Identification and Structural Characterization of a Novel COL3A1 Gene Duplication in a Family With Vascular Ehlers-Danlos Syndrome

Abstract

Background: Vascular Ehlers-Danlos syndrome (vEDS) is caused by alterations in the COL3A1 gene, typically involving missense variants that replace glycine residues. In contrast, short in-frame insertions, deletions, and duplications are rare and pose significant challenges for investigation.

Methods: The histological examination of vascular tissue from a 26-year-old man, who died from a common iliac artery aneurysm and whose mother died at age 60 from an abdominal aortic dissection, strongly suggested a diagnosis of Ehler-Danlos type IV. Ex vivo collagen phenotype assessment, molecular analysis, and in silico structural studies of type III collagen were subsequently performed.

Results: Ex vivo analysis of the patient's fibroblasts revealed altered collagen synthesis, whereas the molecular testing identified a novel 18-nucleotide in-frame duplication (c.2868_2885dup-GGGTCTTGCAGGACCACC) in the COL3A1 gene, resulting in a six-amino acid insertion, p.(Leu958_Gly963dup). Structural investigation indicated that this duplication led to a local perturbation of the collagen triple helix near a metalloproteinase cleavage site.

Conclusion: This study highlights the pathogenic role of a novel in-frame duplication in the COL3A1 gene, demonstrating how this seemingly benign alteration significantly compromises collagen turnover and contributes to the development of vEDS.

Keywords: COL3A1 gene; collagen; duplication; genetic variant; metalloproteinase; vascular Ehlers–Danlos syndrome.

FIGURE 1

(a) (2.5×, scale bar = 500 μm): (1) thrombus within the vascular lumen; (2) media layer; (3) adventitia layer; (4) dissecting hematoma within the media layer; (5) wall degeneration. Figure 1b–e: (20×, scale bar = 100 μm) Four specific stains are displayed: (b) H&E, (c) PASD, (d) SMA, and (e) Silver. The H&E, PASD, and SMA staining reveals an evident wall degeneration characterized by myocyte loss and sclerosis, whereas the silver staining highlights the elastic laminae destruction.

FIGURE 2

6% SDS–PAGE of pepsin‐treated collagen secreted by fibroblasts (M) and retained in cell layer (CL) of a patient affected by vEDS (P) and an age‐matched control (C).

FIGURE 3

The location of the 18 nucleotides duplication in the COL3A1 gene and the corresponding insertion of 6 amino acids into the type III α1 chain are underscored. The MMP cleavage site, shown both as nucleotide and amino acid sequence, is highlighted in grey.

FIGURE 4

Representative conformations: (a) wild type; (b) mutated homotrimer; (c) hetero‐trimers with one mutated chain; (d) heterotrimers with two mutated chains. Colour code: Wild type chain leading A (white), middle B (grey), trailing C (black); mutant chain leading A (yellow), middle B (orange), trailing C (red). In all systems Pro residues are highlighted by van der Waals spheres. Insertions are highlighted as well as transparent grey surfaces. The unique collagenase cleavage site between the Gly and Ile residues is marked with a red bar. The exosite recognized by the hemopexin domain of MMP‐1, crucial for cleaving the collagen triple helix, is highlighted with a blue box and it overlaps with the mutated region. Trajectories analysis: (e) number of interchain hydrogen bonds and (f) number of contacts, average over 500 ns of molecular dynamics simulation; (g) average binding free energy be‐tween two chains and (h) between one chain and the other two. Energies were calculated over 500 ns of molecular dynamics simulation with the MM/GBSA method. Error bars are standard deviations.

FIGURE 5

Molecular dynamics results. (a) For each model: Protein backbone root mean squared deviation (RMSD) in grey the original dataset, in black the same data spline‐smoothed; 500 ns trimers conformation with the Hyp groups highlighted; and root mean squared fluctuation (RMSF) for each chain, where the curves of chain C and B have been displaced by 0.1 and 0.2 nm, respectively. Color code: Wild type (black/white‐grey/grey), mutants (red/yellow/orange). (b) Snapshots corresponding to the maximum deformation of the collagen observed during the simulation: 0.0104 nm⁻¹ for the wild type, and 0.0116 nm⁻¹ for the mutated homotrimer, respectively. Color code: Wild type (black/grey/white), mutant (red/orange/yellow). The movies with the entire dynamics are available as Supporting Information.