Pathogenic variants in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most common known monogenic cause of Parkinson's disease (PD). First identified in 2004, LRRK2 pathogenic variants account for up to ~40% of all PD cases in selected populations, e.g., North African Berber (1). In the PD phenotype-genotype database MDSGene (www.mdsgene.org), ~700 individual LRRK2 pathogenic variant carriers are listed, but clinical information apart from cardinal signs of PD is overall scarce (1).

The majority of the reported LRRK2 pathogenic variant-positive PD patients are of European descent (63%), whereas all other ethnicities comprise ~10% or fewer patients of described pathogenic variant carriers despite clusters in the Ashkenazi Jewish (AJ) and Arab Berber populations (1, 2). Definitely pathogenic variants identified in LRRK2 include p.G2019S, p.R1441C/G/H, p.N1437H, p.Y1699C, and, p.I2020T. Of these, the p.G2019S pathogenic variant is the most common with an estimated frequency of 1% in sporadic and 4% in hereditary PD cases worldwide (3). However, data on many populations are still missing.

LRRK2-linked PD is inherited in an autosomal dominant fashion with reduced, age-dependent penetrance and variable expressivity that differ across ethnicities and geographic regions, indicating that ancestral background or environmental factors contribute to the manifestation and expressivity of pathogenic LRRK2 variants. For example, at the age of 60 years, 60% of Tunisian p.G2019S pathogenic variant carriers manifest PD, compared with only 20% of the Norwegian carriers of the same pathogenic variant (4). Recent studies have suggested mitochondrial involvement in the penetrance of LRRK2-linked PD (5, 6). Environmental factors can also influence penetrance, as recently found in the Tunisian Arab Berber population, where tobacco and black tea use delayed age at onset in LRRK2 pathogenic variant carriers (7).

LRRK2-linked PD is clinically indistinguishable from idiopathic PD, i.e., PD of unknown cause, although the disease course appears to be milder with regard to motor and cognitive functions (8). In this comparatively small subgroup of PD patients with a known etiology, valuable clues can be garnered toward pathophysiological mechanisms and targeted treatments, which may be generalized to idiopathic PD. Currently, there are three substances in clinical trial specifically targeting LRRK2: the LRRK2 kinase inhibitors DNL151 (NCT04557800) and DNL201 (NCT04551534) from Denali and the antisense oligonucleotide BIIB094 (NCT03976349) from Biogen (2). However, no genetic testing guidelines and no clinical trial-ready cohorts exist to date.

To identify LRRK2 pathogenic variant carriers among 10,000 PD patients from 15 countries and to establish a clinical trial-ready cohort, the ROPAD study was initiated (9). The ROPAD study performs genetic testing of pathogenic variants in the LRRK2 and GBA genes and, if negative, carries out genetic screening of 68 genes linked to PD or related movement disorders. Out of the first 1,288 participants who underwent genetic testing in ROPAD, 40 (3.1%) harbored pathogenic or likely pathogenic LRRK2 variants, 109 (8.5%) harbored GBA variants, three (0.2%) had alterations in both genes (9). The ROPAD study is still ongoing and continues to identify LRRK2 pathogenic variant carriers internationally. It gathers only basic clinical data: age at examination, age at onset, cardinal PD symptoms, and 12/33 items of MDS-UPDRS Part III and, thus, does not allow a systematic in-depth characterization of LRRK2 pathogenic variant carriers.

Recently published data from the currently largest multinational prospective study that includes LRRK2 pathogenic variant carriers, i.e., the Parkinson's Progression Markers Initiative (PPMI), provide more phenotypic details on LRRK2 pathogenic variant carriers across international sites (6). However, as the focus of the PPMI study is predominantly on biomarkers, it does not cover environmental factors in-depth, and only includes participants with a PD diagnosis 2 years or less before enrolment who are untreated with PD medication. There remains a gap in combining phenotypic, genetic, and environmental data on LRRK2 pathogenic variant carriers internationally, regardless of the onset of signs and symptoms and their medication status, that LIPAD is aiming to close. The objectives of the LIPAD study are (1) to provide a systematic characterization of PD patients and unaffected carriers with pathogenic variants in the LRRK2 gene; and (2) to enable the investigation of modifiers of penetrance of LRRK2 pathogenic variants using genetic and environmental data.

Here, we describe a protocol and a feasibility study of the first 150 participants and give a brief overview of nested studies within LIPAD. Of note, the LIPAD protocol has also been adapted to take place online due to COVID19 contact restriction measures.

A comprehensive eCRF has been developed, consisting of an investigator-rated (1 h) and a self-rated (1.5-h) part. The questionnaires are listed in Table 1. We used validated versions of questionnaires and scales in the local languages.

The first 10 centers across Germany and five international sites have been initiated with further centers currently undergoing ethical review (Figure 2). The first 150 participants have been enrolled (as of March 25th, 2021); Table 2.

LIPAD is a large-scale international scientific effort focusing on deep phenotyping of LRRK2-linked PD and healthy pathogenic variant carriers, including a comparison with additional relatively frequent genetic forms of PD, and with a future perspective to identify genetic and environmental modifiers of penetrance and expressivity.

