- Email: [email protected]
The cyclooxygenase 2 -765 C promoter allele is a protective factor for Alzheimer's disease
Neuroscience Letters 395 (2006) 240–243
The cyclooxygenase 2 -765 C promoter allele is a protective factor for Alzheimer’s disease Laila Abdullah a,∗ , Ghania Ait-Ghezala a , Fiona Crawford a , Timothy A. Crowell a , Warren W. Barker b , Ranjan Duara b,c , Michael Mullan a b
a Roskamp Institute, 2040 Whitfield Ave. Sarasota, FL, USA Wien Center for Alzheimer’s Disease and Memory Disorders, Mt Sinai Medical Center, Miami Beach, Miami, FL, USA c Miller School of Medicine, University of Miami, Miami, FL, USA
Received 4 October 2005; received in revised form 23 October 2005; accepted 31 October 2005
Abstract The cyclooxygenase-2 enzyme (COX-2) is of particular importance in the inflammatory response and recent findings have demonstrated a considerable role in Alzheimer’s disease (AD) pathogenesis. In order to assess the possible putative role of a COX-2 polymorphism (765 G/C) in AD, we examined its distribution in 161 community-based controls and 168 AD clinic-based cases previously recruited from memory disorder clinics in Tampa and Miami, Florida. There were no significant differences between the two groups in age/age of onset or gender. A significant difference was observed in the distribution of the COX-2 -765 alleles between AD cases and controls (χ2 = 6.565, p = .010; OR = .596; CI = [.401–.888], p = .011), with the frequency of the C allele being higher in controls. In addition, a significant difference was observed for this polymorphism by genotype (χ2 = 6.561, p = .038) and by presence or absence of C+ genotypes (χ2 = 6.207, p = .013; OR = .464, CI = [.351–.885], p = .013). In this sample, the C allele of COX-2 -765 promoter polymorphism is associated with decreased risk of Alzheimer’s disease, a finding which further supports the involvement of COX-2 in AD etiology. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease cyclooxygenase 2; Inflammation
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by cognitive deficit with progressive worsening of memory, and pathologically by the presence of amyloid plaques and neurofibrillary tangles (NFTs) in the brain. A key role for inflammation in AD is supported by studies demonstrating an upregulation of prostaglandins (PGs), inflammatory cytokines, acute phase proteins, and activation of the complement cascade and further supported by the presence of microglia and astrocytes in and around the amyloid plaques [2,7,10,14,15,20,26,30]. The cyclooxygenase (COX) enzymes are the main producers of PGs from arachidonic acid [21,36]. Two distinct COX enzymes have been identified. The constitutively expressed COX-1 enzyme is mainly responsible for housekeeping functions in addition to the PG and thromboxane production in gastric mucosa [33]. By contrast, the inducible COX-2 enzyme has been
∗
Corresponding author. Tel.: +1 813 979 2008; fax: +1 813 979 2248. E-mail address: [email protected] (L. Abdullah).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.10.090
shown to be expressed mostly in central nervous system and inflammatory cells [8,16]. In addition, elevated COX-2 levels are present in neurons from hippocampal pyramidal regions of the AD brain, and correlate with amyloid plaque density and NFTs [11,12,23]. The finding that COX-2 inhibitors have less gastrointestinal toxicity than non-specific COX inhibitors has lead to their widespread use in anti-arthritic therapy [3,18,34]. Additionally, several epidemiological studies have suggested that the use of non-steroidal anti-inflammatory drugs, which include COX-2 inhibitors, may reduce the risk of developing AD [1,9,13]. The gene for COX-2 has been mapped to 1q25 which is located between 1q23 and 1q31, two regions to which genetic linkage to AD has been reported [19,29,31]. Recently, Papafili and colleagues have identified a functional polymorphism in the promoter region of the COX-2 gene; a G to C transversion at position -765 resulting in significantly lower promoter activity [25]. We have investigated this polymorphism for association with AD and find evidence that possession of the rarer “C” allele is associated with decreased risk for AD.
