Background
Mutations in parkin are estimated to account for as much as 50% of familial Parkinson disease (PD) and 18% of sporadic PD. Single heterozygous mutations in parkin in both familial and sporadic cases may also increase susceptibility to PD. To our knowledge, all previous studies have been restricted to PD cases; this is the first study to systematically screen the parkin coding regions and exon deletions and duplications in controls.
Objective
To determine the frequency and spectrum of parkin variants in early-onset PD cases (aged ≤50 years) and controls participating in a familial aggregation study.
Patients and Methods
We sequenced the parkin gene in 101 cases and 105 controls. All cases and controls were also screened for exon deletions and duplications by semiquantitative multiplex polymerase chain reaction.
Results
Thirteen (12.9% [95% confidence interval, 7%-21%]) of the 101 cases had a previously described parkin mutation: 1 was homozygous, 11 were heterozygous, and 1 was a compound heterozygote. The mutations Arg42Pro (exon 2) and Arg275Trp (exon 7) were recurrent. The previously reported synonymous substitution Leu261Leu (c.884A>G) was identified in 4 (3.9%) of 101 cases and 2 (2%) of 105 controls (P = .44). Excluding the synonymous substitution Leu261Leu (heterozygotes), 10 (9.9% [95% confidence interval, 4.6%-17.5%]) carried mutations.
Conclusions
The frequency of mutations among cases that were not selected based on family history of PD is similar to what has previously been reported in sporadic PD. The similar frequency of Leu261Leu in cases and controls suggests it is a normal variant rather than a disease-associated mutation. We confirmed that heterozygous parkin mutations may increase susceptibility for early-onset PD.
Mutations in 4 genes, α-synuclein (PARK1),1,2parkin (PARK2),3DJ-1 (PARK7),4-6 and PINK1 (PARK6),7-11 are associated with early-onset Parkinson disease (EOPD). Parkin mutations account for the majority of familial and sporadic EOPD cases with a known genetic association.3,12 In PD cases with age at onset (AAO) of 45 years or younger, the frequency of parkin mutations is estimated at 49% in cases with a family history of PD13 and 15% in cases without a family history of PD.14 In the only community-based study, the frequency of mutations was 9% in 111 cases with AAO of 50 years or younger.15
In both familial and sporadic cases, homozygous, compound heterozygous, and single heterozygous mutations in parkin have been described. Several studies indicate that heterozygous mutations may increase susceptibility to PD,3,16-19 although this is controversial. The carrier (heterozygous) frequency of parkin alleles in the normal population is unknown. Previous studies have only assessed the frequency of specific parkin mutations and/or variants that were identified in PD cases and have not systematically screened the parkin gene for mutations in normal controls.3,20
We have screened participants in the Genetic Epidemiology of PD Study (GEPD) for variants in DJ-121 and glucocerebrosidase.22 In the present study, to identify variants in the parkin gene, coding exons were sequenced completely in 206 subjects, including 101 EOPD cases and 105 controls from GEPD. All subjects (cases and controls) were also screened for exon deletions and duplications by semiquantitative multiplex polymerase chain reaction (PCR).
Cases were recruited based on AAO of motor signs of 50 years or younger (EOPD), regardless of family history of PD in a first-degree relative. All cases were seen at the Center for Parkinson's Disease and Other Movement Disorders at Columbia University, and EOPD cases were oversampled.23 Duration of PD was calculated as the years from motor onset to enrollment in GEPD. We have previously shown that reliability of reporting the AAO of motor signs of PD was excellent.23 All parkin coding exons were sequenced and analysis of exon deletions and duplications were completed in 101 of 256 EOPD cases from GEPD23 on whom data were complete at the time of the analysis. The current study includes 21 of 33 cases previously screened for parkin variants.24 Analysis was performed without knowledge of these results.
One hundred five of 412 controls from GEPD on whom data were complete were randomly chosen for sequencing of parkin exons and analysis of exon deletions and duplications by semiquantitative multiplex PCR. For PD cases recruited from the Center for Parkinson's Disease and Other Movement Disorders, controls were recruited by random-digit dialing and were frequency matched based on key demographic variables.23 For cases who were residents of Washington Heights, Manhattan, NY, and 65 years or older, controls were recruited from a 50% sample of names and addresses of Medicare recipients provided by Health Care Finance Association. For 18 cases younger than 65 years, controls were recruited from the Northern Manhattan Stroke Study in Washington Heights.25 For the analysis of variants in exon 7, including the synonymous substitution Leu261Leu, an additional 82 Hispanic controls from the 50% Medicare sample were included to enrich the number of Hispanic controls.
