Etude du gène PON1 - Réplication d’études d’association portant sur des gènes candidats impliqu

1. Réplication d’études d’association portant sur des gènes candidats impliqués dans la composante

1.2. Etude du gène PON1

(Voir article : “Association study of the paraoxonase 1 gene with the risk of developing Alzheimer's disease”).

1.2.1. Contexte de l’étude

Comme nous l’avons vu en introduction (paragraphe 6.1.3.2.2), malgré la fonctionnalité des polymorphismes –107G/A, L55M et R192Q, les études ayant recherché un lien entre ces polymorphismes et la MA ont abouti à des résultats contradictoires (Dantoine et al., 2002; Pola et al., 2003; Yamada et al., 2002). Plus récemment, 10 polymorphismes ont été étudiés dans une population composée de familles d’origines ethniques différentes, Caucasienne et Africaine (Erlich et al., 2006). Les polymorphismes -161C/T et rs854565 (Figure 32) ont montré une association avec la MA dans ces 2 populations.

1.2.2. Présentation du travail

Suite à cette dernière étude, nous avons décidé d’évaluer dans notre population cas-témoins de Lille l’impact des polymorphismes –161C/T et rs854565 G/A sur le risque de développer la MA. De plus, au vu de la fonctionnalité des polymorphismes –107G/A, L55M et R192Q, et devant les résultats contradictoires précédemment publiés quant à leur association avec le risque de développer la MA, nous avons également entrepris de les étudier dans notre population cas-témoins de Lille.

1.2.3. Résumé des résultats

Nous avons pu répliquer l’association des polymorphismes –107G/A et rs854565 G/A avec le risque de développer la MA (respectivement OR=1,5 IC95%=[1,0-2,1] p=0,03 et OR=0,5 IC95%=[0,3-0,8] p=0,006). Aucune association n’a été retrouvée pour les polymorphismes –161C/T, L55M et R192Q. L’analyse haplotypique a révélé qu’en ce qui concerne les polymorphismes –107G/A et rs854565 G/A, l’effet principal semble être porté par le polymorphisme rs854565 G/A.

1.2.4. Discussion

Le polymorphisme rs854565 G/A a fait l’objet de 2 analyses dans le cadre d’études d’association. Par 2 fois, il a pu être associé à la MA. Il semblerait donc un bon marqueur de l’effet potentiel de PON1 dans la composante génétique de la MA.

En complément de ce travail publié, nous avons récemment analysé les polymorphismes localisés dans le gène PON1 étudiés dans la GWA de Li et collaborateurs (Li et al., 2008). Le polymorphisme rs854565 ne faisait pas partie des polymorphismes étudiés. Cependant, le polymorphisme rs2299261 A/G a été associé au risque de développer la MA lors de cette GWA. Plus précisément, les individus porteurs des génotypes AG et GG présentent une diminution du risque de développer la MA (OR=0,75 IC95%=[0,61-0,93] p=0,03) par rapport aux individus porteurs du génotype AA. Ainsi, le polymorphisme rs2299261 présente un effet protecteur tout comme le polymorphisme rs854565. Or, de façon intéressante, le polymorphisme rs2299261 est également situé dans l’intron 1. De plus, selon les données de HapMap, ces 2 polymorphismes feraient partie d’un même bloc haplotypique s’étendant de l’intron 1 à l’exon 3 (Figure 33). On peut alors supposer qu’un polymorphisme causal responsable de l’impact du gène PON1 dans la MA soit situé dans cette région du gène.

Toutes ces observations tendent à montrer l’intérêt de cette région du gène PON1 dans la composante génétique de la MA. De plus, il a été montré que des facteurs de régulation du niveau de transcription d’un gène peuvent se fixer dans l’intron 1. Il est alors possible qu’un polymorphisme situé dans l’intron 1 du gène PON1 influe sur son niveau d’expression. Cependant, selon des données du NCBI, cette région du gène regrouperait 98 polymorphismes différents, dont 40% ne sont pas validés. Dans le but d’identifier les polymorphismes réellement présents dans cette région du gène, nous avons entrepris de séquencer la région correspondante (introns 1 et 2, exons 2 et 3) chez 10 individus témoins. Suite à cette analyse, 23 polymorphismes ont été retrouvés dans cette région d’intérêt. Il sera nécessaire d’évaluer l’impact de chacun de ces polymorphismes sur le risque de développer la MA par des études d’association, grâce aux différentes populations disponibles au laboratoire.

