high frequency of germline succinate dehydrogenase mutations in sporadic cervical paragangliomas in...

High Frequency of Germline Succinate DehydrogenaseMutations in Sporadic Cervical Paragangliomas inNorthern Spain: Mitochondrial SuccinateDehydrogenase Structure-Function Relationships andClinical-Pathological Correlations

Jorge Lima,* Talia Feijao,* Andre Ferreira da Silva, Isabel Pereira-Castro, Gregorio Fernandez-Ballester,Valdemar Maximo, Agustin Herrero, Luis Serrano, Manuel Sobrinho-Simoes, and Ginesa Garcia-Rostan

Institute of Molecular Pathology and Immunology of University of Porto (J.L., T.F., A.F.d.S., I.P.-C., V.M., M.S.-S., G.G.-R.)and Medical Faculty (J.L., M.S.-S.), University of Porto, 4200–465 Porto, Portugal; Institute of Molecular and CellularBiology (IBMC) (G.F.-B.), Miguel Hernandez University, Elche, 03202 Alicante, Spain; Department of Pathology (A.H.),School of Medicine, Oviedo University, Oviedo, 33006 Asturias, Spain; Department Structural and Computational Biology(L.S.), European Molecular Biology Laboratory, Heidelberg, 69117 Germany and Systems Biology Programme, Centre forGenomic Regulation, 08003 Barcelona, Spain; and Hospital S. Joao (M.S.-S.), 4202–451 Porto, Portugal

Purpose: Germline SDHB, SDHC, and/or SDHD mutations havebeen reported in familial and apparently sporadic paragangliomas(PGLs). There is, however, some variation in the prevalence, pen-etrance, and phenotypic expression of the succinate dehydrogenase(SDH) mutated gene among different populations. We sought to de-termine whether germline mutations in SDHB, SDHC, and/or SDHDplay a role in cervical PGLs from northern Spain, where this disorderis particularly frequent, and whether there is any difference withrespect to the data published in other populations.

Design: Thirty-six sporadic cervical PGLs and four familial PGLswere investigated by PCR-single-strand conformation polymorphismanalysis and sequencing. Computational biology was applied to ad-dress the structural-conformational changes behind missense muta-tions and, simultaneously, infer the possible consequences in proteinfunction.

Results: Eight sporadic cases (22.2%) carried pathogenic germlinemutations, six of which were in SDHB and two in SDHD. Three

families had mutations in SDHD and one in SDHB. Seven of 11different pathogenic mutations (64%) affected SDHB. Ten mutationswere novel. Missense mutations were primarily found in SDHB andframeshift mutations in SDHD. Missense SDHB mutations seemedto alter the enzymatic activity by hampering the electron transfer.SDH-linked tumors occurred mainly in males (P � 0.0033), occurredat a younger age (P � 0.0001), were usually multifocal (P � 0.0011),and exhibited a larger size (P � 0.0341).

Conclusions: A significant proportion of sporadic cervical PGLsarise as a consequence of intrinsic genetic factors. At variance withprevious reports, SDHB is frequently mutated in sporadic cervicalPGLs and the mutations do not entail a deleterious behavior. There-fore, SDHB genetic testing may be considered in all subjects pre-senting with solitary cervical PGL and no family history. (J ClinEndocrinol Metab 92: 4853–4864, 2007)

CERVICAL PARAGANGLIOMAS (PGLS) represent0.012% of all human tumors and 0.6% of all neoplasms

in the head and neck. Those occurring in the carotid bodyaccount for 60–80% of all cervical PGLs. Other tumor loca-tions include the jugulotympanic paraganglia at the skullbase (18–36%) and the vagal body (3–4%) (1). It is estimatedthat up to 30% of the cervical PGLs are familial, which cor-responds to one of the highest frequencies of inherited sus-ceptibility among human tumors. Although most sporadiccervical PGLs present as a single highly vascularized mass,lacking endocrine activity, multicentric or bilateral tumors

occur in 10% of the cases. In the familial setting, about 40%of the patients display bilateral carotid body tumors. Mul-ticentric PGLs may develop at other head and neck para-ganglia or the sympathoadrenal abdominal paraganglia. Pa-tients with hereditary cervical PGLs may be diagnosedearlier than sporadic cases, with a trend for an earlier age oftumor onset in successive generations (2, 3). Cervical PGLsare mostly benign, slow-growing tumors. Only 2–5% of ca-rotid body or jugulotympanic PGLs and 10–19% of vagalPGLs disclose uncertain malignant potential (4–6).

Familial PGL is a genetically heterogeneous disorder(OMIM 168000), which involves various chromosomal loci.In 1997, linkage analyses in several unrelated Dutch andNorth American PGL families revealed two loci, 11q23.1 and11q13, that segregated with the disease (7). In 2000, Baysal etal. (8) showed that germline loss-of-function mutations inSDHD at 11q23.1 caused familial PGL. Shortly afterward,studies based on direct candidate gene analyses demon-

First Published Online September 11, 2007* J.L. and T.F. contributed equally to this work.Abbreviations: PGL, Paraganglioma; SDH, succinate dehydrogenase;

SSCP, single-strand conformation polymorphism analysis.JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the en-docrine community.

0021-972X/07/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 92(12):4853–4864Printed in U.S.A. Copyright © 2007 by The Endocrine Society

doi: 10.1210/jc.2007-0640

4853

strated that germline mutations in two other genes at chro-mosome 1, SDHB (1p36.13) and SDHC (1q23.3), were alsoinvolved in familial PGL (9, 10). The causative gene mappingat 11q13 still awaits identification.

SDHB, SDHC, and SDHD genes (SDH genes) are nucleargenes that code for the SDHB, SDHC, and SDHD subunits ofthe heterotetrameric mitochondrial enzyme succinate dehy-drogenase (SDH). The SDHB subunit, also known as iron-sulfur protein, is part of the hydrophilic catalytic domain andis highly conserved throughout species. SDHB is directlybound to SDHA, which contains the flavin-adenine dinucle-otide (FAD) prosthetic group and the substrate binding site,and to SDHC and SDHD, which together anchor SDH to themitochondrial inner membrane and provide the ubiquinonebinding site. SDHC and SDHD harbor the heme group towhich the electrons are transferred from the iron-sulfur clus-ters: FES (2Fe-2S), FS4 (4Fe-4S), and F3S (3Fe-4S). These iron-sulfur clusters are located within SDHB and act as redoxcenters, transporting the electrons derived from the FADprosthetic group (reduced on oxidation of succinate) to themembrane soluble transporter ubiquinone, which will thenenter the oxidative phosphorylation (OXPHOS) system(11, 12).

