Identification of novel mutations in the human EXT1 tumor suppressor gene

Hereditary multiple exostoses (EXT) is a genetically heterogeneous bone disorder caused by genes segregating on human chromosomes 8, 11, and 19 and designated EXT1, EXT2 and EXT3, respectively. Recently, the EXT1 gene has been isolated and partially characterized and appears to encode a tumor suppressor gene. We have identified six mutations in the human EXT1 gene from six unrelated multiple exostoses families segregating for the EXT gene on chromosome 8. One of the mutations we detected is the same 1-bp deletion in exon 6 that was previously reported in two independent EXT families. The other five mutations, in exons 1, 6, 9, and the splice junction at the 3′ end of exon 2, are novel. In each case, the mutation is likely to result in a truncated or nonfunctional EXT1 protein. These results corroborate and extend the previous report of mutations in this gene in two EXT families, and provide additional support for the EXT1 gene as the cause of hereditary multiple exostoses in families showing linkage to chromosome 8. Received: 2 August 1996 / Revised: 18 November 1996     

Mutation screening of the EXT1 and EXT2 genes in patients with hereditary multiple exostoses.

Hereditary multiple exostoses (HME), the most frequent of all skeletal dysplasias, is an autosomal dominant disorder characterized by the presence of multiple exostoses localized mainly at the end of long bones. HME is genetically heterogeneous, with at least three loci, on 8q24.1 (EXT1), 11p11-p13 (EXT2), and 19p (EXT3). Both the EXT1 and EXT2 genes have been cloned recently and define a new family of potential tumor suppressor genes. This is the first study in which mutation screening has been performed for both the EXT1 and EXT2 genes prior to any linkage analysis. We have screened 17 probands with the HME phenotype, for alterations in all translated exons and flanking intronic sequences, in the EXT1 and EXT2 genes, by conformation-sensitive gel electrophoresis. We found the disease-causing mutation in 12 families (70%), 7 (41%) of which have EXT1 mutations and 5 (29%) EXT2 mutations. Together with the previously described 1-bp deletion in exon 6, which is present in 2 of our families, we report five new mutations in EXT1. Two are missense mutations in exon 2 (G339D and R340C), and the other three alterations (a nonsense mutation, a frameshift, and a splicing mutation) are likely to result in truncated nonfunctional proteins. Four new mutations are described in EXT2. A missense mutation (D227N) was found in 2 different families; the other three alterations (two nonsense mutations and one frameshift mutation) lead directly or indirectly to premature stop codons. The missense mutations in EXT1 and EXT2 may pinpoint crucial domains in both proteins and therefore give clues for the understanding of the pathophysiology of this skeletal disorder.     

Hereditary multiple exostoses (EXT): mutational studies of familial EXT1 cases and EXT-associated malignancies.

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of cartilage-capped prominences that develop from the growth centers of the long bones. EXT is genetically heterogeneous, with three loci, currently identified on chromosomes 8q24.1, 11p13, and 19q. The EXT1 gene, located on chromosome 8q24.1, has been cloned and is encoded by a 3.4-kb cDNA. Five mutations in the EXT1 gene have been identified--four germ-line mutations, including two unrelated families with the same mutation, and one somatic mutation in a patient with chondrosarcoma. Four of the mutations identified resulted in frameshifts and premature termination codons, while the fifth mutation resulted in a substitution of leucine for arginine. Loss of heterozygosity (LOH) analysis of chondrosarcomas and chondroblastomas revealed multiple LOH events at loci on chromosomes 3q, 8q, 10q, and 19q. One sporadic chondrosarcoma demonstrated LOH for EXT1 and EXT3, while a second underwent LOH for EXT2 and chromosome 10. A third chondrosarcoma underwent LOH for EXT1 and chromosome 3q. These results agree with previous findings that mutations at EXT1 and multiple genetic events that include LOH at other loci may be required for the development of chondrosarcoma.     

One third of Japanese patients with multiple osteochondromas may have mutations in genes other than EXT1 or EXT2

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.     

Refinement of the Multiple Exostoses Locus (EXT2) to a 3-cM Interval on Chromosome 11

Hereditary multiple exostoses (EXT) is an autosomal dominant skeletal disorder characterized by the formation of multiple exostoses on the long bones. EXT is genetically heterogeneous, with at least three loci involved: one (EXT1) in the Langer-Giedion region on 8q23-q24, a second (EXT2) in the pericentromeric region of chromosome 11, and a third (EXT3) on chromosome 19p. In this study, linkage analysis in seven extended EXT families, all linked to the EXT2 locus, refined the localization of the EXT2 gene to a 3-cM region flanked by D11S1355 and D11S1361/D11S554. This implies that the EXT2 gene is located at the short arm of chromosome 11, in band 11p11-p12. The refined localization of EXT2 excludes a number of putative candidate genes located in the pericentromeric region of chromosome 11 and facilitates the process of isolating the EXT2 gene.     

Mutation screening of the EXT genes in patients with hereditary multiple exostoses in Taiwan

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.     

Diminished levels of the putative tumor suppressor proteins EXT1 and EXT2 in exostosis chondrocytes

The EXT family of putative tumor suppressor genes affect endochondral bone growth, and mutations in EXT1 and EXT2 genes cause the autosomal dominant disorder Hereditary Multiple Exostoses (HME). Loss of heterozygosity (LOH) of these genes plays a role in the development of exostoses and chondrosarcomas. In this study, we characterized EXT genes in 11 exostosis chondrocyte strains using LOH and mutational analyses. We also determined subcellular localization and quantitation of EXT1 and EXT2 proteins by immunocytochemistry using antibodies raised against unique peptide epitopes. In an isolated non-HME exostosis, we detected three genetic hits: deletion of one EXT1 gene, a net 21-bp deletion within the other EXT1 gene and a deletion in intron 1 causing loss of gene product. Diminished levels of EXT1 and EXT2 protein were found in 9 (82%) and 5 (45%) exostosis chondrocyte strains, respectively, and 4 (36%) were deficient in levels of both proteins. Although we found mutations in exostosis chondrocytes, mutational analysis alone did not predict all the observed decreases in EXT gene products in exostosis chondrocytes, suggesting additional genetic mutations. Moreover, exostosis chondrocytes exhibit an unusual cellular phenotype characterized by abnormal actin bundles in the cytoplasm. These results suggest that multiple mutational steps are involved in exostosis development and that EXT genes play a role in cell signaling related to chondrocyte cytoskeleton regulation.     

Association of EXT1 and EXT2, hereditary multiple exostoses gene products, in Golgi apparatus

We prepared the specific antibodies for EXT1 and EXT2, hereditary multiple exostoses (HME) gene products, and characterized their expression, subcellular localization, and protein association among EXT members. Biochemical analyses indicate that EXT1 and EXT2 can associate and form homo/hetero-oligomers in vivo with or without HME-linked mutations, EXT1 (R340C) and EXT2 (D227N), when exogenously expressed in COS-7 cells. An immunocytochemical analysis showed that both EXT1 and EXT2 localized in Golgi apparatus, irrespective of HME mutations. An immunohistochemical analysis on developing bones further showed that both EXT1 and EXT2 were concomitantly expressed in hypertrophic chondrocytes of forelimb bones from 1-day-old neonatal mouse, but down-regulated in maturing chondrocytes of developing cartilage from 21-day-old mouse. Taken together with the recent finding that EXTs encode for the glycosyltransferase required for the synthesis of heparan sulfate [Lind, T., Tufaro, F., McCormick, C., Lindahl, U., and Lindholt, K. (1998) J. Biol. Chem. 273, 26265-26268], our results implied a molecular basis that a HME-linked mutation found in EXT genes could interfere the physiological function(s) of EXT homo/hetero-oligomers as glycosyltransferases in the developing bones of HME patients.     

Mutation analysis of hereditary multiple exostoses in the Chinese

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2, the two genes responsible for EXT1 and EXT2, respectively, have been cloned. Recently, three other members of the EXT gene family, named the EXT-like genes (EXTL: EXTL1, EXTL2, and EXTL3), have been isolated. EXT1, EXT2, and the three EXTLs are homologous with one another. We have identified the intron-exon boundaries of EXTL1 and EXTL3 and analyzed EXT1, EXT2, EXTL1, and EXTL3, in 36 Chinese families with EXT, to identify underlying disease-related mutations in the Chinese population. Of the 36 families, five and 12 family groups have mutations in EXT1 and EXT2, respectively. No disease-related mutation has been found in either EXTL1 or EXTL2, although one polymorphism has been detected in EXTL1. Of the 15 different mutations (three families share a common mutation in EXT2), 12 are novel. Most of the mutations are either frameshift or nonsense mutations (12/15). These mutations lead directly or indirectly to premature stop codons, and the mutations generate truncated proteins. This finding is consistent with the hypothesis that the development of EXT is mainly attributable to loss of gene function. Missense mutations are rare in our families, but these mutations may reflect some functionally crucial regions of these proteins. EXT1 is the most frequent single cause of EXT in the Caucasian population in Europe and North America. It accounts for about 40% of cases of EXT. Our study of 36 EXT Chinese families has found that EXT1 seems much less common in the Chinese population, although the frequency of the EXT2 mutation is similar in the Caucasian and Chinese populations. Our findings suggest a possibly different genetic spectrum of this disease in different populations. Electronic Publication     

