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Multiple osteochondromas (MO) is an inherited skeletal disorder, and the molecular mechanism of MO remains elusive. Exome sequencing has high chromosomal coverage and accuracy, and has recently been successfully used to identify pathogenic gene mutations. In this study, exome sequencing followed by Sanger sequencing validation was first used to screen gene mutations in two representative MO patients from a Chinese family. After filtering the data from the 1000 Genome Project and the dbSNP database (build 132), the detected candidate gene mutations were further validated via Sanger sequencing of four other members of the same MO family and 200 unrelated healthy subjects. Immunohistochemisty and multiple sequence alignment were performed to evaluate the importance of the identified causal mutation. A novel frameshift mutation, c.1457insG at codon 486 of exon 6 of EXT1 gene, was identified, which truncated the glycosyltransferase domain of EXT1 gene. Multiple sequence alignment showed that codon 486 of EXT1 gene was highly conserved across various vertebrates. Immunohistochemisty demonstrated that the chondrocytes with functional EXT1 in MO were less than those in extragenetic solitary chondromas. The novel c.1457insG deleterious mutation of EXT1 gene reported in this study expands the causal mutation spectrum of MO, and may be helpful for prenatal genetic screening and early diagnosis of MO. Show less

Long bones are integral components of the limb skeleton. Recent studies have indicated that embryonic long bone development is altered by mutations in Ext genes and consequent heparan sulfate (HS) deficiency, possibly due to changes in activity and distribution of HS-binding/growth plate-associated signaling proteins. Here we asked whether Ext function is continuously required after birth to sustain growth plate function and long bone growth and organization. Compound transgenic Ext1(f/f);Col2CreERT mice were injected with tamoxifen at postnatal day 5 (P5) to ablate Ext1 in cartilage and monitored over time. The Ext1-deficient mice exhibited growth retardation already by 2weeks post-injection, as did their long bones. Mutant growth plates displayed a severe disorganization of chondrocyte columnar organization, a shortened hypertrophic zone with low expression of collagen X and MMP-13, and reduced primary spongiosa accompanied, however, by increased numbers of TRAP-positive osteoclasts at the chondro-osseous border. The mutant epiphyses were abnormal as well. Formation of a secondary ossification center was significantly delayed but interestingly, hypertrophic-like chondrocytes emerged within articular cartilage, similar to those often seen in osteoarthritic joints. Indeed, the cells displayed a large size and round shape, expressed collagen X and MMP-13 and were surrounded by an abundant Perlecan-rich pericellular matrix not seen in control articular chondrocytes. In addition, ectopic cartilaginous outgrowths developed on the lateral side of mutant growth plates over time that resembled exostotic characteristic of children with Hereditary Multiple Exostoses, a syndrome caused by Ext mutations and HS deficiency. In sum, the data do show that Ext1 is continuously required for postnatal growth and organization of long bones as well as their adjacent joints. Ext1 deficiency elicits defects that can occur in human skeletal conditions including trabecular bone loss, osteoarthritis and HME. Show less

The electron conformational and genetic algorithm methods (EC-GA) were integrated for the identification of the pharmacophore group and predicting the anti HIV-1 activity of tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepinone (TIBO) derivatives. To reveal the pharmacophore group, each conformation of all compounds was arranged by electron conformational matrices of congruity. Multiple comparisons of these matrices, within given tolerances for high active and low active TIBO derivatives, allow the identification of the pharmacophore group that refers to the electron conformational submatrix of activity. The effects of conformations, internal and external validation were investigated by four different models based on an ensemble of conformers and a single conformer, both with and without a test set. Model 1 using an ensemble of conformers for the training (39 compounds) and test sets (13 compounds), obtained by the optimum seven parameters, gave satisfactory results (R²(training) = 0.878, R²(test)= 0.910, q² = 0.840, q²(ext1) = 0.926 and q²(ext2) = 0.900). Show less

We present a 19-year-old male with laxity of skin and joints, sparse scalp hair, facial dysmorphism, epilepsy, multiple exostoses, scoliosis, gastroesophageal reflux, cardiovascular defects, and an 8q23.3-q24.22 deletion detected by array comparative genomic hybridization. The patient was previously misdiagnosed as having Ehlers-Danlos syndrome. However, his clinical findings are in fact correlated with trichorhinophalangeal syndrome type II/Langer-Giedion syndrome and Cornelia de Lange syndrome-4. We discuss the genotype-phenotype correlation and the consequence of haploinsufficiency of TRPS1, RAD21, EXT1 and KCNQ3 in this case. Show less

