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Background: Multiple osteochondroma (MO), an autosomal dominant genetic disease, is caused by heterozygous mutations in the EXT1 and EXT2 genes. Approximately 80% of pathogenic mutations are nonsense/missense mutations, small indels, and splicing mutations. Splicing mutations, particularly at the 3' and 5' splice sites, disrupt normal mRNA processing and cause exon skipping or aberrant splicing, ultimately resulting in protein truncation and loss of function. Methods: Polymerase chain reaction (PCR) and Sanger sequencing were applied to detect subtle mutations in a Chinese family with MO, the pathogenicity of a splicing variant was predicted by bioinformatics and further verified using a minigene splicing assay. Results: A novel and heterozygous splicing mutation, c.626 + 2_626 + 5delTAGG, was identified in the EXT2 gene of the proband and the father by PCR and Sanger sequencing, whereas the unaffected mother and brother had wild-type alleles at the same site. Bioinformatics predicted that the 5' splicing site of exon 3 in the EXT2 gene was destroyed due to this mutation. A hybrid minigene splicing assay (HMSA) indicated that the mutation disturbed the normal splicing of the EXT2 gene mRNA and led to a deletion of 79 bp at the 5' end of exon 3, which resulted in aberrant splicing of exon 3 and introduced an earlier stop codon in the EXT2 gene. Conclusion: A novel splicing mutation was identified that produced the MO phenotype through aberrant splicing in a Chinese family. This observation, expands our knowledge of the spectrum of molecular pathogenic mechanisms leading to aberrant mRNA splicing.

Background: Multiple osteochondromas (MO) are an autosomal-dominant disease characterized by the growth of multiple cartilage-capped prominences in the growth plate region of the metaphysis in long and flat bones. Materials and Methods: To detect genetic mutations related to MO, a three-generation Chinese family with MO was evaluated using whole exome sequencing for mutation screening. The candidate pathogenic mutation was validated by Sanger sequencing. Results: A novel frameshift (NM_000401.3:c.1321del:p.Leu441TrpfsTer28) in exon 8 of the exotosin 2 ( EXT2 ) gene was identified in two affected individuals. Codons 441 and 468 in the EXT2 gene are highly conserved among vertebrates as demonstrated by multiple sequence alignment. The c.1321 del C resulted in an amino acid change at codon 441, which generated a premature stop codon at position 468, causing complete loss of the glycosyltransferase domain. Conclusions: A novel frameshift mutation c.1321delC detected in the EXT2 gene may help in prenatal genetic screening and early diagnosis of MO.

Aim: To detect mutations in the EXT1 and EXT2 genes in four Chinese families with hereditary multiple osteochondromas (HMO). HMO is an autosomal dominant disorder characterized by the overgrowth of multiple cartilage-capped bones in the metaphysis of long bones and flat bones. Methods: Polymerase chain reaction-based amplification followed by DNA sequencing of the complete coding sequences of EXT1 and EXT2 was performed for four Chinese families with HMO. Results: The mutant allele was found in six patients: three mutations were found in EXT1 and two in EXT2 . A novel frameshift mutation, which generates a premature stop codon at codon 586 and causes partial loss of the glycosyltransferase domain, was detected in exon 9 of EXT1 (F579Yfs*8). We hypothesize that F579Yfs*8 is a pathogenic mutation. Two novel missense mutations (G339S and V545D) were found in EXT1 . The variant c.1634T>A (V545D) is apparently benign. In addition we found a novel deletion mutation in EXT2 , c.856_864 del TTCCTCCTG, which results in the deletion of 286Phe, 287Leu, and 288Leu, that is likely pathogenic. Finally, we identified a likely benign variant in exon 13 of EXT2 . c.2035-41T>C (rs3740878). Conclusions: We found three novel, potentially pathogenic mutations in EXT1 and EXT2 , including a novel frameshift mutation. More importantly, our study results have expanded the spectrum of EXT mutations conducive to the genetic diagnosis and counseling of patients with HMO.

Aims: Identification of genetic mutations linked to hereditary multiple osteochondromas (HMO) is crucial for understanding the molecular mechanisms leading to disease pathogenesis. In this study, we investigated four patients and eight healthy individuals from a family with HMO. Methods: Clinical HMO data and Sanger sequences of the coding regions of the exostosin glycosyltransferase 1 ( EXT1 ) gene (18q24.11) and the EXT2 gene (11p12) of all 12 members of the family were analyzed. Results: A novel nonsense mutation in the EXT2 gene (c.526C>T; p.Gln176*) was detected, which was present in all four patients but absent in their healthy relatives. This mutation encodes a stop codon that results in a truncated EXT2 protein that consists of only 176 amino acids and lacks the remaining 522 amino acids at its C-terminus, missing the entire glycosyltransferase domain. Conclusions: Association of a truncated EXT2 protein with HMO provides new insights into exostosis pathogenesis, highlighting potential roles of the EXT2 gene and its glycosyltransferase domain. Further research is required to understand the mechanisms underlying the development of exostosis.

