BACKGROUND:T315I mutations of the BCR::ABL1 gene lead to resistance to tyrosine kinase inhibitors (TKIs). This study evaluated the performance of digital droplet polymerase chain reaction (ddPCR) in quantifying T315I mutations and their frequency in Philadelphia chromosome (Ph) positive hematological patients. METHODS:The course of disease and BCR::ABL1 fusion transcripts (e13a2, e14a2 and e1a2) were retrospectively reviewed in 21 patients with acute lymphoblastic leukemia (ALL) and 85 patients with chronic myeloid leukemia (CML). T315I mutation analysis was carried out using ddPCR and the limit of detection was assessed using mutant T315I DNA at varying variant allele fractions. RESULTS:T315I mutations were found in two ALL patients and one CML patient without remission in molecular biology and with mutation burdens of 29.20%, 40.85%, and 3.00%, respectively. The mutation burden of ALL patients was higher than that of CML patients, but there was no significant difference between the two (p-value = 0.0536). The test's limit of detection was 0.02% with a correlation coefficient greater than 0.99 between the expected and actual detection abundances. CONCLUSION:T315I mutations have a high incidence in Ph-positive ALL patients even if the course of disease is short. In molecular biology, T315I mutation detection is indicated for CML patients not in remission.

Background TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. Methods The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. Results Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. Conclusions This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.

Introduction: HCMV infection is widespread in humans. This retrospective study aimed to explore the relationship between human cytomegalovirus (HCMV) glycoprotein B (gB) genotype distribution, viral load, and the demographic and clinical features of symptomatic infants. The detection rate of HCMV in blood and urine samples was also compared. Methodology: Retrospective data from 265 infants who underwent urine HCMV DNA testing were analyzed. The viral load and gB genotype were detected in 91 HCMV positive infants by quantitative fluorescence polymerase chain reaction (PCR) and DNA sequencing, respectively. Results: The positive rate of HCMV infection was 46.04% (122/265) in all infants, and increased rapidly with age. Among the 91 infants investigated, liver function abnormality was the most common diagnosis (34/91, 37.36%), followed by pneumonia (21/91, 23.07%). Sequence analysis of gB yielded two genetic subtypes: the most prevalent gB3 (47/91, 51.65%), followed by gB1 (44/91, 48.35%). The gB3 HCMV infection was more prevalent in infants aged 0-2 months than in infants aged 3-12 months (χ2 = 4.38, p = 0.0364). The data showed that ALT and AST levels were significantly higher in the anti-HCMV IgM+IgG– group than in the anti-HCMV IgM+IgG+ and IgM-IgG+ groups. In addition, this study showed that the detection rate of HCMV DNA in the blood was significantly lower than that in the urine (χ2 = 6.7131, p = 0.0096). Conclusions: This study presents the HCMV infection status of infants and its relationship with their demographic characteristics and clinical manifestations. In addition, this study suggests that urinary PCR is the most appropriate assay for detecting HCMV infections.

BACKGROUND:High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation.OBJECTIVES:This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs).MATERIAL AND METHODS:High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-β1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit.RESULTS:The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-β1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs.CONCLUSIONS:In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.

Abstract Purpose To explore the relationship between human cytomegalovirus (HCMV) glycoprotein B (gB) genotype distribution, virus load and demographic and clinical features in Chinese infants. Methods The retrospective data of 265 infants who underwent urine HCMV DNA test were analyzed. The viral load and gB genotype were further detected in 91 HCMV positive infants by quantitative fluorescence polymerase chain reaction (PCR) and DNA sequencing, respectively. Results The positive rate of HCMV infection was 46.04% (122/265), and it reached the peak at the age of two months (9.62%, 25.81% and 68.57% in infants aged 0–30 days, one month and two months, respectively). As for the 91 infants studied furtherly, the top two clinical diagnoses are hepatitis (34/91, 37.36%) and lower respiratory tract infection (21/91, 23.07%). The most prevalent genotype was gB3 (47/91, 51.65%), followed by gB1 (44/91, 48.35%). The gB3 HCMV infection was more prevalent in infants aged 0–2 months (gB1 = 14, gB3 = 22) than in other infants aged 3–12 months (gB1 = 20, gB3 = 11, χ2 = 4.38, p = 0.0364). The mean level of HCMV DNA was 1.047×104 copies/ml. There was no significant difference in viral load among infants infected with gB1 or gB3 genotype HCMV. The data also showed that the ALT and AST levels in the anti-HCMV IgM + and IgG– group are significantly higher than those in the anti-HCMV IgM + and IgG+, and IgM- and IgG + group. Conclusions This study revealed the infection status and process of HCMV in Chinese infants. In addition, this study suggests that anti-HCMV IgG may play a role in protecting liver function.

