Journal of the Neurological Sciences 337 (2014) 123–128

Contents lists available at ScienceDirect

Journal of the Neurological Sciences

j ourna l homepage: www.e lsev ie r .com/ locate / jns

Hypomethylation of SNCA in blood of patients with sporadicParkinson's disease

San-xi Ai a, Qian Xu a, Ya-cen Hu d, Cheng-yuan Song a, Ji-feng Guo a,b, Lu Shen a,b, Chun-rongWang a, Ri-li Yu a,Xin-xiang Yan a,b, Bei-sha Tang a,b,c,⁎a Department of Neurology, Xiangya Hospital, Central South University, Changsha, Chinab Neurodegenerative Disorders Research Center, Central South University, Changsha, Chinac National Laboratory of Medical Genetics of China, Central South University, Changsha, Chinad Department of Geriatric Neurology, Xiangya Hospital, Central South University, Changsha, China

⁎ Corresponding author at: Department of Neurology,University, Changsha, China. Tel.: +86 731 89757398; fax

E-mail address: bstang7398@163.com (B. Tang).

0022-510X/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.jns.2013.11.033

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 May 2013Received in revised form 16 November 2013Accepted 20 November 2013Available online 1 December 2013

Keywords:Parkinson's diseaseMethylationPBMCsSNCA hypomethylationSNCA intron 1Rep1

SNCA is a pathogenic gene identified in rare familial PD, and over-expression of SNCA was suggested in thepathogenesis of familial and sporadic PD. Rep1 polymorphism of SNCA was associated with susceptibility tosporadic PD and SNCA expression in intro and in vivo. Hypomethylation in SNCA intron-1 was associated withincreased SNCA expression and was observed in postmortem brains of patients with sporadic PD. We studiedthe methylation status of SNCA intron-1, SNCA mRNA levels and Rep1 genotypes in PBMCs of 100 sporadic PDpatients and 95 controls and explored the relationship between DNA methylation, mRNA expression and Rep1genotypes. Hypomethylation of SNCA intron-1 was detected in PBMCs of PD patients, and DNA methylationlevels were associated with Rep1 polymorphism. The shorter allele was associated with higher level of SNCAintron-1 methylation, and genotypes carrying the shorter allele showed significantly higher methylation levelof SNCA intron-1 than genotypes carrying the longer allele. However, SNCA mRNA levels were not associatedwith disease status, Rep1 polymorphism or DNA methylation of SNCA intron-1 in our study.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Parkinson's disease (PD), the secondmost frequent neurodegenera-tive disorder, is pathologically characterized by the progressive loss ofdopaminergic neurons in the substantia nigra. Several genes havebeen identified in rare familial PD cases with Mendelian inheritance,but the etiology of sporadic PD remains obscure [1].

SNCA is a causative gene of rare familial PD, and its encoding protein(alpha-synuclein) is a major component of Lewy bodies, a pathologicalhallmark of PD [1]. Triplication and duplication of SNCAwere identifiedin rare familial PD cases, and increased expression of wild-type SNCAwas reported in postmortem brain tissues of patients with SNCA multi-plications, suggesting that the pathogenic effects of SNCAmultiplicationis possibly mediated by SNCA over-expression [2–5]. SNCA over-expression was also reported in brain tissues of patients with sporadicPD [6–8], suggesting that SNCA over-expression may also contributeto the pathogenesis of sporadic PD.

Xiangya Hospital, Central South: +86 731 84327332.

ights reserved.

Rep1, a polymorphic dinucleotide repeat located ~10 kb upstreamof the translational start site of SNCA, has been associated with PDby multiple studies [9–12]. Studies showed that the longer allele ofSNCA-Rep1 conferred increased risk to develop PD, while the shorterallele is associated with reduced risk for PD. Similar to the effect ofSNCA multiplication, Rep1 variants were reported to have an effect onSNCA-mRNA levels in brains of patients with sporadic PD. Homogenousgenotype of the shorter allele was associatedwith lower levels of SNCA-mRNA compared with genotypes carrying longer alleles [13]. Studies incell cultures and animal models revealed that Rep1 had an effect ontranscriptional activities of SNCA and the longer allele was associatedwith higher levels of SNCA expression relative to the shorter allele[14,15]. All together, these findings suggest that Rep1 allele lengthvariability modulates the susceptibility to PD possibly via regulation ofSNCA expression.

