Neuroscience Letters 375 (2005) 112–116
Australian data and meta-analysis lend support for alpha-synuclein (NACP-Rep1) as a risk factor for Parkinson’s disease George D. Mellicka,∗ , Demetrius M. Maraganoreb , Peter A. Silburna a
Department of Neurology, School of Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Australia b Department of Neurology, Mayo Clinic, Rochester, MN, USA Received 30 July 2004; received in revised form 23 August 2004; accepted 27 October 2004
Abstract It remains unclear whether genetic variants in SNCA (the alpha-synuclein gene) alter risk for sporadic Parkinson’s disease (PD). The polymorphic mixed sequence repeat (NACP-Rep1) in the promoter region of SNCA has been previously examined as a potential susceptibility factor for PD with conflicting results. We report genotype and allele distributions at this locus from 369 PD cases and 370 control subjects of European Australian ancestry, with alleles designated as −1, 0, +1, +2, and +3 as previously described. Allele frequencies designated (0) were less common in Australian cases compared to controls (OR = 0.80, 95% CI 0.62–1.03). Combined analysis including all previously published ancestral European Rep1 data yielded a highly significant association between the 0 allele and a reduced risk for PD (OR = 0.79, 95% CI 0.70–0.89, p = 0.0001). Further study must now proceed to examine in detail this interesting and biologically plausible genetic association. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Alpha-synuclein; Parkinson’s disease; Meta-analysis
The role for alpha-synuclein in the pathogenesis of Parkinson’s disease (PD) is now well established. However, it remains unclear whether genetic variants in SNCA (the alpha-synuclein gene) alter risk for sporadic PD. A polymorphic mixed sequence micro-satellite repeat (designated NACP-Rep1), approximately 10 kb upstream from the SNCA transcription start site, exhibits 5 common allele sizes in human populations. The locus consists of a (TC)x (T)2 (TC)y (TA)2 (CA)w motif, with demonstrated sizerelated expression differences . The most common allele in individuals of European ancestry was designated (+1) in the original article by Xia et al. , with alternative allele designations representing relative micro-satellite sizes; the (0) allele is two base-pairs shorter and the (+2) and (+3) are two and four base-pairs longer than the (+1) allele, respectively. Using this convention, the five alleles observed in human populations are −1, 0, +1, +2 and +3. While the size variability results largely from differences in the (CA)w portion ∗
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of the variable sequence, Farrer and colleagues showed that differences in the (TC)y (TA)2 region also exist . Recent studies demonstrate that alpha-synuclein expression in-vitro relates to the size, rather than the sequence of the Rep1 region . The (+1) allele exhibits a three-fold greater expression relative to the (0) allele, while the (+2) and (+3) alleles show intermediate expressions (1.5- and 2.5-fold increases relative to the (0) allele, respectively) . Six previous studies have reported Rep1-PD association data in sample populations of European ancestry [4,7,9,13,16,18]. Here, we report Rep1 allele frequencies in 369 PD cases and 370 control subjects of European Australian ancestry. The details of our Australian case-control cohort have been published [3,10,12]. Prevalence PD cases were recruited from the Movement Disorders clinic at Princess Alexandra Hospital and the private neurology clinic of one of the authors (PAS). The diagnosis of probable or definite PD was made when the subject had a combination of three of the following features: resting tremor, rigidity, bradykinesia, postural instability; or two of these features with asymmetry in
G.D. Mellick et al. / Neuroscience Letters 375 (2005) 112–116
tremor, rigidity or bradykinesia. Subjects were excluded if they showed no response to l-dopa therapy, if there were features consistent with another akinetic rigid syndrome, if cognitive decline was an early feature in the presentation, or if they were unable to complete a structured questionnaire either independently or with assistance. Controls consisted of 156 patient spouses and 214 unrelated volunteers from patient neighbourhoods and community organisations. All subjects were examined by a Movement Disorders Neurologist. Data pertaining to ethnic background, family history of PD (first-degree relatives with PD) and age at onset of PD symptoms were obtained from study subjects via face-to-face interviews using a structured questionnaire. Rep1 genotyping and allele designations followed those previously described . Briefly, genomic DNA was extracted from blood leukocytes and the appropriate target amplified using the following primer sequences:
