Association of LRRK2 exonic variants with susceptibility to
Parkinson's disease: a case-control study
Author
Ross, Owen A, Soto-Ortolaza, Alexandra I, Heckman, Michael G, Aasly, Jan O, Abahuni,
Nadine, Annesi, Grazia, Bacon, Justin A, Bardien, Soraya, Bozi, Maria, Brice, Alexis, Brighina,
Laura, Van Broeckhoven, Christine, Carr, Jonathan, Chartier-Harlin, Marie-Christine, Dardiotis,
Efthimios, Dickson, Dennis W, Diehl, Nancy N, Elbaz, Alexis, Ferrarese, Carlo, Ferraris,
Alessandro, Fiske, Brian, Gibson, J Mark, Gibson, Rachel, Hadjigeorgiou, Georgios M, Hattori,
Nobutaka, Ioannidis, John PA, Jasinska-Myga, Barbara, Jeon, Beom S, Kim, Yun Joong, Klein,
Christine, Kruger, Rejko, Kyratzi, Elli, Lesage, Suzanne, Lin, Chin-Hsien, Lynch, Timothy,
Maraganore, Demetrius M, Mellick, George D, Mutez, Eugenie, Nilsson, Christer, Opala,
Grzegorz, Park, Sung Sup, Puschmann, Andreas, Quattrone, Aldo, Sharma, Manu, Silburn,
Peter A, Sohn, Young Ho, Stefanis, Leonidas, Tadic, Vera, Theuns, Jessie, Tomiyama,
Hiroyuki, Uitti, Ryan J, Valente, Enza Maria, van de Loo, Simone, Vassilatis, Demetrios K,
Vilarino-Gueell, Cartes, White, Linda R, Wirdefeldt, Karin, Wszolek, Zbigniew K, Wu, Ruey-
Meei, Farrer, Matthew J
Published
2011
Journal Title
Lancet neurology
Version
Accepted Manuscript (AM)
DOI
https://doi.org/10.1016/S1474-4422(11)70175-2
Copyright Statement
© 2011 Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-
NoDerivatives 4.0 International Licence which permits unrestricted, non-commercial use,
distribution and reproduction in any medium, providing that the work is properly cited.
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LRRK2 exonic variants and susceptibility to Parkinson’s disease
Owen A. Ross, PhD
1,*
, Alexandra I. Soto-Ortolaza, BSc
1
, Michael G. Heckman, MS
2
, Jan O.
Aasly, MD
3
, Nadine Abahuni, MD
4
, Grazia Annesi, PhD
5
, Justin A. Bacon, BSc
1
, Soraya
Bardien, PhD
6
, Maria Bozi, MD
7
, Alexis Brice, MD
8,9,10,11
, Laura Brighina, MD, PhD
12
,
Christine Van Broeckhoven, PhD
13,14
, Jonathan Carr, MD
15
, Marie-Christine Chartier-
Harlin, MD
16,17
, Efthimios Dardiotis, MD
18,19
, Dennis W. Dickson, MD
1
, Nancy N. Diehl, BS
2
,
Alexis Elbaz, MD, PhD
20,21
, Carlo Ferrarese, MD, PhD
12
, Alessandro Ferraris, MD, PhD
22
,
Brian Fiske, PhD
23
, J. Mark Gibson, MD
24,†
, Rachel Gibson, PhD
25
, Georgios M.
