Zhou Youyou, Lum Josephine M S, Yeo Gare-Hoon, Kiing Jennifer, Tay Stacey K H, Chong Samuel S
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, and Children's Medical Institute, National University Hospital, Singapore.
Clin Chem. 2006 Aug;52(8):1492-500. doi: 10.1373/clinchem.2006.068593. Epub 2006 Jun 22.
Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is most commonly related to hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene. Southern blot analysis is the most commonly used method for molecular diagnosis of FXS. We describe a simplified strategy based on fluorescent methylation-specific PCR (ms-PCR) and GeneScan analysis for molecular diagnosis of fragile X syndrome.
We used sodium bisulfite treatment to selectively modify genomic DNA from fragile X and normal lymphoblastoid cell lines and from patients. We then performed ms-PCR amplification using fluorescently-labeled primers complementary to modified methylated or unmethylated DNA. Amplification products were resolved by capillary electrophoresis. FMR1 mutational status was determined by a combination of fluorescent peak sizes and patterns on the GeneScan electropherogram.
DNA samples from male and female persons with known NL, PM, and FM FMR1 CGG repeats were analyzed. Each FMR1 genotype produced a unique GeneScan electropherogram pattern, thus providing a way to identify the various disease states. The number of CGG repeats in all NL and PM alleles were determined accurately. Analysis by both the new assay and Southern blot of a family segregating with FXS showed complete concordance between both methods.
This simplified molecular diagnostic test, based on fluorescent methylation-specific PCR, may be a suitable alternative or complement to Southern blot analysis for the diagnosis of FXS.
脆性X综合征（FXS）是遗传性智力障碍最常见的病因，最常与FMR1基因5'非翻译区多态性CGG三核苷酸重复序列的过度扩增和高甲基化有关。Southern印迹分析是FXS分子诊断最常用的方法。我们描述了一种基于荧光甲基化特异性PCR（ms-PCR）和基因扫描分析的简化策略，用于脆性X综合征的分子诊断。
我们使用亚硫酸氢钠处理，选择性地修饰来自脆性X和正常淋巴母细胞系以及患者的基因组DNA。然后，我们使用与修饰的甲基化或未甲基化DNA互补的荧光标记引物进行ms-PCR扩增。扩增产物通过毛细管电泳进行分离。FMR1突变状态通过基因扫描电泳图上荧光峰大小和模式的组合来确定。
分析了已知具有正常长度（NL）、前突变（PM）和全突变（FM）FMR1 CGG重复序列的男性和女性的DNA样本。每种FMR1基因型都产生了独特的基因扫描电泳图模式，从而提供了一种识别各种疾病状态的方法。所有NL和PM等位基因中CGG重复序列的数量都被准确确定。对一个与FXS共分离的家系进行新检测和Southern印迹分析，结果显示两种方法完全一致。
这种基于荧光甲基化特异性PCR的简化分子诊断试验，可能是Southern印迹分析用于FXS诊断的合适替代方法或补充方法。
