Large meta-analysis establishes the ACE insertion-deletion polymorphism as a marker of Alzheimer's disease

Article (PDF Available)inAmerican Journal of Epidemiology 162(4):305-17 · September 2005with8 Reads
DOI: 10.1093/aje/kwi202 · Source: PubMed
Abstract
Apolipoprotein E ε4 (APOE*4) is the only fully established susceptibility allele for Alzheimer's disease. One of the most studied candidates is the insertion (I)/deletion (D) polymorphism (indel) of the gene for angiotensin I-converting enzyme (ACE). This study aimed to clarify its association with Alzheimer's disease. The meta-analysis included 39 samples, comprising 6,037 cases of Alzheimer's disease and 12,099 controls, using mainly primary data. Potential interactions with gender, age, ethnic group, and carrier status of the apolipoprotein E ε4 allele were all examined. D homozygotes were at reduced risk of Alzheimer's disease (odds ratio = 0.81, 95% confidence interval: 0.72, 0.90; corrected p = 0.0004); I homozygotes showed no association with Alzheimer's disease, while heterozygotes were at increased risk. Although there were clear differences among the three ethnic groups examined (North Europeans, South Caucasians, and East Asians), in all groups D homozygotes were at reduced risk. These results confirm the association of the angiotensin I-converting enzyme indel with Alzheimer's disease across diverse populations, although this is probably due to linkage disequilibrium with the true risk factor. Further, in North Europeans, both association and Hardy-Weinberg analysis suggested partial heterosis, that is, an increased risk for heterozygotes, due to a hidden interaction with another, as yet unknown, risk factor. This interaction warrants further investigation.
META-ANALYSIS
Large Meta-Analysis Establishes the ACE Insertion-Deletion Polymorphism as
a Marker of Alzheimer’s Disease
Donald J. Lehmann
1
, Mario Cortina-Borja
2
, Donald R. Warden
1
, A. David Smith
1
, Kristel Sleegers
3
,
Jonathan A. Prince
4
, Cornelia M. van Duijn
3
, and Patrick G. Kehoe
5
1
The Oxford Project to Investigate Memory and Ageing (OPTIMA), Department of Pharmacology, University of Oxford,
Oxford, United Kingdom.
2
Centre for Paediatric Epidemiology and Biostatistics, Institute of Child Health, University College London, London,
United Kingdom.
3
Department of Epidemiology and Biostatistics, Erasmus MC, Rotterdam, the Netherlands.
4
Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, Sweden.
5
Care of the Elderly, Department of Clinical Science at North Bristol, University of Bristol, Frenchay Hospital, Bristol,
United Kingdom.
Received for publication January 5, 2005; accepted for publication March 22, 2005.
Apolipoprotein E e4(APOE*4) is the only fully established susceptibility allele for Alzheimer’s disease. One of
the most studied candidates is the insertion (I )/deletion (D) polymorphism (indel) of the gene for angiotensin
I-converting enzyme (ACE). This study aimed to clarify its association with Alzheimer’s disease. The meta-analysis
included 39 samples, comprising 6,037 cases of Alzheimer’s disease and 12,099 controls, using mainly primary
data. Potential interactions with gender, age, ethnic group, and carrier status of the apolipoprotein E e4 allele were
all examined. D homozygotes were at reduced risk of Alzheimer’s disease (odds ratio ¼ 0.81, 95% confidence
interval: 0.72, 0.90; corrected p ¼ 0.0004); I homozygotes showed no association with Alzheimer’s disease, while
heterozygotes were at increased risk. Although there were clear differences among the three ethnic groups
examined (North Europeans, South Caucasians, and East Asians), in all groups D homozygotes were at reduced
risk. These results confirm the association of the angiotensin I-converting enzyme indel with Alzheimer’s disease
across diverse populations, although this is probably due to linkage disequilibrium with the true risk factor. Further,
in North Europeans, both association and Hardy-Weinberg analysis suggested partial heterosis, that is, an in-
creased risk for heterozygotes, due to a hidden interaction with another, as yet unknown, risk factor. This interac-
tion warrants further investigation.
Alzheimer disease; heterogeneity; meta-analysis
Abbreviations: ACE, angiotensin I-converting enzyme (protein); ACE, angiotensin I-converting enzyme (gene); APOE*4,
apolipoprotein E e4 (allele).
In 1999, Kehoe et al. (1) first proposed an association
with Alzheimer’s disease of the insertion (I )/deletion (D)
polymorphism (indel) in intron 16 of the gene for angiotensin
I-converting enzyme (ACE) on chromosome 17q23. The
enzyme converts angiotensin I to angiotensin II, which is
potently hypertensive. The D allele is associated with raised
plasma levels of the enzyme (2, 3). Kehoe et al. (1) found
that I positives, that is, DI þ II, were at increased risk of
Correspondence to D. J. Lehmann, University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, United Kingdom
(e-mail: donald.lehmann@pharm.ox.ac.uk).
305 Am J Epidemiol 2005;162:305–317
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Alzheimer’s disease. The study comprised three samples,
from Cardiff and London in Britain and Belfast in Northern
Ireland, with strikingly similar results.
Since then, this potential association has been examined
in over 40 samples (4, 5) (table 1) from around the world.
Several studies replicated the report of increased risk for
I positives, just a few contradicted it, and many found no
association with Alzheimer’s disease. One study suggested
an effect of age on the association (6), and another proposed
an influence of gender (7). An association with onset age of
Alzheimer’s disease has also been proposed (8).
