Animal Models of Ehlers-Danlos Syndromes: Phenotype, Pathogenesis, and Translational Potential

Abstract

The Ehlers-Danlos syndromes (EDS) are a group of heritable connective tissues disorders mainly characterized by skin hyperextensibility, joint hypermobility and generalized tissue fragility. Currently, 14 EDS subtypes each with particular phenotypic features are recognized and are caused by genetic defects in 20 different genes. All of these genes are involved in the biosynthesis and/or fibrillogenesis of collagens at some level. Although great progress has been made in elucidating the molecular basis of different EDS subtypes, the pathogenic mechanisms underlying the observed phenotypes remain poorly understood, and consequentially, adequate treatment and management options for these conditions remain scarce. To date, several animal models, mainly mice and zebrafish, have been described with defects in 14 of the 20 hitherto known EDS-associated genes. These models have been instrumental in discerning the functions and roles of the corresponding proteins during development, maturation and repair and in portraying their roles during collagen biosynthesis and/or fibrillogenesis, for some even before their contribution to an EDS phenotype was elucidated. Additionally, extensive phenotypical characterization of these models has shown that they largely phenocopy their human counterparts, with recapitulation of several clinical hallmarks of the corresponding EDS subtype, including dermatological, cardiovascular, musculoskeletal and ocular features, as well as biomechanical and ultrastructural similarities in tissues. In this narrative review, we provide a comprehensive overview of animal models manifesting phenotypes that mimic EDS with a focus on engineered mouse and zebrafish models, and their relevance in past and future EDS research. Additionally, we briefly discuss domestic animals with naturally occurring EDS phenotypes. Collectively, these animal models have only started to reveal glimpses into the pathophysiological aspects associated with EDS and will undoubtably continue to play critical roles in EDS research due to their tremendous potential for pinpointing (common) signaling pathways, unveiling possible therapeutic targets and providing opportunities for preclinical therapeutic interventions.

Keywords: EDS; Ehlers–Danlos syndromes; animal models; mouse; zebrafish.

FIGURE 1

Schematic overview of collagen and glycosaminoglycan (GAG) biosynthesis and collagen fibrillogenesis. Molecules defective in Ehlers-Danlos syndromes (EDS) are highlighted in bold. (A) Fibrillar collagen biosynthesis starts with transcription and translation of pro-α-chains (step 1). Nascent pro-α-chains are heavily post-translationally modified by several proline and lysine hydroxylases and galactosyltransferases (step 2). The association of the C-terminal propeptides of three pro-α-chains, initiates triple helix formation which propagates to the N-terminus in a zipperlike fashion and is assisted by several molecular chaperones (step 3). The trimeric procollagen molecules aggregate laterally, are transported in secretory vesicles and are eventually directed to the extracellular environment (step 4). Removal of the N- and C-propeptides, by ADAMTS-2 and BMP-1/mTLD, respectively, results in the formation of a collagen molecule (step 5) that can then assemble into highly ordered striated fibrils. The tissue-specific assembly of collagen fibrils requires the concerted action of several assisting proteins, categorized as organizers, nucleators and regulators (step 6). At the plasma membrane, fibronectin and integrins serve as organizers of fibril assembly. Some collagens, such as type V collagen, function as nucleators, which initiate immature fibril assembly at the cell surface. Type V collagen co-assembles with type I collagen into heterotypic fibrils with the entire triple helical domain of type V collagen embedded within the fibril, whereas its partially processed N-propeptide domain protrudes to the fibril surface and controls fibrillogenesis by sterically hindering the addition of collagen monomers. The intermediate fibrils are then deposited into the extracellular matrix (ECM). Stabilization of these fibrils is provided by interactions with regulators such as the small leucine-rich proteoglycan (SLRP) decorin, tenascin-X and type XII collagen, which influence the rate of assembly, size and structure of the collagen fibrils. As fibrillogenesis proceeds, fibril growth occurs through linear and lateral fusion of intermediate collagen fibrils which are subsequently stabilized by the formation of covalent intra- and inter-molecular cross-links. (B) GAG biosynthesis starts with the synthesis of a proteoglycan core protein which is subsequently modified by several Golgi-resident enzymes. Initially, a common linker region containing four monosaccharides is formed. Biosynthesis of this tetrasaccharide linker region starts with the stepwise addition of a xylose (Xyl) residue to a specific serine residue of the core protein, catalyzed by xylosyltransferase-I and II (XylT-I/-II). Subsequently, two galactose (Gal) residues are added by galactosyltransferase-I (GalT-I or β4GalT7) and galactosyltransferase-II (GalT-II or β3GalT6). Finally, the addition of a glucuronic acid (GlcA), catalyzed by glucuronosyltransferase-I (GlcAT-I) completes the formation of the linker region. The alternating addition of either N-acetyl-glucosamine (GlcNAc) or N-galactosyl-glucosamine (GalNAc) and GlcA defines the composition of the GAG-chain and subdivides proteoglycans into heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS)/dermatan sulfate (DS) proteoglycans. The GAG-chains are then further modified by epimerization and sulfation. DS synthesis necessitates the epimerization of GlcA towards iduronic acid (IdoA), which is catalyzed by DS epimerases–1 and -2 (DS-epi1 and DS-epi2). Subsequently, dermatan 4-O-sulfotransferase-1 (D4ST1) is able to catalyze 4-O-sulfation of GalNAc, thereby preventing back-epimerization of the adjacent IdoA.

