Molecular Genetic Study Of SOX9 Gene Mutation In Congenital Vertebral Malformations

Background:Campomelic dysplasia is an autosomal dominant skeletal dysplasia caused by SOX9 mutation.It is characterized by congenital shortening and bending of long bone,dysplasia of scapula,stenosis of iliac wing and no mineralization of thoracic pedicle.Due to the respiratory insufficiency related to the hypoplasia of trachea and bronchus,the patients usually die within one year after birth.SOX9 gene mutation is also related with 46,XY sex reversal.However,about 10%of CD patients do not have long bone bending,which is called acampomic CD(ACD).Although it has been confirmed that CD and ACD are caused by the mutation of SOX9,the specific mechanism has not been clarified.Congenital vertebral malformations(CVM)is a skeletal dysplasia which is relatively commonly seen in orthopedics diseases.It is a serious congenital malformation of the spine and one of the main diseases that cause disability in adolescents.Vertebral malformations can be the only phenotype of most CVM patients.Many syndromic CVM patients may only manifest as CVM clinically,without other systemic deformities.CD/ACD patients usually associated with spinal deformity,with a high disability and deformity rate.The limited knowledge of syndromic CVM may result in misdiagnosis of some patients.Therefore,studying the function of SOX9 gene in CVM patients is of great significance to improve the diagnosis accuracy and explore the pathogenesis of SOX9 related skeletal malformations.Objects:1.To investigate SOX9 candidate mutants in CVM patients identified from large scale Whole Exome Sequencing(WES).2.To verify the functional changes of candidate mutants,and explore the underlying pathological mechanism of SOX9 in vitro experiments.3.To provide new directions and ideas for the pathogenesis,diagnosis and targeted treatment of CVM and other skeletal malformations using gene editing mouse model.Methods:A total of 570 CVM patients and 2000 cases without spinal deformity were collected for WES after peripheral genome DNA extraction.The original data obtained from WES were analyzed with Peking Union Medical College Hospital Pipeline(PUMchPipeline,PUMP),which is an in-house developed analytic method for next generation sequencing data.Pathogenic/Likely pathogenicity variants were identified by integrative analysis of both the WES data and the clinical phenotype.The candidate variants were functionally verified by in vitro experiments to confirm their pathogenicity.Finally,the SOX9 gene mutation mouse was successfully constructed by CRISPR/cas9 systerm.The pathological changes of the spine and intervertebral disc of Sox9 gene-editing mice were studied by histopathology.Results:1.In 570 CVM patients,7 patients were found to carry 5 missense mutations of SOX9 with high suspected pathogenicity.They are Ile73Thr,Met113Leu,Ala133Gl,Gly276Cys and Met469Val.Combined the clinical symptoms with bioinformatics analysis,we suspected that patients carried Ile73Thr,Met113Leu and Ala133Gl were likely to be ACD patients,and Gly276Cys and Met469Val were likely to cause CS.2.Through in vitro functional experiments,we confirmed that all of the five mutations were deleterious.Combined with clinical data,WES sequencing data and functional experiments,we proved that CS69,CS900 and CS670 were in line with the molecular diagnosis of ACD.In addition,the Gly276Cys and Met469Val mutations were novel variants.The SOX9 transactivation abilities were significantly decreased,therefore we suppose that these two mutations are new pathogenic mutants leading to CS.3.We successfully constructed Sox9 mutation mouse model by CRISPR/Cas9 technology.We found that the homozygous Sox9del/del(D272del)mice recapitulated the phenotype as the Gly276Cys patient in human.ABH/OG showed that the impaired bone development of caudal vertebraes and the shape of the intervertebral disc.4.Both the variant of D272del and the Gly276Cys located at the K2 domain of SOX9.Because both the mice and our patient showed spinal dysplasia,we suggest that K2 domain may play an important role in the pathogenecity of CS.Conclusion:In this study,7 patients carried five missense mutation were identified.These five variants were confirmed to be deterious through Western blot,Immunofluorescence,luciferase and EMSA in vitro function test.Three patients with CVM received molecular diagnosis of ACD.We successfully constructed D272del mice model by CRISPR/Cas9 systerm,and studied the pathological changes of the spine and intervertebral disc.We suggest that the K2 domain of SOX9 may play a pivotal role in the pathogenesis of CS.