Rare variants in LRRK1 and Parkinson's disease

Abstract

Approximately 20 % of individuals with Parkinson's disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset PD to identify 15 potentially causal variants. Segregation analysis and frequency assessment in 862 PD cases and 1,014 ethnically matched controls highlighted variants in EEF1D and LRRK1 as the best candidates. Mutation screening of the coding regions of these genes in 862 cases and 1,014 controls revealed several novel non-synonymous variants in both genes in cases and controls. An in silico multi-model bioinformatics analysis was used to prioritize identified variants in LRRK1 for functional follow-up. However, protein expression, subcellular localization, and cell viability were not affected by the identified variants. Although it has yet to be proven conclusively that variants in LRRK1 are indeed causative of PD, our data strengthen a possible role for LRRK1 in addition to LRRK2 in the genetic underpinnings of PD but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.

Fig. 1

Pedigree of family used for exome sequencing. Open symbols indicate unaffected family members; affected individuals are denoted by closed symbols. An arrow denotes the proband. Sex was obscured and birth order was altered to protect privacy. A diagonal line indicates a deceased individual

Fig. 2

Location of EEF1D and LRRK1 variants identified in variant screening in relation to known functional domains. An asterisk denotes the variant identified by exome sequencing Variants printed in blue and annotated above the gene were found in cases, variants in green and below the gene were found in controls

Fig. 3

Prediction of pathogenic potential of newly identified variants. a For each variant (colored lines) the predicted score s of an individual algorithm, its reliability r, and the transformed score p(s, r, c) are shown. Variants holding a predicted disease-causing potential (class = 1) were respectively marked with an asterisk. The diverse results among each single algorithm motivated the calculation of one combined score (Pscore), which was adjusted by additional structural analyses (Dscore) resulting in a mutation score (Mscore). The highest scoring variant is p.Tyr1410Asp (Mscore = 0.839), a variant only present in PD cases, followed by the LRRK2 equivalent of Gly2019Ser (Mscore = 0.771), the loss of autophosphorylation mutation Lys1270Trp (Mscore = 0.768), and two variants abolishing kinase activity: Ile1412Thr (Mscore = 0.728) and Lys746Gly (Mscore = 0.723). b Hierarchical clustering with Ward's minimum variance agglomeration method and Euclidean distance matrix shows that three of the novel variants which were only found in individuals with PD (p.Arg631Trp, p.Arg1271His, and p.Tyr1410Asp) cluster with the LRRK1 equivalents of LRRK2 p.Arg1441Gly, p.Tyr1699Cys, p.Gly2019Ser, and p.Ile2020Thr as well as the LRRK1 kinase- and GTP-binding dead amino acid substitutions

Fig. 4

Cellular expression of LRRK1 and mutant variants. a Western blot analysis of myc-tagged LRRK1 expression in SHSY5Y cells with beta actin-loading control. b Analysis of LRRK1 toxicity as measured by MTT assay in SHSY5Y cells. No significant toxicity was associated with wild-type LRRK1, artificial mutations in LRRK1, or disease-associated coding changes. Data is expressed as percentage of untransfected control cells, mean, and standard error measurement displayed. c Immunocytochemistry analysis of myc-tagged LRRK1 constructs. Staining for myc is shown separately and merged. All tagged constructs displayed a diffuse cytoplasmic staining pattern. Scale bar = 20 μm