L. Abdullah et al. / Neuroscience Letters 395 (2006) 240–243
The study subjects were previously recruited under IRB approved studies at the University of South Florida and University of Miami. The AD sample consisted of 168 cases (57.8% female) recruited from Memory Disorder Clinics in Tampa and in Miami, Florida. The community-based control sample consisted of 161 individuals (62.9% female), recruited from a community screening project in the Dade county area of Florida. The mean age of onset of the AD cases was 73.89 (±6.954 S.D.), and the mean age of the controls at the time of the screen was 74.46 (±6.925 S.D.). All the cases and controls considered themselves Caucasians. The AD cases were genetically unrelated and diagnosed using NINCDS-ADRDA criteria for probable AD, however the pathological confirmation on these cases is not yet available [22]. The controls were defined as having MMSE > 27 and scored at or above the 16th percentile on delayed memory and semantic fluency tasks (short-term and long-term recall of items from the MMSE, recall items from a grocery list, and category fluency for animals, fruits, and vegetables) [27]. Additionally, these individuals maintained independent activities of daily living, and were free of active neurologic, or psychiatric disorders or ongoing medical conditions that would potentially interfere with their cognitive performance. DNA was extracted from whole blood using the Pure GeneTM Kit (Gentra Systems). The COX-2 -765G/C polymorphism was analyzed using PCR primers and conditions previously described by Papafili and colleagues.25 In the presence of the G allele, AciI restriction endonuclease digestion of the amplified 306 bp fragment results in 188 bp and 118 bp fragments. All subjects were also genotyped for APOE using previously described conditions [35]. The Pearson χ2 statistic was used to compare gender, HardyWeinberg equilibrium and the genotype and allele distributions between AD cases and controls for the COX-2 -765 G/C polymorphism, and APOE polymorphisms. Age of the controls at the initial cognitive testing was compared to age of onset of AD cases using a t-test for independent samples. Binary logistic regression modeling was used to calculate odds ratios (OR) and associated 95% confidence intervals (CI). To evaluate possible interactions between gender, APOE polymorphism and COX-2 polymorphism (by allele and genotype), multiple hierarchical logistic regression analyses were performed using diagnosis of AD as the dependent variable. Additionally, to determine a possible effect of the COX-2 -765 C allele on age of onset, analysis of variance was carried out for AD cases. A power analysis of these data was conducted using GPower software and revealed a power of 99.9% based on a sample of 329 individuals for a medium effect size. Alpha levels were set at 0.05 and SPSS version 13.1 was used to analyze these data. No significant difference in age/age of onset between controls and AD cases was observed (p > .05). Although there were more females in the control group compared to the cases, the difference did not reach statistical significance (p > .05). For the APOE and COX-2 -765 polymorphisms, the genotype distributions were found to be in Hardy-Weinberg equilibrium for cases, controls, and the entire sample (p > .05). The frequencies of the COX-2 -765 C and G alleles and the three genotypes in our control population (see tables) are similar to the frequencies of the
241
Table 1 Allele frequencies of COX-2 -765 polymorphisma COX-2 -765
Controls
AD cases
G allele C allele 2n
249 (77.3) 73 (22.7) 322 (100)
286 (85.1) 50 (14.9) 336 (100)
χ2 = 6.565, d.f. = 1, p = .010; OR = .596; CI = .401–.888, p = .011 a
Data are expressed as no. (%).
control population (over 70 age group) reported by Cipollone and colleagues (G allele = 72.7%, C allele 27.3%; G/G = 51.3%, G/C = 41.2%, C/C = 7.5%) [35]. As expected from many previous reports, the APOE 4 allele and 4+ genotypes were significantly increased in AD cases when compared to controls (χ2 = 29.098, p < .001). A significant difference was observed in the distribution of COX-2 -765 alleles between AD cases and controls (p < .05, Table 1), with the frequency of the C allele being higher in controls. In addition, a significant difference was observed for this polymorphism by genotype (p = .038, Table 2); given the low occurrence of the C/C genotype in cases and in controls, we conducted the analysis by presence or absence of the C allele in the genotypes. The C+ genotypes were found to be more frequent in controls when compared to AD cases (p = .013, Table 2). However, no interactions were observed between APOE and COX-2 -765 polymorphisms nor between gender and the COX-2 -765 polymorphism on diagnosis of AD (p > .05, data not shown). Furthermore, no interaction was observed between age/age of onset and this polymorphism on the diagnosis of AD (p > .05, data not shown). In this case-control study, we report that the COX-2 -765 C allele appears to confer protection against AD. The initial studies on the functional effects of this polymorphism revealed that the rare C allele is associated with approximately 30% less expression of COX-2 compared to the more commonly occurring G allele [19]. More recently, the C allele has been shown to be associated with reduced risk of myocardial infarction and stroke [5]. Consistent with their genetic findings, the investigators addiTable 2 Genotype frequencies of COX-2 -765 polymorphism and distribution by presence or absence of Ca COX-2 -765 genotype
Controls
AD cases
G/G G/C C/C Total
96 (59.6) 57 (35.4) 8 (5.0) 161 (100)
122 (72.6) 42 (25.0) 4 (2.4) 168 (100)
χ2 = 6.561, d.f. = 2, p = .038 COX-2 -765 C+
Controls
AD cases
C− C+ Total
96 (59.6) 65 (40.4) 161 (100.0)
122 (72.6) 46 (27.4) 168 (100.0)
χ2 = 6.207, d.f. = 1, p = .013; OR = .557, CI = [.351–.885], p = .013 a
Data are expressed as no. (%).
