OBJECTIVE: To develop a multiplex PCR method for a rapid detection of Y chromosome-specific sequences in patients with Turner syndrome. METHODS: Nine genes were selected from various regions of the Y chromosome for designing the primers, which included SRY, TBL1Y, TSPY on the short arm of the Y chromosome, DDX3Y, HSFY1, RPS4Y2 and CDY1 on the long arm of Y chromosome and SHOX in the short arm and SPRY3 in the long arm of the pseudoautosomal region (PAR) of X and Y chromosomes. A multiplex PCR method for the nine genes in Y chromosome was established and optimized. The sensitivity was tested by using different amounts of genomic DNA. A total of 36 patients with Turner syndrome and a patient with male dwarfism with karyotype of 46, X, +mar were examined by the multiplex PCR method for the existence of materials from the Y chromosome. RESULTS: The optimization results of the multiplex PCR reaction system (50 μL) showed that when the final concentration of upstream and downstream of each pair of primers was 0.1 μM, the multiplex PCR reaction of the 9 pairs of primers clearly amplified the target with the expected band size, and there was no non-specific amplification. The bands were clearly visible when the amount of genomic DNA in the multiple PCR reaction system was as low as 1 ng. By using the method, we have examined the 36 patients with Turner syndrome. One patient with Turner syndrome with karyotype of 45,X[40]/47XYY[21] amplified specific seven genes on Y chromosome, 35 patients with Turner syndrome amplified only two target genes SHOX and SPRY3, but not the other seven specific genes on the Y chromosome, which was in keeping with the clinical manifestations of such patients. CONCLUSION This study established a multiplex PCR reaction system with nine genes, which can quickly and accurately screen Y chromosome materials in patients with Turner syndrome. It has the advantages of low cost, simple operation, high specificity and rapid turn-around time, and can be used to detect Turner syndrome patients with Y chromosome material in time. The method has provided a diagnostic basis for preventive gonad resection to prevent malignant gonadal tumors.
Humans ; Male ; Turner Syndrome/genetics* ; Multiplex Polymerase Chain Reaction ; Y Chromosome ; Karyotyping ; DNA Primers ; DNA ; Chromosomes, Human, Y/genetics* ; Transducin/genetics* ; Minor Histocompatibility Antigens ; DEAD-box RNA Helicases/genetics*

OBJECTIVES: To study the association of maternal methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) gene polymorphisms with congenital heart disease (CHD) in offspring. METHODS: A hospital-based case-control study was conducted. The mothers of 683 children with CHD alone who attended Hunan Children's Hospital, from November 2017 to March 2020 were enrolled as the case group, and the mothers of 740 healthy children who attended the same hospital during the same period and did not have any deformity were enrolled as the control group. A questionnaire survey was performed to collect related exposure data, and then venous blood samples (5 mL) were collected from the mothers to detect MTHFD1 and MTHFD2 gene polymorphisms. A multivariate logistic regression analysis was used to evaluate the association of MTHFD1 and MTHFD2 gene polymorphisms with CHD. The four-gamete test in Haploview 4.2 software was used to construct haplotypes and evaluate the association between haplotypes and CHD. The generalized multifactor dimensionality reduction method and logistic regression analysis were used to examine gene-gene interaction and its association with CHD. RESULTS: The multivariate logistic regression analysis showed that maternal MTHFD1 gene polymorphisms at rs11849530 (GA vs AA: OR=1.49; GG vs AA: OR=2.04) andat rs1256142 (GA vs GG: OR=2.34; AA vs GG: OR=3.25) significantly increased the risk of CHD in offspring (P<0.05), while maternal MTHFD1 gene polymorphisms at rs1950902 (AA vs GG: OR=0.57) and MTHFD2 gene polymorphisms at rs1095966 (CA vs CC: OR=0.68) significantly reduced the risk of CHD in offspring (P<0.05). The haplotypes of G-G-G (OR=1.86) and G-A-G (OR=1.35) in mothers significantly increased the risk of CHD in offspring (P<0.05). The gene-gene interaction analyses showed that the first-order interaction between MTHFD1 rs1950902 and MTHFD1 rs2236222 and the second-order interaction involving MTHFD1 rs1950902, MTHFD1 rs1256142, and MTHFD2 rs1095966 might be associated with risk of CHD (P<0.05). CONCLUSIONS Maternal MTHFD1 and MTHFD2 gene polymorphisms and their haplotypes, as well as the interaction between MTHFD1 rs1950902 and MTHFD1 rs2236222 and between MTHFD1 rs1950902, MTHFD1 rs1256142, and MTHFD2 rs1095966, are associated with the risk of CHD in offspring.
Aminohydrolases/genetics* ; Case-Control Studies ; Child ; Female ; Genetic Predisposition to Disease ; Heart Defects, Congenital/genetics* ; Humans ; Methylenetetrahydrofolate Dehydrogenase (NADP)/genetics* ; Minor Histocompatibility Antigens/genetics* ; Mothers ; Multifunctional Enzymes/genetics* ; Polymorphism, Single Nucleotide ; Risk Factors

