Mutations in SDHD, a Mitochondrial Complex II Gene, in Hereditary Paraganglioma

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Science  04 Feb 2000:
Vol. 287, Issue 5454, pp. 848-851
DOI: 10.1126/science.287.5454.848


Hereditary paraganglioma (PGL) is characterized by the development of benign, vascularized tumors in the head and neck. The most common tumor site is the carotid body (CB), a chemoreceptive organ that senses oxygen levels in the blood. Analysis of families carrying thePGL1 gene, described here, revealed germ line mutations in the SDHD gene on chromosome 11q23. SDHD encodes a mitochondrial respiratory chain protein—the small subunit of cytochrome b in succinate-ubiquinone oxidoreductase (cybS). In contrast to expectations based on the inheritance pattern of PGL, the SDHD gene showed no evidence of imprinting. These findings indicate that mitochondria play an important role in the pathogenesis of certain tumors and that cybS plays a role in normal CB physiology.

Regulation of oxygen homeostasis is essential for most organisms (1). In mammals, the CB, a highly vascular small organ located at the bifurcation of the common carotid artery in the neck, plays a major role in acute adaptation to hypoxia (oxygen deprivation) by stimulating the cardiopulmonary system (2). At the cellular level, this adaptation involves activation of a transcription factor, hypoxia-inducible factor–1 (HIF-1), which subsequently leads to a systemic response, including an increase in red blood cell mass, stimulation of new blood vessel growth, and increased ventilation (3). Chronic exposure to hypoxia (e.g., as occurs in individuals dwelling at high altitudes or in those with certain medical conditions, such as cyanotic heart and chronic lung diseases) induces cellular hyperplasia/anaplasia in the CB (4).

The CB is also the most common tumor site for hereditary paraganglioma (PGL), a rare disorder characterized by the development of mostly benign, highly vascular, slow-growing tumors in the head and neck. PGL tumors display cellular hyperplasia/anaplasia (5) in the absence of any hypoxic stimulus. A gene responsible for PGL was mapped to chromosome band 11q23 (PGL1) and remains the only locus confirmed in independent families (6–8).PGL1 is inherited in an autosomal dominant fashion with incomplete penetrance when transmitted through fathers, whereas no disease phenotype occurs when transmitted maternally. This inheritance pattern is observed in all confirmed PGL1 pedigrees and suggests that there is sex-specific epigenetic modification ofPGL1 during gametogenesis, consistent with genomic imprinting (9). This consistent inheritance pattern gives PGL a unique place among the known human genetic disorders with parent-of-origin effects (10). Several features of PGL tumors, including their benign biological behavior, limited organ involvement, and histopathology, are markedly similar to those of chronic hypoxia-stimulated CBs. This led us to hypothesize that the genetic defect in PGL1 involves a critical component in the oxygen-sensing and -signaling pathway.

We previously localized PGL1 to an approximately 1.5-Mb critical interval between D11S1986 and D11S1347 (8). BAC and yeast artificial chromosome (YAC) contig construction and discovery of 16 new simple tandem repeat polymorphisms (STRPs) (11) enabled us to confine PGL1 to an approximate 400-kb region (12) flanked by the recombination breakpoints in families 5 (7) and 12 (8) (Fig. 1).

Figure 1

(left). A summary map of the PGL1 critical region on chromosome 11q23. Two new STRPs confined the critical region to approximately 400 kb.SDHD was localized within the critical region with a BAC and YAC contig. The genomic structure of SDHDis shown schematically at the bottom but is not drawn to scale. Exons are denoted with boxes; introns are denoted with horizontal lines. The solid boxes in exons 1 and 4 represent the 5′– and 3′–untranslated regions, respectively.

