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Functional Consequences of a SDHB Gene Mutation in an Apparently Sporadic Pheochromocytoma

Département de Génétique Moléculaire (A.-P.G.-R., C.R., X.J.), Hôpital Européen Georges Pompidou, Assistance Publique/Hôpitaux de Paris, Paris; Institut National de la Santé et de la Recherche Médicale (INSERM) U36 (J.F., X.J.), Collège de France, Paris; INSERM U393 (P.R., A.R.), Hôpital des Enfants Malades, Paris; Service d’Endocrinologie (V.K.), Hôpital de la Cavale Blanche, Brest, France; and Service d’Hypertension Artérielle (P.-F.P.), Hôpital Européen Georges Pompidou, Paris, France

Address all correspondence and requests for reprints to: Dr. Anne-Paule Gimenez-Roqueplo, Département de Génétique Moléculaire, Hôpital Européen Georges Pompidou, 20–40, rue Leblanc, 75015 Paris, France. E-mail: Anne-Paule.Gimenez{at}hop.egp.ap-hop-paris.fr.

Abstract

Three genes encoding for mitochondrial complex II proteins are linked to hereditary paraganglioma. We have recently shown that an inactivation of the SDHD gene is associated with a complete loss of mitochondrial complex II activity and a stimulation of the angiogenic pathway (Gimenez-Roqueplo, A. P., J. Favier, P. Rustin, J. J. Mourad, P. F. Plouin, P. Corvol, A. Rötig, and X. Jeunemaitre, 2001, Am J Hum Genet 69:1186–1197). Here, we relate the case of a malignant sporadic pheochromocytoma induced by a germline missense mutation of the SDHB gene. Within the tumor, a loss of heterozygosity at chromosome 1pter led to a null SDHB allele and to a complete loss of complex II enzymatic activity. In situ hybridization and immunohistochemistry experiments showed a high expression of hypoxic-angiogenic responsive genes, similar to that previously observed in inherited-SDHD tumors. This observation highlights the role of the complex II mitochondrial genes in the oxygen-sensing pathway and in the regulation of angiogenesis of neural crest-derived tumors.

MAJOR PROGRESS HAS been recently obtained on the molecular genetics of neural crest-derived tumors. It is especially the case for hereditary paragangliomas, which are rare, usually benign, tumors preferentially located on the neck region in the carotid body (MIM 168000, 601650, and 605373). Paragangliomas have been related to mutations of the SDHD, SDHC, and SDHB genes (1, 2, 3). These genes encode three essential proteins of the succinate dehydrogenase complex within the mitochondrial-respiratory chain. The complex II is involved in the Krebs cycle and in the aerobic electron transport chain, which plays a critical role in cellular oxygen sensing (4). It contains four proteins (SDHA, SDHB, SDHC, SDHD) encoded by four nuclear genes (SDHA, SDHB, SDHC, SDHD). The catalytic core is formed by the subunits A (flavoprotein) and B (iron-sulfur protein), whereas the subunits C and D compose the anchorage domain of complex II in the inner mitochondrial membrane.

Because familial neck paragangliomas may be associated in SDHD-inherited kindreds with extra-adrenal and adrenal catecholamine-secreting pheochromocytomas (5, 6), hereditary paraganglioma genes have also been investigated in nonfamilial or sporadic pheochromocytomas. Gimm et al. (7) reported, among 18 sporadic pheochromocytomas, two germline (R38X and IVS1 + 2T>G) and one somatic (P81L) SDHD gene mutations. Two punctual mutations (R90X and P197R) of the SDHB gene were reported in familial pheochromocytomas and one microdeletion among 24 nonfamilial pheochromocytomas (3). More recently, Neuman et al. (8) have found 11 mutations of SDHD and 12 mutations of SDHB in a large cohort of 271 patients with nonsyndromic pheochromocytoma.

In a previous report, we have described the functional consequences of an inactivating mutation of the SDHD gene, which results in a selective loss of complex II electron transfer activity and in activation of the hypoxic/angiogenic pathway revealed by an increase of endothelial PAS domain protein 1 (EPAS1), hypoxia inducible factor 1, and vascular endothelial growth factor (VEGF) expression in inherited tumors (5). Nonetheless, nothing is known about the functional consequences induced by SDHB variants on complex II mitochondrial activity and the hypoxic pathway.

Subject and Methods

Patient

The patient was a 55-yr-old female bearing a nonfamilial malignant pheochromocytoma. The diagnosis was suspected based on the clinical phenotype of uncontrolled hypertension associated with bouts of sweating. It was confirmed by measuring the 24-h urinary metanephrines, which were more than 100-fold the normal values. A highly vascularized adrenal mass was detected by computed tomography and whole-body scintigraphy with metaiodo-benzylguanidine. This voluminous mass, containing necrosis and calcifications, pressed back the right kidney and the liver (Fig. 1A). It extended to the right auricle via the superior cava vein (Fig. 1B). It weighed 350 g and measured 140 mm in diameter at surgery and was associated with pulmonary metastasis. The mother and the father of the patient were deceased, and no familial incidence of pheochromocytoma or paraganglioma was known in the kindred. The blood and tumoral samples were stored in a bank after obtaining informed consent. DNA was extracted from leukocytes and from the tumoral tissue according to standard procedures.

