Rare variants in LRRK1 and Parkinson's disease

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. Electronic supplementary material The online version of this article (doi:10.1007/s10048-013-0383-8) contains supplementary material, which is available to authorized users.


Description of case/control sample
All cases used in variant screening and genotyping were recruited at Paracelsus-Elena Klinik, a hospital specializing in Parkinson's disease (PD), in Kassel, Germany, as well as at the departments of neurology at Wilhelminenspital and Allgemeines Krankenhaus in Vienna, Austria. PD diagnosis was made in accordance with the UK Brain Bank Criteria. Controls belong to a large general population cohort (KORA) based in the region around Augsburg in Southern Germany and have been described previously. [1] KORA-AGE represents a subset of the KORA cohort collected in 2009 as a gender-and age-stratified subsample of the KORA S1-S4 studies comprising participants born before 1944. All individuals taking dopaminergic drugs were excluded from the control sample.

Multiple Sequence Alignment
A multiple sequence alignment was computed using ClustalW based on LRRK1/LRRK2 pairs from the following organisms: Homo sapiens (NP_078928. 3

Multi-model Ensemble
We implemented a multi-model ensemble of prediction algorithms (PolyPhen [2], PolyPhen-2 [3], Phd-SNP [4], SIFT [5], SNPs3D [6], MutationTaster [7] and Pmut [8], each contributing equally). Since each model provides different scoring schemes, their solution space was transformed by a function p computing the probability score of a variant to affect the function of the protein: (1) If a reliability value of the classification was denoted by the algorithm, it was considered in the transformation, otherwise r was set to 1. A low confidence converges the probability score to 0.5, e.g. a non-reliable prediction was scored as . The score distributions of each class were determined by means of an exhaustive set of predictions provided by the algorithms' databases. The probability scores of each algorithm were combined into a single score: (2) Structural Analysis of Mutation LRRK1 amino acid sequences containing annotated protein domains were folded by the multiple-threading approach I-TASSER [9] and the predicted tertiary structures with the highest confidence scores were selected. Mutations were mapped to the peptide structure with the SWISSPDB [10] viewer. Energy minimizations were performed by the NOMAD-Ref algorithm [11] with the conjugate gradient method for the wildtype and the variant structures.
We computed the root mean square deviation (RMSD) between the wildtype peptide and the variant peptide : Based on all variants considered, the RMSD was normalized for each functional domain m and a deviation score was calculated: (4)

Scaling of Prediction Values
To include tertiary structure information, we combined the PScore and the Dscore by means of a weighted harmonic mean to a mutation score: Since ab initio tertiary structure determination is rather inaccurate, we selected , thus giving a higher weight to the prediction ensemble.

Cellular analyses
Reported (p.Lys651Ala and p.Lys1270Trp) [  permeabilized at room temperature for 30 minutes by using a solution of 15% normal goat serum (S1000, Vector) and 0.1% Triton X-100 in DPBS. After washing, cells were incubated overnight at 4°C with the primary antibody. Anti-mouse, secondary antibody (A21124, Alexa Fluor, emission at 568 nm) was used to reveal the primary antibody staining and nuclei were labelled with a 0.05% solution of Hoechst in DPBS before the sealing the coverslips with Fluoromount G mounting medium (Southern Biotech). Images were acquired with a Leica DM5500 B fluorescence microscope.

ONLINE RESOURCES FIGURES
Online Resources Figure 1 Filtering scheme for variants identified by exome sequencing in the two affected family members examined.