Hadjigeorgiou, MD
18,19
, Nobutaka Hattori, MD, PhD
26
, John P.A. Ioannidis, MD, DSc
27,28
,
Barbara Jasinska-Myga, MD, PhD
29
, Beom S. Jeon, MD, PhD
30
, Yun Joong Kim, MD, PhD
31
,
Christine Klein, MD, PhD
32
, Rejko Kruger, MD
33
, Elli Kyratzi, MD
34
, Suzanne Lesage,
PhD
8,9,10
, Chin-Hsien Lin, MD
35
, Timothy Lynch, FRCPI
36
, Demetrius M. Maraganore, MD
37
,
George D. Mellick, PhD
38
, Eugénie Mutez, MD
16,17,39
, Christer Nilsson, MD, PhD
40
,
Grzegorz Opala, MD, PhD
29
, Sung Sup Park, MD
41
, Andreas Puschmann, MD
40,42
, Aldo
Quattrone, MD
43
, Manu Sharma, PhD
33
, Peter A. Silburn, PhD
44
, Young Ho Sohn, MD,
PhD
45
, Leonidas Stefanis, MD
34
, Vera Tadic, MD
32
, Jessie Theuns, PhD
13,14
, Hiroyuki
Tomiyama, MD, PhD
26
, Ryan J. Uitti, MD
48
, Enza Maria Valente, MD, PhD
22
, Simone van de
Loo, PhD
4
, Demetrios K. Vassilatis, PhD
34
, Carles Vilariño-Güell, PhD
46
, Linda R. White,
PhD
47
, Karin Wirdefelt, MD, PhD
48
, Zbigniew K. Wszolek, MD
49
, Ruey-Meei Wu, MD
50
, and
Matthew J. Farrer, PhD
1,46,*
on behalf of the Genetic Epidemiology Of Parkinson’s Disease
(GEOPD) consortium
1
Division of Neurogenetics, Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA
2
Division of Biostatistics, Mayo Clinic, Jacksonville, Florida, USA
3
Department of Neuroscience,
Norwegian University of Science and Technology, Trondheim, Norway
4
Department of Neurology,
Goethe University Frankfurt am Main, Germany
5
Institute of Neurological Sciences, National
Research Council, Cosenza Italy
6
Division of Molecular Biology and Human Genetics, University
of Stellenbosch, Cape Town, South Africa
7
General Hospital of Syros, Syros, Greece
8
Université
Pierre et Marie Curie-Paris6, Centre de Recherche de l’Institut du Cerveau et de la Moelle
épinière, UMR-S975, Paris, France
9
Inserm, U975, Paris, France
10
Cnrs, UMR 7225, Paris,
© 2011 Elsevier Ltd. All rights reserved.
*
Corresponding authors’ contact information: Owen A. Ross PhD, Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road,
Jacksonville, FL 32224, Tel: (904)-953-6280, Fax: (904)-953-7370, ross.owen@mayo.edu. Matt Farrer PhD. Department of Medical
Genetics, University of British Columbia, Brain Research Centre, 2211 Wesbrook Mall, Vancouver, British Columbia, Canada V6T
2B5, Tel: 604.822.7753, Fax: 604.875.3840, mfarrer@can.ubc.ca.
†
In memory of Dr John Mark Gibson (1953–2010)
Statistical Analysis was performed by Michael G. Heckman MS (Mayo Clinic).
Author Contributions
OAR, MJF were the principal investigators and responsible for the concept and design of the study. AIO, JB, OAR, CVG were
responsible for technical aspects of study. MGH, ND were responsible for all analysis. OAR, MJF were responsible for drafting of
manuscript. All authors participated in study design and approach, sample collection, data acquisition, critical revision and final
approval of manuscript.
Conflict of interest
The authors report no conflict of interest
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France
11
AP-HP, Hôpital de la Salpêtrière, Department of Genetics and Cytogenetics, F-75013,
Paris, France
12
Department of Neuroscience-Section of Neurology, University of Milano-Bicocca,
San Gerardo Hospital, Monza, Italy
13
Neurodegenerative Brain Diseases group, Department of
Molecular Genetics, VIB, Antwerpen, Belgium
14
Laboratory of Neurogenetics, Institute Born-
Bunge and University of Antwerp, Antwerpen, Belgium
15
Division of Neurology, University of
Stellenbosch, Cape Town, South Africa
16
University Lille Nord de France, Centre de recherche
Jean-Pierre Aubert, Lille, France
17
INSERM, U837, Lille, France
18
Department of Neurology,
Laboratory of Neurogenetics, Faculty of Medicine, University of Thessaly, Larissa, Greece
19
Institute of Biomedical Research & Technology, CERETETH, Larissa, Greece
20
INSERM,
U708, Neuroepidemiology, F-75005, Paris, France
21
UPMC University of Paris 06, UMR_S708,
Neuroepidemiology, F-75005, Paris, France
22
IRCCS Casa Sollievo della Sofferenza Hospital,
Mendel Laboratory, San Giovanni Rotondo, Italy
23
The Michael J Fox Foundation for Parkinson’s
Research, New York, NY, USA
24
Department of Neurology, Royal Victoria Hospital, Belfast,
Ireland
25
Research and Development, GlaxoSmithKline Pharmaceuticals Ltd., Harlow, England
26
Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
27
Clinical and
Molecular Epidemiology Unit, Department of Hygiene and Epidemiology, University of Ioannina
Medical School, Ioannina, Greece
28
Stanford Prevention Research Center, Stanford University
School of Medicine, Stanford, CA, USA
29
Department of Neurology, Medical University of Silesia,
Katowice, Poland
30
Department of Neurology, Seoul National University Hospital, Seoul 110-744,
South Korea
31
ILSONG Institute of Life Science and Department of Neurology, Hallym University,
Anyang, South Korea
32
