Association Analysis of 27 Single Nucleotide Polymorphisms in a Chinese Population with Essential Tremor

Genetic factors play a major role in essential tremor (ET) pathogenesis. This study aimed to assess variant burden in ET-associated genes in a relatively large Chinese population cohort. We genotyped 27 single nucleotide polymorphisms (SNPs) previously reported to be associated with ET by multiplex PCR amplicon sequencing assay in 488 familial and sporadic ET patients and 514 healthy controls (HCs). Then, we performed allelic and genotypic association test by Pearson chi-square test or Fisher’s exact test. A total of 1002 samples were included in our analysis, consisting of 488 ET patients and 514 sex and age-matched HCs. For rs10937625, the C allele was linked to increased risk of ET (P = 0.019, OR = 1.503, 95% CI = 1.172–1.928). The carriers of the C/C homozygote and C/T heterozygote showed a significantly higher risk of ET, compared with the T/T homozygote under the dominant model (P = 0.019, OR = 1.628, 95% CI = 1.221–2.170). There were no statistically significant differences in the frequency of other SNPs between ET patients and healthy controls. Rs10937625 (STK32B) may increase the risk of ET in eastern Chinese population.


Introduction
Essential tremor (ET) is a common movement disorder. Nearly 1% of population and 4-5% in persons ≥ 65 years old suffered from this disease (Shanker 2019), and the prevalence of ET grows in an exponential manner with aging populations (Louis 2019). Although pathophysiological mechanisms of ET remain unclear, cerebellar oscillator hypothesis and cerebellar decoupling hypothesis have been raised (Erro et al. 2022). One frequently mentioned hypothesis suggests that genetic, epigenetic, and environmental factors work together to cause lesions in the inferior olive nucleus and cerebellum, which lead to abnormal function of circuitry involving the cerebello-thalamo-cortical pathway, and ultimately result in tremor with or without additional soft neurological signs of uncertain clinical significance (Shanker 2019).
Over 50% of ET individuals have family history, suggesting genetic factors play a major role in ET pathogenesis. To date, at least 14 loci and 11 ET pathogenic genes have been described and various risk or protective genetic factors have been discovered. Through linkage analysis, ETM1 (Gulcher et al. 1997), ETM2 (Higgins et al. 1997) and ETM3 (Shatunov et al. 2006) were identified. Whole exome sequencing (WES) and whole genomewide scan (WGS) discovered several candidate genes in ET families, including FUS (Merner et al. 2012), TENM4 (Hor et al. 2015), HTRA2 (Unal Gulsuner et al. 2014), SCN4A , SORT1 , SCN11A (Leng et al. 2017), NOS3, HAPLN4, and USP46 (Liu et al. 2016), CACNA1G, SLIT3, KARS, and KIF5A (Odgerel et al. 2019), andCCDC183, MMP10, andGPR151 (Diez-Fairen et al. 2021). ET is also associated with abnormal GGC repeat expansion in the 5′ region of NOTCH2NLC gene with a detection rate of 1.32-5.58% in Chinese's ET pedigrees (Sun et al. 2020;Yan et al. 2020) but much lower in Caucasian populations 1 3 (Liao et al. 2020a;Yau et al. 2021). In addition, several genome-wide association studies (GWAS) have been conducted for ET (Liao et al. 2022;Müller et al. 2016;Stefansson et al. 2009;Thier et al. 2012). To date, more than 20 possibly related single nucleotide polymorphisms (SNPs) have been reported in previous studies. The Asian population is the largest worldwide and thus makes up a significant fraction of patients with ET globally. Studies have demonstrated both similarities and differences in genetic factors underlying ET in Asian and European individuals, such as the protective role of C allele of rs10937625 in STK32B and risk factor rs7903491 in CTNNA3 (Müller et al. 2016;Xiao et al. 2017;Zhang et al. 2017). However, only part of SNPs reported have been tested in Chinese Han population (Chen et al. 2019;Zhang et al. 2017;Zuo et al. 2010), and due to the limited sample size and high clinical heterogeneity, no definite relationships have been established between the SNPs and ET etiology. Further studies are needed to confirm contributions of these SNPs to the pathogenesis of ET and explore any underlying mechanisms.
To identify SNPs that modulate the risk of ET in the Chinese Han population, we designed a case-control study to evaluate the association between 27 SNPs in 19 genes and ET in an Eastern Chinese patient cohort.

Patients and Controls
A total of 488 patients with ET and 514 healthy control (HC) subjects included in our study were recruited from the Department of Neurology, the Second Affiliated Hospital of Zhejiang University, between January 2015 and September 2020. Two or more experienced neurologists confirmed that all patients fulfill the established guidelines of the Consensus Statement of the Movement Disorders Society for the diagnosis of essential tremor (Bhatia et al. 2018). Patients with psychogenic tremor, hyperthyroidism tremor and other secondary causes of tremor have been excluded in our study samples. HC subjects were recruited through advertisements posted in the hospital. Family history of tremor was excluded in HCs.
This study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University. All the samples were collected after obtaining written informed consent in accordance with the Declaration of Helsinki.
We documented a detailed medical history of patients with ET, and two experienced movement disorders specialists performed a thorough neurological evaluation. The cognitive status was evaluated by the Mini-Mental State Examination. Hamilton Anxiety Scale (HAMA) and Hamilton Depression Scale (HAMD) were used to assess patients' anxiety and depression status.

Gene Selection
A detailed literature search was performed manually to find ET associated genes in the papers published until April 23, 2020 using the key words "essential tremor," "ET," "gene," "variant," "mutation," and "single nucleotide polymorphisms" on PubMed database. All the ET-associated genes selected in our study should be identified in at least one genetic study including linkage studies, WES, WGS, and GWAS studies performed in ET families and sporadic cases. The 27 possible ET-associated genes were listed in Supplementary Table 1.
The primer is designed by Hangzhou Cred Technology Co., Ltd, Tm set at 60 ± 2 °C and GC% at 40 ~ 60%. Primer sequences are provided in Supplementary Table 4. Add a fixed sequencing adapter to each PCR primer. The amplified length is between 50 and 250 bp. No homologous region in genome after blast.
The barcoded PCR were mixed and cleaned by DNA Clean Beads. Concentration of the DNA was determined using Invitrogen™ Qubit™ dsDNA HS Assay Kit and Agilent 2100. We used 20 ng of DNA sample as the template, with the following thermal cycling conditions to amplify targeted loci: 98 °C for 3 min; followed by 20 cycles of 98 °C for 20 s and 60 °C for 4 min; a final extension at 72 °C for 2 min; and an infinite hold at 10 °C. PCR products were purified using DNA Clean Beads. Each targeted amplicon (the beads with purified PCR products) was barcoded and amplified using the following PCR cycling conditions: an initial incubation at 98 °C for 2 min; followed by 6 cycles of 98 °C for 15 s, 58 °C for 15 s, and 72 °C for 30 s; a final extension at 72 °C for 2 min; and an infinite hold at 10 °C. Libraries were purified using DNA Clean Beads and quantified by a Qubit® dsDNA HS Assay Kit, according to the manufacturer's instructions for final normalization to produce equal volumes before sequencing. Sequencing was performed using the Illumina® NovaSeq™ 6000 System (Illumina, San Diego, CA, USA) with a 2 × 150-bp strategy, according to the manufacturer's recommendation.

NGS Data Analysis
The libraries were sequenced by HiSeq Novaseq 6000. The cleaned reads were grouped to each sample based on index primers and then were mapped to the human reference genome (hg19) using Bowtie and SAMtools to create BAM and index files. Alignment data were next subjected to a strategic procedure for variant calling by GATK. All hotspots are checked for the mutation rate in BAMs and added the variants with a high mutation rate (> 0.1) and high depth (> 30 ×) as a complement to GATK detection. The genotype call is expressed as homozygous (allele fraction ≥ 0.8), heterozygous (0.1 ≤ allele fraction < 0.8), and wildtype (allele fraction < 0.1).

Sanger Sequencing
Sanger sequencing was performed on an ABI 3730 sequencer following PCR using the same primers and same samples as for NGS to provide orthogonal confirmation of the three SNPs (rs7033345, rs9652490, and rs2071746) in five samples.

Statistical Analysis
All statistical analyses were performed using SPSS 26.0. Mann-Whitney U test was used to assess differences in age between ET patients and HCs. Chi-square test was used to detect the Hardy-Weinberg equilibrium (HWE), 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 (CI) for each SNP was calculated to assess the relative risk between the SNPs and ET susceptibility according to dominant and recessive models. The obtained P values were corrected by false discovery rate (FDR) test. Power calculations were performed with the Power and Sample Size Calculation (PS) program (Dupont and Plummer 1998). P < 0.05 was considered statistically significant.

Results
A total of 1002 samples were included in our analysis, consisting of 488 ET patients (223 males and 265 females, mean age = 56.85 ± 16.45 years) and 514 HCs (241 males and 273 females, mean age = 56.15 ± 13.79 years). The demographic and clinical characteristics of the ET patients and HCs are listed in Table 1. There were no statistically significant differences found in age (P = 0.059) or gender (P = 0.706) between ET and HC groups.
After correction for multiple comparison analysis, the differences in allele frequency of rs10937625 remained significant. For rs10937625, the C allele was linked to increased risk of ET (P = 0.019, OR = 1.503, 95% CI = 1.172-1.928, Table 2). The carriers of the C/C homozygote and C/T heterozygote showed a significantly higher risk of ET, compared

