The role of B3GNT3 as an oncogene in the growth, invasion and migration of esophageal cancer cells

Purpose: To investigate the role and mechanism of ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). Methods: The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. Results: B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. Conclusion: B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.

Identification of pathogenic variations in two Chinese pedigrees affected with hereditary multiple exostosis / 中华医学遗传学杂志

OBJECTIVE@#To identify pathogenic variations of EXT1 and EXT2 genes in two Chinese pedigrees affected with hereditary multiple exostosis (HME).@*METHODS@#Genomic DNA was extracted from peripheral blood samples using a phenol-chloroform method. PCR and Sanger sequencing was conducted to amplify the exons and the flanking intronic regions of the EXT1 and EXT2 genes.@*RESULTS@#DNA sequencing has revealed a heterozygous missense variation c.812A>G (p.Tyr271Cys) in the exon 1 of EXT1 in pedigree 1, and a heterozygous frameshift variation c.1431dup (p.Ser478Leufs*43) in the exon 6 of EXT1 in the proband from pedigree 2. Both variations have co-segregated with the disease phenotype, which was also consistent with previous report.@*CONCLUSION@#Two heterozygous pathogenic variations underlying HME have been identified. The result has facilitated genetic counseling and prenatal diagnosis for the affected pedigrees.

Analysis of EXT1 and EXT2 gene mutations in two Chinese pedigrees affected with hereditary multiple exostosis / 中华医学遗传学杂志

OBJECTIVE@#To detect EXT1 and EXT2 gene mutations in two pedigrees affected with hereditary multiple exostosis (HME).@*METHODS@#The coding regions and exon/intron boundaries of the EXT1 and EXT2 genes were analyzed by targeted next-generation sequencing (NGS). Suspected mutations were confirmed by Sanger sequencing of the probands, their family members and 200 unrelated healthy controls. Gross deletion was confirmed by quantitative PCR (qPCR) analysis and multiple ligation-dependent probe amplification (MLPA) analysis.@*RESULTS@#Two mutations were detected in the pedigrees, which included EXT2 gene c.337_338insG mutation in pedigree 1 and deletion of entire EXT1 in pedigree 2. Analysis of sequencing data revealed that a novel heterozygous mutation (c.337_338insG) in EXT2 gene in proband 1 and his father. The same mutation was not found among healthy family members and 200 unrelated healthy controls. As shown by NGS and MLPA analysis, proband 2 carried a heterozygous deletion of entire EXT1 gene. The same deletion was also found in her mother by qPCR.@*CONCLUSION@#Mutations of the EXT1 and EXT2 genes probably underlie the HME in both pedigrees. NGS combined with Sanger sequencing, qPCR and MLPA is effective for attaining the diagnosis.

Estudio clínico y molecular en una familia con osteocondromatosis múltiple / Clinical and molecular study in a family with multiple osteochondromatosis

Resumen: Se presentan dos casos de una familia con diagnóstico de osteocondromatosis múltiple, el cual fue confirmado por estudio molecular con mutación sin sentido en heterocigosis c.1219C>T, (p.Gln407Stop) en el gen EXT1. En el primer caso, en un paciente se presentó deformidad de Madelung como hallazgo infrecuente y en el otro caso, condrosarcoma como complicación temida, resaltando la variación intrafamiliar, por lo que se recomienda la evaluación individual e interdisciplinaria. Además, ante una entidad genética debe brindarse el adecuado y oportuno asesoramiento genético familiar a todos sus integrantes.

Abstract: We present two cases of a family with the diagnosis of multiple osteochondromatosis, which was confirmed by molecular study with nonsense in heterozygosis mutation c.1219C>T, (p.Gln407Stop) in the EXT1 gene. In these cases, the Madelung deformity was presented in one patient as an uncommon finding and chondrosarcoma as a feared complication in the other case, highlighting intrafamilial variation, which is why individual and interdisciplinary evaluation is recommended. In addition, before a genetic entity should provide adequate and timely family genetic counseling to all its members.

