Prevalence of angiotensin-converting enzyme methylenetetrahydrofolate reductase Factor v Leiden prothrombin and apolipoprotein e gene polymorphisms in Morocco

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ORIGINAL ARTICLE

Prevalence of angiotensin-converting enzyme,

methylenetetrahydrofolate reductase, Factor V Leiden, prothrombin and apolipoprotein E gene polymorphisms in Morocco

THIERRY PALUKU THEY-THEY

¹

, KHALIL HAMZI

¹

, MOHAMED

TAHA MOUTAWAFIK

¹

, HANANE BELLAYOU

¹

, MARIAME EL MESSAL

²

&

SELLAMA NADIFI

¹

1

Laboratoire de Ge´ne´tique Me´dicale et Pathologie Mole´culaire (LGPM), Faculte´ de Me´decine et de Pharmacie – Casablanca, Casablanca, Morocco, and

²

Laboratoire de Biochimie et Biologie Molećulaire, Groupe de Geńe´tique et Biologie Molećulaire, Faculte´ des Sciences Ain chock, Casablanca, Morocco (Received 28 July 2009; accepted 27 January 2010)

Abstract

Background: Evidence of the influence of genetic risk factors on cardiovascular diseases is more or less

established. These genetic factors are involved in several pathways affecting blood pressure regulation, blood coagulation, homocysteine and lipid metabolisms.

Aim: We evaluated frequencies of five genetic polymorphisms to assess their informativeness as markers

for prospective clinical studies.

Subjects and methods: 182 healthy Moroccan subjects were genotyped for ACE I/D by amplification

alone and by amplification followed by enzymatic digestion for other polymorphisms.

Results: Allele frequencies of ACE ID, MTHFR C677T were 76.6%, 26.9% for D and T alleles,

respectively. APOE polymorphism showed 11.3%, 78.6% and 10.2% for the alleles E2, E3 and E4, respectively. The frequency for FII G20210A polymorphism was around 2.7% for A allele. Our data showed an absence of FVL mutation. Using allele frequencies, genetic distances between Moroccan and other populations revealed an independent variability of these polymorphisms.

Conclusion: These values appear to be influenced by findings in European and African peoples, and may

be considered in assessing the clinical significance of a predisposition to cardiovascular disease.

Keywords: Cardiovascular risk factor, genotyping, Moroccan population

ISSN 0301-4460 print/ISSN 1464-5033 onlineq2010 Informa UK, Ltd.

DOI: 10.3109/03014461003738850

Correspondence: Professor Sellama Nadifi, Laboratoire de Ge´ne´tique Me´dicale et Pathologie Mole´culaire (LGPM), Faculte´

de Me´decine et de Pharmacie – Casablanca, 19, rue Tarik-Ibn-Ziad, BP 9154, 10 000 Casablanca, Morocco.

E-mail: labgenmed@yahoo.fr Ann Hum Biol Downloaded from informahealthcare.com by Michigan University on 11/04/14 For personal use only.

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Introduction

Cardiovascular disease (CVD) is an emerging public health problem not only in developing societies, but also in low- and middle-income countries. Its development involves the interaction of multiple genetic factors and environmental influences (e.g. hypertension, diabetes and smoking). Numerous polymorphisms have been identified within genes encoding peptides directly and indirectly involved in pathways related to CVD etiology. Many of these genetic markers have been associated with CVD in studies evaluating defined patient groups; however, the potential impact of these genetic risk factors, both individually and in combination with one another, on overall health remains unclear. Moreover, polymorphisms in genes encoding for proteins involved in the pathogenesis mechanism of thrombosis have been studied as putative risk factors (Lane and Grant 2000). The phenotype that results from these polymorphisms is characterized by altered protein activity that affects several pathways such as regulation of blood pressure (angiotensin-converting enzyme (ACE) I/D), homocysteine metabolism (5,10 methylenetetrahydrofolate reductase (MTHFR) C677T), blood coagulation (Factor V Leiden (FVL) G1691A and prothrombin or Factor II (FII) G20210A), and lipid metabolism (apolipoprotein E (APOE) E2, E3 and E4).

