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中国循证儿科杂志

中国循证儿科杂志 ›› 2020, Vol. 15 ›› Issue (5): 382-384.

• 论著 • 上一篇    下一篇

同型半胱氨酸修饰甲基-CpG结合蛋白2与自闭症谱系障碍相关

王晔阳, 赵健元   

  1. 复旦大学生命科学院遗传工程国家重点实验室 上海，200438
  • 收稿日期:2020-07-31 修回日期:2020-10-20 出版日期:2020-10-25 发布日期:2020-10-25
  • 通讯作者: 赵健元
  • 基金资助:
     

Homocysteine-modified methyl-CpG binding protein 2 is associated with autism spectrum disorder

WANG Yeyang, ZHAO Jianyuan   

  1.  State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China
  • Received:2020-07-31 Revised:2020-10-20 Online:2020-10-25 Published:2020-10-25
  • Contact: ZHAO Jianyuan
  • Supported by:
     

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摘要： 摘要 目的：探讨高浓度的同型半胱氨酸（Hcy）在自闭症谱系障碍（ASD）发病中的可能机制。方法：ASD患儿和对照组cDNA样本来自本课题组前期研究，实验中的细胞株包括人胚肾上皮细胞系HEK-293T、小鼠神经干细胞系NE-4C和小鼠海马神经元细胞系HT22，实验动物为野生型C57BL/6J小鼠。采用实时荧光定量PCR比较ASD患儿和对照组外周血cDNA中甲硫氨酰-tRNA合成酶（MARS）表达水平；串联亲和纯化法检测与MARS存在互作关系的转录因子；免疫沉淀和液相质谱法研究Hcy对该转录因子的修饰作用以及可能的赖氨酸残基修饰位点；ChIP实验在细胞系和小鼠中验证该转录因子与其下游靶基因启动子的结合受MARS或Hcy的影响；比较ASD患儿和对照组cDNA样本中这些靶基因的转录水平。结果：①ASD患儿的MARS转录水平高于对照组（P＜0.01），MARS与MeCP2互作；②Hcy可修饰到MeCP2的7个赖氨酸残基位点上，以此抑制MeCP2与DNA甲基化CpG结合，使受MeCP2调控的下游神经发育基因GRIN2A、BDNF等转录水平上调（P＜0.01）；③42.9%ASD患儿 GRIN2A、BDNF、EAAT1~4基因转录水平高于对照组均值；ASD患儿EAAT2、EAAT4基因平均转录水平高于对照组（P＜0.05）。结论：高MARS表达水平和Hcy浓度使MeCP2被Hcy修饰，降低了MeCP2蛋白与DNA甲基化CpG的结合活性，从而影响MeCP2下游神经发育基因的转录水平。

 

Abstract: Abstract Objective：To explore the possible mechanism of high homocysteine (Hcy) in the pathogenesis of autism spectrum disorder (ASD). Methods：cDNA samples from children with ASD and the control group were from the preliminary study of the research team. The cell lines in the experiment included human embryonic kidney epithelial cell line HEK-293T, mouse neural stem cell line NE-4C and mouse hippocampal neuron cell line HT22. The experimental animals were wild type C57BL/6J mice. Real-time fluorescence quantitative PCR was used to compare the expression levels of methionyl-tRNA synthetase (MARS) in peripheral blood cDNA of ASD children and the control group. Tandem affinity purification method was used to detect transcription factors that interacted with MARS. Immunoprecipitation and liquid chromatography-mass spectrometry was used to detect the modification of Hcy to the transcription factor and possible lysine residue modification sites. Chromatin immunoprecipitation assay was used to verify that the binding of the transcription factor to its downstream target gene promoters was affected by MARS or Hcy in cell line and mice. The transcription levels of target genes were compared in cDNA samples from children with ASD and the control group. Results： a. The MARS transcription levels of children with ASD were higher than those of the control group (P＜0.01). MARS interacted with MeCP2. b. Hcy modified 7 lysine residues of MeCP2. The modifications of Hcy inhibited the binding of MeCP2 with DNA methylated CpG. The transcription levels of neurodevelopmental genes GRIN2A and BDNF regulated by MeCP2 increased significantly (P＜0.01). c. The transcription levels of GRIN2A, BDNF and EAAT1-4 genes in 42.9% of children with ASD were higher than the average level of the control group. The average transcription levels of EAAT2 and EAAT4 genes in children with ASD were higher than control group (P＜0.05). Conclusion： High MARS expression and high Hcy made MeCP2 become N-homocysteinylation (Hcy modification), which reduced the binding activity of MeCP2 protein and DNA-methylated-CpG, affecting the transcription levels of MeCP2 downstream neurodevelopmental genes.

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