ARTICLE IN PRESS

Parkinsonism and Related Disorders 12 (2006) 420–426

www.elsevier.com/locate/parkreldis

Molecular pathogenesis of Parkinson’s disease: Identification of

mutations in the Parkin gene in Indian patients
Arindam Biswasa, Arnab Guptab, Tufan Naiyaa, Gautami Dasa, Rajarshi Neogib,
Somnath Dattab, Subhas Mukherjeec, Shyamal K. Dasd, Kunal Rayb, Jharna Raya,
a
S.N. Pradhan Centre for Neurosciences, University of Calcutta, 244B, A.J.C. Bose Road, Kolkata 700 020, India
b
Human Genetics & Genomics Division, Indian Institute of Chemical Biology, Kolkata, India
c
Medical College and Hospital, Kolkata, India
d
Bangur Institute of Neurology, Kolkata, India
Received 28 September 2005; received in revised form 4 April 2006; accepted 8 April 2006

Abstract

Parkinson’s disease (PD), the second most common neurodegenerative disorder, affects at least 1% of the population over the age
of 50. However, very little information is available regarding the molecular basis of PD among Indians. Since the largest number of
mutations have been detected in the Parkin gene among all known PD loci, we aim to use Parkin as the candidate gene to assess its
role in PD-related pathogenesis in Indian patients. A total of 138 PD patients, with the mean age of onset being 47714 (age range,
5–77 years), and 100 controls were recruited for the study from eastern India. Parkin mutations were detected by amplification of
exons of the gene along with the flanking splice junctions by polymerase chain reaction, single-stranded conformation
polymorphism and DNA sequencing. A total of 18 nucleotide variants including six novel changes were detected. These include five
missense mutations (Gln34Arg, Arg42Cys, Arg42His, Tyr143Cys and Arg334Cys) detected in eight patients in heterozygous
condition and a homozygous deletion encompassing exons 3 and 4 in two sibs affected with PD. Clinical features of the Parkin
mutants were compared. Among eastern Indian PD patients, mutation in Parkin was identified in 7.24% cases.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Parkin; Parkinson’s disease; PD

1. Introduction face. The prevalence of PD in North America and West

Parkinson’s Disease (PD) is the second most common
neurodegenerative disease after Alzheimer’s disease af-
fecting at least 1% of the population over age 50 [1–3].
The disease is progressive with mean age of onset being
55 years, and its incidence increases with age [4]. The
hallmark of PD is the degeneration of nigrostriatal
dopaminergic pathway. Depletion of brain dopamine
initiates aberrant motor activities including rest tremor,
rigidity, bradykinesia, postural instability and mask
Corresponding author. Tel.: +91 33 2223 2084;
fax: +91 33 2223 3260.
E-mail address: thisisjr@rediffmail.com (J. Ray).
European countries is 150–200 per 100,000 [5] and much
lower in China, Japan and Africa (45–80 per 100,000).
In India, PD affects around 14–27 per 100,000 but the
incidence of the disease is much higher among the Parsis
(328 per 100,000) living in India [6,7]. PD generally
arises as sporadic condition but occasionally inherited in
families both as an autosomal dominant or autosomal
recessive trait. To date, 11 chromosomal loci (PARK1–
PARK11) have been implicated for PD of which six
causal genes have been identified, and one locus
(PARK4), later found to have been assigned based on
false linkage. Three genes a-Synuclein (SNCA), Ubiqui-
tin carboxy-terminal hydrolase L-1 (UCHL-1), LRRK2
and three loci (on chromosomes 2p13, 1p32 and

