Bias in detection of instability of the (C)8 mononucleotide repeat of MSH6 in tumours from HNPCC patients
Bias in detection of instability of the (C)8 mononucleotide repeat of MSH6 in tumours from HNPCC patients
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DOI：
10.1038/sj.onc.1204795
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发表时间：
2001-09-27
期刊：
影响因子：
8
通讯作者：
Morreau, H
中科院分区：
文献类型：
--
作者：
de Leeuw, WJF;van Puijenbroek, M;Morreau, H
最近，我们和其他人报道了 MSH6 外显子 5 中 (C)8 重复的不稳定性，作为 MSH6 种系突变携带者肿瘤中体细胞突变的优先目标。在这里，我们报告说，在 45% 的 MLH1、MSH2 和 MSH6 种系突变携带者肿瘤中，对 PCR 产物进行 DNA 测序分析，结果发现 MSH6 的 (C)8 重复序列没有发生序列变化，且电泳迁移率发生了变化。使用“标准”PCR引物，发现(C)8重复的不稳定频率较高（50-86%），但使用改良的PCR反向引物，在体外PCR扩增过程中通过Taq聚合酶完成腺嘌呤非模板添加的调节，在来自MLH1、MSH2和MSH6突变携带者的轮次中发现明显较低的不稳定频率（分别为6%、13%和40%）。此外，与MLH1、MSH2突变携带者相比，MSH6突变携带者肿瘤中(C)8重复序列不稳定的频率存在显着差异。这些结果可能对检测其他短单核苷酸重复的不稳定性具有重要意义，例如TGF β RII、BAX、IGFRII、PTEN、BRCA2。
Recently, we and others reported instability in the (C)8 repeat in exon 5 of MSH6 as a preferential target for somatic mutations in tumours from MSH6 germline mutation carriers. Here, we report that in 45% of tumours from MLH1, MSH2 and MSH6 germline mutation carriers no sequence change in the (C)8 repeat of MSH6 was found upon DNA sequencing analysis of PCR products with a shift in electrophoresis mobility. Using 'standard' PCR primers a high frequency of instability (50-86%) of the (C)8 repeat was found, but using a modified PCR reverse primer, accomplishing modulation of non-templated addition of adenine during in vitro PCR amplification by the Taq polymerase, a markedly lower frequency of instability was found in turnours from MLH1, MSH2 and MSH6 mutation carriers (6, 13 and 40%, respectively). Furthermore, a significant difference of the frequency of instability of the (C)8 repeat in tumours from MSH6 mutation carriers was found compared to MLH1, MSH2 mutation carriers. These results might have important implications for the detection of instability of other short mononucleotide repeats, e.g. TGF beta RII, BAX, IGFRII, PTEN, BRCA2.