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Alzheimers disease is not associated with the hypertension genetic risk factors PLA2 or G protein 3 either independently or interactively with apolipoprotein e

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American Journal of Medical Genetics (Neuropsychiatric Genetics) 88:465–468 (1999)
Alzheimers Disease is not Associated With the
Hypertension Genetic Risk Factors PLA2 or G
Protein ␤3, Either Independently or Interactively
With Apolipoprotein E
Terrence Town,1* Daniel Paris,1 Timothy A. Parker,1 Amy Kundtz,1 Jun Tan,1 Ranjan Duara,2
Michael Gold,3 Fiona Crawford,1 and Michael Mullan1
1
The Roskamp Institute, Department of Psychiatry, University of South Florida, Tampa, Florida
Mount Sinai Memory Disorder Clinic, University of Miami Medical School, Miami, Florida
3
Department of Neurology, University of South Florida, Tampa, Florida
2
Growing evidence suggests that hypertension and Alzheimers disease (AD) may share
a common etiology. To evaluate the contribution to AD of genetic factors associated
with hypertension, we genotyped clinic and
community-based AD cases and controls for
polymorphisms within the pancreatic PLA2
gene and the G protein ␤3 subunit gene,
both of which are located on chromosome
12. Our results do not support an independent association between either of these
genes and AD. We further assessed the possibility that either of these genes may interact with the apolipoprotein E gene, a known
risk factor for hypertension and AD, on predicting AD. We were unable to find statistical interaction between either the pancreatic PLA2 or G␤3 genes and the apolipoprotein E gene on risk for AD. These results do
not support a shared genetic etiology between hypertension and AD. Possibly, a
clinical association between these diseases
could be due to pathophysiologic interactions. Am. J. Med. Genet. (Neuropsychiatr.
Genet.) 88:465–468, 1999. © 1999 Wiley-Liss, Inc.
KEY WORDS: Alzheimers disease; hypertension; phospholipase A2; G
protein; chromosome 12
Contract grant sponsor: National Institutes of Health; Contract
grant number: AG12412-02.
*Correspondence to: Terrence Town, Roskamp Institute, University of South Florida, 3515 E. Fletcher Ave., Tampa, FL 33613.
E-mail: ttown@hsc.usf.edu
Received 19 June 1998; Accepted 30 December 1998
© 1999 Wiley-Liss, Inc.
INTRODUCTION
Occurrence of hypertension (HTN) has been suggested to be directly associated with risk for vascular
dementia and Alzheimers disease (AD), possibly
through induction of cerebral infarcts, ischaemic subcortical white matter lesions, and blood-brain barrier
dysfunction [Lis and Gaviria, 1997; Skoog, 1997]. Furthermore, in an animal model of HTN, spontaneously
hypertensive rats were shown to develop cognitive and
learning abnormalities when compared with normotensive rats, suggesting that HTN may contribute to the
cognitive impairment associated with AD [Gattu et al.,
1997]. The ⑀4 allele of the apolipoprotein E gene
(APOE) has been consistently implicated as a risk factor for AD and as a predictor of HTN [Pasquier and
Leys, 1997; Sparks, 1997]. Based on such evidence for
a shared etiology between AD and HTN, we investigated a possible genetic association between AD and
two genes on chromosome 12 previously found to be
associated with HTN: pancreatic PLA2 and G protein
␤3 subunit (G␤3). We further assessed a possible interaction between polymorphisms within either of these
genes and APOE on risk for AD.
MATERIALS AND METHODS
Study Subjects
We genotyped community-based control subjects,
community-based AD patients, and clinic-based AD patients. Community-based AD subjects and controls
were recruited as part of a community-based screen for
dementia in the elderly (age 60 or older) in the Miami
area of Florida. Subjects who met criteria for healthy
elderly (must score >26 on the Mini Mental State Examination [MMSE], achieve >10 accurate recalls of
three words in four separate trials, generate >30 category-specific words in 3 min, and achieve a normal
score on the Hamilton depression scale) were included
in the control group. Subjects who did not meet one or
466
Town et al.
