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Intereleukin-10 Promoter Polymorphism in Mild Cognitive Impairment and in Its Clinical Evolution

Intereleukin-10 Promoter Polymorphism in Mild Cognitive Impairment and in Its Clinical Evolution SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2010, Article ID 854527, 5 pages doi:10.4061/2010/854527 Research Article Intereleukin-10 Promoter Polymorphism in Mild Cognitive Impairment and in Its Clinical Evolution 1 1 1 2 Beatrice Arosio, Luigina Mastronardi, Carlo Vergani, and Giorgio Annoni Department of Internal Medicine, Universita ` degli Studi di Milano, Geriatric Unit, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Via Pace 9, 20122 Milano, Italy Department of Internal Medicine and Prevention, Universita ` degli Studi di Milano-Bicocca, Geriatric Clinic, San Gerardo Hospital, Via Pergolesi 33, 20052 Monza, Italy Correspondence should be addressed to Giorgio Annoni, giorgio.annoni@unimib.it Received 1 April 2010; Accepted 17 June 2010 Academic Editor: Diana Paleacu Copyright © 2010 Beatrice Arosio et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Specific proinflammatory alleles are associated with higher risk of Alzheimer disease (AD) in different onset age. The homozygosis for the A allele of−1082 polymorphism (G/A) of interleukin-10 (IL-10) promotes a higher risk of AD and reduced IL-10 generation in peripheral cells after amyloid stimulation. In this paper we analysed genotype and allele frequencies of this polymorphism in 138 subjects with mild cognitive impairment (MCI) diagnosed, respectively, as amnestic (a-MCI) and multiple impaired cognitive domains (mcd-MCI). The genotype frequencies were similar in a-MCI and AD subjects, whereas in mcd-MCI comparable to controls (AA genotype: 50% in a-MCI, 49.2% in AD, 28.7% in mcd-MCI and 31.8% in controls). Consequently, both allele and genotype distributions were significantly different between a-MCI and mcd-MCI (allele: P = .02, genotype: P< .05). These results support the theory that polymorphisms of cytokine genes can affect neurodegeneration and its clinical progression. IL-10 may partly explain the conversion of a-MCI to AD or be a genetic marker of susceptibility. 1. Introduction less consistently, from peripheral cell [7, 8]. Furthermore, an increased intrathecal production of the proinflammatory cytokine TNF-α and a decreased production of the anti- The pathogenic process of Alzheimer’s disease (AD) starts decades before the clinical onset of the disease [1]. During inflammatory cytokine TGF-β have been demonstrated in this preclinical phase, there is a gradual loss of axons and the brain of patients with MCI, suggesting there is a neurons, and at a certain threshold the first symptoms, proinflammatory state in such patients at high risk for AD most often impaired episodic memory, appear. At this stage, [9]. patients do not fulfil the criteria for dementia and may be Moreover, circulating acute phase reactant levels in diagnosed with mild cognitive impairment (MCI). There is middle age predict AD risk in old age and in particular cer- considerable clinical heterogeneity of this pathology since tain functional promoter polymorphisms in cognate genes that modulate inflammation are often found at elevated different clinical patterns can be recognized: amnestic MCI (a-MCI), MCI with multiple impaired cognitive domains frequency among AD cases. (mcd-MCI), and single nonmemory domain MCI [2]. Recently specific risk sets of proinflammatory alleles were identified that characterize AD in different onset age (before Although a-MCI may be the preclinical stage of AD, there is no established method to predict progression to AD in age 65, at ages 65–74, and at older ages) [10]. individuals with MCI. These alleles comprise also the −1082 promoter gene polymorphisms of IL-10 (G/A substitution) [11]. Inflammation is accepted to be a feature of AD [3, 4] and the pathogeneses of neurodegeneration have been at IL-10 maps to chromosome 1 between 1q31 and 1q32 least in part attributed to the release of proinflammatory is highly polymorphic, and its production is correlated cytokines from brain resident cells [5, 6] and, although to biallelic polymorphisms at positions −1082 (G to A), 2 International Journal of Alzheimer’s Disease −819 (T to C), and −592 (A to C). The polymorphism at tical Manual of mental Disorders—4th ed.) and NINCDS- position −1082 lies within an Ets (E-twenty-six specific)- ADRDA (National Institute of Communicative Disorders like recognition site and may affect the binding of this and Stroke-Alzheimer’s Disease and Related Disorders Asso- transcriptional factor and, therefore, alter transcription ciationWorkGroup)criteria[17]. activation; the −1082 A allele correlates with low IL-10 Within MCI who progressed to AD (MCI→ AD), only generation after stimulation of T cells in vitro [12], while two were diagnosed as mcd-MCI at enrolment; all the others polymorphisms at positions −819 and −592 do not seem to were diagnosed as a-MCI. be involved. In order to minimize the risk of possible inflammatory In a previous study, we found that the homozygosis for processes, all subjects were selected in the absence of clinical the A allele of the IL-10 −1082 G/A single nucleotide poly- signs of inflammation (e.g., normal body temperature, no morphism (SNP) was associated with six-fold higher risk of concomitant inflammatory condition) and with normal AD. In the same study, we also analysed the production of IL- blood chemistry (red blood cell sedimentation rate, albumin, 10 in Peripheral Blood Mononuclear Cells (PBMCs) of AD transferring, and C reactive protein plasma levels). patients and age-matched controls after specific stimulation Informed consent was obtained from all subjects and with amyloid peptide, LPS, and Flu. Since the generation the Ethics Committee of both universities approved the of IL-10 was reduced in patients after amyloid stimulation, study, which was conducted according to the Helsinki II we concluded that these specific immune responses may be declaration. This population was matched with AD patients selectively impaired in AD [13]. (n = 63) and nonsdemented sex- and age-matched healthy The aim of this study was to analyse the genotype and controls (n = 63) enrolled for our previous study [13]. allele frequencies of these IL-10 SNPs in 138 subjects with MCI and to compare them with those previously shown in 2.2. Gene Polymorphism Analysis. Whole blood was collected AD and healthy controls (HCs) [13]. by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson Co., Rutherford, NJ). Genomic DNA was extracted by the salting-out method 2. Materials and Methods as described in [18]. The concentration and purity of DNA were determined by spectrophotometric analysis. In order to 2.1. Study Protocol. This study comprised 138 subjects with establish IL-10 genotypes we employed a polymerase chain MCI age 80.37 ± 5.93 years (mean ± standard deviation reaction using sequence-specific primers (PCR-SSPs). The (SD)). All patients were Caucasian, living in Northern sequence in the promoter region of the IL-10 gene (polymor- Italy, and selected from a larger ambulatory population phic positions −1082, −819, and −592) was amplified using sample followed at the Geriatric Unit of the Ospedale the cytokine genotyping tray method (One Lambda, Canoga Maggiore Policlinico IRCCS, University of Milan, Italy and Park, CA, USA). The human β-globin gene was amplified the Geriatric Clinic of the University of Milan-Bicocca, Italy. as an internal control for the genomic DNA preparation. At enrolment, MCI subjects were divided into two groups PCR conditions were indicated by the One Lambda PCR based on cognitive features and diagnosed, respectively, as a- program (OLI-1) and the PCR products were visualised by MCI (30 patients) and mcd-MCI (108 patients). electrophoresis in 2.5% agarose gel. In particular, a-MCI met the criteria described by ApoE genotypes were determined by means of PCR Petersen [14]: subjects with memory impairment only amplification of a 234 base-pair fragment of exon 4 of (>1.5 SD above the age- and education-specific norms) and the ApoE gene, followed by digestion with Cfo1. The no difficulties in any other area of cognitive functions. restriction patterns were revealed by means of 4% agarose mcd-MCIs were subjects diagnosed with impairment in gel electrophoresis [13]. at least two cognitive domains of more than 1 SD below the mean of the respective age- and education-matched population, and with cognitive decline confirmed by the 2.3. Statistical Analysis. Statistical analysis was performed individuals themselves or reliable informants, but in whom with the SPSS statistical package (SPSS version 17, Chicago, no diagnosis of dementia could be achieved. A cut-off score IL). Genotype and allele frequencies in the study groups were