Somatic and Occult Germ-Line Mutations in SDHD, a Mitochondrial Complex II Gene, in Nonfamilial Pheochromocytoma1

[CANCER RESEARCH 60, 6822–6825, December 15, 2000] Advances in Brief

Somatic and Occult Germ-line Mutations in SDHD, a Mitochondrial Complex II Gene, in Nonfamilial Pheochromocytoma1

Oliver Gimm, Mary Armanios, Heather Dziema, Hartmut P. H. Neumann, and Charis Eng2 Clinical Cancer Genetics and Human Cancer Genetics Programs, Comprehensive Cancer Center, and Division of Human Genetics, Department of Internal Medicine, The Ohio State University, Columbus, Ohio 43210 [O. G., M. A., H. D., C. E.]; Medizinische Universita¨tsklinik, Albert-Ludwigs-Universita¨t, D79106 Freiburg, Germany [H. P. H. N.]; and Cancer Research Campaign, Human Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 2QQ, United Kingdom [C. E.]

Abstract most familial forms of pheochromocytoma is well known. Germ-line mutations in the RET proto-oncogene are found in Ͼ98% of MEN 2 Most pheochromocytomas are sporadic but about 10% are thought patients with pheochromocytoma (5, 7), and germ-line mutations in to be hereditary. Although the etiology of most inherited pheochromocy- the tumor suppressor gene VHL are found in Ͼ98% of VHL patients toma is well known, little is known about the etiology of the more common sporadic tumor. Recently, germ-line mutations of SDHD, a mitochondrial with pheochromocytoma (8, 9). In contrast, little is known about the complex II gene, were found in patients with hereditary paraganglioma. etiology of the more common sporadic form of pheochromocytoma. We sought to determine whether SDHD plays a role in the development of Somatic RET mutations have been found in up to 10% of tumors and sporadic pheochromocytomas and performed a mutation and deletion somatic VHL mutations in no more than 2% (10–13). analysis of SDHD. Among 18 samples, we identified 4 heterozygous se- Pheochromocytomas are also referred to as adrenal paraganglio- quence variants (3 germ-line, 1 somatic). One germ-line SDHD mutation mas. Paragangliomas originate from the neural crest-derived paragan- ؉ IVS1 2T>G (absent among 78 control alleles) is predicted to cause glia of the autonomic nervous system. They are identified throughout aberrant splicing. On reinvestigation, this patient was found to have a the body in both adrenal and extra-adrenal sites. Hereditary paragan- tumor of the carotid body, which was likely a paraganglioma. Another gliomas are highly vascularized tumors located in the head and neck. patient with malignant, extra-adrenal pheochromocytoma was found to have germ-line c.34G>A (G12S). However, this sequence variant was also They are most commonly found at the bifurcation of the carotid artery found in 1 of 78 control alleles. The third, germ-line nonsense mutation in the neck, also referred to as the carotid body. Recently, germ-line R38X was found in a patient with extra-adrenal pheochromocytoma. The mutations of SDHD were found in patients with hereditary paragan- only somatic heterozygous mutation, c.242C>T (P81L), has been found in glioma (14). SDHD, located on 11q23 (15), encodes the small subunit the germ line of two families with hereditary paraganglioma and is (cybS) of cytochrome b in the succinate-ubiquinone oxidoreductase conserved among four eukaryotic multicellular organisms. Hence, this complex (mitochondrial complex II). This enzyme complex plays an mutation is most likely of functional significance too. Overall, loss of important role in both the tricarboxylic acid cycle and the aerobic heterozygosity in at least one of the two markers flanking SDHD was respiratory chain of eukaryotic cell mitochondria. SDHD comprises found in 13 tumors (72%). All of the tumors that already harbored intragenic SDHD mutations, whether germ-line or somatic, also had loss four exons and three introns spanning over 19 kb (16). of heterozygosity. Our results indicate that SDHD plays a role in the We sought to determine whether SDHD plays a role in the devel- pathogenesis of pheochromocytoma. Given the minimum estimated germ- opment of sporadic cases of what has been defined as pheochromo- line SDHD mutation frequency of 11% (maximum estimate up to 17%) in cytomas in the German-Polish Registry by analyzing the SDHD gene this set of apparently sporadic pheochromocytoma cases and if these data for sequence variants and microsatellite markers flanking the gene for can be replicated in other populations, our observations might suggest LOH. Our results indicate that SDHD plays a role in the pathogenesis that all such patients be considered for SDHD mutation analysis. of pheochromocytoma.

