摘要
目的
近年来，嗜铬细胞瘤的分子遗传学研究取得了重要进展。在家族性嗜铬细胞瘤中，原癌基因RET(Rearranged during Transfection)点突变和抑癌基因VHL(von Hippel-Lindau)的失活可能分别为多内分泌腺瘤病2型(multiple endocrine neoplasia type 2，MEN 2)和VHL病的致病因素，而单纯家族性嗜铬细胞瘤中RET和VHL基因的突变情况尚不明确。散发性嗜铬细胞瘤发病的分子机制，可能涉及原癌基因的激活、抑癌基因的缺失以及凋亡障碍和端粒酶活性增强等诸多因素。本研究旨在通过检测原癌基因RET和抑癌基因VHL在家族性及散发性嗜铬细胞瘤中的种系突变情况，探讨二者在嗜铬细胞瘤发生中的遗传基因变化。
方法
1、选取2000年至2006年间北京协和医院经手术和病理确诊的嗜铬细胞瘤患者57例，其中MEN—2A型5例，MEN—2B型1例，散发性嗜铬细胞瘤51例。在散发性嗜铬细胞瘤中，肾上腺嗜铬细胞瘤29例，肾上腺外副神经节瘤22例；恶性嗜铬细胞瘤21例，良性嗜铬细胞瘤30例。正常健康受试者16例作为对照组。
2、选取6个家族性嗜铬细胞瘤家系，共29例受检查者，其中包括11名患者，一级亲属13例，二级亲属2例，三级亲属3例(均以先证者为参照对象)。
3、提取上述受试者外周血DNA，采用多聚酶链式反应(polymerase chain reaction，PCR)技术对RET exons 10，11，13，14，15,16进行扩增，PCR产物经纯化后直接测序，以明确其是否存在点突变。
结果
1、在51例散发性嗜铬细胞瘤患者中，4例(4／21，19.1％)恶性嗜铬细胞瘤，4例(4／30，13.3％)良性嗜铬细胞瘤发生RET基因第11外显子的点突变(G1y691Ser)，总突变率为15.7％(8／51)。且恶性嗜铬细胞瘤突变发生率高于良性嗜铬细胞瘤，差异有统计学意义(P=0.036)。
2、在51例散发性嗜铬细胞瘤患者中未发现VHL基因的点突变。
3、在6个家族性嗜铬细胞瘤家系中，家系1的17例受检者，分别检出2例患者(2／2)、10例一级亲属(10／13)、1例二级亲属(1／2)发生RET exon 11的点突变(G1y691Ser)；家系3中仅有1例患者检出RET exon11的点突变(G1y691Ser)。在家族性嗜铬细胞瘤11例患者中，有3例发生RET基因的点突变，突变率为27.3％(3／11)。
4、在6个家族性嗜铬细胞瘤家系中，家系4的2例受检者中1例患者被发现有VHL exon3点突变(712C→T，Arg→Trp)，另1例为一级亲属，未发现基因突变；家系6的3例患者发生VHL exon3点突变(695G→A，Arg→Gln)，余3例为亲属，未发现基因突变。在家族性嗜铬细胞瘤11例患者中，VHL基因的点突变率为45.5％(5／11)。
结论
1、散发性嗜铬细胞瘤中，RET基因的点突变率为15.7％，其中恶性嗜铬细胞瘤高于良性嗜铬细胞瘤；未发现VHL基因的种系突变。
2、家族性嗜铬细胞瘤患者中VHL基因的突变率为45.5％，RET基因的点突变率为27.3％。
3、对于嗜铬细胞瘤患者宜行RET和VHL基因的点突变筛查，有助于临床分型及预后的判断；对有家族史的嗜铬细胞瘤患者的一、二级亲属应行RET和VHL基因的点突变筛查，并对发生突变者进行随访。
研究目的
von Hippel-Lindau基因是一种抑癌基因，定位于3p26-25。现已明确其与VHL病密切相关，约70％的VHL病患者携带有VHL基因的种系突变，但其种系突变在散发性嗜铬细胞瘤中比较少见。在我们前面对51例散发性嗜铬细胞瘤RET和VHL基因突变的检测中亦未发现VHL基因的种系突变，因此本研究的目的是明确抑癌基因VHL的体细胞突变在散发性嗜铬细胞瘤发生情况，并比较良、恶性嗜铬细胞瘤间突变率是否有差别，进而探讨其临床意义。
研究方法
1、选取2000-2006年北京协和医院诊治的40例嗜铬细胞瘤患者，取其外周血及相应嗜铬细胞瘤肿瘤组织。其中，散发性嗜铬细胞瘤34例(位于肾上腺者20例，肾上腺外者14例)，MEN-2A型4例，VHL病2例。
2、采用多聚酶链式反应(polymerase chain reaction，PCR)方法，对嗜铬细胞瘤肿瘤组织DNA分别扩增VHL基因exon1，2，3，并直接测序，明确有无基因突变。
3、选用相应的微卫星Marker，采用杂合缺失(loss of heterozygosity，LOH)检测技术，对嗜铬细胞瘤组织是否发生3号染色体短臂某一片段的缺失及微卫星不稳定性(Microsatellite instability，MSI)进行检测，包括对VHL基因杂合缺失的检测。
4、运用统计学方法进一步明确抑癌基因VHL的体细胞突变与嗜铬细胞瘤的良、恶性及肿瘤的部位是否相关。
研究结果
1、在40例嗜铬细胞瘤患者中，有16例分别发生了第3号染色体短臂上1-5个位点的杂合缺失，突变率为40％；在34例散发性嗜铬细胞瘤患者中，有15例发生了上述某个位点杂合缺失，突变率为44％；3p26.1-24有杂合缺失，与恶性散发性嗜铬细胞瘤相关(P=0.044)；与肿瘤分泌高浓度的去甲肾上腺素(NE)和肾上腺素(E)相关(NE∶P=0.022；E∶P=0.048)。
