Variants in the 3′ End of SLC6A3 in Northwest Han Population with Parkinson's

Parkinson's disease (PD) is one of the most common neurodegenerative disorders in neurology. It is possible that multifactorial and genetic factors are related to its pathogenesis. Recently, there have been reports of SLC6A3 genetic variants leading to PD. However, the role of 3′ end of SLC6A3 in PD is less studied in different ethnic groups. To explore the roles of 3′ end of SLC6A3 in PD development, 17 SNP sites in 3′ end of SLC6A3 were analyzed in 360 PD patients and 392 normal controls of Han population residing in northwest of China. The significant difference of gene type and allele frequencies between the PD and control groups was detected only in rs40184 (P = 0.013 and 0.004, respectively; odds ratio 2.529, 95% confidence interval 1.325–4.827). The genotype and allele frequencies of the other 16 SNP sites were not found to be different between the PD group and the control group. rs2550936, rs3776510, and rs429699 were selected to construct the haplotypes; no significant difference was found in a frequency of 5 haplotypes between the PD group and the control group. These results suggest that the SLC6A3 variant in rs40184 A allele may increase the risk of PD in northwest Han population and may be a biomarker of PD.


Introduction
Parkinson's disease (PD) is a neurodegenerative disease; its prevalence increases with age, and PD influences 1% of the population over 60 years [1,2]. Due to the high disability rate and long course of illness, PD has seriously impacted the quality of life of middle-aged and elderly people. Worldwide, the burden of medical expenses on Parkinson's disease has risen from 2.5 million patients in 1990 to 6.1 million patients in 2016, almost doubled in 26 years [3]. e detail mechanism of the etiology and pathogenesis of PD is still unknown. e evidence from most studies showed that PD was a complex multifactor disease influenced by environmental and genetic factors [4,5], in which genetic factors play a critical role [6][7][8]. Up to now, the genetic susceptibility of Parkinson's disease is mainly focused on the screening of new genes in the PD family and the exploring of susceptible genes in patients. e genetic susceptibility genes in patients are mainly concentrated in the dopamine metabolic system genes, including the catecholamine oxymethyltransferase gene, the monoamine oxidase gene, the dopamine receptor gene, and the dopamine transporter (DAT) gene (gene symbol: SLC6A3), in which the DAT gene plays an essential role in the pathogenesis of PD. DAT is a transmembrane protein which is expressed by the presynaptic dopamine neuron. e main function of DAT is to reuptake dopamine released into the synaptic space and to stop the transmission of information among nerve cells [9,10]. In addition, neurotoxin 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) enters into the synapse cell through DAT. Finally, MPTP destroys the black dopaminergic neurons by oxidative stress.
Since PD is characterized by the selective loss of dopaminergic neurons, genes that affect the expression of dopaminergic neurons may become candidate genes for PD. e evidence from genetic studies revealed that variants in SLC6A3 were associated with PD [11][12][13]. e most extensively studied polymorphism was the variable number of tandem repeats (VNTR) in the 3′untranslated region (UTR) of SLC6A3 gene. However, the results from a number of related studies have not yet reached a consensus on its genetic correlation with PD. Moreover, there are few related studies on other polymorphisms at the 3′ end of SLC6A3 and PD. In order to further investigate the correlation between SLC6A3 and PD in northwest Han population, we performed a variants study in 3′ end of SLC6A3 in 360 PD patients and 392 healthy controls.  [14]. e control group consisted of 392 age-and sex-matched healthy persons (63.7 ± 9.7 years) from the same geographic areas. is study was approved by the Institutional Review Board of the Inner Mongolia Medical University, Hohhot, China. e study obtained the informed consent of all participants.

Genotyping.
Genomic DNA was extracted from leukocytes in a peripheral blood sample using a blood DNA extraction kit (TIANamp Blood DNA kit; TIANGEN BIOTECH, Beijing, China), which was stored at −20°C. Gene typing was performed using the polymerase chain reaction (PCR)/ligase detection reaction assay. Primers were synthesized by HAYU Biological Engineering LTD in Shanghai; the information of the primers is shown in Table 1. e probe for each group of ligase detection reactions consists of one common probe and two discriminating probes for the two types, as shown in Table 2.
e multiplex PCR methods were used to amplify the target DNA sequences. e final volume of PCRs for each subject was 20 μl, which contained 1X PCR buffer, 3.0 mM/ L·MgCl 2 , 2.0 mM/L deoxynucleotide triphosphate, 0.5 μmol/μl primer mix, 5 U/μl Qiagen HotStarTaq Polymerase (QIAGEN, Shenzhen, China), 1X Q-solution, and 50 ng/μl genomic DNA. e thermal cycle was carried out in GeneAmp PCR system 9600 (Norwalk, CT.06859, USA). e initial denaturation was 2 min at 95°C, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 90 s, and extension at 72°C for 1 min. e final extension of 72°C is 10 min. e total volume of the ligase detection reaction is 10 μl for each subject, which contains 1X NEB Taq DNA ligase buffer 1 μl, 2 pmol of each probe mix 1 μl, 5 U/μl Taq DNA ligase 0.05 μl (BIOWING, Jiangsu, China), and 4 μl multi-PCR product. A total of 40 ligase detection reaction cycles were performed under conditions of 92°C for 2 min, 94°C for 15 s, and 50°C for 25 s. e fluorescent products of ligase detection reaction were identified by PRISM 3730 (ABI). e experimental methods are similar to Chang et al. [15].

