Gene Summary

Gene:EXT2; exostosin glycosyltransferase 2
Aliases: SOTV
Summary:This gene encodes one of two glycosyltransferases involved in the chain elongation step of heparan sulfate biosynthesis. Mutations in this gene cause the type II form of multiple exostoses. Alternatively spliced transcript variants encoding different isoforms have been noted for this gene. [provided by RefSeq, Jul 2008]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Source:NCBIAccessed: 18 March, 2015


What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 18 March 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Amino Acid Sequence
  • Cancer DNA
  • Base Sequence
  • Frameshift Mutation
  • Chromosome Mapping
  • Chromosome 8
  • Tumor Suppressor Gene
  • Exons
  • Exostoses, Multiple Hereditary
  • Adolescents
  • Genetic Linkage
  • Gene Deletion
  • Introns
  • Syndecan-1
  • Asian Continental Ancestry Group
  • Genotype
  • Chondrosarcoma
  • Wnt1 Protein
  • Germ-Line Mutation
  • Point Mutation
  • Phenotype
  • Genetic Predisposition
  • Bone Cancer
  • Osteochondroma
  • Missense Mutation
  • Genetic Testing
  • Loss of Heterozygosity
  • Chromosome 11
  • DNA Mutational Analysis
  • Mutation
  • Polymerase Chain Reaction
  • China
  • Single-Stranded Conformational Polymorphism
  • N-Acetylglucosaminyltransferases
  • Childhood Cancer
  • Heparitin Sulfate
  • Chromatography, High Pressure Liquid
  • Chromosome 19
  • DNA Primers
  • exostosin-2
Tag cloud generated 18 March, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (2)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Entity Topic PubMed Papers
Multiple Hereditary ExostosesEXT2 and Multiple Hereditary Exostoses View Publications100
ChondrosarcomaEXT2 mutations in Secondary Chondrosarcoma
Osteochondroma (the most common type of benign bone tumor) is frequently characterised by mutations of EXT1 and EXT2 genes, often sporadic but sometimes germline, as seen in Multiple Hereditary Exostoses. Osteochondromas occasionally undergo neoplastic transformation resulting in secondary chondrosarcoma.
View Publications33

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: EXT2 (cancer-related)

Samuel AM, Costa J, Lindskog DM
Genetic alterations in chondrosarcomas - keys to targeted therapies?
Cell Oncol (Dordr). 2014; 37(2):95-105 [PubMed] Related Publications
BACKGROUND: Chondrosarcomas are malignant tumors of chondrocytes and represent the second most common type of primary bone tumors. Within the context of normal chondrogenesis, this review summarizes results from recent research outlining the key molecular changes that occur during the development of this sarcoma type.
RESULTS: Current data support the notion that a two-hit scenario, common to many tumors, also underlies chondrosarcoma formation. First, early-stage mutations alter the normal proliferation and differentiation of chondrocytes, thereby predisposing them to malignant transformation. These early-stage mutations, found in both benign cartilaginous lesions and chondrosarcomas, include alterations affecting the IHH/PTHrP and IDH1/IDH2 pathways. As they are not observed in malignant cells, mutations in the EXT1 and EXT2 genes are considered early-stage events providing an environment that alters IHH/PTHrP signaling, thereby inducing mutations in adjacent cells. Due to normal cell cycle control that remains active, a low rate of malignant transformation is seen in benign cartilaginous lesions with early-stage mutations. In contrast, late-stage mutations, seen in most malignant chondrosarcomas, appear to induce malignant transformation as they are not found in benign cartilaginous lesions. These late-stage mutations primarily involve cell cycle pathway regulators including p53 and pRB, two genes that are also known to be implicated in numerous other human tumor types.
CONCLUSIONS: Now the key genetic alterations involved in both early and late stages of chondrosarcoma development have been identified, focus should be shifted to the identification of druggable molecular targets for the design of novel chondrosarcoma-specific therapies.

Jones KB, Pacifici M, Hilton MJ
Multiple hereditary exostoses (MHE): elucidating the pathogenesis of a rare skeletal disorder through interdisciplinary research.
Connect Tissue Res. 2014; 55(2):80-8 [PubMed] Related Publications
Abstract An interdisciplinary and international group of clinicians and scientists gathered in Philadelphia, PA, to attend the fourth International Research Conference on Multiple Hereditary Exostoses (MHE), a rare and severe skeletal disorder. MHE is largely caused by autosomal dominant mutations in EXT1 or EXT2, genes encoding Golgi-associated glycosyltransferases responsible for heparan sulfate (HS) synthesis. HS chains are key constituents of cell surface- and extracellular matrix-associated proteoglycans, which are known regulators of skeletal development. MHE affected individuals are HS-deficient, can display skeletal growth retardation and deformities, and consistently develop benign, cartilage-capped bony outgrowths (termed exostoses or osteochondromas) near the growth plates of many skeletal elements. Nearly 2% of patients will have their exostoses progress to malignancy, becoming peripheral chondrosarcomas. Current treatments are limited to the surgical removal of symptomatic exostoses. No definitive treatments have been established to inhibit further formation and growth of exostoses, prevent transition to malignancy, or address other medical problems experienced by MHE patients, including chronic pain. Thus, the goals of the Conference were to assess our current understanding of MHE pathogenesis, identify key gaps in information, envision future therapeutic strategies and discuss ways to test and implement them. This report provides an assessment of the exciting and promising findings in MHE and related fields presented at the Conference and a discussion of the future MHE research directions. The Conference underlined the critical usefulness of gathering experts in several research fields to forge new alliances and identify cross-fertilization areas to benefit both basic and translational biomedical research on the skeleton.

Cao L, Liu F, Kong M, et al.
Novel EXT1 mutation identified in a pedigree with hereditary multiple exostoses.
Oncol Rep. 2014; 31(2):713-8 [PubMed] Related Publications
Hereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors. EXT1 located on chromosome 8q23-q24 and EXT2 located on 11p11-p12 are the main disease-causing genes which are responsible for ~90% of HME cases. Mutations of EXT1 or EXT2 result in insufficient heparan sulfate biosynthesis, which facilitates chondrocyte proliferation, boosts abnormal bone growth of neighboring regions, causes multiple exostoses, and ultimately leads to possible malignant transformation. A family who displayed typical features of HME was enrolled in the present study. Mutation screening by Sanger sequencing identified a novel heterozygous nonsense mutation c.1902C>A (p.Tyr634X) in the EXT1 gene exclusively in all 3 patients, which is located in the glycosyltransferase domain and results in the truncation of 112 amino acids at the C-terminus of the EXT1 protein. Thus, the present study identified a novel disease-causing EXT1 mutation in a pedigree with HME, which provides additional evidence for developing quick and accurate genetic tools for HME diagnosis.

Busse-Wicher M, Wicher KB, Kusche-Gullberg M
The exostosin family: proteins with many functions.
Matrix Biol. 2014; 35:25-33 [PubMed] Related Publications
Heparan sulfates are complex sulfated molecules found in abundance at cell surfaces and in the extracellular matrix. They bind to and influence the activity of a variety of molecules like growth factors, proteases and morphogens and are thus involved in various cell-cell and cell-matrix interactions. The mammalian EXT proteins have glycosyltransferase activities relevant for HS chain polymerization, however their exact role in this process is still confusing. In this review, we summarize current knowledge about the biochemical activities and some proposed functions of the members of the EXT protein family and their roles in human disease.

Kang QL, Xu J, Zhang Z, et al.
Mutation screening for the EXT1 and EXT2 genes in Chinese patients with multiple osteochondromas.
Arch Med Res. 2013; 44(7):542-8 [PubMed] Related Publications
BACKGROUND AND AIMS: Multiple osteochondromas (MO), an autosomal dominant skeletal disease, is characterized by the presence of multiple cartilage-capped bone tumors (exostoses). Two genes with mutations that are most commonly associated with MO have been identified as EXT1 and EXT2, which are Exostosin-1 and Exostosin-2. In this study, a variety of EXT1 and EXT2 gene mutations were identified in ten Chinese families with MO.
METHODS: We investigated ten unrelated Chinese families involving a total of 46 patients who exhibited typical features of MO. The coding exons of EXT1 and EXT2 were sequenced after PCR amplification in ten probands. Radiological investigation was conducted simultaneously.
RESULTS: Nine mutations were identified, five in EXT1 and four in EXT2, of which three were de novo mutations and six were novel mutations. One proband carried mutations in both EXT1 and EXT2 simultaneously, and three probands, including one sporadic case and two familial cases, had no detectable mutations.
CONCLUSIONS: Our findings are useful for extending the mutational spectrum in EXT1 and EXT2 and understanding the genetic basis of MO in Chinese patients.

Wu Y, Xing X, Xu S, et al.
Novel and recurrent mutations in the EXT1 and EXT2 genes in Chinese kindreds with multiple osteochondromas.
J Orthop Res. 2013; 31(9):1492-9 [PubMed] Related Publications
Multiple osteochondromas (MO) is an autosomal dominant hereditary disorder caused by heterozygous germline mutations in the exostonsin-1 (EXT1) or exostosin-2 (EXT2) genes. In this study, we screened mutations in the EXT1/EXT2 genes in four Chinese MO kindreds by direct sequencing. Three point mutations were detected, including a nonsense mutation in the EXT2 gene (c.544C > T) and two splice site mutations in the EXT1 and EXT2 genes, respectively (EXT1: c.1883 + 1G > A and EXT2: c.1173 + 1G > T). Although splice site mutations constitute at least 10% of all mutations that cause MO, there has been limited research on their pathogenic effect on RNA processing due to poor availability of patient RNA samples. In this study, ex vivo and in vivo splicing assays were used to investigate the effect of EXT1 and EXT2 mutations on aberrant splicing at the mRNA level. Our results indicate that identified splice site mutations can cause either cryptic splice site usage or exon skipping.

Sarrión P, Sangorrin A, Urreizti R, et al.
Mutations in the EXT1 and EXT2 genes in Spanish patients with multiple osteochondromas.
Sci Rep. 2013; 3:1346 [PubMed] Free Access to Full Article Related Publications
Multiple osteochondromas is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours. Two causal genes have been identified, EXT1 and EXT2, which account for 65% and 30% of cases, respectively. We have undertaken a mutation analysis of the EXT1 and EXT2 genes in 39 unrelated Spanish patients, most of them with moderate phenotype, and looked for genotype-phenotype correlations. We found the mutant allele in 37 patients, 29 in EXT1 and 8 in EXT2. Five of the EXT1 mutations were deletions identified by MLPA. Two cases of mosaicism were documented. We detected a lower number of exostoses in patients with missense mutation versus other kinds of mutations. In conclusion, we found a mutation in EXT1 or in EXT2 in 95% of the Spanish patients. Eighteen of the mutations were novel.

Waaijer CJ, Winter MG, Reijnders CM, et al.
Intronic deletion and duplication proximal of the EXT1 gene: a novel causative mechanism for multiple osteochondromas.
Genes Chromosomes Cancer. 2013; 52(4):431-6 [PubMed] Related Publications
Multiple osteochondromas (MO) is a syndrome in which benign cartilage-capped neoplasms develop at the surface of the long bones. Most cases are caused by exonic changes in EXT1 or EXT2, but 15% are negative for these changes. Here we report for the first time a family of MO patients with germline genomic alterations at the EXT1 locus without detectable mutations or copy number alterations of EXT exonic sequences. Array-CGH showed an 80.7 kb deletion of Intron 1 of EXT1 and a 68.9 kb duplication proximal of EXT1. We identified a breakpoint between the distal end of the duplicated region and a sequence distal of the deleted region in the first intron. This breakpoint was absent in non-affected family members. The configuration of the breakpoint indicates a direct insertion of the duplicated region into the deletion. However, no other breakpoint was found, which suggests a more complex genomic rearrangement has occurred within the duplicated region. Our results reveal intronic deletion and duplication as a new causative mechanism for MO not detected by conventional diagnostic methods.

Ciavarella M, Coco M, Baorda F, et al.
20 novel point mutations and one large deletion in EXT1 and EXT2 genes: report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis.
Gene. 2013; 515(2):339-48 [PubMed] Related Publications
BACKGROUND: Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although "de novo" cases are not infrequent.
AIM: Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5years.
RESULTS: Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding.
CONCLUSIONS: Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.

Bari MS, Jahangir Alam MM, Chowdhury FR, et al.
Hereditary multiple exostoses causing cord compression.
J Coll Physicians Surg Pak. 2012; 22(12):797-9 [PubMed] Related Publications
Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder characterized by the presence of multiple osseous prominences. It can occur sporadically or within families (22 - 56%). Two genes, EXT1 and EXT2 located respectively at 8q24 and 11p11-p12, have been isolated to cause HME. It can cause gross deformity of limbs and growth disturbance which is quite a common complication. Malignant transformation to chondrosarcoma can also occur. Neurological presentations are rare and usually happened due to direct compression of a peripheral nerve or nerve root or less often the spinal cord. This case is possibly the first case of HME described from Bangladesh, presented with dorsal cord compression. Decompression was done and the complaints of myelopathy were improved.

Niini T, Scheinin I, Lahti L, et al.
Homozygous deletions of cadherin genes in chondrosarcoma-an array comparative genomic hybridization study.
Cancer Genet. 2012; 205(11):588-93 [PubMed] Related Publications
Chondrosarcoma is a malignant bone tumor that is often resistant to chemotherapy and radiotherapy. We applied high resolution oligonucleotide array comparative genomic hybridization to 46 tumor specimens from 44 patients with chondrosarcoma and identified several genes with potential importance for the development of chondrosarcoma. Several homozygous deletions were detected. The tumor suppressor genes CDKN2A and MTAP were each homozygously deleted in four of the cases, and the RB1 gene was homozygously deleted in one. Two homozygous deletions of MTAP did not affect CDKN2A. Deletions were also found to affect genes of the cadherin family, including CDH4 and CDH7, each of which had a targeted homozygous loss in one case, and CDH19, which had a targeted homozygous loss in two cases. Loss of the EXT1 and EXT2 genes was uncommon; EXT1 was homozygously deleted in none and EXT2 in two of the cases, and large heterozygous losses including EXT1 and/or EXT2 were seen in three cases. Targeted gains and amplifications affected the MYC, E2F3, CDK6, PDGFRA, KIT, and PDGFD genes in one case each. The data indicate that chondrosarcomas develop through a combination of genomic imbalances that often affect the RB1 signaling pathway. The inactivation of cadherin genes may also be critical in the pathogenesis of the tumor.