OBJECTIVETo investigate the effect of ASCT2 gene (glutamine transporter) knock-down by shRNA on biological behaviors of colorectal cancer cells.

METHODSshRNA was transfected into colorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine.

RESULTSASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells(P<0.01). MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells (A490) and decrease the number of invasive Lovo and SW480 cells from the membrane (both P<0.01). The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells (both P<0.01).

CONCLUSIONSASCT2 plays an oncogenic role in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.

Amino Acid Transport System ASC ; drug effects ; genetics ; physiology ; Cell Line, Tumor ; physiology ; Cell Proliferation ; genetics ; Colorectal Neoplasms ; genetics ; physiopathology ; Down-Regulation ; drug effects ; Gene Knockdown Techniques ; methods ; Glutamine ; drug effects ; genetics ; physiology ; Humans ; Minor Histocompatibility Antigens ; drug effects ; genetics ; physiology ; Neoplasm Invasiveness ; genetics ; physiopathology ; Oncogenes ; drug effects ; genetics ; RNA, Messenger ; physiology ; RNA, Small Interfering ; pharmacology ; Transfection

PURPOSE: Numerous studies have assessed the association of SP110 gene variants with tuberculosis (TB), but the results were inconsistent. Through a comprehensive review and meta-analysis, our study aimed to clarify the nature of genetic risks contributed by 11 polymorphisms for the development of TB. MATERIALS AND METHODS: Through searching PubMed, web of science, China National Knowledge Infrastructure (CNKI) databases, a total of 11 articles including 13 independent studies were selected. The pooled odd ratios (ORs) along with their corresponding 95% confidence interval (CI) were estimated for allelic comparisons, additive model (homozygote comparisons; heterozygote comparisons), dominant model and recessive model. We also assessed the heterogeneity across the studies and publication bias. RESULTS: The results of combined analysis revealed a significantly increased risk of TB for single nucleotide polymorphism (SNP) rs9061 in all five comparisons (allelic comparisons: OR=1.28, 95% CI=1.14–1.44, p<0.0001; homozygote comparisons: OR=2.84, 95% CI=1.84–4.38, p<0.00001; heterozygote comparisons: OR=1.23, 95% CI=1.05–1.43, p=0.009; dominant model: OR=1.32, 95% CI=1.14–1.53, p=0.0003; recessive model: OR=2.26, 95% CI=1.18–4.34, p=0.01). In subgroup analysis, the risk of TB associated with SNP rs9061 appeared to be increased. Moreover, increased risk of TB was also found in Asian subgroup of SNP rs11556887, while decreased risk of TB appeared in large sample size subgroup of SNP rs1135791. No significant association was observed between other SNPs and the risk of TB. CONCLUSION: Our meta-analysis suggested that the variant of SNP rs9061 might be a risk factor for TB.
Alleles ; Asian Continental Ancestry Group/genetics ; China ; Confidence Intervals ; Genetic Predisposition to Disease ; Heterozygote ; Homozygote ; Humans ; Minor Histocompatibility Antigens/*genetics ; Nuclear Proteins/*genetics ; Odds Ratio ; *Polymorphism, Single Nucleotide ; Risk Factors ; Tuberculosis, Pulmonary/*genetics

OBJECTIVETo investigate the genetic association pattern between single-nucleotide polymorphisms (SNP) of key genes in T regulatory cells signaling pathways and the efficacy of allergic rhinitis (AR) specific immune therapy(SIT).

