Rapid Detection of SNP (c.309T&gt;G) in the MDM2 Gene by the Duplex SmartAmp Method

Enokida, Yasuaki; Shimizu, Kimihiro; Abe, Jun; Lezhava, Alexander; Tanaka, Yuki; Kimura, Yoshinobu; Soma, Takehisa; Hanami, Takeshi; Kawai, Yosuke; Usui, Kengo; Okano, Yasuko; Kakegawa, Seiichi; Ogawa, Hiroomi; Miyamae, Yohei; Miyagi, Yohei; Nakayama, Haruhiko; Ishikawa, Toshihisa; Hayashizaki, Yoshihide; Takeyoshi, Izumi

doi:10.1371/journal.pone.0060151

Cited by 9 publications

(11 citation statements)

References 38 publications

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“…This, in turn, attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans [45]. Asians, including Japanese, have higher frequencies of the 309G allele as compared with African-Americans and Caucasians [46]. It has been reported that this polymorphism in the MDM2 gene is associated with the prognosis for several types of tumors, including lung cancer [47].…”

Section: Discussionmentioning

confidence: 99%

SNP (–617C>A) in ARE-Like Loci of the NRF2 Gene: A New Biomarker for Prognosis of Lung Adenocarcinoma in Japanese Non-Smoking Women

et al. 2013

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PurposeThe transcription factor NRF2 plays a pivotal role in protecting normal cells from external toxic challenges and oxidative stress, whereas it can also endow cancer cells resistance to anticancer drugs. At present little information is available about the genetic polymorphisms of the NRF2 gene and their clinical relevance. We aimed to investigate the single nucleotide polymorphisms in the NRF2 gene as a prognostic biomarker in lung cancer.Experimental DesignWe prepared genomic DNA samples from 387 Japanese patients with primary lung cancer and detected SNP (c.–617C>A; rs6721961) in the ARE-like loci of the human NRF2 gene by the rapid genetic testing method we developed in this study. We then analyzed the association between the SNP in the NRF2 gene and patients’ overall survival.ResultsPatients harboring wild-type (WT) homozygous (c.–617C/C), SNP heterozygous (c.–617C/A), and SNP homozygous (c.–617A/A) alleles numbered 216 (55.8%), 147 (38.0%), and 24 (6.2%), respectively. Multivariate logistic regression models revealed that SNP homozygote (c.–617A/A) was significantly related to gender. Its frequency was four-fold higher in female patients than in males (10.8% female vs 2.7% male) and was associated with female non-smokers with adenocarcinoma. Interestingly, lung cancer patients carrying NRF2 SNP homozygous alleles (c.–617A/A) and the 309T (WT) allele in the MDM2 gene exhibited remarkable survival over 1,700 days after surgical operation (log-rank p = 0.021).ConclusionSNP homozygous (c.–617A/A) alleles in the NRF2 gene are associated with female non-smokers with adenocarcinoma and regarded as a prognostic biomarker for assessing overall survival of patients with lung adenocarcinoma.

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Section: Discussionmentioning

confidence: 99%

SNP (–617C>A) in ARE-Like Loci of the NRF2 Gene: A New Biomarker for Prognosis of Lung Adenocarcinoma in Japanese Non-Smoking Women

et al. 2013

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“…product development. A variety of different SNP detection methods have lately been described [169][170][171]. Kawai et al report about a first Influenza A (H1N1) assay, combining both a reverse transcriptase and a SmartAmp amplification in one-step setup.…”

Section: Various Other Methodsmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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“…The DNA was used for SNP typing of c.309T>G and p53 Arg72Pro polymorphisms. Genotyping of c.309T>G was carried out using the Duplex SmartAmp method as described previously [22]. p53 Arg72Pro was genotyped using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) based on a previous report [23].…”

Section: Snp Genotypingmentioning

confidence: 99%

“…Although our findings need to be validated in prospective studies, c.309T>G would be a useful prognostic marker that is detectable at any stage of diagnosis or treatment and influences the therapeutic strategies. Furthermore, we had already established the Duplex SmartAmp method [22] to detect c.309T>G with a single drop (5 μL) of blood within 40 min from sample collection. If we can make this method more practical, we will detect this SNP more easily and quickly in any clinical situation.…”

Section: Mdm2 Snp309 and Lung Cancer Prognosesmentioning

confidence: 99%

Prognostic potential of the MDM2 309T>G polymorphism in stage I lung adenocarcinoma

et al. 2016

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The MDM 2 protein plays an important role in the regulation of cell proliferation and apoptosis via ubiquitination and proteasome‐mediated degradation of p53. The genetic polymorphism rs2279744 (c.309T>G) of the MDM 2 gene is reportedly associated with susceptibility and/or prognosis in various cancers. In this study, we investigated the risk factors for worse survival in patients with lung adenocarcinoma ( AC ). We examined the association between c.309T>G and the prognosis of lung cancer by retrospectively reviewing 453 lung cancer patients. We studied both, clinicopathological and genetic characteristics, including the c.309T>G, p53 Arg72Pro, EGFR , KRAS , and p53 mutations. Associations between these factors and survival outcome were analyzed using Cox proportional hazards models. The frequencies of MDM 2 polymorphisms were T/T, 20.8%; T/G, 48.6%, and G/G, 30.7%. The overall survival ( OS ) of AC patients with pathological stage I disease and the MDM 2 T/T genotype was significantly shorter than that of those with the T/G or G/G genotypes ( P = 0.02). Multivariate analysis revealed that the MDM 2 T/T genotype was an independent, significant prognostic factor (hazard ratio [ HR ] = 2.23; 95% confidence interval [ CI ]: 1.07–4.65; P = 0.03). The MDM 2 T/T genotype was predictive of poorer survival in a Japanese population. Genotyping for this polymorphism might predict the clinical outcomes of stage I AC patients.

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Rapid Detection of SNP (c.309T>G) in the MDM2 Gene by the Duplex SmartAmp Method

Cited by 9 publications

References 38 publications

SNP (–617C>A) in ARE-Like Loci of the NRF2 Gene: A New Biomarker for Prognosis of Lung Adenocarcinoma in Japanese Non-Smoking Women

SNP (–617C>A) in ARE-Like Loci of the NRF2 Gene: A New Biomarker for Prognosis of Lung Adenocarcinoma in Japanese Non-Smoking Women

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Prognostic potential of the MDM2 309T>G polymorphism in stage I lung adenocarcinoma

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