Please cite this article in press as: Chapuis, J. et al., Association study of the paraoxonase 1 gene with the risk of developing Alzheimer’s

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Brief communication

Association study of the paraoxonase 1 gene with the

risk of developing Alzheimer’s disease

Julien Chapuis, Maud Boscher, Faiza Bensemain, Dominique Cottel,

Philippe Amouyel, Jean-Charles Lambert

∗

INSERM U744, Institut Pasteur de Lille, Universit´e de Lille 2, France

Received 4 January 2007; received in revised form 2 May 2007; accepted 27 May 2007

Abstract

Recently, a region encompassing the promoter and intron 1 of the paraoxonase 1 gene (PON1) have been associated with the risk of developing Alzheimer’s disease (AD) in a large pan-ethnic (Caucasian and African-American) dataset. We attempted to replicate this observation in a large French study of sporadic cases and controls. We confirmed that the proximal promoter and 5sequence of the PON1 gene may harbor unknown functional variant(s) associated with the risk of developing AD.

Keywords: Alzheimer; Paraoxonase; Association; Polymorphism

1. Introduction

Mutations in theAPP, presenilin-1 and presenilin-2 genes have been shown to account for most cases of the early-onset autosomal dominant forms of AD, but only for 2–5% of all the AD cases (Cruts and Van Broeckhoven, 1998; Kamboh, 2004.). The genetics of the late-onset forms of the disease is far more complex and the only unequivocal genetic risk is the␧4 allele of the apolipoprotein E (APOE) gene (Farrer et al., 1997).

Since APOE is a major protein in the control of cholesterol homeostasis in the brain, it has been suggested an involve-ment of the lipid transport system in the etiology of AD. This hypothesis was reinforced by several observations: (i) depletion of brain cholesterol levels reduces the generation of␤-amyloid peptides (A␤), (ii) at the opposite, excess brain cholesterol has been associated with increased formation and deposition of A␤peptides; (iii) cholesterol-lowering drugs may reduce the risk of dementia (Shobab et al., 2005). Con-∗_{Corresponding author at: Unit´e INSERM 744, Institut Pasteur de Lille,} BP 245, 1 rue du professeur Calmette, 59019 Lille C´edex, France. Tel.: +33 3 20 87 73 91; fax: +33 3 20 87 78 94.

E-mail address:jean-charles.lambert@pasteur-lille.fr(J.-C. Lambert).

sequently, it has been suggested that genes involved in brain cholesterol homeostasis may be potential candidate genes for AD and among the hundred or so genes already studied, a non negligible part of them takes place in cholesterol metabolism and transport. However, conversely to APOE, any of them seem to be unequivocal genetic risk of AD.

Among these genes exhibiting controversial association with the risk of developing AD, the human PON locus is of particular interest for several reasons: (i) paraoxonase is a component of the lipid transport system and protect lipids and low density lipoproteins (LDL) from oxidation (Shih et al., 1998); (ii) specific coding or promoter poly-morphisms at the PON locus have been associated with a variety of vascular disorders resulting from atherosclerosis of blood vessels (Serrato and Marian, 1995; Leviev et al., 2001); (iii) several studies observed association of PON poly-morphisms with several neurodegenerative disorders such as Parkinson’s disease (Fong et al., 2005), vascular demen-tia (Helbecque et al., 2004) and of course AD (Scacchi et al., 2003); (iv) lastly, in a large pan-ethnic (Caucasian and African-American) dataset of AD family-based genetic study, a region encompassing the promoter and the three first exons ofPON1 was associated with the risk of developing the disease (Erlich et al., 2006). This region was defined

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2 J. Chapuis et al. / Neurobiology of Aging xxx (2007) xxx–xxx by four SNPs: rs705381 (−161C/T), rs705379 (−107G/A),

rs854565 (intron 1), rs854560 (L55M).

Here, we evaluated the association of these four SNPs on the risk of developing AD in a large French AD spo-radic cases–control study. We also included the rs662 SNP (R192Q): this one has been also controversially associated with dementia and AD.

2. Methods

2.1. Subjects

The French AD and control subjects were all Caucasian (sporadic AD casesn= 629, age at examination = 73.0±8.8 years, age at onset = 69.6±8.7 years, 36.5% male; controls