FIGURE 2

Timeline illustrating the first identification of molecular defects in human EDS (above timeline) and the first description of engineered animal models targeting an EDS-associated gene (below timeline). Human genes associated with EDS without a corresponding animal model are indicated in dark gray. Note that for some human EDS subtypes, biochemical and/or ultrastructural findings preceded the identification of the molecular defect. Mouse models are depicted in light gray and zebrafish models in blue. Bold indicates what was first, either the identification of the human disease gene or the generation of the engineered animal model.

FIGURE 3

Phenotypic characteristics of mouse models with defects in EDS-associated genes. The presence or absence of a phenotype in mice is indicated with “+” or “-”, respectively, when investigated. A detailed description of the murine phenotypes can be found in Supplementary Table 1. The major and minor clinical characteristics in human EDS patients as defined in the International EDS Classification, published in 2017, are indicated with a gray background (Malfait et al., 2017). HI, haploinsufficiency; KO, (homozygous) knockout; TG, transgenic; KI, knock-in; Y, viable; ↓, decreased survival rate.

FIGURE 4

Overview of the phenotypic findings in some mouse models of EDS. (A) Col5a1^+/– mouse model of cEDS. Images adapted from Wenstrup et al. (2006). (B) Col3a1^Tg–G182S mouse model of vEDS. Image adapted from D’hondt et al. (2018). (C) Plod1^–/– mouse model of kEDS-PLOD1. Images adapted from Takaluoma et al. (2007). (D) Dse^–/– mouse model of mcEDS-DSE. Image adapted from Maccarana et al. (2009). (E) Slc39A13-KO mouse model of spEDS-SLC39A13. Images adapted from Fukada et al. (2008). Images depicted in (A,C,D,E) were used under the Creative Commons License and image B with license number 5090710991420.

FIGURE 5

Phenotypic characteristics of zebrafish models with defects in EDS-associated genes. The presence or absence of a phenotype in the zebrafish model is indicated with “+” or “-”, respectively, when investigated. A detailed description of the zebrafish phenotypes can be found in Supplementary Table 2. The major and minor clinical characteristics in humans EDS patients as defined in the International EDS Classification, published in 2017, are indicated with a gray background (Malfait et al., 2017). BCS, brittle cornea syndrome; KO, knockout; KD, (morpholino-based) knockdown; HM, hypomorphic; Y, viable; N, not viable.

FIGURE 6

Overview of the phenotypic findings in zebrafish models of EDS. (A) Adult col1a2^–/– zebrafish model of cvEDS. Images adapted from Gistelinck et al. (2018). (B) Larval b4galt7 morphant, crispant and knockout models of spEDS-B4GALT7. Scale bars: 1 mm. Images adapted from Delbaere et al. (2019a). (C) Adult b3galt6^–/– zebrafish model of spEDS-B3GALT6. Images adapted from Delbaere et al. (2020). (D) Morpholino (MO)-based knockdown of prdm5 in zebrafish larvae. Images adapted from Meani et al. (2009). Images depicted in (A,C,D) were used under the Creative Commons License and image B with license number 5078981221357.