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To explore the genetic basis for a pedigree affected with hereditary multiple osteochondroma (HMO).Peripheral blood samples were collected from the proband and members of his pedigree with informed consent. Following extraction of genomic DNA, all coding exons and flanking intronic sequences (-10 bp) of the EXT1 and EXT2 genes were subjected to targeted capture and next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing.A heterozygous nonsense variant (c.1911CA) was found in exon 10 of the EXT1 gene in the proband and his affected father but not in a healthy sister and normal controls. The variant was classified as a pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (PVS1+PM2+PP1). Bioinformatic analysis predicted that the c.1911CA variant may be disease-causing via nonsense-mediated mRNA decay and anomalous splicing.The c.1911CA variant probably underlay the disease in this pedigree. Discovery of this variant enriched the variant spectrum of HMO.

To detect variants of EXT1 and EXT2 genes among five pedigrees affected with multiple osteochondromas and provide prenatal diagnosis for the families based on the results.The EXT1 and EXT2 genes of the probands were analyzed by targeted next generation sequencing (NGS). Suspected pathological variants were validated by Sanger sequencing in the probands, their family members and 200 unrelated healthy controls. Multiple ligation-dependent probe amplification (MLPA) was used to confirm the presence of gross deletions. Prenatal diagnosis was provided for 2 couples carrying pathogenic or likely pathogenic variants.Five variants were detected in the pedigrees, which included EXT1 exon 2-3 deletion, c.1468dupC (p.Leu490ProfsX31), c.2084delC (p.Pro695LeufsX11), and EXT2 c.187delT (p.Phe63SerfsX29) and c.1362TG (p.Tyr454X). Among these, EXT1 exon 2-3 deletion, c.2084delC (p.Pro695LeufsX11) and EXT2 c.187delT (p.Phe63SerfsX29) were unreported previously. The three novel variants were not found among unaffected members of the pedigree and the 200 healthy controls. Upon prenatal diagnosis, the two fetuses were found to carry the same variants of the the probands.Pathological variants of the EXT1 and EXT2 genes probably underlie the multiple osteochondromas among the 5 pedigrees. Prenatal diagnosis based on the results can effectively reduce the birth of further offspring affected with the disease.

To identify pathogenic variations of EXT1 and EXT2 genes in two Chinese pedigrees affected with hereditary multiple exostosis (HME).Genomic DNA was extracted from peripheral blood samples using a phenol-chloroform method. PCR and Sanger sequencing was conducted to amplify the exons and the flanking intronic regions of the EXT1 and EXT2 genes.DNA sequencing has revealed a heterozygous missense variation c.812AG (p.Tyr271Cys) in the exon 1 of EXT1 in pedigree 1, and a heterozygous frameshift variation c.1431dup (p.Ser478Leufs*43) in the exon 6 of EXT1 in the proband from pedigree 2. Both variations have co-segregated with the disease phenotype, which was also consistent with previous report.Two heterozygous pathogenic variations underlying HME have been identified. The result has facilitated genetic counseling and prenatal diagnosis for the affected pedigrees.

To detect EXT1 and EXT2 gene mutations in two pedigrees affected with hereditary multiple exostosis (HME).The coding regions and exon/intron boundaries of the EXT1 and EXT2 genes were analyzed by targeted next-generation sequencing (NGS). Suspected mutations were confirmed by Sanger sequencing of the probands, their family members and 200 unrelated healthy controls. Gross deletion was confirmed by quantitative PCR (qPCR) analysis and multiple ligation-dependent probe amplification (MLPA) analysis.Two mutations were detected in the pedigrees, which included EXT2 gene c.337_338insG mutation in pedigree 1 and deletion of entire EXT1 in pedigree 2. Analysis of sequencing data revealed that a novel heterozygous mutation (c.337_338insG) in EXT2 gene in proband 1 and his father. The same mutation was not found among healthy family members and 200 unrelated healthy controls. As shown by NGS and MLPA analysis, proband 2 carried a heterozygous deletion of entire EXT1 gene. The same deletion was also found in her mother by qPCR.Mutations of the EXT1 and EXT2 genes probably underlie the HME in both pedigrees. NGS combined with Sanger sequencing, qPCR and MLPA is effective for attaining the diagnosis.

