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The role of genetics in Parkinson’s disease:
a large cohort study in Chinese mainland
population
Yuwen Zhao,
1,2,
* Lixia Qin,
1,2,
* Hongxu Pan,
1
Zhenhua Liu,
1,2
Li Jiang,
1
Ya n H e ,
1
Qian Zeng,
1
Xun Zhou,
1
Xiaoxia Zhou,
1
Yangjie Zhou,
1
Zhenghuan Fang,
3
Zheng Wang,
4
Yaqin Xiang,
1
Honglan Yang,
1
Yige Wang,
1
Kailin Zhang,
1
Rui Zhang,
1
Runcheng He,
1
Xiaoting Zhou,
1
Zhou Zhou,
1
Nannan Yang,
1
Dongxiao Liang,
1
Juan Chen,
1
Xuxiang Zhang,
1
Yao Zhou,
1
Hongli Liu,
1
Penghui Deng,
1
Kun Xu,
1
Ke Xu,
1
Chaojun Zhou,
1
Junfei Zhong,
1
Qian Xu,
1
Qiying Sun,
4
Bin Li,
2
Guihu Zhao,
2
Tao Wang,
5
Ling Chen,
6
Huifang Shang,
7
Weiguo Liu,
8
Piu Chan,
9,10
Zheng Xue,
11
Qing Wang,
12
Li Guo,
13
Xuejing Wang,
14
Changshui Xu,
15
Zhentao Zhang,
16
Tao Chen,
17
Lifang Lei,
18
Hainan Zhang,
19
Chunyu Wang,
19
Jieqiong Tan,
3
Xinxiang Yan,
2
Lu Shen,
1,2
Hong Jiang,
1,2
Zhuohua Zhang,
3
Zhengmao Hu,
3
Kun Xia,
3
Zhenyu Yue,
20
Jinchen Li,
1,2,3,4
Jifeng Guo
1,2,3
and Beisha Tang,
1,2,3,4
*These authors contributed equally to this work.
See Morris (doi:10.1093/brain/awaa185) for a scientific commentary on this article.
This study aimed to determine the mutational spectrum of familial Parkinson’s disease and sporadic early-onset Parkinson’s disease
(sEOPD) in a mainland Chinese population and the clinical features of mutation carriers. We performed multiplex ligation-depend-
ent probe amplification assays and whole-exome sequencing for 1676 unrelated patients with Parkinson’s disease in a mainland
Chinese population, including 192 probands from families with autosomal-recessive Parkinson’s disease, 242 probands from fami-
lies with autosomal-dominant Parkinson’s disease, and 1242 sEOPD patients (age at onset 450). According to standards and
guidelines from the American College of Medical Genetics and Genomics, pathogenic/likely pathogenic variants in 23 known
Parkinson’s disease-associated genes occurred more frequently in the autosomal-recessive Parkinson’s disease cohort (65 of 192,
33.85%) than in the autosomal-dominant Parkinson’s disease cohort (10 of 242, 4.13%) and the sEOPD cohort (57 of 1242,
4.59%), which leads to an overall molecular diagnostic yield of 7.88%(132 of 1676). We found that PRKN was the most fre-
quently mutated gene (n= 83, 4.95%) and present the first evidence of an SNCA duplication and LRRK2 p.N1437D variant in
mainland China. In addition, several novel pathogenic/likely pathogenic variants including LRRK2 (p.V1447M and p.Y1645S),
ATP13A2 (p.R735X and p.A819D), FBXO7 (p.G67E), LRP10 (c.322dupC/p.G109Rfs*51) and TMEM230 (c.429delT/
p.P144Qfs*2) were identified in our cohort. Furthermore, the age at onset of the 132 probands with genetic diagnoses (median,
31.5 years) was about 14.5 years earlier than that of patients without molecular diagnoses (i.e. non-carriers, median 46.0 years).
Specifically, the age at onset of Parkinson’s disease patients with pathogenic/likely pathogenic variants in ATP13A2,PLA2G6,
PRKN,orPINK1 was significantly lower than that of non-carriers, while the age at onset of carriers with other gene pathogenic/
likely pathogenic variants was similar to that of non-carriers. The clinical spectrum of Parkinson’s disease-associated gene carriers
in this mainland Chinese population was similar to that of other populations. We also detected 61 probands with GBA possibly
pathogenic variants (3.64%) and 59 probands with GBA p.L444P (3.52%). These results shed insight into the genetic spectrum
and clinical manifestations of Parkinson’s disease in mainland China and expand the existing repertoire of pathogenic or likely
Received October 3, 2019. Revised March 19, 2020. Accepted April 6, 2020. Advance access publication July 2, 2020
V
CThe Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For permissions, please email: journals.permissions@oup.com
doi:10.1093/brain/awaa167 BRAIN 2020: 143; 2220–2234 |2220
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pathogenic variants involved in known Parkinson’s disease-associated genes. Our data highlight the importance of genetic testing
in Parkinson’s disease patients with age at onset 540 years, especially in those from families with a recessive inheritance pattern,
who may benefit from early diagnosis and treatment.
1 Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
2 National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan 410008,
China
3 Centre for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University,
Changsha, Hunan 410008, China
4 Department of Geriatrics, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
5 Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
Hubei 430022, China
6 Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China
7 Department of Neurology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
8 Department of Neurology, Affiliated Brain Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China
9 Department of Neurobiology, Xuanwu Hospital of Capital Medical University, Beijing 100053, China
10 Parkinson’s Disease Center, Beijing Institute for Brain Disorders, Beijing 100101, China
11 Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
Hubei 430022, China
12 Department of Neurology, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong 510282, China
13 Department of Neurology, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong 510632, China
14 Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450047, China
15 Department of Neurology, Henan provincial people’s hospital, Zhengzhou, Henan 450003, China
16 Department of Neurology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China
17 Department of Neurology, Hainan General Hospital, Haikou, Hainan 570311, China
18 Department of Neurology, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China
19 Department of Neurology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China
20 Departments of Neurology and Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY
10029, USA
*Correspondence to: Beisha Tang
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan
Province 410008, China
E-mail: bstang7398@163.com
*Correspondence may also be addressed to: Jifeng Guo
E-mail: guojifeng2003@163.com
*Jinchen Li
National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South
University, Changsha, Hunan 410008, China
E-mail: lijinchen@csu.edu.cn
Keywords: Parkinson’s disease; age at onset; whole-exome sequencing; disease-associated gene; pathogenic or likely pathogenic
variant
Abbreviations: AAO = age at onset; ADPD = autosomal-dominant Parkinson’s disease; ARPD = autosomal-recessive Parkinson’s
disease; CNV = copy-number variant; P/LP = pathogenic/likely pathogenic; sEOPD = sporadic early-onset Parkinson’s disease;
VUS = variants of uncertain significance
Introduction
As the second most frequent neurodegenerative disorder,
Parkinson’s disease is characterized by motor symptoms,
such as bradykinesia, resting tremor, muscle stiffness, and
postural instability, as well as non-motor symptoms, includ-
ing olfactory dysfunction, sleep disorders, constipation and
dysautonomia, which are due to the loss of neurons in sev-
eral brain areas and may occur before or after the loss of
dopaminergic neurons (He et al.,2018). Parkinson’s disease
is thought to be caused by a combination of ageing, as well
as genetic and environmental risk factors (Guo et al.,2018a;
Lim et al.,2019;Bandres-Ciga et al.,2020), and 5–10%
of patients with Parkinson’s disease have rare Mendelian
Genetic spectrum of Parkinson’s disease in China BRAIN 2020: 143; 2220–2234 |2221
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variants. To date, 23 genes with different degrees of genetic
evidence (Blauwendraat et al.,2019b) have been found to be
mutated in monogenic Parkinson’s disease (defined as
Parkinson’s disease-associated genes in this study), and var-
iants in the glucocerebrosidase (GBA) gene were found to be
an important risk factor in Parkinson’s disease (Sidransky
et al.,2009;Sun et al.,2010;Velez-Pardo et al.,2019). The
23 genes included 10 genes with recessive inheritance pat-
terns and 13 genes with dominant inheritance patterns
(Puschmann, 2013;Singleton and Hardy, 2016,2019;
Sudhaman et al.,2016;Quadri et al.,2018a;Blauwendraat
et al.,2019b).
