ORIGINAL COMMUNICATION
Mutation analysis of Parkin, PINK1 and DJ-1 genes in Chinese
patients with sporadic early onset parkinsonism
Ji-feng Guo • Xue-wei Zhang • Li-luo Nie •
Hai-nan Zhang • Bin Liao • Jing Li • Lei Wang •
Xin-xiang Yan • Bei-sha Tang
Received: 5 October 2009 / Revised: 25 December 2009 / Accepted: 20 January 2010 / Published online: 10 February 2010
 Springer-Verlag 2010
Abstract Early onset parkinsonism (EOP) has been asso-
ciated with mutations in the Parkin, PINK1, and DJ-1 genes.
We studied the prevalence of mutations in all three genes in
127 unrelated Chinese patients with apparently sporadic
EOP using direct sequencing analysis and real-time quanti-
tative PCR analysis assay. There are 16 patients (12.6%) with
mutations of Parkin gene, four patients (3.1%) with muta-
tions of PINK1 gene, and three patients (2.4%) with mutation
of DJ-1 gene. In conclusion, Parkin gene mutation is the
most common pathogenic factor in Chinese patients with
sporadic EOP. Mutations of DJ-1 and PINK1 gene are also
found in Chinese patients with sporadic EOP.
Keywords Parkinson’s disease  Genetics 
Gene mutation  Phenotype
Introduction
Parkinson’s disease (PD) is one of the most frequent
neurodegenerative disorders caused by loss of
dopaminergic neurons in the substantia nigra, which
results in decreased dopamine availability in the striatum.
The etiology of PD has not been fully elucidated. In
recent years, linkage analyses in large families and the
positional cloning of an increasing number of genes that
cause monogenic forms of PD have provided new insight
into the pathogenesis of this disorder. To date, nine of the
13 loci that have already been identified are associated
with the monogenic forms of parkinsonism and related
disorders [1].
Three genes are responsible for autosomal recessive
early onset parkinsonism (AREP): Parkin at PARK2 [2]
(OMIM*602544), PINK1 at PARK6 [3] (OMIM*608309),
and DJ-1 at PARK7 [4] (OMIM#606324). Several studies
indicate that the mutations of these genes contribute not
only to patients with AREP, but also to patients with idi-
opathic Parkinson’s disease (IPD), especially in patients
with apparently sporadic early onset parkinsonism (EOP)
[5–18].
There are some reports that ethnic background of the
patients affects the mutation rate with frequencies of gene
mutations. To our knowledge, there are limited data
regarding the prevalence rate of Parkin, PINK1, and DJ-1
genes mutations in Chinese patients with sporadic EOP.
Herein, we report our results of genetic analysis of Parkin,
PINK1, and DJ-1 genes in a cohort of Chinese patients with
apparently sporadic EOP.
Patients and methods
Patients and controls
Patients were collected from the outpatient neurology
clinics of Xiangya Hospital and the National Lab of
J. Guo and X. Zhang are contributed equally to the study.
J. Guo  X. Zhang  L. Nie  H. Zhang  B. Liao  J. Li 
L. Wang  X. Yan  B. Tang (&)
Department of Neurology, Xiangya Hospital,
Central South University, Changsha 410008,
Hunan, People’s Republic of China
e-mail: bstang7398@yahoo.com.cn
B. Tang
National Laboratory of Medical Genetics of China,
Changsha 410008, Hunan, People’s Republic of China
J. Guo  X. Yan  B. Tang
Neurodegenerative Disorders Research Center,
Central South University, Changsha 410008,
Hunan, People’s Republic of China
123
J Neurol (2010) 257:1170–1175
DOI 10.1007/s00415-010-5485-8

Medical Genetics of China from October 2000 to
November 2008. All patients underwent a standardized
neurological examination by two movement disorder spe-
cialists according to the following criteria [5]: (1) at least
two of the three cardinal motor signs (resting tremor,
bradykinesia, rigidity); (2) excellent response following
L-Dopa therapy; (3) the absence of extensor plantar
reflexes, ophthalmoplegia, early dementia, or early auto-
nomic failure in the family members; (4) the absence of a
family history of PD; and (5) an age at onset B50 years.