2、在40嗜铬细胞瘤患者中，有8例散发性嗜铬细胞瘤观察到VHL基因的杂合缺失，突变率为23.6％(8／34)，其中肾上腺嗜铬细胞瘤6例(6／8，75％)，副神经节瘤2例(2／8，25％)；恶性嗜铬细胞瘤3例(3／8，37.5％)，良性嗜铬细胞瘤5例(5／8，63.5％)。在4例MEN-2A及2例VHL病中，未发现VHL基因的杂合缺失。
结论
在散发性嗜铬细胞瘤中3p26.1-24发生较高频率的杂合缺失，突变率为44％；3p26.1-24杂合缺失的发生可能与恶性嗜铬细胞瘤的发病相关，因而可能成为鉴别嗜铬细胞瘤良恶性的分子遗传学指标之一。
目的
缺氧诱导因子是哺乳动物和人体内细胞存在着的一类介导低氧适应性反应的转录因子。在恶性肿瘤中，它的活性在维持肿瘤的能量代谢、新生血管生成、促成肿瘤增值和转移中起作用。常氧下HIF-1α的降解正是通过pVHL—泛素蛋白酶途径来实现的。本研究的目的是通过对发生von Hipple-Lindau(VHL)基因杂合缺失(loss of heterozygosity，LOH)的嗜铬细胞瘤组织进行免疫组化研究，明确其von Hipple-Lindau蛋白(pVHL)、缺氧诱导因子—1α(hypoxia-inducible factor 1α，HIF-1α)、缺氧诱导因子—2α(hypoxia-inducible factor 2αHIF-2α)及血管内皮生长因子(Vascular endothelial growth factor，VEGF)蛋白表达的变化情况，并对其发生途径作初步探讨。
方法
1、选取2000-2006年在北京协和医院经手术病理证实的40例嗜铬细胞瘤患者，取其嗜铬细胞瘤肿瘤组织。其中，散发性嗜铬细胞瘤34例(位于肾上腺者20例，肾上腺外者14例)，MEN-2A型4例，VHL病2例。正常肾上腺髓质6例。
2、链霉素抗生物素蛋白-过氧化物酶复合物法(SP法)用于HIF-1α，HIF-2α，pVHL、VEGF染色。
3、HIF-1α，HIF-2α，pVHL、VEGF采用半定量计数法进行评分，以染色强度和阳性细胞百分率的评分之和进行判定，0～3为低表达，4～6为高表达。比较正常肾上腺髓质及嗜铬细胞瘤各组之HIF-1α，HIF-2α，pVHL、VEGF间的高、低表达率区别，并比较VHL基因LOH与上述四种蛋白表达的关系。
结果
1、发生VHL基因杂合缺失的肿瘤组织中pVHL的表达低于未发生杂合缺失者和正常肾上腺髓质，差异有统计学意义(P＜0.05)；
2、发生VHL基因杂合缺失的肿瘤组织中HIF-1α的表达高于未发生杂合缺失者，差异有统计学意义(P＜0.05)；且两组HIF-1α的表达均高于正常肾上腺髓质(P＜0.05)；
3、嗜铬细胞瘤中HIF-2α和VEGF的表达均高于正常肾上腺髓质(P＜0.05)，但在嗜铬细胞瘤中发生VHL基因杂合缺失者与未发生杂合缺失者之间二者表达无统计学差异。
结论
在散发性嗜铬细胞瘤中VHL基因的杂合缺失导致pVHL表达减少，从而使HIF-α蛋白蓄积，VEGF表达增加是其可能的发病机制之一，该机制的发生途径主要是通过HIF-1α介导。
OBJECTIVES :
Phaeochromocytomas may occur sporadically, or as part of the inherited cancersyndromes multiple endocrine neoplasia (MEN) type 2, von Hippel-Lindau disease(VHL), and, rarely, in type 1 neurofibromatosis. In MEN 2, germline missensemutations have been found in one of eight codons within exons 10, 11, 13, 14, and 16of the RET proto-oncogene. In VHL, germline mutations within one of the threeexons are responsible for the majority of cases. To determine if mutations similar tothose seen in the germline in MEN 2 or VHL disease play a role in the pathogenesisof sporadic or familial phaeochromocytomas.
METHODS:
