Discussion

Nos résultats semblent suggérer que le polymorphisme –2578C/A n’ait aucun impact sur le risque de développer la MA. De plus, bien que ce polymorphisme ait été associé à une modification du niveau d’expression du VEGF, aucune variation de l’importance des lésions vasculaires dans le cerveau de malades n’a été associée aux allèles du polymorphisme – 2578C/A.

Depuis notre publication, l’impact du polymorphisme –2578C/A sur la MA a été évalué dans une population espagnole regroupant 395 témoins et 362 patients; le polymorphisme –2578C/A n’a pas pu être associé au risque de développer la MA (OR=1,2 IC95%=[0,8-1,9] p=0,44) (Mateo et al., 2006). Afin d’apporter une réponse quant à l’impact du polymorphisme –2578C/A du gène VEGF sur le risque de développer la MA, il est possible de regrouper les données issues des différentes études pour réaliser une méta-analyse (contrairement aux ORs précédemment cités, les ORs de la méta-analyse ne sont pas ajustés sur l’âge, le sexe et l’allèle ε4 de l’APOE) (Figure 31). En combinant les 4 populations étudiées, on observe une augmentation significative du risque de développer la MA chez les individus porteurs du génotype AA par rapport aux individus porteurs du génotype CC (OR=1,23 IC95%=[1,01-1,49] p=0,04). Cependant, cette analyse ne peut pas faire l’objet d’une interprétation rigoureuse. En effet, il existe une grande hétérogénéité entre les populations étudiées (I²=65% p=0,03), l’impact significatif du polymorphisme –2578C/A n’étant retrouvé que dans 2 populations sur 4 (Figure 31). De plus, les 2 populations présentant les effectifs les plus importants sont celles qui ne retrouvent aucune association entre le polymorphisme –2578C/A et la MA. Ceci suggère un biais potentiel dans les 2 études initiales portant sur de petits effectifs.

De plus, nous avons recherché si des polymorphismes localisés sur le gène VEGF ont été analysés dans le cadre des 2 GWA que nous avons précédemment présentées en introduction (paragraphe 6.4.1.3). Seuls 2 polymorphismes (rs3024994 et rs3025047) localisés sur ce gène ont été analysés lors de ces études et aucune association n’a été retrouvée avec le risque de développer la MA. L’absence d’association entre ces Tag-SNPs définissant des blocs haplotypiques et la pathologie semble suggérer que le gène VEGF n’est pas un déterminant génétique de la MA. Cependant, il est peu probable que seuls 2 polymorphismes soient suffisants pour recouvrir l’ensemble de l’information génétique parmi les 140 polymorphismes référencés sur ce gène dans la base de données du NCBI. Egalement, ces 2

polymorphismes ne présentent pas de déséquilibre de liaison avec le polymorphisme – 2578C/A précédemment associé à la MA (respectivement r²=0,06 et r²=0,05). Par conséquent, ils ne reflètent pas l’information portée par le polymorphisme –2578C/A.

Par ailleurs, une analyse complémentaire des données de Del Bo, a été réalisée (Saleheen, 2005). L’impact de la combinaison de 3 polymorphismes –2578C/A, -1190G/A et -1154G/A a été évalué. Suite à cette analyse, il est apparu que l’impact de l’allèle A du polymorphisme –2578 n’est retrouvé que lorsqu’il est associé aux allèles fréquents des polymorphismes –1190G/A et –1154G/A. Ainsi, c’est l’haplotype (–2578A, -1190G, -1154G) qui serait porteur d’un effet délétère sur le risque de développer la MA (OR=3,32 IC95%=[1,56-7,16] p=0,0005). L’ensemble de ces polymorphismes devrait être étudié dans différentes populations afin de mieux évaluer l’impact du gène VEGF dans la génétique de la composante vasculaire de la MA.

