Polymorphisms in the SNCA Gene: Association with the Risk of Development of the Sporadic Form of Parkinson’s Disease and the Level of SNCA Gene Expression in Peripheral Blood of Patients from Russia

Parkinson’s disease (PD) is one of the 
most common human neurodegenerative disorders caused by the loss of dopaminergic neurons in the brain. The α-synuclein (SNCA) gene is one of the most studied genes involved in the pathogenesis of PD. In our study, we 
conducted a genetic analysis of promoter and intron single-nucleotide polymorphisms 
(SNPs) in the SNCA gene. We also 
analyzed the association of genotypes of these SNPs with expression levels of SNCA mRNA. One of four SNPs in the SNCA gene, and the rs2736990 polymorphism, associates 
with the risk of the sporadic form of PD in Russian population. The risk of PD was increased almost twofold 
in carriers of allele C (odds ratios = 1.9, 95% confidence interval: 1.2-2.91, p = 0.003). However, no 
association was found between any of the genotypes of SNPs tested (rs2583988, 
rs2619363, rs2619364 and rs2736990) and alterations in SNCA levels. Our findings support the hypothesis that the rs2736990 
polymorphism is associated with PD. SNPs rs2583988, rs2619363 and rs2619364 in the promoter region of the SNCA gene themselves do not significantly influence the 
expression of SNCA. Most likely, SNCA gene expression is a very complex 
process that is affected by different genetic and epigenetic factors.


Introduction
Parkinson's disease (PD) is one of the most common human neurodegenerative disorders after Alzheimer's disease. PD is morphologically characterized by progressive loss of dopaminergic neurons and in some cases by formation of fibrillar inclusions (Lewy bodies) with αsynuclein (SNCA) as its main component [1]. SNCA acts as a molecular chaperone and regulates protein-protein interactions in cells [2]. According to the latest data, SNCA is required for the normal operation of a pre-synaptic complex by participating in the cyclic release of dopamine from the presynaptic vesicles [3][4][5]. SNCA was the first gene whose association with PD has been shown [6]. Currently, the SNCA gene is one of the most studied genes involved in the pathogenesis of PD. Several point mutations in the SNCA gene and duplications and triplications of the genomic region containing SNCA were revealed. They are very rare and lead to the development of an autosomal dominant form of PD [7][8][9][10][11].
To date, a number of single-nucleotide polymorphisms (SNPs) in SNCA associated with a risk of the sporadic form of the disease have been revealed. According to genome-wide association studies (GWASs), more than five polymorphic variants were found to be associated with the risk of development of PD. One of the first of these SNPs identified in a genome-wide study was rs2736990. An association of this polymorphism with the risk of PD in patients from Europe and North America has been shown [12,13]. However, the risk of disease development did not exceed odds ratio (OR) = 1.4. Thus, further verification of the results of GWASs in other population samples is required.
Polymorphisms in the SNCA Gene: Association with the Risk of Development of the Sporadic Form of Parkinson's Disease and the Level of SNCA Gene Expression in Peripheral Blood of Patients from Russia 209 Furthermore, polymorphic microsatellite Rep1 was found in the promoter region of 10 kb upstream of the SNCA transcription start site [14][15][16]. One of the microsatellite polymorphic variants, Rep1-261, has been associated with an increase in mRNA levels of SNCA in experiments with transgenic mice [17]. At the same time, the presence of the Rep1-259 allele has been demonstrated to correlate with a low level of SNCA mRNA in post mortem brain tissues of patients with PD [18]. It was shown that Rep1 was associated with rs2583988, rs2619363 and rs2619364, and that these polymorphic variants were in a strong linkage disequilibrium (LD: r 2 = 0.99, D' = 1.00) [19,20]. In studies of patients with PD from Europe (Belgium, Germany and Serbia), an association of these polymorphisms with the risk of PD was identified [20][21][22].
Thereby, the aim of our study was to assess the role of rs2736990, rs2583988, rs2619363 and rs2619364 in the determination of genetic risk of development of PD in the Russian population. In addition, an analysis of the association between these SNPs and levels of SNCA transcripts in the peripheral blood of patients with PD from Russia was performed.

