Department of Molecular Genetics, Nichols Institute, Quest Diagnostics, San Juan Capistrano, California 92690, USA.
Genet Med. 2010 Mar;12(3):162-73. doi: 10.1097/GIM.0b013e3181d0d40e.
Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5'-untranslated region. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths >200. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats.
The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. Amplicons are resolved by capillary electrophoresis.
A distinctive pattern of tapering or "stutter" polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths ≥55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. This method also detected the full mutation alleles in DNA isolated from blood spots.
This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. The method distinguishes normal homozygous females from full mutation carrying females. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals.
脆性 X 综合征是由 FMR1 5'非翻译区的 CGG 三核苷酸重复扩展和随后的甲基化引起的。通常需要 Southern 印迹分析来确定重复长度>200 的扩展大小。我们描述了一种基于三链引物聚合酶链反应的方法，使用自动毛细管电泳检测进行定性评估扩展的 CGG 重复。
该测定法使用三链引物聚合酶链反应与 GC 融解试剂结合，并使用 7-脱氮-2-脱氧 GTP 替代 dGTP。通过毛细管电泳分离扩增子。
在携带所有检查的扩增等位基因长度（高达 2000 个 CGG 重复）的男性和女性患者中，毛细管电泳上明显存在独特的渐细或“顿挫”聚合酶链反应扩增模式，可用于区分正常、中间、前突变和全突变等位基因。全突变等位基因在毛细管电泳上显示出另一个迟迁移的扩增子。混合实验表明，检测全突变等位基因的灵敏度低至 1%。在一项针对我们现有的聚合酶链反应平台（重复长度≥55 时进行 Southern 印迹分析）的 1275 个样本一致性研究中，三链引物聚合酶链反应方法对正常、中间、扩展和全突变等位基因的一致性为 100%。该方法还检测到来自血斑的 DNA 中的全突变等位基因。
该测定法提供了对 FMR1 重复状态的准确评估，并有望用于携带者和新生儿筛查。该方法可将正常纯合子女性与全突变携带女性区分开来。虽然该方法不适用于准确的尺寸测量，但它补充了经典的聚合酶链反应方法，并显著减少了实验室中需要进行 Southern 印迹分析的数量，以准确评估所有个体的 FMR1 基因型。
