Exostosin Glycosyltransferase Research Articles

The exostosin glycosyltransferase 1/STAT3 axis is a driver of breast cancer aggressiveness

The epithelial-mesenchymal transition (EMT) program is crucial for transforming carcinoma cells into a partially mesenchymal state, enhancing their chemoresistance, migration, and metastasis. This shift in cell state is tightly regulated by cellular mechanisms that are not yet fully characterized. One intriguing EMT aspect is the rewiring of the proteoglycan landscape, particularly the induction of heparan sulfate proteoglycan (HSPG) biosynthesis. This proteoglycan functions as a co-receptor that accelerates cancer-associated signaling pathways through its negatively-charged residues. However, the precise mechanisms through which EMT governs HSPG biosynthesis and its role in cancer cell plasticity remain elusive. Here, we identified exostosin glycosyltransferase 1 (EXT1), a central enzyme in HSPG biosynthesis, to be selectively upregulated in aggressive tumor subtypes and cancer cell lines, and to function as a key player in breast cancer aggressiveness. Notably, ectopic expression of EXT1 in epithelial cells is sufficient to induce HSPG levels and the expression of known mesenchymal markers, subsequently enhancing EMT features, including cell migration, invasion, and tumor formation. Additionally, EXT1 loss in MDA-MB-231 cells inhibits their aggressiveness-associated traits such as migration, chemoresistance, tumor formation, and metastasis. Our findings reveal that EXT1, through its role in HSPG biosynthesis, governs signal transducer and activator of transcription 3 (STAT3) signaling, a known regulator of cancer cell aggressiveness. Collectively, we present the EXT1/HSPG/STAT3 axis as a central regulator of cancer cell plasticity that directly links proteoglycan synthesis to oncogenic signaling pathways.

Involvement of cell surface glycosaminoglycans in chebulagic acid's and punicalagin's antiviral activities against Coxsackievirus A16 infection

BackgroundCoxsackievirus A16 (CVA16) is responsible for several recent outbreaks of Hand, Foot, and Mouth Disease in the Asia-Pacific region, and there are currently no vaccines or specific treatments available. We have previously identified two tannins, chebulagic acid (CHLA) and punicalagin (PUG), as efficient entry inhibitors against multiple viruses known to engage cell surface glycosaminoglycans (GAGs). Interestingly, these two phytochemicals could also block enterovirus infection by directly inactivating CVA16 virions, which were recently reported to utilize GAGs to mediate its entry. PurposeThe aim of this study is to evaluate the involvement of GAGs in the anti-CVA16 activities of CHLA and PUG. MethodsTo explore a potential mechanistic link, the role of GAGs in promoting CVA16 entry was first confirmed by treating human rhabdomyosarcoma (RD) cells with soluble heparin or GAG lyases including heparinase and chondroitinase. We then performed a combination treatment of CHLA or PUG with the GAG interaction inhibitors to assess whether CHLA's and PUG's anti-CVA16 activities were related to GAG competition. Molecular docking and surface plasmon resonance (SPR) were conducted to analyze the interactions between CHLA, PUG, and CVA16 capsid. Lastly, CRISPR/Cas9 knockout (KO) of the Exostosin glycosyltransferase 1 (EXT1) gene, which encodes a transmembrane glycosyltransferase involved in heparan sulfate biosynthesis, was used to validate the importance of GAGs in CHLA's and PUG's antiviral effects. ResultsIntriguingly, combining GAG inhibition via heparin/GAG lyases treatments with CHLA and PUG revealed that their inhibitory activities against CVA16 infection were overlapping. Further molecular docking analysis indicated that the predicted binding sites of CHLA and PUG on the CVA16 capsid are in proximity to the putative residues recognized for GAG interaction, thus pointing to potential interference with the CVA16-GAG association. SPR analysis also confirmed the direct binding of CHLA and PUG to CVA16 capsid. Finally, RD cells with EXT1 KO decreased CHLA's and PUG's antiviral effect on CVA16 infection. ConclusionAltogether, our results suggest that CHLA and PUG bind to CVA16 capsid and prevent the virus’ interaction with heparan sulfate and chondroitin sulfate for its entry. This study provides mechanistic insight into the antiviral activity of CHLA and PUG against CVA16, which may be helpful for the development of antiviral strategies against the enterovirus.

