Annals of Human Biology, May–June 2006; 33(3): 319–329
ORIGINAL ARTICLE
Haplotype structure of five SNPs within the ACE gene
in the Tunisian population
MAHA REBAÏ, NAJLA KHARRAT, IMEN AYADI, & AHMED REBAÏ
Bioinformatics Unit, Centre of Biotechnology of Sfax, Sfax, Tunisia
(Received 24 November 2005; revised 2 January 2006; accepted 23 January 2006)
Abstract
Background: The Angiotensin-Converting Enzyme (ACE) is a candidate gene in the aetiology of several
common diseases. The study of the haplotype structure of this gene is of interest in diagnosis and
in pharmacogenomics.
Aim: The study investigated the haplotype profile of single nucleotide polymorphisms (SNPs) within the
ACE gene in the Tunisian population and compared it with other populations.
Subjects and methods: Five SNPs (rs1800764, rs4291, rs4309, rs4331, rs4340) covering a region of 15.6 kb
of the ACE gene were typed by PCR-digestion in a sample of 100 healthy subjects.
Results: All SNPs were polymorphic and in Hardy–Weinberg equilibrium. A total of 21 haplotypes were
identified but only eight had a frequency of more than 1%. The four most common haplotypes had
a cumulative frequency of 87.4%. The ‘Yin–Yang’ phenomenon (the two major haplotypes are
complementary at all sites) was found. Linkage disequilibrium between all pairs of loci was
highly significant (p<105). A simple and efficient statistical procedure was used to identify three
important SNPs.
Conclusion: The Tunisian population showed a different haplotype structure from the European one for
the ACE gene and three important SNPs were identified. These will be very helpful in future association
studies in the Tunisian and North African populations.
Keywords: ACE, SNP, Tunisian, haplotype, linkage disequilibrium
Introduction
The analysis of haplotypes for multiple single nucleotide polymorphisms (SNPs) in a gene or
a region of interest is a fundamental step in the process of identification of DNA variants
underlying complex inherited diseases. With the availability of the whole sequence of the
human genome, with more than 10 millions SNPs, and the near-completion of the HapMap
project (International HapMap Consortium 2003, 2005a), it is now possible to target
Correspondence: Ahmed Reba€ı,
E-mail: [email protected]
Centre
of
Biotechnology
of
ISSN 0301–4460 print/ISSN 1464–5033 online ß 2006 Informa UK Ltd.
DOI: 10.1080/03014460600621977
Sfax,
PO
Box
‘K’,
3038
Sfax,
Tunisia.
320
M. Reba€ı et al.
association studies by choosing the most appropriate SNPs based on their polymorphism
features, position within the gene or region of interest and linkage disequilibrium
among them.
The HapMap project, started in 2002 has the objective to provide by 2006, a full
haplotype map of the human genome including 3.5 million SNPs in four populations
of African, European and Asiatic origin (Schmidt 2005). This will provide useful
information for geneticists and medical geneticists who are interested in identifying
haplotypes associated with particular forms of common diseases or drug response and will
open the door for a personalized approach of medicine. One of the major objectives of
haplotype studies is to find haplotype tagging SNPs (Johnson et al. 2001), which are SNPs
that best represent a gene or region of interest. Finding such SNPs (denoted tagSNP or
tSNP and sometimes htSNP) will result in a considerable saving of time and money in
genotyping for association and pharmacogenomics studies (Allen-Brady and Camp 2005;
Camp et al. 2005).
The Angiotensin-Converting Enzyme (ACE) or kininase II (EC 3.4.15.1, MIM 106180)
catalyses the conversion of the angiotensin I to the physiologically active peptide angiotensin
II, which controls fluid–electrolyte balance and systemic blood pressure. A large number of
physiological, pharmacological and genetic studies have shown the importance of ACE and
its inhibition in the pathogenesis and treatment of a variety of cardiovascular and associated
diseases (see Niu et al. 2002 and Scharplatz et al. 2004 for good reviews). Particularly, DNA
variants within the ACE gene were shown to be involved in the aetiology of several common
diseases and in the therapeutic response to several drugs (see the Genetic Association
Database at http://geneticassociationdb.nih.gov for a large list of references; National
Center for Biotechnology Information 2005b).
The ACE gene maps to 17q23 region extending over 45 kb genomic size. The two most
abundant RNA variants of this gene encode two isozymes, the somatic form and the
testicular form that are equally active. The somatic variant is a transcript of 25 exons
(exons 1–25 and corresponding to 20 kb genomic size) while the testicular variant includes
an alternate in-frame exon in the 50 coding region and results in a different N-terminal part
of the protein.
The ACE gene has 243 SNPs in the human SNP database (dbSNP, http://
www.ncbi.nlm.nih.gov/SNP, National Center for Biotechnology Information 2005a) build
124 among which 127 are validated. Among these, 50 SNPs within the coding region or in
close proximity to the ACE gene were typed in the HapMap project, most of them (37
SNPs) are located in the 50 part of the gene (corresponding to the part coding for the somatic
variant). At the current status of the HapMap project (HapMap web site, http://
www.hapmap.org; International HapMap Consortium 2005b), 28 of these 50 SNPs
showed no polymorphism in all studied populations. Keavney et al. (1998) studied the
haplotype structure of 10 SNPs within the ACE gene in the European (British) population.
Zhu et al. (2000) genotyped seven SNPs in the 30 end of ACE in Afro-Caribbean subjects
and evaluated the linkage disequilibrium between them, while Zhu et al. (2001) studied the
association between 13 SNPs within the ACE gene with the plasma ACE concentration and
blood pressure in a Nigerian population sample.
However, several recent studies on linkage disequilibrium structure and haplotype
distribution of SNPs in different populations have shown that results in this field
have limited transferability from one population to another (Nejentsev et al. 2004;
Mueller et al. 2005).
Here we report the results of haplotype analysis with five SNPs in the ACE gene in
a sample of 100 controls from the Tunisian population. Comparison with other populations
321
Haplotype structure of ACE gene SNPs in Tunisia
revealed different features in haplotype structure. Based on linkage disequilibrium among
markers, three SNPs were retained as being the most important.
Materials and methods
Subjects
A sample of 100 healthy unrelated subjects from southern Tunisia (Sfax region) was
recruited for this study. These were chosen randomly from a larger sample of volunteers
having given informed consent and blood samples. There were 50 males and 50 females with
a mean age of 39 years.
Isolation of genomic DNA from peripheral blood
Genomic DNA from the blood sample was extracted using a standard phenol–chloroform
protocol and was stored at 20 C for SNP genotyping.
Genotyping of SNPs
The five SNPs typed on controls were chosen from the dbSNP build 121 (2004) according
to the following conditions: the average distance between SNPs is 5 kb, and the frequency of
the minor allele is more than 5%. We looked preferably for SNPs located in coding and
regulatory regions of the somatic isoform. To allow comparison with other populations,
we used a subset of SNPs from the study of Keavney et al. (1998) and Zhu et al. (2001).
Names, features and positions of the SNPs relative to the gene are given in Table I and
Figure 1.
The ACE ID (insertion/deletion) polymorphism and four biallelic SNPs were typed by
PCR amplification followed by restriction-enzyme digestion. The PCR reactions were
performed using a GenAmp PCR system 9600 thermocycler (Perkin–Elmer). PCR products
Table I. Features of the five ACE SNPs and primer sequences and enzymes used for their genotyping.
SNP
Accession
number
Type
Position
(bp)
ACE3
rs1800764
T/C
3905
ACE4
rs4291
A/T
240
ACE6
rs4309
C/T
5489
ACE7
rs4331
A/G
9618
ACE ID
rs4340
I/D
11 698
Primer sequence
F: 50 ATAGTGTATATAGGGCTTGGTAC30
R: 50 AGAAGATATTTGCAAAGTATGTACTG30
F: 50 ACCATGGCCTGGTGAAGAAGC30
R: 50 CGGCTCTGCCCCTTCTCCTGCGC3
Fint: 50 TGTCACTCCGGAGGCGGGAGGCT30
Rint: 50 GAGAAAGGGCCTCCTCTCTCT30
F: 50 AGTGCACACGGGTCACGATG30
R: 50 CCCCCCGACGCAGGGAGCC30
F: 50 CACACCCTGAAGTACGGCAC30
R: 50 TCCTCCAGCTCCTGGGCAG30
F: 50 CTGGAGACCACTCCCATCCTTTCT30
R: 50 GATGTGGCCATCACATTGGTCAGAT30
Fv: 50 TGGGACCACAGCGCCCGCCACTAC30
Rv: 50 TCGCCAGCCCTCCCATGCCCATAA30
Restriction
enzyme
PstI
XbaI
MspI
HaeII
Position relative to the first base position of the first exon of the gene. For ACE4, Fint and Rint are internal primers
used in nested PCR and for ACE ID Fv and Rv are primers used in homozygotes D/D validation.
