Single cell analysis of dysregulated inducible DNA amplifications of immune response genes in Down syndrome

. Despite knowing for more than 60 years that three copies of chromosome 21 (chr21, T21) is the genetic cause of Down syndrome (DS), we still know relatively little about how T21 drives the vast majority of DS pathophysiology. Over the last several decades, alterations in the response to immune signaling have been identified in T21 cells, mouse models of DS and individuals with DS. Indeed, we have shown that the interferon (IFN) response is constitutively active in T21 cells, leading to an interferonopathy-like state of global immune dysregulation, likely due to the presence of four IFN receptors (IFNRs) in a single locus on chr21. Recently, we identified the IFNR locus as a target of directed transient site-specific gene amplification (TSSG) in response to immune activation. Importantly, we found that while the IFNR cluster is subject to TSSG in healthy cells, this process is impaired in T21 cells. Our work to understand rereplication of the IFNR locus has uncovered that innate immune signaling drives rereplication of a network of immune response genes that may be dysregulated in DS. This project will test the hypothesis that innate immune activation drives a heterogenous program of inducible DNA rereplication of immune response loci which is dysregulated by T21. This project addresses Component 1 of the INCLUDE project, targeted high risk – high reward basic science studies in areas highly relevant to DS and aligns closely with the NIAID interest in examination of immune system dysregulation in Down syndrome and its molecular basis. Dysregulation of the immune system and interferon signaling in T21 is a key contributor to many comorbidities associated with DS and our project will delineate further avenues of dysregulation in the immune response in T21 cells. This work will employ a novel single cell amplicon sequencing approach which we leverage to identify transient changes in copy number for the first time. This work will determine how many immune response genes are amplified during immune activation and how this is dysregulated in T21. Furthermore, we will determine if the innate immune driven rereplication program is conserved in primary mouse T cells and if it is dysregulated in a mouse model of DS. Given the novelty of our observation and hypothesis, this proposal seeks to answer a number of questions regarding the innate immune program in T21 including: 1) How do T21 cells suppress TSSG of the immune response? 2) How many immune response loci are amplified in the same cell at the same time and is this heterogeneity different in T21? 3.) Does T21 initiate a different immune response gene amplification program? Using human cell culture models and a novel amplicon sequencing approach we will comprehensively determine the extent of dysregulation of DNA rereplication of the immune response program in T21. Project Number: 1R21AI190900-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Kelly Sullivan | Institution: UNIVERSITY OF COLORADO DENVER, Aurora, CO | Award Amount: $234,000 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 IVBH-A (56)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19090001