The clinical assessment in LIPAD is close to the one used in the currently largest multinational cohort of genetic PD (PPMI), except for lack of collection of cerebrospinal fluid, but with the addition of an extensive questionnaire on environmental factors (15). Further, there are no restrictions on the age at diagnosis or medication for inclusion in the LIPAD study. Several other multinational prospective studies focusing on pathogenic LRRK2 variants included only a small number of populations (4, 16, 17), revealing an important knowledge gap in multinational, standardized, genetic prospective studies, which LIPAD is aiming to close.

A comprehensive investigation of modifiers of penetrance and expressivity requires assessment of a considerable number of pathogenic variant carriers. Due to the direct enrolment of family members at an international scale, the LIPAD study will enable the analysis of modifiers of penetrance in different populations in one setting. Our recruitment strategy, mainly through the international multicenter ROPAD study, will ensure participation of a large number of LRRK2+/PD+ patients.

With the nested studies, which will be conducted on a subset of participants, we are aiming to provide a more in-depth phenotypic characterization especially of non-affected carriers of LRRK2 pathogenic variants.

Currently, a complication affecting many patient studies across the world are measures imposed on personal visits to contain SARS-CoV-2 in many countries, limiting the possibility for the collection of data and biomaterials. To allow the study to proceed under these circumstances, we adjusted the protocol and procedures to collect the necessary data online. After making an online appointment, patients receive a package with patient information, informed consent forms, and a smell test by post. Then an online appointment with the study team takes place, during which the study is explained, the data is collected and the examination is performed. Study participants send signed informed consent forms and a household dust sample back to the study center by post. Biomaterials will be taken at a general practitioner's office or the study center at a later time point when study visits become possible again. The study will revert to the on-site recruitment as soon as possible after the pandemic restrictions are lifted.

LIPAD aims for a longitudinal follow-up of its study participants over a 10–15-year time period. The exact protocol is currently being developed and potential funding opportunities are being explored.

The studies involving human participants were reviewed and approved by Ethics Committee (EC) of the University of Lübeck (19-065); EC LÄK Hessen (2019-1364-zvBO); EC LÄK Hamburg (MC-002/20); EC LÄK Brandenburg [AS 35(bB)/2020]; EC Würzburg (161/19_z-sc); EC Kiel (B 292/19); EC Marburg (111/20); EC UK IRAS (project ID: 275553, REC reference: 20/NE/011); EC USA IRB tracking number: 20193494; Pavia, Italy (20200048346); Reggio Emilia, Italy (1268/2020/OSS/AUSLRE); Istanbul, Turkey (2020.362.IRB1.144). The patients/participants provided their written informed consent to participate in this study.

Daniela Berg, University Hospital Schleswig-Holstein, Kiel, Germany; Leonor Correia Guedes, Hospital de Santa Maria, Lisbon, Portugal; Ilona Csoti, Natalia Koleva-Alazeh, Gertrudis-Kliniken im Parkinson-Zentrum, Leun-Biskirchen, Germany; Georg Ebersbach, Doreen Gruber, Neurologisches Fachkrankenhaus für Bewegungsstörungen/Parkinson, Beelitz-Heilstätten, Germany; Sibel Ertan, Özgür Öztop Çakmak, Department of Neurology, Koç University School of Medicine, Istanbul, Turkey; Jon Infante, University Hospital Marqués de Valdecilla, Santander, Spain; Stuart Isaacson, Parkinson's Disease and Movement Disorder Center of Boca Raton, Boca Raton, U.S.A.; Christine Klein, Universität zu Lübeck, Lübeck, Germany; Andrea A. Kühn, Friederike Borngräber, Charité University Medicine Berlin, Berlin, Germany; Marcello Merello, Malco Rossi, FLENI Foundation, Buenos Aires, Argentina; Brit Mollenhauer, Paracelsus-Elena-Klinik, Kassel, Germany; David J. Pedrosa, University Hospital Marburg, Marburg, Germany; Kathrin Reetz, University Hospital RWTH Aachen, Aachen, Germany; Esther Sammler, University of Dundee, Dundee, Scotland, UK; Enza Maria Valente, Department of Molecular Medicine, University of Pavia and IRCCS Mondino Foundation, Pavia, Italy; Micol Avenali, IRCCS Mondino Foundation, Neurorehabilitation Unit, Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy; Franco Valzania, Giulia Toschi, Francesco Cavallieri, Azienda USL – IRCCS, Reggio Emilia, Italy; Jens Volkmann, University Hospital Würzburg, Würzburg, Germany; Simone Zittel, Lisa Prilop, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

TU: organization and execution of the research project, design and execution of statistical analysis, and writing the first draft of the manuscript. E-JV and AR: conception and organization of the research project, review and critique of statistical analysis, and review and critique of the manuscript. NS and SS: organization and execution of the research project, review and critique of statistical analysis, and review and critique of the manuscript. VS, XB, HG, TF, AH, NK-A, IC, DG, ESa, EV, FV, SZ, AB, SE, and GG: organization of the research project, review and critique of statistical analysis, and review and critique of the manuscript. PB, AG, ESc, JT, DB, SI, AK, BM, DP, and JV: review and critique of statistical analysis and review and critique of the manuscript. KL: organization of the research project and review and critique of the manuscript. NB, MK, and CK: conception and organization of the research project, design and review and critique of statistical analysis, and review and critique of the manuscript. All authors contributed to the article and approved the submitted version.

VS, XB, HG, TF, AH, PB, and AR were employed by company CENTOGENE GmbH. The authors declare that this study received funding from Centogene GmbH. The funder was involved in the study design, organization of the research project and review and critique of the manuscript.

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