Statistical Analysis.
Statistical analysis was carried out by the Statistical Program for Social Sciences (SPSS version 11.0). Hardy-Weinberg equilibrium of each group was determined by using the chi-squared test. Allele and genotype frequencies between groups were studied using SHEsis software [16]. We use SHEsis software to calculate the coefficient D′ of linkage disequilibrium (LD) and to build haplotypes. A haplotype with a frequency of less than 3% is considered rare and ignored. When D′ is more than 0.8, it is considered that there is a strong linkage disequilibrium. A P value of 0.05 was considered to have statistical significance.

Association Study of TagSNPs and PD.
e genotype and allele frequencies of the 3′ end of SLC6A3 are summarized in Table 3. No deviation from Hardy-Weinberg equilibrium was evident in the PD and control groups (P > 0.05). Statistically significant differences in genotype and allele frequencies were found in the SLC6A3 variant rs40184 between PD (AA 5.6%, AG 47.2%, and GG 47.2%) and controls (AA 2.0%, AG 24.0%, and GG 74.0%). e frequency of the minor A allele was 29.2% in patients and 14.0% in controls.
ere was no significant difference in genotype and allele frequencies for other 16 SNP polymorphism sites in the 3′ end of SLC6A3.

Haplotypes of TagSNPs.
LD plots of the SLC6A3 in the study are shown in Figure 1. e LD was measured among the tagSNPs by the Lewontin standardized disequilibrium coefficient D′ [17]. Adjacent SNPs in strong LD (D′ > 0.8), rs2550936, rs3776510, and rs429699 have strong LD (D′ > 0.99), which were chosen to build the haplotypes for subsequent analyses. A total of 5 haplotypes were formed, and the frequencies of haplotypes are shown in Table 4. No significant difference was found in frequencies of 5 haplotypes between PD and control groups in the Han population.
Here, we conducted a case-control study of 752 participants in the northwest Han population to further investigate the role of the 3′ end of SLC6A3 in the development of PD. We found that only the SLC6A3 variant rs40184 had statistically significant differences in genotypes and alleles, which may be related to PD. e minor A allele of rs40184 may lead to an increased risk of PD in northwest Han Chinese, and it may be a marker of PD. However, the other 16 polymorphic sites were not found to be related to PD. e reason why the other 16 sites had negative results may be the insufficient number of samples in this study and that these 16 sites may be rare genotypes or alleles in the Han population. Haplotype analysis is thought to be much more powerful than singlenucleotide polymorphism sites in correlation studies [27]. Haplotype analysis greatly reduces the number of test samples and control type I errors although this method will increase the incidence of unavoidable type II errors [28,29]. Few studies have reported associations between haplotypes of the 3′ end of SLC6A3 and PD; we built the haplotypes among 3 strong LD tagSNPs, while no difference was found between the PD and the control group.
At present, more than 1,500 genes are known to be closely related to the occurrence of diseases. For a long time, coding areas have been a major research direction of genetic diseases and only a small part of noncoding area has been proved to be a useful component that can help genes to be turned on and off to regulate gene expression. e normal expression of genes cannot be separated from the participation of regulatory elements. Abnormalities in certain regulatory components can also lead to mutations in the corresponding gene-coding regions. erefore, we can assume that the variant rs40184 in the noncoding area of SLC6A3 may lead to differences in susceptibility to PD in our study.
In summary, our findings showed a link between the 3′ end of SLC6A3 gene variant rs40184 and PD in northwest Han population. Given that different populations are genetically heterogeneous and mutations have a specific population frequency, larger sample size studies are needed to confirm the correlation between the 3′ end of SLC6A3 variant and PD in independent larger cohorts and in different geographical origins. Moreover, functional studies of the 3′ end of SLC6A3 need to be carried out to further understand the role of SLC6A3 in PD. Further research is likely to find genetic variations that are risky or protective of PD, which is important for the prevention and treatment of PD.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Peiye Chang, Yongwang Fu, and Ping Zhao are the co-first authors.