DNA methylation is a key epigenetic mechanism involved in theregulation of gene transcription. Methylation refers to the transfer of amethyl group from S-adenosyl methionine to cytosine residues at theCG dinucleotides on the DNA [16]. Recently, hypomethylation of SNCAintron-1 was observed in postmortem brain tissues from patients withsporadic PD and hypomethylation of this region was associated withincreased expression of SNCA in vitro [17,18].

124 S. Ai et al. / Journal of the Neurological Sciences 337 (2014) 123–128

In the present work, we investigated DNA methylation status ofSNCA intron-1, Rep1 genotypes and SNCA-mRNA expression in periph-eral blood mononuclear cells (PBMCs) of patients with sporadic PD.

2. Materials and methods

2.1. Subjects

The study was approved by the Medical Ethics Committee ofXiangya Hospital, Central South University and written consents wereobtained from every participant. 100 sporadic PD patients (mean ± SDage = 62.1 ± 9.6 years, female = 45, male = 55) and 95 controls(mean ± SD age = 61.4 ± 9.8 years, female = 45, male = 50) wereenrolled in this study. PD patients were collected from the Departmentof Neurology, Xiangya Hospital, Central South University; NationalLaboratory of Medical Genetics of China and Neurodegenerative Disor-ders Research Center, Central South University. All patients underwenta standardized neurological examination by two movement disorderspecialists. The clinical diagnosis of PD was established according tothe United Kingdom PD Brain Bank Criteria [19]. All of the patientswere of Chinese Han ethnicity from China. PD clinical stages were eval-uated according to the classification of Hoehn and Yahr (H-Y) [20]. 69patients had received L-dopa treatment before the study. The controlgroupwas composed of age-, gender-, and origin-matched healthy indi-viduals from Health Examine Center of Second Xiangya Hospital. Sub-jects were excluded if they had major organ dysfunction, neurologicaldisease, or family history of movement disorders.

2.2. Isolation of PBMCs

A 30 ml sample of venous peripheral blood was drawn from eachsubject into ethylenediamine tetra-acetate (EDTA) vacutainer tubes.All blood samples from these subjects were drawn in the morningafter an overnight fast. PBMCs were isolated by Ficoll–Hypaque densitygradient centrifugation (Shanghai Hengxin Chemical Reagent Co.,Shanghai, China).

2.3. Nucleotide extraction

Genomic DNAwas extracted from PBMCs using the TIANamp Geno-mic DNA blood kit (Tiangen Biotech, Beijing, China). Total RNA was

Fig. 1. Schematic drawing of the 5′ region of SNCA. A. The translational start site ismarked in exointron-1, and the gray box represents Rep1 (~10 kb upstream of ATG). B. Sequence of amplifiethis region. Sequences with underlines represent predicted transcription factor binding sites (p

isolated by standard Trizol method (Qiagen). DNA and RNA sampleswere stored in−80 °C until experiments.

2.4. Bisulfite sequencing

A 451 bp SNCA intron-1 fragment (−926 to −476) containing 23CpG sites was analyzed by bisulfite sequencing (Fig. 1A). Bisulfiteconversion of genomic DNA was carried out with Epitect Bisulfite Kit(Qiagen). The 451 bp SNCA intron-1 fragment was amplified by chainreaction (PCR) and cloned into the pGEM-T easy vector (Promega,Madison, WI, U.S.A.). At least 10 independent clones were sequencedfor each subject. Primer sequences for PCR were described previously[17]. Quality control was performed by BiQ analyzer (quality controlsoftware for DNA methylation data from bisulfite sequencing). Thelevel of DNA methylation was determined as percentage of methylatedCpG. For each subject, methylation level (methylated CpG/total CpG) atindividual CpG site and the mean methylation level of all the 23 CpGsites were calculated.

