SDHA is a tumor suppressor gene causing paraganglioma

Abstract

Mitochondrial succinate-coenzyme Q reductase (complex II) consists of four subunits, SDHA, SDHB, SDHC and SDHD. Heterozygous germline mutations in SDHB, SDHC, SDHD and SDHAF2 [encoding for succinate dehydrogenase (SDH) complex assembly factor 2] cause hereditary paragangliomas and pheochromocytomas. Surprisingly, no genetic link between SDHA and paraganglioma/pheochromocytoma syndrome has ever been established. We identified a heterozygous germline SDHA mutation, p.Arg589Trp, in a woman suffering from catecholamine-secreting abdominal paraganglioma. The functionality of the SDHA mutant was assessed by studying SDHA, SDHB, HIF-1alpha and CD34 protein expression using immunohistochemistry and by examining the effect of the mutation in a yeast model. Microarray analyses were performed to study gene expression involved in energy metabolism and hypoxic pathways. We also investigated 202 paragangliomas or pheochromocytomas for loss of heterozygosity (LOH) at the SDHA, SDHB, SDHC and SDHD loci by BAC array comparative genomic hybridization. In vivo and in vitro functional studies demonstrated that the SDHA mutation causes a loss of SDH enzymatic activity in tumor tissue and in the yeast model. Immunohistochemistry and transcriptome analyses established that the SDHA mutation causes pseudo-hypoxia, which leads to a subsequent increase in angiogenesis, as other SDHx gene mutations. LOH was detected at the SDHA locus in the patient's tumor but was present in only 4.5% of a large series of paragangliomas and pheochromocytomas. The SDHA gene should be added to the list of genes encoding tricarboxylic acid cycle proteins that act as tumor suppressor genes and can now be considered as a new paraganglioma/pheochromocytoma susceptibility gene.

Figure 1.

Microarray analysis of genes involved in oxidative phosphorylation and glycolysis pathways in SDHA-mutated paraganglioma and other inherited paraganglioma/pheochromocytoma. Unsupervised hierarchical clustering analysis of 69 samples according to the expression of 80 genes (150 probes) involved in oxidative phosphorylation and 23 glycolysis-associated genes (46 probes). Expression profiles are shown as a heat map indicating high (red) and low (green) expression according to a log2-transformed scale. The higher bipartition allows distinguishing VHL (white), SDH (grey), and RET and NF1 (black) tumors. The SDHA-tumor (in yellow, indicated by an asterisk) is classified with SDHx-tumors.

Figure 2.

Identification of the SDHA mutation, associated with LOH at the SDHA locus. (A) Sequencing chromatograms of SDHA in the region of the mutation. Direct sequencing of the patient's DNA extracted from leukocytes (left panel) shows the SDHA heterozygous mutation c.1765C>T. Direct sequencing of the DNA extracted from the patient's tumor tissue (middle panel) and of the cDNA obtained by RT–PCR of RNA extracted from frozen tumor tissue (right panel) show the predominance of the mutant allele (T) in the tumor tissue suggesting an LOH at the SDHA locus. The faint persistence of a WT (C) peak is probably imputable to non-tumor cell contaminations (e.g. endothelial cells and stromal cells). (B) Alignment of SDHA homologues. The sequences surrounding arginine Arg589 (arrow) of human and other animal, plant and bacterial SDHA have been aligned by blastp. The short conserved sequence with Arg589 is enclosed by the box. (C) BAC array CGH analysis reveals 5p, 1p, 22q losses and partial 7p gain in SDHA tumor.

Figure 3.

Functional consequences of the SDHA mutation expressed in yeast. (A) Growth phenotype on respiratory media (minimal glycerol/ethanol) and glycolytic media (minimal glucose). Ten-fold serial dilutions of the WT parent (W303-1A), the SDH1 null mutant (aW303ΔSDH1), the null mutant with an integrated copy of SDH1 (aW303ΔSDH1/ST20) and the null mutant with an integrated copy of the Arg582Trp mutant gene (aW303ΔSDH1/ST19) were plated on the minimal glycerol/ethanol and on the minimal glucose plates. Growth was recorded after 2 days on glucose and 4 days on glycerol/ethanol. (B) Western blot analysis of Sdh1 and OSCP, a subunit of mitochondrial ATP synthase. Mitochondria were prepared from cells grown in rich YPGal (2% galactose, 1% yeast extract and 2% peptone) by the method of Meisinger et al. (34). Mitochondrial proteins (40 µg) were separated on a 12% polyacrylamide gel. They were transferred to a nitrocellulose membrane and probed with polyclonal antibodies against Sdh1 and OSCP, used as a loading control. A rabbit polyclonal antibody was obtained against a fusion protein consisting of the N-terminal half of anthranilate synthase component 1 fused to residues 158–498 of SDH (35). No Sdh1 signal is detected in mitochondria of aW303ΔSDH1/ST19, the transformant harboring a chromosomally integrated copy of the mutant gene. (C) Structure of chicken complex II. The arginine 589 homologue is indicated by the arrow on the ribbon representation of chicken SDHA (in yellow) and is located distal to subunits SDHB (green), SDHC (red) and SDHD (blue). The environment of arginine 589 shows the conserved polar interactions in the structure of WT SDHA that are lost in the Arg582Trp mutant. R590, E588, D560 and D132 of chicken SDHA are homologous to R589, E587, D559 and D131 of the human protein, respectively.

Figure 4.

Expression of SDHA, SDHB and COX-IV in SDHA-mutated paraganglioma compared with other inherited paraganglioma/pheochromocytoma. SDHA-positive immunostaining is observed in the chromaffin cells of SDHB- and RET-related tumors. In the patient's tumor, it is detected solely in blood vessels and not in tumor cells. SDHB protein expression is lost in both SDHA- and SDHB-mutated tumors, whereas it is still present in RET-related pheochromocytomas. Subunit COX-IV of mitochondrial cytochrome c oxidase is expressed at comparable levels in all tumors. Calibration bar: 100 µm.

Figure 5.

Pseudohypoxia and angiogenesis in SDHA-mutated tumor compared with other inherited paraganglioma/pheochromocytoma. (A) HIF1-α nuclear immunostaining is detected in tumors with mutations in either SDHA- or SDHB- but not in RET-related pheochromocytomas. Calibration bar: 100 µm. CD34 immunohistochemistry was performed to evaluate angiogenesis in the SDHA-mutated paraganglioma and compared with other inherited paraganglioma/pheochromocytoma. Histogreen was used as a chromogen for detection (blue labeling). Quantification of vascular density reveals that the SDHA-mutated paraganglioma is highly vascularized, as are other SDHx-related tumors. (B) Unsupervised hierarchical clustering analysis of 69 samples according to the expression of 54 HIF-1 and/or HIF-2 targets (122 probes). Expression profiles are shown as a heat map indicating high (red) and low (green) expression according to a log2-transformed scale. The higher bipartition allows pseudo-hypoxic VHL- (white) and SDHx- (gray) to be distinguished from RET and NF1 (black) tumors. The SDHA paraganglioma (in yellow indicated by an asterisk) is classified with SDHx tumors.