The Impact of Vitamin B12 Deficiency on Nuclear de novo Thymidylate Biosynthesis

描述(申请人提供)：在这些研究中，我们将确定维生素B12在调节核从头DTMP生物合成、维持基因组稳定方面的作用，以及阐明维生素B12在出生缺陷中的潜在机制。由于Shmt1-/+小鼠进食叶酸缺乏的饮食，尿嘧啶在DNA中积累，并对发生神经管缺陷(NTDS)和结肠癌敏感，因此DTMP生物合成中的核损伤是叶酸相关基因病理的基础。最近，SHMT被鉴定为一种支架蛋白，需要在DNA复制和修复位置正确组装从头合成DTMP生物合成复合体。维生素B12缺乏会导致细胞内5-甲基四氢呋喃和DNA中尿嘧啶的积累增加，已经被认为是导致NTDS的原因，但尚未得到证实。这些拟议的研究将确定维生素B12缺乏对从头合成DTMP的影响，并确定这一途径的损害是否有助于维生素B12相关的NTDS。此外，这些研究的结果将通过确定进一步预防产前叶酸和维生素B12相关病理的围产期措施，为未来的临床实践提供信息，并为制定以人群为基础的方法以通过强制强化改善维生素B12状况的决策提供信息。有待研究的两个相互关联和重叠的领域是：1)确定维生素B12缺乏是否会损害细胞核中从头合成胸腺酸(DTMP)的生物合成。这些研究将确定维生素B12缺乏的潜在机制，如MTR抑制导致5-甲基THF不可逆积累，通过以下方式损害核从头合成DTMP：1)破坏用于DTMP合成的多酶复合体或2)耗尽核苷酸生物合成所需的四氢叶酸辅助因子。B)确定嘧啶核苷Du和U是否以dUTP或dTTP的形式差异地掺入核DNA中。这些研究将在维生素B12缺乏的细胞模型中建立核DNA中尿嘧啶的来源(S)，并调和目前文献中关于以dUTP形式并入的核苷的不确定性。目的II.确定维生素B12缺乏是否会增加DTMP生物合成受损小鼠模型中NTDS的发生率。这些研究将利用我们的从头合成DTMP受损的小鼠模型，确定母亲缺乏维生素B12是否会通过饮食与基因的相互作用增加后代的NTD发生率。此外，这些研究将确定维生素B12缺乏是否会损害母体和胎儿基因组的完整性。

DESCRIPTION (provided by applicant): In these studies, we will determine the role of vitamin B12 in regulating nuclear de novo dTMP biosynthesis, maintaining genome stability, as well as elucidate the mechanisms underlying vitamin B12 in birth defects. Impairments nuclear in de novo dTMP biosynthesis underlie folate-associated pathologies in a gene by diet manner, as Shmt1-/+ mice fed a folate-deficient diet accumulate uracil in DNA and are sensitized to developing neural tube defects (NTDs) and colon cancer. Recently, SHMT was identified as a scaffolding protein required for proper assembly of the de novo dTMP biosynthesis complex at sites of DNA replication and repair. Vitamin B12 deficiency results in elevated intracellular 5-methylTHF and uracil accumulation in DNA, and has been suggested but not proved to cause NTDs. These proposed studies will determine the impact of a vitamin B12 deficiency on de novo dTMP biosynthesis and establish whether impairments in this pathway contribute to vitamin B12-associated NTDs. Furthermore, the findings from these studies will inform future clinical practice through identification of additional periconceptional measures to further prevent occurrence of prenatal folate - and vitamin B12-associated pathologies, and inform policy-making decisions in the development of population- based approaches to improve vitamin B12 status through mandated fortification. The two related and overlapping areas to be investigated are: Aim I. a) Determine if vitamin B12 deficiency impairs de novo thymidylate (dTMP) biosynthesis in the nucleus. These studies will determine the potential mechanisms by which vitamin B12 deficiency, as modeled by MTR inhibition leading to an irreversible accumulation of 5-methylTHF, impairs nuclear de novo dTMP biosynthesis via the: 1) disruption of the multi-enzyme complex for dTMP synthesis or 2) depletion of tetrahydrofolate cofactors required for nucleotide biosynthesis. b) Determine if pyrimidine nucleosides, dU and U, are differentially incorporated as either dUTP or dTTP in nuclear DNA. These studies will establish the origin(s) of uracil in nuclear DNA in a cell model of vitamin B12 deficiency, and reconcile the current uncertainty in the literature with respect to the nucleosides that are incorporated as dUTP. Aim II. Determine if vitamin B12 deficiency increases the incidence of NTDs in mouse models of impaired de novo dTMP biosynthesis. These studies will determine if maternal vitamin B12 deficiency increases NTD incidence in offspring through a gene by diet interaction using our mouse model of impaired de novo dTMP biosynthesis. Additionally, these studies will determine if vitamin B12 deficiency compromises genome integrity in maternal and fetal genomes.