Aberrant gene expression from chromosome X in autoimmunity

Autoimmune diseases predominantly affect women. The reason for this sex bias has been historically attributed to 'hormones'. Although sex hormones clearly have some influence on the immune system and autoimmunity, it is becoming increasingly apparent that genetic differences between males and females also contribute to sex related differences in immune function that are likely involved in autoimmune disease. Genetic material in packaged into symmetrically paired chromosomes in the cell with the exception of the sex chromosomes: which in males are XY and in females are XX. The unique male Y chromosome, which confers sex determination, is small and codes for a much smaller set of genes than the X chromosome, which is much larger. In order to compensate for the extra X chromosome present in females compared with males, one of the female X chromosomes is silenced in each cell. This random inactivation of one if the two X chromosomes in each cell results in parity of X chromosome genes in males and females. Interestingly, the process of chromosome inactivation is not completely efficient. Between 10-20% of genes coded on the X chromosome escape the silencing process to some extent, that renders the majority of genes on that X inactive. Thus, for the group of genes escaping inactivation, females would exhibit a greater gene activity because there are two working pairs of genes. The magnitude of the increased gene activity will be contingent on the degree of escape, but if the escape is complete then the elevation in gene activity will be double in females compared with males.

SLE is an instructive autoimmune disease to investigate in the context of the X chromosome because it is nine times more common in females than males. Moreover, preliminary results from separate genetic studies in humans and mice as well as a small study in SLE immune cells provide support for the hypothesis that aberrant X chromosome is associated with the disease.

In this project we will be studying B lymphocytes as these cells are an important immune cell in SLE and other autoimmune diseases. We will primarily employ a technique called single cell RNA sequencing to study the question of X chromosome inactivation in immune cells. In this technique individual cells are isolated and the RNA in the cells is purified, amplified and sequenced. RNA is the material that is immediately derived from genes and reflects the activity of the genes. The purpose of sequencing is RNA is twofold: it provides information about the amount of starting RNA in the cell and secondly, it can tell us about the parental origin of the gene from which it arose. Sequence differences in the gene creating the RNA between the two X chromosomes can be used to distinguish the origin of the RNA, (one X from the person's mother, the other X from the person's father). Thus, RNA sequencing can be used to quantify the parental source of the RNA from each gene in a cell.

If X inactivation is complete, as it will be for most genes present on X, then in each cell the RNA will be derived from only one of the two X chromosomes. If there is some escape from inactivation, then the RNA from the genes will be derived from both of X chromosomes. The amount arising from each chromosome will have some relationship to the extent to which the second X chromosome is active. Single cell RNA sequencing is technically challenging and hence we will use another method using pooled cells (bulk) RNA sequencing as a point of comparison. Bulk RNA sequencing is simpler to undertake, but the results are not as informative or sensitive as single cell sequencing. The RNA sequencing results of B cells from women with SLE and an equal number of healthy controls will be compared to ask a number of questions: which genes escape X inactivation, do more genes escape X inactivation in women with SLE? What function do these genes have? And how might these gene influence the development of SLE?

We will investigate the hypothesis that SLE exhibits excessive loss of X chromosome inactivation (XCI), a physiological process resulting in the elevated expression of a subset of genes coded on the X chromosome in females (compared with males). Evidence supporting this hypothesis includes the increased SLE prevalence in X chromosome polyploidies and murine studies in the Sry-/- model showing segregation of autoimmunity with X chromosome dose rather than hormonal status. To test this hypothesis, we will use single cell (sc)RNA sequencing to quantify the parent-of-origin of X chromosome transcripts using allele-specific expression (ASE) analysis. We will select B cells for analysis as they play a key effector role in SLE (and other autoimmune diseases) and are important therapeutic targets for biologic drugs. The study will comprise analysis of B cells from six females with SLE and six matched healthy controls. Quantifying gene expression from each X chromosome is technically challenging.

scRNA-Seq presents many potential advantages in assessing the chromosomal origin of transcripts at points of heterozygosity. However, transcript dropout and poor representation of low abundance RNA is a concern. To address these issues, a separate aliquot of B cells from each participant will be subject to bulk RNA-Seq. To quantify XCI in bulk data, it is necessary to look for deviation of ASE in individuals with shewed parental X inactivation. We can quantify the latter using the well-established HUMARA assay. Thus, loss of XCI will be quantified by ASE using bulk-RNA- and full-length scRNA-Seq (96 B cells per participant). We can thus generate ranked gene sets showing loss of XCI per individual, comparing variation between cells and between the aggregated scRNA data and bulk data. We will then determine whether there are qualitative and/or quantitative differences between the gene sets escaping XCI in SLE versus controls and the biological pathways implicated.

The two groups of people who might benefit from this project are other researchers and patient groups.

Researchers: The results from this project will have impact on a variety of researchers in the fields of genetics, immunology and biochemistry as described in the academic beneficiaries. Aside from the direct sharing of data from the project in departmental seminars, publications and at conferences, on a broader level we argue that the study will have a more general impact on research.

Students/Post-docs - we will share our workflows and analytical methods in a tutorial format to allow other interested scientist to undertake similar analyses. There will be a link to this information from the Insidegen website and from the manuscripts published. Our workflows and analytical methods may also be collated into a face-to-face 2-day computer-based course for KCL and/or outside students and post-docs. We already have experience in hosting a 2-day Genetic Analysis course for European students.

We will offer a MSc post-graduate project for Jan 2020, to inspire the next generation of students. Information regarding the techniques being used for the project will be incorporated into lectures and tutorials given by the PIs/co-PIs to undergraduate students.

Wider academic community: The study lends emphasis to the role of the X chromosome in disease genetics. It is noted that there is consistent under-reporting of X chromosome genetic signals in genome-wide association studies. The human X chromosome is a similar size to chromosome 7, the X chromosome marker representation on genotyping platforms is on average about half that of chromosome 7, furthermore, we have noted a disproportionate loss of informative markers following quality control of genotyping from X compared with autosomes. By examining the role of X inactivation in the sex bias for SLE and by sharing our research protocols, our findings will both help publicise the importance of the X chromosome in human disease, particularly those showing sexual dimorphism and also allow other researchers to undertake similar studies. We will do this by writing a review article arising from the project and by adding information to the scientific tab of our Insidegen website and on the FaceBook page.

Patients:
The results of our study will also have impact for the patient population. We have already embarked a process of public engagement: we host our own website that provides access to our results: www.insidegen.com. This website also serves a role regarding PPI, but we propose to expand this capacity. We add to basic explanation of SLE to include lay summaries of all of the projects we are undertaking with further details of the techniques being used. There are simple concepts in genetics so that patients and the public can get better understanding of our research and patients can better understand what we do with the samples that they provide us for research. Patients and lay members of the public will be encouraged to provide feedback/ask questions after reading the blog/FaceBook page being set up for the project.

We have recently organised a public engagement meeting with a small PPI group (six patients) at the Wellcome Trust. The feedback was extremely positive. This study investigating the genetics of the female bias in SLE should resonate strongly with patients. The usual explanation for a female bias in SLE that they receive is simply that it is a result of 'female hormones' is rather simplistic and frustrating as the sex hormonal constitution of an individual is not readily alterable. This study focuses on the role of X inactivation in explaining sex bias in SLE. Thus, we argue that the project will receive strong patient support.

We are also in the early stages of setting up a Research Advisory Group for our projects for patients, other researchers and lay members of the public (see communications plan).