Center for Medical Genetics, UZ Brussel, Brussels, Belgium.
J Mol Diagn. 2012 Nov;14(6):560-8. doi: 10.1016/j.jmoldx.2012.05.003. Epub 2012 Aug 23.
This study evaluated a large set of blinded, previously analyzed prenatal DNA samples with a novel, CGG triplet-repeat primed (TP)-PCR assay (Amplidex FMR1 PCR Kit; Asuragen, Austin, TX). This cohort of 67 fetal DNAs contained 18 full mutations (270 to 1100 repeats, including 1 mosaic), 12 premutations (59 to 150 repeats), 9 intermediate mutations (54 to 58 repeats), and 28 normal samples (17 to 50 repeats, including 3 homozygous female samples). TP-PCR accurately identified FMR1 genotypes, ranging from normal to full- mutation alleles, with a 100% specificity (95% CI, 85.0% to 100%) and a 97.4% sensitivity (95% CI, 84.9% to 99.9%) in comparison with Southern blot analysis results. Exact sizing was possible for a spectrum of normal, intermediate, and premutation (up to 150 repeats) alleles, but CGG repeat numbers >200 are only identified as full mutations. All homozygous alleles were correctly resolved. The assay is also able to reproducibly detect a 2.5% premutation and a 3% full-mutation mosaicism in a normal male background, but a large premutation in a full male mutation background was masked when the amount of the latter was >5%. Implementation of this TP-PCR will significantly reduce reflex testing using Southern blot analyses. Additional testing with methylation-informative techniques might still be needed for a few cases with (large) premutations or full mutations.
本研究使用新型 CGG 三核苷酸重复引物（TP）-PCR 检测法（Amplidex FMR1 PCR 试剂盒；Asuragen，奥斯汀，TX），对一组大量经过盲法分析的产前 DNA 样本进行了评估。这组 67 个胎儿 DNA 样本中包含 18 个全突变（270 至 1100 个重复，包括 1 个镶嵌型）、12 个前突变（59 至 150 个重复）、9 个中间突变（54 至 58 个重复）和 28 个正常样本（17 至 50 个重复，包括 3 个纯合女性样本）。TP-PCR 能够准确识别 FMR1 基因型，从正常到全突变等位基因，特异性为 100%（95%CI，85.0%至 100%），敏感性为 97.4%（95%CI，84.9%至 99.9%），与 Southern 印迹分析结果相比。对于正常、中间和前突变（高达 150 个重复）等位基因的范围，可以进行精确的大小测定，但 CGG 重复数>200 仅被鉴定为全突变。所有纯合等位基因均得到正确解析。该检测法还能够在正常男性背景下重复检测到 2.5%的前突变和 3%的全突变镶嵌型，但在全男性突变背景下，如果后者的量>5%，则会掩盖大量的前突变。实施这种 TP-PCR 将显著减少使用 Southern 印迹分析进行的反射性检测。对于一些存在（大）前突变或全突变的病例，可能仍需要使用甲基化信息性技术进行额外检测。
