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Association of the apolipoprotein A-IV 360 glnhis polymorphism with cerebrovascular disease obesity and depression in a Brazilian elderly population.

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merican Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 135B:65 –68 (2005)
Association of the Apolipoprotein A-IV: 360 Gln/His
Polymorphism With Cerebrovascular Disease, Obesity,
and Depression in a Brazilian Elderly Population
T.F. Ejchel,1 L.M.Q. Araújo,1,2 L.R. Ramos,2 M.S. Cendoroglo,2 and Marı́lia de Arruda Cardoso Smith1*
1
Disciplina de Genética, Departamento de Morfologia, Universidade Federal de São Paulo, Escola Paulista de Medicina,
São Paulo, SP, Brazil
2
Disciplina de Geriatria, Departamento de Clı´nica Médica, Universidade Federal de São Paulo, Escola Paulista de Medicina,
São Paulo, SP, Brazil
The identification of genetic polymorphisms as
risk factors for complex diseases can be relevant
for their prevention, diagnosis, and prognosis.
The apolipoprotein A-IV: 360 Gln/His polymorphism was investigated in 383 elderly individuals,
who were participants of a longitudinal study
commenced in 1991. The major morbidities that
affect elderly people, such as cardiovascular diseases, diabetes, low cognitive function, depression, and obesity, were extensively investigated.
DNA was isolated from blood cells, amplified by
PCR, and digested with Fnu4HI. In this population the frequency of the His allele was 0.056
and the genotypes were distributed according
to Hardy–Weinberg equilibrium. Logistic regression analysis showed a significant association
between the presence of His allele and cerebrovascular disease and/or transitory ischemic
attack (odds ratio) (OR ¼ 3.070, P ¼ 0.027), obesity
(OR ¼ 2.241, P ¼ 0.047), and depression (OR ¼ 2.879,
P ¼ 0.005). This study indicates that the presence
of the rare allele in elderly people can play a
significant role in the occurrence of multifactorial
diseases. This is the first study analyzing this
polymorphism in elderly people in Brazil. More
studies should be encouraged to elucidate the
mechanisms involved in these diseases.
ß 2005 Wiley-Liss, Inc.
KEY WORDS:
elderly population; APOA-IV: 360
polymorphism; morbidities; apolipoprotein A-IV
INTRODUCTION
Genetic polymorphism studies are very important to identify
the difference between alleles and to search for their associa-
Grant sponsor: Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP); Grant sponsor: Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (CNPq).
*Correspondence to: Marı́lia de Arruda Cardoso Smith, Ph.D.,
Disciplina de Genética, Departamento de Morfologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua
Botucatu, 740, SP 04023-900, São Paulo, Brazil.
E-mail: macsmith.morf@epm.br
Received 9 November 2004; Accepted 23 December 2004
DOI 10.1002/ajmg.b.30175
ß 2005 Wiley-Liss, Inc.
tion with longevity and the most common diseases affecting
elderly people, such as cardiovascular disease, diabetes, and
low cognitive function. The selection of favorable genotypes
and the low frequency of risk alleles can lead to a successful
aging.
The genetic factors that participate in lipid metabolism
could be the leading key for the susceptibility or resistance to
atherogenesis. It has been proposed that the isoproteins of the
apolipoprotein A-IV gene (APOA-IV) may play different roles
in lipids modulation [Eichner et al., 1989].
APOA-IV polymorphisms have been detected by isoeletric
focusing followed by immunoblotting [Lohse et al., 1990].
DNA sequence analyses have shown that a single nucleotide
substitution of a G to a T in isoform 2 converts glutamine to
histidine in position 360 of the mature protein. The APOA-IV
His polymorphism adds a positive charge in relation to the
APOA-IV-Gln isoprotein, generating a more basic isoprotein,
which is more hydrophobic and has an altered conformation.
These changes may lead to more efficient protein activation
and better lipoprotein binding.
