Case-control study of vascular endothelial growth factor (VEGF) genetic variability in Alzheimer's disease

Neuroscience Letters 401 (2006) 171–173

Case-control study of vascular endothelial growth factor (VEGF) genetic variability in Alzheimer’s disease Ignacio Mateo a , Javier Llorca b , Jon Infante a , Eloy Rodr´ıguez-Rodr´ıguez a , Coro S´anchez-Quintana a , Pascual S´anchez-Juan a , Jos´e Berciano a , Onofre Combarros a,∗ a

Service of Neurology, University Hospital “Marqu´es de Valdecilla” (University of Cantabria), 39008 Santander, Spain b Division of Preventive Medicine, University of Cantabria School of Medicine, 39008 Santander, Spain Received 12 February 2006; received in revised form 4 March 2006; accepted 8 March 2006

Abstract Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and blood vessel function. Recent evidence indicates that VEGF facilitates memory and learning through stimulating angiogenesis and neurogenesis in the rat hippocampal dendate gyrus. Abnormal regulation of VEGF expression has been reported in the pathogenesis of both atherosclerosis and motoneuron degeneration in amyotrophic lateral sclerosis, with low VEGF-producing polymorphisms (−2578 allele A and −634 allele G) conferring increased susceptibility for the development of the disorders. We tested whether these polymorphisms downregulating expression of VEGF might increase the risk of developing Alzheimer’s disease (AD). So, we performed a case-control study in 362 Spanish AD patients and 428 healthy controls. The current study does not demonstrate an association between VEGF (−2578) and VEGF (−634) genotypes or haplotypes and AD. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; VEGF; Polymorphism; Angiogenesis

Recent emphasis on the link between Alzheimer’s disease (AD) and atherosclerosis [6,7] and significant cerebral microvascular pathology (degeneration of endothelial cells and decreased microvascular density) [16], indicate that vascular factors might play a role in the pathogenesis of AD. Vascular endothelial growth factor (VEGF) is a hypoxia-inducible protein that promotes the proliferation and survival of vascular endothelial cells. Abnormal regulation of VEGF expression has now been implicated in several neurodegenerative disorders, in part by impairing neural tissue perfusion [14]. Indeed, reduced VEGF levels in gene-targeted mice caused progressive motor neuron degeneration, reminiscent of human amyotrophic lateral sclerosis (ALS), and treatment with VEGF protected mice against ischemic motoneuron death [9]. In addition, a transgenic mouse model of spinal and bulbar muscular atrophy revealed that androgen receptors containing expanded polyglutamine caused decreased expression of VEGF, thereby contributing to the motor neuron

∗

Corresponding author. Tel.: + 34 942 202 520; fax: +34 942 202 655. E-mail address: [email protected] (O. Combarros).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.03.020

degeneration [13]. Moreover, VEGF is capable of protecting dopaminergic neurons against degeneration in a rat model of Parkinson’s disease [15]. In humans, sequence alterations in the promoter region of the VEGF gene that reduce both VEGF gene transcription and VEGF plasma levels, have been implicated in increasing the risk of developing ALS [9]. VEGF seems to be important for memory and learning. In fact, training in a Morris water maze was associated with rat hippocampal overexpression of VEGF, which leaded to increased angiogenesis and neurogenesis in the hippocampal dentate gyrus and improved cognition [3]. A dowregulation of VEGF production has been recently demonstrated by peripheral immune cells of AD subjects [12], and in a preliminary report, a functional polymorphism within the promoter region (−2578) of the VEGF gene that lowers VEGF expression, has been associated with increased risk of AD [5]. However, in a subsequent study [4], this polymorphism did not confer greater risk for AD, nor modulated the extent of brain vascular lesions in AD. To further elucidate the relationship between VEGF genetic variation and AD, we tested this association in a large group of Spanish patients with AD and controls, by examining the promoter

172

I. Mateo et al. / Neuroscience Letters 401 (2006) 171–173

(−2578) and the 5 -UTR (−634) VEGF polymorphisms, which had previously shown the strongest influence on VEGF activity. The study included 362 AD patients (69% women; mean age at study 75.8 years; S.D. 8.5; range 50–97 years; mean age at onset 72.3 years; S.D. 8.3; range 48–94 years) who met NINCDS/ADRDA criteria for probable AD [10]. All AD cases were defined as sporadic because their family history did not mention any first-degree relative with dementia. AD patients were consecutively admitted to the Department of Neurology, University Hospital “Marqu´es de Valdecilla”, Santander, Spain, from January 1997 to January 2001. The large majority of patients were living in the community and had been referred by their general practitioner; few had been admitted from hospital wards or nursing home facilities. Control subjects were 428 unrelated individuals (69% women; mean age 80.5 years; S.D. 7.8; range 51–100 years) randomly selected from a nursing home. These subjects had complete neurologic and medical examinations that showed that they were free of significant illness and had Mini Mental State Examination scores of 28 or more, which were verified by at least one subsequent annual following-up assessment. The controls arose from the same base population as the cases. The AD and control samples were Caucasians originating from a limited geographical area in northern Spain. All patients and controls were ascertained to have parents and grandparents born in Northern Spain to ensure ethnicity. Consequently, possible confounding effects of the inclusion in the study of members of different ethnic groups have been minimized. Blood samples were taken after written informed consent had been obtained from the subjects or their representatives. The study was approved by the ethical committee of the University Hospital “Marqu´es de Valdecilla”. Genotyping of VEGF (−2578) (rs 699947) and VEGF (−634) (rs 2010963) was performed by a Taq-Man single-nucleotide-polymorphism assay (Applied Biosystems, Warrington, Cheshire, UK) and a ABI PRISM 7000 sequence detection system (Applied Biosystems). Association between dichotomous variables was analyzed with odds ratio, and 95% confidence intervals were estimated by the Cornfield method or the exact method; P-values for association were estimated by Wald test. Hardy–Weinberg equilibrium was tested by chi-squared without continuity correction or Fisher exact tests. Logistic regression was used to obtain adjusted odds ratio estimates. All statistical analysis was performed with the package Stata Intercooled, version 8/SE (Stata Corporation, College Station, Texas, USA). The two-site haplotype frequencies were estimated using the Expectation-Maximization algorithm in the EH software program (http://www.linkage.rockefeller.edu/ott/eh.htm). Control groups for VEGF (−2578) (P = 0.980) and VEGF (−634) (P = 0.684) polymorphisms were within the range of Hardy–Weinberg equilibrium. As shown in Table 1, the distribution of the allele and genotype frequencies of the VEGF (−2578) and (−634) polymorphisms did not differ significantly between AD and control groups. Both VEGF polymorphisms were in significant linkage disequilibrium with each other (P < 0.0001), both in patients and controls. Next, we tested the haplotype frequency differences in the AD group and controls (Table 2),

Table 1 Distribution of VEGF (−2578) and VEGF (−634) polymorphisms in patients and controls Polymorphism

Patients

Controls

OR (95% CI)

P

VEGF (−2578) CC CA AA Total Allele frequency C/A

119 (0.33) 169 (0.47) 74 (0.20) 362 0.56/0.44

130 (0.33) 193 (0.49) 72 (0.18) 395 0.57/0.43

1 (reference) 1.00 (0.70–1.43) 1.19 (0.76–1.88)

0.992 0.441

VEGF (−634) GG GC CC Total Allele frequency G/C

140 (0.39) 169 (0.47) 53 (0.14) 362 0.62/0.38

169 (0.40) 203 (0.47) 56 (0.13) 428 0.63/0.37

1 (reference) 1.03 (0.74–1.44) 1.13 (0.70–1.82)

0.864 0.623

Values in parentheses indicate frequencies; OR: odds ratio adjusted by age, gender and APOE ␧4 status; CI: confidence interval.

