基于TCGA数据库分析FTH1在头颈部鳞状细胞癌中的表达和临床意义

目的 探究重链多肽亚基由铁蛋白重链多肽1(ferritin heavy chain polypeptide 1,FTH1)在头颈部鳞状细胞癌（head and neck squamous cell carcinoma,HNSCC）中的表达和临床意义。方法 基于癌症基因图谱（The Cancer Genome Atlas,TCGA）数据库分析FTH1在HNSCC组织和癌旁组织中的差异表达情况，并分析其表达与肿瘤分期的关系和对总生存的影响。采用Cox比例风险模型评估影响HNSCC患者预后的因素。使用GSEA软件预测FTH1可能参与调控的信号通路。结果 FTH1在HNSCC中高表达，且与肿瘤分期有关（均P<0.05）。FTH1高表达的HNSCC患者总生存率降低（P<0.05）。单因素Cox分析结果显示，高表达的FTH1和肿瘤分期均是影响HNSCC患者的预后因素(均P<0.05)。多因素Cox分析结果显示，肿瘤分期和性别均是影响HNSCC患者的预后因素(均P<0.05)。FTH1的通路富集分析显示，FTH1的高表达样本显著富集到溶酶体、谷胱甘肽代谢、磷酸戊糖途径及癌症通...     

SHP-1 在食管鳞状细胞癌组织中异常低表达的表观遗传学调节机制及其临床意义

目的：探讨食管鳞状细胞癌（esophageal squamous cell carcinoma，ESCC）组织中蛋白酪氨酸磷酸酶1（protein tyrosin phosphatase 1，SHP-1）基因异常低表达的表观遗传学调节机制及其临床意义。方法：所用组织标本均来自河北医科大学第四医院2008-2011 年行食管癌根治术并且病理诊断为ESCC患者的癌组织及相应癌旁组织（距癌灶边缘2 cm以上），共71 例。ESCC细胞株（Eca109、Kyse170、Yes-2）培养完成后，进行甲基化抑制剂5-Aza-dC 或组蛋白去乙酰化酶抑制剂TSA处理，qPCR和Western blotting 实验检测ESCC组织和细胞系中SHP-1 mRNA和蛋白的表达变化，亚硫酸氢盐基因组测序（bisulfite genome sequencing，BGS）法检测ESCC 细胞系中SHP-1 基因启动子区CpG 位点的甲基化频率，甲基化特异性PCR（methylation specific PCR，MSP）技术检测ESCC组织和细胞中SHP-1 启动子区的甲基化状态，应用双荧光素酶报告基因实验检测SHP-1 启动子区CpG岛甲基化对其转录活性影响。分析ESCC组织中SHP-1 甲基化状态分别与临床病理特征和SHP-1 mRNA表达的关系，对组织SHP-1甲基化水平与ESCC患者生存率进行Kaplan-Meier 生存分析和Log-Rank 检验。结果:5-Aza-dC 处理后，SHP-1 mRNA蛋白在3种细胞株中的表达显著上调（均P<0.05），同时其启动子区的甲基化程度均明显降低(均P<0.05）；应用TSA处理细胞株后，SHP-1在各细胞株中的表达情况及甲基化状态无明显改变(P>0.05）；甲基转移酶处理细胞荧光素报告载体活性显著低于未处理细胞荧光素报告载体活性（P<0.05），表明SHP-1 的甲基化可抑制自身的转录。ESCC组织中启动子区的甲基化率明显高于癌旁组织（P<0.05），并与TNM分期、病理分级及淋巴结转移密切有关(P<0.05）；与癌旁组织相比，ESCC组织中SHP-1 mRNA相对表达量显著降低（P<0.05），并与启动子区甲基化有关（P<0.05）；Kaplan-Meier 分析显示，启动子区高甲基化与ESCC患者的不良预后有关（P<0.05）。结论：ESCC组织和细胞株中SHP-1 基因启动子区高甲基化状态可抑制其自身的转录活性，进而导致该基因表达沉默；SHP-1的高甲基化与ESCC患者预后不良有关，SHP-1 的甲基化状态可能成为ESCC患者预后的评估指标。     

