Orru Germano, Coghe Ferdinando, Faa Gavino, Pillai Sara, Manieli Cristina, Montaldo Caterina, Pilia Francesca, Pichiri Giuseppina, Piras Vincenzo, Coni Pierpaolo
Laboratorio Analisi Chimico Cliniche e Microbiologia, Azienda Ospedaliero-Universitaria di Cagliari, Cagliari, Italy.
Diagn Mol Pathol. 2010 Mar;19(1):1-8. doi: 10.1097/PDM.0b013e3181a23bd5.
BRAF is an oncogene that is commonly mutated in both melanomas and papillary thyroid carcinomas (PTCs). Usually, mutations in the codons 600 or 601 lead to constitutive activity in the Ras-mitogen-activated protein kinase pathway and, recently, the BRAF deletion was described as a relevant risk factor for loco-regional PTC lymph node metastasis. For these reasons, BRAF mutations may be considered a key genetic factor for the metastatic progression of PTC and also for other tumors such as melanoma and colon cancer and a new BRAF-specific therapeutic strategy was already suggested. In this report we describe the development of a rapid qualitative fluorescent real-time polymerase chain reaction assay designed for the detection of BRAF deletion using 2 specific molecular beacons. The assay is able to detect in a single tube the homozygous as well the heterozygous genotypes. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific molecular beacons, and the throughput of a multicolor fluorescence detection procedure. This technique, together with an earlier described real-time test specific for V600E and K601E will be useful for research and molecular diagnostic laboratories involved in the study of BRAF-related neoplasia.
BRAF是一种癌基因，在黑色素瘤和甲状腺乳头状癌（PTC）中普遍发生突变。通常，密码子600或601的突变会导致Ras-丝裂原活化蛋白激酶途径的组成性活性，最近，BRAF缺失被描述为PTC局部区域淋巴结转移的一个相关危险因素。基于这些原因，BRAF突变可能被认为是PTC以及其他肿瘤（如黑色素瘤和结肠癌）转移进展的关键遗传因素，并且已经提出了一种新的BRAF特异性治疗策略。在本报告中，我们描述了一种快速定性荧光实时聚合酶链反应检测方法的开发，该方法使用2种特异性分子信标来检测BRAF缺失。该检测方法能够在单个试管中检测纯合子以及杂合子基因型。该程序结合了聚合酶链反应的高灵敏度、等位基因特异性分子信标提供的特异性以及多色荧光检测程序的通量。这项技术，连同先前描述的针对V600E和K601E的实时检测方法，将对参与BRAF相关肿瘤研究的研究和分子诊断实验室有用。
