Analysis And Identification Of Novel Mutation Of EXT Genes In Hereditary Multiple Exostoses

Hereditary Multiple Exostoses(HME),also called Multiple Osteochondromas(MO),is an autosomal-dominant disorder characterized by bone defects and multiple exostoses that form within the perichondrium flanking the growth plates of long bones.A part of HME patients can be short stature,suffer from asymmetric exostoses,or even undergo malignant transformation,turning into chondrosarcomas.Current studies suggest that HME is associated with mutations in EXT1 gene located in 8q24.11-q24.13 and EXT2 gene located in 11q11-12.So far,over 700 kinds of EXT1/EXT2 gene mutations having been found,but there are still some unknown mutations waiting for us to discover.Thus,in this study,we take HME families and rare cases of HME as earriers to analyse and identify novel mutations of EXT1 and EXT2 genes.Part Ⅰ Identification of novel mutation of EXT1 gene in hereditary multiple exostoses pedigree and research of disease-causing gene in a rare case of intra-articular osteochondroma manifesting as loose bodiesObjective:To investigate the EXT1 gene mutation of a Han Chinese HME family,and analyse the disease-causing gene of a rare case of HME combined with intra-articular osteochondroma manifesting as loose bodies.Methods:A three-generation Han Chinese family of HME from Zhejiang was collected in this study.The pedigree chart was drawn.Clinical data and blood DNA samples were collected.Physical and radiographical examinations were performed.And then the features of the disease were summarized.Finally,the mutation screening of EXT1 and EXT2 genes was performed.After genomic DNA was extracted from blood samples,29 primers were designed by Primer 5.0 software.Then PCR was performed to amplify the coding regions and intron-exon boundaries of EXT1 and EXT2 genes.The PCR products were then confirmed by polyacrylamidegel electrophoresis,and finally sequenced by Sanger direct sequencing.In silico analysis was used to analyse possible changes in protein structure that may affect the expression of EXT1 gene and pathogenicity of HME.Besides,a rare case of HME combined with intra-articular osteochondroma manifesting as loose bodies was collected.The clinical feature,diagnosis and treatment methods,radiographical data,and pathologic findings were summarized and analysed.The blood DNA samples of the patient’s family were also extracted to perform mutation screening of EXT genes.Results:After mutation screening,a novel heterozygous nonsense mutation c.1902C>A(p.Tyr634X)in EXT1 gene was identified,which was unreported before.In silico analysis suggested the critical impact of this novel mutation on EXT1 gene function,which would cause morbidity of HME.The patient of HME combined with intra-articular osteochondroma manifesting as loose bodies underwent surgery,and the operation curative effect was quite good.A heterozygous nonsense mutation c.115G>T(p.E39X)in EXT1 gene was detected in this patient and his mother,which was first reported in Chinese.Conclusions:A novel heterozygous nonsense mutation c.1902C>A(p.Tyr634X)in EXT1 gene was the disease-causing mutation of the family from Zhejiang with HME.And another heterozygous nonsense mutation c.115G>T(p.E39X)in EXT1 gene was the disease-causing mutation of the rare case of HME combined with intra-articular osteochondroma manifesting as loose bodies.Part Ⅱ Identification of novel mutation of EXT2 gene in hereditary multiple exostoses pedigree and research of disease-causing gene in a rare case of distal tibiofibular syndesmosis bone bridge formed by osteochondromasObjective:To investigate the EXT2 gene mutation of a Han Chinese HME family,and analyse the disease-causing gene of a rare case of distal tibiofibular syndesmosis bone bridge formed by osteochondromas.Methods:A three-generation Han Chinese family of HME from Jiangxi was collected in this study.The pedigree chart was drawn.Clinical data and blood DNA samples were collected.Physical and radiographical examinations were performed.And then the features of the disease were summarized.Finally,the mutation screening of EXT1 and EXT2 genes was performed.After genomic DNA was extracted from blood samples,31 primers were designed by Primer 5.0 software.Then PCR was performed to amplify the coding regions and intron-exon boundaries of EXT1 and EXT2 genes.The PCR products were sequenced by Sanger direct sequencing.In silico analysis was used to analyse possible changes in protein structure that may affect the expression of EXT2 gene and pathogenicity of HME.Besides,a rare case of distal tibiofibular syndesmosis bone bridge formed by osteochondromas was collected.The clinical feature,diagnosis and treatment methods,radiographical data,and pathologic findings were summarized and analysed.The blood DNA samples of the patient’s family were also extracted to perform mutation screening of EXT genes.Results:After mutation screening,a novel heterozygous nonsense mutation c.67C>T(p.Arg23X)in EXT2 gene was identified,which was unreported before.In silico analysis suggested the critical impact of this novel mutation on EXT2 gene function,which would cause morbidity of HME.The patient of distal tibiofibular syndesmosis bone bridge formed by osteochondromas underwent surgery,and the operation curative effect was quite good.The same heterozygous nonsense mutation c.67C>T(p.Arg23X)in EXT2 gene was also detected in this patient and her family member who suffered from HME.Conclusions:A novel heterozygous nonsense mutation c.67C>T(p.Arg23X)in EXT2 gene was both the disease-causing mutation of the family from Jiangxi with HME and the rare case of distal tibiofibular syndesmosis bone bridge formed by osteochondromas.