2.5. Genotyping of Rep1

The region of SNCA containing Rep1 was amplified by PCR fromgenomic DNA isolated from PBMCs. SNCA Rep1 alleles were sized on ahigh-resolution capillary electrophoresis platform using ABI 3730XL au-tomated sequencer (Applied Biosytems, Inc., Foster City, CA) and allelicsizes were analyzed using GeneScan Version 4.0 software (AppliedBiosytems, Inc., Foster City, CA). The Rep1 alleles (with a size differenceof two nucleotides) were defined according to the length of the PCRproduct. Due to ethnic differences, Rep1 calling may be inconsistentacross studies [10,12,21]. We termed the alleles according to the rulesdetermined by Izumi et al.: allele −2 = 263 bp, allele −1 = 265 bp,allele 0 = 267 bp, allele 1 = 269 bp, and allele 2 = 271 bp. Theprimers were described previously [21].

2.6. cDNA synthesis and real-time quantitative PCR (RT-PCR)

cDNA was synthesized with an input of 2 μg of total RNA using theRevertAid™ First Strand cDNA synthesis Kit (Fermentas, Burlington,Canada). RT-PCR was performed on a ABI 7900 HT Fast Real-time PCRsystem (Applied Biosytems, Inc., Foster City, CA), using the SYBR PremixEx Taq™ real-time PCR Kit (Takara Biotech, Co., Dalian, China). All reac-tionswere run for two times, and duplicate each time. β-Actinwas used

n-2 by ATG (+1). The black boxes represent exon-1 and exon-2, thewhite box representsd fragment of SNCA intron-1 (−926 to−476) and 23 CpG sites (marked with number) inredicted by TESS program).

Fig. 2. Methylation levels of SNCA intron-1 and SNCA-mRNA levels in PBMCs from PDpatients and controls. A. Mean methylation levels of SNCA intron-1 (mean percentagesof methylated CpG of all 23 CpG sites). The mean methylation level was lower in PD pa-tients compared with controls (p = 0.033, linear regression analysis). B. Site specificmethylation levels (percentages of methylated CpG at particular CpG site). PD patients'DNA was hypomethylated compared with controls at CpG sites 6, 8 and 9 (p = 0.036,0.012, 0.029 respectively). * indicates significant difference (p b 0.05).

125S. Ai et al. / Journal of the Neurological Sciences 337 (2014) 123–128

as an internal control. Primer sequences for RT-PCRwere described pre-viously [22]. The mRNA level of SNCA relative to β-actin was deter-mined by the comparative CT method [23].

2.7. Statistical analyses

All statistical analysis was performed using Statistical Packagefor Social Sciences (SPSS, version 17.0). All data was expressed asmean ± SD. Chi-square test was used to analyze data as frequenciesand percentages. Linear regression analysis was performed to assessthe association of mRNA or DNA methylation levels with primary vari-ables (disease status and Rep1 genotypes) as well as secondary vari-ables (gender and age) that may affect DNA methylation and mRNAexpression levels [13,16,22]. For post hoc multiple comparisonsbetween different genotypes, Dunnett T3 test was applied. Pearsoncorrelation was performed for correlation between DNA methylationand mRNA levels. For all analyses, p b 0.05 was considered statisticallysignificant.

3. Results

3.1. Comparison of methylation between patients and controls

Demographic data was summarized in Table 1. There was no signif-icant difference in gender or age distribution between PD patients andcontrols.

First, we compared the DNA methylation levels of SNCA intron-1betweenPDpatients and controls by linear regression analysis adjustingfor Rep1 genotypes, age, and gender. As shown in Fig. 2A and Table 1,the mean methylation level of SNCA intron-1 was lower in the PDpatients compared to controls (p = 0.033). A detailed comparison ofDNAmethylation levels at individual CpG site between PD and controlsshowed that PD patients' DNAwas significantly hypomethylated at CpGsites 6, 8 and 9 (p = 0.036, 0.012, 0.029, Fig. 2B).

3.2. Effect of Rep1 genotypes on DNA methylation

Rep1 allele distribution and frequencieswere listed in Table 2. Therewas no significant difference in allele distribution between PD andcontrols. Since alleles−1 and −2 were rare in both groups, they werenot included in the following analysis.