The association has recently been studied in the longitu-
dinal, observational cohorts of the Oxford Project to Inves-
tigate Memory and Ageing (O PTIMA) and the Rotterdam
Study (9). Both groups found a suggestion of a gender dif-
ference in their results, consistent with a previous report (7).
We therefore undertook a meta-analysis of studies of the
association of the ACE indel with Alzheimer’s disease, strat-
ified by gender, by age, by ethnic group, and by apolipopro-
tein E e4 allele (APOE*4) carrier status, using mainly
primary data, suppl ied by authors.
Although three previous meta-analyses of ACE studies
had been reported (10–12), subsequent studies have now
made further data available. Compared even with the latest
meta-analysis (12), 10 more samples from eight studies
were available to us (9, 13–18, and the above study of the
Oxford Project to Investigate Memory and Ageing). In ad-
dition, all previous meta-analyses were based on published
data, that is, largely unstratified, and were thus limited in
the interpretations they allowed. Using primary data, we
were able to explore more deeply, searching for sources
of heterogeneity. Two previous stratified meta-analyses of
Alzheimer’s disease genes (19, 20), based on primary data,
have both deepened our understanding.
Egger et al. (21) have stressed the need to examine
sources of heterogeneity in meta-analyses of case-control
TABLE 1. Cohorts used in the meta-analysis of the ACE* insertion-deletion polymorphism in Alzheimer’s disease
Reference
Location of
samples
Alzheimer’s
disease (no.)
Controls
(no.)
Alzheimer’s disease
diagnosisy
Stratified
data supplied
and usedz
Alvarez et al. (63) Asturias, Spain 351.0 536.0 NINCDS-ADRDA probable Yes
Buss et al. (40) Hamburg, Germany;
Basel, Switzerland;
and Brescia, Italy
261 306 NINCDS-ADRDA Yes
Camelo et al. (13) Colombia 98 72 NINCDS-ADRDA probable Yes
Carbonell et al. (64) London, United Kingdom 80 65 NINCDS-ADRDA
Chapman et al. (65) Tel Aviv, Israel 49 40 NINCDS-ADRDA probable
Cheng et al. (66) Taipei, Taiwan 173 116 NINCDS-ADRDA probable
Crawford et al. (7) Florida, United States 171 175 NINCDS-ADRDA
Farrer et al. (6) Moscow, Russia 151 206 NINCDS-ADRDA
Farrer et al. (6) Toronto, Canada;
Miami, Florida;
and Florence, Italy
236 169 NINCDS-ADRDA
Ferna
´
ndez-Novoa
et al. (14)
Galicia, Spain 185 117 NINCDS-ADRDA and
DSM-IV
Yes
Hu et al. (67) Japan 132 148 NINCDS-ADRDA probable
Isbir et al. (68) Turkey 35 29 NINCDS-ADRDA probable
Kehoe et al. (11) Bristol and London,
United Kingdom
296 95 CERAD or NINCDS-
ADRDA probable
Yes
Kehoe et al. (1) Cardiff, Wales 198 77 NINCDS-ADRDA probable
Kehoe et al. (1) London, United Kingdom 201 205 NINCDS-ADRDA Yes
Kehoe et al. (1) Belfast, Northern Ireland 209 198 NINCDS-ADRDA probable
and DSM-IV
Yes
Keikhaee et al. (15) Iran 132 107 Yes
Lendon et al. (16) Manchester, England 206 33 NINCDS-ADRDA probable
or CERAD
Yes
Lendon et al. (16) Scotland 167 357 NINCDS-ADRDA Yes
Mattila et al. (69) West Finland 77 67 CERAD or NINCDS-ADRDA
probable
Yes
Monastero et al. (70) Palermo, Italy 149 149 NINCDS-ADRDA probable
Myllykangas et al. (71) Vantaa, Finland 121 75 CERAD
Narain et al. (10) Cambridge, England 107 163 CERAD Yes
Table continues
306 Lehmann et al.
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studies. We therefore divided our analysis into two phases:
first, a broad overall analysis and, second, the detailed
examination of sources of heterogeneity. We aimed to dis-
cover whether the ACE indel is associated with Alzheimer’s
disease and, if so, the nature of that association.
MATERIALS AND METHODS
Phase 1: broad analysis
In April 2003, following PubMed searches, the study of
references and of abstracts, and communication with ex-
perts, we wrote to the authors of all association studies of
the ACE indel and Alzheimer’s disease known to us, 33
studies in all, involving 41 samples (4, 5) (table 1), including
seven samples from unpublished studies. The searches were
updated in November 2004, without further data arising.
For the stratified analyses, we used the raw data or stratified
summaries received from authors on 26 samples. For the
overall analyses, we also used published data, making 39
samples altogether (table 1). Very early onset cases (<50
years) and young controls (<50 years) were excluded,
where identifiable, as were subjects with incomplete data.
Distinct populations were treated separately, where possi-
ble. The large number of controls in the cohort of Sleegers
et al. (9) had all been fully assessed (22). All samples had
been genotyped for the ACE indel, except for the two of
Prince et al. (23, 24), who had used the ACE rs4343 marker,
which is in 85–90 percent linkage disequilibrium with the
indel in North Europeans.
Phase 2: examination of heterogeneity
We examined six possible sources of heterogeneity. We
performed analyses stratified by age, gender, and APOE*4
status. For the age stratifications (<65 years, 65–75 years,
and >75 years), we divided the samples into two groups,
TABLE 1. Continued
Reference
Location of
samples
Alzheimer’s
disease (no.)
