Age acquired skewed X chromosome inactivation is associated with adverse health outcomes in humans
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Evaluation Summary:
Women are a mosaic of two population of cells, one with the paternal X-chromosome and the other with the maternal one in the active state due to random X-chromosome inactivation (XCI) that occurs during embryogenesis. During aging, one of the two populations dominates the other in a significant proportion of women. This skewing of XCI is of unknown etiology and its impact on health remains enigmatic. In this study, Amy L. Robert et al, demonstrate that skewing may not be benign and it is associated with a modest but significant increased risk of cardiovascular disease and cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
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.
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Author Response
Reviewer #3 (Public Review):
Understanding the relevance of skewed X-Chromosome Inactivation (XCI) in women disease susceptibility and development is an intriguing open question. In this manuscript entitled "Age acquired skewed X Chromosome Inactivation is associated with adverse health outcomes in humans" Roberts et al. attempt to characterize this relationship by assaying skewed X-Chromosome Inactivation in >1.500 females from the TwinsUK population cohort. The authors reported an association between skewed XCI and increased cardiovascular risk across the tested population. This association is reinforced by a twin study based on age matched twin pairs discordant in their degree of XCI skewing. This approach is indeed powerful as it controls for age in predicting the cardiovascular disease risk score. The authors …
Author Response
Reviewer #3 (Public Review):
Understanding the relevance of skewed X-Chromosome Inactivation (XCI) in women disease susceptibility and development is an intriguing open question. In this manuscript entitled "Age acquired skewed X Chromosome Inactivation is associated with adverse health outcomes in humans" Roberts et al. attempt to characterize this relationship by assaying skewed X-Chromosome Inactivation in >1.500 females from the TwinsUK population cohort. The authors reported an association between skewed XCI and increased cardiovascular risk across the tested population. This association is reinforced by a twin study based on age matched twin pairs discordant in their degree of XCI skewing. This approach is indeed powerful as it controls for age in predicting the cardiovascular disease risk score. The authors also found an association between skewed XCI and a haematopoietic bias towards the myeloid lineage. Finally, skewed XCI was shown to be predictive of future cancer incidence. -This area of research is timely and of great interest for the community. However, in my opinion, the conclusions of this manuscript are not fully supported by the presented data and some aspects of the data analysis and results need to be extended.
We thank the reviewer for their kind comments on the importance of our study. We do hope they agree that, through addressing the necessary changes outlined above, particularly with regards to the discussion, that the conclusions of the manuscript are now more nuanced and present the work in the broader context of related fields, in particular clonal haematopoiesis. Overall, we hope the reviewer agrees that the conclusions of the manuscript now better reflect the results presented.
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Evaluation Summary:
Women are a mosaic of two population of cells, one with the paternal X-chromosome and the other with the maternal one in the active state due to random X-chromosome inactivation (XCI) that occurs during embryogenesis. During aging, one of the two populations dominates the other in a significant proportion of women. This skewing of XCI is of unknown etiology and its impact on health remains enigmatic. In this study, Amy L. Robert et al, demonstrate that skewing may not be benign and it is associated with a modest but significant increased risk of cardiovascular disease and cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Amy L. Roberts et al. Investigated the health outcomes impacts of age acquired skewing of X-chromosome inactivation (XCI) ratios occurring in blood cells in 1575 females comprised of 423 monozygotic twins pairs, 257 dizygotic twins pairs and 215 singleton. They demonstrate: (i) associating between skewing and age; (ii) skewing was independent of other biological markers of aging such as smoking, telomere length, or DNAm Grim Age acceleration and frailty index (iii). (iv) that skewing was associated with a myeloid bias with an increase monocyte to lymphocyte and neutrophil to lymphocyte ratio; (v)a strong negative association with IL-10 level and skewing; (vi) skewing was associated with increase cardiovascular risk; and that (vi) skewing was predictive of cancer.
Age-associated XCI skewing has been described …
Reviewer #1 (Public Review):
Amy L. Roberts et al. Investigated the health outcomes impacts of age acquired skewing of X-chromosome inactivation (XCI) ratios occurring in blood cells in 1575 females comprised of 423 monozygotic twins pairs, 257 dizygotic twins pairs and 215 singleton. They demonstrate: (i) associating between skewing and age; (ii) skewing was independent of other biological markers of aging such as smoking, telomere length, or DNAm Grim Age acceleration and frailty index (iii). (iv) that skewing was associated with a myeloid bias with an increase monocyte to lymphocyte and neutrophil to lymphocyte ratio; (v)a strong negative association with IL-10 level and skewing; (vi) skewing was associated with increase cardiovascular risk; and that (vi) skewing was predictive of cancer.
Age-associated XCI skewing has been described 25 years ago, yet this phenomenon remains enigmatic in terms of etiology and consequences. The study demonstrates the age-effect on skewing (which is known), but the evolution over time in a sub cohort of subject that were re-sampled. The originality of this study resides in the demonstration that age-associated skewing is associated with health outcomes. The particular strength of the study is the twin component (always aged-matched) and the iterative re-sampling of subgroup of the population study. Despite starting with a large cohort of 1552 individuals, most of the critical observations are made on smaller subsets of subjects: for ASCV risk score the total of subject is 231; IL-10 n=27. Fortunately, for the ASCV score this observation is re-enforced by the twin pairs comparison (N=34). The association with cancer risk is performed on a larger cohort (1417) and controlled for age. However, despite increased risk of Cardiovascular event and cancer, there is no significant association with overall mortality. This indicate that the effect of age-associated skewing is present but modest. The observation that there is a myeloid bias, which is associated with aging, that is more pronounced in subjects with skewing is interesting.