and the overall haplotype frequencies were not statistically different between cases and controls (P = 0.301). There were no major differences in allele, genotype or haplotype frequencies of VEGF polymorphisms between cases and controls for either early-onset (<65 years) or late-onset (≥65 years) subgroups (data not shown). Similarly, no significantly different risk of AD was observed when our data set was stratified by gender or APOE genotype. Polymorphisms within the promoter region (−2578) and 5 -UTR (−634) of the VEGF gene have been shown to have functional significance: alleles −2578 A and −634 G are associated with low VEGF production from lypopolysaccharide stimulated peripheral blood mononuclear cells [2,11]; VEGF serum levels are lower in individual homozygous for either −2578 A or −634 G alleles [1,9]; basic transcriptional activity levels associated with the −634 G allele are lower compared with those seem with the −634 C allele in human glioma cells [2]. These VEGF polymorphisms downregulating expression of VEGF have been shown to increase the susceptibility to certain diseases: the VEGF (−2578) AA genotype acted as a risk factor for atherosclerosis development via association with lower VEGF production [8]; a European collaborative group from Belgium, Sweden and England studied a possible role of VEGF in ALS, and found that “low-VEGF” haplotypes (−2578 A/−1154

Table 2 Estimated haplotype frequencies of the VEGF (−634) and VEGF (−2578) polymorphisms among patients and controls −634

−2578

Patients (n = 353)

Controls (n = 357)

All subjects (n = 710)

G G C C ln(L)a

C A C A

0.1956 0.4249 0.3674 0.0121 −623.86

0.2161 0.4185 0.3618 0.0036 −599.47 P = 0.301

0.2058 0.4216 0.3646 0.0080 −1225.16

a Calculated from the T statistic: 2[ln(L) cases + ln(L)controls − ln(L)all subjects ]; d.f. = 3.

I. Mateo et al. / Neuroscience Letters 401 (2006) 171–173

A/−634 G or −2578 A/−1154 G/−634 G) were associated with lower VEGF plasma levels (due to impaired transcription and translation of several VEGF isoforms) and increased risk of ALS [9]. We speculated that significant overrepresentation of VEGF −2578 A and −634 G alleles among the AD population would suggest that low VEGF production is a risk factor for AD, but none of them showed significant differences in genotype or haplotype differences in our AD and control populations. In an Italian case-control study, Del Bo et al. [5] recently investigated the association of four polymorphisms (−2578, −1198, −1190, and −1154) in the promoter region of the VEGF gene in patients with AD, and found that the −2578 AA genotype was associated with and increased risk for AD; in addition, the haplotype (−2578 A/−1190 G/−1154 G) was overrepresented in AD population. Our study had 85.7% power to detect an OR = 2 as obtained by Del Bo et al. A subsequent study of VEGF (−2578) polymorphism in a large French case-control population failed to replicate such an association with AD [4]. Our negative findings in the Spanish population argue against the hypothesis that the VEGF (−2578) polymorphism is causally related to AD. Still, analyzing additional VEGF polymorphisms with a larger sample size deserves further attention, since supporting evidence for the biological role of VEGF in AD exists.

[5]

[6]

[7]

[8] [9]

[10]

Acknowledgement

[11]

This work was supported by Grant No. PI050005 from Fondo de Investigaci´on Sanitaria.

[12]

References [1] T. Awata, K. Inoue, S. Kurihara, T. Ohkubo, M. Watanabe, K. Inukai, I. Inoue, S. Katayama, A common polymorphism in the 5 -untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes, Diabetes 51 (2002) 1635–1639. [2] T. Awata, S. Kurihara, N. Takata, T. Neda, H. Iizuka, T. Ohkubo, M. Osaki, M. Watanabe, Y. Nakashima, K. Inukai, I. Inoue, I. Kawasaki, K. Mori, S. Yoneya, S. Katayama, Functional VEGF C-634G polymorphism is associated with development of diabetic macular edema and correlated with macular retinal thickness in type 2 diabetes, Biochem. Biophys. Res. Commun. 333 (2005) 679–685. [3] L. Cao, X. Jiao, D.S. Zuzga, Y. Liu, D.M. Fong, D. Young, M.J. During, VEGF links hippocampal activity with neurogenesis, learning and memory, Nat. Genet. 36 (2004) 827–835. [4] J. Chapuis, J. Tian, J. Shi, F. Bensemain, D. Cottel, C. Lendon, P. Amouyel, D. Mann, J.C. Lambert, Association study of the vascular