Feline osteochondromatosis in a 12-year-old feline leukaemia virus-negative cat

Feline osteochondromatosis is a spontaneous osteocartilaginous exostosis associated with feline leukaemia virus (FeLV) infection or due to a frameshift variant in the exostosin glycosyltransferase 1 (EXT1) gene. Osteochondromatosis was diagnosed in an indoor-only, 12-year-old, neutered female, Russian Blue cat. Radiographs revealed bilateral calcified proliferations in the elbow, costochondral and sternochondral joints, which distorted the normal skeletal structure. Grossly, the proliferated joints presented with consistent, rounded masses, causing complete ankylosis. The main histopathological finding was an osteocartilaginous proliferation composed of multiple irregular islands of well-differentiated hyaline cartilage surrounded and delimited by osteoid tissue. Immunohistochemistry of the osteochondromas, bone marrow and mediastinal lymph nodes, using a primary anti-FeLV gp70 antibody, and FeLV proviral DNA real-time polymerase chain reaction on bone marrow were negative. Sequencing of exon 6 of the EXT1 gene was performed and nucleotide BLAST analysis demonstrated the absence of a frameshift variant. This study reports the only case of spontaneous feline osteochondromatosis in an animal more than 10 years old.

Targeting transglutaminase 2 mediated exostosin glycosyltransferase 1 signaling in liver cancer stem cells with acyclic retinoid

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca2+ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs.

Clinical survey of a pedigree with hereditary multiple exostoses and identification of EXT‑2 gene deletion mutation.

The aim of the present study was to report a clinical survey of hereditary multiple exostoses (HME) in a large Chinese pedigree, and the identification of a novel deletion mutation of exostosin glycosyltransferase 2 (EXT-2) gene. A patient with multiple exostoses with huge cartilage-capped tumors in scapula, knees and ankles received surgery in Department of Orthopedics (Shanghai Changhai Hospital). A total of 20 family members were recruited to the study, with seven members (five male; two female) diagnosed as HME. The family members of the patients with HME were examined, clinical data and peripheral blood samples were collected, and their DNA was sequenced. The incidence of HME in this family pedigree was 35%. Exostoses were most frequently in the tibiae with occurrence in six patients, followed by ribs, femurs, radii, fibulae, scapulae and humeri. DNA sequencing of peripheral blood revealed a novel deletion mutation, c.824–826delGCA, in exon 5 of the EXT-2 gene, which was observed in all the patients with HME, but not in the healthy family members. Several characteristics of HME in the pedigree were observed, such as susceptibility of male gender, decreased average age of onset and height and increased severity of clinical symptoms with generations.

Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a serious threat to the swine industry worldwide. Exostosin glycosyltransferase 1 (EXT1), an enzyme involved in the biosynthesis of heparin sulfate, has also been reported to be a host factor essential for a wide variety of pathogens. However, the role of EXT1 in PRRSV infection remains uncharted. Here, we identified that PRRSV infection caused an increase of EXT1 expression. EXT1 knockdown promoted virus infection, whereas its overexpression inhibited virus infection, suggesting an inhibitory function of EXT1 to PRRSV infection. We found that EXT1 had no effects on the attachment, internalization, or release of PRRSV but did restrict viral RNA replication. EXT1 was determined to interact with viral nonstructural protein 3 (nsp3) and nsp5 via its N-terminal cytoplasmic tail and to enhance K48-linked polyubiquitination of these two nsps to promote their degradation. Furthermore, the C-terminal glycosyltransferase activity domain of EXT1 was necessary for nsp3 and nsp5 degradation. We also found that EXT2, a EXT1 homolog, interacted with EXT1 and inhibited PRRSV infection. Similarly, EXT1 effectively restricted porcine epidemic diarrhea virus and porcine enteric alphacoronavirus infection in Vero cells. Taken together, this study reveals that EXT1 may serve as a broad-spectrum host restriction factor and suggests a molecular basis for the potential development of therapeutics against PRRSV infection.