In order to determine the potential effect of Rep1 polymorphism onDNA methylation of SNCA intron-1, we compared DNA methylationlevels among different Rep1 genotypes by linear regression analysisadjusting for disease status, gender and age. Significant difference ofDNA methylation levels was observed between different Rep1 geno-types (p = 0.001, Fig. 3A), and genotypes carrying allele 0 (0/0, 0/1and 0/2) showed significantly higher methylation levels than genotype1/2 (p = 0.044, 0.038, 0.031) and genotype 2/2 (p = 0.009, 0.007,0.006, Table 3). Genotype 1/1 did not show significant difference ofDNA methylation level compared with any of the other genotypes(p N 0.05). We further compared the DNA methylation levels betweendifferent allele carrier statuses of alleles 0, 1 and 2. Significant difference

Table 1Summary of demographic data and levels of SNCAmethylation and mRNA of PD patientsand controls.

Group PD (n = 100) Control (n = 95) p valuea

Gender (male/female) 55/45 50/45 0.740Age (years) 62.1 ± 9.6 61.4 ± 9.8 0.575SNCA methylation levelb(%) 14.22 ± 8.54 16.90 ± 8.66 0.033SNCA mRNA level 0.0013 ± 0.0007 0.0013 ± 0.0008 0.844

a p value of Chi-square test.b p values presented were corrected by linear regression analysis.

of methylation levels was observed between non-carriers of 0 andcarriers of 0 (p = 0.000, Fig. 3B), but not between non-carriers of 2and carriers of 2 (p = 0.442) or between non-carriers of 1 and carriersof 1 (p = 0.984).

3.3. mRNA levels between patients and control

We then compared SNCA-mRNA levels between PD patients andcontrols by linear regression analysis adjusting for gender, age andRep1 genotypes. No significant difference of mRNA levels was foundbetween the two groups (p = 0.844, Fig. 4A).

We evaluated the effects of gender, age, onset age, disease duration,H-Y stage and medication (L-dopa) on methylation and mRNA levels,and none of these factors was associated with methylation levels ormRNA levels (data not shown).

3.4. Effect of Rep1 genotype on mRNA level

We then examined the association of SNCA-mRNA levels withRep1 genotypes. SNCA mRNA levels did not differ between differentRep1 genotypes (p = 0.136, Fig. 4B). And no correlation was observed

Table 2Comparison of the allele frequencies of Rep1 in PD patients and controls.

Group Rep1 allele

−2a −1a 0a 1a 2a

Control (nb=190) 1 (0.5%) 0 (0) 65 (34.2%) 65 (34.2%) 59 (31.1%)PD (nb=200) 0 (0) 2 (1.0%) 72 (36.0%) 64 (32.0%) 62 (31.0%)p valuec 0.487 0.499 0.711 0.643 0.991

a Rep1 nomenclature: −2 = 263 bp, −1 = 265 bp, 0 = 267 bp, 1 = 269 bp, and2 = 271 bp.

b n = 2 ∗ case numbers.c p value of Chi-square test.

Fig. 3. Comparison of mean methylation level of SNCA intron-1 between different Rep1genotypes. A. Comparison of methylation level between 6 genotypes of Rep1. Genotypes0/0, 0/1 and 0/2 were significantly hypermethylated than genotypes 1/2 and 2/2(p b 0.05). B. Comparison of methylation level between different allele 0 carrier statuses.Allele 0 carrier status was significantly hypermethylated than allele 0 non-carrier status(p = 0.000). ** indicates significant difference (p b 0.01).

Fig. 4. A. SNCA-mRNA levels in PBMCs from PD patients and controls. No significantdifference was observed (p = 0.844). B. Comparison of SNCA-mRNA levels betweendifferent genotypes of Rep1. No significant difference was observed (p = 0.136).

126 S. Ai et al. / Journal of the Neurological Sciences 337 (2014) 123–128

between SNCA-mRNA and DNAmethylation levels (p = 0.263, Pearsoncorrelation).

