The Association between Sporadic Alzheimer's Disease and the Human ABCA1 and APOE Gene Polymorphisms in Iranian Population.

Abstract

Apolipoprotein E (APOE), which its ε4 allele has been reported as a risk factor in late onset Alzheimer's disease (AD), is the main cholesterol carrier in the brain. ATP-binding cassette transporter A1 (ABCA1) gene on chromosome 9, which has been known by genome-wide AD linkage study, has an important role in cellular cholesterol efflux. This study determines the association between sporadic AD and the human ABCA1 and APOE gene polymorphisms in Iranian population.
154 AD cases and 162 control subjects from Iranian population were genotyped for APOE genotypes and ABCA1 polymorphism (R219K).
The frequency of ε2ε3 genotype was higher in control subjects comparing AD patients but was not significant (13% versus 5.8%) and ε3ε4 genotype frequency was significantly higher in AD cases comparing with control subjects. APOE-ε2 allele frequency in cases was lower than control subjects but this difference was not significant (4.5% versus 8%). Individuals carrying ε4 allele, developed AD 6.5 times more than non-carriers (OR=6.52, 95%CI=2.63-16.17). There was no significant association between ABCA1 polymorphism and AD.
Unlike other studies, R219K polymorphism was not dependent on gender and APOE-ε4 allele and there was no association between APOE and ABCA1 in AD patients compared to controls.

Full text

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Iranian Red Crescent Medical Journal

Iran Red Crescent Med J 2011; 13(4):256-262 ©Iranian Red Crescent Medical Journal
ORIGINAL ARTICLE
The Association between Sporadic Alzheimer’s Disease
and the Human ABCA1 and APOE Gene Polymorphisms
in Iranian Population


HR Khorram Khorshid1*, E Gozalpour1, K Kamali2, M Ohadi1, M Karimloo3, MH Shahhosseiny4

1Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran,
2Reproductive Biotechnology Research Centre, Avicenna Research Institute (ACECR), Tehran, Iran,
3Epidemiology and Biostatistics Department, University of Social Welfare and Rehabilitation Sciences,
Tehran, Iran
4Microbiology Department, Islamic Azad University, Shahr-e-Qods Branch, Tehran, Iran


Abstract

Background: Apolipoprotein E (APOE), which its ε4 allele has been reported as a risk factor in late onset Alz-
heimer’s disease (AD), is the main cholesterol carrier in the brain. ATP-binding cassette transporter A1 (ABCA1)
gene on chromosome 9, which has been known by genome-wide AD linkage study, has an important role in
cellular cholesterol efflux. This study determines the association between sporadic AD and the human ABCA1
and APOE gene polymorphisms in Iranian population.

Methods: 154 AD cases and 162 control subjects from Iranian population were genotyped for APOE genotypes
and ABCA1 polymorphism (R219K).

Results: The frequency of ε2ε3 genotype was higher in control subjects comparing AD patients but was not
significant (13% versus 5.8%) and ε3ε4 genotype frequency was significantly higher in AD cases comparing with
control subjects. APOE-ε2 allele frequency in cases was lower than control subjects but this difference was not
significant (4.5% versus 8%). Individuals carrying ε4 allele, developed AD 6.5 times more than non-carriers
(OR=6.52, 95%CI=2.63-16.17). There was no significant association between ABCA1 polymorphism and AD.

Conclusion: Unlike other studies, R219K polymorphism was not dependent on gender and APOE-ε4 allele and
there was no association between APOE and ABCA1 in AD patients compared to controls.

Keywords: Alzheimer’s disease; Genetic association; Apolipoprotein E; Polymorphism; ATP-binding cassette
transporter A1; Iran


Introduction

Alzheimer’s disease (AD), which presents progres-
sive cognitive defects such as memory loss, apraxia
and personality changes, is the commonest cause of
dementia in the mid and late ages.1,2 Two neuropath-
ophysiological hallmarks of AD are intracellular neu-
rofibrillary tangles and beta amyloid plaques in brain
blood vessels. As hundreds genes have been known
as the risk factors for late onset AD, the well-known
one is apolipoprotein E gene (APOE) which has been
recognized as the most important risk factor in 65%
of sporadic cases.3 Apolipoprotein E is the main part
of very low density lipoproteins, intermediate density
lipoproteins (IDL), chylomicrons and the main cho-
lesterol carrier in the brain and its synthesis is inde-
pendent in central nervous system (CNS) and lung.
As APOE expression is stimulated by any CNS dam-
ages or diseases, it seems that APOE regulates cho-
lesterol metabolism and distribution in the brain to
repair and stabilize neurons’ membrane and myelin.4-6
Several lines of evidence show that cholesterol me-