METHODSA population of 102 AR patients(Beijing Tongren hospital, from January to Decemeber 2012) caused by simple dust mite received standardized specific immune therapy, who lived in Beijing region was recruited. In immunotherapy before and after 1 years of treatment, the study objects were scored by nasal symptoms score, nasal signs score and total score of daily life distress three indicators to assess the efficacy. A total of 43 reprehensive marker SNP which were in FOXP3, IL-2, TGF-βand EBI3 gene regions and the upstream and downstream 1 000 kb were selected according to the Beijing people database from Hapmap website. The individual genotyping was performed by MassARRAY platform.Plink software was used for statistic analysis.

RESULTSSubgroup analysis for the efficacy evaluation of three indicators displayed that IL-2_rs77468365, FOXP3(rs2280883, rs2232365 and rs3761548) were associated with the improvement of sneezing in nasal symptoms. IL-2_rs77468365, FOXP3(rs2280883, rs2232365 and rs3761548) were associated with the improvement of runny nose in nasal symptoms. TGF-β(rs747857, rs6508975, rs2241715, rs12462166, rs12983775, rs1800470 and rs2317130)and FOXP3(rs2280883, rs2232365 and rs3761548)were associated with the improvement of nasal obstruction in nasal symptoms. FOXP3(rs2280883, rs2232365 and rs3761548)were associated with the improvement of nasal itching in nasal symptoms. IL-2_rs77468365 and FOXP3(rs2280883, rs2232365 and rs3761548) were associated with the overall improvement in nasal symptoms. EBI3_rs670188 and FOXP3(rs2280883, rs2232365, rs3761549, rs3761548 and rs3761547) were associated with the improvement of inferior turbinate mucosa swelling in nasal signs. IL-2_rs77468365, EBI3_rs393581, TGF-β(rs11466359 and rs11466345), FOXP3(rs2280883, rs17847095, rs2232365 and rs3761548)were associated with the improvement of inferior turbinate mucosa color in nasal signs. EBI3(rs393581, rs4740 and rs353702), FOXP3(rs2280883, rs2232365 and rs3761548)were associated with the improvement of water discharge in nasal signs. IL-2_rs77468365, EBI3(rs393581, rs4740 and rs353702), FOXP3( rs2280883, rs2232365 and rs3761548)were associated with the overall improvement in nasal signs. TGF-β(rs12461895, rs2241717 and rs7258445), FOXP3(rs2280883, rs2232365, rs3761549, rs3761548 and rs3761547)were associated with the improvement of life puzzle.

CONCLUSIONThe genetic polymorphism (SNPs) of four important functional candidate genes( FOXP3, IL-2, TGF-βand EBI3) in T regulatory cells signaling pathways were detected in significant correlation with the efficacy of allergic rhinitis specific immune therapy.

Beijing ; Forkhead Transcription Factors ; genetics ; Genotype ; Humans ; Immunotherapy ; Interleukin-2 ; genetics ; Interleukins ; genetics ; Minor Histocompatibility Antigens ; Polymorphism, Single Nucleotide ; Rhinitis, Allergic ; genetics ; therapy ; Signal Transduction ; T-Lymphocytes, Regulatory ; cytology ; Transforming Growth Factor beta1 ; genetics ; Turbinates ; pathology

