Germline Mutations In The RET Proto-oncogene And The Von Hippel-Lindau Disease Tumour Suppressor Gene In Phaeochromocytomas

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.