希腊家族性腺瘤性息肉病队列中APC基因突变筛查的变性高效液相色谱法评估
Evaluation of dHPLC in mutation screening of the APC gene in a Greek FAP cohort.
作者信息
Mihalatos Markos, Apessos Angela, Triantafillidis John K, Kosmidis Paris A, Fountzilas George, Agnantis Niki J, Yannoukakos Drakoulis, Nasioulas George
机构信息
Molecular Biology Research Center HYGEIA, Antonis Papayiannis, Kifissias Ave. & Erythrou Stavrou 4 Str., 15123 Maroussi, Athens, Greece.
出版信息
Anticancer Res. 2003 May-Jun;23(3B):2691-5.
BACKGROUND
Germline mutations in the APC gene predispose to colorectal adenomas leading to cancer in over 80% of patients. A multitude of mutations, dispersed throughout the gene, have been described. We wanted to evaluate the usefulness of denaturing high performance liquid chromatography (dHPLC) for mutation screening.
MATERIALS AND METHODS
Ten amplicons containing 14 mutations in the APC, previously identified by sequencing in 22 FAP patients, were analysed by dHPLC. dHPLC was also used to screen members of a family for a mutation identified in the proband.
RESULTS
We analysed 10 amplicons under a total of 59 temperatures. Successful results were obtained from 51 out of 59 tested temperatures (86.4%). In all cases a different heteroduplex-homoduplex pattern was obtained from mutant DNA in at least two of the temperatures. All 14 mutations identified by sequencing were also detected using dHPLC. Sequence analysis of a large family confirmed the dHPLC results.
CONCLUSION
Using dHPLC we detected all mutations previously identified by sequencing.
背景
APC基因的种系突变易导致结直肠腺瘤，超过80%的患者会发展为癌症。已描述了该基因中分布的多种突变。我们想评估变性高效液相色谱法（dHPLC）用于突变筛查的实用性。
材料与方法
对10个包含APC基因中14个突变的扩增子进行分析，这些突变先前在22例家族性腺瘤性息肉病（FAP）患者中通过测序鉴定，采用dHPLC进行分析。dHPLC还用于筛查一个家族中先证者所鉴定出的突变的成员。
结果
我们在总共59个温度下分析了10个扩增子。在59个测试温度中有51个（86.4%）获得了成功结果。在所有情况下，至少在两个温度下从突变DNA获得了不同的异源双链-同源双链模式。通过测序鉴定的所有14个突变也使用dHPLC检测到。对一个大家庭的序列分析证实了dHPLC结果。
结论
使用dHPLC我们检测到了先前通过测序鉴定的所有突变。
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