Clinical Endocrinology (2008)

68

, 762–768 doi: 10.1111/j.1365-2265.2007.03131.x

© 2008 The Authors

762

Journal compilation © 2008 Blackwell Publishing Ltd

O R I G I N A L A R T I C L E

Blackwell Publishing Ltd

Uncommon clinical presentations of pheochromocytoma and paraganglioma in two different patients affected by two distinct novel VHL germline mutations

Tonino Ercolino*, Lucia Becherini*, Andrea Valeri¶, Michele Maiello*, Maria Sole Gaglianò*, Gabriele Parenti*, Matteo Ramazzotti‡, Elisa Piscitelli*, Lisa Simi†, Pamela Pinzani†, Gabriella Nesi§, Donatella Degl’Innocenti‡, Nico Console¶, Carlo Bergamini¶ and Massimo Mannelli*

*

Department of Clinical Physiopathology, Division of Endocrinology,

†

Division of Clinical Biochemistry,

‡

Department of Biochemistry,

§

Department of Human Pathology and Oncology, University of Florence, Florence, Italy and

¶

General and Vascular Surgical Unit, Azienda Ospadaliera Universitaria Careggi, Florence, Italy

Summary

Context

The von Hippel-Lindau (VHL) syndrome is an inherited

multitumour disorder characterized by clinical heterogeneity and

high penetrance. Pheochromocytoma (Pheo) is present in 10%–15%

of cases and can be isolated or associated with other lesions such as

haemangioblastomas, kidney cysts or cancer and pancreatic lesions.

In VHL patients, Pheos generally secrete norepinephrine and are

located in the adrenals. Extra-adrenal Pheos (paragangliomas, PGLs)

are rare.

Objective

While performing genetic testing in patients affected by

apparently sporadic Pheos or PGLs, we found two novel different

VHL germline mutations in two females who presented with two

distinct very uncommon clinical pictures. One patient was studied

for the presence of an adrenal incidentaloma and the other for the

presence of a neck tumour.

Methods and results

Patients coding regions and exon–intron

boundaries of RET (exons 10, 11, 13–15), VHL, SDHD, SDHB and

SDHC genes were amplified and sequenced. We identified two novel

VHL point mutations: a

L198V

missense mutation in a 32-year-old

female affected by a right adrenal compound and mixed tumour

constituted by an epinephrine secreting Pheo, a ganglioneuroma and

an adrenocortical adenoma, and a

T152I

missense mutation in a 24-

year-old female affected by a left carotid body tumour. No other

lesions were found in the patients or in the VHL mutation positive

relatives.

Conclusions

These cases enlarge the list of VHL mutations and

add new insights in the clinical variability of VHL disease, thus

confirming the importance of genetic testing in patients affected by

apparently sporadic Pheos or PGLs.

(Received 12 July 2007; returned for revision 7 August 2007; finally

revised 27 September 2007; accepted 16 October 2007)

Introduction

von Hippel-Lindau (VHL) disease is an autosomal dominant-

inherited tumour syndrome

1

related to mutations in the VHL

tumour suppressor gene, located on chromosome 3p25-26.

2

The major features of VHL disease are retinal and central nervous

system (CNS) haemangioblastomas, clear cell renal carcinomas and

pheochromocytomas (Pheo).

1–3

The overall frequency of Pheo in

VHL is about 10%–15%,

1–3

but Pheo is often the most frequent

feature in type 2A and 2B families while it is the sole manifestation

in type 2C and typically absent in type 1 kindreds.

1,3,4

All type 2A and 2C families and the vast majority of 2B families

are affected by VHL missense mutations.

4–8

In VHL patients, Pheos

are generally intra-adrenal, often bilateral

1,3,9

and are typically

noradrenaline secreting.

10

Extra-adrenal locations are rare.

We report two patients affected by two distinct novel germline

VHL missense mutations, who present with two very uncommon

clinical pictures.

Patients and methods

Mutation analysis

After obtaining informed consent, DNA was extracted from peri-

pheral blood leucocytes using the commercial kit NucleoSpin Blood

L (Macherey-Nagel, Düren, Germany) following the manufacturer’s

instructions.