ACE plays an important role in blood pressure regulation by hydrolysing angiotensin I into vasoconstrictive angiotensin II and inactivating the vasodilator bradykinin (Erdos and Skidgel 1987). ACE insertion (I)/deletion (D) polymorphism, involving Alu repetitive 287bp long sequence within intron 16, has been strongly associated with interindividual variability of plasma ACE (Rigat et al. 1992). The plasma ACE level of DD subjects has been found to be about twice that of II subjects, while ID subjects have intermediate levels. ACE D and I allele distributions were found at near-equal frequency among healthy Western Eurasians, giving rise to genotype frequencies of 25% (II), 50% (ID), and 25% (DD) (Rigat et al. 1992).

MTHFR catalyses the conversion of 5,10methylenetetrahydrofolate to 5methyltetrahy- drofolate, a cosubstrate for homocysteine remethylation to methionine. A C-to-T substitution at nucleotide 677 that converts a Val222 to Ala222 leads to the synthesis of a thermolabile form of MTHFR with 50% of wild-type gene product activity (Frosst et al. 1995). This polymorphism is only associated with elevated homocysteine levels in cases of low folate intake (Bauduer et al. 2005). The C677T MTHFR polymorphism is associated with low to higher risk of CVD such as myocardial infarction, coronary heart disease and stroke particularly when low folate status results in high homocysteine levels (Casas et al. 2004).

In Western Eurasian population, there is a geographic gradient (north to south increase) with a very high prevalence among Mediterranean countries (Wilcken et al. 2003).

The Factor V gene encodes coagulation Factor V, a plasma glycoprotein that circulates with little or no activity. Activated form Factor V (FVa) serves as an essential protein in the coagulation pathway and acts as a cofactor for the conversion of prothrombin to thrombin by factor Xa (F10). FVL mutation resulting in an arg506-to-gln (R506Q) substitution, abolishes one of three PROC cleavage sites in FV (Bertina et al. 1994). R506Q substitution prevents inactivation of activated factor V by PROC resulting in increased risk of recurrent thrombosis (Ridker et al. 1995). In Europe, 5% of the general population shows this mutation versus 20 – 60% in patients. Heterozygous carriers of the FV G1691A allele have an 8fold increased risk of venous thrombosis, then homozygous expression confers an estimated 50 – 80fold increased risk in venous thrombosis (Caprini et al. 2004).

In the FII prothrombin gene, a G-to-A transition at position 20 210 within the 3prime

untranslated region has been associated with elevated plasma prothrombin levels and an

increased risk of venous thrombosis (Poort et al. 1996). The mutation is associated with

a 1.5 – 4fold increased risk of myocardial infarction. In Europe, the estimated prevalence

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of heterozygous carriers of the 20210A mutation is 2.0% with a south – north gradient increase (1.7 – 3%). The prothrombin variant seems to be very rare in people of Asian and African descent (Rosendaal et al. 1998).

APOE is a glycosylated polymorphic protein, involved in lipid metabolism. The three major isoforms of APOE are E2, E3 and E4. The three isoforms differ in amino acid sequence at two sites, C112R and C158R substitutions. The derived alleles are characterized as E2 (C112/C158), E3 (C112/R158) and E4 (R112/R158). E4 allele results in high LDL cholesterol levels since it is degraded more rapidly than E3. (Donnelly et al. 2008). So, the E3 allele may have a protective role, while the E4 allele may be a risk factor for thrombosis.

Distribution of these five mutations shows an ethnic and geographical variability leading to controversial results as genetic risk factors of CVDs (Ioannidis et al. 2004).

Morocco is a north-western Africa country peopled by Western Eurasian populations extended from Iberia with ethnic diversity (Cavalli-Sforza et al. 1988). This population is divided into Berbers, Arabs, Jews and South-Saharans, with a predominance of the first two sub-groups. Their contribution to the genetic background is certainly evident. However, studies are limited. A strategy to identify polymorphisms associated with common human diseases, such as vascular disease, could be the comparison of genotype frequencies among ethnic healthy populations.