We analysed 51 sporadic phaeochromocytomas and 6 familialphaeochromocytomas for mutations in RET exons 10, 11, 13, 14, 15, and 16, and theentire coding sequence of VHL. Genomic amplicons encompassing exons 10, 11, 13,14, 15, and 16 of the RET proto-oncogene and the entire coding sequence of VHLwere created by the polymerase chain reaction (PCR). PCR products were purifiedand then were analysed by direct sequence analysis.
RESULTS:
Eight of 51 sporadic phaeochromocytomas had RET mutations within exons 11,and which were composed of 4 patients with adrenal phaeochromocytomas and 4patients extra-adrenal phaeochromocytomas. In 8 patients, there were 4 patients withmalignant phaeochromocytomas. In the family, there were 13 cases had RETmutations within exons 11. No VHL mutations were detected in sporadicphaeochromocytomas.And there were 2 families which had point mutation of VHLgene.
CONCLUSION:
The ratio of mutation in the RET proto-oncogene was 15.7% in sporadicpheochromocytomas.And the ratio was higher in malignant pheochromocytomas thanin benign tumors. The mutations of RET and VHL should be detected in patients andtheir first relatives.
OBJECTIVES:
To evaluate whether somatic inactivation of the VHL gene plays a role in sporadicpheochromocytomas.
METHODS:
We examined 34 sporadic pheochromocytomas and 6 familial pheochromocytomasfor loss of heterozygosity (LOH) in chromosome 3p using a microdissectiontechnique. And the von Hippel-Lindau gene locus was analysed. DNA of the tumorwas eatracted , and the entire coding sequence of VHL were created by thepolymerase chain reaction (PCR). PCR products were purified and then were analysedby direct sequence analysis.
RESULTS:
1.16 of 40 pheochromocytomas cases showed loss of heterozygosity on the shortarm of chromosome 3.Significant deletions were found at 3p26.1-24 in malignantpheochromocytomas. And the level of NE and E were higher in tomous which showedloss of heterozygosity than those which did not show loss of heterozygosity.
2. 8 of 34 sporadic pheochromocytomas cases showed loss of heterozygosity oftumor suppressor gene VHL, in which 2 cases were extra-adrenal paragangliomas.
CONCLUSION:
We found significant deletions on the short arm of chromosome 3 in sporadicpheochromocytomas. This suggests that tumor suppressor gene, including VHL geneappears to play a role in the development of sporadic pheochromocytomas.
