Laboratory of Molecular Genetics, Department of Psychological, Humanities and Territorial Sciences, School of Medicine and Health Sciences, G, d'Annunzio University, via dei Vestini 31, Chieti, 66013, Italy.
BMC Med Genet. 2013 Aug 5;14:79. doi: 10.1186/1471-2350-14-79.
Fragile X Syndrome (FXS), the most common cause of familiar mental retardation, is associated in over 99% of cases to an expansion over 200 repeats of a CGG sequence in the 5' UTR of the FMR1 gene (Xq27.3), leading to the hypermethylation of the promoter. Molecular diagnosis of FXS have been so far based on the use of the Southern Blot (SB) analysis, a low throughput and time consuming technique. In order to update the diagnostic approach for FXS, we evaluated the usefulness of the Methylation-Specific Multiplex-Ligation-dependent Probe Amplification assay (MS-MLPA).
The study was carried out by retrospectively analysing 44 male patients, 10 Chorionic Villus Sampling (CVS) samples and 10 females previously analyzed by SB. In addition, a prospective study on 98 male subjects, 20 females and 1 CVS sample was carried out for assessing the feasibility and the impact of MS-MLPA in a routine lab work.
Results provided by both the retrospective and the prospective parts of this study strongly demonstrate the robustness and reproducibility of the MS-MLPA assay, able to correctly detect the methylation status in all normal and full mutation male samples analyzed, including CVS male samples. On the other hand, MS-MLPA analysis on females samples produced unreliable results.
Based on our results, we suggest the necessity of a separate workflow for male and female patients with suspected FXS in the routine diagnostic setting. MS-MLPA, in combination with CGG repeat sizing using a single-tube primed FMR1 PCR, represents a reliable diagnostic protocol in the molecular diagnosis of FXS male patients.
脆性 X 综合征（FXS）是最常见的家族性智力低下的原因，超过 99%的病例与 FMR1 基因（Xq27.3）5'UTR 中 CGG 序列的 200 次以上扩展有关，导致启动子超甲基化。FXS 的分子诊断迄今为止一直基于 Southern Blot（SB）分析的使用，这是一种低通量且耗时的技术。为了更新 FXS 的诊断方法，我们评估了甲基化特异性多重连接依赖性探针扩增检测（MS-MLPA）的有用性。
该研究通过回顾性分析 44 名男性患者、10 例绒毛膜绒毛取样（CVS）样本和 10 名女性患者进行，这些患者之前通过 SB 进行了分析。此外，还对 98 名男性、20 名女性和 1 名 CVS 样本进行了前瞻性研究，以评估 MS-MLPA 在常规实验室工作中的可行性和影响。
本研究回顾性和前瞻性部分的结果均强烈证明了 MS-MLPA 检测的稳健性和可重复性，能够正确检测分析的所有正常和完全突变男性样本的甲基化状态，包括 CVS 男性样本。另一方面，对女性样本的 MS-MLPA 分析产生了不可靠的结果。
基于我们的结果，我们建议在常规诊断环境中对疑似 FXS 的男性和女性患者进行单独的工作流程。MS-MLPA 与使用单管引物 FMR1 PCR 进行 CGG 重复大小测定相结合，代表了 FXS 男性患者分子诊断的可靠诊断方案。