Antituberculosis (anti‐TB) drug‐induced hepatotoxicity may be related to the excessive reactive oxygen species induced by hepatotoxic metabolites. Antioxidant activity involves the nuclear factor erythroid 2‐related factor 2 (Nrf2) signaling pathway. The BTB domain and CNC homologue 1 (Bach1) may compete with Nrf2 for binding to transcriptional enhancers. Elimination of Bach1‐mediated transcriptional repression depends on nuclear exporter exportin 1 (Xpo1). Thus, Xpo1 may indirectly affect antioxidant activity. The present study aimed to examine the role of tag single‐nucleotide polymorphisms in XPO1 in Chinese anti‐TB treatment patients. A 1:2 matched case‐control study was conducted using 314 anti‐TB drug‐induced hepatotoxicity cases and 628 controls. After correcting for weight and hepatoprotectant use, conditional logistic regression analysis showed that patients carrying the AA genotype of rs4430924 in XPO1 were at higher risk of anti‐TB drug‐induced hepatotoxicity than those carrying the GG genotype based on the subgroup of probable cases (adjusted OR, 1.938; 95%CI, 1.035‐3.628; P = .039), and marginally significant differences were also found under the recessive model (P = .048) and the additive model (P = .047). Based on this 1:2 matched case‐control study, the AA genotype of rs4430924 in XPO1 may be associated with higher risk of anti‐TB drug‐induced hepatotoxicity in Chinese anti‐TB treatment patients. Further studies in larger and more varied populations are required to validate this relationship.

Purpose: This study aimed to evaluate Atorvastatin (ATO)-associated hepatotoxicity using prescription sequence symmetry analysis (PSSA), based on a health insurance database of a Chinese population living in Jiangsu Province, China. Methods: Patients prescribed ATO and hepatoprotective drugs in 2017 were identified, and the run-in period was determined based on the "waiting-time" distribution. Adjusted sequence ratio (ASR) and 95% confidence interval (95% CI) were calculated to estimate the risk of ATO-associated hepatotoxicity under different time intervals or based on gender and age stratification. Results: A total of 2,549 patients, with 1,518 filling the ATO prescription first and 1,031 filling the ATO prescription second, were analyzed. After setting the run-in period as 30 days and the time interval as 15, 30, 60, 90, 120, and 180 days, the ASRs were 1.492 (95% CI: 1.367-1.652), 1.399 (95% CI: 1.308-1.508), 1.280 (95% CI: 1.213-1.357), 1.292 (95% CI: 1.234-1.356), 1.278 (95% CI: 1.226-1.336), and 1.274 (95% CI: 1.229-1.323), respectively. No significant difference was observed between different genders and ages (chi(2)=0.161, P=0.688; chi(2)=1.565, P=0.211, respectively). Conclusion: This is the first study conducted in a real-world setting to evaluate the relationship between ATO and hepatotoxicity using the PSSA in a Chinese population. We found a 1.3- to 1.5-fold increase in risk of hepatotoxicity following ATO, with the greater risk occurring within the first 30 days of treatment.

Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal types of cancer within the urinary system. Great efforts have been made to elucidate the pathogeny. However, the molecular mechanism of ccRCC is still not well understood. The aim of this study is to identify key genes in the carcinogenesis and progression of ccRCC. The mRNA microarray dataset GSE53757 was downloaded from the Gene Expression Omnibus database. The GSE53757 dataset contains tumor and matched paracancerous specimens from 72 ccRCC patients with clinical stage I to IV. The linear model of microarray data (limma) package in R language was used to identify differentially expressed genes (DEGs). The protein–protein interaction (PPI) network of the DEGs was constructed using the search tool for the retrieval of interacting genes (STRING). Subsequently, we visualized molecular interaction networks by Cytoscape software and analyzed modules with MCODE. A total of 1,284, 1,416, 1,610 and 1,185 up-regulated genes, and 932, 1,236, 1,006 and 929 down-regulated genes were identified from clinical stage I to IV ccRCC patients, respectively. The overlapping DEGs among the four clinical stages contain 870 up-regulated and 645 down-regulated genes. The enrichment analysis of DEGs in the top module was carried out with DAVID. The results showed the DEGs of the top module were mainly enriched in microtubule-based movement, mitotic cytokinesis and mitotic chromosome condensation. Eleven up-regulated genes and one down-regulated gene were identified as hub genes. Survival analysis showed the high expression of CENPE, KIF20A, KIF4A, MELK, NCAPG, NDC80, NUF2, TOP2A, TPX2 and UBE2C, and low expression of ACADM gene could be involved in the carcinogenesis, invasion or recurrence of ccRCC. Literature retrieval results showed the hub gene NDC80, CENPE and ACADM might be novel targets for the diagnosis, clinical treatment and prognosis of ccRCC. In conclusion, the findings of present study may help us understand the molecular mechanisms underlying the carcinogenesis and progression of ccRCC, and provide potential diagnostic, therapeutic and prognostic biomarkers.

Reactive metabolites from anti‐tuberculosis (anti‐TB) drugs can result in abnormal accumulation of reactive oxygen species (ROS), which plays an important role in anti‐TB drug‐induced liver injury (ATLI). Liver cells could keep the production of ROS in balance by antioxidant activities. The heme oxygenase 1, encoded by the HMOX1 gene and NADH:quinone oxidoreductase 1, encoded by the NQO1 gene are crucial mediators of cellular defense against ROS. The present study aimed to investigate the associations between HMOX1 and NQO1 polymorphisms and ATLI in Chinese anti‐TB treatment population.

Objective: Anti-tuberculosis drug-induced hepatotoxicity (ATDH) is related to hepatotoxic intermediaries, which are detoxified by glutathione S-transferases (GSTs). GSTM1 and GSTT1 are regulated by nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathway, and the BTB domain and CNC homologue 1 (Bach1) could compete with Nrf2 for binding to the DNA. Thus, bach1 may indirectly affect GSTs expression. The present study aimed to examine the role of tagSNPs in BACH1 in a Chinese population-based cohort Methods: A nested case-control study was conducted. Each ATDH case was matched with two controls by age, gender, treatment history, etc. Seven tagSNPs were detected and analysed. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated using a conditional logistic regression model. Results: A total of 290 ATDH cases and 580 controls were included in the present study. Patients carrying GG genotype of rs372883 were at a lower risk of ATDH than with AA genotype (OR = 0.553, 95%CI: 0.357-0.857, P = .008), and significant differences were also found under recessive model (P = .021) and additive model (P = .009). Similar results were also found in the polymorphism of rs1153285 (AA vs. GG, OR = 0.574, 95%CI: 0.360-0.914, P = .019), and under its recessive model (P = .033) and additive model (P = .026). Two haplotypes of A-G-T and C-T-G were identified to be associated with ATDH development Further subgroup analysis also suggested significant association between BACH1 polymorphisms and ATDH among certain and probable hepatotoxicity cases. Conclusions: This is the first study to explore the relationship between tagSNPs of BACH1 and ATDH in a Chinese cohort Based on this cohort, genetic polymorphisms of BACH1 may be associated with susceptibility to ATDH in the Chinese population.

Although an association between obesity and the occurrence of renal cell carcinoma (RCC) has been identified, the mechanism by which obesity functions to increase this risk of cancer remains unclear. Leptin, visfatin, apelin, resistin and adiponectin are peptide hormones secreted by adipocytes; it is considered that these may affect RCC development by exerting effects on proliferation, cell growth and inflammation. The aim of the present study was to investigate the association between the aforementioned adipokine genes and clear cell RCC (CC-RCC). The GSE6344 dataset was downloaded from the Gene Expression Omnibus database, and the relative expression levels of the adipokine genes were analyzed. To verify the results of the mRNA microarray, 77 paired samples of CC-RCC and corresponding adjacent normal tissue were allocated into two groups. The extraction of total RNA was conducted, and the mRNA expression of adipokine genes was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The data from the GSE6344 dataset indicated that the expression of visfatin and apelin was upregulated (P<0.0001 and P<0.01, respectively), and adiponectin was downregulated (P<0.001) in the CC-RCC tissues compared with the adjacent normal tissues. The data from RT-qPCR demonstrated that visfatin and resistin gene expression was increased (P<0.01 and P<0.05, respectively) in the CC-RCC tissues. Furthermore, the mRNA expression level of leptin and adiponectin in the adjacent normal tissue was higher than those in the cancer tissue (P<0.01). The current study verifies that visfatin and adiponectin are associated with an increased risk of CC-RCC, which presents further insights into the molecular mechanisms of CC-RCC tumorigenesis.