Patients
All patients (Russians residing in the European part of Russia) were diagnosed with PD at the Research Center of Neurology, Russian Academy of Medical Sciences (RAMS). All patients with PD were selected and studied according to the international Unified Parkinson's Disease Rating Scale (UPDRS) and Hoehn and Yahr scores [23,24]. The diagnosis of PD was based on the UK PD Brain Bank Criteria [25]. In this work, two groups of patients with PD were studied: 1) Patients with the sporadic form of PD at different stages of the disease (193 persons); 2) Newly diagnosed untreated patients with the sporadic form of PD (Hoehn and Yahr scores 1 -2) (48 persons). In total, we recruited 241 sporadic patients with PD (115 males, 126 females). The mean age ± SD at disease onset was 47.4 ± 13.6 years (30 -74), and the mean age at enrollment was 56.5 ± 13.8 years (32 -83). Two hundred twenty-six neurologically normal age-matched individuals from the same population were studied as controls. All blood samples were collected with the informed consent of the investigated persons. The study was approved by the Ethics Committee of the Research Center of Neurology, RAMS.

Genetic Analysis
Isolation of genomic DNA from leukocytes was performed using the AxyPrepBlood Genomic DNA Mini-prep Kit (Axygen, Tewksbury, MA, USA) according to the manufacturer's recommendations.
To determine the genotypes of SNPs rs2583988, rs2619363, rs2619364 and rs2736990 in the SNCA gene, real-time TaqMan ® PCR assays (ID: C_16258278_10, C_16036895_10, C_3113461_10 and C_3208948_10, respectively) were used (Applied Biosystems, Foster City, CA, USA). The real-time PCR and thermal cycling were performed according to the manufacturer's instructions using StepOnePlus (Applied Biosystems). Each blood sample was analyzed in three independent runs for corrections of differences in sample quality.

Expression Analysis
For expression analysis, all blood samples were taken at 8:00 a.m. before eating and then stored for less than 2 h at +4˚C before isolation of RNA. Isolation of total RNA from whole blood was performed using the ZR Whole-Blood Total RNA Kit™ (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer's recommendations. Reverse transcription was carried out using the RevertAid™ H Minus Reverse Transcriptase kit (Fermentas, Vilnius, Lithuania).
Relative levels of the expression of SNCA gene and reference genes (POLR2F, PSMB6 and PSMA5) were analyzed using TaqMan real-time PCR. Transcripts of reference genes were amplified together with the SNCA transcript to normalize gene expression data. Primers and probes were designed using Beacon Designer 7.02 software ( Table 1).
Real-time reactions were conducted using the ANK-32 machine (Institute for Analytical Instrumentation, St. Petersburg, Russia) and PCR reagents (Syntol, Moscow, Russia). Thermal cycling was performed as follows: 1) 600 s at 95˚C and 2) 45 cycles of 50 s at 95˚C and 20 s at 60˚C.
It is necessary to note that each blood sample was analyzed in three independent runs for corrections of differences in sample quality and reverse transcription efficiency. Normalization was performed on the basis on amplification levels of reference genes. To assess the relative levels of gene expression, the method of comparison of amplification thresholds ΔCt was used [26].

Statistical Analysis
Estimation of OR and statistical significance of differences in allele and genotype distribution were performed using the software GraphPad InStat v 3.06 (GraphPad Software, Inc.).
The Hardy-Weinberg equilibrium calculator was used to calculate the compliance of genotype distribution in a opulation to the Hardy-Weinberg equilibrium using the p  Significance of the differences in expression levels of different genotypes was determined by the Kruskal-Wallis ANOVA method using the software package Statistica for Windows 8.0 (StatSoft, Inc. 2007).

Genetic Analysis
In this study, we analyzed four SNPs, three of which were located in the promoter region of the SNCA gene (rs2619363, rs2619364 and rs2583988); SNP rs2736990 was located in the fourth intron of the same gene. For each studied SNP, the genotype distribution is consistent with the Hardy-Weinberg equilibrium (for rs2583988: χ 2 = 0.23 and p = 0.63; for rs2619363: χ 2 = 0.01 and p = 0.92; for rs2619364: χ 2 = 0.08 and p = 0.77; for rs2736990: χ 2 = 1.32 and p = 0.25). Genotype data are shown in Table 2.
The presented data show no statistically significant differences in the genotype distribution of the three SNPs (rs2583988, rs2619363 and rs2619364), located in the SNCA promoter region, between patients with PD and the population control. However, a statistically significant difference was shown in the genotype distribution of rs2736990 between the studied groups. Genotype CC predominates in patients with PD and the presence of the C allele is associated with an increased risk of the development of PD.