OBJECTIVES:
Pheochromocytoma are catecholamine-producing neuroendocrine tumors arisingfrom chromaffin cells of the adrenal medulla or extra-adrenal paraganglia. Nearly90% of pheochromocytoma are sporadic forms. The mechanism of sporadicpheochromocytoma is not known yet. Therefore, we investigate VEGF,pVHL,HIF-1αand HIF-2αexpression in the tumors which had loss of heterozygosity in VHL geneby immunohistochemical method, combining with the tumors which had no loss ofheterozygosity in VHL gene, to explore the significance of above factors in thegrowth and development of sporadic pheochromocytoma.
METHODS:
1. The specimens of 40 pheochromocytomas operated from 1990 to 2005 wereobtained from PUMC, including 34 cases of sporadic forms (benign 27,malignant9 cases ), 6 cases of familial forms.
2. Immunohistochemistry methods: Envision two steps is used for VEGF, pVHL,HIF-1αand HIF-2αstaining.
3. Semi-quantitative point scores for VEGF, pVHL,HIF-1αand HIF-2αstaining:Calculated as the percentage and intensity of staining: low expression was definedas 0～3 point scores,and high expression was 4～6 point scores.
4. Compare the difference of VEGF, pVHL, HIF-1αand HIF-2αexpresion amongnormal adrenal gland and pheochromocytomas.
RESULTS:
1. pVHL expression was lower in PHEO which had LOH of VHL gene than that ofwhich had no LOH of VHL gene (P＜0.05) .
2. HIF-1αexpression was hgher in PHEO which had LOH of VHL gene than that ofwhich had no LOH of VHL gene (P＜0.05) .
3. HIF-2αand VEGF expression was hgher in PHEO than normal adrenal glandtissue (P＜0.05) .
CONCLUSION:
These results suggest that somatic alterations of the VHL gene may play a role inthe tumorigenesis of some sporadic pheochromocytomas by decreasing the expressionof pVHL and increasing the expression of HIF-1α.
引文
1. Shuin T, Ashida S, Yao M et al. Von Hippel-Lindau disease. Nippon Rinsho, 2000,58: 1448-1454.
2. Smith DP, Houghton C, Ponder BA. Germline mutation of RET codon 883 in two cases of de novo MEN 2B. Oncogene. 1997,15: 1213-1217.
3. Beldjord C, Desclaux-Arramond F, Raffin-Sanson M The RET protooncogene in sporadic pheochromocytomas: frequent MEN 2-like mutations and new molecular defects. J Clin Endocrinol Metab. 1995 Jul; 80(7): 2063-8.
4. Komminoth P, Roth J, Muletta-Feurer S RET proto-oncogene point mutations in sporadic neuroendocrine tumors. J Clin Endocrinol Metab. 1996 Jun;81(6): 2041-6.
5. Chew SL Absence of mutations in the MEN2A region of the ret proto-oncogene in non-MEN 2A phaeochromocytomas. Clin Endocrinol (Oxf). 1995 Jan; 42(1): 17-21.
6. Brauch H, Hoeppner W, Jahnig H Sporadic pheochromocytomas are rarely associated with germline mutations in the vhl tumor suppressor gene or the ret protooncogene. J Clin Endocrinol Metab. 1997 Dec; 82(12): 4101-4.
7. Bar M, Friedman E, Jakobovitz O et al. Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel-Lindau and RET genes. Clin Endocrinol (Oxf), 1997,47: 707-712.
8 Gimenez-Roqueplo AP, Favier J, Rustin P, Rieubland C et al. Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas. Cancer Res. 2003 Sep 1;63(17):5615-21.
9 Kitamura K, Kangawa K, Kawamoto M, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun. 1993; 192: 553-560.
10 Kitamura K, Kangawa K, Kojima M, et al. Complete amino acid sequence of porcine adrenomedullin and clining of cDNA encoding its precursor. FEBS Lett. 1994; 338: 306-10.
11 Ishimitsu T, Kojima M, Kangawa K, et al. Genomic structure of human adrenomedullin gene. Biochem Biophys Res Commun. 1994; 203: 631-9.
12 Hay DL, Smith DM, Adenomedullin receptors: molecular identity and function. Peptides. 2001; 22: 1753-63.
13 Kobayashi H, Yamamoto R, Kitamura K, et al. Selective inhibition of nicotiniccholinergic receptors by proadrenomedullin N-terminal 12 peptide in bovine adrenal chromaffin cells. Brain Res Mol Brain Res. 2001; 87(2): 175-83.