Recently, several genetic-testing studies have been con-
ducted with Parkinson’s disease patients (Bandres-Ciga
et al.,2019;Lin et al., 2019;Tan et al.,2019a;Trinh et al.,
2019;Youn et al.,2019). These studies revealed that the
mutational spectrum does vary across different populations,
even within the same genes, which provided evidence of gen-
etic heterogeneity in Parkinson’s disease due to geographic
and ethnic differences, among the studied populations (Lim
et al., 2019). For example, the p.G2019S variation accounts
for most patients with LRRK2 mutations from the UK or
other European population-based cohorts (Montaut et al.,
2018;Tan et al.,2019b), whereas very few Asian patients
with Parkinson’s disease have been found to carry the
p.G2019S (Lin et al.,2019;Shu et al.,2019;Youn et al.,
2019). In addition to the ethnic and geographical heteroge-
neities of the subjects, other characteristics such as the age at
onset (AAO) and family history of Parkinson’s disease are
also associated with the prevalence of specific mutations
(Alcalay et al.,2010). Most disease-associated genes are first
identified in familial Parkinson’s disease patients (Deng
et al.,2016;Quadri et al.,2018a) and there is evidence that
the earlier the age at onset, the more likely a genetic cause
will be found (Xu et al.,2017;Guo et al.,2018a;Kasten
et al.,2018;Blauwendraat et al.,2020), in addition to the
correlation between age at onset and common polygenic
alleles (Escott-Price et al.,2015;Blauwendraat et al.,2019a).
Several large-scale studies have been conducted to screen
for Parkinson’s disease-associated genes in cohorts based on
UK (Tan et al.,2019b), Norwegian (Gustavsson et al.,
2017), European (Alcalay et al.,2010) and Taiwanese popu-
lations (Lin et al.,2019). In addition, some relatively small,
but more systematic studies were conducted with German
(Trinh et al.,2019) and Korean populations (Youn et al.,
2019). Previously, we performed mutation analysis of
PRKN,PINK1,DJ1,ATP13A2 (Guo et al.,2008),
PLA2G6 (Shi et al.,2011), CHCHD2 (Liu et al.,2015),
RAB39B (Kang et al.,2016), TMEM230 (Yan et al.,2017)
and other genes associated with familial Parkinson’s disease
or early-onset Parkinson’s disease, using a patient cohort
from mainland China. We also applied trio-based whole
exome sequencing to reveal the association between NUS1
and early onset Parkinson’s disease in Han Chinese individu-
als (Guo et al.,2018a). Some possible limitations were asso-
ciated with these previous studies. First, some of these
studies (Lin et al.,2019;Trinh et al.,2019) might have
focused on patients diagnosed as atypical parkinsonism, or
on disease-associated genes associated with other movement
disorders. Second, most of these studies (Alcalay et al.,
2010;Tan et al.,2019b;Youn et al.,2019), including our
previous studies, used techniques such as multigene panel
testing or Sanger sequencing, which only included a certain
set of genes, thereby limiting the comprehensiveness and ex-
tensibility of genetic testing. Therefore, it was necessary to
systematically study the mutational spectrum of the
Parkinson’s disease population in mainland China.
In this study, we comprehensively analysed the mutational
spectrum of known Parkinson’s disease-associated genes for
patients with familial Parkinson’s disease and sporadic early-
onset Parkinson’s disease (sEOPD) in a mainland Chinese
population, using gene dosage analysis and whole-exome
sequencing. First, we systematically identified pathogenic
and likely pathogenic (P/LP) variants of known Parkinson’s
disease-associated genes, including known variants and
novel variants, and summarized and compared the frequency
among patients with autosomal-recessive familial
Parkinson’s disease (ARPD), autosomal-dominant familial
Parkinson’s disease (ADPD), or sEOPD. Second, we com-
pared the frequency of P/LP variants among patients with
different AAOs and among different genes separately, in
order to determine differences in the molecular diagnosis,
and to depict the AAO spectrum for these disease-associated
genes in our population. Third, we generalized the clinical
manifestations of Parkinson’s disease patients with certain
mutated genes important for Parkinson’s disease pathogen-
esis, thereby contributing to genetic counselling, early diag-
nosis, and early intervention.
Materials and methods
Subjects
Participants with sEOPD (AAO 450 years old) or from fami-
lies with ARPD and ADPD were recruited at the Xiangya
Hospital (Central South University) between October 2006 and
January 2019 and at other co-operating hospitals of Parkinson’s
Disease and Movement Disorders Multicenter Database and
Collaborative Network in China (PD-MDCNC, http://pd-
mdcnc.com : 3111/) established by our team. The ARPD fami-
lies were consanguineous, or the probands in these families had
two or more affected siblings but no affected family members
within two consecutive generations, whereas Parkinson’s disease
was diagnosed in ADPD family members for two or more gen-
erations. Patients with sporadic, early-onset Parkinson’s disease
lacked a family history of Parkinson’s disease and had an AAO
of no more than 50 years old. Of them, 15 probands were
found to have certain mutations in specific target genes by real-
time quantitative PCR or Sanger sequencing analysis, in our pre-
vious studies (Tang et al., 2006;Guo et al.,2008,2010;
Shi et al.,2011). All participants fulfilled the UK Parkinson’s
disease Society Brain Bank clinical diagnostic criteria (Gibb and
Lees, 1988) or Movement Disorders Society (MDS) clinical
diagnostic criteria for Parkinson’s disease (Postuma et al.,
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2015). Patients with detectable polynucleotide repeats (SCA3,
SCA2, SCA1, SCA17 etc.) were identified as described in our
previous study (Wang et al.,2009),andexcludedfromthis
study. The study was approved by the Ethics Committee of
Xiangya Hospital (Central South University), and written
informed consent was collected from all participants. Genomic
DNA was prepared from peripheral blood leucocytes according
to standard procedures. A comprehensive dataset of basic demo-
graphic data, including the subjects’ age, gender, family history,
disease duration, and clinical features (including motor and
non-motor manifestations, shown in Supplementary material),
was collected from the Parkinson’s disease patients enrolled in
this study and inputted into the PD-MDCNC.