Controls
(no.)
Alzheimer’s disease
diagnosisy
Stratified
data supplied
and usedz
Narain et al. (10) Oxford, England 26 179 CERAD Yes
Palumbo et al. (72) Italy 96 40 NINCDS-ADRDA probable
Panza et al. (17) South Italy 141 268 NINCDS-ADRDA probable Yes
Perry et al. (41) Alabama, United States
(African Americans)
111 78 NINCDS-ADRDA probable Yes
Prince et al. (23)
(cohort 1);
Kehoe et al. (11)
Sweden 201 161 NINCDS-ADRDA or CERAD Yes
Prince et al. (23)
(cohort 2);
Kehoe et al. (11)
Sweden 168 322 NINCDS-ADRDA Yes
Richard et al.
(cohort 1) (73)
North Europe 421 474 NINCDS-ADRDA probable Yes
Richard et al. (73)
(cohort 2)
North Europe 56 221 NINCDS-ADRDA probable Yes
Scacchi et al. (74) Rome, Italy 80 153 NINCDS-ADRDA probable Yes
Seripa et al. (18) Rome, Italy 97 101 NINCDS-ADRDA probable Yes
Seripa et al. (18) Wisconsin and Kentucky,
United States
102 66 NINCDS-ADRDA probable
and Khachaturian
Yes
Sleegers et al. (9) Netherlands 236 5,889 NINCDS-ADRDA Yes
Wu et al. (75) Beijing, People’s
Republic of China
96 96 DSM-IV Yes
Yang et al. (76) Shanghai, People’s
Republic of China
188 227 NINCDS-ADRDA probable
Zuliani et al. (77) Northeast Italy 35 51 NINCDS-ADRDA probable Yes
OPTIMA* study§ Oxfordshire, England 198 268 CERAD or NINCDS-ADRDA
probable
Yes
Totals 6,037 12,099
* ACE, angiotensin I-converting enzyme gene; OPTIMA, Oxford Project to Investigate Memory and Ageing.
y Based on criteria of the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related
Disorders Association (NINCDS-ADRDA) (26), Diagnostic and Statistical Manual of Mental Disorders: DSM-IV (DSM-IV) (78), Consortium to
Establish a Registry for Alzheimer’s Disease (CERAD) (25), or Khachaturian (79).
z Full data were obtained on 84% of subjects.
§ Unpublished observations.
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according to how the ages of cases had been defined, for
example, at onset (first group) or at examination or death
(second group). We also conducted analyses restricted to
controls and to diagnoses of probable or definite Alzheimer’s
disease according to criteria of the Consortium to Establish
a Registry for Alzheimer’s Disease (CERAD) (25) or of the
National Institut e of Neurological and Communicative Dis-
orders and Stroke–Alzheimer’s Disease and Related Disor-
ders Association (NINCDS-ADRDA) (26) (26 full samples
and two part samples). In separate analyses, we excluded
eight samples where Hardy-Weinberg analysis and genotyp-
ing methods were compatible with mistyping. Underreport-
ing of heterozygotes may occur unless either 5 percent
dimethyl sulfoxide is included in the polymerase chain re-
action (27) or the DD genotype is confirmed by an insertion-
specific second amplification (28). In further analyses,
samples were placed where possible (34 of 39 samples) in
one of three ethnic groups: North European (19 samples);
South Caucasian (11 samples), defined here as from the
Mediterranean or from the Middle East; and East Asian
(four samples), from China or from Japan.
Data analysis and statistical methods
Three methods are commonly used to produce odds ratios
in genetic association studies: method 1, allelic (i.e., D vs. I
in this case); method 2, comparing each genotype with one
other in turn (e.g., DD vs. DI); method 3, comparing each
genotype in turn with the other two combined. All three
methods are subject to bias, the degree of bias depending
on how imperfectly the model fits reality. For instance,
method 1 is best suited to testing a codominant model,
where there is an allelic dose effect (e.g., DD > DI > II).
Method 3, on the other hand, may be used to test either an
I-dominant/D-recessive or a D-dominant/I-recessive model.
We chose to follow the hypothesis-generating study (1) in
using method 3, partly because preliminary examination of
subsequent studies had shown no evidence of codominance.
We also used both methods 2 and 3 to examine heterosis
(heterozygotes at greater or lesser risk than either homozy-
gote). Our main approach, method 3, gives an estimate of
the risk associated with each genotype when compared with
the rest of the population.
Pooled odds ratios were calculated three times, by the
fixed-effects method of Mantel and Haenszel (29), by the
random-effects method of DerSimoni an and Laird (30), and
by the Bayesian random-effects method of Warn et al. (31).
However, as the results of all three methods were very sim-
ilar, only those of the first two are shown (table 2). The
methods of meta-analysis permit the accumulation of results
across independent studies, even where the number of sub-
jects or the ratio of cases to controls varies consider ably, as
here in the cohort of Sleegers et al. (9), for example. The
heterogeneity test was by the method of Armitage (32).
Logistic regression analysis was used to compare odds ra-
tios among ethnic groups. Funnel plots (33) and cumulative
meta-analysis (34) were used to investigate bias. Analyses
were performed using ‘R’ (35) and the package ‘rmeta’
by Thomas Lumley (http://www.cran.r-project.org/src/
contrib/Descriptions/rmeta.html). All tests of significance
were two sided. A Bonferroni corr ection factor of 3 was
applied to all p values by genotype.
RESULTS
Phase 1: broad analysis
Examination of bias.
We found no evidence of publica-
tion bias. Funnel plots for the comparisons most commonly
made, that is, for D homozygotes versus I positives (i.e.,
DI þ II) and for I homozygotes versus D positives, are
shown in figure 1. These plots gave p ¼ 0.19 and p ¼
0.21, respectively. Figure 2 shows the cumulative meta-
analysis after each study from July 1998 to November
2004, for D homozygotes versus I positives, that is, the
comparison made in the hypothesis-generating study of
Kehoe et al. (1). After only four of 39 cohorts had been
studied, the odds ratio by DerSimonian and Lair d (30)
became significantly less than 1 and remained so thereafter.
It changed very little from around 0.8 after 2001.
TABLE 2. Odds ratios of Alzheimer’s disease for each ACE* genotype versus the other
two in the meta-analysisy
Comparisonz
Fixed effects§ Random effects§
Heterogeneity
(p value)§
Odds
ratio
95% confidence
interval
Odds
ratio
95% confidence
interval
DD versus (DI þ II) 0.82 0.76, 0.89 0.81 0.72, 0.90 0.01
DI versus (DD þ II) 1.12 1.04, 1.20 1.12 1.04, 1.22 0.08{
II versus (DD þ DI) 1.07 0.98, 1.17 1.07 0.95, 1.21 0.02
* ACE, angiotensin I-converting enzyme gene.
y All analyses were based on 39 samples, comprising 6,037 cases of Alzheimer’s disease and
12,099 controls.
z D represents the deletion allele; I represents the insertion allele.
§ Fixed-effects odds ratios were by the method of Mantel and Haenszel (29), random-effects
odds ratios by that of DerSimonian and Laird (30), and the heterogeneity test by that of Armitage
(32).
{ Not significant at 0.05.
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Risk of Alzheimer’s disease, by genotype. The odds ra-
tios for each genotype versus the other two are shown in
figures 3, 4, and 5 and are summarized in table 2. D homo-
zygotes were at reduced risk of Alzheimer’s disease, with an
overall odds ratio of 0.81 (95 percent confidence interval:
0.72, 0.90; corrected p ¼ 0.0004) by the method of
DerSimonian and Laird (30). Heterozygotes were at in-
creased risk of Alzheimer’s disease, with an overall odds
ratio of 1.12 (95 percent confidence interval: 1.04, 1.22;
corrected p ¼ 0.006). The odds ratio of Alzheimer’s
disease for I homozygotes versus D positives did not
differ significantly from 1. Two of these three results were
heterogeneous.
Onset age of Alzheimer’s disease. Onset age data were
available from 13 samples, comprising 2,309 case s of
Alzheimer’s disease. Both stratified analyses and linear
regression analysis were performed on these data and also
on a subset of 10 North European samples, comprising
1,822 cases of Alzheimer’s disease. No effect of ACE*I
carrier status on onset age of Alzheimer’s disease and, sur-
prisingly, only a modest effect of APOE*4 carrier status on
onset age were seen (data not shown).
Phase 2: examination of heterogeneity
We examined six potential sources of heterogeneity: age,
gender, race, APOE*4 status, misdiagnosis, and mistyping.
Stratification by gender and by APOE*4 status. No sig-
nificant effects were seen of gender or of APOE*4 status on
the associations with Alzheimer’s disease, nor did stratifi-
cation by either of these two factors remove the heteroge-
neity (data not shown).
Stratification by age. We calculated the odds ratios of
Alzheimer’s disease for ACE*D homozygotes versus I
positives when stratified by age (< 65 years, 65–75 years,
and >75 years). Rather little effect of age was seen on the
association of D homozygotes with Alzheimer’s disease
(data not shown), nor was there any reduction in heteroge-
neity on stratification by age.
Examination of diagnosis. We repeated the overall ana-
lyses of the three genotypes, restricting the data to the 28
samples that could be limited to the more rigorous diagnoses
of probable or definite Alzheimer’s disease according
to criteria of the Consortium to Establish a Registry for
Alzheimer’s Disea se (25) or the National Institut e of
FIGURE 1. Funnel plots of angiotensin I-converting enzyme gene (ACE) studies: A, D homozygotes versus (DI þ II )(p ¼ 0.21); B, I homozygotes
versus (DI þ DD)(p ¼ 0.19). D represents the deletion allele, and I represents the insertion allele. Horizontal lines are 95% confidence intervals;
dashed vertical lines are summary log odds ratios.
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Neurological and Communicative Disorders and Stroke–
Alzheimer’s Disease and Related Disorders Association
(26). The odds ratios were similar to those sho wn in table 2,
that is, significant odds ratios by the method of DerSimoni an
and Laird (30) of 0.77 for D homozygotes and of 1.15 for
heterozygotes and a non significant odds ratio of 1.10 for I
homozygotes. Heterogeneity remained for D homozygotes
(p ¼ 0.005) but was no longer significant (p ¼ 0.16) for
I homozygotes.