more of these criteria were asked to undergo further
examination at the Mount Sinai Memory Disorder
Clinic. Individuals who returned for a complete examination and were diagnosed normal were also included
in the control set; those diagnosed with AD were included in the community-based AD sample; and those
with other dementia diagnoses or pending further investigation were not included in this study. Clinicbased AD cases were drawn from a sample in which
patients were recruited from over 40 clinical centers
throughout the United States. All AD subjects met criteria for probable or possible AD (according to National
Institute of Neurological and Communicative Disorders and Stroke-Alzheimers Disease and Related Disorders Association criteria). Peripheral blood samples
were collected from study subjects, and DNA was extracted from whole blood using the PureGene娂 kit
(Gentra Systems) or by standard phenol-chloroform
methods.
We tested the hypothesis that the clinic-based
sample and the community-based sample would differ
in their genetic profiles for hypertension. This expectation arose because the clinic-based AD sample had
been selected for a therapeutic trial in which HTN/
vascular disease was a strict exclusion criterion
whereas in the community-based sample hypertensives were not excluded if this systemic disorder could
not explain the dementia. We did not find a significant
difference (by genotype, p ⳱ .781; by allele, p ⳱ .453)
in the G␤3 genetic profiles for hypertension between
the clinic- and community-based samples. Therefore,
as there are no known phenotypic differences between
G␤3 and PLA2-associated HTN, we focused on the less
biased community-based AD sample in our PLA2 study.
Control subjects (n ⳱ 167, mean age ⳱ 71.81 ± 7.37,
59.0% female) and community-based AD cases (n ⳱ 77,
mean age ⳱ 75.72 ± 7.58, 53.7% female) included in the
PLA2 genotyping strategy did not differ by gender (p ⳱
.468), but control subjects were significantly younger
than cases (p < .001). For G␤3 genotyping, control subjects (n ⳱ 181, mean age ⳱ 71.02 ± 8.93, 71.7% female), community-based AD cases (n ⳱ 79, mean age
⳱ 74.96 ± 8.43, 50.7% female), and clinic-based AD
cases (n ⳱ 70, mean age ⳱ 74.54 ± 8.33, 76.2% female)
were included. Clinic-based AD cases and controls had
similar gender frequencies (p ⳱ .503), while community-based AD samples and controls did not (p < .01),
yet the gender comparison of the combined AD group to
controls was not significantly different (p ⳱ .057). Both
AD groups were significantly older than controls (p <
.01). All samples (cases and controls) were drawn from
primarily Caucasian and Hispanic populations and no
significant differences (p > .05) were noted between
Caucasians and Hispanics within each sample on G␤3
or PLA2 status.
APOE and G␤3 Polymorphism Genotyping
APOE genotyping was performed according to the
methods of Wenham and colleagues [1991]. G␤3 genotyping was performed as follows: approximately 200 ng
of genomic DNA was used in a total volume of 25 ␮L for
a polymerase chain reaction (PCR) designed to amplify
a 268 bp fragment containing a coding polymorphism
[C825T, Siffert et al., 1998] within exon 10 of the heterotrimeric G protein ␤3 subunit gene. Forward (5⬘TGACCCACTTGCCACCCGTGC-3⬘) and reverse (5⬘GCAGCAGCCAGGGCTGGC-3⬘) oligonucleotides were
used as reported by Siffert and colleagues [1998]. Thermocycling was performed for 35 cycles at 94°C for 1
min, 66°C for 40 sec, and 72°C for 1 min, and a final
extension step at 72°C for 10 min. PCR products were
digested with 1.25 U of BsaJ I (NEB, Beverly, MA),
separated on 2.0% agarose gels, and viewed by UV
transillumination. The uncut PCR product corresponds
to the T/T genotype while complete digestion results in
fragments of 116 bp and 152 bp (C/C genotype).
