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Hereditary multiple exostoses (HME) is a genetically heterogeneous disease characterized by the development of bony protuberances at the ends of all long bones. Genetic analyses have revealed HME to be a multigenic disorder linked to three loci on chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1 and EXT2 genes have been cloned and defined as glycosyltransferases involved in the synthesis of heparan sulfate. EST database analysis has demonstrated additional gene family members, EXT-like genes (EXTL1, EXTL2, and EXTL3), not associated with a HME locus. The mouse homologs of EXT1 and EXT2 have also been cloned and shown to be 99% and 95% identical to their human counterparts, respectively. Here, we report the identification of the mouse EXTL1 gene and show it is 74% identical to the human EXTL1 gene. Expression studies of all three mouse EXT genes throughout various stages of embryonic development were carried out and whole-mount in situ hybridization in the developing limb buds showed high levels of expression of all three EXT genes. However, in situ hybridization of sectioned embryos revealed remarkable differences in expression profiles of EXT1, EXT2, and EXTL1. The identical expression patterns found for the EXT1 and EXT2 genes support the recent observation that both proteins form a glycosyltransferase complex. We suggest a model for exostoses formation based on the involvement of EXT1 and EXT2 in the Indian hedgehog/parathyroid hormone-related peptide (PTHrP) signaling pathway, an important regulator of the chondrocyte maturation process. Show less

Heparan sulfate formation occurs by the copolymerization of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues. Recent studies have shown that these reactions are catalyzed by a copolymerase encoded by EXT1 and EXT2, members of the exostosin family of putative tumor suppressors linked to hereditary multiple exostoses. Previously, we identified a collection of Chinese hamster ovary cell mutants (pgsD) that failed to make heparan sulfate (Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massagué, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2267-2271). Here, we show that pgsD mutants contain mutations that either alter GlcA transferase activity selectively or that affect both GlcNAc and GlcA transferase activities. Expression of EXT1 corrects the deficiencies in the mutants, whereas EXT2 and the related EXT-like cDNAs do not. Analysis of the EXT1 mutant alleles revealed clustered missense mutations in a domain that included a (D/E)X(D/E) motif thought to bind the nucleotide sugar from studies of other transferases. These findings provide insight into the location of the GlcA transferase subdomain of the enzyme and indicate that loss of the GlcA transferase domain may be sufficient to cause hereditary multiple exostoses. Show less

Gain of chromosome arm 8q is a frequent genetic alteration in breast and prostate cancer. Two amplified subregions, 8q21 and 8q23-24, have been identified with comparative genomic hybridization (CGH). We have recently demonstrated that the EIF3S3 (eIF3-p40) gene, located at 8q23, is often amplified and overexpressed in both breast and prostate cancer. Here, we used fluorescence in situ hybridization (FISH) to map the amplified region around EIF3S3 in primary breast cancers and cell lines. The size of the common highly amplified region was about 2.5 Mb between the markers D8S1668 and WI-7959. Next, we analyzed the expression of all expressed sequence tags (ESTs) located within and near this region by RNA slot blot hybridization. In addition to EIF3S3, three anonymous ESTs and EXT1 were found to be highly expressed in cancer cell lines with the amplification at 8q23-q24. However, the anonymous ESTs were located outside the minimal highly amplified region and EXT1 was overexpressed only in one of the cancer cell lines with 8q amplification. Since EIF3S3 was the only consistently overexpressed gene located in the minimal highly amplified region, it is the strongest candidate target gene for 8q23-q24 amplification. Show less

