Screening of mutations in genes that predispose to hereditary paragangliomas and pheochromocytomas

Thirty per cent of the paragangliomas and pheochromocytomas reported are hereditary. Mutations in SDHB, SDHC, SDHD, and more recently SDHAF2 and TMEM127 genes have been described in these hereditary tumors. We looked for mutations in these 5 genes in a series of 269 patients with paragangliomas and/or pheochromocytomas. The SDHB, SDHC, and SDHD genes were analyzed in a series of 269 unrelated index patients with paragangliomas and/or pheochromocytomas using dHPLC screening of point mutations followed by direct sequencing and Multiplex PCR Liquid Chromatography to detect large rearrangements confirmed by quantitative PCR. In a second phase, we adapted Multiplex PCR Liquid Chromatography to the SDHAF2 and TMEM127 genes. This method and direct sequencing were applied to 230 patients without the SDHB, C, D mutations. Of the 269 patients, 44 carried a mutation (16.3%). Thirty-seven different mutations were identified: 18 in SDHB (including 2 large deletions), 8 in SDHD, 6 in SDHC, 5 in TMEM127, and no mutations in SDHAF2. Thirteen mutations have not been published so far. An exhaustive study of the different genes is needed to make possible a familial genetic diagnosis in paraganglioma and pheochromocytoma hereditary syndromes. Although mutations in SDHC and TMEM127 are less frequent than mutations in SDHB and SDHD, they also have less evident clinical feature indicators. Analyzing SDHAF2 must be restricted to familial extra-adrenal paragangliomas. Multiplex PCR Liquid Chromatography is a sensitive, fast, and inexpensive method for screening large rearrangements, which are infrequent in these syndromes.     

Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma

The pheochromocytomas are an important cause of secondary hypertension. Although pheochromocytoma susceptibility may be associated with germline mutations in the tumor-suppressor genes VHL and NF1 and in the proto-oncogene RET, the genetic basis for most cases of nonsyndromic familial pheochromocytoma is unknown. Recently, pheochromocytoma susceptibility has been associated with germline SDHD mutations. Germline SDHD mutations were originally described in hereditary paraganglioma, a dominantly inherited disorder characterized by vascular tumors in the head and the neck, most frequently at the carotid bifurcation. The gene products of two components of succinate dehydrogenase, SDHC and SDHD, anchor the gene products of two other components, SDHA and SDHB, which form the catalytic core, to the inner-mitochondrial membrane. Although mutations in SDHC and in SDHD may cause hereditary paraganglioma, germline SDHA mutations are associated with juvenile encephalopathy, and the phenotypic consequences of SDHB mutations have not been defined. To investigate the genetic causes of pheochromocytoma, we analyzed SDHB and SDHC, in familial and in sporadic cases. Inactivating SDHB mutations were detected in two of the five kindreds with familial pheochromocytoma, two of the three kindreds with pheochromocytoma and paraganglioma susceptibility, and 1 of the 24 cases of sporadic pheochromocytoma. These findings extend the link between mitochondrial dysfunction and tumorigenesis and suggest that germline SDHB mutations are an important cause of pheochromocytoma susceptibility.     

Hereditary paraganglioma/pheochromocytoma and inherited succinate dehydrogenase deficiency

Mitochondrial complex II, or succinate dehydrogenase, is a key enzymatic complex involved in both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation as part of the mitochondrial respiratory chain. Germline succinate dehydrogenase subunit A (SDHA) mutations have been reported in a few patients with a classical mitochondrial neurodegenerative disease. Mutations in the genes encoding the three other succinate dehydrogenase subunits (SDHB, SDHC and SDHD) have been identified in patients affected by familial or 'apparently sporadic' paraganglioma and/or pheochromocytoma, an autosomal inherited cancer-susceptibility syndrome. These discoveries have dramatically changed the work-up and genetic counseling of patients and families with paragangliomas and/or pheochromocytomas. The subsequent identification of germline mutations in the gene encoding fumarase--another TCA cycle enzyme--in a new hereditary form of susceptibility to renal, uterine and cutaneous tumors has highlighted the potential role of the TCA cycle and, more generally, of the mitochondria in cancer.     