Searching for germline mutations of the susceptibility genes in

patients affected by Pheo, we analysed RET (exons 10, 11, 13–15),

VHL (all exons), SDHD (all exons), SDHB (all exons) and SDHC

(all exons) genes.

All the gene coding regions and exon–intron boundaries were

amplified by PCR using the appropriate primers. For exons

Correspondence: Massimo Mannelli, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. Tel.: +39 0554271428; Fax: +39 0554271413; E-mail: m.mannelli@dfc.unifi.it

VHL and clinical presentations

763

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd,

Clinical Endocrinology

,

68

, 762–768

amplification, genomic DNA (200 ng) was denaturated at 95

°

C for

5 min, mixed with 10

×

buffer, 1·5 m

m

MgCl

2

, 0·4 m

m

primers,

200 m

m

dNTP, 1 U Taq polymerase in a final volume of 25

μ

l, and

cycled 30

×

at 95

°

C for 1 min, 58

°

C for 30 s, 72

°

C for 30 s and final

extention at 72

°

C for 7 min. Total PCR products were then purified

using a PCR purification kit (Qiagen, Milan, Italy) following the

protocol instructions and semiquantified in a 2% agarose ethidium

bromide gel using DNA molecular weight (Roche, Indianapolis, IN).

To perform the cycle-sequencing reaction, 5 ng of DNA were then

blended with each primer (0·8

μ

m

) in a Terminator Ready Reaction

Mix containing Big Dye Terminators (Applied Biosystems, Milan,

Italy) and submitted to 1 min at 96

°

C and 25 cycles at 96

°

C for 10 s,

50

°

C for 5 s, 60

°

C for 4 min. A second purification step was

necessary for the Big Dye removal with DyeEx 2·0 Spin Kit (Qiagen).

A 5-

μ

l of marked and purified DNA were submitted to sequencing

analysis with ABI PRISM 310 Genetic Analyser (Applied Biosystems).

Loss of heterozigosity (LOH) analysis

LOH was only determined in DNA extracted from laser microdissected

cells of tumour obtained at surgery from patient 1. For microdissec-

tion of frozen tissue sections, we used the PALM Laser – Microbeam

System (P.A.L.M. Microlaser Technologies AG, Bernried, Germany)

which enables the contact-free isolation of single cells or group of

cells. The sections were mounted onto a polyethylene membrane

slide according to the manufacturer’s instructions. Frozen sections

were fixed with ethanol immediately after cutting in a cryostat,

stained with Haematoxylin/Eosin followed by increasing ethanol

series and air-dried. The microdissected cells were catapulted into

the lid of a 0·5-ml reaction tube using the laser pressure catapulting

technique of the instrument. For the isolation of DNA, 30

μ

l of RLT

Buffer (Qiagen) were applied into the lid. Subsequently the tubes

were centrifuged and the samples extracted by using the DNA micro

kit (Qiagen) following manufacturer’s instructions. An aliquot of

this DNA was used for subsequent PCR and sequencing analysis as

previously reported.

11

Moreover, microsatellite analysis was per-

formed in two different loci according to the consensus physical map

of chromosome 3p. We choose the microsatellites D3S1038 in the

locus 3p26.1-3p25.2 and D3S1297 in the locus 3pter-3p25. The PCR

and sequencing of the microsatellite D3S1038 and D3S1297 were

carried out in a final volume of 25

μ

l containing 50 ng of genomic

DNA. The amplification was performed denaturing at 95

°

C for

5 min, mixed with 10

×

buffer, 1·5 m

m

MgCl

2

, 0·4 m

m

of fluorescent

primers, 200 m

m

dNTP, 1 U Taq polymerase and cycled 25

×

at 95

°

C

for 1 min, 58

°

C for 30 s, 72

°

C for 30 s and final extention at 72

°

C for

7 min. A 2-ul of the fluorescent PCR was added to 12·5

μ

l of formamide

and 0·5

μ

l of ROX size standard and then submitted to sequencing

analysis with ABI PRISM 310 Genetic Analyser (Applied Biosystems).