In this study, we evaluated the prevalence of the above-mentioned mutations in a healthy Moroccan population to provide baseline epidemiological data for future clinical investigations of the polymorphisms in association with CVD or other pathologies.

Materials and methods Samples

Blood samples were collected in tubes containing EDTA from 182 voluntary subjects (94 males and 88 females) of two majority sub-groups, 92 Arabs and 90 Berbers. All study participants were unrelated and were randomly selected. Ethnicity was ascertained by subjects identifying themselves as belonging to one of two major groups (Berbers and Arabs).

Informed consent was obtained before entering the study, which was approved by the ethics committee of our institution.

Genotype analysis

Total genomic DNA was isolated from leukocytes using phenol – chloroform extraction.

MTHFR C677T, FVL G1691A, FII G20210A, and APOE polymorphisms were identified by PCR amplification, with appropriate primers, followed by enzymatic digestion using the technique described by Frosst et al. (1995), Gandrille et al. (1995), Danneberg et al. (1998) and Hixson et al. (1990), respectively.

The ACE ID polymorphism was detected by PCR using conditions described by Rigat et al. (1992). To increase the accuracy of genotyping further, we reanalyzed all samples that were typed initially as a DD genotype using 5% dimethylsulfoxide (DMSO) as reported by Shanmugam et al. (1993). Summaries of our protocols are listed in Table I.

Statistical analysis

Genepop version 3.4 and Phylip version 3.68 software were used to calculate genotype and allele frequencies and population genetic distances, respectively. Deviation from Hardy – Weinberg expectations and investigation of the differences in allele frequencies between the studied ethnicities samples were tested using chi-square with XLSTAT version 2009.4.01.

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TableI.Protocolamplificationsofthefivecardiovasculargeneticmarkers. PolymorphismsPrimersTm(8C)MgCl2(mM)Ampliconsize(bp)EnzymeReferences ACEI/D50-CTGGAGACCACTCCCATCCTTTCT-30572.5490(I);190(D)Rigatetal.1992 50-GATGTGGCCATCACATTCGTCAGAT-30 MTHFRC677T50-TGAAAGGAGAAGGTGTCTGCGGGA-3‘611.5198HinfIFrosstetal.1995 50-GGACGGTGCGGTGAGAGTG-30 FIIG20210A50-TCTAGAAACAGTTGCCTGGC-30601.5486HindIIIDannebergetal.1998 50-ATAGCACTGGGAGCATTGAA*GC-30 FVG1691A50-TCAGGCAGGAACAACACCAT-30561.5241HindIIIGandrilleetal.1995 50-GGTTACTTCAAGGACAAAATACCTGTAAAAGCT-30 ApoE50-ACAGAATTCGCCCCGGCCTGGTACAC-30622244HhaIHixsonetal.1990 50-TAAGCTTGGCACGGCTGTCCAAGGA-30

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Results

Genotype and allele frequencies

Genotype and allele frequencies of these polymorphisms in our Moroccan samples are shown in Table II.

ACE D, MTHFR 677Tand APOE4 alleles are relatively common. The distribution of this variant was according to Hardy – Weinberg equilibrium for MTHFR ( p ¼ 0.457) and FII polymorphisms ( p-value ¼ 1) but not for ApoE ( p-value , 0.001) and ACE polymorphisms ( p-value ¼ 0.0131). For FII G20210A polymorphism A allele frequencies were 1.6% for Arabs and 4.4% for Berbers. But no significant differences was found between the sub- groups ( p-value ¼ 0.962) with A allele frequency around of 2.7%. This study showed absence of FVL mutation. Neither homozygote for A allele was detected for both FII and FVL. The genotype and allele frequencies of the studied polymorphisms are similar in the two sub-groups, Arabs and Berbers, data shown in Table III, and according to sex, males and females, data not shown.