Introduction Materials and Methods

Pheochromocytomas produce and secrete catecholamines and, Patients and DNA Samples. Genomic DNA from tumor and peripheral hence, can cause hypertension. They derive from large pleomorphic blood leukocytes was obtained from 18 unrelated patients (14 female, 4 male) chromaffin cells that most commonly arise from the adrenal medulla. who underwent surgery for sporadic pheochromocytoma at the University of In rare instances, pheochromocytomas are extra-adrenal. Most pheo- Freiburg, Germany. All of the samples were obtained with informed consent. chromocytomas are sporadic but ϳ10% are hereditary (1, 2) and may The patients were selected from a population-based register that currently be found in association with the MEN 23 syndromes or VHL (3–5). holds 170 sporadic pheochromocytomas4. These 18 patients were chosen Isolated familial forms of pheochromocytoma without other associ- because they were the only ones who had paired germ-line DNA and frozen ated clinical features occur even less commonly (6). The etiology of pheochromocytoma in the sample bank. All of the 18 pheochromocytomas were classified as being sporadic because of the absence of germ-line mutations specific for MEN 2 (RET, localized to 10q11.2) and VHL syndrome Received 6/9/00; accepted 10/31/00. The costs of publication of this article were defrayed in part by the payment of page (VHL, 3p25–26; Ref. 5, 8). Furthermore, the patients’ personal and family charges. This article must therefore be hereby marked advertisement in accordance with histories, as well as meticulous physical examinations, biochemical tests, and 18 U.S.C. Section 1734 solely to indicate this fact. imaging studies, were not suggestive of MEN 2, VHL, or NF 1 (3, 13, 17). Of 1 Partially supported by the National Cancer Institute, Bethesda, Maryland (P30CA16058 to Ohio State University Comprehensive Cancer Center) and by generous the 18 tumors, 4 pheochromocytomas were extra-adrenal, 3 of which were gifts from the Brown family in memory of Welton Brown and the Abrams family malignant. Another intra-adrenal pheochromocytoma also was classified as (to C. E.). malignant. Malignancy was operationally defined as local infiltration of adja- 2 To whom requests for reprints should be addressed, at Human Cancer Genetics Program, The Ohio State University, 420 W. 12th Avenue, Room 690C TMRF, Columbus, OH 43210. Phone: (614) 292-2347; Fax: (614) 688-3582 or (614) 688-4245; E-mail: 4 H. P. H. Neumann, S. Gla¨sker, B. U. Bender, T. W. Apel, R. Munk, O. Gimm, H. [email protected]. Dziema, C. Smigielski, K. Zerres, A. Januszewicz, and C. Eng. High frequency of 3 The abbreviations used are: MEN 2, multiple endocrine neoplasia type 2; VHL, von inherited mutations causing pheochromocytoma in a registry-based cohort, submitted for Hippel-Lindau syndrome; LOH, loss of heterozygosity. publication. 6822

