The normal distribution of PRNP codon 129 polymorphism in the Moroccan population (Arabs and Casablanca residents)

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Original article

The normal distribution of PRNP codon 129 polymorphism in the Moroccan population (Arabs and Casablanca residents)

Ge´notypage et distribution du polymorphisme du codon 129 du ge`ne du prion (PRNP) dans un e´chantillon de la population marocaine

(Arabe, Casablancais et Berbe`re)

S. Nadifi

^a,

* , I. Slassi

^c

, K.M. El Hachimi

^b

, B. Gazzaz

^a

, H. Bellayou

^a

, K. Raddaoui

^a

, J.L. Laplanche

^b

aGenetic and Molecular Pathology Laboratory, Medical School, Hassan II University, Casablanca, Morocco

bBiochemistry and Molecular Biology of the Lariboisière Hospital of Paris, 75013 Paris, France

cNeurology Department, CHU Ibn Rochd, Casablanca, Morocco Received 13 June 2007;accepted 22 August 2007

Available online 9 January 2008

Abstract

The common prion protein gene (PRNP) codon 129 polymorphism is a strong susceptibility factor for human prion diseases. In this study, we examined the allelic variation of methionine and valine at codon 129 in 147 subjects representing the normal Moroccan population.

The sharing of the genotype was 57.1% for Methionine-Methionine (MM), 36% for Methionine-Valine (MV), and 6, 8% for Valine-Valine (VV). These results are indeed intermediate between those discovered at the European and Asian populations.

However, and for a better assessment of the risk to develop prion diseases in the Moroccan population, the survey of the frequency of the codon 219 polymorphism is required.

Résumé

Les maladies à prions, humaines ou animales, se caractérisent par l’accumulation dans le système nerveux central d’une forme modifiée de PrP.

Ces particules ont la propriété d’imprimer leur empreinte conformationnelle aux protéines PrP normales avec lesquelles elles rentrent en contact.

Ce transfert « d’information » est à la base du concept de propagation des prions dans un organisme ou interorganisme. La recherche des facteurs génétiques de susceptibilité aux prions, mis en évidence chez l’homme, a montré le rôle clé joué par le gène de la protéine prion, particulièrement un polymorphisme au codon 129 (ATG/GTG, Met/Val) qui module de façon majeure la susceptibilité des individus aux formes acquises et sporadiques de la MCJ. Alors que la distribution des génotypes au codon 129 dans la population générale européenne est en moyenne de met/met 40 %, met/val 50 % et val/val 10 %, celle observée au sein des patients atteints de MCJ sporadique est très différente : met/met 75 %, met/val 15 % et val/val 10 %. Cette différence montre que l’homozygotie au codon 129, particulièrement met/met est un facteur prédisposant à la MCJ, alors que l’hétérozygotie apparaît comme protectrice.

Notre étude a concerné 147 individus pris au hasard dans la population marocaine. Ces sujets sont répartis selon l’origine ethnique en 42 Arabes, 44 Casablancais et 61 d’origine berbère. Le génotypage au codon 129 a été fait par PCR suivie d’une digestion enzymatique.

http://france.elsevier.com/direct/PATBIO/

Disponible en ligne sur www.sciencedirect.com

Pathologie Biologie 56 (2008) 133–136

* Corresponding author.

E-mail address:labgenmed@gmail.com(S. Nadifi).

doi:10.1016/j.patbio.2007.08.011

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La fréquence des met/met retrouvée dans notre étude est plus élevée que celle décrite en Europe. La fréquence de l’allèle met est de 0,75. Ces chiffres sont similaires à ceux que nous avons observés dans les populations libanaises et turques ; il n’existe pas encore de données au niveau du Maghreb (quelques données fragmentaires en Algérie qui montrent aussi une fréquence plus élevée de l’allèle met).

Keywords:PRNP gene; Genetic susceptibility; Polymorphism in codon 129

Mots clés : Gène PRNP ; Polymorphisme codon 129 ; Prédisposition génétique ; Maladie à prion

1. Introduction

Transmissible spongiform encephalopathy’s (TSE), or prion diseases, are critical, neurodegenerative disorders in both humans and animals [1], which can be sporadic (sporadic Creutzfeldt-Jakob disease (sCJD), sporadic fatal insomnia), acquired (kuru, iatrogenic and new variant of Creutzfeldt-Jakob disease (nvCJD), or inherited (familial Creutzfeldt-Jakob disease (fCJD) Gerstmann Straussler Scheinker disease (GSS), fatal familial insomnia (FFI)[2].

Prion diseases are characterized by accumulation of an abnormal, protease resistant form of the host-encoded prion protein. The disease-associated isoforms, PrPSc, and normal PrPc are identical in amino acid composition but differ in their conformational structure. Abnormal particles PrPSc have the property to transmit their three-D print to the normal prion protein PrPc [1]. At least seven polymorphisms have been identified in the human prion protein gene (PRNP) [3], the Methionine/Valine polymorphism at codon 129 is the most widely examined. It is now established that the common polymorphism modifies, the susceptibility, the duration of incubation, the age of onset and the phenotype of prion diseases [1,4–8].