242
L. Abdullah et al. / Neuroscience Letters 395 (2006) 240–243
tionally report that in the patients with G/G genotypes, there is a significant increase in COX-2 staining in atherosclerotic carotid plaques. In addition, an increase in expression of COX-2 and PGE2 levels was also observed in monocytes from patients with G/G genotypes. Furthermore, among cases with a C+ genotype, significantly lower serum C reactive protein (a low grade marker of inflammation) was also reported [5]. Given that cardiovascular disease and cerebrovascular disease may be risk factors for AD, our finding of a reduced risk for AD associated with the C allele is consistent with this vascular connection [4,6,17,24,28]. In addition, given the close proximity of this polymorphism to genes previously shown to be linked to AD on chromosome 1, it is possible that we are detecting a signal from another gene in linkage disequilibrium with this one [19,29,31]. Although our analyses show adequate power, further investigations in different populations are necessary to validate the putative protective role of the C versus G allele of this polymorphism in AD. In addition, our data suggest that the reduction of risk by COX inhibitors against AD may be specific to particular genotypes of this polymorphism, analogous to the observed protection against colorectal adenoma [32]. Acknowledgements We would like to thank Robert and Diane Roskamp for providing the funding for this project. References [1] J.C. Anthony, J.C. Breitner, P.P. Zandi, M.R. Meyer, I. Jurasova, M.C. Norton, S.V. Stone, Reduced prevalence of AD in users of NSAIDs and H2 receptor antagonists: the Cache County study, Neurology 54 (2000) 2066–2071. [2] L. Bergamaschini, S. Canziani, B. Bottasso, M. Cugno, P. Braidotti, A. Agostoni, Alzheimer’s beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner, Clin. Exp. Immunol. 115 (1999) 526–533. [3] I. Bjarnason, A. Macpherson, H. Rotman, J. Schupp, J. Hayllar, A randomized, double-blind, crossover comparative endoscopy study on the gastroduodenal tolerability of a highly specific cyclooxygenase-2 inhibitor, flosulide, and naproxen, Scand. J. Gastroenterol. 32 (1997) 126–130. [4] M.M. Breteler, Vascular involvement in cognitive decline and dementia. Epidemiologic evidence from the Rotterdam Study and the Rotterdam Scan Study, Ann. N.Y. Acad. Sci. 903 (2000) 457–465. [5] F. Cipollone, E. Toniato, S. Martinotti, M. Fazia, A. Iezzi, C. Cuccurullo, B. Pini, S. Ursi, G. Vitullo, M. Averna, M. Arca, A. Montali, F. Campagna, S. Ucchino, F. Spigonardo, S. Taddei, A. Virdis, G. Ciabattoni, A. Notarbartolo, F. Cuccurullo, A. Mezzetti, Identification of New Elements of Plaque Stability (INES) Study Group, A polymorphism in the cyclooxygenase 2 gene as an inherited protective factor against myocardial infarction and stroke, JAMA 291 (2004) 2221–2228. [6] K. Czyzewski, A. Pfeffer, B. Wasiak, E. Luczywek, M. Golebiowski, M. Styczynska, M. Barcikowska, Vascular risk factors in demented elderly: analysis of Alzheimer Clinic materials, Neurol. Neurochir. Pol. 35 (2001) 405–413. [7] D.W. Dickson, J. Farlo, P. Davies, H. Crystal, P. Fuld, S.H. Yen, Alzheimer’s disease. A double-labeling immunohistochemical study of senile plaques, Am. J. Pathol. 132 (1988) 86–101. [8] R.N. Dubois, S.B. Abramson, L. Crofford, R.A. Gupta, L.S. Simon, L.B. Van De Putte, P.E. Lipsky, Cyclooxygenase in biology and disease, FASEB J. 12 (1998) 1063–1073.
[9] M. Etminan, S. Gill, A. Samii, Effect of non-steroidal anti-inflammatory drugs on risk of Alzheimer’s disease: systematic review and metaanalysis of observational studies, BMJ 327 (2003) 128. [10] S.A. Frautschy, F. Yang, M. Irrizarry, B. Hyman, T.C. Saido, K. Hsiao, G.M. Cole, Microglial response to amyloid plaques in APPsw transgenic mice, Am. J. Pathol. 152 (1998) 307–317. [11] L. Ho, C. Pieroni, D. Winger, D.P. Purohit, P.S. Aisen, G.M. Pasinetti, Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer’s disease, J. Neurosci. Res. 57 (1999) 295–303. [12] J.J. Hoozemans, A.J. Rozemuller, I. Janssen, C.J. De Groot, R. Veerhuis, P. Eikelenboom, Cyclooxygenase expression in microglia and neurons in Alzheimer’s disease and control brain, Acta Neuropathol. (Berl.) 101 (2001) 2–8. [13] B.A. Veld, L.J. Launer, M.M. Breteler, A. Hofman, B.H. Stricker, Pharmacologic agents associated with a preventive effect on Alzheimer’s disease: a review of the epidemiologic evidence, Epidemiol. Rev. 24 (2002) 248–268. [14] S. Itagaki, P.L. McGeer, H. Akiyama, S. Zhu, D. Selkoe, Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease, J. Neuroimmunol. 24 (1989) 173–182. [15] N. Iwamoto, K. Kobayashi, K. Kosaka, The formation of prostaglandins in the postmortem cerebral cortex of Alzheimer-type dementia patients, J. Neurol. 