Evaluation of Apolipoprotein e4 allele as Susceptible Factor for Neurodegenerative Diseases among Eastern Indians

Dipanwita Sadhukhan; Smriti Mishra; Priyanka Mukherjee; Atanu Biswas; Subhra Prakash Hui; Tapas Kumar Banerjee; Arindam Biswas

Research Article

Austin Alzheimers J Parkinsons Dis. 2023; 6(2): 1040.

Evaluation of Apolipoprotein e4 allele as Susceptible Factor for Neurodegenerative Diseases among Eastern Indians

Dipanwita Sadhukhan¹; Smriti Mishra¹; Priyanka Mukherjee¹; Atanu Biswas²; Subhra Prakash Hui³; Tapas Kumar Banerjee¹; Arindam Biswas¹

¹Molecular Biology & Clinical Neuroscience Division, National Neurosciences Centre, Calcutta, Kolkata, India

²Institute of Post graduate of Medical Education & Research and Bangur Institute of Neurosciences, Kolkata, India

³S.N Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India

*Corresponding author: Arindam Biswas Molecular Biology & Clinical Neuroscience Division, National Neurosciences Centre, Calcutta, Peerless Hospital (2nd floor) 360, Panchasayar, Kolkata 700094, India. Email: [email protected]

Received: October 18, 2023 Accepted: November 20, 2023 Published: November 27, 2023

Abstract

Background & Objectives: Apolipoprotein E (ApoE) and age have been identified as the major risk factors for several neurodegenerative disorders. Among the three major isoforms (ApoE2, ApoE3 & ApoE4) of APOE, ApoE4 often shows ethnicity dependent association with neurodegenerative diseases. In the present study, we aim to determine the e4 allele/genotype frequency among the different neurodegenerative disorders and their correlation with several demographic and clinical parameters from eastern India.

Methods: A total of 826 individuals were recruited for this study which includes 128 PD-MCI, 144 PDD, 90 DLB, 114 FTD, 94 AD and 256 unrelated neurologically controls from eastern India. Subjects were analysed for APOE genotype (E2, E3 and E4) by PCR-RFLP techniques and Sanger sequencing.

Results: The APOE4 allele was significantly associated with each of the disease subtypes, selected for the study (P=0.016 to 0.000), while e3 allele was predominant among controls. Further stratification of subjects identified significant overrepresentation of (a) positive family history for FTD (P=0.0414) & AD (P=0.029) and (b) early age at onset of for PDD (P=0.0316) and FTD (P=0.0034) among the e4 allele carriers. Furthermore, lowering of BMSE score was also observed among the e4 allele carriers of AD subjects (P=0.0324).

Conclusion: There is a significant association of APOE4 allele with different neurodegenerative diseases influencing lowering of age at onset, BMSE score and positive family history among ethnic Bengali population of eastern India.

Keywords: APOE; Neurodegenerative diseases; Indians

Introduction

The Apolipoprotein E (ApoE) protein has a crucial role in lipid and cholesterol flux in the central nervous system [1]. Among its three major human isoforms, ApoE2, ApoE3, and ApoE4, APOE4 is considered as one of the most potent risk factors for the development of neurological disorders. The presence of two arginine at the 112th and 158th positions in the APOE4 protein provides it with an enhanced lipid binding property with reduced stability rendering pathogeniecity than other isoforms.

Neuronal death in neurodegenerative diseases is often associated with abnormal protein accumulation, immune system activation, neuroinflammation and blood-brain barrier disruption. In Alzheimer’s Disease (AD) pathology, APOE polymorphisms with the combined effect of sex, age, diet, and physical exercise, have a major effect on astrocytic and microglial function and microglial dynamics, synaptic function, amyloid-β load, tau pathology, autophagy, and cell–cell communication [2]. As a result, APOE4 carriers often show a greater Aβ deposition both in quantity and density compared to non-APOE4 carriers. Similarly, in Parkinson’s Disease (PD), an increase in alpha-synuclein aggregate formation is found to be influenced by the APOE4 genotype [3]. However, studies showed incongruities in this regard suggesting that APOE polymorphisms may result in a higher risk of cognitive impairment than motor deterioration. In Lewy body dementia, it also has distinct roles in microglia-associated alpha-synuclein clearance in the midbrain [4]. A number of genetic association studies also highlighted increased disease risk for e4 alleles in frontotemporal dementia [5].