The analysis was performed by using the Genscan software and

analysed by Genotyper software.

Structural analysis

Three-dimensional analysis of the wild-type and the mutated VHL

proteins was performed by SWISS-PROT <http://www.expasy.ch/

cgi-bin/sprot-search-ful>.

Case reports

Patient 1.

A 32-year-old female presented a right adrenal mass at

sonography performed for gallbladder’s stones. An abdominal

computed tomography (CT) confirmed the presence of a solid

hypodense lesion of the right adrenal gland, 3

×

2·5 cm in size, that

showed an inhomogeneous enhancement after contrast injection

and was associated with a cystic lesion. In spite of a completely silent

clinical history, the hormonal analysis revealed elevated levels of

urinary metanephrine (2275

μ

g/24 h, n.v. 52–341) (11·54

μ

mol/24 h,

n.v. 0·26–1·7), normal levels of urinary normetanephrine (407

μ

g/

24 h, v.n. 88–440) (2·26

μ

mol/24 h, n.v. 0·48–2·4) and vanillylmandelic

acid (4·6 mg/24 h, v.n. 1·8–6·7) (23·0

μ

mol/24 h, n.v. 9·0–34·0),

normal levels of basal plasma catecholamines (adrenaline 40 pg/ml,

n.v. 5–125; noradrenaline 90 pg/ml, n.v. 65–400) (adrenaline

218 pmol/l n.v. 27–683; noradrenaline 532 pmol/l, n.v. 355–2185),

elevated level of plasma cortisol (1150 nmol/l, v.n.160–690) and

urinary free cortisol (486 nmol/l, v.n. < 275), normal levels of

androgens and ACTH in the low normal range (11·6 ng/l, v.n. 9–

52). Dexamethasone suppression test showed suppression of plasma

cortisol (76 nmol/l). A

123

I-MIBG scintigraphy showed a high tracer

uptake in the region of the right adrenal gland. The patient was

asymptomatic and the physical examination was negative. Blood

pressure measurement was always normal. After 7 days of doxazosine

treatment, the patient underwent laparoscopic removal of the

right adrenal gland and of the gallbladder. The postsurgical period

was uneventful and all hormonal parameters were normal 2 weeks

after surgery.

Patient 2.

A 24-year-old female was referred to our out-patient

clinic after having been operated for a 2·8-cm left carotid body

tumour. No relevant diseases were present in her past medical

history. Her family history was negative for the presence of neural

crest-derived tumours. Being a student in medicine, she asked for

genetic testing. After having sequenced all the exons of SDHD, SDHB

and SDHC genes, we then analysed the VHL gene.

Patients’ families.

Clinical, laboratory and genetic studies were later

on extended to all the family members who consented to be studied.

Results

Mutation analysis

In patient 1, no mutations were found in RET, SDHD, SDHB and

SDHC genes. In contrast, we identified a novel VHL point mutation

constituted by an heterozygous missense mutation,

L198V

(

594C >

G

), a C to G nucleotide substitution leading to a Leucine to Valine

amino acid change in exon 3 (Fig. 1a).

In patient 2, the analysis of SDHD, SDHB and SDHC genes

showed a normal sequence in all the exons. In contrast, we found

an heterozygous missense mutation

T152I

(

455C > T

), a C to T

nucleotide substitution, leading to a Threonine to Isoleucine amino

acid change in exon 2 of VHL gene (Fig. 2a). Both VHL codons at

the mutated sites are highly conserved through the species and were

not detected in 100 control subjects.

764

T. Ercolino

et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd,

Clinical Endocrinology

,

68

, 762–768

The genetic test performed in the family members, the brother

and the father, of patient 1 showed no mutations (Fig. 1b), while in

patient 2 family, the sister and the mother, were found to carry the

VHL mutation (Fig. 2b).

LOH analysis

We did not find LOH in any of the components of the mixed tumour.