236 (1989) 80–84. [16] W.E. Kaufmann, P.F. Worley, J. Pegg, M. Bremer, P. Isakson, COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 2317–2321. [17] L.H. Kuller, P. Velentgas, J. Barzilay, N.J. Beauchamp, D.H. O’Leary, P.J. Savage, Diabetes mellitus: subclinical cardiovascular disease and risk of incident cardiovascular disease and all-cause mortality, Arterioscler. Thromb. Vasc. Biol. 20 (2000) 823–829. [18] F.L. Lanza, M.F. Rack, T.J. Simon, H. Quan, J.A. Bolognese, M.E. Hoover, F.R. Wilson, S.E. Harper, Specific inhibition of cyclooxygenase2 with MK-0966 is associated with less gastroduodenal damage than either aspirin or ibuprofen, Aliment. Pharmacol. Ther. 13 (1999) 761–767. [19] E. Levy-Lahad, E.M. Wijsman, E. Nemens, L. Anderson, K.A. Goddard, J.L. Weber, T.D. Bird, G.D. Schellenberg, A familial Alzheimer’s disease locus on chromosome 1, Science 269 (1995) 970–973. [20] F. Licastro, L. Parnetti, M.C. Morini, L.J. Davis, D. Cucinotta, A. Gaiti, U. Senin, Acute phase reactant alpha 1-antichymotrypsin is increased in cerebrospinal fluid and serum of patients with probable Alzheimer disease, Alzheimer Dis. Assoc. Disord. 9 (1995) 112–118. [21] J.L. Masferrer, B.S. Zweifel, S.M. Colburn, R.L. Ornberg, D. Salvemini, P. Isakson, K. Seibert, The Role of Cyclooxygenase-2 in Inflammation, Am. J. Ther. 2 (1995) 607–610. [22] G. McKhann, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, 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 (1984) 939–944. [23] A. Oka, S. Takashima, Induction of cyclo-oxygenase 2 in brains of patients with Down’s syndrome and dementia of Alzheimer type: specific localization in affected neurones and axons, Neuroreport 8 (1997) 1161–1164. [24] A. Ott, R.P. Stolk, F. van Harskamp, H.A. Pols, A. Hofman, M.M. Breteler, Diabetes mellitus and the risk of dementia: the Rotterdam Study, Neurology 53 (1999) 1937–1942. [25] A. Papafili, M.R. Hill, D.J. Brull, R.J. McAnulty, R.P. Marshall, S.E. Humphries, G.J. Laurent, Common promoter variant in cyclooxygenase2 represses gene expression: evidence of role in acute-phase inflammatory response, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 1631– 1636. [26] C.J. Pike, B.J. Cummings, C.W. Cotman, Early association of reactive astrocytes with senile plaques in Alzheimer’s disease, Exp. Neurol. 132 (1995) 172–179. [27] R.J. Prineas, J. Demirovic, J.A. Bean, R. Duara, O. Gomez-Marin, D. Loewenstein, S. Sevush, F. Stitt, J. Szapocznik, South Florida Pro-
L. Abdullah et al. / Neuroscience Letters 395 (2006) 240–243 gram on Aging and Health. Assessing the prevalence of Alzheimer’s disease in three ethnic groups, J. Fla. Med. Assoc. 82 (1995) 805– 810. [28] B.M. Psaty, C.D. Furberg, L.H. Kuller, N.O. Borhani, P.M. Rautaharju, D.H. O’Leary, D.E. Bild, J. Robbins, L.P. Fried, C. Reid, Isolated systolic hypertension and subclinical cardiovascular disease in the elderly. Initial findings from the Cardiovascular Health Study, JAMA 268 (1992) 1287–1291. [29] A.J. Saunders, L. Bertram, K. Mullin, A.J. Sampson, K. Latifzai, S. Basu, J. Jones, D. Kinney, L. MacKenzie-Ingano, S. Yu, M.S. Albert, T.J. Moscarillo, R.C. Go, S.S. Bassett, M.J. Daly, N.M. Laird, X. Wang, G. Velicelebi, S.L. Wagner, D.K. Becker, R.E. Tanzi, D. Blacker, Results of a high-resolution genome screen of 437 Alzheimer’s disease families, Hum. Mol. Genet. 12 (2003) 23–32. [30] J.G. Sheng, K. Ito, R.D. Skinner, R.E. Mrak, C.R. Rovnaghi, L.J. Van Eldik, W.S. Griffin, In vivo and in vitro evidence supporting a role for the inflammatory cytokine interleukin-1 as a driving force in Alzheimer pathogenesis, Neurobiol. Aging 17 (1996) 761–766.
243
[31] A. Tay, JA. Squire, H. Goldberg, K. Skorecki, Assignment of the human prostaglandin-endoperoxide synthase 2 (PTGS2) gene to 1q25 by fluorescence in situ hybridization, Genomics 23 (1994) 718–719. [32] C.M. Ulrich, J. Whitton, J.H. Yu, J. Sibert, R. Sparks, J.D. Potter, J. Bigler, PTGS2 (COX-2) -765G > C promoter variant reduces risk of colorectal adenoma among nonusers of nonsteroidal anti-inflammatory drugs, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 616–619. [33] J.R. Vane, Y.S. Bakhle, R.M. Botting, Cyclooxygenases 1 and 2, Annu. Rev. Pharmacol. Toxicol. 38 (1998) 97–120. [34] D.J. Watson, Q. Yu, J.A. Bolognese, A.S. Reicin, T.J. Simon, The upper gastrointestinal safety of rofecoxib vs. NSAIDs: an updated combined analysis, Curr. Med. Res. Opin. 20 (2004) 1539–1548. [35] P.R. Wenham, W.H. Price, G. Blandell, Apolipoprotein E genotyping by one-stage PCR, Lancet 337 (1991) 1158–1159. [36] Y. Zhang, A. Shaffer, J. Portanova, K. Seibert, P.C. Isakson, Inhibition of cyclooxygenase-2 rapidly reverses inflammatory hyperalgesia and prostaglandin E2 production, J. Pharmacol. Exp. Ther. 283 (1997) 1069–1075.