Potential coordination role between O-GlcNAcylation and epigenetics

Dynamic changes of the post-translational O-GlcNAc modification (O-GlcNAcylation) are controlled by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA) in cells. O-GlcNAcylation often occurs on serine (Ser) and threonine (Thr) residues of the specific substrate proteins via the addition of O-GlcNAc group by OGT. It has been known that O-GlcNAcylation is not only involved in many fundamental cellular processes, but also plays an important role in cancer development through various mechanisms. Recently, accumulating data reveal that O-GlcNAcylation at histones or non-histone proteins can lead to the start of the subsequent biological processes, suggesting that O-GlcNAcylation as 'protein code' or 'histone code' may provide recognition platforms or executive instructions for subsequent recruitment of proteins to carry out the specific functions. In this review, we summarize the interaction of O-GlcNAcylation and epigenetic changes, introduce recent research findings that link crosstalk between O-GlcNAcylation and epigenetic changes, and speculate on the potential coordination role of O-GlcNAcylation with epigenetic changes in intracellular biological processes.

Analysis of a multiple osteochondroma case caused by novel splice mutation (c.1164+1G to A) of EXT1 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism.</p><p><b>METHODS</b>The coding regions and their flanking sequences of the EXT1/EXT2 genes were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified by excluding possible single nucleotide polymorphisms and bioinformatics analysis. Transcripts of the EXT1 gene in the proband were analyzed by TA clone-sequencing, with its abundance compared with that of healthy controls.</p><p><b>RESULTS</b>DNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G to A) at the 5'splice sites of intron 3 of the EXT1 gene. The same mutation was not found in the healthy controls. Bioinformatics analysis indicated that the mutation is highly conserved and can lead to skipping of exon 3 or aberrant splicing. TA clone-sequencing indicated that the numbers of transcripts with skipping of exon 3 has significantly increased in the proband (< 0.05) compared with the controls.</p><p><b>CONCLUSION</b>The c.1164+1G to A mutation has resulted in skipping of exon 3 in a proportion of EXT1 gene transcripts. As the result, the number of transcripts with tumor suppressing function is relatively reduced and has ultimately led to the tumors.</p>

A splicing mutation of EXT1 in a Chinese pedigree with hereditary multiple exostoses / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Hereditary multiple exostoses (HME) is an autosomal dominant monogenic disorder of paraplasia ossium. Mutations in EXT1 and EXT2 have been suggested to be responsible for over 70% of HME cases. This study aimed to analyze the clinical features and pathogenic mutations in a Chinese family with HME (6 patients in 24 members of 3 generations) and to review the relative literature regarding mutations in EXT1 and EXT2 in the Chinese population.</p><p><b>METHODS</b>Clinical pedigree dada from a Chinese family of HME were collected and analysed. EXT gene mutations in this pedigree assessed by PCR and sequencing. Pubmed and Wanfang (a Chinese database) were searched for the literature related to gene mutations in Chinese HME patients.</p><p><b>RESULTS</b>In the pedigree analyzed, the age of onset of HME was becoming younger, the disease was becoming more severe, and the number of osteochondromas was increasing, in successive generations. A splicing mutation IVS5+1G>A, first identified in Chinese population, was found in all diseased members of this pedigree. According the currently available literature, EXT1 and EXT2 mutations have been detected in 29% (26/90) and 43% (39/90) Chinese families with HME.</p><p><b>CONCLUSIONS</b>HME starts earlier and becomes more severe and extensive with each successive generation in members of the pedigree analyzed. A splicing mutation, IVS5+1G>A, of EXT1, first identified in Chinese population, may be responsible for HME in the studied pedigree. EXT1 and EXT2 mutation rates may be different between the Chinese and Western populations.</p>

