- Email: [email protected]
No evidence for association of the monocyte chemoattractant protein-1 (−2518) gene polymorphism and Alzheimer's disease
Neuroscience Letters 360 (2004) 25–28 www.elsevier.com/locate/neulet
No evidence for association of the monocyte chemoattractant protein-1 (2 2518) gene polymorphism and Alzheimer’s disease Onofre Combarrosa,*, Jon Infantea, Javier Llorcab, Jose´ Bercianoa a
Service of Neurology, University Hospital ‘Marque´s de Valdecilla’, University of Cantabria, 39008 Santander, Spain b Division of Preventive Medicine, University of Cantabria School of Medicine, 39008 Santander, Spain Received 10 November 2003; accepted 16 January 2004
Abstract Activation of microglia is a central part of the chronic inflammatory process in Alzheimer disease (AD). The monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a role in microglial migration and accumulation at sites of beta-amyloid deposition in senile plaques in the AD brain. A polymorphism in the regulatory region (2 2518) of the MCP-1 gene affects the level of MCP-1 expression, and has been associated with a stronger inflammatory response and higher peripheral tissue damage in chronic inflammatory diseases. We investigated whether the MCP-1 (22518) polymorphism might be responsible for susceptibility to AD in a large Spanish population, utilizing a clinically well-defined group of 328 sporadic AD patients and 315 control subjects. We also examined the combined gene effects between MCP-1 and other proinflammatory cytokine genes such as interleukin-1A (IL-1A) and tumor necrosis factor-a (TNF-a), and the apolipoprotein E (APOE) gene. In the present study, neither the MCP-1 (2 2518) G allele itself nor its interaction with the IL-1A (2 889) allele 2, TNF-a (2850) allele T or APOE e4 allele conferred increased risk for AD. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Monocyte chemoattractant protein-1; Chemokine; Polymorphism
Activation of microglia is a central part of the chronic inflammatory processes associated with amyloid beta (Ab) peptide plaques in Alzheimer’s disease (AD) [21]. Activated microglial cells produce and release potentially toxic products, including proinflammatory cytokines such as interleukin (IL)1A, IL-1B, IL-6 and tumor necrosis factor (TNF)-a, and chemokines such as IL-8, macrophage inflammatory peptide1A, and monocyte chemoattractant protein-1 (MCP-1), which could damage neighboring neurons [6]. For example, IL-1 over-expression in the brain correlates with the degree of progression of senile plaques, the formation of neurofibrillary tangles, and neuronal damage [5]. TNF-a production is also increased in AD brain [15]. The role of chemokines in AD progression is not understood, but likely represents an additional recruitment mechanism for the migration of microglia to Ab deposits associated with senile plaques, followed by a subsequent and persistent activation of microglial cells [17]. Upregulation of the chemokine MCP-1 has been demonstrated in several studies in AD brain tissue * Corresponding author. Tel.: (34-942-202-520; fax: (34-942-202-655. E-mail address: [email protected] (O. Combarros).