OBJECTIVE To detect potential mutations of the EXT1 and EXT2 genes in a pedigree affected with hereditary multiple exostosis (HME). METHODS For a four-generation family with 7 affected individuals from 17 family members,genomic DNA was extracted from peripheral venous blood samples. All exons of the EXT1 and EXT2 genes were screened for potential mutation by PCR and Sanger sequencing. RESULTS A novel heterozygous frameshift mutation c.1202delT (p.I401Tfs*2)was found in exon 4 of the EXT1 gene in the proband and the other 6 affected individuals. The same mutation was not detected among the healthy members from the family. The mutation has given rise a truncated EXT1 protein with loss of 345 amino acids. CONCLUSION A novel frameshift mutation of the EXT1 gene has been identified in a pedigree affected with HME, which has enriched the mutational spectrum of the EXT1 gene and may facilitate genetic counseling and prenatal diagnosis for the family.

To detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism.The coding regions and their flanking sequences of the EXT1/EXT2 genes were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified by excluding possible single nucleotide polymorphisms and bioinformatics analysis. Transcripts of the EXT1 gene in the proband were analyzed by TA clone-sequencing, with its abundance compared with that of healthy controls.DNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G to A) at the 5'splice sites of intron 3 of the EXT1 gene. The same mutation was not found in the healthy controls. Bioinformatics analysis indicated that the mutation is highly conserved and can lead to skipping of exon 3 or aberrant splicing. TA clone-sequencing indicated that the numbers of transcripts with skipping of exon 3 has significantly increased in the proband (0.05) compared with the controls.The c.1164+1G to A mutation has resulted in skipping of exon 3 in a proportion of EXT1 gene transcripts. As the result, the number of transcripts with tumor suppressing function is relatively reduced and has ultimately led to the tumors.

To investigate EXT1 and EXT2 genes mutations in a family with hereditary multiple osteochondromas (HME).A four-generation family with HME from Linyi city of Shandong Province was studied. There were 6 affected individuals among the 17 family members. Physical examination and radiographical evaluations were carried out for all family members. Genomic DNA was extracted from peripheral venous blood and the samples were subjected to mutation screening by PCR of the coding regions of EXT1 and EXT2 genes.The family has featured an autosomal dominant inheritance pattern. Sequencing of the EXT1 and EXT2 genes suggested the causative gene in this family was in linkage with the second exon of EXT2. A c.244delG mutation was detected, which has resulted in a frameshift mutation p.Asp81IlefsX30. The mutation was found in all of the 6 affected individuals but not in normal family members. And the mutation has co-segregated with the phenotype.The mutation c.244delG in the EXT2 gene is the probably the cause of the disease in this family.

To identify the genetic cause for a Chinese Han family affected with hereditary multiple osteochondromas.Two patients, five unaffected relatives of the family and 100 unrelated healthy controls were collected. The coding sequences and intron/exon boundaries of EXT1 gene were amplified with polymerase chain reaction (PCR) and sequenced.A heterozygous c.600GA (p.Trp200X) mutation in exon 1 of the EXT1 gene was detected in the patients. The same mutation was not found in unaffected family members and 100 healthy controls.The hereditary multiple osteochondromas in the family is caused by a nonsense mutation (p.Trp200X) in the EXT1 gene.

To detect the underlying genetic defect in two Chinese families with hereditary multiple exostoses and provide genetic counseling.Potential mutations in EXT1 and EXT2 genes in the probands were detected by direct sequencing of PCR-amplified exons. Suspected mutations were verified in all available family members and 200 unrelated healthy controls.A heterozygous frameshift mutation c.346_356delinsTAT in exon 1 of EXT1 and a heterozygous deletion mutation c.2009-2012del(TCAA) in exon 10 of EXT1 were respectively detected in affected members from the two families. The same mutations were not detected in unaffected members and 200 unrelated healthy controls. No mutations in EXT2 were detected in the two families.Two novel mutations of EXT1 have been detected in association with hereditary multiple exostoses in two Chinese families. Above results have provided a basis for genetic counseling for the two families and expanded the spectrum of EXT1 mutations.

To screen for potential mutations in an ethnic Han Chinese family from Shanxi with hereditary multiple exostoses.Polymerase chain reaction and DNA sequencing were used to screen potential mutations in EXT1 and EXT2 genes.For EXT1 gene, two synonymous mutations (P477P and E587E), three intronic mutations (c.1537 -48AG, c.1721 +203AG and c.1722 -103CG) were detected. For EXT2 gene, five intronic mutations (c.-29 -148AT, c.1080 -18TA, c.1336 -93CT, c.1526 -166CT, and c.1526 -195CT) were identified. Among these, EXT1 P477P, EXT1 E587E and EXT2 c.1080 -18TA are polymorphisms listed by Multiple Osteochondroma Mutation Database, whilst the other 7 sites have not been reported.No mutations have been found among all exons of the EXT1 and EXT2 genes in this family. Linkage analysis is necessary for identifying the cause of this disease.

