Exploring the role of fibronectin in spondylometaphyseal dysplasia

La fibronectine (FN), une glycoprotéine largement exprimée, a été associée à de nombreux processus biologiques fondamentaux, mais n’a jamais été impliquée dans des maladies osseuses. L’identification de mutations dans le gène qui code pour cette protéine chez des patients présentant un sous-type rare de dysplasie spondylométaphysaire (SMD) a révèlé un aspect inexploré de cette protéine. Des études récentes ont montré que ces mutations empêchent la sécrétion de la FN dans les cellules HEK293, mais comment ce défaut contribue mécanistiquement à la pathogenèse de la SMD est encore inconnu. Pour étudier les effets des mutations in vitro, un protocole de différenciation des chondrocytes a été optimisé et testé sur des cellules ATDC5. En outre, pour déterminer si les phénotypes des patients étaient dus à une réduction globale de la FN, des souris simples knock-out (KO) et double KO dépourvues de FN dans le foie et/ou le cartilage ont été générées. Les résultats révèlent que la suppression de la FN dans ces tissus ne nuit ni à la croissance ni à la viabilité. Aucune modification n'a été détectée dans le poids ni dans la longueur du corps des KO par rapport aux souris témoins, et aucune anomalie du squelette n'a été observée sur les radiographies. Ces résultats montrent que la suppression de FN dans ces deux tissus ne conduit pas aux phénotypes observés dans les SMD. De plus, le séquençage du gène FN1 chez des individus soupçonnés d’être atteints de SMD a permis l’identification d’une mutation de novo chez un patient. Cette découverte élargit les caractéristiques cliniques de cette maladie induit par FN1.A widely expressed glycoprotein, fibronectin (FN), has been associated with many basic biological processes, but has never been implicated with skeletal disorders. Identification of mutations in the gene that encodes this protein in patients with a rare subtype of spondylometaphyseal dysplasia (SMD) reveals an unexplored aspect in the field. Recent studies have shown that these mutations impair FN secretion in HEK293 cells, but it is still not known how this defect mechanistically contributes to SMD pathogenesis. To investigate the effects of the mutations in vitro, a chondrocyte differentiation protocol was optimized and tested on ATDC5 cells. Furthermore, to determine if the patients’ phenotypes were caused by a global reduction of FN, single and double knockout (KO) mice that lack FN in the liver and/or the cartilage were generated. Results reveal that deletion of FN in these tissues does not impair growth or viability. No changes were detected in the body weight nor length in the KO compared to control mice, and no skeletal abnormalities were observed in radiographs. These results show that the deletion of FN in these two tissues does not lead to the phenotypes observed in SMD. Additionally, sequencing of the FN1 gene in individuals with suspected SMD identified a de novo mutation in one patient. This finding expands the clinical features of FN1-induced SMD

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

Heterozygous pathogenic variants in the FN1 gene, encoding fibronectin (FN), have recently been shown to be associated with a skeletal disorder in some individuals affected by spondylometaphyseal dysplasia with “corner fractures” (SMD-CF). The most striking feature characterizing SMD-CF is irregularly shaped metaphyses giving the appearance of “corner fractures”. An array of secondary features, including developmental coxa vara, ovoid vertebral bodies and severe scoliosis, may also be present. FN is an important extra cellular matrix component for bone and cartilage development. Here we report five patients affected by this subtype of SMD-CF caused by five novel FN1 missense mutations: p.Cys123Tyr, p.Cys169Tyr, p.Cys213Tyr, p.Cys231Trp and p.Cys258Tyr. All individuals shared a substitution of a cysteine residue, disrupting disulfide bonds in the FN type-I assembly domains located in the N-terminal assembly region. The abnormal metaphyseal ossification and “corner fracture” appearances were the most remarkable clinical feature in these patients. In addition, generalized skeletal fragility with low-trauma bilateral femoral fractures was identified in one patient. Interestingly, the distal femoral changes in this patient healed with skeletal maturation. Our report expands the phenotypic and genetic spectrum of the FN1-related SMD-CF and emphasizes the importance of FN in bone formation and possibly also in the maintenance of bone strength.Peer reviewe

Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations