APC MUTATION ANALYSIS BY CHEMICAL CLEAVAGE OF MISMATCH AND A PROTEIN TRUNCATION ASSAY IN FAMILIAL ADENOMATOUS POLYPOSIS
APC MUTATION ANALYSIS BY CHEMICAL CLEAVAGE OF MISMATCH AND A PROTEIN TRUNCATION ASSAY IN FAMILIAL ADENOMATOUS POLYPOSIS
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DOI：
10.1038/bjc.1994.408
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发表时间：
1994-11-01
影响因子：
8.8
通讯作者：
DUNLOP, MG
中科院分区：
文献类型：
--
作者：
PROSSER, J;CONDIE, A;DUNLOP, MG
总的来说，在文献报道的研究中，只有30%的家族性腺瘤性息肉病（FAP）患者中发现了APC突变。为了确定可检测的APC突变的真实频率，我们开始彻底搜索APC的整个编码区的致病突变的10例患者与经典FAP从苏格兰激酶显示与5 q标记。采用错配分析的化学裂解作为初始筛选技术。通过直接DNA测序证实突变，并通过体外蛋白质合成测定显示产生提前终止密码子。突变导致一个过早的终止密码子，无论是通过碱基取代或移码被确定在9个家庭。虽然其余的亲属与基因内APC标记相关，在Z（max）= 0.0时的lodscore为1.69，但对先证者DNA、RNA和染色体扩散的进一步分析未能检测到任何异常。这是尽管采用单链构象多态性（SSCP）分析，异源双链分析，DNA测序，逆转录-聚合酶链反应（RT-PCR）分析剪接缺陷，蛋白质截短测试涵盖整个APC基因和荧光原位杂交染色体分析（FISH）。这些数据表明，这些FAP激酶中有90%具有APC突变，可通过化学裂解错配检测到，并且所采用的众多其他技术都不能检测其余亲属中的突变。本研究显示了APC体外蛋白截短试验的筛选价值。
Overall, the causative APC mutation has been identified in only 30% of the patients with familial adenomatous polyposis (FAP) who have been included in studies reported in the literature. In order to determine the true frequency of detectable APC mutations, we set out to search exhaustively the entire coding region of APC for causative mutations in ten patients with classical FAP from Scottish kindreds shown to be linked to 5q markers. Chemical cleavage of mismatch analysis was employed as the initial screening technique. Mutations were confirmed by direct DNA sequencing and shown to generate a premature stop codon by an in vitro protein synthesis assay. Mutations resulting in a premature stop codon either by base substitution or by frameshift were identified in nine families. Although the remaining kindred was linked to intragenic APC markers with a lodscore of 1.69 at Z(max) = 0.0, further analysis of DNA, RNA and chromosome spreads from the proband failed to detect any abnormality. This was despite employing single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, reverse transcription-polymerase chain reaction (RT-PCR) analysis for splicing defects, a protein truncation test encompassing the entire APC gene and fluorescent in situ hybridisation chromosome analysis (FISH). These data show that 90% of these FAP kindreds had APC mutations detectable by chemical cleavage of mismatch and that none of the numerous other techniques employed could detect the mutation in the remaining kindred. This study shows the value of screening the APC vitro protein truncation test.