CD39 在头颈部鳞状细胞癌组织中的表达及其预后价值

目的：检测CD39 在头颈鳞状细胞癌（head and neck squamous cell carcinoma,HNSCC）组织中的表达,分析其表达与患者临床病理特征的关系及其预后意义。方法：选用2012 年5 月至2013 年12 月在天津市肿瘤医院接受外科手术的85 例HNSCC患者的组织标本及病例资料,Oncomine 数据库获取的基因芯片,以及HNSCC细胞系SCC15、UM1 和Cal25。在线分析CD39 在HNSCC组织与正常颊黏膜组织转录水平的差异性,用Western blotting 和免疫组化法检测HNSCC组织中CD39 蛋白的表达。采用Spearman’s 检验分析HNSCC组织中CD39 的表达与患者临床病理特征的相关性,Kaplan-Meier 曲线法和Log rank 检验分析HNSCC组织CD39 表达与生存的关系,Cox风险比例回归模型评价CD39 表达与复发风险的关系。结果：CD39 在HNSCC组织的转录水平显著高于正常颊黏膜组织（P<0.01）,其在HNSCC细胞Cal25、SCC-15 和UM1 中均有表达,UM1 细胞中CD39的表达呈地塞米松（dexamethasone, DXM）剂量依赖性。CD39 高表达患者53（62.4%）例,其高表达与术前化疗正相关（r=0.234,P<0.05）,CD39 高表达患者的无复发生存期较低表达组显著缩短（P<0.05）,CD39 高表达是HNSCC 复发的独立风险因素（HR=2.328,95%CI=1.091~4.967;P<0.05）。结论：CD39 在HNSCC中呈DXM诱导性表达和组成性表达,其在癌组织中过表达是HNSCC患者不良预后的独立预测因子,提示其在HNSCC进展过程中可能发挥重要作用。     

基于Oncomine和TCGA数据库分析GABRE在结肠癌组织中的表达及生物学意义

目的：通过挖掘Oncomine和TCGA等数据库信息分析GABRE基因在结肠癌中的表达及其生物学意义。方法：利用Oncomine、TCGA数据库分析GABRE基因在结肠癌组织中的表达及其与患者预后的关系;运用TargetScan、starBase、mirDIP和miRWalk寻找靶向GABRE基因的上游miRNA,并分析其在结肠癌中的表达及其与预后的关系。进一步利用LinkedOmics数据库寻找GABRE共表达基因,并进行GO富集分析及KEGG通路分析。结果：数据库数据分析显示,GABRE在结肠癌组织中高表达且预示着患者预后较差（均P<0.05）。韦恩图显示,hsa-miR-370-3p靶向GABRE,并在正常组织中的表达显著升高（P<0.01）。GABRE基因与OGT、FAM156A基因等表达呈正相关（P<0.05）,与ATP5A1、MPDU1基因等表达呈负相关（P<0.05）。GO生物功能及KEGG通路富集分析提示,GABRE基因可能参与蛋白脱烷基化和周期蛋白依赖性蛋白激酶活性调节等生物学过程,并在牛磺酸代谢和NF-κB信号通路等方面富集。结论：GABRE基因在结肠癌患者中高表达且预示着患者预后较差,提示该基因是结肠癌的诊断和治疗的潜在新靶点。     

基于TCGA数据库构建头颈部鳞癌RNA甲基化调控因子预后模型#br#

目的探讨RNA甲基化调控因子与头颈部鳞癌（HNSCC）患者预后的相关性。方法从TCGA数据库获取HNSCC样本与正常样本的基因表达谱及临床数据，评估43个RNA甲基化调控因子预测预后的价值。使用LASSO回归分析构建预后模型，将患者按风险评分划为高、低风险组，并对模型进行评估。根据高、低风险组差异表达的基因进行GO富集分析和ssGSEA分析。结果HNSCC组织中显著差异表达的RNA甲基化调控因子共有38个。LASSO回归模型中，高风险组的5年生存率低于低风险组（P＜0001）。单因素与多因素COX回归分析结果显示该评分模型是HNSCC患者独立的预后因素（P＜005）。GO富集分析和ssGSEA分析提示高风险组的免疫功能可能受到抑制。结论RNA甲基化调控因子在HNSCC的预后中发挥重要作用。     

基于生物信息学数据分析ATM在胃癌组织中的表达及其临床意义

目的：探讨幽门螺杆菌（Helicobacter pylori, Hp）感染对胃癌细胞共济失调毛细血管扩张突变（ataxia-telangiectasia mutated,ATM）基因表达的影响及其临床意义。方法：从TCGA数据库中获取胃癌相关RNAseq数据,比较ATM基因的表达差 异,分析ATM表达与患者临床病理参数的相关性及预后价值,用Kaplan-Meier法进行生存分析,LinkedOmics数据库分析ATM相 关基因,用R语言进行GO、KEGG富集分析。选用2019年3月至2019年12月贵州医科大学附属医院12例手术切除的胃癌及癌 旁组织标本,以及胃癌细胞系AGS和BGC823,用感染复数40∶1的Hp GZ7菌感染细胞,用免疫组织化学染色法检测胃癌组织中 ATM蛋白的表达,qPCR法检测胃癌组织和细胞中ATM mRNA的表达。结果：TCGA数据显示胃癌和Hp感染胃癌组织中ATM miRNA表达水平均显著高于癌旁组织（均P<0.01);胃癌组织中ATM miRNA表达与患者的T分期、AJCC分期等病理参数呈正相 关（均P<0.05),ATM 高表达时生存率显著降低(P<0.05)。实验检测显示,胃癌组织标本中ATM蛋白的表达水平明显高于癌旁 组织(P<0.01);Hp感染胃癌细胞中ATM miRNA 表达水平显著高于未感染胃癌细胞(P<0.01)。胃癌中ATM基因与NPAT等 12 461个基因呈正相关(P<0.05),与MIF等7 764个基因呈负相关(P<0.05)。GO、KEGG富集分析显示,ATM富集到DNA修复复 合体、癌症中的转录失调等信号通路。结论：ATM基因在胃癌组织中高表达,患者生存率随表达水平的增高而降低,其与患者的 T分期、AJCC分期等病理参数相关,且Hp感染引起ATM表达水平升高可能是Hp引起胃癌的原因之一。     

基于生物信息学数据分析ELTD1在肾透明细胞癌组织中的表达、甲基化水平及其临床意义

目的：探讨表皮生长因子蛛毒素受体7穿膜结构域蛋白1(ELTD1)在肾透明细胞癌（ccRCC）组织中的表达、甲基化水平及其与患者临床病理特征和预后的相关性。方法：通过公共数据库分析ccRCC组织中ELTD1表达和甲基化的差异水平,探讨ELTD1表达水平与患者临床病理特征和预后的相关性。通过TIMER2.0数据库评估ccRCC免疫细胞浸润,筛选ELTD1相关免疫检查点基因,进行GO功能和KEGG通路富集分析,通过基因共表达分析、筛选与ELTD1相关的基因。结果：与癌旁组织比较,ELTD1在ccRCC组织中呈高表达（P<0.05）。TCGA-KIRC队列中,ELTD1的甲基化水平与其表达呈负相关（R=-0.37,P<0.01）。ELTD1转录表达在ccRCC患者年龄、T分期、M分期、临床分期及病理分级组间存在显著差异（均P<0.01）,且高表达ELTD1与较长的OS和PFS密切相关（HR=0.55、0.63,均P<0.01）,ELTD1高表达是ccRCC的独立保护因素。ELTD1表达与B细胞、CD4+T细胞、CD8+T细胞、巨噬细胞和嗜中性粒细胞的免疫浸润呈显著负相关...     

