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

Mutations in PIGS, Encoding a GPI Transamidase, Cause a Neurological Syndrome Ranging from Fetal Akinesia to Epileptic Encephalopathy

Inherited GPI deficiencies (IGDs) are a subset of congenital disorders of glycosylation that are increasingly recognized as a result of advances in whole-exome sequencing (WES) and whole-genome sequencing (WGS). IGDs cause a series of overlapping phenotypes consisting of seizures, dysmorphic features, multiple congenital malformations, and severe intellectual disability. We present a study of six individuals from three unrelated families in which WES or WGS identified bi-allelic phosphatidylinositol glycan class S (PIGS) biosynthesis mutations. Phenotypes included severe global developmental delay, seizures (partly responding to pyridoxine), hypotonia, weakness, ataxia, and dysmorphic facial features. Two of them had compound-heterozygous variants c.108G>A (p.Trp36∗) and c.101T>C (p.Leu34Pro), and two siblings of another family were homozygous for a deletion and insertion leading to p.Thr439_Lys451delinsArgLeuLeu. The third family had two fetuses with multiple joint contractures consistent with fetal akinesia. They were compound heterozygous for c.923A>G (p.Glu308Gly) and c.468+1G>C, a splicing mutation. Flow-cytometry analyses demonstrated that the individuals with PIGS mutations show a GPI-AP deficiency profile. Expression of the p.Trp36∗ variant in PIGS-deficient HEK293 cells revealed only partial restoration of cell-surface GPI-APs. In terms of both biochemistry and phenotype, loss of function of PIGS shares features with PIGT deficiency and other IGDs. This study contributes to the understanding of the GPI-AP biosynthesis pathway by describing the consequences of PIGS disruption in humans and extending the family of IGDs

Early infantile epileptic encephalopathy due to biallelic pathogenic variants in PIGQ : Report of seven new subjects and review of the literature

We investigated seven children from six families to expand the phenotypic spectrum associated with an early infantile epileptic encephalopathy caused by biallelic pathogenic variants in the phosphatidylinositol glycan anchor biosynthesis class Q (PIGQ) gene. The affected children were all identified by clinical or research exome sequencing. Clinical data, including EEGs and MRIs, was comprehensively reviewed and flow cytometry and transfection experiments were performed to investigate PIGQ function. Pathogenic biallelic PIGQ variants were associated with increased mortality. Epileptic seizures, axial hypotonia, developmental delay and multiple congenital anomalies were consistently observed. Seizure onset occurred between 2.5 months and 7 months of age and varied from treatable seizures to recurrent episodes of status epilepticus. Gastrointestinal issues were common and severe, two affected individuals had midgut volvulus requiring surgical correction. Cardiac anomalies including arrythmias were observed. Flow cytometry using granulocytes and fibroblasts from affected individuals showed reduced expression of glycosylphosphatidylinositol (GPI)-anchored proteins. Transfection of wildtype PIGQ cDNA into patient fibroblasts rescued this phenotype. We expand the phenotypic spectrum of PIGQ-related disease and provide the first functional evidence in human cells of defective GPI-anchoring due to pathogenic variants in PIGQ

Early infantile epileptic encephalopathy due to biallelic pathogenic variants inPIGQ: Report of seven new subjects and review of the literature

We investigated seven children from six families to expand the phenotypic spectrum associated with an early infantile epileptic encephalopathy caused by biallelic pathogenic variants in the phosphatidylinositol glycan anchor biosynthesis class Q (PIGQ) gene. The affected children were all identified by clinical or research exome sequencing. Clinical data, including EEGs and MRIs, was comprehensively reviewed and flow cytometry and transfection experiments were performed to investigate PIGQ function. Pathogenic biallelicPIGQvariants were associated with increased mortality. Epileptic seizures, axial hypotonia, developmental delay and multiple congenital anomalies were consistently observed. Seizure onset occurred between 2.5 months and 7 months of age and varied from treatable seizures to recurrent episodes of status epilepticus. Gastrointestinal issues were common and severe, two affected individuals had midgut volvulus requiring surgical correction. Cardiac anomalies including arrythmias were observed. Flow cytometry using granulocytes and fibroblasts from affected individuals showed reduced expression of glycosylphosphatidylinositol (GPI)-anchored proteins. Transfection of wildtypePIGQcDNA into patient fibroblasts rescued this phenotype. We expand the phenotypic spectrum ofPIGQ-related disease and provide the first functional evidence in human cells of defective GPI-anchoring due to pathogenic variants inPIGQ

Mutations in PIGB cause an inherited GPI biosynthesis defect with an axonal neuropathy and metabolic abnormality in severe cases

Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions

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