Periodontal Ehlers-Danlos Syndrome Is Caused by Mutations in C1R and C1S, which Encode Subcomponents C1r and C1s of Complement.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.

Degradation of collagen I by activated C1s in periodontal Ehlers-Danlos Syndrome.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and C1S genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder.

C1R Mutations Trigger Constitutive Complement 1 Activation in Periodontal Ehlers-Danlos Syndrome.

Heterozygous missense or in-frame insertion/deletion mutations in complement 1 subunits C1r and C1s cause periodontal Ehlers-Danlos Syndrome (pEDS), a specific EDS subtype characterized by early severe periodontal destruction and connective tissue abnormalities like easy bruising, pretibial haemosiderotic plaques, and joint hypermobility. We report extensive functional studies of 16 C1R variants associated with pEDS by in-vitro overexpression studies in HEK293T cells followed by western blot, size exclusion chromatography and surface plasmon resonance analyses. Patient-derived skin fibroblasts were analyzed by western blot and Enzyme-linked Immunosorbent Assay (ELISA). Overexpression of C1R variants in HEK293T cells revealed that none of the pEDS variants was integrated into the C1 complex but cause extracellular presence of catalytic C1r/C1s activities. Variants showed domain-specific abnormalities of intracellular processing and secretion with preservation of serine protease function in the supernatant. In contrast to C1r wild type, and with the exception of a C1R missense variant disabling a C1q binding site, pEDS variants had different impact on the cell: retention of C1r fragments inside the cell, secretion of aggregates, or a new C1r cleavage site. Overexpression of C1R variants in HEK293T as well as western blot analyses of patient fibroblasts showed decreased levels of secreted C1r. Importantly, all available patient fibroblasts exhibited activated C1s and activation of externally added C4 in the supernatant while control cell lines secreted proenzyme C1s and showed no increase in C4 activation. The central elements in the pathogenesis of pEDS seem to be the intracellular activation of C1r and/or C1s, and extracellular presence of activated C1s that independently of microbial triggers can activate the classical complement cascade.

Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes.

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.