Parkinson’s disease (PD) is a progressive neurodegenerative disease affecting 1% of people older than 60 years and 4% of people older than 85 years (Kalia and Lang, 2015). With aging of the population, prevalence is expected to exceed 5 million in China by 2030, accounting for almost half of the global number of patients with PD (Li et al., 2019). Parkinson’s disease involves complex pathogenic mechanisms, with advancing age as the greatest risk factor; both environmental and genetic factors also affect disease risk and progression. Approximately 5%–10% of patients develop PD early and display different clinical characteristics from PD in the elderly (Mehanna et al., 2014). Thus, PD is divided into early-onset Parkinson’s disease (EOPD) and late-onset Parkinson’s disease (LOPD) according to the age of onset. There is no unified international standard for defining the age of onset for EOPD. However, age boundaries have a significant impact on the statistical association of genetic mutations to PD. Taking the PRKN gene as an example, several European studies defining EOPD onset age at 45 found that more than 15% of sporadic EOPD patients carried pathogenic mutations in PRKN (Lücking et al., 2000; Periquet et al., 2003), while a Chinese study defining EOPD onset at 50 found only 2.66% of sporadic EOPD patients with such mutations (Zhao et al., 2020). At present, most studies set the age of onset for EOPD at ≤50 years old based on the genetic evidence from previous studies (Tanner et al., 1999). Genetic factors are closely related to EOPD. The earlier the onset age, the greater the possibility of detecting genetic variation (Kasten et al., 2018; Blauwendraat et al., 2020a,b; Zhao et al., 2020). Screening the pathogenic gene mutation spectrum in EOPD patients will help us to understand the pathogenesis of PD and explore targeted treatment strategies.

A growing number of pathogenic genes for PD have been identified and assigned PARK numbers according to their reported order (Lunati et al., 2018). Parkinson’s disease caused by pathogenic variants of these genes comply with Mendelian genetics, and are referred to as monogenic Parkinson’s disease. The rare pathogenic variants of these genes result in 20% of EOPD and no more than 3% of LOPD (Klein and Westenberger, 2012). The PRKN, PINK1 and DJ-1 genes are the three most common genes of autosomal-recessive EOPD, and account for approximately 13% of EOPD occurring before the age of 40 years (Puschmann, 2013). The SNCA, LRRK2 and VPS35 are autosomal-dominant genes with relatively clear pathogenic mechanisms (Lunati et al., 2018; Kluss et al., 2019; Cutillo et al., 2020). The significance of other genes requires further functional validation and replication in larger cohorts and different populations. These genes include PLOG, GBA, LRP10, RIC3, and RAB39b; suggesting a wide mutational spectrum for EOPD. Numerous studies around the world from Germany, Britain, Finland and South Korea, as well as southwest China, western China and Taiwan have revealed the genetic heterogeneity of different regions and races for EOPD (Siitonen et al., 2017; Lin et al., 2019; Tan et al., 2019; Trinh et al., 2019; Youn et al., 2019; Li et al., 2020; Zhao et al., 2020). At present, there is no genetic map of EOPD patients in eastern China.

To provide insights into the mutational profiles of eastern Chinese EOPD patients, this study screened mutations in 24 known PD-related genes through whole exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) in this cohort. In addition, the clinical characteristics of patients with pathogenic/likely pathogenic (P/LP) variants were analyzed to provide a correlation between genotype and phenotype.

This study investigated the genetic causes of EOPD patients in eastern China using WES integrated with MLPA. The results identified 14 patients (9.03%) with P/LP variants in PRKN, PLA2G6, LRRK2, SNCA, CHCHD2, TMEM230 and GIGYF2; the first case of LRRK2 c.6055G > A (p.G2019S) mutation in a Chinese population, and four novel P/LP mutation sites. This revealed some geographical heterogeneities.