Mutation spectrum of EXT1 and EXT2 in the Saudi patients with hereditary multiple exostoses

BackgroundHereditary Multiple Exostoses (HME), also known as Multiple Osteochondromas (MO) is a rare genetic disorder characterized by multiple benign cartilaginous bone tumors, which are caused by mutations in the genes for exostosin glycosyltransferase 1 (EXT1) and exostosin glycosyltransferase 2 (EXT2). The genetic defects have not been studied in the Saudi patients.Aim of studyWe investigated mutation spectrum of EXT1 and EXT2 in 22 patients from 17 unrelated families.MethodsGenomic DNA was extracted from peripheral leucocytes. The coding regions and intron–exon boundaries of both EXT1 and EXT2 genes were screened for mutations by PCR-sequencing analysis. Gross deletions were analyzed by MLPA analysis.ResultsEXT1 mutations were detected in 6 families (35%) and 3 were novel mutations: c.739G > T (p. E247*), c.1319delG (p.R440Lfs*4), and c.1786delA (p.S596Afs*25). EXT2 mutations were detected in 7 families (41%) and 3 were novel mutations: c.541delG (p.D181Ifs*89), c.583delG (p.G195Vfs*75), and a gross deletion of approximately 10 kb including promoter and exon 1. Five patients from different families had no family history and carried de novo mutations (29%, 5/17). No EXT1 and EXT2 mutations were found in the remaining four families. In total, EXT1 and EXT2 mutations were found in 77% (13/17) of Saudi HME patients.ConclusionEXT1 and EXT2 mutations contribute significantly to the pathogenesis of HME in the Saudi population. In contrast to high mutation rate in EXT 1 (65%) and low mutation rate in EXT2 (25%) in other populations, the frequency of EXT2 mutations are much higher (41%) and comparable to that of EXT1 among Saudi patients. De novo mutations are also common and the six novel EXT1/EXT2 mutations further expands the mutation spectrum of HME.

Human Milk Oligosaccharides Mediate the Crosstalk Between Intestinal Epithelial Caco-2 Cells and Lactobacillus Plantarum WCFS1in an In Vitro Model with Intestinal Peristaltic Shear Force

ABSTRACTBackgroundThe intestinal epithelial cells, food molecules, and gut microbiota are continuously exposed to intestinal peristaltic shear force. Shear force may impact the crosstalk of human milk oligosaccharides (hMOs) with commensal bacteria and intestinal epithelial cells.ObjectivesWe investigated how hMOs combined with intestinal peristaltic shear force impact intestinal epithelial cells and crosstalk with a commensal bacterium.MethodsWe applied the Ibidi system to mimic intestinal peristaltic shear force. Caco-2 cells were exposed to a shear force (5 dynes/cm2) for 3 d, and then stimulated with the hMOs, 2′-fucosyllactose (2′-FL), 3-FL, and lacto-N-triose II (LNT2). In separate experiments, Lactobacillus plantarumWCFS1 adhesion to Caco-2 cells was studied with the same hMOs and shear force. Effects were tested on gene expression of glycocalyx-related molecules (glypican 1 [GPC1], hyaluronan synthase 1 [HAS1], HAS2, HAS3, exostosin glycosyltransferase 1 [EXT1], EXT2), defensin β-1 (DEFB1), and tight junction (tight junction protein 1 [TJP1], claudin 3 [CLDN3]) in Caco-2 cells. Protein expression of tight junctions was also quantified.ResultsShear force dramatically decreased gene expression of the main enzymes for making glycosaminoglycan side chains (HAS3 by 43.3% and EXT1 by 68.7%) (P <0.01), but did not affect GPC1 which is the gene responsible for the synthesis of glypican 1 which is a major protein backbone of glycocalyx. Expression of DEFB1, TJP1, and CLDN3 genes was decreased 60.0–94.9% by shear force (P <0.001). The presence of L. plantarumWCFS1 increased GPC1, HAS2, HAS3, and ZO-1 expression by 1.78- to 3.34-fold (P <0.05). Under shear force, all hMOs significantly stimulated DEFB1 and ZO-1, whereas only 3-FL and LNT2 enhanced L. plantarumWCFS1 adhesion by 1.85- to 1.90-fold (P <0.01).Conclusions3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarumWCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs.

Abnormal sodium and water homeostasis in mice with defective heparan sulfate polymerization.

