Kosaki K, McGinniss M J, Veraksa A N, McGinnis W J, Jones K L
Department of Pediatrics, University of California, San Diego, La Jolla, USA.
Am J Med Genet. 1997 Dec 19;73(3):308-13.
The putative promoter region of the SNRPN gene contains a CpG island which is heavily methylated in the maternally derived allele and unmethylated in the paternally derived allele. In patients with Prader-Willi syndrome (PWS) only the methylated allele is present, while in those with Angelman syndrome (AS) only the unmethylated allele is present. The purpose of this paper is to report a polymerase chain reaction (PCR)-based assay to evaluate methylation status of the CpG island of the SNRPN gene and to show that this assay allows rapid diagnosis of PWS and AS. Methylated cytosines in the CpG dinucleotide are resistant to chemical modification by sodium bisulfite. In contrast, bisulfite treatment converts all unmethylated cytosines to uracil. Based on this differential effect, the bisulfite-modified DNA sequence of a methylated allele was successfully distinguished from that of an unmethylated allele using 2 sets of allele-specific primer pairs: a methylated allele-specific primer pair (MET) and an unmethylated allele-specific primer pair (UNMET). Bisulfite-modified DNA from 10 patients with PWS amplified only with the MET pair while modified DNA from 5 patients with AS amplified only with the UNMET pair. Modified DNA from 50 normal unrelated individuals amplified with both primer pairs. In that methylation-specific PCR (MSPCR) can detect all presently testable causes of PWS and AS in a rapid and cost-effective fashion, serious consideration should be given to the use of this test in the initial evaluation of all patients in which PWS or AS is being considered.
SNRPN基因的假定启动子区域包含一个CpG岛，该岛在母源等位基因中高度甲基化，而在父源等位基因中未甲基化。在普拉德-威利综合征（PWS）患者中，仅存在甲基化等位基因，而在天使综合征（AS）患者中，仅存在未甲基化等位基因。本文的目的是报告一种基于聚合酶链反应（PCR）的检测方法，以评估SNRPN基因CpG岛的甲基化状态，并表明该检测方法可快速诊断PWS和AS。CpG二核苷酸中的甲基化胞嘧啶对亚硫酸氢钠的化学修饰具有抗性。相反，亚硫酸氢盐处理可将所有未甲基化的胞嘧啶转化为尿嘧啶。基于这种差异效应，使用两组等位基因特异性引物对成功区分了甲基化等位基因和未甲基化等位基因的亚硫酸氢盐修饰DNA序列：甲基化等位基因特异性引物对（MET）和未甲基化等位基因特异性引物对（UNMET）。10例PWS患者的亚硫酸氢盐修饰DNA仅用MET引物对扩增，而5例AS患者的修饰DNA仅用UNMET引物对扩增。50名正常无关个体的修饰DNA用两组引物对均能扩增。鉴于甲基化特异性PCR（MSPCR）能够以快速且经济高效的方式检测出目前所有可检测的PWS和AS病因，在对所有疑似PWS或AS的患者进行初始评估时，应认真考虑使用该检测方法。