So far, the majority of the studies dealing with germlineSDH mutations in PGL patients have been carried out inNorth European and/or North American populations. Thesestudies, besides revealing an evident familial clustering ofthe disease, with three founder mutations in The Nether-lands, have also shown that a subset of apparently sporadiccervical PGLs harbor germline mutations in SDH genes. Thelatter individuals represent occult familial cases with incom-plete/low penetrance of the mutated allele (13, 14).

Although germline mutations in SDHB and/or SDHDhave been reported in apparently sporadic and familial cer-vical PGLs (70–90% of familial cervical PGLs and �8% ofsporadic cervical PGLs), the overall mutation frequency foreach of these two genes is significantly different. Mutationsin SDHD are considered the major cause of cervical PGLs. Todate, 94 and 69 unique allelic variants have been describedin SDHB and SDHD, respectively (http://chromium.liacs.nl/lovd_sdh) (15). The prevalence of SDHC mutation carri-ers among cervical PGL patients is much lower. Only ninedifferent allelic variants, primarily associated with familialpresentations, have been annotated in SDHC (16).

The accumulated data indicate that there is some variationin the prevalence, penetrance, and phenotypic expression ofthe SDH mutations among different populations. Under-standing the cause/s of these variations is important for thegenetic counseling and proper management of PGL patients.In this study, we sought to determine whether germlinemutations in SDHB, SDHC, and/or SDHD play a role in thenosology of cervical PGLs from northern Spain, where thisdisorder is particularly frequent. We also assessed whetherthere is any difference with respect to the overall mutationprevalence, mutation pattern, and penetrance reported inother populations. Because some missense substitutions mayresult in partially functional mutant proteins that could ac-count for the reduced/incomplete penetrance observed insome SDH mutation carriers, we also addressed the struc-

tural-functional consequences behind punctual, unreportedSDHB mutations.

Patients and MethodsPatients and control cases

Individuals diagnosed with cervical PGL at the Hospital Central deAsturias (Spain), between 1981 and 2005, were identified by retrospec-tively reviewing the clinicopathological records and direct patient/fam-ily interview. Forty unrelated cases (36 sporadic and four inherited) wereenrolled in the study due to the availability of DNA from peripheralblood leukocytes, tumor tissue, and clinical data. All histological diag-noses were reviewed according to established morphological criteria (3,4). Sporadic cases were defined as those having no familial history ofPGLs in the parental and grandparental generations. Disease was con-sidered to be inherited when at least two first-degree relatives, prefer-entially from different generations, or two second-degree relatives wereaffected by PGLs. Patients displaying syndromic features associatedwith von Hippel Lindau disease (VHL), multiple endocrine neoplasiatype 2 (MEN2), or neurofibromatosis type 1 (NF1) were excluded fromthe study population. Only the index patient of each family entered inthe first screening of mutations. Missense mutations were regarded aspathogenetically relevant if not found among 203 anonymous healthyblood donors residing in the same geographic area of the patients.Identification of a putative disease-causing mutation was followed bygenetic testing of all at risk living relatives of the mutation carriers.

Informed consent for genetic testing was obtained from all patients,relatives, and control cases. Processing of samples and of patient infor-mation was in agreement with protocols approved by the correspondingethical committees.

Mutational analysis

Genomic DNA was extracted from peripheral blood leukocytes usinga standard salt-precipitation method (17). Matching tumoral DNA wasextracted from formalin-fixed, paraffin-embedded specimens accordingto a previously described protocol (18). When necessary, tumors weremicrodissected to increase the proportion of neoplastic cells, whichalways represented at least 80% of the total.

The occurrence of SDHB, SDHC, and/or SDHD mutations was eval-uated by PCR-single-strand conformation polymorphism analysis(SSCP). Primers and PCR/SSCP conditions are available on request. Allmobility shifts observed in the SSCP analysis were ratified by repeatingthe PCR-SSCP assay.

Whenever a sample displayed recurrent aberrant SSCP conformers,the respective PCR product was purified using the GFX PCR DNA andgel band purification kit (Amersham Biosciences, Piscataway, NJ). Thepurified target DNA was subjected to automatic sequencing (ABI PRISM3100 genetic analyzer; Applied Biosystems, Foster City, CA), using theBigDye terminator version 3.1 cycle sequencing kit (Applied Biosys-tems). Sense and antisense sequencing was performed for confirmation.All mutations were further verified by PCR and direct sequencing froma new DNA template.

Structural analysis of SDHB mutations

The structure of the human mitochondrial enzyme SDH was modeledat 2.4A resolution (1ZOY.PDB) by means of homology with the recentlysolved crystal structure from porcine heart (19). The porcine structuresorts out the low sequence homology found in the integral membraneanchors of the prokaryotic counterpart (20) and overrides the absence ofthe ubiquinone binding sites, providing a reliable model to study thestructural-conformational changes induced by human mutations as wellas their relationship with human mitochondrial pathologies. Modelingwas performed upon sequence alignment using the BuildModel func-tion implemented in Fold-X (21, 22). During this procedure Fold-Xperforms the mutagenesis while testing different rotamers, allowingneighbor side chains to move. The emerging models undergo additionaloptimization by the Repair function of Fold-X, where those residues thathave bad torsion angles or van der Waals clashes are identified andcorrected.

The structure was edited with Swiss-PDB viewer 3.7 (23) and all the

4854 J Clin Endocrinol Metab, December 2007, 92(12):4853–4864 Lima et al. • Germline SDHB Mutations in Sporadic Paragangliomas

molecular graphics created with Pymol (24). The comparison betweenwild-type and mutated models gives the opportunity to understand thestructural consequences behind punctual mutations.

Statistical analysis

The Fisher’s exact test, ANOVA test, and �2 test with the Yatescorrection were applied in the statistical computing (Statview; SAS Inc.,Cary, NC). P � 0.05 was considered statistically significant.

