Characteristics of DNMT3A mutations in acute myeloid leukemia

BACKGROUND: DNMT3A mutations occur in approximately 20% of AML cases and are associated with changes in DNA methylation. CDKN2B plays an important role in the regulation of hematopoietic progenitor cells and DNMT3A mutation is associated with CDKN2B promoter methylation. We analyzed the characteristics of DNMT3A mutations including their clinical significance in AML and their influence on promoter methylation and CDKN2B expression.METHODS: A total of 142 adults, recently diagnosed with de novo AML, were enrolled in the study. Mutations in DNMT3A, CEBPA, and NPM1 were analyzed by bidirectional Sanger sequencing. We evaluated CDKN2B promoter methylation and expression using pyrosequencing and RT-qPCR.RESULTS: We identified DNMT3A mutations in 19.7% (N=28) of enrolled patients with AML, which increased to 29.5% when analysis was restricted to cytogenetically normal-AML. Mutations were located on exons from 8–23, and the majority, including R882, were found to be present on exon 23. We also identified a novel frameshift mutation, c.1590delC, in AML with biallelic mutation of CEBPA. There was no significant difference in CDKN2B promoter methylation according to the presence or type of DNMT3A mutations. CDKN2B expression inversely correlated with CDKN2B promoter methylation and was significantly higher in AML with R882H mutation in DNMT3A. We demonstrated that DNMT3A mutation was associated with poor AML outcomes, especially in cytogenetically normal-AML. The DNMT3A mutation remained as the independent unfavorable prognostic factor after multivariate analysis.CONCLUSION: We characterized DNMT3A mutations in AML and revealed the association between the DNMT3A mutation and CDKN2B expression and clinical outcome.

Evaluation of Multiplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Sexually Transmitted Infections Using Swab Specimen

Sexually transmitted infections (STIs) are caused by the spread of pathogens via sexual activity and can cause serious complications if left untreated, regardless of their symptoms. Therefore, early diagnosis of STI is important, and molecular diagnostic methods for rapid detection and monitoring are needed. In this study, we evaluated a multiplex polymerase chain reaction (PCR) kit for simultaneously detecting 13 different bacterial, fungal, and viral microorganisms that cause STIs. The kit performance was evaluated for its sensitivity, lot-to-lot variation, and interference in detecting different pathogens. Additionally, its clinical usefulness was evaluated by estimating its sensitivity and specificity for clinical samples. The limit of detection (LOD) was 0.021–50.104 copies for each pathogen. In the tests of lot-to-lot, 100% of positive samples were detected at low concentrations and negative samples all showed negative results. This result confirms that there is no the variation of lot-to-lot. In the test for interference between pathogens, the efficiency of amplification for each pathogen was not significantly reduced and no nonspecific amplification product was formed. We tested 322 vaginal swab samples using the multiplex PCR kit and confirmed that its clinical sensitivity and specificity were 100% for all pathogens. This multiplex PCR kit can be used widely for rapid diagnosis and monitoring of STIs.

Diagnosis of Viral Infection Using Real-time Polymerase Chain Reaction

The laboratory-based diagnosis of viral infection has been evolving over the years, to increase objectivity, accuracy, and sensitivity via the continuous development of various technologies. Cell culture, which is one of the methods used for the diagnosis of viral infection, is a “gold-standard” approach; however, it is time consuming and is associated with a high risk of contamination. To overcome these shortcomings, molecular biology methods, such as conventional polymerase chain reaction (cPCR), real-time PCR, and sequencing, have been used recently for virus diagnosis. Realtime PCR has higher accuracy and sensitivity compared with cPCR. Moreover, realtime PCR can quantify viral nucleic acids by confirming the amplification using the threshold cycle, which is the initial amplification point. Real-time PCR applications for the detection of various types of viruses in clinical settings should be based on the use of appropriate samples, nucleic acid extraction according to virus characteristics, and selection of diagnostic methods using sensitivity and specificity targets. In addition, the implementation of real-time PCR requires to evaluate the performance of the test protocol by measuring sensitivity, specificity, accuracy, and reproducibility. The verified real-time PCR method is an easy, fast, and accurate method for monitoring the diagnosis and treatment outcomes in a clinical setting. In this review, we summarize the characteristics of the typical diagnostic methods for viral infection, especially of the advanced real-time PCR method, to detect human pathogenic viruses.

