Apolipoprotein E Genotype
s Associated with AlzheimerDisease and Concomitant StrokeNajiba Fekih-Mrissa, PhD,*† Sarra Klai, PhD,*‡ Meriem Mrad, PhD,*‡
Malek Mansour, MD,xjj Jamel Zaouali, MD,xjj Nasreddine Gritli, PhD,*{and Ridha Mrissa, MDxjj
From the *Laborato
d’H�ematologie, Hopital
Montfleury, Tunis; †Aca
‡Facult�e des Sciences d
Manar; xService de Neur
tion de Tunis, Montfleury
versit�e de Tunis El M
Universit�e de Monastir, M
Received February 9, 20
June 8, 2013.
Address corresponden
de Biologie Mol�eculaire
Principal d’Instruction d
E-mail: [email protected].
1052-3057/$ - see front
� 2013 by National Str
http://dx.doi.org/10.1
Journal of Stroke and C
Background: The 34 allele of the apolipoprotein E (APOE) gene is awell-characterized
genetic risk factor for Alzheimer disease (AD). The association between stroke and
a higher risk for AD has also been reported. Our study sought to determine the
relationship between the APOE gene and AD and the comorbid risk of stroke.
Methods: The subjects of this study consisted of 48 patientswith AD and 48members
of a control group. All subjects were genotyped for APOE. Results: The results
clearly show a significant increased risk of AD in carriers of the APOE ε3/ε4 geno-
type (P 5 .003, odds ratio [OR] 5 4.1) or ε4 allele (P 5 .001, OR 5 4.2). The risk for
stroke in AD patients was also increased for carriers of the APOE ε3/ε4 genotype
(P 5 .02, OR 5 9.0) and for carriers of the APOE ε4 allele (P 5 .004, OR 5 5.5).
Conclusions: The present study is the first to establish a relationship between
APOE ε4 and concomitant AD and stroke in the Tunisian population. Key Words:
Apolipoprotein E—Alzheimer disease—stroke—risk factor—genotyping.
� 2013 by National Stroke Association
Introduction
Alzheimer disease (AD) is the most common cause of
dementia in elderly patients. It is a progressive neurode-
generative cerebral disease, characterized by a progres-
sive loss of memory and other cognitive functions.
Deposits of proteins in senile plaques and neurofibrillary
ire de Biologie Mol�eculaire, Service
Militaire Principal d’Instruction de Tunis,
d�emie Militaire Fondouk J�edid, Nabeul;
e Tunis, Universit�e de Tunis El Manar, El
ologie, Hopital Militaire Principal d’Instruc-
, Tunis; jjFacult�e deM�edecine de Tunis, Uni-
anar, Tunis; and {Facult�e de Pharmacie,
onastir, Tunisie.
13; revision receivedMay 20, 2013; accepted
ce to Najiba Fekih-Mrissa, PhD, Laboratoire
, Service d’H�ematologie, Hopital Militaire
e Tunis 1008 Montfleury, Tunis, Tunisie.
matter
oke Association
016/j.jstrokecerebrovasdis.2013.06.018
erebrovascular Diseases, Vol. -, No. - (---
tangles, leading to neural dysfunction and the develop-
ment of dementia, have been found in the brains of AD
patients.1
The gene encoding apolipoprotein E (ApoE) has been
suggested to be a risk factor for the disease.2 It is ex-
pressed within the human brain in 3 distinct isoforms,
termed ApoE2, ApoE3, and ApoE4, and they are encoded
by the APOE- 32, 33, and 34 alleles, respectively. A number
of studies have investigated the association of APOE
genetic polymorphisms with AD risk. In particular, the
APOE 34 allele has been identified as a genetic susceptibil-
ity factor for AD in diverse ethnic populations, whereas
the ε2 type is believed to offer protection against AD.2-5
Moreover, an association between stroke and a higher
risk for AD has also been reported.6 Some studies have
previously shown that vascular factors affect the rate of
dementia progression after AD diagnosis.7-9
The present study examined the genetic polymor-
phisms of the APOE gene ( 32, 33, and 34) in a group of
AD individuals and controls. The purpose was to deter-
mine the nature of the relationship between the APOE
gene and the risk of AD and, possibly, concomitant stroke
in AD patients in Tunisian subjects.
), 2013: pp 1-5 1
N. FEKIH-MRISSA ET AL.2
Patients and Methods
Study Population
We studied 48 patients with AD (16 female and 32
male) recruited from the Department of Neurology at
the Military Hospital of Tunis. The mean age was
75.45 years 6 7.14 (SD). The diagnoses of probable AD
were based on the National Institute of Neurological and
Communicative Disorders and the Stroke and Alzheimer’s
disease and Related Disorders Association (NINCDS-
ADRDA) clinical diagnostic criteria (Mckhann et al10 and
Diagnostic and Statistical Manual of Mental Disorders, Fourth
Edition). All participants underwent a complete clinical in-
vestigation, includingmedical history, neurologic and neu-
ropsychological examinations, screening laboratory tests,
and neuroimaging consisting of computed tomography
scan and/or magnetic resonance imaging (MRI). Neuroi-
magingwas used in a systematic manner in the differential
diagnosis of AD as to exclude other possible causes of
dementia such as multi-infarct dementia. In particular,
MRI was used to assess the degree of cortical atrophy
and hippocampal volume in patients with AD as com-
pared with controls. Six of 48 (12.5%) of patients reported
family members with symptoms consistent with dementia
although no family member was available for a rigorous
examination and determination of disease.
Additionally, the control group consisted of 48 age- and
gender-matched subjects with diverse Tunisian origin
similar to that of the patients and chosen based on their
medical history and physical examination. The mean
age was 72.43 years 6 6.64 (SD). Their cognitive function
was assessed using the Mini-Mental State Examination,11
and they did not exhibit signs of dementia. No family his-
tory of AD or dementia was reported in controls. The
study protocol was approved by our ethics committee,
and all subjects of the study or their guardians had given
their fully informed consent.
Laboratory Methods
Genomic DNA was extracted from peripheral blood
leukocytes with DNA extraction kit (QIAmp blood kit;
Qiagen GmbH, Hilden, Germany) according to the man-
ufacturer’s protocol. Amultiplex amplification with bioti-
nylated primers was used to amplify the genomic DNA.
The polymerase chain reaction products then underwent
a reverse hybridization by the use of the GenoType APOE
test that is based on the DNA.STRIP technology (Hain
Lifescience GmbH, Nehren, Germany) and permits the
molecular genetic characterization of the APOE isoforms
32, 33, and 34.
Statistical Analysis
All analyses related to the case–control study were
performed using the Statistical Package for the Social Sci-
ences v.16 (SPSS Inc., Chicago, IL). Data on quantitative
characteristics are expressed as means 6 SD. Differences
between cases and controls were evaluated by using the
chi-square test for qualitative variables and the Student
t test for quantitative variables. In addition, the odds ratio
(OR) and 95% confidence intervals were calculated. Prob-
ability values less than .05 were considered statistically
significant.
Results
The Alzheimer series comprised 48 patients. The aver-
age age of AD subjects (75.45 6 7.14 years) was slightly
higher than that of the control (72.43 6 6.64 years)
(mean difference 5 3 years, 95% confidence interval 5
.22-5.8, P5 .035). Among patients, 16 (33.3%) were female,
10 (20.8%) were smokers, 6 (12.5%) were diabetic, 8
(16.6%) had hypertension, and 6 (12.5%) had a history
of stroke. None of these factors constituted a statistically
significant risk for AD in this study. Clinical variables of
the study subjects and controls are tabulated in Table 1.
The genotype frequencies and allele frequencies were
calculated for patients and controls in Table 2. There
were significant differences in ε3/ε4 genotype frequen-
cies between AD cases and controls (41.6% versus
14.5%; P 5 .003, OR 5 4.1); however, the ε3/ε3 genotype
was more frequent in controls than in patients (68.7%
versus 37.5%; P5 .004, OR5 .27). The ε2/ε3 genotype ap-
peared with equal frequency in both cases and controls
(16.7%), and the ε2/ε2 genotype was absent in both
groups. The genotype ε4/ε4 was not common in patients
and was absent in controls.
The ε4 allele presented with higher frequency in
patients than controls (25% versus 7.3%). Conversely, the
ε3 allelewas seenmore frequently in controls than in cases
(84.3% versus 66.6%) and the distribution of the ε2 allele
was the same in both groups (8.3%). These results clearly
show a significant increased risk of AD in carriers of the
APOE ε3/ε4 genotype (P 5 .003, OR 5 4.1) and ε4 allele
(P 5 .001, OR 5 4.2), whereas the ε3/ε4 genotype and ε3
allele seemed to confer protection against AD (P 5 .004,
OR5 .37). TheAPOE ε2 allele was present equally among
patients and controls and, therefore, had no significance.
