Clinical Characteristics of Pheochromocytoma Patients With Germline Mutations in SDHD
http://www.100md.com
《临床肿瘤学》
the Department of Pathology, Josephine Nefkens Institute, Erasmus MC, University Medical Center, Rotterdam
Department of Pathology, St Radboud University Medical Center, Nijmegen, the Netherlands
Institute of Pathology, Hospital Baden, Baden, Switzerland
ABSTRACT
PURPOSE: We examined the value of SDHD mutation screening in patients presenting with apparently sporadic and familial pheochromocytoma for the identification of SDHD-related pheochromocytomas.
PATIENTS AND METHODS: This retrospective study involved 126 patients with adrenal or extra-adrenal pheochromocytomas, including 24 patients with a family history of multiple endocrine neoplasia 2, von Hippel-Lindau disease, neurofibromatosis type 1, or paraganglioma (PGL). Conformation-dependent gel electrophoresis and sequence determination analysis of germline and tumor DNA were used to identify SDHD alterations. The clinical and molecular characteristics of sporadic and hereditary tumors were compared. We reviewed the literature and compared our results with those from previously published studies.
RESULTS: Pathogenic germline SDHD mutations were identified in three patients: two (2.0%) of the 102 apparently sporadic pheochromocytoma patients and one patient with a family history of PGL. These patients presented with multifocal disease (two of three multifocal patients) or with a single adrenal tumor (one of 82 patients). In the literature, mutations are mostly found in patients 35 years of age or presenting with multifocal or extra-adrenal disease. All patients with an SDHD mutation developed extra-adrenal tumors (pheochromocytomas or PGLs) at presentation or during follow-up.
CONCLUSION: SDHD gene mutations in patients presenting with apparently sporadic adrenal pheochromocytoma are rare. We recommend SDHD mutation screening for patients presenting with a family history of pheochromocytoma or PGL, multiple tumors, isolated adrenal or extra-adrenal pheochromocytomas, and age 35 years. Analysis of SDHD can also help to distinguish synchronous primary tumors from abdominal metastases.
INTRODUCTION
Pheochromocytoma (PCC) is a neuroendocrine tumor, usually arising in the adrenal medulla. Despite its low incidence, the diagnosis of PCC is considered in many clinical situations, because catecholamine secretion by the tumor causes a wide range of symptoms. Furthermore, rapid establishment of the diagnosis is important to prevent life-threatening complications, whereas surgical resection of the tumor is curative in the majority of patients.1,2
Familial PCC is inherited as an autosomal dominant trait alone or as a component of the multiple endocrine neoplasia type 2 (MEN 2) syndrome, von Hippel-Lindau disease (VHL), or neurofibromatosis type 1 (NF1). The remaining 90% of PCCs are classified as sporadic or nonsyndromic. However, Neumann et al3 recently reported the presence of germline mutations in 24% of a large series of apparently sporadic PCC patients. One can thus conclude that, in the general population, more than 24% of PCC patients have a genetic predisposition to this tumor. This recent improvement in recognizing predisposition to PCCs is caused by the finding of germline mutations in succinate dehydrogenase subunit D (SDHD), in patients with familial and apparently sporadic PCC. The SDHD gene was initially identified as a susceptibility gene for the autosomal dominant familial parasympathetic paraganglioma syndrome (PGL1; MIM 168000).4 The gene encodes the small subunit (cybS) of cytochrome b in the mitochondrial enzyme complex II (succinate-ubiquinone oxidoreductase) and plays an important role in both the citric acid cycle and the aerobic respiratory chain.5 It has been demonstrated that germline mutations in SDHC (succinate dehydrogenase subunit C) and SDHB (succinate dehydrogenase subunit B), encoding two other components of complex II, also predispose to hereditary paraganglioma (PGL4 and PGL3, respectively).6,7
Because PCCs and parasympathetic paragangliomas (PGLs) both develop from neural-crest derived tissue, and co-occurrence of both tumors is reported,8 analysis of SDHD as a susceptibility gene for sporadic PCC was performed in seven previous studies, with mutation rates between 0% and 17%.3,9-15 These results are somewhat inconclusive, especially with respect to whether SDHD mutation screening is appropriate for all PCC patients or only for a specific subset of these patients. To determine appropriate indications for genetic screening is clinically important because of psychologic and financial implications.
Regarding parasympathetic PGL,16,17 screening for SDHD mutations in PCCs can be clinically important if it identifies patients who are at risk for developing multiple tumors. Screening potentially improves appropriate follow-up and early diagnosis of multiple tumors. In addition, it would be important to screen first-degree relatives to identify family members who are predisposed and should undergo biochemical and radiographic monitoring for the development of component tumors.
To establish whether screening for SDHD mutations is of value for all PCC patients, we evaluated a series of 126 patients with sporadic and syndrome-related PCCs. We also performed a comparative review of the literature to compare our results with those from previously published studies.
PATIENTS AND METHODS
We collected tumor specimens and normal tissues together with the clinical data of 126 patients with adrenal or extra-adrenal PCC, including 89 patients with clinically benign PCC and 37 patients with a proven malignant tumor. All 126 patients had undergone surgery between 1973 and 2001 at several hospitals in the Netherlands (with consecutive series from the Erasmus MC, Univeristy Medical Center Rotterdam and the University Medical Center Nijmegen; n = 99), the University Hospital in Lille, France, and the University Hospital in Zürich, Switzerland. Patients investigated by Perren et al12 were excluded from this study. The diagnosis of the tumors was confirmed according to standard histopathologic analysis. Clinical (follow-up) data were obtained by review of medical records.
Malignancy was determined either by histologically confirmed distant metastases or a positive [125I]metaiodobenzylguanidine scan (MIBG) outside the adrenal area, with persistent postoperative elevation of catecholamine levels. Ninety-eight patients had localized disease, and so far, after a mean follow-up time of 136 months (range, 11 to 336 months), no metastases have been diagnosed in these patients.
A PCC was considered sporadic if the patient did not harbor a germline mutation specific for MEN 2 and VHL and the patient's personal and family histories were not suggestive of NF1, familial PCC, or hereditary PGL. Information on medical and family histories was obtained by review of the medical records. The presence of multiple tumors was assessed by review of the pathology reports and the radiology reports of octreotide scintigraphy and/or magnetic resonance imaging (MRI).
A total of 144 primary PCCs (adrenal and extra-adrenal) were observed in the 126 patients, of which 134 primary tumors and matched normal tissues were available for analysis. In 14 of these patients, the primary tumor and a metastasis were analyzed. After coupling of the clinical information to the pathology specimen, both patient information and DNA samples were anonymized in accordance with the Erasmus MC guidelines for studies involving patient data and tissues. A collective database of clinical and molecular features was prepared. Patients were classified by presenting diagnosis and genetic background. For each patient, we recorded the age at diagnosis, clinical history, genetic background of the tumor, hormonal activity, the laterality/multifocality of the tumors, and the presence of metastases. Table 1 lists relevant clinical characteristics of the patients.
