Mechanistic insights into the cellular effects of a novel FN1 variant associated with a spondylometaphyseal dysplasia

Abstract

Spondylometaphyseal dysplasia (SMD) is characterized by developmental changes in long bones and vertebrae. It has large phenotypic diversity and multiple genetic causes, including a recent link to novel variants in the extracellular matrix (ECM) protein fibronectin (FN), a regulator of ECM assembly and key link between the ECM and proper cell function. We identified a patient with a unique SMD, similar to SMD with corner fractures. The patient has been followed over 19 years and presents with short stature, genu varum, kyphoscoliosis, and pectus carinatum. Radiography shows metaphyseal changes that resolved over time, vertebral changes, and capitular avascular necrosis. Whole exome sequencing identified a novel heterozygous FN1 variant (p.Cys97Trp). Using mass spectroscopy, mutant FN was detected in plasma and in culture medium of primary dermal fibroblasts isolated from the patient, but mutant protein was much less abundant than wild-type FN. Immunofluorescence and immunoblotting analyses show that mutant fibroblasts assemble significantly lower amounts of FN matrix than wild-type cells, and mutant FN was preferentially retained within the endoplasmic reticulum. This work highlights the importance of FN in skeletal development, and its potential role in the pathogenesis of a subtype of SMD.

Novel fibronectin mutations and expansion of the phenotype in spondylometaphyseal dysplasia with “corner fractures”

“…Novel featured bilateral corner fractures in both femurs and wrist that resolved over time [5]. A similar clinical scenario is typical for metaphyseal anadysplasia, a disease that is characterized by severe metaphyseal changes during growth, which resolve spontaneously upon skeletal maturation [29,30].…”

“…Five out of seven previously reported mutations describe similar cysteine substitutions that affect disulfide bonds in the FN type-I domains (Fig. 3) [4,5]. Only one missense mutation, p.Tyr240Asp, affects an amino acid other than cysteine in the FN type-I domain 5 (Fig.…”

“…Previous in vitro studies testing two cysteine substitutions, p.Cys87Phe and p.Cys260Gly, and the non-cysteine substitution p.Tyr240Asp in HEK293 cells showed that the mutated FN is retained within the cells instead of being secreted [4]. More recently, studies on skin fibroblasts of the patient with a p.Cys97Trp mutation showed that the mutated FN is preferentially retained in the endoplasmic reticulum instead of being secreted thus leading to a reduced production of FN matrix compared to control fibroblasts [5]. Furthermore, this patient also shows decreased levels of plasma FN.…”

“…Previously, COL2A1 mutations were reported in three individuals [2,3]. Only recently, SMD-CF has been linked to mutations in the FN1 gene, encoding fibronectin (FN) [4,5]. FN is a ubiquitous glycoprotein in the extracellular matrix (ECM) with multiple structural and regulatory functions [6,7].…”

“…These include developmental coxa vara, metaphyseal irregularities, scoliosis, and abnormal ossification at the growth plate and secondary ossification sites, which give rise to a "corner-fracture" like phenotype [1,3,4,17]. Only eleven patients in eight families with FN1 mutation-related SMD-CF have been described thus far [4,5] and therefore the complete genetic and phenotypic spectrum of the disease remains to be more thoroughly characterized.…”