One of the major unknown of this study is how much of the effect attributed to XCI skewing is in fact related to true clonal hematopoiesis or CHIP that is associated with both outcomes described in this study and will mascarade by changes in XCI. -
Reviewer #2 (Public Review):
In this manuscript, Roberts et al. explore whether ageing-induced X-chromosome inactivation (XCI) skewing could be a valuable biomarker for adverse disease outcomes in women. XCI is a dosage compensation process resulting in the inactivation of one of the two X chromosomes in females to equalize X-linked gene dosage between XX and XY individuals. XCI is initially established in a random fashion and, as consequence, female tissues have roughly 1:1 ratio of cells that inactive either the paternal or maternal X chromosomes. However, skewing towards one of the two X chromosomes has been noticed, especially in mitotically active tissues, such as the hematopoietic system. This is likely to be caused by fluctuations in hematopoietic cell pool such as stem cell depletion/clonal expansion.
In this study, the authors …
Reviewer #2 (Public Review):
In this manuscript, Roberts et al. explore whether ageing-induced X-chromosome inactivation (XCI) skewing could be a valuable biomarker for adverse disease outcomes in women. XCI is a dosage compensation process resulting in the inactivation of one of the two X chromosomes in females to equalize X-linked gene dosage between XX and XY individuals. XCI is initially established in a random fashion and, as consequence, female tissues have roughly 1:1 ratio of cells that inactive either the paternal or maternal X chromosomes. However, skewing towards one of the two X chromosomes has been noticed, especially in mitotically active tissues, such as the hematopoietic system. This is likely to be caused by fluctuations in hematopoietic cell pool such as stem cell depletion/clonal expansion.
In this study, the authors take advantage of the large TwinsUK cohort of 1,575 female individuals, many of which are mono- or dizygotic twins, for which relevant data (e.g., blood cell counts, molecular markers, clinical information) has been gathered over a period spanning 20 years. XCI skewing was determined by HUMARA, a classical method to measure skewing based on methylation differences between inactive and active X chromosomes in the highly polymorphic Androgen Receptor X-linked gene. The major strength of this study is the large cohort containing genetically identical or similar individuals which is the golden standard for study epigenetic phenomena such as XCI. Despite this, some of the parameters studied by the authors could only be measured in a subset of patients. As the authors, themselves, comment this reduces the statistical power to draw more definitive conclusions.
The data gathered by the authors strengthen previous findings that XCI skewing increases with ageing. This finding is not novel, but this study represents the most comprehensive analysis of age acquired XCI skewing to my knowledge. Interestingly, XCI skewing did not correlate with several hallmarks of biological ageing, but is associated with myeloid bias of the hematopoietic lineage and increased risk of cardiovascular disease or cancer incidence. Their results show the importance of the use of large twin cohorts for epigenetic studies to better understand disease progression.
Whether XCI skewing is a bystander of fluctuations in hematopoietic stem cell pool due to depletion or clonal expansion or itself could have a direct role in disease progression remains unknown. In any case, this study also sets the stage for future functional studies, using animal models, where XCI skewing or hematopoietic stem pool could be manipulated, to better understand the link between XCI skewing and increase propensity to age-driven disorders.
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Reviewer #3 (Public Review):
Understanding the relevance of skewed X-Chromosome Inactivation (XCI) in women disease susceptibility and development is an intriguing open question. In this manuscript entitled "Age acquired skewed X Chromosome Inactivation is associated with adverse health outcomes in humans" Roberts et al. attempt to characterize this relationship by assaying skewed X-Chromosome Inactivation in >1.500 females from the TwinsUK population cohort. The authors reported an association between skewed XCI and increased cardiovascular risk across the tested population. This association is reinforced by a twin study based on age matched twin pairs discordant in their degree of XCI skewing. This approach is indeed powerful as it controls for age in predicting the cardiovascular disease risk score. The authors also found an …
Reviewer #3 (Public Review):
Understanding the relevance of skewed X-Chromosome Inactivation (XCI) in women disease susceptibility and development is an intriguing open question. In this manuscript entitled "Age acquired skewed X Chromosome Inactivation is associated with adverse health outcomes in humans" Roberts et al. attempt to characterize this relationship by assaying skewed X-Chromosome Inactivation in >1.500 females from the TwinsUK population cohort. The authors reported an association between skewed XCI and increased cardiovascular risk across the tested population. This association is reinforced by a twin study based on age matched twin pairs discordant in their degree of XCI skewing. This approach is indeed powerful as it controls for age in predicting the cardiovascular disease risk score. The authors also found an association between skewed XCI and a haematopoietic bias towards the myeloid lineage. Finally, skewed XCI was shown to be predictive of future cancer incidence.
This area of research is timely and of great interest for the community. However, in my opinion, the conclusions of this manuscript are not fully supported by the presented data and some aspects of the data analysis and results need to be extended.
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