Movement Disorders Vol. 16, No. 4, 2001, pp. 705–707 © 2001 Movement Disorder Society Published by Wiley-Liss, Inc.
Brief Report
Genetic Polymorphisms of Superoxide Dismutase in Parkinson’s Disease Federico M. Farin, MD,1 Yolanda Hitosis, BS,1 Sarah E. Hallagan, BS,1 John Kushleika, MS,1 James S. Woods, PhD,1 Patricia S. Janssen, MS,1 Terri Smith-Weller, MN,1 Gary M. Franklin, MD, MPH,1,2 Phillip D. Swanson, MD, PhD,2 and Harvey Checkoway, PhD1* 1
Department of Environmental Health, University of Washington, Seattle, Washington, USA 2 Department of Neurology, University of Washington, Seattle, Washington, USA
Abstract: Oxidative stress reactions may contribute to the pathogenesis of Parkinson’s disease (PD). The superoxide dismutases potentially play significant roles in PD by detoxifying superoxide radical. We developed genomic DNA and cDNAbased sequencing assays to identify genetic variants in the copper/zinc superoxide dismutase (SOD1) and manganese superoxide dismutase (SOD2) genes. No genetic variants were detected in the gene encoding SOD1 in DNA from 45 idiopathic PD cases and 49 controls from a population-based case-control study. However, we identified a previously described polymor-
phism of the mitochondrial targeting sequence consisting of a C47T in exon 2 of SOD2, which results in an alanine to valine substitution. We analyzed this SOD2 variant in DNA from 155 cases and 231 controls from the same study, using an allelespecific fluorogenic 5⬘ nuclease assay, and found no differences in the distributions of allelic frequencies. These results indicate that SOD gene variants do not contribute to PD pathogenesis. © 2001 Movement Disorder Society. Key words: Parkinson’s disease; superoxide dismutase; genetic polymorphisms; oxidative stress
Damage to dopaminergic neurons in the substantia nigra by reactive oxygen species, such as superoxide free radicals, may contribute significantly to Parkinson’s disease (PD) pathogenesis.1 A consistently observed manifestation of oxidative stress in PD is reduced mitochondrial Complex I activity.2,3 Superoxide dismutases (SOD) are metalloenzymes that detoxify superoxide radicals, and are expressed in multiple tissues, including brain. The two main isoenzymes of human SOD are cytoplasmic copper/zinc-dependent (SOD1)4 and mitochondrial manganese-dependent (SOD2). 5,6 SOD2, which is the principal defense against reactive oxygen species in mitochondria, may be especially relevant in PD.
The evidence for SOD2 genetic polymorphisms in PD is limited. The mitochondrial targeting sequence (MTS) of the SOD2 gene is essential for its effective transport. A study in Japan detected a significant allelic difference in PD cases compared with controls (12.1% vs. 19.3%) for a T to C substitution in exon 2 of the SOD2 MTS.7 This polymorphism (C47T), which results in a valine to alanine change, was subsequently related to elevated SOD activity in familial PD.8 However, no associations with SOD2 gene variants were detected in two European studies.9,10 Point mutations in the SOD1 gene have been identified in some patients with familial amyotrophic lateral sclerosis.11,12 To date, familial PD has not been associated with SOD1 mutations.13 In the present study, we compared the genomic sequence of all exons and exon-intron boundaries in the SOD1 and SOD2 genes between idiopathic, nonfamilial PD cases and controls. Furthermore, we investigated the association of PD with the previously described polymorphism in the MTS in exon 2 of SOD2.7
*Correspondence to: Harvey Checkoway, PhD, University of Washington, Department of Environmental Health, Box 357234, Seattle, WA 98195-7234. E-mail:
[email protected] Received 2 May 2000; Revised 19 October 2000; Accepted 20 October 2000 Published online 16 July 2001; DOI 10.1002/mds.1153
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F.M. FARIN ET AL. METHODS
Study Subjects Newly diagnosed idiopathic, unrelated PD cases were identified during 1992–1998 from neurology and general medical practice clinics of Group Health Cooperative (GHC) in western Washington state and the University of Washington Neurology Clinic. Chart reviews for cases not referred by neurologists were reviewed by two of us (G.M.F. and P.D.S.) to verify PD diagnoses, indicated by the presence of at least two of the four cardinal signs of PD: bradykinesia, resting tremor, cogwheel rigidity, and postural reflex impairment. Controls were GHC enrollees without past histories of PD or other progressive neurologic disorders, as determined from chart reviews and subject interviews. The control group was frequency-matched to cases by age in 10-year categories, gender, and original year of GHC enrollment. Study subjects donated two 10-ml tubes of collected venous blood. Genomic DNA was extracted from buffy coat by standard methods. For initial analyses of SOD1 and SOD2 cDNA- and genomic DNA-based sequencing, we identified a sample of 45 cases and 49 controls. After identifying a sufficiently large allellic frequency of the SOD2 mitochondrial target sequence polymorphism, we expanded genotyping analysis to 155 cases and 231 controls to permit a meaningful data analysis. The ultimate case group included 93 men and 62 women, aged 37–88 years (mean 69), and controls included 148 men and 83 women, aged 40–84 years (mean 69). All study subjects were non-Hispanic Caucasians. Study forms and procedures were approved by the Institutional Review Board committees on Human Subjects Research at the University of Washington and the GHC Center for Health Studies. Genotyping SOD1 and SOD2 PCR-based assays Polymerase chain reaction (PCR)-based amplification of the 5 exon regions and corresponding intron-exon boundaries of the human SOD1 gene was performed utilizing previously published specific forward primers (FPs) and reverse primers (RPs).14 Similarly, we conducted PCR amplification of the 5 exons and associated intron/exon boundaries of SOD2, based on published FPs and RPs.15 SOD2 C47T allelic discrimination A TaqMan™-based allelic discrimination assay was developed to identify the C47T polymorphism of the SOD2 gene using an ABI7700 Sequence Detection System (PE Biosystems, Foster City, CA). Genomic DNA