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M. Reba€ı et al.
ACE3
271
+1
−3900
ACE 7
ACE6
ACE 4
−240
Exon1
5394
5617
5489
Exon 8
9485
ACE ID
9643 9914
10001
11689
9618
Exon 15
Exon 16
SNP within 5’UTR
Exonic SNP
Intronic SNP
Figure 1. Positions of the studied SNPs within the ACE gene (The colour version of this figure is
included in the online version of the journal).
were digested with the appropriate enzyme (Table I) and were run on acrylamide gels (10%)
stained with ethidium bromide, and scored by UV visualization. The SNPs ACE3, ACE4,
ACE7 were amplified using the conditions described by Zhu et al. (2001) while the SNP
ACE6 was amplified following the conditions of Keavney et al. (1998). For the ACE ID, no
digestion is needed but the PCR was carried out in two steps; first PCR was performed with
the primers from Keavney et al. (1998). Since the preferential amplification of the D allele
(190 bp) over the I allele (490 bp) have been reported with these primers (Lindpaintner et al.
1995), a second PCR was performed on putative homozygous individuals, i.e. those having
a single D band in first PCR (which may actually be either D/D homozygotes or I/D
heterozygotes). This second PCR uses the primers from Yoshida et al. (1995) and gives
a PCR product of 335 bp only when the I allele is present, thus allowing us to distinguish
I/D from D/D individuals.
Positive and negative controls were used in all PCR and digestion reactions in order to
check for the correct amplification and restriction of the products.
Statistical analysis
The estimation of allele frequency and exact test for Hardy–Weinberg equilibrium was
performed using the Genetic Data Analyses (GDA) program (version 1.1) (Weir 1996).
Inference of haplotypes and their frequencies from genotype data was performed using the
PHASE program (version 2.0.2) (Stephens et al. 2001; Stephens and Donnelly 2003).
Three measures of linkage disequilibrium (LD) between SNPs were then computed from
controls in order to study the LD structure of the gene: gametic disequilibrium from inferred
haplotypes, composite LD from genotype data (Weir 1996) and three-locus LD from
estimated haplotype frequencies based on the Bennett (1954) coefficient. These measures
were standardized as correlation coefficients (r2) so that Nr2 provides a 2 test of LD. The
Haploxt (Abecasis and Cookson 2000) program was used to estimate the LD between pairs
of loci from inferred haplotypes. We calculated composite LD using the GDA program
(Weir 1996). Three locus composite LD were calculated using an Excel worksheet
implementing the formula of Bennett’s coefficient provided by Weir (1996).
We defined a block of LD as a subset of SNPs where three to five haplotypes represent
75–90% of the observed haplotypes in the population (Patil et al. 2001). The determination
of key SNPs was performed using an implementation in R language of the method of
Lin and Altman (2004).
Haplotype frequencies between samples were compared using exact test of population
stratification in GENPOP (Raymond and Rousset 1995).
323
Haplotype structure of ACE gene SNPs in Tunisia
Results and discussion
Distribution of allelic frequencies
The allele frequencies of the five SNPs are given in Table II. No significant deviation from
Hardy–Weinberg equilibrium was found for any of the SNPs and all of them have a good
information content (observed heterozygosities close to 0.5, the maximal value).
The markers ACE3, ACE4 and ACE ID were the most informative.