Despite the fact that the precise function of APOA-IV is not
well known, this protein seems to participate in dietary fat
absorption [Ordovas et al., 1989; Weinberg et al., 1990], in
the transport of triacilglycerol [Otha et al., 1985] and in reverse transport of cholesterol [Duverger et al., 1996]. APOA-IV
also appears to modulate the activity of some enzymes, such
as lecithin cholesterol acyltransferase (LCAT) [Steinmetz
and Utermann, 1985] and cholesteryl ester transfer protein
(CETP) [Gambert et al., 1988]. APOA-IV is also found in
cerebrospinal fluid (CSF) [Koch et al., 2001].
The APOA-IV genotype was associated with changes in
HDL-C and LDL-C levels reflecting fat content changes
in balanced diets, with individuals carrying the His allele
exhibiting a favorable response [Clifton et al., 1997]. In
individuals with type II diabetes, the APOA-IV Gln/His
genotype was associated with a rise in risk of myocardial
infarction, and obesity exacerbated this risk [Rewers et al.,
1994].
A Brazilian study conducted in a general population indicated that three different variants of APOA-IV gene polymorphisms, including the 360 His variant, were associated
with obesity-related traits [Fiengenbaum and Hutz, 2003].
In studies comparing centenarians and adults, and in those
examining Alzheimer disease patients, elderly and general
population, it has been suggested that several variants of
APOA-IV polymorphisms observed in elderly people could be
associated with healthier longevity [Merched et al., 1998; Pepe
et al., 1998].
In our study, we derived the frequency of APOA-IV: 360 Gln/
His polymorphism in an elderly population of a community in
São Paulo. We investigated the association of this polymorphism and major morbidities affecting elderly people.
66
Ejchel et al.
MATERIALS AND METHODS
Population Study
The population that was studied consisted of participants
from the Elderly Longitudinal Study [Ramos et al., 1998]. This
study began in 1991 and originally involved 1,667 people over
the age of 60 living in a community of São Paulo, Brazil. These
subjects were clinically evaluated every 2 years and a sub
sample of 383 individuals was invited to participate in our
study during the 4th wave (2000–2001) of that study.
We informed the participants about the study protocol
and collected information about their previous medical conditions. They were evaluated by a physician and blood samples
were collected for laboratory procedures. The Research Ethics
Committee of UNIFESP approved this study and participants gave us informed consent according to the Helsinki
Declaration.
and Vernier [1991]. In brief, a 127 bp sequence containing the
polymorphic site was amplified by polymerase chain reaction
(PCR) using the primers HT3 (50 -CACCTGCTCCTGCTACTGCTCC-30 ) and HT5 (50 -CCTGAGGGACAAGGTCAACTC-30 ).
After amplification, digestion of PCR products with Fnu4HI
was performed for 3 hr at 378C. Restriction fragment length
polymorphism products were resolved on a 12.5% acrilamide
gel. After electrophoresis, the gels were stained with silver for
fragment visualization.
Gene Frequency
Genotype and allele frequencies were calculated by allele
counting as described by Emery [1986]. Chi-square test was
applied to verify the Hardy–Weinberg equilibrium.
Statistical Analyses
Evaluation of Previous Morbidities
*
*
*
*
*
*
Cardiovascular disease: individuals who reported previous
myocardial infarction (MI), coronary heart disease (CHD),
transitory ischemic attack (TIA), or cerebrovascular disease
(CVD), or were taking specific medication prescribed by
physicians.
Hypertension: people using anti-hypertensive drugs or
with systolic blood pressure above 140 or diastolic above
95 mmHg [adapted from Chobanian et al., 2003; WHO/ISH,
2003].
Type II diabetes: subjects taking insulin or oral medication and those that had fasting glucose equal to or above
126 mg/dl [adapted from The expert committee on the diagnosis and classification of diabetes mellitus, 1997; DECODE
study group, 1999].
Obesity: characterized by a body mass index (BMI) above
27 kg/m2, as specified for persons 65 years of age and older
[adapted from Rolland-Cachera et al., 1991; Lipschitz, 1994;
Kyle et al., 2001].
