Methods

Genomic DNA was obtained from peripheral blood lymphocyte or saliva samples (Oragene^TM DNA Self-Collection Kit, DNA Genotek), by standard protocols. Patients with variants of the GBA risk factor (n = 153), or with variants of genes for which the causal role in AD PD was uncertain, such as GIGYF2 (n = 6), EIF4G1 (n = 2), and c9ORF72 (n = 4), were not included in this study. In addition, we excluded 25 (23 with bi-allelic PRKN mutations and two with bi-allelic PINK1 mutations) of the 1,089 PD index cases who have been screened for autosomal recessive (AR)-PD associated genes [814 by gene panel/exome sequencing (see below) and 275 by direct sequencing of the two most frequent AR PD genes, PRKN and PINK1].

All index cases were genotyped in duplicate for LRRK2 Gly2019ser, by the TaqMan allelic discrimination Assay-By-Design method, in accordance with the manufacturer's instructions, with 8 ng of DNA mixed with the TaqMan Genotyping Master Mix (Thermo Fisher Scientific Inc.) and custom-produced TaqMan SNP genotyping assays [C_63498123_10 (rs34637584), Thermo Fisher Scientific Inc.] on an Applied Biosystems PRISM 7000 sequence detection system (Thermo Fisher Scientific Inc.) or LightCycler® 480 machine (Roche, Life Technologies SAS). All patients found not to carry LRRK2 Gly2019ser were then screened for pathogenic variants of the coding sequences of LRRK2, SNCA, and VPS35, by Sanger sequencing (n = 855), targeted sequencing of a customized next-generation sequencing (NGS) gene panel containing the 22 most prevalent PD-associated genes (n = 404; Supplementary Table 1), or available whole-exome sequencing (n = 410), as previously described (11, 12). We considered known pathogenic mutations of the three genes.

Sanger sequencing was used to confirm variants and co-segregation analyses were performed, where possible. SNCA rearrangements were detected by semi-quantitative multiplex PCR (13) or by the SALSA multiplex ligation-dependent probe amplification method (MLPA, MRC Holland, Amsterdam, the Netherlands; http://www.mlpa.com), according to the manufacturer's instructions.