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doi:10.1016/j.parkreldis.2006.04.005
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A. Biswas et al. / Parkinsonism and Related Disorders 12 (2006) 420–426
2q36-q37) have been implicated in the pathogenesis of
autosomal dominant PD. On the other hand, the locus
1p36 and the three genes Parkin, DJ-1 and PINK1 have
been implicated for autosomal recessive PD [8].
Among all the genes causal to PD, largest number of
mutations have been detected in the Parkin gene
(PARK2 locus), which has been mostly associated with
juvenile PD but has been observed in late-onset PD as
well. Parkin is an E3-ligase, catalyzing the addition of
ubiquitin to specific substrates that targets them for
degradation through ubiquitin–proteasome systems [9].
Parkin has five functional domains, the extreme amino
terminal ubiquitin-like domain (UBL), unique Parkin
domain (UPD) and C-terminal RING1 (really interest-
ing new gene), RING2 and in-between RING (IBR)
domains [10]. Most Parkin mutations are clustered in
the conserved domains of the protein suggesting
impairment of its biological activity. Parkin is a large
gene of estimated size 1.5 Mb and consists of 12 exons
with an open reading frame of 1395 bp, encoding a
protein of 465 amino acids. So far, 109 Parkin mutations
have been reported in Human Genome Mutation
database (http://uwcmml1s.uwcm.ac.uk/uwcm/mg/
search/6802742.html) of which missense and gross
deletion are common (40% and 35%, respectively).
The remaining 25% constitutes all other types (small
deletion, insertion, gene duplication, etc.) of mutations.
Despite extensive studies on the molecular bases of
PD in different population groups, very little informa-
tion is available regarding the molecular pathogenesis
of the disease among Indians [11]. A single report
has demonstrated the absence of known a-synuclein
mutations (Ala30Pro and Ala53Thr), causal to PD, in
Indian patients [12]. To our knowledge, the present
study is the first report on the genetic basis of PD in the
Eastern Indian population using Parkin as a candidate
gene.
2. Materials and methods
2.1. Patients
PD patients were examined in the Movement Dis-
order Clinic, Bangur Institute of Neurology (BIN),
Kolkata, India and Medical College and Hospital,
Kolkata, India. One hundred and thirty-eight pati-
ents having at least two parkinsonism symptoms
(rest tremor, bradykinesia, rigidity and postural in-
stability) were recruited in this study. Only eight of these
patients had any history of PD in the family. One
hundred unrelated controls were selected for this
study with no personal or family history of Parkinson-
ism or any other neurological problems (mean age,
55710 years).
421
2.2. Collection of blood samples and genomic DNA
preparation
Approximately 10 ml of peripheral blood samples
were collected in EDTA with written and informed
consent from PD patients, their family members and
from normal individuals as controls making sure about
adequate understanding by the donors. The experiments
were conducted in accordance with Declaration of
Helsinki. The internal review committee on research
using human subject cleared the project after proper
review as per regulation of Indian Council of Medical
Research. Genomic DNA was prepared from fresh
whole blood by using conventional phenol–chloroform
method [13]. Genomic DNA was dissolved in TE
(10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0).
2.3. Polymerase chain reaction (PCR)
PCR was carried out to amplify exons and adjacent
flanking region in a total volume of 25.0 ml containing
40–100 ng genomic DNA, 0.4 mM of each primer,
0.2 mM of each dNTP, 0.5–1.5 mM of MgCl2 (as
appropriate) and 0.5 unit of Taq polymerase (Invitrogen,
Carlsbad, CA) in a Thermocycler (GeneAmp-9700, PE
Applied Biosystems, Foster City, CA). Annealing
temperatures were calculated on the basis of Tm of the
primer pairs. Primer pairs used for analysis were
previously reported [14], except a few, which were
designed to get better amplification (available on
request). PCR amplified DNA fragments were analyzed
on 6% polyacrylamide gel and visualized by ethidium
bromide staining.
2.4. Mutation and polymorphism detection
(i) Single-stranded conformation polymorphism
(SSCP) analysis: PCR products were heated for
5 min at 94 1C, chilled at 0 1C and then electrophor-
esed on a 12.5% or 15% polyacrylamide gel at 160 V
for 24 h. The gels were then stained using conven-
tional silver staining method [15]. In addition to
manual SSCP, pre-cast gels (Amersham Biosciences
AB, Uppsala, Sweden) were also used as per
manufacturer’s protocol. Electrophoresis was car-
ried out at 600 V for 3 h. DNA sequencing was
carried out in samples showing band shifts com-
pared to the normal controls.
(ii) DNA sequencing: The PCR products, free of
contaminating bands due to non-specific amplifica-
tion, were column purified using Qiagen PCR-
purification kits (Qiagen, Hilden, Germany), and
bi-directional sequencing was done using an Avant
3100 sequencer (Applied Biosystems, Foster City,
CA) with dye-termination technique. Nucleotide
changes were detected by comparing sequence
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422 A. Biswas et al. / Parkinsonism and Related Disorders 12 (2006) 420–426

