Endocrine

, Volume 35, Issue 3, pp 347–355

Mutations and polymorphisms in the SDHB, SDHD, VHL, and RET genes in sporadic and familial pheochromocytomas

Authors

    • Department of SurgeryUniversity Hospital Giessen and Marburg
    • Department of SurgeryPhilipps University Marburg
  • Peter Langer
    • Department of SurgeryUniversity Hospital Giessen and Marburg
  • Nils Habbe
    • Department of SurgeryUniversity Hospital Giessen and Marburg
  • Volker Fendrich
    • Department of SurgeryUniversity Hospital Giessen and Marburg
  • Anette Ramaswamy
    • Department of SurgeryUniversity Hospital Giessen and Marburg
  • Matthias Rothmund
    • Department of SurgeryUniversity Hospital Giessen and Marburg
  • Detlef K. Bartsch
    • Department of SurgeryUniversity Hospital Giessen and Marburg
  • Emily P. Slater
    • Department of SurgeryUniversity Hospital Giessen and Marburg
Original Paper

DOI: 10.1007/s12020-009-9178-y

Cite this article as:
Waldmann, J., Langer, P., Habbe, N. et al. Endocr (2009) 35: 347. doi:10.1007/s12020-009-9178-y

Abstract

The prevalence of germ line mutations within the RET-protooncogene and the tumor suppressor genes SDHB, SDHD, and VHL in pheochromocytomas (PC) varies in recent studies from 12 to 24%, if one look at them collectively. DNA was extracted from frozen tumor tissue as well as from blood leukocytes of 36 PC (26 sporadic/10 MEN2). Exons 1-8 of the SDHB-gene, 1-4 of the SDHD-gene, 1-3 of the VHL-gene, and exons 10, 11, 13, 14, 16 of the RET-gene were amplified by PCR and analyzed by DHPLC with the Transgenomic WAVE®-System. Samples with aberrant wave profiles were subjected to direct sequencing. Genetic aberrations were correlated to clinical characteristics. Germ line mutations in sporadic PC were identified in four patients (11%) whereas somatic mutations were observed in two (5%) patients. Nine coding polymorphisms (PM) were identified in seven (19%) patients. Intronic variants were observed in six (17%) patients and were all located in the SHDB gene. Patients with wild type alleles in all assessed genes were older (53 vs. 37 years, P = 0.007) and presented with an increased tumor size (49 vs. 32 mm, P = 0.003) compared to patients with mutations. Malignant PC revealed multiple (>2) genetic alterations more frequently than benign PC (4/7 vs. 4/29, P = 0.03). Interestingly intronic variants of the SDHB gene occur more frequently in malignant than in benign PC (3/7 vs. 2/29, P = 0.04). The frequency of germ line mutations in sporadic pheochromocytomas was lower in our cohort than previously reported. Polymorphisms of the RET gene are common (17%) and occur in familial and sporadic PC. Multiple genetic alterations including mutations, polymorphisms and intronic variants are more frequently observed in malignant PC.

Keywords

PheochromocytomaGeneticsMutations

Introduction

The traditional role that pheochromocytoma (PC) is called the “10%”-tumor is no longer tenable. About 24% of PC are caused by mutations of the RET-protooncogene, the succinate dehydrogenase subunit B (SDHB)-, the succinate dehydrogenase subunit D (SDHD)-, the Von-Hippel-Lindau (VHL)-, and the NF-1 gene according to a study which has included 271 patients with sporadic PCs [1]. However, the cumulative incidence of germ line mutations in several recent studies intended to be lower: RET (0.4%), SDHB (8.5%), SDHD (2.1%), and VHL (2.5%) [213]. Germ line mutations and hereditary PCs are associated with an increased risk of malignant PC, of recurrent tumors and bilateral disease [14]. These findings emphasize the importance of genetic testing in every patient with PC. Predictive genetic screening and regular screening of members with confirmed mutations in the context of hereditary PC intend an early diagnosis and treatment. Despite the high frequency of germ line mutations, somatic mutations of the genes responsible for hereditary PC and Paraganglioma (PG) have been rarely observed and occur in about 8% [7, 9, 10]. Recently the first somatic SDHB mutation was reported [15]. A part from mutations which predispose for PC secondary genetic alterations may be needed to develop PC. Chromosomal loss of 1p, 3p, 3q, and 11q is frequently observed in sporadic and hereditary PC [8, 1619]. The LOH of VHL, RET, and SDHD mutation is reported in hereditary PC, following the idea of Knudson’s “two-hit” hypothesis. Several pathways have been recently identified which are activated in PC as the hypoxia-driven pathway HIF1-alpha, or the ras-mediated MAPK-pathway but the oncogenesis of PC is still not completely understood [20, 21]. An alternative mutation scanning method based on denaturing high performance liquid chromatography (DHPLC) has been introduced about 10 years ago. The high accuracy and specificity of this technology to identify mutations was documented by numerous reports [22, 23].