Multiplex ligation-dependent probe
amplification and quantification PCR
The Salsa Multiplex Ligation-Dependent Probe Amplification
Kit (P051-c1; MRC) was used to detect exon deletions or dupli-
cations [copy-number variants (CNVs)] of several Parkinson’s
disease-associated genes, including PRKN,PINK1,ATP13A2,
DJ1 and SNCA, according to the manufacturer’s instructions.
Furthermore, patients with SNCA duplications were later con-
firmed by real-time quantitative PCR (Klein et al.,2005).
Whole-exome sequencing and
analysis
Genomic DNA were isolated from peripheral blood leucocytes.
Whole-exome DNA was capture using the SureSelect Human
All Exon Kit V6 (Agilent) and high-throughput sequencing was
conducted using the Illumina 10 platform with an average
123coverage. Paired-end sequence reads were aligned with
the Burrow-Wheeler Aligner (Li, 2014) against the reference
human genome (UCSC hg19). The Picard tool (http://broadinsti
tute.github.io/picard/) was used to remove duplicate reads, gen-
erate the converse format, and index the sequencing data. Base
quality-score recalibration, local realignments around possible
insertions/deletions (indels), variant calling, and filtering were
performed with the Genome Analysis Toolkit (GATK)
(McKenna et al., 2010). ANNOVAR (Wang et al.,2010a;Yang
and Wang, 2015) and VarCards (Li et al., 2018a) was used to
systematically annotate the variants with RefSeq (UCSC hg19),
including gene regions, amino acid alterations, functional effects,
and allele frequencies in East Asian (EAS) population (GnomAD
database, ExAC database, and an in-house generated Chinese
control database, which included 1279 Chinese controls without
neurological disease). Among the recessive disease-associated
genes, the minor allele frequency (MAF) of the variants was lim-
ited to 0.01 for the above population database, whereas the
MAF of variants in dominant disease-associated genes was lim-
ited to 0.001.
Only predicted damaging missense and loss-of-function var-
iants (nonsense variants, frameshift indels, and splicing-site var-
iants) of 23 known Parkinson’s disease-associated genes were
included for further analysis. Non-frameshift indels in the
PRKN gene were also included in our study. Recently, our
group developed the ReVe (Li et al.,2018b) programme, which
was used in this study to predict deleterious missense variants.
Rare damaging variants, including CNVs, single-nucleotide var-
iants (SNVs), and indels, were further classified as pathogenic,
likely pathogenic or of uncertain significance, according to the
standards and guidelines from the American College of Medical
Genetics and Genomics (ACMG) (Richards et al.,2015;Li
et al.,2018b).
We searched the Gene4PD database (http://www.genemed.
tech/gene4pd/), which was recently developed by our group, the
MDSgene database (https://www.mdsgene.org/g4d), and
PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) to determine
which, if any, variants had been reported previously. Sanger
sequencing and multiplex ligation-dependent probe amplifica-
tion (MLPA) were also carried out in cases where affected or
unaffected samples from families who carried P/LP variants
were available, as described in our previous study (Guo et al.,
2018a). The study workflow is shown in Fig. 1.
Statistical analyses
Wilcoxon’s rank-sum test was used to compare the AAO and
mutation statuses of the patients. Linear regression was used to
compare demographic data with covariate adjustment. The rela-
tionship between clinical manifestations and the genetic status
(P/LP variant carriers or non-carriers) was assessed by perform-
ing linear regressions of continuous scores versus the genetic sta-
tus, adjusting for age at entry, disease duration, and gender. We
performed binary logistic regression of the symptom status
(motor complication) against the genetic status to assess correla-
tions between symptom status and genetic status. The Hoehn
and Yahr stage and motor subtype were analysed by ordered lo-
gistic regression and multinomial logistic regression, where the
tremor-dominant group was used as the referent group, respect-
ively. All P-values were two-tailed. Statistical analysis of the
data was conducted using SPSS software, version 24.0.
Data availability
The datasets supporting the results and conclusions of this
manuscript are included within the article and its
Supplementary material. All other datasets used and analysed
during the current study are available from the corresponding
authors on reasonable request.
Results
Cohort description
We included 1676 probands in this study, and they were
divided into the ARPD (n= 192), ADPD (n=242) and
sEOPD cohorts (n= 1242), based on the family history and
AAO. The baseline characteristics of the demographic data,
and the motor and non-motor manifestations of patients
from the ARPD, ADPD, sEOPD cohorts, and entire cohort
are presented in Table 1. Briefly, the mean AAO of pro-
bands from the ARPD, ADPD, and sEOPD cohorts were
45.30 ±16.47, 51.60 ±10.93 and 43.58 ±6.43 years, respect-
ively. The proportions of male subjects in each group were
51.0%, 54.5% and 54.8%, respectively.
We carried out whole-exome sequencing for all 1676
probands. On average, we obtained 11.66 GB clean data
with a mean sequencing depth of 123-fold. Over 99.83%,
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99.72%, or 99.32% of the targeted regions were covered at
least 1-fold, 4-fold, or 10-fold, respectively (Supplementary
Table 1). Specifically, our study focused on known
Parkinson’s disease-associated genes and high-risk gene
GBA (Supplementary Table 2). The workflow of this study
is presented in Fig. 1.
Mutational spectrum of members of
the ARPD cohort
In ARPD families, we detected 37 homozygous variants and
28 compound heterozygous variants of Parkinson’s disease-
associated genes in 65 of 192 probands (33.85%).
Interestingly, the molecular diagnosis was associated with
the AAO of the patients, which showed that the molecular
diagnostic rate was 93.02%, 78.38%, or 60.19% for ARPD
probands with an AAO of 430 years, 40 years, or 50
years, respectively. Of note, we did not detect any P/LP var-
iants in 84 ARPD probands with an AAO 450 years
(Fig. 2A and Supplementary Table 3).
Of the 65 probands with P/LP variants, PRKN was the
most frequently mutated gene, accounting for 50 (26.04%;
of 192 ARPD probands) molecularly diagnosed probands,
including 27 patients with homozygous variants and 23
patients with compound heterozygous variants
(Supplementary Table 3). Among these P/LP variants,
PRKN CNVs were detected in 42 probands (21.88%; of
192 ARPD probands), including 20 patients with homozy-
gous CNVs, eight patients with compound heterozygous
CNVs, and 14 patients with a combination of PRKN CNVs
and SNVs/indels. Exon deletions represented the most preva-
lent type of mutations, especially in exon 3 (n= 18) and
exon 4 (n= 19). The remaining 21 probands carried PRKN
P/PL SNVs/indels. We noted that two known variants,
p.G284R (n=7)andp.M1T (n= 3), were the most frequent
PRKN P/PL SNVs in our ARPD cohort (Supplementary Fig.
1A and Supplementary Table 4A). Interestingly, since we
included patients with loss-of-function variants and non-
frameshift variants of PRKN, we found that two probands
carried a novel PRKN splicing SNV (c.619-1G4C) and two
other probands carried the reported PRKN non-frameshift
deletion (c.968_973del/p.C323_V324del) (Guo et al.,2008),
which were extremely rare variants that were absent from
the East Asian populations of GnomAD exome, GnomAD
genome, the ExAC database, and our in-house Chinese con-
trol cohort. Of note, we found that the splicing mutation,
PRKN c.619-1G4C, caused exon 6 skipping and generated
a truncated protein where exon 6-encoded amino acids were
replaced with an abnormal 28 amino acid sequence followed
by premature stop codons (Supplementary material and
Supplementary Fig. 2D). Besides, PRKN c.968_973del was
found to cause the protein to lose mitochondrial localization
in CCCP-induced mitophagy (Supplementary material and
Figure 1 Workflow of this study. *Standard for allele frequency filtering: MAF 51% for the recessive model, MAF 51&for the dominant
model. Functional-effect predictions: loss-of-function variants or missense variants predicted to be damaging by ReVe. PD = Parkinson’s disease.