All PD case and control probands underwent an evaluation that included a medical history and Unified Parkinson's Disease Rating Scale26 and videotape assessment. The modified Mini-Mental State Examination was administered to all probands in either English or Spanish.27 A valid, reliable, structured family history of PD questionnaire (Family History Information) was administered in English or Spanish to all PD cases and controls, either in person or over the telephone, for ascertainment of PD and other neurologic disease in first-degree relatives.28 The institutional review board at the College of Physicians and Surgeons, Columbia University, approved this study. Informed consent was obtained from all study participants.
Molecular genetic analysis
DNA was isolated from whole blood using standard techniques. All blood and genomic DNA samples were coded on receipt to ensure patient confidentiality. Polymerase chain reaction amplification of parkin exons 1 to 12 was performed in cases and controls. The primers used for PCR amplification of parkin exons and intronic and exonic boundaries and sequencing have been described previously.29,30 Cycle sequencing was performed on the purified PCR product as per the manufacturer's instructions (BigDye; Applied Biosystems, Foster City, Calif). Products were analyzed on an ABI3700 genetic analyzer (Applied Biosystems). Chromatograms were viewed using Sequencher (Gene Codes Corporation, Ann Arbor, Mich) and sequence variants determined. All sequence variants identified in cases and controls were confirmed by analysis in a separate PCR followed by bidirectional sequencing. Sequence variants were classified as polymorphic if their frequency was 1% or greater in ethnically matched controls. These analyses were performed without knowledge of case-control status.
To identify genomic deletions and exon rearrangements in parkin, semiquantitative multiplex PCR was performed as previously described.31 Briefly, Hex-tagged fluorescent-labeled primers for parkin exons were optimised in pooled sets of 4 primer pairs for multiplexing along with an internal control (328-bp PCR product of an unlinked gene transthyretin on chromosome 18). The PCR fragments were analyzed on an ABI3700 genetic analyzer. Electropherograms were viewed and product size and peak heights calculated using Genescan 3.1 and Genotyper software (Applied Biosystems).
Baseline characteristics at the time of enrollment were compared using the t test for continuous variables and the χ2 test or Fisher exact test for categorical variables. First, we compared the demographic characteristics of the subjects who were included in the analysis of parkin variants with those of the subjects who were not. Second, we compared the demographic characteristics of cases and controls who were analyzed for parkin. Third, we compared the demographic and clinical characteristics of cases with and without mutations in the parkin gene. The Wilcoxon rank sum test was used to compare the total modified Mini-Mental State Examination score in PD cases with and without parkin mutations because of the inadequacy of normality assumption of the distribution.
In a separate analysis including an additional 82 Hispanic controls, we compared the frequency of Leu261Leu in cases and controls and performed a stratified analysis by ethnic group to determine whether there was an association with disease status.
Demographic and clinical characteristics of the case-control cohort
At the time of this study, 668 subjects including 256 EOPD cases and 412 controls had been recruited into GEPD. All parkin coding exons were sequenced and analyzed for exon deletions and duplications in a subset of 206 (101 PD cases with AAO≤50 years and 105 controls). Demographic and clinical characteristics of the 206 cases and controls are presented in Table 1. The mean (SD) AAO of 101 cases was 41.1 (7.2) years, disease duration was 11.7 (8.0) years, and total motor score (Unified Parkinson's Disease Rating Scale Part III) was 21.0 (13.00). Compared with the subjects in whom parkin was not sequenced (n = 462), the 206 cases and controls were younger (56.9 vs 63.2 years; P<.001), had completed more years of education (15.6 vs 14.5 years; P<.001), and were more likely to be white (89.3% vs 66.8%; P<.03). Cases in whom the parkin gene was sequenced and analyzed for exon deletions and duplications (n = 101) did not differ significantly from cases who were not analyzed (n = 155) in AAO of PD, age at evaluation, years of education, ethnicity, sex, or family history of PD. Those cases in whom the gene was analyzed had longer duration of PD (11.9 vs 10.0 years; P = .05); however, Hoehn and Yahr score and total motor score on the Unified Parkinson's Disease Rating Scale Part III were similar to nonsequenced cases.