Although APOE plays a significant role in several central nervous system pathologies which are mostly complex in nature, the genetic association data often appear to be inconsistent due to differences in APOE allele frequencies across ethnic populations. In India, a number of genetic studies have been performed in neurodegenerative disorders considering either one or few disease types at a time. However, the results are conflicting and may be influenced by with limited number of study subjects and mutiethnicity. Therefore, the present study aims to evaluate the frequency of APOE isoforms in several neurodegenerative diseases in a relatively larger sample representing the ethnic Bengali population of Eastern India.

Materials and Methods

Study Subjects

A total of 570 patients clinically diagnosed with neurodegenerative disorders [128 Parkinson’s disease with mild cognitive impairment (PD-MCI), 144 Parkinson’s Disease with Dementia (PDD), 90 Dementia with Lewy body (DLB), 114 Frontotemporal Dementia (FTD) and 94 Alzheimer’s Disease (AD)] by clinicians were recruited from Eastern India in this study with their written informed consent as per guidelines of Indian Council of Medical Research (ICMR). Patients were diagnosed following standard diagnosis criteria. In addition, 256 age and ethnicity matched unrelated healthy individuals with no personal or family history of neurodegenerative diseases and/ or any other neurological symptoms were also recruited as controls in the present study from Kolkata. Bengali version of Mini Mental State Examination (BMSE) scale was used for primary tool during evaluation [6]. The diagnostic labelling was done by trained neurologists (AB and TKB). Diagnosis was performed using standard diagnostic criteria (MDS task force criteria for PD-MCI [7] and PDD [8], FTD by FTD Consortium criteria [9], DLB by DLB consortium criterion – fourth consensus [10] and AD by NIA-AA criteria [11]. The demographic details of patients are described in Table 1.

Table 1: Demographic details of studied population.




  
    Category
    Male: Female
    Familial:    Sporadic
    Early onset:    Late onset
    Age at onset    ± SD
    BMSE ± SD
  
  
    PD–MCI (n=128)
    90: 38
    32: 96
    38: 90
    55.54±15.43
    21.95±6.13 
  
  
    PDD (n=144)
    100: 44
    32: 112
    26: 118
    60.89±12.94
    20.23±7.39 
  
  
    DLB (n=90)
    64: 26
    24: 66
    16: 74
    60.65±10.45
    15.60±8.98 
  
  
    FTD (n=114
    76: 38
    20: 94
    24: 90
    55.77±10.41
    15.15±8.67 
  
  
    AD (n=94)
    52: 42
    48: 46
    26: 68
    58.30±9.84
    14.04±6.91
  
  
    CONTROL

      (n=256)
    174: 82
    Not    applicable
    Not    applicable
    Not    applicable
    ≥26
  
  
    *PD - MCI: Parkinson’s    disease with mild cognitive impairment; PDD: Parkison’s disease with    dementia; DLB: Dementia with Lewy body; FTD: Frontotemporal dementia; AD:    Alzheimer’s disease; SD: Standard deviation; Early onset ≤50 years; Late    onset >50 years; BMSE: Bengali version of Mini Mental State Examination.



Table 1: Demographic details of studied population.

Collection of Blood Samples and Genomic DNA Preparation

10 mL of peripheral blood samples were collected in EDTA vials from patients and controls. Genomic DNA was prepared from fresh whole blood by conventional salting out method using sodium perchlorate followed by isopropanol precipitation [12] and dissolved in TE (10 mM Tris− HCl, 0.1 mM EDTA, pH 8.0) and stored at 40C.

Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) Analysis

APOE genotypes were determined by Polymerase Chain Reaction (PCR) and restriction fragment length polymorphism techniques. The amplicon which was generated using specific primer pair was subjected to enzymatic digestions for 3 hours at 37°C with 1 U of the AflIII (New England Biolabs, Ipswich) and HaeII (New England Biolabs, Ipswich) independently. The digested products were separated on a 6% polyacrylamide gel and analysed further after visualization in a Gel documentation system (BIO-RAD, USA).

