Apolipoprotein E Genotype

s Associated with AlzheimerDisease and Concomitant Stroke

Najiba 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: fnajiba@yahoo.fr.

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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