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Cited by 117Parkinson's Disease Mechanisms and TreatmentsNuclear Receptors and SignalingDeep Brain Stimulation and Movement Disorders

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RAB32 Ser71Arg in autosomal dominant Parkinson's disease: linkage, association, and functional analyses

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Gustavsson, Emil K, Follett, Jordan, Trinh, Joanne, Barodia, Sandeep K, Real, Raquel, Liu, Zhiyong, Grant-Peters, Melissa, Fox, Jesse D, Appel-Cresswell, Silke, Stoessl, A Jon, Rajput, Alex, Rajput, Ali H, Auer, Roland, Tilney, Russel, Sturm, Marc, Haack, Tobias B, Lesage, Suzanne, Tesson, Christelle, Brice, Alexis, Vilariño-Güell, Carles, Ryten, Mina, Goldberg, Matthew S, West, Andrew B, Hu, Michele T, Morris, Huw R, Sharma, Manu, Gan-Or, Ziv, Samanci, Bedia, Lis, Pawel, Periñan, Maria Teresa, Amouri, Rim, Ben Sassi, Samia, Hentati, Faycel, Tonelli, Francesca, Alessi, Dario R, Farrer, Matthew J·2024·The Lancet Neurology
DOI 10.1016/s1474-4422(24)00121-2PMC PMC10827257

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Abstract

Background Parkinson's disease is a progressive neurodegenerative disorder with multifactorial causes, among which genetic risk factors play a part. The RAB GTPases are regulators and substrates of LRRK2, and variants in the LRRK2 gene are important risk factors for Parkinson's disease. We aimed to explore genetic variability in RAB GTPases within cases of familial Parkinson's disease. Methods We did whole-exome sequencing in probands from families in Canada and Tunisia with Parkinson's disease without a genetic cause, who were recruited from the Centre for Applied Neurogenetics (Vancouver, BC, Canada), an international consortium that includes people with Parkinson's disease from 36 sites in 24 countries. 61 RAB GTPases were genetically screened, and candidate variants were genotyped in relatives of the probands to assess disease segregation by linkage analysis. Genotyping was also done to assess variant frequencies in individuals with idiopathic Parkinson's disease and controls, matched for age and sex, who were also from the Centre for Applied Neurogenetics but unrelated to the probands or each other. All participants were aged 18 years or older. The sequencing and genotyping findings were validated by case–control association analyses using bioinformatic data obtained from publicly available clinicogenomic databases (AMP-PD, GP2, and 100 000 Genomes Project) and a private German clinical diagnostic database (University of Tübingen). Clinical and pathological findings were summarised and haplotypes were determined. In-vitro studies were done to investigate protein interactions and enzyme activities. Findings Between June 1, 2010, and May 31, 2017, 130 probands from Canada and Tunisia (47 [36%] female and 83 [64%] male; mean age 72·7 years [SD 11·7; range 38–96]; 109 White European ancestry, 18 north African, two east Asian, and one Hispanic] underwent whole-exome sequencing. 15 variants in RAB GTPase genes were identified, of which the RAB32 variant c.213C>G (Ser71Arg) cosegregated with autosomal dominant Parkinson's disease in three families (nine affected individuals; non-parametric linkage Z score=1·95; p=0·03). 2604 unrelated individuals with Parkinson's disease and 344 matched controls were additionally genotyped, and five more people originating from five countries (Canada, Italy, Poland, Turkey, and Tunisia) were identified with the RAB32 variant. From the database searches, in which 6043 individuals with Parkinson's disease and 62 549 controls were included, another eight individuals were identified with the RAB32 variant from four countries (Canada, Germany, UK, and USA). Overall, the association of RAB32 c.213C>G (Ser71Arg) with Parkinson's disease was significant (odds ratio [OR] 13·17, 95% CI 2·15–87·23; p=0·0055; I 2 =99·96%). In the people who had the variant, Parkinson's disease presented at age 54·6 years (SD 12·75, range 31–81, n=16), and two-thirds had a family history of parkinsonism. RAB32 Ser71Arg heterozygotes shared a common haplotype, although penetrance was incomplete. Findings in one individual at autopsy showed sparse neurofibrillary tangle pathology in the midbrain and thalamus, without Lewy body pathology. In functional studies, RAB32 Arg71 activated LRRK2 kinase to a level greater than RAB32 Ser71. Interpretation RAB32 Ser71Arg is a novel genetic risk factor for Parkinson's disease, with reduced penetrance. The variant was found in individuals with Parkinson's disease from multiple ethnic groups, with the same haplotype. In-vitro assays show that RAB32 Arg71 activates LRRK2 kinase, which indicates that genetically distinct causes of familial parkinsonism share the same mechanism. The discovery of RAB32 Ser71Arg also suggests several genetically inherited causes of Parkinson's disease originated to control intracellular immunity. This shared aetiology should be considered in future translational research, while the global epidemiology of RAB32 Ser71Arg needs to be assessed to inform genetic counselling. Funding National Institutes of Health, the Canada Excellence Research Chairs program, Aligning Science Across Parkinson's, the Michael J Fox Foundation for Parkinson's Research, and the UK Medical Research Council.