In allogeneic transplantation, including the B6 anti-BALB.B settings, H60 and H4 are two representative dominant minor histocompatibility antigens that induce strong CD8 T-cell responses. With different distribution patterns, H60 expression is restricted to hematopoietic cells, whereas H4 is ubiquitously expressed. H60-specific CD8 T-cell response has been known to be dominant in most cases of B6 anti-BALB.B allo-responses, except in the case of skin transplantation. To understand the mechanism underlying the subdominance of H60 during allogeneic skin transplantation, we investigated the dynamics of the H60-specific CD8 T cells in B6 mice transplanted with allogeneic BALB.B tail skin. Unexpectedly, longitudinal bioluminescence imaging and flow cytometric analyses revealed that H60-specific CD8 T cells were not always subdominant to H4-specific cells but instead showed a brief dominance before the H4 response became predominant. H60-specific CD8 T cells could expand in the draining lymph node and migrate to the BALB.B allografts, indicating their active participation in the anti-BALB.B allo-response. Enhancing the frequencies of H60-reactive CD8 T cells prior to skin transplantation reversed the immune hierarchy between H60 and H4. Additionally, H60 became predominant when antigen presentation was limited to the direct pathway. However, when antigen presentation was restricted to the indirect pathway, the expansion of H60-specific CD8 T cells was limited, whereas H4-specific CD8 T cells expanded significantly, suggesting that the temporary immunodominance and eventual subdominance of H60 could be due to their reliance on the direct antigen presentation pathway. These results enhance our understanding of the immunodominance phenomenon following allogeneic tissue transplantation.
Animals ; Antigen Presentation ; Antigen-Presenting Cells/immunology/metabolism ; CD8-Positive T-Lymphocytes/*immunology ; Epitopes, T-Lymphocyte/*immunology ; Female ; Graft Rejection/*immunology ; Interferon-gamma ; Lymphocyte Activation/immunology ; Lymphocyte Count ; Mice ; Minor Histocompatibility Antigens/*immunology/metabolism ; *Skin Transplantation ; Transplantation, Homologous

Minor histocompatibility antigens are MHC-bound peptides and contribute to the generation of allo-responses after allogeneic transplantation. H60 is a dominant minor H antigen that induces a strong CD8 T-cell response in MHC-matched allogeneic transplantation settings. Here, we report establishment of a TCR transgenic mouse line named J15, wherein T cells express TCRs specific for H60 in complex with H-2K(b), and different fates of the thymocytes expressing J15 TCRs in various thymic antigenic environments. Thymocytes expressing the J15 TCRs were positively selected and differentiated into CD8+ single positive (SP) cells in the thymus of C57BL/6 mice, wherein the cognate antigen H60 is not expressed. However, thymocytes were negatively selected in thymus tissue where H60 was transgenically expressed under the control of the actin promoter, with double-positive stages of cells being deleted. Despite the ability of the H60H peptide (LTFHYRNL) variant to induce cytotoxic activity from H60-specific CTL lines at ~50% of the activity induced by normal H60 peptides (LTFNYRNL), J15-expressing thymocytes were positively selected in the thymus where the variant H60H was transgenically expressed. These results demonstrate that a single amino-acid change in the H60 epitope peptide influences the fate of thymocytes expressing the cognate TCR.
Actins ; Animals ; Histocompatibility Antigens* ; Histocompatibility* ; Mice ; Mice, Transgenic ; Minor Histocompatibility Antigens ; Peptides ; T-Lymphocytes ; Thymocytes* ; Thymus Gland ; Transplantation, Homologous

OBJECTIVETo investigate the association between single nucleotide polymorphisms (SNPs) in Wnk1 gene and ischemic stroke in Chinese Han population.

METHODSA hospital-based case-control study was carried out. The ischemic stroke group included 294 Chinese Han subjects, who were admitted with non-fatal ischemic stroke in departments of neurology of 5 hospitals in Xinjiang during January 2008 through December 2009. Control group included 314 age and sex-matched Han subjects without an inquired history of stroke, hospitalized in departments of surgery of these 5 hospitals. Ten tagging SNPs (tSNPs) of the Wnk1 gene were genotyped, and the association between these tSNPs and ischemic stroke were evaluated. The tSNPs (rs3858703, rs11611246, rs7305065, rs1990021, rs34408667, rs12309274, rs1012729, rs956868, rs12828016 and rs953361) were determined by the Multiplex SNaPshot platform. The data were analyzed by using t-test, Ξ2-test and logistic regression. Linkage disequilibrium and haplotype were analyzed by Haploview software.