Glycosaminoglycans in the skin interstitium and endothelial surface layer have been shown to be involved in local sodium accumulation without commensurate water retention. Dysfunction of heparan sulfate glycosaminoglycans may therefore disrupt sodium and water homeostasis. In this study, we investigated the effects of combined heterozygous loss of heparan sulfate polymerization genes (exostosin glycosyltransferase 1 and 2; Ext1+/-Ext2+/-) on sodium and water homeostasis. Sodium storage capacity was decreased in Ext1+/-Ext2+/- mice as reflected by a 77% reduction in endothelial surface layer thickness and a lower skin sodium-to-glycosaminoglycan ratio. Also, these mice were characterized by a higher heart rate, increased fluid intake, increased plasma osmolality and a decreased skin water and sodium content, suggesting volume depletion. Upon chronic high sodium intake, the initial volume depletion was restored but no blood pressure increase was observed. Acute hypertonic saline infusion resulted in a distinct blood pressure response: we observed a significant 15% decrease in control mice whereas blood pressure did not change in Ext1+/-Ext2+/- mice. This differential blood pressure response may be explained by the reduced capacity for sodium storage and/or the impaired vasodilation response, as measured by wire myography, which was observed in Ext1+/-Ext2+/- mice. Together, these data demonstrate that defective heparan sulfate glycosaminoglycan synthesis leads to abnormal sodium and water homeostasis and an abnormal response to sodium loading, most likely caused by inadequate capacity for local sodium storage.

A novel splice mutation induces exon skipping of the EXT1 gene in patients with hereditary multiple exostoses.

The molecular mechanism of hereditary multiple exostoses (HME) remains ambiguous and a limited number of studies have investigated the pathogenic mechanism of mutations in patients with HME. In the present study, a novel heterozygous splice mutation (c.1284+2del) in exostosin glycosyltransferase 1 (EXT1) gene was identified in a three-generation family with HME. Bioinformatics and TA clone-sequencing indicated that the splice site mutation would result in exon 4 skipping. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed that the expression levels of wild-type EXT1/EXT2 mRNA in patients with HME were significantly decreased, compared with normal control participants (P<0.05). Abnormal EXT1 transcript lacking exon 4 (EXT1-DEL) and full-length EXT1 mRNA (EXT1-FL) were overexpressed in 293-T cells and Cos-7 cells using lentivirus infection. RT-qPCR demonstrated that the expression level of EXT1-DEL was significantly increased, compared with EXT1-FL (17.032 vs. 6.309, respectively; P<0.05). The protein encoded by EXT1-DEL was detected by western blot analysis, and the level was increased, compared with EXT1-FL protein expression. Immunofluorescence indicated that the protein encoded by EXT1-DEL was located in the cytoplasm of Cos-7 cells, which was consistent with the localization of the EXT1-FL protein. In conclusion, the present study identified a novel splice mutation that causes exon 4 skipping during mRNA splicing and causes reduced expression of EXT1/EXT2. The mutation in EXT1-DEL generated a unique peptide that is located in the cytoplasm in vitro, and it expands the mutation spectrum and provides molecular genetic evidence for a novel pathogenic mechanism of HME.

A novel EXT2 frameshift mutation identified in a family with multiple osteochondromas.

Multiple osteochondromas (MO) is an autosomal inherited disease that is characterized by benign bone tumors. However, the underlying mechanism of MO at a molecular level requires further investigation. The majority of mutations associated with MO occur in the exostosin glycosyltransferase genes (EXT)1 or EXT2. In the present study, the genetic causes of the disease were investigated. Polymerase chain reaction amplification, followed by DNA sequencing of the complete EXT1 and EXT2 coding regions, were conducted in a family with MO (n=5). A novel frameshift mutation in exon 3 of EXT2 (c.660delG) was detected. The production of a defective EXT2 protein, lacking 450 C-terminal amino acid residues is predicted to be caused by the c.660delG mutation, located within the exostosin domain of EXT2. The missing residues contain the exostosin and glycosyltransferase family 64 domains, which are critical for the function of EXT2. The novel c.660delG frameshift mutation in the EXT2 gene extends the etiological understanding of MO and may provide an effective reference for genetic counseling and prenatal diagnosis in this family.

Genome-wide admixture and association study of subclinical atherosclerosis in the Women's Interagency HIV Study (WIHS).