Long-term observations of individuals with the so-called Langer-Giedion (LGS) or tricho-rhino-phalangeal type II (TRPS2) are scarce. We report here a on follow-up of four LGS individuals, including one first described by Andres Giedion in 1969, and review the sparse publications on adults with this syndrome which comprises ectodermal dysplasia, multiple cone-shaped epiphyses prior to puberty, multiple cartilaginous exostoses, and mostly mild intellectual impairment. LGS is caused by deletion of the chromosomal segment 8q24.11-q24.13 containing among others the genes EXT1 and TRPS1. Most patients with TRPS2 are only borderline or mildly cognitively delayed, and few are of normal intelligence. Their practical skills are better than their intellectual capability, and, for this reason and because of their low self-esteem, they are often underestimated. Some patients develop seizures at variable age. Osteomas on processes of cervical vertebrae may cause pressure on cervical nerves or dissection of cerebral arteries. Joint stiffness is observed during childhood and changes later to joint laxity causing instability and proneness to trauma. Perthes disease is not rare. Almost all males become bald at or soon after puberty, and some develop (pseudo) gynecomastia. Growth hormone deficiency was found in a few patients, TSH deficiency so far only in one. Puberty and fertility are diminished, and no instance of transmission of the deletion from a non-mosaic parent to a child has been observed so far. Several affected females had vaginal atresia with consequent hydrometrocolpos. Show less

Recent experimental evidence suggests that transcellular propagation of fibrillar protein aggregates drives the progression of neurodegenerative diseases in a prion-like manner. This phenomenon is now well described in cell and animal models and involves the release of protein aggregates into the extracellular space. Free aggregates then enter neighboring cells to seed further fibrillization. The mechanism by which aggregated extracellular proteins such as tau and α-synuclein bind and enter cells to trigger intracellular fibril formation is unknown. Prior work indicates that prion protein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface to transmit pathologic processes. Here, we find that tau fibril uptake also occurs via HSPG binding. This is blocked in cultured cells and primary neurons by heparin, chlorate, heparinase, and genetic knockdown of a key HSPG synthetic enzyme, Ext1. Interference with tau binding to HSPGs prevents recombinant tau fibrils from inducing intracellular aggregation and blocks transcellular aggregate propagation. In vivo, a heparin mimetic, F6, blocks neuronal uptake of stereotactically injected tau fibrils. Finally, uptake and seeding by α-synuclein fibrils, but not huntingtin fibrils, occurs by the same mechanism as tau. This work suggests a unifying mechanism of cell uptake and propagation for tauopathy and synucleinopathy. Show less

To identify the mutation in the disease gene and provide prenatal diagnosis for a hereditary multiple osteochondromas (HMO) family. The exons of EXT1 gene in the proband with HMO and his family members were amplified by PCR. The products were analyzed by direct sequencing. Prenatal genetic diagnosis was performed by amniocentesis sampling after genotyping the proband. In the family, the affected proband was heterozygous of the mutation of 1476₁₄₇₇delTC in the EXT1 gene, and the proband's father carried the same mutation in part of his somatic cells. No mutation was found in the EXT1 gene of the proband's mother and other 11 siblings of his father. METHODS for molecular diagnosis and prenatal diagnosis of HMO were established and applied to a family of HMO. Show less

Langer-Giedion syndrome (LGS; MIM 150230), also called trichorhinophalangeal syndrome type II (TRPS2), is a contiguous gene syndrome caused by a one-copy deletion in the chromosome 8q23-q24 region, spanning the genes TRPS1 and EXT1. We identified an LGS family with two affected and two unaffected siblings from unaffected parents. To investigate the etiology of recurrence of LGS in this family, array CGH was performed on all family members. We identified a 7.29 Mb interstitial deletion at chromosome region 8q23-q24 in the two affected siblings, but no such deletion in the unaffected family members. However, the mother and one of the two unaffected siblings carried a 1.29 Mb deletion at chromosome region 8q24.1, sharing the distal breakpoint with the larger deleted segment found in the affected siblings. Another unaffected sibling had a 6.0 Mb duplication, sharing the proximal breakpoint of the deletion in the affected siblings. Karyotypic and FISH analyses in the unaffected mother revealed an insertional translocation of 8q23-q24 genomic material into chromosome 13: 46,XX,ins(13;8)(q33;q23q24). This insertional translocation in the mother results in the recurrence of LGS in this family, highlighting the importance of submicroscopic rearrangements in the genetic counseling for LGS. Show less

