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Whole-exome sequencing in early-onset Parkinson’s disease among ethnic Chinese
Journal Pre-proof Whole-exome sequencing in early-onset Parkinson’s disease among ethnic Chinese Nannan Li, Ling Wang, Jinhong Zhang, Eng-King Tan, Junying Li, Jiaxin Peng, Liren Duan, Chaolan Chen, Dong Zhou, Li He, Rong Peng PII:
S0197-4580(19)30452-X
DOI:
https://doi.org/10.1016/j.neurobiolaging.2019.12.023
Reference:
NBA 10749
To appear in:
Neurobiology of Aging
Received Date: 23 August 2019 Revised Date:
19 November 2019
Accepted Date: 27 December 2019
Please cite this article as: Li, N., Wang, L., Zhang, J., Tan, E.-K., Li, J., Peng, J., Duan, L., Chen, C., Zhou, D., He, L., Peng, R., Whole-exome sequencing in early-onset Parkinson’s disease among ethnic Chinese, Neurobiology of Aging (2020), doi: https://doi.org/10.1016/j.neurobiolaging.2019.12.023. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Whole-exome
sequencing
in
early-onset
Parkinson’s
disease
among ethnic Chinese
Nannan Li a, Ling Wang a, Jinhong Zhang b, Eng-King Tan
c,d
, Junying Li a, Jiaxin
Peng a, Liren Duan a, Chaolan Chen a, Dong Zhou a, Li He a, Rong Peng a,*
a
Department of Neurology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
b
Department of Internal Medicine, Wangjiang Hospital, Sichuan University, Chengdu, Sichuan,
China c
Department of Neurology, National Neuroscience Institute, Singapore General Hospital,
Singapore, Singapore d
Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
* Corresponding author at: Department of Neurology, West China Hospital, Sichuan University, No.37 Guoxue Xiang, Chengdu, Sichuan 610041, China. Tel.: +86 28 85423038, fax: +86 28 85582944. E-mail address: [email protected] (R. Peng).
1
1
ABSTRACT
2
Although early-onset Parkinson’s disease (EOPD) has a more penetrant genetic
3
etiology, the genetic architecture of EOPD remains unclear. The objectives of this
4
study were to assess the genetic and clinical features of EOPD among ethnic Chinese
5
from mainland China. Using whole-exome sequencing (WES), we performed genetic
6
analyses of 240 participants including 193 with sporadic and 47 with familial EOPD
7
(age of onset < 50 years). In total, 18 patients (7.5%) harbored pathogenic or likely
8
pathogenic variants in known PD genes. Among these variants, biallelic variants in
9
Parkin and PINK1 were responsible for 4.2% of cases, and rare likely pathogenic
10
variants in LRRK2 (1.7%) also appeared to be a relatively common cause of EOPD.
11
Notably, 7.5% of patients carried risk variants in either LRRK2 or GBA, which should
12
also be considered for EOPD. Nevertheless, 41 patients (17.1%) had rare variants of
13
unknown significance. In conclusion, our findings provide a better understanding of
14
the genetic architecture of PD among ethnic Chinese, and the pathogenicity of
15
numerous rare variants should be further investigated.
16
Keywords: Early-onset Parkinson’s disease; Genetics; Whole-exome sequencing;
17
Clinical features
18 19 20 21 22 23 24 2
1
1. Introduction
2
Parkinson’s disease (PD, OMIM #168600) is one of the most common
3
neurodegenerative disease, and its prevalence increases with age, affecting about 1%
4
of people over 60 years of age (de Lau and Breteler, 2006). Only 5.4% of all patients
5
with PD experience disease onset before 50 years of age, which is usually defined as
6
early-onset PD (EOPD) in the literature (Wickremaratchi et al., 2009). Notably,
7
patients with EOPD have a more penetrant genetic etiology than late-onset PD (LOPD)
8
and thus represent a good cohort to explore the genetics of PD. Elucidating the
9
pathophysiologic mechanisms of EOPD can also facilitate accurate diagnosis and
10
genetic counselling early and promote identification of new therapeutic targets.
11
Causative variants in the autosomal-recessive (AR) PD genes Parkin (PARK2),
12
PINK1 (PARK6), and DJ-1 (PARK7) are common causes of EOPD and account for
13
approximately 13% of these cases (Kilarski et al., 2012). In addition, several other AR
14
genes in EOPD such as ATP13A2, PLA2G6, FBXO7, DNAJC6, SYNJ1, VPS13C have
15
been identified (Schormair et al., 2018; Shen et al., 2018; Taghavi et al., 2018).
16
Moreover, some other known PD genes have also been implicated in EOPD
17
(Gustavsson et al., 2017; Lin et al., 2019; Ylonen et al., 2017). Recently, by
18
integrating whole-exome sequencing (WES) and functional evidence, several novel
19
candidate genes have been nominated in patients with EOPD (Guo et al., 2018; Jansen
20
et al., 2017; Lin et al., 2019), suggesting that a wide spectrum of genetic variants is
21
linked to EOPD. To date, there is still limited detailed data on the genetic architecture
22
of EOPD in ethnic Chinese. 3
1
To address these gaps in knowledge, we performed an extensive WES screening of
2
the common and rare genetic variants causing disease in a relatively large cohort of
3
patients with sporadic and familial EOPD among ethnic Chinese from mainland China.
4
In addition, we also examined the clinical features of these patients to better
5
understand the relationship between genotypes and clinical phenotypes.
6
2. Materials and Methods
7
2.1. Participants
8
A total of 240 participants including 193 patients with sporadic EOPD and 47
9
EOPD probands with a positive familial history (at least 1 family member in 3
10
generations with PD) (age of onset (AAO) < 50 years) were recruited from the
11
Movement Disorders Outpatient Clinic of West China Hospital, Sichuan University
12
(Chengdu, China) from 2004 to 2018. Of the 47 probands, 39 were compatible with
13
an autosomal-dominant (AD) inheritance pattern, and 7 were compatible with AR
14
inheritance or had 1 other affected first-degree relative with PD. All patients were
15
diagnosed with idiopathic PD by two neurologists using the United Kingdom PD
16
Society Brain Bank criteria (Hughes et al., 1992). All participants underwent detailed
17
neurological and neuropsychological assessment. This study was approved by the
18
Ethics Committee of Sichuan University. Written informed consent was obtained
19
from all participants.
20
2.2. Genetic analysis
21
2.2.1. Whole-exome sequencing
22
Genetic testing of all patients was conducted using WES technology. The 4
1
SureSelect Human All Exon v6 Kit from Agilent (Agilent, Santa Clara, CA, USA)
2
was used to capture whole exomes. In brief, libraries were constructed according to
3
the manufacturer's protocols and sequenced on the Illumina HiSeq X10 platform
4
(Illumina, San Diego, CA, USA) using the 150 bp paired-end module. Quality control
5
was applied to the raw data to guarantee the meaningfulness of the downstream
6
analysis. The mean depth of coverage per sample was over 100-fold, with more than
7
99.5% of the targeted regions covered and 98% at least 20-fold. Alignment to the
8
human genome assembly hg19 was carried out using the Burrows-Wheeler Aligner
9
(BWA) (Li and Durbin, 2009), followed by variant calling using the Genome
10
Analysis Toolkit (GATK version 3.7) (McKenna et al., 2010). All variants were
11
annotated with ANNOVAR (Wang et al., 2010). We used a stringent minor allele
12
frequency filter (MAF < 1%) in public databases (1000g, 1000 Genomes,
13
http://www.1000genomes.org;
14
http://gnomad.broadinstitute.org;
15
http://exac.broadinstitute.org), focusing on missense and loss-of-function variants
16
(nonsense, frameshift, splice, and copy number variants (CNVs) called by CNVkit
17
(Talevich et al., 2016)). The sequence variants were also further interpreted and
18
classified according to the American College of Medical Genetics and Genomics
19
(ACMG) recommendations (Richards et al., 2015), during which allele frequency,
20
nucleotide conservation by the Conservation Reserve Enhancement Program (GERP,
21
http://mendel.stanford.edu/sidowlab/downloads/gerp/index.html),
22
prediction
(MutationTaster,
gnomAD, ExAC,
Genome Exome
Aggregation Aggregation
http://www.mutationtaster.org; 5
Database, Consortium,
pathogenicity PolyPhen-2,
1
http://genetics.bwh.harvard.edu/pph2;
2
http://cadd.gs.washington.edu), and inheritance pattern were all further analyzed. The
3
rare variants were defined as variants with a minor allele frequency (MAF) ≤ 0.1%.
4
This analysis did not screen for trinucleotide or hexanucleotide repeat expansions. All
5
240 patient samples were analyzed as singleton exomes rather than as trios (a patient
6
with EOPD and the patient’s parents) or quads (a patient with EOPD, the patient’s
7
parents and a patient’s healthy sibling). Genomic DNA was extracted using standard
8
extraction procedures. Variants considered likely related to the patient’s phenotype
9
were confirmed by Sanger sequencing.
10 11
SIFT,
http://sift.jcvi.org;
and
CADD,
2.2.2. Multiplex ligation-dependent probe amplification and qPCR assays For CNVs called by CNVkit, the multiplex ligation-dependent probe amplification
12
(MPLA)
13
polymerase chain reaction (qPCR) were used to detect these large deletions or
14
duplications. The qPCR analysis was performed and analyzed using the Eco
15
Real-Time PCR System. We presented the primers and reaction conditions for the
16
qPCR in the Supplementary Table S2.
17
2.3. Statistical analysis
(MRC-Holland, Amsterdam, The Netherlands)
and
quantification
18
Statistical analysis was performed using SPSS software version 24.0 (SPSS Inc.,
19
Chicago, IL, USA). The Chi-square test was used to compare proportions between
20
groups. A two-tailed p < 0.05 was considered statistically significant.
