Mechanism of sex difference in severe brain malformations

出生缺陷是新生儿死亡的主要原因，在幸存者中，残疾和反复治疗是许多情况下的结果。这使得美国疾病控制中心（2020年）得出结论，“有必要继续确定出生缺陷的潜在风险因素，确定[它们]在不同群体之间可能存在的差异，并找到预防它们的机会”。这是我们研究的总体目标，目前的项目旨在确定一种特殊的出生缺陷，即无脑畸形，在女性中比在男性中更常见。这些知识将增加我们对这种情况的理解，并有助于防止比目前可能发生的更多病例的发生。当胚胎未来的大脑在受孕后的第三周未能闭合时，就会出现无脑儿。它是最常见和最严重的出生缺陷之一，与相关的脊柱裂缺陷一起构成“神经管缺陷”（NTDs）。平均而言，每1000例怀孕中约有1例受到被忽视热带病的影响，但在世界某些地区，特别是在非洲，这一比例超过了所有怀孕的1%，给家庭和卫生服务带来了巨大负担。女性无脑畸形的发生率是男性的2-3倍，但我们目前还不清楚这种差异是如何产生的。大脑在胚胎性别变得明显之前几周就关闭了（当卵巢或睾丸形成时），因此无脑畸形中的女性过剩不可能是激素。相反，一些科学家认为原因在于不同的性染色体。雌性有两条X染色体，而雄性有一条X染色体和一条y染色体。随着胚胎的发育，两条X染色体上的基因都处于活跃状态（即制造蛋白质）是有害的，因为雌性的剂量是雄性的两倍。因此，雌性细胞使一条X染色体失活，以“平衡”基因剂量。这在发育的早期就开始了，远在大脑关闭之前。X染色体的失活涉及到用一种叫做甲基的小分子“抹灰”它。这关闭了染色体上的大部分基因。然而，与男性细胞相比，这可能会使女性细胞处于劣势。甲基被用于许多其他功能，特别是在快速发育的胚胎中，甲基的缺乏可能会增加女性胚胎发育不良的风险。因此，无脑畸形在女性中更为常见。在这个项目中，我们将检验“X失活假说”。老鼠胚胎可以发育出一个开放的大脑，称为无脑畸形，相当于人类的无脑畸形，女性比男性受影响的要多。老鼠是理想的研究动物模型，因为胚胎可以在大脑关闭的阶段在试管中生长。这样就可以在不需要给孕妇注射或给药的情况下进行治疗。我们对这种方法的使用非常有经验，我们发现减少甲基主要影响女性，而当我们补充甲基时，女性获救，并且与男性有相似的开颅率。我们将用这个系统扩展我们的研究，从最广泛的意义上测试X失活假说，调查由特定药物或缺陷基因引起的NTDs是否也受到甲基短缺的影响。我们将测试不同的叶酸，包括叶酸，以了解从缺陷中“拯救”女性胚胎的最佳补充剂。然后，在研究的第二部分，我们将在分子水平上检查甲基缺乏的影响，了解哪些基因和蛋白质可能受到最大的影响，以及这可能如何干扰胚胎中大脑关闭的过程，导致无脑畸形。总之，这个项目代表了首次尝试完全理解出生缺陷最有趣和最神秘的方面之一：性别之间的重大差异是如何在胚胎发育的早期阶段出现的。

Birth defects are a leading cause of death in newborn babies and, among survivors, disability and repeated medical treatments are the outcomes in a great many cases. This led the Centres for Disease Control in the USA (2020) to conclude there is a "need to continue identifying potential risk factors for birth defects, determining how [they] may differ between groups, and finding opportunities to prevent them". This is the overall goal of our research, and the present project seeks to determine how a particular birth defect, anencephaly, is more common in females than males. This knowledge will add to our understanding of the condition, and help to prevent more cases from occurring than is possible at present.Anencephaly arises when the future brain of the embryo fails to close in the 3rd week after conception. It is one of the commonest and most severe birth defects and, together with the related defect spina bifida, makes up the 'neural tube defects' (NTDs). On average, NTDs affect around 1 in every 1000 pregnancies, but rates in some parts of the world, especially in Africa, exceed 1% of all pregnancies, placing a huge burden on families and health services. Anencephaly is 2-3 times more frequent in females than in males, but we currently do not understand how this difference comes about. The brain closes several weeks before the embryo's sex becomes apparent (when an ovary or testis forms) and so the female excess in anencephaly cannot be hormonal. Instead, some scientists have suggested that the reason is because of the different sex chromosomes.Females have two X chromosomes, whereas males have an X and a Y. As the embryo develops, it is damaging for the genes on both X chromosomes to be active (i.e. making proteins), as females would then have twice the dose as males. So female cells inactivate one X chromosome, to 'even up' the gene dosage. This begins very early in development, well before the brain closes. Inactivation of an X chromosome involves 'plastering' it with small molecules called methyl groups. This switches off most of the genes on the chromosome. However, this could put female cells at a disadvantage compared with male cells. Methyl groups are used for many other functions, especially in the rapidly developing embryo, and a shortage of methyl groups may increase the risk of female embryos undergoing faulty development. Anencephaly is proposed to be more frequent in females for this reason.In this project, we will test the "X inactivation hypothesis". Mouse embryos can develop an open brain, called exencephaly, which is equivalent to anencephaly in humans and affects many more females than males. Mice are an ideal animal model to study, as embryos can be grown in a test-tube at the stage when the brain closes. This allows treatments to be applied without the need to inject or dose the pregnant female. We are very experienced in use of this method, and find that reducing methyl groups affects females mainly, whereas when we replenish methyl groups females are rescued, and have similar rates of open brain as males. We will extend our studies with this system to test the X inactivation hypothesis in its widest sense, investigating whether NTDs that result from particular drugs or faulty genes are also affected by methyl group shortage. We will test different folates, including folic acid, to understand the best supplement to use for 'rescuing' female embryos from defects. Then, in the second part of the study, we will examine the effects of methyl group shortage at the molecular level, understanding which genes and proteins might be most affected, and how this may interfere with the process of brain closure in the embryo, leading to anencephaly.Together, this project represents the first attempt to fully understand one of the most intriguing and mysterious aspects of birth defects: how a major difference between the sexes can arise at such an early stage in an embryo's development.