and in response to experimental stimulation of microglia by Ab peptide or by cytokines, such as IL-1 and TNF-a [6,7,17, 20]. Moreover, there is good evidence indicating that levels of MCP-1 in plasma and cerebrospinal fluid might be candidates as biomarkers for monitoring the inflammatory process in AD [3,18]. Recently, polymorphisms in genes encoding IL-1A, IL1B, IL-6 and TNF-a have been shown to increase the risk for AD, probably by upregulating inflammation in the brain [10]. Since cytokine gene polymorphisms in the regulatory region are related to the amount of cytokine produced, they may account for individual susceptibility to inflammatory conditions. Thus, homozygosity for allele 2 of IL-1A (2889) has been associated with a five-fold increase in production of the cytokine compared with homozygosity for allele 1 [2], and conferred a three-fold increased risk for AD [1,10]. Similarly, the possession of the T allele in the promoter region (2850) of the TNF-a gene increased the risk of AD [9]. A biallelic A/G polymorphism at position 2 2518 of the MCP-1 gene seems to influence the transcriptional activity because cells obtained from GG or AG individuals produce more MCP-1 protein in response to
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.01.035
26
O. Combarros et al. / Neuroscience Letters 360 (2004) 25–28
stimuli such as cytokines, than those isolated from AA individuals [16]. Therefore, it would be reasonable to speculate that individuals bearing G at position 22518 have a stronger inflammatory response with higher tissue damage. In fact, prevalence of these high MCP-1 producing genotypes has been shown to be associated with the risk for premature kidney graft failure [8], predisposition to more severe fibrosis and inflammation in patients with chronic hepatitis C [12], or the risk for severe coronary artery disease [19]. Functional in vitro studies comparing cells from individuals with different MCP-1 (22518) genotypes have been performed with peripheral blood mononuclear cells [16] and activated hepatic stellate cells [12], but the role of this polymorphism in microglial cells has not yet been evaluated. To our knowledge, only one case-control study in a Japanese sample reporting no association of MCP-1 (2 2518) with AD has been published [13]. However, a racial difference was found in the MCP-1 (22518) polymorphism [16], and therefore, it is particularly necessary to study different ethnic groups to establish the relationship between this polymorphism and AD. In the present study, we investigated whether the MCP-1 (2 2518) G variant (AG and GG genotypes) might be responsible for susceptibility to AD in a large Spanish population. We also examined the combined gene effects between MCP-1 and IL-1A and TNF-a. In addition, apolipoprotein E (APOE) genotyping was conducted in the same AD case and control groups to examine any possible association with MCP-1 (2 2518). The study included 328 AD patients (69% women; mean age at the time of study 75.8 years; SD 9.9; range 50–98 years; mean age at onset 72.1 years; SD 8.7; range 48–95 years) who met NINCDS/ADRDA criteria for probable AD [11]. 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 ‘Marque´s de Valdecilla’, Santander, Spain, from January 1997 to June 2000. 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 315 unrelated individuals (71% women; mean age 80.5 years; SD 7.5; range 63–100 years) randomly selected from a nursing home. It is often considered that individuals living in nursing homes do not represent the general population of that particular group age but might represent a subset of people; however, the fact that control genotypes were in Hardy–Weinberg equilibrium argues against a significant bias being introduced by this method of selection. These subjects had complete neurological and medical examinations that showed 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 follow-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. Genotyping of MCP-1 (2 2518), IL-1A (2 889), TNF-a (2 850), and APOE polymorphisms were determined as described previously [1,9,14]. Statistical methods: Association between dichotomous variables was analyzed with odds ratio, and 95% confidence intervals were estimated by the Cornfield method or the Fisher exact method. Means of quantitative variables were compared using the Student’s t-test. P-values were estimated by chi-square or Fisher exact tests. Interrelations were analyzed by stratification. All statistical analysis were performed with the package Stata 8/SE (Stata Corporation, College Station, TX, USA). The distributions of MCP-1 (22518) genotypes were in Hardy–Weinberg equilibrium (P ¼ 0:723). As shown in Table 1, there were no statistical differences in the AD group when compared to controls. When compared to AA genotype, the odds ratio (OR) for the AG genotype was 0.98 (95% CI ¼ 0.70–1.36), and 0.84 (95% CI ¼ 0.43–1.68) for the GG genotype. Similarly, no significantly different risk of AD was observed when our data set was stratified by gender. Next, we studied the synergistic effects between MCP-1 (2 2518) and IL-1A (2 889), TNF-a (2 850), and APOE polymorphisms by logistic regression analysis (Table 2). There was no significant association between the MCP-1 (2 2518) G-carrying (AG þ GG) genotypes and the risk of AD in either IL-1A (2 889) allele 2 (1/2 þ 2/2 genotypes) carriers and TNF-a (2 850) T allele (CT þ TT genotypes) carriers. Our analysis showed the expected association between the APOE e4 allele and AD, but there was no interaction between carriage of MCP-1 (2 2518) G allele and of the APOE e4 allele. We then examined the influence of MCP-1 (2 2518) genotypes on the age at onset of AD, and no significant variation existed among AA (mean age of 71.8, SD 8.8 years), AG (mean age of 72.3, SD 9.0 years), and GG (mean age of 75.3, SD 6.5 years) genotypes. There have been a number of reports of inflammatory gene variants being associated with susceptibility to AD Table 1 MCP-1 (22518) genotype and allele frequencies in AD patients and control subjects n
AD Controls
328 315
Genotype, n (%)
Allele frequency
AA
AG
GG
A
G
195 (59.4) 184 (58.4)
116 (35.4) 112 (35.6)
17 (5.2) 19 (6.0)
0.77 0.76
0.23 0.24
n ¼ number of cases; and AD ¼ Alzheimer’s disease.
O. Combarros et al. / Neuroscience Letters 360 (2004) 25–28
27
Table 2 Odds ratios for Alzheimer’s disease risk according to interaction of MCP-1 (22518) genotypes with IL-1A (2889), TNF-a (2850), and APOE genotypes Genotypes IL-1A (1/2 þ 2/2) 2 2 þ þ TNF-a (CT þ TT) 2 2 þ þ APOE e 4 2 2 þ þ
MCP-1 (AG þ GG)
Alzheimer
Controls
OR (95% Confidence Interval)
P
2 þ 2 þ
87 67 74 48
103 65 58 44
1 (reference) 1.22 (0.76–1.95) 1.51 (0.94–2.42) 1.29 (0.76–2.19)
0.380 0.070 0.314
2 þ 2 þ
152 95 24 26
131 100 29 14
1 (reference) 0.82 (0.56–1.20) 0.71 (0.38–1.34) 1.60 (0.77–3.46)
0.283 0.260 0.179
2 þ 2 þ
94 57 101 76
153 105 31 26
1 (reference) 0.88 (0.57–1.36) 5.30 (3.21–8.84) 4.76 (2.77–8.29)
0.556 ,0.001 ,0.001
MCP-1 (AG þ GG) (2) ¼ monocyte chemokine protein-1 AA; IL-1A (1/2 þ 2/2) (2) ¼ interleukin-1A 1/1; TNF-a (CT þ TT) (2) ¼ tumor necrosis factor-a CC; APOE e4 (2) ¼ no copies of apolipoprotein E e4; and APOE e4 ( þ ) ¼ one or two copies of APOE e4.
[10], but the present study is the first to analyze MCP-1 gene variants in susceptibility to AD in Caucasians. We hypothesized that subjects with the MCP-1 (22518) G allele exhibited a higher level of MCP-1 expression and a higher intensity of migration and activation of microglia in the brain than those without the MCP-1 (22518) G allele, and consequently had an increased risk of AD. Reviewing previous reports, it seems evident that the MCP-1 (22518) G allele frequency demonstrates racial heterogeneity. In fact, while in Japanese [13,14] and Mexican [16] populations a G allele frequency of 64 and 47% has been reported, respectively, in Caucasian American and European populations the G allele frequency ranges from 21 to 29% [4,8,16,19]. With regard to Europe, the MCP-1 (22518) G allele frequency is quite homogeneously distributed. We did not find any association between the MCP-1 (22518) G allele and AD in Spanish subjects, thus confirming a recent negative report conducted in Japanese population [13]. In the present study, interaction of MCP-1 (22518) G allele with IL-1A (2889) allele 2, TNF-a (2850) allele T, and APOE e4 allele did not confer increased risk for AD. In conclusion, although supporting evidence for the biological role of MCP-1 in AD exists, the MCP-1 (2 2518) polymorphism distribution does not differ between patients with AD and controls, whether they are white or Japanese, and therefore, this polymorphism is unlikely to confer genetic susceptibility to AD.
Acknowledgements This work was supported by Grant No. PI020027 from Fondo de Investigacio´n Sanitaria.
