Identification of genomic deletions of the APC gene in familial adenomatous polyposis by two independent quantitative techniques
Identification of genomic deletions of the APC gene in familial adenomatous polyposis by two independent quantitative techniques
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DOI：
10.1089/gte.2004.8.248
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发表时间：
2004-09-01
期刊：
影响因子：
--
通讯作者：
Nordling, M
中科院分区：
文献类型：
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作者：
Meuller, J;Kanter-Smoler, G;Nordling, M
导致家族性腺性息肉病 (FAP) 的 APC（腺性息肉病大肠杆菌）基因中的大量缺失无法通过常规突变检测技术轻易检测到。因此，我们开发了两种独立的定量方法来检测 APC 的大缺失，包括一个或多个外显子。在一个反应中进行多重连接依赖性探针扩增 (MLPA)，以对所有 APC 外显子拷贝数进行初始定量。随后，使用实时定量 PCR (QRT-PCR) 来验证 MLPA 反应中获得的结果。描述了经典 FAP 患者中整个 APC 基因缺失的鉴定。通过两种定量方法检测突变，并通过中期扩散的 FISH（荧光原位杂交）在染色体水平上进一步验证。此外，在两个明显不相关的家族中检测到覆盖 APC 基因外显子 11-13 的大缺失。通过长程 PCR 进一步验证和表征了该缺失。 MLPA 测试可确保对 APC 基因的大量缺失进行灵敏的高通量筛选，并且可以轻松地在 FAP 的诊断测试中实施。
Large deletions in the APC (adenornatous polyposis coli) gene, causing familial adenornatous polyposis (FAP), cannot easily be detected by conventional mutation-detection techniques. Therefore, we have developed two independent quantitative methods for the detection of large deletions, encompassing one or more exons, of APC. Multiplex ligation-dependent probe amplification (MLPA) is performed in one reaction for the initial quantification of all APC exon copy numbers. Subsequently, quantitative real-time PCR (QRT-PCR) is used to verify the results obtained in the MLPA reaction. The identification of a deletion of the whole APC gene in a patient with classical FAP is described. The mutation was detected with the two quantitative methods and further verified on chromosomal level by the use of FISH (fluorescence in situ hybridization) on metaphase spreads. Furthermore, a large deletion covering exons 11-13 of the APC gene was detected in two apparently unrelated families. This deletion was further verified and characterized with long-range PCR. The MLPA test ensures a sensitive high-throughput screening for large deletions of the APC gene and can easily be implemented in the diagnostic testing for FAP.