Aims: Multiple exostoses (MO), also referred to as hereditary multiple exostoses (HME), is an autosomal dominant inherited skeletal disorder that has been found to be associated with mutations in the EXT1 and EXT2 genes. In the present study, we report a Chinese family with HME and our mutational analyses of the EXT1 and EXT2 genes in affected and unaffected individuals., Methods: All exons of the EXT1 and EXT2 genes in seven family members were polymerase chain reaction amplified from blood and sequenced., Results: A heterozygous mutation (c.1056G>T) was identified in exon 2 of the EXT1 gene in the proband and other affected family members; this mutation was not found in the unaffected family members., Discussion: This c.1056G>T mutation is located in the exostosin domain of the EXT1 protein and leads to an amino acid change of Glutamine (Gln) to Histidine (His) at position 352. Homology searches reveal that Gln352 is highly conserved in many species and may play an essential role in the normal function of the EXT1 protein., Conclusions: This study contributes to a better understanding of the genetic basis of HME, expands the known mutational spectrum of EXT1, and provides a reference for genetic counseling and prenatal diagnosis of this family.

Multiple osteochondroma (MO) is an autosomal dominant disease characterized by abnormal skeleton development: one or more exostoses localized mainly at the end of long bones. Three pathogenic gene loci have been identified and cloned: EXT1, 2, and 3. Only EXT1 and 2 mutations were reported to cause MO. Here, we report on a large Chinese family with MO and a disease-causing mutation in EXT. We extracted DNA from peripheral blood samples of 25 family members, 9 with MO. Polymerase chain reaction and direct DNA sequencing of the entire coding regions of EXT1 and 2 for the nine patients revealed a novel pathogenic mutation, insertion of a T in exon 2 (c.72-73 insT) of EXT2. Our results extend the mutational spectrum of MO and can help with genetic counseling and prenatal diagnosis for this family.

Hereditary multiple exostoses (HME) is an autosomal dominantly inherited disorder characterized by multiple benign cartilage-capped exostoses. Clinical manifestation of the disease is heterogenous. Overriding toes, scoliosis, spinal cord compression, and brachydactyly caused by shortening of metatarsals are rare findings. EXT1 and EXT2 are the genes responsible in most HME patients. We have characterized 11 HME families and 6 sporadic cases involving a total of 37 patients and performed mutational analysis of EXT1 and EXT2. Structural modeling of the wild and mutant proteins was also performed. Thirteen mutations were identified, including 8 that are novel. Among the novel mutations in EXT1, c.1004T>G-associated HME exhibited overriding toes and scoliosis, c.1883+2T>A-associated HME exhibited brachydactyly, and c.459_460delCT-associated exostosis arising from vertebra T4 caused spinal cord compression. Our structural predictions revealed four domains in the proteins encoded by EXT1 and EXT2: signalP, transmembrane regions, exostosin, and glyco_transf-64. The mutations truncated either part or whole of the exostosin domain and/or the C terminus of the glyco_transf-64 domain, or occurred within one of the domains. Our results provide new data for genetic diagnosis, identification of presymptomatic carriers, phenotype-genotype correlation, and understanding of the mechanisms of disease.

Hereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by growth of benign multiple exostoses. In our present study, we describe a four-generation Han Chinese kindred with eight members affected by HME. Haplotyping analysis and mutation detection was performed. The results linked the disease-causing gene to the EXT1 locus on chromosome 8. A novel mutation in EXT1, c.1897delC, which cosegregated with the disease phenotype, was detected. To further confirm this mutation, a mismatch primer was designed to introduce a ScaI restriction site into the normal allele by polymerase chain reaction, and the following restriction fragment length polymorphism analysis demonstrated that the mutation was not detected in any unaffected individuals of the family or 100 unrelated Han Chinese control individuals. This mutation leads to a frameshift from codon 633, resulting in a premature termination at codon 642 and loss of the highly conserved C terminal region of the protein. Therefore, this heterozygous mutation must be classified as pathogenic and can be regarded as the cause of HME in this Chinese family.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder most frequently caused by the EXT1 and EXT2 gene mutations resulting in reduction or absence of heparan sulfate (HS) in the exostotic cartilage cap. In this study, we investigated the molecular defects in five Chinese pedigrees with HME by direct sequencing analysis. Two novel EXT1 gene mutations and two novel EXT2 gene mutations were identified in two and three pedigrees, respectively. Of the four mutations identified, the c.651-664delinsTTT and c.680delG mutations in the exon 1 of EXT1 gene would cause frameshift (K218fs and R227fs) and introduce premature stop codon at amino acid site 220 and 251, respectively. The two missense mutations of c.398T > G in exon 2 and c.1016G > A in exon 6 of EXT2 gene result in the Leu133Arg and Cys339Tyr substitution, respectively. As HME is caused by defects in HS synthesis that is a complex process and not fully understood, these naturally occurring EXT mutations may provide important clues to future studies elucidating how EXT proteins contribute to HS biosynthesis.