Clinical evaluation

Consequently to the finding of a VHL mutation, patient 1 was

extensively evaluated for the presence of other VHL-linked lesions.

Ophthalmoscopy as well as brain, thoracic and abdominal Nuclear

Magnetic Resonance (NMR) did not show any of the typically VHL-

associated lesions (retinal or CNS angiomas; endolymphatic sac

tumours; pancreatic or renal cysts; renal carcinoma; cystoadenoma

of the broad ligament).

Patient 2 as well as her mother and sister, both carriers of

the mutation, underwent a complete clinical evaluation,

which included an extensive physical examination, laboratory

tests (urinary metanephrine and normetanephrine, plasma

chromogranin A), an ophthalmoscopic examination and radio-

logical exams (NMR of the brain, CT scan of the thorax and

the abdomen). In each of the three subjects, the clinical examina-

tion, the laboratory tests and the radiological evaluations were

normal.

Histological characterization of the tumours

The histological examination of tumour in patient 1 showed a com-

pound adrenal medullary neoplasm, Pheo and ganglioneuroma,

associated to a cortical adenoma (Fig. 1c) appearing as a distinct

nodule in the context of the mass.

Histological characteristics of tumour in patient 2 showed a

glomus tumour and are shown in Fig. 2.

Fig. 1 (a) Electropherogram of wild-type (upper) and patient 1 (lower) showing the germline mutation L198V in exon 3 of the VHL gene. (b) Pedigree of patient 1 family. (c) Anatomical and histological characteristics of the compound and mixed tumour. S1: areas of pheochromocytoma containing large, polygonal chief cells which exhibit nuclear pleomorphism (Haematoxylin and Eosin stain; original magnification, × 25) S2: Ganglioneuromatous areas in which ganglion cells are admixed with abundant Schwann cells (Haematoxylin and Eosin stain; original magnification, ×60). S3: Adrenal cortical adenoma composed of alveolar clusters of lipid-rich cells (Haematoxylin and Eosin stain; original magnification, ×60).

VHL and clinical presentations

765

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd,

Clinical Endocrinology

,

68

, 762–768

Structural analysis of missense mutations

Structural modelling of the two mutations is shown in Fig. 3.

Discussion

These two VHL mutation positive patients presented with a clinical

picture which, although associated to the presence of a Pheo and a

paraganglioma (PGL), by no way resembled the typical phenotype

of VHL disease. In fact, none of the patients presented with the

typically associated lesions in the CNS, retina, kidney and pancreas,

and although the type 2C form is characterized by the sole presence

of Pheo, the tumours diagnosed in our patients showed very unusual

or unique characteristics.

In patient 1, the adrenal mass was a compound and mixed tumour

presenting not only the association of chromaffin tissue with a

ganglioneuroma but also a distinct adrenocortical adenoma. The

adenoma was probably cortisol secreting, as suggested by the

increased urinary free cortisol, although dexamethasone suppressed

plasma cortisol and signs of chronic hypercortisolism were absent.

Compound

12–18

and/or mixed

19,20

adrenal tumours, although rare,

have been reported in the literature but, to our knowledge, this is

the first report on the association of three different tumours in the

same adrenal in a patient presenting a missense mutation of VHL

gene. Interestingly, recently we reported on a patient affected by a

compound Pheo/ganglioneuroma and an adrenal adenoma in the

contralateral gland, presenting an intronic variant in the VHL gene.

21

In VHL disease, Pheos are characterized by a noradrenergic

phenotype.

10,22

Therefore, an additional surprising finding in patient

1 was the pure adrenergic phenotype of the tumour. Urinary

adrenaline was about seven times higher than the upper limit of

normal while urinary noradrenaline was normal, thus demonstrating

the intratumoural synthesis of adrenaline.

It is impossible to establish whether adrenaline production might

depend on phenyl-ethanol-amine-

N

-methyltransferase (PNMT)

activation by cortisol secreted by the cortical adenoma, but the very

low or even absent PNMT gene expression in VHL Pheos

10

makes

the hypothesis unlikely. Moreover, in normal adrenal medulla

PNMT is maximally activated by adrenal cortisol as demonstrated

by the unchanged adrenaline : noradrenaline ratio in adrenal venous

blood of patients with Cushing’s disease.