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes. Show less

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation. Show less

EXT1 and EXT2 are two genes responsible for the majority of cases of hereditary multiple exostoses (HME), a dominantly inherited bone disorder. In order to develop an efficient screening strategy for mutations in these genes, we performed two independent blind screens of EXT1 and EXT2 in 34 unrelated patients with HME, using denaturing high-performance liquid chromatography (DHPLC) and fluorescent single-strand conformation polymorphism analysis (F-SSCP). The mutation likely to cause HME was found in 29 (85%) of the 34 probands: in 22 of these (76%), the mutation was in EXT1; seven patients (24%) had EXT2 mutations. Nineteen of these disease mutations have not been previously reported. Of the 42 different amplicon variants identified in total in the cohort, 40 were detected by DHPLC and 39 by F-SSCP. This corresponds to mutation detection efficiencies of 95% and 93% respectively. We have also found that we can confidently distinguish between different sequence variants in the same fragment using F-SSCP but not DHPLC. In light of this, and the similarly high sensitivities of the two techniques, we propose to continue screening with F-SSCP. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of exostoses, which are cartilage-capped bony protuberances mainly located on long bones. Two genes, EXT1 and EXT2, and at least one other unidentified gene, are known to be involved in the formation of exostoses. To date, 49 different EXT1 and 25 different EXT2 mutations have been found in EXT patients, and there is evidence that mutations in these two genes are responsible for over 70% of the EXT cases. Among the 49 EXT1 mutations there are 9 nonsense, 21 frameshift, and 5 splice site mutations; 2 in-frame deletions of 1 and 5 amino acids respectively; and 12 missense mutations. For EXT2, 8 nonsense, 11 frameshift, 3 splice site and 3 missense mutations are described. The majority of these mutations are mutations causing loss of function, which is consistent with the presumed tumor suppressor function of the EXT genes. Show less

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. Show less

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase. Show less

Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses. Show less

Hereditary multiple exostoses (HME) is an autosomal dominant condition in which bony outgrowths occur from the juxtaepiphyseal regions of the long bones. In a few percent of cases these exostoses undergo malignant transformation to chondrosarcomas. HME results from mutations in one of two homologous genes, EXT1 and EXT2. These are members of a new gene family that is conserved from Caenorhabditis elegans to higher vertebrates. In humans this family comprises five genes which are most conserved at their C-termini, but they do not contain any discernible functional motifs and their function(s) is unclear. Indirect evidence suggests that EXT proteins are involved in glycosaminoglycan synthesis, act as tumor suppressors and affect hedgehog signaling. One recent study has also reported that these proteins co-purify with glycosyltransferase (GlcA and GlcNAc transferase) activity and on that basis it has been postulated that they are themselves glycosyl-transferases. We performed two-hybrid screens with a fragment of EXT2 from the region that is most highly conserved in the gene family and identified two interacting proteins: the tumor necrosis factor type 1 associated protein and a novel UDP-GalNAc:poly-peptide N -acetylgalactosaminyltransferase. Significantly, both these interactions were abrogated by a disease-causing EXT mutation, indicating that they are important in the etiology of HME. The EXT2-GalNAc-T5 interaction provides the first direct physical link between EXT proteins and known components of glycosamino-glycan synthesis. Show less

We previously demonstrated that metastasis-related tumor suppressor gene(s) may exist on chromosome 8p21-22 on allelotype analysis of early colorectal carcinomas (CRC) with lymph node metastasis. Here, we searched for target gene(s) in this chromosomal region in the UniGene database. The EXTL3 (also called EXTR1) gene was selected as a candidate because of its homology to EXT1 and EXT2, putative tumor suppressor genes. We screened 12 CRC cell lines for mutations by means of polymerase chain reaction (PCR)-single strand conformation polymorphism. Three cell lines showed EXTL3 mutations, all of which were located within exon 3 and caused amino acid substitutions. Reverse transcription-PCR analysis showed that the EXTL3 expression was lacking in 1 of the 12 colorectal cancer cell lines. Although there is still no definitive evidence that EXTL3 is a tumor suppressor gene for CRC, these data suggest that inactivation of the EXTL3 gene may at least offer a selective growth advantage for some CRC cell lines. Show less

Bone development is a highly regulated process sensitive to a wide variety of hormones, inflammatory mediators and growth factors. One of the most common hereditary skeletal dysplasias, hereditary multiple exostoses (HME), is an autosomal dominant disorder characterized by skeletal malformations that manifest as bony, benign tumours near the end of long bones. HME is usually caused by defects in either one of two genes, EXT1 and EXT2, which encode enzymes that catalyse the biosynthesis of heparan sulphate, an important component of the extracellular matrix. Thus, HME-linked bone tumours, like many other skeletal dysplasias, probably result from disruptions in cell surface architecture. However, despite the recent success in unravelling functions for several members of the EXT gene family, significant challenges remain before this knowledge can be used to develop new approaches for the diagnosis and treatment of disease. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominantly inherited disease characterized by the formation of cartilage-capped prominences (exostoses) that develop from the juxtaepiphyseal regions of the long bones. Recently, EXT1 and EXT2 genes were cloned and germline mutations of EXT1 and EXT2 were identified in EXT families. In this study, we performed a mutational analysis of EXT1 and EXT2 genes in eight unrelated Korean EXT families by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing. As a result, we were able to identify one family (SNU-OC3) with the EXT1 mutation and another family (SNU-OC15) with the EXT2 mutation. The EXT1 mutation was a 10-bp deletion at the 3' end of exon 5 (CTAATTTAGg) including the splice site of this exon. The EXT2 mutation identified in the SNU-OC15 family was a missense mutation at codon 85 of exon 2 (TGC-->CGC), resulting in an amino acid change from cysteine to arginine. This missense mutation cosegregated with the disease phenotype in this family, suggesting that it is the disease-causing mutation. These two mutations identified in EXT1 and EXT2 are novel ones. Show less