2224 |BRAIN 2020: 143; 2220–2234 Y. Zhao et al.
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Supplementary Fig. 2E). Both of them supported that they
could be deleterious and cause Parkinson’s disease.
Nevertheless, other unreported P/LP variants still deserve
further replication in larger cohorts or experimental studies
in the future.
We identified five probands carrying homozygous PINK1
P/LP variants and one proband carrying a compound hetero-
zygous PINK1 variant. In addition, another nine probands
were identified with P/LP variants in other recessive
Parkinson’s disease-associated genes, including PLA2G6
(n=6), ATP13A2 (n= 2), and DJ1 (n= 1). Of the
ATP13A2 gene carriers, two novel homozygous variants
(p.R735X and p.A819D) were identified in two consanguin-
eous families, in which two probands were initially diag-
nosed with Parkinson’s disease, and their follow-up
diagnosis (after molecular testing) was considered as
Parkinsonism, due to the presence of a red flag sign (patients
with p.R735X showed pyramidal tract signs) or the clinical
manifestation of absolute exclusion criteria (parkinsonian
features of patients with p.A819D restricted to the lower
limbs for more than 3 years) in MDS clinical-diagnostic cri-
teria for Parkinson’s disease (Postuma et al.,2015).
Mutational spectrum of members of
the ADPD cohort
In the ADPD families, 10 of 242 probands (4.13%) carried
P/LP variants of Parkinson disease-associated genes including
LRRK2 (n=5), SNCA (n= 4), and LRP10 (n=1) (Fig. 2B
and Supplementary Table 3). Five probands (2.07%) carried
Table 1 Summary of clinical features of the Parkinson’s disease patients in this study
Clinical features ARPD ADPD sEOPD All
(n= 192) (n= 242) (n= 1242) (n= 1676)
Age at onset, years 45.30 ±16.47 51.60 ±10.93 43.58 ±6.43 44.94 ±9.31
Male (%) 51.0 54.5 54.8 54.4
Age at assessment, years 53.50 ±14.65 56.93 ±11.22 50.14 ±7.66 51.51 ±9.60
Disease duration, years 8.20 ±7.57 5.33 ±3.95 6.56 ±5.46 6.57 ±5.61
Motor manifestations
UPDRS-Part I score 2.62 ±2.23 2.47 ±2.11 2.41 ±2.10 2.44 ±2.11
UPDRS-Part II score 12.40 ±7.44 11.60 ±7.00 11.87 ±6.77 11.89 ±6.88
UPDRS-Part III score 29.45 ±16.69 26.88 ±15.46 27.33 ±15.96 27.51 ±15.98
Tremor score 4.21 ±4.11 4.32 ±3.97 3.65 ±3.73 3.81 ±3.82
Stiffness score 5.75 ±4.32 5.23 ±4.09 5.65 ±4.28 5.60 ±4.26
Bradykinesia score 10.78 ±6.51 9.88 ±6.47 10.21 ±6.68 10.22 ±6.63
Postural instability score 4.73 ±3.47 3.99 ±3.31 3.99 ±3.14 4.07 ±3.21
Hoehn and Yahr stage (%)
0–1.5 20.3 31.0 32.5 30.9
2 or 2.5 48.4 46.7 43.2 44.3
3 + 31.3 22.3 24.3 24.8
Dyskinesia 17.9 11.7 17.5 16.7
Freezing gait 30.9 27.5 27.3 27.7
Motor subtype (%)
Tremor-dominant 27.10 34.7 24.9 26.6
Intermediate 17.20 16.9 16.9 16.9
PIGD-dominant 55.70 48.4 58.2 56.5
Non-motor manifestations
Cognition: total MMSE score 26.29 ±3.68 26.57 ±3.42 27.29 ±3.00 27.06 ±3.18
PDSS score 120.13 ±22.53 113.41 ±31.04 115.92 ±30.08 116.06 ±29.43
RBDQ-HK score 13.36 ±15.67 15.70 ±17.68 12.73 ±15.55 13.26 ±15.93
ESS score 7.84 ±5.95 7.76 ±6.41 7.24 ±6.08 7.40 ±6.11
HAMD score 5.96 ±5.34 5.99 ±6.35 5.79 ±5.48 5.84 ±5.61
HRS score 21.04 ±5.58 19.37 ±7.09 19.91 ±6.30 19.96 ±6.36
PFS score 45.85 ±18.81 42.97 ±19.56 44.29 ±18.98 44.29 ±19.04
SCOPA-AUT score 7.53 ±6.48 8.66 ±7.36 7.00 ±6.80 7.32 ±6.87
PDQ39 score 33.63 ±28.14 30.02 ±27.09 29.69 ±25.67 30.23 ±26.22
Scores in the last four columns are mean ±SD; gender, motor complication (dyskinesia, wearing-off, freezing gait), motor subtype, restless legs syndrome and constipation are
shown as numbers and proportions (%).Tremor score was measured by adding up score of tremors at rest and action and postural tremor of hands from UPDRS score, while brady-
kinesia score was calculated by score on finger taps, hand movements, rapid alternating movements of hand, and leg agility. And rigidity score was added up by the score on rigidity
of neck, hands and fee. Disease motor subtype was classified as tremor-dominant (TD) phenotype when the ratio of tremor score and postural instability and gait difficulty (PIGD)
score was no less than 1.5, while patients with a ratio of no more than 1.0 were defined to PIGD phenotype and rest of patients belonged to the indeterminate phenotype. ADPD
= PD families with autosomal dominant inheritance; All = ARPD + ADPD + sEOPD; ARPD = PD families with autosomal recessive inheritance; ESS = Epworth Sleepiness Score;
HAMD = 17-item Hamilton Depression Rating Scale; HRS = Hyposmia Rating Scale; PDQ39 = The 39-item Parkinson’s disease Questionnaire; PDSS = Parkinson’s disease sleep
scale; PFS = Parkinson’s disease fatigue scale; RBDQ-HK = REM sleep behaviour disorder questionnaire-Hong Kong; SCOPA-AUT = Scales for Outcomes in Parkinson’s disease-
Autonomic; sEOPD = patients with sporadic Parkinson’s disease with AAO younger than 50 years old; UPDRS = Unified Parkinson’s Disease Rating Scale.
Genetic spectrum of Parkinson’s disease in China BRAIN 2020: 143; 2220–2234 |2225
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Figure 2 Mutational frequencies of known Parkinson’s disease-associated genes in Parkinson’s disease cohorts. Mutational fre-
quencies of all (left) and each (right) known Parkinson’s disease-associated gene in the different cohorts. (A) ARPD cohort. (B) ADPD cohort.
(C) All familial Parkinson’s disease (FPD) cohort. (D) The sEOPD cohort. (E) Entire Parkinson’s disease (PD) cohort. *Variants that were classi-
fied as pathogenic or likely pathogenic according to the standards and guidelines of the ACMG. ‘Pathogenic’ means that the patients had patho-
genic variants in known Parkinson’s disease-associated genes, and ‘likely pathogenic’ means that the patients had likely pathogenic variants in
known Parkinson’s disease-associated genes.
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four LRRK2 missense variants: p.N1437D (n=2),
p.R1441C, p.R1441H, and p.K616R (n=1 each) (Wang
et al.,2010b)(Supplementary Tables 3 and 4A). LRRK2
p.N1437D, a missense variant absent from East Asian popu-
lations of public databases and our in-house control cohort,
was first identified in two families of our cohort; this vari-
ation segregated with the parkinsonism phenotype within
one family, but one unaffected member (a 65-year-old fe-
male) from another family also carried this variant. The
novel p.N1437D variant occurred at the same position as
another pathogenic missense change (p.N1437H), which
was only reported previously in two Norwegian families
(Aasly et al.,2010). Moreover, we performed an in vitro
kinase assay to assess the respective enzymatic properties of
wild-type protein, LRRK2 p.G2019S, p.N1437H, and
p.N1437D. Compared to wild-type protein, LRRK2
p.N1437D and p.N1437H display a significantly higher pro-
portion of LRRK2 kinase compared to wild-type LRRK2
(P50.01), consistent with the property observed for the
p.G2019S LRRK2 pathogenic substitution (Supplementary
material and Supplementary Fig. 2A and B). The increased
enzyme activity caused by the p.N1437D further supported
its pathogenicity. In the family identified with LRRK2
p.R1441C, two unaffected members (a 74-year-old male
and a 71-year-old female) also carried the variant; these two
unaffected carriers may be explained by incomplete pene-
trance as described in a previous report (Haugarvoll et al.,
2008)(Supplementary Fig. 1B). Of the family identified with
LRRK2 p.R1441H, two unaffected members (a 59-year-old
male and a 56-year-old male) carried this variant, which
may also be explained by incomplete penetrance. The
LRRK2 p.R1441C and p.R1441H variants were detected in
two families in the ADPD cohort, indicating that these two
variants had prevalence of 0.12% separately. This finding
was in agreement with other studies showing the rarity of
LRRK2 p.R1441C in European ancestry (0–0.3%)
(Zabetian et al.,2005;Tan et al.,2006;Moller et al.,2008).
We found that four probands carried SNCA P/LP variants,
including three patients with SNCA duplications and one pa-
tient with an unreported missense variant (p.P117S).
Furthermore, Triton-X soluble and insoluble a-synuclein
lysates assay indicated that the p.P117S mutation may also
alter a-synuclein protein solubility (as described in the
Supplementary material and Supplementary Fig. 2C), which
also suggested its pathogenicity. Nevertheless, further func-
tional analyses using animal models is needed, in addition to
the recruitment of more families with this mutation, to valid-
ate the pathogenicity. Of these three families with CNVs,
one of them segregated with the parkinsonism phenotype; in
another two families, two of the probands’ unaffected sib-
lings were identified as having SNCA duplications, but they
currently remain asymptomatic and are 42 and 47 years old,
which is still below the median AAO of 48 years (the pene-
trance remains incomplete even at 79 years) (Nishioka et al.,
2009;Konno et al.,2016). To our knowledge, this study is
the first to identify an SNCA duplication in young-onset or
familial Parkinson’s disease in mainland China, although the
frequency is slightly lower than it was in other populations
(Bonifati, 2014;Schneider and Alcalay, 2017). In addition, an
unreported insertion mutation (c.322dupC/p.G109Rfs*51)
was identified in LRP10, which segregated with parkinsonism
in the corresponding family (Supplementary Fig. 2). As a re-
cently reported disease-associated gene, the pathogenicity of
LRP10 remains controversial (Quadri et al.,2018b), yet the
presence of this deletion mutation in our cohort provides
more evidence of its pathogenicity.
Mutational spectrum of members of
the sEOPD cohort
In the sEOPD cohort, we detected P/LP variants of
Parkinson’s disease-associated genes in 57 of 1242 (4.59%)
patients (Fig. 2D and Supplementary Table 3). We found
that a lower AAO was associated with a higher molecular
diagnosis rate. When the AAO decreased from 40 to 30
years, the molecular diagnosis rate increased from 13.29%
to 42.59%. It was noted that molecular diagnosis was much
more frequently established with the ARPD cohort (n=65of
192; 33.85%) than with the ADPD cohort (n= 10 of 242;
4.13%) and the sEOPD cohort (n= 57 of 1242; 4.59%).
This indicated that the P/LP variants occurred more frequent-
ly in patients with a family history of Parkinson’s disease, es-
pecially those with a pattern of recessive inheritance.
PRKN was also the most frequently mutated gene in the
sEOPD cohort, accounting for 33 (2.66%; of 1242 pro-
bands) of 57 molecularly diagnosed probands. Exon dele-
tions in PRKN also represented the most common mutation
type, especially in exon 3 (n= 19) and exon 2 (n= 16). Exon
3 was further confirmed as a hotspot mutation site between
the ARPD and sEOPD cohorts, similar to findings with
Hispanic populations (exon 3–4 deletions were more com-
mon) (Marder et al.,2010). Furthermore, we noted that the
most frequent variant in PRKN P/PL SNVs was p.C441R
(n= 3), which was reported previously (Supplementary
Table 4A). Probands (26.04%) from the ARPD cohort were
more likely to have biallelic mutations in PRKN than
patients with sEOPD (2.66%) in our cohort.
We detected five (0.40%) P/LP variants in LRRK2, the se-
cond most frequently mutated gene in the sEOPD cohort.
We identified three patients with the LRRK2 p.S1181Y vari-
ant, and the rest of the variants were p.V1447M (n=1), or
p.Y1645S (n= 1). Familial cases with Alzheimer’s disease in-
heritance were more likely to have LRRK2 P/LP variants
than patients from the sEOPD cohort, with relative propor-
tions of 2.07% versus 0.40%, respectively, in our cohorts.
In general, P/LP variants occur more frequently in familial
Parkinson’s disease patients (Di Fonzo et al.,2005;Nichols
et al.,2005)thaninsporadiccases(Gilks et al.,2005).
However, the p.G2019S was not found in our cohort, al-
though it is common in Europe (Bonifati, 2007) and North
Africa (Puschmann, 2013).
Numerous P/LP variants of other genes were also identi-
fied, including GIGYF2 (n=4), PLA2G6 (n=3), VPS13C
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(n=2), RIC3 (n=2), VPS35 (n=1), TMEM230 (n=1),
SNCA (n=1), PINK1 (n=1), LRP10 (n=1), HTRA2
(n=1), FBXO7 (n= 1), and DNAJC13 (n=1)
(Supplementary Table 3). Furthermore, two patients in our
sEOPD cohort were identified with the GIGYF2 p.D1075
missense variant, which is absent from Eastern Asian popu-
lations of public databases and our in-house control cohort.
