OBJECTIVE: To study the effects of FLT3-ITD length on 32D cell proliferation, apoptosis and sensitivity to FLT3 inhibitor, so as to provide references for stepwise therapy of FLT3-ITD mutated acute myeloid leukemia patients. METHODS: Three different FLT3-ITD mutants with same or adjacent insert sites were selected and constructed in an eukaryotic expression vector. FLT3-ITD mutants stably expressed 32D cell strains were selected with the help of lentivirus system and IL3 free cell culture medium. The proliferation and apoptosis of 32D cell strains after AC220 treatment were detected. RESULTS: FLT3-ITD mutants (ITD1, ITD2 and ITD3) stably expressed 32D cell strains were constructed successfully. In the absence of IL3 factor, the proliferation number of ITD1, ITD2 and ITD3 cell strains were mounted up to 2.3 folds, 3.7 folds, and 4.3 folds after 48 hours, respectively. Under the exposure of FLT3 inhibitor AC220, the IC CONCLUSION FLT3-ITD mutant expressed cell strains with longer ITD show higher capacity of proliferation and higher tolerance to AC220 treatment.
Apoptosis ; Cell Proliferation ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Mutation ; Protein Kinase Inhibitors ; Tandem Repeat Sequences ; fms-Like Tyrosine Kinase 3/genetics*

BACKGROUNDDuring the past decades, the incidence of invasive aspergillosis (IA) caused by Aspergillus fumigatus has increased dramatically. The aims of this study were to investigate the susceptibility of clinical isolates of A. fumigatus to triazole and the underlying cyp51A mutations in triazole-resistant A. fumigatus.

METHODSA total of 126 A. fumigatus clinical isolates from 126 patients with proven or probable IA were obtained from four large tertiary hospitals in Nanjing, China, between August 2012 and July 2015. The determination of minimal inhibitory concentrations (MICs) for itraconazole, voriconazole, and posaconazole was performed by broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing reference method.

RESULTSA total of 4 A. fumigatus isolates (3.17%) were confirmed to be itraconazole resistant, with MICs of ≥8 mg/L, and one isolate (0.8%) was confirmed to be voriconazole resistant and posaconazole resistant, with MICs of 4 mg/L and 0.5 mg/L, respectively. We found that two of the 4 isolates of triazole-resistant A. fumigatus had the L98H amino acid substitution in combination with a 34-base pair tandem repeat in the promoter region, one isolate had an M220I mutation, and another itraconazole-resistant isolate did not have a substitution in the cyp51A gene.

CONCLUSIONSThis study shows that triazole-resistant A. fumigatus clinical isolates are present in Nanjing, China, which is a new challenge to the clinical management of IA.

Antifungal Agents ; pharmacology ; Aspergillus fumigatus ; drug effects ; genetics ; China ; Drug Resistance, Fungal ; Itraconazole ; pharmacology ; Microbial Sensitivity Tests ; Promoter Regions, Genetic ; genetics ; Tandem Repeat Sequences ; genetics ; Triazoles ; pharmacology ; Voriconazole ; pharmacology

UNLABELLEDOBJECTICE: To evaluate the application of high-throughput sequencing to sequence the FMS-like Tyrosine Kinase 3 internal tandem duplication (FLT3-ITD) in de novo acute myeloid leukemia (AML) patients with lower allelic ratio FLT3-ITD mutation or more than one ITD, and to analyze the feature of ITD.

METHODSThe genomic DNA of 23 AML patients with positive FLT3-ITD was amplified by PCR, capillary electrophoresis was used to detect the ITD mutation. Then, the FLT3 gene was amplified using primer with different barcode, and the product was analyzed by illumina Miseq, and the results were compared with UCSC database.

RESULTSOut of 23 AML patients, 17 had a single ITD, and 3 had 2 ITDs, and the remaining 3 had 3 ITD detected by capillary electrophoresis. The high-throughput sequencing showed that 17 ITD were the complate duplications of wild-type FLT3, and the remaining 16 ITD were partial duplications in the all 33 ITDs. The same length ITD mutation contained 2 different ITD sequences in one patient with more than one ITD, and the other patient with 2 ITD had the same ITD insertion position. The ITD occurred in the regions from p. Y572 to p. L602 of the FLT3 protein, and all the patient ITD covered one or more amino acid between p. V592 and p. E598.