In addition, clinical data from PCC patients and their SDHD status were extracted from the literature and compared with our results. We also assessed whether genetic testing would have had impact on clinical decision making and follow-up.
DNA Preparation and Single-Stranded Conformational Polymorphism Analysis
Fresh-frozen or formalin-fixed, paraffin-embedded tumor and normal tissues from all patients, including 134 of the 144 tumors, were retrieved from the archives of the pathology departments of the above-mentioned hospitals. Hematoxylin-eosin staining was performed to assess the amount of tumor tissue in the sections. DNA from fresh-frozen tumors was isolated using the D-5000 Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturers' recommendations. DNA extraction from paraffin-embedded tumor and normal tissues or peripheral-blood samples was performed by standard detergent-proteinase K lysis, followed by phenol/chloroform extraction and ethanol precipitation.
The entire open reading frame of the SDHD gene and all exon-intron boundaries were investigated with PCR primers and conditions as described previously.4 PCR amplification of tumor DNA and matched normal DNA was performed in 15-μL reaction mixtures containing 1.5 mmol/L of MgCl2, 10 mmol/L of Tris-HCl, 50 mmol/L of KCl, 0.02 mmol/L of dATP, 0.2 mmol/L of dGTP, dTTP, dCTP each, 0.8 μCi of 32P-dATP (Amersham, Buckinghamshire, United Kingdom), 20 pmol of each sense and antisense primer, and 1 U of Taq DNA polymerase (Amplitaq Gold, Perkin Elmer, Norwalk, CT). The amplification profile consisted of an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 60 seconds. A final extension step was carried out at 72°C for 10 minutes. Electrophoresis of polymerase chain reaction (PCR) products was carried out overnight at 8W on nondenaturing gels, containing 8% polyacrylamide (49:1) and 10% (v/v) glycerol. For the exon 4 amplicons, electrophoresis was performed on 8% polyacrylamide gel without glycerol for 6 hours at +4°C and 20W. The gels were dried and exposed to x-ray film overnight at –70°C. DNA samples from three PGL patients with known germline SDHD mutations D92Y, L95P (both exon 3), and L139P (exon 4) served as positive controls.
DNA Sequencing
For each variant pattern identified by single-stranded conformational polymorphism (SSCP) analysis, two independent genomic DNA samples from the patient's tumor were amplified for direct sequencing with the original primer pair. These PCR products were bi-directionally sequenced using Applied Biosystems Taq DyeDeoxy terminator cycle sequencing (Baseclear, Leiden, the Netherlands).
Statistics
Correlations between a specific SDHD mutation and clinical features were tested by use of the 2 test or an unpaired t test. P values less than .05 were considered statistically significant.
RESULTS
Identification of SDHD Gene Mutations
SSCP analysis revealed four different aberrant patterns, which were present in the tumors and germline DNA of eight patients. We did not detect any somatic SDHD gene alterations in our series of 134 tumors from 126 patients. By sequence analysis, the aberrant patterns, located in exons 2 and 3, were identified as the pathogenic mutations D92Y and L95P and polymorphisms H50R and S68S. D92Y and H50R have been described in PCC patients previously,3,12 whereas L95P has only been reported in patients with PGL so far.18 Tumors from patients with D92Y and L95P showed loss of heterozygosity (LOH) of the wild-type allele, whereas no LOH was observed in the four tumors with the S68S polymorphism and in the adrenal PCC and a lung metastasis from the patient with the H50R variant. Examples of SSCP analysis, the LOH observed herein, and the sequence determination of the SDHD missense mutations are shown in Fig 1.
The specific D92Y missense mutation, known as a Dutch founder mutation,19 was observed in two Dutch patients. Patient A ( Table 2), a 27-year old woman, presented with an apparently sporadic adrenal PCC and later developed a second primary tumor, namely, an extra-adrenal PCC after 25 years. Patient B had a family history of PGL and presented with a mediastinal catecholamine-producing tumor at the age of 38 years. Somatostatin receptor scan imaging revealed a carotid body tumor, and the patient developed multiple extra-adrenal PCCs during the first year of follow-up.
The L95P mutation was found in patient C (25 years of age) with extra-adrenal PCCs at multiple abdominal spots, which was suspected of malignancy. Histopathologic examination did not prove the presence of tumor surrounded by preexistent lymphoid tissue. On SRS imaging, the patient also appeared to have bilateral carotid body tumors. After 12 years of follow-up, the patient is alive and well.
The H50R variant, which is likely a rare polymorphism but possibly increases PCC susceptibility,20 was present in the germline DNA of one patient (0.8%; Patient D). This 32-year-old patient presented with both an adrenal PCC, a pelvic mass, and seemed to have a lung metastasis but had no additional tumors during 7 years of follow-up.
The S68S polymorphism was observed in four patients (3.2%), including three patients with adrenal PCC and one patient with an extra-adrenal tumor.
Altogether, pathogenic SDHD mutations were identified in two (2.0%) of 102 apparently sporadic patients and in the one patient with a family history of PGL. No (somatic) mutations were found in patients with MEN 2, VHL, or NF1.
Patients' Characteristics Associated With SDHD Mutations
Two of the three patients with a germline SDHD mutation presented with multifocal disease. One patient presented with a single adrenal PCC, but this patient also developed an extra-adrenal PCC during follow-up. The mean age of onset in patients harboring a germline mutation was 30 years (range, 25 to 38 years) compared with 47 years (range, 13 to 79 years) in patients without a SDHD mutation (P = .032).
Table 3 shows all publications that report on SDHD mutation analysis in PCC, including the number of mutations and the relevant clinical characteristics. These studies included 412 apparently sporadic and 27 familial PCC patients, either with or without PGL. Altogether, germline SDHD mutations were found in 11 (2.7%) of 412 apparently sporadic patients and in three (33%) of nine patients with a family history of PGL and/or PCC in which MEN 2, VHL, or NF1 was excluded. Only one somatic mutation was found, P81L, which is also known as germline mutation in some PGL families.11 Mutations were not found in MEN 2–, VHL-, or NF1-related PCCs, but were observed in one of five PCC families. The majority (10 of 14) of the SDHD mutations were observed in patients presenting with an extra-adrenal PCC or with multiple tumors. Again, all patients with an SDHD mutation and presenting with a sporadic adrenal tumor developed one or more extra-adrenal tumors (including PGL) during follow-up.
Comparing our data with those from the literature reveals similar clinical features that indicate the likelihood of identifying an SDHD mutation in PCC patients. Overall, these include multifocal presentation (eight of 17; 47%), extra-adrenal location (four of 55; 7.3%), or family history of (extra-adrenal) PCC or PGL (four of 10; 40%). Twelve (72%) of the 17 patients with a mutation were 35 years of age or younger, and 15 (88%) of the 17 patients presented at the age of 40 years or younger. In patients presenting with an adrenal tumor, a younger age of onset ( 35 years) increased the likelihood of an SDHD mutation (6.3% v 1.1% in total subpopulation, based on this study and Neumann et al3). Table 2 shows all PCC patients with a SDHD mutation, including their presenting diagnosis and follow-up.
DISCUSSION
This investigation of SDHD alterations in 126 PCC patients underlines specific clinical features, including multifocal presentation of the tumor, younger age of onset ( 35 years), and a family history of extra-adrenal PCC or PGL, that increase the likelihood of identifying an SDHD mutation in these patients.
Genetic screening can be essential in early diagnosis and prevention of disease. However, considerable debate is ongoing as unnecessary screening has an undesirable psychologic impact on the patients and is not cost effective. One should therefore carefully report on indications that favor genetic testing.21 So far, studies have recognized the fact that SDHD mutations are associated with extra-adrenal PCC,22 but the clinical relevance of SDHD mutation screening has been poorly discussed. A careful review of all current data indicates that specific subgroups of PCC patients could be considered for genetic screening of SDHD. These include patients presenting with multifocal tumors (PCC and/or PGL) independent of their family history of PCC/PGL (50% harbor SDHD mutations) and patients presenting with an extra-adrenal PCC (7% harbor SDHD mutations). Regarding patients presenting with a sporadic adrenal PCC, the overall likelihood of germline SDHD mutations is only 1%. However, younger age of onset ( 35 years) or a family history of PCC or PGL in these patients are two features that increase the likelihood of a mutation to 6.3% (based on this study and Neumann et al3) and 16.7%, respectively. Screening of patients presenting at age 35 years and younger will identify at least 72% of patients with germline SDHD mutations. Screening in apparently sporadic patients older than 35 years and without a family history of PCC/PGL, as well as in patients with sporadic bilateral PCC, seems redundant because mutations in these patients are extremely rare (< 1%).
To justify genetic screening, testing for SDHD mutations should help to improve early diagnosis and prognosis or influence treatment. In PCC patients, early detection is the key factor to reduce morbidity and mortality, and identification of patients that are prone to develop multiple tumors may improve early detection. It is thus of interest to consider to what extent SDHD mutation screening contributes to early diagnosis in PCC patients. Most patients who seem to harbor an SDHD mutation present with multiple tumors, so that the risk of additional tumors is already evident and surveillance will be adjusted. In these cases, genetic screening is of interest to clarify the genetic cause of the disease or to identify positive family members. Additionally, in some patients presenting with multiple abdominal foci on MRI, MIBG, or octreotide scintigraphy, mutation screening may help to differentiate between lymph node metastases and multiple independent synchronous tumors.
Mutations are infrequent in patients presenting with apparently sporadic isolated PCC (up to 6.3% in patients 35 years old), which does not favor genetic testing in these patients. However, an SDHD mutation specifically identifies patients who are prone to develop additional PCC or PGL tumors, a reason to target a specific follow-up strategy to these patients alone (Fig 2). At least 60% (five of eight patients; Tables 2 and 3) of isolated patients with germline SDHD mutations developed metachronous primary tumors (PCC or PGL), and Neumann et al3 estimated a 20% to 30% likelihood of the subsequent development of a parasympathetic PGL. One reason to extend the SDHD screening to patients that present at the age of 40 years or younger is the fact that it will identify almost 90% of patients with germline SDHD mutations, instead of 72% when the cutoff age is 35 years. This will decrease the likelihood of a mutation, but is certainly defendable in the light of the relatively low burden of the disease and the importance for early diagnosis and treatment. Because PCC patients remain in follow-up because of the risk of malignancy, the follow-up management in PCC patients with SDHD mutation needs complementation. Periodic physical and ultrasonographic examinations of the neck or cervical MRI can be performed to detect PGL. Furthermore, we propose MRI imaging of the paravertebral sympathetic chain for the surveillance of (extra-adrenal) PCC. Alternatively, MIBG or octreotide scintigraphy can be used. According to recent studies, [(18)F]-dopamine positron emission tomography may be a superior imaging method to detect both PCC and PGL.23,24
Although the majority of patients presenting with multiple tumors have SDHD mutations, a considerable number of multifocal patients lack a germline SDHD mutation. These patients may harbor a mutation in SDHB.25 Patients with SDHB mutations present more frequently with PCCs (mostly extra-adrenal),3,6,25-27 and SDHD carriers present more frequently with PGL,16,17 but these studies also show that similar features (multifocal presentation, family history of PGL or PCC, extra-adrenal location, or age of onset 35 years) indicate the presence of a germline SDHB mutation. Therefore, when genetic testing is appropriate, both SDHD and SDHB genes should be investigated simultaneously.
Genetic testing can be offered to first-degree relatives of patients with a germline mutation. For appropriate genetic counseling, an estimation of the penetrance and the lifetime risk on PCC and PGL is important. Unfortunately, data on penetrance of the disease or the lifetime risk on PCC or PGL are only poorly established with regard to SDHD and SDHB germline mutations. Examination of available data suggests that the family history of more than 60% of apparently sporadic patients with mutations becomes positive after screening of asymptomatic carriers in their families.3 Follow-up in asymptomatic carriers should probably be proposed at 5 to 10 years of age28 and should comprise physical and ultrasonographic examinations of the neck and exclusion of catecholamine hypersecretion.
When patients have multiple tumor locations at presentation or during follow-up, it can be difficult to distinguish independent primary tumors from metastases or recurrent disease. In patient A ( Table 2), we can regard the second lesion as a primary tumor based on the location of the tumors, the absence of preexistent lymph node tissue, and the otherwise clinically benign behavior. The finding of an SDHD mutation indicates the presence of a second primary tumor in this patient. Patient C was suspected to have a malignant tumor, because of multiple extra-adrenal abdominal spots observed with MIBG scintigraphy. Again, the absence of preexistent lymphoid tissue in combination with the presence of a germline SDHD mutation is suggestive of synchronous para-aortic PCCs.
Patient D harbored the H50R variant, which is shown to occur in 2.8% of apparently healthy individuals, indicating that H50R is a nonpathogenic variant. However, our patient presented with two tumor masses, an adrenal and a pelvic mass, and the relatively young age of onset in this patient also suggested the existence of a genetic predisposition. Further investigations revealed a pulmonal mass, apparently a metastasis of the adrenal mass as shown by similar comparative genomic hybridisation (CGH) profiles of the two samples (data not shown). The pelvic mass was not available for CGH analysis, but the presence of preexistent lymphoid tissue and retention of heterozygosity of the SDHD locus ( Fig 1) suggest that this patient developed one primary PCC with pelvic and pulmonal metastasis. Altogether, this provides additional information that H50R is a nonpathogenic variant.