RESULTSThe rates of alcohol drinking, hypertension ,diabetes and hyperlipidemia in ischemic stroke group were higher than those in control group (37.1% vs 21.0%, 62.9% vs 36.6%, 18.0% vs 6.1% and 36.4% vs 17.5%, respectively, all P<0.01). No significant difference in smoking rate was found between two groups. The genotyping loss rates of all sites were less than 1%. All the tSNPs were examined by Hardy-Weinberg equilibrium test except rs34408667. tSNP rs11611246 in the 4th intron of the Wnk1 gene was significantly associated with ischemic stroke. The distribution frequency of T allele in cases was significantly lower than that in male controls (30.3% vs 35.7%, P =0.046). When the samples were further stratified according to gender, rs11611246 was found to be associated with a reduced risk of ischemic stroke in male cases than in controls. GT and TT genotype frequencies were 43.3% and 7.2% in male cases, 43.1% and 15.2% in male controls, respectively (P=0.038). The T allele was associated with a reduced risk of ischemic stroke, with a per-allele OR of 0.702(95%CI:0.517-0.953, P=0.023) in male cases than in male controls. The significance remained after adjusting the covariates of age (P=0.022), or the covariates of age, BMI, cigarette smoking, alcohol drinking, hypertension, diabetes and hyperlipidemia (P=0.008). No association between other 9 tSNPs and ischemic stroke was noted in Chinese Han subjects.

CONCLUSIONThe polymorphism of rs11611246 on the 4th intron of Wnk1 gene is associated with a reduced risk of ischemic stroke in Chinese Han population and the T allele might be a protective factor for ischemic stroke in male Chinese Hans.

Aged ; Asian Continental Ancestry Group ; genetics ; Brain Ischemia ; genetics ; Female ; Genotype ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Middle Aged ; Minor Histocompatibility Antigens ; Polymorphism, Single Nucleotide ; Protein-Serine-Threonine Kinases ; genetics ; Stroke ; genetics ; WNK Lysine-Deficient Protein Kinase 1

OBJECTIVETo analyze the association of micoRNA-related genes DROSHA single nucleotide polymorphisms (SNP) rs10719 and rs6877842, DICER1 rs3742330and GEMIN4 rs3744741 with prognosis of T-cell lymphoma.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to determine the genotypes of the above 4SNPs and their associations with complete remission (CR) rate and overall survival (OS) in 163 patients with TCL.

RESULTSPatients carrying the rs6877842 CG genotype had a significantly higher CR rate compared with those carrying the CC genotype (OR=0.07, 95% CI 0.01-0.72, P=0.026); the same for patients carrying the DICER1 rs3742330 GG genotype compared with those carrying the GA genotype (OR=0.15, 95% CI 0.02-0.97, P=0.047) or the AA genotype (OR=0.11, 95% CI 0.02-0.71, P=0.020). In addition, patients with the DICER1 rs3742330 GG genotype had a significantly improved OS compared with those carrying the GA (HR=9.02, 95% CI 1.22-66.92, P=0.031) or AA genotype (HR=8.77, 95% CI 1.19-64.67, P=0.033). The other two SNPs of rs10719 and rs3744741 had no significant association with CR or OS.

CONCLUSIONDROSHA rs6877842 and DICER1 rs3742330 were independent factors for TCL CR, and DICER1 rs3742330 was also an independent prognostic factor for TCL OS.

DEAD-box RNA Helicases ; genetics ; Genetic Predisposition to Disease ; Genotype ; Humans ; Lymphoma, T-Cell ; diagnosis ; genetics ; MicroRNAs ; genetics ; Minor Histocompatibility Antigens ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Prognosis ; Ribonuclease III ; genetics ; Ribonucleoproteins, Small Nuclear ; genetics

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.
Animals ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Survival ; drug effects ; ErbB Receptors ; antagonists & inhibitors ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; MCF-7 Cells ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Minor Histocompatibility Antigens ; Mitogen-Activated Protein Kinases ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Transplantation, Heterologous ; Tyrphostins ; pharmacology