Dote Hideaki, Tsukuda Kazunori, Toyooka Shinichi, Yano Masaaki, Pass Harvey I, Shimizu Nobuyoshi
Department of Cancer and Thoracic Surgery, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Oncol Rep. 2004 Feb;11(2):361-3.
BRAF encodes a RAS-regulated serine/threonine kinase that mediates the pathway for cell growth and malignant transformation. Point mutations of BRAF were reported recently in 66% of melanomas, over 30% of thyroid papillary and low-grade ovarian cancers, and a smaller percentage of other human cancers. Mutations in malignant cells were reported to occur only in exons 11 and 15. Among these mutations, BRAF V599E is most frequent and proved to invert its transcript to the dominant active form. To exclude the interference of co-existing normal cells in clinical samples, we developed a new enriched PCR-RFLP assay for detecting mutations of BRAF codon 599 mutation. The sensitivity of this assay was examined to find that one mutant allele among 10(2) wild-type alleles could be detected. We applied this method for 53 cases of primary malignant pleural mesotheliomas (MPMs) and 6 cell lines and found no mutations in these samples. Our results demonstrate that the developed enriched PCR-RFLP is a sensitive assay to detect BRAF codon 599 mutation. However, it may be a rare type of mutation in MPMs. Our new assay is useful and can be applied for screening of BRAF codon 599 mutation in various kinds of clinical samples.
BRAF编码一种RAS调节的丝氨酸/苏氨酸激酶，该激酶介导细胞生长和恶性转化途径。最近报道，66%的黑色素瘤、超过30%的甲状腺乳头状癌和低级别卵巢癌以及较小比例的其他人类癌症中存在BRAF点突变。据报道，恶性细胞中的突变仅发生在外显子11和15中。在这些突变中，BRAF V599E最为常见，并被证明可将其转录物转化为显性活性形式。为了排除临床样本中共存正常细胞的干扰，我们开发了一种新的富集PCR-RFLP检测方法，用于检测BRAF密码子599突变。检测了该检测方法的灵敏度，发现可以检测到10(2)个野生型等位基因中的一个突变等位基因。我们将该方法应用于53例原发性恶性胸膜间皮瘤（MPM）和6个细胞系，发现这些样本中无突变。我们的结果表明，开发的富集PCR-RFLP是检测BRAF密码子599突变的灵敏检测方法。然而，它在MPM中可能是一种罕见的突变类型。我们的新检测方法很有用，可用于各种临床样本中BRAF密码子599突变的筛查。