Heparan sulfate (HS) is an essential component of cell surface and matrix-associated proteoglycans. Due to their sulfation patterns, the HS chains interact with numerous signaling proteins and regulate their distribution and activity on target cells. Many of these proteins, including bone morphogenetic protein family members, are expressed in the growth plate of developing skeletal elements, and several skeletal phenotypes are caused by mutations in those proteins as well as in HS-synthesizing and modifying enzymes. The disease we discuss here is hereditary multiple exostoses (HME), a disorder caused by mutations in HS synthesizing enzymes EXT1 and EXT2, leading to HS deficiency. The exostoses are benign cartilaginous-bony outgrowths, form next to growth plates, can cause growth retardation and deformities, chronic pain and impaired motion, and progress to malignancy in 2-5% of patients. We describe recent advancements on HME pathogenesis and exostosis formation deriving from studies that have determined distribution, activities and roles of signaling proteins in wild-type and HS-deficient cells and tissues. Aberrant distribution of signaling factors combined with aberrant responsiveness of target cells to those same factors appear to be a major culprit in exostosis formation. Insights from these studies suggest plausible and cogent ideas about how HME could be treated in the future. Show less

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. Show less

The gene products of two members of the EXT (exostosin) gene family, EXT1 and EXT2, function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2 (EXT-like 2), one of the three EXTL genes in the human genome that are homologous to EXT1 and EXT2, encodes an N-acetylhexosaminyltransferase. We have demonstrated that EXTL2 terminates chain elongation of GAGs (glycosaminoglycans), and thereby regulates GAG biosynthesis. The abnormal GAG biosynthesis caused by loss of EXTL2 had no effect on normal development or normal adult homoeostasis. Therefore we examined the role of EXTL2 in CCl4 (carbon tetrachloride)-induced liver failure, a model of liver disease. On the fifth day after CCl4 administration, the liver/body weight ratio was significantly smaller for EXTL2-knockout mice than for wild-type mice. Consistent with this observation, hepatocyte proliferation following CCl4 treatment was lower in EXTL2-knockout mice than in wild-type mice. EXTL2-knockout mice experienced less HGF (hepatocyte growth factor)-mediated signalling than wild-type mice specifically because GAG synthesis was altered in these mutant mice. In addition, GAG synthesis in hepatic stellate cells was up-regulated during liver repair in EXTL2-knockout mice. Taken together, the results of the present study indicated that EXTL2-mediated regulation of GAG synthesis was important to the tissue regeneration processes that follow liver injury. Show less

Lymphocyte homing to peripheral lymph nodes (PLNs) is mediated by multistep interactions between lymphocytes and high endothelial venules (HEVs). Heparan sulfate (HS) has been implicated in the presentation of chemokines on the surface of HEVs during this process. However, it remains unclear whether this cell surface presentation is a prerequisite for lymphocyte homing. In this study, we generated conditional knockout (cKO) mice lacking Ext1, which encodes a glycosyltransferase essential for HS synthesis, by crossing Ext1(flox/flox) mice with GlcNAc6ST-2-Cre transgenic mice expressing Cre recombinase in HEVs. Immunohistochemical studies indicated that HS expression was specifically eliminated in PLN HEVs but retained in other blood vessels in the cKO mice. The accumulation of a major secondary lymphoid tissue chemokine, CCL21, on HEVs was also abrogated without affecting CCL21 mRNA levels, indicating that HS presents CCL21 on HEVs in vivo. Notably, a short-term lymphocyte homing assay indicated that lymphocyte homing to PLNs was diminished in the cKO mice by 30-40%. Consistent with this result, contact hypersensitivity responses were also diminished in the cKO mice. The residual lymphocyte homing to PLNs in the cKO mice was dependent on pertussis toxin-sensitive Gi protein signaling, in which lysophosphatidic acid-mediated signaling was partly involved. These results suggest that chemokine presentation by HS on the surface of HEVs facilitates but is not absolutely required for lymphocyte homing. Show less

Some neonates with Down syndrome (DS) are diagnosed with self-regressing transient myeloproliferative disorder (TMD), and 20% to 30% of those progress to acute megakaryoblastic leukemia (AMKL). We performed exome sequencing in 7 TMD/AMKL cases and copy-number analysis in these and 10 additional cases. All TMD/AMKL samples contained GATA1 mutations. No exome-sequenced TMD/AMKL sample had other recurrently mutated genes. However, 2 of 5 TMD cases, and all AMKL cases, showed mutations/deletions other than GATA1, in genes proven as transformation drivers in non-DS leukemia (EZH2, APC, FLT3, JAK1, PARK2-PACRG, EXT1, DLEC1, and SMC3). One patient at the TMD stage revealed 2 clonal expansions with different GATA1 mutations, of which 1 clone had an additional driver mutation. Interestingly, it was the other clone that gave rise to AMKL after accumulating mutations in 7 other genes. Data suggest that GATA1 mutations alone are sufficient for clonal expansions, and additional driver mutations at the TMD stage do not necessarily predict AMKL progression. Later in infancy, leukemic progression requires "third-hit driver" mutations/somatic copy-number alterations found in non-DS leukemias. Putative driver mutations affecting WNT (wingless-related integration site), JAK-STAT (Janus kinase/signal transducer and activator of transcription), or MAPK/PI3K (mitogen-activated kinase/phosphatidylinositol-3 kinase) pathways were found in all cases, aberrant activation of which converges on overexpression of MYC. Show less

