Genç B, Müller-Hartmann H, Zeschnigk M, Deissler H, Schmitz B, Majewski F, von Gontard A, Doerfler W
Institute of Genetics, University of Cologne, D-50931 Köln, Germany.
Nucleic Acids Res. 2000 May 15;28(10):2141-52. doi: 10.1093/nar/28.10.2141.
Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5'-(CGG)(n)-3' repeat in the promoter and 5'-untranslated region (5'-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M- Sss I-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5'-(CGG)(n)-3' repeats and in the levels of methylation in the repeat and the 5'-UTR. In one patient (OEl) with high repeat length hetero-geneity ( n = 15 to >200), shorter repeats (n = 20-80) were methylated or unmethylated, longer repeats ( n = 100-150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5'-CG-3' sequences were found in some repeats and 5'-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M- Sss I-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.
脆性X综合征（FRAXA）在分子水平上的特征是人类X染色体Xq27.3上脆性X智力低下（FMR1）基因的启动子和5'非翻译区（5'-UTR）中天然存在的5'-(CGG)(n)-3'重复序列发生扩增。当该区域扩增时，通常会发生高度甲基化。FMR1启动子的失活和FMR1蛋白的缺失可能是该综合征的病因。通过使用基因组测序方法的亚硫酸氢盐方案，我们确定了健康个体、选定的前突变携带者和FRAXA患者单条染色体上该区域的甲基化模式。在使用未甲基化或M - Sss I预甲基化DNA的对照实验中，已确定该方案能够可靠地将所有胞嘧啶或5-甲基胞嘧啶分别检测为未甲基化或甲基化的核苷酸。对FRAXA患者DNA的分析揭示了5'-(CGG)(n)-3'重复序列长度以及重复序列和5'-UTR中甲基化水平的显著变异性。在一名具有高重复长度异质性（n = 15至>200）的患者（OEl）中，较短的重复序列（n = 20 - 80）甲基化或未甲基化，较长的重复序列（n = 100 - 150）通常完全甲基化，但一个n = 160的重复序列被证明完全未甲基化。在几名FRAXA患者中观察到了这种类型的甲基化镶嵌现象。在健康女性中，如预期的那样，在一些重复序列和5'-UTR中发现了甲基化的5'-CG-3'序列，这些序列来自其中一条X染色体。天然的FMR1启动子对甲基化敏感，这在使用与荧光素酶基因融合的未甲基化或M - Sss I预甲基化FMR1启动子进行转染实验中，以活性作为指标得到了证明。