Homozygosity at codon 129 for methionine is associated with an earlier age at onset and a shorter duration of illness than is heterozygosity; 74% of sporadic cases of CJD and all tested cases of nvCJD have been methionine homozygotes at codon 129[9].

Heterozygosity at codon 129 confers relative resistance to prion diseases. The aim of our study was the characterization of the valine and the methionine allele frequency at codon 129 in 147 individuals representing the standard Moroccan population.

2. Materials and methods 2.1. DNA samples

We analysed DNA samples of 147 healthy Moroccan subjects, including 75 women and 72 men, and the mean age was 24.5 years with extremes of two and 67 years. Peripheral

blood samples were obtained by venipuncture from unrelated healthy volunteer donors. Three groups were studied as follows:

forty-four unrelated individuals Randomly sampled, resident of Casablanca (the industrial capital of Morocco) where the urban growth is maintained by immigration from all parts of Morocco (the urban residents represent a sample of the populations of the whole area from which the immigrants have originated);

forty-two unrelated individuals from the Arab and Moroccan population (Doukala, Chaouia, Haouz, etc.);

sixty-one unrelated Berbers origining from different regions of Morocco (Taroudante, Tafraout, Souss, Amzmiz, Khmis- sat) representing a pooled Moroccan Berber population.

DNA was extracted from leucocytes of the peripheral blood after treatment with proteinase k using a standard phenol- chloroform extraction method.

The polymorphism frequencies of codon 129 in each population genetic analysis was performed at two centers: the medical genetics laboratory of the faculty of medicine and pharmacy of Casablanca, Morocco; and the biochemistry and molecular biology of the Lariboisière hospital of Paris, France.

The PRNP codon 129 genotyping protocols were similar at the two centers (Table 1).

2.2. PCR amplification

PCR primers sequences and location have been reported previously (Sequence accession number NM_000311: PrP mRNA Ref: Kretzschmar et al., 1986).

PCR amplification was carried out in 25ml using 30 ng of target DNA, 15 pmoles of each primer, 200 mM dNTP in 1,5 mm Mgcl2, and 1–2 U of Taq polymerase (Promega). Each sample was subjected to the following amplification conditions:

1 min at 948C, 2 min at 608C annealing temperature and 2 min at 728C during 35 cycles. Ten microliters of PCR products were separated on 2% TBE agarose gel containing 0,5 mg/ml of

Table 1

PRNP codon 129 genotype distribution in the Moroccan population

Studied groups met/met genotype met/val genotype val/val genotype Met Allelic frequency val Allelic frequency

Casablanca resident (n= 44) 28 (63.6%) 13 (29.5%) 3 (6,8) 0.78 0.21

Arab population (n= 42) 18 (42.8%) 21 (50%) 3 (7,1) 0.68 0.32

Berber population (n= 61) 38 (62.2%) 19 (31.1%) 4 (6,5) 0.78 0.22

Total (n= 147) 84 (57.1%) 53 (36) 10 (6,8) 0.75 0.24

S. Nadifi et al. / Pathologie Biologie 56 (2008) 133–136 134

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Ethidium Bromide and the reaction products were directly visualized using UV fluorescence, with 0.5 ng of Hae III digestedfX 174 DNA (as fragment length marker). PCRs were performed in a Gene Amp PCR system 2700, applied biosystem.

2.3. RFLP analysis

Codon 129 polymorphism (ATG/GTG) is explored byTaiI (Fermentas, ACGT/) that we use with the following protocol:

PCR: 10ml, Buffer R+: 1,3ml,TaiI: 0,6ml (2.5U), H2O: 1ml, incubate 2 h at 658C. The whole digestion is loaded on gelTaiI cuts when codon 129 encodes valine.TaiI has also a natural site which serve as internal control and cleaves after nt 657 giving a constant band at 107 bp.The allelic frequencies of M/M polymorphism, the exact test of Hardy Weinberg equilibrium (Guo and Thompson, 1992) and the genotypic disequilibria have been performed using Genepop program.

2.4. Data analysis

A segment of 583 bp was amplified by the polymerase chain reaction (PCR) using Taq polymerase (Promega). The PCR products were digested with the restriction endonuclease Tai I (Fermentas). The digested products were then submitted to electrophoresis in 2% agarose gel. Codon 129 genotypes were determined on the basis of the distinct restriction patterns as seen for each genotype (Fig. 1). Expected sizes: Met/Met:

476 + 107 bp, Met/Val: 476 + 254 + 222 + 107 bp, Val/Val:

254 + 222 + 107 bp. (Fig. 2.).

In case of deletion, the digestion profile is slightly different.

When the met allele carries the deletion, the size of the fragment is 452 bp instead of 476 bp. When the val allele carries the deletion, the size of the fragment is 230 bp instead of 254 bp.

2.5. Statistical study

For a comparison between our own data and those prevailing in the literature, the chi-square test was used.