m6A结合蛋白IGF2BP1在肝细胞癌中的基因调控网络分析

目的 分析m6A阅读器胰岛素样生长因子2-mRNA结合蛋白1（insulin-like growth factor 2 mRNA-binding protein 1，IGF2BP1）在肝细胞癌（hepatocellular carcinoma，HCC）中的表达水平及其对HCC患者预后的影响，并探讨IGF2BP1在HCC发生发展中的作用及其潜在机制。方法 基于5对HCC癌及相应癌旁组织mRNA-seq数据和TCGA数据库LIHC的mRNA-seq数据综合分析IGF2BP1在HCC中的表达情况，同时利用TCGA数据库中343例HCC患者的临床随访资料分析IGF2BP1表达水平对HCC患者总生存期的影响。基于TCGA数据库筛选IGF2BP1的共表达mRNA，并利用m6Avar在线网站预测mRNA的m6A位点及其RNA结合蛋白等信息，最终构建IGF2BP1的基因调控网络。结果 IGF2BP1基因在HCC中表达上调（log2FC HCC转录组数据=10.684，P<0.001；log2FC TCGA-LIHC数据集=7.032，P<0.001）。生存分析显示IGF2BP1低表达的HCC患者中位生存时间为5.84年，IGF2BP1高表达患者为4.44年，高表达患者总生存期缩短（P=0.011）。22个差异表达的mRNA与IGF2BP1存在靶向结合关系，并与其表达水平呈正相关。其中，HMGA2等15个高表达mRNA的HCC患者总生存期缩短。HMGA2、PEG10、CEP55、RHO、CDC6和KIF23基因中的潜在m6A甲基化位点位于mRNA 3'UTR端的miRNA结合区域。结论 IGF2BP1在HCC中高表达且导致患者总生存期缩短。IGF2BP1可能通过m6A甲基化及miRNA抑制作用的方式上调mRNA的表达，促进HCC发生并导致不良预后。     

肝癌增强子-miRNA调控作用对的识别与特征分析

目的：探讨肝癌和正常肝组织中增强子与miRNA形成的调控关系及调控miRNA的增强子的特征,筛选出由增强子调控的差异表达miRNA并探讨其与肝癌治疗靶点的相关性。方法：基于TCGA与FANTOM5 数据库,对肝癌和正常肝组织中共417 个样本的增强子与miRNA进行共表达与3D基因组分析得到调控关系。通过ENCODE数据库中肝癌与正常肝组织的组蛋白修饰与转录因子的ChIP-seq 数据分析调控miRNA的增强子上信号值的差异。筛选由增强子调控的差异表达的miRNA,并对患者的生存期与治疗靶点进行相关性分析。结果：在肝癌与正常肝组织中分别识别增强子-miRNA 作用对93 对和40 对。ChIP-seq 数据比对分析发现,肝癌中组蛋白修饰H3K27ac、H3K4me1 和H3K4me3 信号在调控miRNA增强子区域显著高于不调控miRNA的增强子区域（|rho|>0.3,P<0.05）;多种转录因子在肝癌相关增强子中的富集显著低于正常肝组织（|rho|>0.3,P<0.05）。对增强子调控的miRNA进行差异表达分析,识别了6个与肝癌患者生存相关miRNA（hsa-miR-4664、hsa-miR-5003、hsa-miR-1915、hsa-miR-3619、hsa-miR-4745、hsa-miR-6728）,发现这些miRNA 与87 个用于靶向治疗的基因以及8 个免疫检查点基因显著相关（|rho|>0.1,FDR<0.05）。结论：成功在肝癌中识别出增强子-miRNA调控作用对与调控miRNA的增强子的特征,并筛选出由增强子调控的与肝癌患者生存和治疗靶点相关的miRNA,为后续深入开展肝癌的基础与临床研究提供了参考依据。     