In this study we assessed the frequency of germ line and somatic mutations as well as polymorphisms and intronic variants in patients with PC and PG in the RET, VHL, SDHB, and SDHD gene.

Patients and methods

Patients

A total of 36 of 58 patients with pheochromocytoma, who underwent surgery in our hospital since 1993 were included. Ten of these 36 patients were known to have Multiple Endocrine Neoplasia Type 2 (MEN2) (8 MEN2A, 2 MEN2 B). Twenty-six patients had no family history of PC or PG and were classified as sporadic cases. Twenty-nine PC were classified histologically benign. Four patients showed PC with distant metastases and in another three patients histological criteria as invasion of the tumor capsule or lymphatic and vascular-invasion were recorded.

Diagnostic work-up

A 24-h urine sample was assessed for epinephrine, norepinephrine, metanephrine, and normetanephrine. All patients underwent a CT scan or MRI of the abdomen and a Metaiodobenzoguanidyl (MIBG)-scan.

Surgery

The standard procedure was a laparoscopic transabdominal adrenalectomy. If radiological findings suspected malignancy or distant metastases were present at diagnosis we choose an open adrenalectomy through a thoraco-abdominal approach.

Histology

The tumor size was measured. Invasion of the capsule as well as the invasion of lymphatic and blood vessels were determined. Immunohistochemistry for chromogranin A, synaptophysin, and Ki-67 was obtained in most samples. In all tumor samples a cellularity of at least 85% tumor was confirmed by AR based on HE staining before DNA was extracted.

Analysis of sequence variations

DNA was extracted from frozen tumor tissue as well as from whole blood leukocytes using standard procedures in all patients. Primers were selected and polymerase chain reactions (PCR) for every amplicon were established (see Tables 1 and 2). Exons 1-8 of the SDHB-gene, 1-4 of the SDHD-gene, 1-3 of the VHL-gene, and exons 10, 11, 13, 14, 16 of the RET-gene were amplified by PCR using PanTag polymerase. All PCRs were carried out on a Eppendorf Mastercycler. We focused on the letter RET exons as they have been reported to be hot spots for MEN2 mutations associated with PC and in regard to cost-effectiveness of mutation screening.
Table 1

Primer sequences of each gene with the size of amplicons and GCT-clamps, if used are encountered

Gene

Exon

Primer sequence (5′–3′)

Amplicon (bp)

GCT-Clamp

RET

10

ACA CTG CCC TGG AAA TAT GG

254

GCT

CTC AGA TGT GCT GTT GAG AC

  

RET

11

ATG AGG CAG AGC ATA CGC A

338

 

GAA ATG GGG GCA GAA CAC A

  

RET

13

AAC TTG GGC AAG GCG ATG C

231

 

AGA ACA GGG CTG TAT GGA GC

  

RET

14

AAG ACC CAA GCT GCC TGA C

296

 

GCT GGG TGC AGA GCC ATA T

  

RET

16

AGG GAT AGG GCC TGG CCT T

184

 

TAA CCT CCA CCC CAA GAG

  

VHL

1

CCT CGC CTC CGT TAC AAC GGC CTA

603

 

GCA GGG ACG ATA GCA CGG TC

  

VHL

2

CAC CGG TGT GGC TCT TTA ACA A

264

 

CCA GTT CTC AAT TTT TGC CTG ATG T

  

VHL

3

CTG AGA CCC TAG TCT GCC ACT GAG

266

 

TAC ACT GTT TCA TCT CAG CTT TTG

  

SDHD

1

TGA CCT TGA GCC CTC AGG AAC G

98

 

TCA GGG TGG GAA GAC CCC T

  

SDHD

2

GAT CAT CCT AAT GAC TCT TTC C

208

GCT

AGC AGC AGC GAT GGA GAG AA

  

