Heterozygous THBS2 pathogenic variant causes Ehlers-Danlos syndrome with prominent vascular features in humans and mice

Abstract

Ehlers-Danlos syndromes (EDS) are a group of connective tissue disorders caused by mutations in collagen and collagen-interacting genes. We delineate a novel form of EDS with vascular features through clinical and histopathological phenotyping and genetic studies of a three-generation pedigree, displaying an apparently autosomal dominant phenotype of joint hypermobility and frequent joint dislocations, atrophic scarring, prolonged bleeding time and age-related aortic dilatation and rupture. Coagulation tests as well as platelet counts and function were normal. Reticular dermis displayed highly disorganized collagen fibers and transmission electron microscopy (TEM) revealed abnormally shaped fibroblasts and endothelial cells, with high amount and irregular shape of extracellular matrix (ECM) substance, especially near blood vessels. Genetic analysis unraveled a heterozygous mutation in THBS2 (NM_003247.5:c.2686T>C, p.Cys896Arg). We generated CRISPR/Cas9 knock-in (KI) mice, bearing the heterozygous human mutation in the mouse ortholog. The KI mice demonstrated phenotypic traits correlating with those observed in the human subjects, as evidenced by morphologic, histologic, and TEM analyses, in conjunction with bleeding time assays. Our findings delineate a novel form of human EDS with classical-like elements combined with vascular features, caused by a heterozygous THBS2 missense mutation. We further demonstrate a similar phenotype in heterozygous THBS2^Cys896Arg KI mice, in line with previous studies in Thbs2 homozygous null-mutant mice. Notably, THBS2 encodes Thrombospondin-2, a secreted homotrimeric matricellular protein that directly binds the ECM-shaping Matrix Metalloproteinase 2 (MMP2), mediating its clearance. THBS2 loss-of-function attenuates MMP2 clearance, enhancing MMP2-mediated proteoglycan cleavage, causing ECM abnormalities similar to those seen in the human and mouse disease we describe.

Fig. 1. Pedigree and clinical manifestations.

A Clinical findings (patient III:1) (a–d) joint hypermobility (e) atrophic scarring (f) piezogenic papules. B Family tree: black filling denotes affected individuals heterozygous for the THBS2 NM_003247.5:c.2686T>C p.Cys896Arg variant. Individual I:1 (in gray) passed away at the age of 70 before being clinically or genetically assessed and is likely affected based on the limited available clinical history. C, D MRA of thorax and abdomen demonstrating dilated aortic arch (patient II:1).

Fig. 2. The THBS2 pathogenic variant.

A Sanger sequencing of affected (II-1) and healthy (II-2) individuals. The THBS2 NM_003247.5:c.2686T>C variant is marked by a black rectangle. B Conservation analysis of the locus surrounding the THBS2 p.Cys896Arg variant. Symbols below indicate conservation: asterisk for complete identity; colon for high similarity and blank for none. (Clustal-Omega - https://www.ebi.ac.uk/Tools/msa/clustalo) C 3D model of the crystalized THBS2 protein showing the Cys876-Cys896 disulfide bond eliminated by the THBS2 p.Cys896Arg variant (marked in red) (https://www.rcsb.org/structure/1YO8).

Fig. 3. CRISPR/Cas9 THBS2 p.Cys896Arg heterozygous knock-in mice.

A Schematic representation of the CRISPR/Cas9 modification used to generate knock-in mice with the c.2686T>C variant and Sanger sequencing result from F0 knock-in mice. B An image and a video (C) of a F0 knock-in female mouse having a knot in her tail while keeping calm. Notably, wild type mice cannot have their tails tied in that manner. D Bleeding time assay for wild-type (left) and heterozygote (right) male littermates at the time of experiment termination. All wild-type mice stopped bleeding after less than 4 min while mutant mice bled over 11 min.

Fig. 4. Histology of upper back skin punch biopsies from patient III:1 and an age-matched control.

H&E staining (A control, B patient; X100, insert X400) demonstrate multiple dilated blood vessels with mild perivascular lymphocytic infiltrate in the reticular dermis (dotted arrows) with mucinous material between disorganized collagen fibers (full arrows). Masson’s trichrome staining of the same subjects highlighting the disorganized and thin collagen fibers in the papillary dermis (C – control, D – patient; X400) and, most prominently, in the reticular dermis (E – control, F – patient; X400).