中国人结直肠癌患者碱基切割修复基因胚系突变及与腺瘤样息肉基因体细胞突变的关系研究

The study of the relationship between human mutY homologue germline mutations in colorectal cancers and adenomatous polyposis coli somatic mutations in Chinese population

取42例经过病理确认的结直肠癌患者癌组织及癌旁正常组织,抽提基因组DNA,应用单链构象多态性分析(SSCP)、变性高效液相色谱(DHPLC)分析,结合DNA直接测序,在全基因分析humanmutY homologue(MYH)基因胚系突变的同时,探讨其对结直肠癌细胞adenomatous polyposis coli(APC)基因体细胞突变的影响.结果42例患者中检出6例(14.29%)携带MYH基因的胚系突变,其中3例(7.14%)为MYH基因第2外显子单体型突变c.53C>T/c.74G>A(p.Pro18Leu/p.Gly25 Asp),3例(7.14%)为第12外显子的单碱基替换导致的错义突变c.972G>C(p.Gln324His).初步分析显示,这两种突变在正常对照组的检出较低,仅为3/213(1.41%)和0/59(0%).另一方面4/6例携带MYH基因胚系突变患者的癌组织样本中检出5个APC基因体细胞突变.本文结果提示,散发性结直肠癌患者中频繁检出MYH基因的胚系突变,其存在可能导致细胞DNA氧化损伤修复功能减弱,并使机体细胞APC基因发生突变的风险增高,从而可能参与部分结直肠癌的发生.

Colorectal cancer（CRC） is the 5th top cause of malignant tumor deaths in Chinese population.As familial autosomal dominant for main incidence model,CRC may be caused by multiple gene mutations,for example,adenomatous polyposis coli（APC） gene mutations.However,a variety of recent researches revealed that there was also autosomal recessive genetic model in hereditary CRC,such as two germline mutations of human mutY homologue（MYH） gene existed in part of familial adenomatous polyposis（FAP） patients without APC germline mutations detected in European population.Based on these results,the aim of our study is to detect the germline mutations of the MYH gene in the CRC patients and to explore the relationship with the APC somatic mutations in cancer tissues.Genomic DNA was extracted from the tumor tissue cells and the adjacent normal tissue cells of 42 CRC patients.The diagnosis of CRC had been confirmed by histopathology.According to the structure characteristics of MYH gene,eleven pairs of primers were designed to amplify sixteen exons and some nearby introns in MYH gene.Amplification of the DNA fragment in MYH gene was performed by polymerase chain reaction（PCR） and all PCR products were identified by agarose gels.MYH gene was screened for mutations using single-strand conformation polymorphism（SSCP）,denaturing high-performance liquid chromatography（DHPLC） and DNA sequencing.MYH germline mutations were detected in 6/42 colorectal cancers（14.29%）,including the mutation c.53C〉T/c.74G〉A（p.Pro18Leu/p.Gly25Asp） in exon two in three patients（7.14%）and c.972G〉C（p.Gln324His）in exon twelve in other three ones（7.14%）.However the frequencies of these two kinds of mutations in normal control group were distinctly low,3/213（1.41%）and 0/59（0%） respectively.Among these six CRC patients carrying the germline mutation in MYH gene,five APC somatic mutations had been detected in the cancer tissues of 4 patients during our preliminary research.We considered that the germline mutations of MYH gene frequently detected in sporadic colorectal cancer patients might affect its function of the repair of oxidative DNA damage,and result in the somatic mutations of APC gene,and could play an important role in tumorigenesis of some CRC patients.Therefore,the mutation analysis of MYH gene in CRC may be important for early prevention of these disorders.