*Correspondence: Hamid Reza Khorram Khorshid, MD, PhD,
Genetic Research Centre, University of Social Welfare and
Rehabilitation Sciences, Tehran, Iran. Tel/Fax: +98-21-22180138,
e-mail: hrkk1@uswr.ac.ir
Received: June 1, 2010, Revised: September 10, 2010, Accepted:
November 1, 2010

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tabolism and Aβ deposition are related to each other.
Cholesterol reducing drugs such as statin decrease
brain Aβ level and increase non-amyloidogenic α-
secretase cleavage of amyloid precursor protein and
lead in reduced Aβ deposition.7-10 High cholesterol
diet in transgenic animal models of AD leads in
increased Aβ deposition in the brain.8,11,12 On the
other hand, it was shown that the prevalence of AD
is 60%-70% lower in cholesterol reducing drug
consumers.1,13,14 Thus the risk of AD and Aβ depo-
sition can be altered by factors associated with cho-
lesterol level.
The brain has the highest cholesterol content of
the body (20%) and because of blood brain barrier,
the cholesterol homeostasis is independent from cho-
lesterol level of plasma. Most of the brain cholesterol
is immobilized in the myelin and the remaining is in
neurons, glials and extracellular lipoproteins.6 Every
day, 6-7 mg of excess cholesterol is converted to 24
S-hydroxycholesterol by
transport out of the brain through blood brain barrier
and the gene encoding 24 S-hydroxilase has found to
be associated with the risk of AD.15,16 Cellular choles-
terol is transported out of the cell by a mechanism
called cholesterol efflux in which a membrane-
associated protein, ATP-binding cassette transporter
A1 (ABCA1), transports the cholesterol to high den-
sity lipoproteins (HDL).13 ABCA1 gene is located in
9q31.3 position which has been shown to be linked
with AD by previous studies.17,18 Loss of ABCA1
function causes Tangier’s disease which is character-
ized by absence of HDL, coronary artery disease and
neuropathy. Like APOE, the expression of ABCA1 is
regulated by RXR-LXR heterodimer and there are
some evidences suggesting lack of APOE secretion
from microglia, when ABCA1 is not expressed, so it
seems that ABCA1 can influence APOE and choles-
terol metabolism in the CNS.5,19
In support for a link between AD and cholesterol
metabolism, it has been supposed that ABCA1 poly-
morphisms may influence brain cholesterol homeo-
stasis and risk of developing AD. Association of
ABCA1 polymorphisms and AD has been studied in
different population and there are some positive and
some negative results.7,8,13,15,17,20,21 Raygani et al.
showed that APOE- ε4 allele was a risk factor in de-
veloping AD in Iranian population but the protective
role for APOE- ε2 against AD in this population was
not statistically significant.22 This study determines
the association between sporadic AD and the human
24 S-hydroxilase to
ABCA1 and APOE gene polymorphisms in an Irani-
an population.

