Parkinsonism and Related Disorde
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Received 23 July 2007; received in revised form 28 September 2007; accepted 1 October 2007
Keywords: Parkin; Early onset Parkinson’s disease; Mutation analysis
familial PD aetiology have been identified (SNCA, parkin,
DJ-1, PINK1, LRRK2), and their study has made an
mainly in EOPD [8–16]. Mutations in the parkin gene are
the most common cause of EOPD in many countries and
account for 10–25% of cases. Parkin mutations vary from
ARTICLE IN PRESSpoint mutations to complex rearrangements, including
deletions and/or duplications of one or more exons [17].
Genotype/phenotype correlations have been reported in
1353-8020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.parkreldis.2007.10.003

Corresponding author. Tel.: +39 02 5799 3382; fax: +39 02 5799 3214.
E-mail address: goldwurm@parkinson.it (S. Goldwurm).1. Introduction
Parkinson’s disease (PD) is a common neurodegenera-
tive disorder with a prevalence of 2% in persons 465
years old [1]. It is characterized by resting tremor,
bradykinesia, rigidity and postural instability. Mean age
at onset is 60 years of age, but about 5% of patients have
an early onset PD (EOPD) before 40 years of age [2]. The
aetiology of PD has not been fully elucidated. PD is
generally considered to be the result of the interaction
between genetic and environmental factors [3]. In recent
years, many discoveries have been made concerning the
genetic aspects of this disease. Several genes involved in
important contribution to the understanding of molecular
mechanisms underlying the disease [4].
Parkin was first identified in autosomal recessive-juvenile
Parkinsonism (AR-JP) [5]. AR-JP was originally described
by Yamamura et al. and is characterized by a young age of
onset, prominent foot dystonia, hyperactive tendon re-
flexes, diurnal fluctuation, sleep benefit, an excellent
response to levodopa and frequent and early occurrence
of L-dopa-induced dyskinesias [6]. The gene responsible for
at least some patients with AR-JP was first mapped to
6q25.2-q27 in 1997 by Matsumine et al. [7] and was later
cloned and designated as the parkin gene [5]. Parkin
mutations were identified in many series of idiopathic PD,Abstract
We analysed the parkin gene in a large consecutive series (146) of unrelated early onset Parkinson’s disease (onset p40 years of age)
patients. Twelve cases (8.2%) had homozygous or compound heterozygous point mutations and/or exon rearrangements, while a single
mutation was found in four subjects (2.7%). We identified eight exon rearrangements and nine point mutations, two of which were novel:
c.735delT (p.C212/X224) and c.815C4G (p.C238W). Genotype–phenotype correlation revealed that parkin carriers had features similar
to those of non-carrier early onset Parkinson disease patients.
r 2007 Elsevier Ltd. All rights reserved.Parkin analysis in early
Francesca Sironi
a,b
, Paola Primignani
b
, Mich
Sara Ricca
a,b
, Tiziana Brambilla
a,b
, Angelo A
Anna Zecchinelli
a
, Claudio Mariani
a
, Nicolet
Ioannis U. Isaias
a
, Barbara Garavaglia
d
, Dani
Domenico A. Coviello
b
, Giann
a
Parkinson Institute, Istituti Clin
b
Medical Genetics Laboratory, Foundation IRCCS ‘‘Ospedale
c
Institute of Medical Statistics and Biometry ‘‘G
d
Unit of Molecular Neurogenetics, Neurologicalrs 14 (2008) 326–333
set Parkinson’s disease
a Zini
a
, Sara Tunesi
a,c
, Claudio Ruffmann
a
,
tonini
a
, Silvana Tesei
a
, Margherita Canesi
a
,
Meucci
a
, Giorgio Sacilotto
a
, Roberto Cilia
a
,
Ghezzi
d
, Maurizio Travi
b
, Adriano Decarli
c
,
ezzoli
a
, Stefano Goldwurm
a,
i Perfezionamento, Milan, Italy
ggiore Policlinico, Mangiagalli e Regina Elena’’, Milan, Italy
. Maccaro’’, University of Milan, Milan, Italy
itute C. Besta Foundation IRCCS, Milan, Italy
www.elsevier.com/locate/parkreldis

ARTICLE IN PRESS
Relsome articles where a series of patients were collected from
several clinics [9,15–19]. The clinical features associated to
parkin mutations were the same as those of the first families
described with AR-JP.
The aim of our study was to determine the frequency of
parkin mutations in a large consecutive series of EOPD
patients (onset p40 years of age) collected in a single
Italian centre. In addition, we compared the phenotype of
patients with and without parkin mutations.
2. Materials and methods
2.1. Subjects
We studied 146 consecutive and unrelated patients with EOPD (onset
p40 years of age), who had contributed samples to the DNA Bank of the
Parkinson Institute, Istituti Clinici di Perfezionamento, Milan, Italy
(‘‘Human genetic bank of patients affected by PD and parkinsonisms’’;
http://www.parkinson.it/dnabank.html). In addition, if a parkin mutation
was identified in a proband, we studied all family members presenting with
PD signs and symptoms. In this way, we enrolled four additional siblings
from three families (total of 150 subjects). Data on patient C-0560 has
been previously published [19].
The clinical diagnosis of PD was established according to the UK
Parkinson’s Disease Society Brain Bank criteria [20,21]. The diagnosis of
PD required the presence of bradykinesia and at least one of the following:
resting tremor, rigidity or postural instability; a positive response to
dopaminergic therapy; the absence of atypical features or other causes of
Parkinsonism.
Among the 146 consecutive EOPD patients, 91 were male (62.3%), the
mean age at onset was 35.2 years (range 14–40, S.D.74.8), and the mean
disease duration was 16.56 years (range 4–56, S.D.79.13). Presence of
LRRK2-G2019S mutation was analysed in all subjects, and three patients
were carriers of the G2019S with no family history in their first-degree
relatives [22]. One subject (a member of a possible autosomal dominant
family) was extensively studied and found to be a carrier of another
putative mutation (I1371V) [23]. These LRRK2 mutation carriers were
not excluded from this study. Analysis of PINK1 and DJ-1 gene is
currently ongoing.
With the exception of seven patients, each originating from seven
different countries (Argentina, Albania, Colombia, France, Ireland, Sri
Lanka, Spain), all the affected subjects were Caucasian and of Italian origin.
Patients were classified as ‘‘familial’’ if at least one relative among their
first- and second-degree relatives had a formal diagnosis of PD. The
remaining subjects were classified as ‘‘sporadic’’. The age at which the patient
noticed the first PD symptom was considered to be the age at onset of disease.
Up to three symptoms at onset were collected during medical history. Clinical
diagnosis of dementia was made according to DSM-IV criteria.
Neurological examination was performed by neurologists with
experience in movement disorders and included the Unified Parkinson’s
Disease Rating Scale (UPDRS) [24] and Hoehn and Yahr scale [25] during
therapy (‘‘on’’ status). Clinical data were collected from medical records
by a single neurologist expert in movement disorders (MZ).
Novel parkin sequence variants were tested in 50 controls: elderly
individuals free from neurodegenerative disorders with no family history
of PD (spouses or care-givers of PD cases who contributed to the DNA
Bank: http://www.parkinson.it/dnabank.html). The mean age of the
controls at the time of blood collection was 6477.2.
The study was approved by the local Ethics Committee. Each
participant signed an informed consent prior to participation in the study.
2.2. DNA analysis
F. Sironi et al. / Parkinsonism andGenomic DNA was extracted from peripheral blood according to
standard methods. The 12 exons of the parkin gene were analysed alongwith exon–intron junctions and were amplified as described before [26].
Mutation analysis was performed by denaturating high performance
liquid chromatography (DHPLC) (Transgenomic Inc., Omaha, NE). All
PCR products were mixed with wild type fragments to generate artificial
heterozygous samples.
To optimise sensitivity and specificity of DHPLC, different positive
controls were used to cover the whole size and domains of the amplicons.
When natural controls were not available, site-directed mutagenesis was
used to obtain artificial positive amplicons [27,28].
Melting temperatures for optimal heteroduplex separation were first
determined by using WAVEMAKER
TM
software and then experimen-
tally optimised to ensure covering of the entire amplicon domains. When
necessary, two or more temperatures were used. Samples with a variation
in peak shape were sequenced with both forward and reverse primers by
PCR cycle sequencing (Big Dye Terminator System) and analysed on an
ABI Prism 3100 Genetic Analyzer (PE Applied Biosystems, USA).
Mutations were confirmed on a second independent PCR product
analysis.
Patients with a very young onset disease (p30 years) or with only a
single heterozygous mutation or variant were further investigated for exon
rearrangements. Gene dosage was performed using real-time fluorescence-
based PCR of the 12 exons with AB7300 Realtime PCR System. Exonic
primers were designed using Primer
TM
Express software (PE Applied
Biosystems, USA). Each genomic DNA sample, a control DNA and a
blank control (no-template) were tested in duplicate.
Amplification of 40–50ng of genomic DNA was performed in a final
volume of 50mL containing oligonucleotide primers and SYBR
s
green
PCR Master Mix (ABI-Roche Molecular Biochemicals). The RNaseP
gene (TaqMan
s
RnaseP Control Reagents, VIC
TM
dye, PE Applied
Biosystems) was used as external control gene.
Gene dosage of exons 1, 3 and 6 was also set using 5
0
FAM
fluorescently labelled probes and the TaqMan Universal PCR Master Mix
(ABI-Roche Molecular Biochemicals) using RNaseP as internal control.
The dosage of parkin exons was determined using the 2
DDCt
method. All
positive results were confirmed at least twice.
Consequences of DNA variants were deduced from published parkin
gene sequence (GenBank accession no.: NM_004562).
2.3. Statistical analysis
Statistical analyses were performed using the SAS package version 9.1
(SAS Institute, Inc., Cary, NC). We used the w
2
test or the Fisher’s exact
test when appropriate for comparison of proportions and t-test for
comparison of means. UPDRS p-value was adjusted for disease duration
from onset using linear regression analysis and Hoehn/Yahr p-value was
adjusted for disease duration from onset using multiple logistic regression.
3. Results
3.1. Description of parkin mutations
We identified several nucleotide substitutions and exon
rearrangements (Table 1).
Mutation analysis identified nine point mutations; seven
of these have been previously reported and are known as
pathogenic changes (p.R42P, p.M192L, p.T240M,
p.C253Y, p.R275W, p.E409X, p.R402C), while the other
two (c.735delT and c.815C4G) are novel. The single base
deletion c.735delT was found in one affected patient in
association with the known p.R275W mutation and
resulted in a premature stop-codon that caused the trun-
cation of more than half of the protein (p.C212/X224). We
ated Disorders 14 (2008) 326–333 327found two unrelated compound heterozygous patients
carrying the c.815C4G (p.C238W) missense mutation in