Cardiovascular disease (CVD) is a major comorbidity among HIV-infected individuals. Common carotid artery intima-media thickness (cCIMT) is a valid and reliable subclinical measure of atherosclerosis and is known to predict CVD. We performed genome-wide association (GWA) and admixture analysis among 682 HIV-positive and 288 HIV-negative Black, non-Hispanic women from the Women’s Interagency HIV study (WIHS) cohort using a combined and stratified analysis approach. We found some suggestive associations but none of the SNPs reached genome-wide statistical significance in our GWAS analysis. The top GWAS SNPs were rs2280828 in the region intergenic to mediator complex subunit 30 and exostosin glycosyltransferase 1 (MED30 | EXT1) among all women, rs2907092 in the catenin delta 2 (CTNND2) gene among HIV-positive women, and rs7529733 in the region intergenic to family with sequence similarity 5, member C and regulator of G-protein signaling 18 (FAM5C | RGS18) genes among HIV-negative women. The most significant local European ancestry associations were in the region intergenic to the zinc finger and SCAN domain containing 5D gene and NADH: ubiquinone oxidoreductase complex assembly factor 1 (ZSCAN5D | NDUF1) pseudogene on chromosome 19 among all women, in the region intergenic to vomeronasal 1 receptor 6 pseudogene and zinc finger protein 845 (VN1R6P | ZNF845) gene on chromosome 19 among HIV-positive women, and in the region intergenic to the SEC23-interacting protein and phosphatidic acid phosphatase type 2 domain containing 1A (SEC23IP | PPAPDC1A) genes located on chromosome 10 among HIV-negative women. A number of previously identified SNP associations with cCIMT were also observed and included rs2572204 in the ryanodine receptor 3 (RYR3) and an admixture region in the secretion-regulating guanine nucleotide exchange factor (SERGEF) gene. We report several SNPs and gene regions in the GWAS and admixture analysis, some of which are common across HIV-positive and HIV-negative women as demonstrated using meta-analysis, and also across the two analytic approaches (i.e., GWA and admixture). These findings suggest that local European ancestry plays an important role in genetic associations of cCIMT among black women from WIHS along with other environmental factors that are related to CVD and may also be triggered by HIV. These findings warrant confirmation in independent samples.

Circulating microRNAs as Biomarkers for Pediatric Astrocytomas

Since MicroRNAs (miRNAs) are potent regulators of gene expression, their expression and function alterations are associated with different types of cancer, including pediatric astrocytoma. Since the secretion of miRNAs by tumors into corporal fluids has made it possible to identify biomarkers in cancer, their deter mination in pediatric astrocytoma is vital. In order to gain insight into the mechanisms controlled by miRNAs in these neoplasms, we tested the expression of miRNAs 130a, 145, 335, 1303, and let-7g-3p by qPCR in tumors and blood serum from pediatric patients with astrocytoma. The data was analyzed with the DIANA-miRPath v3.0 platform. The data represented expression changes of all mirRNAs tested in both tumors and blood serum, which strongly suggest their use as circulating biomarkers for astrocytic tumors. The bioinformatic analysis -with DIANA-miRPath v3.0- showed the involvement of these miRNAs in extracellular matrix (ECM)-receptor interaction and proteoglycans in cancer, which control many hallmarks of cancer. In fact, the expression of the proteoglycan syndecan 4 (SDC4) and that of its biosynthetic enzymes, Exostosin Glycosyltransferase 1 (EXT1) and Xylosyltransferase 1 (XYLT1), were altered in pediatric astrocytoma. Our results highlight the role of microRNAs in the biology of pediatric astrocytoma and demonstrated for the first time the potential use of some circulating microRNAs as non-invasive biomarkers for this type of tumors, particularly miRs 130a, 145, and335.

Familial solitary chondrosarcoma resulting from germline EXT2 mutation.

Germline mutations of EXT2, encoding Exostosin Glycosyltransferase 2, are associated with multiple osteochondromas (MO), an autosomal dominant disease characterized by the development of multiple peripheral cartilaginous benign tumors with a weak risk of malignant transformation. We report here a family with a remarkable clinical presentation characterized by the development of isolated chondrosarcomas, mostly located in ribs. Comparative analysis of exomes from two third-degree affected relatives led us to identify a single common disruptive variation, corresponding to a stop mutation (c.237G > A, p.Trp79*; (NM_000401.3); c.138G > A, p.Trp46*; (NM_207122.1)) within exon 2 of the EXT2 gene. Interestingly, no obvious sign of MO was detected in affected members by radiological examination. This report shows that germline mutations of EXT2 can result, not only in the development of multiple benign osteochondromas, but also in the development of isolated malignant cartilaginous tumors including central tumors, and that the presence of germline EXT2 mutation should be considered in patients suspected to have an inherited predisposition to chondrosarcoma, even in the absence of MO. © 2016 Wiley Periodicals, Inc.