View larger version (106K): [in this window] [in a new window]   Figure 1. SDHB-induced pheochromocytoma A. Abdominal CT-scan. The arrows indicate a voluminous heterogeneous tumor with necrosis. B, Thoracic CT scan. The arrow points out the tumoral extension in the right auricle of the heart.

The SDHD and SDHC genes were sequenced as previously described (1, 3). The eight exons of the SDHB gene (GenBank, http://www.ncbi.nlm.gov/GenBank/; accession nos. U17296, U17880, U17881, U17882, U17883, U17884, U17885, and U17886) were amplified by PCR according to the conditions described by Astuti et al. (3). Annealing temperatures of 52 C were used, except for the exons 1 and 4 for which they were of 56 C and 50 C, respectively. The resulting PCR products were directly sequenced using an ABI 3700 fluorescence sequencer (PE Applied Biosystems, Courtaboeuf, France). A TaqI restriction enzyme digestion was used to screen the R46Q mutation in normal control chromosomes.

A search for a loss of heterozygosity (LOH) at the SDHB locus was performed using tumoral and peripheral DNA from SDHB-inherited pheochromocytoma tissue and from blood. Eleven fluorescent microsatellite markers (D1S243, D1S468, D1S2694, D1S244, D1S2667, D1S507, D1S199, D1S478, D1S2674, D1S2749, and D1S513) were analyzed. These markers lie on a 60-cM chromosomal region between 1p34.3 and 1p36.33 (telomere) and cover the SDHB locus (1p36.13). Germline and tumor DNA were amplified and analyzed with an ABI 3700 instrument (PE Applied Biosystems). The regional LOH at 11q23-qter (SDHD locus) was also tested as previously described (5).

Functional studies

Succinate cytochrome c reductase activities were measured spectrophometrically on SDHB-mutated pheochromocytoma tissue homogenates, as described elsewhere (5). The expression of five genes (EPAS1, VEGF, VEGFR-1, VEGFR-2, and neuron-specific enolase) encoding angiogenic and hypoxia-induced factors was studied in SDHB-mutated pheochromocytoma tissue by immunohistochemistry and in situ hybridization with procedures previously described (5, 9).

Results

SDHB mutation analysis

The direct sequencing of the eight exons of the SDHB gene in germline DNA showed a heterozygous G to A nucleotide transition in exon 2, changing the arginine to a glutamine at position 46 (R46Q) of the mature protein (Fig. 2A, electrophoregram b). This missense mutation replaced a highly conserved residue present in the SDHB coding sequences of species such as Escherichia coli, yeast, Drosophila melanogaster, and rat. A TaqI restriction enzyme digestion was used to screen the R46Q mutation, which was not found in 358 normal control chromosomes. Sequencing of the complete sequence of the SDHD and SDHC genes did not detect associated mutations. No normal SDHB allele was detected in somatic tumor DNA (Fig. 2A, electrophoregram c), suggesting a loss of the wild-type allele in the tumor. Loss of heterozygosity was studied with the analysis of 11 microsatellite markers at the SDHB locus on germline and tumoral DNA. A LOH was detected between D1S468 and D1S513, demonstrating a terminal deletion of 1p chromosome (Fig. 2B). No LOH was observed on SDHD locus (11q23 region).

View larger version (17K): [in this window] [in a new window]   Figure 2. Germline and somatic SDHB gene analysis. A, Sequence analysis of exon 2 of the SDHB gene in a control wild-type DNA (a), DNA extracted from leukocytes of affected subject (b), and DNA extracted from tumor (c). B, LOH analysis of 11 microsatellite markers at chromosome 1pter to 1p34 in SDHB-inherited pheochromocytoma. The white and black boxes represent noninformative markers (homozygosity) and LOH, respectively.

To study the functional consequences of this SDHB inactivating mutation, we have tested the activity of complex II in the mitochondrial respiratory chain by enzymatic experiments on pheochromocytoma tissue. We found that succinate dehydrogenase activity was abolished in the SDHB-inherited pheochromocytoma. The malonate-sensitive succinate cytochrome c reductase activity was measured at less than 0.01 nmol/min·mg protein, whereas a normal antimycin-sensitive quinol cytochrome c reductase (QCCR) activity (320 nmol/min·mg protein) was detected.

We have studied the expression of mRNAs encoding angiogenic and hypoxia-induced factors in tumoral tissue. We observed a high expression of EPAS1 and VEGF in tumor cells and of VEGF receptors VEGFR-1 and -2 and EPAS1 in vascular endothelial cells (Fig. 3). This stimulation of the angiogenic pathway was associated in our patient with a spectacular tumor vascularization and tumor growth.

