Straniero Letizia, Rimoldi Valeria, Melistaccio Giada, Di Fonzo Alessio, Pezzoli Gianni, Duga Stefano, Asselta Rosanna
Department of Biomedical Sciences, Humanitas University, Via Rita Levi Montalcini 4, 20090, Pieve Emanuele, Milan, Italy.
IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, Dino Ferrari Center, Neuroscience Section, Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.
Parkinsonism Relat Disord. 2020 Nov;80:138-141. doi: 10.1016/j.parkreldis.2020.09.036. Epub 2020 Sep 22.
Deleterious variants in the GBA gene confer a 2- to 20-fold increased risk of Parkinson's disease (PD) and are associated with a more severe disease course. The presence of a highly-similar pseudogene complicates genetic screening, both by Sanger and next-generation sequencing (NGS). Among PD-associated GBA variants, four missense substitutions (p.L444P, p.N370S, p.T369M, p.E326K) account for the majority of cases. Here, we aimed at developing an allele-specific PCR (AS-PCR) able to concomitantly detect the most common PD-related GBA variants.
A multiplex PCR assay was designed using allele-specific oligonucleotides that distinguish the gene from the pseudogene and can exclusively amplify the variant alleles. Primer sequences and molarity, and thermal profiles were empirically optimized. The assay was validated on 4016 DNAs extracted by standard salting-out and previously analyzed by whole-exome sequencing (WES) followed by Sanger validation.
AS-PCRs performed on 4016 DNAs detected 103 variants; among them, 97 were true positives and 6 false positives. When comparing the results with the original WES data, for the "difficult" p.L444P, the number of false positives was 2/9 and 18/24 for multiplex-AS-PCR and WES, respectively. As we could have missed some p.L444P alleles by NGS, we verified the test performance on 50 DNAs from Sanger-validated p.L444P heterozygotes. All samples tested correctly.
We set up and validated a rapid and inexpensive test for screening large cohorts of individuals for variants conferring a significant PD risk. This screening method is particularly interesting to identify patients who could benefit most from early access to personalized therapies.
GBA基因中的有害变异会使帕金森病（PD）的发病风险增加2至20倍，并与更严重的病程相关。一个高度相似的假基因的存在使通过桑格测序和下一代测序（NGS）进行的基因筛查变得复杂。在与PD相关的GBA变异中，四个错义替换（p.L444P、p.N370S、p.T369M、p.E326K）占了大多数病例。在此，我们旨在开发一种等位基因特异性PCR（AS-PCR），能够同时检测最常见的与PD相关的GBA变异。
设计了一种多重PCR检测方法，使用等位基因特异性寡核苷酸来区分该基因与假基因，并能专门扩增变异等位基因。通过实验优化了引物序列、摩尔浓度和热循环参数。该检测方法在通过标准盐析法提取并先前经全外显子测序（WES）分析后再经桑格测序验证的4016份DNA上进行了验证。
对4016份DNA进行的AS-PCR检测到103个变异；其中，97个为真阳性，6个为假阳性。将结果与原始WES数据进行比较时，对于“难检测的”p.L444P，多重AS-PCR和WES的假阳性数量分别为2/9和18/24。由于我们可能通过NGS遗漏了一些p.L444P等位基因，我们在来自经桑格测序验证的p.L444P杂合子的50份DNA上验证了检测性能。所有测试样本结果均正确。
我们建立并验证了一种快速且廉价的检测方法，用于筛查大量个体中赋予显著PD风险的变异。这种筛查方法对于识别那些可能从早期获得个性化治疗中获益最大的患者特别有意义。