Table 1 Summary of clinical features paraganglioma, and was scheduled for surgery. The IVS1ϩ2TϾG Cases with Cases without variant was not found among 78 control alleles. Another patient, a germ-line germ-line 17-year-old female, was found to have germ-line missense mutation Clinical feature SDHD variant SDHD variants G12S (c.34GϾA, exon 1; Fig. 1A). Clinically, she had an extra- a No. of cases 314adrenal, intra-abdominal pheochromocytoma. After surgery, cate- Age at presentation (yrs) 17–65 17–75 Multifocal presentation 1 1 cholamine levels were normal but increased again. At 21 years of age, Intra-adrenal disease at presentation 2 13 she underwent reoperation because of recurrent extra-adrenal, intra- Extra-adrenal disease at presentation 2 2 abdominal pheochromocytoma. Because there was nodal involvement a Single case with somatic mutation in tumor removed from clinical summary. within the jugular fossa, this pheochromocytoma was classified as malignant. We then analyzed 78 control alleles and found this sequence variant in 1 allele from one patient with intestinal lipoma. This cent tissues and/or metastatic disease. Relevant clinical information is sum- patient’s general practitioner was contacted, and the clinical absence marized in Table 1. of pheochromocytoma and paraganglioma was confirmed. A third Genomic DNA from peripheral blood leukocytes from a region-matched, patient, a 33-year-old female, was found to have germ-line nonsense race-matched control group was available. This group consisted of 39 individ- Ͼ uals who were admitted to the Department of General Surgery, Halle, Ger- mutation R38X (c.112C T, exon 2; Fig. 1C). Clinically, she had two many, for nonpheochromocytoma-related diseases. Informed consent was extra-adrenal pheochromocytomas, one intra-abdominal tumor, and given in all of the cases. one thoracic tumor. DNA extraction was performed using the QIAamp tissue kit (Qiagen, Santa The only somatic mutation c.242CϾT, P81L (exon 3; Fig. 1D) was Clarita, CA) according to the manufacturer’s instruction. Mutation Analysis. PCR amplification was carried out in 1 ϫ PCR buffer (Qiagen) that contained 200 ␮mol/liter deoxynucleotide triphosphate, 0.6 ␮mol/liter of each primer (14), 2.5 units of Taq polymerase (Qiagen), and 100–200 ng of tumor DNA template in a 50 ␮l volume. PCR conditions were: one cycle of 15 min at 95°C; 35 cycles of 1 min at 95°C; 1 min at 58°C; 1 min at 72°C; followed by one cycle of 10 min at 72°C. PCR amplicons were gel (Bio-Rad Laboratories, Hercules, CA) and column (Wizard PCR Prep; Promega, Madison, WI) purified and subjected to semi- automated sequencing using the above primers, dye terminator technology, and the ABI377xl or PE3700. If sequencing revealed a variant, the corresponding leukocyte DNA was examined in the same manner to determine whether the sequence variant was somatic or germ-line. Each sample with a sequence variant was subjected to repeat mutation analysis that used a separate PCR reaction. LOH Analysis. All of the PCR reactions were carried out using 0.6 ␮M each of forward and reverse primer in 1 ϫ PCR buffer (Qiagen), 4.5 mM ϫ MgCl2 (Qiagen), 1 Q-buffer (Qiagen), 2.5 units of HotStarTaq polymerase (Qiagen), and 200 ␮M deoxynucleotide triphosphate (Life Technologies, Inc., Gaithersburg, MD) in a final volume of 50 ␮l. Reactions were subjected to 35 cycles of 94°C for 1 min, 55–60°C for 1 min, and 72°C for 1 min followed by 10 min at 72°C. LOH analysis was performed as described previously (18) using the two markers flanking SDHD, D11S1347 (telomeric) and D11S1986 (centromeric). Both forward primers were 5Ј-labeled with either HEX or 6-FAM fluorescent dye (Research Genetics, Huntsville, AL). PCR reactions were carried out as described above and separated by electrophoresis through 6% denaturing polyacrylamide gels using an Applied Biosystems model 377 automated DNA sequencer (Applied Biosystems and Perkin-Elmer Corp., Ridgeway, CA). The results were analyzed by automated fluorescence detection using the GeneScan collection and analysis software (GeneScan; Applied Biosystems, Norwalk, CT). Scoring of LOH was performed by inspection of the GeneScan analysis output. A double peak, observed in the microsatellite marker that was amplified from DNA extracted from the germ-line sample, indicated heterozygosity. A single peak in DNA extracted and amplified from the corresponding tumor sample indicated a loss of one allele. If normal cells were admixed with tumor cells, a ratio of 1.5:1 or greater of the germ-line DNA peak:tumor DNA peak was operationally defined as LOH.

Results SDHD Mutation Analysis in Pheochromocytomas. Among 18 samples, we identified four heterozygous sequence variants in four distinct samples (Fig. 1). Three of these were germ-line, and the remaining one was somatic. A germ-line SDHD mutation IVS1ϩ2TϾG (Fig. 1B) was detected in a 39-year-old male. Although RNA was not available, this variant is predicted to cause aberrant splicing. We then contacted this patient’s general practitioner, who Fig. 1. Somatic and germ-line SDHD sequence variants (denoted with arrowheads). The affected codon and the amino acids are depicted below the chromatograms. Note that reported that this patient, at 43 years of age, had recently been the mutant chromatograms appear apparently heterozygous despite demonstration of LOH diagnosed with a tumor of the carotid body, which was likely a of the wild-type allele likely because of admixed normal tissue contamination. 6823

dilemma because it has been found in one regional control who did not have any evidence of neuroendocrine tumors at the age of 72. Baysal et al. (14), on the other hand, did not note G12S in their 200 normal control chromosomes. Nonetheless, we can postulate either that this is a pathogenic but low-penetrance germ-line missense mutation that predisposes to pheochromocytoma or that this variant could be a very rare polymorphism. Codon 12 is conserved between Homo sapiens and the domestic cattle (14). In that position, an alteration from a very small to a larger neutral and polar amino acid might have some consequence; although without direct functional assay, it is open to speculation. Given that the corresponding tumor had LOH of the wild-type allele also lends some support that G12S may be somehow pathogenic. Nonetheless, the conservative estimate of occult germ- line SDHD mutations that are likely pathogenic would be 11% if we discount the G12S variant (see below). The LOH frequency at the SDHD locus on 11q23 is worthy of note, as well. LOH in pheochromocytomas has been reported on 1p (42– 71%), 3p25 (16–56%), 17p (24%), and 22q (31–53%; Refs. 19–22). Fig. 2. LOH analysis in pheochromocytomas; A, illustrative examples of LOH (arrows) However, LOH on chromosome 11 in pheochromocytomas has not at D11S1986 (left panel), retention of heterozygosity (ROH) and noninformativity (NI)at D11S1347 (right panel). GL, germline; Tu, tumor; B, summary of LOH data for the 18 been reported to date. We found LOH in at least one of the two tumor-germ-line pairs. f, LOH; Ⅺ, retention of heterozygosity; and o, noninformativity. investigated markers on 11q23 in 72% of pheochromocytomas, a significant frequency. Compared with this high frequency of LOH, the frequency of functional mutations in SDHD (17%) is relatively low. found to occur in a heterozygous state in a pheochromocytoma from The 6% somatic mutation frequency in SDHD, however, is compara- a 42-year-old female. This missense mutation was not found in ble with that of RET and VHL mutations. We suspect that in the corresponding germ-line DNA. tumors with apparent hemizygous deletion, other epigenetic mecha- LOH Analysis. Overall, LOH (Fig. 2) in at least one of the two nisms, e.g., methylation of CpG islands in this region, may also play 11q23 markers was found in 13 tumors (72%). LOH of both markers a role in silencing SDHD similar to the inactivation of 14–3-3␴ in was found in 3 tumors (17%). The remaining 10 tumors showed LOH of one or the other 11q23 marker. All of the tumors already harboring breast cancer (23). Because the promoter of SDHD has not been intragenic SDHD mutations also showed LOH. identified yet (16), no further attempt to address this question has been undertaken. Another explanation of our LOH data is that the LOH Discussion actually reflects deletion of genes that neighbor SDHD and not SDHD itself. In our series of 18 apparently sporadic pheochromocytoma sam- The molecular basis for sporadic pheochromocytomas has been ples, we identified one (6%) somatic SDHD mutation and three (17%) somewhat elusive. Allelotyping