Examination of genotyping. There were 12 instances of
Hardy-Weinberg disequilibrium in individual studies and
four in the six groups of pooled cases and controls analyzed
by race (table 3). We therefore excluded the eight samples
where mistyping was considered possible, that is, underre-
porting of heterozygotes, taking into account both genotyp-
ing methods and Hardy-Weinberg analysis. We then
repeated the overall analyses with the remaining 31 sam-
ples. Th e odds ratios were similar to those in table 2, that is,
significant odds ratios of 0.81 for D homozygotes and of
1.12 for heterozygotes and a nonsignificant odds ratio
of 1.09 for I homozygotes. Heterogeneity remained for
I homozygotes (p ¼ 0.02) but was no longer significant
for D homozygotes (p ¼ 0.19).
Ethnic stratification. The samples were divided into three
ethnic groups, where possible: North European (19 sam-
ples), South Caucasian (Mediterranean and Middle Eastern,
11 samples), and East Asian (Chinese and Japanese, four
samples). This removed nearly all heterogen eity (table 3).
Table 3 shows clear differences among the three ethnic
groups. There was no overlap whatsoever in allelic frequen-
cies among the three groups. North Europeans and East
Asians differed in the odds ratios for D homozygotes versus
I positives (p ¼ 0.0008) and for I homozygotes versus
D positives (p ¼ 0.0005). South Caucasians also differed
from East Asians in the odds ratio for D homozygotes ver-
sus I positives (p ¼ 0.0003). D homozygotes were at re-
duced risk in all groups, but particularly in East Asians .
Heterozygotes were at increased risk in North Europeans.
I homozygotes were at higher risk in East Asians, except
by Bayesian analysis. We further examined heterosis
FIGURE 2. Cumulative meta-analysis of the odds ratio of Alzheimer’s disease for ACE*D homozygotes versus I positives. D represents the
deletion allele, and I represents the insertion allele. Horizontal lines are 95% confidence intervals; the vertical dotted line is the summary odds ratio.
OPTIMA, Oxford Project to Investigate Memory and Ageing; ACE, angiotensin I-converting enzyme gene. Additional study information is given in
table 1.
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(heterozygotes at greater risk) in North Europeans, by com-
paring DI with DD and with II in turn. We found that het-
erozygotes were at higher risk than D homozygotes (odds
ratio ¼ 1.25, 95 percent confidence interval: 1.08, 1.45) but
not significantly higher than I homozygotes (odds ratio ¼
1.08, 95 percent confidence interval: 0.95, 1.22).
DISCUSSION
This was a large meta-analysis, by the standards of
Alzheimer’s disease genetics, with 39 samples, comprising
6,037 cases of Alzheimer’s disease and 12,099 elderly con-
trols (table 1). We found no evidence of publication bias.
ACE*D homozygotes were at reduced risk of Alzheimer’s
disease (table 2) in each of the three main ethnic groups
examined (table 3). I homozygotes were neutral as regards
Alzheimer’s disease risk (table 2), except in East Asians, in
whom I homozygotes may be at higher risk (table 3). Het-
erozygotes were at increased risk of Alzheim er’s disease
overall (ta ble 2) and mainly in North Europeans (table 3).
This example of heterosis will be discussed below. Very
similar results were achieved by three methods: fixed ef-
fects, random effects, and Bayesian random effects (re fer
to Materials and Methods and table 2). Only genot ypic com-
parisons were made (refer to Materials and Methods), which
showed that there was no allelic dose effect, except in East
Asians (table 3), and that thus allelic comparisons would
have proved less informative.
These findings of odds ratios close to 1, even though
significantly different from 1 (tables 2 and 3), suggest either
linkage disequilibrium with the true risk factor or confound-
ing by a hidden interaction or both. The secon d possibility is
discussed below. Regarding the former, Kehoe et al. (1) in
their study of ACE haplotypes gave evidence that the indel
may not be the actual risk factor but rather in linkage dis-
equilibrium with a nearby functional polymorphism that is
the true risk factor. ACE haplotype analysis has also proved
of value with other phenotypes, such as enzyme levels (36),
insulin levels, myocardial infarction, and obesity (37). Fu-
ture Alzheimer’s disease studies may do better to examine
polymorphisms in the regulatory regions, rather than the
indel.
FIGURE 3. Odds ratios of Alzheimer’s disease for ACE*D homozygotes versus I positives. D represents the deletion allele, and I represents the
insertion allele. Horizontal lines are 95% confidence intervals. OPTIMA, Oxford Project to Investigate Memory and Ageing; ACE, angiotensin
I-converting enzyme gene. Additional study information is given in table 1.
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The overall results were consistent with those of Kehoe
et al. (1) from 1999 and also with those of the recent meta-
analyses of Kehoe et al. (11) from 2003 and of Elkins et al.
(12) from 2004. The latter report (12) also suggested an age
difference, however, although actual ages were not available
to them, only mean cohort ages, and they did not take into
account the different ways of defining case ages (refer to
Materials and Methods), a strongly confounding factor. Be-
cause of the latter problem, we cannot exclude the possibil-
ity that the association might be stronger in younger cases,
for example, in those aged less than 65 years at onset or less
than 75 years at death.
Heterogeneity
Our overall results showed heterogeneity (table 2). Het-
erogeneity may be due to differences in sample selection
(e.g., in age, gender, or diagnosis) or in methods (e.g., of
genotyping), or it may be due to real differences in popula-
tions (e.g., in race) or in interactions with other risk factors
(e.g., APOE * 4). We examined all of these potential sources
of heterogeneity. We found little effect of age, gender, or
APOE*4 status, thus confounding our initial hypothesis.
