Kiss N B, Geli J, Lundberg F, Avci C, Velazquez-Fernandez D, Hashemi J, Weber G, Höög A, Ekström T J, Bäckdahl M, Larsson C
Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital-Solna, CMM L8:01; SE-171 76 Stockholm, Sweden.
Endocr Relat Cancer. 2008 Jun;15(2):609-21. doi: 10.1677/ERC-07-0285.
Pheochromocytomas and abdominal extra-adrenal paragangliomas are related to endocrine tumors of the sympathetic nervous system. Studies in animal models have shown that inactivation of the products of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene locus, p16INK4A and p14ARF, promotes the development of pheochromocytoma, especially in malignant form. The present study evaluated the involvement of CDKN2A in human pheochromocytomas and abdominal extra-adrenal paragangliomas from 55 patients. Promoter methylation was assessed using quantitative Pyrosequencing and methylation-specific PCR, and mRNA expression was measured by quantitative real-time PCR. For p16, western blot analysis and sequencing were also performed. succinate dehydrogenase complex subunit B (SDHB) sequencing analysis included extra-adrenal paragangliomas, all tumors classified as malignant, and cases diagnosed at 30 years or younger. The p16INK4A promoter was heavily methylated in a subset of paragangliomas, and this was significantly associated with malignancy (P<0.0043) and SDHB mutation (P<0.002). p16INK4A mRNA expression showed moderate suppression in malignant cases (P<0.05). In contrast, very little p14ARF promoter methylation was seen and there was no significant difference in p14ARF expression between tumors and normal samples. The p16 protein expression was reduced in 16 tumors, and sequence variations were observed in four tumors including the missense mutation A57V and the single nucleotide polymorphism (SNP) A148T. The results suggest that p16INK4A, and not p14ARF, is a subject of frequent involvement in these tumors. Importantly, hypermethylation of the p16INK4A promoter was significantly associated with malignancy and metastasis, and SDHB gene mutations. This finding suggests an etiological link and could provide a clinical utility for diagnostic purposes.
嗜铬细胞瘤和腹部肾上腺外副神经节瘤是与交感神经系统内分泌肿瘤相关的疾病。动物模型研究表明，细胞周期蛋白依赖性激酶抑制剂2A（CDKN2A）基因座的产物p16INK4A和p14ARF失活会促进嗜铬细胞瘤的发生发展，尤其是恶性嗜铬细胞瘤。本研究评估了55例患者的人嗜铬细胞瘤和腹部肾上腺外副神经节瘤中CDKN2A的参与情况。使用定量焦磷酸测序和甲基化特异性PCR评估启动子甲基化，并通过定量实时PCR测量mRNA表达。对于p16，还进行了蛋白质印迹分析和测序。琥珀酸脱氢酶复合物亚基B（SDHB）测序分析包括肾上腺外副神经节瘤、所有分类为恶性的肿瘤以及30岁及以下诊断的病例。p16INK4A启动子在一部分副神经节瘤中高度甲基化，这与恶性肿瘤（P<0.0043）和SDHB突变（P<0.002）显著相关。p16INK4A mRNA表达在恶性病例中显示出中度抑制（P<0.05）。相比之下，p14ARF启动子甲基化很少见，肿瘤与正常样本之间的p14ARF表达没有显著差异。16例肿瘤中p16蛋白表达降低，在4例肿瘤中观察到序列变异，包括错义突变A57V和单核苷酸多态性（SNP）A148T。结果表明，p16INK4A而非p14ARF经常参与这些肿瘤的发生。重要的是，p16INK4A启动子的高甲基化与恶性肿瘤、转移以及SDHB基因突变显著相关。这一发现提示了病因学联系，并可为诊断目的提供临床应用价值。