Novel mutation of EXT2 identified in a large family with multiple osteochondromas.

Multiple osteochondromas (MO), also known as hereditary multiple exostoses, is an autosomal dominant bone disorder. Mutations in exostosin glycosyl transferase-1 (EXT1) and exostosin glycosyl transferase-2 (EXT2), including missense, nonsense, frameshift and splice-site mutations, account for up to 80% of reported cases. The proteins EXT1 and EXT2 form a hetero-oligomeric complex that functions in heparan sulfate proteoglycan biosynthesis. A heterozygous EXT2 mutation, c.939+1G>T, was identified in a five-generation 33-member MO family, and was present in all 13 affected members. The mutation results in deletion of exon 5 in the mRNA, producing a frameshift that leads to a premature termination codon. The present study extends the mutational spectrum of EXT2.

Gene expression profiling of the 8q22-24 position in human breast cancer: TSPYL5, MTDH, ATAD2 and CCNE2 genes are implicated in oncogenesis, while WISP1 and EXT1 genes may predict a risk of metastasis.

Gene expression profiling has been suggested to predict breast cancer outcome. The prognostic value of the 8q22-24 position in breast cancer remains to be elucidated. The present study evaluated expression patterns of the genes located at this position in metastatic and non-metastatic breast cancer. A total of 85 patients with recurrent/metastatic (n=15) and non-metastatic (n=70) early-stage, estrogen receptor-positive and lymph node-negative breast tumors were included. In addition, 15 normal breast tissue samples were used as controls. Demographic and clinical features were recorded. Subsequently, mRNA copy numbers of exostosin glycosyltransferase 1 (EXT1), WNT1 inducible signaling pathway protein 1 (WISP1), ATPase family, AAA domain containing 2 (ATAD2), TSP-like 5 (TSPYL5), metadherin (MTDH) and cyclin E2 (CCNE2) genes were measured by reverse transcription-quantitative polymerase chain reaction assay. The expression of EXT1 and WISP1 exhibited a significant decline in the metastatic breast cancer group compared to the control (P=0.015 and P=0.012, respectively). The expression of TSPYL5, MTDH and ATAD2 was significantly decreased in the metastatic (P=0.002, P=0.018 and P=0.016, respectively) and non-metastatic (P=0.038, P=0.045 and P=0.000, respectively) breast cancer groups compared with the control. The expression of CCNE2 in the metastatic and non-metastatic breast cancer groups was significantly increased compared with the control (P=0.002 and P=0.001, respectively). WISP1 expression demonstrated a correlation with patient age and tumor size, and TSPYL5 expression was correlated with lymphovascular invasion. None of the genes investigated exhibited any correlation with stage and grade of disease. The TSPYL5, MTDH, ATAD2 and CCNE2 genes may be implicated in the pathogenesis of human breast cancer, while the WISP1 and EXT1 genes may have the potential to serve as promising indicators of the risk of metastasis. However, further studies are required to validate these results.

Assessing the general population frequency of rare coding variants in the EXT1 and EXT2 genes previously implicated in hereditary multiple exostoses

Hereditary multiple exostoses (HME) is a rare childhood-onset skeletal disease linked to mutations in exostosin glycosyltransferase 1 (EXT1) or 2 (EXT2). Patients are heterozygous for either an EXT1 or EXT2 mutation, and it is widely assumed that exostosis formation and associated defects, such as growth retardation and skeletal deformities, require loss-of-heterozygosity or a second hit in affected cells. However, the relevance and phenotypic impact of many presumed pathogenic EXT variants remain uncertain. We extracted all amino acid-altering (missense) and loss of function (LoF; nonsense, frameshift, or splice-site) variants from the Exome Aggregation Consortium (ExAC), a large population-based repository of exome sequence data from diverse ancestries that has screened out severe pediatric disease, to assess the overall mutation spectrum of predicted protein-damaging variants across these two genes in the general population. We then determined whether clinically-identified, presumably pathogenic variants implicated in HME exist among healthy individuals. We found six EXT1 and four EXT2 missense mutations in ExAC, suggesting that these mutations have either been misclassified as pathogenic or are not fully penetrant. Furthermore, EXT1 is heavily selectively constrained, while EXT2 is more tolerant to protein-damaging variants, especially at its C-terminus, possibly explaining the genotype–phenotype correlation that EXT1 variants usually result in more severe disease. In conclusion, population-based exome data is a useful filter for determining whether clinically detected variants are likely pathogenic, as well as revealing biological insight into rare disease genes such as EXT1 and EXT2.