Genetic distances

Nei’s genetic distance (D) was calculated using the allele frequencies between the Moroccan population and those of Mediterranean, European and African origin. The Tunisian population showed, in general, a genetic similarity to the Moroccan population (D ¼ 0.006328) for all markers, except for the FV Leiden. In contrast, the Turkish population showed a distant genetic distance (0.030940) from the Moroccan population.

The ACE I/D and FV Leiden polymorphisms seemed closer genetically to the African populations (D ¼ 0.000794 and D ¼ 0.000000, respectively), but more distant from the European populations (D ¼ 0.123781 and D ¼ 0.001269). For MTHFR C677T, F II G20210A and Apo E polymorphisms, the Moroccan populations were genetically closer to Europe (D ¼ 0.001367, D ¼ 0.000009 and D ¼ 0.000000, respectively), and much further

Table II. Allele frequencies of the polymorphisms and the observed/expected genotypes of the five genes among 182 Moroccan healthy individuals.

Polymorphism Allele Frequency Genotype % observed % expected HWE

ACE D/I D 0.765 DD 62.1 58.7

I 0.235 DI 29.1 35.9 0.0131**

II 8.8 5.4

MTHFR 677CT C 0.731 CC 52.2 53.3

T 0.269 CT 41.8 39.5 0.457*

TT 6.0 7.2

FII 20210GA G 0.973 GG 94.5 94.6

A 0.027 GA 5.5 5.4 1*

AA 0 0

FV1691GA G 1.00 GG – –

A 0.00 GA – – –

AA – –

APOE E2 0.113 E2E2 3.3 1.3

E3 0.786 E3E3 67.6 61.7 0.00**

E4 0.102 E4E4 2.2 1

E2E3 11 17.7

E2E4 4.9 2.3

E3E4 11 16

HWE, Hardy – Weinberg equilibrium; *p-value in HWE; **p-value not in HWE.

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away from Africa (D ¼ 0.052661, 0.000170 and D ¼ 0.006135, respectively). Generally, our finding showed that these polymorphisms varied independently in the Moroccan population. The results are shown in Tables IV and V.

Discussion

The aim of our study was to assess the frequency of polymorphisms in ACE I/D, MTHFR C677T, Apo E, FII G20210A and FV Leiden genes in a sample from the Moroccan population. This was, to our knowledge, the first study involving several genetic factors considered as risk factors for CVD in this population. The distribution of this variant was according to Hardy – Weinberg equilibrium for MTHFR and FII polymorphisms but not for ApoE and ACE polymorphisms. No significant differences were observed in genotype or allele frequency between Berbers and Arabs. Our data were in agreement with a previous study in the Moroccan population, which also revealed a close genetic similarity between Berbers and Arabic speakers and suggested that Arabs and Berbers from the Western areas of Maghreb share common ancestors, probably the first inhabitants of North Africa (Coudray et al. 2004). A recent study, using classical genetic markers analysis, showed no genetic

Table III. Frequencies of genotypes and alleles in Moroccan ethnic group.

