¹International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, Cedex 08, France

²Institute of Neuropathology, Universitiy Hospital, 8091 Zürich, Switzerland

³Institute of Neuropathology, University of Münster, 48129 Münster, Germany

⁴Department of Pathology, Hadassah University Hospital, 91120 Jerusalem, Israel

⁵Laboratory of Neuropathology, Victor Segalen University, 33076 Bordeaux, France

⁶Division of Pathology, University Hospital, Federal University of Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil

⁷Laboratory of Neuropathology, University Hospital, 69003 Lyon, France

⁸Department of Neurosurgery, University Hospital, 8091 Zürich, Switzerland

Correspondence to: H Ohgaki, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, Cedex 08, France. E-mail: ohagaki@iarc.fr

Hereditary paraganglioma of the head and neck is associated with germline mutations in the SDHD gene, which encodes a mitochondrial respiratory chain protein. Paragangliomas of the central nervous system are very rare, occur almost exclusively in the cauda equina of the spinal cord and are considered non-familial. In the present study, we screened 22 apparently sporadic paragangliomas of the cauda equina for SDHD mutations. One spinal paraganglioma and similar cerebellar tumours that developed 22 years later in the same patient contained a missense mutation at codon 12 (GGTAGT, GlySer) and a silent mutation at codon 68 (AGCAGT, SerSer). There was no family history of paragangliomas but DNA from white blood cells of this patient showed the same sequence alterations, indicating the presence of a germline mutation. All other cases of spinal paraganglioma had the wild-type SDHD sequence, except one case with a silent mutation at codon 68 (AGCAGT, SerSer). This is the first observation indicating that inherited SDHD mutations may occasionally cause the development of paragangliomas in the central nervous system. Oncogene (2001) 20, 5084-5086.

SDHD; germline mutation; paraganglioma; cauda equina

Paragangliomas are neoplasms of ubiquitous distribution in the human body, and originate from the neural crest (Brodeur and Shimada, 1998). Up to 50% of cases are familial (Grufferman et al., 1980; van der Mey et al., 1989). Hereditary paragangliomas are benign, slow-growing tumors of parasympathetic ganglia. They typically develop in the carotid body located at the bifurcation of the common carotid artery, a small chemoreceptive organ that senses oxygen levels in the blood, but also develop in the jugular foramen, the vagal nerve and the middle ear (van Baars et al., 1982). Inheritance occurs as an autosomal dominant trait with incomplete penetrance. The disease phenotype develops only if transmitted through the father, suggestive of involvement of genetic imprinting (van der Mey et al., 1989).

Linkage studies have revealed two distinct loci on chromosome 11q, suggesting heterogeneity of this disease. The most common locus is in the chromosomal region 11q22.3-q23, and another locus is in the 11q13.1 region (Heutink et al., 1992, 1994; Mariman et al., 1995). The SDHD gene located at 11q23 was recently shown to be mutated in the germline of families with hereditary paragangliomas (Baysal et al., 2000). SDHD encodes the small subunit of cytochrome b (cybS) in succinate-ubiquinone oxidoreductase (mitochondrial complex II), which is involved in the Krebs cycle (tricarboxylic acid cycle) and in the aerobic electron transport chain (Scheffler, 1998). More recently, the third locus involved in hereditary paragangliomas (PGL3) has been identified (Niemann et al., 1999). Recent analyses by Niemann and Muller (2000) demonstrated a germline mutation in the SDHC gene on this locus in patients from a family with the non-maternally imprinted hereditary paragangliomas.

Paragangliomas of the central nervous system (CNS) are almost exclusively located at the lower end of the spinal canal, in the region of the cauda equina (Soffer and Scheithauer, 2000). To date, no association has been reported between paragangliomas of cauda equina and those at other sites (Soffer and Scheithauer, 2000). In contrast to paragangliomas of the head and neck, there have been no reports of familial occurrence of cauda equina paragangliomas.

In this study, we assessed whether mutational inactivation of SDHD is involved in the development of paragangliomas of the CNS. SSCP followed by DNA sequencing revealed that one paraganglioma of the cauda equina and a similar cerebellar lesion from the same patient contained a missense mutation at codon 12 (GGTAGT, GlySer, Figure 1) and a silent mutation at codon 68 (AGCAGT, SerSer). DNA from white blood cells of this patient showed the same sequence alterations (Figure 1), indicating that this is a germline mutation.

The other tumors had the wild-type SDHD sequence, except for one biopsy with a silent mutation at codon 68 (AGCAGT, SerSer). Thus, there was no somatic SDHD miscoding mutation in any of the sporadic paragangliomas of the cauda equina examined in this study, suggesting that this gene does not play a role in the development of these neoplasms. It remains to be shown whether this is also true for the other, as yet unidentified locus on chromosome 11q13.1 or SDHC gene associated with familial paragangliomas (Niemann and Muller, 2000).

The patient with an SDHD germline mutation was admitted at the age of 39 years with a 1-year history of back pain radiating to both thighs. Radiological diagnostic procedures revealed a tumor in the cauda equina region. Following surgical resection, the patient was disease-free but presented 22 years later with a 2-year history of ataxic gait and recent episodes of nausea. MRI revealed a local recurrence in the cauda equina and multiple cerebellar lesions, some of the latter being cystic. The patient was operated for both the cerebellar and cauda equina tumors. The spinal and cerebellar lesions were diagnosed as paragangliomas. Details of the neuroimaging and histopathology of the primary and the recurrent spinal tumor as well as the cerebellar neoplasms have been previously reported (Strommer et al., 1995). Six years later, the patient is in satisfactory condition, although follow-up neuroimaging has revealed several small, slowly growing nodular, contrast-enhancing lesions in the cerebellum at the former operation site. The patient is married and has five children; one son developed a neurological disease shortly after birth, with epilepsy and mental retardation. All other children are healthy, and there is no family history of neoplastic disease.