In dbSNP, only the allelic frequencies of the SNPs ACE6 and ACE7 were available (0.607
and 0.499 for C and A alleles, respectively). These values are significantly different from
those estimated in our sample (p < 0.005). In the HapMap project, only data for the SNP
ACE6 were available for European, Chinese, Japanese and Nigerian population samples.
Frequencies of C allele in these four populations are given in Table III. We see that
the frequency in our population is significantly different from all other populations
(all p < 0.001), being midway between the frequencies in European and African populations.
Note also that the heterozygosity in our population is midway between those of African and
European samples of the HapMap project.
Haplotypes analysis
Twenty one haplotypes were inferred but only eight have a frequency greater than 1%
(Table IV). Cumulative frequency of these eight haplotypes was 90%. The four first
Table II. Allelic frequencies of the five SNPs in a sample of 100 controls from the Tunisian population.
SNP
ACE3
ACE4
ACE6
ACE7
ACE ID
Frequency
Observed H
Expected H
p-value of HWE
C: 0.57; T: 0.43
A: 0.66; T: 0.34
C: 0.75; T: 0.25
A: 0.695; G: 0.305
I: 0.325; D: 0.675
0.440
0.460
0.340
0.390
0.450
0.493
0.451
0.377
0.426
0.441
0.21
0.97
0.21
0.27
0.96
H, Heterozygosity. Observed H is the ratio of number of heterozygotes to total number of individuals. Expected H
is calculated as one minus sum of squares of allele frequencies. HWE, Hardy–Weinberg equilibrium. Exact p-values
for HWE were calculated using the permutation procedure in GDA (Weir 1996).
Table III. Frequency of the C allele of the SNP ACE6 according to the HapMap project and comparison with
the present study.
Population
Chinese
Japanese
European
Present study
Nigerian
n
Frequency
H (%)
2 test
p-value
45
0.244
44.4
65.74
<106
43
0.488
55.8
18.66
1.6 105
60
0.542
41.7
14.77
1.2 104
100
0.75
34.0
–
–
60
0.917
13.3
18.56
2.2 104
n is the number of individuals genotyped, H is the observed heterozygosity in % and 2 test is the Chi-square test
for comparison of allele frequency in that population to allele frequency in the population in the present study.
324
M. Reba€ı et al.
Table IV. Haplotype frequencies of five SNPs in Tunisian and British population samples.
Haplotypes
CTCAD
TATGI
CACAD
TACAD
CTCGI
TACAI
TACGI
CACAI
Present study
(n ¼ 100)
Keavney et al. (1998)
study (n ¼ 555)
2 test
p-value
0.295
0.227
0.201
0.151
0.033
0.022
0.019
0.011
0.298
0.360
0.076
0.095
0.008
0.005
0.050
0.003
0.01
13.84
30.57
5.63
10.16
7.81
3.60
2.37
0.93
0.0002
<106
0.017
0.0014
0.0051
0.057
0.123
2 test is the Chi-square test for comparison of haplotype frequency between the present study and that of
Keavney et al. (1998).
haplotypes (CTCAD, TATGI, CACAD, TACAD) are the most frequent and represent
87.4% of the haplotypes, suggesting that the five SNPs belong to a single block of LD.
The two most frequent haplotypes, CTCAD and TATGI, complement each other at all
sites. This phenomenon, known as ‘Ying–Yang haplotypes’, has been reported by Zhang
et al. (2003) for many genes in the human genome and seems to be a general characteristic
of eukaryotic genomes. According to the estimation of these authors, this phenomenon
would be present in 75–85% of the human genomic regions. The Ying–Yang phenomenon
was also observed in the ACE gene in the study of Keavney et al. (1998) for the 10 SNPs
studied.
We have recomputed haplotype frequencies for the five SNPs studied here, from the data
of Keavney et al. (1998) on 10 SNPs. We noticed that the number of the observed
haplotypes in our population (21 haplotypes) is larger than that found in the study
of Keavney et al. (19 haplotypes). Fourteen haplotypes were common to both studies. It can
be seen from Table IV that the order of the four most frequent haplotypes is different in the
two studies. The most discordant haplotypes among those that are common are TATGI
(14% more frequent in the British population) and CACAD (12.5% more frequent in our
population), which are complementary for all SNPs except ACE4. Exact test of population
differentiation based on haplotype counts revealed a significant difference in haplotype
structure of the two populations (p ¼ 0.006). Haplotype diversities (calculated as one minus
the sum of squares of haplotype frequencies) were, respectively, 0.7992 0.0085 (standard
error) and 0.7672 0.0057, indicating that diversity is significantly larger in our population.