References [1] O. Combarros, M. Sa´nchez-Guerra, J. Infante, J. Llorca, J. Berciano, Gene dose-dependent association of interleukin-1A (2889) allele 2 polymorphism with Alzheimer’s disease, J. Neurol. 249 (2002) 1242– 1245. [2] R. Dominici, M. Cattaneo, G. Malferrari, D. Archi, C. Mariani, L.M.E. Grimaldi, I. Biunno, Cloning and functional analysis of the allelic polymorphism in the transcription regulatory region of interleukin-1a, Immunogenetics 54 (2002) 82–86. [3] D. Galimbetti, N. Schoonenboom, E. Scarpini, P. Scheltens, Chemokines in serum and cerebrospinal fluid of Alzheimer’s disease patients, Ann. Neurol. 53 (2003) 547–548. [4] M.F. Gonza´lez-Escribano, B. Torres, F. Aguilar, R. Rodrı´guez, A. Garcı´a, A. Valenzuela, A. Nu´n˜ez-Rolda´n, MCP-1 promoter polymorphism in Spanish patients with rheumatoid arthritis, Hum. Immunol. 64 (2003) 741–744. [5] W.S.T. Griffin, R.E. Mrak, Interleukin-1 in the genesis and progression of and risk for development of neuronal degeneration in Alzheimer’s disease, J. Leukoc. Biol. 72 (2002) 233 –238. [6] U.K. Hanisch, Microglia as a source and target of cytokines, Glia 40 (2002) 140–155. [7] K.A. Harkness, J.D. Sussman, G.A.B. Davies-Jones, J. Greenwood, M.N. Woodroofe, Cytokine regulation of MCP-1 expression in brain and retinal microvascular endothelial cells, J. Neuroimmunol. 142 (2003) 1–9. [8] B. Kru¨ger, B. Schro¨pel, R. Ashkan, B. Marder, C. Zu¨lke, B. Murphy, B.K. Kra¨mer, M. Fischereder, A monocyte chemoattractant protein-1 (MCP-1) polymorphism and outcome after renal transplantation, J. Am. Soc. Nephrol. 13 (2002) 2585–2589. [9] S.M. McCusker, M.D. Curran, K.B. Dynan, C.D. McCullagh, D. Urquhart, C.C. Patterson, S.P. McIlroy, A.P. Passmore, Association between polymorphism in regulatory region of gene encoding tumour necrosis factor a and risk of Alzheimer’s disease and vascular dementia: a case-control study, Lancet 357 (2001) 436–439. [10] P.L. McGeer, E.G. McGeer, Polymorphisms in inflammatory genes and the risk of Alzheimer disease, Arch. Neurol. 58 (2001) 1790– 1792. [11] 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
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O. Combarros et al. / Neuroscience Letters 360 (2004) 25–28 Health and Human Services Task Force on Alzheimer’s Disease, Neurology 34 (1984) 934–944. M. Mu¨hlbauer, A.K. Bosserhoff, A. Hartmann, W.E. Thasler, T.S. Weiss, H. Herfarth, G. Lock, J. Scho¨lmerich, C. Hellerbrand, A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease, Gastroenterology 125 (2003) 1085–1093. M. Nishimura, S. Kuno, I. Mizuta, M. Ohta, H. Maruyama, R. Kaji, H. Kawakami, Influence of monocyte chemoattractant protein 1 gene polymorphism on age at onset of sporadic Parkinson’s disease, Mov. Disord. 18 (2003) 953–955. K. Omori, J.J. Kazama, J. Song, S. Goto, T. Takada, N. Saito, M. Sakatsume, I. Narita, F. Gejyo, Association of the MCP-1 gene polymorphism A 2 2518G with carpal-tunnel syndrome in hemodialysis patients, Amyloid 9 (2002) 175 –182. R.T. Perry, J.S. Collins, H. Wiener, R. Acton, R.C.P. Go, The role of TNF and its receptors in Alzheimer’s disease, Neurobiol. Aging 22 (2001) 873–883. B.H. Rovin, L. Lu, R. Saxena, A novel polymorphism in the MCP-1
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gene regulatory region that influences MCP-1 expression, Biochem. Biophys. Res. Commun. 259 (1999) 344–348. W.J. Streit, J.R. Conde, J.K. Harrison, Chemokines and Alzheimer’s disease, Neurobiol. Aging 22 (2001) 909– 913. Y.X. Sun, L. Minthon, A. Wallmark, S. Warkentin, K. Blennow, S. Janciauskiene, Inflammatory markers in matched plasma and cerebrospinal fluid from patients with Alzheimer’s disease, Dement. Geriatr. Cogn. Disord. 16 (2003) 136–144. C. Szalai, J. Duba, Z. Proha´szka, A. Kalina, T. Szabo´, B. Nagy, L. Horva´th, A. Csa´sza´r, Involvement of polymorphisms in the chemokine system in the susceptibility for coronary artery disease (CAD). Coincidence of elevated Lp(a) and MCP-1 22518 G/G genotype in CAD patients, Atherosclerosis 158 (2001) 233–239. D.G. Walker, L.F. Lue, T.G. Beach, Gene expression profiling of amyloid beta peptide-stimulated human post-mortem brain microglia, Neurobiol. Aging 22 (2001) 957–966. T. Wyss-Coray, L. Mucke, Inflammation in neurodegenerative disease. A double-edged sword, Neuron 35 (2002) 419 –432.
