Characterization of molecular mechanisms in hypermobile Ehlers-Danlos syndrome via transcriptome analysis

Summary: Abstract Body: Ehlers-Danlos Syndrome (EDS) is a group of genetic connective tissue disorders characterized by hyperextensible skin, joint hypermobility and cutaneous fragility. Caused primarily by mutations in genes related to collagen and its biogenesis, EDS remains challenging to study due to the lack of reliable in vivo and in vitro models, particularly for the most common type, hypermobility EDS (hEDS). Genetic causes of hEDS are poorly understood. Therefore, recapitulating the hEDS phenotype in a reliable 3D model opens doors to phenotype characterization and causative mutation validation. We developed an in vitro cell-based model of hEDS by generating 3D skin equivalents using hEDS human fibroblasts embedded in thiolated-hyaluronan (HA-SH) fibrin gel scaffolds. These skin equivalents, derived from either primary hEDS fibroblasts or fibroblasts differentiated from hEDS induced pluripotent stem cells, exhibited disorganized collagen fibrils in the extracellular matrix, resembling those in hEDS patient skin biopsies. Spatial transcriptomics of our 3D skin equivalents revealed a distinct biomolecular phenotype of hEDS; including deficiencies in PLOD1 and NFKB2. PLOD1 encodes lysyl-hydroxylase 1 (LH1), essential for fibrillar collagen assembly, while NFKB2 encodes a component of the NFκB complex. Both gene deficiencies were validated via qPCR analysis of fibroblast monolayer cultures from all patients in our cohort (n=18). Immunofluorescent staining has preliminarily validated the deficiency in LH1 on the protein level. Our findings suggest that PLOD1 is regulated via NFκB signaling, and any mutation(s) upstream of this signaling pathway provides a possible link between connective tissue and immunological symptoms of hEDS. CUT&RUN and western blot validations are underway. Parker Jesberg^{1, 2}, Jeevan Mann¹, Nicole Diette¹, Kalpana Velmurugan¹, Ellen Elias³, Maryna Pavlova¹, Igor Kogut¹, Ganna Bilousova¹ 1. Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States. 2. Bioengineering, University of Colorado Anschutz Medical Campus, Aurora, CO, United States. 3. Children's Hospital Colorado, Aurora, CO, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics