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MCP-1 in Alzheimer’s disease patients: A-2518G polymorphism and serum levels
Neurobiology of Aging 25 (2004) 1169–1173
MCP-1 in Alzheimer’s disease patients: A-2518G polymorphism and serum levels Chiara Fenoglio a , Daniela Galimberti a,∗ , Carlo Lovati b , Ilaria Guidi a , Alberto Gatti a , Sergio Fogliarino c , Marco Tiriticco a , Claudio Mariani b , Gianluigi Forloni c , Carla Pettenati d , Pierluigi Baron a , Giancarlo Conti a , Nereo Bresolin a , Elio Scarpini a a
Department of Neurological Sciences, “Dino Ferrari” Center, University of Milan, IRCCS Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122 Milan, Italy b Department of Neurology, University of Milan, Ospedale L. Sacco, Milan, Italy c Department of Neuroscience, Instituto di Ricerche Farmacologiche M. Negri, Via Eritrea 62, 20157 Milan, Italy d Centro Regionale Alzheimer, U.O. Neurologia, Ospedale Passirana-Rho, Milan, Italy Received 12 August 2003; received in revised form 6 November 2003; accepted 13 November 2003
Abstract MCP-1 levels are increased in CSF of patients with Alzheimer’s disease (AD) compared with controls, suggesting a role in the development of dementia. Recently, a biallelic A/G polymorphism in the MCP-1 promoter at position −2518 has been found, influencing the level of MCP-1 expression in response to an inflammatory stimulus. The distribution of the A-2518G SNP was determined in 269 AD patients and in 203 healthy age matched controls, showing no differences between the two groups. On the contrary, a significant increase of MCP-1 serum levels in AD patients carrying at least one G mutated allele was observed. Moreover, the highest peaks of MCP-1 serum levels were present in patients carrying two G alleles. Stratifying by ApoE genotype, gender or age at onset, no differences in both allele frequency and MCP-1 serum concentration were observed. The A-2518G polymorphism in MCP-1 gene does not seem to be a risk factor for the development of AD, but its presence correlates with higher levels of serum MCP-1, which can contribute to increase the inflammatory process occurring in AD. © 2004 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Polymorphism; Chemokines; Monocyte chemoattractant protein-1; MCP-1; Genetics
1. Introduction Immunoreactivity for a number of chemokines, as well as for their related receptors, has been demonstrated in resident cells of the CNS, and upregulation of some of them is associated with Alzheimer’s disease (AD) pathological changes [17]. Chemokines are supposed to play a relevant role in the pathogenesis of the inflammatory process occurring during the development of the pathology, because of their chemotactic activity on brain phagocytes [11]. In particular, monocyte chemotactic protein-1 (MCP-1), which has been demonstrated to be essential for monocyte recruitment in inflammatory models in vivo, as MCP-1−/− mice are specifically unable to recruit monocytes [10], was found immunohistochemically in mature, but not in immature, senile plaques and in reactive microglia of brain tissues from ∗
Corresponding author. Tel.: +39-02-55033858; fax: +39-02-50320430. E-mail address: [email protected] (D. Galimberti).