Age at onset spectrum
We found that the median AAO of patients with molecular
diagnosis (median 31.50 years; average 32.27 years) was ap-
proximately 14.5 years earlier than the median AAO of
patients without molecular diagnosis (median 46.00 years;
average 46.02 years) across the entire Parkinson’s disease co-
hort (Fig. 3C and Supplementary Table 5). These results
indicated that the median AAO was significantly lower in
Parkinson’s disease patients with P/LP variants, especially in
patients with a family history (Fig. 3A). The median AAO
ranking in carriers in the FPD cohort with P/LP variants in
known Parkinson’s disease-associated genes was similar to
that in the entire Parkinson’s disease cohort, except for
LRRK2 (Fig. 3A). The median AAO ranking in carriers in
the sEOPD cohort with P/LP variants in these Parkinson’s
disease-associated genes was consistent across the entire
Parkinson’s disease cohort, except for PLA2G6 (Fig. 3B).
The subtle difference of median AAO ranking in patients
with LRRK2 P/LP variants may be caused by late-onset
Parkinson’s disease with reported LRRK2 P/LP variants in
the ADPD cohort, which would be consistent with previous
studies.
These results indicated that the top five genes in our
cohorts were autosomal-recessive genes (Fig. 3C and
Supplementary Table 5), which is in line with the opinion
that recessive genes usually exist in patients with early AAO
(Kasten et al.,2018). Specifically, the median AAOs of sub-
jects with ATP13A2,PLA2G6,PRKN,andPINK1 P/LP
variants were significantly lower than those of non-carriers,
whereas the median AAOs of subjects with VPS13C and
RIC3 P/LP variants were slightly lower and the median
AAOs of subjects with LRRK2,SNCA,andGIGYF2 P/LP
variants were similar to those of non-carriers (Fig. 3C).
Clinical manifestations in subjects
with Parkinson’s disease-associated
gene variants
We then analysed associations between five Parkinson’s dis-
ease-associated genes and phenotypes based on genes with
P/LP variants in five or more patients. We found that car-
riers of PRKN,PLA2G6,andPINK1 P/LP variants had ear-
lier AAOs than non-carriers, after adjusting for gender and
disease duration, and carriers of PRKN and PINK1 P/LP
variants also had longer disease durations than non-carriers,
after adjusting for age at assessment and gender (Table 2;
comparison of all clinical features shown in Supplementary
Table 6A). PRKN P/LP variant carriers associated with
milder motor manifestations, especially in terms of stiffness,
milder autonomic dysfunction, and lighter depression, but
they were associated with severe sleep disorders, a higher
rate of dyskinesias, and a lower rate of wearing-off than
non-carriers, after adjusting for age at assessment, gender
and disease duration. PLA2G6 P/LP-variant carriers showed
more severe motor symptoms, especially in terms of postural
instability symptoms, more severe autonomic dysfunction,
Figure 3 AAO spectrum of Parkinson’s disease-associated genes. Spectrum of AAO in patients with P/LP variants of each Parkinson’s
disease-associated gene (only gene with no less than two patients carried was included in) and in patients without P/LP variants of known
Parkinson’s disease-associated genes: (A) Familial Parkinson’s disease (FPD) cohort. (B) The sEOPD cohort. (C) Entire Parkinson’s disease (PD)
cohort. Comparison of AAO in all patients with P/LP variants of known Parkinson’s disease-associated genes and patients without P/LP variants
in known Parkinson’s disease-associated genes. (D) FPD cohort. (E) sEOPD cohort. (F) Entire Parkinson’s disease cohort. *The dashed red line
refers to the median AAO of all patients with P/LP variants in known Parkinson’s disease-associated genes, whereas the dashed grey line refers
to the median AAO of patients without P/LP variants in known Parkinson’s disease-associated genes in the corresponding cohort.
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and tended to have a higher rate of freezing gait problems
(Table 2 and Supplementary Table 6A). We did not find any
differences in clinical manifestations between LRRK2 and
SNCA P/LP-variant carriers and non-carriers (Table 2 and
Supplementary Table 6B). Specifically, we then individually
analysed the SNCA duplication carriers, and found that they
showed more severe cognitive dysfunction, depression, fa-
tigue, and automatic dysfunction. We also further assessed
the cognitive function (Montreal Cognitive Assessment) of
three probands with SNCA duplication, and they scored 23
(duration = 1 year), 16 (duration = 3.5 years), and 11
(duration = 11 years), respectively. Our results showed that
the clinical manifestations of subjects with Parkinson’s dis-
ease-associated genes were similar with those of other popu-
lations or studies (Kasten et al.,2018;Trinh et al.,2018).
Potential variations meriting further
study
We further identified variants of uncertain significance
(VUSs) and heterozygous variants of autosomal-recessive
Table 2 Differential clinical features between carriers of P/LP variants in Parkinson’s disease-associated genes and
mutation-negative patients
Mutation carriers Clinical features Non-carriers (n= 1544) Carriers Beta P-value
PRKN carriers n= 83 biallelic Age at onset, years 46.02 ±8.25 29.07 ±8.78 –0.372 50.001
a
Age at assessment, years 52.25 ±8.92 41.43 ±11.49 –0.360 50.001
a
Disease duration, years 6.24 ±5.16 12.36 ±9.24 0.353 50.001
b
Motor manifestations
UPDRS-Part II score 11.97 ±6.87 10.02 ±6.76 –0.104 50.001
Stiffness score 5.61 ±4.25 4.99 ±4.32 –0.054 0.045
Dyskinesia (%) 16.2 22.2 0.190 0.007
Non-motor manifestations
PDSS score 115.77 ±29.69 122.46 ±26.93 0.082 0.010
RBDQ-HK score 13.43 ±16.02 9.85 ±12.21 –0.072 0.024
HAMD score 5.87 ±5.62 4.57 ±5.15 –0.095 0.003
SCOPA-AUT score 7.39 ±6.90 4.24 ±5.09 –0.121 50.001
PDQ39 score 30.11 ±26.00 28.50 ±29.07 –0.107 50.001
PLA2G6 carriers n= 9 biallelic Age at onset, years 46.02 ±8.25 25.67 ±10.43 –0.187 50.001
a
Age at assessment, years 52.25 ±8.92 30.56 ±12.61 –0.173 50.001
a
Disease duration, years 6.24 ±5.16 4.89 ±4.14 0.057 0.015
b
Motor manifestations
UPDRS-Part I score 2.44 ±2.08 3.33 ±3.08 0.060 0.018
UPDRS-Part II score 11.97 ±6.87 15.89 ±9.06 0.086 50.001
Postural instability score 4.06 ±3.20 6.56 ±5.22 0.101 50.001
Hoehn and Yahr stage 0.053 0.045
0–1.5 (%) 32.0 22.2
2 or 2.5 (%) 43.8 44.4
3 + (%) 24.2 33.3
Freezing gait (%) 27.3 77.8 –1.223 0.001
Non-motor manifestations
SCOPA-AUT score 7.39 ±6.90 18.00 ±9.90 0.075 0.018
PDQ39 score 30.11 ±26.00 77.00 ±19.80 0.069 0.016
PINK1 carriers n= 7 biallelic Age at onset, years 46.02 ±8.25 29.00 ±9.42 –0.124 50.001
a
Age at assessment, years 52.25 ±8.92 40.86 ±7.49 –0.115 50.001
a
Disease duration, years 6.24 ±5.16 11.86 ±7.56 0.108 50.001
b
PDQ39 score 30.11 ±26.00 60.25 ±14.66 0.058 0.044
SNCA dup carriers n= 3 heterozygous Cognition: total MMSE score 27.03 ±3.19 23.00 ±7.07 –0.070 0.014
HAMD score 5.87 ±5.62 15.50 ±9.19 0.081 0.007
PFS score 44.28 ±18.92 70.00 ±14.14 0.074 0.035
SCOPA-AUT score 7.39 ±6.90 14.00 ±1.41 0.064 0.043
Only clinical manifestations with significant differences between mutational carriers and non-carriers are shown, and the complete information is provided in the Supplementary
material. Non-carriers were patients without any Parkinson’s disease associated genes P/LP variants. Mutation-negative = patients without any pathogenic or likely pathogenic
variants of Parkinson’s disease-associated genes. Scores in the first four columns are mean ±SD, except for gender, initial symptoms, motor subtype, constipation and RLS,
which are shown as nor proportions (%). Increasing scores and increasing beta-values for motor and non-motor variables are associated with worse symptoms, except for the
Mini-Mental State Examination (MMSE) test scores. Increasing scores for the MMSE test are associated with better cognition. HAMD = 17-item Hamilton Depression Rating Scale;
PDQ39 = The 39-item Parkinson’s disease Questionnaire; PDSS = Parkinson’s disease sleep scale; PFS = Parkinson’s disease fatigue scale; RBDQ-HK = REM sleep behaviour
disorder questionnaire-Hong Kong; SCOPA-AUT = Scales for Outcomes in Parkinson’s disease-Autonomic; UPDRS = Unified Parkinson’s Disease Rating Scale.
