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Monocyte chemoattractant protein-1 (MCP-1) gene polymorphism and risk of Alzheimer's disease in Italians
Experimental Gerontology 39 (2004) 1249–1252 www.elsevier.com/locate/expgero
Short report
Monocyte chemoattractant protein-1 (MCP-1) gene polymorphism and risk of Alzheimer’s disease in Italians Roberto Pola*, Andrea Flex, Eleonora Gaetani, Anna S. Proia, Pierangelo Papaleo, Angela Di Giorgio, Giuseppe Straface, Giovanni Pecorini, Michele Serricchio, Paolo Pola Laboratory of Vascular Biology and Genetics, Department of Medicine, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy Received 24 February 2004; received in revised form 3 May 2004; accepted 4 May 2004 Available online 31 May 2004
Abstract Monocyte chemoattractant protein-1 (MCP-1) is a key molecule for monocyte chemotaxis and tissue extravasation and for the modulation of leukocyte function during inflammation. Upregulation of MCP-1 may occur in the brain of subjects affected by Alzheimer’s disease (AD) and MCP-1 levels in plasma and cerebrospinal fluid have been proposed as biological markers for the inflammatory process that accompanies AD pathogenesis. Importantly, serum levels and biological activity of MCP-1 protein are strongly influenced by a single nucleotide polymorphism occurring at position 2 2518 of the MCP-1 gene promoter. A recent study has investigated the possible association between this gene polymorphism and AD in a Spanish population, with negative results. Here, we performed a case –control study to test whether the risk for AD might be influenced by the 2 2518 A/G polymorphism of the MCP-1 gene in an ethnically homogeneous Italian population. The GG genotype and the G allele of the MCP-1 gene polymorphism were significantly more common in the AD group than in control individuals ðP , 0:0001Þ: A logistic regression analysis indicated that the GG genotype was an independent risk factor for AD in our population. This effect was not influenced by the presence of the APOE 14 high-risk allele, nor by the presence of other gene variations associated with a proinflammatory phenotype. These findings indicate that the 22518 A/G polymorphism of the MCP-1 gene is associated with AD in Italians and confirm that inflammatory gene variations may be important contributors in the development and progression of neurodegenerative disorders. q 2004 Elsevier Inc. All rights reserved. Keywords: MCP-1; Gene polymorphism; Inflammation; Alzheimer’s disease
1. Introduction A considerable body of evidence supports the notion that various inflammatory mediators, including cytokines, chemokines, and adhesion molecules, are involved in the initiation and progression of neurodegeneration in Alzheimer’s disease (AD) (Lee et al., 2002). Indeed, in this pathologic condition, the initial injurious inflammatory stimulus is thought to be the deposition of insoluble extracellular aggregates of amyloid-beta (A-beta) fibrils, that leads to an innate host response characterized by the upregulation of inflammatory mediators and the accumulation of activated microglia (Dickson et al., 1988). In this scenario, it appears obvious that molecules that are able to * Corresponding author. Address: Istituto di Patologia Speciale Medica, Laboratory of Vascular Biology and Genetics, L.go A. Gemelli, 8, 00168 Rome, Italy. Tel.: þ39-06-30154518; fax: þ39-06-35500486. E-mail address: [email protected] (R. Pola). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.05.001
modulate recruitment, migration, accumulation, and activation of monocytes into sites of tissue injury may be crucial for the initiation of the inflammatory process responsible for neurodegeneration (Luster, 1998). A key molecule for monocyte chemotaxis and tissue extravasation and for modulation of leukocyte function during inflammation is the monocyte chemoattractant protein-1 (MCP-1), a potent chemokine for which an important contribution in the pathogenesis of autoimmune diseases, chronic inflammatory disorders, and neuroinflammatory phenomena has already been proposed (Luster, 1998; Izikson et al., 2002; Muhlbauer et al., 2003; Gerard and Rollins, 2001). The potential role of MCP-1 in the pathogenesis of AD is supported by the fact that MCP-1 may be upregulated in the brains of subjects affected by AD and that plasma and cerebrospinal fluid MCP-1 levels might be used as biological markers of the inflammatory process that accompanies the development of AD (Sun et al., 2003).