The exostosin (EXT) genes encode glycosyltransferases required for glycosaminoglycan chain polymerization in the biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutations in the tumor suppressor genes EXT1 and EXT2 disturb HSPG biosynthesis and cause multiple osteochondroma (MO). How EXT1 and EXT2 traffic within the Golgi complex is not clear. Here, we show that Rotini (Rti), the Drosophila GOLPH3, regulates the retrograde trafficking of EXTs. A reduction in Rti shifts the steady-state distribution of EXTs to the trans-Golgi. These accumulated EXTs tend to be degraded and their re-entrance towards the route for polymerizing GAG chains is disengaged. Conversely, EXTs are mislocalized towards the transitional endoplasmic reticulum/cis-Golgi when Rti is overexpressed. Both loss of function and overexpression of rti result in incomplete HSPGs and perturb Hedgehog signaling. Consistent with Drosophila, GOLPH3 modulates the dynamic retention and protein stability of EXT1/2 in mammalian species. Our data demonstrate that GOLPH3 modulates the activities of EXTs, thus implicating a putative role for GOLPH3 in the formation of MO. Show less

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. Show less

Heparan sulfate is perhaps the most complex polysaccharide known from animals. The basic repeating disaccharide is extensively modified by sulfation and uronic acid epimerization. Despite this, the fine structure of heparan sulfate is remarkably consistent with a particular cell type. This suggests that the synthesis of heparan sulfate is tightly controlled. Although genomics has identified the enzymes involved in glycosaminoglycan synthesis in a number of vertebrates and invertebrates, the regulation of the process is not understood. Moreover, the localization of the various enzymes in the Golgi apparatus has not been carried out in a detailed way using high-resolution microscopy. We have begun this process, using well-known markers for the various Golgi compartments, coupled with the use of characterized antibodies and cDNA expression. Laser scanning confocal microscopy coupled with line scanning provides high-quality resolution of the distribution of enzymes. The EXT2 protein, which when combined as heterodimers with EXT1 comprises the major polymerase in heparan sulfate synthesis, has been studied in depth. All the data are consistent with a cis-Golgi distribution and provide a starting point to establish whether all the enzymes are clustered in a multimolecular complex or are distributed through the various compartments of the Golgi apparatus. Show less

Langer-Giedion syndrome (LGS) is a contiguous gene syndrome caused by a hemizygous deletion on chromosome 8q23.3-q24.11 involving TRPS1 and EXT1 genes. We report on a girl with LGS phenotype and a 7.5 Mb interstitial deletion at chromosome 8q23.3-q24.13. Array-comparative genomic hybridization (a-CGH) revealed a deletion encompassing only the EXT1 and not the TRPS1 gene. Even though the deletion of TRPS1 and EXT1 genes is responsible for craniofacial and skeletal features of LGS, there have been previous reports of patients with LGS phenotype and 8q24 deletions leaving the TRPS1 gene intact. To our knowledge, this is the third such case. Our patient differs from previously reported LGS patients without TRPS1 gene deletion in that she has the typical LGS facial dysmorphism and skeletal abnormalities. However, the girl is of normal height and has only a mild developmental delay. Additionally, she has dyslalia and premature adrenarche classified as Tanner stage 3 premature pubarche which have not yet been described as features of LGS. We examine the molecular breakpoints and phenotypes of our patient and previously reported cases. Show less

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. 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. 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. 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. Show less