FREQUENCY OF PARKIN MUTATIONS IN CASES AND CONTROLS
Thirteen (12.9%) (95% confidence interval [CI], 7%-21%) of the 101 cases had a previously described parkin mutation; 1 was homozygous, 11 were heterozygous, and 1 was a compound heterozygote (Table 2). Five different point mutations and 3 different exon rearrangements were identified (Table 2). Point mutations included 3 missense mutations, 1 synonymous substitution, and 1 splice mutation. Exon deletions were found in 3 different exons (exons 3, 4, and 5). Fifty-four percent (7/13) of the variants identified in cases were found in exons encoding functional domains including the ubiquitin domain (exons 2 and 3) and RING1 domain (exon 7). The mutations Arg275Trp, Arg42Pro, and the synonymous substitution Leu261Leu were recurrent; 2 cases carried Arg275Trp, 2 carried Arg42Pro, and 4, including 1 compound heterozygote, carried Leu261Leu (3 Hispanic subjects, 1 white non-Hispanic subject). The synonymous substitution Leu261Leu was identified in 2% (2/105) of controls (1 Hispanic subject and 1 African American subject). To our knowledge, all previously published studies have evaluated the Leu261Leu allele frequency only in white non-Hispanic control subjects. Because we identified the synonymous substitution in Hispanic and white non-Hispanic subjects and our pool of Hispanic controls was limited (n = 4), we added 82 Hispanic controls from the 50% Health Care Finance Association sample to the existing control group (n = 105). We found that 13% (11/86) of Hispanic controls carried the synonymous substitution Leu261Leu (Table 3). The allele frequency was higher in Hispanic cases and controls combined, 14.4% (14/97), compared with all other ethnicities, including white non-Hispanic, African American, and Asian cases and controls combined (1.1% [2/191]) (P<.001).
FREQUENCY OF PARKIN POLYMORPHISMS
The previously reported parkin polymorphisms, IVS3-20C>T, IVS2 + 25T>C, IVS7-35A>G, Ser167Asn, Asp394Asn, and Val380Leu, were observed at a similar frequency in cases and controls and were not significantly associated with PD (data not shown).
CLINICAL CHARACTERISTICS OF CASES WITH AND WITHOUT PARKIN MUTATIONS
Demographic and clinical characteristics of 91 cases without parkin mutations and 10 cases (excluding 3 cases with the synonymous substitution Leu261Leu) with mutations are presented in Table 4. Information on history of PD in first-degree relatives was available for 97 of 101 cases and 9 of 11 parkin mutation carriers. The frequency of parkin mutations was 15.4% (2/13) in cases with a family history of PD in a first-degree relative, compared with 8.3% (7/84) in those without a family history of PD (P = .35). The 2 cases with parkin mutations who reported a family history of PD in a first-degree relative had AAOs of 32 and 38 years, respectively. One of the probands, the only homozygote (AAO, 38 years), had 2 siblings with PD (AAOs, 26 and 30 years). The frequency of parkin mutations (n = 10) was 50% (1/2) in cases with AAO of 20 years and younger, 33% (2/6) in cases with AAO of 21 to 30 years, 9% (3/33) in cases with AAO of 31 to 40 years, and 7% (4/60) in cases with AAO of 41 to 50 years. Noncarriers and carriers did not differ in terms of first symptom reported (rest tremor, bradykinesia, rigidity, or gait impairment) nor did they differ in terms of current use of levodopa/carbidopa or dopamine agonists. None of the cases reported hallucinations or cognitive impairment preceding motor signs. Cases were not specifically queried about sleep benefit or sensitivity to levodopa in GEPD.
The prevalence of parkin mutations in 101 cases with AAO younger than 50 years was 9.9% (95% CI, 4.9%-17.5%) (excluding 3 heterozygous cases with the synonymous substitution Leu261Leu) and 8.3% (95% CI, 3.4%- 16.4%) among those cases who did not report a family history of PD in a first-degree relative, which is within the range (9%-18%) reported in other series comprising primarily sporadic cases.14,15,32 The frequency of parkin mutations in cases with AAO younger than 45 years in our study was 11.1% (95% CI, 4.6%-21.6%), which is very similar to the 15% frequency reported by Periquet et al14 in patients with isolated early-onset parkinsonism (AAO, <45 years). Phenotypic differences were not observed between cases with and without parkin mutations in this study; however, detailed psychiatric and cognitive assessments were not performed. The synonymous substitution Leu261Leu (c.884A>G), which has been previously described as a mutation,24,33 was identified with similar frequency in cases and controls, suggesting that it is a common variant rather than a disease-associated mutation. The frequency of this variant appeared to be higher in Hispanic subjects than in non-Hispanic subjects. Our current sample size was insufficient to assess definitively the possibility that the synonymous substitution Leu261Leu is a susceptibility allele, and functional data are not available to determine pathogenicity. Based on the allele frequencies we observed in subjects of Hispanic ethnicity, a sample size of 934 (467 cases and 467 controls) would have been required to detect a statistically significant difference (80% power, α = .05).
To our knowledge, this is the first study to describe complete sequencing of parkin coding exons and analysis of exon deletions and duplications by semiquantitative PCR in cases and controls. Several studies have reported heterozygous parkin mutations in both EOPD and late-onset PD.18,20,24,29,32-40 Currently, the identification of heterozygous mutations in patients with PD is controversial, and it is unknown whether heterozygous mutations alone are pathogenic or whether additional mutations have been missed in screening the parkin gene in these studies. Our current study helps clarify this issue by screening controls and finding that heterozygous mutations are absent. The absence of control subjects carrying parkin mutations suggests that heterozygous parkin mutations may increase susceptibility for EOPD. There are few functional studies to support the hypothesis that parkin heterozygous mutations are pathogenic. However, 1 study41 has demonstrated that 2 RING finger 1 mutations (R256C and R275W) are dominant gain-of-function mutations, which is consistent with the identification of heterozygous mutations in several studies and supports the hypothesis that parkin heterozygous mutations are pathogenic. Other strengths of this study include that all cases and controls from GEPD were administered reliable, structured neurologic examinations and family history of PD interviews. The identification of possible ethnic differences in allele frequencies for the Leu261Leu variant highlights the importance of well-characterized multiethnic samples in determining disease susceptibility alleles and in diagnostic screening.
Correspondence: Lorraine N. Clark, PhD, Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (lc654@columbia.edu).
Accepted for Publication: October 12, 2005.
Author Contributions: Drs Clark and Marder were responsible for data validation; Drs Marder, Harris, Louis, Cote, Andrews, Fahn, Waters, Ford, and Frucht and Ms Mejia-Santana, for patient information. Study concept and design: Clark, Fahn, Ottman, and Marder. Acquisition of data: Clark, Afridi, Karlins, Mejia-Santana, Harris, Cote, Andrews, Fahn, Waters, Ford, Frucht, Ottman, and Marder. Analysis and interpretation of data: Clark, Afridi, Wang, Louis, Fahn, Ottman, and Marder. Drafting of the manuscript: Clark, Wang, Mejia-Santana, Andrews, and Marder. Critical revision of the manuscript for important intellectual content: Clark, Afridi, Karlins, Wang, Mejia-Santana, Harris, Louis, Cote, Fahn, Waters, Ford, Frucht, Ottman, and Marder. Statistical analysis: Wang, Louis, and Ottman. Obtained funding: Clark, Ottman, and Marder. Administrative, technical, and material support: Clark, Afridi, Karlins, Mejia-Santana, Harris, Andrews, Ford, and Marder. Study supervision: Clark and Fahn.
Funding/Support: This study was supported by grants by NS36630 (Dr Marder), RR00645, AG07232, NS39422, and NS29993 from the National Institutes of Health and the Parkinson's Disease Foundation (Dr Clark).
Acknowledgment: We appreciate the assistance of Richard Mayeux, MD, MSc, Ralph Sacco, MD, and Paul Greene, MD, for the recruitment of patients. We also thank Christine Klein, MD, and Katja Hedrich, PhD.
1.Polymeropoulos
MHLavedan
CLeroy
E
et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease.
