The contribution folate and vitamin B12 genes to disease.

分子病理学部分的研究重点是定义基因的变化，这些基因是常见疾病(如癌症和出生缺陷)遗传易感性的基础。叶酸和维生素B12代谢的变化与肿瘤形成、出生缺陷和认知能力下降有关。叶酸和维生素B12基因也与DNA甲基化和正常大脑功能有关。我们正在寻找与叶酸、蛋氨酸和同型半胱氨酸代谢相关的基因变异。患有癌症或脊柱裂(神经管缺陷的一种形式)的患者将接受这些变异的测试。在疾病患者中发现的较高频率的变异将有助于我们识别与风险相关的基因。 在过去，我们发现TCN2和TCN2R这两个基因的变异似乎会影响怀孕期间血液中维生素B12的水平。此外，TCN2R的变异与神经管缺陷的风险有关。这些发现可能与出生缺陷有关，也可能有助于解释为什么一些老年人会因维生素B12缺乏而变得贫血并出现神经症状。我们还发现，母亲携带第二个基因MTHFD1的特定变异，生下患有神经管缺陷的孩子的风险增加50%。在所有神经管缺陷中，这种以前未被描述的变异可能要负责多达25%。人口中大约五分之一的人携带这些风险因素之一。我们最近确定，这种特殊的变异也是胎盘早剥的危险因素，胎盘早剥是流产的常见原因，也是妊娠中期流产的常见原因。我们已经在实验室合成了这些基因的副本，目前正在使用一个实验系统来准确确定这些变体如何改变这些蛋白质的功能。 我们已经测试了比更多的变异基因，这些变异可能会扰乱叶酸代谢，因此与生下患有神经管缺陷的孩子的风险增加有关。这是通过对大量患有神经管缺陷的家庭和未受影响的对照组的1200多个单核苷酸多态进行基因分型进行的。这项大型实验使我们能够排除这份清单上的大多数基因。对大约12个基因的结果表明，它们与神经管缺陷有关。在过去的一年里，我们进行了第二系列实验，以确定在第一阶段发现的基因是否确实与神经管缺陷有关。这些数据目前正在分析中。 其中一个基因已经通过了我们的第二阶段验证。这种基因会产生一种与维生素B12结合的蛋白质，并将其从血液输送到组织中。我们已经公布的数据表明，这种转运蛋白的几个变种与生下患有NTD的孩子的风险有关。虽然我们现在知道哪些变异与风险有关，但我们还不知道它们是否真的导致了风险，或者与改变蛋白质功能的其他变异有关。为了筛选更多的变异，我们对大量个体中包含这种转运蛋白基因的DNA进行了测序。这项测序实验在该基因中发现了一些以前未被识别的变异。我们测量了这些变体对转运蛋白功能的影响。测试的大多数变种似乎对受体的功能没有不良影响。然而，我们发现，在健康的大样本中，有几个变异与维生素B12水平的变化有关。我们计划在大量老年人样本中测试这些变异，以确定这些变化是否与人类疾病状况有关。当我们在孕妇身上进行同样的测试时，这种变异与维生素B12水平的下降有关。这一结果与相同变异和妊娠不良结局之间的关联是一致的。 我们还进行了旨在确定叶酸、维生素B12和DNA甲基化之间关系的实验。这些实验很难在人体上进行。我们使用斑马鱼作为这些研究的模式生物，并制作了涵盖重要发育阶段的斑马鱼基因组全基因组甲基化图谱。我们还对斑马鱼中叶酸/维生素B12途径的基因进行了表征。在这项工作中，我们能够将预测的功能分配给一些尚未表征的基因。我们还发现，叶酸代谢的中断会导致斑马鱼的发育缺陷。通过使用多种方法，我们得出结论，这些缺陷是由于缺乏细胞分裂，而不是发育信号的变化。这项工作的完成将使我们能够干扰斑马鱼发育中的这些基因，并观察这些干扰是如何影响早期发育的。 我们工作的一个好处是，对叶酸维生素B12代谢途径中基因的功能的详细了解将增加我们对神经管缺陷的了解，并可能有助于指导营养补充领域的公共卫生政策。

Research in the Molecular Pathogenesis Section is focused on defining changes in the genes that underlie inherited susceptibilities to common diseases such as cancer and birth defects. Changes in folate and vitamin B12 metabolism are associated with tumor formation, birth defects and cognitive decline. Folate and vitamin B12 genes are also involved in the methylation of DNA and normal brain function. We are searching for genetic variants in genes related to folate, methionine and homocysteine metabolism. Individuals affected with cancer or spina bifida (one form of neural tube defects) will be tested for these variants. Variants found at higher frequency in individuals with disease will help us identify genes associated with risk. In the past we found that variants in two genes, TCN2 and TCN2R, appear to affect the levels of vitamin B12 in the blood during pregnancy. In addition, variants in TCN2R are associated the risk of neural tube defects. These findings may be related to birth defects and also may help to explain why some elderly individuals become anemic and suffer neurological symptoms from vitamin B12 deficiency. We also found that mothers carrying a specific variant in a second gene, MTHFD1, have a 50% increased risk bearing a child with a neural tube defect. This previously un-described variant may be responsible for up to 25% of all neural tube defects. Approximately one in five individuals in the population carry one of these risk factors. We recently determined that this particular variant was also an risk factor for placental abruption a common cause of miscarriage and for miscarriages that occur in the second trimester. We have synthesized copies of these genes in the laboratory and are currently using an experimental system to determine exactly how these variants alter the function of these proteins. We