Genetic and environmental determinants of age-acquired skewed X-inactivation and escape from X-inactivation

In humans, females carry two copies of the X chromosome and males one X and one Y chromosome. To maintain a consistent level of gene expression between the sexes, in every cell females silence or 'inactivate' one of their two X chromosomes. Most females randomly silence one X in each cell so that at a tissue level each X is silenced 50% of the time. Functionally, this means that cells in the same body are utilizing different DNA depending on which chromosome X is silenced in a given cell.

However, as women age, one of the two X's starts to predominate; 35% of women over 60 have the same X chromosome silenced in >90% of their cells (termed 'skewed X-inactivation'), and the proportion of women with preferential silencing of the same chromosome X increases with age. It is not known why this age-associated preferential silencing happens, however it is associated with biomarkers of cancer and is seen in individuals with autoimmune diseases such as systemic lupus and thyroid disease. Intriguingly, twin-studies have shown that age-associated preferential silencing is heritable, suggesting an individual's genetics interact with the ageing process to promote cells silencing one X or the other. In this study we will investigate the genetic basis of age-associated chromosome X preferential silencing in order to determine what genetic factors drive this process and whether the same genetic factors are implicated in risk of cancer and autoimmune disease. As the UK population ages, it is a public health priority to identify factors that contribute to healthy ageing and develop biomarkers that monitor healthy ageing and predict future disease. We will explore the relationship between preferential silencing and ageing phenotypes such as frailty and disease incidence to determine if preferential silencing can act as a biomarker of healthy ageing in women.

Some genes on the silenced (or 'inactive') X chromosome are still turned on - they escape inactivation. Genes on the silenced X have a critical role in female biology; women with only one X chromosome (and therefore absolutely no expression of genes from a second chromosome) have Turners syndrome, which includes dysfunction of various organ systems. Genes expressed from the silenced X have a higher dosage in females than males which is thought to underlie differences in disease prevalence between men and women. For example, several of the genes known to escape X-inactivation are tumor-suppressor genes and the extra dosage from the silenced X chromosome has a protective effect - overall women develop less cancer than men. Conversely, several genes implicated in the female-biased auto-immune diseases Systemic Lupus Erythematosus and Rheumatoid Arthritis are expressed from the silenced X chromosome. In this case, it is thought that women may produce too much of these genes which pre-disposes them to developing disease.

Understanding how genes on the silenced X are controlled, and how this varies in different contexts, is critical to understanding how these genes contribute to disease. For example, tissue-limited escape would indicate which tissue is important for mediating a gene's effect on disease. It is currently estimated that 12-20% of genes on the silenced X can be turned on and that their strength of escape varies across individuals. In this study we will perform deep sequencing of the genes on chromosome X to determine which genes escape, how the strength of escape varies across unrelated individuals, between identical twins (thereby removing variability due to genetics), and across tissues. We will focus on autoimmune disease relevant tissues as these have been linked to escape, as well as looking in existing multi-tissue datasets of blood, fat and skin. Finally, we will investigate how escape varies with age both cross-sectionally in the autoimmune tissues and longitudinally over 10 years in a whole blood dataset.

The fundamental aim of this project is to understand the role of genetics and the environment in age-acquired skewed X-inactivation and in regulating the expression of genes on Chromosome X in females.

This project will assay skewed X-inactivation in 4000 ageing female twins from the deeply-phenotyped TwinsUK cohort. We will investigate the relationship between skewing and environmental factors, and determine if skewing can act as a biomarker for healthy ageing or disease prediction. Age-acquired skewing is heritable - we will utilize three strategies to explore its genetic basis. Classical twin modeling will be used to identify Gene x Environment interactions on the heritability of skewing. We will perform a GWAS in 4000 twins to identify individual loci associated with skewing. Genome-wide genetic correlation methods will be used to identify shared genetic architecture underlying skewing and autoimmune diseases or cancer.

This project will comprehensively investigate escape from X-inactivation by deeply sequencing chromosome X transcripts in autoimmune relevant cell-types obtained from 50 female twins and by re-purposing existing large multi-tissue and longitudinal 'omic datasets from up to 850 deeply phenotyped female twins. This will allow investigation of which genes escape X-inactivation, and importantly how escape varies across individuals, across twin pairs, across tissues and over time.

Identification of regulatory variants (eQTLs and splice eQTLs) have been critical to interpretation, and eventual translation, of GWAS signals. Most eQTL studies do not include Chromosome X, and those that do have not taken the skewed status of the individuals into account, which may bias eQTL tests. This project will identify eQTLs on Chromosome X in a large multi-tissue dataset after accounting for skewed X-inactivation, and use these eQTLs to interpret GWAS loci.

Pharma and biotechnology companies are increasingly turning to genetics to identify and prioritize drug targets. The biological insights we will generate can be used by industry to develop novel products to address translational opportunities. The insights we generate on female-specific biology (both related to ageing processes and gene expression) can potentially be used to stratify monitoring or treatment and thereby increase effectiveness.

The release of our unique dataset will benefit both academic and industrial researchers. The twins already have thousands of biomedical and social phenotypes held in the TwinsUK database, and have been profiled with a wide range of 'omics (epigenetics, microbiome, immune-omics, metabolomics, glycomics, proteomics). All data generated in this proposal will be deposited in the TwinsUK Database and will be accessible to bona fide academic and industrial researchers to link to previously collected phenotypes, including those beyond the expertise of the proposed research team.

The broader economic and social impact will be manifest through:

Economic: Benefits to pharma and biotechnology companies able to exploit translational opportunities with to develop novel approaches and therapies that build on the datasets and biology we detect.

Social: Improved effectiveness of public services (NHS) and improved health outcomes if the biological insights result in better ways of predicting, treating or preventing disease. Improved health outcomes will generate better quality of life and personal economic impact for individuals.

Society: Interactive displays on chromosome X biology and inactivation may interest museums, particularly the Science Museum in London which TwinsUK works with on a regular basis. King's College London is a partner in the new Science Gallery London (https://london.sciencegallery.com/ opening in 2018 on the King's College London site, and we will submit proposals for relevant future seasons (current season is 'Blood').