Background:SEMA3B is a candidate tumor suppressor gene,which plays an essential role in tumorigenesis and progression of a wide variety of cancer. Aims:To explore preliminarily the influence of DNA methylation on regulation of SEMA3B gene expression in gastric cancer. Methods:Real-time PCR was used to determine the expression of SEMA3B mRNA in six human gastric cancer cell lines(SGC7901,AGS,MGC803,BGC823,MKN45,and HGC27),one gastric epithelial cell line(GES-1),and 41 gastric cancer tissue and paired adjacent noncancerous tissue specimens. Methylation specific PCR was used to detect the promoter methylation of SEMA3B gene,and correlation between methylation of SEMA3B gene and clinicopathological characteristics of gastric cancer was analyzed. After treated with a demethylation agent,5-Aza-dC(10μmol/L),for 72 hours,the methylation and mRNA expression of SEMA3B gene were re-examined in above-mentioned cell lines. Results:SEMA3B mRNA expression was significantly lower in six gastric cancer cell lines and gastric cancer tissues than that in GES-1 cells(P <0. 001)and paired adjacent noncancerous tissues(P <0. 01). Promoter methylation of SEMA3B gene could be detected in all six gastric cancer cell lines but not GES-1 cells. Frequency of SEMA3B gene methylation was significantly higher in gastric cancer tissues than in paired adjacent noncancerous tissues (67. 6% vs. 32. 4%,P <0. 01),and the frequency was correlated with differentiation and lymph node metastasis of gastric cancer(P<0. 05). After treated with 5-Aza-dC,hypermethylation of SEMA3B gene in 5 gastric cancer cell lines was decreased,accompanied by up-regulation of SEMA3B mRNA expression. Conclusions:Hypermethylation in promoter of SEMA3B gene might be one of the mechanisms accounting for the reduced expression of SEMA3B,and being involved in tumorigenesis and progression of gastric cancer. SEMA3B might be a potential biomarker for diagnosis and prognostic assessment for gastric cancer and a promising epigenetic therapeutic target.

Background:Because of its non-invasiveness,direct inspection,and high detection rate,capsule endoscopy(CE) has been accepted as the first-line examination for diagnosis of obscure gastrointestinal bleeding(OGIB). However,no matter the result of CE is positive or negative,it is unable to accurately predict the occurrence of rebleeding. Aims:To preliminarily investigate the related risk factors of rebleeding in OGIB patients with positive or negative CE for reducing the rebleeding rate. Methods:One hundred and sixteen OGIB patients undergone CE and with follow-up data from October 2009 to October 2013 at the First Affiliated Hospital of Soochow University were recruited,the rebleeding rate of patients with positive and negative CE,and the risk factors of rebleeding were analyzed. Results:CE diagnostic rate was 56. 9% , and the overall rebleeding rate was 37. 9% . The rebleeding rate in CE positive patients was significantly higher than that in CE negative patients(48. 5% vs. 24. 0% ,P < 0. 01). Male,age ≥50 years,hypertension,accumulated bleeding ≥500 mL within 3 months before CE were the independent risk factors of increase in rebleeding rate in CE positive patients. Age≥50 years,abnormal blood coagulation,without specific treatment were the independent risk factors of increase in rebleeding rate in CE negative patients. Conclusions:Followed-up should be performed in OGIB patients with risk factors of rebleeding for at least 24 months after CE. Repeated examination can be avoided in OGIB patients without risk factors.

Background:The incidence of inflammatory bowel disease(IBD)is increasing recently. However,the pathogenesis has not been fully clarified. Aims:To investigate the expressions and significance of interleukin-22( IL-22),matrix metalloproteinase-9(MMP-9)and macrophage migration inhibitory factor(MIF)in peripheral blood of patients with IBD. Methods:A total of 80 patients with IBD admitted from May 2011 to Nov. 2014 at the First Affiliated Hospital of Soochow University were enrolled,in which 43 cases were Crohn’s disease(CD),37 cases were ulcerative colitis(UC). Forty healthy subjects were served as normal controls. Peripheral levels of IL-22,MMP-9 and MIF were detected by ELISA. Multivariate Logistic regression model was used to analyze IL-22,MMP-9 and MIF in active CD and UC and ROC curve was used to evaluate the diagnostic performance of these markers for screening of active CD and UC. Results:Compared with normal control group,peripheral levels of IL-22,MMP-9 and MIF increased significantly in CD and UC groups(P <0. 05),while no significant difference was found between CD and UC groups(P > 0. 05). Peripheral levels of IL-22, MMP-9 and MIF in active CD and UC were significantly higher than those in remission stage(P < 0. 05). For screening of active IBD,the area under ROC curve(AUC)of combined detection of IL-22 and MMP-9(0. 853 for CD,0. 867 for UC) was superior to that of IL-22,MMP-9 or MIF only(0. 747,0. 770 and 0. 699 for CD,0. 774,0. 815 and 0. 761 for UC). Conclusions:Peripheral levels of IL-22,MMP-9 and MIF increase markedly in IBD patients,which are correlated closely with the activity of IBD. Combined detection of IL-22 and MMP-9 might greatly increase the accuracy for screening of active IBD.