Many studies have, in fact, reported a greater genetic diversity among the old African
population than among Europeans (Gabriel et al. 2002). Due to its special geographic
location in the Mediterranean basin, Tunisia, whose indigenous inhabitants were African
Berbers, has been subject, during its recent history (from 814 BC to 1830), to many
conquests by several populations, including Phoenicians, Romans, Arabs, Vandals and
Ottomans (Julien 2003). This may have resulted in a particular genetic composition,
in between African and Caucasian populations.
Linkage disequilibrium analysis
Table V gives the r2 measures of pairwise LD (haplotypic and composite). Significant LD
was found for all SNP pairs. All p-values of 2 test were <0.0001. The strongest LD was
Haplotype structure of ACE gene SNPs in Tunisia
325
Table V. Gametic and composite linkage disequilibrium r2 coefficients between five SNPs.
SNP
Gametic r2
Composite r2
0.389
0.354
0.320
0.366
0.134
0.114
0.132
0.675
0.575
0.783
0.417
0.379
0.303
0.315
0.201
0.134
0.140
0.706
0.599
0.785
ACE3–ACE4
ACE3–ACE6
ACE3–ACE7
ACE3–ACE ID
ACE4–ACE6
ACE4–ACE7
ACE4–ACE ID
ACE6–ACE7
ACE6–ACE ID
ACE7–ACE ID
See text for definition of r2 coefficients.
1
0.9
rs= −0.5
0.8
Haplotypic LD
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2000
4000
6000
8000 10000 12000 14000 16000
Physical distance (bases)
Figure 2. Relation between linkage disequilibrium and physical distance for the SNPs studied.
observed between SNPs ACE7 and ACE ID (r2 ¼ 0.783). The correlation between
haplotypic and composite r2 values is very high (r ¼ 0.99, p < 0.001). This correlation
provides a measure of the precision of haplotype inference.
In Figure 2 we plotted haplotypic LD measure r2 against physical distance. The relation
between these two measures is non-linear. Spearman rank correlation was rs ¼ 0.50,
indicating that physical distance explains only 50% of the LD variation between loci.
In particular, a strong LD is observed between two distant loci (ACE6 and ACE ID)
whereas a weak LD is observed between two close loci (ACE3 and ACE4). This relation
is similar to that found in genome-wide studies; for example, Abecasis et al. (2001) reported
a correlation of rs ¼ 0.49 from LD data between 127 SNPs in three genomic regions.
Fitting a polynomial quadratic model to our data allows us to predict that useful LD
(r2 > 0.1) will exist on average between loci that are 8 kb apart in the ACE gene region.
We also calculated the LD between three adjacent loci based on the Bennett (1954)
coefficient. Among the combinations tested, only the SNPs ACE6–ACE7–ACE ID showed
326
M. Reba€ı et al.
a significant LD ( p ¼ 0.011), suggesting that those markers constitute a block of LD.
In fact, LD between three loci is rarely found in the human genome; a 5 kb region on
chromosome 12 is the only genome region that has been reported to harbour loci with high
three-point LD (Meng et al. 2003).
Identification of key SNP
In order to find the most important SNP among the five studied, we first calculated the
number of representative SNP based on the formula of Nyholt (2004):
Neff ¼ 1 þ ðL 1Þð1 VarðÞ=LÞ
where L is the total number of SNP and Var() is the variance of eigenvalues of the matrix
of pairwise LD coefficients. We found Neff ¼ 3.4, indicating that three SNPs among the five
studied might be enough to represent the gene region studied. In order to identify the three
most representative SNPs we used the method Lin and Altman (2004), which eliminates
one by one the least informative SNPs, i.e. those SNPs that are associated (after varimax
rotation) with the eigenvectors having the smallest eigenvalues of the LD matrix. The two
least important eigenvectors have eigenvalues of 0.01 and 0.22 and are, respectively,
associated with ACE7 and ACE6 SNPs. The key SNPs that are identified by this approach
are thus ACE3, ACE4 and ACE ID. It is appealing that the two SNPs removed are those
located in coding exons (but are non-synonymous) of the gene while two of the three kept
are located in the 50 regulatory region. ACE ID polymorphism, located in the middle of the
gene (intron 16), is known as being one of the most important markers of the ACE gene.
Many studies have associated this polymorphism with the levels of circulating enzyme
or cardiovascular pathophysiologies (see the Genetic Association Database web site,
http://geneticassociationdb.nih.gov). The most important marker (associated with the
highest eigenvalue, 3.46) identified in this study is ACE3 located in the 50 regulatory
region of the gene, at about 4 kb upstream of the first codon of the gene. This SNP may
become a key marker for future association studies.
Haplotype diversity calculated with these three SNPs is close to its value calculated with
all five SNPs (0.762 vs. 0.799), indicating that the loss of information is minor.
Conclusion
We have studied the haplotype structure of the ACE gene in a sample of the Tunisian
population using five SNPs within this gene: ACE3 (rs1800764), ACE4 (rs4291), ACE6
(rs4309), ACE7 (rs4331) and ACE ID (rs4340).
The statistical analysis of the genotypes of these SNPs allowed us to show that there is
a significant difference in haplotype distribution between our population and the British
population studied by Keavney et al. (1998). The Yin–Yang phenomenon was found in both
studies.
Strong LD was found among all SNPs studied, covering a region of about 16 kb within the
ACE gene. Among these, three SNPs were identified as key SNPs based on a simple
statistical procedure: ACE3 (rs1800764), ACE4 (rs4291) and ACE ID (rs4340). These key
SNPs will be very valuable for future effective association studies of the ACE gene
polymorphisms with diseases or pharmacogenetic studies in the Tunisian and similar North
African populations.
Haplotype structure of ACE gene SNPs in Tunisia
327
Acknowledgements
This work was supported by the Ministry of Scientific Research, Technology and Human
Resources, Tunisia.
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Zhu X, Bouzekri N, Southam L, Cooper RS, Adeyemo A, McKenzie CA, Luke A, Chen G, Elston RC, Ward R.
2001. Linkage and association analysis of angiotensin I converting enzyme (ACE) gene polymorphisms with
ACE concentration and blood pressure. Am J Hum Genet 68:1139–1148.
Zhu X, McKenzie CA, Forrester T, Nickerson DA, Broeckel U, Schunkert H, Doering A, Jacob HJ, Cooper RS,
Rieder MJ. 2000. Localization of a small genomic region associated with elevated ACE. Am J Hum Genet
67:1144–1153.
Résumé. Arrie`re plan: Le gène de l’enzyme de conversion de l’angiotensine (ECA) peut être
potentiellement impliqué dans l’étiologie de plusieurs maladies courantes. L’étude de la structure de son
haplotype est donc utile pour la diagnose comme pour les recherches pharmacogénomiques.
Objectif: Etudier le profile haplotypique des polymorphismes de nucléotides uniques (PNU) dans le gène
ECA de la population tunisienne et le comparer à d’autres populations. Trois PNU importants pour
leur emploi dans les futures études d’association ont été identifiés.
Sujets et me´thodes: Cinq PNU (rs1800764, rs4291, rs4309, rs4331, rs4340) couvrant une région de 15,6
kb du gène ECA ont été typés par PCR-RFLP dans un échantillon de 100 personnes en bonne santé.
Re´sultats: Tous les PNU étaient polymorphiques et en équilibre de Hardy-Weinberg. Un total de 21
haplotypes ont été identifiés, mais seulement huit avaient une fréquence supérieure à 1%. Les quatre
haplotypes les plus communs avaient une fréquence cumulée de 87,4%. On a rencontré le phénomène
‘‘Yin-Yang ’’ (les deux haplotypes majeurs sont complémentaires à tous les sites). Le déséquilibre de
linkage entre toutes les paires de loci était hautement significatif (p < 10–5). Une méthode statistique
simple et efficace a été utilisée pour identifier trois PNU importants.