Low cognitive function: individuals with a mini-mental
state examination (MMSE) score below 24. Cognitive function was evaluated by the MMSE screening process, which
was adapted to the Brazilian population taking literacy into
account [Bertolucci et al., 1994a,b]. A score of less than
24 (out of 30) has 80%–90% sensitivity and 80% specificity
for discriminating low cognition level from normal subjects
[Tombaugh and McIntyre, 1992; Third report of NCEP,
2001].
Depression: characterized by score above 5 in a Brazilian
validity version of the Older American’s Resources and
Services (OARS) [adapted from Blay et al., 1988].
Although some studies showed that self-reported past
history and medical records usually are concordant for selected
medical conditions in the elderly [Bush et al., 1989], past
history was only accepted when there was also evidence in
physical exams, ECG, CT-scan, or physician’s report.
DNA Extraction
Total blood was collected in tubes containing 0.1% EDTA and
genomic DNA was isolated using modified procedures from
Lahiri and Nurnberger [1991].
Genotyping
The APOA-IV: 360 Gln/His polymorphism was analyzed by
procedures modified from those of Tenkanen [1989] and Hixson
Statistical analyses were performed using the SPSS 10.0
software. Allele frequencies of APOA-IV polymorphism were
estimated by the gene-counting method. To test the effect of the
allele in each disease we applied logistic regression using sex
and age as co-variables in the models (confidence interval of
95% and significant when P < 0.05).
RESULTS
The frequency of His allele in this population was 0.056.
Genotype distributions were according to Hardy–Weinberg
equilibrium. We found 39 heterozygous and 2 homozygous
individuals for this allele.
The characteristics of the population are described in Table I.
The genotypes were dichotomized for the presence of the His
allele for statistical reasons. We found that the presence of His
allele was associated with a higher frequency of CVD/TIA,
depression, and obesity (P < 0.05). The other diseases listed
in Table I did not show significant differences between the
groups.
Logistic regression analysis for each disease in relation to
the presence or absence of His allele, gender, and age as covariables were performed. Diseases with significant association are described in Table II. We noticed that His allele
presence increased the risk of CVD/TIA by approximately
three times. His allele presence also doubled the risk of obesity.
Obesity is mostly related to female sex and aging. Depression
was found to be almost three times more frequent amongst His
carriers. The other diseases analyzed did not show increased
risk due to the presence or absence of this allele.
TABLE I. Population Characteristics According to the
Polymorphism of Apolipoprotein A-IV:360 Gln/His
(Dichotomized for the Presence of the His Allele)
N (female/male)
Age in years SD
CVD/TIA
MI/CHD
Hypertension
Diabetes
Low cognitive function
Depression
Obesity
360 Gln/Gln
360 His/þ
P value
342 (234/108)
79.8 5.33
6.77%
15.81%
84.46%
64.22%
8.90%
17.98%
39.43%
41 (28/13)
79.7 5.28
17.14%
17.14%
78.05%
56.10%
0%
37.84%
58.62%
0.987
0.926
0.025*
0.781
0.293
0.308
0.052
0.005*
0.046*
CVD, cerebrovascular disease; TIA, transitory ischemic attack; MI,
myocardial infarction; CHD, coronary heart disease; SD, standard deviation.
*Significant difference (P < 0.05).
APOA-IV: 360 Polymorphism and Morbidities in an Elderly Population
TABLE II. Results of the Logistic Regression Analysis
for Diseases With Significant Association With the His
Allele in the Population Studied (Dichotomized for
the Presence of the His Allele)
Disease
CVD/TIA
Obesity
Depression
Variables
P
OR
Presence/absence of His Allele
Gender (female/male)
Age
Presence/absence of His Allele
Gender (female/male)
Age
Presence/absence of His Allele
Gender (female/male)
Age
0.027*
0.448
0.300
0.047*
0.012*
0.010*
0.005*
0.065
0.366
3.070
0.727
1.040
2.241
1.936
0.940
2.879
1.767
1.023
CVD, cerebrovascular disease; TIA, transitory ischemic attack; OR, odds
ratio.