Both diagnostic and genotyping differences contributed
some heterogeneity. The former was mainly due to the di-
agnosis of ‘possible Alzheim er’s disease, reported to have
a poor record of confirmation at autopsy (38). The latter was
largely due to certain genotyping methods that may lead to
underreporting of heterozyg otes (27, 28), as shown by
Hardy-Weinberg analysis.
However, the greatest source of heterogeneity was
ethnic differences. Ethnic stratification removed nearly all
heterogeneity (table 3). There were clear contrasts among
the three ethnic groups examined (North Europeans, South
Caucasians, and East Asians) in allelic frequenc ies and in
odds ratios (table 3). The latter might be due to differences
in linkage disequilibrium with the true risk polymorphism
(see above). Some of these ethnic differences had previously
been pointed out by Panza et al. (17, 39), by Buss et al. (40),
and by Elkins et al. (12). These contrasts highlight the
FIGURE 4. Odds ratios of Alzheimer’s disease for ACE heterozygotes versus all homozygotes. Horizontal lines are 95% confidence intervals.
OPTIMA, Oxford Project to Investigate Memory and Ageing; ACE, angiotensin I-converting enzyme gene. Additional study information is given in
table 1.
312 Lehmann et al.
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dangers of combining ethnic groups within a single cohort,
even North and South Europeans, as indicated by Panza
et al. (17, 39). Unfortunately, there was only one cohort of
African origin, from the United States (41). Thus, no con-
clusions could be drawn about Africans.
Heterosis
Deviations from Hardy-Weinberg equilibrium are com-
mon in studies of the ACE indel, as previously pointed out
by Buss et al. (40). This may be due to either mistyping or
heterosis. Several cases of such disequilibrium were indeed
compatible with mistyping. Most examples of disequilib-
rium in North Europeans, however, were of an excess of
heterozygotes in Alzheim er’s disease, which requires an-
other explanation. We believe that explanation is heterosis.
Heterosis occurs when heterozygotes show either a greater
or lesser association with a given trait than does either group
of homozygotes. In this case, in North Europeans, both an
excess of heterozygotes in Alzheimer’s disease and an as-
sociation of heterozygotes with Alzheimer’s disease were
found. Comings and MacMurray (42) have suggested three
explanations for heterosis, of which their second can be
applied here. This proposes that heterosis may follow from
the inadvertent combination of two unlike subsets due to
a hidden interaction with another, unknown risk factor. To
test this proposal, we constructed a mathematical model
with two equal subsets based on a median split of an
unknown factor X (table 4). The model illustrates how,
although heterozygotes may have intermediate odds ratios
in each subset, they can emerge with the highest odds ratio
when the subsets are combined (i.e., heterosis). The overall
results of this illustrative model (table 4) were very close to
those of the present meta-analysis (table 2). We should
stress, however, that heterosis was found only in North
Europeans. Moreover, although heterozygotes were at
greater risk than D homozygotes (odds ratio ¼ 1.25, 95 per-
cent confidence interval: 1.08, 1.45), they were not at sig-
nificantly higher risk than I homozygotes (odds ratio ¼ 1.08,
95 percent confidence interval: 0.95, 1.22). Thus, partial
heterosis better describes this case.
FIGURE 5. Odds ratios of Alzheimer’s disease for ACE*I homozygotes versus D positives. D represents the deletion allele, and I represents the
insertion allele. Horizontal lines are 95% confidence intervals. OPTIMA, Oxford Project to Investigate Memory and Ageing; ACE, angiotensin
I-converting enzyme gene. Additional study information is given in table 1.
ACE Gene in Alzheimer’s Disease 313
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A hidden interaction
Partial heterosis in North Europeans suggests an interac-
tion with another risk factor for Alzheim er’s disease, an-
other potential source of heterogeneity. Candidates include
cardiovascular risk factors, since many also contribute to
Alzheimer’s disease risk and since the ACE indel has been
associated with cardiovascular risk (43–47).
Biologic background
Full discussion of the biologic basis of these associations
is outside the scope of this mainly analytical study. How-
ever, we note the associations of the ACE indel and of
nearby polymorphisms with various phenotypes, including
cardiovascular risk (37, 43–47). In addition, ACE protein
levels are raised in certain brain regions with Alzheimer’s
disease (48–50), the enzyme has been reported to modify
b-amyloid aggregation (51), ACE inhibitors have been re-
ported to maintain memory in aged rats (52) and in human
patients (53), and brain-penetrating ACE inhibitors have
been associated with both reduced incidence of Alzheimer’s
disease in elderly hypertensive patients (54) and less cogni-
tive decline in cases with mild-to-moderate Alzheimer’s
disease (55). Sleegers et al. (9) have recently provided ev-
idence from magnetic resonance imaging of increased atro-
phy in both the hippocampus and amygdala of nondemented
women homozygous for the ACE*I allele, together with
a modest but significant increase in risk of Alzheimer’s
disease, independent of vascular factors. The D allele is
associated with raised plasma levels of ACE (2), but that
association is thought to be due to linkage disequilibrium
with other nearby polymorphisms (3, 36). There is an
TABLE 3. Odds ratios of Alzheimer’s disease for each ACE* genotypey in the meta-analysis, by ethnic group
North Europeans
South Caucasians
(Mediterranean and
Middle East)
East Asians
(China and Japan)
Alzheimer’s disease cases
(no.)/controls (no.) 3,380/9,361 1,350/1,591 589/587
D frequencies in controls
(% (95% CI*)) 53.0 52.2, 53.7 63.4 61.7, 65.0 41.3 38.5, 44.1
DD versus (DI þ II)
(OR*,z (95% CI)) 0.86 (a)§ 0.71, 1.05 0.81 0.70, 0.95 0.45 0.32, 0.63
0.81 (b)§ 0.70, 0.95
DI versus (DD þ II)
(ORz (95% CI)) 1.11 (a)§ 0.97, 1.28 1.11 0.95, 1.30 0.98 0.78, 1.25
1.15 (b)§ 1.04, 1.28
II versus (DD þ DI)
(ORz (95% CI)) 0.99 (a)§ 0.85, 1.16 1.12 0.86, 1.46 1.51 1.19, 1.91
0.99 (b)§ 0.88, 1.12
HW* excess (p value)
Alzheimer’s disease Heterozygotes (0.0009) Homozygotes (0.01) Equilibrium
Controls Equilibrium Homozygotes (0.02) Homozygotes (0.003)
* ACE, angiotensin I-converting enzyme gene; CI, confidence interval; OR, odds ratio; HW, Hardy-Weinberg.