CONCLUSIONIllumina Miseq can analyze the sequence of ITDs precisely and accurately. ITD mutation varies widely, but the hotspots are concentrated.

Alleles ; DNA Mutational Analysis ; Genotype ; High-Throughput Nucleotide Sequencing ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Polymerase Chain Reaction ; Tandem Repeat Sequences ; fms-Like Tyrosine Kinase 3 ; genetics

Adult ; Female ; Humans ; Leukemia, Myeloid, Acute ; enzymology ; genetics ; Mutation ; genetics ; Niacinamide ; adverse effects ; analogs & derivatives ; Phenylurea Compounds ; adverse effects ; Tandem Repeat Sequences ; genetics ; Thyroiditis ; chemically induced ; enzymology ; genetics ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVE: To establish a 15-plex rapid STR multiplex amplification system. METHODS: Fourteen auto-chromosome loci and one sex-chromosome were selected to compare the situations of allelic losses and nonspecific amplication under different conditions. FastStart Taq DNA polymerase and DNA standard sample 9947A were used during amplification and optimization process.15-plex rapid STR amplification system was achieved by performing various experiments including selection of amplification conditions and the volume of DNA polymerase, adjustment of inter-locus balance, optimization of rapid amplification, screening of reaction buffers, selection of reaction volume, and a variety of additives. RESULTS: Using 10 μL rapid PCR system, including 1 ng DNA templates, 0.4 μL polymerase and 10xFastStart high fidelity reaction buffer, a complete and well-balance DNA profile of 15 STR loci for standard genomic DNA was obtained in 32 minutes, without the allele drop-out and non-specific amplicons. Meanwhile, 5% glycerinum, 0.01% gelatin, 0.05% gelatin and 5 mmol/L ammonium sulfate could be used as the reactive additive during the amplification procedure. CONCLUSION The 15-plex rapid STR multiplex amplification system can be used to decrease reaction time and enhance sample throughput.
Alleles ; Chromosome Mapping ; DNA/genetics* ; DNA Fingerprinting/methods* ; Forensic Genetics/methods* ; Humans ; Microsatellite Repeats/genetics* ; Polymerase Chain Reaction/methods* ; Racial Groups/genetics* ; Sensitivity and Specificity ; Tandem Repeat Sequences

OBJECTIVE: To explore the feasibility of detecting of Y-STR of fetal DNA in maternal plasma using Ion Torrent PGM™ System. METHODS: A total of 16 fetal DNA samples from maternal plasmas (8 cases from 38 weeks gestational age and 8 ones from 12 weeks) were prepared and a multiplex assay with 7 STR loci (DYS390, DYS391, DYS393, DYS438, DYS437, DYS456, DYS635) was designed for multiplex-PCR amplification. Using Ion Torrent PGM™ System, the results of Y-STR sequences and capillary electrophoresis were obtained and compared. RESULTS: Y-STR specific alleles were detected in the maternal plasma of all the pregnant women having male babies of second and third trimester, which were higher than that detected by capillary electrophoresis. Consistent Y-STR genotypes were observed between fetal DNA from maternal plasma and genomic DNA from the newborn babies. CONCLUSION Based on Ion Torrent PGM™ System, the prenatal Y-STR detection method may provide a high-sensitive and high-throughput choice for prenatal STR detection in forensic testing.
Alleles ; Chromosomes, Human, Y/genetics* ; DNA/blood* ; Family ; Female ; Fetal Blood/chemistry* ; Genotype ; Haplotypes ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Pregnancy ; Sensitivity and Specificity ; Sex Determination Analysis ; Tandem Repeat Sequences/genetics*

OBJECTIVETo review the common genotyping techniques of Chlamydia trachomatis in terms of their principles, characteristics, applications and limitations.

DATA SOURCESData used in this review were mainly from English literatures of PubMed database. The search terms were "Chlamydia trachomatis" and "genotyping". Meanwhile, data from World Health Organization were also cited.

STUDY SELECTIONOriginal articles and reviews relevant to present review's theme were selected.

