Objective:b> Glycogen storage disease type IX (GSD-IX) is a rare primary glucose metabolism abnormality caused by phosphorylase kinase deficiency and a series of pathogenic gene mutations. The clinical characteristics, gene analysis, and functional verification of a mutation in a child with hepatomegaly are summarized here to clarify the pathogenic cause of the disease. Methods:b> The clinical data of a child with GSD-IX was collected. Peripheral blood from the child and his parents was collected for genomic DNA extraction. The patient's gene diagnosis was performed by second-generation sequencing. The suspected mutations were verified by Sanger sequencing and bioinformatics analysis. The suspected splicing mutations were verified in vivo by RT-PCR and first-generation sequencing. Results:b> Hepatomegaly, transaminitis, and hypertriglyceridemia were present in children. Liver biopsy pathological examination results indicated glycogen storage disease. Gene sequencing revealed that the child had a c.285 + 2_285 + 5delTAGG hemizygous mutation in the PHKA2 gene. Sanger sequencing verification showed that the mother of the child was heterozygous and the father of the child was of the wild type. Software such as HSF3.1 and ESEfinder predicted that the gene mutation affected splicing. RT-PCR of peripheral blood from children and his mother confirmed that the mutation had caused the skipping of exon 3 during the constitutive splicing of the PHKA2 gene. Conclusion:b> The hemizygous mutation in the PHKA2 gene (c.285 + 2_285 + 5delTAGG) is the pathogenic cause of the patient's disease. The detection of the novel mutation site enriches the mutation spectrum of the PHKA2 gene and serves as a basis for the family's genetic counseling.
Child ; Humans ; Exons ; Glycogen Storage Disease/genetics* ; Hepatomegaly/genetics* ; Mutation ; Phosphorylase Kinase/genetics* ; Male ; Female

OBJECTIVE: To explore the genetic etiology of a patient with glycogen storage diseases. METHODS: Clinical data of child and his parents were collected. The genes associated with glycogen storage diseases were subjected to high-throughput sequencing to screen the variants. Candidate variant was validated by Sanger sequencing. Pathogenicity of the variant was predicted by bioinformatic analysis. RESULTS: High-throughput sequencing results showed that the boy has carried a hemizygous c.749C>T (p.S250L) variant of the PHKA2 gene. Sanger sequencing verified the results and confirmed that it was inherited from his mother. This variant was unreported previously and predicted to be pathogenic by bioinformatic analysis. CONCLUSION The patient was diagnosed with glycogen storage disease type IXa due to a novel c.749C>T (p.S250L) hemizygous variant of the PHKA2 gene. High-throughput sequencing can facilitate timely and accurate differential diagnosis of glycogen storage disease type IXa.
Child ; Family ; Genetic Testing ; Glycogen Storage Disease/pathology* ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Male ; Mutation ; Phosphorylase Kinase/genetics*

PURPOSE: To study the efficacy of capecitabine or S-1 plus oxaliplatin (CAPOX or SOX) for treating thymidine phosphorylase (TP)- or dihydropyrimidine dehydrogenase (DPD)-positive advanced gastric cancer.MATERIALS AND METHODS: Eighty-six patients with stage IIIC to IV gastric cancer were assessed for TP and DPD expression by immunohistochemistry. The association between CAPOX or SOX efficacy and TP/DPD expression was retrospectively analyzed.RESULTS: There were no significant differences in the objective remission rate (ORR, 52.27% vs. 47.62%; P>0.05), disease control rate (72.73% vs. 73.81%, P>0.05), progression-free survival (hazard ratio [HR], 1.119; 95% confidence interval [CI], 0.739–1.741; P=0.586), and overall survival (OS; HR, 0.855; 95% CI, 0.481–1.511; P=0.588) between CAPOX and SOX. A higher number of stage IV patients showed TP positivity, while DPD-positive patients predominantly showed intestinal type of gastric cancer. In TP-positive patients, the ORRs associated with CAPOX and SOX treatments were 57.14% and 38.10%, respectively; OS was better with CAPOX than with SOX (HR, 0.447; 95% CI, 0.179–0.978; P=0.046). Among DPD-positive patients, the SOX treatment-associated ORR (60.87%) was significantly higher than the CAPOX treatment-associated ORR (43.48%). Furthermore, SOX treatment resulted in better OS than did CAPOX treatment (HR, 2.020; 95% CI, 1.019–4.837; P=0.049).CONCLUSIONS: No significant difference in clinical efficacy was found between CAPOX and SOX. TP-positive patients might respond better to CAPOX while DPD-positive patients may respond better to SOX. Our findings might serve as a guide for personalized chemotherapy for gastric cancer.
Capecitabine ; Dihydrouracil Dehydrogenase (NADP) ; Disease-Free Survival ; Drug Therapy ; Humans ; Immunohistochemistry ; Retrospective Studies ; Stomach Neoplasms ; Thymidine Phosphorylase ; Thymidine ; Treatment Outcome

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V⁺) and uninfected (V⁻) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V⁺ compared with V⁻ isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V⁺ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V⁺ and V⁻ isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.
Glucose-6-Phosphate Isomerase ; Glycogen Phosphorylase ; Heat-Shock Proteins ; Host-Parasite Interactions ; Malate Dehydrogenase ; Metabolism ; Polymerase Chain Reaction ; Proteome ; Reticuloendotheliosis virus ; Ribosomal Proteins ; RNA, Double-Stranded ; RNA, Messenger ; Trichomonas vaginalis* ; Trichomonas* ; Triose-Phosphate Isomerase ; Virulence

<b>OBJECTIVEb>To observe the efficacy and safety of chemotherapy regimens oxaliplatin combined with capecitabine (CAPOX) or oxaliplatin combined with tegafur, gimeracil and oteracil potassium capsules (S-1)(SOX), and to investigate the value of expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) proteins in tumor tissue for predicting the efficacy of CAPOX and SOX regimens in advanced gastric cancer patients.

<b>METHODSb>A total of 107 newly-diagnosed, stage Ⅲc/Ⅳ gastric cancer patients (no surgical indication, ECOG performance scores 0-2 and expected survival time ≥3 months) were recruited with 101 patients evaluated. The patients were randomly divided into two groups. One was study group in which the patients received CAPOX regimen. The other was control group received SOX regimen. Each patient received four cycles, at least two cycles chemotherapy every three weeks and followed up until death or lost. Tumor biopsies were obtained by gastroscopy for immunohistochemical examination of the expression of TP and DPD proteins before chemotherapy. Response rate (ORR), overall survival (OS) and time to tumor progression (TTP) of the patients were assessed.

<b>RESULTSb>The objective response rate (ORR) of the study and control groups was 49.0% (5/51) vs. 46.0% (23/50), respectively (P>0.05). The overall survival (OS) was 357.36±24.69 days in the study group and 349.87±22.63 days in the control group, and the time-to-progression (TTP) was 216.75±19.32 days in the study group and 220.54±18.47 days in the control group (P>0.05 for both). Stratified analysis showed that the ORR of TP-positive patients in the study group was significantly higher than that in the control group (72.0 % vs. 41.7 %, P=0.032). There was no significant difference in ORR between the TP-negative patients in the study and control groups (26.9% vs. 50.0%, P=0.087), while the ORR of DPD-positive patients in the control group was significantly higher than that of the study group (51.9% vs. 34.6%, P=0.046). There was no significant difference in the ORR between DPD-negative patients in the study and control groups (64.0% vs. 39.1%, P=0.084). The follow-up showed that the OS (378.42±22.56 days) and TTP (271.77±24.92 days) in the TP-positive patients of the study group were significantly longer than those of the control group (OS: 326.57±19.84 days, and TTP: 229.13±22.68 days)( P<0.05). The OS was 371.25±23.97 days and TTP was 264.66±21.36 days in the DPD-positive patients of control group, significantly longer than those of the study group (OS: 334.73±21.47days, and TTP: 208.58±20.70 days) (P<0.05). But there was no significant difference in the OS and TTP between the TP- and DPD-negative patients in the two groups (P>0.05). In respect of adverse events, both the rates of hematological and non-hematological toxicities were low and similar between the two groups (P>0.05), and well-tolerated by the patients.

<b>CONCLUSIONSb>Both CAPOX and SOX regimens are effective chemotherapeutic protocols in treatment of patients with advanced gastric cancer. The expression levels of TP and DPD in tumor tissue can be used as a predictive factor for the efficacy of capecitabine or tegafur, gimeracil and oteracil potassium capsules combined with oxaliplatin regimens. CAPOX chemotherapy regimen is more suitable for the TP-positive gastric cancer patients, and SOX regimen is more suitable for the DPS-positive gastric cancer patients.

Antineoplastic Agents ; adverse effects ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Capecitabine ; administration & dosage ; adverse effects ; Capsules ; Dihydrouracil Dehydrogenase (NADP) ; metabolism ; Disease Progression ; Humans ; Neoplasm Proteins ; metabolism ; Organoplatinum Compounds ; administration & dosage ; adverse effects ; Oxonic Acid ; administration & dosage ; adverse effects ; Pyridines ; administration & dosage ; adverse effects ; Stomach Neoplasms ; drug therapy ; metabolism ; mortality ; pathology ; Tegafur ; administration & dosage ; adverse effects ; Thymidine Phosphorylase ; metabolism

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is characterized by significant gastrointestinal dysmotility. Early and long-term nutritional therapy is highly recommended. We report a case of MNGIE in a patient who was undergoing long-term nutrition therapy. He was diagnosed with a serious symptom of fatty liver and hyperlipidemia complications, along with homozygous mutation of the thymidine phosphorylase (TYMP) gene (c.217G > A). To our knowledge, this is the first report of such a case. Herein, we describe preventive measures for the aforementioned complications and mitochondrial disease-specific nutritional therapy.
Fatty Liver ; Humans ; Hyperlipidemias ; Nutrition Therapy* ; Thymidine Phosphorylase

<b>OBJECTIVEb>To explore the changes of anticancer efficiency of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-Fu) in colorectal cancer cell line HT29 and LS174T cells after transfection of thymidine phosphorylase (TP) cDNA with a lentiviral vector.

<b>METHODSb>TP cDNA was transfected into human colorectal cancer cell lines HT29 and LS174T with the lentiviral vector pLenti6.3-MCS-IRES2-EGFP, and the transfection efficiency of the two cell lines passed 5 generations was analyzed by flow cytometry. The expression of TP protein and the relative quantitative expression of TP mRNA in these 2 cell lines were detected by Western blot and RT-PCR, respectively. The 50% inhibitory concentration (IC50) of 5'-DFUR and 5-Fu in both HT29 and LS174T parent cells and TP-transfected cells were assessed by MTS assay. Finally, the concentration of converted 5-Fu was detected by high performance liquid chromatography (HPLC) either in the medium containing a series of concentrations of 5'-DFUR, in which HT29/HT29-TP or LS174T/LS174T-TP cells were cultured, or in the cell culture lysates.

<b>RESULTSb>The HT29 and LS174T cells transfected with human TP cDNA were monitored for 5 generations, and their transfection efficiency was about 95.0%. Immunohistochemical staining showed that both the parent cells and TP-transfected HT29 and LS174T cells were TP-positive, while vector-transfected cells were TP-negative. Western blotting showed that the TP protein expression in HT29-TP and LS174T-TP cells were significantly increased compared with that in their parents cells. The relative quantity (RQ) values of TP mRNA in HT29-TP and LS174T-TP cells were 8.45 ± 0.15 and 2 615.02 ± 253.97, respectively, which were significantly higher than that in their parents cells (P < 0.01). The IC50 values of 5'-DFUR on HT29-TP cells and its parents cell were (14.33 ± 0.74) µmol/L and (707.66 ± 5.66) µmol/L, respectively (P < 0.05), while (0.59 ± 0.11) µmol/L in LS174T-TP cells and (239.20 ± 21.83) µmol/L in its parent cells, respectively (P < 0.05). The IC50 values of 5-Fu of HT29-TP cells and its parents cells were (5.42 ± 0.75) µmol/L and (14.19 ± 0.97) µmol/L, respectively (P < 0.05), while (4.41 ± 0.96)µmol/L in LS174T-TP cells and (16.42 ± 2.12)µmol/L in its parents cells, respectively (P < 0.05). The HPLC results showed that the 5-Fu concentration detected from media contained a series of concentrations of 5'-DFUR for culturing HT29-TP and LS174T-TP cells were 12.2 to 28.7-folds and 13.1 to 23.6-folds, respectively, higher than that in their parents cells, (P < 0.01 for all). Otherwise, just a little of 5-Fu was detected in the two TP-transfected cells lysate, about 0.9% to 4.2% of 5-Fu detected in the media of the same cultured cells, whereas no 5-Fu was detected in the two parent cell lysates.

