Citation: Ritelli, M.; Chiarelli, N.; Cinquina, V.; Zoppi, N.; Bertini, V.; Venturini, M.; Colombi, M. RNA-Seq of Dermal Fibroblasts from Patients with Hypermobile Ehlers–Danlos Syndrome and Hypermobility Spectrum Disorders Supports Their Categorization as a Single Entity with Involvement of Extracellular Matrix Degrading and Proinflammatory Pathomechanisms. Cells 2022, 11, 4040. https://doi.org/10.3390/ cells11244040 Academic Editors: Michela Pozzobon, Victor Carriel Araya and Sebastián San Martín Received: 10 November 2022 Accepted: 12 December 2022 Published: 14 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article RNA-Seq of Dermal Fibroblasts from Patients with Hypermobile Ehlers–Danlos Syndrome and Hypermobility Spectrum Disorders Supports Their Categorization as a Single Entity with Involvement of Extracellular Matrix Degrading and Proinflammatory Pathomechanisms Marco Ritelli 1,† , Nicola Chiarelli 1,† , Valeria Cinquina 1 , Nicoletta Zoppi 1 , Valeria Bertini 1 , Marina Venturini 2 and Marina Colombi 1, * 1 Division of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia, 25121 Brescia, Italy 2 Division of Dermatology, Department of Clinical and Experimental Sciences, Spedali Civili University Hospital Brescia, 25121 Brescia, Italy * Correspondence: marina.colombi@unibs.it; Tel.: +39-030-3717240 † These authors contributed equally to this work. Abstract: Hypermobile Ehlers–Danlos syndrome (hEDS) and hypermobility spectrum disorders (HSD) are clinically overlapping connective tissue disorders of unknown etiology and without any validated diagnostic biomarker and specific therapies. Herein, we in-depth characterized the cellular phenotype and gene expression profile of hEDS and HSD dermal fibroblasts by immunofluorescence, amplicon-based RNA-seq, and qPCR. We demonstrated that both cell types show a common cellular trait, i.e., generalized extracellular matrix (ECM) disarray, myofibroblast differentiation, and dys- regulated gene expression. Functional enrichment and pathway analyses clustered gene expression changes in different biological networks that are likely relevant for the disease pathophysiology. Specifically, the complex gene expression dysregulation (mainly involving growth factors, structural ECM components, ECM-modifying enzymes, cytoskeletal proteins, and different signal transducers), is expected to perturb many ECM-related processes including cell adhesion, migration, proliferation, and differentiation. Based on these findings, we propose a disease model in which an unbalanced ECM remodeling triggers a vicious cycle with a synergistic contribution of ECM degradation products and proinflammatory mediators leading to a functional impairment of different connective tissues reflecting the multisystemic presentation of hEDS/HSD patients. Our results offer many promising clues for translational research aimed to define molecular bases, diagnostic biomarkers, and specific therapies for these challenging connective tissue disorders. Keywords: cytokines; extracellular matrix; hypermobile Ehlers–Danlos syndrome; hypermobility spectrum disorders; inflammation; matrix metalloproteinases; myofibroblasts; RNA-seq 1. Introduction The Ehlers–Danlos syndromes (EDS) represent a group of 14 heritable connective tissue disorders (HCTDs), the majority of which result from alterations in genes encoding collagens and enzymes involved in their biogenesis or in the extracellular matrix (ECM) homeostasis [1]. The most common form of these disorders is hypermobile EDS (hEDS) that is characterized by a marked variable phenotype and high rate of chronic disability and remains the unique variant without an identified molecular cause. Hence, hEDS is diagnosed only clinically based on a set of more stringent criteria presented in 2017 by the International EDS Consortium [2]. hEDS is currently defined by the simultaneous presence of generalized joint hypermobility (gJHM) according to an age-specific Beighton score (BS) Cells 2022, 11, 4040. https://doi.org/10.3390/cells11244040 https://www.mdpi.com/journal/cells