• BMB Reports
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• 제29권5호
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• pp.442-447
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• 1996

Mutations in codon 12, 13 and 61 of one of the three ras genes, H-ras, K-ras and N-ras, convert these genes into active oncogenes. The presence of H-ras gene mutations have important prognostic implications in various cancers. In this study, the H-ras gene mutations were investigated by two-step PCRRFLP in patients with bladder and stomach cancer. For the control experiments, T24 and SK2 cell lines were used. In a total of 36 bladder cancer patient cases, five (13.9%) mutations were found by this method. Of these, point 12 mutations were two (5.6%) cases and point 61 mutations were three (8.3%) cases. On the other hand, H-ras mutation was not found in 29 cases of stomach cancer. The results of the mutated H-ras gene confirmed by direct sequencing analysis were correlated well with PCR analysis. From the sensitivity test, the H-ras mutation was found to have about 0.2% of mutated DNA mingled in normal DNA. In conclusion, the H-ras mutation has a higher clinical Significance in bladder cancer than stomach cancer. Moreover the two-step PCR-RFLP method is sensitive, rapid and relatively simple for clinical work in detecting H-ras point mutations.

• Journal of Yeungnam Medical Science
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• 제16권1호
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• pp.114-118
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• 1999

LHON은 사립체 DNA의 점돌연변이에 의해서 유발되며 11778, 3460, 14484의 세 부위가 주된 사립체 DNA 점돌연변이의 위치로 알려져 있다. 이에 저자들은 점진적인 시력 저하를 호소하면서 사립체 DNA 분석 결과 11778 점돌연변이가 확인된 LHON환자 1례를 경험하였기에 문헌고찰과 함께 보고하는 바이다.

• BMB Reports
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• 제46권5호
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• pp.270-275
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• 2013

It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry.

• 한국임상수의학회지
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• 제19권2호
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• pp.195-198
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• 2002

개에서 자연적으로 발생한 12예의 종양에 대해, 종양 억제 유전자 p53의 변이와의 관계를 확인해 보았다. 종양조직에서 일반적인 방법으로 DNA를 추출하여, PCR 기법으로 p53을 증폭하여 염기서열을 확인한 결과, 개의 유선암종 예에서 exon 8의 codon 285에서 CCT $\longrightarrow$ TCT (proline $\longrightarrow$ serine)로 점변이 된 것이 확인되었다. 또한 악성 비만세포 종양 예에서도 exon 8의 codon 249에서 AGT $\longrightarrow$AGC로 점변이 된 것이 확인되었으나 silent point mutation (serine)으로 판명되었다. 이상의 결과를 토대로 개의 유선암종과 악성 비만세포 종양에서 종양억제 유전자 p53의 변이가 확인되었으며, 이는 종양의 형성과 관련된 p53의 역할이나 종양의 치료 및 예후 판정에 p53 을 활용하는 연구의 초석이 되리라 사료되며, 차후 이 유전자에 대한 광범위한 연구가 지속되어야 하리라 생각된다.

• Journal of Gastric Cancer
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• 제3권3호
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• pp.145-150
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• 2003

Purpose: The p53 tumor suppressor gene is believed to play a pivotal role in preventing the uncontrolled cellular growth characteristic of cancer. Mutation of the p53 gene represent one of the most common genetic alterations in human cancers, and the acquisition of such defects is strongly associated with tumor progression and metastasis. The aim of this study was to evaluate the relation between p53 immunoreactivity and the mutation of p53 gene in gastric adenocarcinoma obtained by laser capture microscope. Materials and Methods: Formalin fixed paraffin embedded tissue specimens were obtained from 20 patients who underwent surgery for gastric cancer. According to UICC TNM system, 3 of the cases were Ia, 2 cases II, 4 cases IIIa, 5 cases IIIb, and 6 cases IV. Results: Immunohistochemical staining revealed eight cases as negative (less than $10\%$), twelve cases as postive (more than $10\%$). The locations of mutations were as follows; 7 cases had point mutation at exon 4, and 3 cases point mutation at exon 8. There was no mutation at exon 5, 6, 7 and 9. The mutation was observed in 1 case out of 8 p53 oncoprotein negative cases, and 7 cases out of 12 p53 positive cases. The mutation was more common in p53 positive cases (P<0.05), However, there was no significant correlation between p53 mutation observed by DNA sequencing after laser capture microdissection and expression of p53 oncoprotein. Conclusion: These result suggest that he expression of p53 oncoprotein not to be related to the mutation of p53 gene at exons 4 through 9 in gastric cancer.

• 한국발생생물학회지:발생과생식
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• 제4권1호
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• pp.61-66
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• 2000

본 연구에서는 조기 난소 부전증 환자를 대상으로 난포 자극 호르몬 수용체 유전자의 돌연변이와 발현 양상을 분석하였다. 돌연변이 분석을 위해 환자의 말초혈액에서 genomic DNA를 분리하고 nucleotide 566을 포함하고 있는 exon 7에 특이적인 primer쌍을 이용하여 중합효소 연쇄 반응을 시행하였다. 전기 영동으로 반응산물을 확인한 다음, 돌연변이 여부를 조사하기 위하여 제한효소 절단분석 (Restriction Fragment Length Polymorphism, RFLP)을 시행한 결과, 대조군과 조기 난소 부전증 환자군 모두에서 돌연변이를 관찰할 수 없었다. 난포 자극 호르몬 수용체의 발현양상을 확인하기 위해 시험관아기 시술과정 중 난자 채취과정에서 얻어진 황체화 과립세포에서 total RNA를 추출하여 역전사 중합효소 연쇄 반응을 시행하였다. 반응 산물을 전기 영동하여 발현양상을 비교해 본 결과, 대조군에 비해 조기 난소 부전증 환자군에서 난포 자극 호르몬 수용체 유전자의 발현이 다소 낮은 것으로 확인되었다. 이상의 결과로 보아 조기 난소 부전증 환자에서 난포 자극 호르몬 수용체 유전자의 돌연변이는 발견할 수 없었으며 난포 자극 호르몬 수용체 유전자의 발현이 대조군에 비해 낮아 과배란 유도시에 생식소 자극 호르몬에 대해 저적응증을 보이며 난포형성과정에도 장애를 받는 것으로 사료된다.