Loss of heterozygosity in chondrosarcomas for markers linked to hereditary multiple exostoses loci on chromosomes 8 and 11.

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant condition characterized by growth of multiple benign cartilage-capped tumors. EXT greatly increases the relative risk to develop chondrosarcoma, although most chondrosarcomas are sporadic. This observation suggests that, like the genes responsible for retinoblastoma and other dominantly inherited cancer susceptibility disorders, the genes that cause EXT may have tumor-suppressor function and may play a role in the pathogenesis of the related sporadic tumors. To investigate this hypothesis, we evaluated chondrosarcomas for loss of constitutional heterozygosity (LOH) at polymorphic loci linked to three recently identified genomic regions containing genes involved in EXT. LOH for markers linked to EXT1 on chromosome 8 was detected in a chondrosarcoma that arose in a man with EXT. Four of 17 sporadic tumors showed LOH for markers linked to EXT1, and 7 showed LOH for markers linked to EXT2 on chromosome 11. In all, LOH was observed for markers linked to EXT1 or EXT2 in 44% of the 18 tumors, whereas heterozygosity was retained for markers on 19p linked to EXT3. These findings support the hypothesis that genes on 8q and the pericentromeric region of 11 have tumor-suppressor function and play a role in the development of chondrosarcomas.     

Mutational screening of EXT1 and EXT2 genes in Polish patients with hereditary multiple exostoses

Hereditary multiple exostoses (HME) also known as multiple osteochondromas represent one of the most frequent bone tumor disorder in humans. Its clinical presentation is characterized by the presence of multiple benign cartilage-capped tumors located most commonly in the juxta-epiphyseal portions of long bones. HME are usually inherited in autosomal dominant manner, however de novo mutations can also occur. In most patients, the disease is caused by alterations in the EXT1 and EXT2 genes. In this study we investigated 33 unrelated Polish probands with the clinical and radiological diagnosis of HME by means of Sanger sequencing and MLPA for all coding exons of EXT1 and EXT2. We demonstrated EXT1 and EXT2 heterozygous mutations in 18 (54.6 %) and ten (30.3 %) probands respectively, which represents a total of 28 (84.9 %) index cases. Sequencing allowed for the detection of causative changes in 26 (78.8 %) probands, whereas MLPA showed intragenic deletions in two (6.1 %) further cases (15 mutations represented novel changes). Our paper is the first report on the results of exhaustive mutational screening of both EXT1/EXT2 genes in Polish patients. The proportion of EXT1/EXT2 mutations in our group was similar to other Caucasian cohorts. However, we found that EXT1 lesions in Polish patients cluster in exons 1 and 2 (55.6 % of all EXT1 mutations). This important finding should lead to the optimization of cost-effectiveness rate of HME diagnostic testing. Therefore, the diagnostic algorithm for HME should include EXT1 sequencing (starting with exons 1–2), followed by EXT2 sequencing, and MLPA/qPCR for intragenic copy number changes.     

A novel mutation in the EXT2 gene identified in two unrelated Chinese families with hereditary multiple exostoses

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.     

Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.     

Transgenic expression of the EXT2 gene in developing chondrocytes enhances the synthesis of heparan sulfate and bone formation in mice