studies have revealed regions of LOH germ-line SDHD variants. The only somatic mutation, P81L, is almost (above). Despite relatively high frequencies of LOH in the VHL gene certainly pathogenic. It has also been found in the germ-line of two region (3p25), Ͻ2% of sporadic pheochromocytomas carry somatic families with hereditary paraganglioma (14). This missense mutation VHL mutations. Similarly, somatic RET mutations have been shown is highly conserved in four eukaryotic multicellular organisms, and to occur in 0–10% of sporadic pheochromocytomas (10, 12, 24). the proline-leucine substitution in that position would be predicted to Although our data need to be confirmed in a larger series, they alter cybS conformation (14). This tumor with somatic P81L also has strongly argue for a role of SDHD in the pathogenesis of sporadic LOH of the remaining wild-type allele, which lends additional support pheochromocytoma. It was of particular interest that we found not that the missense mutation is pathogenic and that SDHD is a tumor only a somatic mutation but also occult germ-line variants, suggesting suppressor gene that plays some role in the pathogenesis of sporadic a familial predisposition. Among prior series of apparently sporadic pheochromocytoma. pheochromocytoma presentations, occult germ-line mutations in VHL The nonsense germ-line R38X mutation is almost certainly patho- and RET were found only in ϳ5% and Ͻ2%, respectively (10, 13). genic because it would likely result in a very short nonfunctional Thus, the minimum estimate of occult germ-line SDHD mutation protein. This mutation was also found by Baysal et al. (14) in the germ frequency of 11% (17% if the variant of unknown significance is line of one family with hereditary paraganglioma. The germ-line considered) found in these patients is significant. This mutation fre- ϩ Ͼ IVS1 2T G mutation occurs at one of the highly conserved con- quency is clinically important in the context that a preliminary survey sensus splice sequences and is almost certainly functionally signifi- of the entire Registry, which comprises ϳ100 nonfamilial pheochro- cant inasmuch as it would be predicted to result in aberrant splicing. mocytoma cases, also reveals an occult germ-line SDHD mutation Unfortunately, despite great efforts, no RNA is available. This germ- frequency of 10%.5 From this current study alone, it is not known line variant has not been found in 78 regional control alleles, and whether germ-line SDHD mutations in isolated pheochromocytoma Baysal et al. (14) did not note the presence of this variant in 200 of cases would predict for the development of extra-adrenal disease or their control chromosomes either. That this patient developed a ca- whether they could also predispose to a familial pheochromocytoma rotid body tumor, almost certainly a paraganglioma, 4 years subse- only syndrome. Of interest, among the Registry cases as a whole, 70% quent to his diagnosis of isolated pheochromocytoma and that the of those with germ-line SDHD mutations had intra-adrenal pheochro- tumor had LOH of the remaining allele lend strong support that this mocytomas only, and 30% had extra-adrenal disease.5 Families with germ-line variant is actually an occult germ-line SDHD mutation that predisposed to both the pheochromocytoma and the carotid body 5 H. P. H. Neumann, S. Gla¨sker, B. U. Bender, T. W. Apel, R. Munk, O. Gimm, H. tumor. Finally, the G12S germ-line variant that occurred in a teenager Dziema, A. Januszewicz, and C. Eng. Occult germline SDHD mutations in a registry- with adrenal and extra-adrenal pheochromocytoma is somewhat of a based pheochromocytoma cohort, manuscript in preparation. 6824

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2000 American Association for Cancer Research. Somatic and Occult Germ-line Mutations in SDHD, a Mitochondrial Complex II Gene, in Nonfamilial Pheochromocytoma

Oliver Gimm, Mary Armanios, Heather Dziema, et al.

Cancer Res 2000;60:6822-6825.

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