Background: Hereditary multiple exostosis (HME) is an autosomal dominant bone disorder, characterized by short stature and the presence of multiple benign tumors mainly at the ends of long bones. HME is genetically heterogeneous with two known genes on 8q24 (EXT1) and 11p11 (EXT2), and a third minor locus mapped to 19p (EXT3). The majority of EXT1 and EXT2 mutations result in premature protein truncation and loss of function., Materials and Methods: We analyzed two autosomal dominant HME families of Indian origin. Linkage analysis using fluorescently labeled microsatellite markers at the candidate gene regions was performed. Mutation analysis was carried out by bidirectional sequencing of purified PCR products., Results: We found linkage in one family to EXT1 and in the other family to EXT2. Mutation screening in the EXT1 gene revealed a novel frameshift mutation, a single base deletion in exon 1 (c.142delC). This mutation segregated in all affected members and was absent in the unaffected family members and 60 unrelated controls. In the second family, a previously unreported stop mutation, the substitution c.817C>T, was observed in the EXT2 gene in all affected members and in none of the unaffected family members and 90 unrelated controls., Conclusions: Our findings expand the mutation spectrum of EXT1 and EXT2 and highlight the genetic and phenotypic heterogeneity of HME.

Multiple osteochondromas (MO; also referred to as hereditary multiple exostoses [HME] in the literature) is an autosomal dominant disorder characterized by benign, cartilage-capped bone tumors that grow from the metaphyses of long bones. Two genes are associated with this disease: EXT1 on 8q24.11-q24.13 and EXT2 on 11p12-p11. Mutations in EXT1 and EXT2 are found in 54-96% of patients with MO and are generally more frequent in EXT1 than in EXT2. We previously studied 43 Japanese families with MO using single-strand conformation polymorphism analysis for EXT1 and EXT2, and reported 23 families (54%) with mutations and 20 families (46%) with no mutations in these genes. Among the families with mutations, 17 families (40%) had mutations in EXT1, and 6 families (14%) had mutations in EXT2. Here we examined the same 43 Japanese families using denaturing high-performance liquid chromatography as an alternative technique. We detected five mutations, three of which are novel, in seven families in addition to the previously described mutations. In summary, we detected mutations in EXT1 or EXT2 in 30 (70%) out of 43 families. Our result suggests the presence of other gene(s) responsible for MO, at least in Japanese patients.

Hereditary multiple exostoses (HME) is an autosomal-dominant disorder characterized by the presence of bony outgrowths on the long bones. In this report, we describe a Chinese family with HME. Linkage analysis and mutation detection were performed. Linkage with the EXT2 was established in this family. A novel mutation, EXT2 c239-244delG, was identified. Mutation analysis in a family with HME allows for genetic counseling and prenatal diagnosis.

Multiple osteochondromas (MO) is an autosomal-dominant inherited disorder. The two genes responsible (EXT1 and EXT2) have been identified. We investigated 12 MO families for phenotype details and the genetic basis by cosegregation and mutation analysis (seven novel pathogenic mutations [five frameshift, one splice site, and one gross deletion] and one novel missense polymorphism). We found EXT1 to be responsible in seven families (19 affected members) and EXT2 in four families (17 affected members). One family remains undetermined. We found a tendency to a more severe phenotype in EXT1 families. As a novel finding, we could identify a single parameter (ulna/height ratio) that separates EXT1 family from EXT2 family in our series.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by benign bone tumors. In this report, we describe two unrelated Chinese families with HME. Linkage analysis and mutation detection was performed. Clinical analysis was also performed for some affected individual in both families. Linkage with the EXT2 was established in both families. A novel mutation, c505 G > T, was identified in both families. Further allelic heterogeneity of EXT2 was demonstrated by the intrafamilial and interfamilial variability in clinical phenotype.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by growth of benign bone tumors. This genetically heterozygous disease comprises three chromosomal loci: the EXT1 gene on chromosome 8q23-q24, EXT2 on 11p11-p13, and EXT3 on 19p. Both EXT1 and EXT2 have been cloned and defined as a new family of potential tumor suppressor genes in previous work. However, no studies have been conducted in the Taiwanese population. To determine if previous results can also be applied to the Taiwanese, we analyzed 5 Taiwanese probands with clinical features of HME: 1 of them is a sporadic case, and the others are familial cases. Linkage studies were performed in the familial cases before the mutation analysis to determine to which of the three EXT chromosomes these cases could be assigned. Our results showed that one proband is linked to the EXT1 locus and three are linked to the EXT2 locus; the sporadic case was subsequently found to involve EXT1. We then identified four new mutations that have not been found in other races: two in EXT1--frameshift (K218fsX247) and nonsense (Y468X) mutations and two in EXT2-missense (R223P) and nonsense (Y394X) mutations. Our results indicate that in familial cases, linkage analysis can prove useful for preimplantation genetic diagnosis.