Schroeder Christopher, Walter Michael, Berg Daniela, Leitner Petra, Bauer Peter, Kohl Zacharias, Winkler Jürgen, Riess Olaf, Bonin Michael
Department of Medical Genetics, University of Tübingen, Tübingen, Germany.
J Mol Diagn. 2008 May;10(3):217-24. doi: 10.2353/jmoldx.2008.070100. Epub 2008 Apr 10.
To date, 11 gene loci that contribute to familial Parkinson's disease (PD) are known. Of these, mutations in six genes have been identified, allowing genetic testing and more accurate phenotypic characterization of genetically defined disease subtypes. In particular, mutations in Parkin, DJ-1, and Pink1 genes are associated with autosomal recessive PD and may also play a major role in early onset PD (EOPD). However, genetic testing for sequence alterations in these genes remains laborious. Therefore, our aim was to develop a flexible, rapid, high-throughput screening procedure using matrix-assisted laser desorption ionization/time of flight technology and homogeneous mass cleave assays. Using this novel approach, we screened all 27 coding exons of the Parkin, DJ-1, and Pink1 genes in 31 patients with EOPD, a total of 367,195 nucleotides. Four positive controls with known autosomal recessive PD mutations that had previously been screened by denaturing high performance liquid chromatography in combination with sequencing were also tested. All known alterations were detected by matrix-assisted laser desorption ionization/time of flight mass spectrometer, as well as additional polymorphisms in formerly unscreened regions. Overall, two previously described mutations in three patients with EOPD, 27 known polymorphisms with 386 occurrences, and eight unknown variants with 21 occurrences were detected. In total, we identified 410 sequence alterations in 31 patients with EOPD. In conclusion, this is the first study using matrix-assisted laser desorption ionization/time of flight mass spectrometry and homogeneous mass cleave assay for high-throughput mutation screening.
迄今为止，已知有11个基因位点与家族性帕金森病（PD）相关。其中，已鉴定出6个基因的突变，这使得基因检测以及对基因定义的疾病亚型进行更准确的表型特征分析成为可能。特别是，Parkin、DJ-1和Pink1基因的突变与常染色体隐性帕金森病相关，并且可能在早发性帕金森病（EOPD）中也起主要作用。然而，对这些基因中的序列改变进行基因检测仍然很费力。因此，我们的目标是开发一种灵活、快速、高通量的筛选程序，使用基质辅助激光解吸电离/飞行时间技术和均相质量裂解分析。使用这种新方法，我们对31例早发性帕金森病患者的Parkin、DJ-1和Pink1基因的所有27个编码外显子进行了筛选，共计367,195个核苷酸。还对4个已知常染色体隐性帕金森病突变的阳性对照进行了检测，这些对照先前已通过变性高效液相色谱结合测序进行了筛选。所有已知的改变都通过基质辅助激光解吸电离/飞行时间质谱仪检测到，以及在以前未筛选区域的额外多态性。总体而言，在3例早发性帕金森病患者中检测到2个先前描述的突变、27个已知多态性（共出现386次）以及8个未知变体（共出现21次）。我们总共在31例早发性帕金森病患者中鉴定出410个序列改变。总之，这是第一项使用基质辅助激光解吸电离/飞行时间质谱和均相质量裂解分析进行高通量突变筛选的研究。