14 Gagel RF. ret protooncogene mutations and endocrine neoplasia-a story ntertwined with neural crest differentiation. Endocrinology. 1996 May; 137(5): 1509-11.
15 Hoff AO, Cote GJ, Gagel RF. Multiple endocrine neoplasias. Annu Rev Physiol. 2000;62:377-411.
16 Takahashi M, Buma Y, Iwamoto T et al. Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene. 1988 Nov;3(5):571-8.
17 Takahashi M, Buma Y, Hiai H. Isolation of ret proto-oncogene cDNA with an amino-terminal signal sequence. Oncogene. 1989 Jun;4(6):805-6.
18 Robledo M, Gil L, Pollan M, et al Polymorphisms G691S/S904S of RET as genetic modifiers of MEN 2A. Cancer Res. 2003 Apr 15;63(8): 1814-7.
19 Zedenius J, Wallin G, Hamberger B, et al Somatic and MEN 2A de novo mutations identified in the RET proto-oncogene by screening of sporadic MTC:s. Hum Mol Genet. 1994 Aug;3(8): 1259-62.
20 Hofstra RM, Landsvater RM, Ceccherini I,et al A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature. 1994 Jan 27;367(6461):375-6.
21 Komminoth P, Kunz E, Hiort O,et al Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing. Am J Pathol. 1994 Oct;145(4):922-9.
22 Eng C, Smith DP, Mulligan LM, et al. Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Hum Mol Genet. 1994 Feb;3(2):237-41.
23 Lindor NM, Honchel R, Khosla S, et al. Mutations in the RET protooncogene in sporadic pheochromocytomas. J Clin Endocrinol Metab. 1995 Feb;80(2):627-9.
24 Yoshimoto K, Tanaka C, Hamaguchi S, et al. Tumor-specific mutations in the tyrosine kinase domain of the RET proto-oncogene in pheochromocytomas of sporadic type. Endocr J. 1995 Apr;42(2):265-70.
1. Hofstra RM, Stelwagen T, Stulp RP, et al Extensive mutation scanning of RET in sporadic medullary thyroid carcinoma and of RET and VHL in sporadic pheochromocytoma reveals involvement of these genes in only a minority of cases. J Clin Endocrinol Metab. 1996 Aug;81(8):2881-4.
2. Foster K, Crossey PA, Cairns P, et al Molecular genetic investigation of sporadic renal cell carcinoma: analysis of allele loss on chromosomes 3p, 5q, 11p, 17 and 22. Br J Cancer. 1994 Feb;69(2):230-4.
3. Waber PG, Lee NK, Nisen PD. Frequent allelic loss at chromosome arm 3p is distinct from genetic alterations of the Von-Hippel Lindau tumor suppressor gene in head and neck cancer. Oncogene. 1996 Jan 18;12(2):365-9.
4. Kanno H, Shuin T, Kondo K, et al Somatic mutations of the von Hippel-Lindau tumor suppressor gene and loss of heterozygosity on chromosome 3p in human glial tumors. Cancer Res. 1997 Mar 15;57(6):1035-8.
5. Vargas MP, Zhuang Z, Wang C, er al Loss of heterozygosity on the short arm of chromosomes 1 and 3 in sporadic pheochromocytoma and extra-adrenal paraganglioma. Hum Pathol. 1997 Apr;28(4):411-5.
6. Prowse AH, Webster AR, Richards FM, et al Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors. Am J Hum Genet. 1997 Apr;60(4):765-71.
7. Opocher G, Schiavi F, Vettori A, et al Fine analysis of the short arm of chromosome 1 in sporadic and familial pheochromocytoma. Clin Endocrinol (Oxf). 2003 Dec;59(6):707-15.
8. Brauch H, Hoeppner W, Jahnig H,et al Sporadic pheochromocytomas are rarely associated with germline mutations in the vhl tumor suppressor gene or the ret protooncogene. J Clin Endocrinol Metab. 1997 Dec; 82(12): 4101-4.
9. Bar M, Friedman E, Jakobovitz O, et al Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel-Lindau and RET genes. Clin Endocrino 1 (Oxf), 1997,47: 707-712.