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

The tricho-rhino-phalangeal syndrome type II (TRPS II, or Langer-Giedion syndrome) is an example of contiguous gene syndromes, as it comprises the clinical features of two autosomal dominant diseases, TRPS I and a form of multiple cartilaginous exostoses caused by mutations in the EXT1 gene. We have constructed a contig of cosmid, lambda-phage, PAC, and YAC clones, which covers the entire TRPS I critical region. Using these clones we identified a novel submicroscopic deletion in a TRPS I patient and refined the proximal border of the minimal TRPS1 gene region by precisely mapping the inversion breakpoint of another patient. As a first step towards a complete inventory of genes in the Langer-Giedion syndrome chromosome region (LGCR) with the ultimate aim to identify the TRPS1 gene, we analyzed 23 human expressed sequence tags (ESTs) and four genes (EIF3S3, RAD21, OPG, CXIV) which had been assigned to human 8q24.1. Our analyses indicate that the LGCR is gene-poor, because none of the ESTs and genes map to the minimal TRPS1 gene region and only two of these genes, RAD21 and EIF3S3, are located within the shortest region of deletion overlap of TRPS II patients. Two genes, OPG and CXIV, which are deleted only in some patients with TRPS II may contribute to the clinical variability of this syndrome. Show less

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. Show less

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. Show less

Osteochondroma most frequently arises sporadically and as a solitary lesion, but also may arise as multiple lesions characterizing the autosomal dominant disorder hereditary multiple exostoses (HME) and the contiguous gene syndromes Langer-Giedion and DEFECT-11 syndromes. HME is genetically heterogeneous with association of three loci including 8q24.1 (EXT1), 11p11-12 (EXT2), and 19p (EXT3). Constitutional chromosomal microdeletions of 8q24.1 and 11p11-12 are features of the Langer-Giedion and DEFECT-11 syndromes, respectively. Cytogenetic studies of osteochondroma are rare. Cytogenetic analysis was performed on 34 osteochondroma specimens from 22 patients with sporadic lesions and 4 patients with HME utilizing standard methodologies. Fluorescence in situ hybridization with chromosome specific probes was performed on three cases to define structural rearrangements further. Clonal abnormalities were detected in ten cases. Notably, deletion of 11p11-13 was observed in one case (a sporadic tumor) and loss or rearrangement of 8q22-24.1 in eight cases (seven sporadic and one hereditary tumor). These findings: 1) confirm previous observations of 8q24.1 karyotypic anomalies in sporadic osteochondroma, 2) reveal the presence of somatic chromosomal anomalies in hereditary osteochondromata, 3) suggest that similar to hereditary lesions, sporadic osteochondromas also are genetically heterogeneic (involvement of both 8q24.1 and 11p11-12), and 4) support the hypothesis that loss or mutation of EXT1 and EXT2, two putative tumor suppressor genes, may be important in the pathogenesis of sporadic as well as hereditary osteochondromata. Show less

Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1-3. Here, we report the purification of a protein from bovine serum that harbored the D-glucuronyl (GlcA) and N-acetyl-D-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis (McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P., and Tufaro, F. (1998) Nat. Genet. 19, 158-161), was shown to elevate the low GlcA and GlcNAc transferase levels of mutant cells. Thus at least two members of the EXT family of tumor suppressors encode glycosyltransferases involved in the chain elongation step of HS biosynthesis. Show less

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors (exostoses). Besides suffering complications caused by the pressure of these exostoses on the surrounding tissues, EXT patients are at an increased risk for malignant chondrosarcoma, which may develop from an exostosis. EXT is genetically heterogeneous, and three loci have been identified so far: EXT1, on chromosome 8q23-q24; EXT2, on 11p11-p12; and EXT3, on the short arm of chromosome 19. The EXT1 and EXT2 genes were cloned recently, and they were shown to be homologous. We have now analyzed the EXT1 and EXT2 genes, in 26 EXT families originating from nine countries, to identify the underlying disease-causing mutation. Of the 26 families, 10 families had an EXT1 mutation, and 10 had an EXT2 mutation. Twelve of these mutations have never been described before. In addition, we have reviewed all EXT1 and EXT2 mutations reported so far, to determine the nature, frequency, and distribution of mutations that cause EXT. From this analysis, we conclude that mutations in either the EXT1 or the EXT2 gene are responsible for the majority of EXT cases. Most of the mutations in EXT1 and EXT2 cause premature termination of the EXT proteins, whereas missense mutations are rare. The development is thus mainly due to loss of function of the EXT genes, consistent with the hypothesis that the EXT genes have a tumor- suppressor function. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by growth of benign bone tumors. Three chromosomal loci have been implicated in this genetically heterogeneous disease: EXT1 at 8q24, EXT2 at 11p13, and EXT3 on 19p. EXT1 and EXT2 were recently cloned. We evaluated 34 families with EXT to estimate the proportion of disease attributable to EXT1, EXT2, and EXT3 and to investigate the spectrum of EXT1 mutations. Linkage analyses combined with heterogeneity testing provides strong evidence in favor of linkage of disease to both chromosomes 8 and 11, but does not support evidence of linkage to chromosome 19 in this data set. The 11 EXT1 exons were PCR-amplified and sequenced in all 11 isolated cases and in 20 of the 23 familial cases. Twelve different novel EXT1 mutations were detected, including 5 frame-shift deletions or insertions, 1 codon deletion, and 6 single base-pair substitutions distributed across 8 of the exons. Only 2 of the mutations were detected in more than one family. Three mutations affect sites in which alterations were previously reported. Nonchain-terminating missense mutations were identified in codons 280 and 340, both coding for conserved arginine residues. These residues may be crucial to the function of this protein. Although the prevalence of EXT has been estimated to be approximately 1/50,000 individuals, the disease has been reported to occur much more frequently in the Chamorro natives on Guam. Our detection of an EXT1 mutation in one Chamorro subject will allow investigation of a possible founder effect in this population. Combined mutational and heterogeneity analyses in this set of families with multiple exostoses suggest that 66% of our total sample, including 45% of isolated and 77% of familial cases, are attributable to abnormalities in EXT1. Show less

Two homologous genes, EXT1 and EXT2, responsible for the development of benign multiple cartilagenous bone tumors (exostoses) on the long bones, have been identified in the past 2 years. Several arguments have been provided to support the hypothesis that these genes have tumor suppressor activity and that loss of function of these genes may contribute to the development of bone tumors. The recent identification of two EXT-like genes, EXTL1 and EXTL2, homologous to the EXT genes and to each other, revealed the existence of a larger family of genes. We now report the identification of a homologous EST (EST01365), not derived from the known EXT and EXTL genes, indicating the existence of one additional member of this gene family. We characterized this third EXT-like gene, EXTL3, and compared it with the other four members of the EXT-EXTL family. In view of its putative tumor suppressor function, the EXTL3 gene can be considered a candidate gene for the breast cancer locus on chromosome 8p12-p22. Show less

Hereditary multiple exostoses (EXT) is a genetically heterogeneous, autosomal dominant skeletal disorder. The gene for EXT1 maps to human chromosome 8q24.1 and encodes an evolutionary conserved protein that is a member of a multigene family. The mouse homolog of human EXT1 protein is 99% similar to its human counterpart. Here, we present the expression profiles of the mouse EXT1 gene. EXT1 mRNA is initially expressed at 6.5 days post-coitum (d.p.c.), which coincides with gastrulation of the mouse embryo. Whole mount in situ hybridization with 10.5 to 12.5 d.p.c. mouse embryos showed a high level of expression of EXT1 mRNA in developing limb buds. Epitope tagging experiments revealed the endoplasmic reticulum localization of EXT1 protein. This localization was consistent with a hydrophobic stretch of amino acids present at the N-terminal end of the EXT1 protein. These results provide novel information on the function of EXT1 and the etiology of hereditary multiple exostoses. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder that is characterized by the appearance of multiple outgrowths of the long bones (exostoses) at their epiphyses. Genetical heterogeneities have segregated at least on chromosome 8, 11, and 19 and been designated EXT1, EXT2, and EXT3, respectively. Recently, the responsible genes for EXT1 and EXT2 have been isolated and appeared to define a structurally related gene family. In the present study, we have identified novel genes which share significant sequence homologies with the EXT genes. The predicted protein products of the novel EXT-related genes, EXTR and EXTR2 (for EXT-related genes 1 and 2), consist of 919 and 330 amino acid residues, respectively. These genes were transcribed ubiquitously in various tissues. Based on PCR-assisted analyses of both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel, EXTR1 was localized to the chromosome 8p21 region, where loss of heterozygosity has been frequently observed in various tumors, and EXTR2 was assigned to the chromosome 1p21 region, where osteopetrosis, a dominant hereditary disease of bone, has been mapped by genetic linkage analysis, implying that the protein products of these two EXT-related genes, as well as of the EXT genes, have potential tumor suppressor activity. Show less

The rare fragile site is a specific point on a chromosome that is expressed as an isochromatid gap or break under certain conditions of cell culture and is inherited in a Mendelian codominant fashion. Five folate-sensitive fragile sites were cloned, and the molecular basis of fragile site mutation was shown to be a new class of mutation, called dynamic mutation, resulting from an allelic expansion of (CCG)n repeats. The mechanism responsible for other types of rare fragile sites, i.e., distamycin A-inducible and BrdU-requiring, is unknown, although cytogenetic studies suggested that these fragile sites play a mechanistic role in breakage and recombination and may also be integration and modification sites of foreign viral DNA genomes. A distamycin A-inducible fragile site, FRA8E, is mapped to 8q24.1 in which various loci implicated in genomic instability are located. Here we identified a YAC clone spanning both FRA8E and the hereditary multiple exostosis (EXT1) gene, using fluorescence in situ hybridization (FISH) analysis of a yeast artificial chromosome (YAC) contig. By using P1 clones as probes, the FRA8E locus was further localized to a 400-kb region including the EXT1 gene. Furthermore, the integration and amplification site of human papillomavirus 16 DNA in the ASCC (argyrophil small cell carcinoma) cells were shown not to coincide with FRA8E, but to be involved in an extensively broad genomic region of 8q24.1, including the c-myc gene. Show less

Hedgehog (Hh) proteins act through both short-range and long-range signalling to pattern tissues during invertebrate and vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects are not understood. Here we identify a new Drosophila gene, named tout-velu, that is required for diffusion of Hedgehog. Characterization of tout-velu shows that it encodes an integral membrane protein that belongs to the EXT gene family. Members of this family are involved in the human multiple exostoses syndrome, which affects bone morphogenesis. Our results, together with the previous characterization of the role of Indian Hedgehog in bone morphogenesis, lead us to propose that the multiple exostoses syndrome is associated with abnormal diffusion of Hedgehog proteins. These results show the existence of a new conserved mechanism required for diffusion of Hedgehog. Show less

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p (refs 6-9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization. Show less

Hereditary multiple exostose(EXT) is an autosomal dominant disorder of skeletal system. Three genetic loci have been identified at 8q24.1(EXT1), 11p11(EXT2) and 19p(EXT3) respectively. In this paper, EXT2 gene was cloned with positional cloning and homologous screening. SSCP and sequencing analysis have been done in 37 EXT patients who came from 20 EXT families, 2 mutations of insertion were tested in 2 patients. This confirmed that the gene cloned in this paper was EXT2 gene which locus at 11p11. Additionally EXT4 gene was cloned with homologous screening and located at 1p36.1 with FISH in this paper. Show less

Hereditary predisposition to multiple exostoses is a genetically heterogeneous disease. Recently, we have reported the identification of the EXT1 gene on human chromosome 8. We have now isolated a cDNA clone from a human adult lung cDNA library and have determined the genomic organization and promoter structure of the EXT1 gene. The gene is composed of 11 exons, ranging from 90 to 1735 bp, and spans approximately 350 kb of genomic DNA. Sequence analysis of the promoter region revealed the presence of a CpG island containing GC and CAAT boxes, but no TATA box. Such a promoter is characteristic for housekeeping genes. This finding is in good agreement with the ubiquitous expression of the EXT1 gene. Show less