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Recently, it has been shown that serum levels and biological activity of the MCP-1 protein may be regulated by a single nucleotide polymorphism occurring at position 2 2518 of the MCP-1 gene promoter (Jibiki et al., 2001; Rovin et al., 1999). Interestingly, several studies have demonstrated that this gene variation is clinically important, as it is significantly associated with a number of immune, inflammatory, and cardiovascular diseases (Gonzalez-Escribano et al., 2003; Szalai et al., 2001a,b). Very recently, the role of this gene polymorphism has been investigated also in AD (Combarros et al., 2004). In this study, performed on subjects coming from a geographically limited area of Northern Spain, no evidence of a significant association between the 2 2518 A/G polymorphism of the MCP-1 gene and AD has been found. However, many association studies in AD have provided controversial results, depending on the ethnic background of the studied population, as well as on its demographic and clinical characteristics. Therefore, the role of the MCP-1 2 2518 A/G polymorphism in AD remains to be elucidated. In this case –control study, we investigated whether the 2 2518 A/G polymorphism of the MCP-1 gene may influence the risk of AD in an ethnically homogeneous Italian population.
2. Methods A total of 141 patients with sporadic AD were studied (mean age 77.6 ^ 5.4 years, male:female ratio 58:83). Diagnosis of dementia was performed according to the DSM-III criteria (Nussbaum et al., 1992) and diagnosis of probable AD was made in accordance with the NINCDSADRDA guidelines (McKhann et al., 1984). All patients underwent brain imaging evaluation by CT scan, structured interview, formal neuro-psychological testing, and minimental state examination (MMSE). The Hachinski ischemic score (HIS) was also used to aid in distinguishing between AD and multi-infarct dementia (MID) (Hachinski et al., 1975). Patients were recruited among subjects consecutively admitted to the Departments of Geriatric Medicine and Internal Medicine of the A. Gemelli University Hospital of Rome, Italy, from November 2000 to October 2001 and from January to June 2002. Control subjects were 206 ageand sex-matched individuals, admitted to the same departments in the same period of time, not affected by dementia. The presence of cognitive deterioration in control subjects was clinically and instrumentally excluded by MMSE and CT scan of the brain. The mean age of controls at the time of assessment was 76.3 ^ 6.8 years. The male:female ratio in controls was 99:107. All patients and controls were Caucasians from central and southern Italy and belonged to independent pedigrees. Subjects with MID, suspected mixed dementia (MID and AD), or dementia of metabolic origin were excluded. In both patient and control groups, subjects affected by tumors, chronic inflammatory diseases,
and autoimmune diseases were excluded as well. The study protocol was accepted by the Ethics Committee of our University Hospital. DNA was extracted from peripheral blood and assayed for the detection of the polymorphisms of the MCP-1 (2 2518 A/G), APOE, interleukin-6 ( – 174 G/C), and ICAM-1 (469 E/K) genes, as previously described (Aguilar et al., 2001; Hixons and Vernier, 1990; Pola et al., 2002, 2003). Demographic and clinical data between groups were compared by x2 - or t-test. Genotype and allele frequencies were compared by x2 -test. To estimate the association between genotypes and AD, a logistic regression model was used. Odds ratios were calculated with 95% confidence interval. All analyses were done by using Intercooled STATA 6.0 for Windows (Statistics/Data Analysis, Stata Corporation, College Station, Texas, USA). Statistical significance was established at P , 0:05:
3. Results Demographic and clinical characteristics of AD and control subjects are shown in Table 1. There were no significant differences between groups in terms of age and sex. Concomitant pathologic conditions were hypertension, hypercholesterolemia, diabetes, and cardiovascular diseases. Among these, hypertension and cardiovascular disorders were significantly more frequent in control subjects than in AD patients ðP , 0:001Þ: In contrast, hypercholesterolemia was significantly more common in subjects affected by AD ðP , 0:01Þ: The genotype and allele distribution of MCP-1 in AD and control subjects is presented in Table 2. Genotypes were in Hardy– Weinberg equilibrium. In the 141 patients with sporadic AD, the genotype distribution was 59 AA, 52 AG, 30 GG, and was significantly different from that observed in the 206 control subjects (114 AA, 81 AG, 11 GG) ðP , 0:0001Þ: In particular, the frequency of the GG genotype was almost four times higher in patients with AD than in control individuals (21.3 versus 5.4%). Similarly, the G allele was significantly more frequent in AD patients than in controls (39.7 versus 25.0%, P , 0:0001). When a logistic regression model was used, in order to adjust for possible confounding factors (Table 3), we found Table 1 Demographic and clinical characteristics of AD subjects and controls