ResultsPatient and tumor characteristics

Among the 48 patients diagnosed with PGL, 36 (75%) hadno family history and 12 (25%) inherited the disease. Theclinical and pathological features (Table 1) closely paralleledthose described in most larger series (3, 4). Apparently spo-radic carotid body tumors was the only group in which ahigher prevalence of males (five of eight, 63%) was detected.In the familial group, nearly 38% of the patients with carotidbody PGLs showed bilateral, synchronous, or metachronoustumors. Tympanic PGLs were detected only in sporadic pre-sentations (P � 0.0213), displayed a smaller size (average 0.8cm), and occurred preferentially in females (nine of 10).

In agreement with the reported nonfunctional status ofmost cervical PGLs, none of the patients involved in thisstudy had increased levels of catecholamine secretion. Mi-croscopically, consistent with the neuroendocrine nature ofnormal paraganglia, the neoplastic chief cells showed strong-moderate immunoreactivity for chromogranin-A, neuron-specific enolase, and/or synaptophysin, whereas the non-

neoplastic sustentacular cells embracing the chief cellsdemonstrated weak positivity for S-100.

The mean follow-up time was similar in both groups (�7yr). All the sporadic patients who developed a tumor recur-rence had undergone resection of a single jugular (two offive) or jugulotympanic (three of five) PGL. Up to the lastfollow-up performed in this cohort, no patient had evidencedlocal and/or distant metastases. Only one familial patientbearing extra-adrenal PGLs died of the disease. It is note-worthy that 73% of the patients had lived near the sea levelor at low altitudes.

Molecular genetics

Twenty-five of the 40 PGL patients (62.5%) initially en-rolled in the germline mutational screening revealed aber-rant SSCP conformers. The type of alteration, exonic/introniclocation, altered nucleotide position, specific nucleotidechange, and corresponding amino acid substitution are sum-marized in Table 2.

Half of the patients (20 of 40) carried germline codingsequence variants [13 in SDHB (65%), five in SDHD (25%),and two in both genes (10%)]. Noncoding sequence alter-ations were limited to SDHB (13 patients, 32.5%) and SDHC(two patients, 5%). Nine of the patients who displayed theintronic variant IVS2–36G�T at SDHB harbored a concom-itant exonic variant (A6A) in SDHB.

Frameshift coding alterations were more likely to appearin SDHD (80%) and missense alterations in SDHB (62.5%).

TABLE 1. Clinical features of 48 PGL patients analyzed for SDHB, SDHC, and SDHD germline mutations

Clinical features All patients(n � 48)

Familial cases(n � 12)a

Sporadic cases(n � 36) P valueb

GenderFemale 29 (60) 4 (33) 25 (70) 0.041Male 19 (40) 8 (67) 11 (30)

Mean age at diagnosis, yr 49 41 52 0.0338Tumor focality

Single PGL 41 (85) 5 (42) 36 (100) �0.0001Multiple PGLs 7 (15) 7 (58) 0

Mean tumor size (cm) 3.3 4 2.8 0.0343Tumor originc,d

Carotid body 16 (29.5) 8 (44)d 8 (22) 0.119Jugular glomus 10 (18.5) 4 (22) 6 (17) n.s.s.Tympanic bulb 10 (18.5) 0 10 (28) 0.0213Jugulotympanic paraganglia 10 (18.5) 1 (6) 9 (25) 0.137Vagal body 6 (11) 3 (17) 3 (8) n.s.s.Extraadrenal paraganglia 2 (4) 2 (11) 0 0.106

Total number of tumors 54 18 36Mean follow-up time, months (range) 81 (4–300) 86 (4–300) 79 (26–239) n.s.s.Recurrence 11 (23) 6 (50) 5 (14) 0.0176Local and/or distant metastases 0 0 0Altitude with respect to sea levele

Near sea level (1–50 m) 18 (37.5) 4 (33) 14 (39) n.s.s.Low-level altitudes (�50 to � 300 m) 17 (35) 6 (50) 11 (30) n.s.s.Medium-level altitudes (�300 to � 700 m) 8 (17) 2 (17) 6 (17) n.s.s.High-level altitudes (�700 to � 1000 m) 5 (10.5) 0 5 (14) n.s.s.

The numbers in parentheses indicate percentage values. n.s.s., Not statistically significant.a Includes the proband of each pedigree and all the relatives who underwent surgery for PGL.b Comparison of the clinical features in a cohort of 12 familial and 36 sporadic cases diagnosed with PGL. P � 0.05 are considered statistically

significant. P values between 0.05 and 0.2 indicate a trend for an association. P � 0.2 are not statistically significant (n.s.s.).c In the group of familial patients, all the tumors that originated in different paraganglia within a single individual were indicated.d Bilateral carotid body tumors were counted only once.e Patients were divided into four categories according to the altitude of their residential area with respect to sea level.

Lima et al. • Germline SDHB Mutations in Sporadic Paragangliomas J Clin Endocrinol Metab, December 2007, 92(12):4853–4864 4855

The ratio of different nonsynonymous to different synony-mous sequence variants was 3:1 for SDHD and 5:3 for SDHB.Most of the missense sequence variants were transitions andinvolved residues highly conserved among species (87.5%,seven of eight). The only missense transversion (R17L inSDHD) observed in this cohort affected an amino acidconserved in mammals but not in bacteria. Six of nineSDHB (67%) (C93R, C186Y, P197S, P254L, L180L, andc.312InsCACTGCA), one SDHC (100%) (IVS1 � 13 InsTG),and three of seven SDHD (43%) (R17L, c.383–386InsT, andc.120–127DelCCCAGAAT) sequence alterations had notbeen previously reported (http://chromium.liacs.nl/lovd_sdh) (15). Sequence alterations in the SDHD gene were

roughly equally distributed throughout all four exons,whereas those in the SDHB gene tended to cluster in exon 1(50%) followed by exon 4 (19%) and were not seen in exons2 and 8.

Four coding sequence variants were present in the chro-mosomes of healthy individuals: G12S (three of 186; 1.6%),H50R (three of 274; 1%), and S68S (9 of 252; 3.6%) for SDHDand A6A (two of 182; 1%) for SDHB. The two intronic se-quence variants, IVS2–36G�T in SDHB and IVS1 � 13 InsTGin SDHC, detected in 15 PGL patients, were also found in12.8% (52 of 406) and 6.3% (12 of 192) healthy control cases,respectively. All six alterations will be designated hereafteras polymorphisms.

