Identification And Characterization Of Novel Hereditary Multiple Exostoses-associated Mutations

Background: Hereditary multiple osteochondroma(HME)is an autosomal single gene dominant b one dysplasia,more than 70% of which is caused by EXT1 and EXT2 mutations.According to the datas of multiple osteochondroma mutation database,more than 700 mutation sites hav e been found in EXT1 and EXT2 genes.Both EXT1 and EXT2 can encode type II transmembrane glycoproteins and participate in the biosynthesis of HS.It is reported that the loss of heterozygosity or compound heterozygosity mutation of EXT1 and EXT2 genes will lead to the decrease of HS synthesis,resulting in the occurrence of HME.But the specific pathogenic mechanism is not completely clear.Objective: In order to expand the pathogenic mutation spectrum of HME and provide clinical genetic counsel ing for patients.The members of 5independent consanguineous families with HME were perfo rmed in whole exome sequencing,and the results of whole exome sequencing were analyzed by us to screen the pathogenic mutations.Methods: The blood samples of HME family members were collected and sequenced with whole exome sequencing.The sequencing resu lts were analyzed to screen the disease-related mutant genes.The selected mutation sites were verified by Sanger sequencing.Silico analysis was used to predict the change of m RNA level caused by mutation.To verify this prediction,we analyzed the m RNA o f the gene about the mutations of the patient by RT-PCR and TA cloning.Further,the EXT2 m RNA of HME patients was compared with that of healthy controls.At the same time,in order to explore whether the function of the mutant gene changed at the cellular level,the plasmid associated with EXT2 was constructed by us,and whether the subcellular localization of EXT2 t runcated protein(Protein encoded by abnormally spliced EXT2 transcripts)was consistent with that of EXT2 normal protein(Protein encoded by the full-length coding sequence of EXT2)was explored by cellular immune-fluorescence assay,and whether the amoun t of HS synthesized by normal EXT2 protein and EXT2 truncated protein was explored by ELISA experiment.Results: After whole exome sequencing being performed in 5 independent consanguineous HME families(family I-V),we found that the pathogenic mutations in 4 HME families were EXT1(c.1776C>G,c.G600A)and EXT2(c.G1286 A,c.1173+2T>A).A novel heterozygous splice site mutation(c.1173+2T>G)in EXT2 gene was detected by WES in a third-generation pedigree V with three diseased individuals.This novel mutation caused exon 7 of EXT2 m RNA to be skipped out,resulting in a shift of the codon reading frame of Arg360,caus ing an early termination codon to appear after the synthesis of 43 new amino acids.The results of cellular immunofluorescence showed that there was no difference in the subcellular localization of EXT2 truncated protein and EXT2 normal protein,both of them were co-located with Golgi marker GM130.In the ELISA assay,it was found that compared with the normal EXT2 protein,the HS synthesized by EXT2 truncated protein was significantly decreased,which affected the regulation of chondrocyte proliferation an d differentiation Conclusions: Most of the pathogenic genes in these 5 independent consanguineous HME families are related to EXT1(c.1776C>G,c.G600A)and EXT2(c.G1286 A,c.1173+2T>A).The heterozygous splicing mutation of c.1173+2T>A in EXT2 gene is the main cause of HME in family V,and a novel pathogenic mutation of HME.The splicing mutation leads to the skipping of exon 7 of EXT2 gene transcript,resulting in abnormal EXT2 transcripts,which may be mediated by nonsense m RNA degradation,resulting in abnormal EXT2 m RNA degradation that can not be detected.EXT2 truncated protein and EXT2 normal protein,but both of them were co-localized with Golgi marker GM130.Therefore,the cause of the disease in this family is not caused by the abnormal accumulation of abnormal proteins in the endoplasmic reticulum.In the correct subcellular localization,compared with EXT2 normal protein,EXT2 truncated protein due to the deletion of Glycosyl Transferase Family 64 domain,resulting in a significant decrease in HS synthesis activity.These evid ences suggest that the novel c.1173+2T>A mutation led to the low expression of EXT2 m RNA and the decrease of HS synthesis activity,which leads to the decrease of the total amount of HS synthesis,which is the basis of the pathogenesis of HME in the family V.