The cyclooxygenase 2 -765 C promoter allele is a protective factor for Alzheimer’s disease Laila Abdullah a,∗ , Ghania Ait-Ghezala a , Fiona Crawford a , Timothy A. Crowell a , Warren W. Barker b , Ranjan Duara b,c , Michael Mullan a b
a Roskamp Institute, 2040 Whitfield Ave. Sarasota, FL, USA Wien Center for Alzheimer’s Disease and Memory Disorders, Mt Sinai Medical Center, Miami Beach, Miami, FL, USA c Miller School of Medicine, University of Miami, Miami, FL, USA
Received 4 October 2005; received in revised form 23 October 2005; accepted 31 October 2005
Abstract The cyclooxygenase-2 enzyme (COX-2) is of particular importance in the inflammatory response and recent findings have demonstrated a considerable role in Alzheimer’s disease (AD) pathogenesis. In order to assess the possible putative role of a COX-2 polymorphism (765 G/C) in AD, we examined its distribution in 161 community-based controls and 168 AD clinic-based cases previously recruited from memory disorder clinics in Tampa and Miami, Florida. There were no significant differences between the two groups in age/age of onset or gender. A significant difference was observed in the distribution of the COX-2 -765 alleles between AD cases and controls (χ2 = 6.565, p = .010; OR = .596; CI = [.401–.888], p = .011), with the frequency of the C allele being higher in controls. In addition, a significant difference was observed for this polymorphism by genotype (χ2 = 6.561, p = .038) and by presence or absence of C+ genotypes (χ2 = 6.207, p = .013; OR = .464, CI = [.351–.885], p = .013). In this sample, the C allele of COX-2 -765 promoter polymorphism is associated with decreased risk of Alzheimer’s disease, a finding which further supports the involvement of COX-2 in AD etiology. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease cyclooxygenase 2; Inflammation
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by cognitive deficit with progressive worsening of memory, and pathologically by the presence of amyloid plaques and neurofibrillary tangles (NFTs) in the brain. A key role for inflammation in AD is supported by studies demonstrating an upregulation of prostaglandins (PGs), inflammatory cytokines, acute phase proteins, and activation of the complement cascade and further supported by the presence of microglia and astrocytes in and around the amyloid plaques [2,7,10,14,15,20,26,30]. The cyclooxygenase (COX) enzymes are the main producers of PGs from arachidonic acid [21,36]. Two distinct COX enzymes have been identified. The constitutively expressed COX-1 enzyme is mainly responsible for housekeeping functions in addition to the PG and thromboxane production in gastric mucosa [33]. By contrast, the inducible COX-2 enzyme has been
∗
Corresponding author. Tel.: +1 813 979 2008; fax: +1 813 979 2248. E-mail address: [email protected] (L. Abdullah).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.10.090
shown to be expressed mostly in central nervous system and inflammatory cells [8,16]. In addition, elevated COX-2 levels are present in neurons from hippocampal pyramidal regions of the AD brain, and correlate with amyloid plaque density and NFTs [11,12,23]. The finding that COX-2 inhibitors have less gastrointestinal toxicity than non-specific COX inhibitors has lead to their widespread use in anti-arthritic therapy [3,18,34]. Additionally, several epidemiological studies have suggested that the use of non-steroidal anti-inflammatory drugs, which include COX-2 inhibitors, may reduce the risk of developing AD [1,9,13]. The gene for COX-2 has been mapped to 1q25 which is located between 1q23 and 1q31, two regions to which genetic linkage to AD has been reported [19,29,31]. Recently, Papafili and colleagues have identified a functional polymorphism in the promoter region of the COX-2 gene; a G to C transversion at position -765 resulting in significantly lower promoter activity [25]. We have investigated this polymorphism for association with AD and find evidence that possession of the rarer “C” allele is associated with decreased risk for AD.