Table 1

Allele and genotype distribution in the AD case and control populations

% (n) pâ OR [95% CI]^b^,^c p^d ptrend^c^,ê Controls (n= 663) AD cases (n= 601) rs705381 (−161C/T) C 73.5% (975) 75.5% (908) n.s. T 26.5% (351) 24.6% (294) CC 54.3% (360) 57.2% (344) n.s. Ref. CT 38.5% (255) 36.6% (220) 0.9 [0.7–1.2] n.s. n.s. TT 7.2% (48) 6.2% (37) 0.8 [0.5–1.3] n.s. % (n) pa OR [95% CI]b,c pd ptrendc,e Controls (n= 667) AD cases (n= 623) rs705379 (−107G/A) G 54.0% (721) 49.5% (617) 0.02 A 46.0% (613) 50.5% (629) GG 28.6% (191) 22.8% (142) 0.04 Ref. GA 50.8% (339) 53.4% (333) 1.3 [1.0–1.7] 0.09 0.02 AA 20.5% (137) 23.8% (148) 1.5 [1.0–2.1] 0.03 % (n) pâ OR [95% CI]^b^,^c p^d ptrend^c^,ê Controls (n= 669) AD cases (n= 629) rs 854565 G 67.3% (901) 72.6% (913) 0.004 A 32.7% (437) 27.4% (345) GG 45.7% (306) 52.3% (329) 0.01 Ref. GA 43.2% (289) 40.5% (255) 0.8 [0.6–1.0] 0.06 0.004 AA 11.1% (74) 7.1% (45) 0.5 [0.3–0.8] 0.006 % (n) pa OR [95% CI]b,c pd ptrendc,e Controls (n= 663) AD cases (n= 601) rs854560 (L55M) M 65.3% (866) 62.0% (745) 0.08 L 34.7% (460) 38.0% (457) MM 42.4% (281) 39.1% (235) n.s. Ref. ML 45.8% (304) 45.8% (275) 1.2 [0.9–1.5] n.s. 0.08 LL 11.8% (78) 15.1% (91) 1.3 [0.9–1.9] n.s. % (n) pâ OR [95% CI]^b^,^c p^d ptrend^c^,ê Controls (n= 663) AD cases (n= 601) rs662 (R192Q) Q 73.9% (980) 72.0% (866) n.s. R 26.1% (346) 28.0% (336) QQ 54.9% (364) 51.4% (309) n.s. Ref. QR 38.0% (252) 41.3% (248) 1.1 [0.9–1.5] n.s. n.s. RR 7.1% (47) 7.3% (44) 1.1 [0.7–1.8] n.s.

aPearson’sχ2test for respectively allelic and genotypic distribution. b Odds ratio and 95% confidence intervals OR [95% CI].

cAdjusted on age, gender and␧4 allele of APOE. d p-Value for odds ratio.

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J. Chapuis et al. / Neurobiology of Aging xxx (2007) xxx–xxx 3 Table 2

Haplotype (rs705379 (−107G/A), rs854565 and rs854560 (L55M)) distribution in the AD case and control populations Haplotype rs705379/rs854565/rs854560 (−107) (L55M) Controls (n= 1326) (%) AD cases (n= 1202) (%) OR [95% CI]a,b p (1) G A M 31.3 26.2 Ref. Ref. (2) A G L 27.4 29.8 1.3 [1.1–1.6] 0.01 (3) A G M 17.1 19.2 1.3 [1.0–1.7] 0.01 (4) G G M 15.5 14.5 1.1 [0.9–1.4] ns (5) G G L 7.3 8.4 1.4 [1.0–1.9] 0.05 Global test:p= 0.05.

aOdds ratio and 95% confidence intervals OR [95% CI].

b Adjusted on age, gender and␧4 allele of APOE Haplotype assessed with the maximum of invidious (663 controls and 601 cases). n= 669, age at examination = 73.1±8.5 years, 36.3% male).

A diagnosis of probable AD was established according to DSM-III-R and NINCDS-ADRDA criteria. Caucasian con-trols were defined as subjects without DMS-III-R dementia criteria, with integrity of cognitive function and with a MMS score≥25. Controls were recruited from retirement homes or from electoral rolls (altruistic volunteers). Each individual or next of kin gave informed consent. Control subjects with a family history of dementia were excluded.

2.2. Genotyping

Genotypes of the rs705381 (−161C/T), rs84565, rs854560 (L55M) and rs662 (R192Q) SNPs were deter-mined by PCR then enzymatic digestion as follow: rs705381 (–161C/T) (forward 5

-GGCCGACCAGGTGC-ACAGAA-3_{, reverse 5}

-CCGACTGGACTAGGCACCTA-3_{, HhaI), rs854565 (forward 5}

-AAATGCAGCATCAGTA-GCTT-3_, _reverse ₅_{-ATACACAAGGCCTGCAT-GA-3}_,

MnlI), rs854560 (L55M) (forward 5

-GGATCCACATC-CTGCAATAA-3_{, reverse 5}

-GGGTATACAGAAAGCCTA-AG-3_{, NlaIII) and rs662 (R192Q) (forward 5}

-TATTGTT-GCTGTGG-GACCTGAG-3_{, reverse 5}

-CACGCTAAAC-CCAAATACATCTC-3_{, DpnI). The rs705379 (}−107G/A) SNP was assessed by an amplification refractory mutation system (ARMS) according to the procedure described by Bergmeier et al. (2004).