Section of Clinical and Molecular Neurogenetics at the Department of
Neurology, University of Luebeck, Germany
33
Department for Neurodegenerative Diseases,
Hertie-Institute for Clinical Brain Research and German Center for Neurodegenerative Diseases
(DZNE), University of Tübingen, Germany
34
Divisions of Basic Neurosciences and Cell Biology,
Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece
35
Department of Neurology, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin, Taiwan
36
Dublin Neurological Institute at the Mater Misericordiae University Hospital, and Conway
Institute of Biomolecular & Biomedical Research, University College Dublin, Ireland
37
Department
of Neurology, Mayo Clinic, Rochester, Minnesota, USA
38
Eskitis Institute for Cell and Molecular
Therapies, Griffith University, Queensland, Australia
39
Centre Hospitalier Regional Universitaire
de Lille, 59037 Lille, France
40
Department of Clinical Science, Section of Geriatric Psychiatry,
Lund University, Sweden
41
Department of Laboratory Medicine, Seoul National University
Hospital, Seoul 110-744, South Korea
42
Department of Neurology, Lund, Skåne University
Hospital, Sweden
43
Department of Medical Sciences, Institute of Neurology, University Magna
Graecia, and Neuroimaging Research Unit, National Research Council, Catanzaro, Italy
44
University of Queensland, Centre for Clinical Research, Royal Brisbane Hospital, Australia
45
Department of Neurology, Yonsei University College of Medicine, Seoul, South Korea
46
Department of Medical Genetics, University of British Columbia, Vancouver, V5Z 4H4 BC,
Canada
47
University Hospital and NTNU, Trondheim, Norway
48
Department of Clinical
Neuroscience and Department of Medical Epidemiology and Biostatistics, Karolinska Institutet,
Stockholm, Sweden
49
Department of Neurology, Mayo Clinic, Jacksonville, Florida, USA
50
Department of Neurology, National Taiwan University Hospital, College of Medicine, National
Taiwan University, Taipei, Taiwan
Abstract
Background—Leucine-rich repeat kinase 2 (LRRK2) is known to harbor highly penetrant
mutations linked to familial parkinsonism. However, its full polymorphic variability in
relationship to Parkinson’s disease (PD) risk has not been systematically assessed.
Methods—We examined the frequency pathogenicity of 121 exonic LRRK2 variants in three
ethnic series (Caucasian [N=12,590], Asian [N=2,338] and Arab-Berber [N=612]) consisting of
Ross et al. Page 2
8,611 patients and 6,929 control subjects from 23 separate sites of the Genetic Epidemiology of
Parkinson’s Disease Consortium.
Findings—Excluding carriers of previously known pathogenic mutations, new independent risk
associations were found for polymorphic variants in Caucasian (p.M1646T, OR: 1.43, 95% CI:
1.15 – 1.78, P=0.0012) and Asian (p.A419V, OR: 2.27, 95% CI: 1.35 – 3.83, P=0.0011)
populations. In addition, a protective haplotype was observed at >5% frequency (p.N551K-
p.R1398H-p.K1423K) in the Caucasian and Asian series’, with a similar finding in the small
Arab-Berber series that requires further study (combined 3-series OR: 0.82, 95% CI: 0.72 – 0.94,
P=0.0043). Of the two previously reported Asian risk variants p.G2385R was found to be
associated with disease (OR: 1.73, 95% CI: 1.20 – 2.49, P=0.0026) but no association was
observed for p.R1628P (OR: 0.62, 95% CI: 0.36 – 1.07, P=0.087). Also in the Arab-Berber series,
p.Y2189C showed potential evidence of risk association with PD (OR: 4.48, 95% CI: 1.33 –
15.09, P=0.012). Of note, two variants (p.I1371V and p.T2356I) which have been previously
proposed as pathogenic were observed in patient and control subjects at the same frequency.
Interpretation—LRRK2 offers an example where multiple rare and common genetic variants in
the same gene have independent effects on disease risk. Lrrk2, and the pathway in which it
functions, is important in the etiology and pathogenesis of a greater proportion of patients with PD
than previously believed.
Funding—The present study and original funding for the GEO-PD Consortium was supported by
grants from Michael J. Fox Foundation. Studies at individual sites were supported by a number of
funding agencies world-wide.
Keywords
Parkinson disease; LRRK2; genetics
INTRODUCTION
Parkinson’s disease (PD) is generally considered a late-onset sporadic disorder.
Nevertheless, genetic insights have helped to define the molecular etiology and have
provided new models to develop neuroprotective interventions. Mutations of the leucine-rich
repeat kinase 2 gene (LRRK2; Lrrk2) are now recognized as the most frequent genetic
determinant of familial and sporadic PD
1
. The LRRK2 gene (51 exons) encodes a protein
(2527 amino acid; Lrrk2) which has five conserved domains: including a Roc (Ras in
complex proteins; Rab GTPase) and a catalytic core common to both tyrosine and serine/
threonine kinases.