Age (years ^ SD) Male:female Hypertension, n (%) Hypercholesterolemia, n (%) Diabetes, n (%) Cardiovascular diseases, n (%)
AD ðn ¼ 141Þ
Controls ðn ¼ 206Þ
P
77.6 ^ 5.4 58:83 45 (31.9%) 55 (39.0%) 14 (9.9%) 20 (14.2%)
76.3 ^ 6.8 99:107 104 (50.5%) 58 (28.2%) 33 (16.0%) 60 (29.1%)
0.068 0.103 0.001 0.034 0.103 0.006
R. Pola et al. / Experimental Gerontology 39 (2004) 1249–1252 Table 2 MCP-1 genotype and allele distribution between groups AD ðn ¼ 141Þ Genotype A/A A/G G/G Allele A G
Controls ðn ¼ 206Þ
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Table 4 ORs for AD risk according to interaction of MCP-1 genotypes with APOE, IL-6, and ICAM-1 genotypes P MCP-1 AD Controls OR (95% CI) GG
59 (41.8%) 52 (36.9%) 30 (21.3%)
114 (55.4%) 81 (39.3%) 11 (5.3%)
170 (60.3%) 112 (39.7%)
309 (75.0%) 103 (25.0%)
,0.0001
,0.0001
that the GG genotype of the MCP-1 gene polymorphism was an independent risk factor for AD in our population, as patients carrying the GG genotype had a risk more than five times higher to develop AD than AA homozygous individuals [OR 5.5 (95% CI, 2.3 –12.9; P , 0:0001]. Then, we studied, by logistic regression analysis, the synergistic effect between MCP-1 and the possession of the high-risk 14 allele of the apolipoprotein E (APOE) gene. We found that the GG genotype of the MCP-1 gene is associated with increased risk of AD both in the absence [OR 2.0 (95% CI 1.1 – 3.5), P ¼ 0:009] and the presence [OR 8.5 (95% CI 3.6 –20.3), P , 0:0001] of the APOE 14 allele (Table 4). Next, we evaluated whether the MCP-1 2 2518 A/G gene polymorphism interacts with other pro-inflammatory genotypes that have been associated with AD in our population, such as the IL-6 -174 G/C and the ICAM-1 469 E/K gene polymorphisms (Pola et al., 2002, 2003). We found that the MCP-1 GG genotype is significantly associated with AD independently on the presence or the absence of the IL-6 and ICAM-1 high-risk genotypes (Table 4).
4. Discussion AD is characterized by the activation of a focal inflammatory reaction, that occurs as secondary phenomenon following deposition of A-beta in the parenchyma (Bothwell and Giniger, 2000). In this setting, local production of cytokines and chemokines and upregulation of pro-inflammatory molecules are events of crucial pathogenetic importance (Lee et al., 2002). In recent years, there has been increasing appreciation of the fact that function and activity of several inflammatory mediators may be genetically determined. A number of variations have Table 3 Risk factors for AD based on logistic regression analysis
G/G genotype Hypertension Hypercholesterolemia Diabetes Cardiovascular diseases
P
OR
95% CI
P
5.5 0.3 1.5 0.7 0.4
2.3 –12.9 0.2 –0.6 0.9 –2.7 0.3 –1.7 0.2 –0.8
,0.0001 0.001 0.087 0.554 0.013
APOE 14 2 2 þ þ
2 þ þ 2
29 51 31 30
99 82 10 15
2.0 (1.1–3.5) 0.009 8.5 (3.6–20.3) ,0.0001 5.7 (2.6–12.6) ,0.0001
IL-6 (GG þ GC) 2 2 þ þ
2 þ þ 2
7 12 70 52
41 25 67 73
2.8 (0.9–8.5) 0.05 5.7 (2.3–13.6) ,0.0001 4.2 (1.7–10.5) 0.002
ICAM-1 (EE þ EK) 2 2 þ þ
2 þ þ 2
11 20 62 48
36 26 66 78
3.4 (1.2–9.8) 3.3 (1.5–7.3) 2.7 (1.1–6.4)
0.01 0.03 0.01
been identified in genes encoding for both pro-inflammatory and anti-inflammatory molecules and their functional and clinical importance has been largely investigated. The gene mutation investigated in this study has a crucial influence on the serum levels of the MCP-1 protein (Rovin et al., 1999), with potentially important clinical implications. This is also suggested by a number of clinical reports that have demonstrated the association between the 2 2518 A/G polymorphism of the MCP-1 gene and severe pathologic conditions, such as rheumatoid arthritis, coronary artery disease, and asthma (Gonzalez-Escribano et al., 2003; Szalai et al., 2001a,b). Here, we show that this gene polymorphism is an independent risk factor for AD in Italians. In our population, the occurrence of AD was 5.5 times more common in patients homozygous for the G allele than in subjects carrying the AA genotype. The independent association between AD and the MCP-1 gene polymorphism was confirmed also after correction for other genetic risk factors for AD, such as the presence of the APOE 14 