Mechanism of sex difference in severe brain malformations

出生缺陷是新生儿死亡的主要原因，在许多情况下，幸存者的残疾和反复治疗是其结果。这导致美国疾病控制中心（2020年）得出结论，“需要继续识别出生缺陷的潜在风险因素，确定[它们]在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个特定的出生缺陷，无脑儿，是如何在女性比男性更常见。这些知识将增加我们对这种情况的了解，并有助于防止更多的病例发生比目前可能的。无脑畸形出现时，胚胎的未来大脑未能在受孕后第3周关闭。它是最常见和最严重的出生缺陷之一，与相关的缺陷脊柱裂一起，构成了“神经管缺陷”（NTD）。平均而言，NTD影响每1000例妊娠中约有1例，但在世界某些地区，特别是非洲，NTD的发生率超过所有妊娠的1%，给家庭和卫生服务带来巨大负担。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。大脑在胚胎的性别变得明显（当卵巢或睾丸形成时）前几周关闭，因此无脑畸形中的女性过剩不可能是激素。相反，一些科学家认为这是因为不同的性染色体。女性有两个X染色体，而男性有一个X和一个Y。随着胚胎的发育，两条X染色体上的基因活跃（即制造蛋白质）是有害的，因为女性的剂量是男性的两倍。因此，女性细胞中有一条X染色体，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染色体的失活涉及用称为甲基的小分子“取代”它。这关闭了染色体上的大多数基因。然而，这可能使女性细胞与男性细胞相比处于不利地位。甲基还用于许多其他功能，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常见。在这个项目中，我们将检验“X染色体失活假说”。小鼠胚胎可以发育出一个开放的大脑，称为露脑畸形，这相当于人类的无脑畸形，并且影响女性比男性更多。小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。这允许在不需要注射或给药怀孕雌性的情况下应用治疗。我们在使用这种方法方面非常有经验，并且发现减少甲基主要影响女性，而当我们补充甲基时，女性被拯救，并且与男性具有相似的脑开放率。我们将用这个系统扩展我们的研究，以测试最广泛意义上的X失活假说，调查由特定药物或缺陷基因引起的NTD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，以了解用于“拯救”女性胚胎缺陷的最佳补充剂。接下来，在研究的第二部分，我们将在分子水平上研究甲基缺乏的影响，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大脑闭合的过程，导致无脑畸形。总之，这个项目代表了第一次尝试全面了解出生缺陷最有趣和神秘的方面之一：两性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.