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54:643-649, 1994
Summary
The apolipoprotein E gene (APOE) has three common alleles (2, 3, and 4) that determine six genotypes in the
general population. In this study, we examined 77 patients with late-onset Alzheimer disease (AD), along with
an equal number of age- and sex-matched controls, for an association with the APOE-4 allele. We show that
the frequency of this allele among AD patients was significantly higher than that among the control population
(.351 vs. .130, P = .000006). The genotype frequencies also differed between the two groups (P = .0002), with
the APOE-4/3 genotype being the most common in the AD group and the APOE-3/3 being the most
common in the control group. In the AD group, homozygosity for 4 was found in nine individuals, whereas
none was found in the control group. The odds ratio for AD, when associated with one or two 4 alleles, was
4.6 (95% confidence interval [CI] 1.9-12.3), while the odds ratio for AD, when associated with heterozygosity
for APOE-4, was 3.6 (95% CI 1.5-9.8). Finally, the median age at onset among the AD patients decreased from
83 to 78 to 74 years as the number of APOE-4 alleles increased from 0 to 1 to 2, respectively (test for trend, P
= .001). Our data, which are in agreement with recent reports, suggest that the APOE-4 allele is associated
with AD and that this allelic variant may be an important risk factor for susceptibility to AD in the general
Tsai et al.
644
matter et al. (1993) demonstrated an association between the APOE-s4 allele and late-onset familial AD.
When combined, these data suggest a possible role for
the APOE gene, and in particular one specific isoform,
in the pathogenesis of amyloid fibril formation in AD.
In the present study, we present the association of the
most common APOE alleles and genotypes with AD
(predominately late onset) among 77 pairs of cases and
age- and sex-matched controls.
Subjects, Material, and Methods
Subjects
Patients and controls for this study were selected
from the Mayo Clinic Alzheimer's Disease Patient Registry (ADPR). The ADPR attempts to identify and
enroll all newly diagnosed cases of dementia that present to the Mayo Clinic's Division of Community Internal Medicine (DCIM). For each new dementia case
enrolled, the next age- and sex-matched, cognitively
normal person presenting to the DCIM for routine general medical evaluation is recruited as a normal control.
This sampling frame provides a community-based sample of patients that is relatively free from biases inherent
in referral populations. Controls selected from this
sampling frame are neither unusually healthy nor unhealthy but are representative of older persons seeking
routine general health care. All ADPR registrants receive a general medical examination (GME), comprehensive neurologic exam, and rigorous neuropsychologic evaluations. As part of the blood draw for GME
routine studies, an additional 15-cc sample is obtained
from those registrants consenting to provide DNA for
research purposes.
Potential dementia cases receive computed-tomography or magnetic-resonance-imaging studies and additional laboratory studies as indicated. Dementia diagnoses are based on the Diagnostic and Statistical
Manual of Mental Disorders, Third Edition, Revised
(American Psychiatric Association 1987). AD and vascular dementia diagnoses follow National Institute of
Neurological Diseases and Stroke/Alzheimer's Disease
and Related Disorders Association guidelines
(McKhann et al. 1984) and the California Alzheimer's
Disease Diagnostic and Treatment Centers criteria
(Chui et al. 1992), respectively. Diagnoses are established by consensus of the ADPR investigators, who
include a geriatrician, two behavioral neurologists, and
two neuropsychologists. Patients are followed longitudinally to death, and an autopsy is obtained for all patients from whom patient or family consent has been
High-molecular-weight DNA was isolated from peripheral blood leukocytes (buffy coat) by using an Applied Biosystem 340A DNA Extractor (Applied Biosytems, Foster City, CA) according to the manufacturer's
instructions. DNA was amplified by utilizing a PCR
thermal cycler (Perkin Elmer Cetus) along with oligonucleotide primers APOE-A (5'-CGGGCACGGCTGTCCAAGGAG-3') and APOE-C (5'CACGCGGCCCTGTTCCACGAG-3'). Each amplification reaction
contained 250 ng of genomic DNA, 20 pmol of each
primer, 10% dimethylsulfoxide, 200 ,uM of each
dNTP, and 0.25 pJ of Taq DNA polymerase in a final
volume of 25 pl. Reaction conditions included denaturation for 30 s at 940C, annealing for 30 s at 650C, and
extension for 30 s at 720C, for 30 cycles. The PCR
products were digested with HhaI, and the fragments
were separated by electrophoresis on an 8% polyacrylamide nondenaturing gel. After electrophoresis, the gel
was treated with ethidium bromide for 30 min, and
DNA fragments were visualized by UV illumination.
APOE genotypes for the case and control groups were
determined in a blinded fashion by scoring for a unique
combination of fragment sizes, as described by Hixon
and Vernier (1990).
Statistical Method
645
Table I
Comparison of APOE Genotypes and Allele Frequencies
between AD Cases and Controls
% IN
AD Groupa
Control Groupa
P-valueb
Genotype:
84/84
.........
4/3
&4/&2
.....
83/83
83/F2
2/2
11.7
40.3
6.5
35.1
6.5
0
22.1
39
57.1
15.6
1.3 J
84 .35.1
.58.4
6.5
13.0 1
76.0
11.0
.0002
Allele:
83
.00002
aN = 77.
b
Results
At the time of these analyses, there were 554 community-based patients with cognitive impairment and their
matched controls enrolled in the ADPR. This included
382 women and 172 men. There were 135 patients with
probable or possible AD, 68 patients with probable or
possible vascular dementia, 18 with dementia from
other causes such as Parkinson disease, and 53 subjects
with mild cognitive impairment that may represent preclinical dementia (Flicker et al. 1991).