Kang Z, Peng F, Ling T
Mutation screening of EXT genes in Chinese patients with multiple osteochondromas.
Gene. 2012; 506(2):298-300 [PubMed] Related Publications
Multiple osteochondromas (MO), a dominantly inherited genetic disorder, is characterized by the presence of multiple osteochondromas in the long bones. EXT1 and EXT2 are the causative genes in most MO patients. We have characterized 9 MO families and 1 sporadic case involving a total of 25 patients. The coding exons of EXT1 and EXT2 were screened in 10 probands affected with MO. In five of the 10 probands novel pathogenic mutations have been identified: two in EXT1 and three in EXT2. Four probands carried recurrent mutations and one proband had no detectable mutation. Our study extends the mutational spectrum in EXT1 and EXT2 and will facilitate the deep understanding of the pathophysiology of the disease.

Wang X, Li L, Li J, et al.
Pathogenic gene screening and mutation detection in a Chinese family with multiple osteochondroma.
Genet Test Mol Biomarkers. 2012; 16(7):827-32 [PubMed] Free Access to Full Article Related Publications
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.

Kyriazoglou AI, Dimitriadis E, Arnogiannaki N, et al.
Similar cytogenetic findings in two synchronous secondary peripheral chondrosarcomas in a patient with multiple osteochondromas.
Cancer Genet. 2011; 204(12):677-81 [PubMed] Related Publications
Secondary peripheral chondrosarcoma is a malignant chondroid tumor arising in a benign precursor, either an osteochondroma or an enchondroma. Multiple osteochondromas syndrome (MO) is an autosomal dominant skeletal disorder associated with bony growths in the form of osteochondromas that occasionally undergo malignant transformation to secondary peripheral chondrosarcomas. We describe the genetic examination of three secondary peripheral chondrosarcomas that had arisen synchronously from osteochondromas in a patient with MO by chromosome banding, high resolution chromosomal comparative genomic hybridization, and mutation analysis of the EXT1 and EXT2 genes. In two of the tumors (the third was not genetically informative), very similar chromosome abnormalities were found, indicating that they must somehow be part of the same neoplastic process in spite of being anatomically distinct.

Wiweger MI, Zhao Z, van Merkesteyn RJ, et al.
HSPG-deficient zebrafish uncovers dental aspect of multiple osteochondromas.
PLoS One. 2012; 7(1):e29734 [PubMed] Free Access to Full Article Related Publications
Multiple Osteochondromas (MO; previously known as multiple hereditary exostosis) is an autosomal dominant genetic condition that is characterized by the formation of cartilaginous bone tumours (osteochondromas) at multiple sites in the skeleton, secondary bursa formation and impingement of nerves, tendons and vessels, bone curving, and short stature. MO is also known to be associated with arthritis, general pain, scarring and occasional malignant transformation of osteochondroma into secondary peripheral chondrosarcoma. MO patients present additional complains but the relevance of those in relation to the syndromal background needs validation. Mutations in two enzymes that are required during heparan sulphate synthesis (EXT1 or EXT2) are known to cause MO. Previously, we have used zebrafish which harbour mutations in ext2 as a model for MO and shown that ext2⁻/⁻ fish have skeletal defects that resemble those seen in osteochondromas. Here we analyse dental defects present in ext2⁻/⁻ fish. Histological analysis reveals that ext2⁻/⁻ fish have very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of ext2⁻/⁻ fish have a single tooth at the end of the 5(th) pharyngeal arch, whereas wild-type fish develop three teeth, located in the middle of the pharyngeal arch. ext2⁻/⁻ teeth have abnormal morphology (they were shorter and thicker than in the WT) and patchy ossification at the tooth base. Deformities such as split crowns and enamel lesions were found in 20% of ext2⁺/⁻ adults. The tooth morphology in ext2⁻/⁻ was partially rescued by FGF8 administered locally (bead implants). Our findings from zebrafish model were validated in a dental survey that was conducted with assistance of the MHE Research Foundation. The presence of the malformed and/or displaced teeth with abnormal enamel was declared by half of the respondents indicating that MO might indeed be also associated with dental problems.