In summary, early detection of PCCs is important to improve prognosis and can be achieved by the appropriate follow-up management in patients at risk. SDHD mutation analysis specifically identifies patients who are susceptible to develop multiple PCCs and PGLs. Because surveillance is already continued in most PCC patients because of the risk of malignancy, an adjusted surveillance strategy needs to be targeted to mutation-positive patients. The subsequent identification of mutation carriers in family members will further improve early detection of PCC and PGL. We have demonstrated that SDHD gene mutations in patients with apparently sporadic, adrenal PCC are rare, and therefore, screening for SDHD mutations in these patients is redundant. However, SDHD mutation screening is appropriate for patients presenting with a family history of PCC or PGL, multiple tumors, or isolated adrenal or extra-adrenal PCC and age 35 years. The results of this study, correlating SDHD mutations with clinical features of PCC patients, will hopefully contribute to improving appropriate genetic screening for patients who are at risk of developing multiple PCCs and PGLs. We propose a decision tree for the identification of SDH-related PCCs as shown in Fig 2.
Acknowledgment
We thank W.W. De Herder, MD, Department of Internal Medicine, Section of Endocrinology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands, for helpful discussion. We are also grateful to Frank van der Panne, Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center, for help in the generation of the figures.
Authors' Disclosures of Potential The authors indicated no potential conflicts of interest.
NOTES
Supported by the Vanderes Foundation (grant No. 60), Breda, the Netherlands.
H.D. and F.H.v.N. contributed equally to this work.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Kenady DE, McGrath PC, Sloan DA, et al: Diagnosis and management of pheochromocytoma. Curr Opin Oncol 9:61-67, 1997
Walther MM, Keiser HR, Linehan WM: Pheochromocytoma: Evaluation, diagnosis, and treatment. World J Urol 17:35-39, 1999
Neumann HP, Bausch B, McWhinney SR, et al: Germ-line mutations in nonsyndromic pheochromocytoma. N Engl J Med 346:1459-1466, 2002
Baysal BE, Ferrell RE, Willett-Brozick JE, et al: Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287:848-851, 2000
Hirawake H, Taniwaki M, Tamura A, et al: Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase. Biochim Biophys Acta 1412:295-300, 1999
Astuti D, Latif F, Dallol A, et al: Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet 69:49-54, 2001
Niemann S, Muller U: Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet 26:268-270, 2000
Sato T, Saito H, Yoshinaga K, et al: Concurrence of carotid body tumor and pheochromocytoma. Cancer 34:1787-1795, 1974
Astuti D, Douglas F, Lennard TW, et al: Germline SDHD mutation in familial phaeochromocytoma. Lancet 357:1181-1182, 2001
Cascon A, Ruiz-Llorente S, Cebrian A, et al: Identification of novel SDHD mutations in patients with phaeochromocytoma and/or paraganglioma. Eur J Hum Genet 10:457-461, 2002
Gimm O, Armanios M, Dziema H, et al: Somatic and occult germ-line mutations in SDHD, a mitochondrial complex II gene, in nonfamilial pheochromocytoma. Cancer Res 60:6822-6825, 2000
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
Kytola S, Nord B, Elder EE, et al: Alterations of the SDHD gene locus in midgut carcinoids, Merkel cell carcinomas, pheochromocytomas, and abdominal paragangliomas. Genes Chromosomes Cancer 34:325-332, 2002
Aguiar RC, Cox G, Pomeroy SL, et al: Analysis of the SDHD gene, the susceptibility gene for familial paraganglioma syndrome (PGL1), in pheochromocytomas. J Clin Endocrinol Metab 86:2890-2894, 2001
Bauters C, Vantyghem MC, Leteurtre E, et al: Hereditary pheochromocytomas and paragangliomas: A study of five susceptibility genes. J Med Genet 40:e75, 2003
Dannenberg H, Dinjens WN, Abbou M, et al: Frequent germ-line succinate dehydrogenase subunit D gene mutations in patients with apparently sporadic parasympathetic paraganglioma. Clin Cancer Res 8:2061-2066, 2002
Baysal BE, Willett-Brozick JE, Lawrence EC, et al: Prevalence of SDHB, SDHC, and SDHD germline mutations in clinic patients with head and neck paragangliomas. J Med Genet 39:178-183, 2002
Baysal BE: Hereditary paraganglioma targets diverse paraganglia. J Med Genet 39:617-622, 2002
Taschner PE, Jansen JC, Baysal BE, et al: Nearly all hereditary paragangliomas in the Netherlands are caused by two founder mutations in the SDHD gene. Genes Chromosomes Cancer 31:274-281, 2001
Cascon A, Ruiz-Llorente S, Cebrian A, et al: G12S and H50R variations are polymorphisms in the SDHD gene. Genes Chromosomes Cancer 37:220-221, 2003
American Society of Clinical Oncology policy statement update: Genetic testing for cancer susceptibility. J Clin Oncol 21:2397-2406, 2003
Eng C, Kiuru M, Fernandez MJ, et al: A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat Rev Cancer 3:193-202, 2003
Ilias I, Yu J, Carrasquillo JA, et al: Superiority of 6-[18F]-fluorodopamine positron emission tomography versus [131I]-metaiodobenzylguanidine scintigraphy in the localization of metastatic pheochromocytoma. J Clin Endocrinol Metab 88:4083-4087, 2003
Hoegerle S, Nitzsche E, Altehoefer C, et al: Pheochromocytomas: Detection with 18F DOPA whole body PET: Initial results. Radiology 222:507-512, 2002
Benn DE, Croxson MS, Tucker K, et al: Novel succinate dehydrogenase subunit B (SDHB) mutations in familial pheochromocytomas and paragangliomas, but an absence of somatic SDHB mutations in sporadic pheochromocytomas. Oncogene 22:1358-1364, 2003
Cascon A, Cebrian A, Ruiz-Llorente S, et al: SDHB mutation analysis in familial and sporadic phaeochromocytoma identifies a novel mutation. J Med Genet 39:E64, 2002
Young AL, Baysal BE, Deb A, et al: Familial malignant catecholamine-secreting paraganglioma with prolonged survival associated with mutation in the succinate dehydrogenase B gene. J Clin Endocrinol Metab 87:4101-4105, 2002
Renard L, Godfraind C, Boon LM, et al: A novel mutation in the SDHD gene in a family with inherited paragangliomas–implications of genetic diagnosis for follow-up and treatment. Head Neck 25:146-151, 2003(Hilde Dannenberg, Francie)
Department of Pathology, St Radboud University Medical Center, Nijmegen, the Netherlands
Institute of Pathology, Hospital Baden, Baden, Switzerland
ABSTRACT
PURPOSE: We examined the value of SDHD mutation screening in patients presenting with apparently sporadic and familial pheochromocytoma for the identification of SDHD-related pheochromocytomas.