Analysis of mRNA Levels of SNCA Transcripts
We investigated whether the relative mRNA levels of the SNCA gene depend on the genotypes of rs2583988, rs2619363, rs2619364 and rs2736990 in patients with PD with early stages of the disorder. The results are summarized in Table 3.
Carriers of the rare TT genotype (rs2583988 and rs2619363) and GG genotype (rs2619364) have higher relative mRNA levels of SNCA gene compared with more common genotypes of these SNPs ( Table 3) in patients with PD. On the other hand, it was found that the TT genotype of rs2736990 is associated with lower relative expression levels of SNCA mRNA than genotypes CC and CT. However, the observed differences are not statistically significant.
It should be noted that the data on the relative expression levels for rs2583988 and rs2619364 coincide. Such coincidence is observed in genotype frequencies of these polymorphisms ( Table 2). Perhaps this is because these two SNPs are in complete linkage disequilibrium.

Discussion
We carried out a genotype analysis of rs2736990, for which an association with the development of PD, has been repeatedly shown. Our findings are consistent with the GWAS observations [13,28,29]. As can be seen from Table 2, we succeeded in showing an association of rs2736990 in the SNCA gene with the risk of the sporadic form of PD in the Russian population.
We obtained OR = 1.9 (95% CI: 1.24 -2.91, p = 0.003), which reflects an increased risk of PD almost twofold in carriers of allele C. These findings are also supported by results of genome-wide studies by Simón-Sánchez and Edwards (OR = 1.29 at p = 0.01, OR = 1.27 at p = 6.17 × 10 -13 and OR = 1.23 and p = 2.24 × 10 -16), obtained in large groups of patients with PD [12,30,31]. Furthermore, during analysis of the expression levels of SNCA transcripts, it was shown that these levels were higher in carriers with genotypes CC and CT in rs2736990 than that in patients carrying the TT genotype,   although the differences were not statistically significant. As mentioned above, rs2736990 is located in the center of the fourth intron of the SNCA gene. Studies of patients with PD from Germany and Japan demonstrated that there was a linkage between intron 4 and the 5'-and 3'untranslated regions (UTRs) of SNCA [32,33]. Perhaps rs2736990 is linked with the functionally significant variant in the 3' or 5' UTRs of the gene that may influence the expression of SNCA. Rs2583988, rs2619363 and rs2619364, located in the SNCA gene and linked with Rep1, were also genotyped in this study [20,21]. Our study showed no association of these SNPs and the development of PD in Russian patients (OR = 0.88 -0.91, 95% CI: 0.45 -1.74, p = 0.73 -0.87). These data are consistent with the investigation of Irish patients with PD, where no association among rs2583988, rs2619363 and rs2619364 and the development of PD was found (OR = 1.14 -1.18, 95% CI: 0.76 -1.78) [19]. However, higher relative levels of SNCA mRNA were observed in PD patients carrying the TT genotype (for rs2583988 and rs2619363) and the GG genotype (for rs2619364) in peripheral blood compared with carriers of the C allele in rs2583988, G-in rs2619363 and A-in rs2619364. Although no association of any change in the expression levels of the SNCA gene with SNPs rs2583988 and rs2619363 was found in the work by Linnertz, at the same time, an association between changes in SNCA gene expression and Rep1, linked with these polymorphisms, was demonstrated [18]. It seems like that the three SNPs in the promoter region of the SNCA gene have an insignificant effect on the expression of SNCA.
It is possible that rs2736990 or/and an unknown linked causal sequence variant may regulate transcription of SNCA either directly or indirectly through coordination with transcriptional enhancers and repressors. The results of our study improved our understanding of the contribution of genetic variants within the SNCA locus to spo-radic PD. Better knowledge of the molecular mechanisms that modulate SNCA gene expression, may lead to novel therapeutic approaches based on changes in SNCA levels. Further, this SNP rs2736990 in SNCA gene may be included into the panel of biomarkers for the assessment of the individual risk of PD development.

Acknowledgements
This work was supported by the Russian Foundation for Basic Research (projects no. 12-04-31091, 12-04-01183а), as well as by programs of the Russian Academy of Sciences (Molecular and Cellular Biology, Fundamental Sciences for Medicine), state contracts (no. 16