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Figures

Figure 1. RAB32 Ser71Arg co-segregates with Parkinson’s disease. A) Simplified pedigrees for three families presenting the RAB32 Ser71Arg mutation. Probands are represented by an arrow and a diagonal line indicates deceased subjects. Patients diagnosed with Parkinson’s disease have black filled symbols, patients with a grey filled symbols had tremors but no diagnosis. Heterozygote mutation carriers (M) and wild-type (wt) genotypes are indicated with corresponding age (* age at death) and age at onset of disease.
Figure 1. RAB32 Ser71Arg co-segregates with Parkinson’s disease. A) Simplified pedigrees for three families presenting the RAB32 Ser71Arg mutation. Probands are represented by an arrow and a diagonal line indicates deceased subjects. Patients diagnosed with Parkinson’s disease have black filled symbols, patients with a grey filled symbols had tremors but no diagnosis. Heterozygote mutation carriers (M) and wild-type (wt) genotypes are indicated with corresponding age (* age at death) and age at onset of disease.CC BY · PubMed Central
Figure 2. Overview of the AlphaFold model of Rab32 (Ser71Arg) and LRRK2(350–550) protein complex (Rab32 Arg71 marked in red) (A), with detailed view of electrostatic (B) and hydrophobic (C) interactions predicted to be crucial for the protein-protein binding. (D) Overlay of 2 different rotamers of the Rab32 Arg71 residue, showing the rotamer (in brown) with a possible electrostatic interaction between Arg71 and LRRK2 Asp478. All models showed a binding interface consistent with interfaces identified for RAB29 and RAB38, with a hydrophobic patch formed by Met402, Leu403, Leu406 and Met407 of LRRK2, interacting with Phe62, Val60, Trp80 and Val94 of RAB32 (B). Electrostatic interactions (C) were predicted to form between the following pairs of residues (LRRK2 – RAB32): Arg399 – Asp61, Lys451 – Glu98 and Asp478 – Arg76, with additional salt bridges predicted between Arg361 – Glu86, Asp392 – Arg55 and Lys439 – Asp61.
Figure 2. Overview of the AlphaFold model of Rab32 (Ser71Arg) and LRRK2(350–550) protein complex (Rab32 Arg71 marked in red) (A), with detailed view of electrostatic (B) and hydrophobic (C) interactions predicted to be crucial for the protein-protein binding. (D) Overlay of 2 different rotamers of the Rab32 Arg71 residue, showing the rotamer (in brown) with a possible electrostatic interaction between Arg71 and LRRK2 Asp478. All models showed a binding interface consistent with interfaces identified for RAB29 and RAB38, with a hydrophobic patch formed by Met402, Leu403, Leu406 and Met407 of LRRK2, interacting with Phe62, Val60, Trp80 and Val94 of RAB32 (B). Electrostatic interactions (C) were predicted to form between the following pairs of residues (LRRK2 – RAB32): Arg399 – Asp61, Lys451 – Glu98 and Asp478 – Arg76, with additional salt bridges predicted between Arg361 – Glu86, Asp392 – Arg55 and Lys439 – Asp61.CC BY · PubMed Central
Figure 3: Quantitative immunoblotting analysis of the cellular kinase activity of wild-type LRRK2 in the presence of Rab32 (Ser71 wild-type or Arg71 mutant) or Rab29. HEK293 cells were co-transfected with wild-type LRRK2 and either HA-empty vector (−) or the indicated variants of HA-tagged Rab32 or HA-tagged Rab29, and treated +/− MLi-2 (200 nM, 2 hrs) or DMSO control (0.1% v/v) prior to lysis. Quantitation of phosphorylated Rab10-T73, phosphorylated LRRK2-S1292 and phosphorylated LRRK2-S935 normalized to total Rab10 and total LRRK2 respectively is shown. Error bars indicate mean with SD from two independent experiments, each performed in duplicate.
Figure 3: Quantitative immunoblotting analysis of the cellular kinase activity of wild-type LRRK2 in the presence of Rab32 (Ser71 wild-type or Arg71 mutant) or Rab29. HEK293 cells were co-transfected with wild-type LRRK2 and either HA-empty vector (−) or the indicated variants of HA-tagged Rab32 or HA-tagged Rab29, and treated +/− MLi-2 (200 nM, 2 hrs) or DMSO control (0.1% v/v) prior to lysis. Quantitation of phosphorylated Rab10-T73, phosphorylated LRRK2-S1292 and phosphorylated LRRK2-S935 normalized to total Rab10 and total LRRK2 respectively is shown. Error bars indicate mean with SD from two independent experiments, each performed in duplicate.CC BY · PubMed Central

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Authors
Gustavsson, E. K., Follett, J., Trinh, J., Barodia, S. K., Real, R., Liu, Z., Grant-Peters, M., Fox, J. D., Appel-Cresswell, S., Stoessl, A. J., Rajput, A., Rajput, A. H., Auer, R., Tilney, R., Sturm, M., Haack, T. B., Lesage, S., Tesson, C., Brice, A., Vilariño-Güell, C., Ryten, M., Goldberg, M. S., West, A. B., Hu, M. T., Morris, H. R., Sharma, M., Gan-Or, Z., Samanci, B., Lis, P., Periñan, M. T., Amouri, R., Ben Sassi, S., Hentati, F., Tonelli, F., Alessi, D. R., & Farrer, M. J.
Affiliations
  • University of British Columbia (CA)
  • University of Alabama at Birmingham (US)
  • Aligning Science Across Parkinson's (US)
  • Saskatchewan Health Authority (CA)
  • UCL Queen Square Institute of Neurology (GB)
  • University of Tübingen (DE)
  • Centre National de la Recherche Scientifique (FR)
  • UCL Biomedical Research Centre (GB)
  • Duke University (US)
  • University of Oxford (GB)
  • Hôpital La Rabta (TN)
  • University of Dundee (GB)
Corresponding author
Farrer, Matthew J
ORCID
Venue
The Lancet Neurology
Publisher
Elsevier BV
Published
2024-06
Volume
23
Issue
6
Pages
603-614
ISSN
1474-4422 (ISSN-L)
DOI
10.1016/s1474-4422(24)00121-2
PMC
PMC10827257
OpenAlex
W4394695501
Cited by
114
References
42
Citations by year
2026: 28 · 2025: 51 · 2024: 35
FWCI
30.50
Type
journal article
Language
en
Open access
Licence
Locations
  • The Lancet Neurology (publishedVersion, cc-by)
  • medRxiv (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • UCL Discovery (University College London) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
Funders
  • Canadian Institutes of Health Research
  • UKRI Medical Research Council
  • Aligning Science Across Parkinson's
Topics
  • Health Sciences › Medicine › Clinical Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Receptors and Signaling
  • Health Sciences › Medicine › Clinical Neurology › Deep Brain Stimulation and Movement Disorders
Keywords
Parkinson's disease, Rab32, Rab GTPases, autosomal dominant inheritance, familial Parkinson's disease, genetic linkage, genetic association, LRRK2, haplotype analysis, protein-protein interaction, genetic risk factors, exome sequencing, case-control association, haplotype phasing

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