The geographic distribution of the ACE II genotype: A novel finding

Abstract

Angiotensin converting enzyme (ACE) gene polymorphism insertion (I) or deletion (D) has been widely studied in different populations, and linked to various functional effects and associated with common diseases. The purpose of the present study was to investigate the relationship between the ACE I/D frequency in different populations and geographic location; ACE I/D allele frequency in the Lebanese population and ACE II genotype contribution to the geographic trend were also identified. Five hundred and seventy healthy volunteers were recruited from the Lebanese population. Genomic DNA was extracted from buccal cells, and amplified by polymerase chain reaction; products were then identified by gel electrophoresis. The frequencies of the different ACE I/D genotypes were determined and tested for Hardy-Weinberg equilibrium (HWE). To assess the relationship between ACE I/D frequency and geographic location, and to identify how the Lebanese population contributes to the geographic trend in ACE I/D frequencies, Eurasian population samples and Asians were incorporated in the analyses from the literature. The frequency of the I allele in the Lebanese population was 27% and the corresponding II genotype was at a frequency of 7.37% (in HWE; P=0.979). The ACE I allele and genotype frequencies show an association with longitude, with frequencies increasing eastwards and westwards from the Middle East.

Full text

The geographic distribution of the ACE II genotype:
a novel finding
Y. B. SAAB
1
,
2
*, P. R. G A R D
2
AND A. D. J. OVERALL
2
1
School of Pharmacy, Lebanese American University, Byblos, Lebanon
2
School of Pharmacy & Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK
(Received 8 August 2007 and in revised form 2 November 2007 )
Summary
Angiotensin converting enzyme (ACE) gene polymorphism insertion (I) or deletion (D) has been
widely studied in different populations, and linked to various functional effects and associated with
common diseases. The purpose of the present study was to investigate the relationship between the
ACE I/D frequency in different populations and geo graphic location; ACE I/D allele frequency
in the Lebanese population and ACE II genotype contribution to the geographic trend were also
identified. Five hundred and seventy healthy volunteers were recruited from the Lebanese
population. Genomic DNA was extracted from buccal cells, and amplified by polymerase chain
reaction; products were then identified by gel electrophoresis. The frequencies of the different ACE
I/D genotypes were determined and tested for Hardy–Weinberg equilibrium (HWE). To assess the
relationship between ACE I/D frequency and geographic location, and to identify how the Lebanese
population contributes to the geographic trend in ACE I/D frequencies, Eurasian population
samples and Asians were incorporated in the analyses from the literature. The frequency of the
I allele in the Lebanese populati on was 27% and the corresponding II genotype was at a frequency
of 7
.
37% (in HWE; P=0
.
979). The ACE I allele and genotype frequencies show an association with
longitude, with frequencies increasing eastwards and westwards from the Middle East.
1. Introduction
Angiotensin converting enzyme (ACE) is a
membrane-bound dipeptidyl carboxypeptidase ecto-
enzyme that is expressed both peripherally and in the
central nervous system. ACE is mainly responsible
for the production of angiotensin II, a potent vaso-
constrictor, and the inactivation of the potent vaso-
dilator bradykinin. Because of its broad-ranging
effects on vascular homeostasis, ACE has become
a candidate for association studies with common dis-
eases. The ACE gene maps to chromosome 17q23,
spans 21 kb, and comprises 26 exons and 25 introns
(Hubert et al., 1991); the GenBank accession number
is AC002345 or AF118569 (www.ncbi.nlm.nih.gov).
To date, 259 polymorphisms have been reported in
the ACE gene (www.ncbi.nlm.nih.gov) , with the I/D
polymorphism, first reported by Rigat et al. (1990),
attracting the most interest. This I/D polymorphism is
defined by the presence (insertion; I) or absence (de-
letion; D) of a 287 base pair (bp) Alu repeat sequence
in intron 16 (Rieder et al., 1999). The ACE I/D poly-
morphism has been linked to various functional ef-
fects, for example the DD genotype being associated
with high plasma ACE levels in addition to numerous
diseases. One of the most extensively studied associa-
tions is with cardiovascular diseases, including myo-
cardial infarction (Cambien et al., 1992; Nakai et al.,
1994), left ventricular hypertrophy and dysfunction
(Schunkert et al., 1994), dilated cardiomyopathy
(Raynolds et al., 1993; Harn et al., 1995) hypertrophic
cardiomyopathy (Marian et al., 1993), carotid thick-
ening (Castellano et al., 1995; Kauma et al., 1996),
venous thrombosis (Philipp et al., 1998), nephropathy
* Corresponding author. School of Pharmacy, Lebanese American
University, Byblos, Lebanon, P. O. Box: 36 F 19. Telephone:
+961 9 547254 (ext. 2312). Fax:+961 9 547256. e-mail: ysaab@
lau.edu.lb
Genet. Res., Camb. (2007), 89, pp. 259–267. f 2007 Cambridge University Press
259
doi:10.1017/S0016672307009019 Printed in the United Kingdom