Statistical Analysis

Demographic and cognitive parameters were analysed using Mann Whitney U test and Fisher exact test.

Frequency of APOE alleles among neurodegenerative disorder subgroups was calculated with healthy controls as a reference group, using Fisher’s exact test.

Relative Risk was calculated using the MedCalc statistical software. The probability level of ≤0.05 was considered statistically significant.

Results

Frequency of APOE4 in Disease Subgroups

The APOE SNPs, rs429358 and rs7412, which encode for the polymorphisms ε2, ε3, and ε4 were screened in 570 patients with various neurodegenerative disorders against 256 ethnically matched controls. All APOE alleles were within Hardy-Weinberg equilibrium in both the patients and control groups. Table 2 shows the allele and genotype frequencies of APOE variants in different disease types. For the APOE4 allele, a significant association with PD-MCI, PDD, DLB, FTD and AD were observed in our study cohorts (P=0.016 for PD-MCI and 0.000 for rest). The highest relative risk was observed for the AD group (RR=5.59; 95% CI: 4.44 – 7.60; P=<0.0001) followed by DLB (RR=3.89), FTD (RR=2.54), PDD (RR=2.53) and PD-MCI (RR=1.79) (Table 3). In contrast, the e3 allele was the predominant one in control group.

Table 2: Allele & Genotype frequency of APOE among different neurodegenerative diseases.




  
    Study    Subjects
    Genotype    frequency
    Allele    frequency
    P-Value
    Odds ratio    (95% CI)
  
  
    E2/E3
    E2/E4
    E3/E3
    E3/E4
    E4/E4
    E2
    E3
    E4
     
      Referent
  
  
    CONTROL

      (n=256)
    32

      (0.1250)
    0

      (0.00)
    186

      (0.7265)
    34

      (0.1328)
    4

      (0.0156)
    32

      (0.0625)
    438

      (0.8554)
    42

      (0.0820)
  
  
    PD-MCI

      (n=128)
    2

      (0.0156)
    4

      (0.0312)
    92

      (0.7187)
    28

      (0.2187)
    2

      (0.0156)
    6

      (0.0234)
    214

      (0.8359)
    36

      (0.1406)
    E4 vs Others: 0.016
    1.81    (1.14-2.94)
  
  
    PDD

      (n=144)
    6

      (0.0416)
    0

      (0.0)
    84

      (0.5833)
    48

      (0.3333)
    6

      (0.0416)
    6

      (0.0208)
    222

      (0.7708)
    60

      (0.2083)
    E4 vs Others: 0.000
    2.94    (1.93-4.50)
  
  
    DLB

      (n=90)
    4

      (0.0444)
    0

      (0.0)
    34

      (0.3777)
    32

      (0.3555)
    20

      (0.2222)
    4

      (0.0222)
    104

      (0.5777)
    72

      (0.4000)
    E4 vs Others: 0.000
    7.46    (4.83-11.52)
  
  
    FTD

      (n=114)
    8

      (0.0655)
    2

      (0.0163)
    68

      (0.5573)
    38

      (0.3114)
    6

      (0.0491)
    10

      (0.0409)
    182

      (0.7459)
    52

      (0.2131)
    E4 vs Others: 0.000
    3.03 (1.95-    4.70)
  
  
    AD

      (n=94)
    2

      (0.0212)
    2

      (0.0212)
    14

      (0.1489)
    62

      (0.6595)
    14

      (0.1489)
    4

      (0.0212)
    92

      (0.4839)
    92

      (0.4893)
    E4 vs Others: 0.000
    10.72    (7.00-16.42)



Table 2: Allele & Genotype frequency of APOE among different neurodegenerative diseases.

Table 3: Frequency of APOE4 carriers among different neurodegenerative disorders.