Zhu HY, Hu YL, Yang Y, et al.
Mutation analysis and prenatal diagnosis of EXT1 gene mutations in Chinese patients with multiple osteochondromas.
Chin Med J (Engl). 2011; 124(19):3054-7 [PubMed] Related Publications
BACKGROUND: Multiple osteochondromas (MO), an inherited autosomal dominant disorder, is characterized by the presence of multiple exostoses on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes which encode glycosyltransferases implicated in heparin sulfate biosynthesis.
METHODS: In this study, efforts were made to identify the underlying disease-causing mutations in patients from two MO families in China.
RESULTS: Two novel EXT1 gene mutations were identified and no mutation was found in EXT2 gene. The mutation c.497T > A in exon 1 of the EXT1 gene was cosegregated with the disease phenotype in family 1 and formed a stop codon at amino acid site 166. The fetus of the proband was diagnosed negative. In family 2, the mutation c.1430-1431delCC in exon 6 of the EXT1 gene would cause frameshift and introduce a premature stop codon after the reading frame being open for 42 amino acids. The fetus of this family inherited this mutation from the father.
CONCLUSIONS: Mutation analysis of two MO families in this study demonstrates its further application in MO genetic counseling and prenatal diagnosis.

Jennes I, Zuntini M, Mees K, et al.
Identification and functional characterization of the human EXT1 promoter region.
Gene. 2012; 492(1):148-59 [PubMed] Related Publications
BACKGROUND: Mutations in Exostosin-1 (EXT1) or Exostosin-2 (EXT2) cause the autosomal dominant disorder multiple osteochondromas (MO). This disease is mainly characterized by the appearance of multiple cartilage-capped protuberances arising from children's metaphyses and is known to display clinical inter- and intrafamilial variations. EXT1 and EXT2 are both tumor suppressor genes encoding proteins that function as glycosyltransferases, catalyzing the biosynthesis of heparan sulfate. At present, however, very little is known about the regulation of these genes. Two of the most intriguing questions concerning the pathogenesis of MO are how disruption of a ubiquitously expressed gene causes this cartilage-specific disease and how the clinical intrafamilial variation can be explained. Since mutations in the EXT1 gene are responsible for ~65% of the MO families with known causal mutation, our aim was to isolate and characterize the EXT1 promoter region to elucidate the transcriptional regulation of this tumor suppressor gene.
METHODS: In the present study, luciferase reporter gene assays were used to experimentally confirm the in silico predicted EXT1 core promoter region. Subsequently, we evaluated the effect of single nucleotide polymorphisms (SNP's) on EXT1 promoter activity and transcription factor binding using luciferase assays, electrophoretic mobility shift assays (EMSA), and enzyme-linked immunosorbent assays (ELISA). Finally, a genotype-phenotype study was performed with the aim to identify one or more genetic modifiers influencing the clinical expression of MO.
RESULTS: Transient transfection of HEK293 cells with a series of luciferase reporter constructs mapped the EXT1 core promoter at approximately -917 bp upstream of the EXT1 start codon, within a 123 bp region. This region is conserved in mammals and located within a CpG-island containing a CAAT- and a GT-box. A polymorphic G/C-SNP at -1158 bp (rs34016643) was demonstrated to be located in a USF1 transcription factor binding site, which is lost with the presence of the C-allele resulting in a ~56% increase in EXT1 promoter activity. A genotype-phenotype study was suggestive for association of the C-allele with shorter stature, but also with a smaller number of osteochondromas.
CONCLUSIONS: We provide for the first time insight into the molecular regulation of EXT1. Although a larger patient population will be necessary for statistical significance, our data suggest the polymorphism rs34016643, in close proximity of the EXT1 promoter, to be a potential regulatory SNP, which could be a primary modifier that might explain part of the clinical variation observed in MO patients.

de Andrea CE, Hogendoorn PC
Epiphyseal growth plate and secondary peripheral chondrosarcoma: the neighbours matter.
J Pathol. 2012; 226(2):219-28 [PubMed] Related Publications
Chondrocytes interact with their neighbours through their cartilaginous extracellular matrix (ECM). Chondrocyte-matrix interactions compensate the lack of cell-cell contact and are modulated by proteoglycans and other molecules. The epiphyseal growth plate is a highly organized tissue responsible for long bone elongation. The growth plate is regulated by gradients of morphogens that are established by proteoglycans. Morphogens diffuse across the ECM, creating short- and long-range signalling that lead to the formation of a polarized tissue. Mutations affecting genes that modulate cell-matrix interactions are linked to several human disorders. Homozygous mutations of EXT1/EXT2 result in reduced synthesis and shortened heparan sulphate chains on both cell surface and matrix proteoglycans. This disrupts the diffusion gradients of morphogens and signal transduction in the epiphyseal growth plate, contributing to loss of cell polarity and osteochondroma formation. Osteochondromas are cartilage-capped bony projections arising from the metaphyses of endochondral bones adjacent to the growth plate. The osteochondroma cap is formed by cells with homozygous mutation of EXT1/EXT2 and committed stem cells/wild-type chondrocytes. Osteochondroma serves as a niche (a permissive environment), which facilitates the committed stem cells/wild-type chondrocytes to acquire secondary genetic changes to form a secondary peripheral chondrosarcoma. In such a scenario, the micro-environment is the site of the initiating processes that ultimately lead to cancer.

