• Genomics & Informatics
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• 제12권2호
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• pp.50-57
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• 2014

We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at $30{\times}$ sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than $1,000{\times}$ coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening.

• 대한화학회지
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• 제48권1호
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• pp.33-38
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• 2004

두경부 편평 세포암종(HNSCC: head and neck squamous cell carcinoma) 의 발생과 관련하여 p53 종양 억제 유전자 (tumor suppressor gene) 의 돌연변이는 높은 비율로 나타나는 것으로 보고 되고 있다. 단국대학교 병원에서 두경부 종양으로 진단 받고 수술 받은 환자의 조직 50개를 대상으로 p53 종양 억제 유전자의 exon 5-8 까지의 영역에서 DNA를 추출하여 PCR-SSCP(polymerase chain reaction single strand conformational polymorphism) 방법과 DHPLC(denaturing high performance liquid chromatography) 방법으로 p53체세포 돌연변이(somatic mutation)를 비교 분석하였다. 그 결과 SSCP 분석 방법은 16개(32%), DHPLC 분석 방법은 17개(34%) 를 검출하였고 그 중 SSCP와 DHPLC 분석 방법 모두 exon 8번에서 결실(deletion) 형태의 돌연변이를 확인하였으며 최종적으로 자동 염기 서열 분석기(automatic DNA sequencer) 를 통하여 모든 돌연변이를 확인하였다. DHPLC 분석방법이 SSCP 방법보다 분석 시간이나 노력이 덜 소모되며 보다 더 정확한 돌연변이 검출 방법임을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1053-1055
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• 2016

Background: Polycythemia rubra vera (PV), being a primary polycythemia, is caused by neoplastic proliferation of erythroid, megakaryocytic and granulocytic lineages which result in panmyelosis. PV patients have a somatic acquired mutation in the Janus kinase (JAK2) pathway, rendering cell proliferation independent of the normal regulatory mechanisms that regulate erythropoiesis. The rational of this study was to determine the prevalence of the JAK-2 V617F mutation in Pakistani patients with PV. Materials and Methods: In this cross sectional study, 26 patients with PV were enrolled from January 2010 to December 2014. Patients were diagnosed based on WHO criteria for PV. All were screened for G-T point mutation (V617F) in the JAK2 gene on chromosome 9 by an allele specific PCR. Results: The mean age was $53.4{\pm}9.31years$ (range 36-72) and the male to female ratio was 2:1. The frequency of JAK2 V617F positivity in our PV patients was found to be 92.3%. Overall 30.7% of patients were asymptomatic and remaining 69.3% presented with symptomatic disease. The mean hemoglobin was $18.1{\pm}1.9g/dl$ with the mean hematocrit of $55.6{\pm}8.3%$. The mean total leukocyte count was $12.8{\pm}7.1{\times}10^9/l$ and the platelet count was $511{\pm}341.9{\times}10^9/l$. A positive correlation of JAK2 V617F mutation was established with high TLC count (P=0.01). No correlation of JAK2 V617F could be established with age or gender (P>0.05). Conclusions: The JAK2 V617F mutation frequency in our PV patients was similar to those reported internationally. Screening for the mutation in all suspected PV cases could be beneficial in differentiating patients with reactive and clonal erythrocytosis.

• Molecules and Cells
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• 제28권1호
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• pp.43-48
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• 2009

By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25.1 in C. elegans, defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.

• 환경생물
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• 제29권4호
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• pp.373-378
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• 2011