21
3. Results
22
The mean age at onset of patients with sporadic EOPD was 40.9 ± 6.5 years, and 6
1
53.4% were men, while the mean age at onset of patients with familial EOPD was
2
41.5 ± 6.5 years, and 51.1 % were men. A total of 77 participants (77/240, 32.1%)
3
harbored 77 variants in 18 genes (Table 1): 18 patients (18/240, 7.5%) harbored 24
4
pathogenic or likely pathogenic variants in the AR PD genes (Parkin, PINK1,
5
PLA2G6, and SYNJ1) and the AD PD genes (LRRK2, SNCA, and CHCHD2); 18
6
patients (18/240, 7.5%) carried risk variants in either LRRK2 or GBA; 41 patients
7
(41/240, 17.1%) had 46 rare variants of uncertain significance; the remaining 163
8
patients (163/240, 67.9%) did not have identifiable genetic variants. Supplementary
9
Table S1 presents all of the genetic information.
10
3.1. Pathogenic or likely pathogenic variants
11
3.1.1. Autosomal-recessive PD genes (Parkin, PINK1, PLA2G6, and SYNJ1)
12
Parkin was the most prevalent causative gene in the 240 patients with EOPD. Of
13
the 9 Parkin biallelic variants (homozygous or compound heterozygous variants)
14
carriers (9/240, 3.8%): 1 had a homozygous variant (p.C441R) from a consanguineous
15
family, 8 had compound heterozygous variants (Table 2, Supplementary Table S1). In
16
addition, 3 had single heterozygous variants (exon 3 duplication, exons 2-4
17
duplication, exons 3-4 deletion). Since the prevalence of Parkin single heterozygosity
18
was similar to the rate reported in healthy controls (Malek et al., 2016), therefore
19
patients with single heterozygous variants in recessive genes were analyzed as
20
non-carriers. Exon rearrangements constituted the most common type of variants,
21
especially between exons 2 and 7. Interestingly, 1 patient had a novel frameshift
22
variant in exon 4 of Parkin, p.V164Yfs*13, which was determined to be pathogenic 7
1
using the ACMG recommendations. 4 patients had previously reported missense
2
variants: 2 had p.M1T, 1 had p.G284R, and 2 had p.C441R. However, there was no
3
significant difference in the frequency of biallelic variants in patients with EOPD with
4
a positive family history (2/47, 4.3%) and patients without a family history (7/193,
5
3.6%) (p = 1.00). These patients carrying biallelic variants in Parkin exhibited a
6
typical and generally mild phenotype, a good response to levodopa, slow disease
7
progression, and no cognitive impairment (Table 2).
8
We also identified 1 patient carrying previously reported compound heterozygous
9
pathogenic variants in PINK1 (p.R492* and c.1488+1G>A) (1/240, 0.4%) (Table 2,
10
Supplementary Table S1). The female carrier of biallelic PINK1 variants exhibited
11
very mild symptoms accompanying lower limb dystonia, an excellent response to a
12
low dose of levodopa (100 mg/qd), and very slow disease progression (Table 2).
13
However, no pathogenic or likely pathogenic variant was found in DJ-1.
14
In PLA2G6, 1 sporadic patient had compound heterozygosity for a novel p.R693G
15
variant and the previously reported p.D331Y variant classified as likely pathogenic.
16
The carrier of PLA2G6 variants had an earlier age at onset (25 years), rapid disease
17
progression with a good response to levodopa, early onset of treated-induced
18
dyskinesias, marked postural impairment, depression, no dystonia, normal cognitive
19
function, and a normal brain MRI (Table 2). For SYNJ1, compound heterozygosity for
20
a novel deletion of exon 3 variant and the previously reported p.A551V variant from 1
21
sporadic case were classified as likely pathogenic. This patient with biallelic variants
22
of SYNJ1 had the typical PD phenotype without seizures and cognitive decline (Table 8
1
2).
2
3.1.2. Autosomal-dominant PD genes (LRRK2, SNCA, and CHCHD2)
3
We did not find the p.G2019S variant in LRRK2, which is the most common
4
pathogenic variant in European-descent populations and is rare in Asian populations.
5
However, 4 patients carried 4 rare heterozygous variants (p.R1067Q, p.T1410M,
6
p.N1437D, p.G1451S) as likely pathogenic variants (4/240, 1.7%) (Table 2,
7
Supplementary Table S1). The p.R1067Q (Paisan-Ruiz et al., 2008; Yang et al., 2019;
8
Youn et al., 2019) and p.T1410M (Bozi et al., 2014; Ross et al., 2011) variants had
9
previously been reported, while the p.N1437D, and p.G1451S variants were novel.
10
The p.R1067Q variant is located in the leucine-rich repeat (LRR) domain of LRRK2,
11
and the p.T1410M, p.N1437D, and p.G1451S variants are located in the ROC domain
12
of LRRK2 (Fig. 1). The mean age at onset for the 4 patients carrying rare likely
13
pathogenic variants in LRRK2 was 36.3 ± 2.2 years (range 34-39) (Table 2). They
14
exhibited typical PD symptoms, a slightly slower disease progression, responded well
15
to levodopa treatment, and were without atypical signs (Table 2). In SNCA, the known
16
pathogenic p.A53T variant was identified in 1 patient with familial EOPD with an
17
onset at 31 years of age (Table 2). An additional proband had heterozygous likely
18
pathogenic p.T61I variant in CHCHD2 (Table 2).
19
3.2. Risk variants
20
We identified two common genetic susceptibility variants in LRRK2 among Asian
21
patients with PD, p.R1628P carried by 8 patients and p.G2385R carried by 3 patients,
22
and a low-frequency genetic susceptibility variant for PD, LRRK2 p.A419V carried by 9
1
2 patients (13/240, 5.4%). In GBA, we identified 5 heterozygous carriers (5/240, 2.1%)
2
of rare GBA variants: 1 carried c.115+1G>A (IVS2+1), 1 carried p.G241R (p.G202R),
3
2 carried p.D448H (p.D409H), 1 carried p.L483P (p.L444P) (Table 2, Supplementary
4
Table S1). Notably, these 4 rare variants had been previously identified as being
5
pathogenic in Gaucher disease (GD) and as genetic risk factors for PD in the
6
heterozygous state (Malek et al., 2018). The mean age at onset of the 5 patients who
7
carried known risk variants in GBA was 40.6 ± 5.4 years (range 32-46) (Table 2).
8
Considering the rarity of GBA variants, these data might not be suitable for comparing
9
the age of onset between carriers and non-carriers of risk variants in GBA. They had
10
relatively shorter disease duration (3.2 ± 0.8, range 2-4), and 1 out of 5 patients
11
exhibited cognitive impairment (Mini-Mental State Examination score < 24) (Table
12
2).
13
3.3. Variants of uncertain significance
14
In addition to these (likely) pathogenic and risk variants, 41 patients (41/240,
15
17.1%) had 46 rare variants in some known PD genes (1 in SNCA, 5 in LRRK2, 7 in
16
GIGYF2, 1 in HTRA2, 3 in VPS35, 5 in EIF4G1, 4 in DNAJC13, and 8 in VPS13C)
17
and in other genes (3 in GBA, 4 in MAPT, 2 in GCH1, 1 in SLC41A1, and 2 in SPG11)
18
(Table 1, Supplementary Table S1), which were categorized as being of uncertain
19
significance. Previous studies have shown that MAPT (Nalls et al., 2019; Nalls et al.,
20
2014; Pihlstrom et al., 2013), GCH1 (Mencacci et al., 2014; Nalls et al., 2019; Nalls
21
et al., 2014), SLC41A1 (Lin et al., 2014), and SPG11 (Anheim et al., 2009;
22
Guidubaldi et al., 2011) were associated with PD. Notably, 1 patient with sporadic 10
1
EOPD had a heterozygous p.L1209P variant in GIGYF2 that was previously reported
2
to be associated with LOPD (Guella et al., 2011) and a novel rare heterozygous
3
p.I358T variant in EIF4G1, and the patient had an age at onset of 41 years, typical
4
symptoms and a good response to levodopa (Table 2). More importantly, 33 of these
5
rare variants (33/46, 71.7%) were novel (Table 1, Supplementary Table S1).
6
4. Discussion
7
We performed an extensive WES screening of the common and rare genetic
8
variants causing disease in a relatively large cohort of patients with sporadic and
9
familial EOPD among ethnic Chinese from mainland China. In total, 18 patients
10
(7.5%) harbored pathogenic or likely pathogenic variants in Parkin, PINK1, PLA2G6,
11
SYNJ1, LRRK2, SNCA, and CHCHD2. Notably, 7.5% of patients carried a risk variant
12
in either LRRK2 or GBA, which should also be considered for patients with EOPD.
13
However, 41 patients (17.1%) had rare variants of uncertain significance. Using WES,
14
our study represents an extensive examination of the genetic etiology of EOPD among
15
ethnic Chinese from mainland China, and the results revealed different genetic
16
backgrounds.
17
Parkin was the most common causative gene in EOPD. In our cohort, biallelic
18
Parkin variants accounted for 3.8% of patients with EOPD. Our findings was
19
comparable with those of other WES studies in Taiwanese populations (Lin et al.,
20
2019), which estimated that 2.6% patients with EOPD had biallelic Parkin variants,
21
and in predominantly Caucasian populations with a frequency of 1.3-2.8%
22
(Gustavsson et al., 2017; Schormair et al., 2018; Tan et al., 2019). Moreover, we also 11
1
found that the trend for biallelic Parkin variants was more common in familial (4.3%)
2
than in sporadic (3.6%) EOPD, although this was not significantly different.
3
Importantly, the novel p.V164Yfs*13 variant in exon 4 of Parkin was detected and
4
was classified as a pathogenic variant that may result in an absence of protein by
5
nonsense mRNA decay and cause disease. PINK1 is the second most frequent gene
6
involved in EOPD. Only 1 patient (0.4%) carried known biallelic PINK1 variants in
7
our series, whereas no variant was found in the rarer DJ-1 gene. Similarly, a recent
8
WES study in Taiwanese populations have not found any patients carrying biallelic
9
variants in PINK1 or DJ-1 (Lin et al., 2019). Furthermore, Parkin and PINK1 have
10
been shown to be involved in the same cellular pathway (Pickrell and Youle, 2015),
11
and these patients carrying biallelic variants in Parkin or PINK also exhibited a
12
similar PD phenotype. Taken together, pathogenic or likely pathogenic biallelic
13
variants in Parkin and PINK1 were common causes of EOPD in our population and
14
accounted for 4.2% of these patients.
15
For other AR genes in EOPD, we also identified 4 likely pathogenic variants in
16
PLA2G6 (p.D331Y and a novel p.R693G) and SYNJ1 (a novel deletion of exon 3 and
17
p.A551V) from 2 patients (0.8%). Regarding PLA2G6, previous studies have reported
18
the homozygous state or compound heterozygosity of the p. D331Y variant in several
19
patients with early-onset parkinsonism in Asian populations (Lin et al., 2019; Lu et al.,
20
2012; Shi et al., 2011; Xie et al., 2015). Combining these previous studies with the
21
ExAC and gnomAD databases, the p. D331Y variant may be specific for Asian
22
populations. Interestingly, compound heterozygosity for a novel p.R693G variant and 12
1
the previously reported p.D331Y variant in PLA2G6 were identified in a sporadic case
2
with some clinical and imaging features that are compatible with those of carriers of
3
the homozygous p.D331Y variant (Lin et al., 2019; Shi et al., 2011; Xie et al., 2015).
4
Recently, p.A551V in SYNJ1 was identified in a single heterozygous state in a patient
5
with EOPD from Taiwan (Chen et al., 2015). In the present study, compound
6
heterozygosity for a novel deletion of exon 3 variant and the previously reported
7
p.A551V variant in SYNJ1 were found in a patient with sporadic EOPD who exhibited
8
a typical PD phenotype. Taken together, our findings suggest that other AR genes
9
such as SYNJ1 and PLA2G6 should also be considered for patients with EOPD in
10
ethnic Chinese.
11
Although variants in LRRK2 more commonly result in typical late-onset PD, they
12
can also cause PD at a much younger age (Trinh et al., 2018). In a large UK
13
population-based cohort, the LRRK2 p.G2019S variant was more common in
14
young-onset patients (2.2%) than in late-onset patients (0.4%) (Tan et al., 2019).
15
However, we did not find the p.G2019S variant in LRRK2 because this variant is rare
16
in Asian populations. Evidence is emerging that rare variants of Mendelian genes for
17
PD may also play a role in sporadic PD (Spataro et al., 2015; Yang et al., 2019) and
18
EOPD (Youn et al., 2019). In Korean patients with EOPD, LRRK2 is the known PD
19
gene with the most common rare variants (Youn et al., 2019). In our cohort, 4 rare
20
variants in LRRK2 were also classified as likely pathogenic variants (1.7%), including
21
the previously reported p.R1067Q and p.T1410M variants, and two novel p.N1437D
22
and p.G1451S variants. The p.R1067Q variant in the LRR domain of LRRK2 might 13
1
alter domain stability (Cardona et al., 2014). However, no functional evidence was
2
found for other three variants in LRRK2, so these variants should be studied further.
3
Moreover, 4 patients carried these rare variants in LRRK2 had an earlier mean age at
4
onset of 36.3 years compared with that of all LRRK2 variant carriers in a recent
5
MDSGene systematic review (57.0 years) (Trinh et al., 2018). Therefore, rare likely
6
pathogenic variants in LRRK2 (1.7%) also appeared to be relatively common causes
7
of EOPD.
8
LRRK2 is known as pleomorphic risk loci, at which rare coding variants can cause
9
PD and more common variants can also increase the risk for disease (Blauwendraat et
10
al., 2019b). In LRRK2, we also identified 8 patients carrying p.R1628P, 3 patients
11
carrying p.G2385R, and 2 patients carrying p. A419V (5.4%), which are common
12
(p.R1628P and p.G2385R) and low-frequency (p.A419V) genetic risk variants among
13
Asian patients with PD (Li et al., 2015; Li et al., 2012; Tan et al., 2010). In addition,
14
biallelic variants in GBA cause GD, while heterozygous variants are currently
15
considered the most important risk factor for developing PD (Schapira, 2015).
16
Notably, 5 patients (2.1%) carried known risk variants in GBA in our EOPD cohort.
17
Our observations are in line with the rate of known risk variants in GBA reported for
18
Finnish patients with EOPD (2.8%) (Ylonen et al., 2017) and Czech patients with
19
EOPD (5%) (Schormair et al., 2018). The L483P variant is the most common variant
20
detected in GBA in cases with PD from China (Chen et al., 2014). In our series, only
21
1 patient was found to carry the L483P variant (0.4%), which is also consistent with
22
that in a Taiwanese cohort (0.6%) (Lin et al., 2019). Moreover, previous studies have 14
1
suggested that patients who carried GBA risk variants had an earlier age at onset and
2
more frequent cognitive impairment (Blauwendraat et al., 2019a; Davis et al., 2016;
3
Malek et al., 2018; Mata et al., 2016; Sidransky et al., 2009). Among our patients with
4
EOPD with risk variants in GBA, the mean age at onset was 40.6 years, and 1 out of
5
the 5 carriers had cognitive impairment at the early stage of the disease. To
6
summarize, risk variants in LRRK2 and GBA also constitute an important predisposing
7
risk factor for developing EOPD.
8
Surprisingly, 41 patients (17.1%) carried rare variants of uncertain significance in
9
some known PD-related genes, which remain unexplained. Recently, a study in
10
Chinese patients with sporadic PD have also suggested that rare variants in AD PD
11
genes were associated with increased risk of PD (Yang et al., 2019). However, since
12
the relationship of many of these genes to PD is heavily debated (such as GIGYF2,
13
HTRA2, EIF4G1, and DNAJC13) (Blauwendraat et al., 2019b; Deng et al., 2018),
14
further studies that include larger sample sizes and systematic functional evaluations
15
are warranted. Importantly, the remaining 163 patients (67.9%) did not have
16
identifiable genetic variants. Actually, some variants may have been overlooked
17
because of the limitations of WES technology. Taken together, it is obvious
18
that much remains to be done.
19
In conclusion, 18 patients (7.5%) carried pathogenic or likely pathogenic variants in
20
known PD genes in our patients with EOPD. Additionally, 7.5% of patients carried
21
risk variants in either LRRK2 or GBA, which should also be considered for EOPD
22
patients. Nevertheless, 41 patients (17.1%) had rare variants of uncertain significance. 15
1
Therefore, our findings provide a better understanding of the genetic architecture of
2
PD among ethnic Chinese, and the pathogenicity of numerous rare variants should be
3
further investigated.
4
Disclosure
5 6
The authors have no actual or potential conflicts of interest. Acknowledgements
7
The authors would like to thank the patients for their generous participations. The
8
authors also thank Wenjun Chen, Xingkai An, Yan Wu, Zijuan Zhang, Xueye Mao,
9
Xueli Chang, Lan Cheng, Zhongjiao Lu, Dongmei Zhao, Qiao Liao, Wenjuan Yu, and
10
Xiaoyi Sun for clinical data collection. This work was supported by the National
11
Natural Science Foundation of China (81801272), China Postdoctoral Science
12
Foundation (2017M610605), Fundamental Research Funds for the Central
13
Universities (2018FZ0029 and 2018HH0077), Postdoctoral Research Foundation of
14
Sichuan University (2018SCU12029), and Post-Doctor Research Project, West China
15
Hospital, Sichuan University (2018HXBH085).
16
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Genetic variants of PARK genes in Korean patients with early-onset Parkinson's disease. Neurobiol. Aging. 75, 224.e229-224.e215.
Fig. 1. A schematic view of the LRRK2 protein with different functional domains. Variants found in present study were indicated by arrows and novel variants were highlighted in red. Abbreviations: ARM, Armadillo; ANK, Ankyrin; LRR, Leucine rich repeat; ROC, Ras in complex protein; COR, Carboxyl-terminal of ROC; MAPKKK, Mitogen-activated protein kinase kinase kinase.
