Clinical Validation of the Unparalleled Sensitivity of the Novel Allele-Discriminating Priming System Technology-Based EGFR Mutation Assay in Patients with Operable Non-Small Cell Lung Cancer.

PURPOSE: Recently, we developed allele-discriminating priming system (ADPS) technology. This method increases the sensitivity of conventional quantitative polymerase chain reaction up to 100 folds, with limit of detection, 0.01%, with reinforced specificity. This prospective study aimed to develop and validate the accuracy of ADPS epidermal growth factor receptor (EGFR) Mutation Test Kit using clinical specimens. MATERIALS AND METHODS: In total 189 formalin-fixed paraffin-embedded tumor tissues resected from patients with non-small cell lung cancer were used to perform a comparative evaluation of the ADPS EGFR Mutation Test Kit versus the cobas EGFR Mutation Test v2, which is the current gold standard. When the two methods had inconsistent results, next-generation sequencing-based CancerSCAN was utilized as a referee. RESULTS: The overall agreement of the two methods was 97.4% (93.9%-99.1%); the positive percent agreement, 95.0% (88.7%-98.4%); and the negative percent agreement, 100.0% (95.9%-100.0%). EGFR mutations were detected at a frequency of 50.3% using the ADPS EGFR Mutation Test Kit and 52.9% using the cobas EGFR Mutation Test v2. There were 10 discrepant mutation calls between the two methods. CancerSCAN reproduced eight ADPS results. In two cases, mutant allele fraction was ultra-low at 0.02% and 0.06%, which are significantly below the limit of detection of the cobas assay and CancerSCAN. Based on the EGFR genotyping by ADPS, the treatment options could be switched in five patients. CONCLUSION: The highly sensitive and specific ADPS EGFR Mutation Test Kit would be useful in detecting the patients who have lung cancer with EGFR mutation, and can benefit from the EGFR targeted therapy.

Modified Taq DNA Polymerase for Allele-Specific Ultra-Sensitive Detection of Genetic Variants.

Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.

Transcriptome-metabolome-wide association study (TMWAS) in rats revealed a potential carcinogenic effect of DEHP in thyroid associated with eicosanoids.

The incidence of thyroid cancer (TC) has increased considerably in the last few decades. Environmental factors, including plasticizers, are recognized as potential risks leading to thyroid cancer in humans. In this study, we used a transcriptome-metabolome-wide association study to find the unidentified carcinogenic mechanism of di-2-ethylhexyl phthalate (DEHP) in thyroid and biomarkers for non-invasive diagnosis. Rats were treated with different doses of DEHP (0, 0.3, 3, 30, 150 mg DEHP/kg bw/day) for 13 weeks. Then, the thyroids were processed for Ki67 staining and RNA-seq. Also, 17-h urine samples were collected for high-resolution metabolomics analysis. After a high dose of DEHP exposure, the terminal body weights and the thyroid and parathyroid glands weights were not altered. However, the liver weights and numbers of Ki67-positive cells were increased. Further, multivariate statistical analysis revealed that metabolic shifts were considerably altered above 30 mg DEHP/kg bw/day. In RNA-seq analysis, some cancer-related genes were altered, including 18 upregulated and 9 downregulated transcripts. These cancer transcripts and whole metabolome data were integrated to uncover thyroid cancer-related metabolic pathways, which revealed that cancer-related transcripts had a network structure linked to eicosanoids such as leukotriene D4 and prostaglandin. In brief, our study demonstrated that DEHP can induce thyroid hyperplasia through the eicosanoid-associated pathway, providing further insight into the mechanism of DEHP-associated thyroid cancer.

Comprehensive analysis of transcriptomic changes induced by low and high doses of bisphenol A in HepG2 spheroids in vitro and rat liver in vivo.

Bisphenol A (BPA), a synthetic monomer commonly included in the daily products, has a structure similar to the estrogen receptor agonist. Therefore BPA has been anticipated to interfere with the hormone metabolisms and cause diverse pathological conditions. But the effects of BPA on the genetic landscapes of liver or hepatic cells have not been fully established. Gene expressional changes induced by low- or high-dose of BPA were evaluated in 3D cultured human hepatoma cells (HepG2 spheroids) in vitro at 0, 0.5, 5 and 200 µM and liver of rats exposed to BPA at 0, 0.5 and 250 mg/kg for 90 days in vivo. Functional enrichment analysis, pathway activity measurement and network analysis were performed using BPA-responsive genes. Treatment with BPA changed a lot of gene expressions in both HepG2 spheroids and rat livers depending on doses of BPA. Functional enrichment and pathway analysis show that lipid or steroid metabolism-related functions were altered by BPA in both HepG2 spheroids and livers of rats. Lipid metabolism-related functions altered by BPA formed a large cluster encompassing lipid biosynthesis, steroid metabolic process and cholesterol regulation process. It was also observed that distribution of pathway activities was correlated between HepG2 spheroids and rat livers at low-dose of BPA. Distance distribution in protein-protein interaction network also evidenced the closeness of BPA-responsive genes to metabolism pathways which include lipid metabolism. Collectively, we demonstrated that BPA greatly influenced overall gene expression and biological functions in both human hepatoma spheroids and rat liver, in which lipid- or steroid metabolism-associated genes were significantly altered by the exposure to BPA.

Low Dose Exposure to Di-2-Ethylhexylphthalate in Juvenile Rats Alters the Expression of Genes Related with Thyroid Hormone Regulation.