No evidence for association of the monocyte chemoattractant protein-1 (2 2518) gene polymorphism and Alzheimer’s disease Onofre Combarrosa,*, Jon Infantea, Javier Llorcab, Jose´ Bercianoa a
Service of Neurology, University Hospital ‘Marque´s de Valdecilla’, University of Cantabria, 39008 Santander, Spain b Division of Preventive Medicine, University of Cantabria School of Medicine, 39008 Santander, Spain Received 10 November 2003; accepted 16 January 2004
Abstract Activation of microglia is a central part of the chronic inflammatory process in Alzheimer disease (AD). The monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a role in microglial migration and accumulation at sites of beta-amyloid deposition in senile plaques in the AD brain. A polymorphism in the regulatory region (2 2518) of the MCP-1 gene affects the level of MCP-1 expression, and has been associated with a stronger inflammatory response and higher peripheral tissue damage in chronic inflammatory diseases. We investigated whether the MCP-1 (22518) polymorphism might be responsible for susceptibility to AD in a large Spanish population, utilizing a clinically well-defined group of 328 sporadic AD patients and 315 control subjects. We also examined the combined gene effects between MCP-1 and other proinflammatory cytokine genes such as interleukin-1A (IL-1A) and tumor necrosis factor-a (TNF-a), and the apolipoprotein E (APOE) gene. In the present study, neither the MCP-1 (2 2518) G allele itself nor its interaction with the IL-1A (2 889) allele 2, TNF-a (2850) allele T or APOE e4 allele conferred increased risk for AD. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Monocyte chemoattractant protein-1; Chemokine; Polymorphism
Activation of microglia is a central part of the chronic inflammatory processes associated with amyloid beta (Ab) peptide plaques in Alzheimer’s disease (AD) [21]. Activated microglial cells produce and release potentially toxic products, including proinflammatory cytokines such as interleukin (IL)1A, IL-1B, IL-6 and tumor necrosis factor (TNF)-a, and chemokines such as IL-8, macrophage inflammatory peptide1A, and monocyte chemoattractant protein-1 (MCP-1), which could damage neighboring neurons [6]. For example, IL-1 over-expression in the brain correlates with the degree of progression of senile plaques, the formation of neurofibrillary tangles, and neuronal damage [5]. TNF-a production is also increased in AD brain [15]. The role of chemokines in AD progression is not understood, but likely represents an additional recruitment mechanism for the migration of microglia to Ab deposits associated with senile plaques, followed by a subsequent and persistent activation of microglial cells [17]. Upregulation of the chemokine MCP-1 has been demonstrated in several studies in AD brain tissue * Corresponding author. Tel.: (34-942-202-520; fax: (34-942-202-655. E-mail address: [email protected] (O. Combarros).