obtained in the chromatogram with the normal
Parkin gene sequence (GenBank Accession No.
AB009973) using pair-wise BLAST [16].
3. Results
One hundred and thirty eight PD patients with mean
age of onset 47714 (age range, 5–77 years) from 134
unrelated families and 100 controls were recruited in this
study. Sixty-two patients (45%) had an age of onset
o50 years (age range, 5–50 years with mean age of
onset, 40710) and the remaining 76 (55%) patients had
an age of onset 450 years (age range, 51–86 years with
mean age of onset, 6278.5). Among the patients, 107
(77%) represent sporadic cases, 8 (6%) confirmed
familial cases and 23 (17%) have a history of other
neurological problems in the family including dystonia,
Fig. 1. Location of mutations in the Parkin gene. The Parkin gene,
consisting of 12 exons, is shown schematically. The hatched regions
show location of various motifs in the protein as indicated. All the
point mutations (boxed), SNPs and rare variants are shown. Novel
changes are italicized.
tremor, essential tremor and psychiatric illness. A total
of 18 nucleotide variants were detected in the Parkin
gene (Fig. 1), which were evaluated for their potential
association with PD. Control samples were screened to
identify nucleotide variants found in the patients by bi-
directional sequencing except one (c.1514 C4A in exon
12) at 30 UTR which was analyzed by Hha I RFLP
analysis.
3.1. Identification of mutations in the Parkin gene
Among nine sequence variants, including three novel
and six reported changes detected in the Parkin gene
coding sequence (Tables 1 and 2), five missense
mutations (Table 1) were found only in the patients
under study but none in 100 control individuals selected
based on lack of any neurological symptoms. Among
three residues of Parkin bearing novel variants, two
residues (Gln34 and Arg42) were found to be conserved
in rat (NP_064478.1), mouse (NP_057903.1), chicken
(XP_419615) and fugu fish (AAS79348) while Tyr143
was conserved in the first three species and bovine
(BAB70670). The reported variant, residue Arg334, was
conserved in rat and mouse. The conservation status of
Gln34, Arg42 and Arg334 in bovine homologue (partial
submission) is not known. The conservation of these
amino acids suggests importance of the residues and
potential for functional aberration when mutated. A
total of six PD patients harbored these variants in
heterozygous condition (Table 3). One of the patients
(male, age of onset 58 years) was determined to be
double heterozygote for Arg42His and Arg334Cys.
However, his two asymptomatic sibs (brother, 70 years
and sister, 50 years) had both the variants suggesting

Table 1
Parkin mutations in Indian patients

Nucleotide Amino acid Exons Patients Status Parkin domain Comments

changea change (n ¼ 138)

c.202 A4G Gln34Arg 2 3 Novel Ubiquitin-like domain Likely to affect 26S

proteasome binding
c.225 C4T Arg42Cys 2 2 Novel Ubiquitin-like domain Proline at the 42nd residue
impairs binding of 26S
proteasome [23]

c.226 G4A Arg42His 2 1 Reported [11] Ubiquitin-like domain

c.529 A4G Tyr143Cys 4 1 Novel Unique Parkin domain Likely to affect the linker
region
c.1101 C4T Arg334Cys 9 2 Reported [17] IBR domain Likely to affect E2-enzyme–
substrate binding

Exon deletion — 3&4 2 Reported [27] Unique Parkin domain Truncated protein, likely to be
rapidly degraded

