Study On The Genetic Mutations Of Succinate Dehydrogenase In Pheochromocytoma

ObjetivePheochromocytoma is an important cause of secondary hypertension, whose pathogenesis is yet to be clarified. It is still difficult to distinguish malignant pheochromocytoma from benign tumors at early stage. With the development of molecular biological technique, it was found germline mutations in succinate dehydrogenase B and D(SDHB and SDHD) gene, cause pheochromocytoma. The aim of this study was to detect SDHB, SDHD gene mutation among 63 cases of pheochromocytoma in PUMCH, in order to explore the possible pathogenesis of pheochromocytoma and, in the meanwhile providing early diagnosis guidance of pheochromocytoma at the gene level.Subject and Method1. 63 patients were diagnosed as pheochromocytoma from November 2003 to April 2006 in PUMCH, including 41 cases of pheochromocytoma, 22 cases of extra-adrenal paraganglioma, and among those 63 patients 15 cases were familial pheochromocytoma. 20 cases of healthy people as normal control. SDHB, SDHD gene mutations were detected in 63 patients, 22 kindreds of familial pheochromocytoma and 20 normal controls.2. Twelve pairs of primers were used to amplify eight exons of SDHB gene and four exons of SDHD gene. Combinding of PCR-SSCP and PCR products directly sequence to detect gene mutation.Result1. Two cases have SDHB gene mutations, both of them were patients with sporadic extra-adrenal paraganglioma, the frequency of mutations in SDHB was 3.2%(2/63). There were two kind of germ-line mutations in SDHB, which present in exon 7. One is missense mutation: g.692 G＞A, causing amino acid Arg231His; another is deletion mutation: g.760delT, causing amino acid Cys254Val, 258X. Both of them were not reported worldwide according to our search in the MEDLINE database.2. No germ-line mutation in SDHD was identified in 63 patients.Conclusion1. In our study, we detected two novel germ-line mutation in SDHB in in patients with extra-adrenal paraganglioma, the frequency of mutations in SDHB was 3.2%, we did not detect any mutation in SDHD gene.2. Detect SDHB gene mutation may have significance in diagnosed extra-adrenal paraganglioma at early stage. ObjetiveThe pathogenesis of pheochromocytoma is still unkonwn. It was considered that inactivation of one or more tumor suppressor genes was associated with tumorigenesis. According to Knudson's classical two-hit hypothesis, inactivation of tumor suppressor genes was due to one allele mutation and another loss of heterozygosity (LOH), which led to tumorigenesis. It was reported that SDHB,SDHD gene might contribute to pathogenesis in pheochromocytoma. In this study we detected if chromosome 1p35-p36(SDHB locus) and 11q23(SDHD locus) LOH occurred in patients with pheochromocytoma and, if it occurred, whether there was high frequency of LOH which could be used as a genetic marker to differentiate malignant pheochromocytoma from benign ones.Subject and Method1. The specimens of 40 pheochromocytomas were obtained in PUMCH from November 2003 to April 2006.2. DNA was extracted from tumor tissue and leukocytes. Nine polymorphic microsatellite markers were selected to spanning chromosome 1p35-p36 and 11q23 LOH (four for 1p and five for 11q). With ³²p-α-dCTP cooperated into the reaction volume, PCR amplification of both normal and tumor tissue was performed. The PCR production was analyzed by electrophoresis through a 6% denaturing polyacrylamide gel (PAGE). The gels were dried and exposed to X-ray film with intensifying screen. LOH was defined as a reduction of≥50% in allelic ratio between the tumor specmen and normal DNAsample from the same patient.Result1. Of 40 pheochromocytoma, 15 tumors were shown SDHB-LOH, 10 of them corresponding to extra-adrenal paraganglioma, 5 of them corresponding to adrenal pheochromocytoma. 1 patient had microsatellite instability (MSI) in this region, SDHB-LOH/MSI were not detected in 19 tumors, 5 patients were noninformative, so the frequency of SDHB-LOH was 42.9%(15/35). The frequency of SDHB-LOH was significantly higher in extra-adrenal paraganglioma, than in adrenal ones (P＜0.01). D1S3669 was the high frequency of LOH at SDHB.2. SDHD-LOH was found in 2 cases (5.0%, 2/40), both of them corresponding to malignant extra-adrenal paraganglioma, 2 of 40 patients had SDHD-MSI: one was familial bilateral pheochromocytoma recurrence after operation, another was left adrenal malignant pheochromocytoma.Conclusion1. SDHB-LOH was significantly associated with ectopic site, therefore it could be used as a potential genetic marker to detect extra-adrenal paraganglioma,.2. Detect SDHB and SDHD-LOH/MSI might have significance in diagnosed extra-adrenal malignant paraganglioma, at early stage. ObjetiveThe genetic change of mitochondrial succinate dehydrogenase (SDH) gene can interfere with the mitochondrial respiratory chain, resulting in hypoxia. In hypoxic conditions, hypoxia-inducible factor (HIF) can regulate the expression of vascular endothelial growth factor (VEGF) leading to neoangiogenesis, which is relevant to the growth and metastasis of tumors. We investigate the expression of Hypoxia-inducible factor-1α(HIF-1α),HIF-2α, VEGF and VEGFRs (Flt-1,Flk-1) in pheochromocytom and normal adrenal medulla tissue by immunohistochemical method, to explore the difference protein expression of above factors in patients with or without SDHB,SDHD genetic change.Subject and Method1. 40 patients were diagnosed as phcochromocytoma from November 2003 to April 2006 in PUMCH (male 20, female 20), 8 cases of normal adrenal medulla as normal controls.2. Investigate the protein expression of HIF-1α,HIF-2α, VEGF and Flt-1,Flk-1 in patients with pheochromocytoma by immunohistochemical method, to explore the difference protein expression of above factors in patients with or without SDHB,SDHD genetic change and, compare the protein expression of HIF-1α,HIF-2α, VEGF and Flt-1,Flk-1 with clinical features of pheochromocytoma.ResultImmunohistochemical analysis shown:1. In normal adrenal medulla tissue, there were none or low expression of HIF-1αand HIF-2α, and low expression of VEGF and VEGFRs.2. In pheochromocytoma, VEGF,Flt-1 and Flk-1 were expressed in the cytoplasm of tumor cells and endothelial cells of vessels. HIF-1αand HIF-2αwere expressed in the cytoplasm and nuclear of tumor cells. 3. The protein expression of HIF-1α,HIF-2α(EPAS1), VEGF and Flt-1,Flk-1 was significantly higher in patients with SDHB,SDHD gene mutation/LOH/MSI than those without.4. The higher expression of HIF-1α,HIF-2α(EPAS1), VEGF and Fit-1,Flk-1 was associated with extra-adrenal paraganglioma, malignant pheochromocytoma or pheochromocytoma with necrosis.ConclusionThe genetic change of SDHB,SDHD can interfere with the mitochondrial respiratory chain, made a high protein expression of HIF-1α,HIF-2α(EPAS1), VEGF and VEGFRs, which involved in neoangiogenesis. So the genetic change of SDH was participated in the pathogenesis of pheochromocytoma.