Panagopoulos I, Lassen C, Kristoffersson U, Aman P
Department of Clinical Genetics, University Hospital, Lund, Sweden.
Hum Mutat. 1999;14(1):71-9. doi: 10.1002/(SICI)1098-1004(1999)14:1<71::AID-HUMU9>3.0.CO;2-5.
Fragile X syndrome is associated with the expansion of the number of CGG trinucleotide tandem repeats at the 5' untranslated region of the FMR1 gene. The number of CGG trinucleotide repeats in normal individuals ranges between 5 and 50, in asymptomatic carrier individuals it ranges between 50 and 200, and in affected individuals it is more than 200 CGG repeats. In addition, in affected individuals the cytosine residues in the CGG repeats and the adjacent CpG island are methylated and the FMR1 gene is transcriptionally inactive. The most common diagnostic method for the detection of the syndrome is Southern blot analysis. Methods based on the polymerase chain reaction (PCR) could facilitate the rapid screening of large numbers of individuals by accurately determining the number of CGG repeats. Current PCR techniques for amplification of CGG repeats are, however, inefficient and unreliable because of their 100% C+G composition. Thus, most of the described PCR protocols require subsequent Southern blot analysis and autoradiography. We present a novel PCR approach for the diagnosis of fragile X syndrome based on the methylation-sensitive conversion of C residues to U by bisulfite on single-strand DNA and subsequent amplification of the antisense strand with specific primers. A PCR with primers for methylated C residues will amplify the CpG dinucleotide region upstream to CGG repeats exclusively in affected males. As a result of extensive mismatch between primers and bisulfite-treated DNA, no PCR fragments will be obtained in normal and transmitting males. Moreover, the bisulfite treatment dramatically reduces the C+G component of the region; thus, the high Tm and the strong secondary structures are no longer obstacles for PCR amplification. In normal and carrier individuals, UUG repeats (previously 3'-CCG-5') in the antisense strand can easily be amplified and visualized on a gel by ethidium bromide staining. We applied our method on 25 males previously diagnosed by Southern blot analysis. All the samples were easily and accurately diagnosed. The method has considerable advantages compared with other diagnostic tests for fragile X syndrome.
脆性X综合征与FMR1基因5'非翻译区CGG三核苷酸串联重复序列数量的扩增有关。正常个体中CGG三核苷酸重复序列的数量在5至50之间，无症状携带者个体中其数量在50至200之间，而患病个体中CGG重复序列超过200个。此外，在患病个体中，CGG重复序列中的胞嘧啶残基和相邻的CpG岛发生甲基化，FMR1基因转录失活。检测该综合征最常用的诊断方法是Southern印迹分析。基于聚合酶链反应（PCR）的方法可以通过准确确定CGG重复序列的数量，便于对大量个体进行快速筛查。然而，由于其100%的C+G组成，目前用于扩增CGG重复序列的PCR技术效率低下且不可靠。因此，大多数所述的PCR方案都需要后续的Southern印迹分析和放射自显影。我们提出了一种用于诊断脆性X综合征的新型PCR方法，该方法基于亚硫酸氢盐将单链DNA上的C残基甲基化敏感地转化为U，随后用特异性引物扩增反义链。用针对甲基化C残基的引物进行PCR，将仅在患病男性中扩增CGG重复序列上游的CpG二核苷酸区域。由于引物与亚硫酸氢盐处理的DNA之间存在广泛错配，正常男性和传递男性中不会获得PCR片段化。此外，亚硫酸氢盐处理显著降低了该区域的C+G成分；因此，高解链温度和强二级结构不再是PCR扩增的障碍。在正常个体和携带者个体中，反义链中的UUG重复序列（以前为3'-CCG-5'）可以很容易地扩增出来，并通过溴化乙锭染色在凝胶上可视化。我们将我们的方法应用于25名先前通过Southern印迹分析诊断的男性。所有样本都能轻松、准确地诊断。与其他脆性X综合征诊断测试相比，该方法具有相当大的优势。