肝细胞癌中ChREBP基因CpG岛甲基化与mRNA表达的相关性

目的 检测肝细胞癌中ChREBP基因CpG岛甲基化水平及mRNA表达水平,并探讨两者的相关性。方法 收集肝细胞癌及癌旁冰冻组织90例,采用亚硫酸氢钠处理结合克隆测序检测ChREBP基因CpG岛内29个CpG位点的甲基化水平;采用荧光定量PCR检测其中31对新鲜冰冻组织中ChREBP基因mRNA表达水平,分析甲基化水平与mRNA表达之间的关系。结果 ChREBP基因的5、6、7、14号CpG位点甲基化水平在肝细胞癌中显著低于其配对的癌旁组织（P<0.05）。其余CpG位点及整体甲基化在肝细胞癌与癌旁组织中差异无统计学意义（均P>0.05）。15、18、20、23、26、29号CpG位点在≥50岁年龄组的甲基化水平均高于<50岁年龄组（均P<0.05）。肝细胞癌中ChREBP基因mRNA表达水平低于癌旁组织（P=0.003）,但与甲基化无显著相关性（P>0.05）。结论 肝细胞癌中ChREBP基因mRNA表达水平低于癌旁组织,但这一改变可能不是通过影响DNA的甲基化水平实现的。     

Ⅱ相代谢酶基因多态性与广西肝癌发生的关系

目的　探讨Ⅱ相代谢酶谷胱甘肽硫转移酶M 1、T 1(GSTM 1、GSTT1)及微粒体环氧化物水解酶 (mEH )基因多态性与广西肝癌易感性的关系 ,以及基因与基因间的相互作用。方法　采用多重PCR、PCR RFLP技术 ,对广西地区 10 5例肝癌患者及 15 1例健康对照的GSTM 1、GSTT 1、mEH基因型进行检测。结果　GSTM 1基因缺失率在病例组与对照组中分别为 64 .76%和5 0 .99% ,两者比较有显著性差异 (P <0 .0 5 ,OR =1.77) ;病例组GSTT 1基因缺失率 (4 0 .95 % )高于对照组 (3 3 .11% ) ,mEH 3种基因型频率在病例组分别为 2 7.62 %、2 1.90 %、5 0 .48% ,对照组则分别为 2 1.19%、3 4.44 %、44 .3 7% ,两组比较无显著性差异 (P >0 .0 5 ) ;GSTM 1、T 1基因同时缺失的个体患肝癌的危险性增大了 1.2 2倍。结论　GSTM 1、T1基因同时缺失是肝癌的易感因素 ,可作为肝癌高危人群筛选的标记物。     

Expression of seven-in-absentia homologue 1 and hypoxia-inducible factor 1 alpha: Novel prognostic factors of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an EBV-associated cancer. We analysed Siah1 expression as well as LMP1 and HIF1α expression by immuno-histochemical staining in 74 NPC biopsy specimens and found that the expression of Siah1 was significantly correlated with advanced tumour status and stage. Moreover, Siah1-positive and HIF1α-positive cases had significantly worse prognoses. The expression score for LMP1 was remarkably correlated with that of Siah1, whereas there was little correlation between LMP1 expression and the other markers evaluated. This is the first study to evaluate the pattern and clinical significance of Siah1 and HIF1α expression in NPC, and such an evaluation is valuable for identifying those patients at a high risk for a poor prognosis.     

Upregulated long non-coding RNA AFAP1-AS1 expression is associated with progression and poor prognosis of nasopharyngeal carcinoma

Altered expression of long noncoding RNAs (lncRNAs) associated with human carcinogenesis. We performed a cDNA microarray analysis of lncRNA expression in 12 cases of nasopharyngeal carcinoma (NPC) and 4 non-tumor nasopharyngeal epitheliums. One lncRNA, actin filament associated protein 1 antisense RNA1 (AFAP1-AS1), was identified and selected for further study. AFAP1-AS1 expression was upregulated in NPC and associated with NPC metastasis and poor prognosis. In vitro experiments demonstrated that AFAP1-AS1 knockdown significantly inhibited the NPC cell migration and invasive capability. AFAP1-AS1 knockdown also increased AFAP1 protein expression. Proteomic and bioinformatics analyses suggested that AFAP1-AS1 affected the expression of several small GTPase family members and molecules in the actin cytokeratin signaling pathway. AFAP1-AS1 promoted cancer cell metastasis via regulation of actin filament integrity. AFAP1-AS1 might be a potential novel marker that can predict cancer patient prognosis and as a potential therapeutic target for NPC.     