Vining NC, Done S, Glass IA, et al.
EXT2-positive multiple hereditary osteochondromas with some features suggestive of metachondromatosis.
Skeletal Radiol. 2012; 41(5):607-10 [PubMed] Related Publications
Metachondromatosis (MC) and hereditary multiple osteochondromas (HMO) are thought to be distinct disorders, each with characteristic x-ray and clinical features. Radiographic differences are the current mainstay of differential diagnosis. Both disorders are autosomal dominant, but the majority of patients with HMO have mutations in EXT-1 or EXT 2 genes. The genetic defect in MC is unknown, although recent studies indicate a possible identifiable mutation. The cancer risk in HMO is thought to be greater than in MC, although the small number of cases make such conjecture imprecise. The purpose of this report is to review existing literature and examine whether radiographic findings in HMO and MC can be reliable as a stand-alone means of differential diagnosis. Three members of a multi-generational family with an autosomal dominant exostosis syndrome were studied by clinical examination and complete skeletal survey. The roentgenographic characteristics of all osteochondromas were analyzed. The father underwent gene sequencing for EXT-1 and EXT-2, which revealed a novel EXT-2 mutation. Typical radiographic and clinical findings of both HMO and MC were seen throughout the family as well as in individuals. These family study findings contradict many of the long-standing clinical and x-ray diagnostic criteria for differentiating MC from HMO. The phenotypic crossover between the two conditions in this family, and results of genetic analysis, suggest that in the absence of a definitive genetic diagnosis, radiographic and clinical diagnosis of past and future cases HMO and MC may not be as reliable as previously assumed.

de Andrea CE, Reijnders CM, Kroon HM, et al.
Secondary peripheral chondrosarcoma evolving from osteochondroma as a result of outgrowth of cells with functional EXT.
Oncogene. 2012; 31(9):1095-104 [PubMed] Related Publications
Secondary peripheral chondrosarcoma is the result of malignant transformation of a pre-existing osteochondroma, the most common benign bone tumor. Osteochondromas are caused by genetic abnormalities in EXT1 or EXT2: homozygous deletion of EXT1 characterizes sporadic osteochondromas (non-familial/solitary), and germline mutations in EXT1 or EXT2 combined with loss of heterozygosity define hereditary multiple osteochondromas. While cells with homozygous inactivation of EXT and wild-type cells shape osteochondromas, the cellular composition of secondary peripheral chondrosarcomas and the role of EXT in their formation have remained unclear. We report using a targeted-tiling-resolution oligo-array-CGH (array comparative genomic hybridization) that homozygous deletions of EXT1 or EXT2 are much less frequently detected (2/17, 12%) in sporadic secondary peripheral chondrosarcomas than expected based on the assumption that they originate in sporadic osteochondromas, in which homozygous inactivation of EXT1 is found in ~80% of our cases. FISH with an EXT1 probe confirmed that, unlike sporadic osteochondromas, cells from sporadic secondary peripheral chondrosarcomas predominantly retained one (hemizygous deleted loci) or both copies (wild-type) of the EXT1 locus. By immunohistochemistry, we confirm the presence of cells with dysfunctional EXT1 in sporadic osteochondromas and show cells with functional EXT1 in sporadic secondary peripheral chondrosarcomas. These immuno results were verified in osteochondromas and secondary peripheral chondrosarcomas in the setting of hereditary multiple osteochondromas. Our data therefore point to a model of oncogenesis in which the osteochondroma creates a niche in which wild-type cells with functional EXT are predisposed to acquire other mutations giving rise to secondary peripheral chondrosarcoma, indicating that EXT-independent mechanisms are involved in the pathogenesis of secondary peripheral chondrosarcoma.