ticles and combined with the Australian data in a preliminary combined analysis. Genotype and allele distributions between cases and controls were compared using standard χ2 statistics, univariate odds ratios (ORs) and logistic regression analyses. A forest plot and Breslow-Day test was used to assess heterogeneity in the OR estimates derived from the various studies. A funnel plot was performed to graphically probe for the presence of publication bias. The Rep1 genotype and allele distributions observed in Australian cases and controls are presented in Table 1. Identical genotype and allele frequencies (all within 1%) were obtained when our sample was restricted to (1) subjects with entirely British ancestry (2) PD cases with onset age >45y or (3) PD cases without a first degree relative with PD. Genotype and allele frequencies did not differ between the spouse and volunteer control groups (p-values >0.1). For both control groups the frequency of genotypes carrying the (0) allele, and (0) allele frequencies themselves, were less than that seen in PD cases (although none of these comparisons achieved statistical significance). Overall, the allele frequencies seen in our Australian controls is very similar to that seen in the combined data for controls from all other studies (0 allele frequencies of 26.5 and 27.8%, respectively). We observed an under representation of the 0 allele in PD cases (OR = 0.895%, CI = 0.62–1.03). This is similar to all previous studies [4,7,8,13,16,18]; although due to sample size considerations in most studies, statistically significant differences failed to be detected. Logistic regression analysis
[Hex]5 -CCTGGCATATTTGATTGCAA-3 (forward primer); 5 -GACTGGCCCAAGATTAACCA-3 (reverse primer). The size of the Hex-labelled PCR products were gel resolved using standard fragment analysis procedures using an Applied Biosystems 3700 system. Literature searching using PubMed was used to identify six studies previously reporting Rep1 genotypes in PD cases and controls of European ancestry. Data pertaining to allele frequencies at the Rep1 locus were extracted from these ar-
Table 1 Alpha-synuclein NACP-Rep1 genotype and allele distributions in Australian PD cases and controlsa Current study Genotypes
Cases (n = 369)
Controls (n = 370)
Cases (n = 738)
+1+1 +1+2 +1+3 0+1 0+2 00 +2+2 No 0s One 0 Two 0s No +1s One +1 Two +1s
181 (49.1%) 38 (10.3%) 1 (0.3%) 113 (30.6%) 14 (3.8%) 18 (4.9%) 4 (1.1%) 224 (60.7%) 127 (34.4%) 18 (4.9%) 36 (9.8%) 152 (41.2%) 181 (49.0%)
172 (46.5%) 27 (7.3%) 0 (0%) 123 (33.2%) 17 (4.6%) 28 (7.6%) 3 (0.8%) 202 (54.6%) 140 (37.8%) 28 (7.6%) 48 (13.0%) 150 (40.5%) 172 (46.5%)
1 1.44 (0.82–2.52) N.D.c 0.92 (0.65–1.30) 0.78 (0.36–1.69) 0.60 (0.31–1.16) N.D. 1 0.85 (0.61–1.16) 0.56 (0.29–1.08) 1 1.45 (0.87–2.41) 1.42 (0.86–2.35)
−1 0 +1 +2 +3 +1 0 Others
0 (0%) 0 (0%) 163 (22.1%) 196 (26.5%) 514 (69.6%) 494 (66.8%) 60 (8.1%) 50 (6.8%) 1 (0.1%) 0 (0%) 514 (69.6%) 494 (66.8%) 163 (22.1%) 196 (26.5%) 61 (8.3%) 50 (6.8%) (χ2 = 4.5, d.f. = 2, p = 0.10)
Controls (n = 740)
1 0.80 (0.62–1.03) 1.17 (0.78–1.77)
Combined analysis of allele frequencies including data from all published studies of subjects of European ancestry [4,7,9,13,16,18] Alleles Cases n = 3127 (%) Controls n = 2411 (%) OR (95%CI) +1 0 Others
2170 711 246
69.4 22.7 7.9
1592 661 158
66 27.4 6.5
1 0.79 (0.70–0.89) 1.14 (0.92–1.42)
(χ2 = 17.7, d.f. = 2, p = 0.0001) Cases (163 females; 206 males); average age = 67 ± 10 years (onset 60 ± 10 years); 46 claimed a first degree relative with PD; 34 had an onset age before age 45 years. Controls (257 females; 113 males); age = 64 ± 11 years. 76.5% of PD cases and 86.5% of control subjects claimed an entirely British ancestry. The remainder of subjects were of European Caucasian ancestry; although 10.8% of cases and 2.7% of controls could not define ethnicity further and 1.4% of cases and 0.8% controls claimed some degree of non-European ancestry. b Genotype ORs were adjusted for age and gender using binary logistic regression modelling. c N.D.: not determined. a
G.D. Mellick et al. / Neuroscience Letters 375 (2005) 112–116
Fig. 1. Association between SNCA Rep1 zero allele and Parkinson’s disease in subjects of European ancestry. Odds ratios (OR) and 95% confidence interval (95% CI) for (0) allele versus (+1) allele.
