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目的探讨变性高效液相色谱(DHPLC)-测序技术在早期检测乳腺癌BRCA1基因突变的临床意义。方法用聚合酶链反应(PCR)扩增BRCA1基因,该扩增产物经琼脂糖凝胶电泳鉴定后,直接进行DHPLC分析;有峰型改变的样本作测序分析,以确认突变。结果从124例乳腺癌患者中共发现9种突变。包括7种错义突变,其中,1例Stop598,1例A807E合并E809L,1例A807E,2例Y856H,1例T967S,2例I1170F,1例R1203G;一种存在于一号内含子的突变,即IVS101-10 T>C 17例;一种2号外显子的5’非翻译区突变,即A118T 2例。此外,在乳腺癌标本和正常对照标本中都发现有几处常见的单核苷酸多态性位点。结论本研究确定了9种乳腺癌患者可能发生的突变类型,为今后临床应用DHPLC对高危人群BRCA1基因进行早期基因检测提供了9个位点。DHPLC-测序技术可快速、方便地区分带有碱基替换和小片段缺失的DNA片段,是一种可用于早期临床诊断乳腺癌BRCA1基因改变的高效、灵敏和操作简便的方法。
Objective To investigate the clinical significance of denaturing high performance liquid chromatography (DHPLC)-sequencing in the early detection of BRCA1 gene mutation in breast cancer. Methods The BRCA1 gene was amplified by polymerase chain reaction (PCR). The amplified product was identified by agarose gel electrophoresis and directly analyzed by DHPLC. The samples with the change of peak shape were sequenced to confirm the mutation. Results A total of 9 mutations were found in 124 patients with breast cancer. Including seven kinds of missense mutations, including one Stop598, one A807E with E809L, one with A807E, two with Y856H, one with T967S, two with I1170F and one with R1203G; a mutation in intron one , Ie IVS101-10 T> C17 cases; a 5 ’untranslated region mutation of exon 2, ie, A118T 2 cases. In addition, several common SNP sites were found in both breast and normal controls. Conclusion This study identified possible mutations in nine breast cancer patients and provided 9 sites for early detection of BRCA1 gene in high-risk population by clinical application of DHPLC. DHPLC-sequencing technology can quickly and conveniently distinguish DNA fragments with base replacement and small fragment deletion, which is an efficient, sensitive and easy-to-use method for early clinical diagnosis of BRCA1 gene mutation in breast cancer.