3. Results

The distribution of codon 129 polymorphic was determined in a total of 147 unrelated individuals. In this work, were focused on two objectives: first, to study the 129 codon genotype frequency of Moroccan population since the genetic structure of the North African populations is not explored yet, and its relationships with other populations from different countries previously studied and, second, to assess the degree of interactions and genes exchange between Arabs and Berbers, since it is evidently due to their different origins.

We analyzed three groups: (44) Casablanca residents from the industrial capital of Morocco, (42) Arabs and (61) Berbers using de 129 codon polymorphism performed by simple PCR and enzymatic cleavage. Allelic frequencies, Hardy–Weinberg equilibrium, and heterozygosity were evaluated.

Fig. 1. Restriction patterns obtained after digestion of PRNP with the endonuclease Tai I.

Fig. 2. Codon 129 frequency in different Morocco population.

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Morocco is a northwestern African country peopled by Caucasoid populations that extended from Iberia to Morocco 20 000–70 000 BC, a late Palaeolithic culture called Ibero–

Marusian, (Cavalli-Sforza et al. 1994). The contribution of Arabs and Berbers to the genetic background of Moroccan population is certainly evident. Berbers represent the first Caucasian population that peopled Morocco. They are believed to have their local ancestors among Capsian Mesolithic’s and their ‘‘Neolithic’’

descendants, possibly with genetic contribution from the important Neolithic migration from Near East[10].

The Arabs have their origin in the Arabian Peninsula; they have conquered North Africa (Egypt, Tunisia, Algeria, and Morocco) between seventh and eleventh century during the Islamic expansion. Furthermore, important trade routes were responsible for gene flow between sub-Saharan Africa and Morocco. All these factors enhanced admixture between populations existing in Morocco.

Our result of the genotype was 57,1% for Methionine- Methionine (MM), 36% for Methionine-Valine (MV), and 6,8% for Valine-Valine (VV). Curiously, when we analyze each population, we note that the frequency met/met is higher among the Berber population 62.2% against 53.5 among the non- Berber ones. The X2 is 0.2564.

These results are indeed intermediate between those discovered among the European and those discovered among the Asian populations. The Met allele frequency is higher in Asia than in Europe, however prion diseases are more frequent in Europe than in Asia (Table 2).

We suppose this is due to the frequency of a protective polymorphism in the Asian populations: the codon 219 polymorphism[11,12]In Japan, heterozygosity at PRNP 219 Glu/Lys polymorphism is associated with resistance to sCJD [13].

Will it mean that the Moroccan population is more predisposed than Europeans to develop prion diseases? Does it mean on the contrary, that this population is rather protected like the Asians, by the codon 219 polymorphism? A survey of the codon 219 polymorphism in the Moroccan population is therefore necessary to answer this question.

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[12] Shibuya S, Higuchi J, Shin RW, Tateishi J, Kitamoto T. Codon 219 Lys allele of PRNP is not found in sporadic Creutzfeldt-Jakob disease. Ann Neurol 1998;43:826–8.

[13] Shibuya S, Higuchi J, Shin RW, Tateishi J, Kitamoto T. Protective prion protein polymorphism against sporadic Creutzfeldt-Jakob disease. Lancet 1998;351:419.

[14] Tsai MT, Su YC, Chen YH, Chen CH. Lack of evidence to support the association of the human prion gene with schizophrenia. Mol Psychiatry 2001;6:74–8.

[15] Jeong BH, Nam JH, Lee KH et al. Polymorphisms of the prion protein gene (PRNP) in a Korean population. J Hum Genet. 2004 May 18 (Epub ahead of print).

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[17] Laplanche JL, Delasnerie-Laupretre N, Brandel JP, et al. Molecular genetics of prion disease in France. Neurology 1994;44:2347–51.

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Neurology 2000;55:593–5.

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Table 2

Reported PRNP codon 129 genotype and allele frequencies in various populations

Population Numb MM MV VV M V Reference

Taiwan 100 97 (97%) 3 (3%) 0 (0%) 0,985 0.015 Tsai MT et al. 2001[14]

Korea 529 499 (94,33%) 29 (5,48%) 1 (0,19%) 0,971 0.029 Jeong BH, 2004[15]

Japan 179 165 (92%) 14 (8%) 0 (0%) 0,961 0.039 Doh-ura K et al. 1991[11]

Morocco 147 84 (57.1%) 53 (36%) 10 (6,8%) 0,75 0.24 Our data

France 161 63 (39%) 82 (51%) 16 (10%) 0,645 0.354 Deslys JP et al. 1994[16]

Laplanche JL et al. 1994[17]

UK 406 164 (40%) 196 (48%) 46 (11%) 0,645 0.355 Collinge J et al. 1991[4]

Spain 546 231 (42%) 239 (44%) 76 (14%) 0,642 0.358 Combarros O et al. 2000[18]

Republic of Ireland 203 69 (34%) 114 (56%) 20 (10%) 0,621 0.379 Nurmi MH et al. 2003[19]

M = Methionine, V = Valine.