Mutation analysis of EXT2 gene in a family with hereditary multiple exostosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate EXT1 and EXT2 genes mutations in a family with hereditary multiple osteochondromas (HME).</p><p><b>METHODS</b>A four-generation family with HME from Linyi city of Shandong Province was studied. There were 6 affected individuals among the 17 family members. Physical examination and radiographical evaluations were carried out for all family members. Genomic DNA was extracted from peripheral venous blood and the samples were subjected to mutation screening by PCR of the coding regions of EXT1 and EXT2 genes.</p><p><b>RESULTS</b>The family has featured an autosomal dominant inheritance pattern. Sequencing of the EXT1 and EXT2 genes suggested the causative gene in this family was in linkage with the second exon of EXT2. A c.244delG mutation was detected, which has resulted in a frameshift mutation p.Asp81IlefsX30. The mutation was found in all of the 6 affected individuals but not in normal family members. And the mutation has co-segregated with the phenotype.</p><p><b>CONCLUSION</b>The mutation c.244delG in the EXT2 gene is the probably the cause of the disease in this family.</p>

Clinical characteristics of hereditary multiple exostoses: a retrospective study of mainland chinese cases in recent 23 years / 华中科技大学学报（医学）（英德文版）

Hereditary multiple exostoses (HME) are an autosomal dominant skeletal disease with wide variations in clinical manifestations among different ethnic groups. This study investigated the epidemiology, clinical presentations, pathogenetic features and treatment strategies of HME in mainland China. We searched and reviewed the related cases published since 1990 by searching electronic databases, namely SinoMed database, Wanfang database, CNKI, Web of Science and PubMed as well as Google search engines. A total of 1051 cases of HME (male-to-female ratio 1.5:1) were investigated and the diagnosis was made in 83% before the age of 10 years. Approximately 96% patients had a family history. Long bones, ribs, scapula and pelvis were the frequently affected sites. Most patients were asymptomatic with multiple palpable masses. Common complications included angular deformities, impingement on neighbouring tissues and impaired articular function. Chondrosarcomas transformation occurred in 2% Chinese cases. Among the cases examined, about 18% had mutations in EXT1 and 28% in EXT2. Frameshift, nonsense and missense mutations represented the majority of HME-causing mutations. Diagnosis of HME was made based on the clinical presentations and radiological documentations. Most patients needed no treatment. Surgical treatment was often directed to remove symptomatic exostoses, particularly those of suspected malignancy degeneration, and correction of skeletal deformities. This study shows some variance from current literature regarding other ethnic populations and may provide valuable baseline assessment of the natural history of HME in mainland China.

Mutation analysis of EXT genes in two pedigrees with hereditary multiple exostoses / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the underlying genetic defect in two Chinese families with hereditary multiple exostoses and provide genetic counseling.</p><p><b>METHODS</b>Potential mutations in EXT1 and EXT2 genes in the probands were detected by direct sequencing of PCR-amplified exons. Suspected mutations were verified in all available family members and 200 unrelated healthy controls.</p><p><b>RESULTS</b>A heterozygous frameshift mutation c.346_356delinsTAT in exon 1 of EXT1 and a heterozygous deletion mutation c.2009-2012del(TCAA) in exon 10 of EXT1 were respectively detected in affected members from the two families. The same mutations were not detected in unaffected members and 200 unrelated healthy controls. No mutations in EXT2 were detected in the two families.</p><p><b>CONCLUSION</b>Two novel mutations of EXT1 have been detected in association with hereditary multiple exostoses in two Chinese families. Above results have provided a basis for genetic counseling for the two families and expanded the spectrum of EXT1 mutations.</p>

Genetic diagnosis for a Chinese Han family with hereditary multiple osteochondromas / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the genetic cause for a Chinese Han family affected with hereditary multiple osteochondromas.</p><p><b>METHODS</b>Two patients, five unaffected relatives of the family and 100 unrelated healthy controls were collected. The coding sequences and intron/exon boundaries of EXT1 gene were amplified with polymerase chain reaction (PCR) and sequenced.</p><p><b>RESULTS</b>A heterozygous c.600G>A (p.Trp200X) mutation in exon 1 of the EXT1 gene was detected in the patients. The same mutation was not found in unaffected family members and 100 healthy controls.</p><p><b>CONCLUSION</b>The hereditary multiple osteochondromas in the family is caused by a nonsense mutation (p.Trp200X) in the EXT1 gene.</p>

