Download Functional Consequences of a SDHB Gene Mutation in an

0013-7227/02/$15.00/0
Printed in U.S.A.
The Journal of Clinical Endocrinology & Metabolism 87(10):4771– 4774
Copyright © 2002 by The Endocrine Society
doi: 10.1210/jc.2002-020525
Functional Consequences of a SDHB Gene Mutation in
an Apparently Sporadic Pheochromocytoma
ANNE-PAULE GIMENEZ-ROQUEPLO, JUDITH FAVIER, PIERRE RUSTIN, CLAUDINE RIEUBLAND,
VÉRONIQUE KERLAN, PIERRE-FRANÇOIS PLOUIN, AGNÈS RÖTIG, AND XAVIER JEUNEMAITRE
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
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
M
AJOR 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 (1–3). 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
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. (J Clin Endocrinol Metab 87: 4771– 4774, 2002)
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.
Genetic analysis
Abbreviations: EPAS1, Endothelial PAS domain protein 1; LOH, loss
of heterozygosity; VEGF, vascular endothelial growth factor.
The SDHD and SDHC genes were sequenced as previously described
(1, 3). The eight exons of the SDHB gene (GenBank, http://www.ncbi.
4771
4772
J Clin Endocrinol Metab, October 2002, 87(10):4771– 4774
Gimenez-Roqueplo et al. • SDHB Gene and Pheochromocytoma
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).
Mitochondrial and oxygen-sensing functional studies
FIG. 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.
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
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䡠mg protein, whereas a normal
antimycin-sensitive quinol cytochrome c reductase (QCCR)
activity (320 nmol/min䡠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.
Discussion
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
Gimenez-Roqueplo et al. • SDHB Gene and Pheochromocytoma
J Clin Endocrinol Metab, October 2002, 87(10):4771– 4774 4773
FIG. 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.
FIG. 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 ␮m.
4774 J Clin Endocrinol Metab, October 2002, 87(10):4771– 4774
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 hotspot 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 (11–13). 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 SDHBmutated 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
Gimenez-Roqueplo et al. • SDHB Gene and Pheochromocytoma
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
Received April 3, 2002. Accepted June 25, 2002.
Address all correspondence and requests for reprints to: Dr. AnnePaule Gimenez-Roqueplo, Département de Génétique Moléculaire, Hôpital Européen Georges Pompidou, 20 – 40, rue Leblanc, 75015 Paris,
France. E-mail: [email protected].
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.
References
1. Baysal BE, Ferrell RE, Willett-Brozick JE, Lawrence EC, Myssiorek D, Bosch
A, Van Der Mey A, Taschner PEM, Rubinstein WS, Myers EN, Richard III
CW, Cornelisse CJ, Devilee P, Devlin B 2000 Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287:848 – 851
2. Niemann S, Müller U 2000 Mutations in SDHC cause autosomal dominant
paraganglioma, type 3. Nat Genet 26:268 –270
3. Astuti D, Latif F, Dallol A, Dahia PL, Douglas F, George E, Skoldberg
Husebye ES, Eng C, Maher ER 2001 Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibilty to familial pheochromocytoma
and to familial paraganglioma. Am J Hum Genet 69:49 –54 2002 [Published
Erratum in Am J Hum Genet 70:565]
4. Chandel NS, Schumacker PT 2000 Cellular oxygen sensing by mitochondria:
old question, new insight. J Appl Physiol 88:1880 –1889
5. Gimenez-Roqueplo AP, Favier J, Rustin P, Mourad JJ, Plouin PF, Corvol P,
Rötig A, Jeunemaitre X 2001 The R22X mutation of the SDHD gene in hereditary paraganglioma abolishes the enzymatic activity of complex II in the
mitochondrial respiratory chain and activates the hypoxia pathway. Am J Hum
Genet 69:1186 –1197
6. Astuti D, Douglas F, Lennard TWJ, Aligianis IA, Woodward ER, Evans GR,
Eng C, Latif F, Maher ER 2001 Germline SDHD mutation in familial phaeochromocytoma. Lancet 357:1181–1182
7. Gimm O, Armanios M, Dziema H, Neumann HP, Eng C 2000 Somatic and
occult germ line mutations in SDHD, a mitochondrial complex II gene, in
nonfamilial pheochromocytoma. Cancer Res 60:6822– 6825
8. Neumann H PH, Bausch B, McWhinney SR, Bender BA, Gimm O, Franke
G, Schipper J, Klisch J, Altehoefer C, Zerres K, Januszewicz A, Eng C 2002
Germ-line mutations in nonsyndromic pheochromocytoma. N Engl J Med
346:1459 –1466
9. Sibony M, Commo F, Callard P, Gasc JM 1995 Enhancement of mRNA in situ
hybridization signal by microwave heating. Lab Invest 73:586 –591
10. Cecchini G, Schröder I, Gunsalus RP, Maklashina E 2002 Succinate dehydrogenase and fumarate reductase from Escherichia coli. Biochim Biophys Acta
1553:140 –157
11. Vargas MP, Zhuang Z, Wang C, Votmeyer A, Linehan WM, Merino MJ 1997
Loss of heterozygosity on the short arm of chromosomes 1 and 3 in sporadic
pheochromocytoma and extra-adrenal paraganglioma. Hum Pathol 28:
411– 415
12. Dannenberg H, Speel EJ, Zhao J, Saremaslani P, van Der Harst E, Roth J,
Heitz PU, Bonjer HJ, Dinjens WN, Mooi WJ, Komminoth P and de Krijger
RR 2000 Losses of chromosomes 1p and 3q are early genetic events in the
development of sporadic pheochromocytomas. Am J Pathol 157:353–359
13. Benn DE, Dwight T, Richardson AL, Delbridge L, Bambach CP, Stowasser
M, Gordon RD, Marsh DJ, Robinson BG 2000 Sporadic and familial pheochromocytomas are associated with loss of at least two discrete intervals on
chromosome 1p. Cancer Res 60:7048 –7051