叶酸代谢障碍神经管畸形动物模型的基因组拷贝数变异研究

Study on the copy number variations of neural tube defect animal model with folate dysmetabolism

目的 检测甲氨蝶呤（MTX）诱导叶酸代谢障碍神经管畸形（NTDs）小鼠胚胎神经组织的基因组拷贝数变异（CNVs）,探讨叶酸代谢障碍与NTDs发生的分子遗传学机制.方法 采用本实验室已建立的MTX诱导的叶酸代谢障碍NTDs C57BL/6J小鼠模型,于孕第7.5天采用MTX干预,收集孕鼠血清及NTDs小鼠胚胎神经组织;用NimbleGen高分辨率微阵列比较基因组杂交（array-CGH）芯片对NTDs小鼠胚胎神经组织进行全基因组CNVs分析,反转录（RT）-PCR验证新发现的CNVs;用液相色谱串联质谱法（LC-MS/MS）与酶学方法检测孕鼠血清中叶酸与其相关代谢产物水平及二氢叶酸还原酶（DHFR）活性.结果 array-CGH分析发现,NTD小鼠胚胎神经组织全基因组存在3个高可信度CNVs,分别位于XqE3、XqA1.1-qA2和XqA1.1染色体,RT-PCR验证了这3个CNVs的存在.与对照组相比,MTX干预后,NTDs孕鼠血清中5-甲基四氢叶酸、5-甲酰四氢叶酸、S-腺苷甲硫氨酸水平及DHFR活性明显降低,差异均有统计学意义（P均＜0.05）.结论 MTX诱导的NTDs小鼠胚胎神经组织中存在明显的CNVs,叶酸代谢紊乱可通过CNVs导致小鼠胚胎神经管发育障碍.

Objective To detect the genomic copy number variations（CNVs） of mice embryonic neural tissue with neural tube defects（NTDs） induced by methotrexate（MTX） ,and investigate the molecular genetic mechanisms between folic acid metabolism disorders and NTDs pathogenesis. Methods C57BL/6J NTD mice model was induced by MTX on gestational day 7.5, and the maternal serum and NTD embryonic neural tissues were collected ; the array-comparative genomic hybrktization（array-CGH） assay was utilized to analyze the whole genomic CNVs in NTD embryonic neural tissues;reverse transcription polymerase chain reaction（RT-PCR） was used to confirm the positive results;the maternal serum concentrations of folic acid and related metabolites and dihydrofolate reductase （DHFR） activity were detected by liquid chromatography-tandem mass spectrometry and enzymatic methods, respectively. Results Array- CGH and RT-PCR results showed the 3 high confidence CNVs on XqE3 ,XqA1.1-qA2 and XqA1.1 in the NTD embryonic neural tissues. The NTD maternal serum concentrations of 5-methyhetrahydrofolate,5-formyhetrahydrofolic acid,S-adeno- syl methionine and DHFR activity were reduced significantly compared with the control group, and there were statistical differences （ all P 〈 0.05 ）. Conclusions There are obvious CNVs in embryonic neural tissue of NTD mice induced by MTX and folic acid dysmetabolism might cause mice embryonic neural tube developmental disorders through CNVs.