23

The finding of normal adrenaline plasma levels suggests an intense

intratumoural conversion of adrenaline into metanephrine and

explains the silent clinical picture. Moreover, the finding that

metanephrine was the only abnormal marker of chromaffin pathology

reaffirms that the differential measurement of plasma or urinary

metanephrines is the laboratory test to be used in the diagnosis of

Pheo.

24

Fig. 2 (a) Electropherogram of wild-type (upper) and patient 2 (lower) showing the germline mutation T152I in exon 2 of the VHL gene. (b) Pedigree of patient 2 family. (c) Histological characterization of the glomus tumour: S1: Glomus tumour showing the nested growth (‘zellballen’) with delicate fibrovascular septae (Haematoxylin and Eosin stain; original magnification, ×60) S2: The chief cells react strongly and diffusely with chromogranin (immunoperoxidase and Haematoxylin counterstain; original magnification, ×120). S3: The sustentacular cells are highlighted by S-100 protein (immunoperoxidase and Haematoxylin counterstain; original magnification, ×120).

766

T. Ercolino

et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd,

Clinical Endocrinology

,

68

, 762–768

As no other living member of the family tested positive for the

mutation, it is impossible to determine whether patient 1 presented

with a

de novo

mutation. Her mother died at the age of 51 years of

a lung carcinoma.

Patient 2 was studied 1 year after the removal of a left carotid body

tumour. Head/neck PGLs are typically found in SDHx-mutated

patients,

11,25,26

but the genetic analysis of SDHD, SDHB and SDHC

were negative. To exclude the presence of large deletions in

SDHx

genes we also performed MLPA analysis which gave negative results

(data not shown).

Abdominal PGLs are rarely found in VHL disease and head/neck

PGLs are even rarer.

1

One peculiarity of this patient is that the

glomus tumour was the only lesion present. An additional peculiarity

is the absence of any VHL lesions in the two family members who

are positive for the same VHL mutation. In fact, VHL disease is

characterized by a high penetrance,

27

but the sister and the mother,

who is 48-years-old, were disease free. The patient and her mutated

relatives are regularly evaluated once a year by ophtalmoscopy, head,

neck and abdominal NMR as well as urinary metanephrines and

plasma chromogranin A measurement. The result was disease free

at the last evaluation. These findings might suggest that the VHL

T152I

mutation is characterized by a low penetrance.

We have no functional data to assess that these novel mutations

are responsible for the development of these tumours. The LOH

analysis did not help us in clarifying this issue. In fact, we did not

find LOH in any of the components of the mixed tumour, but it is

known that not all the VHL-linked tumours show LOH.

28

Other

events, such as hypermethylation or point mutations of the promoter

might cause allelic loss of function as well as additional somatic

heterozygous mutations occurring in the tumour cells.

However, structural analysis of the VHL protein suggests that

the mutations might indeed have functional relevance. Both the

missense mutations,

L198V

and

T152I

, are located in the

β

domain

of the VHL gene. The

β

domain consists of seven-stranded

β

sandwich,

residues 63–154, and an

α

helix, residues 193–204. The T152 is

located in the loop connecting the

β

and

α

domains, and could be

important for the alteration of the protein conformation, while the

L198 is located in a very important region for the elongin C binding

(Fig. 3). Moreover, both VHL codons at the mutated sites are highly

conserved through the species, the mutations were not detected in

more than 100 control subjects and amino acid changes in VHL

codon 198 (

L198Q

and

L198R

) have been described. Specifically, the

L198Q mutation has been associated with Pheo.

29,30

The clinical diagnosis of VHL disease stems on the recognition of

the disease-linked lesions which drive to the genetic analysis. Our

study started from the analysis of the susceptibility genes in patients

affected by apparently sporadic Pheos or PGLs and thus permitted

us to detect some uncommon VHL-related clinical presentations

which otherwise would have been gone unrecognized.