Objective To investigate the effect of tandem mass spectrometry and gas chromatography-mass spectrometry to make prenatal diagnosis of methylmalonic acidemia (MMA) by detecting organic acid and acylcarnitine in amniotic fluid.Methods From October 11,2007 to December 20,2014,131 pregnant women with MMA proband received prenatal diagnosis of MMA in Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (case group).Another 120 cases of pregnant women for conventional prenatal diagnosis at the same period were as control group.The pregnant women of two groups had the amniocentesis at 16 to 20 weeks of gestation.The levels of propionylcarnitine(C3) and acetylcarnitine(C2)in amniotic fluid were detected by tandem mass spectrometry.The methylmalonic acid and methylcitrate acid were detected by gas chromatography-mass spectrometry.MMA gene of cells in amniotic fluid of eighty fetuses with proband clearly diagnosed were detected by gene testing.Data were analyzed by Wilcoxon test.Results In case group,29 fetuses were found positive for higher level of C3,C3/C2,methylmalonic acid and methylcitrate acid compared with normal reference value,and the detected rate of fetal MMA was 22.1％(29/131).The levels of C3 and C3 / C2 in amniotic fluid of these 29 cases were higher than those in control group[8.13(2.42-16.70) vs 1.04(0.52-3.40) μmol/L,Z =-8.313; 0.77(0.30-1.79) vs 0.10(0.05-0.22),Z=-8.374; P ＜ 0.05 respectively].The levels of methylmalonic acid and methylcitrate acid were also higher[9.13(1.68-61.78) vs 0.00(0.00-1.31) mmol/mol Crea,Z=-11.348; 0.58(0.00-1.90) vs 0.05(0.00-0.52) mmol/mol Crea,Z=-6.632,P ＜ 0.05 respectively].For the other 102 cases in case group,the levels of C3,C3/C2,methylmalonic acid and methylcitrate acid were not higher than normal reference value,and were similar to those in control group (P ＞ 0.05); while they were lower than those of positive MMA fetuses (all P ＜ 0.05).Among 29 positive fetuses,16 fetuses were detected MMA gene,five were diagnosed as MUT forms of MMA and 11 were MMACHC forms of MMA.In 102 MMA negative fetuses,64 fetuses were detected MMA gene,44 were found one mutant site and 20 were found no gene mutation.The coincidence rate between gene detecting and mass spectrometry was 100％(80/80).Conclusions Mass spectrometry could be used to measure the C3,methylmalonic acid and methylcitrate acid levels in amniotic fluid of pregnant women with MMA proband to make prenatal diagnosis.

Objective: To investigate the effect of pseudodeficiency alleles on the newborn screening of glycogen storage disease type Ⅱ(GSDⅡ) by using afluorometric enzymatic assay to determine acid α-glucosidase (GAA) activity in dried blood spot (DBS). Methods: A total of 30 507 newborns′ DBSs, obtained from Newborn Screening Center of Xinhua Hospital Shanghai Jiao Tong University School of Medicine from May to December 2017, were screened for GSD Ⅱ by fluorometric enzymatic assay of GAA activity. The suspected positive DBSs after the first and second screening were directly analyzed by Sanger sequencing of GAA to confirm the diagnosis. Retrospective analysis of 3 172 controls without GSDⅡand 36 GSD Ⅱ patients were conducted to investigate the carrier status of pseudodeficiency alleles. Statistical analysis of frequency of pseudodeficiency alleles were carried out by Chi-square test or Fisher exact probability test. Results: GAA activity of 30 507 newborns showed a positively skewed distribution.Twenty-nine cases of newborns, suspected to be GSDⅡwere confirmed to be normal with genetic analysis of the original DBSs. Among the 29 suspected positive cases, 24 cases were homozygous for pseudodeficiency alleles c.[1726A/A; 2065A/A], and the other 5 cases were c.[1726G/A; 2065G/A] heterozygote. The frequency of c.1726G>Ahomozygote in 3 172 non-GSD Ⅱcontrols was 2.08% (66/3 172), and c.1726G>A homozygote occurred in allelic conjunction with c.2065G>Ahomozygote. Frequency of c.[1726A; 2065A] haplotype in 3 172 controls was 3.2%(206/6 344). Frequency of c.[1726A/A; 2065A/A] homozygote in 36 GSDⅡpatients (16.67%, 6/36) was significantly higher than that in non-GSD Ⅱcontrols(2.08%, 66/3 172) (χ²=34.517, P<0.001). Conclusions Pseudodeficiency alleles show a high frequency in Chinese, which leads to a high false positive rate in the newborns screening of GSDⅡ.The afterword genetic analysis of the original DBS after the GAA activity screening could reduce the effect of pseudodeficiency alleles on the newborns screening of GSDⅡ.

OBJECTIVETo provide prenatal diagnosis for a pregnant woman who had given birth to a child with Fanconi anemia with combined next-generation sequencing (NGS) and Sanger sequencing.

METHODSFor the affected child, potential mutations of the FANCA gene were analyzed with NGS. Suspected mutation was verified with Sanger sequencing. For prenatal diagnosis, genomic DNA was extracted from cultured fetal amniotic fluid cells and subjected to analysis of the same mutations.

RESULTSA low-frequency frameshifting mutation c.989_995del7 (p.H330LfsX2, inherited from his father) and a truncating mutation c.3971C>T (p.P1324L, inherited from his mother) have been identified in the affected child and considered to be pathogenic. The two mutations were subsequently verified by Sanger sequencing. Upon prenatal diagnosis, the fetus was found to carry two mutations.

CONCLUSIONThe combined next-generation sequencing and Sanger sequencing can reduce the time for diagnosis and identify subtypes of Fanconi anemia and the mutational sites, which has enabled reliable prenatal diagnosis of this disease.

Adult ; Base Sequence ; Child, Preschool ; DNA Mutational Analysis ; Fanconi Anemia ; diagnosis ; genetics ; Fanconi Anemia Complementation Group A Protein ; genetics ; Female ; Fetal Diseases ; diagnosis ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Polymorphism, Single Nucleotide ; Pregnancy ; Prenatal Diagnosis

Objective To investigate the characteristics of glycogen storage disease type IV (GSD IV) clinically, in laboratory tests and in gene mutation. Methods The clinical manifestations, biochemical indexes, activity of chitotriosidase, and the follow-up of the treatment in 5 cases of GSD IV were analyzed. Results Five patients (3 boys and 2 girls) aged 4 months - 5 years presented hepatosplenomegaly and elevated liver enzyme levels for 2 months at hospital visit. Two patients had motor developmental delay and weakness but their creatine kinase (CK) level were normal. Glycogen storage and liver fibrosis were observed in the liver biopsy in 4 patients. Target sequencing found that all 5 children carried the complex heterozygous mutation of the GBE1 gene with 2 reported mutations(p.R515C,p.R524Q)and 7 novel mutations.The novel mutation contains 5 missense mutations (p.I460T, p.F76S, p.F538V, p.L650R, p.W455R), one insertion mutations (c.141_142insGCGC), and one large fragment deletion (exon 3-7). Therefore, diagnosis of liver type of GSD IV was confirmed in those children. Two patients died of liver cirrhosis. The liver transplantation was performed due to liver cirrhosis in one patient whose chitotriosidase activity increased obviously before transplantation and decreased significantly after the transplantation and liver enzyme levels were returned to normal 4 months after transplantation. In the other two patients their growth and liver enzyme levels were normal;one had not received special treatments while the other was treated with raw corn starch and level of chitotriosidase was normal. Conclusions The clinical manifestations of GSD IV are heterogeneous. Target sequencing can be used for fast and noninvasive diagnosis of GSD IV. Chitotriosidase activity is useful in the prognosis assessment for GSD IV.

Objective To explore the value of the combination of homocysteine analysis, liquid chromatography tandem mass spectrometry(LC-MS/MS)and gas chromatography mass spectrometry(GC/MS)in the prenatal diagnosis of combined methylmalonic acidemia and homocystinuria(cblC defect)in amniotic fluid.Methods This is a retrospective study of 187 cases of pregnancies that came to our hospital for prenatal diagnosis between 2014/01-2017/03,among which 78 cases′probands were cblC defect patients and 109 cases′probands were not organic academia patients(control group).Amniotic fluid samples from pregnant women were obtained at 16 -24 weeks of gestation.Propionylcarnitine(C3)and acetylcarnitine (C2)were measured by LC-MS/MS, methylmalonic acid and methylcitric acid were analyzed by GC /MS, and homocysteine was determined by fluorescence polarization immunoassay.Some pregnancies received MMACHC gene sequencing with cultured cells from amniotic fluid.Data were analyzed using Mann-Whitney U and Kruskal-Wallis H tests.Results Among those 78 pregnant women whose probands were diagnosed to be cblC defect,24 cases were diagnosed to be cblC defect(positive group)and 54 pregnant women were diagnosed to be negative(negative group).In positive group, levels of homocysteine, C3, C3/C2, methylmalonic acid and methylcitric acid were all significantly higher than their normal reference ranges, negative group and control group(P values are 0.00).Cases that were diagnosed to be cblC defect by MMACHC gene sequencing were all turned out to be positive in the tests of the above metabolites in amniotic fluid.Cases with negative results of the metabolites were all excluded to be cblC defect by gene sequencing. Besides,2 cases of pregnancies were diagnosed to be positive by homocysteine and mass spectrometric analysis while only one mutation were detected by gene sequencing.Conclusions The combination of homocysteine, LC-MS/MS and GC/MS analysis in amniotic fluid turns out to be reliable for prenatal diagnosis of cblC defect,which may further cover the defect of prenatal diagnosis of those pregnancies whose probands′gene mutation is unknown.