Effects of Xixin decoction on enzymes related to O-GlcNAc glycosylation of tau proteins in the brain of rats with sporadic Alzheimer's disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of Xixin decoction (XXD) on O-GlcNAc transferase (OGT) and O-GlcNAc glycosidase in O-GlcNAc glycosylation of tau proteins in the brain of rats with sporadic Alzheimer's disease (SAD) and explore the possible mechanism.</p><p><b>METHODS</b>Male SD rats were randomly divided into sham-operated group, model group, donepezil group, and low-, moderate-, and high-dose XXD groups. After treatment and behavioral test, the rats were sacrificed for detecting the expressions of OGT and O-GlcNAc glycosidase in the brain using immunohistochemistry and Western blotting.</p><p><b>RESULTS</b>XXD significantly enhanced the expressions of OGT in the hippocampus of SAD rats and lowered the expression of O-GlcNAc glycosidase (P<0.05 or 0.01). OGT and O-GlcNAc glycosidase expressions showed no significant differences between the model group and donepezil group (P>0.05).</p><p><b>CONCLUSION</b>XXD can regulate the expression of OGT and O-GlcNAc glycosidase to enhance O-GlcNAc glycosylation of tau proteins in the hippocampus of SAD rats.</p>

Screening for EXT1 and EXT2 gene mutations in a ethnic Han Chinese family from Shanxi with hereditary multiple exostoses / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen for potential mutations in an ethnic Han Chinese family from Shanxi with hereditary multiple exostoses.</p><p><b>METHODS</b>Polymerase chain reaction and DNA sequencing were used to screen potential mutations in EXT1 and EXT2 genes.</p><p><b>RESULTS</b>For EXT1 gene, two synonymous mutations (P477P and E587E), three intronic mutations (c.1537 -48A>G, c.1721 +203A>G and c.1722 -103C>G) were detected. For EXT2 gene, five intronic mutations (c.-29 -148A>T, c.1080 -18T>A, c.1336 -93C>T, c.1526 -166C>T, and c.1526 -195C>T) were identified. Among these, EXT1 P477P, EXT1 E587E and EXT2 c.1080 -18T>A are polymorphisms listed by Multiple Osteochondroma Mutation Database, whilst the other 7 sites have not been reported.</p><p><b>CONCLUSION</b>No mutations have been found among all exons of the EXT1 and EXT2 genes in this family. Linkage analysis is necessary for identifying the cause of this disease.</p>

Acute UV Irradiation Increases Heparan Sulfate Proteoglycan Levels in Human Skin

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.

Mutation analysis and prenatal diagnosis of EXT1 gene mutations in Chinese patients with multiple osteochondromas / 中华医学杂志(英文版)

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

Clinical and mutation analysis of a Chinese family with muscle eye brain disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the clinical feature of a Chinese family with muscle-eye-brain disease (MEB) and the mutation of protein O-linked-mannose beta-1, 2-N-acetylglucosaminyltransferase 1 gene (POMGNT1).</p><p><b>METHODS</b>Clinical data of the proband and his family members were collected. Genomic DNA from the patient and his parents was extracted using standard procedures from the peripheral blood leukocytes. Polymerase chain reaction and DNA direct sequencing were employed to analyze all of the exons to determine the mutation, and the relationship between genotype and phenotype was analyzed.</p><p><b>RESULTS</b>The proband was diagnosed as floppy baby, presented with delayed psychomotor development and myopathic face. His serum creatine kinase (CK) level elevated moderately and brain MRI showed cerebral and cerebellar gyrus abnormalities with white matter signal intensity changes, cerebellar cysts and cerebellar and brain stem hypoplasia, consistent with congenital muscular dystrophy with eye brain disorder. Further test with DNA detected a compound heterozygous mutation of c.1896 1 G to C before exon 22 which may induce splicing error, and missense mutation c.1319T to G, p.L440R in exon 16. Both parents had a heterozygous mutation at the mutation sites.</p><p><b>CONCLUSION</b>According to our study, the family is diagnosed as MEB. The proband carried compound heterozygous mutations in the POMGNT1 gene, and his parents are heterozygous carriers, which is consistent with autosomal recessive inheritance. The child is definitely diagnosed as having muscle eye brain disease.</p>