22
Table 1 Summary of the variants found in our patients with EODP Patients
Patients
(likely) Gene
Total
Total
variants
patients
Patients carrying (likely)
Risk
carrying
VUS
pathogenic
variants
risk
(novel)
Pathogenic
Inheritance
carrying
variants (novel)
VUS variants
variants
SNCA (PARK1/4)
AD
2
2
1 (0)
1
0
0
1 (1)
1
Parkin (PARK2)
AR
12
9
12 (1)
9
0
0
0 (0)
0
PINK1 (PARK6)
AR
2
1
2 (0)
1
0
0
0 (0)
0
LRRK2 (PARK8)
AD
12
22
4 (2)
4
3
13
5 (4)
5
GIGYF2 (PARK11)
AD
7
8
0 (0)
0
0
0
7 (5)
8
HTRA2 (PARK13)
AD
1
1
0 (0)
0
0
0
1 (0)
1
PLA2G6 (PARK14)
AR
2
1
2 (1)
1
0
0
0 (0)
0
VPS35 (PARK17)
AD
3
3
0 (0)
0
0
0
3 (3)
3
EIF4G1 (PARK18)
AD
5
5
0 (0)
0
0
0
5 (5)
5
SYNJ1 (PARK20)
AR
2
1
2 (1)
1
0
0
0 (0)
0
DNAJC13 (PARK21)
AD
4
4
0 (0)
0
0
0
4 (3)
4
CHCHD2 (PARK22)
AD
1
1
1 (0)
1
0
0
0 (0)
0
VPS13C (PARK23)
AR
8
4
0 (0)
0
0
0
8 (8)
4
GBA
-
7
8
0 (0)
0
4
5
3 (0)
3
MAPT
-
4
4
0 (0)
0
0
0
4 (2)
4
GCH1
-
2
2
0 (0)
0
0
0
2 (1)
2
SLC41A1
-
1
1
0 (0)
0
0
0
1 (0)
1
SPG11
-
2
1
0 (0)
0
0
0
2 (1)
1
Total
-
77
77
24 (5)
18
7
18
46 (33)
41
Key: EOPD, early-onset Parkinson’s disease EOPD; VUS, variants of uncertain significance; AR, autosomal-recessive; AD, autosomal-dominant.
23
Table 2 Genetic and clinical features of patients with EOPD carrying PD-related variants Patient ID
Gene
Inheritance
Variant
ACMG classification
Sex
AAO (y)
Disease duration
Family history
IS
PI
Response to levodopa
Progression
H&Y
UPDRS III
MMSE
Depression
Dystonia
Motor fluctuations
Dyskinesia
3
Parkin
AR
p.C441R (hom)
Pathogenic
F
34
7
-
B
+
Good
Fast
2.5
48
26
+
-
-
+
4
Parkin
AR
Ex5-6del (het)/Ex5del (het)
Pathogenic/Pathogenic
M
31
10
-
T
+
Not good
Slow
2.5
38
26
-
-
+
+
5
Parkin
AR
p.M1T (het)/p.G284R (het)
Pathogenic/Likely pathogenic
F
36
30
-
B
+
Good
Slow
4
61
27
+
-
+
+
6
Parkin
AR
p.M1T (het)/Ex7del (het)
Pathogenic/Pathogenic
F
42
7
-
T
+
Good
Slow
2.5
17
28
-
-
+
+
7
Parkin
AR
Ex2del (het)/Ex3del (het)
Pathogenic/Pathogenic
F
30
7
-
T
-
Good
Slow
2
7
29
-
-
-
-
8
Parkin
AR
Ex2del (het)/p.C174_V175del
Pathogenic/Likely pathogenic
M
32
7
+
T
-
Not treated
Slow
2
12
29
-
-
-
-
9
Parkin
AR
Ex2del (het)/p.C441R (het)
Pathogenic/Pathogenic
F
35
10
-
T
-
Good
Slow
2.5
20
30
-
-
+
+
10
Parkin
AR
p.V164Yfs*13 (het)/Ex5del (het)
Pathogenic/Pathogenic
M
34
6
-
B
-
Not treated
Slow
2
28
28
-
-
-
-
11
Parkin
AR
Ex3-7del (het)/Ex9del (het)
Pathogenic/Pathogenic
M
33
7
+
T
+
Good
Fast
2.5
52
28
-
+
-
-
12
PINK1
AR
p.R492* (het)/p.1488+1G>A (het)
Pathogenic/Pathogenic
F
15
5
-
T
-
Good
Slow
2
14
30
-
+
-
-
44
PLA2G6
AR
p.D331Y (het)/p.R693G (het)
Likely pathogenic/Likely pathogenic
F
25
4
-
T
+
Good
Fast
3
46
28
+
-
+
+
52
SYNJ1
AR
Ex3del (het)/p.A551V (het)
Likely pathogenic/Likely pathogenic
F
48
1
-
T
-
Good
Fast
1.5
9
29
-
-
-
-
13
LRRK2
AD
p.R1067Q (het)
Likely pathogenic
F
39
1
-
T
-
Good
Fast
1.5
11
30
-
-
+
-
14
LRRK2
AD
p.T1410M (het)
Likely pathogenic
M
39
15
-
T
+
Good
Slow
3
70
26
+
-
+
-
15
LRRK2
AD
p.N1437D (het)
Likely pathogenic
F
37
16
+
T
+
Good
Slow
3
37
28
-
-
+
-
16
LRRK2
AD
p.G1451S (het)
Likely pathogenic
M
34
4
-
T
+
Good
Fast
3
44
30
-
-
+
-
1
SNCA
AD
p.A53T (het)
Pathogenic
M
31
5
+
B
-
Good
Slow
2
30
28
-
-
+
-
57
CHCHD2
AD
p.T61I (het)
Likely pathogenic
F
45
7
+
B
-
Good
Slow
1.5
11
29
-
-
-
-
62
GBA
-
c.115+1G>A (het)
Risk factor
F
41
4
+
B
-
Good
Slow
1.5
12
29
-
-
-
-
63
GBA
-
p.G241R (p.G202R) (het)
Risk factor
M
44
3
+
B
+
Good
Fast
2.5
25
24
-
-
+
-
64
GBA
-
p.D448H (p.D409H) (het)
Risk factor
M
32
4
-
B
+
Not treated
Fast
2.5
41
30
-
-
-
65
GBA
-
p.D448H (p.D409H) (het)
Risk factor
M
40
2
+
T
-
Not treated
Fast
2
31
26
-
-
-
-
66
GBA
-
p.L483P (p.L444P) (het)
Risk factor
F
46
3
-
B
-
Good
Fast
2
25
21
-
-
-
-
AD/AD
p.L1209P (het)/p.I358T (het)
VUS/VUS
M
41
10
-
T
-
Good
Slow
2
16
26
-
-
-
-
40
GIGYF2/ EIF4G1
Key: EOPD, early-onset Parkinson’s disease; PD, Parkinson’s disease; AR, autosomal-recessive; AD, autosomal-dominant; hom, homozygous; het, heterozygous; ACMG, American College of Medical Genetics and Genomics; AAO, age at onset; IS, initial symptoms; PI, postural instability; H&Y, Hoehn and Yahr stage; UPDRS III, the motor section of the Unified Parkinson's Disease Rating Scale; MMSE, Mini Mental State Examination, VUS, variants of uncertain significance. Bold indicates novel variations. The fast disease progression is defined as a mean annual rate of progression of more than 5 points on the motor section of the Unified Parkinson’s Disease Rating
24
25
Highlights
18 patients (7.5%) carried pathogenic or likely pathogenic variants in known PD genes.
(likely) pathogenic variants in Parkin and PINK1 were responsible for 4.2% of the patients.
Rare likely pathogenic variants in LRRK2 (1.7%) also appeared to be a relatively common cause of EOPD.
7.5% of patients carried risk variants in either LRRK2 or GBA.
Author Contributions Conceptualization: R. Peng. Formal analysis: N.N. Li. Investigation: N.N. Li, L. Wang. Resources: R. Peng, J.H. Zhang, J.Y. Li, J.X. Peng, L.R. Duan, C.L. Chen, D. Zhou, L. He. Data Curation: N.N. Li, L. Wang, R. Peng. Writing-Original Draft: N.N. Li. Writing-Review & Editing: E.K. Tan, N.N. Li, R. Peng. Supervision: R. Peng.
S0197-4580(19)30452-X
DOI:
https://doi.org/10.1016/j.neurobiolaging.2019.12.023
Reference:
NBA 10749
To appear in:
Neurobiology of Aging
Received Date: 23 August 2019 Revised Date:
19 November 2019
Accepted Date: 27 December 2019
Please cite this article as: Li, N., Wang, L., Zhang, J., Tan, E.-K., Li, J., Peng, J., Duan, L., Chen, C., Zhou, D., He, L., Peng, R., Whole-exome sequencing in early-onset Parkinson’s disease among ethnic Chinese, Neurobiology of Aging (2020), doi: https://doi.org/10.1016/j.neurobiolaging.2019.12.023. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Whole-exome
sequencing
in
early-onset
Parkinson’s
disease
among ethnic Chinese
Nannan Li a, Ling Wang a, Jinhong Zhang b, Eng-King Tan
c,d
, Junying Li a, Jiaxin
Peng a, Liren Duan a, Chaolan Chen a, Dong Zhou a, Li He a, Rong Peng a,*
a
Department of Neurology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
b
Department of Internal Medicine, Wangjiang Hospital, Sichuan University, Chengdu, Sichuan,
China c
Department of Neurology, National Neuroscience Institute, Singapore General Hospital,
Singapore, Singapore d
Duke-National University of Singapore Graduate Medical School, Singapore, Singapore
* Corresponding author at: Department of Neurology, West China Hospital, Sichuan University, No.37 Guoxue Xiang, Chengdu, Sichuan 610041, China. Tel.: +86 28 85423038, fax: +86 28 85582944. E-mail address: [email protected] (R. Peng).
1
1
ABSTRACT
2
Although early-onset Parkinson’s disease (EOPD) has a more penetrant genetic
3
etiology, the genetic architecture of EOPD remains unclear. The objectives of this
4
study were to assess the genetic and clinical features of EOPD among ethnic Chinese
5
from mainland China. Using whole-exome sequencing (WES), we performed genetic
6
analyses of 240 participants including 193 with sporadic and 47 with familial EOPD
7
(age of onset < 50 years). In total, 18 patients (7.5%) harbored pathogenic or likely
8
pathogenic variants in known PD genes. Among these variants, biallelic variants in
9
Parkin and PINK1 were responsible for 4.2% of cases, and rare likely pathogenic
10
variants in LRRK2 (1.7%) also appeared to be a relatively common cause of EOPD.