PATIENTS AND METHODS: This retrospective study involved 126 patients with adrenal or extra-adrenal pheochromocytomas, including 24 patients with a family history of multiple endocrine neoplasia 2, von Hippel-Lindau disease, neurofibromatosis type 1, or paraganglioma (PGL). Conformation-dependent gel electrophoresis and sequence determination analysis of germline and tumor DNA were used to identify SDHD alterations. The clinical and molecular characteristics of sporadic and hereditary tumors were compared. We reviewed the literature and compared our results with those from previously published studies.
RESULTS: Pathogenic germline SDHD mutations were identified in three patients: two (2.0%) of the 102 apparently sporadic pheochromocytoma patients and one patient with a family history of PGL. These patients presented with multifocal disease (two of three multifocal patients) or with a single adrenal tumor (one of 82 patients). In the literature, mutations are mostly found in patients 35 years of age or presenting with multifocal or extra-adrenal disease. All patients with an SDHD mutation developed extra-adrenal tumors (pheochromocytomas or PGLs) at presentation or during follow-up.
CONCLUSION: SDHD gene mutations in patients presenting with apparently sporadic adrenal pheochromocytoma are rare. We recommend SDHD mutation screening for patients presenting with a family history of pheochromocytoma or PGL, multiple tumors, isolated adrenal or extra-adrenal pheochromocytomas, and age 35 years. Analysis of SDHD can also help to distinguish synchronous primary tumors from abdominal metastases.
INTRODUCTION
Pheochromocytoma (PCC) is a neuroendocrine tumor, usually arising in the adrenal medulla. Despite its low incidence, the diagnosis of PCC is considered in many clinical situations, because catecholamine secretion by the tumor causes a wide range of symptoms. Furthermore, rapid establishment of the diagnosis is important to prevent life-threatening complications, whereas surgical resection of the tumor is curative in the majority of patients.1,2
Familial PCC is inherited as an autosomal dominant trait alone or as a component of the multiple endocrine neoplasia type 2 (MEN 2) syndrome, von Hippel-Lindau disease (VHL), or neurofibromatosis type 1 (NF1). The remaining 90% of PCCs are classified as sporadic or nonsyndromic. However, Neumann et al3 recently reported the presence of germline mutations in 24% of a large series of apparently sporadic PCC patients. One can thus conclude that, in the general population, more than 24% of PCC patients have a genetic predisposition to this tumor. This recent improvement in recognizing predisposition to PCCs is caused by the finding of germline mutations in succinate dehydrogenase subunit D (SDHD), in patients with familial and apparently sporadic PCC. The SDHD gene was initially identified as a susceptibility gene for the autosomal dominant familial parasympathetic paraganglioma syndrome (PGL1; MIM 168000).4 The gene encodes the small subunit (cybS) of cytochrome b in the mitochondrial enzyme complex II (succinate-ubiquinone oxidoreductase) and plays an important role in both the citric acid cycle and the aerobic respiratory chain.5 It has been demonstrated that germline mutations in SDHC (succinate dehydrogenase subunit C) and SDHB (succinate dehydrogenase subunit B), encoding two other components of complex II, also predispose to hereditary paraganglioma (PGL4 and PGL3, respectively).6,7
Because PCCs and parasympathetic paragangliomas (PGLs) both develop from neural-crest derived tissue, and co-occurrence of both tumors is reported,8 analysis of SDHD as a susceptibility gene for sporadic PCC was performed in seven previous studies, with mutation rates between 0% and 17%.3,9-15 These results are somewhat inconclusive, especially with respect to whether SDHD mutation screening is appropriate for all PCC patients or only for a specific subset of these patients. To determine appropriate indications for genetic screening is clinically important because of psychologic and financial implications.
Regarding parasympathetic PGL,16,17 screening for SDHD mutations in PCCs can be clinically important if it identifies patients who are at risk for developing multiple tumors. Screening potentially improves appropriate follow-up and early diagnosis of multiple tumors. In addition, it would be important to screen first-degree relatives to identify family members who are predisposed and should undergo biochemical and radiographic monitoring for the development of component tumors.
To establish whether screening for SDHD mutations is of value for all PCC patients, we evaluated a series of 126 patients with sporadic and syndrome-related PCCs. We also performed a comparative review of the literature to compare our results with those from previously published studies.
PATIENTS AND METHODS
We collected tumor specimens and normal tissues together with the clinical data of 126 patients with adrenal or extra-adrenal PCC, including 89 patients with clinically benign PCC and 37 patients with a proven malignant tumor. All 126 patients had undergone surgery between 1973 and 2001 at several hospitals in the Netherlands (with consecutive series from the Erasmus MC, Univeristy Medical Center Rotterdam and the University Medical Center Nijmegen; n = 99), the University Hospital in Lille, France, and the University Hospital in Zürich, Switzerland. Patients investigated by Perren et al12 were excluded from this study. The diagnosis of the tumors was confirmed according to standard histopathologic analysis. Clinical (follow-up) data were obtained by review of medical records.
Malignancy was determined either by histologically confirmed distant metastases or a positive [125I]metaiodobenzylguanidine scan (MIBG) outside the adrenal area, with persistent postoperative elevation of catecholamine levels. Ninety-eight patients had localized disease, and so far, after a mean follow-up time of 136 months (range, 11 to 336 months), no metastases have been diagnosed in these patients.
A PCC was considered sporadic if the patient did not harbor a germline mutation specific for MEN 2 and VHL and the patient's personal and family histories were not suggestive of NF1, familial PCC, or hereditary PGL. Information on medical and family histories was obtained by review of the medical records. The presence of multiple tumors was assessed by review of the pathology reports and the radiology reports of octreotide scintigraphy and/or magnetic resonance imaging (MRI).
A total of 144 primary PCCs (adrenal and extra-adrenal) were observed in the 126 patients, of which 134 primary tumors and matched normal tissues were available for analysis. In 14 of these patients, the primary tumor and a metastasis were analyzed. After coupling of the clinical information to the pathology specimen, both patient information and DNA samples were anonymized in accordance with the Erasmus MC guidelines for studies involving patient data and tissues. A collective database of clinical and molecular features was prepared. Patients were classified by presenting diagnosis and genetic background. For each patient, we recorded the age at diagnosis, clinical history, genetic background of the tumor, hormonal activity, the laterality/multifocality of the tumors, and the presence of metastases. Table 1 lists relevant clinical characteristics of the patients.