Science 1997;2762045- 2047
PubMedGoogle ScholarCrossref 3.Hedrich
KEskelson
CWilmot
B
et al. Distribution, type, and origin of
Parkin mutations: review and case studies.
Mov Disord 2004;191146- 1157
PubMedGoogle ScholarCrossref 4.Bonifati
VRizzu
Pvan Baren
MJ
et al. Mutations in the
DJ-1 gene associated with autosomal recessive early-onset parkinsonism.
Science 2003;299256- 259
PubMedGoogle ScholarCrossref 5.Abou-Sleiman
PHealy
DQuinn
N
et al. The role of pathogenic
DJ-1 mutations in Parkinson's disease.
Ann Neurol 2003;54283- 286
PubMedGoogle ScholarCrossref 6.Hague
SRogaeva
EHernandez
D
et al. Early-onset Parkinson's disease caused by a compound heterozygous
DJ-1 mutation.
Ann Neurol 2003;54271- 274
Google ScholarCrossref 7.Valente
EMAbou-Sleiman
PMCaputo
V
et al. Hereditary early-onset Parkinson's disease caused by mutations in
PINK1.
Science 2004;3041158- 1160
PubMedGoogle ScholarCrossref 9.Healy
DGAbou-Sleiman
PMAhmadi
KR
et al. The gene responsible for
PARK6 Parkinson's disease,
PINK1, does not influence common forms of parkinsonism.
Ann Neurol 2004;56329- 335
PubMedGoogle ScholarCrossref 10.Rohe
CFMontagna
PBreedveld
G
et al. Homozygous
PINK1 C-terminus mutation causing early-onset parkinsonism.
Ann Neurol 2004;56427- 431
PubMedGoogle ScholarCrossref 11.Valente
EMSalvi
SIalongo
T
et al.
PINK1 mutations are associated with sporadic early-onset parkinsonism.
Ann Neurol 2004;56336- 341
PubMedGoogle ScholarCrossref 12.Dekker
MCBonifati
VVan Duijn
CM Parkinson's disease: piecing together a genetic jigsaw.
Brain 2003;126(pt 8)1722- 1733
Google ScholarCrossref 13.Lucking
CBDurr
ABonifati
V
et al. Association between early-onset Parkinson's disease and mutations in the
parkin gene: French Parkinson's Disease Genetics Study Group.
N Engl J Med 2000;3421560- 1567
PubMedGoogle ScholarCrossref 14.Periquet
MLatouche
MLohmann
E
et al.
Parkin mutations are frequent in patients with isolated early-onset parkinsonism.
Brain 2003;1261271- 1278
PubMedGoogle ScholarCrossref 15.Kann
MJacobs
HMohrmann
K
et al. Role of
parkin mutations in 111 community-based patients with early-onset parkinsonism.
Ann Neurol 2002;51621- 625
PubMedGoogle ScholarCrossref 16.Hilker
RKlein
CGhaemi
M
et al. Positron emission tomographic analysis of the nigrostriatal dopaminergic system in familial parkinsonism associated with mutations in the
parkin gene.
Ann Neurol 2001;49367- 376
PubMedGoogle ScholarCrossref 17.Hilker
RKlein
CHedrich
K
et al. The striatal dopaminergic deficit is dependent on the number of mutant alleles in a family with mutations in the
parkin gene: evidence for enzymatic
parkin function in humans.
Neurosci Lett 2002;32350- 54
PubMedGoogle ScholarCrossref 18.Lohmann
EPeriquet
MBonifati
V
et al. How much phenotypic variation can be attributed to
parkin genotype?
Ann Neurol 2003;54176- 185
PubMedGoogle ScholarCrossref 19.Munhoz
RPSa
DSRogaeva
E
et al. Clinical findings in a large family with a
parkin ex3delta40 mutation.
Arch Neurol 2004;61701- 704
PubMedGoogle ScholarCrossref 20.Lincoln
SJMaraganore
DMLesnick
TG
et al.
Parkin variants in North American Parkinson's disease: cases and controls.
Mov Disord 2003;181306- 1311
PubMedGoogle ScholarCrossref 21.Clark
LNAfridi
SMejia-Santana
H
et al. Analysis of an early-onset Parkinson's disease cohort for
DJ-1 mutations.