L. Abdullah et al. / Neuroscience Letters 395 (2006) 240–243
The study subjects were previously recruited under IRB approved studies at the University of South Florida and University of Miami. The AD sample consisted of 168 cases (57.8% female) recruited from Memory Disorder Clinics in Tampa and in Miami, Florida. The community-based control sample consisted of 161 individuals (62.9% female), recruited from a community screening project in the Dade county area of Florida. The mean age of onset of the AD cases was 73.89 (±6.954 S.D.), and the mean age of the controls at the time of the screen was 74.46 (±6.925 S.D.). All the cases and controls considered themselves Caucasians. The AD cases were genetically unrelated and diagnosed using NINCDS-ADRDA criteria for probable AD, however the pathological confirmation on these cases is not yet available [22]. The controls were defined as having MMSE > 27 and scored at or above the 16th percentile on delayed memory and semantic fluency tasks (short-term and long-term recall of items from the MMSE, recall items from a grocery list, and category fluency for animals, fruits, and vegetables) [27]. Additionally, these individuals maintained independent activities of daily living, and were free of active neurologic, or psychiatric disorders or ongoing medical conditions that would potentially interfere with their cognitive performance. DNA was extracted from whole blood using the Pure GeneTM Kit (Gentra Systems). The COX-2 -765G/C polymorphism was analyzed using PCR primers and conditions previously described by Papafili and colleagues.25 In the presence of the G allele, AciI restriction endonuclease digestion of the amplified 306 bp fragment results in 188 bp and 118 bp fragments. All subjects were also genotyped for APOE using previously described conditions [35]. The Pearson χ2 statistic was used to compare gender, HardyWeinberg equilibrium and the genotype and allele distributions between AD cases and controls for the COX-2 -765 G/C polymorphism, and APOE polymorphisms. Age of the controls at the initial cognitive testing was compared to age of onset of AD cases using a t-test for independent samples. Binary logistic regression modeling was used to calculate odds ratios (OR) and associated 95% confidence intervals (CI). To evaluate possible interactions between gender, APOE polymorphism and COX-2 polymorphism (by allele and genotype), multiple hierarchical logistic regression analyses were performed using diagnosis of AD as the dependent variable. Additionally, to determine a possible effect of the COX-2 -765 C allele on age of onset, analysis of variance was carried out for AD cases. A power analysis of these data was conducted using GPower software and revealed a power of 99.9% based on a sample of 329 individuals for a medium effect size. Alpha levels were set at 0.05 and SPSS version 13.1 was used to analyze these data. No significant difference in age/age of onset between controls and AD cases was observed (p > .05). Although there were more females in the control group compared to the cases, the difference did not reach statistical significance (p > .05). For the APOE and COX-2 -765 polymorphisms, the genotype distributions were found to be in Hardy-Weinberg equilibrium for cases, controls, and the entire sample (p > .05). The frequencies of the COX-2 -765 C and G alleles and the three genotypes in our control population (see tables) are similar to the frequencies of the
241
Table 1 Allele frequencies of COX-2 -765 polymorphisma COX-2 -765
Controls
AD cases
G allele C allele 2n
249 (77.3) 73 (22.7) 322 (100)
286 (85.1) 50 (14.9) 336 (100)
χ2 = 6.565, d.f. = 1, p = .010; OR = .596; CI = .401–.888, p = .011 a
Data are expressed as no. (%).
control population (over 70 age group) reported by Cipollone and colleagues (G allele = 72.7%, C allele 27.3%; G/G = 51.3%, G/C = 41.2%, C/C = 7.5%) [35]. As expected from many previous reports, the APOE 4 allele and 4+ genotypes were significantly increased in AD cases when compared to controls (χ2 = 29.098, p < .001). A significant difference was observed in the distribution of COX-2 -765 alleles between AD cases and controls (p < .05, Table 1), with the frequency of the C allele being higher in controls. In addition, a significant difference was observed for this polymorphism by genotype (p = .038, Table 2); given the low occurrence of the C/C genotype in cases and in controls, we conducted the analysis by presence or absence of the C allele in the genotypes. The C+ genotypes were found to be more frequent in controls when compared to AD cases (p = .013, Table 2). However, no interactions were observed between APOE and COX-2 -765 polymorphisms nor between gender and the COX-2 -765 polymorphism on diagnosis of AD (p > .05, data not shown). Furthermore, no interaction was observed between age/age of onset and this polymorphism on the diagnosis of AD (p > .05, data not shown). In this case-control study, we report that the COX-2 -765 C allele appears to confer protection against AD. The initial studies on the functional effects of this polymorphism revealed that the rare C allele is associated with approximately 30% less expression of COX-2 compared to the more commonly occurring G allele [19]. More recently, the C allele has been shown to be associated with reduced risk of myocardial infarction and stroke [5]. Consistent with their genetic findings, the investigators addiTable 2 Genotype frequencies of COX-2 -765 polymorphism and distribution by presence or absence of Ca COX-2 -765 genotype
Controls
AD cases
G/G G/C C/C Total
96 (59.6) 57 (35.4) 8 (5.0) 161 (100)
122 (72.6) 42 (25.0) 4 (2.4) 168 (100)
χ2 = 6.561, d.f. = 2, p = .038 COX-2 -765 C+
Controls
AD cases
C− C+ Total
96 (59.6) 65 (40.4) 161 (100.0)
122 (72.6) 46 (27.4) 168 (100.0)
χ2 = 6.207, d.f. = 1, p = .013; OR = .557, CI = [.351–.885], p = .013 a
Data are expressed as no. (%).
