Gene Nutrient Interactions in Neural Tube Defects

描述(申请人提供)：我们建议阐明脱氧尿苷三磷酸(DUTP)掺入DNA的机制(S)、营养和遗传决定因素，以及它在神经管闭合缺陷(NTDS)病因学中的作用。依赖叶酸和维生素B12(B12)的一碳代谢障碍(OCM)与包括NTDS在内的常见病理有关。最近，我们发现，叶酸依赖的从头合成胸腺酸(DTMP)生物合成障碍通过产生丝氨酸羟甲基转移酶1(SHMT)缺陷的小鼠而导致NTDS。SHMT1是已报道的唯一一种叶酸依赖酶，其破坏导致叶酸反应性NTDS，这为NTDS提供了DNA中从头合成胸腺酸(DTMP)和尿嘧啶积累的证据。最近，另一些人发现，核糖核苷酸还原酶(RNR)催化的UDP到dUDP的转化与叶酸依赖的dTDP合成竞争，以调节dUTP掺入DNA。本文描述的实验将检验最重要的假设，即RNR介导的dUDP合成与叶酸依赖的dTDP合成(通过从头合成DTMP生物合成和DTMP激酶(TMPK))竞争来调节dUTP掺入DNA，这种相互作用是叶酸和维生素B12相关NTD发病的基础。为了支持这一假设，初步数据显示，母体饮食脱氧尿苷(DU)可以挽救叶酸缺乏的Shmt1+/-DAMs中的NTDS，而饮食尿苷导致wt小鼠的NTDS，而不依赖于饮食中的叶酸。这一建议将文献中不同的观察结果整合到一个共同的机制和途径中，包括P53、RNR、叶酸和维生素B12与NTDS相关。这一结果将建立NTDS的途径，并为未来人类和人群预防叶酸和B12相关病理包括NTDS的研究提供信息。目的I.确定维生素B12缺乏是否会损害wt和shmt1+/-小鼠核DTMP的生物合成并改变NTD的发病率。这些研究将确定饮食中叶酸和B12在核DTMP生物合成和NTD发病机制中的作用，并阐明相关的机制。目的II.确定TMPK是否改变WT和Shmt1+/-小鼠NTD的发病率。这些研究将证实，叶酸和B12代谢下游从头合成DTMP的中断会导致NTDS。目的III.确定RNR在DNA中尿嘧啶蓄积和NTD发病机制中的作用。这一目标挑战了当前的教条，即DNA中尿嘧啶的积累是由于DTMP合成受阻而导致的dUTP“误结合”造成的。这些研究将确定P53和RNR的表达是否独立于叶酸影响DNA中的尿嘧啶水平和NTD发生率，以及Shmt1基因是否改变这些结果。目的IV.通过代谢中间产物的饮食挽救，验证NTD发病的遗传和代谢机制。我们将确定母体膳食Du预防Shmt1+/-小鼠NTDS的机制和效果，以及母体膳食尿苷导致NTDS的机制和剂量。

DESCRIPTION (provided by applicant): We propose to elucidate the mechanism(s) and the nutritional and genetic determinants of deoxyuridine triphosphate (dUTP) incorporation into DNA, and its role in the etiology of neural tube closure defects (NTDs). Impairments in folate- and vitamin B12 (B12)-dependent one-carbon metabolism (OCM) are associated with common pathologies, including NTDs. Recently, we discovered that impaired folate-dependent de novo thymidylate (dTMP) biosynthesis causes NTDs in mice by generating serine hydroxymethytransferase 1 (SHMT)-deficient mice. SHMT1 is the only reported folate-dependent enzyme whose disruption causes folate-responsive NTDs, which provides evidence that de novo thymidylate (dTMP) biosynthesis and uracil accumulation in DNA underlies NTDs. Recently, others discovered that the ribonucleotide reductase (RNR)- catalyzed conversion of UDP to dUDP competes with folate dependent dTDP synthesis to regulate dUTP incorporation into DNA. The experiments described herein will test the overarching hypothesis that RNR-mediated dUDP synthesis competes with folate-dependent dTDP synthesis (via de novo dTMP biosynthesis & the enzyme dTMP kinase (TMPK)) to regulate dUTP incorporation into DNA, and that this interaction underlies folate and vitamin-B12-associated NTD pathogenesis. In support of this hypothesis, preliminary data show that maternal dietary deoxyuridine (dU) rescues NTDs in folate-deficient Shmt1+/- dams, whereas dietary uridine causes NTDs in wt mice, independent of dietary folate. This proposal integrates disparate observations in the literature, including that p53, RNR, folate and vitamin B12 are associated with NTDs, into a common mechanism and pathway. The results will establish the pathway for NTDs and inform future human and population studies for the prevention of folate- and B12-associated pathologies including NTDs. Aim I. Determine if vitamin B12 deficiency impairs nuclear dTMP biosynthesis and modifies NTD incidence in wt and Shmt1+/- mice. These studies will establish the role of dietary folate and B12 in nuclear dTMP biosynthesis and NTD pathogenesis, and clarify the associated mechanisms. Aim II. Determine if TMPK modifies NTD incidence in wt and Shmt1+/- mice. These studies will confirm that that disruption of de novo dTMP biosynthesis downstream of folate and B12 metabolism causes NTDs. Aim III. Determine the role of RNR in uracil accumulation in DNA and NTD pathogenesis in mice. This aim challenges the current dogma that uracil accumulation in DNA is caused by dUTP "misincorporation" due to impaired dTMP synthesis. These studies will determine if p53 and RNR expression affects uracil levels in DNA and NTD incidence independent of folate, and if the Shmt1 genotype modifies these outcomes. Aim IV. Validate the genetic and metabolic mechanisms of NTD pathogenesis by dietary rescue with metabolic intermediates. We will determine the mechanism and efficacy of maternal dietary dU in preventing NTDs in Shmt1+/- mice, and the mechanism and dose of maternal dietary uridine that causes NTDs.