  
    Diagnosis
    Subjects (n)
    E4 Carriers (n)
    % of Subjects
    **P value
    Relative Risk (95% CI)
    P value
  
  
    PD-MCI
    128
    34
    26.56%
    0.0081
    1.79    (1.186–2.699)
    0.0055
  
  
    PDD
    144
    54
    37.5%
    <0.00001.
    2.53    (1.760–3.626)
    <0.0001
  
  
    DLB
    90
    52
    57.78%
    <0.00001.
    3.89    (2.763–5.482)
    <0.0001
  
  
    AD
    94
    78
    82.98%
    <0.00001.
    5.59    (4.110–7.602)
    <0.0001
  
  
    FTD
    122
    46
    37.70%
    <0.00001.
    2.54    (1.752–3.683)
    <0.0001
  
  
    Controls
    256
    38
    14.84%
    References
    References
    References
  
  
    **Fisher exact test was calculated considering healthy controls as    reference group; Relative risk was calculated using MedCalC software.



Table 3: Frequency of APOE4 carriers among different neurodegenerative disorders.

Comparison of Demographic Features and Cognitive Status

Next, we compared the demographic features like age at disease onset, positive family history, and gender between carriers and non-carriers of APOE E4 variant in each of the disease subtype. A significant lowering of age at onset for PDD (56.85 years vs 63.08 years; P=0.0316) and FTD (54.09 years vs 58.79 years; P=0.0034) groups was observed for E4 carriers, while the presence of positive family history for FTD (P=0.0414) and AD cases (P=0.029) show significant correlation for the same (Table 4).

Table 4: Comparison of clinical parameters between E4 carriers and E4 noncarriers.




  
    A General    Comparison Between E4 carriers and non-carrier’s cases
  
  
    PD-MCI    (n=128)
    E4 carriers    (n=34)
    E4    non-carriers (n= 94)
    P value
  
  
    Age at disease onset (years), mean ± SD
    54.68±18.81
    55.04±14.48
    0.4972
  
  
    Positive Family History, n (%)
    10(29.41%)
    22(23.40%)
    0.4956
  
  
    Male, n (%)
    28(82.35%)
    62(65.95%)
    0.0831
  
  
    BMSE, mean ± SD
    18.41±7.33
    21.43±3.6
    0.238
  
  
    PDD (n= 144)
    E4 carriers    (n=54)
    E4    non-carriers (n=90)
    P value
  
  
    Age at disease onset (years), mean ± SD
    56.85±12.19
    63.08±11.99
    0.0316
  
  
    Positive Family History, n (%)
    12(22.22%)
    20(22.22%)
    1
  
  
    Male, n (%)
    34(62.96%)
    66(73.33%)
    0.1976
  
  
    BMSE, mean ± SD
    18±9.18
    21.04±7.13
    0.526
  
  
    DLB (n= 90)
    E4 carriers    (n=38)
    E4    non-carriers (n=52)
    P value
  
  
    Age at disease onset (years), mean ± SD
    60.95±11.41
    60.19±9.58
    0.9362
  
  
    Positive Family History, n (%)
    14(26.92%)
    10(26.31%)
    1
  
  
    Male, n (%)
    38(73.08%)
    26(68.42%)
    0.6455
  
  
    BMSE, mean ± SD
    13.69±9.37
    17.17±8.9
    0.2168
  
  
    FTD (n= 122)
    E4 carriers    (n=46)
    E4    non-carriers (n=76)
    P value
  
  
    Age at disease onset (years), mean ± SD
    54.09±8.78
    58.79±6.47
    0.0034
  
  
    Positive Family History, n (%)
    12(26.09%)
    8(10.53%)
    0.0414
  
  
    Male, n (%)
    32(69.57%)
    44(57.89%)
    0.2484
  
  
    BMSE, mean ± SD
    14.83±8.37
    18.33±7.76
    0.2318
  
  
    AD (n= 94)
    E4 carriers    (n=78)
    E4    non-carriers (n=16)
    P value
  
  
    Age at disease onset (years), mean ± SD
    58.88±10.00
    55.75±9.29
    0.1565
  
  
    Positive Family History, n (%)
    44(56.4%)
    4(25%)
    0.029
  
  
    Male, n (%)
    42(53.84%)
    10(62.5%)
    0.59
  
  
    BMSE, mean ± SD
    8.67±4.85
    14±6.93
    0.0324
  
  
    BMSE: Bengali version of Mini Mental State Examination



Table 4: Comparison of clinical parameters between E4 carriers and E4 noncarriers.