Phthalates widely used in the manufacture of plastics have deeply penetrated into our everyday lives. Recently, a concern over the toxicity of phthalates on thyroid, has been raised but in most of cases, the doses employed were unrealistically high. To investigate the effects of phthalates on thyroid, we investigated the effects of the repeated oral exposure to low to high doses (0.3, 3, 30 and 150 mg/kg) di-2-ethylhexylphthalate (DEHP) from weaning to maturity for 90 days in juvenile rats on the thyroid. The histological examination revealed that DEHP significantly induced hyperplasia in the thyroid from the doses of 30 mg/kg, which was confirmed with Ki67 staining. In line with this finding, increased mRNA expression of thyrotropin releasing hormone (Trh) was observed in the thyroid of female at 0.3 mg/kg and 150 mg/kg as determined by RNAseq analysis. Moreover, significantly increased expression of parathyroid hormone (Pth) in the female at 0.3 mg/kg, and thyroglobulin (Tg) and thyroid hormone responsive (Thrsp) in the male at 0.3 mg/kg were noted in the blood, of which changes were substantially attenuated at 150 m/kg, alluding the meaningful effects of low dose DEHP on the thyroid hormone regulation. Urinary excretion of mono-2-ethylhexyl-phthalate (MEHP), a major metabolite of DEHP was determined to be 4.10 and 12.26 ppb in male, 6.65 and 324 ppb in female at 0.3 and 30 mg/kg DEHP, respectively, which fell within reported human urine levels. Collectively, these results suggest a potential adverse effects of low dose phthalates on the thyroid.

Inhibition of hepatitis B virus replication by a dNTPase-dependent function of the host restriction factor SAMHD1.

SAMHD1 is a cellular protein that possesses dNTPase activity and inhibits retroviruses and DNA viruses through the depletion of cellular dNTPs. However, recent evidence suggests the existence of alternative or additional mechanisms that involve novel nuclease activities. Hepatitis B virus is a DNA virus but resembles retroviruses in that its DNA genome is synthesized via reverse transcription of an RNA transcript. SAMHD1 was shown to inhibit the expression and replication of a transfected HBV DNA. We further investigated the antiviral mechanisms in a newly developed infection assay. Our data indicated that SAMHD1 exerts a profound antiviral effect. In addition, unlike previous findings, our results demonstrate the essential role of SAMHD1 dNTPase. SAMHD1 did not affect virion-derived cccDNA and gene expression but specifically inhibited viral DNA synthesis. These results indicate that SAMHD1 inhibits HBV replication at the reverse transcription step, most likely through the depletion of cellular dNTPs.

Inhibition of hepatitis B virus replication by ligand-mediated activation of RNase L.

RNase L is a cellular endoribonuclease that is activated by 2',5'-linked oligoadenylates (2-5A), which are unique and specific ligands synthesized by a family of interferon-inducible, dsRNA-activated enzymes named oligoadenylate synthetases. In the typical antiviral pathway, activated RNase L degrades viral and cellular RNAs, thus limiting viral replication and spread. Although the antiviral activity of RNase L has been demonstrated for several RNA viruses, there is little evidence regarding its role against DNA viruses. In the present study, the potential antiviral activity of RNase L against hepatitis B virus (HBV) was explored utilizing the recently reported infection protocol based on human hepatoma HepG2 cells stably complemented with the virus entry factor NTCP. Viral replication and expression in this cell type was markedly inhibited by poly(I:C)- or 2-5A-mediated activation of RNase L; however, the inhibition was significantly reversed by RNase L knockdown. Further analysis in HBV1.2-transfected Huh-7 hepatoma cells indicated that the antiviral activity of RNase L depends on its ribonuclease function. We also provide evidence for the specific roles of OAS family members in this process. These results suggest that HBV replication can be regulated through interferon-mediated RNA decay pathways and that activation of these host antiviral factors may represent a novel therapeutic strategy for HBV infection.

PKR-dependent mechanisms of interferon-α for inhibiting hepatitis B virus replication.

Interferon-α (IFN-α) inhibits the replication of hepatitis B virus (HBV) in vivo and in vitro, but the molecular mechanism of this inhibition has been elusive. We found that while HBV replication in transfected human hepatoma Huh-7 cell was severely inhibited by IFN-α treatment as reported previously, this inhibition was markedly impaired in the cell in which the expression of IFN-inducible, double-stranded RNA-dependent protein kinase (PKR) was stably and specifically suppressed through RNA-interference. Intracellular level of viral capsids was down-regulated likewise in a PKR-dependent manner, whereas that of HBV transcripts including the viral RNA pregenome was not affected by IFN-α treatment. Ectopic expression of PKR also resulted in the reduction of viral capsids with concomitant increase of phosphorylated eIF2α. These results suggested that PKR functions as a key mediator of IFN-α in opposing HBV replication, most likely through the inhibition of protein synthesis.

Population database on nine STR loci of the AmpFlSTR Profiler kit in Koreans.

Allele frequencies for nine STR loci included in the AmpFlSTR Profiler Kit (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820), were obtained from a sample of 1206 unrelated individuals living in the central region of Korea.

Population genetics of nine STR loci: TH01, TPOX, CSF1PO, vWA, FESFPS, F13A01, D13S317, D7S820 and D16S539 in a Korean population.

Allele frequencies for nine STR loci namely, TH01, TPOX, CSF1PO, vWA, FESFPS, F13A01, D13S317, D7S820 and D16S539 were obtained from a sample of 437 unrelated individuals living in Chungcheong-do, South Korea.