Ethnic groups

Arabs Berbers

Genotypes and allele n¼92 % n¼90 %

ACE

DD 59 64.1 54 60

ID 26 28.3 26 28.9

II 7 7.6 10 11.1

ALLELE D 0.78 0.74

MTHFR C677T

CC 51 55.4 44 48.9

CT 34 37 42 46.7

TT 7 7.6 4 4.4

ALLELE T 0.26 0.27

FII G20210A

GG 89 96.7 83 92.2

GA 3 2.2 7 7.8

AA 0 0 0 0

ALLELE A 0.016 0.044

FV G16191A

GG 92 100 90 100

GA 0 0 0 0

AA 0 0 0 0

ALLELE A 0.00 0.00

APO E

E2/E2 3 3.3 3 3.3

E3/E3 61 66.3 62 68.9

E4/E4 4 4.3 0 0

E2/E3 11 11.9 9 10

E2/E4 3 3.3 6 6.7

E3/E4 10 10.9 10 11.1

E2 0.11 0.11

E3 0.78 0.79

E4 0.11 0.10

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TableIV.Allelefrequenciesofthepolymorphismforthefivegenesindifferentpopulations. Genes PopulationsACEDMTHFR677TFII20210AFV1691AApoEE4ApoEE2References Morocco0.7650.2690.0210.0000.1000.113Ourstudy Tunisian0.8500.1800.0250.0600.0810.073Bouazizetal.2004 Lebanon0.6200.1900.0100.0140.0880.072Gialerakietal.2008 Turkey0.480.130.070.090.280.13Gialerakietal.2008 Spain0.5200.3390.0400.0350.1260.064Gialerakietal.2008 Greece0.630.400.030.050.0790.061Gialerakietal.2008 CentralEurope0.49–0.520.300.017–0.030.0480.10–0.190.08–0.13Gialerakietal.2008 Africa0.61–0.740.030.0030.000.2910.081Bayoumietal.2006; Wilckenetal.2003 Rosendaaletal.1998 Ann Hum Biol Downloaded from informahealthcare.com by Michigan University on 11/04/14 For personal use only.

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differentiation between Berbers and Arabs from North Africa, suggesting an ancient Middle Eastern contribution (probably Caspian) to the genetic background of Berbers or the Berber origin of Arab populations of North Africa (Fadhlaoui-Zid et al. 2009). Although there is no evidence of a common ancestry or major immigration waves between Berbers and Arabs Moroccan populations (Cavalli-Sforza et al. 1988). Berbers represent the first Western Eurasian population that peopled Morocco. They are believed to have their local ancestors among Caspian Mesolithic people and their ‘Neolithic’ descendants, possibly with a genetic contribution from the important Neolithic migration from the Near East (Cavalli-Sforza et al. 1994). The Arabs have their origin in the Arabian Peninsula, conquering North Africa (Egypt, Tunisia, Algeria and Morocco) between 7th and 11th century during the Islamic expansion. The similarity in genotype and allele frequencies of the two population samples studied suggests that there might have been admixture of gene pools among them.

This present study showed a smaller increase in D allele frequency compared with a prior study on Arab and African populations (Bayoumi et al. 2006). In other Western Eurasians populations, D allele frequencies were moderate (0.46 – 0.55), and low in Asian populations (0.29 – 0.45). Study of genetically isolated populations such as those of Indian origin, has suggested an African origin of the polymorphism ACE I/D that moved out of Africa with Paleolithic migrations 100 000 years ago (Cavalli-Sforza et al. 1988). At a molecular level, this insertion of a 287 bp Alu repeat into intron 16 of the ACE gene, probably occurred a few millions years ago, during the evolution of primates (Jurka 2004). The high prevalence of D allele ACE in this study suggests important gene flow between South-Saharan Africa and Morocco probably through important trade routes. Indeed, the Nei’s genetic distances for this marker, showed that the Moroccan population was genetically closer to the population of African origin and very distant from that of European origin. However, deviation from Hardy – Weinberg expected values could probably be explained by genotyping assay errors, some ID heterozygotes being mistyped as DD homozygotes because of preferential amplification of the D allele over the I allele using the original method (Rodriguez et al. 2009).

For the MTHFR C677T variant, the frequency of TT genotype seemed to be intermediate, comparable to that seen in European countries, and South-Saharan populations (Wilcken et al. 2003). However, for this marker, genetic distances revealed a closer genetic similarity between Moroccan populations with European descent than those of African origin. This polymorphism is only associated with elevated homocysteine levels in cases of low folate intake (Bauduer and Lacombe 2005). In this study, folate intake was not assessed but should be taken into account in association studies with disease.

Low frequency observed in prothrombin mutation in this study was in agreement with a previous study in Moroccan populations (Mathonnet et al. 2002). These results suppose the influence of the European population, probably on Andalusian migrations rather than African, in the enrichment of the genetic bound pool to the polymorphism of the FII G20210A in Moroccan populations. This hypothesis seems to be confirmed by genetic distances which showed a closer genetic affinity between Moroccan and European

Table V. Nei’s genetic distance (D) using the allele frequencies between Moroccan population and those of Mediterranean, European and African origin.