of 1 SD was applied, which is less severe than that used compared using the χ -test. P< .05 was taken as the cut-off for a-MCI, in order to obtain higher diagnostic sensitivity for statistical significance. even though diagnostic specificity was reduced. Because the presence of more than one cognitive deficit and frequently initial impairment in Lawton’s instrumental activities of daily 3. Results living also characterized mcd-MCI, it may be mistaken for dementia; thus, a less severe criterion (>1 SD) allows better 3.1. Distribution of IL-10 Genotypes in MCI Subjects. The differentiation between mcd-MCI and dementia [15]. genotype and allele frequencies of the biallelic polymor- At this time, 74 patients out of the 138 completed a phism at position −1082 are reported in Table 1. This four-year follow-up and 24 were diagnosed with AD, 22 SNP alters transcriptional activation with a gene dosage- with vascular dementia (MCI→ VD), and 28 with stable related effect, so GG genotype correlates with high, GA MCI [16]. Subjects who developed AD during follow-up with intermediate, and AA with low IL-10 production after were required to meet the DSM IV (Diagnostic and Statis- stimulation of T cells in vitro [12]. International Journal of Alzheimer’s Disease 3 Table 1: Distribution of genotype and allele frequencies of −1082 (G/A) SNP in Alzheimer’s disease patients (AD), control subjects (CT), and mild cognitive impairment patients (MCI). GG (H) GA (M) AA (L) G A AD 4 (6.4%) 28 (44.4%) 31 (49.2%) 36 (28.6%) 90 (71.4%) CT 14 (22.2%) 29 (46%) 20 (31.8%) 57 (45.2%) 69 (54.8%) MCI 21 (15.2%) 71 (51.4%) 46 (33.3%) 113 (40.9%) 163 (59.1%) Genotype: χ 9.480, d.f. 4; P = .05. Allele: χ 8.257, d.f. 2; P = .02. Table 2: Distribution of genotype and allele frequencies of −1082 (G/A) SNP in amnestic MCI (a-MCI) and multiple cognitive domains MCI patients (mcd-MCI). GG (H) GA (M) AA (L) G A a-MCI 1 (3.3%) 14 (46.7%) 15 (50%) 16 (26.7%) 44 (73.3%) mcd-MCI 20 (18.5%) 57 (52.8%) 31 (28.7%) 97 (44.9%) 119 (55.1%) Genotype: χ 6.927, d.f. 2; P< .05. Allele: χ 5.729, d.f. 1; P = .02. Table 3: Distribution of genotype and allele frequencies of −1082 (G/A) SNP in MCIs that remain stable, progressed to AD (MCI→ AD), andprogressedtoVD(MCI→ VD). GG (H) GA (M) AA (L) G A MCI stable 8 (28.6%) 12 (42.8%) 8 (28.6%) 28 (50%) 28 (50%) MCI→ AD 2 (8.3%) 12 (50%) 10 (41.7%) 16 (33.3%) 32 (66.7%) MCI→ VD 5 (22.7%) 11 (50%) 6 (27.3%) 21 (47.7%) 23 (52.3%) Genotype distribution compared percentages: χ 15.604, d.f. 4; P = .004. Allele distributions compared percentages: χ 6.661, d.f. 2; P< .05. As previously described [13], AD patients show a signif- the presence of ε4 allele in 40% of cases and, during follow- icant higher frequency of the −1082A low producer allele, up, in 54% of MCI→ AD and 39% in stable MCI. The ApoE4 which skews the genotype distribution in AD compared to status is an independent risk factor for AD [13]. HC, with a significant decrease of −1082 GG high producer genotype. 3.3. Follow-Up. After a 4-year follow-up 24 MCI progressed MCI subjects as a whole had an intermediate pattern to AD (MCI→ AD) [16] and 22 progressed to vascular between AD and HC subjects, the percentages of G allele and dementia (MCI→ VD). Table 3 shows −1082 SNP distribu- GG genotype being 40.9% and 15.2%, while the percentage tions in MCI progressing and not progressing to AD (stable of A allele and AA genotype being 59.1% and 33.3%, MCI). respectively, (allele: P = .02, genotype: P = .05) (Table 1). In MCI→ AD both A allele and AA genotype were higher It is interesting to note that the genotype frequencies of than in stable MCI and in MCI→ VD. the −1082 SNP in a-MCI subjects were similar to those of Due to the limited number of patients that completed AD subjects, whereas those of mcd-MCI were comparable the follow-up period, the data reached the statistical signifi- to HC (AA genotype 50% in a-MCI and 49.2% in AD; cances only comparing genotype and allele percentage (allele: 28.7% and 31.8% in mcd-MCI and HC, resp.) (Table 2). P< .05, genotype P = .004). Consequently, the allele and genotype distributions were significantly different between a-MCI and mcd-MCI (allele: P = .02, genotype: P< .05). 