Mov Disord 2004;19796- 800
PubMedGoogle ScholarCrossref 22.Clark
LNNicolai
AAfridi
S
et al. Pilot association study of the beta-glucocerebrosidase N370S allele and Parkinson's disease in subjects of Jewish ethnicity.
Mov Disord 2005;20100- 103
PubMedGoogle ScholarCrossref 24.Hedrich
KMarder
KHarris
J
et al. Evaluation of 50 probands with early-onset Parkinson's disease for
Parkin mutations.
Neurology 2002;581239- 1246
PubMedGoogle ScholarCrossref 25.Sacco
RLBoden-Albala
BGan
R
et al. Stroke incidence among white, black, and Hispanic residents of an urban community: the Northern Manhattan Stroke Study.
Am J Epidemiol 1998;147259- 268
PubMedGoogle ScholarCrossref 26.Fahn
SMarsden
CDCalne
D Recent Developments in Parkinson's Disease. Florham Park, NJ: Macmillan Healthcare Information; 1987
29.West
APeriquet
MLincoln
S
et al. Complex relationship between
Parkin mutations and Parkinson disease.
Am J Med Genet 2002;114584- 591
PubMedGoogle ScholarCrossref 30.Pigullo
SDe Luca
ABarone
P
et al. Mutational analysis of
parkin gene by denaturing high-performance liquid chromatography (DHPLC) in essential tremor.
Parkinsonism Relat Disord 2004;10357- 362
PubMedGoogle ScholarCrossref 31.Lucking
CBBrice
A Semiquantitative PCR for the detection of exon rearrangements in the
Parkin gene.
Methods Mol Biol 2003;21713- 26
PubMedGoogle Scholar 32.Poorkaj
PNutt
JGJames
D
et al.
Parkin mutation analysis in clinic patients with early-onset Parkinson's disease.
Am J Med Genet A 2005;139A56
PubMedGoogle ScholarCrossref 33.Oliveira
SAScott
WKMartin
ER
et al.
Parkin mutations and susceptibility alleles in late-onset Parkinson's disease.
Ann Neurol 2003;53624- 629
PubMedGoogle ScholarCrossref 34.Abbas
NLucking
CBRicard
S
et al. A wide variety of mutations in the
parkin gene are responsible for autosomal recessive parkinsonism in Europe: French Parkinson's Disease Genetics Study Group and the European Consortium on Genetic Susceptibility in Parkinson's Disease.
Hum Mol Genet 1999;8567- 574
PubMedGoogle ScholarCrossref 35.Foroud
TUniacke
SKLiu
L
et al. Heterozygosity for a mutation in the
parkin gene leads to later onset Parkinson disease.
Neurology 2003;60796- 801
PubMedGoogle ScholarCrossref 36.Hedrich
KDjarmati
ASchafer
N
et al. DJ-1 (
PARK7) mutations are less frequent than
Parkin (
PARK2) mutations in early-onset Parkinson disease.
Neurology 2004;62389- 394
PubMedGoogle ScholarCrossref 37.Bertoli-Avella
AMGiroud-Benitez
JLAkyol
A
et al. Novel
parkin mutations detected in patients with early-onset Parkinson's disease.
Mov Disord 2005;20424- 431
PubMedGoogle ScholarCrossref 38.Hedrich
KPramstaller
PPStubke
K
et al. Premutations in the
FMR1 gene as a modifying factor in
Parkin-associated Parkinson's disease?
Mov Disord 2005;201060- 1062
PubMedGoogle ScholarCrossref 39.Khan
NLHorta
WEunson
L
et al.
Parkin disease in a Brazilian kindred: manifesting heterozygotes and clinical follow-up over 10 years.
Mov Disord 2005;20479- 484
PubMedGoogle ScholarCrossref 40.Wu
RMBounds
RLincoln
S
et al.
Parkin mutations and early-onset parkinsonism in a Taiwanese cohort.
Arch Neurol 2005;6282- 87
PubMedGoogle ScholarCrossref 41.Cookson
MRLockhart
PJMcLendon
C
et al.
RING finger 1 mutations in
Parkin produce altered localization of the protein.
Hum Mol Genet 2003;122957- 2965
PubMedGoogle ScholarCrossref