<b>CONCLUSIONSb>Transfection of TP cDNA into colorectal cancer cell lines HT29 and LS174T with lentiviral vector can improve the expression of both TP mRNA and TP protein levels in passaged cells, enhance the conversion of 5-Fu from 5'-DFUR in the medium, and result in an enhanced anticancer effect on these two cell lines.

Antineoplastic Agents ; pharmacology ; Cell Line ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; DNA, Complementary ; Floxuridine ; pharmacology ; Fluorouracil ; Genetic Vectors ; HT29 Cells ; Humans ; RNA, Messenger ; Thymidine Phosphorylase ; genetics ; metabolism ; Transfection

Glycogen storage disease type IX (GSD IX) is caused by a defect in phosphorylase b kinase (PhK) that results from mutations in the PHKA2, PHKB, and PHKG2 genes. Patients usually manifest recurrent ketotic hypoglycemia with growth delay, but some may present simple hepatomegaly. Although GSD IX is one of the most common causes of GSDs, its biochemical and genetic diagnosis has been problematic due to its rarity, phenotypic overlap with other types of GSDs, and genetic heterogeneities. In our report, a 22-month-old boy with GSD IX is described. No other manifestations were evident except for hepatomegaly. His growth and development also have been proceeding normally. Diagnosed was made by histologic examination, an enzyme assay, and genetic testing with known c.3210_3212del (p.Arg1070del) mutation in PHKA2 gene.
Child* ; Diagnosis ; Enzyme Assays ; Genetic Heterogeneity ; Genetic Testing ; Glycogen Storage Disease ; Glycogen* ; Growth and Development ; Hepatomegaly* ; Humans ; Hypoglycemia ; Infant ; Male ; Phosphorylase Kinase

Glycogen storage disease type V (GSD-V) is the most common disorder of muscle glycogenosis with characteristic clinical and laboratory findings. A 32-yr-old woman complained of exercise intolerance and myoglobulinuria since early adolescence. She reported several episodes of second-wind phenomenon. Physical examination did not show any neurological abnormality, including fixed muscle weakness or atrophy. Serum creatine kinase level was 1,161 IU/L at rest. The result of the non-ischemic forearm exercise test was compatible with GSD-V. Mutation analysis identified the compound heterozygous mutations of the PYGM, p.D510fs and p.F710del, which has not yet been reported in Korea. The present case recognizes that detail clinical and laboratory analysis is the first step in the diagnosis of GSD-V.
Adult ; Base Sequence ; Creatine Kinase/blood ; Exons ; Female ; Frameshift Mutation ; Gene Deletion ; Genotype ; Glycogen Phosphorylase, Muscle Form/genetics ; Glycogen Storage Disease Type V/*diagnosis/genetics/pathology ; Humans ; Pedigree ; Sequence Analysis, DNA

AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid (MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a (GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β (IRβ), protein kinase B (also known as Akt), and glycogen synthase kinase-3β (GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet (HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.
Animals ; Diabetes Mellitus ; drug therapy ; enzymology ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Enzyme Inhibitors ; administration & dosage ; Glycogen ; metabolism ; Glycogen Phosphorylase ; antagonists & inhibitors ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Insulin ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Signal Transduction ; drug effects ; Triterpenes ; administration & dosage