Brief communication

Association study of the vascular endothelial growth factor gene

with the risk of developing Alzheimer’s disease

Julien Chapuis

, Jinzhou Tian

_{, Jing Shi}

_{, Faiza Bensemain}

, Dominique Cottel

,

Corinne Lendon

, Philippe Amouyel

, David Mann

, Jean-Charles Lambert

aUnit´e INSERM 508, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 245, 59019 Lille C´edex, France bClinical Neuroscience Research Group, University of Manchester, Greater Manchester Neurosciences Centre,

Hope Hospital, Stott Lane, Salford, M6 8HD, UK

cDepartment of Molecular Psychiatry, Division of Neuroscience, Queen Elizabeth Psychiatry Hospital, University of Birmingham, Birmingham, B15 2QZ, UK

dDepartment of Care of the Elderly, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China

Received 30 May 2005; received in revised form 19 July 2005; accepted 20 July 2005 Available online 8 September 2005

Abstract

Numerous observations indicate that cerebrovascular dysfunction contributes to cognitive decline and neurodegeneration in AD. Converging evidence points to a pivotal role for vascular endothelial growth factor (VEGF) in neuronal protection, and the lack of activity of this in neurodegenerative disorders. The VEGF gene is located at 6p21.3, a site several studies have shown to have significant linkage with AD, and a functional polymorphism within the VEGF promoter may alter the risk of developing AD. We assessed the potential impact of this polymorphism on the risk of developing AD in a large French case-control population, and investigated its association with the severity of brain vascular lesions (arteriosclerosis, white matter loss and cerebral amyloid angiopathy) in several brain regions (frontal, temporal, parietal and occipital cortex) in AD.

No association of the VEGF promoter polymorphism with the risk of developing AD was observed. No relationship between this polymor-phism and vascular pathological changes in AD was detected. Our data indicate that although this polymorpolymor-phism is functional, it does not confer greater risk for AD, nor modulate the extent of vascular pathology.

© 2005 Published by Elsevier Inc.

Keywords: Alzheimer; VEGF; Polymorphism; Promoter; Association study; Brain; White matter lesion; Arteriosclerosis; Cerebral angiopathy amyloid

1. Introduction

Alzheimer’s disease (AD) is a progressive neurodegenera-tive disorder that occurs predominantly in later life. The main pathological features of AD are neurofibrillary tangle and senile plaque formation, caused by the progressive deposi-tion of amyloid␤protein (A␤) in the brain, composed mainly of 39–43 amino acid peptides generated through proteolytic cleavage of the A␤precursor protein (APP).

Variations in four genes have been directly linked to the pathogenesis of AD. Mutations in APP, presenilin 1 (PS-1) ∗_{Corresponding author. 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).

or presenilin 2 (PS-2) genes account for most cases of early-onset, autosomal dominant, familial AD (FAD), but these only constitute 2% of all AD cases. The genetics of the late-onset form of the disease is far more complex with only the ␧4 allele of the apolipoprotein E gene (APOE) having so far been identified as playing a major causative role[9].

Numerous loci have been brought to the fore by genome scans. Amongst these, there is evidence for a weak linkage to chromosome 6, in the vicinity of the major histocompat-ibility complex (MCH) at 6p21.3[2,17]. This has led to the investigation of several candidate genes at this locus. To date, the human leukocyte antigen gene (HLA)-A2 allele[12,27], and variations in the regulatory region of the tumour necro-sis factor alpha (TNF-alpha) gene[4], have been reported

0197-4580/$ – see front matter © 2005 Published by Elsevier Inc. doi:10.1016/j.neurobiolaging.2005.07.013

to modestly to increase the risk of developing AD. However, conflicting data has not allowed the definite exclusion or con-firmation of the impact of either of these loci on AD risk

[1,13], and it is possible that these associations may result from a gene in linkage with the HLA-A2 allele or TNF-alpha promoter polymorphisms. Against this background, we have recently excludedNOTCH4as a genetic determi-nant of AD[15]. However, there are still numerous genes of interest located within the MCH 6p21.3 locus, such as

HLA-DR3,CREBL1, the receptor for advanced glycosylation end-products (RAGE), or vascular endothelial growth factor (VEGF), which can be considered as candidate genes for AD. In this report, we have investigated the potential involve-ment ofVEGFsince the rational for this as a candidate gene is strong:

(i) There is increasing evidence for an important role of vascular network damage in AD (for example, insuffi-cient vascularity and reduced cerebral perfusion)[24]. (ii) VEGF is an angiogenic cytokine, involved in neuronal

survival, neuroprotection, regeneration, growth, differ-entiation and axonal outgrowth[21].

(iii) Abnormal regulation of VEGF expression has been reported in AD pathogenesis. Particularly, VEGF immunoreactivity is enhanced in AD neocortex, being located in reactive astrocytes and perivascular amyloid deposits[14].