Mutations in the C. elegans Succinate Dehydrogenase Iron-Sulfur Subunit Promote Superoxide Generation and Premature Aging

The mitochondrial succinate dehydrogenase (SDH) is an iron-sulfur flavoenzyme linking the Krebs cycle and the mitochondrial respiratory chain. Mutations in the human SDHB, SDHC and SDHD genes are responsible for the development of paraganglioma and pheochromocytoma, tumors of the head and neck or the adrenal medulla, respectively. In recent years, SDH has become recognized as a source of reactive oxygen species, which may contribute to tumorigenesis. We have developed a Caenorhabditis elegans model to investigate the molecular and catalytic effects of mutations in the sdhb-1 gene, which encodes the SDH iron-sulfur subunit. We created mutations in Pro211; this residue is located near the site of ubiquinone reduction and is conserved in human SDHB (Pro197), where it is associated with tumorigenesis. Mutant phenotypes ranged from relatively benign to lethal and were characterized by hypersensitivity to oxidative stress, a shortened life span, impaired respiration and overproduction of superoxide. Our data suggest that the SDH ubiquinone-binding site can become a source of superoxide and that the pathological consequences of SDH mutations can be mitigated with antioxidants, such as ascorbate and N-acetyl-l-cysteine. Our work leads to a better understanding of the relationship between genotype and phenotype in respiratory chain mutations and of the mechanisms of aging and tumorigenesis.     

Prevalence and spectrum of SDHx mutations in pheochromocytoma and paraganglioma in patients from Belgium: an update

Since the early 2000s, the prevalence and spectrum of mutations in genes encoding subunits of succinate dehydrogenase (SDHx) were reported in large cohorts of patients with pheochromocytoma (PC) and paraganglioma (PGL) from most Western countries. Unfortunately, in Belgium, no equivalent work was performed thus far. Therefore, the aim of the work was to look for mutations in SDHx genes and genotype-phenotype correlations in patients with PC and/or PGL from Belgium. Screening of the coding parts of SDHx genes and deletion search were performed in all patients with PC and/or PGL referred to the -Cliniques Universitaires Saint-Luc from 05/2003 to 05/2011. Genetic screening was performed in 59 unrelated head and neck (hn)PGLs (8 fami-lial) and 53 PCs (7 extra-adrenal; 3 metastatic). In hnPGLs, 10 different SDHD mutations (3 substitutions, 5 deletions, 2 splice site mutations) were detected in 16 patients, including 7 familial cases and 9 apparently sporadic cases. In the same subset, we found 8 different SDHB mutations (5 substitutions, 1 splice site mutation, 1 deletion, 1 duplication) in 10 patients with sporadic hnPGL without evidence of malignancy. No SDHx mutation was detected in patients harboring PCs and no SDHC mutation whatsoever. In conclusion, in our multicentric database of PC-PGLs from Belgium, (i) the prevalence of SDHx mutations was high in hnPGLs (44% in the whole subset, 37% of apparently sporadic cases); (ii) in sporadic cases, the prevalence of SDHB mutations was high (20%), similar to that of SDHD (18%); and (iii) no SDHx mutation was found in a subset of mostly adrenal, benign PCs.     

Malignant Pheochromocytoma Secreting Vasoactive Intestinal Peptide and Response to Sunitinib: A Case Report and Literature Review

ObjectiveMalignant pheochromocytoma is rare and may be sporadic or have a genetic basis. Vasoactive intestinal peptide (VIP)-secreting pheochromocytoma has rarely been described in the literature, and treatment remains challenging in the absence of well-controlled randomized trials. The hypoxia-inducible factor – vascular endothelial growth factor axis has been implicated in pheochromocytoma when associated with germline Von-Hippel-Lindau (VHL) or succinate dehydrogenase (SDH) mutations, suggesting potential clinical activity of sunitinib in this setting.MethodsWe present a case report of a patient with a VIP-secreting malignant pheochromocytoma manifested as severe watery diarrhea, with an exquisite clinical response to sunitinib. We review this rare clinical entity and the potential role of sunitinib in this context.ResultsA 51-year-old male initially presented with a pheochromocytoma causing symptoms related to norepinephrine excess. He underwent adrenalectomy, which resulted in complete resolution of his symptoms. Three years later, he developed multifocal metastatic disease from his primary tumor, showing immunohistochemical evidence of VIP production accompanied by severe watery diarrhea and hypokalemia. The patient had a rapid, complete, and durable clinical response to sunitinib, but with only a minor radiological response and without significant toxicity. Genetic testing was negative for germline mutations in VHL, SDHB, SDHC, SDHD, transmembrane protein 127 (TMEM127) and for neurofibromatosis type 1 (NF-1).ConclusionTo the best of our knowledge, this is the first report of a case of malignant VIP-producing pheochromocytoma that was responsive to sunitinib.     

Évaluation fonctionnelle par TEP 18F-FDopa des paragangliomes et phéochromocytomes non métastatiques : impact de la localisation lésionnelle et du statut génétique

ObjectiveParaganglioma (PGL) and pheochromocytoma (PCC) are neuroendocrine tumors most often benign associated with hereditary syndromes in about 30% of cases. This study aims to define the impact of tumor location and patient genotype on the clinical value of ¹⁸F-FDopa PET by assessing in detail the false negative occurrences.Patients and methodsA retrospective study was conducted on a cohort of 53 cases with non-metastatic sporadic or inherited PGL/PCC (SDHx or VHL related syndromes), investigated with ¹⁸F-FDopa PET.ResultsOverall detection sensitivity of ¹⁸F-FDopa PET was 88%. Seventy-three lesions were found using this technique, including 49 head-and-neck PGL (HNP), two thoracic PGL (1 sympathetic and 1 parasympathetic), eight extra-adrenal retroperitoneal PGL and 15 PCC. The 10 missed lesions were seven extra-adrenal abdominal PGL (2 SDHB, 2 SDHD), two HNP (1 sporadic, 1 SDHD) and one PCC (1 SDHD).Conclusion¹⁸F-FDopa PET is a sensitive technique for the evaluation of non-metastatic head and neck and adrenal PGLs. Exploration of extra-adrenal retroperitoneal PGL associated with SDHB or SDHD syndrome is the main limitation of this technique, encouraging the use of alternative functional imaging modalities like FDG-PET. Negativity of ¹⁸F-FDopa PET in the initial assessment of a PGL should prompt to search for a SDHx mutation.     

Ubiquinone-binding site mutations in the Saccharomyces cerevisiae succinate dehydrogenase generate superoxide and lead to the accumulation of succinate

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo.We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.     

SDH mutations in cancer

The SDHA, SDHB, SDHC, SDHD genes encode the four subunits of succinate dehydrogenase (SDH; mitochondrial complex II), a mitochondrial enzyme involved in two essential energy-producing metabolic processes of the cell, the Krebs cycle and the electron transport chain. Germline loss-of-function mutations in any of the SDH genes or assembly factor (SDHAF2) cause hereditary paraganglioma/phaeochromocytoma syndrome (HPGL/PCC) through a mechanism which is largely unknown. Owing to the central function of SDH in cellular energy metabolism it is important to understand its role in tumor suppression. Here is reported an overview of genetics, clinical and molecular progress recently performed in understanding the basis of HPGL/PCC tumorigenesis.     

Early-onset renal cell carcinoma as a novel extraparaganglial component of SDHB-associated heritable paraganglioma

Hereditary paraganglioma syndrome has recently been shown to be caused by germline heterozygous mutations in three (SDHB, SDHC, and SDHD) of the four genes that encode mitochondrial succinate dehydrogenase. Extraparaganglial component neoplasias have never been previously documented. In a population-based registry of symptomatic presentations of phaeochromocytoma/paraganglioma comprising 352 registrants, among whom 16 unrelated registrants were SDHB mutation positive, one family with germline SDHB mutation c.847-50delTCTC had two members with renal cell carcinoma (RCC), of solid histology, at ages 24 and 26 years. Both also had paraganglioma. A registry of early-onset RCCs revealed a family comprising a son with clear-cell RCC and his mother with a cardiac tumor, both with the germline SDHB R27X mutation. The cardiac tumor proved to be a paraganglioma. All RCCs showed loss of the remaining wild-type allele. Our observations suggest that germline SDHB mutations can predispose to early-onset kidney cancers in addition to paragangliomas and carry implications for medical surveillance.     

New insights in the genetics of adrenocortical tumors, pheochromocytomas and paragangliomas.

