Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia;
Center for Molecular Studies, J.C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC;
Clin Chem. 2014 Jul;60(7):963-73. doi: 10.1373/clinchem.2013.217331. Epub 2014 Apr 28.
Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1) CpG island 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females.
We describe methylation specific-quantitative melt analysis (MS-QMA) that targets 10 CpG sites, with 9 within FMR1 intron 1, to screen for FXS from birth in both sexes. The novel method combines the qualitative strengths of high-resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. Its performance was assessed in 312 control (CGG <40), 143 premutation (PM) (CGG 56-170), 197 full mutation (FM) (CGG 200-2000), and 33 CGG size and methylation mosaic samples.
In male and female newborn blood spots, MS-QMA differentiated FM from control alleles, with sensitivity, specificity, and positive and negative predictive values between 92% and 100%. In venous blood of FM females between 6 and 35 years of age, MS-QMA correlated most strongly with verbal IQ impairment (P = 0.002). In the larger cohort of males and females, MS-QMA correlated with reference methods Southern blot and MALDI-TOF mass spectrometry (P < 0.05), but was not significantly correlated with age. Unmethylated alleles in high-functioning FM and PM males determined by both reference methods were also unmethylated by MS-QMA.
MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes.
标准脆性 X 综合征（FXS）诊断测试针对脆性 X 智力低下 1 号（FMR1）CGG 扩展 5'的 CpG 岛的甲基化，可以用于从出生开始预测男性疾病的严重程度，但不能用于女性。
我们描述了甲基化特异性定量熔解分析（MS-QMA），该方法针对 10 个 CpG 位点，其中 9 个位于 FMR1 内含子 1 内，用于在两性中从出生开始筛查 FXS。该新方法结合了高分辨率熔解的定性优势和高通量、定量实时 PCR 标准曲线，在单次测定中提供了 DNA 甲基化的准确定量。在 312 个对照（CGG <40）、143 个前突变（PM）（CGG 56-170）、197 个全突变（FM）（CGG 200-2000）和 33 个 CGG 大小和甲基化嵌合体样本中评估了其性能。
在男性和女性新生儿血斑中，MS-QMA 区分了 FM 与对照等位基因，其敏感性、特异性、阳性和阴性预测值在 92%至 100%之间。在 6 至 35 岁的 FM 女性静脉血中，MS-QMA 与言语智商损伤相关性最强（P=0.002）。在更大的男性和女性队列中，MS-QMA 与参考方法 Southern blot 和 MALDI-TOF 质谱（P<0.05）相关，但与年龄无显著相关性。通过两种参考方法确定的高功能 FM 和 PM 男性中的未甲基化等位基因也通过 MS-QMA 未甲基化。
MS-QMA 立即应用于 FXS 诊断，其定量甲基化输出具有在两性中用于预后的潜力。
