Assessment of three essential tremor genetic loci in sporadic Parkinson's disease in Eastern China

Abstract Objective The aim of this study was to investigate potential genetic overlap between essential tremor and Parkinson's disease in a cohort of 825 subjects from an Eastern Chinese population. Methods A total of 441 Parkinson's disease patients and 384 healthy controls were recruited. The MassARRAY System was used to detect three essential tremor‐related single nucleotide polymorphisms. Odds ratio (OR) and 95% confidential interval (CI) were calculated to assess the relationship between polymorphisms and Parkinson's disease susceptibility. Results Our results demonstrated that the odds ratios of rs3794087 of SLC1A2, rs9652490 of LINGO1, and rs17590046 of PPARGC1A were 0.71 (95% CI = 0.55‐0.91), 0.99 (95% CI = 0.78‐1.26), and 0.88 (95% CI = 0.62‐1.25), respectively. Conclusion An essential tremor SNP (rs3794087 of SLC1A2) is associated with a decreased risk of PD in the Eastern Han Chinese population, while rs9652490 (LINGO1) and rs17590046 (PPARGC1A) do not show an association.

variants (LINGO1 rs9652490; SLC1A2 rs3794087; and PPARGC1A rs17590046) that were previously reported to be associated with ET. Among them, LINGO1 rs9652490 and SLC1A2 rs3794087 were two lead single nucleotide polymorphisms of previous small genome-wide association studies in ET. 7-9 PPARGC1A rs17590046 was also proved to be associated with essential tremor recently. 10 But they showed controversial associations in PD studies.

| Study participants
Parkinson's disease patients were recruited and diagnosed by two movement disorder specialists from outpatient clinics according to the diagnostic criteria provided by the Movement Disorder Society. 11 Patients with secondary causes of parkinsonism such as vascular, drug-induced, and toxin-induced, and other neurodegenerative diseases such as progressive supranuclear palsy, multiple system atrophy, essential tremor, and Wilson's disease were excluded. Unrelated healthy control subjects were enrolled from local communities and evaluated by two movement disorder specialists for exclusion of PD. Written informed consents were signed by all participants. This study was reviewed and approved by the Medical Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine in accordance with the Declaration of Helsinki.

| DNA preparation and genotyping
Two milliliter blood samples were collected from all subjects. DNA was extracted by a plant DNA rapid extraction kit (BioTeKe Corporation). Genotyping assays were designed using AssayDesigner 3.1. The following three single nucleotide polymorphisms (SNPs) were tested: rs3794087 of SLC1A2, rs9652490 of LINGO1, and rs17590046 of PPARGC1A. Agena MassARRAY Typer 4.0 (Agena, Inc) was used to determine the genotypes of these three SNPs in all subjects.

| Statistical analysis
Student's t test was used to assess differences in age between PD patients and controls. Chi-square test was used to detect the HWE (Hardy-Weinberg equilibrium), gender, and allele distribution differences between the two groups. Pearson chi-square test or Fisher's exact test was performed in risk analysis, and an OR with a 95% confidence interval (95% CI) for each SNP was calculated according to dominant and recessive models. Cochran-Armitage trend test was conducted to evaluate the OR of the additive model. The strength of polymorphisms and PD susceptibility was assessed by OR. The genetic power calculations were performed in Quanto. 12 All other statistical analyses were performed in IBM SPSS Statistics 23.0. A two-tailed P < .05 was considered significant for all analyses.

| RE SULTS
A total of 441 patients with PD (male/female: 235/206; age: 60.5 ± 11.1 years) and 384 unrelated healthy control participants (male/female: 197/187; age: 59.8 ± 8.3 years) who were natives of east China were included in this study. There were no significant differences in gender or age distribution between patients and controls (Table 1). High genotyping qualities were acquired in all genotyping reactions. All control genotype frequencies in all of the studied polymorphisms were in HWE. In genotypic analysis, the OR of dominant and recessive models of the SNP rs3794087 (SLC1A2) was 0.70 (95% CI = 0.51-0.95, P = .023) and 0.47 (95% CI = 0.24-0.93, P = .028), respectively. And the powers of dominant and recessive models were more than 0.6 ( Table 2 and Table 3). In allelic analysis, the OR of T allele of rs3794087 was 0.71 (95%CI = 0.55-0.91, P = .007). No significant difference was found in SNPs of LINGO1 and PPARGC1A.

| D ISCUSS I ON
The association between essential tremor and Parkinson's disease has been a subject of long-standing debates. [13][14][15] A study by Jankovic and colleagues was the first to suggest that some patients with essential tremor have a genetically increased risk for PD. 13 Later, Koller et al 14 argued that frequency of a family history of ET was higher among PD patients than control subjects but lacked statistically significant difference. Rajput et al 16  of rs9652490 (LINGO1) and rs17590046 (PPARGC1A) between PD patients and healthy controls.
The leucine-rich repeat and Ig domain containing 1 gene (LINGO1) was reported as the first genetic evidence of a link between ET and PD by Wszolek and colleagues. 17 The SNP of rs9652490 is a part of the Nogo receptor complex, which is involved in inhibition of oligodendrocyte differentiation, axonal myelination and regeneration, and dopaminergic neuronal survival. 18 Many studies have been performed to elucidate the role of LINGO1 in ET and PD. Among them, it is worth noting that one study performed in Chinese patients found the G allele of rs9652490 has a protective effect on Parkinson's disease, 19 whereas original studies found this allele to be risky for essential tremor. 8 A meta-analysis suggested a protective role for the rs9652490GG genotype in PD (HR = 0.70, P = .028). 20 However, a study of the rs9652490 SNP in an Austrian sample of PD patients and healthy subjects failed to demonstrate a link between PD or the tremor-based subgroup of PD. 18 Given these conflicting results, the role of LINGO1 in PD and ET needs to be further explored.
Solute carrier family 1-glial affinity glutamate transporter-member 2 (SLC1A2) encodes the major glutamate reuptake transporter EAAT2 of the brain, which removes glutamate from the synaptic cleft. It is notable that EAAT2 is strongly expressed in the inferior olive, which is implicated in rhythm generation in ET, but not in the substantia nigra. The SNP rs3794087 of SLC1A2 was found to be associated with ET in a GWAS study from a European population, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), or PGC-1a, regulates mitochondrial biogenesis and suppresses oxidative stress. 25 Accumulation of misfolded proteins has been shown to influence neuronal survival and vulnerability in PD. 26 It is also reported that PGC-1a plays a protective role against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity, which has been shown to be a causative factor of PD. 27,28 Rs17590046 in PPARGC1A was found to be significantly associated with increased risk of ET through a GWAS in an European population. 10 Recently, a study in Singapore verified the role of rs17590046 in ET patients with Asian ancestry. 29 Zhang et al 30  PD patients. They found that none of these variants including rs17590046 were significantly associated with PD. 31 Our study also failed to identify a relationship between rs17590046 and PD.

| CON CLUS ION
In conclusion, our data show that the ET polymorphism, rs3794087 (SLC1A2), reduces the risk for PD in the Eastern Chinese population and that the other two polymorphisms do not show an association with PD. Due to the limited sample size and racial heterogeneity of our cohort, additional studies may help to further illuminate the relationship between PD and these variants.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.