• The Korean Journal of Physiology and Pharmacology
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• 제7권3호
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• pp.157-162
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• 2003

Our earlier studies found a significant correlation between the activities of ranitidine N-oxidation catalyzed by hepatic flavin-containing monooxygenase (FMO) and the presence of mutations in exon 4 (E158K) and exon 7 (E308G) of the FMO3 gene in Korean volunteers. However, caffeine N-1 demethylation (which is also partially catalyzed by FMO) was not significantly correlated with these FMO3 mutations. In this study, we examined another common mutation (V257M) in exon 6 of FMO3 gene. The V257M variant, which is caused by a point mutation (G769A), was commonly observed (13.21% allele frequency) in our subjects (n=159). This point mutation causes a substitution of $Val^{257}$ to $Met^{257}$, with transformation of the secondary structure. The presence of this mutant allele correlated significantly with a reduction in caffeine N-1-demethylating activity, but was not correlated with the activity of N-oxidation of ranitidine. In a family study, the low FMO activity observed in a person heterozygous for a nonsense mutation in exon 4 (G148X) and heterozygous for missense mutation in exon 6 (V257M) of FMO3 was attributed to the mutations. Our results suggest that various point mutations in the coding regions of FMO3 may influence FMO3 activity according to the probe substrates of varying chemical structure that correlate with each mutation on the FMO3 gene.

• Genomics & Informatics
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• 제6권3호
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• pp.142-146
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• 2008

In order to understand the protein functions that are related to disease, it is important to detect the correlation between amino acid mutations and disease. Many mutation studies about disease-related proteins have been carried out through molecular biology techniques, such as vector design, protein engineering, and protein crystallization. However, experimental protein mutation studies are time-consuming, be it in vivo or in vitro. We therefore performed a bioinformatic analysis of known disease-related mutations and their protein structure changes in order to analyze the correlation between mutation and disease. For this study, we selected 111 diseases that were related to 175 proteins from the PDB database and 710 mutations that were found in the protein structures. The mutations were acquired from the Human Gene Mutation Database (HGMD). We selected point mutations, excluding only insertions or deletions, for detecting structural changes. To detect a structural change by mutation, we analyzed not only the structural properties (distance of pocket and mutation, pocket size, surface size, and stability), but also the physico-chemical properties (weight, instability, isoelectric point (IEP), and GRAVY score) for the 710 mutations. We detected that the distance between the pocket and disease-related mutation lay within $20\;{\AA}$ (98.5%, 700 proteins). We found that there was no significant correlation between structural stability and disease-causing mutations or between hydrophobicity changes and critical mutations. For large-scale mutational analysis of disease-causing mutations, our bioinformatics approach, using 710 structural mutations, called "Structural Mutatomics," can help researchers to detect disease-specific mutations and to understand the biological functions of disease-related proteins.

• Bulletin of the Korean Chemical Society
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• 제27권9호
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• pp.1346-1352
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• 2006

We describe an effective method of microchip electrophoresis (ME) based on single strand conformation poly-morphism (SSCP) analysis to rapidly detect the point mutation, Leu72Met, in a human obesity gene. The 207-bp dsDNA in the Leu72Met region, an estimate of the child obesity DNA mutant, was amplified by polymerase chain reaction (PCR) and submitted to a conventional glass microchip analysis with a sieving matrix of 1.75% poly(vinylpyrrolidone) (Mr 1 300 000), 1.0% poly(ethyleneoxide) (Mr 600 000) and 5.0% w/w glycerol. When combined with base stacking (BS) with hydroxide ions, the SSCP-ME provided rapid analysis as well as sensitive detection. The detection sensitivity was effectively enhanced in the OH- concentration range of 0.01-0.025 M NaOH. The sensitivity and speed of this ME-based SSCP methodology for the rapid detection of Leu72Met point mutations makes this an attractive method for diagnosing childhood obesity in a clinical diagnostic laboratory.

• Clinical and Experimental Reproductive Medicine
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• 제25권3호
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• pp.281-286
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• 1998

Premature ovarian failure is a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women younger than 40 years. Many causes of premature ovarian failure were reported, including genetic abnormalities, enzymatic defects, defects in gonadotropin secretion or action, autoimmune disorders, physical and idiopathic causes. Recently, Finnish group reported a point mutation in the follicle stimulating hormone (FSH) receptor gene in premature ovarian failure patients. But it was reported that the group from United States could not find any mutation in FSH receptor gene. So we analysed C566T point mutation of FSH receptor gene using restriction fragment length polymorphism (RFLP) and compared the result between premature ovarian failure patient with idiopathic and known causes. But we did not find 556C${\rightarrow}$T mutation in the FSH receptor gene in both groups. These findings suggest that the missense mutation in the human FSH receptor gene reported in Finnish women with premature ovarian failure is uncommon in Korean women with premature ovarian failure.