Long-range PCR facilitates the identification of PMS2-specific mutations
Long-range PCR facilitates the identification of PMS2-specific mutations
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DOI：
10.1002/humu.20318
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发表时间：
2006-05-01
期刊：
影响因子：
3.9
通讯作者：
de la Chapelle, A
中科院分区：
文献类型：
--
作者：
Clendenning, M;Hampel, H;de la Chapelle, A
DNA错配修复基因“减数分裂后分离增加2”（PMS2）中的突变与遗传性非息肉病性结直肠癌（HNPCC； Lynch综合征）的易感性有关。一个高度同源的PMS2假基因大家族的存在使得以前对PMS2进行测序的尝试非常困难。在这里，我们描述了一种利用远程PCR作为优先扩增PMS2而不是假基因的新方法。其次，外显子特异性扩增，从稀释的远程产品使我们获得一个干净的序列，显示没有证据的假基因污染。该方法已被用于筛选一组患者，这些患者的肿瘤经免疫组化检测PMS2蛋白为阴性，且在MLH1基因内未显示任何突变。对30例结直肠癌和子宫内膜癌患者的PMS2基因进行测序，发现了10个新的序列变化以及17个先前发现的序列变化。总的来说，在41个家族中有11个检测到假定的病理突变。其中5个新突变为c.705+1G >t、c.736-741del6ins11、c.862_863del、c.1688G >t和c.2007-IG > a。我们得出结论，在选定的Lynch综合征和Lynch综合征样患者中检测PMS2突变是可行和可取的。
Mutations within the DNA mismatch repair gene, "postmeiotic segregation increased 2" (PMS2), have been associated with a predisposition to hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome). The presence of a large family of highly homologous PMS2 pseudogenes has made previous attempts to sequence PMS2 very difficult. Here, we describe a novel method that utilizes long-range PCR as a way to preferentially amplify PMS2 and not the pseudogenes. A second, exon-specific, amplification from diluted long-range products enables us to obtain a clean sequence that shows no evidence of pseudogene contamination. This method has been used to screen a cohort of patients whose tumors were negative for the PMS2 protein by immunohistochemistry and had not shown any mutations within the MLH1 gene. Sequencing of the PMS2 gene from 30 colorectal and I I endometrial cancer patients identified 10 novel sequence changes as well as 17 sequence changes that had previously been identified. In total, putative pathologic mutations were detected in 11 of the 41 families. Among these were five novel mutations, c.705+1G > T, c.736-741del6ins11, c.862_863del, c.1688G > T, and c.2007-IG > A. We conclude that PMS2 mutation detection in selected Lynch syndrome and Lynch syndrome-like patients is both feasible and desirable.