10. Bender BU, Gutsche M, Glasker S,et al Differential genetic alterations in von Hippel-Lindau syndrome-associated and sporadic pheochromocytomas.J Clin Endocrinol Metab. 2000 Dec;85(12):4568-74.
11. Whaley JM, Naglich J, Gelbert L,et al Germ-line mutations in the von Hippel-Lindau tumor-suppressor gene are similar to somatic von Hippel-Lindau aberrations in sporadic renal cell carcinoma. Am J Hum Genet. 1994 Dec;55(6): 1092-102. Erratum in: Am J Hum Genet 1995 Jan;56(1):356.
12. Hamano K, Esumi M, Igarashi H, et al Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene in sporadic renal cell carcinoma. J Urol. 2002 Feb;167(2 Pt 1):713-7.
13. van der Harst E, de Krijger RR, Dinjens WN,et al Germline mutations in the vhl gene in patients presenting with phaeochromocytomas. Int J Cancer. 1998 Jul 29;77(3):337-40.
14. Crossey PA, Richards FM, Foster K,et al Identification of intragenic mutations in the von Hippel-Lindau disease tumour suppressor gene and correlation with disease phenotype. Hum Mol Genet. 1994 Aug;3(8): 1303-8.
15. Zbar B, Kishida T, Chen F,et al Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan. Hum Mutat. 1996;8(4):348-57.
16. Hobbs MR, Pole AR, Pidwirny GN, et al Hyperparathyroidism-jaw tumor syndrome: the HRPT2 locus is within a 0.7-cM region on chromosome 1q.Am J Hum Genet. 1999 Feb;64(2):518-25.
17. Villablanca A, Farnebo F, Teh BT, et al Genetic and clinical characterization of sporadic cystic parathyroid tumours. Clin Endocrinol (Oxf). 2002 Feb;56(2):261-9.
18. Kitamura Y, Shimizu K, Tanaka S, et al Allelotyping of anaplastic thyroid carcinoma: frequent allelic losses on 1q,9p, 11, 17, 19p, and 22q.Genes Chromosomes Cancer. 2000 Mar;27(3):244-51.
19. Peczkowska M, Januszewicz A. Multiple endocrine neoplasia type 2.Fam Cancer. 2005;4(1):25-36. Review.
20. Maher ER. Von Hippel-Lindau disease. Curr Mol Med. 2004 Dec;4(8):833-42. Review.
21. Simpson NE, Kidd KK, Goodfellow PJ, et al. Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage.Nature. 1987 Aug 6-12;328(6130):528-30.
22. Mathew CG, Chin KS, Easton DF,et al A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature. 1987 Aug 6-12;328(6130):527-8.
23. Seizinger BR, Rouleau GA, Ozelius LJ,et al Genetic linkage of von Recklinghausen neurofibromatosis to the nerve growth factor receptor gene. Cell. 1987 Jun 5;49(5):589-94.
24. Latif F, Tory K, Gnarra J,et al Identification of the von Hippel-Lindau disease tumor suppressor gene. Science. 1993 May28;260(5112):1317-20.
25. Zeiger MA, Zbar B, Keiser H,et alLoss of heterozygosity on the short arm of chromosome 3 in sporadic, von Hippel-Lindau disease-associated, and familial pheochromocytoma. Genes Chromosomes Cancer. 1995 Jul; 13(3): 151-6.
26. Khosla S, Patel VM, Hay ID,et al Loss of heterozygosity suggests multiple genetic alterations in pheochromocytomas and medullary thyroid carcinomas. J Clin Invest. 1991 May;87(5):1691-9.
27.Gnarra JR, Tory K, Weng Y, et al Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat Genet. 1994 May;7(1):85-90.
28. Tory K, Brauch H, Linehan M,et al Specific genetic change in tumors associated with von Hippel-Lindau disease. J Natl Cancer Inst. 1989 Jul 19;81(14):1097-101.
29. Koch CA, Huang SC, Zhuang Z,et al Somatic VHL gene deletion and point mutation in MEN 2A-associated pheochromocytoma. Oncogene. 2002 Jan 17;21(3):479-82.
30. Prowse AH, Webster AR, Richards FM,et al Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors. Am J Hum Genet. 1997 Apr;60(4):765-71.
1. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992 Dec; 12(12):5447-54.
2. Warnecke C, Zaborowska Z, Kurreck J,et al Differentiating the functional role of hypoxia-inducible factor (HIF)-lalpha and HIF-2alpha (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2alpha target gene in Hep3B and Kelly cells. FASEB J. 2004 Sep; 18(12): 1462-4.
3. Volm M,Koomagi R,Mattern J.Prognostic value of vascular endothelial growth factor and its receptor Flt-1 in squamous cell lung cancer.Int J Cancer, 1997,74:64-68
4. Matterm J, Koomagi R, Volm M. Coexpression of VEGF and bFGF in human epidernoid lung carcinoma is associated with increased vessel density.Anticancer Res.1997,17(3C):2249-2252
5．曾正陪 多内分泌腺瘤病见：史轶蘩 主编 协和内分泌和代谢学．北京：科学出版社，1999：1606—1621．
6. Shuin T, Ashida S, Yao M et al. Von Hippel-Lindau disease Nippon Rinsho, 2000, 58: 1448—1454.
7. Tian H, McKnight SL, Russell DW. Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev. 1997 Jan 1;11(1):72-82.
8. Wiesener MS, Turley H, Allen WE, et al Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-lalpha. Blood. 1998 Oct 1;92(7):2260-8.
9. Hu CJ, Wang LY, Chodosh LA, et al Differential roles of hypoxia-inducible factor lalpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol. 2003 Dec;23(24):9361-74.
10. Park SK, Dadak AM, Haase VH, et al Hypoxia-induced gene expression occurs solely through the action of hypoxia-inducible factor 1alpha (HIF-1alpha): role of cytoplasmic trapping ofHIF-2alpha. Mol Cell Biol. 2003 Jul;23(14):4959-71.
11. Lando D, Gorman JJ, Whitelaw ML, et al Oxygen-dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation. Eur J Biochem. 2003 Mar;270(5):781-90.
12. Latif F, Tory K, Gnarra J, et al Identification of the von Hippel-Lindau disease tumor suppressor gene.Science. 1993 May 28;260(5112): 1317-20.
13. Kim WY, Kaelin WG. Role of VHL gene mutation in human cancer. J Clin Oncol. 2004 Dec 15;22(24):4991-5004.
14. Tanimoto K, Makino Y, Pereira T, et al Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein. EMBO J. 2000 Aug 15;19(16):4298-309.
15. Yuan Y, Hilliard G, Ferguson T, et al Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem. 2003 May 2;278(18):15911-6.
16. Lando D, Peet DJ, Whelan DA,et al Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch.Science. 2002 Feb 1;295(5556):858-61.
1. Shuin T, Ashida S, Yao M et al.Von Hippel-Lindau disease Nippon Rinsho, 2000, 58: 1448-1454.
2. Smith DP, Houghton C, Ponder BA. Germline mutation of RET codon 883 in two cases of de novo MEN 2B. Oncogene. 1997, 15: 1213-1217.
3. Beldjord C, Desclaux-Arramond F, Raffin-Sanson M The RET protooncogene in sporadic pheochromocytomas: frequent MEN 2-like mutations and new molecular defects. J Clin Endocrinol Metab. 1995 Jul; 80(7): 2063-8.
4. Komminoth P, Roth J, Muletta-Feurer S RET proto-oncogene point mutations in sporadic neuroendocrine tumors. J Clin Endocrinol Metab. 1996 Jun; 81(6): 2041-6.
5. Chew SL Absence of mutations in the MEN2A region of the ret proto-oncogene in non-MEN 2A phaeochromocytomas. Clin Endocrinol (Oxf). 1995 Jan; 42(1): 17-21.
6. Brauch H, Hoeppner W, Jahnig H Sporadic pheochromocytomas are rarely associated with germline mutations in the vhl tumor suppressor gene or the ret protooncogene. J Clin Endocrinol Metab. 1997 Dec; 82(12): 4101-4.
7. Bar M, Friedman E, Jakobovitz O et al. Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel-Lindau and RET genes. Clin Endocrinol (Oxf), 1997,47: 707-712.