allele and the carriage of specific high-risk IL-6 and ICAM-1 genotypes. Our results are consistent with the hypothesis that inflammation and inflammatory mediators are crucial in the pathogenesis of neurodegenerative disorders. Experimental findings demonstrate that MCP-1 is present in AD lesions and that A-beta is able to stimulate MCP-1 production in neonatal astrocytes, that also migrate and accumulate at sites of A-beta deposition in response to MCP-1 stimulation (Wyss-Coray et al., 2003). In addition, a recent clinical report has suggested that plasma levels of MCP-1 and other inflammatory molecules might be useful bio-markers of the inflammatory process in individuals affected by AD (Sun et al., 2003). On the other hand, a recent study has found no association between the MCP-1 2 2518 A/G gene polymorphism and AD
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in a population composed of subjects coming from the Northern Spain’s region of Cantabria. These controversial findings may depend on several reasons, such as differences in the ethnic background of the studied populations, as well as in other clinical and demographic parameters. Indeed, it is well known that ethnicity and population structure may strongly influence the role of genetic risk factors in neurodegenerative diseases. The distribution of several gene variations may be greatly different among European countries, as well as among different areas in the same country. Also, differences in the inclusion and exclusion criteria may influence the study results, especially if subjects affected by chronic inflammatory diseases, autoimmune diseases, or undiagnosed cancers are not excluded from studies investigating pro-inflammatory gene variations. Both this investigation and that of Combarros and coll. are casecontrol studies and a possible survival bias cannot be excluded for the group of patients with AD. In both studies, control subjects were recruited among hospitalized individuals and may not be representative of the general population. Finally, it cannot be excluded a role played by genes that are in linkage disequilibrium with the MCP-1 gene. In particular, MCP-1 is part of a cluster also including the MCP-2, MCP-3, MIP-1 alpha, and CCL11 genes, on chromosome 17q11.2. This chromosomal region also contains a further chemokine gene cluster that includes MPIF-1, HCC-2, HCC-1, LEC, and RANTES. It is also possible that one or more of these genes are in linkage disequilibrium with the MCP-1 gene in some populations but not in others, thus influencing the results of these association studies. In conclusion, we found that, in an Italian population, the 2 2518 A/G polymorphism of the MCP-1 gene promoter is a risk factor for AD. Our findings suggest that this gene polymorphism may be clinically important and confirm a role for MCP-1 in the pathophysiology of neurodegenerative diseases, with potentially important therapeutic implications.
Acknowledgements We are grateful to Prof. Roberto Bernabei and the staff of the Department of Geriatric Medicine of the A. Gemelli University Hospital for their help in the recruitment of subjects affected by AD.
References Aguilar, F., Gonzalez-Escribano, M.F., Sanchez-Roman, J., Nunez-Roldan, A., 2001. MCP-1 promoter polymorphism in Spanish patients with systemic lupus erythematosus. Tissue Antigens 58, 335 –338. Bothwell, M., Giniger, E., 2000. Alzheimer’s disease: neurodevelopment converges with neurodegeneration. Cell 102, 271–273. Combarros, O., Infante, J., Llorca, J., Berciano, J., 2004. No evidence for association of the monocyte chemoattractant protein-1 (22518) gene polymorphism and Alzheimer’s disease. Neurosci. Lett. 360, 25 –28.
Dickson, D.W., Farlo, J., Davies, P., Crystal, H., Fuld, P., Yen, S.H., 1988. Alzheimer’s disease double-labeling immunohistochemical study of senile plaques. Am. J. Pathol. 132, 86 –101. Gerard, C., Rollins, B.J., 2001. Chemokines and disease. Nat. Immunol. 2, 108 –115. Gonzalez-Escribano, M.F., Torres, B., Aguilar, F., Rodriguez, R., Garcia, A., Valenzuela, A., Nunez-Roldan, A., 2003. MCP-1 promoter polymorphism in Spanish patients with rheumatoid arthritis. Hum. Immunol. 64, 741 –744. Hachinski, V.C., Iliff, L.D., Zilhka, E., Du Boulay, G.H., McAllister, V.L., Marshall, J., Russell, R.W., Symon, L., 1975. Cerebral blood flow in dementia. Arch. Neurol. 32, 632–637. Hixons, J.E., Vernier, D.T., 1990. Restriction isotyping of human apolipoprotein E by gene amplification and clavage with HhaI. Lipid Res. 31, 545 –548. Izikson, L., Klein, R.S., Luster, A.D., Weiner, H.L., 2002. Targeting monocyte recruitment in CNS autoimmune disease. Clin. Immunol. 