Furthermore, a polymorphism (rs699947) within the

VEGFpromoter has been very recently reported to increase the risk of developing AD[6]. Collectively, all these data have led us to assess the impact of the rs699947 polymorphism in a large population of French AD patients. Furthermore, we have analysed the impact of this polymorphism on vari-ous pathological hallmarks of the disease, particularly those relating to or responsible for vascular damage such as arte-riosclerosis (ART), cerebral amyloid angiopathy (CAA) and white matter loss (ML), in different brain regions in AD.

2. Materials and methods 2.1. Population

The French AD and control samples were Cau-casian (AD cases n= 601, age = 72.4±7.2 years, age at onset = 69.5±7.4 years, 39.5% male; controls n= 631, age = 72.5±7.9 years, 36% male). An early age at onset was defined as≤65 years. A diagnosis of probable AD was established according to DSM-III-R and NINCDS-ADRDA criteria. Caucasian controls were recruited in defined as sub-jects without DMS-III-R dementia criteria, with integrity of cognitive function and with a MMS score≥25. Presence of family history of dementia was considered as a criteria of exclusion. Controls were recruited either in retirement home or from electoral rolls (altruistic volunteers). Each individual or next of kin gave informed consent.

2.2. AD brain samples

AD brains were obtained at autopsy from 114 patients with early- and late-onset sporadic AD accessioned from Greater Manchester region of United Kingdom during years 1986–2001 (mean age at death = 73.1±9.1 years old; mean age at onset = 65.9±10.3 years old; 51% male). All patients were of Caucasian ethnic group. Pathological diagnoses were made in accordance with CERAD neuropathological criteria for AD. All patients were at Braak stages 5 or 6 at time of death. Formalin fixed blocks of frontal (Brodmann area (BA) 8/9), temporal (BA 21/22), occipital (BA 17/18) and parietal (BA 39/40) cortex were similarly and routinely embedded in paraffin wax and consecutive sections, cut at a thickness of 6 mm, were mounted onto 3-aminopropyltriethoxysilane coated slides. Sections were stained by Weigert’s haema-toxylin and eosin method to assess the severity of CAA and ART, and by Luxol fast blue to assess the severity of ML as an index of white matter damage. The severity of CAA, the degree of ART, and the extent of ML were assessed semi-quantitatively in each of the four cortical areas, as described elsewhere, and scores were summed across regions to provide a combined severity score for each patient[22].

2.3. Genotyping

The rs699947 genotypes were determined by the genomic DNA amplification of a 187 bp fragment using the forward primer 5_{-AACCTAGCACCTCCACCAAA-3 and reverse}

primer 5_{-GTCAGTCTGATTATCCACCC-3and by abglII}

digestion after separation on a 3% agarose gel. The allele and genotype distributions were considered different between the AD and control populations whenp< 0.05.

3. Results

We assessed the impact of the rs699947 polymorphism on the risk of developing AD. The polymorphism distribution was in Hardy-weinberg equilibrium in both AD and control populations.

In contrast to a previous report[6], no association with AD was observed in our French population (Table 1). No significant statistical interaction was observed between the ␧4 allele of theAPOE gene, age, gender and the rs699947 polymorphism. No effect of the rs699947 polymorphism on ART, CAA and ML was detected irrespective of brain area studied (Table 2).

4. Discussion

Recently, much importance has been placed on the co-morbidity of AD and cerebrovascular disease [7,11], the link between AD and ART[3,18], the cognitive impairment associated with CAA[10,25], cerebral microvascular

patho-Table 1

Allele and genotype distribution in the AD case and control populations

n Allele distribution (%)a Genotype distribution (%)a,b

C A CC AC AA

AD cases 630 668 (0.53) 592 (0.47) 186 (0.30) 296 (0.47) 148 (0.23)

Control 664 688 (0.52) 640 (0.48) 182 (0.27) 324 (0.49) 158 (0.24)

aNot significant (Pearsonχ2-test).

b OR (AA + AC vs. CC) = 1.1, 95% CI [0.9–1.4],p< 0.39 adjusted on age and gender.

logical changes [8] and deficient clearance of A␤ across the blood-brain barrier in AD[5,19]. All of these observa-tions indicate that a vascular disorder might be an important pathogenetic feature of the process leading to chronic neu-rodegeneration in AD [28]. In terms of AD susceptibility genes, it is therefore reasonable to hypothesize that the genes coding for proteins involved in development and maintenance of the vascular network, might be good candidates in deter-mining or modifying the risk of developing AD.

VEGF, a major inducer of angiogenesis could be one of these candidates. A hypothetical loss of a VEGF-mediated neuroprotective effect has been proposed as a cause of neu-rodegenerative disorders[20]. While VEGF may be overex-pressed in AD brain, in order to enhance neovascularization and compensate for hypoperfusion linked to cerebrovascular pathologies involving endothelial degeneration and/or CAA, most of the VEGF expressed might be strongly and inertly bound to A␤deposits without producing biological effect. Theoretically, this could result in a local deficiency of soluble VEGF (around amyloid plaques), reinforcing hypoperfusion, as has been suggested by Yang et al.[26].

A correlation between VEGF levels and particular at-risk genotypes and alleles has been associated with normal and pathological in vivo and in vitro conditions[16]. The−2578 (rs699947) VEGF promoter polymorphism lowers VEGF expression. Hence, the level of VEGF expression could be a determinant in the pathogenesis of AD. Recently, Del Bo et al. investigated whether genetic variability within the VEGF

promoter might represent a risk factor for AD. These work-ers reported that−2578 (rs699947) A-allele was associated with an increased risk of developing AD[6]. Two other poly-morphisms were studied, but neither exhibited an association with AD. Following this result, we tested the association of the rs699947 polymorphism with the risk of developing AD in a large French case-control population, but failed to repli-cate such an association. It is important to note that when compared with the initial report of De Bo et al.[6], the minor allele frequency is similar in our AD population (0.48 versus 0.49) but not in the control one (0.48 versus 0.41). This dif-ference may explain in part the discrepancy between the two studies. It is not unusual to observe such variations between different European populations, for instance as observed for the APOE␧2/␧3/␧4 polymorphisms[23]. Nevertheless, we also tested the possibility that the rs699947 may be asso-ciated with lower cognitive performances, but we did not observe such an association when studying the MMS score in our control population (AA = 28.0±1.2; AC = 27.9±1.4 and CC = 28.0±1.6). Furthermore, we also looked for a potential impact of the rs699947 polymorphism on patho-logical hallmarks of AD relating to vascular damage, such as ML, ART or CAA. We were unable to detect an association of the rs699947 A-allele with the severity of any of these lesions.

In conclusion our data indicate that this polymorphism in VEGF promoter, although functional, is not associated with increased risk of developing AD.

Table 2

Effect of the rs699947 polymorphism on ART, ML and CAA in several brain regions in AD

n Frontal^a Parietal^a Temporal^a Occipital^a Total^a

ART CC 37 2.2±2.8 2.9±2.6 1.9±2.4 3.3±2.6 10.2±8.1 AC 24 2.6±2.1 2.4±2.3 2.6±2.0 3.3±2.4 10.9±6.1 AA 15 1.9±2.1 2.7±2.1 1.8±2.0 3.5±2.7 9.9±7.0 ML CC 37 0.3±0.6 0.7±0.9 0.3±0.6 0.9±0.9 2.3±2.2 AC 24 0.3±0.8 0.3±0.7 0.2±0.8 0.7±1.2 1.3±1.9 AA 15 0.3±0.6 0.6±0.8 0.3±0.6 0.6±0.6 1.7±1.6 CAA CC 37 1.7±0.8 1.7±0.9 1.7±0.7 2.2±0.8 7.4±2.7 AC 24 1.9±0.7 1.9±0.9 1.8±0.7 2.1±1.0 7.4±2.8 AA 15 1.6±0.6 1.9±0.9 1.6±0.6 2.3±1.0 7.1±2.9

References

[1] Araria-Goumidi L, Lambert JC, Cottel D, Amouyel P, Chartier-Harlin MC. No association of the HLA-A2 allele with Alzheimer’s disease. Neurosci Lett 2002;335:75–8.

[2] Blacker D, Bertram L, Saunders AJ, et al. Results of a high-resolution genome screen of 437 Alzheimer’s disease families. Hum Mol Genet 2003;12:23–32.

[3] Casserly I, Topol E. Convergence of atherosclerosis and Alzheimer’s disease: inflammation, cholesterol, and misfolded proteins. Lancet 2004;363:1139–46.

[4] Collins JS, Perry RT, Watson Jr B, et al. Association of a haplo-type for tumor necrosis factor in siblings with late-onset Alzheimer disease: the NIMH Alzheimer disease genetics initiative. Am J Med Genet 2000;96:823–30.

[5] Deane R, Wu Z, Sagare A, et al. LRP/amyloid beta-peptide inter-action mediates differential brain efflux of Abeta isoforms. Neuron 2004;43:333–44.

[6] Del Bo R, Scarlato M, Ghezzi S, et al. Vascular endothelial growth factor gene variability is associated with increased risk for AD. Ann Neurol 2005;57:373–80.

[7] de la Torre JC. Alzheimer’s disease is a vasocognopathy: a new term to describe its nature. Neurol Res 2004;26:517–24.

[8] Farkas E, Luiten PG. Cerebral microvascular pathology in aging and Alzheimer’s disease. Prog Neurobiol 2001;64:575–611.

[9] Farrer LA, Cupples LA, Haines JL, et al. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer disease meta analysis consortium. JAMA 1997;278:1349–56.

[10] Greenberg SM, Gurol ME, Rosand J, Smith EE.

Amy-loid angiopathy-related vascular cognitive impairment. Stroke 2004;35:2616–9.

[11] Gorelick PB. Risk factors for vascular dementia and Alzheimer dis-ease. Stroke 2004;35:2620–2.

[12] Harris JM, Cumming AM, Craddock N, St Clair D, Lendon CL. Human leucocyte antigen-A2 increases risk of Alzheimer’s disease but does not affect age of onset in a Scottish population. Neurosci Lett 2000;294:37–40.

[13] Infante J, Llorca J, Berciano J, Combarros O. No synergistic effect between -850 tumor necrosis factor-alpha promoter polymorphism and apolipoprotein E epsilon 4 allele in Alzheimer’s disease. Neu-rosci Lett 2002;328:71–3.

[14] Kalaria RN, Cohen DL, Premkumar DR, Nag S, LaManna JC, Lust WD. Vascular endothelial growth factor in Alzheimer’s

dis-ease and experimental cerebral ischemia. Brain Res Mol Brain Res 1998;62:101–5.

[15] Lambert JC, Mann D, Harris J, Araria-Goumidi L, Chartier-Harlin MC, Cottel D, et al. Association study of Notch 4 polymor-phisms with Alzheimer’s disease. J Neurol Neurosurg Psychiatry 2004;75:377–81.

[16] Lambrechts D, Storkebaum E, Morimoto M, et al. VEGF is a modi-fier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet 2003;34:383–94. [17] Myers A, Wavrant De-Vrieze F, Holmans P, et al. Full genome

screen for Alzheimer disease: stage II analysis. Am J Med Genet 2002;114:235–44.

[18] Roher AE, Esh C, Rahman A, Kokjohn TA, Beach TG. Atherosclero-sis of cerebral arteries in Alzheimer disease. Stroke 2004;35:2623–7. [19] Shibata M, Yamada S, Kumar SR, et al. Clearance of Alzheimer’s amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest 2000;106:1489–99. [20] Storkebaum E, Carmeliet P. VEGF: a critical player in

neurodegen-eration. J Clin Invest 2004;113(1):14–8.

[21] Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, et al. VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest 2003;111:1843–51.

[22] Tian J, Shi J, Bailey K, Mann DM. Relationships between arte-riosclerosis, cerebral amyloid angiopathy and myelin loss from cere-bral cortical white matter in Alzheimer’s disease. Neuropathol Appl Neurobiol 2004;30:46–56.

[23] Tiret L, de Knijff P, Menzel HJ, Ehnholm C, Nicaud V, Havekes LM. ApoE polymorphism and predisposition to coronary heart dis-ease in youths of different European populations. The EARS Study. European Atherosclerosis Research Study. Arterioscler Thromb 1994;14:1617–24.

[24] Vagnucci Jr AH, Li WW. Alzheimer’s disease and angiogenesis. Lancet 2003;361:605–8.

[25] Vinters HV, Farag ES. Amyloidosis of cerebral arteries. Adv Neurol 2003;92:105–12.

[26] Yang SP, Bae DG, Kang HJ, Gwag BJ, Gho YS, Chae CB. Co-accumulation of vascular endothelial growth factor with beta-amyloid in the brain of patients with Alzheimer’s disease. Neurobiol Aging 2004;25:283–90.

[27] Zareparsi S, James DM, Kaye JA, Bird TD, Schellenberg GD, Payami H. HLA-A2 homozygosity but not heterozygosity is associ-ated with Alzheimer disease. Neurology 2002;58:973–5.

[28] Zlokovic BV. Neurovascular mechanisms of Alzheimer’s neurode-generation. Trends Neurosci 2005;28:202–8.