Pancreatic PLA2 Polymorphism Genotyping
PCR was designed to amplify a 430-bp fragment containing a Taq I dimorphic site within the first intron of
the pancreatic PLA2 gene. This Taq I dimorphism was
previously identified by Frossard and Lestringant using a southern blot technique [1995]. Because of the
difficulty in amplifying this fragment directly from
some samples, a nested PCR strategy was used. Forward (5⬘-GAGTACAGTGGTGCGATCTC-3⬘) and reverse (5⬘-CCATTCTCCTGCCTCAGCCT-3⬘) oligonucleotides were used with ∼200 ng of genomic DNA in a
TABLE I. G␤3 Polymorphism Frequencies*
Clinicbased AD
C/C
29 (41.4%)
T/C
33 (47.1%)
T/T
8 (11.4%)
n ⳱ 70
␹2 ⳱ .385,**
p ⳱ .825
91 (65.0%)
C
T
49 (35.0%)
n ⳱ 140
␹2 ⳱ .230,**
p ⳱ .632
ORs with
95% CIs
Communitybased AD
ORs with
95% CIs
Combined
AD
ORs with
95% CIs
1.52
[.51–4.57]
1.17
[.39–3.54]
1
35 (44.3%)
1.11
[.46–2.69]
1.07
[.44–2.55]
1
64 (43.0%)
1.24
[.58–2.67]
1.09
[.51–2.33]
1
72 (39.8%)
1.12
[.78–1.61]
1
227 (62.7%)
1.26
[.76–2.09]
1
33 (41.8%)
11 (13.9%)
n ⳱ 79
␹2 ⳱ .486,**
p ⳱ .784
103 (65.2%)
55 (34.8%)
n ⳱ 158
␹2 ⳱ .294,**
p ⳱ .588
1.05
[.69–1.60]
1
*Control ages and AD ages of onset ⱖ 60 years; OR ⳱ odds ratio; CI ⳱ confidence interval.
**Comparison made to control.
66 (44.3%)
19 (12.8%)
n ⳱ 149
␹2 ⳱ .400,**
p ⳱ .819
194 (65.1%)
104 (34.9%)
n ⳱ 298
␹2 ⳱ .406,**
p ⳱ .524
Controls
83 (45.9%)
26 (14.4%)
n ⳱ 181
135 (37.3%)
n ⳱ 362
PLA2, G␤3 Genes, and Alzheimer’s Disease
TABLE II. PLA2 Polymorphism Frequencies*
Communitybased AD
A1/A1
A1/A2
A2/A2
13 (16.9%)
36 (46.8%)
28 (36.4%)
n ⳱ 77
A1
A2
62 (40.3%)
92 (59.7%)
n ⳱ 154
Controls
36 (21.6%)
87 (52.1%)
44 (26.3%)
n ⳱ 167
␹2 ⳱ 2.622,
p ⳱ .270
159 (47.6%)
175 (52.4%)
n ⳱ 334
␹2 ⳱ 2.306,
p ⳱ .129
ORs with
95% CIs
1
1.15 [.52–2.55]
1.46 [.62–3.44]
1
1.21 [.79–1.85]
*Control ages and AD ages of onset ⱖ60 years; OR ⳱ odds ratio; CI ⳱
confidence interval.
total volume of 25 ␮L. Thermocycler conditions were as
follows: 94°C for 10 min followed by 35 cycles of 94°C
for 30 sec, 60°C for 30 sec, and 72°C for 20 sec, and a
final extension step at 72°C for 10 min. One microliter
of this product was then used in a 25 ␮L PCR with
forward (5⬘-AGTGGTGCGATCTCGGCTCA-3⬘) and reverse (same as above) oligonucleotides to yield the 430
bp fragment. Thermocycler conditions were as follows:
94°C for 10 min followed by 30 cycles of 94°C for 30 sec,
63°C for 30 sec, and 72°C for 30 sec, and a final extension step at 72°C for 10 min. PCR products were digested with 1.5 U of Taq I (Promega), separated on
2.0% agarose gels, and viewed by UV transillumination. The uncut PCR product corresponds to the A1/A1
genotype while complete digestion results in fragments
of 263 bp and 167 bp (A2/A2 gentotype).
Statistical Analysis
The likelihood ratio ␹2 statistic was used to compare
genotype and allelic distributions, as well as gender
distributions, between control subjects and AD cases. A
t-test for independent samples was used to compare
control ages with AD ages of onset. Multiple logistic
regression models were used to assess odds ratios
(ORs) for each genotype or allele, as well as to assess
possible interaction between either the PLA2 polymorphism or the G␤3 polymorphism and APOE genotype.