Jennes I, de Jong D, Mees K, et al.
Breakpoint characterization of large deletions in EXT1 or EXT2 in 10 multiple osteochondromas families.
BMC Med Genet. 2011; 12:85 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Osteochondromas (cartilage-capped bone tumors) are by far the most commonly treated of all primary benign bone tumors (50%). In 15% of cases, these tumors occur in the context of a hereditary syndrome called multiple osteochondromas (MO), an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped bone tumors at children's metaphyses. MO is caused by various mutations in EXT1 or EXT2, whereby large genomic deletions (single-or multi-exonic) are responsible for up to 8% of MO-cases.
METHODS: Here we report on the first molecular characterization of ten large EXT1- and EXT2-deletions in MO-patients. Deletions were initially identified using MLPA or FISH analysis and were subsequently characterized using an MO-specific tiling path array, allele-specific PCR-amplification and sequencing analysis.
RESULTS: Within the set of ten large deletions, the deleted regions ranged from 2.7 to 260 kb. One EXT2 exon 8 deletion was found to be recurrent. All breakpoints were located outside the coding exons of EXT1 and EXT2. Non-allelic homologous recombination (NAHR) mediated by Alu-sequences, microhomology mediated replication dependent recombination (MMRDR) and non-homologous end-joining (NHEJ) were hypothesized as the causal mechanisms in different deletions.
CONCLUSIONS: Molecular characterization of EXT1- and EXT2-deletion breakpoints in MO-patients indicates that NAHR between Alu-sequences as well as NHEJ are causal and that the majority of these deletions are nonrecurring. These observations emphasize once more the huge genetic variability which is characteristic for MO. To our knowledge, this is the first study characterizing large genomic deletions in EXT1 and EXT2.

Bowen ME, Boyden ED, Holm IA, et al.
Loss-of-function mutations in PTPN11 cause metachondromatosis, but not Ollier disease or Maffucci syndrome.
PLoS Genet. 2011; 7(4):e1002050 [PubMed] Free Access to Full Article Related Publications
Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome.

de Andrea CE, Prins FA, Wiweger MI, Hogendoorn PC
Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.
J Pathol. 2011; 224(2):160-8 [PubMed] Related Publications
Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (β3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no gradient formation. Most chondrocytes from human osteochondromas showed normal PEI staining. However, approximately 10% of tumour chondrocytes were similar to those found in the dak mutant (e.g., lack of PEI gradients). The cells in the reduced PEI-stained areas are likely associated with loss-of-function mutations in the EXT genes, and they might contribute to tumour initiation by disrupting the gradients.

Stancheva-Ivanova MK, Wuyts W, van Hul E, et al.
Clinical and molecular studies of EXT1/EXT2 in Bulgaria.
J Inherit Metab Dis. 2011; 34(4):917-21 [PubMed] Related Publications
EXT1/EXT2-CDG (Multiple cartilagineous exostoses, hereditary multiple osteochondroma (MO); OMIM 133700/133701) are common defects of O-xylosylglycan glycosylation. The diagnostic criteria are at least two osteochondromas of the juxta-epiphyseal region of long bones with in the majority of cases a positive family history and/or mutation in one of the EXT genes. The authors report data on clinical symptoms and complications of 23 patients (from 16 families), discussing the family history, age of diagnosis, new clinical and molecular data. Fifteen mutations and large deletions, of which nine are new, were detected in the EXT1 and EXT2 gene by sequence analysis, FISH and MLPA analysis.

Szuhai K, Jennes I, de Jong D, et al.
Tiling resolution array-CGH shows that somatic mosaic deletion of the EXT gene is causative in EXT gene mutation negative multiple osteochondromas patients.
Hum Mutat. 2011; 32(2):E2036-49 [PubMed] Related Publications
Multiple osteochondromas (MO) is a hereditary skeletal disorder characterized by the presence of cartilage capped bony outgrowths at bone surface. Causative mutations in EXT1 or EXT2 genes have been described in 85-90 % of MO cases. However, in about 10-15 % of the MO cases, genomic alterations can not be detected, implying the potential role of other alterations. We have designed a custom-made Agilent oligonucleotide-based microarray, containing 44,000 probes, with tiling coverage of EXT1/2 genes and addition of 68 genes involved in heparan sulfate biosynthesis and other related pathways. Out of the 17 patient samples with previously undetected mutations, a low level of deletion of the EXT1 gene in about 10-15% of the blood cells was detected in two patients and mosaic deletion of the EXT2 was detected in one patient. Here we show that for the first time somatic mosaicism with large genomic deletions as the underlying mechanism in MO formation was identified. We propose that the existence of mosaic mutations and not alterations of other heparan sulfate biosynthesis related genes play a significant role in the development of MO in patients who are tested negative for mutations in Exostosins.

Pei Y, Wang Y, Huang W, et al.
Novel mutations of EXT1 and EXT2 genes among families and sporadic cases with multiple exostoses.
Genet Test Mol Biomarkers. 2010; 14(6):865-72 [PubMed] Related Publications
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.