View larger version (109K): [in this window] [in a new window]   Figure 3. Gene expression patterns in SDHB-inherited sporadic pheochromocytoma determined by in situ hybridization and immunohistochemistry. EPAS1 transcripts (A) are detected in tumor cells and, at a higher level, in vascular endothelial cells (arrows), whereas VEGF mRNA is strongly expressed in tumor cells (B and E) but not in the blood vessel wall (arrows). In a mirror image to their ligand’s expression pattern, VEGFR-1 (C and F) and VEGFR-2 (D) are specifically expressed in endothelial cells (arrows). Note the expression of neuron-specific enolase (G), as a marker for neuroendocrine differentiation of tumor cells. The in situ hybridization signals are visualized either under dark field (A–D; signal visible as white dots) or bright field illumination (E and F; labeling detected by black dots). Immunostaining (G) is revealed by a brown coloration. Scale bars, 100 µm.

In this study, we report the identification of a new germline mutation of the SDHB gene and its functional consequences, in an apparently sporadic malignant pheochromocytoma.

After the first description by Astuti et al. (3) of an exon 6 frameshift deletion in the SDHB gene of one patient and the recent report of 9 different mutations of SDHB gene in 12 subjects with nonsyndromic pheochromocytoma (8), the discovery of a new germline SDHB mutation in a patient harboring an apparently sporadic pheochromocytoma confirms the important role of this mitochondrial gene in the genetic determination of pheochromocytoma. The arginine at position 46, which is conserved in different species (3), seems to be an important residue. It is located near the ferredoxin domain of the protein (between codon 54 and codon 119), containing the [2Fe-2S] cluster, which participates in electron transfer between the quinol pool and the flavin (10). The Arg⁴⁶, cationic residue, could thus be important for the physical organization of the iron-sulfur clusters of the protein. Interestingly, this residue has been found as mutated in glycine in two patients with pheochromocytoma (8) and in glutamine in our patient. Whether it might represent a hot-spot site of mutation at the SDHB gene in pheochromocytomas will require further larger studies.

We have previously surveyed the consequences of an inactivating SDHD germline mutation associated with a loss of wild-type allele, which participates in hereditary paraganglioma development (5). The direct consequence of the SDHD mutation was a complete loss of electron transport chain complex II activity in mitochondria. We have observed the same result in the SDHB mutated pheochromocytoma. Altogether, our data show that both the anchor (SDHD) and the catalytic (SDHB) subunits are necessary for the enzymatic activity of complex II. In addition, the second consequence induced by SDHD mutation was an activation of angiogenesis through the increased expression of hypoxia-responsive genes in tumoral tissues. As in SDHD-induced tumors, we have also observed on SDHB-induced pheochromocytoma an activation of angiogenic pathway.

The R46Q SDHB-inherited pheochromocytoma was characterized by its malignant presentation. It is interesting to observe that our mutation was associated with a malignant pheochromocytoma, whereas a frameshift mutation within exon 6 of the SDHB gene was associated with a benign pheochromocytoma in the case reported by Astuti et al. (4). The loss of the wild-type allele in the tumor might be responsible for this difference because no LOH was present in the benign case. A high incidence of allelic loss of chromosome 1p has been reported in pheochromocytomas (11, 12, 13). Benn et al. (13) have suggested that the chromosome 1p contains three possible distinct regions of common somatic loss involved in the tumorigenesis of pheochromocytoma. In the inherited-pheochromocytoma described here, these three regions, PC1 (from D1S243 to D1S 244), PC2 (D1S228 to D1S507), and PC3 (D1S507 toward the centromere) were deleted. They contain candidate regions for putative tumor suppressor loci implicated in several cancer such as neuroblastoma (13). It will be of interest to determine the precise role of 1p in SDHB and SDHD-inherited pheochromocytomas.

In conclusion, we demonstrate for the first time in SDHB-mutated pheochromocytoma a dramatic disturbance of mitochondrial and hypoxia pathways similar to those induced by a SDHD gene mutation. These findings highlight the importance of inactivation of mitochondrial genes in neural crest-derived tumors in general and in pheochromocytomas in particular. Further molecular studies on different large collections of sporadic pheochromocytomas will be essential to evaluate the frequency of germline and somatic mutations of complex II mitochondrial genes and of associated LOH. Analysis of these genetic events might also give insight on the risk of recurrence and/or malignancy of these tumors.

Acknowledgments

Footnotes

This study was supported by Institut National de la Santé et de la Recherche Médicale and by Projet Hospitalier de Recherche Clinique Grant AOM 95201 for the Cortico et Medullosurrénale: Etude des Tumeurs Endocrines Network. J.F. is a recipient of a fellowship from la Ligue contre le cancer.

Abbreviations: EPAS1, Endothelial PAS domain protein 1; LOH, loss of heterozygosity; VEGF, vascular endothelial growth factor.

Received April 3, 2002.

Accepted June 25, 2002.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gimenez-Roqueplo, A.-P. Articles by Jeunemaitre, X. Search for Related Content PubMed PubMed Citation Articles by Gimenez-Roqueplo, A.-P. Articles by Jeunemaitre, X.