103, 125–131. Jibiki, T., Terai, M., Shima, M., Ogawa, A., Hamada, H., Kanazawa, M., Yamamoto, S., Oana, S., Kohno, Y., 2001. Monocyte chemoattractant protein 1 gene regulatory region polymorphism and serum levels of monocyte chemoattractant protein 1 in Japanese patients with Kawasaki disease. Arthritis Rheum. 44, 2211–2212. Lee, Y.B., Nagai, A., Kim, S.U., 2002. Cytokines, chemokines, and cytokine receptors in human microglia. J. Neurosci. Res. 69, 94–103. Luster, A.D., 1998. Chemokines-chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338, 436– 445. McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D., Stadlan, E.M., 1984. Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA work group under the auspices of department of health and human services task force on Alzheimer’s disease. Neurology 34, 939– 944. Muhlbauer, M., Bosserhoff, A.K., Hartmann, A., Thasler, W.E., Weiss, T.S., Herfarth, H., Lock, G., Scholmerich, J., Hellerbrand, C., 2003. A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease. Gastroenterology 125, 1085–1093. Nussbaum, M., Treves, T.A., Korczyn, A.D., 1992. DSM-III-R criteria for primary degenerative dementia and multi-infarct dementia. Alz. Dis. Assoc. Disord. 6, 111–118. Pola, R., Flex, A., Gaetani, E., Dal Lago, A., Gerardino, L., Pola, P., Bernabei, R., 2002. The -174 G/C polymorphism of the interleukin-6 gene promoter is associated with Alzheimer’s disease in an Italian population. Neuroreport 13, 1645– 1647. Pola, R., Flex, A., Gaetani, E., Santoliquido, A., Serricchio, M., Pola, P., Bernabei, R., 2003. Intercellular adhesion molecule-1 K469E gene polymorphism and Alzheimer’s disease. Neurobiol. Aging 24, 385 –387. Rovin, B., Lu, L., Saxena, R., 1999. A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem. Biophys. Res. Commun. 259, 344–348. Sun, Y.X., Minthon, L., Wallmark, A., Warkentin, S., Blennow, K., Janciauskiene, S., 2003. Inflammatory markers in matched plasma and cerebrospinal fluid from patients with Alzheimer’s disease. Dement. Geriatr. Cogn. Disord. 16, 136–144. Szalai, C., Duba, J., Prohaszka, Z., Kalina, A., Szabo, T., Nagy, B., Horvath, L., Csaszar, A., 2001. 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, 233–239. Szalai, C., Kozma, G.T., Nagy, A., Bojszko, A., Krikovszky, D., Szabo, T., Falus, A., 2001. Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and severity. J. Allergy Clin. Immunol. 108, 375–381. Wyss-Coray, T., Loike, J.D., Brionne, T.C., Lu, E., Anankov, R., Yan, F., Silverstein, S.C., Husemann, J., 2003. Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nat. Med. 9, 453– 457.
Short report
Monocyte chemoattractant protein-1 (MCP-1) gene polymorphism and risk of Alzheimer’s disease in Italians Roberto Pola*, Andrea Flex, Eleonora Gaetani, Anna S. Proia, Pierangelo Papaleo, Angela Di Giorgio, Giuseppe Straface, Giovanni Pecorini, Michele Serricchio, Paolo Pola Laboratory of Vascular Biology and Genetics, Department of Medicine, A. Gemelli University Hospital, Universita` Cattolica del Sacro Cuore School of Medicine, Rome, Italy Received 24 February 2004; received in revised form 3 May 2004; accepted 4 May 2004 Available online 31 May 2004
Abstract Monocyte chemoattractant protein-1 (MCP-1) is a key molecule for monocyte chemotaxis and tissue extravasation and for the modulation of leukocyte function during inflammation. Upregulation of MCP-1 may occur in the brain of subjects affected by Alzheimer’s disease (AD) and MCP-1 levels in plasma and cerebrospinal fluid have been proposed as biological markers for the inflammatory process that accompanies AD pathogenesis. Importantly, serum levels and biological activity of MCP-1 protein are strongly influenced by a single nucleotide polymorphism occurring at position 2 2518 of the MCP-1 gene promoter. A recent study has investigated the possible association between this gene polymorphism and AD in a Spanish population, with negative results. Here, we performed a case –control study to test whether the risk for AD might be influenced by the 2 2518 A/G polymorphism of the MCP-1 gene in an ethnically homogeneous Italian population. The GG genotype and the G allele of the MCP-1 gene polymorphism were significantly more common in the AD group than in control individuals ðP , 0:0001Þ: A logistic regression analysis indicated that the GG genotype was an independent risk factor for AD in our population. This effect was not influenced by the presence of the APOE 14 high-risk allele, nor by the presence of other gene variations associated with a proinflammatory phenotype. These findings indicate that the 22518 A/G polymorphism of the MCP-1 gene is associated with AD in Italians and confirm that inflammatory gene variations may be important contributors in the development and progression of neurodegenerative disorders. q 2004 Elsevier Inc. All rights reserved. Keywords: MCP-1; Gene polymorphism; Inflammation; Alzheimer’s disease
1. Introduction A considerable body of evidence supports the notion that various inflammatory mediators, including cytokines, chemokines, and adhesion molecules, are involved in the initiation and progression of neurodegeneration in Alzheimer’s disease (AD) (Lee et al., 2002). Indeed, in this pathologic condition, the initial injurious inflammatory stimulus is thought to be the deposition of insoluble extracellular aggregates of amyloid-beta (A-beta) fibrils, that leads to an innate host response characterized by the upregulation of inflammatory mediators and the accumulation of activated microglia (Dickson et al., 1988). In this scenario, it appears obvious that molecules that are able to * Corresponding author. Address: Istituto di Patologia Speciale Medica, Laboratory of Vascular Biology and Genetics, L.go A. Gemelli, 8, 00168 Rome, Italy. Tel.: þ39-06-30154518; fax: þ39-06-35500486. E-mail address: [email protected] (R. Pola). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.05.001
modulate recruitment, migration, accumulation, and activation of monocytes into sites of tissue injury may be crucial for the initiation of the inflammatory process responsible for neurodegeneration (Luster, 1998). A key molecule for monocyte chemotaxis and tissue extravasation and for modulation of leukocyte function during inflammation is the monocyte chemoattractant protein-1 (MCP-1), a potent chemokine for which an important contribution in the pathogenesis of autoimmune diseases, chronic inflammatory disorders, and neuroinflammatory phenomena has already been proposed (Luster, 1998; Izikson et al., 2002; Muhlbauer et al., 2003; Gerard and Rollins, 2001). The potential role of MCP-1 in the pathogenesis of AD is supported by the fact that MCP-1 may be upregulated in the brains of subjects affected by AD and that plasma and cerebrospinal fluid MCP-1 levels might be used as biological markers of the inflammatory process that accompanies the development of AD (Sun et al., 2003).
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Recently, it has been shown that serum levels and biological activity of the MCP-1 protein may be regulated by a single nucleotide polymorphism occurring at position 2 2518 of the MCP-1 gene promoter (Jibiki et al., 2001; Rovin et al., 1999). Interestingly, several studies have demonstrated that this gene variation is clinically important, as it is significantly associated with a number of immune, inflammatory, and cardiovascular diseases (Gonzalez-Escribano et al., 2003; Szalai et al., 2001a,b). Very recently, the role of this gene polymorphism has been investigated also in AD (Combarros et al., 2004). In this study, performed on subjects coming from a geographically limited area of Northern Spain, no evidence of a significant association between the 2 2518 A/G polymorphism of the MCP-1 gene and AD has been found. However, many association studies in AD have provided controversial results, depending on the ethnic background of the studied population, as well as on its demographic and clinical characteristics. Therefore, the role of the MCP-1 2 2518 A/G polymorphism in AD remains to be elucidated. In this case –control study, we investigated whether the 2 2518 A/G polymorphism of the MCP-1 gene may influence the risk of AD in an ethnically homogeneous Italian population.
2. Methods A total of 141 patients with sporadic AD were studied (mean age 77.6 ^ 5.4 years, male:female ratio 58:83). Diagnosis of dementia was performed according to the DSM-III criteria (Nussbaum et al., 1992) and diagnosis of probable AD was made in accordance with the NINCDSADRDA guidelines (McKhann et al., 1984). All patients underwent brain imaging evaluation by CT scan, structured interview, formal neuro-psychological testing, and minimental state examination (MMSE). The Hachinski ischemic score (HIS) was also used to aid in distinguishing between AD and multi-infarct dementia (MID) (Hachinski et al., 1975). Patients were recruited among subjects consecutively admitted to the Departments of Geriatric Medicine and Internal Medicine of the A. Gemelli University Hospital of Rome, Italy, from November 2000 to October 2001 and from January to June 2002. Control subjects were 206 ageand sex-matched individuals, admitted to the same departments in the same period of time, not affected by dementia. The presence of cognitive deterioration in control subjects was clinically and instrumentally excluded by MMSE and CT scan of the brain. The mean age of controls at the time of assessment was 76.3 ^ 6.8 years. The male:female ratio in controls was 99:107. All patients and controls were Caucasians from central and southern Italy and belonged to independent pedigrees. Subjects with MID, suspected mixed dementia (MID and AD), or dementia of metabolic origin were excluded. In both patient and control groups, subjects affected by tumors, chronic inflammatory diseases,