Materials and Methods

This case and control study involved 154 AD cases
(mean age=78.55±7.80 years) and 162 control sub-
jects (mean age=77.14±6.95 years) in which AD cas-
es recruited using DSM IV criteria and control sub-
jects were included if they were older than 65 years
old with no known neuropsychiatry disorders. The
informed consent was signed by all of them or their
legal guardians. The criterion for inclusion as a case
was the diagnosis of AD diagnosed by the expert psy-
chiatrist and lacking any neurologic or psychiatric
disorders for the control group. Subjects were exclud-
ed if they had any family history of dementia or neu-
rologic diseases. AD and control subjects were re-
cruited from Alzheimer’s society of Iran and Geriatric
centers of Farzanegan, Mehrvarzan, Shayestegan,
Kahrizak, Hasheminejhad and Rheumatism Center in
Tehran, Iran from 2007 to 2008. The information re-
garding the age, sex, ethnicity, job and education
were asked and recorded and finally 5 ml of peripher-
al blood sample was collected in tubes containing 200
µl of 0.5 M EDTA. Genomic DNA was extracted
from peripheral blood leukocytes using salting-out
method. APOE was genotyped by PCR-RFLP method
which had been described by Wenham et al.23 DNA
was amplified by polymerase chain reaction (PCR)
using forward primer: 5´-TCC AAG GAG CTG CAG
GCG GCG CA-3´; and Reverse primer: 5´-ACA
GAA TCC GCC CCG GCC TGG TAC ACT GCC A-
3´. The 227 bp PCR products were digested by Hha I
(10 U/µl, Fermentas) and loaded on a 12% poly-
acrylamide gel for electrophoresis; finally the gels
were stained using silver staining method.
To genotype ABCA1 in AD cases and control sub-
jects, a part of exon 7 of ABCA1 was amplified by
polymerase chain reaction (PCR) using forward pri-
mer: 5´-CCT GTC ATT GTG CCT TGT G -3´; and
reverse primer: 5´-GGA TTG GCT TCA GGA TGT
C -3´. The 372 bp PCR product was digested by Sty I
(10 U/µl, Fermentas) and loaded on an 8% poly-
acrylamide gel for electrophoresis; finally the gels
were stained by silver staining method.
APOE and ABCA1 alleles and genotypes fre-
quencies were analyzed through logistic regression,
χ2 or Fisher’s Exact tests. Statistical significance
was assumed when p value was less than 0.05. The
statistical analysis and the odd ratios (OR) were de-

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258
termined using SPSS software (versian13, Chicago,
IL, USA) and free online epidemiological software
of Open Epi (2.2.1).

Results

Distribution of age, sex, jobs, educational level and
genetic background was almost the same in both
groups, so there was no need to use any method for
adjustment of cases and controls (Table 1). The mean
age and number of females were slightly higher in
patients compared to control subjects. Our data
showed that the highest frequency of AD was ob-
served in housewives and the lowest was among
farmers. People with academic education had the
lowest frequency among patients and illiterate indi-
viduals had the most frequency. The samples were
consisted of 5 Iranian genetic backgrounds in which
Fars was the most common population.
The frequencies of APOE genotypes and alleles in
AD cases and control subjects were shown in Table 2.
The frequency of ε2ε2 genotype in control subjects
was lower than that in AD cases but it was not signif-
icant (p=0.444). The distribution of ε2ε3 genotype
was not significantly different in both groups (13% in
controls versus 5.8% in AD, p=0.128) and OR was
found to be 0.53 (95%CI=0.23-1.21). The genotype
frequency of ε3ε3 was higher in control subjects
compared with patients (Reference Group). The ε3ε4
genotype frequency in AD cases was significantly
higher than that in control group (20.8% versus 3.7%,
p=0.001).The distribution of ε2ε4 genotype was the
same in both groups and different distribution of ε4ε4
genotype in the groups was not significant (2% versus 0,
Table 1: Comparison of mean age, sex, jobs, education levels and genetic backgrounds between AD cases and
control subjects.
Parameter
AD patients
(No.=154)
Age 78.55±7.80a
Sex (M/F)b 63/91

Jobs Own business 23.4%
Worker 9.2%
Farmer 3.2%
Employee 8.4%


Education
levels Secondary school 16.2%
Diploma 11.1%
Academic 1.9%

Genetic back-
ground Turk 25.3%
Kurd 3.9%
Lor 0.7%
Gilak and Mazani 9.1%
a Mean±SD, b Male/Female

Table 2: The genotype and allele frequencies were compared between AD cases and control subjects.
Genotype/Allele Alzheimer
No.=154 No.=162
Genotype
ε3ε3 69.5% 82.1%
ε2ε2 1.3% 0.6%
ε2ε3 5.8% 13%
ε2ε4 0.6% 0.6%
ε3ε4 20.8% 3.7%
ε4ε4 2% 0
Allele
ε3 82.8% 90.1%
ε2 4.5% 8%
ε4 12.7% 1.9%
a Reference Group.