in the Australian data also suggests that PD risk (as measured by OR) reduces with increasing dose of 0 allele (see Table 1). Given the similarity between our allele data and that seen in previously reported Rep1 studies, a simple combined analysis of all 3127 PD and 2411 control alleles between PD cases and controls was performed. Fig. 1 presents a forest plot summarising the data from the current study and those from previously published studies. Our analysis revealed a highly significant difference between PD and control groups (p = 0.0001), with the (+1) allele over-represented and the (0) allele under-represented in PD cases (see Table 1). There was no evidence for heterogeneity in the OR between studies (Breslow-Day test χ2 = 6.96, d.f. = 6, p = 0.33). A funnel plot, which shows the distribution of ORs versus the respective error in these measurements, is presented in Fig. 2. This plot is marginally asymmetrical making it difficult to exclude the possibility of publication bias operating here. Functionally relevant genetic variations in and around SNCA are extremely plausible genetic risk factors for sporadic PD. SNCA mutations [9,14] and dosage abnormalities  cause familial PD. Moreover, extensive evidence now implicates alpha-synuclein protein aggregation as a key event in the pathogenesis of sporadic PD. Recent studies clearly demonstrate that Rep1 genotype influences SNCA promoter-drive gene expression. Chiba-Falek et al. used luciferase reporter constructs in SH-SY5Y cells to show that promoters carrying the Rep1 (+1) allele drive three-fold greater expression compared to those carrying the (0) allele . These in-vitro data support the hypothesis that individuals carrying Rep1(0) alleles exhibit a reduced alphasynuclein expression. Given that over-expression of alphasynuclein is thought to contribute to the neurodegeneration
observed in familial PD and in animal models of parkinsonism, an extension to this speculation is that the (0) allele may convey protection against PD. The current genetic association result is consistent with this hypothesis. Further support for this hypothesis comes from our data that suggest a trend to reduced OR with increasing (0) allele dose (see Table 1). There is close agreement between studies examining subjects of European ancestry as to the allele frequencies at the Rep1 locus. Moreover, the odds ratios observed in our Australian sample are remarkably similar to those derived from previously published Rep1 studies in subjects of European
Fig. 2. Funnel plot assessing publication bias in studies of the SNCA Rep1 association with Parkinson’s disease. Odds ratios (OR) for (0) allele versus (+1) allele; SE(ln OR) represents the standard error in the natural logarithm of the OR of individual studies.