y D represents the deletion allele; I represents the insertion allele.
z All odds ratios shown were by the random-effects method of DerSimonian and Laird (30), which is generally more conservative than the fixed-
effects method; all significant odds ratios were very closely replicated by the Bayesian random-effects method of Warn et al. (31), except for the
II odds ratio for East Asians, whose result was 1.53 (95% CI: 0.96, 2.54) by the Bayesian method.
§ Heterogeneity was seen only in North European cohorts; it was removed upon exclusion of two cohorts with possible genotyping problems.
Results for North Europeans are shown for all 19 cohorts (a) and after the removal of those two cohorts (b). North Europeans versus East Asians:
OR for DD versus (DI þ II), p ¼ 0.0008; OR for II versus (DD þ DI), p ¼ 0.0005. South Caucasians versus East Asians: OR for DD versus (DI þ II),
p ¼ 0.0003.
TABLE 4. Model to show how heterosis may follow from the
combination of two unlike subsets
Model
Odds ratios for each ACE*
genotype versus both others
DD DI II
Low factor Xy 0.50 1.17 1.59
High factor Xy 1.24 1.09 0.63
All 0.82 1.13 1.05
Present meta-analysisz 0.81 1.12 1.07
* ACE, angiotensin I-converting enzyme gene.
y Factor X is hypothesized to interact with the ACE insertion-
deletion polymorphism in Alzheimer’s disease risk; the two subsets
(‘‘low’’ and ‘‘high’’) are based on a median split of values of factor X.
The model was devised to give opposing results in each subset,
while maintaining Hardy-Weinberg equilibrium, but to produce overall
results similar to those of this meta-analysis.
z See table 2.
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apparent conflict between the benefits of ACE inhibitor
therapy and the associations of the indel with Alzheimer’s
disease risk and pathology and with plasma levels of ACE.
However, the influence of ACE polymorphisms on brain
levels of the enzyme is not yet known and cannot be as-
sumed to reflect the levels in plasma. This subject warrants
fuller discussion than is possible here. A more detailed dis-
cussion is given by Kehoe (56), although it will need to be
reconsidered and perhaps revised in the light of any inter-
acting factor or factors that may emerge in future studies.
Limitations of this meta-analysis
The lack of information from some authors was a limita-
tion, but we obtained full data on 84 percent of subjects. The
quality of diagnosis and of genotyping varied among stud-
ies, but our overall results were not changed when these
questions were taken into account. There was considerable
heterogeneity in our initial results. However, we found the
main sources of that heterogeneity and were able to remove
it. Since cardiovascular factors are strong candidates for
a potential interaction with ACE variants, the lack of data
available to us on those factors was a limitation, which we
hope will be overcome by future studies. Cardiovascular
factors might also have influenced our results through
selective mortality. However, D homozygotes have only
a very slightly increased risk of cardiovascular complica-
tions (43–47, 57) and little evidence of any association with
reduced life span (57–62). Thus, the influence of selective
mortality, if any, is likely to be very small and not enough to
provide an alternative explanation for our findings.
Conclusions
We suggest that this large stud y has established the ACE
indel as a marker of Alzheimer’s disease risk, since we
found no evidence of bias, we found and removed the main
sources of heterogeneity, we excluded various potential in-
teractions, and significant results remained in each of the
three ethnic groups examined. The indel, however, is prob-
ably in linkage disequilibrium with the true risk polymor-
phism (11). There were marked contrasts among the three
ethnic groups studied (table 3). We also found evidence of
an interaction with another risk factor in North Europeans.
Potential interactions with other factors than age, gender,
and APOE*4 status should be examined, particularly inter-
actions with cardiovascular risk factors.
ACKNOWLEDGMENTS
Financial support was received from Bristol Myers
Squibb, Phytopharm plc, the Medical Research Council,
the Norman Collisson Foundation, and the Takaya ma
Foundation. Dr. P. G. Kehoe is supported by a Gestetner
Foundation Fellowship.
The authors thank all those groups who have undertaken
ACE/Alzheimer’s disease association studies, thereby pro-
viding the data for these analyses. They especially thank
those authors who generously responded to requests for
primary data: Dr. V. Alvarez, Dr. H. Arboleda, Dr.
A. Capurso, Dr. L. Cook, Dr. M. M. Esiri, Dr. R. Fellin,
Dr. L. Ferna
´
ndez-Novoa, Dr. U. Finkh, Dr. R. C. P. Go, Dr.