In conclusion, the present paper enlarges the list of VHL mutations

and adds new insights in the clinical variability of VHL disease, thus

confirming the importance of genetic testing in patients affected by

apparently sporadic Pheos or PGLs.

Acknowledgements

This work was supported by grants from MIUR (Ministero

dell’Istruzione, dell’Università e della Ricerca) (prot. 2004069534),

from the Department of Pathophysiology, University of Florence and

by an unrestricted grant from Villa Gisella (Florence, Italy).

M. Mannelli is member of the ENS@T (European Network for the

Study of Adrenal Tumours).

References

1 Lonser, R.R., Glenn, G.M., Walther, M.M., Chew, E.Y., Libutti, S.K.,Linehan, W.M. & Oldfield, E.H. (2003) von Hippel-Lindau disease.

Lancet

,

361

, 2059–2067.2 Latif, F., Tory, K., Gnarra, J., Yao, M., Duh, F.M., Orcutt, M.L.,

Stackhouse, T., Kuzmin, I., Modi, W., Geil, L., Schmidt, L., Zhou, F.,

Fig. 3 Schematic representation of the three-dimensional structure of the VHL protein and the VHL complex with the mutations. Panel (a) shows the VHL protein structure with both mutations indicated as red wireframes with sidechains of wild-type residues. Ribbon colours show the binding domains of VHL: yellow for elongin C, blue for elongin B, green for chaperonin TRiC and pink for HIF-1. Panel (b) shows the VHL structure in complex with elongin B, elongin C and a fragment of HIF-1. The location of the two mutants T152I and L198V is indicated as red wireframes with sidechains of wild-type residues.

VHL and clinical presentations

767

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd,

Clinical Endocrinology, 68, 762–768

Li, H., Wie, M.H., Chen, F., Glenn, G., Choyke, P., Walther, M.M.,Wenig, Y., Duan, D.S.R., Dean, M., Glavac, D., Richards, F.M.,Crossey, P.A., Fergusson-Smith, M.A., Le Paslier, D., Chumakov, I.,Cohen, D., Chinault, C., Maher, E.R., Linehan, W.M., Zbar, B. &Lerman, M.I. (1993) Identification of the von Hippel-Lindau diseasetumor suppressor gene. Science, 260, 1317–1320.

3 Maher, E.R. & Kaelin, W.G. Jr (1997) von Hippel-Lindau disease.Medicine, 76, 85–105.

4 Hes, F.J., Hoeppener, J.W.M. & Lips, C.J.M. (2003) Pheochromocytomain von Hippel-Lindau disease. Journal of Clinical Endocrinology andMetabolism, 88, 969–974.

5 Crossey, P.A., Eng, C., Ginalska-Malinowska, M., Lennard, T.W.J.,Sampson, J.R., Ponder, B.A.J. & Maher, E.R. (1995) Molecular geneticdiagnosis of von Hippel-Lindau disease in familial phaeochromo-cytoma. Journal of Medical Genetics, 32, 885–886.

6 Brauch, H., Kishida, T., Glavac, D., Chen, F., Pausch, F., Hofler, H.,Latif, F., Lerman, M.I., Zbar, B. & Neumann, H.P.H. (1995) vonHippel-Lindau (VHL) disease with pheochromocytoma in the blackregion of Germany: evidence for a founder effect. Human Genetics,95, 551–556.

7 Maher, E.R., Webster, A.R., Richards, F.M., Green, J.S., Crossey, P.A.,Payne, S.J. & Moore, A.T. (1996) Phenotypic expression in vonHippel-Lindau disease: correlation with germline VHL gene mutations.Journal of Medical Genetics, 33, 328–332.

8 Woodward, E.R. & Maher, E.R. (2006) von Hippel-Lindau diseaseand endocrine tumour susceptibility. Human Molecular Genetics, 13,415–425.