The proportion of familial PD in this study (5.2%) was lower than in the study conducted in southwest (19.6%) of China (Li et al., 2020). However, the proportion of patients carrying P/LP variants in this study was a bit higher than the study in southwest (7.5%). Additionally, the proportion of patients carrying the P/LP variants in the sporadic EOPD cohort in southcentral of China was only 4.59% (Zhao et al., 2020). The most common mutated gene across all three studies was PRKN, as it accounted for nearly 50.0% of all patients with identifiable mutations. The most common form of pathogenic mutation in PRKN was exon rearrangement with compound heterozygous mutation. We also found that the mean age of onset for patients with PRKN mutations was 15 years earlier than that of EOPD patients without mutations, and this is consistent with previous reports (Kasten et al., 2018; Zhao et al., 2020). In addition to PRKN, an LP variant in PLA2G6 was also detected. The age of onset for this patient was 35 years, which was consistent with a previous report that patients with ARPD-related gene mutations had an early age of onset (Kasten et al., 2018). Previous studies have reported that patients with PRKN, PLA2G6, PINK1 and ATP13A2 mutations showed significantly earlier onset than patients with VPS13C and RIC3 variants, while those with LRRK2, SNCA and GIGYF2 mutations generally showed later onset (Zhao et al., 2020). Therefore, genetic testing can be helpful in diagnosing EOPD when symptoms occur before 40 years of age, especially for those with a family history of ARPD. In this regard, the MLPA technology is particularly useful in detecting exon rearrangement variations in EOPD patients, and should be considered for routine use in clinics.

It has been reported that the mutation frequency of PINK1 is secondary to PRKN, and occurs in 3.7% of AR-EOPD patients (Hernandez et al., 2016). However, pathogenic variants of PINK1 were not detected in our cohort, and one patient carrying the PINK1 variant was detected in the study of southwest and southcentral regions, respectively. A compound heterozygous variant of PLA2G6 (c.1634A > G, c.991G > T) was detected, which is the only mutation in a recessive gene detected in our study, aside from PRKN. The 33-year-old patient suffered from dystonia of the left foot, and developed dyskinesia after two years despite responding well to levodopa in early treatment, which was consistent with the clinical manifestation of dystonia-Parkinson’s syndrome caused by PLA2G6 (Paisan-Ruiz et al., 2009). The study in southwest China also detected one compound heterozygous variant of PLA2G6 (Li et al., 2020). The southcentral study detected three compound heterozygous variants of PLA2G6 in sporadic EOPD, and was found to be the second most frequent mutation in the ARPD cohort (Zhao et al., 2020). It seems that the mutation frequency of PLA2G6 in the Chinese population is higher than PINK1 and DJ-1, and most of them exist in the compound heterozygous state. Moreover, several single heterozygous mutations of PLA2G6 detected in Chinese PD patients were associated with impaired PLA2G6 phospholipase activity (Gui et al., 2013). Of particular interest is mutations in PLA2G6 have been linked to several neurological diseases including infantile neuroaxonal dystrophy (INAD) (Khateeb et al., 2006), neurodegeneration with brain iron accumulation (NBIA) (Morgan et al., 2006), hereditary spastic paraplegia (HSP) (Koh et al., 2019a) and PD, but the underlying mechanism is still unknown. Thus, further research on the complexity of gene-gene and gene-environment interactions will be required to unveil the role of PLA2G6 in the Chinese population.

Four novel mutations were detected in this cohort, all of which were SNVs of ADPD related genes. Two non-sense mutations, c.297C > A (p.Y99X) of CHCHD2 and c.4C > T (p.Q2X) of TMEM230, resulted in premature termination codons, which may produce truncated proteins and caused the disease through haploinsufficiency effect or non-sense mediated mRNA decay (NMD). TMEM230 is a recently discovered PD-associated gene and encodes membrane transporter 230. The physiological function of this protein is unclear; but it can be found in vesicle structures in dopaminergic neurons of the substantia nigra and Purkinje neurons of the cerebellum; and may participate in the transport and circulation of synaptic vesicles, autophagy, neurotoxicity, and Golgi secretion. Several PD-linked TMEM230 mutations have been reported in North American (Deng et al., 2016), Caucasian (Giri et al., 2017), and Chinese populations (Fan et al., 2017; Yang et al., 2017). However, functional studies are still required to determine the role of these variants. The CHCHD2 protein is located in the mitochondrial intermembrane space and is highly expressed in dopaminergic neurons of the substantia nigra. This protein is involved in the mitochondrial-mediated apoptotic pathway and regulates the function of complex IV in the mitochondrial respiratory chain (Imai et al., 2019). Abl2 kinase phosphorylation of CHCHD2 at Tyr99 is involved in the regulation of complex IV (Aras et al., 2017); the c.297C > A (p.Y99X) mutation identified in our study may be pathogenic by affecting CHCHD2 phosphorylation. There is a high linkage disequilibrium between the two mutations of DNAJC13, c.2578C > T (p.R860C) and c.2564A > G (p.N855S), which were reported to be the pathogenic variants of a large four-generation ADPD family (Vilariño-Güell et al., 2014). There may be synergism between these two mutations, affecting the endosomal transport and sorting function of the DNAJC13 protein, and participating in the deposition of α-synuclein aggregates and Lewy bodies in specific brain regions (Vilariño-Güell et al., 2014). Point mutations in SNCA may upregulate α-synuclein expression (Cronin et al., 2009), alter its solubility (Porcari et al., 2015), promote early oligomerization and accelerate α-synuclein fibrillation (Mohite et al., 2018). The c.14T > C (p.M5T) mutation in SNCA detected in our study may result in two possible pathogenic mechanisms. The first may be a gain-of-function (GoF) effect, resulting in increased oligomerization of α-synuclein, while the second may be a dominant negative (DN) effect, with compensatory over-expression of wild-type single-stranded DNA of the heterozygous variant. However, functional studies will be required to validate the pathogenic role of SNCA c.14T > C (p.M5T). The clinical manifestation of this patient is consistent with the phenotypic characteristics of missense mutation in SNCA: the age of onset is late (50 years old), the progress is rapid, with the development of obvious cognitive impairment (MMSE 24 points, MoCA 17 points) and mental symptoms (hallucinations, spousal infidelity delusions) after 2 years.

