Mechanism of sex difference in severe brain malformations

Lead Research Organisation: University College London
Department Name: Institute of Child Health

Abstract

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.

Technical Summary

Sex differences in disease frequency and severity are common, and can arise before sex differentiation in the embryo. Anencephaly is a major congenital brain defect resulting from failed neural tube closure in the 3rd embryonic week. It affects 0.2-5 per 1000 births and is markedly more common in females than males. In mice, exencephaly - the forerunner of anencephaly - also shows a female bias, indicating a general mammalian phenomenon. The X chromosome inactivation (XCI) hypothesis views heavy usage of methyl groups in female cells as creating an 'epigenetic sink' that compromises other methylation events in the embryo. This proposal will: (i) test the XCI hypothesis, to determine why mouse exencephaly is predominant in females, and is hindbrain-specific, compared with males where it is forebrain-specific; (ii) determine the underlying molecular mechanisms by which methyl group availability impacts cranial neural tube closure. Brain closure will be studied in whole mouse embryo culture, which has already yielded pilot data consistent with the XCI hypothesis. Variations in sex chromosome constitution (XXY, XO) will provide a direct test of the XCI hypothesis while assessment of teratogens (e.g. valproic acid), and single NTD-causing genes, will reveal the generalisability of the XCI mechanism. Folates will be compared for preventive effects. The molecular analysis will involve assessment of downstream methylation targets in the embryo: DNA, where a methylation array will be used, and protein where methylation events involving cytoskeletal actin and tubulin will be studied. We will test whether methylation is critical for hindbrain neural crest emigration, that could underlie the female brain closure defect. Genes that 'escape' X inactivation will be evaluated for possible protection of females against forebrain defects. This project aims to resolve a long-standing knowledge gap in birth defects research, that has strong clinical implications.

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