Skewed X Chromosome Inactivation: A sex-specific biomarker of immune system ageing

Ageing is a heterogenous process which increases the susceptibility to adverse health outcomes, including frailty.

This is thought to be partly attributable to immune system deterioration but the precise mechanisms are incompletely understood. Defining biomarkers of immune system deterioration could significantly advance our understanding of healthy ageing.

X chromosome inactivation (XCI) is the mammalian process which equalises the gene dosage between the X and Y chromosomes.

During development, either the maternal or paternal X chromosome in each 46, XX cell is chosen at random to be transcriptionally silenced.

The XCI status of a cell is stable and clonally inherited by all daughter cells giving an expected 1:1 ratio across a tissue.

However, some females display a “skewed” pattern of XCI (XCI-skew) in which >75% of cells in a tissue have the same X inactivated. Age acquired XCI-skew refers to increasing XCI-skew with age and is particularly prevalent in mitotically active blood.

As XCI-skew reflects changes in the proliferation of the underlying haematopoietic stem cells, it offers a unique lens through which to study age-related haematopoietic shifts in humans.

Importantly, though XCI-skew shares some similarity with clonal haematopoiesis of indeterminate potential (CHIP), these two traits are not mutually inclusive, and XCI-skew is far more prevalent, affecting a third of females over 60. The consequences of age acquired XCI-skew – from the molecular to clinical levels - remain largely unknown.

Our recent work, using a subset of the TwinsUK population cohort (n=1,575, median age=61), defined XCI-skew as an immune cell phenotype with adverse health outcomes.

We demonstrated that XCI-skew correlates with cardiovascular disease risk and predicts future cancer diagnosis but is independent of other molecular markers of ageing such as telomere length shortening.

Using whole blood data, XCI-skew correlates with increased monocyte counts and myeloid-to-lymphoid ratios, and reduced IL-10 levels, suggesting changes in immune cell composition and functional consequences.

Crucially, we identified 27% of identical twin pairs in TwinsUK as discordant for XCI-skew status, representing genetically identical individuals who have aged discordantly for an immune cell phenotype with known epidemiological associations.

We hypothesise that age-acquired XCI-skew in blood could be utilised as a biomarker for immune ageing, and the overarching aim of this grant is to elucidate the pathways which contribute to age-related development of frailty and cognitive decline through utilising XCI-skew.

We will assess the changes to the immune system that are captured and caused by XCI-skew in ageing females, and how this sex-specific cellular phenotype correlates with measures of ill-health, physical frailty, and cognitive decline. Our aims are to: 1) Define the functional effects of XCI-skew on TLR7/8 and NLRP3 through ex vivo immune challenges.

Using 25 pairs of identical twins (50 individuals) who are discordant for XCI-skew, thus perfectly controlling for age and genotypic effects in pairwise analyses, we will test the hypothesis that XCI-skew affects inflammatory responses to immune challenge.

We will target the TLR7/8 and NLRP3 pathways, which are known to be implicated in age-related dysregulated cytokine production, using the TruCulture ex vivo system and measure cytokine levels using Cytometic Bead Array. 2) Explore differential cellular and transcriptomic changes accompanying XCI-skew across the immune system.

Using 10 pairs of identical twins (20 individuals) who are discordant for XCI-skew, thus perfectly controlling for age and genotypic effects, we will test the hypotheses that XCI-skew captures differential gene expression within distinct immune cell populations, and compositional changes across and within immune cell types.

We will capture up to 10,000 unselected PBMCs from each participant using the 10X Chromium Gene Expression single cell sequencing technology.

We will use dimensional reduction for cell clustering and identification before employing linear mixed effects models for statistical analyses.

These analyses will utilise powerful intra-twin study designs, which control against inherited genetic differences, a known source of heterogeneity in single cell studies 3) Characterise the phenotypic effects of XCI-skew in a cross-sectional population study.

We will utilise the full TwinsUK XCI dataset (n=1,575) and employ multivariate mixed effects regression models to assess correlations between XCI-skew and extensive phenotypic measures of frailty, cognitive decline, and ill-health.

Ultimately, we aim to investigate a prevalent and understudied sex-specific cellular phenotype to better understand how age-related changes to the immune system predisposes to adverse health outcomes, including frailty.