The patient group was subsequently examined for as-
sociations of genotype with incidence of stroke. Stroke
was diagnosed through a neurologic exam that noted
the sudden onset of clinical signs of focal neurologic def-
icit. Cerebral infarct was confirmed with computed to-
mography and/or MRI. Overall, 6 AD patients had
a stroke compared with 42 who had no indication of
stroke. In all, 5 of 6 AD/stroke patients (83.3%) were
ε3/ε4 carriers versus 15 of 42 (35.7%) patients who did
not develop a stroke (P 5 .02, OR 5 9.0). Furthermore,
the ε3/ε3 genotype was frequent in the group of patients
(42.8%) who did not have any episode of stroke as com-
pared with patients with stroke antecedent, none of
whom were ε3/ε3 carriers. This genotype seems to confer
Table 1. Comparison of clinical variables in Alzheimer patients and controls
Clinical variable Patients (N 5 48), n (%) Controls (N 5 48), n (%) OR (95% CI) P value
Gender
Female 16 (33.3) 12 (25)
Male 32 (66.6) 36 (75)
Mean age 6 SD (y) 75.45 6 7.14 72.43 6 6.64 3.0* (.22-5.8) .035
Tobacco use 10 (20.8) 12 (25) .79 (.30-2.0) .85
Diabetes 6 (12.5) 5 (10.4) 1.2 (.35-4.3) 1.0
Hypertension 8 (16.6) 6 (12.5) 1.4 (.45-4.4) .77
Stroke 6 (12.5) 3 (6.25) 2.1 (.50-9.1) .48
Abbreviations: CI, confidence interval; OR, odds ratio.
*Difference in the mean values.
APOLIPOPROTEIN E GENOTYPES WITH AD AND CONCOMITANT STROKE 3
protection against stroke in AD patients (P 5 .04). The
APOE ε4 allele was shown to be a significant risk factor
for developing stroke in AD patients (P 5 .004,
OR5 5.5). TheAPOE ε3 allele was of borderline statistical
significance as protection from developing stroke in AD
patients (P 5 .05, OR 5 .30; see Table 3).
Discussion
We must recognize several limitations in our study. Al-
though the patients and controls were gender and age
matched and were referred from all regions of Tunisia,
the number of subjects was small and our findings would
benefit from confirmation with a larger cohort. Further-
more, although only an autopsy can definitively confirm
adiagnosis ofAD, the combination of careful history taking,
conscientious examination of clinical features, neuropsy-
chological cognitive tests, and neuroimaging can signifi-
cantly increase the preciseness of clinical diagnosis and
permits a reliable selection of candidates in a study of AD.
Despite the small number, this sample, including pa-
tients and controls, was a good representation of
a cross-section of our population as were referred from
all regions of Tunisia.
Table 2. Distribution of APOE genotypes a
Patients, n (%) Con
Genotypes APOE* (N 5 48) (Nε2/ε3 8 (16.6)
ε3/ε3 (p) 18 (37.5) 3
ε3/ε4 (s) 20 (41.7)
ε4/ε4 2 (4.17)
Alleles APOE (N 5 96) (N
ε2 8 (8.3)
ε3 (p) 64 (66.6) 8
ε4 (s) 24 (25)
Abbreviations: CI, confidence interval; OR, odds ratio; p, protective eff
Significant P values are in bold.
*There were no carriers of the ε2/ε2 or ε2/ε4 genotype.
The well-established association between the APOE ε4
and AD was also demonstrated in this study of Tunisian
patients. Recently, the associations between stroke and
a higher risk for AD have been reported also.6 It has,
therefore, become increasingly relevant to study the rela-
tionship between stroke and APOE alleles in AD.
The results presented in this study show significant dif-
ferences in ε3/ε4 genotype frequencies between patients
and controls. This genotype is significantly associated
with the disease (P 5 .003, OR 5 4.1). However, the
ε3/ε3 genotype is more frequently found in controls
than in patients (68.7% versus 37.5%) and seems to confer
protection in AD (P 5 .004, OR 5 .27).
Our findings clearly demonstrate that the APOE ε4 al-
lele frequency is significantly higher in AD subjects than
in the control group (25% versus 7.3%) in the Tunisian
population. Our data show that APOE ε4 increased the
risk for AD (P 5 .001, OR 5 4.2). This is consistent with
previous studies demonstrating an association between
APOE polymorphisms and AD in Caucasian, Asian, and
other ethnic groups around the world.12,13 In addition,
the APOE ε3 allele frequency is higher in controls
(84.3%) than in the patient group. This allele seems to be
negatively associated with the disease (P 5 .004), and
nd alleles in AD and control subjects
trols, n (%) OR (95% CI) P value
5 48)
8 (16.6) 1.0 (.34-2.9) 1
3 (68.7) .27 (.12-.63) .0047 (14.5) 4.1 (1.6-11) .003
0 .5
5 96)
8 (8.3) 1.0 (.3-2.7) 1
1 (84.3) .37 (.18-.74) .0047 (7.3) 4.2 (1.7-10) .001
ect; s, susceptibility to AD.