and in response to experimental stimulation of microglia by Ab peptide or by cytokines, such as IL-1 and TNF-a [6,7,17, 20]. Moreover, there is good evidence indicating that levels of MCP-1 in plasma and cerebrospinal fluid might be candidates as biomarkers for monitoring the inflammatory process in AD [3,18]. Recently, polymorphisms in genes encoding IL-1A, IL1B, IL-6 and TNF-a have been shown to increase the risk for AD, probably by upregulating inflammation in the brain [10]. Since cytokine gene polymorphisms in the regulatory region are related to the amount of cytokine produced, they may account for individual susceptibility to inflammatory conditions. Thus, homozygosity for allele 2 of IL-1A (2889) has been associated with a five-fold increase in production of the cytokine compared with homozygosity for allele 1 [2], and conferred a three-fold increased risk for AD [1,10]. Similarly, the possession of the T allele in the promoter region (2850) of the TNF-a gene increased the risk of AD [9]. A biallelic A/G polymorphism at position 2 2518 of the MCP-1 gene seems to influence the transcriptional activity because cells obtained from GG or AG individuals produce more MCP-1 protein in response to
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.01.035
26
O. Combarros et al. / Neuroscience Letters 360 (2004) 25–28
stimuli such as cytokines, than those isolated from AA individuals [16]. Therefore, it would be reasonable to speculate that individuals bearing G at position 22518 have a stronger inflammatory response with higher tissue damage. In fact, prevalence of these high MCP-1 producing genotypes has been shown to be associated with the risk for premature kidney graft failure [8], predisposition to more severe fibrosis and inflammation in patients with chronic hepatitis C [12], or the risk for severe coronary artery disease [19]. Functional in vitro studies comparing cells from individuals with different MCP-1 (22518) genotypes have been performed with peripheral blood mononuclear cells [16] and activated hepatic stellate cells [12], but the role of this polymorphism in microglial cells has not yet been evaluated. To our knowledge, only one case-control study in a Japanese sample reporting no association of MCP-1 (2 2518) with AD has been published [13]. However, a racial difference was found in the MCP-1 (22518) polymorphism [16], and therefore, it is particularly necessary to study different ethnic groups to establish the relationship between this polymorphism and AD. In the present study, we investigated whether the MCP-1 (2 2518) G variant (AG and GG genotypes) might be responsible for susceptibility to AD in a large Spanish population. We also examined the combined gene effects between MCP-1 and IL-1A and TNF-a. In addition, apolipoprotein E (APOE) genotyping was conducted in the same AD case and control groups to examine any possible association with MCP-1 (2 2518). The study included 328 AD patients (69% women; mean age at the time of study 75.8 years; SD 9.9; range 50–98 years; mean age at onset 72.1 years; SD 8.7; range 48–95 years) who met NINCDS/ADRDA criteria for probable AD [11]. 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 ‘Marque´s de Valdecilla’, Santander, Spain, from January 1997 to June 2000. 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 315 unrelated individuals (71% women; mean age 80.5 years; SD 7.5; range 63–100 years) randomly selected from a nursing home. It is often considered that individuals living in nursing homes do not represent the general population of that particular group age but might represent a subset of people; however, the fact that control genotypes were in Hardy–Weinberg equilibrium argues against a significant bias being introduced by this method of selection. These subjects had complete neurological and medical examinations that showed 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 follow-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. Genotyping of MCP-1 (2 2518), IL-1A (2 889), TNF-a (2 850), and APOE polymorphisms were determined as described previously [1,9,14]. Statistical methods: Association between dichotomous variables was analyzed with odds ratio, and 95% confidence intervals were estimated by the Cornfield method or the Fisher exact method. Means of quantitative variables were compared using the Student’s t-test. P-values were estimated by chi-square or Fisher exact tests. Interrelations were analyzed by stratification. All statistical analysis were performed with the package Stata 8/SE (Stata Corporation, College Station, TX, USA). The distributions of MCP-1 (22518) genotypes were in Hardy–Weinberg equilibrium (P ¼ 0:723). As shown in Table 1, there were no statistical differences in the AD group when compared to controls. When compared to AA genotype, the odds ratio (OR) for the AG genotype was 0.98 (95% CI ¼ 0.70–1.36), and 0.84 (95% CI ¼ 0.43–1.68) for the GG genotype. Similarly, no significantly different risk of AD was observed when our data set was stratified by gender. Next, we studied the synergistic effects between MCP-1 (2 2518) and IL-1A (2 889), TNF-a (2 850), and APOE polymorphisms by logistic regression analysis (Table 2). There was no significant association between the MCP-1 (2 2518) G-carrying (AG þ GG) genotypes and the risk of AD in either IL-1A (2 889) allele 2 (1/2 þ 2/2 genotypes) carriers and TNF-a (2 850) T allele (CT þ TT genotypes) carriers. Our analysis showed the expected association between the APOE e4 allele and AD, but there was no interaction between carriage of MCP-1 (2 2518) G allele and of the APOE e4 allele. We then examined the influence of MCP-1 (2 2518) genotypes on the age at onset of AD, and no significant variation existed among AA (mean age of 71.8, SD 8.8 years), AG (mean age of 72.3, SD 9.0 years), and GG (mean age of 75.3, SD 6.5 years) genotypes. There have been a number of reports of inflammatory gene variants being associated with susceptibility to AD Table 1 MCP-1 (22518) genotype and allele frequencies in AD patients and control subjects n
AD Controls
328 315
Genotype, n (%)
Allele frequency
AA
AG
GG
A
G
195 (59.4) 184 (58.4)
116 (35.4) 112 (35.6)
17 (5.2) 19 (6.0)
0.77 0.76
0.23 0.24
n ¼ number of cases; and AD ¼ Alzheimer’s disease.