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ancestry [4,7,9,13,16,18] (see Fig. 1). However, there is some variation in the reported base-pair sizes corresponding to the various allele designations, which has lead to some confusion in previous interpretations of these data. It is clear from careful examination of each data set and aligning the most common alleles in each case, that inconsistencies in allele designation have occurred previously. This needs to be addressed in any future collaborative re-analysis of Rep1 data in PD. Our preliminary combined analysis of allele frequencies was considered appropriate given this excellent consistency in allele frequencies and ORs between studies. Rep1 allele distributions in Asians are somewhat different and were, therefore, not included in this simple combined analysis. However, a significant association between Rep1 and PD is also seen in this group [6,17,19]. While the effect size of this Rep1-PD association is relatively small, it is important to note that recent data support similar sized effects operating for polymorphic variants in other PD-related genes such as UCHL1 (OR = 0.84 for 18Y variant carriers)  and MAPT (OR = 0.73 for non-H1 haplotype carriers) [5,10] Several limitations exist in our data. First, our controls are a loosely matched convenience sample. We conducted binary logistic regression analysis, adjusting ORs for age and gender in an attempt to minimise limitations in this mis-matching. Unfortunately, it was not possible to combine genotype data from all previously published studies or to include potential confounding variables in a more vigorous regression analysis of this data, although this would be desirable. Secondly, potential genetic stratification and other forms of confounding must always be considered in case-control studies of out-bred populations; however, our genotype and allele frequency data is robust even when stratified for ethnicity, age-at-onset and family history of PD. Third, although Rep1 itself appears to have functional relevance, we are yet to examine other genetic variants in and around SNCA for association with PD. Fourth, our simple meta-analysis should be considered a preliminary result because selection criteria for cases and controls and genotyping methods may not have been equivalent in the various studies included. Finally, we must consider the possibility of a publication bias operating here given that we only combined data from published reports and that we may have unavoidably excluded important unpublished data that does not show results consistent with an association finding. The funnel plot (Fig. 2) is relatively symmetrical, although this is somewhat difficult to assess given the small number of studies published to date. None-the-less, given the similarities in the allele frequencies and ORs between our sample and those previously published, and given the functional plausibility of the association, our analysis strongly supports Rep1 as a genetic risk factor of PD. Our result highlights the need for formal collaborative pooled analyses of the alpha-synuclein locus as a susceptibility factor in sporadic PD. Such analyses have various advantages including increased power, an ability to investigate subgroups, and an opportunity to standard-
ise clinical definitions, selection criteria, genotyping calling and statistical methods . Given that an excess of alphasynuclein leads directly to PD  and reduced expression appears to reduce risk, it also seems appropriate that strategies targeting alpha-synuclein expression now be explored as potential treatments for PD.
Acknowledgement Dr Mellick is supported by the Geriatric Medical Foundation of Queensland.
References  O. Chiba-Falek, R.L. Nussbaum, Effect of allelic variation at the NACP-Rep1 repeat upstream of the alpha-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system, Hum. Mol. Genet. 10 (2001) 3101–3109.  O. Chiba-Falek, J.W. Touchman, R.L. Nussbaum, Functional analysis of intra-allelic variation at NACP-Rep1 in the alpha-synuclein gene, Hum. Genet. 113 (2003) 426–431.  Y. Deng, B. Newman, M.P. Dunne, P.A. Silburn, G.D. Mellick, Further evidence that interactions between CYP2D6 and pesticide exposure increase risk for Parkinson’s disease, Ann. Neurol. 55 (2004) 897.  M. Farrer, D.M. Maraganore, P. Lockhart, A. Singleton, T.G. Lesnick, M. de Andrade, A. West, R. de Silva, J. Hardy, D. Hernandez, alpha-synuclein gene haplotypes are associated with Parkinson’s disease, Hum. Mol. Genet. 10 (2001) 1847–1851.  D.G. Healy, P.M. Abou-Sleiman, A.J. Lees, J.P. Casas, N. Quinn, K. Bhatia, A.D. Hingorani, N.W. Wood, Tau gene and Parkinson’s disease: a case-control study and meta-analysis, J. Neurol. Neurosurg. Psychiatry 75 (2004) 962–965.  Y. Izumi, H. Morino, M. Oda, H. Maruyama, F. Udaka, M. Kameyama, S. Nakamura, H. Kawakami, Genetic studies in Parkinson’s disease with an alpha-synuclein/NACP gene polymorphism in Japan, Neurosci. Lett. 300 (2001) 125–127.  N. Khan, E. Graham, P. Dixon, C. Morris, A. Mander, D. Clayton, J. Vaughan, N. Quinn, A. Lees, S. Daniel, N. Wood, R. de Silva, Parkinson’s disease is not associated with the combined alphasynuclein/apolipoprotein E susceptibility genotype, Ann. Neurol. 49 (2001) 665–668.  R. Kruger, W. Kuhn, T. Muller, D. Woitalla, M. Graeber, S. Kosel, H. Przuntek, J.T. Epplen, L. Schols, O. Riess, Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease, Nat. Genet. 18 (1998) 106–108.  R. Kruger, A.M. Vieira-Saecker, W. Kuhn, D. Berg, T. Muller, N. Kuhnl, G.A. Fuchs, A. Storch, M. Hungs, D. Woitalla, H. Przuntek, J.T. Epplen, L. Schols, O. Riess, Increased susceptibility to sporadic Parkinson’s disease by a certain combined alpha-synuclein/apolipoprotein E genotype, Ann. Neurol. 45 (1999) 611–617.  J.B. Kwok, E. Teber, C. Loy, M. Hallupp, G. Nicholson, G.D. Mellick, D.D. Buchanan, P.A. Silburn, P.R. Schofield, Tau promoter haplotypes regulate transcription and are associated with Parkinson’s disease, Ann. Neurol. 55 (2004) 329–334.  D.M. Maraganore, T.G. Lesnick, A. Elbaz, M.C. Chartier-Harlin, T. Gasser, R. Kruger, N. Hattori, G.D. Mellick, A. Quattrone, J. Satoh, T. Toda, J. Wang, J.P. Ioannidis, M. De Andrade, W.A. Rocca, UCHL1 is a Parkinson’s disease susceptibility gene, Ann. Neurol. 55 (2004) 512–521.