4. Discussion

PD is a progressive neurodegenerative disease and the majorsymptoms of PD arise from the degeneration of dopaminergic neuronsin substantia nigra pars compacta. Because of difficult access to braintissues, PD related biomarkers in accessible tissues have been thefocus of researchers. We chose to use PBMCs for studies because somePD-specific biochemical alterations observed in postmortem brains, in-cluding mitochondrial complex deficiency [24], decreased proteasomeactivity and increased caspase activity [25], and altered content of dopa-mine and dopamine transporters [26–28], were also detected in PBMCsof PD patients, suggesting that PBMCsmay reflect some disease-relatedpathogenesis. Furthermore, decreased mRNA levels of NURR-1, a gene

Table 3Comparison of SNCA methylation levels between different Rep1 genotypes.

Rep1 genotype Control PD SNCA methylationlevela (%)

p valueb

0/0 (n = 30c) 12 18 17.6 ± 8.5 0.0010/1 (n = 38c) 23 15 17.6 ± 9.40/2 (n = 37c) 18 19 18.4 ± 10.81/1 (n = 24c) 12 12 15.6 ± 8.41/2 (n = 43c) 18 25 12.1 ± 5.62/2 (n = 20c) 11 9 10.6 ± 4.8

a SNCA methylation level represents mean methylation level of SNCA intron-1.b p value presented was derived from the comparison of DNA methylation between

the six genotypes above by linear regression analysis, adjusting for disease status, genderand age.

c No significant difference of genotype distribution between patients and controls(p N 0.05).

involved in dopaminergic neurogenesis and pathogenesis of PD wereobserved in PBMCs of patients with sporadic PD [29,30]. IncreasedSNCA mRNA was detected in PBMCs of patients with sporadic PD [22].Consistent with the association between SNCA expression in brainsand Rep1 variants [13], SNCA protein (alpha-synuclein) levels inPBMCs were also associated with Rep1 polymorphisms [31]. A recentstudy identified concordant methylation alterations in brain tissuesand PBMCs of patientswith PD, further lending support to the feasibilityof using PBMCs for studies [32].

In the present work, we found that SNCA intron-1 was hypo-methylated in PBMCs of patients with sporadic PD compared with con-trols. This result is consistent with previous studies which showedhypomethylation of the same region in postmortem brains of patientswith sporadic PD [17,18]. Admittedly, such a small difference ofmethyl-ation level (~3%) may not be a useful biomarker for PD diagnosis, butthe previous study in postmortem brains showed similarly small differ-ence of methylation level between PD patients and controls (~5%) [17].Thus, this study can be taken as an indication that alterations of themethylation level of SNCA intron-1 in PBMCs may reflect the changesof methylation in brains of patients with sporadic PD. Furthermore,studies in cell cultures showed that 40% (Hela cells) or 20% (293 cells)decrease of methylation levels of SNCA intron-1 could induce increasedSNCA-mRNA expressions [17,18]. Given the low baseline methylationlevels of SNCA intron-1 (16.9% in controls), the ~3% difference of meth-ylation amounts to a 16% decrease of methylation in PD patientscompared with controls. Thus the modest hypomethylation observedin PD patients may have an effect on the regulation of SNCA expressionaccording to these researches. In addition, nearly all CpG sites werelocated within the predicted TF binding sites (Fig. 1B), suggesting thatDNA methylation of this region, especially CpG sites 6, 8, 9 whichwere found to be significantly hypomethylated in PD patients, mayhave an effect on SNCA transcription.

Genetic and environmental factors may be involved in the alter-ations of DNA methylation. In this study, we evaluated the effects of