Our aim was to investigate the genotoxicity of ambient air in the Krak$\acute{o}$w area after Fukushima Nuclear Power Plant (NPP) accident and compare with results from Chernobyl fallout. For the detection of ambient air genotoxicity the technique for screening gene mutation frequency in somatic cells of the $Tradescantia$ stamen hairs ($Trad$-SH assay) was used. Since 11th of March 2011 (Fukushima NPP accident), several pots containing at least 15 shoots of bioindicating plants were exposed to ambient air at 2 sites in the Krak$\acute{o}$w surrounding area, one in the city center, and about 100 pots in a control site (in the glasshouse of the Institute of Nuclear Physics) Continuous screening of mutations was performed. Progenies of 371,090 cells exposed were analyzed. Mutation frequency obtained in the first 10 days has shown a mean control level (GMF*100=$0.06{\pm}0.01$). At scoring period related to influence of a potential Fukushima fallout, a significant increase of gene mutation frequencies above the control level was observed at each site in the range, 0.10~0.33 depending on the location, (mean value for all sites GMF*100=$0.19{\pm}0.05$) that was associated with a strong expression of toxic effects. In the reported studies following the Chernobyl NPP accident monitoring $in$ $situ$ of the ambient air genotoxicity was performed in the period since April $29^{th}$ till June $3^{rd}$ 1986 also with Trad-SH bioindicator. In general, mutation frequency increases due to Chernobyl fallout(GMF*100=$0.43{\pm}0.02$) were corresponding to fluctuation of radioactivity in the air reported from physical measures, and to published reports about increase in chromosome aberration levels. Although, recent data obtained from monitoring of the ambient air quality in the Krak$\acute{o}$w and surroundings are lower when compared to results reported after Chernobyl NPP accident, though results express a significant increase above the control level and also are corresponding with increased air radioactivity reported from physical measurements. Statistically significant in comparison to control increase in gene mutation rates and more prolonged than that after Chernobyl fallout increase of GMF was observed during the period following the Fukushima NPP failure.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2004년도 춘계학술대회
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• pp.47-60
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• 2004

Higher plants can be valuable genetic assay systems for monitoring environmental pollutants and evaluating their biological toxicity. Two assays are considered ideal for in situ monitoring and testing of soil, airborne and aqueous mutagenic agents; the Tradescantia stamen hair assay for somatic cell mutations and the Tradescantia micronucleus assay for chromosome aberrations. Both assays can be used for in vivo and in vitro testing of mutagens. Since higher plant systems are now recognized as excellent indicators and have unique advantages over in situ monitoring and screening, higher plant systems could be accepted by regulatory authorities as an alternative first-tier assay system for the detection of possible genetic damages resulting from the pollutants or chemicals used and produced by industrial sectors. It has been concluded that potential mutagen and carcinogen such as the heavy metals among indoor air particulates, volatile compounds in the working places, soil, and water pollutants contribute to the overall health risk. This contribution can be considerable under certain circumstances. It is therefore important to identify the level of genotoxic activity in the environment and to relate it to the biomarkers of a health risk in humans. The results from the higher plant bioassays could make a significant contribution to assessing the risks of pollutants and protecting the public from agents that can cause mutation and/or cancer. The plant bioassays, which are relatively inexpensive and easy to handle, are recommended for the scientists who are interested in monitoring pollutants and evaluating their environmental toxicity to living organisms.

• 한국환경생물학회:학술대회논문집
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• 한국환경생물학회 2004년도 학술대회
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• pp.1-15
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• 2004

Higher plants can be valuable genetic assay systems for monitoring environmental pollutants and evaluating their biological toxicity. Two assays are considered ideal for in situ monitoring and testing of soil, airborne and aqueous mutagenic agents; the Tradescantia stamen hair assay for somatic cell mutations and the Tradescantia micronucleus assay for chromosome aberrations. Both assays can be used for in vivo and in vitro testing of mutagens. Since higher plant systems are now recognized as excellent indicators and have unique advantages over in situ monitoring and screening, higher plant systems could be accepted by regulatory authorities as an alternative first-tier assay system for the detection of possible genetic damages resulting from the pollutants or chemicals used and produced by industrial sectors. It has been concluded that potential mutagen and carcinogen such as the heavy metals among indoor air particulates, volatile compounds in the working places, soil, and water pollutants contribute to the overall health risk. This contribution can be considerable under certain circumstances. It is therefore important to identify the level of genotoxic activity in the environment and to relate it to the biomarkers of a health risk in humans. The results from the higher plant bioassays could make a significant contribution to assessing the risks of pollutants and protecting the public firom agents that can cause mutation anuor cancer. The plant bioassays, which are relatively inexpensive and easy to handle, are recommended for the scientists who are interested in monitoring pollutants and evaluating their environmental toxicity to living organisms.

• Asian Pacific Journal of Cancer Prevention
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• 제17권12호
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• pp.5147-5152
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• 2016

Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes.