American Journal of Medical Genetics 129A:44–50 (2004)
parkin Mutation Analysis in Clinic Patients With
Early-Onset Parkinson’s Disease
P. Poorkaj,
1,2
J.G. Nutt,
3
D. James,
3,4
S. Gancher,
5
T.D. Bird,
1,6,7
E. Steinbart,
1,6
G.D. Schellenberg,
1,2,8
and Haydeh Payami
3,4
*
1
Geriatric Research Education Clinical Center, Veterans Affairs Puget SoundHealth Care System, Seattle Division, Seattle, Washington
2
Division of Gerontology and Geriatric Medicine, University of Washington, Seattle, Washington
3
Department of Neurology, Oregon Health & Science University, Portland, Oregon
4
Division of Genetic Disorders, Wadsworth Center, Albany, New York
5
Kaiser Permanante, Portland, Oregon
6
Department of Neurology, University of Washington, Seattle, Washington
7
Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington
8
Departments of Neurology, and Pharmacology, University of Washington, Seattle, Washington
parkinmutations are themost common identified
cause of Parkinson’s disease (PD). It has been
suggested that patients with young-onset PD be
screened for parkin mutations as a part of their
clinical work-up. The aim of this study was to
assess parkin mutation frequency in a clinical
setting, correlate genotype with phenotype, and
evaluate the current justification for clinical
parkin testing. Patients were selected from a
movement disorder clinic based on diagnosis of
PDandonsetage
40years.parkinwasgenotyped
by sequence and dosage analysis for all 12 exons.
Key relatives and controls were screened for
identified mutations. Mutations were found in 7/
39 patients. Two patients were compound hetero-
zygous; five were heterozygous. Mutations
included deletions in exons 2, 3, and 8, duplica-
tions in exons 2–4, and 9, and P437L substitution.
Seventy-eight percent of mutations were dele-
tions/multiplications. A novel substitution (R402W)
was found in one patient and in one control.
None of the point mutations found in patients
were detected in 96 controls. parkin phenotypes
were consistent with idiopathic PD. In conclu-
sion, parkin mutations are common in the clinic
setting: 10% of PD patients had early-onset and
18% of them had parkin mutations. However, if
parkin is recessive, only 5% of early-onset cases
whohadcompoundmutations couldbe attributed
to this locus. Mutation frequencywas 0.12 (95% CI
0.04–0.19). parkin cases can present as typical
idiopathic PD, distinguishable only by molecular
testing. Seventy percent of parkin cases were
heterozygous. It is unclear whether heterozygous
mutations are pathogenic. parkin-based diagno-
sis andcounseling require abetterunderstanding
of the mode of inheritance, penetrance, and
carrier frequencies.
2004 Wiley-Liss, Inc.
KEY WORDS: Parkinson’s disease; parkin; clin-
ical testing; gene dosage
INTRODUCTION
Once believed to be non-genetic, Parkinson’s disease (PD)
has proved to be a heterogeneous disorder with a significant
genetic component. In the past 7 years, more than 10 genetic
loci have been linked to PD [Gwinn-Hardy, 2002; Kruger et al.,
2002], four of which have been identified, and many of the
mutations have been characterized. Mutations in a-synuclein
[Polymeropoulos et al., 1997] and ubiquitin carboxy-terminal
hydrolase L1 [Leroy et al., 1998] cause autosomal dominant
PD. Mutations in parkin [Kitada et al., 1998] and DJ-1
[Bonifati et al., 2003] are associated with early-onset recessive
PD. parkin is themost common single-gene cause of PD known
to date.
Localized on chromosome 6 (PARK2 on 6q25-27), parkin is a
large gene with 12 exons and a 1,395 base-pair (bp) open
reading frame [Kitada et al., 1998]. The protein product,
Parkin, functions as an ubiquitin E3 ligase in the proteosome
degradation pathway [Shimura et al., 2000]. Over 80 parkin
mutations have been identified, spanning all exons. The
mutations include point mutations, as well as deletions and
duplications that range in size from a few bp to multiple whole
exons [West et al., 2002]. DNA sequence analysis can detect
only a subset of mutations. Quantitative gene-dosage analysis
is required for detection of heterozygous and compound
deletions and multiplications, which comprise 60–80% of all
parkinmutations [Hedrich et al., 2001; Kann et al., 2002;West
et al., 2002; Foroud et al., 2003; Oliveira et al., 2003].
parkin was originally discovered as the genetic locus for
autosomal recessive juvenile parkinsonism, a rare disorder
that is characterized by onset <20 years, slow disease pro-
gression, and marked responsiveness to levodopa. The recent
expansion of parkin screening to the more common forms of
PD has uncovered mutations in patients and families with
varied clinical and pathological phenotypes, including families
with seemingly autosomal dominant inheritance [Klein et al.,
2000; Maruyama et al., 2000; Lucking et al., 2001; Kobayashi
et al., 2003], and an autopsy-confirmed parkin family with
Lewy bodies [Farrer et al., 2001]. The age of onset range has
also been expanded, from juvenile and early-onset to beyond
age 70 [Lucking et al., 2000; Foroud et al., 2003; Oliveira et al.,
2003]. Although parkin mutations were presumed recessive,
Grant sponsor: National Institutes of Health; Grant number:
RO1 NS36960; Grant sponsor: Veterans’ Administration
(PADRECC).
*Correspondence to: Dr. Haydeh Payami, Genomics Institute,
New York State Department of Health Wadsworth Center, 120
New Scotland Ave., Albany, NY 12208.
E-mail: hpayami@wadsworth.org
Received 9 July 2003; Accepted 9 December 2003
DOI 10.1002/ajmg.a.30157

2004 Wiley-Liss, Inc.

recent evidence suggests that heterozygous mutations may
also be pathogenic, or may confer increased susceptibility to
typical late-onset PD [Farrer et al., 2001; Hedrich et al., 2002;
Hilker et al., 2002; West et al., 2002; Oliveira et al., 2003].
The increasing number of reports of parkin mutations in
patients with common forms of PD suggests a wider involve-
ment of parkin in PD than was once believed. These reports
raise questions concerning theprevalence ofparkinmutations,
and whether patients with idiopathic PD, at least those with
early-onset, should be screened for parkin as a part of their
clinical work-up [Khan et al., 2003]. Analysis of parkin
mutations is complex and expensive. Routine clinical genetic
testing would not be justified if mutations were rare in the
clinical setting. Many studies report that parkin mutations
are common in early-onset PD. For example, in one study of
early-onset PD, 49% of families with recessive inheritance and
18% of isolated cases had parkin mutations [Lucking et al.,
2000].Most published estimates, however, are based on special
populations selected for genetic studies (e.g., consanguineous
families, sib-pairs, and early-onset recessive families in which
parkin mutations are most prevalent); hence, the mutation
rates may have been inflated by selection bias [Kitada et al.,
1998; Lucking et al., 2000; Foroud et al., 2003; Oliveira et al.,
2003]. A community-based study found parkin mutations in
9% of German patients with onset age <50 years [Kann et al.,
2002]. The single clinic-based study published to date reports
a carrier frequency of 4%, but the authors did not perform
gene-dosage studies, and could thus havemissed a substantial
number of heterozygous and compound deletions and duplica-
tions [Chen et al., 2003]. Here, we report the results of a
comprehensive parkin mutation analysis in a clinical setting.
We studied every clinic patient who had the diagnosis of
idiopathic PD with early age at onset, and who agreed to
participate in the study, regardless of family history. We
sequenced all 12 exons and performed quantitative gene-
dosage analysis for all exons, to detect homozygous, hetero-
zygous, and compound deletions, multiplications, and point
mutations. This study represents a first step in assessing the
justification for parkin screening in the clinical settings.
MATERIALS AND METHODS
Patients
Study subjects were patients who were seen by neurologists
at the movement disorder clinic at Oregon Health & Science
University (OHSU), and had received a clinical diagnosis of PD
according to the British Parkinson’s Disease Brain Bank
criteria except that family historywas not an exclusion criteria
[Hughes et al., 1992]. The majority of patients are living, thus
lack pathological confirmation.
Subjectswere initially identified and invited to join the study
solely on the basis of having the diagnosis of PD, regardless of
age at onset or family history. Informed consent was obtained
with Institutional Review Board (IRB) approval from OHSU.
Each subject was asked to provide a family history and a blood
sample for DNA preparation. Family history was obtained
using a self-administered questionnaire, and if positive for PD
or other neurological disorders, they were verified by neuro-
logical examination of the family members or by review of
existing medical records. Key family members were identified
and enrolled with IRB-approved informed consent. Ages at
onset were recorded upon entering the study and were cross-
checked against the earlier medical records. Early-onset was
defined as
40 years because parkin mutations are predomi-
nantly found in this group [Lucking et al., 2000].
Four hundred forty two patients with diagnosis of PD
were enrolled in the study and donated DNA (431 Caucasian,
1 Asian, 1 African-American, 3 Hispanic, 5 native American,
1 mixed ethnicity). For 43 subjects, age at onset was un-
ambiguously establishedat or beforeage 40.Four of 43 subjects
had insufficient DNA hence were excluded. parkin analysis
included 39 subjects (22 male, 17 female), representing all
consenting clinic patients with diagnosis of PD, early-onset,
and sufficient DNA. We did not investigate patients with later
onsets because at the time the literature suggested that parkin
mutationswerenot involved in late-onsetdisease [Oliveri etal.,
2001]. All 39 subjects analyzedwereCaucasian, residing in the
Pacific Northwest. Their ages at onset ranged from 7 to 40
years (32.6 8.1 years). Their ages at enrollment/blood draw
ranged from 36 to 81 years (52.5 9.9 years).
Controls
Ninety-six unaffected Caucasian volunteers were used as
control subjects. Like the patients, controls were Caucasians
from the Pacific Northwest. They were ascertained and
enrolled under protocols approved by the IRB of theUniversity
of Washington. Their ages at enrollment and blood draw
ranged from 18 to 72 years (37.9 12.1 years).
parkin DNA Sequence Analysis
To identifypointmutations,we sequencedbothDNAstrands
of all 12 exons of theparkin gene.GenomicDNA frombloodwas
used. Primer pairs for amplification and sequencing have been
described previously [Kitada et al., 1998]. Exons along with
50–100 bp of flanking intronic sequences were PCR-amplified,
agarose gel-purified (Gene-clean III, Bio101), and directly
sequenced by dye-terminator cycle sequencing (ABI, Big-Dye)
using an ABI377 sequencer. Nucleotide changes that had been
found in patients were screened for in controls, by sequencing
the entire exon, or by a restriction assay for the specific change.
Gene-Dosage Assays
To identify exon deletions and duplications, we analyzed
genedosageusing real-timefluorescence-basedPCR (ABI7700
SequenceDetector). Amplification of subject genomicDNAwas
performed using fluorescently labeled probes (5
0
FAM or VIC,
3
0
TAMRA) andTaqmanUniversal PCRMix (ABI) [Maruyama
et al., 2000; Tsuang et al., 2002]. parkin exon amplifications
were multiplexed under standard conditions with an 84 bp
fragment of a single-copy human b-actin gene (Genbank
accession number XM_004814) as an internal control. A stan-
dard curve was generated for each parkin exon and for b-actin
using 0, 5, 15, 55, and 220 ng of control human genomic DNA.
Thenumber of PCRcycles required before theABI7700detects
each parkin exon product (CT value) was plotted against
the corresponding exon standard curve, thus calculating the
relative parkin copy number. The copy number for each exon
was normalized to the single-copy actin gene within each
multiplexed reaction and to a normal control reference
individual, allowing an estimate of the number of copies of
parkin. Optimal threshold levels for each primer set were
maintained between plate analyses. All controls and samples
were analyzed in triplicate.
R402W Restriction Digest Assay
Because we found a novel substitution, R402W, in one
patient, we developed a restriction digest assay to detect it.
A BsiWI site encompassing the mutated nucleotide was
artificially introduced, by changing the underlined nucleotides
in the reverse detection primer: R402W R 5
0
-GGTTTCTTT-
GGAGGCTGCTTCCGTAC-3
0
. PCR amplification with the
11F/R402W R primer set amplifies a 156 bp product, where
the normal A allele digests withBsiWI, yielding two fragments
of 22 and 134 nt, and the mutant T allele does not digest. PCR
parkin Gene Testing in Clinic Setting 45