Centre for Genetic Disorders, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Department of Neurosurgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Indian J Med Res. 2020 Nov;152(5):498-507. doi: 10.4103/ijmr.IJMR_730_18.
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.
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).
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.
帕金森病（PD）是一种影响运动的运动障碍。已经确定了 24 个以上的基因座和 28 个相关基因与这种疾病有关。本研究说明了两个候选基因亮氨酸丰富重复激酶 2（LRRK2）和 parkin RBR E3 泛素蛋白连接酶（PRKN）在 PD 患者中的作用，并对其进行了计算机模拟和体外特征分析。
共纳入 145 例散发性 PD 病例和 120 例种族匹配的健康对照者，并获得其知情同意。通过直接对 LRRK2 的靶向外显子和 PRKN 的所有侧翼内含子的 DNA 进行测序，对致病性 PRKN 变体进行突变筛选。通过荧光染料四甲基罗丹明甲酯（TMRM）测量致病性 PRKN 变体对药物（MG-132）诱导的线粒体膜电位（△ΨM）丧失的影响。
在 LRRK2 和 PRKN 中分别鉴定出 12 种和 20 种遗传变异。有趣的是，7 种外显子 LRRK2 变异中有 5 种是同义的。在对照者中的进一步评估证实了两种如此的 p.Y1527 和 p.V1615 非常罕见。在 PRKN 中的致病性错义变异（如计算机模拟预测）中，选择了两种（p.R42H 和 p.A82E）进行体外功能研究，结果显示与野生型相比，TMRM 的荧光强度降低，而另一种则没有。
在所研究的患者队列中，约 6.2%的病例（9/145）发现杂合状态下存在致病性（如计算机模拟预测）错义变异，但 LRRK2 则很少见。LRRK2 的两种罕见同义变异（p.Y1527 和 p.V1615）的存在可能支持密码子偏倚现象。所选 PRKN 变异的功能特征表明，p.R42H 导致线粒体膜电位（△ΨM）破坏，使细胞更容易受到细胞应激的影响。