In addition, clinical data from PCC patients and their SDHD status were extracted from the literature and compared with our results. We also assessed whether genetic testing would have had impact on clinical decision making and follow-up.
DNA Preparation and Single-Stranded Conformational Polymorphism Analysis
Fresh-frozen or formalin-fixed, paraffin-embedded tumor and normal tissues from all patients, including 134 of the 144 tumors, were retrieved from the archives of the pathology departments of the above-mentioned hospitals. Hematoxylin-eosin staining was performed to assess the amount of tumor tissue in the sections. DNA from fresh-frozen tumors was isolated using the D-5000 Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturers' recommendations. DNA extraction from paraffin-embedded tumor and normal tissues or peripheral-blood samples was performed by standard detergent-proteinase K lysis, followed by phenol/chloroform extraction and ethanol precipitation.
The entire open reading frame of the SDHD gene and all exon-intron boundaries were investigated with PCR primers and conditions as described previously.4 PCR amplification of tumor DNA and matched normal DNA was performed in 15-μL reaction mixtures containing 1.5 mmol/L of MgCl2, 10 mmol/L of Tris-HCl, 50 mmol/L of KCl, 0.02 mmol/L of dATP, 0.2 mmol/L of dGTP, dTTP, dCTP each, 0.8 μCi of 32P-dATP (Amersham, Buckinghamshire, United Kingdom), 20 pmol of each sense and antisense primer, and 1 U of Taq DNA polymerase (Amplitaq Gold, Perkin Elmer, Norwalk, CT). The amplification profile consisted of an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 60 seconds. A final extension step was carried out at 72°C for 10 minutes. Electrophoresis of polymerase chain reaction (PCR) products was carried out overnight at 8W on nondenaturing gels, containing 8% polyacrylamide (49:1) and 10% (v/v) glycerol. For the exon 4 amplicons, electrophoresis was performed on 8% polyacrylamide gel without glycerol for 6 hours at +4°C and 20W. The gels were dried and exposed to x-ray film overnight at –70°C. DNA samples from three PGL patients with known germline SDHD mutations D92Y, L95P (both exon 3), and L139P (exon 4) served as positive controls.
DNA Sequencing
For each variant pattern identified by single-stranded conformational polymorphism (SSCP) analysis, two independent genomic DNA samples from the patient's tumor were amplified for direct sequencing with the original primer pair. These PCR products were bi-directionally sequenced using Applied Biosystems Taq DyeDeoxy terminator cycle sequencing (Baseclear, Leiden, the Netherlands).
Statistics
Correlations between a specific SDHD mutation and clinical features were tested by use of the 2 test or an unpaired t test. P values less than .05 were considered statistically significant.
RESULTS
Identification of SDHD Gene Mutations
SSCP analysis revealed four different aberrant patterns, which were present in the tumors and germline DNA of eight patients. We did not detect any somatic SDHD gene alterations in our series of 134 tumors from 126 patients. By sequence analysis, the aberrant patterns, located in exons 2 and 3, were identified as the pathogenic mutations D92Y and L95P and polymorphisms H50R and S68S. D92Y and H50R have been described in PCC patients previously,3,12 whereas L95P has only been reported in patients with PGL so far.18 Tumors from patients with D92Y and L95P showed loss of heterozygosity (LOH) of the wild-type allele, whereas no LOH was observed in the four tumors with the S68S polymorphism and in the adrenal PCC and a lung metastasis from the patient with the H50R variant. Examples of SSCP analysis, the LOH observed herein, and the sequence determination of the SDHD missense mutations are shown in Fig 1.
The specific D92Y missense mutation, known as a Dutch founder mutation,19 was observed in two Dutch patients. Patient A ( Table 2), a 27-year old woman, presented with an apparently sporadic adrenal PCC and later developed a second primary tumor, namely, an extra-adrenal PCC after 25 years. Patient B had a family history of PGL and presented with a mediastinal catecholamine-producing tumor at the age of 38 years. Somatostatin receptor scan imaging revealed a carotid body tumor, and the patient developed multiple extra-adrenal PCCs during the first year of follow-up.
The L95P mutation was found in patient C (25 years of age) with extra-adrenal PCCs at multiple abdominal spots, which was suspected of malignancy. Histopathologic examination did not prove the presence of tumor surrounded by preexistent lymphoid tissue. On SRS imaging, the patient also appeared to have bilateral carotid body tumors. After 12 years of follow-up, the patient is alive and well.
The H50R variant, which is likely a rare polymorphism but possibly increases PCC susceptibility,20 was present in the germline DNA of one patient (0.8%; Patient D). This 32-year-old patient presented with both an adrenal PCC, a pelvic mass, and seemed to have a lung metastasis but had no additional tumors during 7 years of follow-up.
The S68S polymorphism was observed in four patients (3.2%), including three patients with adrenal PCC and one patient with an extra-adrenal tumor.
Altogether, pathogenic SDHD mutations were identified in two (2.0%) of 102 apparently sporadic patients and in the one patient with a family history of PGL. No (somatic) mutations were found in patients with MEN 2, VHL, or NF1.
Patients' Characteristics Associated With SDHD Mutations
Two of the three patients with a germline SDHD mutation presented with multifocal disease. One patient presented with a single adrenal PCC, but this patient also developed an extra-adrenal PCC during follow-up. The mean age of onset in patients harboring a germline mutation was 30 years (range, 25 to 38 years) compared with 47 years (range, 13 to 79 years) in patients without a SDHD mutation (P = .032).
Table 3 shows all publications that report on SDHD mutation analysis in PCC, including the number of mutations and the relevant clinical characteristics. These studies included 412 apparently sporadic and 27 familial PCC patients, either with or without PGL. Altogether, germline SDHD mutations were found in 11 (2.7%) of 412 apparently sporadic patients and in three (33%) of nine patients with a family history of PGL and/or PCC in which MEN 2, VHL, or NF1 was excluded. Only one somatic mutation was found, P81L, which is also known as germline mutation in some PGL families.11 Mutations were not found in MEN 2–, VHL-, or NF1-related PCCs, but were observed in one of five PCC families. The majority (10 of 14) of the SDHD mutations were observed in patients presenting with an extra-adrenal PCC or with multiple tumors. Again, all patients with an SDHD mutation and presenting with a sporadic adrenal tumor developed one or more extra-adrenal tumors (including PGL) during follow-up.