Hereditary multiple exostoses (HME), a dominantly inherited disorder characterized by multiple cartilaginous tumors, is caused by mutations in the gene for, EXT1 or EXT2. Recent studies have revealed that EXT1 and EXT2 are required for the biosynthesis of heparan sulfate and exert maximal transferase activity as a complex. The Drosophila homologue of EXT1 (tout-velu) regulates the movement and signaling of Hedgehog protein, which plays an important role in the regulation of chondrocyte differentiation and bone development. In this study, to investigate the biological role of EXT2 in bone development in vivo and the pathological role of HME mutations in the development of exostoses, we generated transgenic mice expressing EXT2 or mutant EXT2 in developing chondrocytes. Histological analyses and micro-CT scanning showed that the biosynthesis of heparan sulfate and the formation of trabeculae were upregulated in EXT2-transgenic mice, but not in mutant EXT2-transgenic mice. The expression of EXT1 is concomitantly upregulated in EXT2-transgenic and even mutant EXT2-transgenic mice, suggesting an interactive regulation of EXT1 and EXT2 expression. These findings support that the EXT2 gene encodes an essential component of the glycosyltransferase complex required for the biosynthesis of heparan sulfate, which may eventually modulate the signaling involved in bone formation.     

The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis

The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.     

An extension of the admixture test for the study of genetic heterogeneity in hereditary multiple exostoses

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the presence of multiple cartilage-capped exostoses in the juxta-epiphyseal regions of the long bones. EXT is heterogeneous with at least three different locations currently having been identified on chromosomes 8, 11 and 19. We have tested a series of 29 EXT families for possible linkage to the three disease loci and estimated the probability of linkage of the disease to each locus in our series, by using an extension of the admixture test, which makes modelling of heterogeneous monogenic disease feasible. The maximum likelihood was obtained for proportions of 44%, 28% and 28% of families being linked to chromosome 8, 11 and 19, respectively. The a posteriori probability of linkage of the disease to EXT1, EXT2 and EXT3 was greater than 80% for 8/29, 5/29 and 3/29 families, respectively, and did not give evidence of a fourth locus for the disease. The present approach can be generalized to the investigation of genetic heterogeneity in other monogenic diseases, as it simultaneously estimates the location of each disease gene and the proportion of families linked to each locus. Received: 28 May 1996 / Revised: 7 October 1996     

Genetic heterogeneity in families with hereditary multiple exostoses

We have carried out a linkage analysis on 11 families segregating gene(s) for hereditary multiple exostoses (EXT). Four highly informative, short tandem-repeat (STR) markers that have been physically mapped to an interval surrounding the Langer-Giedion chromosomal region (8q24.11-q24.13) were used in a multipoint linkage analysis. Significant evidence for linkage of EXT with genetic heterogeneity was found. A model of heterogeneity with linkage of the disease gene to the STR markers in 70% of the families (with a 95% confidence interval of 26%–96%) produced a maximum LOD score of 8.11, with the most likely position of EXT between D8S85 and D8S199. Thus there are at least two genes that are capable of causing hereditary multiple exostoses, one in the Langer-Giedion region and one at another, unlinked location.     

Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the <Emphasis Type="Italic">EXT1</Emphasis> gene

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at θ = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.     

EXT-mutation analysis and loss of heterozygosity in sporadic and hereditary osteochondromas and secondary chondrosarcomas.

Osteochondromas occur as sporadic solitary lesions or as multiple lesions, characterizing the hereditary multiple exostoses syndrome (EXT). Approximately 15% of all chondrosarcomas arise within the cartilaginous cap of an osteochondroma. EXT is genetically heterogeneous, and two genes, EXT1 and EXT2, located on 8q24 and 11p11-p12, respectively, have been cloned. It is still unclear whether osteochondroma is a developmental disorder or a true neoplasm. Furthermore, it is unclear whether inactivation of both alleles of an EXT gene, according to the tumor-suppressor model, is required for osteochondroma development, or whether a single EXT germline mutation acts in a dominant negative way. We therefore studied loss of heterozygosity and DNA ploidy in eight sporadic and six hereditary osteochondromas. EXT1- and EXT2-mutation analysis was performed in a total of 34 sporadic and hereditary osteochondromas and secondary peripheral chondrosarcomas. We demonstrated osteochondroma to be a true neoplasm, since aneuploidy was found in 4 of 10 osteochondromas. Furthermore, LOH was almost exclusively found at the EXT1 locus in 5 of 14 osteochondromas. Four novel constitutional cDNA alterations were detected in exon 1 of EXT1. Two patients with multiple osteochondromas demonstrated a germline mutation combined with loss of the remaining wild-type allele in three osteochondromas, indicating that, in cartilaginous cells of the growth plate, inactivation of both copies of the EXT1 gene is required for osteochondroma formation in hereditary cases. In contrast, no somatic EXT1 cDNA alterations were found in sporadic osteochondromas. No mutations were found in the EXT2 gene.