TABLE 2. SDHB, SDHC, and/or SDHD sequence variants in sporadic and familial cervical PGLs: distribution according to tumor origin

Tumor origin I.D.

SDHDa SDHBa SDHCa

Carriersb Altitude(m)c

Exon(E),

Intron(I)

Nucleotide changeAminoacid

change

Exon(E),

Intron(I)

Nucleotidechange

Aminoacid

change

Exon(E),

Intron(I)

Nucleotidechange

Aminoacid

change

Sporadic casesCarotid body PGLs 4 E-2 c.149A�G H50R NA 8

18 E-6 c.589C�T P197S 2 673I-2 IVS2–36 G�T

28 E-1 c.50G>T R17L E-4 c.300T�C S100Sd 2 23229 E-1 c.34G�A G12S NA 232

E-3 c.204C�T S68Sd

40 E-7 c.761C>T P254L 2 55743 E-1 c.18C�A A6Ad NA 395

I-2 IVS2–36 G�TJugular PGLs 31 E-1 c.18C�A A6Ad NA 850

I-2 IVS2–36 G�T3 I-1 IVS1�13 InsTG NA 838

JugulotympanicPGLs

1 E-1 c.18C�A A6Ad NA 280

I-2 IVS2–36 G�T9 I-2 IVS2–36 G�T NA 232

12 E-5 c.540G>A L180Ld,e ND 97913 E-3 c.277T>C C93R 3 5016 E-1 c.18C�A A6Ad NA 1

I-2 IVS2–36 G�T66 E-4 c.312

InsCACTGCA2 300

73 I-2 IVS2–36 G�T NA 1Tympanic PGLs 7 I-2 IVS2–36 G�T NA 838

8 I-2 IVS2–36 G�T NA 26074 E-1 c.18C�A A6Ad NA 400

I-2 IVS2–36 G�T77 E-1 c.18C�A A6Ad NA 5

I-2 IVS2–36 G�TVagal PGLs 14 E-6 c.557G>A C186Y 1 8

76 E-4 c.337–340 Del GACT 1 15Familial casesf

Jugular PGL 54 E-4 c.293G�A C98Y 7 6E-1 c.18C�A A6Ad

I-2 IVS2–36 G�TMultiple head and

neck PGLs26 E-4 c.386–387 InsT E-1 c.18C�A A6Ad 7 232

I-2 IVS2–36 G�TMultiple head and

neck PGLs55 E-4 c.337–340 Del GACT I-1 IVS1�13 Ins TG 5 80

Multiple head andneck PGLs

37 E-2 c.120–127DelCCCAGAAT

5 467

Nucleotide sequences from both the sense and antisense orientation were compared with wild-type GenBank reference accession no.AB006202/NM_003002 (SDHD), U17248/NM_003000 (SDHB), NM_003001 (SDHC) and Ensembl gene no. ENSG00000204370 (SDHD),ENSG00000117118 (SDHB), ENSG00000143252 (SDHC). I.D., Patient identification number; IVS, intronic sequence variant; Ins, insertion;Del, deletion; NA, not applicable; ND, not determined.

a Exon/intron number with altered nucleotide position/s and corresponding amino acid substitution. Mutations not previously reported arein boldface. Sequence variants found in the control population are in italics.

b Includes the proband and all the phenotypically affected and unaffected relatives harboring the pathogenic germline mutation.c Altitude with respect to sea level. Patients were divided into four categories according to the altitude of their residential area: near sea

level (1–50 m); low-level altitudes (�50 to � 300 m); medium-level altitudes (�300 to � 700 m); and high-level altitudes (�700 to � 1000 m).d Synonymous mutations.e This mutation occurred in the last nucleotide of exon 5 and led to abnormal splicing of the SDHB primary transcript.f Only the index case of each pedigree is included.


After excluding the aforementioned polymorphisms, itemerged a trend for an association of pathogenic missensemutations with SDHB (five of six, 83%) and frameshift mu-tations with SDHD (four of five, 80%) (P � 0.0801).

Sporadic paragangliomas

Sixteen patients (44.4%) carried germline coding alter-ations [12 (75%) in SDHB, three (19%) in SDHD and one (6%)in both genes] (Table 2). The only germline alteration ob-served in SDHC was an intronic polymorphism (IVS1 � 13InsTG).

A total of 13 different germline coding alterations wereidentified, including seven nonsynonymous/missense alter-ations (54%), four synonymous alterations (31%), and twoframeshift alterations (15%). All these germline alterationswere confirmed at the somatic level and no additional so-matic changes were observed. Figure 1 shows the SSCP pat-terns and sequencing chromatograms of representativeSDHD and SDHB mutation carriers.

Most of the coding alterations (89%) were point mutations.One of those point mutations (L180L), involving a G�Atransition in the last nucleotide of SDHB exon 5, resulted inalternative splicing of the SDHB primary transcript. To dem-onstrate that L180L could be an unreported splice site mu-tation and thus a pathogenic mutation, we extracted RNAfrom peripheral blood leukocytes and synthesized cDNA. Bycombining the same forward primer with two reverse prim-ers, one specific for the wild-type transcript and the otherspecific for the mutated, we observed PCR-amplificationonly with the wild-type reverse primer, suggesting an ab-normal splicing of the mutated allele (Fig. 2A). To furtherverify the splicing, a fragment encompassing part of exon 5

up to exon 7 was amplified and sequenced. The chromato-gram showed the peak corresponding only to the wild-typeallele (G) and not two superimposed peaks (G and A) as itwould be expected if both alleles were correctly transcribedand spliced (Fig. 2B).

FIG. 1. Representative SSCP patterns of missense SDHB and SDHD mutations detected in apparently sporadic cervical PGLs. A, C, and Dcorrespond with carotid body PGLs. B shows a jugulotympanic PGL. Next to each hematoxylin and eosin section are the sequence chromatogramsthat emerged from the shifted bands that showed up in the SSCP gel (arrow at index lane). The migration pattern of our target DNA wascompared with the electrophoretic mobility of a known wild-type sequence (WT lane). The nucleotide change is indicated within each sequencetrace (arrow). Phenotypically unaffected at risk relatives of mutation carriers were investigated for the mutation found in the index case.Pedigrees are shown on each panel.