Comparing our data with those from the literature reveals similar clinical features that indicate the likelihood of identifying an SDHD mutation in PCC patients. Overall, these include multifocal presentation (eight of 17; 47%), extra-adrenal location (four of 55; 7.3%), or family history of (extra-adrenal) PCC or PGL (four of 10; 40%). Twelve (72%) of the 17 patients with a mutation were 35 years of age or younger, and 15 (88%) of the 17 patients presented at the age of 40 years or younger. In patients presenting with an adrenal tumor, a younger age of onset ( 35 years) increased the likelihood of an SDHD mutation (6.3% v 1.1% in total subpopulation, based on this study and Neumann et al3). Table 2 shows all PCC patients with a SDHD mutation, including their presenting diagnosis and follow-up.
DISCUSSION
This investigation of SDHD alterations in 126 PCC patients underlines specific clinical features, including multifocal presentation of the tumor, younger age of onset ( 35 years), and a family history of extra-adrenal PCC or PGL, that increase the likelihood of identifying an SDHD mutation in these patients.
Genetic screening can be essential in early diagnosis and prevention of disease. However, considerable debate is ongoing as unnecessary screening has an undesirable psychologic impact on the patients and is not cost effective. One should therefore carefully report on indications that favor genetic testing.21 So far, studies have recognized the fact that SDHD mutations are associated with extra-adrenal PCC,22 but the clinical relevance of SDHD mutation screening has been poorly discussed. A careful review of all current data indicates that specific subgroups of PCC patients could be considered for genetic screening of SDHD. These include patients presenting with multifocal tumors (PCC and/or PGL) independent of their family history of PCC/PGL (50% harbor SDHD mutations) and patients presenting with an extra-adrenal PCC (7% harbor SDHD mutations). Regarding patients presenting with a sporadic adrenal PCC, the overall likelihood of germline SDHD mutations is only 1%. However, younger age of onset ( 35 years) or a family history of PCC or PGL in these patients are two features that increase the likelihood of a mutation to 6.3% (based on this study and Neumann et al3) and 16.7%, respectively. Screening of patients presenting at age 35 years and younger will identify at least 72% of patients with germline SDHD mutations. Screening in apparently sporadic patients older than 35 years and without a family history of PCC/PGL, as well as in patients with sporadic bilateral PCC, seems redundant because mutations in these patients are extremely rare (< 1%).
To justify genetic screening, testing for SDHD mutations should help to improve early diagnosis and prognosis or influence treatment. In PCC patients, early detection is the key factor to reduce morbidity and mortality, and identification of patients that are prone to develop multiple tumors may improve early detection. It is thus of interest to consider to what extent SDHD mutation screening contributes to early diagnosis in PCC patients. Most patients who seem to harbor an SDHD mutation present with multiple tumors, so that the risk of additional tumors is already evident and surveillance will be adjusted. In these cases, genetic screening is of interest to clarify the genetic cause of the disease or to identify positive family members. Additionally, in some patients presenting with multiple abdominal foci on MRI, MIBG, or octreotide scintigraphy, mutation screening may help to differentiate between lymph node metastases and multiple independent synchronous tumors.
Mutations are infrequent in patients presenting with apparently sporadic isolated PCC (up to 6.3% in patients 35 years old), which does not favor genetic testing in these patients. However, an SDHD mutation specifically identifies patients who are prone to develop additional PCC or PGL tumors, a reason to target a specific follow-up strategy to these patients alone (Fig 2). At least 60% (five of eight patients; Tables 2 and 3) of isolated patients with germline SDHD mutations developed metachronous primary tumors (PCC or PGL), and Neumann et al3 estimated a 20% to 30% likelihood of the subsequent development of a parasympathetic PGL. One reason to extend the SDHD screening to patients that present at the age of 40 years or younger is the fact that it will identify almost 90% of patients with germline SDHD mutations, instead of 72% when the cutoff age is 35 years. This will decrease the likelihood of a mutation, but is certainly defendable in the light of the relatively low burden of the disease and the importance for early diagnosis and treatment. Because PCC patients remain in follow-up because of the risk of malignancy, the follow-up management in PCC patients with SDHD mutation needs complementation. Periodic physical and ultrasonographic examinations of the neck or cervical MRI can be performed to detect PGL. Furthermore, we propose MRI imaging of the paravertebral sympathetic chain for the surveillance of (extra-adrenal) PCC. Alternatively, MIBG or octreotide scintigraphy can be used. According to recent studies, [(18)F]-dopamine positron emission tomography may be a superior imaging method to detect both PCC and PGL.23,24
Although the majority of patients presenting with multiple tumors have SDHD mutations, a considerable number of multifocal patients lack a germline SDHD mutation. These patients may harbor a mutation in SDHB.25 Patients with SDHB mutations present more frequently with PCCs (mostly extra-adrenal),3,6,25-27 and SDHD carriers present more frequently with PGL,16,17 but these studies also show that similar features (multifocal presentation, family history of PGL or PCC, extra-adrenal location, or age of onset 35 years) indicate the presence of a germline SDHB mutation. Therefore, when genetic testing is appropriate, both SDHD and SDHB genes should be investigated simultaneously.
Genetic testing can be offered to first-degree relatives of patients with a germline mutation. For appropriate genetic counseling, an estimation of the penetrance and the lifetime risk on PCC and PGL is important. Unfortunately, data on penetrance of the disease or the lifetime risk on PCC or PGL are only poorly established with regard to SDHD and SDHB germline mutations. Examination of available data suggests that the family history of more than 60% of apparently sporadic patients with mutations becomes positive after screening of asymptomatic carriers in their families.3 Follow-up in asymptomatic carriers should probably be proposed at 5 to 10 years of age28 and should comprise physical and ultrasonographic examinations of the neck and exclusion of catecholamine hypersecretion.
When patients have multiple tumor locations at presentation or during follow-up, it can be difficult to distinguish independent primary tumors from metastases or recurrent disease. In patient A ( Table 2), we can regard the second lesion as a primary tumor based on the location of the tumors, the absence of preexistent lymph node tissue, and the otherwise clinically benign behavior. The finding of an SDHD mutation indicates the presence of a second primary tumor in this patient. Patient C was suspected to have a malignant tumor, because of multiple extra-adrenal abdominal spots observed with MIBG scintigraphy. Again, the absence of preexistent lymphoid tissue in combination with the presence of a germline SDHD mutation is suggestive of synchronous para-aortic PCCs.
Patient D harbored the H50R variant, which is shown to occur in 2.8% of apparently healthy individuals, indicating that H50R is a nonpathogenic variant. However, our patient presented with two tumor masses, an adrenal and a pelvic mass, and the relatively young age of onset in this patient also suggested the existence of a genetic predisposition. Further investigations revealed a pulmonal mass, apparently a metastasis of the adrenal mass as shown by similar comparative genomic hybridisation (CGH) profiles of the two samples (data not shown). The pelvic mass was not available for CGH analysis, but the presence of preexistent lymphoid tissue and retention of heterozygosity of the SDHD locus ( Fig 1) suggest that this patient developed one primary PCC with pelvic and pulmonal metastasis. Altogether, this provides additional information that H50R is a nonpathogenic variant.
