NEW INSIGHTS IN BONE BIOLOGY FROM EXOME SEQUENCING OF RARE SKELETAL DISEASES

Whole exome sequencing (WES) is a powerful tool to identify new molecules involved in skeletal homeostasis. In particular we used WES to establish the molecular diagnosis of two particular skeletal diseases: osteopetrosis and the acrofrontofacionasal dysostosis 1 (AFFND1). The osteopetroses are a group of rare bone diseases characterized by increased bone density due to the failure in bone resorption. Due to their genetic heterogeneity, WES represents a valuable strategy to identify the genetic defect. We analyzed osteopetrotic patients with autosomal dominant osteopetrosis (ADO) and autosomal recessive osteopetrosis (ARO), which is the most severe form. In our cohort we performed molecular diagnosis of 4 ADOI or ADOII patients that carried mutations in the LRP5 and CLCN7 genes, respectively. The analysis of ARO patients confirmed TCIRG1 as the most frequently mutated gene, identified mutations in the other known ARO genes and in genes very rarely associated with osteopetrosis, namely FERMT3 and USB1. Of note, we demonstrated the causative role of four deep intronic mutations in TCIRG1 gene and two different synonymous changes in the TCIRG1 and CLCN7 genes in the pathogenesis of the disease. In addition, WES helped in the differential diagnosis in a patient who was found to bear a mutation in the FAM20C gene, known to cause Raine syndrome. Regarding AFFND1, this is an extremely rare syndrome, comprising facial and skeletal abnormalities, short stature and intellectual disability. WES found a novel truncating mutation in the neuroblastoma-amplified sequence (NBAS) gene in two Indian patients (c.6237-3C>G). This mutation impaired NBAS functions in HEK293T cells overexpressing the truncated NBAS protein. Furthermore, we demonstrated that NBAS expression in mouse embryos was compatible with a role in bone and brain development and that the depletion of endogenous z-nbas in fish embryos resulted in defective morphogenesis of chondrogenic cranial skeletal elements. Overall, we provided evidence supporting the hypothesis of a causative role of the mutated NBAS gene in the pathogenesis of AFFND1. In conclusion, we effectively exploited WES in the genetic diagnosis of rare skeletal diseases. We also highlighted potential limitations of this approach, specifically with respect to deep intronic mutations and synonymous changes, and underlined the importance to complement WES with analysis at the transcript level and functional validation, when possible.