From this ADPR pool, a sample of 77 clinically probable AD patients and their age- and sex-matched controls were utilized for the present analyses. All casecontrol pairs were selected on the basis of (1) the
patient having a diagnosis of probable AD, (2) availability of a blood sample on the patient, and (3) availability
of a blood sample for the matched control. From each
group of 77, there were 57 women and 20 men, consistent with the actual gender distribution of AD in our
population (Kokmen et al. 1989). The mean age at the
time the DNA sample was obtained was 80.5 (SD = 8.9)
years for the cases and 80.0 (SD = 9.1) years for the
controls. Mean age at estimated onset of AD in the
cases was 78.7 (range 47-95) years. Six of the 77 cases
had onset at <65 years of age, and thus the study population is composed primarily of late-onset (defined as
age >65 years) AD cases. With regard to family history,
12 controls and 20 cases had at least 1 family member
(first or second degree) with dementia.
controls (57%). The three allele frequencies also differed significantly between the case and control groups
(P = .00002), with the 84 allele frequency significantly
higher among cases than among controls (.351 vs. .130,
P = .000006) (table 1). Of significance, 9 (11.7%) of the
77 cases were homozygous for the 84 allele, whereas
none of the controls were determined to be 84 homozygotes.
Table 2 compares the genotypes for paired AD and
control individuals. Compared with the control group,
the odds ratio for the association of AD with one or
two 84 alleles was 4.6 (95% CI 1.9-12.3). Although the
odds ratio for the association of AD with homozygosity
for 84 was undefined (7/0), the lower 95% confidence
limit was 1.4. On the other hand, the odds ratio for
individuals heterozygous for 84 was 3.6 (95% CI 1.5-9.8).
The association of APOE alleles with AD was also
examined separately for males and females. For males,
the APOE allele frequencies were .05 for 82, .525 for
83, and .425 for 84; for females, the frequencies were
.07, .605, and .325, respectively. These three frequencies did not differ significantly between males and females (P = .53), and, likewise, the frequency of 84 alone
also did not differ significantly between these two
groups (P = .26). Furthermore, the odds ratio for the
association of AD with one or two 84 alleles among
males (6.0) did not significantly differ from that for
females (4.0).
To further explore the relative contributions of each
of the three APOE alleles to the relative risk of AD, we
fit several conditional logistic regression models. We
Table 2
Comparison of Genotypes for Paired AD Cases and Controls
AD GENOTYPE
CONTROL GENOTYPE
84/84 ...................
4/ * ...................
*/*
.......................
Total ...................
84/84
0
2
257
9
84/ *
*/ *
TOTAL
0
11
0
7
36
32
0
20
57
77
25
Tsai et al.
646
Table 3
Logistic Model Odds Ratio Predictions
Genotype
c3/e3
e2/2
2/3
2/4
3/4
4/4
a
..........
..........
..........
..........
Model la
Model 2"
1.0
.71
.85
3.61
1.0
1.0
1.0
4.39
4.39
19.26
4.28
..........
18.30
..........
a4
log(ORij) = ai + a,;= % -.1686; a3 .0; 3and
=
b log(OR) = OX; X
1.4535.
1.4791.
4 ALLELES
2
1
0
...........
...........
...........
74.1
78.1
82.7
53.1
47.7
50.5
86.8
94.2
95.3
.001
647
Table 5
Comparison of Apo-E4 Frequencies among Recent Publications
e4 FREQUENCY
(%)
STUDY
Strittmatter et al.
(1993) ..................
Saunders et al.
(1993a) .................
Control
AD
No.
All
All
ADI
TYPE
LOAD
Familial (clinical)
(83)
50
16b
EOAD
LOAD
LOAD
LOAD
LOAD
LOAD
LOAD
Familial
Familial
Sporadic (autopsy)
Sporadic (clinical)
Sporadic (clinical)
Sporadic (clinical)
Sporadic (clinical)
(16)
(36)
(176)
(69)
19
42
40
36
38
34
35
10,c 16b
61
30
(45)
20
57
30
43
MEAN
AGE
47.2
66.1
43
12c
14
11
75.1
33
j1d
13d
10
14
80.6
both the odds ratio and allele frequencies can vary dramatically, as demonstrated by Mayeux et al. (1993). In
our study, the control population was derived from a
community-based patient population, and although the
control population was not specifically matched for
ethnicity, they were derived from the same community
as our cases and were matched for age and sex.
In addition to detecting a higher 4 allele frequency
among AD patients, our study confirms two other important observations that have been made, namely, the
effect of 4 allele dosage on disease susceptibility and
age at onset (Corder et al. 1993; Mayeux et al. 1993;
Poirier et al. 1993). With regard to the first of these, our
data suggest that the risk of having late-onset AD in the
general population increases with increasing dosage of
the 84 allele. We find that individuals with one or two
4 alleles are 4.6 times more likely to have late-onset
AD than those without an 84 allele. Individuals who are
heterozygous for 4 have a 3.6-fold increase in risk.
This amount of risk for heterozygotes is similar both to
that predicted by our logistic regression models and to
that observed by Mayeux et al. (1993). Because there
were no 84/84 controls, we could not directly estimate
the odds ratio for the homozygous 84/84 genotype.
However, on the basis of the logistic model, we estimate an odds ratio of 18.3 for homozygotes, which is
also similar to that estimated by Mayeux et al. (1993).
Furthermore, we estimate that the risk of AD increases
Tsai et al.
648
Acknowledgments
We thank Karen Erwin for her excellent secretarial support. This project was supported in part by National Institute
on Aging grants AG 08031 and AG 06786.
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