Detection of exostosin glycosyltransferase gene mutations in patients with non-hereditary osteochondromas of the mandibular condyle.

Exostosin glycosyltransferase (EXT) 1 and EXT2 have been identified as causative genes in osteochondroma; however, it is not known whether these genes are also involved in condylar osteochondromas. The aim of this study was to identify EXT1 and EXT2 mutations in patients with non-hereditary osteochondromas of the mandibular condyle. DNA was obtained from resected tissues (cartilage cap) of 12 patients with solitary condylar osteochondromas. The exons, 3',5'-untranslated regions and intron-exon boundaries of EXT1 and EXT2 were amplified by polymerase chain reaction and the products were sequenced directly. Through direct sequencing, four genetic variations of EXT1 in 4 cases and three variations of EXT2 in 5 cases were identified. The intronic alteration of the EXT2 gene, occurring in 2 cases, was novel, whereas the other alterations had been previously reported. Nonsense somatic mutations were detected in tumor DNA. Our study extended the mutational spectrum in EXT1 and EXT2 and may facilitate a better understanding of the pathophysiology of condylar osteochondromas.

Identification of a novel frameshift mutation of the EXT2 gene in a family with multiple osteochondroma.

Multiple osteochondroma (MO), also known as multiple hereditary exostoses, is an autosomal dominant skeletal disorder with characteristic multiple cartilage-capped tumours (osteochondromas or exostoses) growing outward from the metaphyseal region of the long tubular bones. Mutations in exostosin glycosyltransferase 1 (EXT1) or EXT2 are the most commonly associated mutations with MO and are responsible for 70–95% of cases. In the present study, a genetic analysis was performed on a large family with MO using polymerase chain reaction and direct DNA sequencing of the entire coding regions of EXT1 and EXT2. Sanger sequencing identified a novel heterozygous frameshift mutation, c.119_120delCT (p.Thr40ArgfsX15), in exon 2 of the EXT2 gene in the proband and all other affected individuals, while this deleterious mutation was not detected in the healthy family members and normal controls. The c.119_120delCT mutation is located in the transmembrane region of the EXT2 protein and results in a truncated EXT2 protein lacking 665 amino acids at the C-terminus, which includes the critical exostosin and glycosyltransferase family 64 domains. Thus, the present study identified a novel causative frameshift mutation in EXT2 from a large MO family. This study is useful for extending the known mutational spectrum of EXT2, for understanding the genetic basis of MO in the patients studied, and for further application of mutation screening in the genetic counseling and subsequent prenatal diagnosis of this family.

Heparan Sulfate Proteoglycans Regulate Fgf Signaling and Cell Polarity during Collective Cell Migration

Collective cell migration is a highly regulated morphogenetic movement during embryonic development and cancer invasion that involves the precise orchestration and integration of cell-autonomous mechanisms and environmental signals. Coordinated lateral line primordium migration is controlled by the regulation of chemokine receptors via compartmentalized Wnt/β-catenin and fibroblast growth factor (Fgf) signaling. Analysis of mutations in two exostosin glycosyltransferase genes (extl3 and ext2) revealed that loss of heparan sulfate (HS) chains results in a failure of collective cell migration due to enhanced Fgf ligand diffusion and loss of Fgf signal transduction. Consequently, Wnt/β-catenin signaling is activated ectopically, resulting in the subsequent loss of the chemokine receptor cxcr7b. Disruption of HS proteoglycan (HSPG) function induces extensive, random filopodia formation, demonstrating that HSPGs are involved in maintaining cell polarity in collectively migrating cells. The HSPGs themselves are regulated by the Wnt/β-catenin and Fgf pathways and thus are integral components of the regulatory network that coordinates collective cell migration with organ specification and morphogenesis.