脆性X综合征FMRl基因CpG岛的甲基化程度分析

Methylation analysis of CpG island DNA of FMR1 gene in the fragile X syndrome

目的建立用甲基化敏感性限制性内切酶定量聚合酶链反应（methylation-sensitiverestrictionenzymes-basedquantitativePCR，MSRE-qPCR）分析FMRl基因CpG岛甲基化程度的方法，并探讨其对脆性X综合征的诊断价值。方法以常规PCR初筛存在FMRl基因5。（CGG）n异常扩增的30例智力低下男童和20名母亲作为研究对象，用EagI酶消化DNA样品，针对FMRl基因CpG岛设计引物，定量PCR扩增EagI酶切前、后DNA，用2△△Ct法计算CpG岛甲基化程度；以Southern印迹杂交确诊的3例患儿和正常体检男、女各30例DNA样品为质控样本，从而建立优化的MSRE-qPCR方法。结果确立了正常甲基化、部分异常甲基化、全甲基化的区间值，并明确30例常规PCR初筛异常患儿中3例存在部分甲基化，27例为全甲基化，其中3例经Southern印迹杂交验证；13例母亲处于正常甲基化，7例存在异常甲基化。结论MSRE-qPCR可以对FMRl基因CpG岛的甲基化程度进行快速可靠分析，为脆性x综合征的分子诊断提供新的策略。

Objective To establish a method of methylation-sensitive restriction enzymes-based quantitative PCR （MSRE-qPCR） for analysis of CpG island DNA ofFMR1 gene, and to assess its value for molecular diagnosis of fragile X syndrome. Methods Thirty boys with mental retardation and abnormat repeats of 5＇ （CGG）n in the FMR1 gene and 20 mothers were analyzed by conventional PCR screening. Eag I was used to digest genomic DNA, and qPCR was performed to amplify CpG island in the FMR1 gene using both undigested and digested templates. Raw Ct values were obtained through quantitative PCR amplification. The degree of CpG island methylation was calculated by 2 zxzxc,. The result of MSRE-qPCR was verified by Southern blotting. 30 healthy females and 30 healthy males were used as controls to optimize the established MSRE-qPCR method. Results The ranges of 2 zx/,c, value for normal methylation, partial methylation and full methylation were determined. Among the 30 patients, 3 were found to have partial methylation of CpG island of the FMR1 gene, and 27 were found to have full methylation （3/30 results were verified by Southern blotting）. Only 7 mothers were found abnormal methylation of CpG island of FMR1 gene, whilst the remaining 13 mothers were normal.. Conclusion MSRE-qPCR is a quick and reliable method for quantitative analysis of CpG island methylation status in FMR1 gene, which may provide a new strategy for the diagnosis of fragile X syndrome.