127S. Ai et al. / Journal of the Neurological Sciences 337 (2014) 123–128

Rep1 polymorphism andmedication (L-dopa) on themethylation levelsof SNCA intron-1.We did not observe an effect of L-dopa on themethyl-ation levels of SNCA intron-1. Rep1 genotypes were demonstrated to beassociated with methylation levels of SNCA intron-1. Genotypes carry-ing allele 0 were significantly hypermethylated compared to genotypes1/2 and 2/2. Allele 0 carrier status was associated with higher level ofmethylation compared with 0 non-carrier status. Together, this studyshowed that the shorter allele was associated with higher methylationlevel of SNCA intron-1, although the Rep1 is distant from the CpGislands in SNCA intron-1. Recent studies showed that DNAmethylationmight be regulated by genetic variants through unknown mechanisms[33–35]. For example, a genome-wide association study by Zhanget al. showed that, DNAmethylationwas frequently heritable and thou-sands of SNPs were associated with methylation of specific CpG sites.The regulation of methylation could be cis-acting or trans-acting, andthe associated SNPs could be distant (thousands of base pairs) fromthe CpG locus. In fact, most (87.9%) associated SNPs were more than2 kb away from the CpG positions [34]. The most compelling evidenceof the association between SNPs and DNA methylation is the IGF2/H19locus, the methylation of which is associated with SNPs in cis [33].Thus, our data is concordant with previous studies and further studiesexploring the mechanism underlying the association between Rep1polymorphism and methylation of SNCA intron-1 are warranted. Theassociation of Rep1with sporadic PDwas reported in large-scale collab-orative analysis [12], but as reviewed by Farrer et al. [9], there was dis-parity in association findings between small studies with potentialbiases. In this study, we failed to determine any allele frequency dis-crepancy between PDpatients and controls, possibly due to the relative-ly small study samples included.

We compared the SNCA mRNA expression in PD patients andcontrols, and no significant difference was observed. Previous studieson SNCA-mRNA levels in PD patients and controls showed conflictingresults [22,36]. Experimental confounding factors, especially contami-nation of red blood cells and platelet, may explain the different resultsamong studies [37,38]. Since Rep1 variants were reported to be associ-ated with SNCA-mRNA levels, the evenly distributed Rep1 allelesbetween PD patients and controls in our study may also be a reasonfor the negative result. Furthermore, we did not observe any associationbetween SNCAmRNA expression in PBMCs and Rep1 polymorphism ormethylation of SNCA intron-1. This finding was not consistent with theprevious studies which indicated the association between SNCA mRNAlevels and Rep1 polymorphism in brain tissues and cell cultures [13,17],and the association between SNCA mRNA levels and methylation ofSNCA intron-1 in vitro [14]. Tissue specific regulation of SNCA transcrip-tion may explain the conflicting results and other mechanisms may beinvolved in the regulation of SNCA mRNA level in PBMCs.

Taken together, our results revealed hypomethylation of SNCAintron-1 in PBMCs of patients with PD and confirmed the effect ofRep1 on DNA methylation of SNCA. These findings may contribute tobetter understanding of the mechanisms underlying the associationsbetween methylation and PD.

Declaration of interest

No conflict of interest exits in the submission of this manuscript.

Acknowledgments

The authors would like to thank all the patients and control subjectsfor participation in this study. This work was supported by grants fromthe NSFC-NIH joint program (81361120404), the Major State Basic Re-search Development Program of China (973 Program) (2011CB510000),the key program of the National Natural Science Foundation of China(81130021), and the program of the National Natural Science Foundationof China (30971035).

References

[1] Shulman JM, De Jager PL, Feany MB. Parkinson's disease: genetics and pathogenesis.Annu Rev Pathol 2011;6:193–222.

[2] Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, et al. alpha-Synuclein locus triplication causes Parkinson's disease. Science Oct 312003;302(5646):841.

[3] Farrer M, Kachergus J, Forno L, Lincoln S, Wang DS, Hulihan M, et al. Comparison ofkindreds with parkinsonism and alpha-synuclein genomic multiplications. AnnNeurol Feb 2004;55(2):174–9.

[4] Chartier-Harlin MC, Kachergus J, Roumier C, Mouroux V, Douay X, Lincoln S, et al.alpha-Synuclein locus duplication as a cause of familial Parkinson's disease. LancetSep 25-Oct 1 2004;364(9440):1167–9.

[5] Ross OA, Braithwaite AT, Skipper LM, Kachergus J, Hulihan MM, Middleton FA, et al.Genomic investigation of alpha-synuclein multiplication and parkinsonism. AnnNeurol Jun 2008;63(6):743–50.