Table 3. Association of APOE genotypes with stroke in AD subjects
Patients (N 5 48) OR (95% CI) P value
Genotypes APOE Stroke (n 5 6), n (%) No ATCD of stroke (n 5 42),
n (%)
ε2/ε3 0 8 (19.0) .25
ε3/ε3 (p) 0 18 (42.8) .04ε3/ε4 (s) 5 (83.3) 15 (35.7) 9.0 (.9-84.3) .02ε4/ε4 1 (16.6) 1 (2.37) 8.2 (.44-152.5) .1
Alleles APOE (N 5 12), n (%) (N 5 84), n (%)
ε2 0 8 (9.52) .26
ε3 (p) 5 (41.6) 59 (70.2) .30 (.087-1.0) .05
ε4 (s) 7 (58.3) 17 (20.2) 5.5 (1.55-19.5) .004
Abbreviations: CI, confidence interval; OR, odds ratio; p, protective effect; s, susceptibility to stroke in AD patients.
Significant P values are in bold.
N. FEKIH-MRISSA ET AL.4
this finding is in concordance with another study
conducted in Tunisia.14,15
The frequencies of the ε2, ε3 and ε4 APOE alleles are
highly variable in different populations. Globally, APOE
ε3 is the most frequently occurring allele, constituting
60%-90% of the allelic variation, whereas APOE ε2 is
found in 7%-8% of individuals and APOE ε4 is present
in 14%-16% of the population.12,16,17
The allelic distributions of our control group were sim-
ilar to the frequencies found in previous studies conduct-
ed on Tunisian, French,18 Spanish,19 and Moroccan
populations.20 However, these values are significantly
different from the reported values in southern Italian,21,22
Irish,23 and Finnish populations.24 This difference may be
because of the peculiar ethnical and geographic character-
istics of Tunisia and also to distinct gene–environment in-
teractions.25 A study by Henn et al26 of genetic diversity
in North Africa found that populations retain a unique
signature of early Maghrebi ancestry. Moreover, he con-
cludes that these groups are not homogeneous and dis-
play diverse combinations of several distinct ancestries
(Maghrebi, European, Near Eastern, and western sub-
Saharan Africa.)
We have also described a relationship between stoke
and APOE ε4 carrier status in AD patients. Our results
indicate that stroke occurs in AD patients with higher
frequency for carriers of the ε3/ε4 genotype than for
any other genotype. Moreover, 83.3% of ε3/ε4 carriers
had a stroke compared with only 35.7% of those car-
riers with no stroke antecedent. The APOE ε4 allele
also increases the chances of developing AD. The sig-
nificant frequency of stroke in ε4 carriers of AD pa-
tients indicates that APOE ε4 allele increases both the
susceptibility of AD and the occurrence of stroke in
these subjects (P 5 .004). The presence of vascular
risk factors in AD has been reported previously, but
fewer studies have focused on the interaction between
stroke and the presence of an APOE ε4 allele among
AD patients.
The results of the Rotterdam study showed a link be-
tween vascular events and poor cognitive performance,
regardless of age and education.27
Other studies based on autopsies have reported that the
APOE ε4 allele is associated with both greater AD patho-
logic progression and greater vascular burden such as
small-vessel arteriolosclerosis and other microvascular
changes.28 Recently, some authors had reported interac-
tions between vascular factors and the APOE ε4 allele in
predicting cognitive decline among AD patients.9
Another recent report described interactions between
vascular factors and APOE ε4 carrier status. This suggests
that, among individuals with a history of stroke, progres-
sion of AD is influenced by APOE ε4 carrier status and
varies by time after AD diagnosis.8 Thus, having an
APOE ε4 allele not only leads to more vascular pathology
but also may lead to more Alzheimer pathology.
Conclusion
To our knowledge, this study is the first attempt to in-
vestigate the association of the APOE gene and stroke in
AD patients in the Tunisian population. We showed that
the APOE ε4 allele increases the susceptibility to AD
and the occurrence of stroke in these subjects; however,
future studies in a larger sample will be helpful to better
understand the association of APOE polymorphisms with
AD and the risk of stroke in AD patients of ε4 carriers.
Acknowledgment: We would like to thank Dr Christian
Winchell for his precious help in correcting this article.
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