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was amplified with oligonucleotide primers 5⬘-GGC TGT GCT TTC TCG TCT TCA-3⬘ (FP) and 5⬘-TCG GTG ACG TTC AGG TTG TTC-3⬘ (RP). Fluorescently labeled oligonucleotide probes, containing a 5⬘-reporter and a 3⬘-quencher dye, were designed: 5⬘-6FAM-CTG GCT CCG GCT TTG GGG TAT C-TAMRA-3⬘ (wild type) and 5⬘-VIC-CAG ATA CCC CAA AAC CGG AGC CAG-TAMRA-3⬘ (mutant). Data Analysis Hardy-Weinberg equilibrium of genotypes was tested by Chi-square analysis. Associations between SOD genotype and PD were estimated as odds ratios (OR) and 95% confidence intervals (95% CI). Analyses were performed using the SPSS-7.5 statistical package (Chicago, IL). RESULTS SOD1 Analyses No variants were detected in either exons or intron/exon boundaries of SOD1 of the 45 case and 49 control samples tested. SOD2 Analyses The only gene variant detected by the cDNA- and genomic DNA-based sequencing assays, and the TaqMan™-based allelic discrimination assay was the C47T polymorphism of the SOD2 gene. Genotypes were in Hardy-Weinberg equilibrium among 155 cases (Chisquare ⳱ 0.002, P > 0.99) and 231 controls (Chi-square ⳱1.60, P ⳱ 0.46). Comparative results for the cases and controls are summarized in Table 1. The corresponding odds ratios (95%confidence intervals) for the CT and CC genotypes were, respectively, 0.90 (0.54–1.50) and 1.14 (0.63–2.05). There was no association for carrying the T variant allele, either CT or TT (odds ratio 0.97, 95% confidence interval 0.60–1.58). DISCUSSION Human SOD2 is a homotetrameric enzyme that protects mitochondria against oxygen-mediated free radical TABLE 1. Allele frequencies of the C47T polymorphism of the mitochondrial targeting sequence of the SOD2 gene in Parkinson’s disease cases and controls Cases
Controls
Genotype
No.
%
No.
%
Odds ratio
95% CI
CC CT TT CT or TT
35 77 43 120
22.6 49.7 27.7 77.4
51 125 55 180
22.1 54.1 23.8 77.9
1.00 0.90 1.14 0.97
— 0.54–1.50 0.63–2.05 0.60–1.58
SOD2, manganese superoxide dismutase. CI, confidence interval.
GENETIC POLYMORPHISMS OF SOD IN PARKINSON’S DISEASE damage. We did not detect the exon 3 T4997C point mutation in the SOD2 gene that encodes for a replacement of the hydrophobic Ile58 with the hydrophilic Thr58. The change resulting from this variant, which has been found in two of six cDNA libraries of cells,15 destablizes the four-helix bundle, leading to decreased enzyme activity and reduced tetramer stability.16,17 We did find the C47T variant in exon 2 of SOD2 in our case and control samples. Contrary to a study in Japan,8 but consistent with studies of Caucasians in Europe,9,10 we did not observe an association of the C47T polymorphism with PD. Discrepant findings among studies may be explained by ethnic differences in allelic frequencies between Asian and Caucasian populations. Population stratification within studies is also a complicating feature of association studies; for example, we were only able to characterize our study sample broadly as non-Hispanic Caucasians. We found no SOD1 variants in samples from either cases or controls. Sequencing of a larger number of samples might detect some mutant forms, but it seems unlikely that a strong relation with PD would be discernible, given the absence of prior evidence for SOD1 polymorphisms.13 It should be appreciated that the literature on SOD polymorphisms in PD has been limited by relatively small sample sizes: 63 and 107 cases in the two European studies,9,10 83 cases in the Japanese study,7 and 155 cases in the present study. A considerably larger sample size, approximately 500 cases and 500 controls, would be needed to reach a reliable statistical conclusion of no association for a polymorphism that occurs with a frequency ranging from 10% to 25% in the normal population. An oxidative stress model of PD pathogenesis provides plausible theoretical support for relations between SOD gene variants and risk of idiopathic PD. Nevertheless, our findings indicate no causal links. Acknowledgments: This research was supported by National Institute of Environmental Health Sciences grants ES04696 and ES0733. The authors are grateful to the Group Health Cooperative and University of Washington neurologists for referring cases. Particular thanks are owed to Drs. Robert Gotshall, Ann Hunt, Eric Kraus, Richard Mesher, Steve Pugh, Bruce Ranson, Ali Samii, Timothy Scearce, Kurt Seifert, Ken Uchini, and Thurman Wheeler. We also appreciate Mr. Alex Cohen for data analysis, Dr. Paola Costa-Mallen for helpful
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comments on an earlier draft, and Ms. Michael Montemurro for manuscript preparation.
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