(Schmidt & Ritz, 1997) and coronary restenosis after
stent implantation (Amant et al., 1997). Currently, the
best evidence for an association with the ACE I/D
polymorphism is with arterial hypertension in men
(Fornage et al., 1988 ; Higaki et al., 2000); one study
of male carriers of the DD genotype showed a 1
.
6-fold
increase in risk (O’Donnell et al., 1998). It has also
been suggested that the ACE I/D polymorphism may
contribute to an individual’s susceptibility to affective
disorders and the onset of action of antidepressant
therapies (Arinami et al., 1996 ; Baghai et al., 2001,
2004, 2005; Gard et al., 2004 ; Saab et al., 2007 b).
The ACE DD genotype was also found to be a sig-
nificant risk factor for children with congenital
renal malformations going on to develop progressive
renal failure (Hohenfellner et al., 2001). Also, it
was found that in patients with lupus nephritis,
the ACE I/D genotype was associated with the se-
verity of the disease and a poor prognosis (Guan et al.,
1997).
Saab et al. (2004) have typed the ACE I/D gene
polymorphism in the Lebanese population, where
the homozygous II genotype accounted for 8 % of the
sample – an incidence that was found to be atypi cally
low relative to European and East Asian populations.
These preliminary results suggested that the ACE II
genotype frequency might vary according to a geo-
graphic trend, as has been postulated for other Alu
insertion polymorphisms (Stoneking et al., 1997).
The objective of the present study was to determine
whether the ACE I/D gene polymorphism frequency
did indeed correlate with geographic distance and
then identify whether the ACE gene can be considered
as a genetic marker for the past demography of
human populations.
2. Materials and methods
(i) ACE genotype frequency determination
in Lebanese subjects
A total of 570 healthy volunteers were recruited from
the Lebanese population. Included were non-obese
subjects (body mass index (BMI) <29
.
5kg/m
2
) with
no history or clinical evidence of diabetes, cardio-
vascular problems, hypertension, renal insufficiency
and/or depression. All study subjects are of Lebanese
origin, and were living in Lebanon at the tim e of
study. Exclusion criteria were set to achieve parity
with other studies.
(ii) Sample collection and DNA extraction
Each volunteer was instructed to give a DNA sample
from the cheek using a cheek swab. The sample was
used for DNA extraction. DNA was extracted using
a protocol described by Saab et al. (2007b).
(iii) ACE I/D gene polymorphism genoty ping
The presence of the insertion/deletion allele in intron
16 of the ACE gene was detected using the method
of Rigat et al. (1990) with some modifications (Sery
et al., 2001). The sequence of the sense oligonucleo-
tide primer is 5k-CTG GAG ACC ACT CCC ATC
CTT TCT-3k and the antisense primer 5k-GAT GTG
GCC ATC ACA TTC GTC AGA T-3k. Polymerase
chain reaction was performed in a final volume of
25 ml containing 50 mM KCl, 10 mM Tris-HCl,
pH 8
.
4, 5 U/ml MgCl
2
,0
.
5 mM of each dNTP, 0
.
6U
Taq DNA polymerase, 0
.
2 mM of each primer, and
3 ml of DNA solut ion. PCR products were separated
and sized by electrophoresis on a 2
.
5% agarose gel
and visualized directly with ethidium bromide staining.
The insertion allele manifested as a 490 bp band, and
the deletion allele was visualized as a 190 bp band.
Because of the possibility of preferential amplification
of the D fragment in relation to the I fragment, re-
sulting in mistyping of I/D as DD genotype, all DD
genotypes were confirmed (Odawara et al., 1997).
(iv) ACE gene geographic mapping
We identified literature, published in English between
1984 and 2006, reporting ACE I/D gene polymor-
phisms. The extracted data are summarized and tabu-
lated in Table 2. Excluded were studies of a small
sample size (<48), studies where the subjects’ origins
were unknown and where subjects were known to be
suffering from a disease.
The exception was the Kuwait sample, which com-
prises 48 individuals suffering from nephropathy. This
sample was included due to the shortage of available
samples from the Middle East, but app ears to display
allele and genotype frequencies consistent with those
observed in the region. In addition, the genotypes did
not appear to be inconsistent with Hardy–Weinberg
expectations.
(v) Statistical analysis
Statistical analyses were performed using SPSS
version 12 for Window s. The study samples’ allele
and genotype frequencies were estimated by the gene
counting method. The agreement with Hardy–
Weinberg equilibrium of the observed genotypic dis-
tribution for the ACE I/D alleles was tested using
Fisher exact tests. A P value of <0
.
05 was considered
statistically significant.
Genetic distances were estimated assuming that
differences in allele frequency distributions between
populations were due to drift. Pairwise distances were
calculated as d=ln (1 – F
ST
) (Weir, 1996). Nei’s
genetic distances (Nei & Feldman, 1972) were also
calculated for the purpose of constructing neighbour-
joining trees (Saitou & Nei, 1987) using the
Y. B. Saab et al. 260

