Nigro J M, Takahashi M A, Ginzinger D G, Law M, Passe S, Jenkins R B, Aldape K
Department of Pathology, University of California-San Francisco, San Francisco, CA 94143, USA.
Am J Pathol. 2001 Apr;158(4):1253-62. doi: 10.1016/S0002-9440(10)64076-X.
The combined loss of chromosomes 1p and 19q has recently emerged as a genetic predictor of chemosensitivity in anaplastic oligodendrogliomas. Here, we describe a strategy that uses a novel method of real-time quantitative polymerase chain reaction, quantitative microsatellite analysis (QuMA), for the molecular analysis of 1p and 19q loss in oligodendrogliomas and oligoastrocytomas in archival routinely processed paraffin material. QuMA is performed on the ABI 7700 and based on amplifications of microsatellite loci that contain (CA)n repeats where the repeat itself is the target for hybridization by the fluorescently labeled probe. This single probe can therefore be used to determine copy number of microsatellite loci spread throughout the human genome. In genomic DNA prepared from paraffin-embedded brain tumor specimens, QuMA detected combined loss of 1p and 19q in 64% (21 of 32) of oligodendrogliomas and 67% (6 of 9) of oligoastrocytomas. We validate the use of QuMA as a reliable method to detect copy number by showing concordance between QuMA and fluorescence in situ hybridization at 37 of 45 chromosomal arms tested. These results indicate that QuMA is an accurate, high-throughput assay for the detection of copy number at multiple loci; as many as 31 loci of an individual tumor can be analyzed on a 96-well plate in a single 2-hour run. In addition, it has advantages over standard allelic imbalance/loss of heterozygosity assays in that all loci are potentially informative, paired normal tissue is not required, and gain can be distinguished from loss. QuMA may therefore be a powerful molecular tool to expedite the genotypic analysis of human gliomas in a clinical setting for diagnostic/prognostic purposes.
1p和19q染色体联合缺失最近已成为间变性少突胶质细胞瘤化疗敏感性的遗传预测指标。在此，我们描述了一种策略，该策略使用一种新型实时定量聚合酶链反应方法——定量微卫星分析（QuMA），用于对存档的常规处理石蜡材料中的少突胶质细胞瘤和少突星形细胞瘤进行1p和19q缺失的分子分析。QuMA在ABI 7700上进行，基于对包含（CA）n重复序列的微卫星位点的扩增，其中重复序列本身是荧光标记探针杂交的靶标。因此，这一单探针可用于确定遍布人类基因组的微卫星位点的拷贝数。在从石蜡包埋的脑肿瘤标本中制备的基因组DNA中，QuMA在64%（32例中的21例）的少突胶质细胞瘤和67%（9例中的6例）的少突星形细胞瘤中检测到1p和19q联合缺失。我们通过显示在45个测试染色体臂中的37个上QuMA与荧光原位杂交结果的一致性，验证了QuMA作为检测拷贝数可靠方法的实用性。这些结果表明，QuMA是一种准确、高通量的检测多个位点拷贝数的方法；在一次2小时的运行中，在96孔板上可对单个肿瘤的多达31个位点进行分析。此外，与标准的等位基因不平衡/杂合性缺失检测相比，它具有优势，因为所有位点都可能提供信息，不需要配对的正常组织，并且可以区分扩增和缺失。因此，QuMA可能是一种强大的分子工具，可在临床环境中加快对人类胶质瘤进行诊断/预后目的的基因分型分析。