Age and gender were included in each logistic regression model as controlling factors, and while age was a
significant (p < .05) term across all analyses, gender
was not (p > .05). Odds ratios and corresponding 95%
confidence intervals (CIs) were calculated according to
standard methods. Alpha levels were set at .05 for each
analysis. Each of these analyses was performed using
SPSS for Windows, release 7.5.1. Power analyses were
carried out using Power and Precision, release 1.0.
RESULTS
As shown in Table I, we did not detect an association
between the G␤3 polymorphism and AD, either by genotype or by allele. We did find the expected APOE ⑀4
elevation when comparing each of the AD groups
shown in Table I with controls, both by allele (p < .001)
and by genotype (p < .01, data not shown). Possible
interaction between APOE and the G␤3 polymorphism
on prediction of AD was assessed, and no significant
467
interactive term was found when including clinic-based
AD patients (p ⳱ .974), community-based AD cases (p
⳱ .904), or the combined group (p ⳱ .948) in the model.
We went on to assess a possible association between
the PLA2 polymorphism and community-based AD
cases, and were unable to find such an association (see
Table II). We did find the expected APOE ⑀4 increase,
again, when comparing the groups shown in Table II
(by allele, p < .001; by genotype, p ⳱ .001, data not
shown). We were unable to find interaction between
APOE and the PLA2 polymorphism on risk for AD (p ⳱
.833).
DISCUSSION
Occurrence of HTN has been suggested to be directly
associated with risk for vascular dementia and AD,
though the mechanism underlying such an association
remains unclear [Skoog, 1996; Lis and Gaviria, 1997].
We investigated a possible genetic etiologic link between HTN and AD, and genotyped AD cases and controls for polymorphisms within the pancreatic PLA2
and G␤3 genes. Both of these genes had previously
been found to be strongly associated with HTN, and the
G␤3 polymorphism was further shown to differentially
effect G protein activity [Frossard and Lestringant,
1995; Siffert et al., 1998]. Additionally, both of these
polymorphisms map to chromosome 12, where other
groups have found linkage to AD [Pericak-Vance et al.,
1997; Rogaeva et al., 1998].
We did not detect an association between either of
these polymorphisms and AD, either independently or
synergistically with APOE. Thus, our results do not
support a genetic etiologic link between HTN and AD.
Statistical power to detect a clinically relevant difference (a disease allele difference of 12% between AD
cases and controls, on the order of that observed with
APOE ⑀4) between either the G␤3 or PLA2 polymorphisms and AD was calculated; our G␤3 and PLA2
polymorphism data sets reveal 91 and 71% power, respectively. Further, power to detect a significant interaction (where each term explains 5% of disease variance in a multiple regression model) between either the
G␤3 or PLA2 polymorphisms and APOE on prediction
of AD was calculated, and these analyses reveal 99 and
96% power, respectively. Thus, overall likelihood of a
type II error in detecting clinically relevant differences
in genetic profiles between cases and controls in our
data set is minimal.
Yet, our apparent lack of association between HTN
genetic risk factors and AD may be due to a relatively
low information content of the polymorphisms examined in this study. Additionally, it is possible that, by
examining an AD sample, we have selected a specific
subset of hypertensive individuals who are etiologically
distinct from hypertensive cases at large. To examine
this hypothesis, the study of further candidate genes
for HTN in AD is warranted. If the results with these
HTN genetic risk factors are indicative of no genetic
etiologic link between HTN and AD, it is possible that
the physiologic consequences of HTN themselves contribute to AD. Alternatively, aspects of the AD process
itself (such as the possible consequences of ␤-amyloidenhanced vasoconstriction) could lead to HTN.
468
Town et al.
ACKNOWLEDGMENTS
We are grateful to Mr. and Mrs. Robert Roskamp for
their generous support which helped to make this work
possible. We would like to thank Danielle Fallin for her
assistance in G␤3 genotyping.
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