Age acquired skewed X chromosome inactivation is associated with adverse health outcomes in humans

Abstract

Background: Ageing is a heterogenous process characterised by cellular and molecular hallmarks, including changes to haematopoietic stem cells and is a primary risk factor for chronic diseases. X chromosome inactivation (XCI) randomly transcriptionally silences either the maternal or paternal X in each cell of 46, XX females to balance the gene expression with 46, XY males. Age acquired XCI-skew describes the preferential selection of cells across a tissue resulting in an imbalance of XCI, which is particularly prevalent in blood tissues of ageing females, and yet its clinical consequences are unknown.

Methods: We assayed XCI in 1575 females from the TwinsUK population cohort using DNA extracted from whole blood. We employed prospective, cross-sectional, and intra-twin study designs to characterise the relationship of XCI-skew with molecular and cellular measures of ageing, cardiovascular disease risk, and cancer diagnosis.

Results: We demonstrate that XCI-skew is independent of traditional markers of biological ageing and is associated with a haematopoietic bias towards the myeloid lineage. Using an atherosclerotic cardiovascular disease risk score, which captures traditional risk factors, XCI-skew is associated with an increased cardiovascular disease risk both cross-sectionally and within XCI-skew discordant twin pairs. In a prospective 10 year follow-up study, XCI-skew is predictive of future cancer incidence.

Conclusions: Our study demonstrates that age acquired XCI-skew captures changes to the haematopoietic stem cell population and has clinical potential as a unique biomarker of chronic disease risk.

Funding: KSS acknowledges funding from the Medical Research Council [MR/M004422/1 and MR/R023131/1]. JTB acknowledges funding from the ESRC [ES/N000404/1]. MM acknowledges funding from the National Institute for Health Research (NIHR)-funded BioResource, Clinical Research Facility and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. TwinsUK is funded by the Wellcome Trust, Medical Research Council, European Union, Chronic Disease Research Foundation (CDRF), Zoe Global Ltd and the National Institute for Health Research (NIHR)-funded BioResource, Clinical Research Facility and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London.

Keywords: X chromosome inactivation; cancer; cardiovascular disease; epidemiology; genetics; genomics; global health; haematopoiesis; human.

Figure 1.. Measuring XCI with the HUMARA assay.

The Human Androgen Receptor Assay (HUMARA) uses methylation-sensitive restriction enzyme digest and PCR to measure skewed X-inactivation. The assay estimates XCI-skew by comparing the relative abundance of allele specific fragments from a mock digest to a methylation-sensitive HpaII digest in which only the alleles from the inactive X are amplified. Representative examples are displayed of fragment analysis of the PCR products for samples with random XCI (top), skewed XCI (middle), and extreme skewed XCI (bottom). The x-axis shows the size, and the y-axis represents the abundance, of the PCR products, respectively. The left panel shows the PCR products after a mock digest with water, resulting in amplification of both alleles regardless of chromosomal inactivation. The right panel shows the PCR products after a restriction enzyme digest with methylation-sensitive enzyme HpaII, resulting in amplification of only the alleles deriving from inactive chromosomes. For each sample, the ratio of the HpaII digested allele products (Rh = allele 1/allele 2) is divided by the ratio of the Mock digest allele products (Rm = allele 1/allele 2) to create a Normalized Ratio (Rn). The XCI percentage is then calculated using the formula [Rn/(Rn +1)] * 100. Images were generated using the Microsatellite Analysis Software on the Thermo Fisher Cloud.

Figure 1—figure supplement 1.. A histogram showing the calculation of the XCI-skew categorical variable from the normalised distribution of XCI scores across the TwinsUK cohort (n=1575).

Figure 1—figure supplement 2.. A flowchart of sample processing and inclusion criteria for each results group.

Figure 2.. Age acquired XCI-skew across age groups and time.