SDHD

3

CTT TTA TGA ATC TGG TCC TTT TTG

236

GCT

CAA CTA TAT TTG GAA TTG CTA

  

SDHD

4

TGA TGT TAT GAT TTT TTC TTT TTC T

224

 

CAA TTC TTC AAA GTA TGA AGT CA

  

SDHB

1

CGG AGA GCG ACC TCG GGG T

185

 

CAT CAG CTC CAG GCA GTC T

  

SDHB

2

GTT TAT ATC CAG CGT TAC ATC

297

 

GGA TGT GAA AAG CAT GTC CCT

  

SDHB

3

CTG AGA AGA CCA AAT GGA TAA G

240

 

GAC CAC AAG TAT CTG GAG CC

  

SDHB

4

GGA GGA TCC AGA AGA AAG TA

281

 

GTA ACA CAC ATA GCA CTG CC

  

SDHB

5

GCT GAG GTG ATG ATG GAA TCTG

248

 

CAC ACT CCT GGC AAT CAT CT

  

SDHB

6

CAC TGA CCC CAA AGT AAC A

200

 

CCT CAG AAT GGC TGG CTT AC

  

SDHB

7

GAG CTT TGA GTT GAG CCA GG

243

 

GCG TGT CAG CTC TGA GGC AG

  

SDHB

8

GGA CAC TGA ACC AGC TGA GG

219

 

GCT CTG AGC TGG TTA TAA ATC

  

bp base pairs

Table 2

Cycle conditions for the amplification of genomic PCR fragments for the assessed genes are shown

Amplicon

DN 96°C

DN 96°C

Annealing

Ext 72°C

Ext 72°C

Cycles

RET Ex 10

5

0.5

66

0.5

10

35

RET Ex 11

5

1

61

1

10

35

RET Ex 13

5

0.5

62

0.5

10

30

RET Ex 14

5

1

62

1

10

35

RET Ex 16

5

0.5

62

0.5

10

30

VHL Ex 1

5

0.5

61

0.5

10

35

VHL Ex 2

5

0.5

61

0.5

10

35

VHL Ex 3

5

0.5

61

0.5

10

35

SDHB Ex 1

5

0.5

58

0.5

10

35

SDHB Ex 2

5

0.5

56

0.5

10

35

SDHB Ex 3

5

0.5

60

0.5

10

35

SDHB Ex 4

5

0.5

60

0.5

10

35

SDHB Ex 5

5

0.5

60

0.5

10

35

SDHB Ex 6

5

0.5

55

0.5

10

35

SDHB Ex 7

5

1

63

1

10

30

SDHB Ex 8

5

0.5

58

0.5

10

35

SDHD Ex 1

5

1

60

1

10

35

SDHD Ex 2

5

1

63

1

10

35

SDHD Ex 3

5

1

62

1

10

35

SDHD Ex 4

5

1

58

1

10

35

DN denaturing, Ext extension

As recommended the melting profile of each amplicon was predicted using the computer program WAVE Maker 5.1 (Transgenomic, Omaha, Nebraska, USA). In some amplicons GCT-clamped primers were needed to prevent complete melt. Occasionally if the calculated conditions failed to produce well outlined peaks they were empirically modified. All analyzing conditions are displayed in Table 3. Prior to the WAVE-analysis hetero-duplex formation of an equal amount of the patients sample and a wild type sample was obtained in a PCR system: 95°C for 5 min, followed by a declining temperature T–1°C for 1 min until the sample reaches a temperature of 60°C. Samples with aberrant wave profiles were subjected to direct sequencing with the ABI310 sequencing analyzer according to standard procedures.
Table 3

Details of the DHPLC analysis are listed regarding the temperature, the percentage of buffer B, the estimated running time, and the time shift

Amplicon

Temp (°C) DHPLC

Runtime

Time shift

RET Ex 10

64.7

2.5

0

RET Ex 11

64.1

2.5

0

RET Ex 13

62.3

2.5

0

RET Ex 14

62.0/65.8

2.5

−0.5/+1.5

RET Ex 16

58.6

2.5

0

VHL Ex 1

65.9

2.5

0

VHL Ex 2

58.2

2.5

+0.5

VHL Ex 3

63.9

2.5

0

SDHB Ex 1

63.4

2.5

0

SDHB Ex 2

55.5

2.5

0

SDHB Ex 3

56.5

2.5

0

SDHB Ex 4

58.2

2.5

0

SDHB Ex 5

56.7

2.5

0

SDHB Ex 6

58.2

2.5

0

SDHB Ex 7

62.0

2.5

0

SDHB Ex 8

56.8

2.5

0

SDHD Ex 1

65.8

2.5

0

SDHD Ex 2

61.8

2.5

0

SDHD Ex 3

62.0

2.5

0

SDHD Ex 4

60.0

2.5

+1

temp temperature

This study was performed according to the guidelines of the local ethic committee.

