Poster Presentation 43rd Lorne Genome Conference 2022

Exploring the effects of X chromosome gene expression in RA using single cell RNA sequencing (#115)

Lachlan G Gray 1 2 , Jose Alquicira 2 3 , Seyhan Yazar 2 , Joseph Powell 1 2 , Sara Ballouz 1 2
  1. Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia
  2. Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia
  3. Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia

Rheumatoid arthritis (RA) is an autoimmune disease which disproportionately affects females. In concert with sex hormones, X-linked genes perturb key signalling pathways in immune cell types and may contribute towards sex bias in autoimmune disease. It has been proposed that incomplete or escape from X chromosome inactivation (XCI) contributes towards heightened immune responses and breakdown of self-tolerance.

 

To test the influence of X-linked genes on RA, we analysed single-cell RNA-sequencing data from PBMCs on a subset of female individuals with RA (n=10) from the OneK1K dataset. We first assessed differences in cell-type proportion, and then performed differential expression analysis in a cell-type specific manner with an age matched set of controls (n=23). We subsequently tested for the overlap of significantly differentially expressed and X-linked genes to measure enrichment for X-escapers, immune genes and signalling pathways.

 

Approximately 1,300 cells were sequenced per individual, and 30 immune cell-types and subtypes identified. We observed significant differences in cell-type proportions of CD14+ Monocytes and CD4 CTL (T-cells) between cases and controls, suggesting roles for these cell- types in RA aetiology. Pseudobulking and applying edgeR-LRT returned fewer significant genes per cell-type (FDR<0.05, mean=2.7±3.8 DEGs) compared to the single-cell approach (FDR<0.05, mean=24.2±49.8 DEGs) but with high concordance (>0.98%). By ranking genes by FDR and log2 fold change, we see chromosome X gene enrichment in B memory cells (Wilcoxon p = 0.05). Interestingly, we found enrichment of X escape genes in RA relevant cells including, B memory cells (Wilcoxon p = 0.009) and T regulatory (Treg) cells (Wilcoxon p = 0.039). These RA DEGs were enriched for immune functions (p = 1.53x10-9), suggesting a role in activating RA pro-inflammatory signalling pathways.

 

Through analysing the transcriptome at a single cell resolution, we have identified inflammatory pathways which are unbalanced by X-linked genes, in particular XCI escape genes.