Single Cell Sequencing and TCR Analysis for Undiagnosed Rare Disease

Abstract

An undiagnosed rare disease is a disease that has not been previously described due to an unknown causal agent. It is an important first step in understanding and identifying the pathogenesis to treat the undiagnosed female patient who suffers from systemic inflammatory symptoms and chronic colitis. As a result of whole-genome sequencing, we confirm that the FOXP3, the master regulator gene of Treg, is deleted on a single X chromosome (heterozygous deletion) and Skewed X Chromosome Inactivation (XCI) pattern in the patient might lead to the disease phenotype. In this paper, we conducted single-cell RNA sequencing(scRNA-seq) along with TCR analysis to identify the disease causality. First, we annotate CD4 subgroups after clustering the scRNA-seq. then we observed that the overall amount of memory population has decreased compared to the control group. On the other hand, the overall amount of effector T cells has increased. As a result of detailed analysis on the effector cell group, it is observed that CMVand Influenza-specific Th1 population has increased. Simultaneously, immature natural regulatory T cell(nTreg) population and conversion of induced regulatory T cell(iTreg) were observed in the sample. It suggests that there was dysregulation of immune homeostasis and regulatory system on immune cells. Further studies such as cell-fate maps are required to verify the hypothesis of pathogenesis mechanism, which Treg dysfunctions cause the disease phenotype on the patient. scRNA-seq Analysis allows us to have a comprehensive analysis of the immune system and the antigenspecific host defense system at a single-cell level. Also, this approach suggests indirect information about how to specific immune cells could be involved in disease.