Allelic loss of the long arm of chromosome 11 is frequent in neuroendocrine tumors (NET) of different organs. However, the MEN1 gene on 11q13 is mutated only in a subset of NET and allelic losses on 11q frequently extend to the telomere. In this genetic region lies the tumor suppressor gene SDHD which is associated with hereditary paragangliomas (PGL1). We sought to determine whether SDHD plays a role in the development of sporadic NET. By mutation and deletion analysis of SDHD we were unable to detect any SDHD mutation in 45 NET of the lung, gastrointestinal tract, pancreas or parathyroid. However, we found allelic deletions in 20 to 50% of all tumors but parathyroid adenomas. Furthermore, we found heterozygous germline variants in 2/8 paragangliomas. A first case of variant c.149 A>G (H50R) was found in a patient with an extra-adrenal pheochromocytoma, the other variant c.34 G>A (G12S) in a patient with a paratracheal paraganglioma, C-cell hyperplasia of the thyroid and hyperplasia of ACTH-producing cells of the pituitary gland. Both variants were absent in 93 controls. Our results demonstrate that somatic SDHD mutations are rare in sporadic NET. However, LOH alone could lead to a complete loss of function since SDHD is an imprinted gene. Furthermore, we describe two germline variants possibly causing hereditary paragangliomas.
Tumors of the diffuse neuroendocrine system are defined by the common expression of neuroendocrine markers and share certain morphologic features. They encompass neuroendocrine tumors (NET) of the lung and gastrointestinal tract (carcinoids), endocrine pancreatic tumors (EPT), parathyroid adenomas (PTA), pheochromocytomas (PCC) and paragangliomas (PGA). The genetic background of the sporadic forms of these tumors is not well established, in contrast to the familial forms which are associated with the MEN1, MEN2 or VHL syndromes. Several studies on different types of sporadic NET have shown frequent allelic loss of the long arm of chromosome 11. More specifically, LOH and CGH studies have demonstrated a loss of 11q in 26 to 53% of PTA, 28% of PCC, 25 to 80% of NET of the lung and gastrointestinal tract and 28 to 69% of EPT (Table 1). In addition, the allelic loss has been shown to extend to the telomere of 11q in all types of NET investigated (Table 1). These findings point towards a tumor suppressor gene located on 11q involved in the tumorigenesis of sporadic NET with the MEN1 gene on 11q13 representing a candidate. However, we and others could demonstrate that only a subset of EPT, PTA and PCC carries mutations of the MEN1 gene and that these tumors exhibit a 2–3-fold higher frequency of 11q13 LOH when compared to mutation rates (Table 1). Other studies and our own data showing isolated chromosomal losses more telomeric of the MEN1 gene also indicate that there might be other tumor suppressor genes involved in the initiation and progression of NET (Eubanks et al., 1994; Chakrabarti et al., 1998; Gortz et al., 1999; Rigaud et al., 2001). One of the regions of interest which has been narrowed down is 11q23 (Rigaud et al., 2001). It harbors the tumor suppressor gene succinate dehydrogenase subunit D (SDHD) (Hirawake et al., 1999) which acts as hydrophobic membrane anchor for the catalytically active subunits of cytochrome II. In addition, it participates in electron transport and interacts with quinones (Scheffler, 1998). Indirect evidence is in favor for a tumor suppressor function of SDHD in endocrine tumors: It is responsible for familial PGA type 1 (PGL1, OMIM 168000); it is mutated in 10% of apparently sporadic PCC (Gimm et al., 2000) and it seems to be selectively imprinted in some neuroendocrine tissues (van der Mey et al., 1989).
The aim of the present study was to investigate a possible role of SDHD inactivation in different types of sporadic NET of different organ locations. We therefore examined 62 sporadic NET for SDHD mutations by PCR/SSCP and allelic deletions.
Somatic SDHD mutations were undetectable in 21 EPT, 14 NET of the gastrointestinal tract and the lung, nine PCC, eight PGA and 10 PTA (Table 2). The only somatic SDHD mutation reported to date was found in 1/18 sporadic PCC (5%) (Gimm et al., 2000). However, a definite answer about the role of SDHD in these tumors cannot be given. The disease phenotype of familial PGL1 caused by SDHD mutations is inherited as an autosomal dominant trait with incomplete penetrance when transmitted through fathers whereas no disease phenotype occurs when transmitted maternally. This inheritance pattern is consistent with genomic imprinting of the maternal allele of the SDHD gene (van der Mey et al., 1989). However, allelic expression analysis in fetal brain and lymphomatoid cell lines revealed biallelic expression (Malik et al., 2000). Recently, SDHD was shown to be monoallelically expressed in paragangliomas (Badenhop et al., 2001). If SDHD is also a monoallelically expressed tumor suppressor in other endocrine tissues, it is predisposed to gene inactivation through a single genetic ‘hit’ by loss of the expressed paternal allele, leading to a cell devoid of SDHD activity. With the exception of PTA, which exhibited neither SDHD mutations nor LOH, the examined neuroendocrine tumor types showed allelic losses of the SDHD locus in 20 to 57% (Table 2, Figure 1). Thirty-four out of 44 (77%) pairs of tumor and germline DNA were informative for at least one of the two markers. Ongoing work is examining whether there is selective allelic loss of the non imprinted allele and whether SDHD expression is completely lost in these tumors.
