Methylation mosaicism of 5′-(CGG)n-3′ repeats in fragile X, premutation and normal individuals
Methylation mosaicism of 5′-(CGG)n-3′ repeats in fragile X, premutation and normal individuals
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DOI：
10.1093/nar/28.10.2141
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发表时间：
2000-05-15
影响因子：
14.9
通讯作者：
Doerfler, W
中科院分区：
文献类型：
--
作者：
Genç, B;Müller-Hartmann, H;Doerfler, W
脆性X综合征（FRAXA）在分子水平上的特征在于人染色体Xq 27，3上脆性X智力低下（FMR 1）基因的启动子和5 '-非翻译区（5'-UTR）中天然存在的5 '-（CGG）（n）-3'重复序列的扩增。当扩增时，该区域通常是高甲基化的。FMR 1启动子的失活和FMR 1蛋白的缺失是该综合征的可能原因。通过使用基因组测序方法的亚硫酸氢盐协议，我们已经确定了健康个体和选定的前突变携带者和FRAXA患者的单染色体上该区域的甲基化模式。在使用未甲基化或M-Sssl-预甲基化DNA的对照实验中，该方案已被确定为分别可靠地检测作为未甲基化或甲基化核苷酸的所有胞苷或5-甲基胞苷。对FRAXA患者DNA的分析揭示了5 '-（CGG）（n）-3'重复序列的长度以及重复序列和5 '-UTR中甲基化水平的相当大的变异性。（n = 15至>200），较短的重复序列（n = 20-80）被甲基化或未甲基化，较长的重复序列（n = 100-150）通常被完全甲基化，但一个n = 160的重复序列被证明是完全未甲基化的。在几个FRAXA患者中观察到这种类型的甲基化镶嵌现象。在健康女性中，在一些重复序列和5 '-UTR中发现了甲基化的5'-CG-3'序列，正如对来自X染色体之一的序列的预期。天然FMR 1启动子是甲基化敏感的，如通过使用未甲基化或M-SssI-预甲基化的FMR 1启动子融合到荧光素酶基因作为活性指示剂的转染实验中的活性损失所证明的。
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-Sssl-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 (OEI) with high repeat length heterogeneity (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-SssI-premethylated FMR1 promoter fused to the luciferase gene as an activity Indicator.