and autoimmune diseases were excluded as well. The study protocol was accepted by the Ethics Committee of our University Hospital. DNA was extracted from peripheral blood and assayed for the detection of the polymorphisms of the MCP-1 (2 2518 A/G), APOE, interleukin-6 ( – 174 G/C), and ICAM-1 (469 E/K) genes, as previously described (Aguilar et al., 2001; Hixons and Vernier, 1990; Pola et al., 2002, 2003). Demographic and clinical data between groups were compared by x2 - or t-test. Genotype and allele frequencies were compared by x2 -test. To estimate the association between genotypes and AD, a logistic regression model was used. Odds ratios were calculated with 95% confidence interval. All analyses were done by using Intercooled STATA 6.0 for Windows (Statistics/Data Analysis, Stata Corporation, College Station, Texas, USA). Statistical significance was established at P , 0:05:
3. Results Demographic and clinical characteristics of AD and control subjects are shown in Table 1. There were no significant differences between groups in terms of age and sex. Concomitant pathologic conditions were hypertension, hypercholesterolemia, diabetes, and cardiovascular diseases. Among these, hypertension and cardiovascular disorders were significantly more frequent in control subjects than in AD patients ðP , 0:001Þ: In contrast, hypercholesterolemia was significantly more common in subjects affected by AD ðP , 0:01Þ: The genotype and allele distribution of MCP-1 in AD and control subjects is presented in Table 2. Genotypes were in Hardy– Weinberg equilibrium. In the 141 patients with sporadic AD, the genotype distribution was 59 AA, 52 AG, 30 GG, and was significantly different from that observed in the 206 control subjects (114 AA, 81 AG, 11 GG) ðP , 0:0001Þ: In particular, the frequency of the GG genotype was almost four times higher in patients with AD than in control individuals (21.3 versus 5.4%). Similarly, the G allele was significantly more frequent in AD patients than in controls (39.7 versus 25.0%, P , 0:0001). When a logistic regression model was used, in order to adjust for possible confounding factors (Table 3), we found Table 1 Demographic and clinical characteristics of AD subjects and controls
Age (years ^ SD) Male:female Hypertension, n (%) Hypercholesterolemia, n (%) Diabetes, n (%) Cardiovascular diseases, n (%)
AD ðn ¼ 141Þ
Controls ðn ¼ 206Þ
P
77.6 ^ 5.4 58:83 45 (31.9%) 55 (39.0%) 14 (9.9%) 20 (14.2%)
76.3 ^ 6.8 99:107 104 (50.5%) 58 (28.2%) 33 (16.0%) 60 (29.1%)
0.068 0.103 0.001 0.034 0.103 0.006
R. Pola et al. / Experimental Gerontology 39 (2004) 1249–1252 Table 2 MCP-1 genotype and allele distribution between groups AD ðn ¼ 141Þ Genotype A/A A/G G/G Allele A G
Controls ðn ¼ 206Þ
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Table 4 ORs for AD risk according to interaction of MCP-1 genotypes with APOE, IL-6, and ICAM-1 genotypes P MCP-1 AD Controls OR (95% CI) GG
59 (41.8%) 52 (36.9%) 30 (21.3%)
114 (55.4%) 81 (39.3%) 11 (5.3%)
170 (60.3%) 112 (39.7%)
309 (75.0%) 103 (25.0%)
,0.0001
,0.0001
that the GG genotype of the MCP-1 gene polymorphism was an independent risk factor for AD in our population, as patients carrying the GG genotype had a risk more than five times higher to develop AD than AA homozygous individuals [OR 5.5 (95% CI, 2.3 –12.9; P , 0:0001]. Then, we studied, by logistic regression analysis, the synergistic effect between MCP-1 and the possession of the high-risk 14 allele of the apolipoprotein E (APOE) gene. We found that the GG genotype of the MCP-1 gene is associated with increased risk of AD both in the absence [OR 2.0 (95% CI 1.1 – 3.5), P ¼ 0:009] and the presence [OR 8.5 (95% CI 3.6 –20.3), P , 0:0001] of the APOE 14 allele (Table 4). Next, we evaluated whether the MCP-1 2 2518 A/G gene polymorphism interacts with other pro-inflammatory genotypes that have been associated with AD in our population, such as the IL-6 -174 G/C and the ICAM-1 469 E/K gene polymorphisms (Pola et al., 2002, 2003). We found that the MCP-1 GG genotype is significantly associated with AD independently on the presence or the absence of the IL-6 and ICAM-1 high-risk genotypes (Table 4).
4. Discussion AD is characterized by the activation of a focal inflammatory reaction, that occurs as secondary phenomenon following deposition of A-beta in the parenchyma (Bothwell and Giniger, 2000). In this setting, local production of cytokines and chemokines and upregulation of pro-inflammatory molecules are events of crucial pathogenetic importance (Lee et al., 2002). In recent years, there has been increasing appreciation of the fact that function and activity of several inflammatory mediators may be genetically determined. A number of variations have Table 3 Risk factors for AD based on logistic regression analysis
G/G genotype Hypertension Hypercholesterolemia Diabetes Cardiovascular diseases
P
OR
95% CI
P
5.5 0.3 1.5 0.7 0.4
2.3 –12.9 0.2 –0.6 0.9 –2.7 0.3 –1.7 0.2 –0.8
,0.0001 0.001 0.087 0.554 0.013
APOE 14 2 2 þ þ
2 þ þ 2
29 51 31 30
99 82 10 15
2.0 (1.1–3.5) 0.009 8.5 (3.6–20.3) ,0.0001 5.7 (2.6–12.6) ,0.0001
IL-6 (GG þ GC) 2 2 þ þ
2 þ þ 2
7 12 70 52
41 25 67 73
2.8 (0.9–8.5) 0.05 5.7 (2.3–13.6) ,0.0001 4.2 (1.7–10.5) 0.002
ICAM-1 (EE þ EK) 2 2 þ þ
2 þ þ 2
11 20 62 48
36 26 66 78
3.4 (1.2–9.8) 3.3 (1.5–7.3) 2.7 (1.1–6.4)
0.01 0.03 0.01
been identified in genes encoding for both pro-inflammatory and anti-inflammatory molecules and their functional and clinical importance has been largely investigated. The gene mutation investigated in this study has a crucial influence on the serum levels of the MCP-1 protein (Rovin et al., 1999), with potentially important clinical implications. This is also suggested by a number of clinical reports that have demonstrated the association between the 2 2518 A/G polymorphism of the MCP-1 gene and severe pathologic conditions, such as rheumatoid arthritis, coronary artery disease, and asthma (Gonzalez-Escribano et al., 2003; Szalai et al., 2001a,b). Here, we show that this gene polymorphism is an independent risk factor for AD in Italians. In our population, the occurrence of AD was 5.5 times more common in patients homozygous for the G allele than in subjects carrying the AA genotype. The independent association between AD and the MCP-1 gene polymorphism was confirmed also after correction for other genetic risk factors for AD, such as the presence of the APOE 14 allele and the carriage of specific high-risk IL-6 and ICAM-1 genotypes. Our results are consistent with the hypothesis that inflammation and inflammatory mediators are crucial in the pathogenesis of neurodegenerative disorders. Experimental findings demonstrate that MCP-1 is present in AD lesions and that A-beta is able to stimulate MCP-1 production in neonatal astrocytes, that also migrate and accumulate at sites of A-beta deposition in response to MCP-1 stimulation (Wyss-Coray et al., 2003). In addition, a recent clinical report has suggested that plasma levels of MCP-1 and other inflammatory molecules might be useful bio-markers of the inflammatory process in individuals affected by AD (Sun et al., 2003). On the other hand, a recent study has found no association between the MCP-1 2 2518 A/G gene polymorphism and AD
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in a population composed of subjects coming from the Northern Spain’s region of Cantabria. These controversial findings may depend on several reasons, such as differences in the ethnic background of the studied populations, as well as in other clinical and demographic parameters. Indeed, it is well known that ethnicity and population structure may strongly influence the role of genetic risk factors in neurodegenerative diseases. The distribution of several gene variations may be greatly different among European countries, as well as among different areas in the same country. Also, differences in the inclusion and exclusion criteria may influence the study results, especially if subjects affected by chronic inflammatory diseases, autoimmune diseases, or undiagnosed cancers are not excluded from studies investigating pro-inflammatory gene variations. Both this investigation and that of Combarros and coll. are casecontrol studies and a possible survival bias cannot be excluded for the group of patients with AD. In both studies, control subjects were recruited among hospitalized individuals and may not be representative of the general population. Finally, it cannot be excluded a role played by genes that are in linkage disequilibrium with the MCP-1 gene. In particular, MCP-1 is part of a cluster also including the MCP-2, MCP-3, MIP-1 alpha, and CCL11 genes, on chromosome 17q11.2. This chromosomal region also contains a further chemokine gene cluster that includes MPIF-1, HCC-2, HCC-1, LEC, and RANTES. It is also possible that one or more of these genes are in linkage disequilibrium with the MCP-1 gene in some populations but not in others, thus influencing the results of these association studies. In conclusion, we found that, in an Italian population, the 2 2518 A/G polymorphism of the MCP-1 gene promoter is a risk factor for AD. Our findings suggest that this gene polymorphism may be clinically important and confirm a role for MCP-1 in the pathophysiology of neurodegenerative diseases, with potentially important therapeutic implications.
Acknowledgements We are grateful to Prof. Roberto Bernabei and the staff of the Department of Geriatric Medicine of the A. Gemelli University Hospital for their help in the recruitment of subjects affected by AD.
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