9 Walther, M.M., Reiter, R., Keiser, H.R., Choyke, P.L., Venzon, D.,Hurley, K., Gnarra, J.R., Reynolds, J.C., Glenn, G.M., Zbar, B. &Linehan, W.M. (1999) Clinical and genetic characterization ofpheochromocytoma in von Hippel-Lindau families: comparison withsporadic pheochromocytoma gives insight into the natural historyof pheochromocytoma. Journal of Urology, 162, 659–664.

10 Eisehofer, G., Walther, M.M., Huynh, T.T., Li, S.T., Bornstein, S.,Vortmeyer, A., Mannelli, M., Goldstein, D.S., Linehan, W.M.,Lenders, J.W.M. & Pacak, K. (2001) Pheochromocytomas in vonHippel-Lindau syndrome and multiple endocrine neoplasia type 2display distinct biochemical and clinical phenotypes. Journal ofClinical Endocrinology and Metabolism, 86, 1999–2008.

11 Simi, L., Sestini, R., Ferruzzi, P., Gaglianò, M.S., Genuini, F.,Ma scalchi, M., Guerrini, L., Pratesi, C., Pinzani, P., Nesi, G., Ercolino, T.,Genuardi, M. & Mannelli, M. (2006) Phenotype variability of neuralcrest-derived tumours in six Italian families segregating the samefounder SDHD mutation Q109X. Journal of Medical Genetics, 42,e52.

12 Contreras, L.N., Budd, D., Yen, T.S., Thomas, C. & Tyrrell, J.B. (1991)Adrenal ganglioneuroma–pheochromocytoma secreting vasoactiveintestinal polypeptide. The Western Journal of Medicine, 154, 334–337.

13 Kimura, N., Watanabe, T., Fukase, M., Wakita, A., Noshiro, T. &Kimura, I. (2002) Neurofibromin and NF1 gene analysis in compositepheochromocytoma and tumors associated with von Recklinghausen’sdisease. Modern Pathology, 15, 183–188.

14 King-Yin, L. & Chung-Yau, L. (1999) Composite pheocromocytoma–ganglioneuroma of the adrenal gland: an uncommon entity withdistinctive clinicopathologic features. Endocrine Pathology, 10, 343–352.

15 Nakagawara, A., Ikeda, K., Tsuneyoshi, M., Daimaru, Y. & Enjoji, M.(1985) Malignant pheochromocytoma with ganglioneuroblastomaelements in a patient with von Recklinghausen’s disease. Cancer, 55,2794–2798.

16 Franquemont, D.W., Mills, S.E. & Lack, E.E. (1994) Immunohisto-chemical detection of neuroblastomatous foci in composite adrenalpheochromocytoma–neuroblastoma. American Journal of ClinicalPathology, 102, 163–170.

17 Candanedo-Gonzalez, F.A., Alvarado-Cabrero, I., Gamboa-Dominguez,A., Cerbulo-Vazquez, A., Lopez-Romero, R., Bornstein-Quevedo, L.& Salcedo-Vargas, M. (2001) Sporadic type composite pheochromo-cytoma with neuroblastoma: clinicomorphologic, DNA content andret gene analysis. Endocrine Pathology, 12, 343–350.

18 Miettinen, M. & Saari, A. (1988) Pheochromocytoma combinedwith malignant schwannoma: unusual neoplasm of the adrenalmedulla. Ultrastructural Pathology, 12, 513–527.

19 Juarez, D., Brown, R.W., Ostrowski, M., Reardon, M.J., Lechago, J.& Truong, L.D. (1999) Pheochromocytoma associated withneuroendocrine carcinoma. A new type of composite pheochromo-cytoma. Archives of Pathology and Laboratory Medicine, 123, 1274–1279.

20 Pathmanathan, N. & Murali, R. (2003) Composite phaeochromocytomawith intratumoural metastatic squamous cell carcinoma. Pathology,35, 263–265.

21 Bernini, G.P., Moretti, A., Mannelli, M., Ercolino, T., Bardini, M.,Caramella, D., Taurino, C. & Solvetti, A. (2005) Unique associationof non-functioning pheochromocytoma, ganglioneuroma, adrenalcortical adenoma, hepatic and vertebral haemangiomas in a patientwith a new intronic variant in the VHL gene. Journal of EndocrinologicalInvestigation, 28, 1032–1037.