0197-4580/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2003.11.008
five patients with AD. This suggests that MCP-1-related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques [8]. Studies carried out in Japanese population indicate that MCP-1 levels in plasma increase with aging [7]. Besides, MCP-1 concentration is selectively increased in cerebrospinal fluid (CSF) of patients with HIV-associated dementia (HAD), and, notably, MCP-1 correlates with the severity of dementia [9]. HAD, as well as other neurodegenerative disorders, including AD, shows similar features, in particular inflammation, monocyte/macrophage recruitment and glial activation [3]. In this regard, it has been recently demonstrated that MCP-1 levels are higher in CSF of patients with AD than in controls, suggesting a role on phagocytic cells within the brain during the development of dementia [2,14]. Recently, a single nucleotide polymorphism (SNP) has been found in the MCP-1 gene regulatory region at position −2518 (A-2518G), that influences the level of MCP-1
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expression in response to an inflammatory stimulus. A reporter gene under control of the MCP-1 distal regulatory region was shown to be induced by interleukin-1 to a greater extent when the allele at −2518 was G and monocytes from individuals carrying a G allele produced more MCP-1 after treatment with IL-1 than monocytes from A/A homozygous individuals [13]. Moreover, European-American men who possess A-2518G allele has higher MCP-1 levels than those who lack this allele [3]. These evidences lead to the hypotesis that MCP-1 levels increase in G allele carriers may in part explain the different severity of pathological conditions among individuals who are affected by the same inflammatory disease. A case-control study comparing patients with coronary artery disease (CAD) and healthy controls showed that the prevalence of A-2518G carriers was significantly higher among CAD patients than controls, thus indicating that this polymorphism may be implicated with the susceptibility to develop the disease [15]. On the basis of these studies, suggesting the potential importance of MCP-1 in the pathogenesis of AD and the effect exerted by the presence of the A-2518G allele, the distribution of the A-2518G polymorphism in 269 patients with AD as well as in 203 healthy subjects was analyzed, in order to determine whether it could influence the susceptibility to develop the disease, both by itself or in combination with the known risk factor for the development of AD, apolipoproteinE (ApoE) ε4 allele [5]. The concentration of MCP-1 in serum was next evaluated, to test the ability of the mutated allele to influence the circulating levels of this chemokine.
2. Materials and methods 2.1. Subjects Between January 2001 and January 2003, 269 Italian AD patients (191 women and 78 men, mean age at disease onset 75 years) were consecutively recruited at Alzheimer Units of Ospedale Maggiore Policlinico (Milan), Ospedale L. Sacco (Milan) and Ospedale Passirana (Rho). All patients underwent a standard battery of examinations, including medical history, physical and neurological examination, screening laboratory tests, neurocognitive evaluation, brain magnetic resonance imaging (MRI) or computed tomography (CT) and, if indicated, positron emission computed tomography (PET). Dementia severity was assessed by the Clinical Dementia Rating (CDR) and the Mini Mental Scale Examination (MMSE). Disease duration was defined as the time in years between the first symptoms (by history) and the clinical diagnosis. The diagnosis of probable (n = 231) or possible (n = 38) AD was made by exclusion according to NINCDS-ADRDA criteria [12]. Patients were divided into those with early disease onset (EOAD; <65 years; 30 patients), and those with late onset (LOAD; ≥65 years; 239 patients).
The control group consisted of 203 subjects matched for ethnic background and age (110 women and 93 men, mean age 72 years). 175 of them were seen at the Alzheimer Units of the Ospedale Maggiore Policlinico (Milan) and the Ospedale L. Sacco (Milan) for subjective memory complaints, not confirmed by the subsequent neuropsychological analysis. Patients with mild cognitive impairment were not included in this group. All these control subjects had not developed dementia after a follow up period of 6 months. The remaining control subjects included 28 healthy age-matched volunteers recruited at the Ospedale Maggiore Policlinico. An informed consent to participate in this study was given by all individuals or their caregivers. Both patients and controls were genotyped for the ApoE status. Serum samples were available from 122 patients with probable AD and 83 controls. All the information about patients and controls are summarized in Table 1. 2.2. Laboratory methods 2.2.1. DNA analysis High molecular weight DNA was isolated from whole blood using a Flexigene Kit (Qiagen, Hildren, Gemany) as described by the manufacturer. The following polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay: • MCP-1 A-2518G: amplification with specific primers produces a 234 bp product. Digestion with PvuII yields 159 and 75 bp fragments when G is at position −2518, as described by Szalai et al. [15]. • ApoE: DNA was amplified using specific primers and then digested with HhaI, as previously described [1]. 2.2.2. MCP-1 determination MCP-1 was measured with a human specific ELISA kit, based on the quantitative sandwich enzyme immunoassay technique. The sensitivity of this assay was 10 pg/ml. 2.2.3. Statistical analysis Statistical analysis was performed using the Sigma Stat 2.0 software. Allelic and genotypic frequencies were Table 1 Characteristics of AD patients and controls
No. of patients Gender (M:F) Mean age (years) (range) Mean disease duration (years) (range) Mean MMSE score (range) LOAD (≥65 years) EOAD (<65 years) ApoE ε4 carriers ApoE ε4 non-carriers
AD
Controls
269 78:191 75 (51–101) 3 (0.5–10)
203 93:110 72 (46–96)
18.6 (3–27) 239/269 30/269 106/269 163/269
28.2 (27–30)
31/203 172/203
C. Fenoglio et al. / Neurobiology of Aging 25 (2004) 1169–1173
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obtained by direct counting. Hardy–Weinberg equilibrium was tested by using a χ2 goodness of fit test. Fisher’s exact test was used to test for differences in allele distributions between the groups. MCP-1 levels were compared using the Mann–Whitney U-test. Spearman test was used for correlations with clinical data. Statistical significance was set at P < 0.05.
