应用变性高效液相色谱检测31例家族性腺瘤性息肉病家系结肠腺瘤性息肉病基因突变

目的 应用变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)技术检测我国家族性腺瘤性息肉病(familial adenomatons polyposis,FAP)家系的结肠腺瘤性息肉病(adenoinatous pelyposis coli,APC)基因变异特征,研究其病因机制.方法 采集31个家系的先证者、患者和家系成员的外周血淋巴细胞,抽提DNA并以降落式PCR扩增APC基因各外显子和启动子.基因突变检测先由DHPLC进行筛选,发现异常峰者进行测序鉴定并TA克隆鉴定,结果与网络数据进行比对.结果 31个家系中共有15个家系检出了12种不同的突变类型,FAP家系APC基因的突变检出率为48.39%.发现了4种新的突变及3例不同的内含子突变.4个新的突变分别位于255、677、1192、1403密码子,均为移码突变.证明了DHPLC能检出APC基因的突变.在APC基因的突变中,移码突变占86.67%,无义突变占13.33%,说明移码突变是中国人APC基因突变的主要方式.在突变位点上,第15外显子突变最常见,约占86.67%.结论 FAP家系APC基因的突变检出率为48.39%,发现了4种新的导致蛋白编码改变的突变.证实中国人FAP家系中APC基因突变位点以第15外显子最常见,类型以移码突变为主.

Detection of adenomatous polyposis coli gene mutations in 31 familial adenomatous polyposis families by using denaturing high performance liquid chromatography

OBJECTIVE: To analyze the adenomatous polyposis coli (APC) gene mutations in familial adenomatous polyposis (FAP) in Chinese. METHODS: DNA was extracted from blood samples taken from 31 FAP families, and all exons of the APC gene were amplified with touch-down PCR. APC gene mutations were screened by denaturing high performance liquid chromatography followed by sequencing if abnormal profile was detected. RESULTS: Twelve categories of APC gene mutations were found in 15 FAP families (48.39%) including 4 novel mutations in coding region and 3 mutations in introns. The 4 novel mutations in coding region were frameshift mutations and located in codons 255, 677, 1192 and 1403 respectively. Most mutations were clustered in exon 15 of APC gene leading to frameshift and accounted for 86.67%. Others were nonsense mutations (13.33%). CONCLUSION: The mutation rate of the APC gene in this group of Chinese FAP families was about 48.39%, and 4 novel mutations were detected. Frameshift mutation was the major mutation type in Chinese FAP and mainly located in exon 15.