Multiple tumors are observed in 4-10% of sporadic and in 32-50% of hereditary head and neck paragangliomas (Grufferman et al., 1980; Spector et al., 1975; van Baars et al., 1982). In contrast, there has been no report of multiple paragangliomas of the spinal cord. The patient with a SDHD germline mutation in this study developed a paraganglioma of the cauda equina and, 22 years later, of the cerebellum. The cerebellar tumors manifesting 22 years after the initial surgical resection were first considered metastatic (Strommer et al., 1995). However, in the light of the present finding of a germline SDHD mutation, the possibility of second primary neoplasms should also be considered. The first spinal neoplasm was localized, well encapsulated and apparently completely removed (Strommer et al., 1995). Considering the slow growth of this tumor type, the second spinal tumour could be a late recurrent lesion but again, a second primary neoplasm at this preferred site cannot be ruled out.

The present study shows that SDHD germline mutations are not restricted to familial paragangliomas of the head and neck but may occasionally cause the development of paragangliomas in the cauda equina and other CNS sites. Contrary to previous observations, this study also shows that a small fraction of cauda equina paragangliomas may develop in families with a germline SDHD mutation.

Hereditary paragangliomas occur as an autosomal dominant trait with incomplete penetrance and manifest only when transmitted through the father. The molecular mechanism of this pattern of inheritance is not yet fully understood. One possible explanation is genomic imprinting of the maternal allele during female oogenesis (van der Mey et al., 1989). However, a recent study by Baysal et al. (2000) showed biallelic SDHD expression in lymphoblastoid cells from affected individuals as well as in three independent normal fetal brain and one fetal kidney samples. Furthermore, SDHD mutations were always found in the paternal allele while loss of heterozygosity (LOH) at the SDHD locus was present in the maternal allele of all nine hereditary paragangliomas analysed to date (Baysal et al., 2000). Despite the possibility of cell type-specific monoallelic SDHD expression in paraganglionic cells, there is no direct evidence of genomic imprinting in hereditary paragangliomas (Baysal et al., 2000). The father of our patient with cauda equina paraganglioma did not suffer from CNS or head and neck paraganglioma; one son with neurological disease may be affected, but a DNA sample was not available for genetic analysis.

Tumors from families with hereditary paragangliomas often show exclusive loss of the normal maternal allele at 11q23 (Baysal et al., 1997; van Schothorst et al., 1998). Germline missense mutations in paternal alleles and subsequent somatic loss of maternal wild-type alleles are consistent with the hypothesis of inactivation of a tumor suppressor gene (Knudson, 1971). The paragangliomas from our case with a germline mutation showed both mutated and wild-type bases, possibly due to contamination of the sample with non-neoplastic cells.

The SDHD gene encodes the small subunit (cybS) of cytochrome b in succinate-ubiquinone oxidoreductase (also termed mitochondrial complex II), an important enzyme involved in the Krebs cycle and in the aerobic electron transport chain (Scheffler, 1998). Mitochondrial complex II catalyzes the oxidation of succinate to fumarate (succinate dehydrogenase: SDH) and transfers its reducing equivalent to ubiquinone (Hägerhäll, 1997; Scheffler, 1998). The catalytic core of mitochondrial complex II consists of a flavoprotein and an iron-sulfur protein, which are highly conserved, while those of cybS show little similarity to those among species (Hirawake et al., 1997). However, cybS acts as a hydrophobic membrane-anchor protein (Hägerhäll and Hederstedt, 1996; Oyedotun and Lemire, 1999) and is essential for the interaction between the mitochondrial complex II and quinone species (Shenoy et al., 1997). The SDHD germline mutations so far identified in hereditary paragangliomas occurred in codons 36, 38, 81, 92 and 102 and were either nonsense mutations leading to premature stop codons or missense mutations that could significantly alter the conformation of cybS (Baysal et al., 2000). The germline mutation found in this study (GlySer at codon 12) has not been reported before. It remains to be shown whether the type of mutation plays a role in the site-specific development of paragangliomas.

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Figure 1 DNA sequencing autoradiographs of an identical GA transition mutation at codon 12 of the SDHD gene in DNA from a paraganglioma of the cauda equina and from white blood cells from the same patient, indicating a germline mutation. Paragangliomas of the cauda equina were obtained from the Department of Pathology, University Hospital, Zurich, Switzerland (five cases), the Institute of Neuropathology, University of Münster, Germany (five cases), the Department of Pathology, Hadassah University Hospital, Israel (three cases), the Department of Pathology, University of Sao Paulo, Brazil (three cases), the Laboratory of Neuropathology, Victor Segalen University, Bordeaux, France (three cases), and the Laboratory of Neuropathology, Neurology Hospital, Lyon, France (three cases). The mean age of patients was 52 years (range 13-73 years), and 12 patients were males and 10 were females. Diagnoses of paragangliomas were confirmed immunohistochemically using antibodies to the neuronal marker proteins synaptophysin, chromogranin A and S-100 (Soffer and Scheithauer, 2000). DNA was extracted from paraffin sections as described previously (Watanabe et al., 1996). Pre-screening for mutations in exons 1-4 of the SDHD gene was carried out by PCR-SSCP analysis, using the primers reported previously (Baysal et al., 2000). Direct DNA sequencing was carried out for samples that displayed mobility shifts as previously described (Reis et al., 2000)