(A) A histogram displaying the age distribution of the TwinsUK HUMARA cohort (age range: 19–99; median age = 61). (B) The proportions of individuals (y-axis) in each of three XCI-skew categories across increasing age groups (x-axis) are shown (N=1575). (C) A Sankey plot shows the longitudinal changes to XCI in 31 individuals across two measures 15–17 years apart. Colours indicate XCI at visit 1, axis 1 displays the age group of individuals at visit 1, and axis 2 displays XCI at visit 2.

Figure 3.. Age acquired XCI-skew and markers of biological ageing and blood cell counts.

Box plots representing the results of the linear regression mixed effects models to assess (A) Smoking status (p=0.33, N_{never_smoker} = 879; N_{ever_smoker} = 673) and (B) obesity (p=0.88, N_{not_obese} = 726; N_obese = 165) after correcting for age with XCI as the dependent variable, and (C) frailty index (p=0.59, N_{ranodm XCI} = 398; n_{Skewed XCI} = 177; N_{extreme skew} = 36), after correcting for age and BMI, (D) Leukocyte telomere length shortening (p=0.9, N_{ranodm XCI} = 278; n_{Skewed XCI} = 103; N_{extreme skew} = 16) after correcting for age and smoking status, and (E) DNAm GrimAge acceleration (p=0.22, N_{ranodm XCI} = 101; n_{Skewed XCI} = 30; N_{extreme skew} = 6), with XCI-skew as the dependent variable. All boxplots display the median and IQR, and have the residuals of the models on the y-axis. (F) A forest plot of associations with data-matched Complete Blood Count data (top panel) and Myeloid-to-lymphoid ratios (bottom) with effect size and lower and upper confidence intervals indicated. Associations were tested with XCI-skew as an independent variable after controlling for age, BMI, seasonality, and smoking status as fixed effects in a linear mixed effects model (N_{ranodm XCI} = 445; n_{Skewed XCI} = 183; N_{extreme skew} = 43). The significance threshold after Bonferroni correction was p<0.005 and p<0.023 to account for multiple testing across the 10 tests and 2 tests, respectively.

Figure 3—figure supplement 1.. Age acquired XCI-skew and Cytokine and CRP measures.

Using a linear regression mixed effects model to control for batch effects, age, seasonality, family structure and zygosity, XCI-skew was assessed for its association with (A) IL-1β (p=0.02); (B) IL-6 (p=0.41); (C) IL-10 (p=0.0008); (D) TNF (p=0.61); (E) CRP (p=0.41). All boxplots display the median and IQR, and have the residuals of the models on the y-axis. The significance threshold after Bonferroni correction for 5 tests was p=0.01. The sample sizes are (A-D) N_{ranodm XCI}=23; n_{Skewed XCI} = 4; (E) N_{ranodm XCI}=121; n_{Skewed XCI} = 38; N_{extreme skew} = 6.

Figure 4.. Age acquired XCI-skew and cardiovascular disease risk score.

(A) Using a linear regression mixed effects model to control for BMI, monocyte count, relatedness and zygosity, XCI-skew is associated with increased atherosclerotic cardiovascular disease risk score (N_{ranodm XCI} = 155; n_{Skewed XCI} = 56; N_{extreme skew} = 17; p=0.01). The boxplot displays the median and IQR. (B) Using age-matched twin pairs discordant for their XCI-skew status (N_pairs = 34), XCI-skew is associated with increased atherosclerotic cardiovascular disease risk score in the intra-twin analysis (one-sided paired samples Wilcoxon test; p=0.025).

Figure 5.. Prospective study of XCI-skew and future cancer diagnosis.

Using a Cox regression analysis and controlling for age, relatedness and zygosity, XCI-skew is predictive of future cancer incidence in a 10 year follow-up (p=0.012; HR = 1.95). Each cancer event represents the first cancer diagnosis (excluding non-melanoma skin cancer) in each individual. A Kaplan-Meier plot (top) and cumulative events (bottom) of cancer diagnosis in individuals with XCI-skew (N=469) and random XCI (N=948) are shown. 2.9% (28/948) of individuals with random XCI, and 6.4% (30/469) of individuals with XCI-skew, developed cancer in the 10 years follow-up.