RESULTSDifferent genotyping techniques were applied on different occasions according to their characteristics, especially in epidemiological studies worldwide, which pushed the study of Chlamydia trachomatis forward greatly. In addition, summaries of some epidemiological studies by genotyping were also included in this work for reference and comparison.

CONCLUSIONSA clear understanding of common genotyping techniques could be helpful to genotype C. trachomatis more appropriately and effectively. Furthermore, more studies on the association of genotypes of Chlamydia trachomatis with clinical manifestations should be performed.

Chlamydia trachomatis ; genetics ; Genotype ; Multilocus Sequence Typing ; Polymerase Chain Reaction ; Tandem Repeat Sequences ; genetics

This study was aimed to explore whether multiple common gene mutations of leukemia synergistically involved in acute promyelocytic leukemia (APL) pathogenesis, and to investigate their relevance to clinical features, cytogenetics and molecular risk stratification. 84 specimens of admitted de novo APL patients from February 2005 to October 2010 were collected, the gene mutations of bone marrow mononuclear cells and clinical features of mutation-positive patients were analyzed by genomic DNA-PCR. The results indicated that the prevalence of mutations was 60.7% (51/84), in which the mutations with the highest incidence were found as FLT3-ITD, reaching 27.4% (23/84). Next, there were 12 cases WT1 mutation, 9 for FLT3-TKD, 7 for TET2, 5 for N-RAS, 4 for ASXL1, 2 for EZH2 mutation and 1 positive case in MLL-PTD, IDH1 and CBL mutation respectively. No mutation was found in other JAK1, DNMT3, c-Kit, NPM1, IDH2, RUNX1 and JAK2 (V617F) common leukemia-related genes. Combined analysis with clinical data demonstrated that the patients with FLT3-ITD mutation displayed higher white blood cell counts, while the patients with N-RAS mutation showed lower platelet counts. Overall survival of these patients was obviously shorten as compared with patients with wild-type. This difference between mutant and wild-type of all above mentioned cases was statistically significant (P < 0.05). The difference between APL with simple t (15;17) and additional abnormal karyotype was not statistically significant. It is concluded that the FLT3-ITD mutation is recurrent genetic change in APL, and together with N-RAS mutation indicates poor prognosis. Additional abnormal karyotype does not associate with prognosis of APL.
Adolescent ; Adult ; Aged ; Child ; DNA Mutational Analysis ; DNA-Binding Proteins ; genetics ; Enhancer of Zeste Homolog 2 Protein ; Female ; Genes, ras ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Polycomb Repressive Complex 2 ; genetics ; Prognosis ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Repressor Proteins ; genetics ; Tandem Repeat Sequences ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics

Yeast flocculation is described as a reversible, asexual and calcium dependent process, in which cells adhere to form flocs by interaction of specific cell surface proteins named flocculins on yeast cells with mannose residues present on the cell wall of adjacent yeast cells. Yeast flocculation provides a very economical and convenient pathway for separation of yeast cells from the fermentation broth or removal of heavy metal ions from effluent. A large number of tandem repeats have been found in genes encoding flocculins, which not only have great regulatory effect on the structure and function of flocculins, generating the diversity of flocculation characteristics, but lead to genetic instability in flocculation as well for driving slippage and recombination reactions within and between FLO genes. Here, the research progress in effect of variation of tandem repeats in FLO genes on flocculation characteristics and genetic stability were reviewed to direct and promote the controllable application of flocculation in industrial fermentation process and environmental remediation.
Fermentation ; Flocculation ; Mannose ; Membrane Proteins ; genetics ; Saccharomyces cerevisiae ; genetics ; growth & development ; Saccharomyces cerevisiae Proteins ; genetics ; Tandem Repeat Sequences

OBJECTIVETo report on a rare allele FGA-13 identified in Guangdong Han population.

METHODSThe rare allele was detected by PCR-STR and DNA sequencing.

RESULTSThe core repeat sequence of rare allele FGA-OL is [TTTC]₃[TTTT][TTCT][CTTT]₅ [CTCC][TTCC]₂, which has been determined as FGA-13.

CONCLUSIONThe rare allele FGA-13 is also present in Guangdong Han population. This is significant for personal identification and paternity testing.

Alleles ; Asian Continental Ancestry Group ; genetics ; Female ; Genotype ; Humans ; Male ; Tandem Repeat Sequences