GENDIST and NEIGHBOR programs in PHYLIP
3.65 (Felsenstein, 1993) and the genetic data analysis
package GDA (Lewis & Zaykin, 2001). In most cases,
specific geographic locations relating to the samples
were not specified in the literature, so geographic
distances were taken as pairwise distances (in kilo-
metres) betw een capital cities of the country in ques-
tion. To identify whether any co rrelation exists
between the two matrices (genetic and geographic
distances), a Mantel test was performed (10 000 per-
mutations, using the Pearson correlation coefficient)
using XLSTAT (Kovach Computing Services, 2007).
3. Results
(i) Subjects’ demog raphic characteristics
A total of 570 Lebanese subjects were included in the
study, which aimed to determine the ACE gene I/D
polymorphism prevalence in the Lebanese popu-
lation. The study samples con sisted of 51
.
9% and
48
.
1% males and females, respectively. The mean age
was 28
.
63 years (range 18–69 years) and the average
BMI was 23
.
04 kg/m
2
(range 17
.
15–28
.
41 kg/m
2
).
(ii) ACE genotype distribution in Lebanese and
Hardy–Weinberg equilibrium
The detailed distribution of the ACE genotypes in the
Lebanese population is depicted in Table 1. The
prevalence of the D allele was 73%, and the II geno-
type accounted for 7
.
37%. Genotype frequencies
were found to be in Hardy–Weinberg equilibrium
(P=0
.
743, Fisher exact test, 10 000 permutations).
(iii) ACE II genotype prevalence among different
populations
The ACE allele frequencies of different populations
retrieved from the literature, along with that of this
study’s finding, are depicted in Table 2. The results
suggest that the ACE II genotype frequency decreas es
according to a geographic trend from northern
Europe to southern Europe, and on to the Medi-
terranean region. Moreover, moving geographically
eastward, the II genotype prevalence appears to
increase progressively. The results of the Mantel test
show a reasonable and significant correlation between
geographic and genetic distances between the popu-
lations (r=0
.
478984, P<0
.
0001). On further analysis,
it appears that this is largely influenced by II fre-
quency and longitude correlation (R
2
=0
.
727), where
the II genotype frequency declines on moving from
Europe to the Middle East, followed by an increase
moving eastwards to Asia (Fig. 1). There was no
meaningful relationship between II genotype fre-
quency and latitude (R
2
=0
.
027). For the correlation
with longitude, the quadratic relationship proved
to be more significant than the linear relationship
(starting with a GLM maximal model: II frequency=
b
0
+b
1
x
1
2
+b
2
x
2
, where x is the degrees east of
Greenwich, UK and the b parameters were estimated
by maximum likelihood; removing the quadratic
term resulted in a significant difference between the
deviance values of the two models (Crawley, 1993),
where P=0
.
0001). The correlation between I allele
frequency and longitude gave a slightly weaker re-
lationship than that with the II gen otype (R
2
=0
.
54).
In brief, the II genotype had an average frequency of
23% in northern Europe, 20% in the UK, 15 % in
Spain, 14 % in north Italy, 12 % in south Italy, 7% in
Lebanon, 6% in the United Arab Emirates (UAE),
2% in Kuwait, and then an average of 35 % in China
and 45% in Japan.
Fig. 2 shows the neighbour-joinin g tree relating all
36 population samples using Nei’s genetic distance
(Nei & Feldman, 1972). Because the ancestral state of
the Alu insertion polymorphisms is considered to be
the absence of the insertion, the tree could be rooted
using a hypothetical outgroup consisting of individ-
uals fixed for the D allele. The multiple popul ation
samples of identical country of origin were averaged
to condense the tree.
4. Discussion
(i) ACE I/D genotype distribution
According to a meta-analysis of 145 studies with
49 959 subjects, the overall prevalence of the D allele
Table 1. ACE I/D observed genotypes/allele frequencies in the Lebanese
population compared with expected genotypes
Genotype
Observed
genotype
N (%)
Observed
allele
frequency
Expected
genotype
N (%)
Chi-square
P
II 42 (7
.
37) I: 0
.
27 41 (7
.
29) 0
.
979
ID 219 (38
.
42) D: 0
.
73 225 (39
.
42)
DD 309 (54
.
21) 304 (53
.
29)
All 570 (100) 570 (100)
The geographic distribution of the ACE II genotype: a novel finding 261

was 54
.
0%. The II, ID and DD genotype fre-
quencies were 22
.
5%, 47
.
0% and 30
.
5%, respectively
(Staessen et al., 1997). Ethnicity was a major deter-
minant of the D and I allele frequencies as the preva-
lence of the D allele was 39
.
1% in Asians, 56
.
2% in
Caucasians and 60
.
3% in blacks (Staessen et al.,
1997). In the present study, the D allele had a fre-
quency of 73
.
42%, which is consistent with the other
two Middle Eastern populations (Kuwait and UAE)
in being amongst the highest recorded.
(ii) ACE I/D gene polymorphism: a genetic marker
The average ACE II genotype frequency in control
subjects in different populations of different countries
was thoroughly examined and compared. Never-
theless, we accept that the comparison of the allele
and genotype frequencies with other published
studies has to be considered with some caution since
Table 2. ACE II genotype frequency in different populations/countries
Country Study authors
Year of
publication
No. of
subjects
ACE II
genotype
frequency (%)
Sweden Kurland et al. 2001 59 27
Denmark Bladbjerg et al. 1999 199 23
United Kingdom Kehoe et al. 1999 386 23
United Kingdom Steeds et al. 2001 507 22
United Kingdom Narain et al. 2000 342 18
Netherland Hosoi et al. 1996 61 20
Hungary Barkai et al. 2005 120 27
Belgium Gu et al. 1994 109 19
Germany Ebert et al. 2005 145 23
Germany Filler et al. 2001 200 18
France Blanche et al. 2001 560 18
France Girerd et al. 1998 340 17
Spain Alvarez et al. 1999 400 15
Spain Coll et al. 2003 133 15
Italy Di Pasquale et al. 2005 684 18
Italy Panza et al. 2002 252 13
Turkey Tanriverdi et al. 2005 102 24
Turkey Serdaroglu et al. 2005 287 22
Turkey Bedir et al. 1999 143 13
Lebanon Saab et al. Current Study 570 7
Kuwait Al-Eisa et al. 2001 48 2
United Arab Emirates Saeed et al. 2005 130 6
India Patil et al. 2005 300 26
China Thomas et al. 2001 119 33
China Ohishi et al. 1994 175 37
China Young et al. 1998 183 39
China Iwai et al. 1994 122 41
China Yan et al. 2005 352 41
Korea Ryu et al. 2002 167 34
Korea Um et al. 2003 613 37
Taiwan Lee & Tsai 2002 750 47
Japan Katoh et al. 2005 270 41
Japan Odawara et al. 1997 248 42
Japan Mannami et al. 2001 3657 43
Japan Maguchi et al. 1996 84 48
Japan Ishigami et al. 1995 87 51
0.6
0.5
0.4
0.3
0.2
0.1
0
0 50 100 150
East Coordinates
II Genotype Frequency
Fig. 1. Plot of ACE II genotype frequencies and
coordinates east of Greenwich, UK. For the linear
relationship R
2
=0
.
599; for the quadratic, R
2
=0
.
727
.
The Lebanese population is circled.
Y. B. Saab et al. 262