a
Adjusting for gender and disease duration at entry.
b
Adjusting for gender and age at entry.
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genes in the 23 Parkinson’s disease-associated genes, which
may be worth investigating further. First, we identified 68
probands carrying VUSs, including 26 VUSs in POLG,13
VUSs in DNAJC13 and 13 VUSs in LRRK2. The other
VUSs were identified in EIF4G1,GIGYF2,LRP10,SNCA,
RIC3,TMEM230 and VPS35. We did not observe signifi-
cant differences in the AAO, gender, and proportion of
patients with a family history of Parkinson’s disease between
patients with VUSs and patients without P/LP variants and
VUSs. Further exploration and periodical reassessment may
be necessary to investigate whether any of the VUSs play
critical roles in Parkinson’s disease (Supplementary Tables
4B and 7). Second, 195 patients carried heterozygous var-
iants of autosomal-recessive Parkinson’s disease-associated
genes. Of note, eight patients carried two heterozygous var-
iants from two different recessive inheritance genes
(Supplementary Table 4C). Some studies have supported
that the oligogenic inheritance of rare Mendelian variants
may be important in Parkinson’s disease (Tang et al.,2006;
Lubbe et al.,2016), but this has not been replicated on a
large scale, which needs further exploration. Of all of these
heterozygous variants, variants in VPS13C were the most
common (n=70),followedbyPRKN variants (n=49).
Mutational frequency of GBA in all
cohorts
In the ARPD families, we detected six possibly pathogenic
variants of GBA in six families (n= 6 of 192; 3.15%) and
two VUSs in two other families. In the ADPD families, we
detected 10 possibly pathogenic variants in 10 families
(n= 10 of 242; 4.13%) and two VUSs in two other families.
In the sEOPD cohort, we detected 53 variants: 45 patients
carried possibly pathogenic variants (n= 45 of 1242; 3.62%)
(Fig. 4 and Supplementary Table 4D), whereas eight patients
carried VUSs. Of note, no homozygous or compound hetero-
zygous GBA mutations was found in our cohorts.
Further, we also found 59 probands who carried p.L444P,
including two probands from ARPD families (n= 2 of 192;
1.04%), nine probands from ADPD families (n=9 of 242;
3.72%), and 48 patients from the sEOPD cohort (n=48 of
1,242; 3.86%), respectively. This gives p.L444P an overall
frequency of 3.52% (59 of 1676) in the entire FPD and
sEOPD cohort (Fig. 4). However, we did not find any
patients that carry p.N370S in our cohort.
Next, we analysed the relationship between GBA possibly
pathogenic variants and phenotypes, and the relationship be-
tween GBA p.L444P and phenotypes (Supplementary
Table 8). We found that patients with GBA possibly patho-
genic variants were associated with more severe rapid eye
movement sleep disorder, depression and autonomic dys-
function. Moreover, patients with GBA p.L444P were asso-
ciated with younger age at onset, more severe motor
symptoms, slighter tremor symptoms, more severe stiffness
symptoms, more severe rapid eye movement sleep disorder,
and autonomic dysfunction.
Discussion
With the development of precision medicine, an improved
understanding of the genetic mechanism underlying
Parkinson’s disease is becoming increasingly important
(Charvin et al., 2018). In this study, we determined the muta-
tional spectrum of 23 known Parkinson’s disease-associated
genes in purely clinically diagnosed Parkinson’s disease
patients with an early AAO or with a family history of
Parkinson’s disease, with a population from mainland China.
Our results indicated that Parkinson’s disease patients with
an AAO of 540 years, especially those from autosomal-re-
cessive families, may benefit from genetic counselling, and we
identified patients with familial or early-onset Parkinson’s
disease who progressed relatively faster or slower.
Above all, this study enabled us to systematically and
comprehensively estimate the prevalence of P/LP variants in
populations from mainland China with early-onset and fa-
milial Parkinson’s disease. As the most common recurrent
gene, PRKN P/LP variants were identified in 4.95% of all
patients (26.04% in the ARPD cohort and 2.66% in the
sEOPD cohort). In addition, LRRK2 p.G2019S accounts for
3–6% of familial Parkinson’s disease cases in European pop-
ulations and nearly 14% in Ashkenazi Jews (Shu et al.,
2019), but this variant was absent from our cohort, and 11
other variants were identified (0.60% in the entire
Parkinson’s disease cohort, 2.07% in the ADPD cohort, and
0.40% in the sEOPD cohort), which was similar to previous
data for Korean (1.43% in early-onset Parkinson’s disease)
(Youn et al.,2019) and Taiwanese populations (2.9% in
patients with autosomal-dominant inheritance of parkinson-
ism) (Lin et al.,2019). We also identified PLA2G6 P/LP var-
iants in six probands from the ARPD cohort and three
probands in the sEOPD cohort, which suggested that
PLA2G6 was indispensable for molecular diagnosis in the
mainland Chinese population, especially among patients
with recessive inheritance or early-onset Parkinson’s disease.
We not only confirmed that PRKN,LRRK2 and PLA2G6
variants were most frequently found in Parkinson’s disease
cohorts, but our findings also implied that these genes
should be screened for in patients from mainland China. In
addition, our findings showed that families with dominant
gene P/LP variants did not always segregate with the pheno-
type successfully, but recessive families almost always did;
this may be due to the incomplete penetrance of dominant
genes and the late AAO of the carriers. Specifically, GBA
possibly pathogenic variants were identified in 3.64% (61 of
1676) of all patients, whereas GBA p.L444P was identified
in 3.52% of all patients. The previous studies have also
shown that heterozygous functional coding variants of GBA
were identified in 4–15% of Parkinson’s disease patients
from different populations (Sidransky et al., 2009;Lesage
et al., 2011;Gan-Or et al., 2015a;Blauwendraat et al.,
2020). However, the pathogenicity of GBA possibly patho-
genic variants still needs further validation using functional
experiments or larger cohorts, especially the loss-of-function
variants. Of note, though some genetic variants of low
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confidence genes (GIGYF2,HTRA2,TMEM230,
DNAJC13,LRP10 and RIC3) were evaluated as P/LP or
VUS variants according to the same criteria of high confi-
dence genes, the pathogenicity of low-confidence genes still
needs to be replicated in larger multi-ethnic populations and
further functional validations.
The clinical spectrum of Parkinson’s disease-associated
genes P/LP-variant subjects in our study was similar to the
Figure 4 Mutational frequencies and AAO spectrum of GBA gene in the Parkinson’s disease cohorts. Mutational frequencies of
GBA possibly pathogenic (PP) variants and p.L444P variant in the different cohorts, spectrum of AAO in patients with GBA variants and p.L444P
variant. (A) All familial Parkinson’s disease (FPD) cohort. (B) The sEOPD cohort. (C) Entire Parkinson’s disease (PD) cohort. *The dashed red
line refers to the median AAO of patients without GBA possibly pathogenic variants, GBA p.L444P and P/LP variants in known Parkinson’s dis-
ease-associated genes in the corresponding cohort. And P-value was adjusted for gender and disease duration at entry.
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subjects reported for other populations (Kasten et al., 2017;
Guo et al.,2018b), suggesting that the pathogenicity of P/LP
variants in Parkinson’s disease-associated genes may conceal
population differences in patients with different clinical phe-
notypes. In our study, we found that carriers of PRKN,
PLA2G6 and PINK1 variants had an earlier AAO, that
PRKN variants were associated with less severe motor
symptoms, and that PLA2G6 was associated with more se-
vere motor symptoms, especially in terms of postural in-
stability symptoms and freezing gait problems. The patients
with an SNCA duplication in our cohorts showed worse
cognitive dysfunction, more severe depression and were
more prone to freezing gait. No special clinical manifesta-
tions were found in patients with LRRK2 P/LP variants,
which may indicate that LRRK2-linked Parkinson’s disease
has a similar clinical course to typical Parkinson’s disease
patients. The clinical symptoms found in patients with GBA
L444P were similar to other studies (Zhang et al.,2018;
Yahalom et al.,2019); however, motor symptoms of GBA
L444P carriers are not significantly more severe compared
to the non-carriers, and there is no cognitive decline, which
may be due to the early-onset and relatively shorter disease
duration of the carriers. Further prospective studies may be
needed to confirm these associations. However, no differen-
ces of motor symptoms were found in GBA possibly patho-
genic variants, which may further indicate that certain
variants may not be relevant to the risk or phenotype of
Parkinson’s disease. Further functional experiments are
needed to verify this. Moreover, other studies have also sup-
ported that GBA variants are associated with rapid eye
movement sleep behaviour disorder (Gan-Or et al.,2015b;
Gamez-Valero et al.,2018), which may also warrant further
mechanistic studies. The similarity with other populations
suggests that the associations between mutated genotypes
and phenotypes in other populations could also be applied
to our own populations; however, given the small samples
of certain groups, further replication in a larger cohort is
also needed to verify this viewpoint.
In this study, we also detected VUSs of dominant genes
and heterozygous variants of recessive genes. The potential
value of VUSs and heterozygous variants of recessive genes
are presently uncertain; thus, they should be studied further
to verify their contributions to Parkinson’s disease. In line
with the rapid pace of increasing knowledge of genetic
causes of diseases, reassessing uncertain findings may im-
prove current interpretations, as more data would become
available through follow-up research, replication, or func-
tional testing in the future. In this context, it is important to
realize that whole-exome sequencing data should be consid-
ered a sustainable source of potential diagnostic clues, and
should be re-examined at appropriate intervals, especially
when the initial results are ambiguous (Liu et al., 2019).
This study represents a comprehensive and systematic
screening of Parkinson’s disease-associated genes in early-
onset and familial Parkinson’s disease patients from main-
land China, although the current study has some limitations.
First, we only focused on known Parkinson’s disease-
associated genes, not susceptibility genes, risk loci, or new
candidate genes, which may play important roles in early-
onset Parkinson’s disease and familial Parkinson’s disease.
Second, we screened for SNVs/indels by whole-exome
sequencing and screened for CNVs in PRKN,PINK1,
ATP13A2,DJ1,andSNCA by multiplex ligation-dependent
probe amplification; yet we did not explore other unknown
CNVs, structural variants, or other complex genetic abnor-
malities, which may need further exploration by whole-gen-
ome sequencing or even third-generation sequencing. Third,
we only analysed coding region variations, but not non-cod-
ing regions such as regulatory elements, which may play im-
portant roles in the pathogenicity of Parkinson’s disease.
Lastly, we tried our best to collect data from family mem-
bers of P/LP variants probands, but incomplete data for sev-
eral families were collected, as they were settled in other
places and may have lost touch with the probands, or
refused to undergo genetic testing. Briefly, along with identi-
fying pathogenic CNVs of known Parkinson’s disease-associ-
ated genes, susceptibility genes, and risk loci, and
discovering new Parkinson’s disease-associated genes, it
should also be possible to improve the molecular diagnostics
of Parkinson’s disease patients in the future (Liu et al.,
2019).
Conclusion
In conclusion, we conducted the most systematic survey of
the mutational spectrum of sEOPD and familial Parkinson’s
disease patients in a mainland Chinese population. Our data
indicate that Parkinson’s disease patients with an AAO of
540 years may benefit from genetic counselling, especially
those from families with a recessive inheritance pattern. Our
findings also expand the existing repertoire of known var-
iants in Parkinson’s disease-associated genes and emphasized
the potential of genetic testing to accurately guide patients to
relevant clinical trials and targeted therapies. The similarity
of clinical spectrum with those of other populations sug-
gested that some findings from correlation studies of geno-
types and phenotypes in other populations can also be
applied to our own populations.
Acknowledgements
We are indebted to the participation of the patients and their
family members in this study.
Funding
This study was supported by grants from the National Key
Plan for Scientific Research and Development of China grants
(Grant No. 2016YFC1306000, No. 2017YFC0909101,
No.2017YFC0840100 and 2017YFC0840104), the National
Natural Science Foundation of China (Grant No. 81430023),
the Central Public-Interest Scientific Institution Basal Research
2232 |BRAIN 2020: 143; 2220–2234 Y. Zhao et al.
Downloaded from https://academic.oup.com/brain/article/143/7/2220/5866165 by Southern Medical University user on 14 October 2020
Fund of Chinese Academy of Medical Sciences (Grant No.
2018-12M-HL-025), the Young Elite Scientist Sponsorship
Program by CAST (Grant No. 2018QNRC001), the
Innovation-Driven Project of Central South University (Grant
No. 20180033040004), Hunan Science Funds for
Distinguished Young Scholar (Grant No. 2017JJ1037), the in-
novative team program from Department of Science &
Technology of Hunan Province (Grant No. 2019RS1010),
and the innovation-driven team project from Central South
University (Grant No. 2020CX016).
Competing interests
The authors report no competing interests.
Supplementary material
Supplementary material is available at Brain online.
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