Recent advances in the molecular genetic of adrenal tumors give new insights in the pathophysiology of these neoplasms in both hereditary and sporadic cases. The practice of genetic counselling in patients with adrenal tumors have been recently changed by the identification and the understanding of new specific hereditary cancer susceptibility syndromes. In the case of sporadic adrenocortical tumors these progress also offer new prognosis predictors.The genetic predisposition to adrenocortical cancer in children has been well established in the Li-Fraumeni and Beckewith-Wiedeman syndromes due to germline p53 mutation located at 17p13 and dysregulation of the imprinted IGF-2 locus at 11p15, respectively. Adrenocortical tumors are also observed in Multiple Endocrine Neoplasia type I syndrome. Cushing's syndrome due to primary pigmented nodular adrenocortical disease have been observed in patients with germline PRKAR1A inactivating mutations. Interestingly allelic loss at 17p13 and 11p15 have been observed in sporadic adrenocortical cancer and somatic PRKAR1A mutations in secreting adrenocortical adenomas. The potential interest of these finding for the diagnosis of these tumors will be discussed. In the case of pheochromocytoma and paraganglioma, the demonstration that three genes encoding three succinate dehydrogenase subunits (SDHD, SDHB, SDHC), belonging to the complex II of the respiratory chain in the mitochondria, are involved in the genetics of familial and especially in apparently sporadic phaeochromocytomas have dramatically modified our practice. Up to date, four diagnosis of familal disease (multiple endocrine neoplasia type II, von Hippel Lindau disease, neurofibromatosis type 1 and hereditary paraganglioma) should be discussed and causative mutations in six different phaechomocytoma susceptibility genes (RET, VHL, NF1, SDHB, SDHD, SDHC) could be identified. In this review, we will perform an update compiling these new clinical, genetic and functional data recently published. We will suggest guidelines for the practice of the phaeochomocytoma genetic testing in the patients and their families, and for an early detection of tumors in the patients or in individuals determined to be at-risk of disease by the presymptomatic genetic testing.     

High throughput proteomic and metabolic profiling identified target correction of metabolic abnormalities as a novel therapeutic approach in head and neck paraganglioma

Head and neck paragangliomas (HNPGLs) are rare neoplasms that represent difficult treatment paradigms in neurotology. Germline mutations in genes encoding succinate dehydrogenase (SDH) are the cause of nearly all familial HNPGLs. However, the molecular mechanisms underlying tumorigenesis remain unclear. Mutational analysis identified 6 out of 14 HNPGLs harboring clinicopathologic SDH gene mutations. The SDHB gene was most frequently mutated in these patients, and western blot showed loss of SDHB protein in tumors with SDHB mutations. The paraganglioma cell line (PGL-626) was established from a sample that harbored a missense SDHB mutation (c.649C > T). Spectrometric analysis using tandem mass tags identified 151 proteins significantly differentially expressed in HNPGLs compared with normal nerves. Bioinformatics analyses confirmed the high level of enrichment of oxidative phosphorylation and metabolism pathways in HNPGLs. The mitochondrial complex subunits NDUFA2, NDUFA10, and NDUFA4, showed the most significantly increased expression and were localized predominantly in the cytoplasm of PGL-626 cells. The mitochondrial complex I inhibitor metformin exerted dose-dependent inhibitory effects on PGL-626 cells via cooperative down-regulation of NDUFA2, 4, and 10, with a significant decrease in the levels of reactive oxygen species and mitochondrial membrane potential. Further metabolomic analysis of PGL-626 cells showed that metabolites involved in central carbon metabolism in cancer and sphingolipid signaling pathways, pantothenate and CoA biosynthesis, and tryptophan and carbon metabolism were significantly altered after metformin treatment. Thus, this study provides insights into the molecular mechanisms underlying HNPGL tumorigenesis and identifies target correction of metabolic abnormalities as a novel therapeutic approach for this disease.     

Mitochondrial and Nuclear Genes of Mitochondrial Components in Cancer

Although the observation of aerobic glycolysis of tumor cells by Otto v. Warburg had demonstrated abnormalities of mitochondrial energy metabolism in cancer decades ago, there was no clear evidence for a functional role of mutant mitochondrial proteins in cancer development until the early years of the 21^st century. In the year 2000, a major breakthrough was achieved by the observation, that several genes coding for subunits of the respiratory chain (ETC) complex II, succinate dehydrogenase (SDH) are tumor suppressor genes in heritable paragangliomas, fulfilling Knudson’s classical two-hit hypothesis. A functional inactivation of both alleles by germline mutations and chromosomal losses in the tumor tissue was found in the patients. Later, SDH mutations were also identified in sporadic paragangliomas and pheochromocytomas. Genes of the mitochondrial ATP-synthase and of mitochondrial iron homeostasis have been implicated in cancer development at the level of cell culture and mouse experiments. In contrast to the well established role of some nuclear SDH genes, a functional impact of the mitochondrial genome itself (mtDNA) in cancer development remains unclear. Nevertheless, the extremely high frequency of mtDNA mutations in solid tumors raises the question, whether this small circular genome might be applicable to early cancer detection. This is a meaningful approach, especially in cancers, which tend to spread tumor cells early into bodily fluids or faeces, which can be screened by non-invasive methods.     