FIG. 2. Abnormal splicing of the mutated allele bearing the G�Atransition at nucleotide 540 of SDHB. A, A PCR product of 246 bp wasobtained using a reverse primer specific for the wild-type primarytranscript (lane A). No amplification was observed with a reverseprimer specifically devised for the mutant allele (lane B). Lane Crepresents an internal PCR control with primers annealing in exons4 and 7 (455 bp). Right margin, 1 kb plus ladder (lane M). B, To furtherconfirm the splicing depicted in 2A, a fragment encompassing part ofexon 5 up to exon 7 was amplified and sequenced. The correspondingchromatogram shows only the wild-type peak (G), where two super-imposed peaks (G and A) should be present if both alleles are correctlytranscribed and spliced.


The intronic polymorphism IVS2–36G�T in SDHB was inlinkage disequilibrium with a coding polymorphism (A6A)in exon 1 of SDHB in six patients and associated with apathogenic mutation (P197S) in exon 6 of SDHB in one pa-tient. In four additional patients, the IVS2–36G�T was theonly change detected. Similarly, two polymorphisms atSDHD (G12S and S68S) arose associated in one patient.

Structural biology studies revealed that all the SDHB mis-sense mutations are predicted to alter the enzymatic activityby disrupting the anchoring to the FES, FS4, and F3S clusterspresent within the catalytic core of SDH. Some also mayaffect the interaction with other subunits of mitochondrialSDH such as flavoprotein (SDHA). Of note, the P197S mu-tation could hamper the binding of the ubiquinone. Themutation is placed in a pocket surrounded by hydrophobicand aromatic residues. The polar group of serine could di-

rectly clash with the ubiquinone ring, could hydrogen bondto W201, or both, breaking the binding of the ubiquinone tochain B and thus avoiding electron transfer. In addition, theadoption of dihedrals slightly forced for serine could inducea small local conformational change that, in turn, could affectC196, one of the three cysteines that link 3Fe-4S, and thendisrupt cluster binding. The C93R mutation breaks the liga-tion of the cluster 2Fe-2S because this cysteine participates inthe binding to the prosthetic group. The mutation places a bigand charged amino acid that is oriented to FAD-bindingprotein (chain A). The arginine side chain clashes with H99,A102 (both in chain A), and even the FAD group, thus ham-pering the chain A-chain B complex association. The C186Ymutation breaks the binding to the cofactor 4Fe-4S. The sidechain of tyrosine also clashes with backbone and side chainof L188 and with A210. The P254L mutation must carry a

FIG. 3. Detailed illustration of the structural consequences of missense mutations in the iron-sulfur protein (SDHB). The wild-type (wt)conformation is shown on the left and the mutant conformation on the right. Chain B, green backbone in wt, cyan backbone in mutants. ChainA, light green backbone in wt and mutant conformation. The FAD-binding protein (chain A), when affected, was represented in light magentafor both wt and mutant. A through E show mutations C93R, C98Y, C186Y, P197S, and P254L, respectively (see text for details). The figurewas made with Pymol (http://www.pymol.org).


conformational reorganization of the loop because dihedralangles adopted by proline are forbidden for leucine. Thisconformational change could affect the contiguous C253 andthus the binding of the cluster 4Fe-4S, in which is directlyimplicated the C253. The side chain of leucine also clasheswith backbone and side chain of L111 and T110. Addition-ally, the P254L mutation could also clash with C191, whichin turn affects the C192 and binding to the 4Fe-4S cluster.Figure 3 illustrates the predicted structural-functional con-sequences behind the pathogenic missense SDHB mutationsfound in this study. Conformational changes induced byR17L mutation in SDHD structure could not be analyzedbecause it is located in the signal peptide region, and nostructural information was available. The mutation may af-fect protein transport to the membrane and/or membraneinsertion.

Sporadic cases evidenced only two frameshift mutations, andboth led to altered proteins with premature stop codons thatremoved, depending on the mutation, half of exon 4 fromSDHD (c.337–340DelGACT)(p.Asp113MetfsX21) or more thanhalf of the mature SDHB protein, including half of exon 4 andexons 5–8 (c.312InsCACTGCA)(p.Ile105HisfsX16).

In a second round of analysis, 19 relatives of sporadicpatients were investigated for the putative disease-causingmutation found in the respective proband. Genetic screeningidentified five phenotypically unaffected SDHB mutationcarriers and one double, SDHD and SDHB, mutation carrier(Table 2). By the time we closed the study, all six mutatedrelatives were free of disease.

Sporadic cases harboring germline mutations disclosedfeatures significantly different from those observed in in-dividuals without germline mutations (Table 3). Irrespec-tive of the affected gene, the mutation carriers were mainlymales (P � 0.040), with a lower mean age at diagnosis (P �0.0012). Curiously, SDHB-mutation carriers were youngerthan SDHD-mutation carriers. The mutated tumors were

larger and affected primarily the vagal body. Despite allthe SDHD-linked and half of the SDHB-linked cervicalPGL patients lived near the sea level or at low altitudes,the residential altitudes were not significantly differentbetween patients with and without germline mutations(62.5 vs. 71%, respectively, lived near the sea level or at lowaltitudes).

Familial paragangliomas

All four families (F1-F4) carried germline mutations (Table2). The genealogical trees are depicted in Fig. 4. All but oneof the SDHD mutation carriers in whom we could trace theparental origin received the mutation from the father. In linewith published data, the only individual who inherited themutation from the mother remained tumor free by the age of31 yr.

In F1, the index case underwent surgery at the age of 36yr for a left vagal body PGL and 1 yr later for a right carotidbody PGL. His uncle had been affected by multiple PGLs,being diagnosed at the age of 45 yr with bilateral carotid bodyPGL, left vagal PGL, and left jugulotympanic PGL. These twopatients shared a novel frameshift mutation in SDHD (c.386–387InsT), which leads to a cDNA with a stop codon 90 bpdownstream of the original one, resulting in a protein 30amino acids larger (p.Leu129PhefsX62). Both patients alsoshowed the aforementioned linkage between the IVS2–36G�T and A6A in SDHB. Before closing this study, twomutation carriers, brothers of the index case, a 27-yr-oldfemale and a 46-yr-old male, underwent surgery for a carotidbody PGL and a vagal PGL, respectively. Three phenotyp-ically unaffected relatives also revealed the c.386–387InsTmutation.