242
L. Abdullah et al. / Neuroscience Letters 395 (2006) 240–243
tionally report that in the patients with G/G genotypes, there is a significant increase in COX-2 staining in atherosclerotic carotid plaques. In addition, an increase in expression of COX-2 and PGE2 levels was also observed in monocytes from patients with G/G genotypes. Furthermore, among cases with a C+ genotype, significantly lower serum C reactive protein (a low grade marker of inflammation) was also reported [5]. Given that cardiovascular disease and cerebrovascular disease may be risk factors for AD, our finding of a reduced risk for AD associated with the C allele is consistent with this vascular connection [4,6,17,24,28]. In addition, given the close proximity of this polymorphism to genes previously shown to be linked to AD on chromosome 1, it is possible that we are detecting a signal from another gene in linkage disequilibrium with this one [19,29,31]. Although our analyses show adequate power, further investigations in different populations are necessary to validate the putative protective role of the C versus G allele of this polymorphism in AD. In addition, our data suggest that the reduction of risk by COX inhibitors against AD may be specific to particular genotypes of this polymorphism, analogous to the observed protection against colorectal adenoma [32]. Acknowledgements We would like to thank Robert and Diane Roskamp for providing the funding for this project. References [1] J.C. Anthony, J.C. Breitner, P.P. Zandi, M.R. Meyer, I. Jurasova, M.C. Norton, S.V. Stone, Reduced prevalence of AD in users of NSAIDs and H2 receptor antagonists: the Cache County study, Neurology 54 (2000) 2066–2071. [2] L. Bergamaschini, S. Canziani, B. Bottasso, M. Cugno, P. Braidotti, A. Agostoni, Alzheimer’s beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner, Clin. Exp. Immunol. 115 (1999) 526–533. [3] I. Bjarnason, A. Macpherson, H. Rotman, J. Schupp, J. Hayllar, A randomized, double-blind, crossover comparative endoscopy study on the gastroduodenal tolerability of a highly specific cyclooxygenase-2 inhibitor, flosulide, and naproxen, Scand. J. Gastroenterol. 32 (1997) 126–130. [4] M.M. Breteler, Vascular involvement in cognitive decline and dementia. Epidemiologic evidence from the Rotterdam Study and the Rotterdam Scan Study, Ann. N.Y. Acad. Sci. 903 (2000) 457–465. [5] F. Cipollone, E. Toniato, S. Martinotti, M. Fazia, A. Iezzi, C. Cuccurullo, B. Pini, S. Ursi, G. Vitullo, M. Averna, M. Arca, A. Montali, F. Campagna, S. Ucchino, F. Spigonardo, S. Taddei, A. Virdis, G. Ciabattoni, A. Notarbartolo, F. Cuccurullo, A. Mezzetti, Identification of New Elements of Plaque Stability (INES) Study Group, A polymorphism in the cyclooxygenase 2 gene as an inherited protective factor against myocardial infarction and stroke, JAMA 291 (2004) 2221–2228. [6] K. Czyzewski, A. Pfeffer, B. Wasiak, E. Luczywek, M. Golebiowski, M. Styczynska, M. Barcikowska, Vascular risk factors in demented elderly: analysis of Alzheimer Clinic materials, Neurol. Neurochir. Pol. 35 (2001) 405–413. [7] D.W. Dickson, J. Farlo, P. Davies, H. Crystal, P. Fuld, S.H. Yen, Alzheimer’s disease. A double-labeling immunohistochemical study of senile plaques, Am. J. Pathol. 132 (1988) 86–101. [8] R.N. Dubois, S.B. Abramson, L. Crofford, R.A. Gupta, L.S. Simon, L.B. Van De Putte, P.E. Lipsky, Cyclooxygenase in biology and disease, FASEB J. 12 (1998) 1063–1073.
[9] M. Etminan, S. Gill, A. Samii, Effect of non-steroidal anti-inflammatory drugs on risk of Alzheimer’s disease: systematic review and metaanalysis of observational studies, BMJ 327 (2003) 128. [10] S.A. Frautschy, F. Yang, M. Irrizarry, B. Hyman, T.C. Saido, K. Hsiao, G.M. Cole, Microglial response to amyloid plaques in APPsw transgenic mice, Am. J. Pathol. 152 (1998) 307–317. [11] L. Ho, C. Pieroni, D. Winger, D.P. Purohit, P.S. Aisen, G.M. Pasinetti, Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer’s disease, J. Neurosci. Res. 57 (1999) 295–303. [12] J.J. Hoozemans, A.J. Rozemuller, I. Janssen, C.J. De Groot, R. Veerhuis, P. Eikelenboom, Cyclooxygenase expression in microglia and neurons in Alzheimer’s disease and control brain, Acta Neuropathol. (Berl.) 101 (2001) 2–8. [13] B.A. Veld, L.J. Launer, M.M. Breteler, A. Hofman, B.H. Stricker, Pharmacologic agents associated with a preventive effect on Alzheimer’s disease: a review of the epidemiologic evidence, Epidemiol. Rev. 24 (2002) 248–268. [14] S. Itagaki, P.L. McGeer, H. Akiyama, S. Zhu, D. Selkoe, Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease, J. Neuroimmunol. 24 (1989) 173–182. [15] N. Iwamoto, K. Kobayashi, K. Kosaka, The formation of prostaglandins in the postmortem cerebral cortex of Alzheimer-type dementia patients, J. Neurol. 