11
Notably, 7.5% of patients carried risk variants in either LRRK2 or GBA, which should
12
also be considered for EOPD. Nevertheless, 41 patients (17.1%) had rare variants of
13
unknown significance. In conclusion, our findings provide a better understanding of
14
the genetic architecture of PD among ethnic Chinese, and the pathogenicity of
15
numerous rare variants should be further investigated.
16
Keywords: Early-onset Parkinson’s disease; Genetics; Whole-exome sequencing;
17
Clinical features
18 19 20 21 22 23 24 2
1
1. Introduction
2
Parkinson’s disease (PD, OMIM #168600) is one of the most common
3
neurodegenerative disease, and its prevalence increases with age, affecting about 1%
4
of people over 60 years of age (de Lau and Breteler, 2006). Only 5.4% of all patients
5
with PD experience disease onset before 50 years of age, which is usually defined as
6
early-onset PD (EOPD) in the literature (Wickremaratchi et al., 2009). Notably,
7
patients with EOPD have a more penetrant genetic etiology than late-onset PD (LOPD)
8
and thus represent a good cohort to explore the genetics of PD. Elucidating the
9
pathophysiologic mechanisms of EOPD can also facilitate accurate diagnosis and
10
genetic counselling early and promote identification of new therapeutic targets.
11
Causative variants in the autosomal-recessive (AR) PD genes Parkin (PARK2),
12
PINK1 (PARK6), and DJ-1 (PARK7) are common causes of EOPD and account for
13
approximately 13% of these cases (Kilarski et al., 2012). In addition, several other AR
14
genes in EOPD such as ATP13A2, PLA2G6, FBXO7, DNAJC6, SYNJ1, VPS13C have
15
been identified (Schormair et al., 2018; Shen et al., 2018; Taghavi et al., 2018).
16
Moreover, some other known PD genes have also been implicated in EOPD
17
(Gustavsson et al., 2017; Lin et al., 2019; Ylonen et al., 2017). Recently, by
18
integrating whole-exome sequencing (WES) and functional evidence, several novel
19
candidate genes have been nominated in patients with EOPD (Guo et al., 2018; Jansen
20
et al., 2017; Lin et al., 2019), suggesting that a wide spectrum of genetic variants is
21
linked to EOPD. To date, there is still limited detailed data on the genetic architecture
22
of EOPD in ethnic Chinese. 3
1
To address these gaps in knowledge, we performed an extensive WES screening of
2
the common and rare genetic variants causing disease in a relatively large cohort of
3
patients with sporadic and familial EOPD among ethnic Chinese from mainland China.
4
In addition, we also examined the clinical features of these patients to better
5
understand the relationship between genotypes and clinical phenotypes.
6
2. Materials and Methods
7
2.1. Participants
8
A total of 240 participants including 193 patients with sporadic EOPD and 47
9
EOPD probands with a positive familial history (at least 1 family member in 3
10
generations with PD) (age of onset (AAO) < 50 years) were recruited from the
11
Movement Disorders Outpatient Clinic of West China Hospital, Sichuan University
12
(Chengdu, China) from 2004 to 2018. Of the 47 probands, 39 were compatible with
13
an autosomal-dominant (AD) inheritance pattern, and 7 were compatible with AR
14
inheritance or had 1 other affected first-degree relative with PD. All patients were
15
diagnosed with idiopathic PD by two neurologists using the United Kingdom PD
16
Society Brain Bank criteria (Hughes et al., 1992). All participants underwent detailed
17
neurological and neuropsychological assessment. This study was approved by the
18
Ethics Committee of Sichuan University. Written informed consent was obtained
19
from all participants.
20
2.2. Genetic analysis
21
2.2.1. Whole-exome sequencing
22
Genetic testing of all patients was conducted using WES technology. The 4
1
SureSelect Human All Exon v6 Kit from Agilent (Agilent, Santa Clara, CA, USA)
2
was used to capture whole exomes. In brief, libraries were constructed according to
3
the manufacturer's protocols and sequenced on the Illumina HiSeq X10 platform
4
(Illumina, San Diego, CA, USA) using the 150 bp paired-end module. Quality control
5
was applied to the raw data to guarantee the meaningfulness of the downstream
6
analysis. The mean depth of coverage per sample was over 100-fold, with more than
7
99.5% of the targeted regions covered and 98% at least 20-fold. Alignment to the
8
human genome assembly hg19 was carried out using the Burrows-Wheeler Aligner
9
(BWA) (Li and Durbin, 2009), followed by variant calling using the Genome
10
Analysis Toolkit (GATK version 3.7) (McKenna et al., 2010). All variants were
11
annotated with ANNOVAR (Wang et al., 2010). We used a stringent minor allele
12
frequency filter (MAF < 1%) in public databases (1000g, 1000 Genomes,
13
http://www.1000genomes.org;
14
http://gnomad.broadinstitute.org;
15
http://exac.broadinstitute.org), focusing on missense and loss-of-function variants
16
(nonsense, frameshift, splice, and copy number variants (CNVs) called by CNVkit
17
(Talevich et al., 2016)). The sequence variants were also further interpreted and
18
classified according to the American College of Medical Genetics and Genomics
19
(ACMG) recommendations (Richards et al., 2015), during which allele frequency,
20
nucleotide conservation by the Conservation Reserve Enhancement Program (GERP,
21
http://mendel.stanford.edu/sidowlab/downloads/gerp/index.html),
22
prediction
(MutationTaster,
gnomAD, ExAC,
Genome Exome
Aggregation Aggregation
http://www.mutationtaster.org; 5
Database, Consortium,
pathogenicity PolyPhen-2,
1
http://genetics.bwh.harvard.edu/pph2;
2
http://cadd.gs.washington.edu), and inheritance pattern were all further analyzed. The
3
rare variants were defined as variants with a minor allele frequency (MAF) ≤ 0.1%.
4
This analysis did not screen for trinucleotide or hexanucleotide repeat expansions. All
5
240 patient samples were analyzed as singleton exomes rather than as trios (a patient
6
with EOPD and the patient’s parents) or quads (a patient with EOPD, the patient’s
7
parents and a patient’s healthy sibling). Genomic DNA was extracted using standard
8
extraction procedures. Variants considered likely related to the patient’s phenotype
9
were confirmed by Sanger sequencing.
10 11
SIFT,
http://sift.jcvi.org;
and
CADD,
2.2.2. Multiplex ligation-dependent probe amplification and qPCR assays For CNVs called by CNVkit, the multiplex ligation-dependent probe amplification
12
(MPLA)
13
polymerase chain reaction (qPCR) were used to detect these large deletions or
14
duplications. The qPCR analysis was performed and analyzed using the Eco
15
Real-Time PCR System. We presented the primers and reaction conditions for the
16
qPCR in the Supplementary Table S2.
17
2.3. Statistical analysis
(MRC-Holland, Amsterdam, The Netherlands)
and
quantification
18
Statistical analysis was performed using SPSS software version 24.0 (SPSS Inc.,
19
Chicago, IL, USA). The Chi-square test was used to compare proportions between
20
groups. A two-tailed p < 0.05 was considered statistically significant.
21
3. Results
22
The mean age at onset of patients with sporadic EOPD was 40.9 ± 6.5 years, and 6
1
53.4% were men, while the mean age at onset of patients with familial EOPD was
2
41.5 ± 6.5 years, and 51.1 % were men. A total of 77 participants (77/240, 32.1%)
3
harbored 77 variants in 18 genes (Table 1): 18 patients (18/240, 7.5%) harbored 24
4
pathogenic or likely pathogenic variants in the AR PD genes (Parkin, PINK1,
5
PLA2G6, and SYNJ1) and the AD PD genes (LRRK2, SNCA, and CHCHD2); 18
6
patients (18/240, 7.5%) carried risk variants in either LRRK2 or GBA; 41 patients
7
(41/240, 17.1%) had 46 rare variants of uncertain significance; the remaining 163
8
patients (163/240, 67.9%) did not have identifiable genetic variants. Supplementary
9
Table S1 presents all of the genetic information.
10
3.1. Pathogenic or likely pathogenic variants
11
3.1.1. Autosomal-recessive PD genes (Parkin, PINK1, PLA2G6, and SYNJ1)
12
Parkin was the most prevalent causative gene in the 240 patients with EOPD. Of
13
the 9 Parkin biallelic variants (homozygous or compound heterozygous variants)
14
carriers (9/240, 3.8%): 1 had a homozygous variant (p.C441R) from a consanguineous
15
family, 8 had compound heterozygous variants (Table 2, Supplementary Table S1). In
16
addition, 3 had single heterozygous variants (exon 3 duplication, exons 2-4
17
duplication, exons 3-4 deletion). Since the prevalence of Parkin single heterozygosity
18
was similar to the rate reported in healthy controls (Malek et al., 2016), therefore
19
patients with single heterozygous variants in recessive genes were analyzed as
20
non-carriers. Exon rearrangements constituted the most common type of variants,
21
especially between exons 2 and 7. Interestingly, 1 patient had a novel frameshift
22
variant in exon 4 of Parkin, p.V164Yfs*13, which was determined to be pathogenic 7
1
using the ACMG recommendations. 4 patients had previously reported missense
2
variants: 2 had p.M1T, 1 had p.G284R, and 2 had p.C441R. However, there was no
3
significant difference in the frequency of biallelic variants in patients with EOPD with
4
a positive family history (2/47, 4.3%) and patients without a family history (7/193,
5
3.6%) (p = 1.00). These patients carrying biallelic variants in Parkin exhibited a
6
typical and generally mild phenotype, a good response to levodopa, slow disease
7
progression, and no cognitive impairment (Table 2).
