Urology Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran.
J Cell Physiol. 2020 Oct;235(10):6954-6968. doi: 10.1002/jcp.29591. Epub 2020 Feb 4.
Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.
循环游离 DNA（cfDNA）已被认为是一种诊断来源，可以追踪癌症中的遗传和表观遗传改变。我们旨在研究组织和患有甲状腺乳头状癌（PTC）和甲状腺结节的患者的游离循环 DNA 样本中 RASSF1 和 SLC5A8 基因启动子区域的除甲基化状态以外的突变。通过 ARMS-蝎实时聚合酶链反应方法在 57 例 PTC 和 45 例甲状腺结节病例中研究 BRAF 突变。通过甲基化特异性高分辨率熔解曲线分析分析 RASSF1 和 SLC5A8 启动子区域的甲基化状态。在 57 例 PTC 组织样本中发现 39 例（68.4％）存在 BRAF 突变，而在 33 例（49.1％）cfDNA 病例中检测到该突变。cfDNA 中 BRAF 突变在转移性和非转移性 PTC 病例之间的频率差异有统计学意义（33 例 PTC 病例中的 22 例与 34 例甲状腺结节样本中的 5 例）。在 cfDNA 和组织 DNA 中，SLC5A8 的三个启动子区域和 RASSF1 的近端启动子区域的甲基化水平在 PTC 和甲状腺结节病例之间均有显著差异。此外，这两个基因在组织 DNA 中的甲基化状态反映在 cfDNA 中观察到的甲基化状态。本研究证实 BRAF 突变更有利于鉴别甲状腺乳头状癌和甲状腺结节。另一方面，RASSF1 和 SLC5A8 基因近端启动子区域的高甲基化状态对这种鉴别具有更高的敏感性和更可接受的特异性。
