焦磷酸测序法检测结直肠癌患者K—ras基因外显子2第12和13密码子点突变  

Genotyping of K-ras exon 2 codons 12 and 13 mutations in colorectal cancer by pyrosequencing

作　　者：谢国化[1] 姚晓虹[2] 吴萍[3] 沈立松[1] 

机构地区：[1]上海交通大学医学院附属新华医院检验科,200092 [2]上海交通大学医学院附属新华医院病理科,200092 [3]上海交通大学医学院附属新华医院普外科,200092

出　　处：《中华检验医学杂志》2012年第7期585-592,共8页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（81101848）;上海市科委重点项目资助课题（10411950400）

摘　　要：目的探讨焦磷酸测序法检测结直肠癌患者肿瘤组织K．ras基因外显子2第12和13密码子点突变方法的临床应用价值。方法以已知K—ras基因突变的结直肠癌细胞株SW480、DLD-1和野生型细胞株HT．29DNA作为测序模板检验焦磷酸测序法的准确性。对含不同比例（2％、3％、5％、10％、20％、30％和50％）结直肠癌细胞株K．ras基因的DNA混合样本采用焦磷酸测序法进行基因突变率检测，并与Sanger测序结果平行进行Fisher精确检验比较，评价其灵敏度。同时用焦磷酸测序法检测分析30份临床结直肠癌患者石蜡包埋组织中K—ras基因第12和13密码子突变。结果当混合已知突变类型的结直肠癌细胞株K—ras基因的DNA样本突变DNA比例在5％和10％浓度时，Sanger测序法检出K—ras基因突变率分别为33．3％（4／12）和58．3％（7／12），焦磷酸测序法分别为91．7％（11／12）和100％（12／12），且2种方法检出K—ras基因突变率的差异有统计学意义（P〈0．05）。此外，用焦磷酸测序法从30例结直肠癌患者石蜡包埋组织标本中检出K—ra$基因外显子2第12和13密码子突变10例，均为杂合型突变，突变率为33．3％（10／30）。最常见的突变类型为G〉A转换[50％（5／10）]和G〉T颠换[（30％（3／10）]。结论焦磷酸测序法检测结直肠癌K—ras基因外显子2第12和13密码子突变具有敏感、准确的优点，可用于临床个体化治疗中肿瘤基因突变检测。（中华检验医学杂志．2012，35：585-592、Objective To investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical coloreetal cancer tissues. Methods Genomic DNA, extracted from K-ras mutant cell lines SW480 （homozygous, c. 35G 〉 T）, DLD-1 （heterozygous, c. 38G 〉 A） and wild-type HT-29, was first used as the sequencing template respectively to test the accuracy of pyrosequeneing methodology. The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2%, 3%, 5% , 10% , 20% , 30% and 50%, the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples. The pyrosequeneing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test. Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at eodons 12 and 13 on DNA isolated from a panel of 30 eoloreetal cancer samples derived from clinical formalin-fixed and paraffin embedded （FFPE） tissues. Results Cancer cell lines with known K-ras mutations （ SW480 and DLD-1 ） were readily detectable by pyrosequeneing-based analysis. When the proportions of mutant colorectal cancer cell line DNA were 5% and 10% content, the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3% （4/12） and 58. 3% （7/12） respectively, whereas the mutation rates detected by pyrosequencing- based assay were 91.7% （ 11/12 ） and 100% （ 12/12 ） respectively, there were significant differences between those two sequencing methodology （P 〈 O. 05 ）. Furthermore, we found 10 patients with K-ras exon 2 point mutations at codons 12 and 13 by pyrosequencing-based assay from 30 colorectal cancer FFPE tissues, the point mutation rate was 33.3% （ 10/30 ） and all of the mutations determined were heterozygous. The codon 12 was most frequently affected [30% （9/30）J. Mutations with the highest frequency wer

关 键 词：结直肠肿瘤 基因 ras 外显子 突变 序列分析 DNA 

分 类 号：R735.3[医药卫生—肿瘤]