Pathogenic LRRK2 variability has been identified by sequencing of probands with familial
parkinsonism, with results confirmed and occasionally extended within community and/or
clinically-based patient-control series
2–6
. Seven definite pathogenic mutations (Lrrk2
p.N1437H, p.R1441C/G/H, p.Y1699C, p.G2019S, and p.I2020T) have been described
7, 8
.
These mutations may be relatively frequent in patients from specific ethnicities, although
still rare in ethnically-matched control subjects. Lrrk2 p.R1441G is found in more than 8%
of patients originating in the Basque region of Northern Spain
9
, whereas Lrrk2 p.G2019S is
found in 30% of Arab-Berber patients with PD
10, 11
. LRRK2 polymorphisms (>1% minor
allele frequency) have also been associated with PD in Asia, for which the estimated
attributable risk is often dependent on the specific ethnicity. Lrrk2 p.R1628P and p.G2385R
are each found in 3–4% of individuals of Chinese descent and increase the risk of PD by
approximately two-fold
12–15
.
Ross et al. Page 3
However, the large majority of LRRK2 variants have not been systematically studied. It is
possible that LRRK2 may harbor many more variants that are important for determining PD
pathogenicity and clinical risk. To address this possibility, with the Genetic Epidemiology of
Parkinson’s Disease (GEO-PD) consortium we have examined frequency of 121 LRRK2
exonic variants in 15,540 subjects including 8,611 patients with PD, and assessed their role
in disease susceptibility.
METHODS
Participants
The GEO-PD consortium includes investigators from 35 sites representing 22 countries, and
six continents. All GEO-PD sites were invited to participate in this study. A total of 23 sites
representing 15 countries and 5 continents agreed to participate in the current study and
contributed clinical data for a total of 15,540 individuals (8,611 patients with PD and 6,929
controls). The Caucasian series consisted of 6,995 PD cases and 5,595 controls, the Asian
series consisted of 1,376 PD cases and 962 controls, and the Arab-Berber series consisted of
240 PD cases and 372 controls. Patients were diagnosed using either the Gelb or the United
Kingdom Parkinson’s Disease Brain Bank (the exclusion criterion “>1 affected relative” was
not included). Controls were collected at each site as unrelated healthy individuals (not all
controls would have been given a detailed neurological examination but would have been
asked about prior diagnosis of a neurological disorder or family history). Demographics for
each series are shown in Table 1 and the sample size breakdown from each site is provided
in Supplemental Table 1. All human biological samples were collected, fulfilling requested
ethical approvals, and used in accord with the terms of subjects’ informed consent.
Genotyping
LRRK2 exonic variants were identified through searches of available literature up to April
1
st
2010, personal communications of Consortium members and from unpublished data
(Table 2). Genotyping was performed on a Sequenom MassArray iPLEX platform (San
Diego, CA) at the Mayo Clinic Florida laboratory of Neurogenetics (except for the groups
from Paris, France, and Belgium who supplied genotype data and positive control genomic
DNA
2, 3
); all primer sequences are provided in Supplemental Table 2. In total 8 iPLEX
variant combinations were used to incorporate 123 LRRK2 coding variants (Table 2).
Positive control DNA was included for each variant; where positive genomic control DNA
was unavailable a synthetic positive control DNA sequence was generated by a mismatch
primer PCR method. A chi-square test followed by Bonferroni correction was used to test
for deviation from Hardy Weinberg equilibrium (HWE) in controls for each site. Direct
DNA sequencing was employed to confirm genotyping for all variants with a frequency
below 0.3% (n<50).
Statistical Analysis
All analyses were performed separately for the Caucasian, Asian and Arab-Berber series’.
For common variants with a minor allele frequency (MAF) of 0.5% or greater, single variant
associations with PD were evaluated utilizing fixed effects logistic regression models, where
genotypes were dichotomized as presence versus absence of the minor allele (dominant
model) due to the fact that LRRK2 mutations cause an autosomal dominantly inherited form
of PD and also given the lack of rare homozygotes for many of the variants; additive models
were also examined. Models were adjusted for site in the Asian and Caucasian series’.
Sensitivity of results to the use of random effects models was also examined
16
. Odds ratios
(ORs) and 95% confidence intervals (CIs) were estimated. Between-site heterogeneity was
assessed using likelihood ratio tests for variant by site interaction in logistic regression
Ross et al. Page 4
