The New p.F1700L LRRK2 Variant Causes Parkinson's Disease by Extensively Increasing Kinase Activity (2023)

Variants in LRRK2 represent the most frequent cause of clinically classical monogenic Parkinson's disease (PD).1 Altered LRRK2 protein function boosts neuroinflammation, impairs vesicle trafficking, and affects ciliogenesis within the striatum.2 Because a relevant fraction of the more than 1000 identified LRRK2 variants3 is not pathogenic, determining pathogenicity for single variants is crucial, particularly because LRRK2 kinase inhibitors have entered phase 3 trials.4 Notably, we have already established an analytic workflow to determine kinase activity and decipher the pathogenicity of single LRRK2 variants in vitro5 and in vivo.6 In this letter, we report on a 74-year-old male patient from northern Germany with advanced typical PD (Unified Parkinson's Disease Rating Scale Part III: 33/108 points, Hoehn and Yahr stage 3–4) without relevant tremor. The age at onset was 67 years. The disease course was slowly progressive, he experienced a good response to dopaminergic therapy, and dementia was absent. Family history was suggestive of autosomal dominant inheritance, with the father and two brothers also diagnosed with PD. However, the father was already deceased, one brother was not available for examination, and the second brother died early after giving blood for genetic investigation. Both brothers carried a new p.F1700L (NM_198578.4: c.5098T>C) variant in LRRK2, initially detected by gene panel analysis in the deceased brother and further investigated by Sanger sequencing. The variant is rated as a variant of uncertain significance according to the criteria of the American College of Medical Genetics (assessed by Franklin: https://franklin.genoox.com/) and is not listed in gnomAD (https://gnomad.broadinstitute.org/). In silico prediction suggested pathogenicity based on a Combined Annotation Dependent Depletion (CADD) score (https://cadd.gs.washington.edu/) of 27.3. The variant is located within the C terminus of the Ras of complex protein B scaffolding domain.5 We investigated LRRK2 kinase pathway activity in a heterologous transient overexpression system in HEK293 cells as described previously5 (Fig. 1A). We then analyzed LRRK2 activity in vivo in fresh peripheral blood, simultaneously collected from a p.F1700L carrier and a sex-matched healthy control subject (Fig. 1B). The resulting LRRK2-dependent pRab10Thr73 phosphorylation level mirrors LRRK2 kinase activation status.6 Both experiments demonstrated significant LRRK2 kinase hyperactivation because of the p.F1700L variant. Notably, p.F1700L demonstrated LRRK2 kinase hyperactivation similar to the neighboring p.Y1699C variant, which has the highest degree of kinase activity among all LRRK2 variants investigated thus far in the HEK293 assay (Fig. 1A). Moreover, we found a 7- to 8-fold increase in Rab10 phosphorylation levels in vivo (Fig. 1B). Together, we provide robust evidence for the pathogenicity of the newly identified p.F1700L variant in LRRK2, demonstrating that this substitution is among the variants with the highest kinase activity of all LRRK2 variants investigated to date and impacts protein function more profoundly than, for example, the frequent p.G2019S variant.5, 6 Thus, carriers of a p.F1700L variant should be included in clinical trials targeting LRRK2 kinase activity already initiated or soon commencing.4 Finally, we confirmed the applicability and usefulness of the applied assays to determine the pathogenicity of LRRK2 variants of uncertain significance. Further studies should focus on an association between the degree of kinase activity and penetrance, disease onset, and disease severity. We thank the participants for agreeing to undergo the examination for research purposes. Moreover, we thank the technical support of the MRC Protein Phosphorylation and Ubiquitylation Unit Reagents and Services, as well as the tissue culture team. This study was approved by the ethics committee of the University of Lübeck and performed according to the Declaration of Helsinki. (1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the first draft, B. Review and Critique. M.B.: 1A, 1B, 1C, 2C, 3A, 3B. N.P.: 1B, 1C, 2A, 2B, 3B. S.S.: 1B, 1C, 3B. F.H.: 1B, 1C, 3B. G.M.: 1B, 1C, 3B. J.U.: 1B, 1C, 3B. K.L.: 1A, 1B, 1C, 3B. C.K.: 1A, 1B, 1C, 3B. D.R.A.: 1A, 1B, 1C, 3B. J.H.: 1A, 1B, 1C, 3B. E.S.: 1A, 1B, 1C, 2A, 2B, 3A, 3B. Max Borsche: Employment, University of Lübeck and University Hospital Schleswig-Holstein, Campus Lübeck; Honoraria, Ipsen Pharma; Grants, German Research Foundation (DFG)-funded Clinician Scientist School, University of Lübeck. Neringa Pratuseviciute: Employment, Carnegie Trust–funded PhD student, University of Dundee. Susen Schaake: Employment, University of Lübeck and University Hospital Schleswig-Holstein, Campus Lübeck. Frauke Hinrichs: Employment, University of Lübeck and University Hospital Schleswig-Holstein, Campus Lübeck. Gabriel Morel: Employment, Undergraduate placement student at University of Dundee. Jan Uter: Employment, University of Lübeck and University Hospital Schleswig-Holstein, Campus Lübeck. Katja Lohmann: Employment, University of Lübeck; Grants, German Research Foundation (DFG), Parkinson's foundation, Damp Foundation, The Michael J. Fox Foundation (MJFF; GP2 project). Christine Klein: Consultancies, Centogene, Retromer Therapeutics; Employment, University of Lübeck and University Hospital Schleswig-Holstein, Campus Lübeck; Honoraria, Desitin and Bial; Royalties, Oxford University Press; Grants, DFG, MJFF, Aligning Science Across Parkinson's. Dario R. Alessi: Employment, University of Dundee; Grants, Medical Research Council UK. Johann Hagenah: Employment, Westküstenklinikum Heide. Esther Sammler: Employment, University of Dundee, UK; Honoraria, Bial, MJFF; Grants, MJFF, Chief Scientist Office, Scotland, UK (personal fellowship). The data presented in this study can be received from the corresponding author upon reasonable request.