O. Combarros et al. / Neuroscience Letters 360 (2004) 25–28
27
Table 2 Odds ratios for Alzheimer’s disease risk according to interaction of MCP-1 (22518) genotypes with IL-1A (2889), TNF-a (2850), and APOE genotypes Genotypes IL-1A (1/2 þ 2/2) 2 2 þ þ TNF-a (CT þ TT) 2 2 þ þ APOE e 4 2 2 þ þ
MCP-1 (AG þ GG)
Alzheimer
Controls
OR (95% Confidence Interval)
P
2 þ 2 þ
87 67 74 48
103 65 58 44
1 (reference) 1.22 (0.76–1.95) 1.51 (0.94–2.42) 1.29 (0.76–2.19)
0.380 0.070 0.314
2 þ 2 þ
152 95 24 26
131 100 29 14
1 (reference) 0.82 (0.56–1.20) 0.71 (0.38–1.34) 1.60 (0.77–3.46)
0.283 0.260 0.179
2 þ 2 þ
94 57 101 76
153 105 31 26
1 (reference) 0.88 (0.57–1.36) 5.30 (3.21–8.84) 4.76 (2.77–8.29)
0.556 ,0.001 ,0.001
MCP-1 (AG þ GG) (2) ¼ monocyte chemokine protein-1 AA; IL-1A (1/2 þ 2/2) (2) ¼ interleukin-1A 1/1; TNF-a (CT þ TT) (2) ¼ tumor necrosis factor-a CC; APOE e4 (2) ¼ no copies of apolipoprotein E e4; and APOE e4 ( þ ) ¼ one or two copies of APOE e4.
[10], but the present study is the first to analyze MCP-1 gene variants in susceptibility to AD in Caucasians. We hypothesized that subjects with the MCP-1 (22518) G allele exhibited a higher level of MCP-1 expression and a higher intensity of migration and activation of microglia in the brain than those without the MCP-1 (22518) G allele, and consequently had an increased risk of AD. Reviewing previous reports, it seems evident that the MCP-1 (22518) G allele frequency demonstrates racial heterogeneity. In fact, while in Japanese [13,14] and Mexican [16] populations a G allele frequency of 64 and 47% has been reported, respectively, in Caucasian American and European populations the G allele frequency ranges from 21 to 29% [4,8,16,19]. With regard to Europe, the MCP-1 (22518) G allele frequency is quite homogeneously distributed. We did not find any association between the MCP-1 (22518) G allele and AD in Spanish subjects, thus confirming a recent negative report conducted in Japanese population [13]. In the present study, interaction of MCP-1 (22518) G allele with IL-1A (2889) allele 2, TNF-a (2850) allele T, and APOE e4 allele did not confer increased risk for AD. In conclusion, although supporting evidence for the biological role of MCP-1 in AD exists, the MCP-1 (2 2518) polymorphism distribution does not differ between patients with AD and controls, whether they are white or Japanese, and therefore, this polymorphism is unlikely to confer genetic susceptibility to AD.
Acknowledgements This work was supported by Grant No. PI020027 from Fondo de Investigacio´n Sanitaria.
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