4. Discussion The same SNP is linked with two other SNPs at positions −819 and −592. They combine with microsatellite alleles to A “cytokine cycle” has been proposed where [25] the form haplotypes where the difference in IL-10 production anti-inflammatory cytokines (IL-4, IL-10, and IL-13) reg- ulate β-amyloid-induced microglial/macrophage inflamma- is mainly accounted by the −1082 SNP [19, 20]. The genotype and allele frequencies of −819 and −592 SNPs were tory responses and modify the microglial activity sur- distributed similarly in our samples (data not shown). rounding amyloid neuritic plaques [26]. These cytokines can inhibit the induction of IL-1, TNF-α,and MCP-1 3.2. Distribution of Apolipoprotein E Genotype in MCI in differentiated human monocytes and, above all, IL- Subjects. The frequency of ApoE ε4 in our sample was in 10 causes dose-dependent inhibition of the IL-6 secretion line with the data already published [21–24]. In particular induced by β-amyloid in these cells and in murine microglia genotyping of our MCI patients globally considered revealed [25]. 4 International Journal of Alzheimer’s Disease In a previous paper, we described not only a significantly receptor antagonist in Alzheimer’s disease,” Dementia and Geriatric Cognitive Disorders, vol. 12, no. 5, pp. 314–317, 2001. higher percentage of IL-10 −1082 AA low-producing geno- [7] P.Bermejo,S.Mart´ın-Aragon, ´ J. Bened´ı et al., “Differences type among AD cases, but also a reduced IL-10 generation in of peripheral inflammatory markers between mild cognitive peripheral blood mononuclear cells from these patients after impairment and Alzheimer’s disease,” Immunology Letters, vol. β-amyloid stimulation [13]. 117, no. 2, pp. 198–202, 2008. Interestingly a report on Italian centenarians, who are [8] K.Bonotis,E.Krikki,V.Holeva,C.Aggouridaki,V.Costa,and clearly less prone than younger persons to age-related S. Baloyannis, “Systemic immune aberrations in Alzheimer’s diseases, showed that extreme longevity is significantly disease patients,” Journal of Neuroimmunology, vol. 193, no. 1- associated with the high IL-10-producing genotypes [27]. 2, pp. 183–187, 2008. In the present study, the allele frequencies of −1082 SNP [9] S. Ray, M. Britschgi, C. Herbert et al., “Classification and in a-MCI subjects were similar to those of AD patients, prediction of clinical Alzheimer’s diagnosis based on plasma whereas those of mcd-MCI were comparable to HC (the signaling proteins,” Nature Medicine, vol. 13, no. 11, pp. 1359– 1362, 2007. frequencies of the low-producer AA genotype were 50% and [10] F. Licastro, E. Porcellini, C. Caruso, D. Lio, and E. H. Corder, 28.7%, in a-MCI and mcd-MCI, resp.). “Genetic risk profiles for Alzheimer’s disease: integration of It is to note that, after an adequate period of follow- APOE genotype and variants that up-regulate inflammation,” up, the twenty-four a-MCI subjects that progressed to AD Neurobiology of Aging, vol. 28, no. 11, pp. 1637–1643, 2007. showed a higher percentage of AA carriers (41.7%) compared [11] D. Lio, F. Licastro, L. Scola et al., “Interleukin-10 promoter to those of MCI that remain stable (28.6%) and compared to polymorphism in sporadic Alzheimer’s disease,” Genes and those progressed in vascular dementia (27.3%). The similar Immunity, vol. 4, no. 3, pp. 234–238, 2003. genotype distribution of this IL-10 SNP in AD and a-MCI [12] J. M. Kim, C. I. Brannan, N. G. Copeland, N. A. Jenkins, T. but not in mcd-MCI and the data retrospectively obtained A. Khan, and K. W. Moore, “Structure of the mouse IL-10 after the follow-up suggest that it is potentially involved in gene and chromosomal localization of the mouse and human the conversion of a-MCI to AD. genes,” Journal of Immunology, vol. 148, no. 11, pp. 3618–3623, However, our results support the theory that the overall 1992. [13] B. Arosio, D. Trabattoni, L. Galimberti et al., “Interleukin- risk of developing AD may be governed by a multifactorial 10 and interleukin-6 gene polymorphisms as risk factors for “susceptibility profile” and that polymorphisms of cytokine Alzheimer’s disease,” Neurobiology of Aging,vol. 25, no.8,pp. genes can affect neurodegeneration and its clinical progres- 1009–1015, 2004. sion. [14] R. C. Petersen, “Mild cognitive impairment as a diagnostic In addiction, IL-10 may partly explain the conversion of entity,” Journal of Internal Medicine, vol. 256, no. 3, pp. 183– a-MCI to AD or, at least, be a genetic marker of susceptibility 194, 2004. [28]. [15] M. Zanetti, C. Ballabio, C. Abbate, C. Cutaia, C. Vergani, Therefore, it is extremely relevant to closely define and L. Bergamaschini, “Mild cognitive impairment subtypes intrinsic (i.e., genetic) individual risk profiles in prevention and vascular dementia in community-dwelling elderly people: and treatment trials. The finding that the set of gene variants a 3-year follow-up study,” Journal of the American Geriatrics in innate immunity associated with earlier onset predicted Society, vol. 54, no. 4, pp. 580–586, 2006. rapid clinical progression suggests that interventions to [16] O. Hansson, H. Zetterberg, P. Buchhave, E. Londos, K. Blennow, and L. Minthon, “Association between CSF control inflammation might be useful especially for relatively biomarkers and incipient Alzheimer’s disease in patients younger cases to delay disease progression. with mild cognitive impairment: a follow-up study,” Lancet Neurology, vol. 5, no. 3, pp. 228–234, 2006. [17] G. McKhann, D. Drachman, and M. Folstein, “Clinical diag- References nosis of Alzheimer’s disease: report of the NINCDS-ADRDA work group under the auspices of Department of Health [1] J. L. Price and J. C. Morris, “Tangles and plaques in nonde- mented aging and “preclinical” Alzheimer’s disease,” Annals of and Human Services Task Force on Alzheimer’s disease,” Neurology, vol. 34, no. 7, pp. 939–944, 1984. Neurology, vol. 45, no. 3, pp. 358–368, 1999. [2] R. C. Petersen, R. Doody, A. Kurz et al., “Current concepts in [18] S. A. Miller, D. D. Dykes, and H. F. Polesky, “A simple salting out procedure for extracting DNA from human nucleated mild cognitive impairment,” Archives of Neurology, vol. 58, no. 12, pp. 1985–1992, 2001. cells,” Nucleic Acids Research, vol. 16, no. 3, p. 1215, 1988. [3] H. W. Querfurth and F. M. LaFerla, “Alzheimer’s disease,” The [19] J. Eskdale, P. Wordsworth, S. Bowman, M. Field, and G. 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Berlau,M.M.Corrada,E.Head, andC.H.Kawas,“ApoE dementia,” Journal of Clinical Immunology,vol. 19, no.4,pp. 223–230, 1999. ε2 is associated with intact cognition but increased Alzheimer pathology in the oldest old,” Neurology, vol. 72, no. 9, pp. 829– [6] E. Tarkowski, A.-M. Liljeroth, A. Nilsson, L. Minthon, and K. Blennow, “Decreased levels of intrathecal interleukin 1 834, 2009. International Journal of Alzheimer’s Disease 5 [22] G. Bu, “Apolipoprotein E and its receptors in Alzheimer’s disease: pathways, pathogenesis and therapy,” Nature Reviews Neuroscience, vol. 10, no. 5, pp. 333–344, 2009. [23] P. P. Singh, M. Singh, and S. S. Mastana, “APOE distribution in world populations with new data from India and the UK,” Annals of Human Biology, vol. 33, no. 3, pp. 279–308, 2006. [24] J. Kim, J. M. Basak, and D. M. Holtzman, “The role of apolipoprotein E in Alzheimer’s disease,” Neuron, vol. 63, no. 3, pp. 287–303, 2009. [25] A. M. Szczepanik, S. Funes, W. Petko, and G. E. 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Intereleukin-10 Promoter Polymorphism in Mild Cognitive Impairment and in Its Clinical Evolution

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Copyright © 2010 Beatrice Arosio et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2010, Article ID 854527, 5 pages doi:10.4061/2010/854527 Research Article Intereleukin-10 Promoter Polymorphism in Mild Cognitive Impairment and in Its Clinical Evolution 1 1 1 2 Beatrice Arosio, Luigina Mastronardi, Carlo Vergani, and Giorgio Annoni Department of Internal Medicine, Universita ` degli Studi di Milano, Geriatric Unit, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Via Pace 9, 20122 Milano, Italy Department of Internal Medicine and Prevention, Universita ` degli Studi di Milano-Bicocca, Geriatric Clinic, San Gerardo Hospital, Via Pergolesi 33, 20052 Monza, Italy Correspondence should be addressed to Giorgio Annoni, giorgio.annoni@unimib.it Received 1 April 2010; Accepted 17 June 2010 Academic Editor: Diana Paleacu Copyright © 2010 Beatrice Arosio et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Specific proinflammatory alleles are associated with higher risk of Alzheimer disease (AD) in different onset age. The homozygosis for the A allele of−1082 polymorphism (G/A) of interleukin-10 (IL-10) promotes a higher risk of AD and reduced IL-10 generation in peripheral cells after amyloid stimulation. In this paper we analysed genotype and allele frequencies of this polymorphism in 138 subjects with mild cognitive impairment (MCI) diagnosed, respectively, as amnestic (a-MCI) and multiple impaired cognitive domains (mcd-MCI). The genotype frequencies were similar in a-MCI and AD subjects, whereas in mcd-MCI comparable to controls (AA genotype: 50% in a-MCI, 49.2% in AD, 28.7% in mcd-MCI and 31.8% in controls). Consequently, both allele and genotype distributions were significantly different between a-MCI and mcd-MCI (allele: P = .02, genotype: P< .05). These results support the theory that polymorphisms of cytokine genes can affect neurodegeneration and its clinical progression. IL-10 may partly explain the conversion of a-MCI to AD or be a genetic marker of susceptibility. 