Discussion
Genetic factors play an important role in the pathogenesis of ET. However, no genetic testing results can be dependent on diagnosis, prognostic evaluation, and genetic counseling of ET. The positive detection rate of reported gene variants in ET patients is very low, and no high frequency causative genes have been identified, so ET is regarded as a group of genetically heterogeneous spectrum diseases. ET-related genetic research has discovered an increasing number of new variants and loci through linkage studies, WES, WGS, and GWAS studies and has validated them in different populations, which can help clinicians to have a better understanding of the disease and lay foundation for elucidation of genotype-phenotype relationships and further in-depth research on the pathogenesis of ET. In our case-control study, we demonstrated that the SNP rs10937625 in STK32B may modulate the risk of ET in Han population from eastern China.
This study suggests that the STK32B rs10937625 variant may be associated with ET in an eastern Chinese population. The STK32B gene (OMIM 119,530), located in chromosome 4p16.2, encodes a serine-threonine protein kinase.   The encoded protein is primarily expressed in kidney, but its effect is still largely unknown. Overexpression of this gene in human cerebellar DAOY cells significantly enriched pathways such as axon guidance, calcium ion transmembrane transport, and olfactory transduction. We also identified previously implicated ET genes whose expression is dysregulated through the overexpression of STK32B, suggesting that overexpressed STK32B may have relevant downstream effects (Liao et al. 2020b). It physically interact with PHF21A, a member of the BRAF-histone deacetylase repression complex (BHC), which is highly expressed in human fetal brain during neurodevelopment (Klajn et al. 2009). PHF21A mutations have been associated with intellectual disability, craniofacial anomalies by autism spectrum disorder, epilepsy, hypotonia, and neurobehavioral problems (Kim et al. 2019).
We found that C allele in rs10937625 (T > C) increases the risk of ET (OR = 1.455), and C allele also has a higher frequency in familial ET than sporadic ET but not statistically significant (P = 0.133), indicating its possible adverse effect in ET genetic etiology.
In 2016, a GWAS from European population identified that C allele of rs10937625 in the STK32B gene acts as a protective factor of ET and ET patients overexpressed STK32B in cerebellar tissue, compared with healthy controls (Müller et al. 2016). There are two related studies in the Han Chinese population after that. One study performed in sporadic Parkinson's disease (PD) patients indicated no association between rs10937625 and PD (Shi et al. 2018). A case-control study including 218 ET patients and 315 healthy controls supported that the C allele of this locus was a protective factor for ET (Zhang et al. 2017), which was contrary to ours. Xiao et al. reported that there were no associations between rs10937625 and ET in a cohort from Singapore (Xiao et al. 2017). A meta-analyses revealed a marginal association between the STK32B rs10937625 and ET (OR = 0.80; I 2 = 61%; 95% CI = 0.65-0.99) (Siokas et al. 2020).
As mentioned above, former researches indicated that rs10937625 C have no effect on ET risk or be a protective factor for ET. The reasons for different results include ethnic differences (not only between European and Asian, but also people from different regions of Asia), sample sizes, and demographic characteristics. From previous candidate gene association studies we can see conflicting results and lack of replication are very common for many candidate genes, which raises the need for collaborative multiethnic studies. Therefore, independent replication analysis is required to validate the role of this locus in ET risk. Despite the high prevalence of ET in the population, many patients with mild symptoms do not come to clinic. Replication analysis may take 5 years or more to implement and requires collaboration between centers. After all, the number of participants included in this study (488 essential tremor patients and 514 healthy controls) was not fewer than that included in several previous independent case control studies. The allele frequencies of SNPs in previous case-control studies were reviewed and listed in Supplementary Table 3. This study has some limitations. Not all the SNPs attain sufficient power. Associations of low impact are hard to detect in our study because of the limited sample size. And our samples are mainly from eastern China's provinces; therefore, the results may not be representative of other countries and regions.

Conclusions
In summary, this case control study shows rs10937625 (STK32B) might increase the risk of ET in Chinese population. Our data provides further evidence for the genetic etiology of ET.

Acknowledgements
We would like to thank all of the patients for their participation in this study.

Author Contributions
All authors contributed to the study conception and design. Lanxiao Cao, statistical analysis and original draft preparation; Luyan Gu, statistical analysis and interpretation of data for the work; Jiali Pu, design of the work; Dayao Lv, Jun Tian, Xinzhen Yin, Ting Gao, Zhe Song, Jinyu Lu, Gaohua Zhao and Baorong Zhang, acquisition of data for the work; Ya-ping Yan, design of the work and revising the work critically for important intellectual content; Guo-Hua Zhao, design of the work and revision. All authors read and approved the final manuscript.

Data Availability
The datasets generated during the current study are not publicly available because they are a part of ongoing research. Still, they are available from the corresponding author on reasonable request.

Declarations
Ethics Approval This study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University. All the samples were collected after obtaining written informed consent in accordance with the Declaration of Helsinki.
Informed Consent This article does not contain any previously unpublished studies that would require informed consent.