As mentioned in Table 1, irrespective of APOE4 status a lowering of BMSE score i.e <25 (referring overall cognitive status) was observed in our patient cohort with different clinical diagnosis. However, after further stratification of subjects according to E4 isoform followed by an intra-group comparison, a significant lowering in BMSE score for AD (P=0.0324) but not for the other conditions was observed between E4 carriers and non-carriers.

Discussion

Identification of population-specific genetic risk variants in complex neurodegenerative diseases has long been reported in different studies. However, the major gene of interest is APOE across the world population showing ethnicity and disease specific association. In this present study, we observed an overrepresentation of APOE e4 allele in AD, PD, PD-MCI, DLB and FTD cases in ethnic Bengali population. Our data corroborates with previous Indian reports on PD, MCI and AD but is at variance with the other studies on DLB, FTD and FTLD (Table 5).

Table 5: Frequency distribution of APOE4 allele as reported in Indian studies on neurodegeneration.




  
    Disease    sub-types
    Geographical    Location
    Study    subjects
    Frequency of e4 allele
    Comments
    References
  
  
    PD
    East India

      Kolkata
    Patients:302

      Controls:302
    Patients:    0.015

      Controls:    0.084
    e4 allele increased the disease risk
    [19]
  
  
    North India

      (Delhi)
    Patients:70

      Controls: 100
    Patients:    0.160

      Controls:    0.080
    e4 allele increased the disease risk
    [20]
  
  
    MCI
    South India

      (Bangalore)
    Patients: 87

      Control: 138
    Patients:    0.285

      Controls:    0.177
    e4 allele increased the disease risk
    [15]
  
  
    DLB
    South India

      (Bangalore)
    Patients: 12

      Controls: 138
    Patients:    0.208

      Controls:    0.177
    The APOE e4 allele remained ineffectual in modulating disease risk
    [15]
  
  
    FTD
    South India

      (Bangalore)
    Patients: 127

      Controls: 138
    Patients:    0.158

      Controls:    0.177
    Not associated with disease risk
    [15]
  
  
    FTLD
    East India

      (Kolkata)
    Patient: 81

      Controls: 269
    Patients:    0.148

      Controls:    0.106
    Not associated with disease risk
    [21]
  
  
    DLB & FTD
    South India

      (Bangalore)
    Patients: 44

      Controls: 195
    Patients:    0.079

      Control:0.079
    Not associated with disease risk
    [16]
  
  
    AD
    Western India    (Mumbai)
    Patients: 49

      Controls: 100
    Patients:    0.190

      Controls:    0.085
    Associated with AD
    [22]
  
  
    North India    (Delhi)
    Patients: 29

      Controls: 76
    Patients:    0.290

      Controls:    0.092
    Associated with AD
    [23]
  
  
    North India    (Delhi)
    Patients: 74

      Controls: 113
    Patients:    0.270

      Controls:    0.066
    Associated with AD
    [24]
  
  
    South India    (Bangalore)
    Patients: 243

      Controls: 164
    Patients:    0.240

      Controls:    0.076
    Associated with AD
    [25]
  
  
    North India    (Delhi)
    Patients: 70

      Controls: 75
    Patients:    0.314

      Controls:    0.073
    Associated with AD
    [26]
  
  
    North India

      (Chandigarh)
    Patients: 44

      Controls: 46
    Patients:    0.390

      Controls:    0.000
    Increased CSF tau protein levels in AD    patients with APOE-e4 allele
    [27]
  
  
    South India    (Bangalore)
    Patients: 137

      Controls: 195
    Patients:    0.210

      Controls:    0.079
    Associated with AD
    [16]
  
  
    North India    (Delhi)
    Patients: 14

      Controls: 46
    Patients:    0.430

      Controls:    0.110
    Associated with AD
    [28]
  
  
    South India    (Bangalore)
    Patients: 209

      Controls: 193
    Patients:    0.469

      Controls:    0.166
    Associated with AD
    [29]
  
  
    East India    (Kolkata)
    Patients: 115

      Controls: 162
    Patients:    0.096

      Controls:    0.105
    APOE-e4 & C allele of rs1800795 together presented with early    onset
    [30]
  
  
    South India

      (Bangalore)
    Control: 156

      Patient:138
    Patients:    0.394

      Controls:    0.177
    The APOE-e4 allele increased the risk for AD
    [15]



Table 5: Frequency distribution of APOE4 allele as reported in Indian studies on neurodegeneration.

