Periodic Reporting for period 1 - XX-Health (X-chromosome biology and immune health in females)
Reporting period: 2022-11-01 to 2025-04-30
Females have a higher risk for autoimmune disease (e.g. lupus) and lower risk of mortality from infectious disease (e.g. COVID-19) than males, reflecting a more robust immune response in females against both self-antigens (autoimmunity) and non-self-antigens (infections). Whereas the cause of sex bias in immunity is unresolved, several lines of evidence suggest that genes on the X-chromosome that ‘escape’ the female-specific process of X-inactivation (XCI) may result in a more active immune response in females. However, investigating the contribution of X-chromosome biology to immune responses is complicated by the unique biology of the X-chromosome in females.
Whereas every cell in a female has two X-chromosomes, one of the two X-chromosomes in each cell is randomly inactivated, a process called X-inactivation (XCI). As XCI is random, different cells in the same female can inactivate the maternal (Xm) or paternal X-chromosome (Xp) resulting in the property of ‘mosaicism’. In addition, different ratios of Xm and Xp may become inactivated in cells of a given tissue resulting in skewed X-inactivation (sXCI). Mosaicism and skewing make the study of X-inactivation in humans very challenging. However, some females inactivate the same X-chromosome in all their cells (complete XCI, cXCI), removing the confounding effect of mosaicism, and offering a powerful genetic system in which to dissect XCI in T-cell biology.
In XX-Health, we will develop a novel method to allow screening for cXCI in biobanks of 1000’s of blood samples from healthy females. Then using blood from cXCI females we will study the role of genes escaping X-inactivation in T-cell biology. This will involve the use of state-of-the-art genomics including single-cell sequencing and long-read sequencing, as well as genetic editing of specific alleles on the X-chromosomes in the primary immune cells from cXCI females. In addition, our study will allow us to investigate the origin of skewed X-inactivation itself and test if sXCI is associated with auto-immune disorders as has often been reported by others.
Sex-bias in COVID-19 mortality has highlighted the importance of sex as a contributor to disease risk. The technical and conceptual advances delivered by XX-Health will make a seminal contribution to our understanding of this poorly understood component of human health.
Whereas every cell in a female has two X-chromosomes, one of the two X-chromosomes in each cell is randomly inactivated, a process called X-inactivation (XCI). As XCI is random, different cells in the same female can inactivate the maternal (Xm) or paternal X-chromosome (Xp) resulting in the property of ‘mosaicism’. In addition, different ratios of Xm and Xp may become inactivated in cells of a given tissue resulting in skewed X-inactivation (sXCI). Mosaicism and skewing make the study of X-inactivation in humans very challenging. However, some females inactivate the same X-chromosome in all their cells (complete XCI, cXCI), removing the confounding effect of mosaicism, and offering a powerful genetic system in which to dissect XCI in T-cell biology.
In XX-Health, we will develop a novel method to allow screening for cXCI in biobanks of 1000’s of blood samples from healthy females. Then using blood from cXCI females we will study the role of genes escaping X-inactivation in T-cell biology. This will involve the use of state-of-the-art genomics including single-cell sequencing and long-read sequencing, as well as genetic editing of specific alleles on the X-chromosomes in the primary immune cells from cXCI females. In addition, our study will allow us to investigate the origin of skewed X-inactivation itself and test if sXCI is associated with auto-immune disorders as has often been reported by others.
Sex-bias in COVID-19 mortality has highlighted the importance of sex as a contributor to disease risk. The technical and conceptual advances delivered by XX-Health will make a seminal contribution to our understanding of this poorly understood component of human health.
We are currently approaching the halfway point in the project and have already established a novel technique that allows for high-throughput screening of skewed X-inactivation in human blood samples. This approach allows accurate determination of skewing from as little as 20ng of DNA, which is important, given that many archived blood samples are of poor quality or of vanishingly small quantities. Further, our approach is cheap, requires only basic laboratory equipment and expertise and can be quickly ported to a clinical setting. To date we have screened over 1000 umbilical cord blood samples for skewing and found support for the presence of completed skewed (cXCI) females in the general female population. Using these females we have begun to investigate the origin of cXCI and found highly divergent causes of cXCI between individuals. However, additional genomic analyses with so-called long-read sequencing and more cXCI females are required before firm conclusions regarding the genetic origins of cXCI can be reported. To further validate our analysis of skewed XCI in biobanked material in Sweden, we have also performed extensive bioinformatics analysis of genomic data from females in publicly available databases, identifying additional cXCI females not detected in previous studies. We have also established many of the more technically challenging approaches, such as single-cell sequencing and genetic editing of primary human immune cells that will be required for functionally dissecting the contribution of the inactive X-chromosome to T-cell biology in females.
Our development of a novel method for accurate low-cost, low-tech and high-throughput screening of skewed XCI progresses well beyond the state of the art. Not only is this technology critical to the successful implementation of our own research, but we also expect its uptake as the gold-standard within the field upon publication. Moreover, its robustness and simplicity may also facilitate its translation into the clinical setting, where female carriers of X-linked disorders are often tested for skewed X-inactivation, but in an unregulated and inconsistent way. We are currently in the process of patenting this technique prior to its academic publication By the end of the project, we will have used this approach to reveal the true frequency of skewed XCI in the general female population. Whereas the potential causes of cXCI are many, our large cohort if cXCI females and access to both neonatal and longitudinal samples will give us unprecedented power to conclusively identify the genetic origin of cXCI in at least some females, and act as a springboard for future studies in the fundamental molecular process governing XCI.