Results

Patients

The median age of the 36 patients was 46 years (20–78) and the median tumor size was 35 mm (9–130). The 10 MEN2 A patients were diagnosed by a median age of 31 (21–62) and therefore at younger age compared to non-syndromic patients. Three of ten patients with MEN2A present with bilateral PC. In total three patients presented with sporadic paraganglioma.

Mutations

Fourteen germ line mutations were detected in 13 patients: 12 patients with RET mutations, one with a VHL mutation (L188P), and one MEN2A patient with an additional SDHB mutation (S163P). Two somatic mutations were identified in the VHL (G144X) and the SDHD (M1V) gene. All 10 patients with formerly known RET mutations were identified by DHPLC and confirmed by direct sequencing. In two patients with sporadic PC, RET mutations were identified. A 59-years-old female patient (P21) with a M918T mutation in exon 16 of the RET gene was presented with a relatively small tumor (40 mm) on the right side and had a negative family history. A 33-years-old male (P36) revealed a RET mutation in exon 11 (S649P). He had several operations due to a recurrent cervical paraganglioma which has metastasized to the lung and lymph nodes. In one patient we detected a VHL mutation L188P (P34). This male patient was diagnosed at the age of 20 years and presented with severe hypertension and a 40 mm PC of the right adrenal gland. The somatic VHL mutation G144X was detected in a 48-years-old male with a MEN1 syndrome which was reported previously [24, 25] with a germ line mutation in the Menin gene K119X. The somatic SDHD mutation M1V was found in the patient (P36) with the RET mutation (S649P) who was additionally harboring a RET PM (G691S). One male patient revealed a polymorphism in exon 5 of the SDHB gene S163P and interestingly was also harboring an RET germ line mutation (P15) (see Fig. 2). Although patient 21 displayed mild features of MEN2b, neither patient 21 nor patient 34 had a positive family history.

Polymorphisms in coding regions

We detected nine polymorphisms in seven patients: three times the PM G691S in RET exon 11, three times the L769L in RET exon 13, and the S836S in RET exon 14. One patient displayed both (P23). The patient (P34) with the VHL mutation had also the polymorphism S69S of the SDHD gene. All three patients with the PM G691S revealed also a RET germ line mutation in exon 11.

Intronic variants

These were only found in the SDHB gene in six patients in Introns 2, 4, and 5 (see Table 4, Figs. 1, 2).
Table 4

Result overview of the present study

Gene

n (G/S)

Mutations

n (G/S)

Polymorphism

n (G/S)

Intronic variants

RET

12/0

C634R, C634Y S649L

8/0

L769L, S836S G691S

0/0

 

VHL

1/1

L168P/G144X

0/0

 

0/0

 

SDHB

1/0

S163P

0/0

 

0/6

I2 G > A –33, I2 G > T +33, I4 T > C-38,-74, G > A –59, I5 G > A +33, I5 T > C +74

SDHD

0/1

M1V

1/0

S69S

0/0

 

n

14/2

 

9/0

 

0/6

 

G germ line, S somatic, n number

https://static-content.springer.com/image/art%3A10.1007%2Fs12020-009-9178-y/MediaObjects/12020_2009_9178_Fig1_HTML.gif
Fig. 1

Intronic variant ISV2-36 G > T was identified in five patients. On the top the DHPLC analysis is shown with the single peak of the wild type’s electropherogram and the four peaks demonstrating the two homo- and two heteroduplexes of the mutant allele. The electropherogram shows the corresponding sequence

https://static-content.springer.com/image/art%3A10.1007%2Fs12020-009-9178-y/MediaObjects/12020_2009_9178_Fig2_HTML.gif
Fig. 2

a Mutation C634Y with the PM G691S; DHPLC electropherograms showing the Wild type (WT) and the Mutated amplicon (M). b Mutation C634R; DHPLC electropherograms showing the Wild type (WT) and the Mutated amplicon (M)

Multiple genetic aberrations

Eight patients revealed more than one aberrant WAVE-profile which correspond to mutations, coding polymorphisms, and intronic variants in the direct sequencing as shown in Table 5.
Table 5

Clinical characteristics and results of the mutation analysis in 36 patients with pheochromocytoma

ID

D/Syndrome

Sex

FH

Age

Size/site

Mutation

Ki-67 (%)

Follow-up

1

Pheo/MEN 2a

f

pos

46

ND/l

RET C634Y

<1

NED

6

Pheo/MEN2a

f

pos

26

30,25/r;31/l

RET C634R

1

NED

14

Pheo/MEN-2a

m

pos

45

30,25,35/l

RET C634R

<1

NED

15

Pheo/MEN-2a

m

pos

31

40/l

RET C634R + G691S, SDHB S163P

<1

NED

20

Pheo/MEN2a

f

neg

62

30/r

RET C634R + G691S

<1

NED

24

Pheo/MEN2a

f

pos

59

15,20/l

RET C634R

<1

NED

26

Pheo/MEN2a

m

pos

21

25/r; 20/l

RET C634Y

<1

NED

27

Pheo/MEN2a

m

pos

36

24/r

RET C634R, RET Pol L769L

<1

NED

3

Pheo mal/MEN2b

m

pos

30

15,15,15/r

RET T M918T

<1

DED

22

Pheo/MEN2b

m

neg

28

15,10,12/l

RET M918T

<1

NED

4

Pheo/MEN1

m

pos

48

40/l

Menin K119X; VHL G144X (T)

ND

NED

8

Pheo mal

f

neg

78

50/r

SDHB ISV2 + 33 G > A (T), SDHB ISV236 G > T (T)

<1

DUC(MI)

10

Pheo mal

f

neg

65

90/l

SDHB ISV4 + 38, + 74 T > C (T); +59G > A (T)

ND

NED

23

Pheo mal

f

neg

44

45/l

RET Pol L769L, RET Pol S926S, SDHB ISV2 +33 G > A, ISV5 -74 T > C (T)

<1

NED

29

Pheo mal

f

neg

22

40/l

RET Pol S926S

<1

NED

33

Pheo mal

m

neg

42

75/l

WT

1

AWD

36

Paragangliom mal

m

neg

33

 

RET S649P + G691S, SDHD M1V (T)

10

AWD

32

Paragangliom

f

neg

47

30/l

WT

1

NED

35

Paragangliom

f

pos

48

35/r

WT

1

NED

2

Pheo

m

neg

40

50/l

WT

<1

Lost

5

Pheo

m

neg

54

45/l

WT

ND

NED

7

Pheo

f

neg

78

47/r

WT

<1

DUC

9

Pheo

f

neg

62

82/r

WT

<1

DUC

11

Pheo

f

neg

55

80/r

SDHB ISV5 -33 ho G > A (T)

<1

NED

12

Pheo

m

neg

63

35/r

WT

<1

NED

13

Pheo

f

neg

46

35/r

SDHB ISV5 -33 G > A (T)

<1

NED

16

Pheo

f

neg

60

60/l

WT

ND

NED

17

Pheo

f

neg

65

35/r

WT

ND

Lost

18

Pheo

m

neg

53

50/r

WT

ND

NED

19

Pheo

m

neg

37

35/r

WT

<1

NED

21

Pheo

f

neg

59

40/r

RET M918T

ND

NED

25

Pheo

m

neg

52

15/l

RET Pol S926S

<1

NED

28

Pheo

f

neg

71

60/l

WT

<1

NED

30

Pheo

f

neg

35

60/r

WT

1

Lost

31

Pheo

f

neg

54

39/r

WT

ND

NED

34

Pheo

m

neg

20

40/r

SDHD Pol S69S, VHL L188P

5

NED

T tumor, Pheo pheochromocytoma, pos positive, neg negative, WT wild type, r right, l left, Mut mutation, Pol polymorphism, NED no evidence for disease, AWD alive with disease, DUC died of unrelated causes, ISV intronic variant

Prevalence of intronic SDHB variants

The intronic variants ISV4 an ISV5 were not identified in 30 healthy controls.

Correlation with clinical data

Fifteen patients had wild type alleles in all four genes screened for mutations. We compared them with either patients who revealed mutations (14), polymorphisms (9) or intronic variants (7).