Control subjects
(No.=162)
77.14±6.95
63/99
56.2%
21.0%
8.6%
3.1%
11.1%

43.2%
29.6%
12.3%
9.3%
5.6%

63.6%
25.3%
1.8%
2.5%
6.8%
P value
0.091
0.714


0.938
Housewife 55.8%
Illiterate
Primary school
41.6%
29.2%


0.427
Fars 61.0%

0.490
ControlP valueOdds Ratio
Rf a
0.444
0.128
0.439
0.001
0.182
2.48 (0.22-27.8)
0.53 (0.23-1.21)
1.24 (0.08-20.1)
6.52 (2.63-16.17)
undefined
Rf a
0.243
0.001
0.67 (0.34-1.32)
7.44 (3.1-17.9)

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259
p=0.182).
The APOE-ε4 allele frequency was significantly
higher in AD cases compared with control subjects
(12.7% versus 1.9%, p=0.001). Comparing allele fre-
quency in APOE- ε4 allele carriers with non-carriers,
OR was found to be 6.52 (95%CI=2.63-16.17). The
frequency of APOE-ε3 allele in patients was lower
than that in control group (Reference Group). Despite
of higher APOE-ε2 allele frequency in AD cases
compared with control subjects, this difference was
not statistically significant (p=0.243 and OR=0.67,
95%CI=0.34-1.32) (Table 2).
Table 3 shows APOE genotype and allele frequen-
cies distributed by sex groups. ε2ε3 genotype fre-
quency in control subjects was higher than AD sub-
jects in men and women group (p>0.05). The geno-
type frequency of ε3ε4 in AD cases was higher than
control subjects in both male and female groups but it
was significant just in female group (p=0.001). The
frequency of APOE-ε4 allele in patients was signifi-
cantly higher than control subjects in both males and
females with different OR [males: p=0.002, OR=8.3
(1.86-37); females: p=0.001, OR=5.59 (2.07-15.05)].
The genotypes and alleles of ABCA1 gene was
compared in two groups of AD cases and controls. As
it has been summarized in Table 4, the GG genotype
frequency was not different between AD cases and
controls (Reference Group). Comparing GA and AA
genotype frequencies, there was no significant differ-
ence between AD cases and control subjects (GA:
p=0.451 and AA: p=0.696). No significant difference
was observed between allele frequencies in cases and
controls (p=0.592). Examining data stratified by gen-
der, neither female AD cases nor male AD cases
showed significant genotype and allele frequencies
compared with female and male controls.
Furthermore, stratification of data by ε4 allele of
APOE, which had been genotyped for the AD cases
and control subjects in the previous study, did not
change the results. ABCA1 genotypes and alleles of
AD and control subjects were compared between ε4
carriers and ε4 non-carriers (Table 5). Distribution of
GG, GA and AA genotypes were not significantly
different between AD cases and control subjects of ε4
carriers and non-ε4 carriers.

Discussion

According to this study, APOE-ε4 allele is a risk
factor for developing late onset AD in Iranian popula-
tion like many other populations.24-29 Although ε2ε3
genotype seems to play a protective role against AD
but the protective role of APOE- ε2 allele has not
Table 3: APOE genotype and allele frequencies distributed by sex.
Genotype/Allele Female
ApoE genotypes
ε3/ε3 Rf a
ε2/ε3 P=0.522, OR=0.663 (0.22-1.75)
ε3/ε4 P=0.001, OR=7.86 (2.58-23.9)
ε4/ε4 No data
APOE alleles
ε3 Rf a
ε4 P=0.001, OR=5.59 (2.07-15.05)
ε2 P=0.157, OR=0.46 (0.17-1.19)
a Reference Group

Table 4: Genotypes and allele frequencies in AD cases and control subjects by sex.
Total

Genotype (No.=154) (No.=162)
GG 33.1% 37% Rfa
GA 48.7% 45.1% 0.451
AA 18.2% 17.9% 0.696
Allele
G 57.5% 59.6% 0.592
A 42.5% 40.4%
a Reference Group
Male

Rfa
P=0.104, OR=0.23 (0.05-1.13)
P=0.080, OR=4.7 (0.96-22.8)
P=0.319, OR= undefined

Rfa
P=0.002, OR=8.3 (1.86-37)
P=0.878, OR=0.8 (0.29-2.24)
Female
AD
(No.=91)
9.6%
0.4%
16.4%
Male
AD
(No.=63)
23.8%
55.6%
0.6%
AD Control P valueControl
(No.=99)
39.4%
40.4%
20.2%
P valueControl
(No.=63)
33.3%
52.4%
14.3%

59.5%
40.5%
P value
Rf
0.803
0.487
Rf
0.341
0.197
59.6%
40.4%
61.5%
38.5%
0.699 51.6%
48.4%
0.205

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Khorram Khorshid et al.