PDK-1 regulates lactate production in hypoxia and is associated with poor prognosis in head and neck squamous cancer

Here we describe the expression and function of a HIF-1-regulated protein pyruvate dehydrogenase kinase-1 (PDK-1) in head and neck squamous cancer (HNSCC). Using RNAi to downregulate hypoxia-inducible PDK-1, we found that lactate and pyruvate excretion after 16-48 h of hypoxia was suppressed to normoxic levels. This indicates that PDK-1 plays an important role in maintaining glycolysis. Knockdown had no effect on proliferation or survival under hypoxia. The immunohistochemical expression of PDK-1 was assessed in 140 cases of HNSCC. PDK-1 expression was not expressed in normal tissues but was upregulated in HNSCC and found to be predominantly cytoplasmic with occasional strong focal nuclear expression. It was strongly related to poor outcome (P=0.005 split by median). These results indicate that HIF regulation of PDK-1 has a key role in maintaining lactate production in human cancer and that the investigation of PDK-1 inhibitors should be investigated for antitumour effects.     

Methylation Profiles of BRCA1, RASSF1A and GSTP1 in Vietnamese Women with Breast Cancer

Objective: This study investigated the DNA promoter methylation profiles of BRCA1, RASSF1A and GSTP1 genes,both individually and in an integrative manner in order to clarify their correlation with clinicopathological parameters ofbreast cancer from Vietnamese patients, and establish new potential integrative methylation biomarkers for breast cancerdetection. Material and methods: The methylation frequencies of BRCA1, RASSF1A and GSTP1 were analyzed bymethylation specific polymerase chain reaction (MSP) in 70 specimens of breast carcinomas and 79 pairs of tumor andmatched adjacent normal tissues from breast cancer patients. Results: All the three analyzed genes showed a concordanceconcerning their promoter methylation in tumor and adjacent normal tissue. The methylation of BRCA1, RASSF1Aand GSTP1 was found in 58.23 %, 74.68 % and 59.49 % of tumor tissues and 51.90 %, 63.29 % and 35.44 % ofcorresponding adjacent tissues, respectively. When each gene was assessed individually, only the methylation ofGSTP1 was significantly associated with tumor tissues (p=0.003). However, the methylation frequency of at least one ofthe three genes and the methylation frequency of all the three genes both showed significant association with tumor(p=0.008 and p=0.04, respectively). The methylation of BRCA1 was found to be significantly associated with tumorgrade (p=0.01). Conclusion: This study emphasized that the panel of the three genes BRCA1, RASSF1A and GSTP1can be further developed as potential biomarkers in diagnosis and classification of breast cancer in Vietnamese women.     

大、小鼠摄入AFB_1和羧乙基锗倍半氧化物体内超氧化物歧化酶变化研究

本实验通过观察摄入ＡＦＢ１及Ｇｅ－１３２后Ｗｉｓｔａｒ大鼠和昆明小鼠体内ＳＯＤ活力的变化，进一步了解ＡＦＢ１代谢途径和Ｇｅ－１３２与自由基的关系。实验表明，摄入ＡＦＢ１的ＳＯＤ活力受到抑制，而摄入Ｇｅ－１３２后ＳＯＤ活力得到提高，且一次性摄入ＡＦＢ１后ＳＯＤ活力最终得到恢复，其中大鼠对ＡＦＢ１敏感性相对较大。     