Nucleotide numbers are based on reported Parkin mRNA (Accession no. AB009973).
The mutations presented in the table were not detected in 100 controls examined.
a
Genotype: All the five point mutations were observed to be heterozygous while the deletion mutation was found in homozygous condition.
 ARTICLE IN PRESS
A. Biswas et al. / Parkinsonism and Related Disorders 12 (2006) 420–426 423

Table 2
Parkin gene variants: polymorphisms and rare variants

Nucleotide change Amino acid change Number of chromosomes (Allele frequency) Status Population

Patient (n ¼ 276) Controls (n ¼ 200)

IVS2+25 T4C NA 35 (0.13) 41 (0.205) rs2075923 European [18]

Japanesea
IVS2+35 G4A NA 0 (0.00) 2 (0.01) Reported Indian [11]
IVS3-20 T4C NA 12 (0.04) 16 (0.08) rs4709583 European [18]
Chinese [19]
b
IVS7-35 G4A NA 20 (0.07) ND rs3765474 European [18]
Japanesea
IVS7-68 C4Gb NA 18 (0.06) ND rs3765475 Japanesea
IVS8–20_–16del CTGCT NA 1 (0.003) 0 (0.00) Novel —
IVS8–21_–17del TCTGC NA 1 (0.003) 0 (0.00) Reported [17]
IVS8-22A4G NA 1 (0.003) ND Novel —
c.1118G4A Ala339Ala 1 (0.003) 0 (0.00) Reported Indian [11]
c.1514C4A 30 UTR 2 (0.007) 3 (0.015) Novel —
a
Populations studied as reported in dbSNP.
b
These polymorphisms are in linkage disequilibrium; NA, not applicable; ND, not determined; rs#—taken from dbSNP.

Table 3
Patients with Parkin mutations and their clinical features

Patienta Mutationb Duration Type of tremor Bradykinesia Rigidity Postural Dystonia/dyskinesia

(years) instability

PR187 (M/58) Gln34Arg 2 – + + – Dystonia in right foot

and toe
PR252 (M/48) Gln34Arg 4 Rest and action + + (Whole – –
body)
PR280 (M/38) Gln34Arg 2 Rest + + – –
PR204 (M/58) Arg42His 3 Rest tremor + + + Orofacial dyskinesia
Arg334Cys (left side4right
side)
PR500 (F/50) Arg334Cys 4 Rest and action + + + Drug-induced
dyskinesia
PR11 (M/40) Tyr143Cys 14 Action + – + Dystonia in foot and leg
PR546 (F/31) Exon 3–4 del 16 Rest and action + + + Drug-induced
(whole body) dyskinesia
PR488 (F/31) Exon 3–4 del 7 Rest and action + + + Drug-induced
(whole body) dyskinesia

Two individuals who visited the clinic with mild rest tremor and suspected to have early sign of PD were found to harbor a novel Parkin variant
(Arg42Cys).
a
Sex and age of onset of the patients are shown in parentheses.
b
All the patients were heterozygous for Parkin missense mutations, including PR204 who was inferred to harbor both the mutations (Arg42His
and Arg334Cys) in one chromosome.

that most likely the patient was not a compound
heterozygote but harbored both variants in a single
chromosome. Segregation of these variants could not be
examined further by haplotype analysis due to the lack
of availability of other family members. The Arg334Cys
mutation was also detected in another patient (female,
age of onset 50 years) as well as in heterozygous
condition, which was not present in two of her
offsprings (son and daughter). This patient did not
harbor Arg42His mutation, instead had a 5-base
deletion in intron 8 (IVS8 21 to 17) (Table 2).
Interestingly, a previous study [17] described a PD
patient homozygous for both Arg334Cys and the 5-base
deletion but the ethnic background was not mentioned
though the cohort contained an Indian family. Another
5-base deletion in intron 8 (IVS8 20 to 16) over-
lapping with the above-mentioned 5-base deletion was
detected in one of 138 patients (Table 2). None of the
100 controls harbored these 5-base deletions. The role of
these 5-base deletions in Parkin gene, if any, is not clear.
A homozygous deletion of exon 3 and 4 was found in
two sibs affected with PD (Fig. 2). These patients could
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424 A. Biswas et al. / Parkinsonism and Related Disorders 12 (2006) 420–426
Fig. 2. Homozygous deletion in a family affected with Parkinson’s
Disease. PCR was done using genomic DNA of two PD-affected sibs;
their normal sister and obligate carrier mother for amplification of
Parkin exons. The PCR products were analyzed by polyacrylamide gel
electrophoresis (6%). The upper panel shows the pedigree, and the
lower panel shows the PAGE analysis. While the exons 2–5 of the
normal sister (II-3) and the mother (I-2) could be amplified, no PCR
products were detected for exons 3 and 4 of the proband (II-1) and her
affected sister (II-2).
be either homozygous for a single deletion event or
heterozygous for two different rearrangements, which
could not be distinguished based on the experimental
strategy used. Two individuals who visited the clinic
with mild rest tremor and suspected to have early sign of
PD were found to harbor a novel Parkin variant
(Arg42Cys) that qualified as potential mutation since
(a) the residue is conserved through evolution, (b) the
change of amino acid is non-conservative, and (c) the
mutation is not present in 200 normal chromosomes
examined.
3.2. Polymorphism and rare variants in Parkin gene
In addition to the six potential mutations, other
nucleotide variants identified in the patients were not
likely to be causal to PD since the changes were either
located in introns (but not splice junctions) or repre-
sented a silent change (Ala339Ala in exon 9) which
would not create any cryptic splice site to produce a
mutant protein. Similarly, a nucleotide change (c.1514
C4A in exon 12) was detected in homozygous
condition in 30 untranslated region (30 UTR) of Parkin
gene in only one of the PD patients. This nucleotide
change was also identified in three controls in hetero-
zygous state (Table 2) and thus may not be pathogenic.
In addition, two polymorphic missense changes (Ser167-
Asn and Val380Leu), reported in dbSNP (rs1801474 and
rs1801582, respectively) were also detected in some of
the patients and controls. These two SNPs are not
included in Table 2 since only a few samples have yet
been analyzed reliably to determine allele frequencies.
The allele frequencies of some of the nucleotide variants
(novel and reported), identified only in Indian popula-
tion, were very low (0.003–0.015) and are rare variants.
However, all the identified polymorphic variants (allele
frequency 0.04–0.205) are also reported to be present in
other population groups (e.g. European, Japanese, and
Chinese) [18–19]. The two heterozygous changes of
intron 7 of Parkin gene were observed to be in linkage
disequilibrium.
3.3. Clinical features in patients carrying Parkin
mutation
Among 138 patients, only 10 were identified to harbor
mutation in Parkin gene. The patients bearing Parkin
mutations had age range of 31–60 years with mean age
of onset 47711. The disease started with rest tremor in
majority of the patients. They responded well with
levodopa treatment. The disease progression was
observed to be slow in all patients except PR204,
PR252 and PR500. Among all the patients harboring
Parkin mutations, in case of only two mutations the
phenotypes were observed to be broadly similar in
patients harboring the same variant allele: (a) Two
patients (PR204 and PR500) bearing Arg334Cys muta-
tion were observed to have all four listed features of
parkinsonism including dyskinesia with some difference
as noted in Table 3. (b) Two sibs (PR548 and PR488)
with the homozygous deletion at exons 3–4 had
strikingly similar phenotype. For example, both the
patients presented with severe rest tremor of the whole
body at 31 years of age and gradually developed
bradykinesia, rigidity, postural instability, and drug-
induced dyskinesia. In addition, the older sib manifested
several secondary features including slurring of speech
and loss of memory. However, multiple patients sharing
the Gln34Arg mutation were observed to have con-
siderable variation in their phenotypes.
4. Discussion
Our results demonstrate the occurrence of Parkin
mutation in 7.24% (10/138) of our PD patient pool.
Analysis of Parkin mutations in PD patients has
revealed that the molecular basis of this disease is the
loss of Parkin-E3 enzyme function in the ubiquitin–
proteasome pathway, which may result in the accumula-
tion of Parkin substrates in neurons [20–22]. Five
missense mutations identified in our patients are located
in the important functional domains of Parkin, i.e.,
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A. Biswas et al. / Parkinsonism and Related Disorders 12 (2006) 420–426
three in UBL domain, one in UPD domain, and one in
IBR domain—thus likely to be involved in the impair-
ment of Parkin function. The mutation (Arg334Cys) in
IBR domain has been already reported for the causation
of PD [17]. Some of our patients harbor mutations at
Arg42 position that might impair proteasome-mediated
degradative pathway. NMR study demonstrated that
Parkin UBL domain binds the Rpn 10 subunit of 26S
proteasomes via the region that includes residue 42 [23].
Interestingly, none of the 200 control chromosomes
examined harbored the 5-base deletions—the potential
role of these variants, if any, in PD require to be
evaluated by functional analyses.
We compared Parkinsonian features and special
features (Table 3) in patients carrying Parkin mutations.
Although our patients had Parkinsonian features (rest
tremor, bradykinesia, rigidity, postural instability) they
demonstrated significant phenotypic heterogeneity with
respect to age of onset, symmetrical presentation,
disease progression, disease severity and drug response
(drug induced dyskinesia) irrespective of type or
location of mutations.
All the characterized mutations, excepting the dele-
tion, were observed in the patients in heterozygous
condition. Some of the unidentified second mutation
might be deletion mutations, reported to represent 35%
of Parkin defects as per the Human Genome Mutation
database (http://uwcmml1s.uwcm.ac.uk/uwcm/mg/
search/6802742.html). However, our present PCR-based
strategy would be refractory to the heterozygous gross
deletion mutations. Studies are in progress to character-
ize such mutation. However, recent reports suggest that
Parkin-mediated PD may not necessarily occur exclu-
sively through autosomal recessive inheritance. Foroud
et al. [24] identified Parkin mutations in 103 individuals
among 559 PD patients. Of these, 41 individuals had
mutation in both alleles, whereas 62 individuals had
mutation in a single allele. Individuals with two Parkin
mutations had earlier age of onset (41.3 years) as
compared to individuals having mutation in one allele
(56.3 years). So, it has been suggested that in addition to
the classical autosomal recessive inheritance, pseudo-
dominance, dominant negative and haploinsufficiency
could cause the disease [25]. The data converging from
diverse experimental approaches seem to favor haploin-
sufficiency model. 18F-Dopa PET scanning in a family
with Parkin mutations has revealed that carriers of
Parkin mutations have a sufficiently lower dopaminergic
input to the striatum compared to controls and might be
presumed to have preclinical PD [26].
Also, the phenotype of the patients carrying Parkin
mutation now includes juvenile (o21 years), early (o45
years) as well as late-onset (445 years) disease [17]. The
common feature of the disease among Parkin mutants
appears to be slow disease progression and sustained
response to levodopa.
425
In conclusion, we have identified six mutations, three
novel and three reported, and six SNPs in sporadic cases
of PD of eastern Indian population. Gln34Arg appears
to be the prevalent Parkin mutation (3/10; 30%) in our
patient pool.
Acknowledgements
The authors are thankful to the patients for partici-
pating in the study. The study has been supported by a
grant from Council of Scientific and Industrial Research
(CSIR), Government of India (to J.R.) and a CSIR
project CMM-0016 (to K.R.). The authors are grateful
to Prof. T.N. Roy for supporting the collaboration
between BIN and University of Calcutta, Kolkata, West
Bengal.
Note added to the proof
Recently another Indian study has been reported in this
Journal on Parkin mutations in the Parkinson’s disease
(doi: 10.1016/j.parkreldis.2005.12.004) E pub: February
22, 2006.
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