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

Abstract

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

“…It is indeed important to note that, on the other hand, homozygous deficiencies of early CP components are the strongest genetic risk factors for developing the autoimmune Systemic Lupus, but not periodontitis (18). Therefore, loss of control of complement activation is one possible component of the disease mechanism, as suggested by a parallel study performed on pEDS variants affecting C1R (19). Studies on patient fibroblasts would be very useful to definitively validate or invalidate this hypothesis, but we do not have access yet to these very scarce samples.…”

“…Studies on patient fibroblasts would be very useful to definitively validate or invalidate this hypothesis, but we do not have access yet to these very scarce samples. It would be indeed interesting to look at a potential constitutive C4 cleavage in pEDS patient fibroblasts SN, for patients affected by C1S mutation, by adding exogenous C4 and looking at its cleavage rate, as shown for some pEDS patients affected by a C1R mutation (19). Since the C1s pEDS variants cannot efficiently cleave C4, C4 cleavage would require an intermediate step of Fg40-mediated activation of full-length C1s, produced by the C1SWT allele.…”

The Relationship between Autism and Ehlers-Danlos Syndromes/Hypermobility Spectrum Disorders

“…The majority of gene mutations associated with the rarer forms of EDS involves fibrillar collagens, proteins that modify collagen, or enzymes involved in collagen processing [ 13 ]. Interestingly, one type of EDS, known as the periodontal type, is associated with variants in two different complement genes involved in the innate immune system, leading to chronic activation of the complement system independent of microbial triggers [ 34 ]. Although C1R and C1S are not collagen-related genes per se, synthesis of types I and III procollagen is nevertheless impaired in this form of EDS, suggesting upstream effects [ 35 ].…”

Periodontal Disease Associated with Genetic Disorders

“…Experimental evidence suggests that it is linked to secretion or release of active C1r serine protease in the extracellular space. This mechanism may cause gingival hyperinflammation in response to mild biofilm accumulation, and subsequently rapidly progressing periodontal destruction [68].…”