Although several studies have documented the role of leptin receptor gene polymorphisms in cancers, the association between leptin receptor gene polymorphisms and renal cell carcinoma (RCC) remains unknown. The aim of this study was to develop a high-resolution melting (HRM) approach for genotyping single nucleotide polymorphisms of leptin receptor gene on the LightCycler 480, and to explore the relation between polymorphisms of the leptin receptor gene and RCC. The study population consisted of 83 patients with renal cell carcinoma and 161 healthy control subjects. The Lys109Arg (A/G) and Gln223Arg (A/G) polymorphisms of leptin receptor gene were examined with HRM assay. Direct DNA sequencing and PCR-restriction fragment length polymorphisms were used as a reference method for genotyping Lys109Arg and Gln223Arg, respectively. Three genotypes of Lys109Arg or Gln223Arg were clearly distinguishable from the melting curve shapes with HRM assay. The data also showed the results of the direct DNA sequencing or PCR-restriction fragment length polymorphisms analysis were in complete concordance to genotyping results obtained by HRM (kappa=1.0). In addition, the data showed the G-G haplotype frequency was higher (p<0.05), and that the A-G (p<0.001) and G-A (p<0.01) haplotypes frequencies were lower in the RCC than controls. We developed a rapid, low cost, high-throughput and reliable single-tube technology for genotyping Lys109Arg and Gln223Arg polymorphisms. In addition, our data suggest that Lys109Arg and Gln223Arg gene polymorphisms are associated with RCC in Chinese Han studied population.

Idiopathic pulmonary fibrosis (IPF) is a rare, progressive, and lethal interstitial lung disease with unknown etiology. Divergent observations have suggested that genetic factors contribute to IPF susceptibility. This study investigated the relationship between human leukocyte antigen (HLA), cytokine gene polymorphisms, and IPF in a Chinese Han population. The gene polymorphisms of HLA-A, -B, -DRB1, tumor necrosis factor alpha [TNF-α (−308 A/G)], transforming growth factor beta [TGF-β1 (+869 T/C)], interleukin 10 [IL-10 (−592 C/A, −819 T/C, and −1082 G/A)], and interferon gamma [IFN-γ (+874 T/A)] were detected in 102 individuals with IPF and 266 unrelated normal controls using PCR with sequence-specific primers and a high-resolution melt (HRM) approach. The data showed that there was no difference in HLA allele frequencies between the IPF and control groups. However, the data showed the frequency of HLA-A*02-DRB1*04 haplotype in the IPF group was significantly higher than that in the control group [odds ratio (OR) = 4.69, 95% confidence interval (CI) = 1.82–12.08, p < 0.001]. In addition, no differences in the allele and genotype distributions of the cytokines were found between the IPF and control groups (p > 0.01). Our findings suggest that there is an association between specific HLA haplotype and IPF genetic susceptibility and that the genetic variability of some cytokines may not be involved in the pathogenesis of IPF.

OBJECTIVE:To analyse the mutation of ADAR gene in a pedigree with dyschromatosis symmetrical hereditaria (DSH).METHODS:A pedigree of DSH was investigated. Mutation scanning was carried out by PCR and direct sequencing. ADAR gene of 50 normal people was also sequenced as control. Through CBMdisc and PubMed, the mutations of ADAR gene were summarized.RESULTS:A novel mutation of c.2447G > A was found in all patients with DSH, but was not found in normal individuals in this DSH family and 50 unrelated controls. There were 64 mutations in ADAR gene.CONCLUSION:A deletion mutation (c.2447G > A) in the ADAR gene has been detected in this DSH family, which is probably one of the molecular bases of the pathogenesis of the disease. Author have summarized a total of 64 mutations in the ADAR gene by previous reports and speculate that the mutation hotspots of ADAR gene might be located in the tRNA-specific and double-stranded RNA adenosine deaminase (ADEAMc) domain.