Nahhas Fatimah A, Monroe Thomas J, Prior Thomas W, Botma Patricia I, Fang Jin, Snyder Pamela J, Talbott Sandi L, Feldman Gerald L
University Laboratories-Molecular Genetics Diagnostic Laboratory, Detroit Medical Center, Detroit, Michigan 48201, USA.
Genet Test Mol Biomarkers. 2012 Mar;16(3):187-92. doi: 10.1089/gtmb.2011.0128. Epub 2011 Oct 12.
Fragile X syndrome (FXS) is caused by the absence of a functional fragile X mental retardation protein (FMRP). In most cases, the molecular mutation is an expansion and consequent methylation of the CGG trinucleotide repeat in the 5' end of the FMR1 gene. Polymerase chain reaction (PCR)-based assays that overcome the limitations of amplifying >100-150 CGG repeats have been designed. One such product, Human FMR1 PCR Reagents, can detect expanded mutation alleles without determining methylation status. We used this assay to amplify 70 clinical samples previously tested in three clinical laboratories, including 28 full mutation alleles, 17 premutation alleles, 6 gray zone alleles, and 21 normal samples (51 normal alleles including 5 homozygous females). The results were concordant with previously reported results. All full and premutation alleles were identifiable: repeat sizes are not assigned when the CGG repeat number is >200 and all full and premutation alleles were scored in the same category using this assay. All normal and gray zone alleles were within 0-1 repeat of their previously reported allele sizes. This method identified a mosaic premutation/full mutation pattern in 12/21 samples previously identified as full mutation only and in 5/7 samples previously reported as mosaic premutation/full mutation. These results demonstrate that this assay provides comparable results to the combination of PCR/Southern blot methodologies. Additional issues such as technologist time, reagent costs, turnaround times, and sample requirements are comparable to the PCR/Southern blotting assays currently utilized; however, methylation status cannot be determined using this assay. It is likely that PCR-only based assays will eventually replace previous methods for FXS and that Southern blotting or another methylation assay will only be utilized when determination of methylation status is necessary. This type of assay may also be utilized for other nucleotide expansion disorders.
脆性X综合征（FXS）是由功能性脆性X智力低下蛋白（FMRP）缺失引起的。在大多数情况下，分子突变是FMR1基因5'端CGG三核苷酸重复序列的扩增及随后的甲基化。已经设计出基于聚合酶链反应（PCR）的检测方法，克服了扩增>100 - 150个CGG重复序列的局限性。其中一种产品，人FMR1 PCR试剂，可检测扩增的突变等位基因，而无需确定甲基化状态。我们使用该检测方法对之前在三个临床实验室检测过的70个临床样本进行扩增，包括28个全突变等位基因、17个前突变等位基因、6个灰色区域等位基因和21个正常样本（51个正常等位基因，包括5名纯合女性）。结果与先前报道的结果一致。所有全突变和前突变等位基因均可识别：当CGG重复数>200时不指定重复大小，使用该检测方法所有全突变和前突变等位基因被归为同一类别。所有正常和灰色区域等位基因与其先前报道的等位基因大小相差在0 - 1个重复范围内。该方法在之前仅鉴定为全突变的12/21样本以及先前报道为嵌合前突变/全突变的5/7样本中鉴定出嵌合前突变/全突变模式。这些结果表明，该检测方法与PCR/ Southern印迹方法组合提供的结果相当。技术人员时间、试剂成本、周转时间和样本要求等其他问题与目前使用的PCR/ Southern印迹检测方法相当；然而，使用该检测方法无法确定甲基化状态。基于仅PCR的检测方法最终可能会取代之前用于FXS的方法，只有在需要确定甲基化状态时才会使用Southern印迹或其他甲基化检测方法。这种类型的检测方法也可用于其他核苷酸扩增疾病。