[6] Chiba-Falek O, Lopez GJ, Nussbaum RL. Levels of alpha-synuclein mRNA in sporadicParkinson disease patients. Mov Disord Oct 2006;21(10):1703–8.

[7] Grundemann J, Schlaudraff F, Haeckel O, Liss B. Elevated alpha-synuclein mRNAlevels in individual UV-laser-microdissected dopaminergic substantia nigra neuronsin idiopathic Parkinson's disease. Nucleic Acids Res Apr 2008;36(7):e38.

[8] Grundemann J, Schlaudraff F, Liss B. UV-laser microdissection and mRNA expressionanalysis of individual neurons from postmortem Parkinson's disease brains.Methods Mol Biol 2011;755:363–74.

[9] Farrer M, Maraganore DM, Lockhart P, Singleton A, Lesnick TG, de Andrade M, et al.alpha-Synuclein gene haplotypes are associated with Parkinson's disease. Hum MolGenet Aug 15 2001;10(17):1847–51.

[10] Mizuta I, NishimuraM,Mizuta E, Yamasaki S, OhtaM, Kuno S.Meta-analysis of alphasynuclein/NACP polymorphism in Parkinson's disease in Japan. J Neurol NeurosurgPsychiatry Sep 2002;73(3):350.

[11] Mellick GD, Maraganore DM, Silburn PA. Australian data and meta-analysis lendsupport for alpha-synuclein (NACP-Rep1) as a risk factor for Parkinson's disease.Neurosci Lett Feb 28 2005;375(2):112–6.

[12] Maraganore DM, de Andrade M, Elbaz A, Farrer MJ, Ioannidis JP, Kruger R, et al.Collaborative analysis of alpha-synuclein gene promoter variability and Parkinsondisease. JAMA Aug 9 2006;296(6):661–70.

[13] Linnertz C, Saucier L, Ge D, Cronin KD, Burke JR, Browndyke JN, et al. Geneticregulation of alpha-synuclein mRNA expression in various human brain tissues.PLoS One 2009;4(10):e7480.

[14] Chiba-Falek O, Nussbaum RL. Effect of allelic variation at the NACP-Rep1 repeatupstream of the alpha-synuclein gene (SNCA) on transcription in a cell culture lucif-erase reporter system. Hum Mol Genet Dec 15 2001;10(26):3101–9.

[15] Cronin KD, Ge D, Manninger P, Linnertz C, Rossoshek A, Orrison BM, et al.Expansion of the Parkinson disease-associated SNCA-Rep1 allele upregulateshuman alpha-synuclein in transgenic mouse brain. Hum Mol Genet Sep 12009;18(17):3274–85.

[16] Jaenisch R, Bird A. Epigenetic regulation of gene expression: how thegenome integrates intrinsic and environmental signals. Nat Genet Mar 2003(33Suppl.):245–54.

[17] Jowaed A, Schmitt I, Kaut O, Wullner U. Methylation regulates alpha-synuclein ex-pression and is decreased in Parkinson's disease patients' brains. J Neurosci May 52010;30(18):6355–9.

[18] Matsumoto L, Takuma H, Tamaoka A, Kurisaki H, Date H, Tsuji S, et al. CpG demeth-ylation enhances alpha-synuclein expression and affects the pathogenesis ofParkinson's disease. PLoS One 2010;5(11):e15522.

[19] Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathicParkinson's disease: a clinico-pathological study of 100 cases. J Neurol NeurosurgPsychiatry Mar 1992;55(3):181–4.

[20] Hoehn MM, Yahr MD. Parkinsonism: onset, progression and mortality. NeurologyMay 1967;17(5):427–42.

[21] Izumi Y, Morino H, Oda M, Maruyama H, Udaka F, Kameyama M, et al. Geneticstudies in Parkinson's disease with an alpha-synuclein/NACP gene polymorphismin Japan. Neurosci Lett Mar 9 2001;300(2):125–7.

[22] Kim S, Jeon BS, Heo C, Im PS, Ahn TB, Seo JH, et al. Alpha-synuclein induces apoptosisby altered expression in human peripheral lymphocyte in Parkinson's disease.FASEB J Oct 2004;18(13):1615–7.

