Halder Tamali, Raj Janak, Sharma Vivek, Das Parimal
Centre for Genetic Disorders, Faculty of Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India.
Department of Neurosurgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India.
Neurosci Lett. 2015 Sep 25;605:29-33. doi: 10.1016/j.neulet.2015.08.021. Epub 2015 Aug 14.
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.
已知PINK1功能丧失突变会导致常染色体隐性早发性帕金森症，约占帕金森病（PD）病例的6.5%。最近，在印度东部也发现PINK1会引发帕金森病（PD）。本研究旨在探究其在印度北部PD患者中的作用。该研究共纳入了106名PD患者和60名种族匹配的健康对照。通过对覆盖所有外显子和外显子 - 内含子边界的PCR扩增子进行直接DNA序列分析，对所有患者进行PINK1突变筛查。通过对10个随机选择的包含变异扩增子的TA克隆进行DNA测序，再次确认了所鉴定的新变异。通过用与N'末端GFP融合的PINK1野生型和突变型（通过定点诱变创建）cDNA构建体转染COS - 7细胞系，然后进行蛋白质印迹分析，对突变蛋白进行体外功能测定。鉴定出两个潜在致病性变异，其中一个是新变异（p.Q267X），另外6个为明显非致病性变异。蛋白质印迹分析显示，在p.Q267X突变体中产生了约55kDa的截短型PINK1融合蛋白，而野生型PINK1融合蛋白的分子量为82/93kDa（GFP的分子量约为27kDa）。我们的研究得出结论，从印度北部人群的PD患者中1.8%（2/106）的发生率可以看出，PINK1变异在印度引发帕金森病（PD）中很常见。本文报道的PINK1新的纯合变异（c.799C>T）可能是该患者疾病表现的原因。然而，未来的研究将有助于了解这种过早的PINK1蛋白（p.Q267X）如何应对细胞应激从而导致PD病理生理学的功能机制。