In F2, the index case underwent surgery at the age of 42yr for a right carotid body PGL and a right jugular PGL. Twoyears later, a left carotid body PGL was removed in the same

TABLE 3. Correlation between pathogenic SDHD and/or SDHB mutations and clinical features in 36 sporadic head and neck PGLpatientsa

Phenotype-genotype features SDHDmutation P valueb SDHB

mutation P valueb SDHD and/orSDHB mutation P valueb No

mutation

GenderFemale 0 0.064 3 (50) n.s.s. 3 (37.5) 0.040 22 (79)Male 2 (100)c 3 (50) 5 (62.5) 6 (21)

Mean age at diagnosis, yr 51 n.s.s. 35 0.0012 39 0.0012 56Mean tumor size (cm) 4 n.s.s. 3.5 n.s.s. 3.7 0.162 2.6Tumor origin 0.072 0.137 0.045

Carotid body 1 (50)c n.s.s. 2 (33) n.s.s. 3 (35.5) n.s.s. 5 (18)Jugular glomus 6 (21)Tympanic bulb 10 (36)Jugulotympanic paraganglia 3 (50) n.s.s. 3 (37.5) n.s.s. 6 (21)Vagal body 1 (50) 0.131 1 (17) n.s.s. 2 (25) 0.117 1 (4)

Mean follow-up time, months (range) 63 (46–80) 85 (44–208) 80 (44–208) 79 (26–239)Recurrences 0 n.s.s. 1 (17) n.s.s. 1 (12.5) n.s.s. 4 (14)Altitude with respect to sea leveld n.s.s. n.s.s. n.s.s.

Near sea level (1–50 m) 1 (50) 2 (33) 3 (37.5) 11 (39)Low-level altitudes (�50 to � 300 m) 1 (50) 1 (17) 2 (25) 9 (32)Medium-level altitudes (�300 to � 700 m) 2 (33) 2 (25) 4 (14)High-level altitudes (�700 to � 1000 m) 1 (17) 1 (12.5) 4 (14)

The numbers in parentheses indicate percentage values.a Only putative disease-causing mutations in SDHD (n � 1), SDHB (n � 6), or both genes (n � 1) were computed in the statistical analysis.

Coding and noncoding polymorphisms were excluded.b All of the P values were calculated with respect to the nonmutated PGL cases (last column). P � 0.05 is considered statistically significant.

P values between 0.05 and 0.2 indicate a trend for an association. P � 0.2 is not statistically significant (n.s.s.).c Patient 28 exhibited a missense substituion in SDHD and a silent mutation in SDHB (S100S).d Patients were divided into four categories according to the altitude of their residential area with respect to sea level.


patient. A brother was also diagnosed of bilateral carotidbody PGLs at the age of 42 yr. Both presented a frameshiftmutation in SDHD (c.337–340DelGACT), which results in apremature stop codon (p.Asp113MetfsX21) and thus in atruncated protein lacking the last 26 amino acids. This mu-

tation was also detected in three other family members that,so far, have not developed the disease. In addition to thec.337–340DelGACT SDHD mutation, the index case also pre-sented the SDHC IVS1 � 13 InsTG intronic variant.

In F3, the index case underwent surgery twice, at the age

FIG. 4. The four families analyzed for SDHB, SDHC and SDHD mutations are shown. Each panel includes the histological appearance ofdifferent types of PGLs removed on that family (see text for details), a SSCP gel with all the relatives tested for the mutation demonstratedin the index case, the genealogical trees, and the corresponding chromatograms. Aberrant mobility shifts resulting from single-nucleotidesubstitutions (F-4), small deletions (F-2 and F-3), or insertions (F-1) are indicated with arrows within each SSCP gel. Beneath each gel laneappears the case number assigned in the respective pedigree.


of 40 yr for a left carotid body PGL and 15 yr later for a rightjugular PGL. A brother was diagnosed of carotid body PGLat the age of 60 yr. Both exhibited an unreported frameshiftmutation in SDHD (c.120–127DelCCCAGAAT), which re-sults in a premature stop codon (p.Ile40MetfsX25) that re-moves 92 amino acids. Three phenotypically unaffected rel-atives also carried the mutation.

In F4, the index case was diagnosed with a right jugularPGL at the age of 39 yr. His mother had developed a carotidbody and a jugulotympanic PGL, being first treated at the ageof 43 yr. The other two diseased members were a brother ofthe index case, deceased at the age of 33 yr, who presentedwith multiple extraadrenal PGLs 2 yr before and a cousin ofthe index case, who, at the age of 38 yr, underwent surgeryfor an extraadrenal PGL. These four patients harbored amissense mutation (C98Y) in a highly conserved residue ofSDHB. They also showed the polymorphisms IVS2–36G�Tand A6A in SDHB. Three disease-free relatives also disclosedthe C98Y mutation. Structural modeling revealed that C98Ymutation may disrupt the enzymatic activity of SDHB be-cause the C98 residue participates in the prosthetic groupbinding. The mutation hampers the interaction with SDHAand breaks the binding to cluster 2Fe-2S. The Y98 aromaticring clashes with the 2Fe-2S cluster and also with the neigh-boring S100, C93 (side chain and backbone), and R94 back-bone (Fig. 3B).

The germline mutations found in these four families wereconfirmed at the somatic level. No additional somatic mu-tations were detected in any of the affected family members.

Fifty-three percent of SDHD and 43% of SDHB mutationcarriers were disease free at the last follow-up annotated inour familial registry. Despite some of the nonaffected at risk

carriers might be too young to have clinical manifestationsand may develop the disease in their lifetime, our findingssuggest incomplete/reduced penetrance of the mutated al-leles. Of note, three families had lived near sea level (SDHB-C98Y) or at low average altitudes (SDHD-c.386–387InsT andSDHD-c.337–340DelGACT).

Genotype-phenotype correlation

To evaluate the impact of our results on the routine clinicalmanagement of cervical PGL patients, the phenotype of pa-tients with germline mutations was compared with that ofpatients without germline mutations (Table 4). SDH-mutatedtumors occurred predominantly in males (P � 0.0033), oc-curred at a younger age (P � 0.0001), were usually multifocal(P � 0.0011), and exhibited a larger average size (P � 0.0341).When comparing familial PGLs with occult familial cases, weobserved that the former group was significantly associatedwith tumor multiplicity (P � 0.014).