[23] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-timequantitative PCR and the 2−ΔΔCT method. Methods Dec 2001;25(4):402–8.

[24] Barroso N, Campos Y, Huertas R, Esteban J, Molina JA, Alonso A, et al. Respiratorychain enzyme activities in lymphocytes from untreated patients with Parkinsondisease. Clin Chem Apr 1993;39(4):667–9.

[25] Blandini F, Sinforiani E, Pacchetti C, Samuele A, Bazzini E, Zangaglia R, et al. Periph-eral proteasome and caspase activity in Parkinson disease and Alzheimer disease.Neurology Feb 28 2006;66(4):529–34.

[26] Caronti B, Tanda G, Colosimo C, Ruggieri S, Calderaro C, Palladini G, et al. Reduceddopamine in peripheral blood lymphocytes in Parkinson's disease. NeuroreportSep 29 1999;10(14):2907–10.

[27] Barbanti P, Fabbrini G, Ricci A, Cerbo R, Bronzetti E, Caronti B, et al. Increased expres-sion of dopamine receptors on lymphocytes in Parkinson's disease. Mov Disord Sep1999;14(5):764–71.

[28] Pellicano C, Buttarelli FR, Circella A, Tiple D, Giovannelli M, Benincasa D, et al.Dopamine transporter immunoreactivity in peripheral blood lymphocytesdiscriminates Parkinson's disease from essential tremor. J Neural Transm Jul2007;114(7):935–8.

[29] Chu Y, Le W, Kompoliti K, Jankovic J, Mufson EJ, Kordower JH. Nurr1 in Parkinson'sdisease and related disorders. J Comp Neurol Jan 20 2006;494(3):495–514.

128 S. Ai et al. / Journal of the Neurological Sciences 337 (2014) 123–128

[30] Le W, Pan T, Huang M, Xu P, Xie W, ZhuW, et al. Decreased NURR1 gene expressionin patients with Parkinson's disease. J Neurol Sci Oct 15 2008;273(1–2):29–33.

[31] Fuchs J, Tichopad A, Golub Y,MunzM, Schweitzer KJ, Wolf B, et al. Genetic variabilityin the SNCA gene influences alpha-synuclein levels in the blood and brain. FASEB JMay 2008;22(5):1327–34.

[32] Masliah E, Dumaop W, Galasko D, Desplats P. Distinctive patterns of DNAmethylation associated with Parkinson disease: identification of concordantepigenetic changes in brain and peripheral blood leukocytes. Epigenetics Aug2013;1:8(10).

[33] Heijmans BT, Kremer D, Tobi EW, Boomsma DI, Slagboom PE. Heritable ratherthan age-related environmental and stochastic factors dominate variationin DNA methylation of the human IGF2/H19 locus. Hum Mol Genet Mar 12007;16(5):547–54.

[34] Zhang D, Cheng L, Badner JA, Chen C, Chen Q, LuoW, et al. Genetic control of individ-ual differences in gene-specific methylation in human brain. Am J Hum Genet Mar12 2010;86(3):411–9.

[35] Lienert F,Wirbelauer C, Som I, Dean A,Mohn F, Schubeler D. Identification of geneticelements that autonomously determine DNA methylation states. Nat Genet Nov2011;43(11):1091–7.

[36] Tan EK, Chandran VR, Fook-Chong S, Shen H, Yew K, Teoh ML, et al. alpha-SynucleinmRNA expression in sporadic Parkinson's disease. Mov DisordMay 2005;20(5):620–3.

[37] Barbour R, Kling K, Anderson JP, Banducci K, Cole T, Diep L, et al. Red blood cells arethe major source of alpha-synuclein in blood. Neurodegener Dis 2008;5(2):55–9.

[38] Shi M, Zabetian CP, Hancock AM, Ginghina C, Hong Z, Yearout D, et al. Significanceand confounders of peripheral DJ-1 and alpha-synuclein in Parkinson's disease.Neurosci Lett Aug 9 2010;480(1):78–82.