American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:367 –369 (2007) Brief Research Communication Cyclooxygenase-2 Polymorphisms in Parkinson’s Disease Anna Håkansson,1* Olle Bergman,1 Cecilia Chrapkowska,1 Lars Westberg,1 Andrea Carmine Belin,3 Olof Sydow,4 Bo Johnels,2 Lars Olson,3 Björn Holmberg,2 and Hans Nissbrandt1 1 Department of Pharmacology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden 2 Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Göteborg University, Stockholm, Sweden 3 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 4 Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden Accumulating evidence indicate that cyclooxygenase-2 (COX-2) is of pathophysiological importance for the neurodegeneration in Parkinson’s disease (PD). For example, in a large epidemiological study, use of NSAIDs was associated with a lower risk of PD. Genetic variants of the COX-2 gene might therefore influence the risk of developing the disease. The genotype distribution of four common single nucleotide polymorphisms (SNPs) in the COX-2 gene (rs689466:A496G, rs20417:G926C, rs5277:G3050C, rs5275:C8473T) was analyzed in PD patients and control subjects in a Swedish population. No differences could be seen between the PD-patient and controls regarding the A496G, G926C, and G3050C SNPs, but the allele frequency of the C8473T SNP was found to differ when male patients were compared to controls (P ¼ 0.007). In females no difference could be seen between PD-patients and controls. In conclusion, the results suggest a possible influence of the COX-2 C8473T SNP in PD, although it only seems to be of importance in men. ß 2006 Wiley-Liss, Inc. KEY WORDS: neurodegeneration; inflammation; COX-2 gene Please cite this article as follows: Håkansson A, Bergman O, Chrapkowska C, Westberg L, Belin AC, Sydow O, Johnels B, Olson L, Holmberg B, Nissbrandt H. 2007. Cyclooxygenase-2 Polymorphisms in Parkinson’s Disease. Am J Med Genet Part B 144B:367–369. Cyclooxygenase (COX) is a key enzyme in the production of prostaglandins, prostacyclins, and thromboxanes. The enzyme exists as constitutive and inducible isoforms; COX-1 and COX-2. The latter is the inducible form and is expressed in response to growth factors, cytokines and other pro-inflammatory molecules. However, in the central nervous system COX-2 is also expressed under normal conditions and contributes to many fundamental brain functions [Minghetti, 2004]. Accumulating evidence indicate that COX-2 is of pathophysiological importance for the neurodegeneration in Parkinson’s disease (PD). COX-2 immunoreactivity could be seen in neuromelanized neurons in the substantia nigra in midbrain sections from PD patients but not in controls [Teismann et al., 2003]. Furthermore, arachidonic acid-stimulated synthesis of the prostaglandin PGE2 has been shown to be higher in postmortem samples of brain tissue from patients with PD than in brain tissue from control subjects [Mattammal et al., 1995]. It has also been shown that substantia nigra neurons in COX-2 gene knockout mice are less vulnerable to MPTP toxicity compared to wild-type mice [Feng et al., 2002] and that JNKmediated induction of cyclooxygenase-2 is required for neurodegeneration in the MPTP-model [Hunot et al., 2004]. COX is the main target of non-steroidal anti-inflammatory drugs (NSAIDs). In a large epidemiological study, use of NSAIDs was associated with a lower risk of PD [Chen et al., 2003]. In the follow-up study, however, an association was only seen for the NSAID ibuprofen [Chen et al., 2005]. In addition, pretreatment of mice or rat with NSAIDs preferentially acting on COX-2 inhibits the deleterious effect of MPTP or 6-OHDA on dopaminergic neurons [Teismann and Ferger, 2001; SanchezPernaute et al., 2004]. Several single nucleotide polymorphisms (SNPs) have been identified in the COX-2 gene, which is located on human chromosome 1. In a previous study an association was found between a SNP (G926C) in the COX-2 gene and Alzheimer’s disease [Abdullah et al., 2006]. The aim of the present study was to investigate the possible association between four common COX-2 SNPs and PD. METHODS Subjects Grant sponsor: The Swedish Research Council; Grant sponsor: Åhlén’s Foundation; Grant sponsor: The Swedish Parkinson Foundation; Grant sponsor: The Swedish Brain Power Initiative; Grant sponsor: The Swedish Brain Foundation; Grant sponsor: Hållstens Forskningsstiftelse; Grant sponsor: Björn Oscarssons stiftelse; Grant sponsor: Karolinska Institutet Funds. *Correspondence to: Anna Håkansson, M.Sc., Department of Pharmacology, Sahlgrenska Academy at Göteborg University, P.O. Box 431, S 405 30 Göteborg, Sweden. E-mail: firstname.lastname@example.org Received 27 March 2006; Accepted 31 August 2006 DOI 10.1002/ajmg.b.30449 ß 2006 Wiley-Liss, Inc. Patients with PD (n ¼ 278) were recruited at Sahlgrenska Hospital, Göteborg (n ¼ 105, mean age 68 years, mean age of onset 59 years) and Karolinska University Hospital, Stockholm (n ¼ 173, mean age 68 years, mean age of onset 60 years). The patients fulfilled the PDS Brain Bank criteria for idiopathic PD [Daniel and Lees, 1993], except that presence of more than one relative with PD was not an exclusion criterion. Control subjects (n ¼ 313) were aged matched volunteers from Göteborg (n ¼ 171, mean age 69 years) and healthy volunteers from Stockholm (n ¼ 142, mean age 42 years). Among the patients 108 were females and 170 were males, whereas the control population consisted of 163 females, 121 males, and 368 Håkansson et al. 29 individuals with unknown gender (all from Stockholm). In more than 95% of patients and controls both parents were of Caucasian origin. All subjects included in the study had provided informed consent, and the study was approved by the ethical committees at Göteborg University and Karolinska Institutet. Molecular Methods In this study the allele frequencies of four different SNPs (rs689466:A496G, rs20417:G926C, rs5277:G3050C, rs5275: C8473T) in the gene encoding COX-2 were investigated (the nomenclature of the SNPs refers to their position within GenBank accession no. D28235). The DNA concentration in the individual patient and control samples was estimated by fluorimetry (SPECTRAmax GEMINI fluorescence microplate reader, Molecular Devices, Sunnyvale, CA) and a quantitation reagent (PicoGreen dsDNA, Molecular Probes, Leiden, The Netherlands). The samples were diluted to 5 ng/ml, requantified and adjusted if necessary. Finally, equal amounts of each sample were pooled into three DNA pools, one consisting of 161 male PD-patients, one of 104 female PDpatients and one of 169 control individuals. (During the construction of the DNA pools some of the samples had to be excluded because of too low DNA-concentration.) DNA was amplified by PCR and allele quantification of the pooled samples and genotyping of individual samples were performed using pyrosequencing (PSQ 96MA and the PSQ 96 SNP Reagent Kit, Pyrosequencing AB, Uppsala, Sweden). A total of 20 ml of PCR product was used for pyrosequencing in accordance with the manufacturer’s instructions. Because genotyping was not successful in a few subjects, there are differences between the total number of included individuals and the number of individuals genotyped. Statistics Fisher’s exact test and the Chi-squared test were performed for comparing allele and genotype frequencies, respectively. Odds ratios and their 95% confidence intervals were calculated for the comparisons. Hardy–Weinberg equilibrium was evaluated for the genotyped polymorphisms using a Chi-square goodness-of-fit test. RESULTS For three of the SNPs (A496G, G3050C, C8473T) the allele frequency was first investigated by allele quantification in the three DNA pools. No differences could be seen between the PD-patient pools and control pool regarding the A496G SNP (minor allele frequency [MAF] data from allele quantification: controls: G ¼ 22%, male patients: G ¼ 19%, female patients: G ¼ 20%) or the G3050C SNP (controls: G ¼ 26%, male patients: G ¼ 25%, female patients: G ¼ 25%), but the allele frequency of the C8473T SNP was found to differ when the pool with male patients was compared to the control pool. To obtain precise allele frequencies, all patient samples were genotyped for the C8437T SNP (see Table I). In this step 142 additional controls were included. The allele frequency was found to be different between the group of all PD-patients and the controls (P ¼ 0.01). However, when the patient group was divided into men and women a difference in allele frequency could only be detected between the male PD-patients and the controls (P ¼ 0.007) (because there were no difference in allele frequency between males and females in the control group, frequencies between all controls and the female and