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. Show less

The genetic pathways of aggressive changes of bone tumors are still poorly understood. It is very important to analyze DNA copy number alterations (DCNAs), to identify the molecular events in the step of progression to the aggressive change of bone tissue. Genome-wide array-based comparative genomic hybridization (array CGH) was used to investigate DCNAs of 14 samples from 13 aggressive bone tumors, such as giant cell tumors (GCTs) and osteosarcoma (OS), etc. Primary aggressive bone tumors had copy number gains of 17.8±12.7% in the genome, and losses of 17.3±11.4% in 287 target clones (threshold for each DCNA: ≦085, 1.15≦). Genetic unstable cases, which were defined by the total DCNAs aberration ≧30%, were identified in 9 of 13 patients (3 of 7 GCTs and all malignant tumors). High-level amplification of TGFβ2, CCND3, WI-6509, SHGC-5557, TCL1A, CREBBP, HIC1, THRA, AFM217YD10, LAMA3, RUNX1 and D22S543, were commonly observed in aggressive bone tumors. On the other hand, NRAS, D2S447, RAF1, ROBO1, MYB, MOS, FGFR2, HRAS, D13S319, D13S327, D18S552, YES1 and DCC, were commonly low. We compared genetic instability between a primary OS and its metastatic site in Case #13. Metastatic lesion showed increased 9 DCNAs of remarkable change (m/p ratio ≧1.3 folds), compared to a primary lesion. D1S214, D1S1635, EXT1, AFM137XA11, 8 M16/SP6, CCND2, IGH, 282 M15/SP6, HIC1 and LAMA3, were overexpressed. We gave attention to HIC1 (17p13.3), which was common high amplification in this series. Our results may provide several entry points for the identification of candidate genes associated with aggressive change of bone tumors. Especially, the locus 17p11-13 including HIC1 close to p53 was common high amplification in this series and review of the literature. Show less

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. Show less

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. Show less

The enchondromatosis include a heterogeneous group of congenital disorders characterized by the presence of multiple enchondromas associated with musculoskeletal malformations and the main complication is the risk of malignant transformation to chondrosarcoma. The hereditary multiple exostosis is an entity with autonomus dominant inheritance pattern, characterized by having multiple exostosis capped benign cartilage and heterogeneous clinical manifestations. Mutations of EXT1 and EXT2 genes have been cloned and are responsible for over 80% of the cases. We report a case of a six years old female with a diagnosis of hereditary multiple exostosis, that has been multidisciplinary assessed at our institution being the second case study in the Medical Genetics Unit of the Universidad de Los Andes; the clinical and genetic aspects, the differential diagnosis with Oilier disease and Maffucci syndrome were reviewed. Show less

Inherited defects in the ability to catabolize glycosaminoglycans result in lysosomal storage disorders known as mucopolysaccharidoses (MPS), causing severe pathology, particularly in the brain. Enzyme replacement therapy has been used to treat mucopolysaccharidoses; however, neuropathology has remained refractory to this approach. To test directly whether substrate reduction might be feasible for treating MPS disease, we developed a genetic model for substrate reduction therapy by crossing MPS IIIa mice with animals partially deficient in heparan sulfate biosynthesis due to heterozygosity in Ext1 and Ext2, genes that encode the copolymerase required for heparan sulfate chain assembly. Reduction of heparan sulfate by 30-50% using this genetic strategy ameliorated the amount of disease-specific biomarker and pathology in multiple tissues, including the brain. In addition, we were able to demonstrate that substrate reduction therapy can improve the efficacy of enzyme replacement therapy in cell culture and in mice. These results provide proof of principle that targeted inhibition of heparan sulfate biosynthetic enzymes together with enzyme replacement might prove beneficial for treating mucopolysaccharidoses. Show less

Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model. We generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate. This is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression. Show less

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. Show less

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. Show less

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling. Show less

In this work, the EC-GA method, a hybrid 4D-QSAR approach that combines the electron conformational (EC) and genetic algorithm optimization (GA) methods, was applied in order to explain pharmacophore (Pha) and predict anti-HIV-1 activity by studying 115 compounds in the class of 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio) thymine (HEPT) derivatives as non-nucleoside reverse transcriptase inhibitors (NNRTIs). The series of NNRTIs were partitioned into four training and test sets from which corresponding quantitative structure-activity relationship (QSAR) models were constructed. Analysis of the four QSAR models suggests that the three models generated from the training and test sets used in previous works yielded comparable results with those of previous studies. Model 4, the data set of which was partitioned randomly into two training and test sets with 11 descriptors, including electronical and geometrical parameters, showed good statistics both in the regression (r2(training) )= 0.867, r2test = 0.923) and cross-validation (q (2) = 0.811, q2(ext1) = 0.909, q2(ext2) = 0.909) for the training set of 80 compounds and the test set of 27 compounds. The prediction of the anti-HIV-1 activity of HEPT compounds by means of the EC-GA method allowed for a quantitatively consistent QSAR model. In addition, eight novel compounds never tested experimentally have been designed theoretically using model 4. Show less