have tested more than 64 additional genes for variants that might perturb folate metabolism and therefore be associated with an increased risk of having a child with an neural tube defect. This was carried out by genotyping more than 1,200 single nucleotide polymorphisms in a large number of families affected with neural tube defects and unaffected controls. This large experiment has allowed us to exclude most of the genes on this list. Results for approximately a dozen genes suggest that they are associated with neural tube defects. Over the past year we have carried out second series of experiments to determine if the genes identified in the first stage of these are definitively associated with neural tube defects. These data are currently being analyzed. One of these genes has already passed through our stage 2 validation. This gene produces a protein that binds vitamin B12 and transports it from the blood into the tissues. We have published data demonstrating that several variants in this transporter are associated with a risk of having a child with an NTD. While we now know which variants are associated with risk, we do not yet know if they are actually causing the risk or are linked to additional variants that change the function of the protein. To screen for additional variants, we sequenced the DNA containing this transporter gene in a large number of individuals. This sequencing experiment uncovered a number of previously unidentified variants in this gene. We measured the impact of these variants on the function of the transporter. The majority of variants tested do not appear to have an adverse effect of the function of the receptor. However, we have found that several of the variants are associated with changes in vitamin B12 levels in a large sample of healthy individuals. We plan to test these variants in a large sample of elderly individuals to determine if these changes are associated with human disease conditions. When we perform the same test in pregnant women, the variant is associated with decreased levels of vitamin B12. This result is consistent with the association of the same variant and adverse outcomes in pregnancy. We have also carried out experiments aimed at determining the relationship between folate, vitamin B12 and DNA methylation. These experiments are difficult to carry out in humans. We have used zebrafish as a model organism for these studies and have produced a whole genome methylation map of the zebrafish genome covering important developmental stages. We have also characterized the genes in the folate/ vitamin B12 pathway in zebrafish. During this work we were able to assign a predicted function to a number of genes that had not been characterized. We also discovered that the disruption of folate metabolism produces developmental defects in zebrafish. By using a number of methods, we conclude that the defects are due to a lack of cell division not a change in developmental signaling. Completion of this work will allow us disrupt these genes in developing zebrafish and observe how these disruptions affect early development. A benefit of our work is that a detailed knowledge of the function of the the genes in the folate vitamin B12 metabolic pathways will add to our understanding of neural tube defects and potentially help guide public health policy in the area of nutritional supplementation.