Farmiloe Grace, Bejczy Veronika, Tabolacci Elisabetta, Willemsen Rob, Jacobs Frank
Evolutionary Neurogenomics, University of Amsterdam, Swammerdam Institute for Life Sciences, Science Park 904, Amsterdam, 1098XH, The Netherlands.
Department of Life Sciences and Public Health, Section of Genomic Medicine, Catholic University, Rome, Italy.
J Neurodev Disord. 2025 Apr 26;17(1):22. doi: 10.1186/s11689-025-09609-5.
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the expansion of a CGG repeat in the 5'UTR of the FMR1 (fragile X messenger ribonucleoprotein 1) gene. Healthy individuals possess a repeat 30-55 CGG units in length. Once the CGG repeat exceeds 200 copies it triggers methylation at the locus. This methylation covers the FMR1 promoter region and silences expression of the gene and the production of FMRP (fragile X messenger ribonucleoprotein). The loss of FMRP is responsible for a number of pathologies including neurodevelopmental delay and autism spectrum disorder. Methylation of the expanded repeat in the FMR1 locus is the causal factor for FXS, however it is not known why the expanded repeat triggers this epigenetic change or how exactly DNA methylation is established. Intriguingly, genetic engineering of expanded CGG repeats of over 300 copies in the FMR1 locus in mice remains unmethylated. Also in humans, in very rare cases, individuals can have an FMR1 CGG expansion > 200 copies but the locus remains unmethylated. These unmethylated full mutation (UFM) individuals give us a rare opportunity to investigate the mechanism of FMR1 promoter methylation.
Fibroblasts were obtained from a healthy control, an FXS patient and two unmethylated full expansion carriers. RNA was extracted and comparative transcriptomic analysis was performed on all samples. Whole genome sequencing was carried out on DNA from the two UFM carriers and the results analysed to investigate DNA variants that could explain the observed differences in gene expression.
Our analyses focused on genes involved in epigenetic modification. We show that Tet methylcytosine dioxygenase 3 (TET3), a gene involved in DNA methylation, is significantly downregulated in UFM carriers compared to healthy controls or FXS patient derived cells. Genomic analyses reveal a number of rare variants present in the TET3 locus in UFM carriers when compared to the reference genome. However, no clear modifying TET3 variants were identified.
Our results suggest that TET3 is a candidate factor responsible for the lack of methylation of the expanded FMR1 locus. Further analyses are needed to further elucidate this relationship, however given its potential to directly interact with CGG repeats and its ambiguous role in 5-hydroxy-methylation of CG containing sequences, TET3 is a strong candidate for further exploration.
脆性X综合征（FXS）是一种神经发育障碍，由FMR1（脆性X信使核糖核蛋白1）基因5'非翻译区的CGG重复序列扩增引起。健康个体的该重复序列长度为30 - 55个CGG单位。一旦CGG重复序列超过200个拷贝，就会在该位点引发甲基化。这种甲基化覆盖FMR1启动子区域，使基因表达沉默并导致脆性X信使核糖核蛋白（FMRP）的产生缺失。FMRP的缺失导致多种病理状况，包括神经发育迟缓与自闭症谱系障碍。FMR1位点扩增重复序列的甲基化是FXS的致病因素，但尚不清楚为何扩增的重复序列会引发这种表观遗传变化，以及DNA甲基化究竟是如何建立的。有趣的是，在小鼠中FMR1位点超过300个拷贝的扩增CGG重复序列的基因工程改造后仍未发生甲基化。在人类中同样如此，在非常罕见的情况下，个体的FMR1 CGG扩增序列>200个拷贝，但该位点仍未甲基化。这些未甲基化的全突变（UFM）个体为我们研究FMR1启动子甲基化机制提供了难得的机会。
从一名健康对照、一名FXS患者以及两名未甲基化的全扩增携带者获取成纤维细胞。提取RNA并对所有样本进行比较转录组分析。对两名UFM携带者的DNA进行全基因组测序，并分析结果以研究可能解释观察到的基因表达差异的DNA变异。
我们的分析聚焦于参与表观遗传修饰的基因。我们发现，与健康对照或FXS患者来源的细胞相比，参与DNA甲基化的Tet甲基胞嘧啶双加氧酶3（TET3）基因在UFM携带者中显著下调。基因组分析显示，与参考基因组相比，UFM携带者的TET3位点存在一些罕见变异。然而，未鉴定出明确的TET3修饰变异。
我们的结果表明，TET3是导致FMR1位点扩增序列缺乏甲基化的候选因素。需要进一步分析以进一步阐明这种关系，然而鉴于其与CGG重复序列直接相互作用的潜力以及在含CG序列的5 - 羟甲基化中的模糊作用，TET3是进一步探索的有力候选对象。