8
We also identified 1 patient carrying previously reported compound heterozygous
9
pathogenic variants in PINK1 (p.R492* and c.1488+1G>A) (1/240, 0.4%) (Table 2,
10
Supplementary Table S1). The female carrier of biallelic PINK1 variants exhibited
11
very mild symptoms accompanying lower limb dystonia, an excellent response to a
12
low dose of levodopa (100 mg/qd), and very slow disease progression (Table 2).
13
However, no pathogenic or likely pathogenic variant was found in DJ-1.
14
In PLA2G6, 1 sporadic patient had compound heterozygosity for a novel p.R693G
15
variant and the previously reported p.D331Y variant classified as likely pathogenic.
16
The carrier of PLA2G6 variants had an earlier age at onset (25 years), rapid disease
17
progression with a good response to levodopa, early onset of treated-induced
18
dyskinesias, marked postural impairment, depression, no dystonia, normal cognitive
19
function, and a normal brain MRI (Table 2). For SYNJ1, compound heterozygosity for
20
a novel deletion of exon 3 variant and the previously reported p.A551V variant from 1
21
sporadic case were classified as likely pathogenic. This patient with biallelic variants
22
of SYNJ1 had the typical PD phenotype without seizures and cognitive decline (Table 8
1
2).
2
3.1.2. Autosomal-dominant PD genes (LRRK2, SNCA, and CHCHD2)
3
We did not find the p.G2019S variant in LRRK2, which is the most common
4
pathogenic variant in European-descent populations and is rare in Asian populations.
5
However, 4 patients carried 4 rare heterozygous variants (p.R1067Q, p.T1410M,
6
p.N1437D, p.G1451S) as likely pathogenic variants (4/240, 1.7%) (Table 2,
7
Supplementary Table S1). The p.R1067Q (Paisan-Ruiz et al., 2008; Yang et al., 2019;
8
Youn et al., 2019) and p.T1410M (Bozi et al., 2014; Ross et al., 2011) variants had
9
previously been reported, while the p.N1437D, and p.G1451S variants were novel.
10
The p.R1067Q variant is located in the leucine-rich repeat (LRR) domain of LRRK2,
11
and the p.T1410M, p.N1437D, and p.G1451S variants are located in the ROC domain
12
of LRRK2 (Fig. 1). The mean age at onset for the 4 patients carrying rare likely
13
pathogenic variants in LRRK2 was 36.3 ± 2.2 years (range 34-39) (Table 2). They
14
exhibited typical PD symptoms, a slightly slower disease progression, responded well
15
to levodopa treatment, and were without atypical signs (Table 2). In SNCA, the known
16
pathogenic p.A53T variant was identified in 1 patient with familial EOPD with an
17
onset at 31 years of age (Table 2). An additional proband had heterozygous likely
18
pathogenic p.T61I variant in CHCHD2 (Table 2).
19
3.2. Risk variants
20
We identified two common genetic susceptibility variants in LRRK2 among Asian
21
patients with PD, p.R1628P carried by 8 patients and p.G2385R carried by 3 patients,
22
and a low-frequency genetic susceptibility variant for PD, LRRK2 p.A419V carried by 9
1
2 patients (13/240, 5.4%). In GBA, we identified 5 heterozygous carriers (5/240, 2.1%)
2
of rare GBA variants: 1 carried c.115+1G>A (IVS2+1), 1 carried p.G241R (p.G202R),
3
2 carried p.D448H (p.D409H), 1 carried p.L483P (p.L444P) (Table 2, Supplementary
4
Table S1). Notably, these 4 rare variants had been previously identified as being
5
pathogenic in Gaucher disease (GD) and as genetic risk factors for PD in the
6
heterozygous state (Malek et al., 2018). The mean age at onset of the 5 patients who
7
carried known risk variants in GBA was 40.6 ± 5.4 years (range 32-46) (Table 2).
8
Considering the rarity of GBA variants, these data might not be suitable for comparing
9
the age of onset between carriers and non-carriers of risk variants in GBA. They had
10
relatively shorter disease duration (3.2 ± 0.8, range 2-4), and 1 out of 5 patients
11
exhibited cognitive impairment (Mini-Mental State Examination score < 24) (Table
12
2).
13
3.3. Variants of uncertain significance
14
In addition to these (likely) pathogenic and risk variants, 41 patients (41/240,
15
17.1%) had 46 rare variants in some known PD genes (1 in SNCA, 5 in LRRK2, 7 in
16
GIGYF2, 1 in HTRA2, 3 in VPS35, 5 in EIF4G1, 4 in DNAJC13, and 8 in VPS13C)
17
and in other genes (3 in GBA, 4 in MAPT, 2 in GCH1, 1 in SLC41A1, and 2 in SPG11)
18
(Table 1, Supplementary Table S1), which were categorized as being of uncertain
19
significance. Previous studies have shown that MAPT (Nalls et al., 2019; Nalls et al.,
20
2014; Pihlstrom et al., 2013), GCH1 (Mencacci et al., 2014; Nalls et al., 2019; Nalls
21
et al., 2014), SLC41A1 (Lin et al., 2014), and SPG11 (Anheim et al., 2009;
22
Guidubaldi et al., 2011) were associated with PD. Notably, 1 patient with sporadic 10
1
EOPD had a heterozygous p.L1209P variant in GIGYF2 that was previously reported
2
to be associated with LOPD (Guella et al., 2011) and a novel rare heterozygous
3
p.I358T variant in EIF4G1, and the patient had an age at onset of 41 years, typical
4
symptoms and a good response to levodopa (Table 2). More importantly, 33 of these
5
rare variants (33/46, 71.7%) were novel (Table 1, Supplementary Table S1).
6
4. Discussion
7
We performed an extensive WES screening of the common and rare genetic
8
variants causing disease in a relatively large cohort of patients with sporadic and
9
familial EOPD among ethnic Chinese from mainland China. In total, 18 patients
10
(7.5%) harbored pathogenic or likely pathogenic variants in Parkin, PINK1, PLA2G6,
11
SYNJ1, LRRK2, SNCA, and CHCHD2. Notably, 7.5% of patients carried a risk variant
12
in either LRRK2 or GBA, which should also be considered for patients with EOPD.
13
However, 41 patients (17.1%) had rare variants of uncertain significance. Using WES,
14
our study represents an extensive examination of the genetic etiology of EOPD among
15
ethnic Chinese from mainland China, and the results revealed different genetic
16
backgrounds.
17
Parkin was the most common causative gene in EOPD. In our cohort, biallelic
18
Parkin variants accounted for 3.8% of patients with EOPD. Our findings was
19
comparable with those of other WES studies in Taiwanese populations (Lin et al.,
20
2019), which estimated that 2.6% patients with EOPD had biallelic Parkin variants,
21
and in predominantly Caucasian populations with a frequency of 1.3-2.8%
22
(Gustavsson et al., 2017; Schormair et al., 2018; Tan et al., 2019). Moreover, we also 11
1
found that the trend for biallelic Parkin variants was more common in familial (4.3%)
2
than in sporadic (3.6%) EOPD, although this was not significantly different.
3
Importantly, the novel p.V164Yfs*13 variant in exon 4 of Parkin was detected and
4
was classified as a pathogenic variant that may result in an absence of protein by
5
nonsense mRNA decay and cause disease. PINK1 is the second most frequent gene
6
involved in EOPD. Only 1 patient (0.4%) carried known biallelic PINK1 variants in
7
our series, whereas no variant was found in the rarer DJ-1 gene. Similarly, a recent
8
WES study in Taiwanese populations have not found any patients carrying biallelic
9
variants in PINK1 or DJ-1 (Lin et al., 2019). Furthermore, Parkin and PINK1 have
10
been shown to be involved in the same cellular pathway (Pickrell and Youle, 2015),
11
and these patients carrying biallelic variants in Parkin or PINK also exhibited a
12
similar PD phenotype. Taken together, pathogenic or likely pathogenic biallelic
13
variants in Parkin and PINK1 were common causes of EOPD in our population and
14
accounted for 4.2% of these patients.
15
For other AR genes in EOPD, we also identified 4 likely pathogenic variants in
16
PLA2G6 (p.D331Y and a novel p.R693G) and SYNJ1 (a novel deletion of exon 3 and
17
p.A551V) from 2 patients (0.8%). Regarding PLA2G6, previous studies have reported
18
the homozygous state or compound heterozygosity of the p. D331Y variant in several
19
patients with early-onset parkinsonism in Asian populations (Lin et al., 2019; Lu et al.,
20
2012; Shi et al., 2011; Xie et al., 2015). Combining these previous studies with the
21
ExAC and gnomAD databases, the p. D331Y variant may be specific for Asian
22
populations. Interestingly, compound heterozygosity for a novel p.R693G variant and 12
1
the previously reported p.D331Y variant in PLA2G6 were identified in a sporadic case
2
with some clinical and imaging features that are compatible with those of carriers of
3
the homozygous p.D331Y variant (Lin et al., 2019; Shi et al., 2011; Xie et al., 2015).
4
Recently, p.A551V in SYNJ1 was identified in a single heterozygous state in a patient
5
with EOPD from Taiwan (Chen et al., 2015). In the present study, compound
6
heterozygosity for a novel deletion of exon 3 variant and the previously reported
7
p.A551V variant in SYNJ1 were found in a patient with sporadic EOPD who exhibited
8
a typical PD phenotype. Taken together, our findings suggest that other AR genes
9
such as SYNJ1 and PLA2G6 should also be considered for patients with EOPD in
10
ethnic Chinese.