To identify the gene causing hereditary multiple exostoses in a Chinese pedigree.Linkage analysis was carried out in the family using microsatellite markers close linkage to the EXT1 and EXT2 genes to define the candidate gene. Then the whole coding sequence and the intron-exon boundaries of the candidate gene were amplified and sequenced.The disease-causing gene of the family was linked to the EXT2 gene. A nonsense mutation of 536GA in exon3 of the EXT2 gene was detected, which was co-segregated with the disease phenotype. The mutation resulted in a stop codon in codon 180. A nonpenetrant case was found in the family.The mutation 536GA in the EXT2 gene is the disease-causing mutation in the pedigree with hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by formation of benign cartilage-capped tumors (exostoses), typically located at the juxtaepiphyseal regions of long bones. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2 have been cloned and are responsible for over 80% of cases. A Chinese family with HME has been analyzed in the present study.Linkage analysis was firstly performed to determine which of the three EXT genes could be the candidate gene, then mutation screening by PCR and direct sequencing was carried out.A novel nonsense mutation (c.1006CT) in exon 6 of EXT2, which converts the codon CAA (Gln) to the stop codon (TAA) (Gln336X), was identified. Next, prenatal diagnosis was performed and the pregnancy was determined to be normal.A new EXT2 nonsense mutation was found in a Chinese family with hereditary multipe exostoses. The information was used for a case of prenatal diagnosis.

To develop a new denaturing high performance liquid chromatograph (DHPLC)-based method to screen patients with EXT gene mutation and to study the gene mutation in three families with multiple exostoses.All the exons of EXT gene, including the intro-exon boundaries, were amplified by PCR. Linkage analysis and DHPLC screening were carried out to identify the mutations. DNA sequencing was used to confirm the mutations.Two known splice site mutations, IVS2+1 G to A and IVS7+1 G to T, and two SNPs have been detected in EXT2 or EXT1 gene.The transversions of IVS2+1 G to A and IVS7+1 G to T in EXT2 gene are suggested to be the disease-causing mutations and the DHPLC is a high throughout, sensitive, simple, quick, economical method to screen gene mutation in hereditary multiple exostosis.

To study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation.Polymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation.By DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before. Mismatch primer amplification and restriction fragment length polymorphism analysis suggested that this mutation was not detected in the normal control.The mutation 1633-26(C--A) may be the disease-causing mutation in this patient with multiple exostoses.

To identify the gene causing hereditary multiple exostoses in a Chinese pedigree.Linkage analysis was carried out in the family using microsatellite markers on chromosome 8, 11 and 19 respectively. To detect the mutation, the whole coding sequence and the intron-exon boundaries of the candidate gene were amplified and sequenced. The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify the mutated mRNA.The disease-causing gene of the family was linked to the EXT2 locus on chromosome 11. A mutation IVS2+1GA was detected in EXT2 and resulting in 221 bp deletion from 316 to 536 of coding sequence(CDS), which was co-segregated with the disease phenotype. This change led to deletion from codon 106 to codon 178 and subsequent 2 nucleotides, producing a frameshift and truncated protein of 125 aa.The mutation IVS2+1GA is the disease-causing mutation in the Chinese pedigree with hereditary multiple exostoses.

To investigate further the genetic basis of hereditary multiple exostoses (EXT) and provide useful information for gene diagnosis of the disease.Polymerase chain reaction-single strand conformation polymorphism was used to examine the entire coding regions of EXT(1) gene on chromosome 8 and EXT(2) gene on chromosome 11 for mutation in thirty EXT families. Mutations were further identified by sequencing.Two frameshift mutations were identified in two unrelated EXT families. One was the deletion of one base(T) in exon 6 of the EXT(1) gene, and the other was the deletion of four bases (tgtt) in exon 2 of the EXT(2) gene. Both of the mutations resulted in a frameshift and premature termination of translation.EXT is a genetically heterogeneous bone disorder caused by the mutation of EXT tumor suppressor gene. These results could be directly applied in the genetic counseling and prenatal genetic diagnosis of EXT.