H. Gurling, Dr. N. Helbecque, Dr. M. R. Keikhaee, Dr.
C. L. Lendon, Dr. S. Lovestone, Dr. D. M. Mann, Dr. K. M.
Mattila, Dr. S. P. McIlroy, Dr. L. Myllykangas, Dr. F. Panza,
Dr. R. T. Perry, Dr. D. C. Rubinsztein, Dr. R. Scacchi,
Dr. D. Seripa, Dr. H. Weiner, Dr. C. Wu, and Dr. G. Zuliani.
The authors are particularly grateful to Dr. U. Finkh and
colleagues for highlighting the issue of Hardy-Weinberg
disequilibrium in ACE studies (40) and to them and to
Dr. A. Capurso and collea gues (17, 39) for pointing out the
ethnic differences in ACE indel frequencies. The authors
also warmly thank Dr. M. Pembrey for advice on genetics,
Dr. J. Marchini for advice on statistics, C. A. Aldridge
for administrative support, M. G. Lehmann for help with
mathematical calculations, and Dr. J. Wu for Chinese
translations.
Conflict of interest: none declared.
Note added in proof: Two further association studies
have been published since submission of this article: Ko
¨
lsch
et al. (Neurosci Lett 2005;377:37–9) and Zhang et al.
(Dement Geriatr Cogn Disord 2005;20:52–6).
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Am J Epidemiol 2005;162:305–317
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    • "To explore the plausible pathophysiological role of ACE in contributing to AD, the genetic marker of the ACE I/D, an I (insertion) or D (deletion) of the Alu element located in Intron 16 has also been extensively examined in association with the risk for AD. The I allele of the ACE gene has been indicated as a potential susceptibility marker for AD in many previous studies [29][30][31][32]. However, some contradicting results keep the previous conclusions as pending [33]. "
    Full-text · Article · Aug 2016 · Neurobiology of Aging
    • "Amongst individuals carrying the ε4 risk allele , the next risk predictor was being ≥77 years old. Of these eldest individuals, the next differentiator was carrying one or more of the minor allele for rs4343 in ACE, an AD-risk gene [48,49]. The fourth differentiator of this subgroup was being homozygous for the major allele of rs8053211 in ATP2C2, a gene associated with dyslexia and other language traits [50,51], as carriers of one or two copies of the minor allele had a higher risk for diagnosis of AD. "
    [Show abstract] [Hide abstract] ABSTRACT: Heritability of Alzheimer's disease (AD) is estimated at 74% and genetic contributors have been widely sought. The ε4 allele of apolipoprotein E (APOE) remains the strongest common risk factor for AD, with numerous other common variants contributing only modest risk for disease. Variability in clinical presentation of AD, which is typically amnestic (AmnAD) but can less commonly involve visuospatial, language and/or dysexecutive syndromes (atypical or AtAD), further complicates genetic analyses. Taking a multi-locus approach may increase the ability to identify individuals at highest risk for any AD syndrome. In this study, we sought to develop and investigate the utility of a multi-variant genetic risk assessment on a cohort of phenotypically heterogeneous patients with sporadic AD clinical diagnoses. We genotyped 75 variants in our cohort and, using a two-staged study design, we developed a 17-marker AD risk score in a Discovery cohort (n = 59 cases, n = 133 controls) then assessed its utility in a second Validation cohort (n = 126 cases, n = 150 controls). We also performed a data-driven decision tree analysis to identify genetic and/or demographic criteria that are most useful for accurately differentiating all AD cases from controls. We confirmed APOE ε4 as a strong risk factor for AD. A 17-marker risk panel predicted AD significantly better than APOE genotype alone (P < 0.00001) in the Discovery cohort, but not in the Validation cohort. In decision tree analyses, we found that APOE best differentiated cases from controls only in AmnAD but not AtAD. In AtAD, HFE SNP rs1799945 was the strongest predictor of disease; variation in HFE has previously been implicated in AD risk in non-ε4 carriers. Our study suggests that APOE ε4 remains the best predictor of broad AD risk when compared to multiple other genetic factors with modest effects, that phenotypic heterogeneity in broad AD can complicate simple polygenic risk modeling, and supports the association between HFE and AD risk in individuals without APOE ε4.
    Full-text · Article · Mar 2015
    • "However, more recent haplotype studies and Genome-Wide Association Studies (GWASs) have not supported the role of ACE as a genetic risk factor for AD. Previous meta-analyses have suggested, however, the possibility that ACE may mediate risk via epistatic interactions with other genetic risk variants (Lehmann et al., 2005). Recently it was shown that the variants in the ABO blood group locus, independently and when combined with ACE variants, explained a greater proportion of the population variance of ACE in plasma than ACE alone (Terao et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: The ABO blood group locus was recently found to contribute independently and via interactions with angiotensin-converting enzyme (ACE) gene variation to plasma levels of ACE. Variation in ACE has previously been not only implicated as individually conferring susceptibility for Alzheimer's disease (AD) but also proposed to confer risk via interactions with other as yet unknown genes. More recently, larger studies have not supported ACE as a risk factor for AD, whereas the role of ACE pathway in AD has come under increased levels of scrutiny with respect to various aspects of AD pathology and possible therapies. We explored the potential combined involvement of ABO and ACE variations in the genetic susceptibility of 2067 AD cases compared with 1376 nondemented elderly. Including the effects of ABO haplotype did not provide any evidence for the genetic association of ACE with AD. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jan 2015
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