AD and PD cases are often been categorized into early-onset or late-onset cases, as age is an independent risk factor for both. Recent evidences suggest that although the APOE2 allele decreases late onset AD (LOAD) risk, the expression of one and two E4 allele increases the disease risk to threefold and 9 to 15-fold, respectively [13]. However, no such findings were observed here which may be due to recruitment of only late-onset AD cases and small number of non-E4 carrying AD patients in study cohort as well. In contrast, APOE4 allele carriers showed an early age at onset for FTD and PDD cohort [5]. Our present data for PDD is consistent with a meta-analysis study in 2006 [14]. However, negative association was reported in an early report from South India on FTD [15,16]. The small number of samples and genetic divergence may be probable reasons for lack of consistency in the nature of association between two studies from India. The south Indian population is representative of the original Dravidians from Central and South India while Eastern Indians belong to the Indo‑European (IE) lineage [17].

Evidences suggest that positive family history in neurodegenerative disorders explains the part of individual differences in risk and progression for the diseases and thus it is often correlated with genetic variants to determine its influence in disease pathology. Here, an intra group comparative study describing the correlation between APOE4 polymorphism and family history in different diseases as summarised in Table 4, revealed a link only for AD and FTD suggesting its potential for these diseases among the Eastern Indians. However, the absence of such in rest suggests the role of additional genes with equal or greater significance.

The progression of neurodegeneration is always accompanied by different degrees of cognitive decline. Diseases like AD, DLB, FTD manifest cognitive failure as an initial symptom while PD-MCI and PDD are developed in few PD patients over time. A number of earlier reports, on the basis of frequency differences between cases and controls, already stated the E4 allele to be correlated with higher prevalence of dementia in synuclenopathies and AD [13,18]. However, when we compared the raw scores according to genotype, except for AD, no other diseases showed differences in BMSE scores between carriers and non-carriers among the patient cohort. Our data is concordant with other national and international reports for AD, while it needs further confirmatory studies considering scores and further sub domain analysis to make a definite conclusion.

Conclusion

In conclusion, our findings demonstrate that the APOE4 allele increases the risk of AD, PDD, PD-MCI, DLB and FTD among ethnic Bengali populations from Eastern India. Additionally, it has a negative impact on cognitive status for AD while influencing the early age at onset for PDD and FTD. A positive correlation between family history and APOE4 was also identified for AD and FTD. It would also be interesting to study and correlate the impaired cognitive subdomains in different disease types with APOE genotypes in future studies.

Author Statements

Ethical Considerations

The study was approved by the Institutional Ethical Committee at Institute of Post Graduate Medical Education & Research, Kolkata and National Neurosciences Centre Calcutta, Kolkata. The Research Ethics Board expressed no ethical objections to the submitted research plan. All participants provided written consent to participate in the study after a full explanation of the study procedures.

Funding

Supported by grants from the Department of Science & Technology, Govt. of India, under Cognitive Science Research Initiative Programme (DST/CSRI-PDF/2021/12) and (SR/CSRI/PDF-32/2014 and DST/CSRI-P/2017/22).

Conflict of Interest

The authors declare no conflicts of interest.

Acknowledgement

The authors thank the patient, family members, and healthy individuals who participated in the study.

Data Availability Statement

The data described in this study are available from the corresponding author upon reasonable request.

References

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Citation: Sadhukhan D, Mishra S, Mukherjee P, Biswas A, Hui SP, et al. Evaluation of Apolipoprotein e4 allele as Susceptible Factor for Neurodegenerative Diseases among Eastern Indians. Austin Alzheimers J Parkinsons Dis. 2023; 6(2): 1040.

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