2.3. Statistical analysis

The SAS software release 8.1 was used (SAS Institute, Cary, NC). Univariate analysis was performed using Pear-son’s χ2 test. Association of the different SNPs with the risk of developing AD was assessed by a multiple logis-tic regression model adjusted for age, gender and␧4 allele of APOE gene (using a co-dominant model). Interactions between APOE andPON1 polymorphisms were tested by logistic regression. The potential impact of these polymor-phisms on age at onset was assessed using a general linear model. THESIAS software was used to investigate the asso-ciation of haplotypes with AD. The objective of the thesias software is to performed haplotype-based association anal-ysis in unrelated individuals. This program is based on the maximum likelihood model described byStram et al. (2003)

and is linked to the SEM algorithm (Tregouet and Tiret, 2004).

3. Results

We assessed the impact of the rs705381 (−161C/T), rs705379 (−107G/A), rs854565, rs854560 (L55M) and rs662 (R192Q) SNPs on the risk of developing AD. Their genotype distributions were in Hardy–Weinberg equilibrium in the control population. No association of the rs705381 (−161C/T), rs854560 (L55M) and rs662 (R192Q) polymor-phisms with AD was observed in our French population (Table 1). Conversely, both allelic and genotypic distribu-tions of the rs705379 (−107G/A) and rs854565 SNPs were significantly different between the AD cases and control pop-ulations (Table 1). After adjustment for age, gender and␧4 allele of the APOEgene, a significant and dose-dependent decrease in the risk of AD among carriers of one or two copies of the rs854565 A allele was observed as compared with those with no copies of this allele (Table 1,pfor trend = 0.004). At a lesser extent, an increase in the risk of AD among carriers of at least one copy of the rs705379 (−107) A allele was detected as compared with those with no copies of this allele (Table 1). For these polymorphisms neither association with age at onset nor interaction with the␧4 allele ofAPOEgene was observed.

Using a stepwise conditional logistic regression including the fivePON1 SNPs, age, gender and the ␧2/␧3/␧4 APOE alleles, only the rs854565 A and␧4 alleles finally appeared to be significantly associated with the risk of developing AD in this model. This observation suggests that this SNP is asso-ciated with the main effect we observed in thePON1locus, as confirmed by haplotype analyses (Table 2). The common allele between the three haplotypes (2, 3 and 5) associated with increased risk of developing AD, is the G allele of SNP rs854565 (Table 2).

4. Discussion

In this study, we observed significant evidence of associ-ations between polymorphisms in thePON1gene and AD in a large French study of sporadic AD cases and controls. We

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Table 3

Linkage disequilibrium assessed in Controls between the five polymor-phisms (D_and_r2)

support the recent results reporting that proximal promoter and 5 _{sequence of the}_PON1_{gene may harbor biologically}

important variant(s) associated with the risk of developing AD in African-American and Caucasians (Erlich et al., 2006). We observed that the main association was carried by the rs854565 SNP within intron 1. This SNP was also associ-ated with AD in the large pan-ethnic study (Erlich et al., 2006). However, in contrast to this previous report, we did not observed an association of the –161C/T SNP with AD. This discrepancy was likely due to difference in linkage dise-quilibrium (LD) between these two SNPs in both populations. In the French population, only the –107G/A which presents the more important LD with the rs854565 (r²= 0.41) was associated with AD in our study (Table 3) while the –161C/T was not in LD with the rs854565 SNP (r²= 0.17). Finally, even the rs662 (R192Q) was inconstantly described to be associated with the risk of developing AD (Helbecque et al., 2004; Scacchi et al., 2003; Pola et al., 2003; Dantoine et al., 2002), we did not observe such an association in your study; result in accordance with a meta-analysis in Cau-casians (http://www/alzforum.org/res/com/gen/alzgen).

In conclusion, evidence for association between the risk of developing AD and a very narrow portion of thePON1 gene seems to exist (including promoter and intron 1), an in-depth and systematic analysis of this region may be realized. However, at least 62 SNPs (validated or not) are currently listed in this region in the National Center for Biotechnology Information (NCBI) database and 42 of them may be able to modify a transcriptional factor binding site as indicated by the use of the genomatix database (http://www.genomatix.de). As a consequence, even if our findings and recent works highlight the importance of further investigation of the role of PON1in AD, only important and ambitious genotyping efforts in combination with systematic replication in indepen-dent populations and with functional analyses of intermediate phenotypes will help determine whether thePON1gene is a genetic determinant of AD.

Disclosure statement

All authors disclose any actual or potential conflicts of interest including any financial, personal or other relation-ships with other people or organizations within 3 years of beginning the work submitted that could inappropriately influence (bias) their work. Appropriate approval and pro-cedures were used concerning human subjects.

Acknowledgement

Julien Chapuis was supported by the French Ministry of Research.

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2. Réplication d’études d’association portant sur un gène candidat (GAB2) issu d’une

Dans le document Identification de déterminants génétiques impliqués dans la composante vasculaire de la MA, par analyses transcriptomiques, génétiques et moléculaires (Page 98-105)