Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based detection of microsatellite instabilities in coding DNA sequences: A novel approach to identify DNA-mismatch repair-deficient cancer cells
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based detection of microsatellite instabilities in coding DNA sequences: A novel approach to identify DNA-mismatch repair-deficient cancer cells
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DOI：
10.1373/49.4.552
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发表时间：
2003-04-01
影响因子：
9.3
通讯作者：
Becker, CM
中科院分区：
文献类型：
--
作者：
Bonk, T;Humeny, A;Becker, CM
背景：DNA错配修复系统中的遗传缺陷导致微卫星重复单位的丢失或增加，通常被称为微卫星不稳定性(MSI)。关键基因编码区的MSI参与了DNA错配修复缺陷型癌症的发病，特别是与遗传性非息肉病性结直肠癌综合征(HNPCC)相关的癌症。因此，MSI分型越来越多地被用于指导HNPC的分子诊断。方法：我们使用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF-MS)来鉴定MSI+肿瘤病变相关基因编码序列中单核苷酸重复序列中的MSI。结果：MALDI-TOF-MS在肿瘤组织和MSI+结直肠癌细胞系的GART、AC1、TGFBR2、MSH3和MSH6基因中检测到MSI，而在MSI对照组织中未检测到MSI。与全长微卫星相比，通过分析代表插入或缺失的峰的单个光谱中的峰积分比，可以相对量化MSI。基于MALDI-TOF-MS的基因分型结果通过常规DNA测序和电泳法得到证实。结论：由于其可靠性高、运行时间短、成本低，该半定量方法是一种有效的替代方法，尤其适用于肿瘤病变中MSI的高通量诊断。(C)2003年美国临床化学协会。
Background: Inherited defects in the DNA mismatch repair system lead to increased loss or gain of repeat units in microsatellites, commonly referred to as microsatellite instability (MSI). MSIs in coding regions of critical genes contribute to the pathogenesis of DNA-mismatch repair-deficient cancers, particularly those associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). MSI typing is therefore increasingly used to guide the molecular diagnosis of HNPCC.Methods: We used matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify MSIs in mononucleotide repeats within the coding sequences of genes relevant to the pathogenesis of MSI+ neoplastic lesions. After a primer extension reaction of PCR products encompassing the microsatellites, the molecular masses of the extension products were determined by MALDI-TOF-MS.Results: MSIs were detected by MALDI-TOF-MS in the GART, AC1, TGFBR2, MSH3, and MSH6 genes in neoplastic tissues and MSI+ colorectal cancer cell lines but not in MSI- control tissues. The analysis of peak-integral ratios in a single spectrum of the peaks representing insertions or deletions compared with the full-length microsatellites allowed relative quantification of MSIs. MALDI-TOF-MS-based genotyping results were confirmed by conventional DNA sequencing and electrophoresis.Conclusions: Because of its reliability, short run times, and low costs, this semiquantitative procedure represents an effective alternative, in particular for diagnostic high-throughput typing of MSIs in neoplastic lesions. (C) 2003 American Association for Clinical Chemistry.