Populations

Morocco Tunisian Lebanon Greece Africa Spain Central Europe Turkey

D* 0 0.006328 0.006912 0.009483 0.014123 0.017700 0.018507 0.030940

D*: Increasing genetic distances based on the five genetic markers.

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populations, compared to Mediterranean and African populations, but very distant to the Turkish population. For the FV Leiden mutation, this study showed absence of this mutation and was in agreement with previous reports (Mathonnet et al. 2002). The distribution of FVL was clearly population-dependent. The frequency is high in Europeans and is absent among South-Saharan Africans (Rahimy et al. 1998), and population of ancient Asian origin. It has been estimated that both FVL and variant 20210A of prothrombin gene with founder effect, arose 21 000 – 34 000 years ago, i.e. after the separation of non-Africans from Africans and after the evolutionary divergence of white and Asians (Zivelin et al. 1998).

Absence of FVL in the Moroccan population, who are thought to represent the Western Eurasian group of pre-Neolithic individualization, suggests limited mixing with populations migrating toward Western Europe through the ages and that the latter mutation appeared as a first event. Indeed, the absence of Ottoman Empire influence could explain the difference in the prevalence of FVL between Morocco (0% for FVL) and other North African country such as Algeria (1.3% for FVL) and Tunisia (3.5% for FVL) (Bouaziz et al. 2004). This fact suggests strong evidence of the genetic influence of a non-Western Eurasian population such as African who show absence of FVL, on the Moroccan genetic pool. By Nei’s genetic distances, for the five markers, the Moroccan population was very genetically distant from the Turkish population compared to other populations and very close, for FV marker, to the African population and far distant from the European and Mediterranean populations.

In the present study, the Apo E allele distribution in the healthy Moroccan population was in agreement with previous study (Lahlali-Kacemi et al. 2002). The deviation in Hardy – Weinberg equilibrium might be explained by chance or include residual technical, and other unknown or unrecognized sequence variation confounding SNP assays. A south-to-north gradient in APOE4 distribution has been described for Africans, highlighted by prevalence rates in South Africa. The prevalence of the E4 allele reported here (0.10) was intermediate between that level and the low rate established for other Arab communities (Saidi et al.

2007). These differences in APOE4 rates between Moroccan (North African), African and other Arab communities can be explained by the admixture with native Africans and Arabs who invaded North Africa in the seventh and eighth centuries AD. In fact, Nei’s genetic distances, for this marker, showed that the Moroccan population was more similar genetically to the European population, followed by eastern populations (Lebanon, Greece), Africa and more distant from the Turkish population. APOE3 is the most frequent in all the human groups, especially in populations with a long-established agricultural economy like those of the Mediterranean basin. The frequency of Apo E4, the ancestral allele, remains higher in populations where an economy of foraging still exists, or food supply was (or was until the recent past) scarce and sporadically available (Corbo and Scacchi 1999).

Analysis of our data suggests genetic heterogeneity of the Moroccan population with a large influence of European population, and a modest contribution of South-Saharan population. This is in agreement with a previous study that reported that the Moroccan population was geographically and genetically intermediate between European populations, with principal component, and South-Saharan population with a contribution of about 20%

(Coudray et al. 2004).

In conclusion, our finding still needs to be correlated with the clinical implication through further studies within the Moroccan population. Therefore, it is highly recommended that studies be conducted in this population based on these five gene polymorphisms correlating the allele frequencies with various clinical entities such as CVD. This study from a Moroccan population will serve as a template for future investigation of the prevalence of these genetic markers in larger population samples along with the associated clinical diagnosis.

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Acknowledgements

We thank the participants who readily agreed to take part in our study.

Declaration of interest: This study was initiated by the STROKE project, supported by the Hassan II Academy of Science and Technology. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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