Table 5: ABCA1 genotypes and alleles distribution in APOE ε4 carriers and APOE ε4 non-carriers.
APOE ε4 carriers
Genotype
Frequency (No.=7) (No.=36)
GG 42.9% 25%
GA 42.9% 58.3%
AA 14.2% 16.7%
Allele Frequency
G 64.3% 54.2%
A 35.7% 45.8%
a Reference Group, b Fischer exact test p value
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260
demonstrated in this study and it may be proved by a
larger sample size.
The risk of developing AD in individuals with
ε2ε3 genotype is about 0.53 (95%CI=0.23-1.21)
compared with individuals without this genotype so
ε2ε3 genotype seems to be protective against AD
whereas protective role of ε2 allele has not demon-
strated in Iranian population yet. APOE-ε4 allele car-
riers develops AD, 6.5 times more than non-carriers
(6.52, 95%CI=2.63-16.17). This allele’s risk seems
different in males and females. Different OR for ε4
allele in men and women indicates that risk of AD in
male APOE-ε4 allele carriers (OR=8.421, CI=1.894-
37.44) is higher than female carriers (OR=5.846,
CI=2.173-15.73), so it seems that despite the age-
dependant and dosage-dependent manner of this al-
lele which were investigated in Iranian population by
Raygani et al.,22 it may act in a sex-dependent way as
well. As three patients were observed with ε4ε4 geno-
type, it was not possible to assess the dosage-
dependent action of ε4 allele in this study.
As the study groups were similar based on poten-
tial confounders (age, sex, genetic background, job
and education), it can be assumed that the results are
mainly unbiased. There was no reliable history or ev-
idence for the time of AD onset, so we couldn't eval-
uate the effect of different genotypes or alleles on the
age of onset in the AD subjects. In an autopsy-based
study, the frequency of ε4 allele and ε4ε4, ε 3ε4 geno-
types were 40%, 16.5% and 43.2% in AD patients
and 16%, 2.2 and 20.9% in the control group.30 In a
group of African Americans AD patients, a signifi-
cantly increased risk of AD was associated with two
ε4 alleles or one ε4 allele when compared to ε 3ε3
genotype.31 In our study, the frequencies of ε4 allele
and ε4ε4, ε3ε4 genotypes were lower than results of
Raygani et al.,22 but the proportion of them was the
same and the results of two studies in Iranian popula-
tion are consistent. No significant association was
found between ε2 allele or related genotypes and AD
but it sounds to work as protective factor for AD;
however this finding, should be confirmed in further
studies with a larger sample size .
In initial studies, it had been shown that ABCA1
expression can affect the level of APOE expression.
Increased amyloid deposition in ABCA1 null mice
and position of this gene on chromosome 9, which
was linked to AD, were another evidence to confirm
ABCA1 gene as a target gene for AD association
studies.3,5 In 2003, Wollmer reported that A allele of
ABCA1 causes 1.7 years delay at onset of AD and A
allele carriers had 33% lower cholesterol in cerebro-
spinal fluid, than non-carriers of this allele.15 Signif-
icant increased frequency of A allele in control sub-
jects compared with AD cases in Chinese population
was reported by Wang et al. in 2007, while Sunder
et al. reported that AD incidence is 1.5 times more
frequent in A allele female carriers compared with
female non-carriers in white American popula-
tion.8,13
In this study, ABCA1 and its association with AD
was studied in Iranian population for the first time.
No association was observed between genotypes and
alleles of ABCA1and AD. In 2006, Shibata et al. re-
ported that ABCA1-A allele frequency was signifi-
cantly higher in APOE-ε4 carriers compared with ε4
non-carriers.7 Therefore it was decided to investigate
about this finding, but stratification by gender and
APOE-ε4 allele did not change the result and there
was no association between genotypes and alleles of
ABCA1 and the risk of AD among APOE-ε4 carriers
and non-carriers.

Acknowledgment

We express our sincere thanks and gratitude to all Alz-
heimer's and control persons or their families for their
kindly participation in this study. We also thank Iran
Alzheimer Association for their sincere collaborations.

APOE ε4 non-carriers
Controls
(No.=155)
36.8%
45.2%
18%
Controls Patients P valuePatients
(No.=118)
35.6%
45.8%
18.6%
P value
Rfa
0.618b
1b
Rf
0.866
0.854
0.485
64.3%
35.7%
54.2%
45.8%
0.836