Discussion

The results of this study reinforce the involvement of germ-line mitochondrial SDH mutations in the etiology of a signif-icant number of apparently sporadic cervical PGLs. Taking intoaccount only those alterations that were absent in healthy con-trols, we observed that 22.2% of the apparently sporadic cer-vical PGLs were most probably the consequence of pathogenicgermline SDHB and/or SDHD mutations. In agreement withprevious studies (see [email protected]/lovd_sdh/and [email protected]/lovd_sdh/), the sequence vari-ants A6A in SDHB and H50R, G12S, and S68S in SDHD werealso found in healthy blood donors, supporting their catego-

TABLE 4. SDH genotype-phenotype correlation in 48 patients with PGL: comparison between familial cases and occult familialconditions

Phenotype-genotypefeatures

Familialcases

(n � 12)a

Sporadic cases (n � 36)Familial cases vs.

sporadic cases withgermline mutations

P valuec

Mutated cases vs.nonmutated cases

P valuec

Withgermline

mutations(n � 8)b

Withoutgermline

mutations(n � 28)

Mean age at diagnosis, yr 41 39 56 n.s.s. �0.0001Mean tumor size (cm) 4 3.7 2.6 n.s.s. 0.0341Gender

Female 4 (33) 3 (37.5) 22 (79) n.s.s. 0.0033Male 8 (67) 5 (62.5) 6 (21)

Single tumor 5 (42) 8 (100) 28 (100) 0.014 0.0011Multiple tumors 7 (58) 0 0PGL location

Head and neck 10 (83) 8 (100) 28 (100) n.s.s. 0.168Abdominal extraadrenal 2 (17) 0 0

Recurrences 6 (50) 1 (12.5) 4 (14) 0.157 0.162Altitude with respect to sea leveld

Near sea level (1–50 m) 4 (33) 3 (37.5) 11 (39) n.s.s. n.s.s.Low-level altitudes (�50 to � 300 m) 6 (50) 2 (25) 9 (32)Medium-level altitudes (�300 to � 700 m) 2 (17) 2 (25) 4 (14)High-level altitudes (�700 to � 1000 m) 0 1 (12.5) 4 (14)

The numbers in parentheses indicate percentage values.a Includes the proband of each pedigree and all of the relatives harboring the mutation who underwent surgery for PGL.b Only putative disease-causing mutations at SDHD (n � 1), SDHB (n � 6), or both genes (n � 1) were computed in the statistical analysis.

Coding and noncoding polymorphisms were excluded.c P � 0.05 is considered statistically significant. P values between 0.05 and 0.2 indicate a trend for an association. P � 0.2 is not statistically

significant (n.s.s.).d Patients were divided into four categories according to the altitude of their residential area with respect to sea level.


rization as nondeleterious polymorphisms. In contrast withprevious reports, we did not detect the SDHB S100S alterationamong our healthy control cases. Such discrepancy might berelated with different allele frequencies in different popula-tions. Because the S100S alteration has been shown in otherhealthy populations and does not result in amino acid change,we consider that it is probably nondeleterious.

In five sporadic SDH mutation carriers, at least one phe-notypically unaffected relative harbored a mutation identicalwith that shown in the proband, excluding the possibility ofconsidering those alterations as de novo germline mutations.This observation supports the term of occult familial condi-tion given to those sporadic patients who carry pathogenicgermline mutations. Of note, the proportion of occult familialcases in our series (22.2%) is higher than the heritabilityestimates recorded thus far for sporadic presentations inmost European and North American series of head and neckPGLs without a founder effect (0–9%) (16, 25–29). A studyperformed in 23 sporadic cervical PGLs from Australia re-vealed a similar percentage (17%) of occult familial condi-tions; however, half of the patients with germline mutationsdisclosed the SDHD P81L mutation, which has been associ-ated with a founder effect (13, 30, 31). Single ancestral hap-lotypes are determinant in cervical PGLs occurring in TheNetherlands, where about 36% of the sporadic cases seem toarise as a consequence of three founder/ancestral mutationsin SDHD (D92Y, L95P, and L139P) (28, 32). According to thereported absence of a founder/ancestral effect in SDHBamong low-altitude inhabitants, in our series we did notobserve founder mutations in the SDHB gene.

Another surprising finding of our study was the differencein the mutation frequency of SDHB and SDHD genes, de-pending on the existence or not of familial disease history.Whereas occult familial cervical PGLs were mainly associ-ated with SDHB mutations, familial cases with proven dis-ease history were mostly associated with SDHD mutations.The causes for the different mutation pattern are unknown.Intrinsic and/or extrinsic modifiers might play a yet unde-termined role in the sporadic population analyzed in thisstudy. In addition, the low residential altitudes in 69% of thesporadic cases might have contributed to the high frequencyof SDHB mutation carriers. It is believed that environmentaloxygen, which is determined by geographical elevations,exerts a major modifier role on the prevalence, penetrance,and phenotypic expression of SDH mutations (14, 33). Lowaltitudes may reduce gene penetrance and consequently re-lax the natural selection against SDH mutations, which aretherefore able to remain in the population gene pool. Dif-ferent population-based studies in Dutch inhabitants livingnear the sea level have shown that, whereas the frequency ofgermline SDHD mutations increases with successive gener-ations through genetic drift, the likelihood of developingcervical PGLs among at risk mutation carriers does not in-crease on the same proportion (28, 32).

Our results support the previous observations that germ-line SDHC mutations are rare among apparently sporadiccervical PGLs (see [email protected]/lovd_sdh/)(16, 25, 26, 34, 35). In this study, germline SDHB mutationsemerged as the leading cause of cervical PGLs. Twelve of 40sporadic and familial patients (30%) disclosed pathogenic

mutations and 58% were in SDHB. Moreover, seven of the 11different pathogenic mutations (64%) found in our studywere located in SDHB. These findings appear to argueagainst putative anatomical preferences by different SDHsubunits as has been formerly suggested (13, 14, 27, 29). Mostof the annotated SDHD mutation carriers have been associ-ated with cervical PGLs, contributing to the belief that SDHDdysfunction is the key event in PGLs developing in the headand neck region. Here we provide genetic evidence support-ing the involvement of the SDHB subunit in a significantnumber of cervical PGLs. Whether differences, other thanfounder/ancestral haplotypes, inherent to the populationsanalyzed thus far may play a role in the preference of onegene over the other still awaits clarification.