Conclusion: La population tunisienne présente une structure haplotypique différente de celle des
européens pour le gène ECA et trois PNU importants ont été identifiés. Ils seront très utiles pour les
futures études d’association dans les populations tunisienne et nord-africaines.
Zusammenfassung. Hintergrund: Das Angiotensin-Converting Enzym (ACE) ist ein Kandidatengen
in der Ätiologie verschiedener häufiger Krankheiten. Die Untersuchung der Haplotypstruktur dieses
Gens ist bei der Diagnostik und der Pharmakogenomik von Interesse.
Ziel: Die Studie untersuchte das Haplotypprofil von Einzelnukleotidpolymorphismen (single nucleotide
polymorphisms, SNPs) innerhalb des ACE-Gens in der Tunesischen Bevölkerung und verglich es mit
anderen Populationen. Drei für den späteren Gebrauch in Assoziationsstudien bedeutsame SNPs
wurden identifiziert.
Probanden und Methoden: Fünf SNPs (rs1800764, rs4291, rs4309, rs4331, rs4340), die sich über eine
Region von 15,6 kb des ACE-Gens erstrecken, wurden mit PCR-RFLP in einer Stichprobe von 100
gesunden Probanden typisiert.
Haplotype structure of ACE gene SNPs in Tunisia
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Ergebnisse: Alle SNPs waren polymorph und im Hardy–Weinberg Gleichgewicht. Insgesamt 21
Haplotypen wurden identifiziert, aber nur acht traten in einer Häufigkeit von über 1% auf. Die vier
häufigsten Haplotypen hatten eine kumulative Häufigkeit von 87,4%. Das ‘Yin–Yang’-Phänomen
wurde gefunden (die beiden wichtigsten Haplotypen sind allseits komplementär). Das
Kopplungsungleichgewicht zwischen den jeweils gepaarten Loci war hochsignifikant (p < 105). Ein
einfaches und effizientes statistisches Verfahren wurde genutzt, um drei bedeutsame SNPs zu
identifizieren.
Zusammenfassung: Die Tunesische Bevölkerung zeigte eine gegenüber der Europäischen unterschiedliche
Haplotypstruktur des ACE-Gens, und es wurden drei bedeutsame SNPs identifiziert. Diese werden
für spätere Assoziationsstudien bei Tunesischen und anderen Nordafrikanischen Populationen
hilfreich sein.
Resumen Antecedentes: El enzima convertidor de la angiotensina (ECA) es un gen candidato en la
etiologı́a de varias enfermedades comunes. El estudio de la estructura haplotı́pica de este gen es de
interés en diagnóstico y farmacogenómica.
Objetivos: El estudio investigó el perfil haplotı́pico de los polimorfismos de nucleótidos simples (SNPs)
dentro del gen ECA en la población de Túnez, y lo comparó con otras poblaciones. Se identificaron tres
importantes SNPs para su futuro uso en estudios de asociación.
Sujetos y me´todos: Se tiparon cinco SNPs (rs1800764, rs4291, rs4309, rs4331, rs4340), que cubren una
región de 15,6 kb del gen ECA, utilizando PCR-RFLP en una muestra de 100 sujetos sanos.
Resultados: Todos los SNPs fueron polimórficos y estaban en equilibrio de Hardy-Weinberg. Se
identificaron un total de 21 haplotipos, pero solo ocho presentaban una frecuencia mayor de 1%. Los
cuatro haplotipos más comunes presentaban una frecuencia acumulada del 87,4%. Se observó el
fenómeno ‘‘Yin-Yang’’ (dos haplotipos mayores son complementarios en todos los sitios). El
desequilibrio de ligamiento entre todos los pares de loci fue altamente significativo ( p<105). Se utilizó
un procedimiento estadı́stico simple y eficiente para identificar tres SNPs importantes.
Conclusio´n: La población de Túnez mostró una estructura haplotı́pica diferente de la europea para el
gen ECA y se identificaron tres SNPs importantes, los cuales serán de gran ayuda en futuros estudios de
asociación en poblaciones de Túnez y del norte de África.