In summary, early detection of PCCs is important to improve prognosis and can be achieved by the appropriate follow-up management in patients at risk. SDHD mutation analysis specifically identifies patients who are susceptible to develop multiple PCCs and PGLs. Because surveillance is already continued in most PCC patients because of the risk of malignancy, an adjusted surveillance strategy needs to be targeted to mutation-positive patients. The subsequent identification of mutation carriers in family members will further improve early detection of PCC and PGL. We have demonstrated that SDHD gene mutations in patients with apparently sporadic, adrenal PCC are rare, and therefore, screening for SDHD mutations in these patients is redundant. However, SDHD mutation screening is appropriate for patients presenting with a family history of PCC or PGL, multiple tumors, or isolated adrenal or extra-adrenal PCC and age 35 years. The results of this study, correlating SDHD mutations with clinical features of PCC patients, will hopefully contribute to improving appropriate genetic screening for patients who are at risk of developing multiple PCCs and PGLs. We propose a decision tree for the identification of SDH-related PCCs as shown in Fig 2.
Acknowledgment
We thank W.W. De Herder, MD, Department of Internal Medicine, Section of Endocrinology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands, for helpful discussion. We are also grateful to Frank van der Panne, Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center, for help in the generation of the figures.
Authors' Disclosures of Potential The authors indicated no potential conflicts of interest.
NOTES
Supported by the Vanderes Foundation (grant No. 60), Breda, the Netherlands.
H.D. and F.H.v.N. contributed equally to this work.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Kenady DE, McGrath PC, Sloan DA, et al: Diagnosis and management of pheochromocytoma. Curr Opin Oncol 9:61-67, 1997
Walther MM, Keiser HR, Linehan WM: Pheochromocytoma: Evaluation, diagnosis, and treatment. World J Urol 17:35-39, 1999
Neumann HP, Bausch B, McWhinney SR, et al: Germ-line mutations in nonsyndromic pheochromocytoma. N Engl J Med 346:1459-1466, 2002
Baysal BE, Ferrell RE, Willett-Brozick JE, et al: Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287:848-851, 2000
Hirawake H, Taniwaki M, Tamura A, et al: Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase. Biochim Biophys Acta 1412:295-300, 1999
Astuti D, Latif F, Dallol A, et al: Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet 69:49-54, 2001
Niemann S, Muller U: Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet 26:268-270, 2000
Sato T, Saito H, Yoshinaga K, et al: Concurrence of carotid body tumor and pheochromocytoma. Cancer 34:1787-1795, 1974
Astuti D, Douglas F, Lennard TW, et al: Germline SDHD mutation in familial phaeochromocytoma. Lancet 357:1181-1182, 2001
Cascon A, Ruiz-Llorente S, Cebrian A, et al: Identification of novel SDHD mutations in patients with phaeochromocytoma and/or paraganglioma. Eur J Hum Genet 10:457-461, 2002
Gimm O, Armanios M, Dziema H, et al: Somatic and occult germ-line mutations in SDHD, a mitochondrial complex II gene, in nonfamilial pheochromocytoma. Cancer Res 60:6822-6825, 2000
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
Kytola S, Nord B, Elder EE, et al: Alterations of the SDHD gene locus in midgut carcinoids, Merkel cell carcinomas, pheochromocytomas, and abdominal paragangliomas. Genes Chromosomes Cancer 34:325-332, 2002
Aguiar RC, Cox G, Pomeroy SL, et al: Analysis of the SDHD gene, the susceptibility gene for familial paraganglioma syndrome (PGL1), in pheochromocytomas. J Clin Endocrinol Metab 86:2890-2894, 2001
Bauters C, Vantyghem MC, Leteurtre E, et al: Hereditary pheochromocytomas and paragangliomas: A study of five susceptibility genes. J Med Genet 40:e75, 2003
Dannenberg H, Dinjens WN, Abbou M, et al: Frequent germ-line succinate dehydrogenase subunit D gene mutations in patients with apparently sporadic parasympathetic paraganglioma. Clin Cancer Res 8:2061-2066, 2002
Baysal BE, Willett-Brozick JE, Lawrence EC, et al: Prevalence of SDHB, SDHC, and SDHD germline mutations in clinic patients with head and neck paragangliomas. J Med Genet 39:178-183, 2002
Baysal BE: Hereditary paraganglioma targets diverse paraganglia. J Med Genet 39:617-622, 2002
Taschner PE, Jansen JC, Baysal BE, et al: Nearly all hereditary paragangliomas in the Netherlands are caused by two founder mutations in the SDHD gene. Genes Chromosomes Cancer 31:274-281, 2001
Cascon A, Ruiz-Llorente S, Cebrian A, et al: G12S and H50R variations are polymorphisms in the SDHD gene. Genes Chromosomes Cancer 37:220-221, 2003
American Society of Clinical Oncology policy statement update: Genetic testing for cancer susceptibility. J Clin Oncol 21:2397-2406, 2003
Eng C, Kiuru M, Fernandez MJ, et al: A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat Rev Cancer 3:193-202, 2003
Ilias I, Yu J, Carrasquillo JA, et al: Superiority of 6-[18F]-fluorodopamine positron emission tomography versus [131I]-metaiodobenzylguanidine scintigraphy in the localization of metastatic pheochromocytoma. J Clin Endocrinol Metab 88:4083-4087, 2003
Hoegerle S, Nitzsche E, Altehoefer C, et al: Pheochromocytomas: Detection with 18F DOPA whole body PET: Initial results. Radiology 222:507-512, 2002
Benn DE, Croxson MS, Tucker K, et al: Novel succinate dehydrogenase subunit B (SDHB) mutations in familial pheochromocytomas and paragangliomas, but an absence of somatic SDHB mutations in sporadic pheochromocytomas. Oncogene 22:1358-1364, 2003
Cascon A, Cebrian A, Ruiz-Llorente S, et al: SDHB mutation analysis in familial and sporadic phaeochromocytoma identifies a novel mutation. J Med Genet 39:E64, 2002
Young AL, Baysal BE, Deb A, et al: Familial malignant catecholamine-secreting paraganglioma with prolonged survival associated with mutation in the succinate dehydrogenase B gene. J Clin Endocrinol Metab 87:4101-4105, 2002
Renard L, Godfraind C, Boon LM, et al: A novel mutation in the SDHD gene in a family with inherited paragangliomas–implications of genetic diagnosis for follow-up and treatment. Head Neck 25:146-151, 2003(Hilde Dannenberg, Francie)