*Significant difference (P < 0.05, a ¼ 0.05), confidence interval 95%.
DISCUSSION
We studied the APOA-IV: 360 Gln/His polymorphisms in a
Brazilian elderly population ranging from 66 to 97 years old,
investigating the frequency of each allele and associations with
some diseases. This is the first study examining these polymorphisms and their association with the most common
diseases affecting elderly people in Brazil. The majority of
studies are carried out with a wider age range, but it is important to emphasize that studies conducted in an older population can demonstrate specific issues concerning the aging
process.
In our population the His allele frequency was 0.056 and the
genotypes distribution were in accordance to Hardy–Weinberg
equilibrium. We neither observed significant differences related to age and sex between the groups nor did we observe that
APOA-IV Gln/His polymorphism is associated with myocardial infarction and/or coronary heart disease, hypertension,
diabetes, or low cognitive function. It should be noted that
there was no His carrier subject with low cognitive function in
our population, which could be due to the fact that the number
of His carriers is small.
We found an association between His allele presence and
cerebrovascular disease and/or transitory ischemic attack,
obesity, and depression in our population. The mechanisms
involved in these diseases could be related to differences in
fatty acid metabolism in His carriers. Impaired fatty acid
metabolism is involved in the occurrence of many diseases,
such as depression and cardiovascular disease [Horrobin and
Bennett, 1999]. The APOA-IV: 360 His isoform could have a
role in brain metabolism that induces susceptibility to these
diseases.
Data in literature shows that APOA-IV has a role in food
intake regulation in response to the presence of dietary fat as a
satiety signal in the CNS [Tso et al., 2001]. APOA-IV Gln and
His alleles seem to act in different manners in this response.
Intervention studies have demonstrated that homozygous
subjects for Gln have a better response than His carriers to
weight loss treatment as a method of reducing cardiovascular
disease risk, and this response is better still in diabetics
[Heilbronn et al., 2000].
The lipoprotein metabolism in the brain is not well known
yet, but synthesis, remodeling, and redistribution of lipids in
the brain should occur in this compartment since intact
particles of lipoprotein cannot cross the blood–brain barrier
[Koch et al., 2001]. It is not known in which form HDL precursors are synthesized in the brain and how remodeling is
done in the CSF. Some enzymes that have their activity
modulated by APOA-IV, such as LCAT and CETP, mediate
67
HDL remodeling in the blood, but in the brain these pathways
are still obscure.
The physiological role of LCAT in vivo was determined in
LCAT-deficient individuals that accumulated cholesterol in
peripheral tissues and developed atherosclerosis prematurely
and showed CNS alterations [Warden et al., 1989]. Some
authors reported that LCAT is active in the brain and is colocalized with APOA-IV [Demeester et al., 2000]. APOA-IV and
APOA-I are the most efficient activators of LCAT in plasma
[Peelman et al., 1998]. The APOA-IV His isoform could activate
LCAT in the brain in a way that causes a cumulative impairment of fatty acid metabolism leading to a higher prevalence of
some diseases in elderly people, such as those seen in our study.
There is a decrease of desaturase activity in the brain with
aging [Bourre et al., 1990]. Desaturases are enzymes that
participate in dietary fatty acid metabolism and regulate the
formation of important mood modulating factors. Anomalies
described for desaturases could have a fundamental role in the
development of many multifactorial diseases, such as depression and cardiovascular disease [Horrobin and Bennett, 1999].
Aging changes combined with the presence of risk alleles in
some individuals could lead to greater risk for these diseases.
Our study showed that the presence of the His allele in
elderly people is associated with CVD/TIA, obesity, and depression. A better understanding of this polymorphism’s role
in multifactorial diseases could help in their prevention,
diagnosis, and prognosis. The mechanisms involved in these
associations have to be better elucidated.
ACKNOWLEDGMENTS
The authors are grateful to Prof. Dr. Clovis Araújo Peres and
his staff for statistical analysis assistance, to the Brazilian
Elderly Longitudinal Study team, to Bianca Borsatto–Galera
for technical support.
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