Finally, we report here the first case of LRRK2 c.6055G > A (p.G2019S) mutation in a Chinese population. This is a 48-year-old man with no family history of PD. His symptoms are characterized by rigidity and bradykinesia of both lower limbs with no obvious tremor; increased axial muscular tension; Pisa syndrome; few non-motor symptoms; slow disease progression and good response to piribedil. These are consistent with the clinical phenotype of Ashkenazi Jews with LRRK2 c.6055G > A (p.G2019S) mutations where carriers had longer disease duration; lower extremity onset; postural instability and gait difficulty; persistent response to levodopa for more than 5 years (Alcalay et al., 2013). The mutation frequency of this locus is high in North African Arabs and German Jews, but the positive rate in Eurasian populations is only 0.1%–4% (Lesage et al., 2006; Ozelius et al., 2006). Our detection of the first Chinese case with this mutation appears to be in line with these reported statistics; reinforcing the need for a deeper understanding of these population differences and their implications for diagnosis and treatment strategies.

There are several limitations in this study: first, the first identified case of LRRK2 c.6055G > A (p.G2019S) mutation in the Chinese population can benefit from more in-depth genetic analysis. Second, the four novel P/LP variants require functional experiments to clarify their pathogenic mechanism. Finally, we only detected rare mutations in the coding region and splicing site of known pathogenic genes; evaluation of non-coding regions may reveal more pathogenic variants. With whole genome sequencing combined with MLPA technology, this may become less challenging, and comprehensive mapping of the mutation spectrum of PD will be possible.

The mutation spectrum of EOPD patients in eastern China displayed different characteristics compared to previously reported populations. Larger scale epidemiological investigations across centers and regions, as well as in-depth mechanistic studies, will further improve our understanding of the etiology of EOPD. The EOPD subtyping system with a combination of clinical markers such as clinical phenotype, imaging and abnormal protein expression will facilitate early diagnosis, guide individualized treatment, and be an important means to realize clinical translation of genetic research.

According to national legislation/guidelines, specifically the Administrative Regulations of the People’s Republic of China on Human Genetic Resources (http://www.gov.cn/zhengce/content/2019-06/10/content_5398829.htm, http://english.www.gov.cn/policies/latest_releases/2019/06/10/content_281476708945462.htm), no additional raw data are available at this time. Data of this project can be accessed after an approved application to the China National Genebank (CNGB, https://db.cngb.org/cnsa/). Please refer to https://db.cngb.org/, or email: CNGBdb@cngb.org for detailed application guidance. The accession code CNP0002762 should be included in the application.

The studies involving human participants were reviewed and approved by the Ethics Committee of Nanjing Medical University. The patients/participants provided their written informed consent to participate in this study.

PH and JG: conceptualization. YZ, QZ, and PH: data curation and formal analysis. PH, LL, and JR: investigation. PH: writing – original draft. JG, BT, and WL: supervision and writing – review and editing. WL: funding acquisition. BT and WL: resources. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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