins bind to several cell-surface receptors to deliver lipids and also to hydrophobic Aβ peptide, which is thought to initiate toxic events that lead to synaptic dysfunction and neurodegeneration in AD.21 APOE isoforms differentially regulate Aβ aggregation and clearance in the brain, and have distinct functions in regulating brain lipid transport, glucose metabolism, neuronal signalling, neuroinflammation, and mitochondrial function.21,22 Immunohistological evidence demonstrates that APOE is co-deposited in senile plaques in the brains of AD patients.23 The Aβ deposition in the form of senile plaques is more abundant in APOEε4 carriers compared with noncarriers.24-26 Furthermore, APOEε4 carriers have lower cerebrospinal fluid (CSF) Aβ42 level27,28 yet the relationship between plasma Aβ and APOEε4 remains inconclusive.29-32

In the present report, we studied the correlations between genetic risk factor APOEε4 with plasma amyloid beta level in both aMCI and AD.

 

 

Subjects in this study were patients who visited memory clinic of Siloam Hospital Lippo Village from September 2013 to September 2014. We included all subjects who fulfilled inclusion criteria: 1) age >60 years old, 2) able to speak Bahasa Indonesia or English (premorbid), 3) able to read and write (premorbid). We excluded subjects with impaired renal function and subjects with major vascular lesions or other structural lesions in magnetic resonance imaging (MRI).

Subjects were categorized into three clinical diagnoses: normal aging, amnestic mild cognitive impairment, and AD. We performed physical, neurological, and neurobehavioral examinations including mini mental status examination (MMSE), montreal cognitive assessment (MoCA), and consortium to establish a registry of Alzheimer’s disease (CERAD). We used AD-8 questionnaire and instrumental activity daily living (IADL) to determine impairments on activities of daily living. We determined severity of AD using Clinical Dementia Rating Scale (CDRS) and Global deterioration Scale (GDS). MRI test was executed on subjects who had stroke history or suspected to have other causes of dementia.

We performed blood test to examine ureum, creatinine, APOEe4 with restriction fragment length polymorphism, plasma Aβ40 level, and plasma Aβ42 level. The concentration of Aβ peptides (Aβ40 and Aβ42) in plasma was measured using commercially available sandwich ELISA assay (IBL International GmbH, Hamburg, Germany) which has been shown to be sensitive enough 1.56–100 pg/mL.

All data analyses were performed using SPSS statistics (IBM Corp. Released 2011, version 20 Armonk, NY, US). The measures were expressed as mean ± standard deviation. We evaluated the association between two qualitative variables using chi square test and the association between qualitative variable and quantitative variable using t test. We performed the correlation test and logistic regression. A probability value less than 5% was considered statistically significant. The protocol of this study has been approved by Mochtar Riady Institute of Nanotechnology (No. 083/MRIN-EC/09/2014). We obtained written informed consent from all patients.

 

 

A total of 80 subjects in memory clinic were evaluated in which 65 patients among them met our inclusion criteria. We excluded five subjects due to a major infarct in MRI findings (one subject) and the impairment of renal function (four subjects). Sixty subjects were eligible for further step analyzing apo-E polymorphism and plasma amyloid beta (Aβ40 and Aβ42 level). Diagnosis is divided into three groups: AD = 20, aMCI = 17, and normal cognition = 23 subjects. Forty two subjects were women (70%) and unlike common demographic profile in Indonesia, half of the subjects had high educational level (>12 years of formal education) (Table 1).

 

 

There is a significant association between APOEe4 genotype with cognitive function, particularly in HC versus AD group (Table 2). Subjects carrying the ε4 allele were more likely to have AD by 3.9 times compared to non-carriers. This likelihood is even higher in subjects with age-group ≤70 years (OR=13).

 

 

Level of Aβ40 and Aβ42 plasma in AD is the highest compared to aMCI and HC, while the Aβ42/Aβ40 ratio in this group is the lowest (Table 3). We found a significant difference between the mean of plasma Aβ40 in HC versus AD group (p<0.05).

 

 

We analyzed the mean difference of plasma Aβ level in APOEε4(+) carrier compared to noncarrier and found it significant in level of Aβ42 plasma (Table 4). When we divided the subjects into two groups (higher level of Aβ and lower level of Aβ) based on certain cut off points, we found significant correlation between APOEε4(+) and higher Aβ42 plasma level (Table 5).

 

 

 

 

 

 

The ε4 allele of APOE as the strongest genetic risk factor for AD has been confirmed by genomewide association studies.33,34 The presence of this allele is associated with increased risk for both early-onset AD and late-onset AD.35,36 To our knowledge, investigation about the role of APOE ε4 in AD incidence among Indonesian has not been established. In population-based studies, the APOE ε4–AD association was weaker among African Americans (ε4/ε4, OR=5.7) and Hispanics (ε4/ε4, OR=2.2) and was stronger in Japanese people (ε4/ε4, OR=33.1) compared with Caucasian cases (ε4/ε4, OR=12.5).37 In this study, the role of APOEε4 as strong genetic risk factor had been proven; showing an increased risk of up to fourfold for carrier APOEε4 carriers to have AD than non-carriers. This risk is increased into 13-fold for subjects with age group ≤70 years, indicating that APOEε4 confers dramatically increased risk of development of AD with an earlier age of onset.

The investigation of plasma Aβ species offers advantage over conventional method for measuring Aβ level in the brain and CSF. Obtaining and analyzing plasma level is relatively inexpensive, minimally invasive, and can easily be performed at multiple time points. Therefore, a plasma-based biomarker for early detection and diagnosis of AD would be ideal. The utility of plasma Aβ as a potential AD biomarker had been assessed in previous studies, but the results have been inconsistent.38-41 It is well established that CSF Aβ42 level decreased in conjunction with the cognitive decline, it has been postulated that plasma Aβ42 may decrease similarly.42 Low plasma Aβ42 at baseline was associated with cognitive decline occurring during follow up.43 We found no statistical significance of differences in plasma Aβ42 levels and Aβ42/Aβ40 ratio among AD, MCI, and HC groups, but there is a tendency that both are lower in aMCI and AD subjects than in HC subjects. Interestingly, plasma Aβ40 is much higher in AD subjects compared to HC and aMCI subjects. Previous studies showed that Aβ levels varied highly in brains of patients with AD, and those with massive amyloid deposition contained predominantly Aβ40. In some patients with sporadic AD, high plasma levels of Aβ40 may contribute to the accumulation of Aβ40 in preexisting plaques.44

We found a significant correlation between APOEε4+ and high plasma Aβ42 level which more prominent in aMCI and AD groups. The APOEε4 has been associated with reduced Aβ clearance from the brain45 and plasma46 and with impaired tight junction integrity.47 Our data suggest that APOEε4 influences Aβ42 levels, but not Aβ40 level. Some studies have shown association between ε4 allele with increased fibrillar Aβ22 and decreased soluble plasma Aβ42 in a dosedependent manner.30 Discrepancies between our results and those reported by others may result from differences in patient populations, the use of different enzyme-linked immunosorbent assays and platforms, and timing of sample collection in relation to the stage of disease progression.

In conclusion, work summarized in this research highlights evidence for the association between APOEε4, plasma Aβ level, and cognitive decline. APOEε4 is a strong genetic risk factor for AD among Indonesian people, particularly in age group ≤70 years. APOEε4 seems to accelerate the rate and progression of AD through Aβ- dependent pathways. The exact mechanisms by which APOEε4 regulates Aβ aggregation and deposition require further investigation.

 

 

 

 

 

 

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