8. Le Hir H, Colucci-D'Amato LG, Charlet-Berguerand N et al.High levels of tyrosine phosphorylated proto-ret in sporadic phenochromocytomas. Cancer Res, 2000, 60: 1365-1370.
9. Califano D, Rizzo C, D'Alessio A et al. Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC 12 cells. J Biol Chem , 2000, 275: 19297-19305.
10. Castilla-Guerra L, Moreno AM, Fernandez-Moreno MC et al.Expression and prognostic value of c-erbB-2 oncogene product in human phaeochromocytomas. Histopathology, 1997. 31: 144-149.
11. e Krijger RR, van der Harst E, van der Ham F, et al. Prognostic value of p53, bcl-2, and c-erbB-2 protein expression in phaeochromocytomas. J Pathol, 1999, 188:51-55.
12. Richard S, Beroud C, Joly D, et al. Von Hippel-Lindau disease and renal cancer: 10 years of genetic progress. Prog Urol, 1998, 8: 330-339.
13. Yoshida M, Ashida S, Kondo K, et al. Germ-line mutation analysis in patients with von Hippel-Lindau disease in Japan:an extended study of 77 families. Jpn J Cancer Res, 2000, 91: 204-212.
14. Brauch H, Kishida T, Glavac D, et al. Von Hippel-Lindau (VHL) disease with pheochromocytoma in the Black Forest region of Germany: evidence for a founder effect. Hum Genet, 1995, 95: 551 -556.
15. Duerr EM, Gimm O, Neuberg DS, et al. Differences in allelic distribution of two polymorphisms in the VHL-associated gene CUL2 in pheochromocytoma patients without somatic CUL2 mutations. J Clin Endocrinol Metab , 1999, 84: 3207-3211.
16. Gutmann DH, Geist RT, Rose K et al. Loss of neurofibromatosis type I (NF1) gene expression in pheochromocytomas from patients without NF1. Genes Chromosomes Cance r, 1995,13:104-109.
17. Aguiar RC, Dahia PL, Sill H et al. Deletion analysis of the p16 tumour suppressor gene in phaeochromocytomas. Clin Endocrinol (Oxf), 1996, 45: 93-96.
18. Wang DG, Johnston CF, Marley JJ, et al. Expression of the apoptosis-suppressing gene BCL-2 in pheochromocytoma is associated with the expression of C-MYC. J Clin Endocrinol Metab, 1997. 82:1949-1952.
19. Shay JW, Wright WE. et al. Telomerase activity in human cancer. Curr Opin Oncol, 1996, 8:66-71.
20.Kubota Y, Nakada T, Sasagawa I, et al. Elevated levels of telomerase activity in malignant pheochromocytoma. Cancer, 1998, 82: 176-179.
21..Aguiar RC , Cox G, Pomeroy SL , et al. Analysis of the SDHD gene ,the susceptibility gene for familial paraganglioma syndrome (PGL1) , in pheochromocytomas. J Clin Endocrinol Metab ,2001 ,86 :2890-2894.
22.Benn DE , Croxson MS , Tucker K, et al. Novel succinate dehydrogenase subunit B (SDHB) mutations in familial pheochromocytomas and paragangliomas , but an absence of somatic SDHB mutations in sporadic pheochromocytomas. Oncogene ,2003 ,22 : 1358-1364.
23.Baysal BE , Willett-Brozick JE , Lawrence EC , et al . Prevalence of SDHB , SDHC , and SDHD germline mutations in clinic patients with head and neck paragangliomas. J Med Genet ,2002 ,39 :178-183.
24.Baysal BE , Ferrell RE ,Willett2Brozick JE , et al . Mutations in SDHD ,a mitochondrial complex II gene , in hereditary paraganglioma. Science ,2000,287:848-851.
25.Neumann HP , Bausch B , McWhinney SR. Germline mutations in non-syndromic pheochromocytoma. N Engl J Med ,2002 ,346 :1459-1466.
26.Kytola S , Nord B , Elder EE, et al. Alterations of the SDHD gene locus in midgut carcinoids , Merkel cell carcinomas , pheochromocytomas , and abdominal paragangliomas. Genes Chrom Cancer ,2002 ,34 :325-332.
27.Astuti D , Douglas F , Lennard TW, et al. Germline SDHD mutation in familial phaeochromocytoma. Lancet ,2001 ,357 :1181-1182.