Expressed sequence tag (EST) gene content mapping of transcripts (13) revealed a high density of transcripts in the 400-kbPGL1 critical region and in its close vicinity (11). A database search using BLAST with one of the ESTs in the critical region, EST229158 (14), identified a complete match to a genomic sequence (GenBank accession number AB026906) containing the exons of the succinate-ubiquinone oxidoreductase subunit D gene (SDHD). SDHD was previously mapped to chromosome band 11q23 by fluorescence in situ hybridization (FISH) (15) and its genomic structure has been characterized (16). We confirmed the location of SDHDin the PGL1 critical region by mapping it back to the BAC and YAC contigs, and placed it close to the telomeric exclusion border. SDHD spans over 19 kb at the genomic level, and its translated region consists of four exons of 52, 117, 145, and 163 base pairs (bp).

SDHD encodes the small subunit (cybS) of cytochrome b in succinate-ubiquinone oxidoreductase (mitochondrial complex II). Mitochondrial complex II is involved in the Krebs cycle and in the aerobic electron transport chain (17). It contains four proteins. The catalytic core consists of a flavoprotein and an iron-sulfur protein; these proteins are anchored to the mitochondrial inner membrane by the large subunit of cytochrome b (cybL) and cybS, which together comprise the heme-protein cytochrome b (18).

Because it has been postulated that the mitochondrial electron transport chain plays a critical role in oxygen sensing and signaling (19), we evaluated SDHD as a candidate gene for PGL1. To test for germ line mutations in SDHD, we initially selected five families (families 3, 5, 7, 8, and 11) who showed significant linkage to PGL1 and demonstrated distinct disease haplotypes (7, 8). Using two affected or carrier individuals from each family, we amplified each exon and its splice sites by primers designed from the flanking intronic sequences and subjected each amplicon to single-strand conformational polymorphism (SSCP) analysis (20). The SSCP analysis yielded at least one aberrant conformer that cosegregated with the disease chromosome in each family. Families 5 and 7 had the same aberrant conformer, whereas family 3 had a distinct aberrant conformer in exon 3. Family 11 had an aberrant conformer in exon 2 and family 8 showed distinct aberrant conformers in exons 2 and 3.

Direct sequencing of each amplicon revealed the sequence alterations responsible for the SSCP conformers (Fig. 2). Families 8 and 11 have nonsense mutations at codon 36 and codon 38, respectively, creating premature stop codons. Both stop codons are located within the mitochondrial signal peptide and presumably block production of mature cybS. Family 3 has a missense mutation, changing His102→Leu102. Families 5 and 7 have a missense mutation that changes Pro81→Leu81. We previously described extensive haplotype sharing among families 12, 24, and 26 (8). These families also show extensive haplotype sharing with family 7 and, as expected, they all have the same missense mutation. Finally, direct sequencing of SDHD from individuals who carry the Dutch founder mutation (21) revealed a missense mutation that changes Asp92→Tyr92. All of the missense mutations replace amino acids conserved in four eukaryotic multicellular organisms (Fig. 3). His102 is located in a region thought to harbor an axial ligand for heme in theEscherichia coli enzyme (22). The other two missense mutations result in nonconservative amino acid substitutions that could dramatically alter cybS conformation. None of the mutations has been observed in more than 200 normal control chromosomes. The mutations cosegregate with the disease phenotype in all affected individuals. The mutations are also inherited in a Mendelian fashion by all at-risk (paternal inheritance) and not-at-risk (imprinted maternal inheritance) carrier individuals as identified by haplotype analysis.

Figure 2

(right). The sequencing chromatograms show the alterations (denoted with arrowheads) responsible for the five major PGL1 mutations. The affected codons and the amino acids are depicted below the chromatograms.

Figure 3

Alignment of cybS amino acid sequences fromHomo sapiens, Bos taurus (domestic cattle),Ascaris suum (a parasitic nematode), andCaenorhabditis elegans, identified by BLASTP analysis of the Swissprot database, revealed conserved amino acids (denoted with asterisks) and was performed with Clustalw 1.7 (available The numbers indicate the mutated amino acids (in bold) in PGL families (Fig. 2) (32). The horizontal bars denote the amino acids with codons spanning exon-exon boundaries in the human genomic sequence. The vertical arrow denotes the amino terminus of the mature cybS.