236 (1989) 80–84. [16] W.E. Kaufmann, P.F. Worley, J. Pegg, M. Bremer, P. Isakson, COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 2317–2321. [17] L.H. Kuller, P. Velentgas, J. Barzilay, N.J. Beauchamp, D.H. O’Leary, P.J. Savage, Diabetes mellitus: subclinical cardiovascular disease and risk of incident cardiovascular disease and all-cause mortality, Arterioscler. Thromb. Vasc. Biol. 20 (2000) 823–829. [18] F.L. Lanza, M.F. Rack, T.J. Simon, H. Quan, J.A. Bolognese, M.E. Hoover, F.R. Wilson, S.E. Harper, Specific inhibition of cyclooxygenase2 with MK-0966 is associated with less gastroduodenal damage than either aspirin or ibuprofen, Aliment. Pharmacol. Ther. 13 (1999) 761–767. [19] E. Levy-Lahad, E.M. Wijsman, E. Nemens, L. Anderson, K.A. Goddard, J.L. Weber, T.D. Bird, G.D. Schellenberg, A familial Alzheimer’s disease locus on chromosome 1, Science 269 (1995) 970–973. [20] F. Licastro, L. Parnetti, M.C. Morini, L.J. Davis, D. Cucinotta, A. Gaiti, U. Senin, Acute phase reactant alpha 1-antichymotrypsin is increased in cerebrospinal fluid and serum of patients with probable Alzheimer disease, Alzheimer Dis. Assoc. Disord. 9 (1995) 112–118. [21] J.L. Masferrer, B.S. Zweifel, S.M. Colburn, R.L. Ornberg, D. Salvemini, P. Isakson, K. Seibert, The Role of Cyclooxygenase-2 in Inflammation, Am. J. Ther. 2 (1995) 607–610. [22] G. McKhann, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, 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 (1984) 939–944. [23] A. Oka, S. Takashima, Induction of cyclo-oxygenase 2 in brains of patients with Down’s syndrome and dementia of Alzheimer type: specific localization in affected neurones and axons, Neuroreport 8 (1997) 1161–1164. [24] A. Ott, R.P. Stolk, F. van Harskamp, H.A. Pols, A. Hofman, M.M. Breteler, Diabetes mellitus and the risk of dementia: the Rotterdam Study, Neurology 53 (1999) 1937–1942. [25] A. Papafili, M.R. Hill, D.J. Brull, R.J. McAnulty, R.P. Marshall, S.E. Humphries, G.J. Laurent, Common promoter variant in cyclooxygenase2 represses gene expression: evidence of role in acute-phase inflammatory response, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 1631– 1636. [26] C.J. Pike, B.J. Cummings, C.W. Cotman, Early association of reactive astrocytes with senile plaques in Alzheimer’s disease, Exp. Neurol. 132 (1995) 172–179. [27] R.J. Prineas, J. Demirovic, J.A. Bean, R. Duara, O. Gomez-Marin, D. Loewenstein, S. Sevush, F. Stitt, J. Szapocznik, South Florida Pro-
L. Abdullah et al. / Neuroscience Letters 395 (2006) 240–243 gram on Aging and Health. Assessing the prevalence of Alzheimer’s disease in three ethnic groups, J. Fla. Med. Assoc. 82 (1995) 805– 810. [28] B.M. Psaty, C.D. Furberg, L.H. Kuller, N.O. Borhani, P.M. Rautaharju, D.H. O’Leary, D.E. Bild, J. Robbins, L.P. Fried, C. Reid, Isolated systolic hypertension and subclinical cardiovascular disease in the elderly. Initial findings from the Cardiovascular Health Study, JAMA 268 (1992) 1287–1291. [29] A.J. Saunders, L. Bertram, K. Mullin, A.J. Sampson, K. Latifzai, S. Basu, J. Jones, D. Kinney, L. MacKenzie-Ingano, S. Yu, M.S. Albert, T.J. Moscarillo, R.C. Go, S.S. Bassett, M.J. Daly, N.M. Laird, X. Wang, G. Velicelebi, S.L. Wagner, D.K. Becker, R.E. Tanzi, D. Blacker, Results of a high-resolution genome screen of 437 Alzheimer’s disease families, Hum. Mol. Genet. 12 (2003) 23–32. [30] J.G. Sheng, K. Ito, R.D. Skinner, R.E. Mrak, C.R. Rovnaghi, L.J. Van Eldik, W.S. Griffin, In vivo and in vitro evidence supporting a role for the inflammatory cytokine interleukin-1 as a driving force in Alzheimer pathogenesis, Neurobiol. Aging 17 (1996) 761–766.
243
[31] A. Tay, JA. Squire, H. Goldberg, K. Skorecki, Assignment of the human prostaglandin-endoperoxide synthase 2 (PTGS2) gene to 1q25 by fluorescence in situ hybridization, Genomics 23 (1994) 718–719. [32] C.M. Ulrich, J. Whitton, J.H. Yu, J. Sibert, R. Sparks, J.D. Potter, J. Bigler, PTGS2 (COX-2) -765G > C promoter variant reduces risk of colorectal adenoma among nonusers of nonsteroidal anti-inflammatory drugs, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 616–619. [33] J.R. Vane, Y.S. Bakhle, R.M. Botting, Cyclooxygenases 1 and 2, Annu. Rev. Pharmacol. Toxicol. 38 (1998) 97–120. [34] D.J. Watson, Q. Yu, J.A. Bolognese, A.S. Reicin, T.J. Simon, The upper gastrointestinal safety of rofecoxib vs. NSAIDs: an updated combined analysis, Curr. Med. Res. Opin. 20 (2004) 1539–1548. [35] P.R. Wenham, W.H. Price, G. Blandell, Apolipoprotein E genotyping by one-stage PCR, Lancet 337 (1991) 1158–1159. [36] Y. Zhang, A. Shaffer, J. Portanova, K. Seibert, P.C. Isakson, Inhibition of cyclooxygenase-2 rapidly reverses inflammatory hyperalgesia and prostaglandin E2 production, J. Pharmacol. Exp. Ther. 283 (1997) 1069–1075.