A decade (2001-2010) of genetic testing for pheochromocytoma and paraganglioma

The identification of 9 susceptibility genes for paraganglioma/pheochromocytoma between 2001 and 2010 has led to the development of routine genetic tests. To study the evolution in genetic screening for paraganglioma/pheochromocytoma over the past decade, we carried out a retrospective study on the tests performed in our laboratory from January 2001 to December 2010. A genetic test for paraganglioma/pheochromocytoma was assessed for 2 499 subjects, 1 620 index cases, and 879 presymptomatic familial genetic tests. A germline mutation in a PGL/PCC susceptibility gene was identified in 363 index cases (22.4%): 269 in SDHx genes (137 in SDHB, 100 in SDHD, 30 in SDHC, 2 in SDHA), 64 in VHL, 23 in RET, and 7 in TMEM127. A presymptomatic paraganglioma/pheochromocytoma test was positive in 427 subjects. Advances in molecular screening techniques led to an increase in the total number of mutation-carriers diagnosed each year. Overall, during the last decade, our laboratory identified a germline mutation in 44.7% of patients with a suspect hereditary PGL/PCC and in 8% of patients with an apparently sporadic PGL/PCC. During the past decade, the discoveries of new paraganglioma/pheochromocytoma susceptibility genes and the subsequent progress of molecular screening techniques have enabled us to diagnose a hereditary paraganglioma/pheochromocytoma in about 22% of patients tested in routine practice. This genetic testing is of major importance for the follow-up of affected patients and for the genetic counselling of their families.     

Hereditäre Paragangliome

Hereditary paragangliomas/pheochromocytomas are inherited as autosomal dominant traits. Three types, i. e. PGL1, PGL2, and PGL3 can be distinguished. They are caused by mutations of the genes SDHD, SDHC, and SDHB, which encode components of mitochondrial complex II (succinate-ubiquinone reductase, SDH) of the respiratory chain. “Loss of heterozygosity” (LOH) of the region harbouring the disease gene is found in all 3 types of paragangliomas. LOH results in functional loss of SDH, the accumulation of succinate and of reactive oxygen species. As a consequence, hypoxia-dependent metabolic pathways are induced which appear to trigger tumorigenesis. PGL3 and PGL4 can be caused by germ-line mutations in either the paternal or the maternal copy of the respective disease gene, i. e. SDHC and SDHB. In contrast, PGL1 only occurs after paternal transmission of a mutation in SDHD. This observation can be explained by partial inactivation (“imprinting”) of the maternal SDHD-gene and induction of hypoxia-dependent genes, which in turn favour non-disjunction and loss of chromosome 11.     

Differences in Clinical Manifestations and Tumor Features Between Metastatic Pheochromocytoma/Paraganglioma Patients With and Without Germline SDHB Mutation

ObjectiveTo compare metastatic pheochromocytoma/paraganglioma (MPP) patients with germline SDHB mutations (SDHB MPP) and without SDHB mutations (non-SDHB MPP) in terms of baseline clinical manifestations, tumor characteristics, and outcomes.MethodsClinical data were retrospectively reviewed in 101 MPP patients, including 34 SDHB MPP patients and 61 non-SDHB MPP patients.ResultsSDHB MPP patients presented at a younger age at onset, diagnosis, or metastasis (25 ± 16 vs 36 ± 14, 28 ± 17 vs 38 ± 15, and 31 ± 17 vs 44 ± 14 years old, respectively, P < .01 for all) than non-SDHB patients. Compared with their non-SDHB counterparts, SDHB patients were more likely to have paragangliomas (83% vs 47%, P < .05), synchronous metastases (44% vs 23%, P < .05), bone metastases (80% vs 48%, P < .01), and a shorter progression-free survival (3 years vs 5 years, P < .01). The Ki-67 index was higher in SDHB tumors (P < .05). The 5- and 10-year survival rates were 79% and 74%, respectively, in all patients. Seventeen patients died from MPP, and the time from metastasis to death in patients who had received systemic therapy was significantly longer than in those who had not (3.1 ± 1.5 vs 1.4 ± 0.7 years, P < .01).ConclusionCompared with MPP patients without SDHB mutations, MPP patients with SDHB mutations were younger at onset, diagnosis, or metastasis; had a higher incidence of synchronous metastases, higher ratio of paraganglioma, and higher Ki-67 index; had a shorter postoperative progression-free survival; and were more likely to develop bone metastasis or sole liver metastasis. Our results suggest that patients with SDHB mutations should be identified early and monitored regularly to achieve optimal clinical outcomes.