1. Introduction less consistently, from peripheral cell [7, 8]. Furthermore, an increased intrathecal production of the proinflammatory cytokine TNF-α and a decreased production of the anti- The pathogenic process of Alzheimer’s disease (AD) starts decades before the clinical onset of the disease [1]. During inflammatory cytokine TGF-β have been demonstrated in this preclinical phase, there is a gradual loss of axons and the brain of patients with MCI, suggesting there is a neurons, and at a certain threshold the first symptoms, proinflammatory state in such patients at high risk for AD most often impaired episodic memory, appear. At this stage, [9]. patients do not fulfil the criteria for dementia and may be Moreover, circulating acute phase reactant levels in diagnosed with mild cognitive impairment (MCI). There is middle age predict AD risk in old age and in particular cer- considerable clinical heterogeneity of this pathology since tain functional promoter polymorphisms in cognate genes that modulate inflammation are often found at elevated different clinical patterns can be recognized: amnestic MCI (a-MCI), MCI with multiple impaired cognitive domains frequency among AD cases. (mcd-MCI), and single nonmemory domain MCI [2]. Recently specific risk sets of proinflammatory alleles were identified that characterize AD in different onset age (before Although a-MCI may be the preclinical stage of AD, there is no established method to predict progression to AD in age 65, at ages 65–74, and at older ages) [10]. individuals with MCI. These alleles comprise also the −1082 promoter gene polymorphisms of IL-10 (G/A substitution) [11]. Inflammation is accepted to be a feature of AD [3, 4] and the pathogeneses of neurodegeneration have been at IL-10 maps to chromosome 1 between 1q31 and 1q32 least in part attributed to the release of proinflammatory is highly polymorphic, and its production is correlated cytokines from brain resident cells [5, 6] and, although to biallelic polymorphisms at positions −1082 (G to A), 2 International Journal of Alzheimer’s Disease −819 (T to C), and −592 (A to C). The polymorphism at tical Manual of mental Disorders—4th ed.) and NINCDS- position −1082 lies within an Ets (E-twenty-six specific)- ADRDA (National Institute of Communicative Disorders like recognition site and may affect the binding of this and Stroke-Alzheimer’s Disease and Related Disorders Asso- transcriptional factor and, therefore, alter transcription ciationWorkGroup)criteria[17]. activation; the −1082 A allele correlates with low IL-10 Within MCI who progressed to AD (MCI→ AD), only generation after stimulation of T cells in vitro [12], while two were diagnosed as mcd-MCI at enrolment; all the others polymorphisms at positions −819 and −592 do not seem to were diagnosed as a-MCI. be involved. In order to minimize the risk of possible inflammatory In a previous study, we found that the homozygosis for processes, all subjects were selected in the absence of clinical the A allele of the IL-10 −1082 G/A single nucleotide poly- signs of inflammation (e.g., normal body temperature, no morphism (SNP) was associated with six-fold higher risk of concomitant inflammatory condition) and with normal AD. In the same study, we also analysed the production of IL- blood chemistry (red blood cell sedimentation rate, albumin, 10 in Peripheral Blood Mononuclear Cells (PBMCs) of AD transferring, and C reactive protein plasma levels). patients and age-matched controls after specific stimulation Informed consent was obtained from all subjects and with amyloid peptide, LPS, and Flu. Since the generation the Ethics Committee of both universities approved the of IL-10 was reduced in patients after amyloid stimulation, study, which was conducted according to the Helsinki II we concluded that these specific immune responses may be declaration. This population was matched with AD patients selectively impaired in AD [13]. (n = 63) and nonsdemented sex- and age-matched healthy The aim of this study was to analyse the genotype and controls (n = 63) enrolled for our previous study [13]. allele frequencies of these IL-10 SNPs in 138 subjects with MCI and to compare them with those previously shown in 2.2. Gene Polymorphism Analysis. Whole blood was collected AD and healthy controls (HCs) [13]. by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson Co., Rutherford, NJ). Genomic DNA was extracted by the salting-out method 2. Materials and Methods as described in [18]. The concentration and purity of DNA were determined by spectrophotometric analysis. In order to 2.1. Study Protocol. This study comprised 138 subjects with establish IL-10 genotypes we employed a polymerase chain MCI age 80.37 ± 5.93 years (mean ± standard deviation reaction using sequence-specific primers (PCR-SSPs). The (SD)). All patients were Caucasian, living in Northern sequence in the promoter region of the IL-10 gene (polymor- Italy, and selected from a larger ambulatory population phic positions −1082, −819, and −592) was amplified using sample followed at the Geriatric Unit of the Ospedale the cytokine genotyping tray method (One Lambda, Canoga Maggiore Policlinico IRCCS, University of Milan, Italy and Park, CA, USA). The human β-globin gene was amplified the Geriatric Clinic of the University of Milan-Bicocca, Italy. as an internal control for the genomic DNA preparation. At enrolment, MCI subjects were divided into two groups PCR conditions were indicated by the One Lambda PCR based on cognitive features and diagnosed, respectively, as a- program (OLI-1) and the PCR products were visualised by MCI (30 patients) and mcd-MCI (108 patients). electrophoresis in 2.5% agarose gel. In particular, a-MCI met the criteria described by ApoE genotypes were determined by means of PCR Petersen [14]: subjects with memory impairment only amplification of a 234 base-pair fragment of exon 4 of (>1.5 SD above the age- and education-specific norms) and the ApoE gene, followed by digestion with Cfo1. The no difficulties in any other area of cognitive functions. restriction patterns were revealed by means of 4% agarose mcd-MCIs were subjects diagnosed with impairment in gel electrophoresis [13]. at least two cognitive domains of more than 1 SD below the mean of the respective age- and education-matched population, and with cognitive decline confirmed by the 2.3. Statistical Analysis. Statistical analysis was performed individuals themselves or reliable informants, but in whom with the SPSS statistical package (SPSS version 17, Chicago, no diagnosis of dementia could be achieved. A cut-off score IL). Genotype and allele frequencies in the study groups were of 1 SD was applied, which is less severe than that used compared using the χ -test. P< .05 was taken as the cut-off for a-MCI, in order to obtain higher diagnostic sensitivity for statistical significance. even though diagnostic specificity was reduced. Because the presence of more than one cognitive deficit and frequently initial impairment in Lawton’s instrumental activities of daily 3. Results living also characterized mcd-MCI, it may be mistaken for dementia; thus, a less severe criterion (>1 SD) allows better 3.1. Distribution of IL-10 Genotypes in MCI Subjects. The differentiation between mcd-MCI and dementia [15]. genotype and allele frequencies of the biallelic polymor- At this time, 74 patients out of the 138 completed a phism at position −1082 are reported in Table 1. This four-year follow-up and 24 were diagnosed with AD, 22 SNP alters transcriptional activation with a gene dosage- with vascular dementia (MCI→ VD), and 28 with stable related effect, so GG genotype correlates with high, GA MCI [16]. Subjects who developed AD during follow-up with intermediate, and AA with low IL-10 production after were required to meet the DSM IV (Diagnostic and Statis- stimulation of T cells in vitro [12]. International Journal of Alzheimer’s Disease 3 Table 1: Distribution of genotype and allele frequencies of −1082 (G/A) SNP in Alzheimer’s disease patients (AD), control subjects (CT), and mild cognitive impairment patients (MCI). GG (H) GA (M) AA (L) G A AD 4 (6.4%) 28 (44.4%) 31 (49.2%) 36 (28.6%) 90 (71.4%) CT 14 (22.2%) 29 (46%) 20 (31.8%) 57 (45.2%) 69 (54.8%) MCI 21 (15.2%) 71 (51.4%) 46 (33.3%) 113 (40.9%) 163 (59.1%) Genotype: χ 9.480, d.f. 4; P = .05. Allele: χ 8.257, d.f. 2; P = .02. Table 