3. Results The MCP-1 allele and genotype frequencies for the study groups of AD patients and healthy controls are reported in Table 2. The two populations were in Hardy–Weinberg equilibrium. The frequency of the A-2518G SNP in Italian healthy subjects was similar to the one reported for Caucasians [13]. No statistically significant differences in the distribution of the A-2518G allelic frequency between AD patients and controls were shown (P > 0.05, Table 2). Patients in the limited group diagnosed as “possible” AD (n = 38) did not show a different genetic profile compared with those with “probable” AD (n = 231). In particular, 16 of them were carriers of the mutation (15 heterozygous and 1 homozygous for the G allele), with a mutated allele frequency of 0.22. Stratifying by ApoE genotype, gender or age at onset, no differences in allele frequency were observed as well (P > 0.05, not shown). No significant differences between mean MCP-1 levels in AD compared with controls were shown (930.1±24.9 pg/ml versus 900.4 ± 33.6 pg/ml; P > 0.05). Stratifying AD patients by the presence of ε4 allele or by gender, no differences in mean MCP-1 concentrations were observed. On the contrary, stratifying for the presence of the mutated A-2518G allele, a significant increase of MCP-1 mean levels in serum of AD patients was observed (957.3 ± 28 pg/ml versus 871.4 ± 39.3 pg/ml; P = 0.03; Fig. 1), while in healthy subjects carrying the mutated allele a slight but not significant increase occurred (932.4 ± 513.6 pg/ml versus 899.7 ± 58.8 pg/ml; P > 0.05; Fig. 2). Moreover, in patients carrying two mutated alleles, the highest peaks of MCP-1 levels in serum were shown (P > 0.05, Fig. 2). Table 2 MCP-1 genotype and allele distribution between groups Frequency (n)
AD patients
Controls
Genotypes A/A A/G G/G
0.56 (151) 0.38 (102) 0.06 (16)
0.52 (106) 0.41 (84) 0.07 (13)
Alleles A G
0.75 (404) 0.25 (134)
0.73 (296) 0.27 (110)
Fig. 1. MCP-1 mean values±S.E.M. in serum from AD patients (n = 122) and healthy subjects (CON; n = 83) stratified according to the presence of the mutated allele. ∗ P = 0.03.
Fig. 2. MCP-1 mean values±S.E.M. in serum from AD patients (n = 122) stratified according to the MCP-1 genotype. P > 0.05.
No correlations between age, MMSE score or disease duration at time of serum sampling and MCP-1 levels were evidenced.
