Dermal fibroblasts derived from types I and IV Ehlers-Danlos syndrome (EDS) patients, carrying mutations in COL5A1 and COL3A1 genes, respectively, synthesize aberrant types V and III collagen (COLL) and show defective organization of these proteins into the extracellular matrix (ECM) and high reduction of their functional receptor, the α₂β₁ integrin, compared with control fibroblasts. EDS cells also show reduced levels of fibronectin (FN) in the culture medium and lack an FN fibrillar network. Finally, EDS cells prevalently organize α_vβ₃ integrin instead of α₅β₁ integrin. The α_vβ₃ integrin, distributed on the whole EDS cell surface, shows FN binding and assembly properties when the cells are treated with purified FN. Treatment of EDS cells with purified COLLV or COLLIII, but not with FN, restores the control phenotype (COLL⁺, FN⁺, α_vβ₃^–, α₅β₁⁺, α₂β₁⁺). Function-blocking antibodies to COLLV, COLLIII, or α₂β₁ integrin induce in control fibroblasts an EDS-like phenotype (COLL^–, FN^–, α_vβ₃⁺, α₅β₁^–, α₂β₁^–). These results show that in human fibroblasts α₂β₁ integrin organization and function are controlled by its ligand, and that the α₂β₁-COLL interaction, in turn, regulates FN integrin receptor recruitment: high α₂β₁ integrin levels induce α₅β₁ integrin organization, while low α₂β₁ integrin levels lead to α_vβ₃ integrin organization.

Background: Classic Ehlers–Danlos syndrome (cEDS) is a rare autosomal dominant connective tissue disorder that is primarily characterized by skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. A recent study demonstrated that more than 90% of patients who satisfy all of these major criteria harbor a type V collagen (COLLV) defect. Methods: This cohort included 40 patients with cEDS who were clinically diagnosed according to the Villefranche nosology. The flowchart that was adopted for mutation detection consisted of sequencing the COL5A1 gene and, if no mutation was detected, COL5A2 analysis. In the negative patients the presence of large genomic rearrangements in COL5A1 was investigated using MLPA, and positive results were confirmed via SNP-array analysis. Results: We report the clinical and molecular characterization of 40 patients from 28 families, consisting of 14 pediatric patients and 26 adults. A family history of cEDS was present in 9 patients. The majority of the patients fulfilled all the major diagnostic criteria for cEDS; atrophic scars were absent in 2 females, skin hyperextensibility was not detected in a male and joint hypermobility was negative in 8 patients (20% of the entire cohort). Wide inter- and intra-familial phenotypic heterogeneity was observed. We identified causal mutations with a detection rate of approximately 93%. In 25/28 probands, COL5A1 or COL5A2 mutations were detected. Twenty-one mutations were in the COL5A1 gene, 18 of which were novel (2 recurrent). Of these, 16 mutations led to nonsense-mediated mRNA decay (NMD) and to COLLV haploinsufficiency and 5 mutations were structural. Two novel COL5A2 splice mutations were detected in patients with the most severe phenotypes. The known p. (Arg312Cys) mutation in the COL1A1 gene was identified in one patient with vascular-like cEDS. Conclusions: Our findings highlight that the three major criteria for cEDS are useful and sufficient for cEDS clinical diagnosis in the large majority of the patients. The borderline patients for whom these criteria fail can be diagnosed when minor signs of connective tissue diseases and family history are present and when genetic testing reveals a defect in COLLV. Our data also confirm that COL5A1 and COL5A2 are the major, if not the only, genes involved in cEDS.