Evaluation of the Real-Q HCV Quantification Kit / 대한임상미생물학회지

BACKGROUND: Hepatitis C virus (HCV) RNA quantification is necessary for predicting the therapeutic response and assessing treatment results in patients with chronic HCV infection. Recently, real-time PCR technology for HCV RNA quantification displayed good linearity within the dynamic range. Thus, it is gradually replacing branched-DNA (bDNA) and PCR- hybridization assays. In this study, we evaluated the performance of the Real-QTM HCV quantification kit (biosewoom. Inc., Seoul, Korea) developed in Korea. METHODS: We evaluated the HCV quantification kit for detection limit, specificity, linearity, accuracy, and recovery rate of HCV RNA standard material. The results were analyzed for a correlation with those of Cobas Amplicor HCV Monitor 2.0. RESULTS: The HCV quantification kit showed a high recovery rate of HCV RNA standard material of various concentrations and amplication of HCV RNA equally in all genotypes. Hepatitis B virus and human immunodeficiency virus showed no cross-reactivity with HCV. Within-run and between-run coefficients of variation (CV) were 9.52~15.84% and 9.40~17.53%, respectively. Between-day coefficients of variation were 11.62~18.04%, and detection limit was 44 IU/mL. It showed a good correlation with Cobas Amplicor HCV Monitor 2.0 (R2=0.8954). CONCLUSION: The Real-Q HCV quantification kit showed a good specificity, sensitivity, linearity, and accuracy; therefore, we propose that it is fully adequate for monitoring antiviral therapy in patients with chronic HCV infection.

Performance Evaluation of TaqMan Probe Method for BK Virus DNA Quantification by Real-time Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: We evaluated the performance of a newly developed real-time polymerase chain reaction (PCR) method using TaqMan probe (TP) and internal control (IC) for quantitation of BK virus (BKV) DNA. METHODS: PCR primers and TP were targeted for the VP1 of BKV and 300 bp-region of VP1 was cloned to prepare a standard DNA. Threshold cycles (Ct) of IC was set at 33+/-3. The recovery rates, precision, linearity, and limit of detection (LOD) were measured using the standard DNA. To correlate TP with previous hybridization probe (HP) method, Ct of those were compared using 35 HP-positive and 15 HP-negative specimens, and the interpretation agreement was analyzed in 63 consecutive clinical specimens including 32 urines and 31 plasmas. Fifty-three53 specimens measured for IC were analyzed for positive rates and levels of BKV according to Ct of IC. RESULTS: The average recovery rate was 101.1% and intra-assay and inter-assay coefficiency variations were 0.017~0.059 and 0.036, respectively, with the specimens of 3 log/mL, and 0.041~0.063 and 0.045, respectively, with the specimens of 6 log/mL. LOD was 183 copies/mL and linearity range was 2.7 log- 12 log/mL. Ct of TP were correlated with those of HP with the function of y=0.8912x+0.3164 (R2=0.9062). Among 63 clinical specimens, 16 were positive in TP and 12 were positive in HP with an agreement of 90.4%. Ct of IC were over 36 in 31 specimens (22 urines and 9 plasmas), of which BKV DNA was much higher in 7 (22.5%) BKV-positive specimens (5.9+/-1.7 log/mL) than in 4 (18.1%) BKV-positive specimens (3.9+/-1.0 log/mL) of 22 having Ct of IC < or =36.; 5.9+/-1.7 vs. 3.9+/-1.0 log/mL. CONCLUSION: TP warrants to be a reliable method for quantification of BKV. IC seemed to be essential to differentiate false-negative results or underestimation of BKV in clinical specimens, especially in urine.