[13]

[14] [15]

[16]

173

endothelial growth factor gene with the risk of developing Alzheimer’s disease, Neurobiol. Aging, in press. R. Del Bo, M. Scarlato, S. Ghezzi, F.M. Boneschi, C. Fenoglio, S. Galbiati, R. Virgilio, D. Galimberti, G. Galimberti, M. Crimi, C. Ferrarese, E. Scarpini, N. Bresolin, G.P. Comi, Vascular endothelial growth factor gene variability is associated with increased risk for AD, Ann. Neurol. 57 (2005) 373–380. A. Hofman, A. Ott, M.M.B. Breteler, M.L. Bots, A.J.C. Slooter, F. van Harskamp, C.N. van Duijn, C. Van Broeckhoven, D.E. Grobbee, Atherosclerosis, apolipoprotein E, and prevalence of dementia and Alzheimer’s disease in the Rotterdam Study, Lancet 349 (1997) 151–154. L.S. Honig, W. Kukull, R. Mayeux, Atherosclerosis and AD: analysis of data from the US National Alzheimer’s Coordinating Center, Neurology 64 (2005) 494–500. W.M. Howell, S. Ali, M.J. Rose-Zerilli, S. Ye, VEGF polymorphisms and severity of atherosclerosis, J. Med. Genet. 42 (2005) 485–490. D. Lambrechts, E. Storkebaum, M. Morimoto, J. Del-Favero, F. Desmet, S.L. Marklund, S. Wyns, V. Thijs, J. Andersson, I. van Marion, A. AlChalabi, S. Bornes, R. Musson, V. Hansen, L. Beckman, R. Adolfsson, H.S. Pall, H. Prats, S. Vermeire, P. Rutgeerts, S. Katayama, T. Awata, N. Leigh, L. Lang-Lazdunski, M. Dewerchin, C. Shaws, L. Moons, R. Vlietinck, K.E. Morrison, W. Robberecht, C. Van Broeckhoven, D. Collen, P.M. Andersen, P. Carmeliet, VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death, Nat. Genet. 34 (2003) 383–394. G. McKhaan, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, Clinical diagnosis of Alzheimer’s disease: report of the NINCDA-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease, Neurology 34 (1984) 934–944. M. Shahbazi, A.A. Fryer, V. Pravica, I.J. Brogan, H.M. Ramsay, I.V. Hutchinson, P.N. Harden, Vascular endothelial growth factor gene polymorphisms are associated with acute renal allograft rejection, J. Am. Soc. Nephrol. 13 (2002) 260–264. S.B. Solerte, E. Ferrari, G. Cuzzoni, E. Locatelli, A. Giustina, M. Zamboni, N. Schifino, M. Rondanelli, C. Gazzaruso, M. Fioravanti, Decreased release of the angiogenic peptide vascular endothelial growth factor in Alzheimer’s disease: recovering effect with insulin and DHEA sulfate, Dement. Geriatr. Cogn. Disord. 19 (2005) 1–10. B.L. Sopher, P.S. Thomas Jr., M.A. LaFevre-Bernt, I.E. Holm, S.A. Wilke, C.B. Ware, L.W. Jin, R.T. Libby, L.M. Ellerby, A.R. La Spada, Androgen receptor YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration, Neuron 41 (2004) 687–699. E. Storkebaum, P. Carmeliet, VEGF: a critical player in neurodegeneration, J. Clin. Invest. 113 (2004) 14–18. T. Yasuhara, T. Shingo, K. Kobayashi, A. Takeuchi, A. Yano, K. Muraoka, T. Matsui, Y. Miyoshi, H. Hamada, I. Date, Neuroprotective effects of vascular endothelial growth factor (VEGF) upon dopaminergic neurons in a rat model of Parkinson’s disease, Eur. J. Neurosci. 19 (2004) 1494–1504. B.V. Zlokovic, Neurovascular mechanisms of Alzheimer’s neurodegeneration, Trends Neurosci. 28 (2005) 202–208.