Although we studied clinically sporadic NET, we detected two amino acid variants of the SDHD gene in tumor and germline DNA of two patients, indicating a heritable tumor predisposition (Figure 2). They were found in one patient (PGA8) suffering from a para-adrenal sympathic PGA (para-adrenal pheochromocytoma) and in one patient (PGA3) with a cervical PGA among a total of eight patients with PGA (25%). The former patient exhibited an SDHD variant H50R in exon 2 which has not yet been described while the G12S variant in exon 1 found in the other patient has recently been described in another pheochromocytoma patient (Gimm et al., 2000). The phenotype of our patient (PGA3) with a G12S germline variation was exceptional, consisting of a paratracheal PGA, C-cell hyperplasia of the thyroid and hyperplasia of ACTH producing cells of the pituitary suggestive of a predisposition to neuroendocrine tumors. History taking revealed these two patients having no family background of PGA or of other NET. In order to elucidate whether these SDHD variants represent polymorphisms or mutations, we examined blood samples of 93 unrelated control persons (186 control alleles) without a history of endocrine tumors and did not detect any SDHD variant in exons 1 and 2. While Baysal and collaborators did not find these two variants in 200 control chromosomes (Baysal et al., 2000, Gimm et al. (2000) described the G12S variant in one patient with a pheochromocytoma and in 1/78 control alleles. Our data provide further evidence that this G12S variant is a mutation causing familial PGA and PCC. Both PGA and PCC are tumors that have been underdiagnosed in the past and the complex inheritance pattern due to imprinting as well as the incomplete penetrance makes it even more difficult to clinically recognize familial forms.
The fact that the tumor PGA3 showed retention of heterozygosity of both flanking polymorphic markers is intriguing since most of the examined tumors from PGL1 patients showed LOH of the wildtype allele, including the PCC patient with the same germline sequence variant G12S. Apart from technical problems, i.e. admixture of non-neoplastic tissue in the tumor DNA, which we would like to exclude due to careful microdissection, there are at least three possible explanations for this finding. First, there might be only a small allelic loss spanning neither of the two adjacent markers. Second, one allele might be inactivated by epigenetic mechanisms as methylation (Baysal et al., 1999, 2000) or third and most probably, one allele is the imprinted maternal allele (Badenhop et al., 2001).
SDHD is unusual among tumor suppressor genes in that it encodes the small subunit of cytochrome b (CybS), a mitochondrial protein. Cytochrome b is part of the mitochondrial complex II (succinate-ubiquinone oxidoreductase) which is involved in the citric acid cycle (Hagerhall, 1997). CybS acts together with CybL as hydrophobic membrane anchor for the catalytically active flavoprotein and iron-sulfur subunits. In addition, it participates in electron transport and interaction with quinones (Scheffler, 1998). The sequence variant of PGA3 in exon 1 changes Gly to a larger neutral and hydrophilic Ser in exon 1. This could potentially impair correct localization of the protein as the amino-terminal sequence directs it to mitochondrial and the appropriate mitochondrial compartment (Koehler, 2000; Pfanner, 2000). In PGA8, His is replaced by Arg. A specific function of this region is not known but this amino acid change could result in a conformational change of the protein. To date no functional analysis exists that examined the cellular effect of these amino acid variants.
In conclusion we demonstrate the lack of somatic SDHD mutations in NET of the lung, gastrointestinal trace, pancreas and parathyroids. However, LOH rates ranging from 20 to 50% except in PGA, do not exclude a role of SDHD in the examined tumors, as the gene is potentially imprinted in these tissues. Studies regarding the SDHD expression in NET will clarify this issue.
We detected two germline sequence variants in PGA which potentially are mutations. As the familial nature of this disease is difficult to recognize clinically, screening of PGA and PCC patients for SDHD mutations seems to be mandatory.
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Supported by Swiss Cancer League grant SKL-997-02-2000 and Swiss National Science Foundation grant 31-618845.00.
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Perren, A., Barghorn, A., Schmid, S. et al. Absence of somatic SDHD mutations in sporadic neuroendocrine tumors and detection of two germline variants in paraganglioma patients. Oncogene 21, 7605–7608 (2002). https://doi.org/10.1038/sj.onc.1205812
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