Reijnders CM, Waaijer CJ, Hamilton A, et al.
No haploinsufficiency but loss of heterozygosity for EXT in multiple osteochondromas.
Am J Pathol. 2010; 177(4):1946-57 [PubMed] Free Access to Full Article Related Publications
Multiple osteochondromas (MO) is an autosomal dominant disorder caused by germline mutations in EXT1 and/or EXT2. In contrast, solitary osteochondroma (SO) is nonhereditary. Products of the EXT gene are involved in heparan sulfate (HS) biosynthesis. In this study, we investigated whether osteochondromas arise via either loss of heterozygosity (2 hits) or haploinsufficiency. An in vitro three-dimensional chondrogenic pellet model was used to compare heterozygous bone marrow-derived mesenchymal stem cells (MSCs EXT(wt/-)) of MO patients with normal MSCs and the corresponding tumor specimens (presumed EXT(-/-)). We demonstrated a second hit in EXT in five of eight osteochondromas. HS chain length and structure, in vitro chondrogenesis, and EXT expression levels were identical in both EXT(wt/-) and normal MSCs. Immunohistochemistry for HS, HS proteoglycans, and HS-dependent signaling pathways (eg, TGF-β/BMP, Wnt, and PTHLH) also showed no differences. The cartilaginous cap of osteochondroma contained a mixture of HS-positive and HS-negative cells. Because a heterozygous EXT mutation does not affect chondrogenesis, EXT, HS, or downstream signaling pathways in MSCs, our results refute the haploinsufficiency theory. We found a second hit in 63% of analyzed osteochondromas, supporting the hypothesis that osteochondromas arise via loss of heterozygosity. The detection of the second hit may depend on the ratio of HS-positive (normal) versus HS-negative (mutated) cells in the cartilaginous cap of the osteochondroma.

Zuntini M, Salvatore M, Pedrini E, et al.
MicroRNA profiling of multiple osteochondromas: identification of disease-specific and normal cartilage signatures.
Clin Genet. 2010; 78(6):507-16 [PubMed] Related Publications
Multiple osteochondroma (MO) is a rare skeletal disease characterized by the formation of multiple benign cartilage-capped bone tumors; in 1-5% of patients, a malignant transformation into peripheral chondrosarcoma may occur. This disorder is characterized by a large spectrum of germline mutations scattered along EXT1/EXT2 genes, the presence of a significant percentage of patients without alterations in EXT genes, and a large phenotypic variability. The molecular basis of MO genetic and clinical heterogeneity, including the causes underlying malignant transformation, is currently unknown. This leads to the lack of appropriate diagnostic/prognostic markers as well as of therapeutic options. Recently, specific microRNAs (miRNAs) were reported to be involved in chondrogenesis and inflammatory cartilage diseases. We therefore hypothesized a role for microRNAs in cartilaginous tumors and investigated microRNA expression in osteochondroma and normal cartilage tissues to evaluate whether they could affect osteochondromas onset and/or clinical manifestations. Our results indicate that miRNAs differentially expressed in MO samples may hamper the molecular signaling responsible for normal differentiation of chondrocytes, contributing to pathogenesis and clinical outcome. Although further studies are needed to validate our observations and to identify targets of miRNAs, this is the first study reporting on miRNA expression in growth plate and its comparison with pathological conditions.

Matsumoto K, Irie F, Mackem S, Yamaguchi Y
A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses.
Proc Natl Acad Sci U S A. 2010; 107(24):10932-7 [PubMed] Free Access to Full Article Related Publications
Multiple hereditary exostoses (MHE) is one of the most common skeletal dysplasias, exhibiting the formation of multiple cartilage-capped bony protrusions (osteochondroma) and characteristic bone deformities. Individuals with MHE carry heterozygous loss-of-function mutations in Ext1 or Ext2, genes which together encode an enzyme essential for heparan sulfate synthesis. Despite the identification of causative genes, the pathogenesis of MHE remains unclear, especially with regard to whether osteochondroma results from loss of heterozygosity of the Ext genes. Hampering elucidation of the pathogenic mechanism of MHE, both Ext1(+/-) and Ext2(+/-) heterozygous mutant mice, which mimic the genetic status of human MHE, are highly resistant to osteochondroma formation, especially in long bones. To address these issues, we created a mouse model in which Ext1 is stochastically inactivated in a chondrocyte-specific manner. We show that these mice develop multiple osteochondromas and characteristic bone deformities in a pattern and a frequency that are almost identical to those of human MHE, suggesting a role for Ext1 LOH in MHE. Surprisingly, however, genotyping and fate mapping analyses reveal that chondrocytes constituting osteochondromas are mixtures of mutant and wild-type cells. Moreover, osteochondromas do not possess many typical neoplastic properties. Together, our results suggest that inactivation of Ext1 in a small fraction of chondrocytes is sufficient for the development of osteochondromas and other skeletal defects associated with MHE. Because the observed osteochondromas in our mouse model do not arise from clonal growth of chondrocytes, they cannot be considered true neoplasms.

de Andrea CE, Wiweger M, Prins F, et al.
Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma.
Lab Invest. 2010; 90(7):1091-101 [PubMed] Related Publications
Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture ('mosaic') of normal lining (EXT(+/-) or EXT(wt/wt)) and EXT(-/-) cells in the cartilaginous cap of osteochondromas.

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