2: Distribution of genotype and allele frequencies of −1082 (G/A) SNP in amnestic MCI (a-MCI) and multiple cognitive domains MCI patients (mcd-MCI). GG (H) GA (M) AA (L) G A a-MCI 1 (3.3%) 14 (46.7%) 15 (50%) 16 (26.7%) 44 (73.3%) mcd-MCI 20 (18.5%) 57 (52.8%) 31 (28.7%) 97 (44.9%) 119 (55.1%) Genotype: χ 6.927, d.f. 2; P< .05. Allele: χ 5.729, d.f. 1; P = .02. Table 3: Distribution of genotype and allele frequencies of −1082 (G/A) SNP in MCIs that remain stable, progressed to AD (MCI→ AD), andprogressedtoVD(MCI→ VD). GG (H) GA (M) AA (L) G A MCI stable 8 (28.6%) 12 (42.8%) 8 (28.6%) 28 (50%) 28 (50%) MCI→ AD 2 (8.3%) 12 (50%) 10 (41.7%) 16 (33.3%) 32 (66.7%) MCI→ VD 5 (22.7%) 11 (50%) 6 (27.3%) 21 (47.7%) 23 (52.3%) Genotype distribution compared percentages: χ 15.604, d.f. 4; P = .004. Allele distributions compared percentages: χ 6.661, d.f. 2; P< .05. As previously described [13], AD patients show a signif- the presence of ε4 allele in 40% of cases and, during follow- icant higher frequency of the −1082A low producer allele, up, in 54% of MCI→ AD and 39% in stable MCI. The ApoE4 which skews the genotype distribution in AD compared to status is an independent risk factor for AD [13]. HC, with a significant decrease of −1082 GG high producer genotype. 3.3. Follow-Up. After a 4-year follow-up 24 MCI progressed MCI subjects as a whole had an intermediate pattern to AD (MCI→ AD) [16] and 22 progressed to vascular between AD and HC subjects, the percentages of G allele and dementia (MCI→ VD). Table 3 shows −1082 SNP distribu- GG genotype being 40.9% and 15.2%, while the percentage tions in MCI progressing and not progressing to AD (stable of A allele and AA genotype being 59.1% and 33.3%, MCI). respectively, (allele: P = .02, genotype: P = .05) (Table 1). In MCI→ AD both A allele and AA genotype were higher It is interesting to note that the genotype frequencies of than in stable MCI and in MCI→ VD. the −1082 SNP in a-MCI subjects were similar to those of Due to the limited number of patients that completed AD subjects, whereas those of mcd-MCI were comparable the follow-up period, the data reached the statistical signifi- to HC (AA genotype 50% in a-MCI and 49.2% in AD; cances only comparing genotype and allele percentage (allele: 28.7% and 31.8% in mcd-MCI and HC, resp.) (Table 2). P< .05, genotype P = .004). Consequently, the allele and genotype distributions were significantly different between a-MCI and mcd-MCI (allele: P = .02, genotype: P< .05). 4. Discussion The same SNP is linked with two other SNPs at positions −819 and −592. They combine with microsatellite alleles to A “cytokine cycle” has been proposed where [25] the form haplotypes where the difference in IL-10 production anti-inflammatory cytokines (IL-4, IL-10, and IL-13) reg- ulate β-amyloid-induced microglial/macrophage inflamma- is mainly accounted by the −1082 SNP [19, 20]. The genotype and allele frequencies of −819 and −592 SNPs were tory responses and modify the microglial activity sur- distributed similarly in our samples (data not shown). rounding amyloid neuritic plaques [26]. These cytokines can inhibit the induction of IL-1, TNF-α,and MCP-1 3.2. Distribution of Apolipoprotein E Genotype in MCI in differentiated human monocytes and, above all, IL- Subjects. The frequency of ApoE ε4 in our sample was in 10 causes dose-dependent inhibition of the IL-6 secretion line with the data already published [21–24]. In particular induced by β-amyloid in these cells and in murine microglia genotyping of our MCI patients globally considered revealed [25]. 4 International Journal of Alzheimer’s Disease In a previous paper, we described not only a significantly receptor antagonist in Alzheimer’s disease,” Dementia and Geriatric Cognitive Disorders, vol. 12, no. 5, pp. 314–317, 2001. higher percentage of IL-10 −1082 AA low-producing geno- [7] P.Bermejo,S.Mart´ın-Aragon, ´ J. Bened´ı et al., “Differences type among AD cases, but also a reduced IL-10 generation in of peripheral inflammatory markers between mild cognitive peripheral blood mononuclear cells from these patients after impairment and Alzheimer’s disease,” Immunology Letters, vol. β-amyloid stimulation [13]. 117, no. 2, pp. 198–202, 2008. Interestingly a report on Italian centenarians, who are [8] K.Bonotis,E.Krikki,V.Holeva,C.Aggouridaki,V.Costa,and clearly less prone than younger persons to age-related S. 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