22 Eisenhofer, G., Huynh, T.T., Pacak, K., Brouwers, F.M., Walther, M.M.,Linehan, W.M., Munson, P.J., Mannelli, M., Goldstein, D.S. &Elkahloun, A.G. (2004) Distinct gene expression profile in nore-pinephrine and epinephrine producing hereditary and sporadicpheochromocytomas: activation of hypoxia-driven angiogenicpathways in von Hippel-Lindau syndrome. Endocrine-Related Cancer,11, 897–911.

23 Mannelli, M., Lanzillotti, R., Pupilli, C., Ianni, L., Conti, A. & Serio,M. (1994) Adrenal medulla secretion in Cushing’s syndrome. Journalof Clinical Endocrinology and Metabolism, 78, 1331–1335.

24 Lenders, J.W., Pacak, K., Walther, M.M., Linehan, W.M., Mannelli, M.,Friberg, P., Kaiser, H.R., Goldstein, D.S. & Eisenhofer, G. (2002)Biochemical diagnosis of pheochromocytoma: which test is best?Journal of the American Medical Association, 287, 1427–1434.

25 Baysal, B.E., Willett-Brozick, J.E., Lawrence, E.C., Drovdlic, C.M.,Savul, S.A., McLeod, D.R., Yee, H.A., Brackmann, D.E., Slattery, W.H.3rd, Myers, E.N., Ferrell, R.E. & Rubistein, W.S. (2002) Prevalenceof SDHB, SDHC, and SDHD germline mutations in clinic patientswith head and neck paragangliomas. Journal of Medical Genetics, 39,178–183.

26 Favier, J., Brière, J.J., Strompf, L., Amar, L., Filali, M., Jeunemaitre, X.,Rustin, P. & Gimenez-Requeplo, A.P. (2005) Hereditary paraganglioma/pheochromocytoma and inherited succinate dehydrogenasedeficiency. Hormone Research, 63, 171–179.

27 Hes, F.J., Lips, C.J. & van der Luijt, R.T. (2001) Clinical managementof von Hippel-Lindau (VHL) disease. The Netherlands Journal ofMedicine, 59, 225–234.

28 Glaesker, S., Bender, B.U., Apel, T.W., van Velthoven, V., Mulligan, L.M.,Zentner, J. & Neumann, H.P.H. (2001) Reconsideration of biallelicinactivation of the VHL tumour suppressor gene in hemangioblastomasof the central nervous system. Journal of Neurology, Neurosurgery andPsychiatry, 70, 644–648.

29 Neumann, H.P., Bausch, B., McWhinney, S.R., Bender, B.U., Gimm, O.,Franke, G., Schipper, J., Klisch, J., Altehoefer, C., Zerres, K., Januszewicz,A., Eng, C., Smith, W.M., Munk, R., Manz, T., Glaesker, S., Apel, T.W.,

768 T. Ercolino et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Clinical Endocrinology, 68, 762–768

Treier, M., Reineke, M., Walz, M.K., Hoang-Vu, C., Brauckhoff, M.,Klein-Franke, A., Klose, P., Schmidt, H., Maier-Woelfle, M.,Peczkowska, M., Szmigielski, C., Eng, C. & Freiburg-Warsaw-ColumbusPheochromocytoma Study Group (2002) Germ-line mutations innonsyndromic pheochromocytoma. New England Journal ofMedicine, 346, 1459–1466.

30 Whaley, J.M., Naglich, J., Gelbert, L., Hsia, Y.E., Lamiell, J.M., Green, J.S.,Collins, D., Neumann, H.P., Laidlaw, J., Li, F.P., Li, F.P., Klein-Szanto,A.J.P., Seizinger, B.R. & Kley, N. (1994) Germ-line mutations in thevon Hippel-Lindau tumor-suppressor gene are similar to somaticvon Hippel-Lindau aberrations in sporadic renal cell carcinoma.American Journal of Human Genetics, 55, 1092–1102.