11
Although variants in LRRK2 more commonly result in typical late-onset PD, they
12
can also cause PD at a much younger age (Trinh et al., 2018). In a large UK
13
population-based cohort, the LRRK2 p.G2019S variant was more common in
14
young-onset patients (2.2%) than in late-onset patients (0.4%) (Tan et al., 2019).
15
However, we did not find the p.G2019S variant in LRRK2 because this variant is rare
16
in Asian populations. Evidence is emerging that rare variants of Mendelian genes for
17
PD may also play a role in sporadic PD (Spataro et al., 2015; Yang et al., 2019) and
18
EOPD (Youn et al., 2019). In Korean patients with EOPD, LRRK2 is the known PD
19
gene with the most common rare variants (Youn et al., 2019). In our cohort, 4 rare
20
variants in LRRK2 were also classified as likely pathogenic variants (1.7%), including
21
the previously reported p.R1067Q and p.T1410M variants, and two novel p.N1437D
22
and p.G1451S variants. The p.R1067Q variant in the LRR domain of LRRK2 might 13
1
alter domain stability (Cardona et al., 2014). However, no functional evidence was
2
found for other three variants in LRRK2, so these variants should be studied further.
3
Moreover, 4 patients carried these rare variants in LRRK2 had an earlier mean age at
4
onset of 36.3 years compared with that of all LRRK2 variant carriers in a recent
5
MDSGene systematic review (57.0 years) (Trinh et al., 2018). Therefore, rare likely
6
pathogenic variants in LRRK2 (1.7%) also appeared to be relatively common causes
7
of EOPD.
8
LRRK2 is known as pleomorphic risk loci, at which rare coding variants can cause
9
PD and more common variants can also increase the risk for disease (Blauwendraat et
10
al., 2019b). In LRRK2, we also identified 8 patients carrying p.R1628P, 3 patients
11
carrying p.G2385R, and 2 patients carrying p. A419V (5.4%), which are common
12
(p.R1628P and p.G2385R) and low-frequency (p.A419V) genetic risk variants among
13
Asian patients with PD (Li et al., 2015; Li et al., 2012; Tan et al., 2010). In addition,
14
biallelic variants in GBA cause GD, while heterozygous variants are currently
15
considered the most important risk factor for developing PD (Schapira, 2015).
16
Notably, 5 patients (2.1%) carried known risk variants in GBA in our EOPD cohort.
17
Our observations are in line with the rate of known risk variants in GBA reported for
18
Finnish patients with EOPD (2.8%) (Ylonen et al., 2017) and Czech patients with
19
EOPD (5%) (Schormair et al., 2018). The L483P variant is the most common variant
20
detected in GBA in cases with PD from China (Chen et al., 2014). In our series, only
21
1 patient was found to carry the L483P variant (0.4%), which is also consistent with
22
that in a Taiwanese cohort (0.6%) (Lin et al., 2019). Moreover, previous studies have 14
1
suggested that patients who carried GBA risk variants had an earlier age at onset and
2
more frequent cognitive impairment (Blauwendraat et al., 2019a; Davis et al., 2016;
3
Malek et al., 2018; Mata et al., 2016; Sidransky et al., 2009). Among our patients with
4
EOPD with risk variants in GBA, the mean age at onset was 40.6 years, and 1 out of
5
the 5 carriers had cognitive impairment at the early stage of the disease. To
6
summarize, risk variants in LRRK2 and GBA also constitute an important predisposing
7
risk factor for developing EOPD.
8
Surprisingly, 41 patients (17.1%) carried rare variants of uncertain significance in
9
some known PD-related genes, which remain unexplained. Recently, a study in
10
Chinese patients with sporadic PD have also suggested that rare variants in AD PD
11
genes were associated with increased risk of PD (Yang et al., 2019). However, since
12
the relationship of many of these genes to PD is heavily debated (such as GIGYF2,
13
HTRA2, EIF4G1, and DNAJC13) (Blauwendraat et al., 2019b; Deng et al., 2018),
14
further studies that include larger sample sizes and systematic functional evaluations
15
are warranted. Importantly, the remaining 163 patients (67.9%) did not have
16
identifiable genetic variants. Actually, some variants may have been overlooked
17
because of the limitations of WES technology. Taken together, it is obvious
18
that much remains to be done.
19
In conclusion, 18 patients (7.5%) carried pathogenic or likely pathogenic variants in
20
known PD genes in our patients with EOPD. Additionally, 7.5% of patients carried
21
risk variants in either LRRK2 or GBA, which should also be considered for EOPD
22
patients. Nevertheless, 41 patients (17.1%) had rare variants of uncertain significance. 15
1
Therefore, our findings provide a better understanding of the genetic architecture of
2
PD among ethnic Chinese, and the pathogenicity of numerous rare variants should be
3
further investigated.
4
Disclosure
5 6
The authors have no actual or potential conflicts of interest. Acknowledgements
7
The authors would like to thank the patients for their generous participations. The
8
authors also thank Wenjun Chen, Xingkai An, Yan Wu, Zijuan Zhang, Xueye Mao,
9
Xueli Chang, Lan Cheng, Zhongjiao Lu, Dongmei Zhao, Qiao Liao, Wenjuan Yu, and
10
Xiaoyi Sun for clinical data collection. This work was supported by the National
11
Natural Science Foundation of China (81801272), China Postdoctoral Science
12
Foundation (2017M610605), Fundamental Research Funds for the Central
13
Universities (2018FZ0029 and 2018HH0077), Postdoctoral Research Foundation of
14
Sichuan University (2018SCU12029), and Post-Doctor Research Project, West China
15
Hospital, Sichuan University (2018HXBH085).
16
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genes for Parkinson's disease contribute to the sporadic forms of the disease. Hum. Mol. Genet. 24, 2023-2034. Taghavi, S., Chaouni, R., Tafakhori, A., Azcona, L.J., Firouzabadi, S.G., Omrani, M.D., Jamshidi, J., Emamalizadeh, B., Shahidi, G.A., Ahmadi, M., Habibi, S.A.H., Ahmadifard, A., Fazeli, A., Motallebi, M., Petramfar, P., Askarpour, S., Askarpour, S., Shahmohammadibeni, H.A., Shahmohammadibeni, N., Eftekhari, H., Shafiei Zarneh, A.E., Mohammadihosseinabad, S., Khorrami, M., Najmi, S., Chitsaz, A., Shokraeian, P., Ehsanbakhsh, H., Rezaeidian, J., Ebrahimi Rad, R., Madadi, F., Andarva, M., Alehabib, E., Atakhorrami, M., Mortazavi, S.E., Azimzadeh, Z., Bayat, M., Besharati, A.M., Harati-Ghavi, M.A., Omidvari, S., Dehghani-Tafti, Z., Mohammadi, F., Mohammad Hossein Pour, B., Noorollahi Moghaddam, H., Esmaili Shandiz, E., Habibi, A., Taherian-Esfahani, Z., Darvish, H., Paisan-Ruiz, C., 2018. A Clinical and Molecular Genetic Study of 50 Families with Autosomal Recessive Parkinsonism Revealed Known and Novel Gene Mutations. Mol. Neurobiol. 55, 3477-3489. Talevich, E., Shain, A.H., Botton, T., Bastian, B.C., 2016. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing. PLoS Comput. Biol. 12, e1004873. Tan, E.K., Peng, R., Teo, Y.Y., Tan, L.C., Angeles, D., Ho, P., Chen, M.L., Lin, C.H., Mao, X.Y., Chang, X.L., Prakash, K.M., Liu, J.J., Au, W.L., Le, W.D., Jankovic, J., Burgunder, J.M., Zhao, Y., Wu, R.M., 2010. Multiple LRRK2 variants modulate risk of Parkinson disease: a Chinese multicenter study. Hum. Mutat. 31, 561-568. Tan, M.M.X., Malek, N., Lawton, M.A., Hubbard, L., Pittman, A.M., Joseph, T., Hehir, J., Swallow, D.M.A., Grosset, K.A., Marrinan, S.L., Bajaj, N., Barker, R.A., Burn, D.J., Bresner, C., Foltynie, T., Hardy, J., Wood, N., Ben-Shlomo, Y., Grosset, D.G., Williams, N.M., Morris, H.R., 2019. Genetic analysis of Mendelian mutations in a large UK population-based Parkinson's disease study. Brain. Trinh, J., Zeldenrust, F.M.J., Huang, J., Kasten, M., Schaake, S., Petkovic, S., Madoev, H., Grunewald, A., Almuammar, S., Konig, I.R., Lill, C.M., Lohmann, K., Klein, C., Marras, C., 2018. Genotype-phenotype relations for the Parkinson's disease genes SNCA, LRRK2, VPS35: MDSGene systematic review. Mov. Disord. 33, 1857-1870. Wang, K., Li, M., Hakonarson, H., 2010. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164. Wickremaratchi, M.M., Perera, D., O'Loghlen, C., Sastry, D., Morgan, E., Jones, A., Edwards, P., Robertson, N.P., Butler, C., Morris, H.R., Ben-Shlomo, Y., 2009. Prevalence and age of onset of Parkinson's disease in Cardiff: a community based cross sectional study and meta-analysis. J. Neurol. Neurosurg. Psychiatry. 80, 805-807. Xie, F., Cen, Z., Ouyang, Z., Wu, S., Xiao, J., Luo, W., 2015. Homozygous p.D331Y mutation in PLA2G6 in two patients with pure autosomal-recessive early-onset parkinsonism: further evidence of a fourth phenotype of PLA2G6-associated neurodegeneration. Parkinsonism Relat. Disord. 21, 420-422. Yang, N., Zhao, Y., Liu, Z., Zhang, R., He, Y., Zhou, Y., Xu, Q., Sun, Q., Yan, X., Guo, J., Tang, B., 2019. Systematically analyzing rare variants of autosomal-dominant genes for sporadic Parkinson's disease in a Chinese cohort. Neurobiol. Aging. 76, 215.e211-215.e217. Ylonen, S., Siitonen, A., Nalls, M.A., Ylikotila, P., Autere, J., Eerola-Rautio, J., Gibbs, R., Hiltunen, M., Tienari, P.J., Soininen, H., Singleton, A.B., Majamaa, K., 2017. Genetic risk factors in Finnish patients with Parkinson's disease. Parkinsonism Relat. Disord. 45, 39-43. Youn, J., Lee, C., Oh, E., Park, J., Kim, J.S., Kim, H.T., Cho, J.W., Park, W.Y., Jang, W., Ki, C.S., 2019. 21
Genetic variants of PARK genes in Korean patients with early-onset Parkinson's disease. Neurobiol. Aging. 75, 224.e229-224.e215.
Fig. 1. A schematic view of the LRRK2 protein with different functional domains. Variants found in present study were indicated by arrows and novel variants were highlighted in red. Abbreviations: ARM, Armadillo; ANK, Ankyrin; LRR, Leucine rich repeat; ROC, Ras in complex protein; COR, Carboxyl-terminal of ROC; MAPKKK, Mitogen-activated protein kinase kinase kinase.
22
Table 1 Summary of the variants found in our patients with EODP Patients
Patients
(likely) Gene
Total
Total
variants
patients
Patients carrying (likely)
Risk
carrying
VUS
pathogenic
variants
risk
(novel)
Pathogenic
Inheritance
carrying
variants (novel)
VUS variants
variants
SNCA (PARK1/4)
AD
2
2
1 (0)
1
0
0
1 (1)
1
Parkin (PARK2)
AR
12
9
12 (1)
9
0
0
0 (0)
0
PINK1 (PARK6)
AR
2
1
2 (0)
1
0
0
0 (0)
0
LRRK2 (PARK8)
AD
12
22
4 (2)
4
3
13
5 (4)
5
GIGYF2 (PARK11)
AD
7
8
0 (0)
0
0
0
7 (5)
8
HTRA2 (PARK13)
AD
1
1
0 (0)
0
0
0
1 (0)
1
PLA2G6 (PARK14)
AR
2
1
2 (1)
1
0
0
0 (0)
0
VPS35 (PARK17)
AD
3
3
0 (0)
0
0
0
3 (3)
3
EIF4G1 (PARK18)
AD
5
5
0 (0)
0
0
0
5 (5)
5
SYNJ1 (PARK20)
AR
2
1
2 (1)
1
0
0
0 (0)
0
DNAJC13 (PARK21)
AD
4
4
0 (0)
0
0
0
4 (3)
4
CHCHD2 (PARK22)
AD
1
1
1 (0)
1
0
0
0 (0)
0
VPS13C (PARK23)
AR
8
4
0 (0)
0
0
0
8 (8)
4
GBA
-
7
8
0 (0)
0
4
5
3 (0)
3
MAPT
-
4
4
0 (0)
0
0
0
4 (2)
4
GCH1
-
2
2
0 (0)
0
0
0
2 (1)
2
SLC41A1
-
1
1
0 (0)
0
0
0
1 (0)
1
SPG11
-
2
1
0 (0)
0
0
0
2 (1)
1
Total
-
77
77
24 (5)
18
7
18
46 (33)
41
Key: EOPD, early-onset Parkinson’s disease EOPD; VUS, variants of uncertain significance; AR, autosomal-recessive; AD, autosomal-dominant.
23
Table 2 Genetic and clinical features of patients with EOPD carrying PD-related variants Patient ID
Gene
Inheritance
Variant
ACMG classification
Sex
AAO (y)
Disease duration
Family history
IS
PI
Response to levodopa
Progression
H&Y
UPDRS III
MMSE
Depression
Dystonia
Motor fluctuations
Dyskinesia
3
Parkin
AR
p.C441R (hom)
Pathogenic
F
34
7
-
B
+
Good
Fast
2.5
48
26
+
-
-
+
4
Parkin
AR
Ex5-6del (het)/Ex5del (het)
Pathogenic/Pathogenic
M
31
10
-
T
+
Not good
Slow
2.5
38
26
-
-
+
+
5
Parkin
AR
p.M1T (het)/p.G284R (het)
Pathogenic/Likely pathogenic
F
36
30
-
B
+
Good
Slow
4
61
27
+
-
+
+
6
Parkin
AR
p.M1T (het)/Ex7del (het)
Pathogenic/Pathogenic
F
42
7
-
T
+
Good
Slow
2.5
17
28
-
-
+
+
7
Parkin
AR
Ex2del (het)/Ex3del (het)
Pathogenic/Pathogenic
F
30
7
-
T
-
Good
Slow
2
7
29
-
-
-
-
8
Parkin
AR
Ex2del (het)/p.C174_V175del
Pathogenic/Likely pathogenic
M
32
7
+
T
-
Not treated
Slow
2
12
29
-
-
-
-
9
Parkin
AR
Ex2del (het)/p.C441R (het)
Pathogenic/Pathogenic
F
35
10
-
T
-
Good
Slow
2.5
20
30
-
-
+
+
10
Parkin
AR
p.V164Yfs*13 (het)/Ex5del (het)
Pathogenic/Pathogenic
M
34
6
-
B
-
Not treated
Slow
2
28
28
-
-
-
-
11
Parkin
AR
Ex3-7del (het)/Ex9del (het)
Pathogenic/Pathogenic
M
33
7
+
T
+
Good
Fast
2.5
52
28
-
+
-
-
12
PINK1
AR
p.R492* (het)/p.1488+1G>A (het)
Pathogenic/Pathogenic
F
15
5
-
T
-
Good
Slow
2
14
30
-
+
-
-
44
PLA2G6
AR
p.D331Y (het)/p.R693G (het)
Likely pathogenic/Likely pathogenic
F
25
4
-
T
+
Good
Fast
3
46
28
+
-
+
+
52
SYNJ1
AR
Ex3del (het)/p.A551V (het)
Likely pathogenic/Likely pathogenic
F
48
1
-
T
-
Good
Fast
1.5
9
29
-
-
-
-
13
LRRK2
AD
p.R1067Q (het)
Likely pathogenic
F
39
1
-
T
-
Good
Fast
1.5
11
30
-
-
+
-
14
LRRK2
AD
p.T1410M (het)
Likely pathogenic
M
39
15
-
T
+
Good
Slow
3
70
26
+
-
+
-
15
LRRK2
AD
p.N1437D (het)
Likely pathogenic
F
37
16
+
T
+
Good
Slow
3
37
28
-
-
+
-
16
LRRK2
AD
p.G1451S (het)
Likely pathogenic
M
34
4
-
T
+
Good
Fast
3
44
30
-
-
+
-
1
SNCA
AD
p.A53T (het)
Pathogenic
M
31
5
+
B
-
Good
Slow
2
30
28
-
-
+
-
57
CHCHD2
AD
p.T61I (het)
Likely pathogenic
F
45
7
+
B
-
Good
Slow
1.5
11
29
-
-
-
-
62
GBA
-
c.115+1G>A (het)
Risk factor
F
41
4
+
B
-
Good
Slow
1.5
12
29
-
-
-
-
63
GBA
-
p.G241R (p.G202R) (het)
Risk factor
M
44
3
+
B
+
Good
Fast
2.5
25
24
-
-
+
-
64
GBA
-
p.D448H (p.D409H) (het)
Risk factor
M
32
4
-
B
+
Not treated
Fast
2.5
41
30
-
-
-
65
GBA
-
p.D448H (p.D409H) (het)
Risk factor
M
40
2
+
T
-
Not treated
Fast
2
31
26
-
-
-
-
66
GBA
-
p.L483P (p.L444P) (het)
Risk factor
F
46
3
-
B
-
Good
Fast
2
25
21
-
-
-
-
AD/AD
p.L1209P (het)/p.I358T (het)
VUS/VUS
M
41
10
-
T
-
Good
Slow
2
16
26
-
-
-
-
40
GIGYF2/ EIF4G1
Key: EOPD, early-onset Parkinson’s disease; PD, Parkinson’s disease; AR, autosomal-recessive; AD, autosomal-dominant; hom, homozygous; het, heterozygous; ACMG, American College of Medical Genetics and Genomics; AAO, age at onset; IS, initial symptoms; PI, postural instability; H&Y, Hoehn and Yahr stage; UPDRS III, the motor section of the Unified Parkinson's Disease Rating Scale; MMSE, Mini Mental State Examination, VUS, variants of uncertain significance. Bold indicates novel variations. The fast disease progression is defined as a mean annual rate of progression of more than 5 points on the motor section of the Unified Parkinson’s Disease Rating
24
25
Highlights
18 patients (7.5%) carried pathogenic or likely pathogenic variants in known PD genes.
(likely) pathogenic variants in Parkin and PINK1 were responsible for 4.2% of the patients.
Rare likely pathogenic variants in LRRK2 (1.7%) also appeared to be a relatively common cause of EOPD.
7.5% of patients carried risk variants in either LRRK2 or GBA.
Author Contributions Conceptualization: R. Peng. Formal analysis: N.N. Li. Investigation: N.N. Li, L. Wang. Resources: R. Peng, J.H. Zhang, J.Y. Li, J.X. Peng, L.R. Duan, C.L. Chen, D. Zhou, L. He. Data Curation: N.N. Li, L. Wang, R. Peng. Writing-Original Draft: N.N. Li. Writing-Review & Editing: E.K. Tan, N.N. Li, R. Peng. Supervision: R. Peng.