published data might have been generated using
slightly diff erent methodologies, and thus there is a
possibility of discrepancies in genotype classification
(Ueda et al., 1996) – in particular, since some geno-
typing methodologies misclassify ID heterozygotes
as DD homozygotes. Although such a misclassi-
fication can result in deviations from Hardy–
Weinberg equilib rium, which was not observed in our
study, it was considered prudent to base our analysis
on the II genotype frequencies in addition to the
I allele frequencies. Indeed, it may be for this reason
that the stronger correlation with longitude was
observed with the genotype data than the allele fre-
quency scores.
Genetic polymorphisms have often been found to
show geographic clines, many of which have been put
to great use in offering insights into the historical
movements of peoples around the globe since at least
as far back as Neolithic times (Cavalli-Sforza et al.,
1993; Barbujani et al., 1998). Such interpretations
of clines are not without their critics (e.g. Richards &
Sykes, 1998) and care is required not to over-interpret
geographic patterns when they emerge. In particular,
little confidence can be placed in the timing of popu-
lation movement. Broader conclusions, such as iden-
tifying the origin of a particular polymorphism on the
basis of its relative frequency, are less controversial;
and this is more so with Alu elements, which are
considered to be highly stable polymorphisms, where
deletion of newly inserted elements is a rare event
(Stoneking et al., 1997). Low frequencies of the
insertion are therefore indicative of the ancestral
state, and African populations tend to have not only
the lowest frequency of the insertion (Bayoumi et al.,
2006) but also the greatest variation in frequen cy
(Stoneking et al., 1997). On this basis it would appear
that the ALU deletion within the ACE gene was,
of the populations studied here, Middle Eastern in
origin. Given that the human migration out of Africa
is likely to have journeyed through the Middle East
before migrating east and west, it is to be expected
that the Lebanese population should be ancestral
with regard to the ACE polymorphism and to have
a relatively lower frequency of the insertion allele ;
this is borne out in the frequency–coordinate corre-
lation analysis in Fig. 1, where a significant quadratic
relationship was observed between both the I allele
frequency (not shown) and II genotype frequency and
the coordinates east of Greenwich, UK. The picture is
less clear in the tree reconstruction in Fig. 2. Although
the Middle Eastern populations appear quite distinct
from both European and Asian populations, these
latter groups are not well resolved, most likely due to
the fact that only a single locus has been investigated
for these populations.
In the analysis of modern human origins, genetic
maps demonstrating allelic clines have been quite
revealing. Classical attempts to distinguish distinct
ancestries of human subgroups (Cavalli-Sforza et al.,
1996) have been quite succ essful in employing classi-
cal genetic markers, such as the different gene fre-
quencies of A and B blood antigens. Consequently,
ABO blood groups have been used as a genetic
marker to differentiate human subgroups, on the
basis of their distinct demographic histories. It is also
considered that the frequency of an allele is likely to
be higher at its place of origin as well as in the region
where selective factors favour it. ABO gene fre-
quencies again offer an example of a gene that follows
this trend (Cavalli-Sforza et al., 1996). The gradient
of decreasing frequencies has also been shown with
haplotypes V and VI (Lucotte et al., 2001). Mapping
ACE I/D polymorphism genotype frequencies from
both this study and those of other authors on to a
geographic map, shows the ACE gene to have a geo-
graphic trend of expansion consistent with what is
known about the migration of modern Hom o sapiens
out of Africa, thus qualifying the ACE gene as
another useful marker tool for studying prehistoric
human demography. It remains to be seen, however,
whether disorders associated with the ACE gene
show geographic trends corresponding with the major
polymorphisms, including the Alu I/D.
Netherland
Taiwan
Japan
Korea
China
Denmark
UK
Belgium
France
Spain
Lebanon
Kuwait
UAE
Italy
Turkey
Germany
India
Hungary
Sweden
Fig. 2. Neighbour-joining tree of population relationships.
The tree is rooted by a hypothetical ancestral population
fixed for the ACE D allele. UAE, United Arab Emirates.
The geographic distribution of the ACE II genotype: a novel finding 263

5. Conclusion
In summary, in view of the reported associations of
ACE gene polymorphisms and different diseases,
ACE genotypes were assessed in the Leba nese popu-
lation. The II genotype frequency was 7
.
37%. Com-
paring this study’s finding with that of other studies
in different populations, the ACE gene can be con-
sidered a useful genetic marker for gaining an insight
into the historical migrations of human populations,
in particular the frequency cline of the wild-type D
allele. Future pharmacogenetic studies are likely to
reveal the natural selection for this gene’s geographic
variation and the pharmacological role of this enzyme
in different populations.
We acknowledge the Lebanese American University (LAU)
for funding the project. We thank Dr Hussam Atat,
Mr Bechara Mfarej and LAU Pharm D graduates of 2004
and 2005 for their assistance in the sample collection and
laboratory work.
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