4. Discussion The presence of the SNP A-2518G in MCP-1 promoter region, according to presented results, does not seem to be a risk factor for the development of AD, whereas the presence of the mutated allele correlates significantly with higher levels of MCP-1 in serum of patients. This polymorphism is known to affect the rate of MCP-1 expression [13], but the molecular basis for the effect of the A-2518G allele on MCP-1 transcription is at present unknown. It does not alter the known transcription factor binding sites of the MCP-1 distal regulatory region, but may affect a previously unidentified site [16]. In the two populations studied, the effect
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exerted by the mutation occurred mostly on AD patients, even though there was the same tendency also in controls. A possible explanation for such stronger effect in patients than in healthy subjects may be the interaction with other activating factors specifically expressed only during AD development. The effect of the G allele appears to be dose-dependent, as cells from individuals homozygous for G produce more MCP-1 than those from G/A heterozygous [13]. This effect has been confirmed directly in serum of our patients carrying two mutated alleles, in which the highest peaks of MCP-1 levels were observed. However, these data are not statistically significant, probably due to the low frequency of the G/G genotype in the population studied. The evaluation of MCP-1 levels in cerebrospinal fluid (CSF), which reflect directly the amount of inflammatory processes into the brain, would be also very useful. In this regard, MCP-1 has been previously determined in CSF of 10 out of 269 patients considered in this study [2]. MCP-1 mean concentration in CSF of these patients is increased compared with the corresponding serum levels, with the highest values in four carriers of the A-2518G SNP. Although data obtained in this very small group of subjects are not conclusive, they suggest that this mutation exerts its effect also on CSF. These results underline the importance of MCP-1 in inflammatory processes occurring in AD, although it has been demonstrated that MCP-1 expression alone is not sufficient for activation of mononuclear phagocytes, but leads to an enhanced inflammatory response in the presence of additional stimuli [4]. A number of observations suggest basic molecular differences in chemokine biology according to race [6]. The G/A polymorphism demonstrates racial heterogeneity. The G allele frequency is increased in Asian and Mexican population, compared to Caucasian and African American populations [13]. At present, no evidences of MCP-1 different levels due to racial heterogeneity have been reported, although an increase in MCP-1 serum levels in elderly has been shown in healthy Japanese subjects [6]. In the Italian population considered, there was no correlation between age and MCP-1 concentrations in serum. Besides, Gonzalez et al. [3] found an association between MCP-1 levels and the presence of the SNP in healthy American males, but analysis of healthy males in our Italian population failed to replicate these results (data not shown). In conclusion, the A-2518G polymorphism in MCP-1 gene, either by itself or interacting with the ⑀4 allele, which represents a known risk factor for the development of AD [5], does not seem to be a risk factor for sporadic AD, but its presence correlates, in a significant way in AD patients, with a higher level of serum MCP-1. This can contribute to increase the inflammatory process occurring in AD. However, further studies in larger and different ethnical populations are certainly needed. Besides, both the possible involvement of other factors of the cytokine/chemokine network and the complex interactions with other genetic
and environmental factors should be investigated in the next future.
Acknowledgments This work was supported by grants from Associazione “Amici del Centro Dino Ferrari”, CARIPLO and Monzino Foundations, IRCCS Ospedale Maggiore Milano and Centre of Excellence for Neurodegenerative Diseases of the University of Milan. References [1] Del Bo R, Comi GP, Bresolin N, Castelli E, Conti E, Degiuli A, et al. The apolipoprotein Eε4 allele causes a faster decline of cognitive performances in Down’s syndrome subjects. J Neurol Sci 1997;145:87–91. [2] Galimberti D, Schoonenboom N, Scarpini E, Scheltens P. On behalf of the DIAR Group. Chemokines in serum and CSF of Alzheimer’s disease patients. Ann Neurol 2003;53:547–8. [3] Gonzalez E, Rovin BH, Sen L, Cooke G, Dhanda R, Mummidi S. HIV-1 infection and AIDS dementia are influenced by a mutant MCP-1 allele linked to increased monocyte infiltration of tissues and MCP-1 levels. PNAS 2002;99:13795–800. [4] Gunn MD, Nelken NA, Liao X, Williams LT. Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation. J Immunol 1997;158:376–83. [5] Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993;261:921–3. [6] Hizawa N, Yamaguchi E, Furuya K, Jinushi E, Ito A, Kawakami Y. The role of the CC chemokine receptor 2 gene polymorphism V64I (CCR2-64I) in sarcoidosis in a Japanese population. Am J Respir Crit Care Med 1999;159:2021–3. [7] Inadera H, Egashira K, Takemoto M, Ouchi Y, Matsushima K. Increase in circulating levels of monocyte chemoattractant protein-1 with aging. J Interferon Cytokine Res 1999;19:1179–82. [8] Ishizuka K, Kimura T, Igata-yi R Katsuragi S, Takamatsu J, Miyakawa T. Identification of monocyte chemoattractant protein1 in senile plaques and reactive microglia of Alzheimer’s disease. Psychiatry Clin Neurosci 1997;51:135–8. [9] Kelder W, McArthur JC, Nance-Sproson T, McClernon D, Griffin DE. -Chemokines MCP-1 and RANTES are selectively increased in cerebrospinal fluid of patients with human immunodeficiency virusassociated dementia. Ann Neurol 1998;44:831–5. [10] Lu B, Rutledge BJ, Gu L, Fiorillo J, Lukacs NW. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J Exp Med 1998;187:601–8. [11] Luster AD. Chemokines-chemotactic cytokines that mediate inflammation. N Engl J Med 1998;338:436–45. [12] McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. 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 1984;34:939–44. [13] Rovin HR, Lu L, Saxena R. A novel polymorphism in the MCP1 gene regulatory region that influences MCP-1 expression. BBRC 1999;259:344–8. [14] Sun YX, Minthon L, Wallmark A, Warkentin S, Blennow K, Janciauskiene S. Inflammatory markers in matched plasma and
C. Fenoglio et al. / Neurobiology of Aging 25 (2004) 1169–1173 cerebrospinal fluid from patients with Alzheimer’s disease. Dement Geriatr Cogn Disord 2003;16:136–44. [15] Szalai C, Duba J, Prohàszka Z, Kalina A, Szabò T, Nagy B. Involvement of polymorphisms in the chemokine system in the susceptibility for coronary artery disease (CAD). Coincidence of elevated Lp(a) and MCP-1 −2518 G/G genotype in CAD patients. Atherosclerosis 2001;158:233–9.
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[16] Ueda A, Ishigatzubo Y, Okubo T, Yoshimura T. Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. Cooperation of the two NF-kappaB sites and NF-kappaB/Rel subunit specificity. J Biol Chem 1997;272:31092–9. [17] Xia MQ, Hyman BT. Chemokines/chemokine receptors in the central nervous system and Alzheimer’s disease. J Neurovirol 1999;5:32– 41.
MCP-1 in Alzheimer’s disease patients: A-2518G polymorphism and serum levels Chiara Fenoglio a , Daniela Galimberti a,∗ , Carlo Lovati b , Ilaria Guidi a , Alberto Gatti a , Sergio Fogliarino c , Marco Tiriticco a , Claudio Mariani b , Gianluigi Forloni c , Carla Pettenati d , Pierluigi Baron a , Giancarlo Conti a , Nereo Bresolin a , Elio Scarpini a a
Department of Neurological Sciences, “Dino Ferrari” Center, University of Milan, IRCCS Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122 Milan, Italy b Department of Neurology, University of Milan, Ospedale L. Sacco, Milan, Italy c Department of Neuroscience, Instituto di Ricerche Farmacologiche M. Negri, Via Eritrea 62, 20157 Milan, Italy d Centro Regionale Alzheimer, U.O. Neurologia, Ospedale Passirana-Rho, Milan, Italy Received 12 August 2003; received in revised form 6 November 2003; accepted 13 November 2003
Abstract MCP-1 levels are increased in CSF of patients with Alzheimer’s disease (AD) compared with controls, suggesting a role in the development of dementia. Recently, a biallelic A/G polymorphism in the MCP-1 promoter at position −2518 has been found, influencing the level of MCP-1 expression in response to an inflammatory stimulus. The distribution of the A-2518G SNP was determined in 269 AD patients and in 203 healthy age matched controls, showing no differences between the two groups. On the contrary, a significant increase of MCP-1 serum levels in AD patients carrying at least one G mutated allele was observed. Moreover, the highest peaks of MCP-1 serum levels were present in patients carrying two G alleles. Stratifying by ApoE genotype, gender or age at onset, no differences in both allele frequency and MCP-1 serum concentration were observed. The A-2518G polymorphism in MCP-1 gene does not seem to be a risk factor for the development of AD, but its presence correlates with higher levels of serum MCP-1, which can contribute to increase the inflammatory process occurring in AD. © 2004 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Polymorphism; Chemokines; Monocyte chemoattractant protein-1; MCP-1; Genetics
1. Introduction Immunoreactivity for a number of chemokines, as well as for their related receptors, has been demonstrated in resident cells of the CNS, and upregulation of some of them is associated with Alzheimer’s disease (AD) pathological changes [17]. Chemokines are supposed to play a relevant role in the pathogenesis of the inflammatory process occurring during the development of the pathology, because of their chemotactic activity on brain phagocytes [11]. In particular, monocyte chemotactic protein-1 (MCP-1), which has been demonstrated to be essential for monocyte recruitment in inflammatory models in vivo, as MCP-1−/− mice are specifically unable to recruit monocytes [10], was found immunohistochemically in mature, but not in immature, senile plaques and in reactive microglia of brain tissues from ∗
Corresponding author. Tel.: +39-02-55033858; fax: +39-02-50320430. E-mail address: [email protected] (D. Galimberti).