新基因NBEAL1的克隆、表达、纯化及其与胶质瘤病理分级的相关性

目的：构建新基因neurobeachin like 1（NBEAL1）的表达载体,研究NBEAL1 的表达与胶质瘤病理分级的相关性。方法：提取人脑胶质瘤细胞U251的总RNA,构建pGEX-KG/NBEAL1表达载体,转化大肠杆菌BL21,IPTG诱导NBEAL1重组蛋白的表达,GST亲和纯化,Western blotting鉴定重组蛋白的纯度;以免疫组化法用纯化蛋白制备的单克隆抗体分析NBEAL1表达与胶质瘤病理分级的相关性。结果：NBEAL1基因片段被成功地克隆入pGEXKG表达载体,测序证实克隆片段序列正确。NBEAL1融合蛋白在大肠杆菌BL21包涵体中表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度95%以上。Western blotting证明所纯化的蛋白含有GST标签与NBEAL1肽段。免疫组化法检测显示NBEAL1蛋白的表达在正常脑组织中较低,而在低级别的脑胶质瘤组织中表达明显上调,但随着恶性程度增加NBEAL1的表达程度降低,两者呈负相关。结论：成功地克隆、表达及纯化NBEAL1蛋白,NBEAL1蛋白在人脑胶质瘤组织中的表达与其恶性程度呈负相关。     

Triplex Polymerase Chain Reaction with Confronting Two-Pair Primers (PCR-CTPP) for NQO1 C609T,GSTM1 and GSTT1 Polymorphisms: the Most Convenient Genotyping Method

The polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a time-saving and inexpensive ‍genotyping method, which is applicable for most single nucleotide polymorphisms (SNPs). To date, we have established ‍PCR-CTPP conditions for tens of SNPs, including duplex genotyping. This paper introduces triplex PCR-CTPP to ‍simultaneously genotype three functional polymorphisms of carcinogen-detoxifying enzymes, NQO1 C609T, GSTM1 ‍null, and GSTT1 null, all of which are reported to have a significant association with smoking-related cancers. We ‍applied this method for 241 non-cancer patients to demonstrate the performance. Among the subjects, the genotype ‍frequency of NQO1 C609T was 35.7% for CC, 44.4% for CT and 19.9% for TT. The null type frequencies of GSTM1 ‍and GSTT1 were 53.4% and 44.0%, respectively. Their distributions were similar to those reported for Japanese by ‍other studies. This is the first paper reporting the success of triplex PCR-CTPP. The polymorphisms applied are ‍useful examples, which could be adopted not only for research purposes, but also for risk assessment of individuals ‍exposed to carcinogenic substances, such as smokers. This convenient genotyping approach has advantages for ‍application in cancer prevention, especially in the Asian Pacific region.     

Multiplex PCR with Confronting Two-pair Primers for CYP1A1 Ile462Val,GSTM1, GSTT1, and NQO1 C609T

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an effective genotyping method ‍for single nucleotide polymorphisms (SNPs) in aspects of reducing time and costs for analysis. So far we have ‍established PCR-CTPP conditions for tens of SNPs, including a triplex genotyping (Kawase et al., 2003). In the ‍present study we report a quadruplex PCR-CTPP to genotype simultaneously four functional polymorphisms of ‍carcinogen-metabolizing enzymes, CYP1A1 Ile462Val, GSTM1 null, GSTT1 null and NQO1 C609T, which were ‍reported that they have significant associations with smoking-related cancers. We applied this method for 475 health ‍check-up examinees to demonstrate the performance. Among the subjects, the genotype frequency of CYP1A1 ‍Ile462Val was 56.8% for Ile/Ile, 38.1% for Ile/Val and 5.1% for Val/Val. The null type frequencies of GSTM1 and ‍GSTT1 were 52.8% and 49.9%, respectively. And the genotype frequency of NQO1 C609T was 41.9% for C/C, ‍41.3% for C/T and 16.8% for T/T. Their distributions were similar to those reported for Japanese by other studies. ‍To the best of our awareness, this is the first paper that reports the success in quadruplex PCR-CTPP. The applied ‍polymorphisms are useful ones, which would be adopted not only for research purposes, but also for risk assessment ‍of individuals exposed to carcinogenic substances. This convenient genotyping would be applied for cancer prevention ‍especially in Asian Pacific regions, where expensive genotyping methods are hardly available.