Identification & characterization of leucine-rich repeat kinase 2 & parkin RBR E3 ubiquitin protein ligase variants in patients with Parkinson's disease.

BACKGROUND & OBJECTIVES: Parkinson's disease (PD) is a motor disorder that affects movement. More than 24 loci and 28 associated genes have been identified to be associated with this disease. The present study accounts for the contribution of two candidates, leucine-rich repeat kinase 2 ( LRRK2) and parkin RBR E3 ubiquitin protein ligase ( PRKN) in the PD patients, and their characterization in silico and in vitro. METHODS: A total of 145 sporadic PD cases and 120 ethnically matched healthy controls were enrolled with their informed consent. Mutation screening was performed by direct DNA sequencing of the targeted exons of LRRK2 and all exons flanking introns of PRKN. The effect of the pathogenic PRKN variants on a drug (MG-132) induced loss of mitochondrial membrane potential (△ΨM) was measured by a fluorescent dye tetramethylrhodamine methyl ester (TMRM). RESULTS: Twelve and 20 genetic variants were identified in LRRK2 and PRKN, respectively. Interestingly, five out of seven exonic LRRK2 variants were synonymous. Further assessment in controls confirmed the rarity of two such p.Y1527 and p.V1615. Among the pathogenic missense variations (as predicted in silico) in PRKN, two were selected (p.R42H and p.A82E) for their functional study in vitro, which revealed the reduced fluorescence intensity of TMRM as compared to wild type, in case of p.R42H but not the other. INTERPRETATION & CONCLUSIONS: About 6.2 per cent of the cases (9/145) in the studied patient cohort were found to carry pathogenic (as predicted in silico) missense variations in PRKN in heterozygous condition but not in case of LRRK2 which was rare. The presence of two rare synonymous variants of LRRK2 (p.Y1527 and p.V1615) may support the phenomenon of codon bias. Functional characterization of selected PRKN variations revealed p.R42H to cause disruption of mitochondrial membrane potential (△ΨM) rendering cells more susceptible to cellular stress.

Novel P-TEN-induced putative kinase 1 (PINK1) variant in Indian Parkinson's disease patient.

Loss-of-function mutation in PINK1 is known for causing autosomal recessive early onset Parkinsonism accounting approximately 6.5% of PD cases. Recently, PINK1 has also been shown to cause Parkinson's disease (PD) in eastern India. Present study is aimed to see its contribution in north-Indian PD patients. A total of 106 PD patients and 60 ethnically matched healthy controls were included in the study. All the patients were screened for mutation in PINK1 by direct DNA sequence analysis of the PCR amplicons covering all exons and exon-intron boundaries. Identified novel variant was reconfirmed by DNA sequencing of 10 randomly selected TA clones containing the variant amplicon. In vitro functional assay of the mutant protein was performed by transfecting COS-7 cell line with wild type and mutant (created by site-directed-mutagenesis) cDNA construct of PINK1 fused to N' terminal GFP followed by western blot analysis. Two potentially pathogenic, one being novel (p.Q267X) and 6 other apparently non-pathogenic variants were identified. Western blot analysis reveals production of truncated PINK1 fusion protein of ∼55kDa in p.Q267X mutant instead of 82/93kDa of wild type PINK1 fusion protein (molecular weight of GFP is ∼27kDa). Our study concludes that PINK1 variants are prevalent for causing Parkinson's disease (PD) in India, as revealed by the occurrence of 1.8% (2/106) in PD patients from north Indian population. The novel homozygous variant of PINK1 (c.799C>T) reported here is the plausible cause for disease manifestation in this patient. Future study, however, would be helpful to understand the functional mechanism how this premature PINK1 protein (p.Q267X) responds to cellular stress leading to the PD pathophysiology.