We assessed allele-specific expression of SDHD to determine whether the lack of maternal disease transmission in PGL1 is caused by paternal monoallelic imprinted gene expression. We detected biallelic expression (23) in lymphoblastoid cell lines from affected and imprinted carrier individuals and in adult brain tissue as well as in fetal tissues from the brain and kidney (Fig. 4). Thus these results cannot explain why children of affected mothers do not develop PGL. Unlike most endogenously imprinted genes, SDHD is not located in an imprinted genomic domain (24). This observation has prompted the hypothesis that PGL1 mutations cause ectopic imprinting (7, 8). Given the single base pair mutations in SDHD and its biallelic expression in lymphoblastoid cells from affected and imprinted individuals, this hypothesis seems unlikely. Although the precise mechanism of the imprinted inheritance pattern in PGL1 remains to be determined, monoallelic expression of SDHD may be confined to the CB and other paraganglionic cells, similar to the brain-limited imprinting of UBE3A of Angelman syndrome (25).

Figure 4

Biallelic expression of humanSDHD. (A) Three independent fetal brain samples and one fetal kidney sample show expression of both alleles as detected by RT-PCR–restriction enzyme digestion analysis with Spe I. A homozygous brain sample is included in the right lane as a control for restriction enzyme digestion. (B) Two independent adult brain samples show biallelic expression. The method is the same as described in (A). A homozygous sample is included in the right lane as a control for restriction enzyme digestion. The numerals 1 and 2 at right denote the two alleles distinguished by the restriction enzyme digestion. (C) Lymphoblastoid cell lines from both PGL-affected and -imprinted carriers show biallelic expression. The mutation in family 5 was used for RT-PCR–restriction enzyme digestion analysis with Msp I. A lymphoblastoid cell line from a normal individual is included in the right lane as a control for restriction enzyme digestion. The mutant and the normal alleles are denoted at right by the numerals 1 and 2, respectively.

Tumors from PGL1 families often show loss of heterozygosity with exclusive loss of the normal maternal chromosome at 11q23 (7, 26). Our analysis of 11 hereditary tumors, nine of which were informative at STRPs within 500 kb ofSDHD, confirmed loss of maternal alleles in all nine cases. In four cases, including the two uninformative ones, the availability of highly enriched tumor cell populations by fluorescence-activated cell sorting allowed us to demonstrate that the mutated allele ofSDHD was retained and that the normal wild-typeSDHD allele was completely lost in the tumor (27). Germ line loss-of-function mutations in the paternal alleles and subsequent somatic loss of normal maternal alleles suggest that SDHD functions as a tumor suppressor gene at the cellular level and needs two events for inactivation. PGL is unusual among hereditary tumor syndromes in that the defective gene encodes a mitochondrial protein. The growth advantage conferred by the loss of maternal chromosomes indicates that imprinting is not absolute in the normal CB or that there is secondary relaxation of imprinting at the maternal allele during tumorigenesis.

On the basis of the phenotypic similarity between PGL tumors and the normal CB exposed to chronic hypoxia, we hypothesize that cybS is a critical component of the oxygen-sensing system of paraganglionic tissue, and that its loss may lead to chronic hypoxic stimulation and cellular proliferation. Hypoxic stimulation has also been implicated as an etiologic factor in von Hippel–Lindau disease tumors, where there is constitutive activation of HIF-1 (28). It is well known that solid tumors are hypoxic relative to normal tissue. This hypoxia not only affects gene expression patterns but it can also be an important prognostic factor and may make tumors resistant to radiation and certain forms of chemotherapy (29). Thus, hypoxic stimulation may provide a selective advantage for tumor cells (30). Interestingly, the SDHD-containing region on chromosome 11q23 (7) is a common site of somatic deletions in bladder, breast, cervical, stomach, lung, ovary, and nasopharyngeal carcinomas, as well as melanoma (31). Identification of SDHD as the gene responsible for PGL will likely increase our understanding of tumorigenesis and may also have future therapeutic potential.

  • * To whom correspondence should be addressed. E-mail: baysalbe{at}


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