the male group were compared). The fourth SNP (G926C) could not be investigated by allele quantification in the DNA pools as a first screen, because its position in the DNA-chain made it impossible to create a reliable assay for allele quantification. Instead we directly genotyped 105 patients and 171 controls, but were not able to find any significant differences in allele or genotype frequencies (controls: GG ¼ 79%, GC ¼ 20%, CC ¼ 1%; PD-patients: GG ¼ 70%, GC ¼ 29%, CC ¼ 1%). The genotype distribution of the C8437T and the G926C SNPs was found to be in Hardy–Weinberg equilibrium in the investigated populations. DISCUSSION The C8473T SNP in exon 10 of the COX-2 gene was found to be related to PD in our Swedish population. However, the difference in allele frequency could only be detected between men with PD and control subjects. The C8473T SNP is located in the 30 -UTR region of the gene at nt427 downstream from the stop codon and this locus is within a functional region which could alter gene expression through both message stability and translational efficiency in vitro [Cok and Morrison, 2001]. The region is characterized by multiple repeats of AU-rich elements, which are also found in several other genes encoding inflammatory mediators (cytokines and protooncogenes), whose mRNA is very unstable [Caput et al., 1986]. Several studies have reported an association between the C8473T SNP TABLE I. Distributions of Genotype and Allele Frequencies of the C8473T SNP in the COX-2 Gene Genotype CC Controls PD-patients (all) P* OR* (95% CI) 25 (8.1%) 37 (13.7%) 0.03 CC vs. any T 1,80 (1.05–3.07) CT 135 (43.8%) 126 (46.7%) TT 148 (48.1%) 107 (39.6%) Allele C 185 (30.0%) 200 (37.0%) T 431 (70.0%) 340 (63.0%) PD-patients (male) P** 22 (13.4%) 0.02 OR** (95% CI) CC vs. any T 1.75 (0.96–3.22) 83 (50.6%) TT vs. any C 1.41 (1.01–1.96) 0.01 C vs. T 1.37 (1.07–1.75) 59 (36.0%) 127 (38.7%) 0.007 201 (61.3%) PD-patients (female) P*** 16 (10.1%) 0.6 OR*** (95% CI) CC vs. any T 1.27 (0.66–2.45) 73 (45.9%) TT vs. any C 1.65 (1.12–2.43) 70 (44.0%) C vs. T 1.47 (1.11–1.95) 73 (34.4%) TT vs. any C 1.18 (0.80–1.73) 0.3 C vs. T 1.22 (0.88–1.71) 139 (65.6%) *P and *OR: P-values and odds ratios obtained from comparisons of allele and genotype distributions between all patients with PD and controls. **P and **OR: P-values and odds ratios obtained from comparisons of allele and genotype distributions between male patients with PD and controls. ***P and ***OR: P-values and odds ratios obtained from comparisons of allele and genotype distributions between female patients with PD and controls. COX-2 and Parkinson’s Disease and disease, further supporting that the variation might be of functional importance, although the results vary regarding which allele that is ‘‘disease-causing’’ and ‘‘protective’’ [Campa et al., 2004; Cox et al., 2004; Hu et al., 2005; Sorensen et al., 2005; Langsenlehner et al., 2006; Park et al., 2006]. One of the most frequently investigated variations in the COX-2 gene is the G926C SNP in the promoter region. This variant has been implicated in transcription alteration of the gene and an increase in the levels of the C-reactive protein [Papafili et al., 2002]. In the present study, neither the G926C SNP nor the other investigated COX-2 promoter SNP, A496G, was found to be associated with PD. The G3050C SNP in exon 3 was also studied in the present work, but no association with PD could be seen. In most studies PD is more common among men than women [Bower et al., 1999; Baldereschi et al., 2000]. Neuroprotective effects exerted by estrogen have been suggested to be of importance for this difference in prevalence [Westberg et al., 2004]. Previous data indicate that the gene encoding the inflammatory mediator interleukin-6 might mediate some of the neuroprotective effects of estrogen [Hakansson et al., 2005]. Interestingly, estrogens were recently reported to reduce lipopolysaccharide-stimulated expression of COX-2 in microglial cells [Vegeto et al., 2001; Baker et al., 2004], pointing to a putative reason for a gender specific association. Considering this finding, a COX-2 gene polymorphism association with PD in women and but not in men would be anticipated rather than the opposite. However, one should be aware of that estrogen is produced, and is of physiological importance, also in men. 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