Albert Amberger

The identification of a pathogenic variant (PV) in one of the mismatch repair (MMR) genes confirms the diagnosis of Lynch syndrome (LS). Hence, the correct classification of MMR gene variants is of utmost importance for appropriate counselling, surveillance, and treatment of LS patients and their families. In 7/200 unrelated Tyrolean-suspected LS patients, we identified the rare variant MLH1:c.836T>G. Clinical and tumor data strongly indicate that this founder variant is associated with an increased risk for early-onset LS-associated tumors. We also demonstrate that the variant leads to aberrant mRNA splicing. However, the splice effect’s leakiness together with the small effect of the amino acid change p.(Val297Gly) encoded by the residual full-length transcripts in a functional assay preclude its formal classification as (likely) PV according to internationally accepted variant interpretation guidelines. The family histories of the carriers suggest that the obstacles to classify the variant as (likely) PV may be related with a reduced penetrance. Nonetheless, and despite the formal classification of MLH1:c.836T>G as a variant of uncertain significance, we show that carriers should undergo cancer surveillance and predictive testing should be offered to relatives. This variant illustrates the need for an improved classification framework for appropriate categorization of lower-penetrance alleles.

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.