These PD patients came from Hunan, Hubei, Jiangxi,
Sichuan and Chongqing province of China. One hundred
twenty-seven (86 men, 41 women) unrelated patients with
EOP were included in this study. The mean age in the
patient group was 44.7 ± 8.1 years (ranging from 21 to 63)
and the mean age at onset was 40.0 ± 8.4 years (ranging
from 19 to 50). A control group of 100 (69 men, 31
women) healthy Chinese individuals from the same geo-
graphic areas was obtained and the mean age was
48.6 ± 6.5 years (ranging from 26 to 70). After obtaining
informed consent, genomic DNA was extracted from
peripheral blood obtained from affected and unaffected
family members. DNA from the 100 normal Chinese
controls was also investigated.
Mutation analysis
To detect small sequence alterations as well as exon rear-
rangements in the Parkin gene, genomic sequencing of all
coding exons and exon–intron boundaries, and real-time
quantitative PCR were performed. Then we successively
used direct sequencing and real-time quantitative PCR
analysis to test for PINK1, DJ-1 genes mutations in those
patients without Parkin gene mutation or only with one
heterozygous Parkin gene mutation.
Small sequence alterations analysis: Primer sequences
and conditions for amplification are available as described
[2–4]. Each PCR product was purified and directly
sequenced in both forward and reverse directions on an
ABI 3100 automated sequencer (Applied Biosystems,
Foster City, CA, USA). Alignment and analysis was car-
ried out with DNAStar (GenBank accession numbers of
Parkin, PINK1, DJ-1 are AB009973, BC008188, and
AB053323, respectively). In all cases in which a novel
variant was discovered in a patient sample, segregation
within families was evaluated and 100 unrelated controls
were screened by the restriction-enzyme analysis or direct
DNA sequencing.
Exon rearrangement analysis: Exon rearrangement
analysis was performed through real-time quantitative PCR
using the 7900HT Fast Real-Time PCR System (ABI
company) and SYBR Green I (TaKaRa company) as
intercalation dye as described previously [19]. In brief, the
Beta globin gene was co-amplified with each individual
exon and served as an internal standard. All samples were
tested in triplicate, and at least one positive and one neg-
ative control were included in every plate (96-well plates).
The initial copy numbers of the objective gene (Parkin,
PINK1, DJ-1 genes) and Beta globin gene were calculated
according to the standard curve. Using the Beta globin
gene as an internal control provided a relative ratio: con-
centration of each exon to concentration of Beta globin
gene. A ratio between 0.8 and 1.2 was considered normal, a
heterozygous deletion was expected at a ratio between 0.4
and 0.6, and a heterozygous duplication between 1.3 and
1.7. All detected gene dosage variations were confirmed in
at least triplicate.
Results
Genetic results
We found four heterozygous Parkin gene mutations using
direct sequencing in three patients (Table 1). Three muta-
tions of IVS1-39G ? T, IVS9 ? 18C ? T and c.712delT
have not been reported previously. Using real-time quan-
titative PCR analysis, 16 patients were found to carry exon
deletions in the Parkin gene. Through familial segregation
analysis, ten patients were identified with Parkin mutations
in homozygous or in compound heterozygous state, and six
affected subjects remained heterozygous, because no other
mutation or exon rearrangements were detected (Table 1).
Three patients were found to carry heterozygous point
mutations in the PINK1 gene using direct sequencing
(Table 1). One mutation of c.C832G had not been reported
previously. Using real-time quantitative PCR analysis, a
novel heterozygous rearrangement mutation (exon3-8del)
in the PINK1 gene was found in one patient (Table 1).
No mutation in DJ-1 gene was found using direct
sequencing. Using real-time quantitative PCR analysis,
three patients were found to carry heterozygous deletion of
exon 2 (Table 1).
All novel Parkin, PINK1 and DJ-1 mutations were
ascertained co-segregation with phenotype and not present
in 200 normal Chinese chromosomes.
Clinical features
There were 16 patients (12.6%) that carried Parkin gene
mutations, four patients (3.1%) carried PINK1 gene
mutations, and three patients (2.4%) carried DJ-1
gene mutations. Detailed clinical features of these patients
with mutations are listed in Tables 1 and 2.
J Neurol (2010) 257:1170–1175 1171
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