The EXT2 gene mutation in a family with hereditary multiple exostoses / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the gene causing hereditary multiple exostoses in a Chinese pedigree.</p><p><b>METHODS</b>Linkage analysis was carried out in the family using microsatellite markers close linkage to the EXT1 and EXT2 genes to define the candidate gene. Then the whole coding sequence and the intron-exon boundaries of the candidate gene were amplified and sequenced.</p><p><b>RESULTS</b>The disease-causing gene of the family was linked to the EXT2 gene. A nonsense mutation of 536G>A in exon3 of the EXT2 gene was detected, which was co-segregated with the disease phenotype. The mutation resulted in a stop codon in codon 180. A nonpenetrant case was found in the family.</p><p><b>CONCLUSION</b>The mutation 536G>A in the EXT2 gene is the disease-causing mutation in the pedigree with hereditary multiple exostoses.</p>

Short hairpin RNA targeting N-acetylglucosaminyltransferase V inhibits the proliferation of prostate cancer PC-3 cell line / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of short hairpin RNA (shRNA) targeting N-acetylglucosaminyltransferase V (GnT-V) on the proliferation of prostate cancer PC-3 cells.</p><p><b>METHODS</b>Lipofectamine 2000 was used to transfect the recombinant plasmids carrying the shRNA targeting GnT-V gene into PC-3 cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of GnT-V, and CCK-8 assay was used to measure the cell proliferation after the transfection.</p><p><b>RESULTS</b>The recombinant plasmids were successfully transfected into PC-3 cells, resulting in a reduction of GnT-V mRNA expression by 73%. The proliferation of PC-3 cells was significantly inhibited after the transfection.</p><p><b>CONCLUSION</b>The shRNA targeting GnT-V gene can reduce the expression of GnT-V mRNA and inhibit the proliferation of PC-3 cells in vitro.</p>

A new EXT2 mutation in a Chinese family with hereditary multiple exostoses / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by formation of benign cartilage-capped tumors (exostoses), typically located at the juxtaepiphyseal regions of long bones. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2 have been cloned and are responsible for over 80% of cases. A Chinese family with HME has been analyzed in the present study.</p><p><b>METHODS</b>Linkage analysis was firstly performed to determine which of the three EXT genes could be the candidate gene, then mutation screening by PCR and direct sequencing was carried out.</p><p><b>RESULTS</b>A novel nonsense mutation (c.1006C>T) in exon 6 of EXT2, which converts the codon CAA (Gln) to the stop codon (TAA) (Gln336X), was identified. Next, prenatal diagnosis was performed and the pregnancy was determined to be normal.</p><p><b>CONCLUSION</b>A new EXT2 nonsense mutation was found in a Chinese family with hereditary multipe exostoses. The information was used for a case of prenatal diagnosis.</p>

Biosynthesis of chitooligosaccharides by recombinant Escherichia coli / 生物工程学报

Acetyl-N-glucosaminyltransferase gene (nodC) was successfully cloned to Escherichia coli from Mesorhizobium loti. The recombinant E. coli harboring nodC gene was able to synthesize chitooligosaccharides (COs) in MMYNG medium. In optimized condition, a yield of 526 mg/L was obtained after 26 h cultivation in 10 L bioreactor. COs concentration reached up to 4.5% of the cell dry weight. The COs products were purified by charcoal adsorption and Bio-gel P4 chromatography, penta-N-acetylchitopentaose (m/z, 1034[M + H]+) and tetra-N-acetylchitopentaose (m/z, 831 [M + H]+) were identified as the dominating COs product using the method of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS).