Parkin is the most common causative gene in a cohort of mainland Chinese patients with sporadic early‐onset Parkinson's disease

Abstract Introduction Genetic mutations associated with early‐onset Parkinson's disease (EOPD) vary widely among different ethnicities. We detected the genes associated with EOPD in a Chinese cohort using next‐generation sequencing (NGS) combined with multiplex ligation‐dependent probe amplification (MLPA) and analyzed the phenotypic characteristics of the mutation carriers. Methods Cohort of 23 sporadic EOPD patients (onset age ≤ 45 years) were recruited. Genetic causes were identified by a targeted NGS panel containing 136 known extrapyramidal disease‐causative genes. Multiplications or deletions of PD‐causing genes were detected using the MLPA method. Demographic and clinical data were obtained, analyzed, and compared between patients with and those without Parkin gene variants. Results We identified 14 pathogenic or likely pathogenic variants (12 in Parkin, 1 in LRRK2, and 1 in VPS13C) in 10 patients (43.5%) and 8 rare variants of uncertain significance in 9 patients (39.1%). Parkin (34.8%) was the most common causative gene among our patients cohort, and exon deletion (62.5%) was the main type of variant. Patients with Parkin mutations had a younger age of onset, longer delay in diagnosis, slower disease progression, higher frequency of hyperreflexia, fatigue, and less hyposmia compared to patients without Parkin mutations. Conclusion Our results revealed a higher prevalence of Parkin mutations in Chinese sporadic EOPD patients, and notably, exon deletion was the most common type of mutation. EOPD patients with Parkin mutations showed unique clinical characteristics.


| INTRODUC TI ON
Parkinson's disease (PD) is the second most common neurodegenerative disease. It is characterized by bradykinesia, rest tremor, rigidity, postural instability, and a progressive loss of dopaminergic neurons. Even though approximately 5%-10% of PD cases have genetic association (Arkinson & Walden, 2018;Lunati, Lesage, & Brice, 2018), the early-onset PD (EOPD) has a significantly higher probability of finding a genetic cause (Lunati et al., 2018). Previous studies have reported various causative genes in patients with EOPD with diverse detection rates ranging from 3.7% to 16.6% (Youn et al., 2019). Multigene panel tests and whole-exome sequencing might be effective in identifying genetic variants in Chinese patients with EOPD as shown previously among patients from Korea (Youn et al., 2019), Germany (Trinh et al., 2019), India (Pandey, Tomar, Kumar, Dinesh, & Thelma, 2019), Finland (Siitonen et al., 2017), and United Kingdom (Sandor et al., 2017) and so on. Several studies have reported novel heterozygous mutation in Parkin gene using target region capture and high-throughput sequencing in a single Chinese family with EOPD (Fang, Mao, Zhu, & Li, 2019;Huang et al., 2019;Shi et al., 2018). In recent findings, a clinical and genetic study of early-onset and familial Parkinsonism in Taiwan including on 324 patients with EOPD and 247 Parkinsonism pedigree provides a better understanding of the genetic architecture of PD in eastern Asia and broadens the clinical spectrum of PD-causing mutations (Lin, Chen, et al., 2019). Recently, domestic scholars provide a better understanding of the genetic architecture of PD among ethnic Chinese using whole-exome sequencing (Li et al., 2020).
Nevertheless, the research subjects and detection methods of these studies were heterogeneous. In order to provide more data characterizing the genetic basis of mainland Chinese EOPD, we performed next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) in 23 Chinese patients with sporadic EOPD.
Parkin (PARK2; OMIM#600116) encodes the E3 ubiquitin ligase, which plays an important role in mitochondrial quality control and turnover. Numerous variants throughout Parkin are linked to autosomal recessive PD (Arkinson & Walden, 2018). Parkin mutations are the most common cause of recessively inherited monogenic PD, accounting for about 8.6% of PD cases with disease onset below the age of 45 years (Kilarski et al., 2012). Younger age, lack of tremor, prominent gait impairment, and the involvement of lower extremities at the time of disease onset were the main characteristics. In addition, a delayed diagnosis was more frequently reported in patients with Parkin-related EOPD Ruiz-Lopez et al., 2019). Indeed, in the absence of genetic testing results, it is difficult to distinguish Parkin-PD patients from EOPD patients without monogenic cause solely on clinical grounds . To better depict the profile of Parkin-PD patients, in the present study, we performed a comprehensive clinical evaluation of EOPD patients and compared patients with and without Parkin mutations.

| Subjects and clinical assessments
Unrelated patients, from outpatient and ward, with EOPD from September 2018 to October 2019 were recruited. The diagnosis of PD was based on the UK Brain Bank diagnostic criteria, and the patients with age of onset less than 45 years were categorized under EOPD. The study was approved by the Ethical Committees of Peking University First Hospital, and an informed written consent was taken from all the participants.
We obtained basic demographic and clinical data, including sex, age, age of onset, disease duration, family history, and current medication. Unified Parkinson disease rating scale (UPDRS) and Hoehn and Yahr (H-Y) stage were used for the clinical evaluation of both motor and nonmotor features during "off" state. Mini-mental Status Examination (MMSE) was used for memory and cognitive assessment. We evaluated rapid eye movement (REM) sleep behavior disorder (RBD) using the RBD single-question screen (RBDSQ). Patients with scores greater than 5, based on RBDSQ, were categorized as having probable RBD (Stiasny-Kolster et al., 2015). Olfaction testing was performed using the China-Germany version Sniffin' Sticks' test (Pinkhardt et al., 2019). All the patients completed clinical evaluation, except one. Levodopa equivalent daily dose (LEDD) was calculated by an established dose equivalence method (Tomlinson et al., 2010).

| Genetic tests
Genomic DNA was extracted from patients' peripheral blood samples using standard procedures, and NGS was performed.
Genetic analysis, using a designed gene panel covering 136 most prevalent extrapyramidal disease-associated genes, was performed (Additional file 1: Table S1). Sanger sequencing with specific primers was performed to confirm the variants detected by NGS. We further performed the MLPA assay (MRC-Holland,  (Richards et al., 2015). Pedigree analysis of the variants was performed among the available family members.

| Statistical analysis
Descriptive and analytical statistics methods were applied using software SPSS 25.0. Data were presented as mean ± standard deviation (SD). Demographic data and clinical characteristics were compared between EOPD patients with or without Parkin gene variants.
Quantitative data such as age at onset and disease duration was analyzed by Student's t test. Frequency data was compared between groups by chi-square (χ 2 ) test or Fisher exact probability test. p < .05 was considered statistically significant.

| Patient characteristics
Unrelated sporadic EOPD patients (23), with 35.48 ± 8.41 years being the mean age at onset and 7.48 ± 6.10 years as mean disease duration, were recruited into this study. Demographic and clinical variables of the subjects are shown in (Additional file 2: Table S2).
None of the EOPD patients suffered from cognitive impairment.
All the patients responded well to levodopa but 43.5% were complicated by peak-dose dyskinesia. Six patients underwent bilateral TA B L E 1 Genetic information of patients with EOPD genes carrying variants of uncertain significance and known mutations subthalamic nucleus deep brain stimulation (DBS), all with good surgical outcomes.

| Genetic results
NGS was performed in all of 23 unrelated EOPD patients (Table 1) (Li et al., 2020). A wide variation was prevalent depending on the studied population, methods used, and the age of the subjects under study. In spite of the relatively small sample size, the efficiency of NGS was used to investigate the frequency of potentially pathogenic known PD genes mutations in EOPD, thus making NGS as an important clinical diagnostic tool in EOPD.

| D ISCUSS I ON
It has been reported that Parkin mutations are highly diverse, and they include missense mutations and nonsense mutations, frameshifts, rearrangements with exon deletion or multiplications (Lunati et al., 2018). The systematic MDSGene review showed the following mutation types: the most common structural variants (43.2%) followed by missense mutations in 22.3% and frameshift mutations in 16.5% cases (Kasten et al., 2018). Our data show exon deletions (62.5%) as the most common types of Parkin variants, and the locations of these exon rearrangements in the Parkin gene were detected in exons 2-9. But, we did not found the deletion of exons 1,10-12.
The results indicate that exon dosage mutations in Parkin gene might be the main cause for sporadic EOPD, similar to the reports among other Chinese studies (Guo et al., 2010(Guo et al., , 2015Lin, Zeng, et al., 2019).
Tang BS group (Guo et al., 2010) reported that 12.6% of the patients with sporadic EOPD (onset age ≤ 50) carried Parkin exon deletions by real-time quantitative PCR analysis in 2010 and later another study in 2015 revealed 11.4% of EOPD patients (onset age ≤ 45) with Parkin exon rearrangements by MLPA assay, deletion being the main mutational cause, especially in exon 2-5 (Guo et al., 2015).
A recent study by Lin, Zeng, et al. (2019) also showed exon copy number variations (CNVs) as relatively common in the Parkin gene (6.7%). Therefore, exon dosage mutation of Parkin is quite common in Chinese EOPD. Traditionally, CNVs were detected by real-time quantitative PCR or MLPA, and however, recently NGS is useful for CNV detection because coding regions are enriched for causal genetic variation (Kadalayil et al., 2015). In our study, we initially suspected six patients with exon CNVs in the Parkin gene, as detected by NGS, which were then further confirmed by MLPA assay. MLPA assay is a practical and effective method for screening large deletions and duplications, particularly in small sample sizes (Wildförster & Dekomien, 2009

| CON CLUS ION
In conclusion, this study presents a systemic genetic analysis of a Chinese cohort to elucidate the genetic architecture of sporadic EOPD. Our results revealed a higher prevalence of Parkin mutations in Chinese patients with sporadic EOPD and emphasized its unique clinical characteristics. Considering the relative small size of the current cohort, studies with larger populations are needed to further elucidate genetic dispositions among Chinese patients.

ACK N OWLED G M ENTS
We thank all patients for their participation in this study. This work was funded by the UULM-PUHSC-Joint-Center (PKU2017ZC001-4).

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest in this work.  TA B L E 2 Demographic and clinical data of the enrolled patients with EOPD drafted the manuscript. Y.Y and Z.W were responsible for the critical revision of the manuscript. E.P and L.Z approved the final version of the manuscript. Y.Y and Z.W should be considered joint senior authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.