G.D. Mellick et al. / Neuroscience Letters 375 (2005) 112–116
 G.D. Mellick, P.A. Silburn, J.A. Prince, A.J. Brookes, A novel screen for nuclear mitochondrial gene associations with Parkinson’s disease, J. Neural. Transm. 111 (2004) 191–199.  A. Parsian, B. Racette, Z.H. Zhang, S. Chakraverty, M. Rundle, A. Goate, J.S. Perlmutter, Mutation, sequence analysis, and association studies of alpha-synuclein in Parkinson’s disease, Neurology 51 (1998) 1757–1759.  M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C. Duvoisin, G. Di Iorio, L.I. Golbe, R.L. Nussbaum, Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease, Science 276 (1997) 2045–2047.  A.B. Singleton, M. Farrer, J. Johnson, A. Singleton, S. Hague, J. Kachergus, M. Hulihan, T. Peuralinna, A. Dutra, R. Nussbaum, S. Lincoln, A. Crawley, M. Hanson, D. Maraganore, C. Adler, M.R. Cookson, M. Muenter, M. Baptista, D. Miller, J. Blancato, J. Hardy, K. Gwinn-Hardy, Alpha-synuclein locus triplication causes Parkinson’s disease, Science 302 (2003) 841.  P. Spadafora, G. Annesi, A.A. Pasqua, P. Serra, I.C. Ciro Candiano, S. Carrideo, P. Tarantino, D. Civitelli, E.V. De Marco, G. Nicoletti,
F. Annesi, A. Quattrone, NACP-REP1 polymorphism is not involved in Parkinson’s disease: a case-control study in a population sample from southern Italy, Neurosci. Lett. 351 (2003) 75–78. E.K. Tan, A. Chai, Y.Y. Teo, Y. Zhao, C. Tan, H. Shen, V.R. Chandran, M.L. Teoh, Y. Yih, R. Pavanni, M.C. Wong, K. Puvan, Y.L. Lo, E. Yap, Alpha-synuclein haplotypes implicated in risk of Parkinson’s disease, Neurology 62 (2004) 128–131. E.K. Tan, T. Matsuura, S. Nagamitsu, M. Khajavi, J. Jankovic, T. Ashizawa, Polymorphism of NACP-Rep1 in Parkinson’s disease: an etiologic link with essential tremor? Neurology 54 (2000) 1195–1198. E.K. Tan, C. Tan, H. Shen, A. Chai, S.Y. Lum, M.L. Teoh, Y. Yih, M.C. Wong, Y. Zhao, Alpha synuclein promoter and risk of Parkinson’s disease: microsatellite and allelic size variability, Neurosci. Lett. 336 (2003) 70–72. Y. Xia, H.A. Rohan de Silva, B.L. Rosi, L.H. Yamaoka, J.B. Rimmler, M.A. Pericak-Vance, A.D. Roses, X. Chen, E. Masliah, R. DeTeresa, A. Iwai, M. Sundsmo, R.G. Thomas, C.R. Hofstetter, E. Gregory, L.A. Hansen, R. Katzman, L.J. Thal, T. Saitoh, Genetic studies in Alzheimer’s disease with an NACP/alpha-synuclein polymorphism, Ann. Neurol. 40 (1996) 207–215.