0197-4580/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2003.11.008
five patients with AD. This suggests that MCP-1-related inflammatory events induced by reactive microglia contribute to the maturation of senile plaques [8]. Studies carried out in Japanese population indicate that MCP-1 levels in plasma increase with aging [7]. Besides, MCP-1 concentration is selectively increased in cerebrospinal fluid (CSF) of patients with HIV-associated dementia (HAD), and, notably, MCP-1 correlates with the severity of dementia [9]. HAD, as well as other neurodegenerative disorders, including AD, shows similar features, in particular inflammation, monocyte/macrophage recruitment and glial activation [3]. In this regard, it has been recently demonstrated that MCP-1 levels are higher in CSF of patients with AD than in controls, suggesting a role on phagocytic cells within the brain during the development of dementia [2,14]. Recently, a single nucleotide polymorphism (SNP) has been found in the MCP-1 gene regulatory region at position −2518 (A-2518G), that influences the level of MCP-1
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expression in response to an inflammatory stimulus. A reporter gene under control of the MCP-1 distal regulatory region was shown to be induced by interleukin-1 to a greater extent when the allele at −2518 was G and monocytes from individuals carrying a G allele produced more MCP-1 after treatment with IL-1 than monocytes from A/A homozygous individuals [13]. Moreover, European-American men who possess A-2518G allele has higher MCP-1 levels than those who lack this allele [3]. These evidences lead to the hypotesis that MCP-1 levels increase in G allele carriers may in part explain the different severity of pathological conditions among individuals who are affected by the same inflammatory disease. A case-control study comparing patients with coronary artery disease (CAD) and healthy controls showed that the prevalence of A-2518G carriers was significantly higher among CAD patients than controls, thus indicating that this polymorphism may be implicated with the susceptibility to develop the disease [15]. On the basis of these studies, suggesting the potential importance of MCP-1 in the pathogenesis of AD and the effect exerted by the presence of the A-2518G allele, the distribution of the A-2518G polymorphism in 269 patients with AD as well as in 203 healthy subjects was analyzed, in order to determine whether it could influence the susceptibility to develop the disease, both by itself or in combination with the known risk factor for the development of AD, apolipoproteinE (ApoE) ε4 allele [5]. The concentration of MCP-1 in serum was next evaluated, to test the ability of the mutated allele to influence the circulating levels of this chemokine.
2. Materials and methods 2.1. Subjects Between January 2001 and January 2003, 269 Italian AD patients (191 women and 78 men, mean age at disease onset 75 years) were consecutively recruited at Alzheimer Units of Ospedale Maggiore Policlinico (Milan), Ospedale L. Sacco (Milan) and Ospedale Passirana (Rho). All patients underwent a standard battery of examinations, including medical history, physical and neurological examination, screening laboratory tests, neurocognitive evaluation, brain magnetic resonance imaging (MRI) or computed tomography (CT) and, if indicated, positron emission computed tomography (PET). Dementia severity was assessed by the Clinical Dementia Rating (CDR) and the Mini Mental Scale Examination (MMSE). Disease duration was defined as the time in years between the first symptoms (by history) and the clinical diagnosis. The diagnosis of probable (n = 231) or possible (n = 38) AD was made by exclusion according to NINCDS-ADRDA criteria [12]. Patients were divided into those with early disease onset (EOAD; <65 years; 30 patients), and those with late onset (LOAD; ≥65 years; 239 patients).
The control group consisted of 203 subjects matched for ethnic background and age (110 women and 93 men, mean age 72 years). 175 of them were seen at the Alzheimer Units of the Ospedale Maggiore Policlinico (Milan) and the Ospedale L. Sacco (Milan) for subjective memory complaints, not confirmed by the subsequent neuropsychological analysis. Patients with mild cognitive impairment were not included in this group. All these control subjects had not developed dementia after a follow up period of 6 months. The remaining control subjects included 28 healthy age-matched volunteers recruited at the Ospedale Maggiore Policlinico. An informed consent to participate in this study was given by all individuals or their caregivers. Both patients and controls were genotyped for the ApoE status. Serum samples were available from 122 patients with probable AD and 83 controls. All the information about patients and controls are summarized in Table 1. 2.2. Laboratory methods 2.2.1. DNA analysis High molecular weight DNA was isolated from whole blood using a Flexigene Kit (Qiagen, Hildren, Gemany) as described by the manufacturer. The following polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay: • MCP-1 A-2518G: amplification with specific primers produces a 234 bp product. Digestion with PvuII yields 159 and 75 bp fragments when G is at position −2518, as described by Szalai et al. [15]. • ApoE: DNA was amplified using specific primers and then digested with HhaI, as previously described [1]. 2.2.2. MCP-1 determination MCP-1 was measured with a human specific ELISA kit, based on the quantitative sandwich enzyme immunoassay technique. The sensitivity of this assay was 10 pg/ml. 2.2.3. Statistical analysis Statistical analysis was performed using the Sigma Stat 2.0 software. Allelic and genotypic frequencies were Table 1 Characteristics of AD patients and controls
No. of patients Gender (M:F) Mean age (years) (range) Mean disease duration (years) (range) Mean MMSE score (range) LOAD (≥65 years) EOAD (<65 years) ApoE ε4 carriers ApoE ε4 non-carriers
AD
Controls
269 78:191 75 (51–101) 3 (0.5–10)
203 93:110 72 (46–96)
18.6 (3–27) 239/269 30/269 106/269 163/269
28.2 (27–30)
31/203 172/203
C. Fenoglio et al. / Neurobiology of Aging 25 (2004) 1169–1173
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obtained by direct counting. Hardy–Weinberg equilibrium was tested by using a χ2 goodness of fit test. Fisher’s exact test was used to test for differences in allele distributions between the groups. MCP-1 levels were compared using the Mann–Whitney U-test. Spearman test was used for correlations with clinical data. Statistical significance was set at P < 0.05.
3. Results The MCP-1 allele and genotype frequencies for the study groups of AD patients and healthy controls are reported in Table 2. The two populations were in Hardy–Weinberg equilibrium. The frequency of the A-2518G SNP in Italian healthy subjects was similar to the one reported for Caucasians [13]. No statistically significant differences in the distribution of the A-2518G allelic frequency between AD patients and controls were shown (P > 0.05, Table 2). Patients in the limited group diagnosed as “possible” AD (n = 38) did not show a different genetic profile compared with those with “probable” AD (n = 231). In particular, 16 of them were carriers of the mutation (15 heterozygous and 1 homozygous for the G allele), with a mutated allele frequency of 0.22. Stratifying by ApoE genotype, gender or age at onset, no differences in allele frequency were observed as well (P > 0.05, not shown). No significant differences between mean MCP-1 levels in AD compared with controls were shown (930.1±24.9 pg/ml versus 900.4 ± 33.6 pg/ml; P > 0.05). Stratifying AD patients by the presence of ε4 allele or by gender, no differences in mean MCP-1 concentrations were observed. On the contrary, stratifying for the presence of the mutated A-2518G allele, a significant increase of MCP-1 mean levels in serum of AD patients was observed (957.3 ± 28 pg/ml versus 871.4 ± 39.3 pg/ml; P = 0.03; Fig. 1), while in healthy subjects carrying the mutated allele a slight but not significant increase occurred (932.4 ± 513.6 pg/ml versus 899.7 ± 58.8 pg/ml; P > 0.05; Fig. 2). Moreover, in patients carrying two mutated alleles, the highest peaks of MCP-1 levels in serum were shown (P > 0.05, Fig. 2). Table 2 MCP-1 genotype and allele distribution between groups Frequency (n)
AD patients
Controls
Genotypes A/A A/G G/G
0.56 (151) 0.38 (102) 0.06 (16)
0.52 (106) 0.41 (84) 0.07 (13)
Alleles A G
0.75 (404) 0.25 (134)
0.73 (296) 0.27 (110)
Fig. 1. MCP-1 mean values±S.E.M. in serum from AD patients (n = 122) and healthy subjects (CON; n = 83) stratified according to the presence of the mutated allele. ∗ P = 0.03.
Fig. 2. MCP-1 mean values±S.E.M. in serum from AD patients (n = 122) stratified according to the MCP-1 genotype. P > 0.05.
No correlations between age, MMSE score or disease duration at time of serum sampling and MCP-1 levels were evidenced.
4. Discussion The presence of the SNP A-2518G in MCP-1 promoter region, according to presented results, does not seem to be a risk factor for the development of AD, whereas the presence of the mutated allele correlates significantly with higher levels of MCP-1 in serum of patients. This polymorphism is known to affect the rate of MCP-1 expression [13], but the molecular basis for the effect of the A-2518G allele on MCP-1 transcription is at present unknown. It does not alter the known transcription factor binding sites of the MCP-1 distal regulatory region, but may affect a previously unidentified site [16]. In the two populations studied, the effect
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exerted by the mutation occurred mostly on AD patients, even though there was the same tendency also in controls. A possible explanation for such stronger effect in patients than in healthy subjects may be the interaction with other activating factors specifically expressed only during AD development. The effect of the G allele appears to be dose-dependent, as cells from individuals homozygous for G produce more MCP-1 than those from G/A heterozygous [13]. This effect has been confirmed directly in serum of our patients carrying two mutated alleles, in which the highest peaks of MCP-1 levels were observed. However, these data are not statistically significant, probably due to the low frequency of the G/G genotype in the population studied. The evaluation of MCP-1 levels in cerebrospinal fluid (CSF), which reflect directly the amount of inflammatory processes into the brain, would be also very useful. In this regard, MCP-1 has been previously determined in CSF of 10 out of 269 patients considered in this study [2]. MCP-1 mean concentration in CSF of these patients is increased compared with the corresponding serum levels, with the highest values in four carriers of the A-2518G SNP. Although data obtained in this very small group of subjects are not conclusive, they suggest that this mutation exerts its effect also on CSF. These results underline the importance of MCP-1 in inflammatory processes occurring in AD, although it has been demonstrated that MCP-1 expression alone is not sufficient for activation of mononuclear phagocytes, but leads to an enhanced inflammatory response in the presence of additional stimuli [4]. A number of observations suggest basic molecular differences in chemokine biology according to race [6]. The G/A polymorphism demonstrates racial heterogeneity. The G allele frequency is increased in Asian and Mexican population, compared to Caucasian and African American populations [13]. At present, no evidences of MCP-1 different levels due to racial heterogeneity have been reported, although an increase in MCP-1 serum levels in elderly has been shown in healthy Japanese subjects [6]. In the Italian population considered, there was no correlation between age and MCP-1 concentrations in serum. Besides, Gonzalez et al. [3] found an association between MCP-1 levels and the presence of the SNP in healthy American males, but analysis of healthy males in our Italian population failed to replicate these results (data not shown). In conclusion, the A-2518G polymorphism in MCP-1 gene, either by itself or interacting with the ⑀4 allele, which represents a known risk factor for the development of AD [5], does not seem to be a risk factor for sporadic AD, but its presence correlates, in a significant way in AD patients, with a higher level of serum MCP-1. This can contribute to increase the inflammatory process occurring in AD. However, further studies in larger and different ethnical populations are certainly needed. Besides, both the possible involvement of other factors of the cytokine/chemokine network and the complex interactions with other genetic
and environmental factors should be investigated in the next future.
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