We found no evidence for distant metastases and/or ex-traparaganglial malignancies in patients with germlineSDHB mutations, who were free of disease after an averagefollow-up period of 7 yr. This observation is in contrast withprevious data showing an increased likelihood for tumorrecurrence, distant metastases, and/or early onset of renalcell carcinoma or papillary thyroid carcinoma among pa-tients with germline SDHB mutations (29, 36–39). However,most of these patients had developed intraabdominal ex-traadrenal PGLs, which tend to be the most aggressive typeof PGL (3, 4). In our series, the only two patients who un-derwent surgery for an intraabdominal extraadrenal PGLbelonged to the single family with a SDHB point mutation,and one of them developed multiple extraadrenal PGLs,dying shortly afterward of disease. Strikingly, most of thereported patients harboring malignant SDHB-mutatedPGLs, with follow-up information available, had prolongedsurvival periods, apparently not significantly different fromthose observed in patients without germline mutations thathad metastases at first intervention or during the follow-up(29, 36, 40). Taking all these findings together, it is likely thatthe hormonal and developmental milieu (head and neck vs.abdominal paraganglia) within which the tumor growsmight exert a considerable influence over its molecular bi-ology and natural history and thus be more important thanthe affected gene (SDHD vs. SDHB).

Our study adds a number of new SDHB and SDHD mu-tations to those already described (35). In contrast with pre-vious reports, nonsense mutations were not detected amongSDHD-linked cervical PGL patients who primarily carriedframeshift mutations. In line with published data, missensesubstitutions were the most common type of mutationamong SDHB-linked cervical PGLs. Both frameshift and mis-sense mutations were predicted to result in profound con-formational changes in mitochondrial SDH structure, whichmost likely result in severe SDH dysfunction. All the patho-genic missense SDHB mutations (C93R, C98Y, P197S, C186Y,and P254L), by breaking the binding to the iron-sulfur clus-ters, may interfere in the electron transfer through the FES,FS4, and F3S clusters present within the catalytic core ofSDH. In addition, C93R and C98Y may inhibit the interactionwith SDHA and P197L may affect ubiquinone binding,which would also hinder electron entry in the OXPHOSsystem. Several other lines of evidence provide additionalindirect support to the functional relevance of the aforemen-tioned missense SDHB mutations. First, the prevalence of


missense mutations in this study was higher than the back-ground mutation frequency of nonfunctional alterations ob-served in the genome of tumor cells. Second, the residuesaffected by those mutations are highly conserved duringevolution. Third, C93R, C98Y, P197S, C186Y, and P254L werenot found in healthy control individuals recruited from thegeneral population. Fourth, the C98Y mutation cosegregatedwith disease phenotype.

In this study we also demonstrate that occult familial casesand familial cases with proven disease history have a com-mon clinicopathological signature, which distinguishes themfrom truly sporadic cervical PGL patients without germlineSDH mutations. The only feature that distinguished trulyfamilial from occult familial cases was tumor multifocality,which occurred more often in familial cases with provendisease history. Indeed, it has been proposed to considerbilateral or multifocal PGLs as a phenotypical marker forinherited predisposition.

In conclusion, this study highlights the clinical usefulnessof genetic testing in all subjects presenting with solitary cer-vical PGL and no family history. We provide genetic evi-dence that associates germline SDHB mutations with thepathobiology of a significant number of apparently sporadiccervical PGLs. Moreover, at variance with intraabdominalextraadrenal PGLs, SDHB dysfunction in PGLs of the headand neck region does not appear to entail a deleterious be-havior. Our results support the option of offering genetictesting to all at-risk living relatives of sporadic and familialpatients harboring a germline mutation. To enable an earlydetection of affected paraganglia and thus avoid the seriousmorbidity of surgery in advanced head and neck PGLs, theasymptomatic SDH-mutation carriers should probably beenrolled in a surveillance program.

Acknowledgments

We acknowledge the Fundacao para a Ciencia e a Tecnologia forfunding this project included in the thesis defended by Jorge Lima at theMedical Faculty of Porto University in December 2006. We thank thepatients and their relatives for their stimulating cooperation with thisproject. We are also grateful for the outstanding contribution of theBlood and Tissue Bank of the Cruz Roja in Oviedo (Asturias), the BloodBank at Hospital Universitario Central de Asturias, and the followingclinicians: Dr. Carlos Suarez, Hospital Universitario Central de Asturias;Dr. Eulalia Porras, Hospital Universitario Puerta del Mar; Dr. CarlosEscobar, Hospital Universitario Morales Meseguer; Dr. Jose Maria Anda,Hospital Santiago Apostol; Dr. Albino Alonso, Hospital Santiago Apos-tol; Dr. Manel Manos, Hospital Universitario de Bellvitge; Dr. JavierYetano, Hospital Galdakao, Osakidetza; Dr. Ramon Malluguiza, Hos-pital General de Elda; Dr. Ignacio Alvarez, Hospital de Leon; Dr. JoseRamon Garcia Villar, Hospital Pontevedra; Dr. Jaime Marco, HospitalClinico Universitario de Valencia; Dr. Manuel Claver Hospital GeneralYague; Dr. Manuel Atienza, Hospital General de Albacete; and Dr. HugoGalera, Hospital Virgen del Rocio. We also thank the technician Mariadel Mar Eiroa Teijero for dealing with blood samples shipment.

Received March 21, 2007. Accepted September 4, 2007.Address all correspondence and requests for reprints to: Ginesa Gar-

cia-Rostan, M.D., Ph.D., Dr. Alfredo Martinez, No. 3, 3°B, 33005 Oviedo-Asturias, Spain. E-mail: [email protected].

This work was supported by Fundacao para a Ciencia e a Tecnologiaof Portugal Grants POCTI/SAU-OBS/61945/2004 (to G.G.-R.) andPOCTI/CBO/43944/2001 (to J.L.) and Editorial Planeta of Spain (toI.P.-C.).

Disclosure Statement: The authors have nothing to disclose.

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high frequency of germline succinate dehydrogenase mutations in sporadic cervical paragangliomas in...

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