Tumor size and age of diagnosis

Patients without any genetic aberration in the assessed genes were older (53 vs. 37 years, P = 0.003) and presented with larger tumors (49 vs. 32 mm, P = 0.007) when compared to patients with mutations. There were no differences between patients without mutations and polymorphisms. Patients with polymorphisms tend to have larger tumors (50 vs. 32, P = 0.055) and were older (51 vs. 37, P = 0.10) when compared to patients harboring germ line mutations.

Multiple tumors

All five patients (P4, P6, P14, P22, and P24) who demonstrated multiple tumors at histology harboring RET germ line mutations.

Ki-67

Twenty-nine PC were stained for Ki-67 by IHC. In most PC Ki-67 index was 1% (5) or lower (22). Two PC/PG showed higher Ki-67 indices with 10% (P36) and 5% in a sporadic PC in a 20-years-old patient (P34) revealing an SDHD PM S69S and a VHL mutation. Four of six malignant PC showed no increased Ki-67 indices.

Malignancy

Six of seven malignant PC were sporadic cases without a positive family history. Only one patient revealed a germ line mutation in the RET protooncogene (S649P + G691S) whereas genetic changes including polymorphisms and intronic variants were frequently found in four of seven malignant PC.

Interestingly three of seven patients with malignant PC revealed intronic variants in the SDHB gene, which was found to be less frequent in benign PC (3/7 vs. 2/29, P = 0.04).

Multiple aberrations (>2) were more frequently observed in malignant than in benign PC (4/7 vs. 4/29, P = 0.03).

There were no statistical differences between side, gender, and catecholamine concentration between the groups.

Discussion

The prevalence of RET mutations of 30% in our patients was higher than reported in the literature which relies on a selection bias due to our institution. If we exclude the 10 patients with family history we found an incidence of 7.8% which is similar to a mean incidence of 4.5% (0–21) reported the literature [1, 6, 9, 10]. Germ line mutations in the SDHB and SDHD gene were not detected which is consistent with the low frequency in these genes of approximately 4% (4–22) [1, 2, 6, 13]. In one patient with a paraganglioma we identified a somatic SDHD mutation M1V which is in order with the literature (1%) [26]. VHL mutations are found in 6.5% as a germ line and in 4.5% as a somatic mutation which is similar to our cohort having a frequency of 4% each [1, 6, 7, 9]. In recent studies single nucleotide polymorphisms (SNP) were described for RET, SDHB, SDHD, and VHL genes. The frequency of polymorphisms in a normal population and their role in tumorigenesis and clinical behavior of tumors, associated with the corresponding hereditary tumor syndromes, varies among these genes. Recently the polymorphism G691S in exon 11 of the RET gene was reported to be associated with earlier manifestation of medullary thyroid cancer in MEN2A patients and with familial C-cell hyperplasia [27, 28]. However, we identified the polymorphism G691S in 3/36 patients, all three had germ line mutations in RET exon 11 and were diagnosed by years of age 30, 33, and 59. Also the polymorphism L769L is reported to act as a modifier in medullary thyroid cancer [29]. Three of 36 (8%) or 3/10 (33%) MEN2 A patients revealed this polymorphism. The polymorphism S836S was detected in 3/26 (11%) patients with sporadic pheochromocytoma whereas recent studies report a frequency of only 3.7% [30] in a normal population. However, we do not know if these findings are randomly in this small cohort of 36 patients. Nevertheless, synonymous amino acid changes might not only cause altered rates of transcription but also, due to changed mRNA structure and stability, translational efficiency may be affected. The SNP (G691S) changes the nonpolar amino acid glycine into the polar amino acid serine. This alteration can substantially alter protein processing, folding, subcellular localization, or other functional properties. This principle has to kept in mind regarding the SDHB polymorphism S163P, which was detected in two patients: one with an MEN2 A syndrome and one with a malignant PC. Intronic variants have been found in six patients all within the SDHB gene (see Table 4). The frequencies in a normal population are only known for ISV +33 (11%) and ISV-36 (3%) according to the Leiden Open Variation Database (http://chromium.liacs.nl). Despite being located in nontranscribed regions, these types of SNPs can have substantial functional effect by altering splice or branch sites or, if located in or near the promoter region, transcription. In this study intronic variants were more frequently in malignant than in benign PC (4/7 vs. 2/29, P = 0.04).

We showed that malignant PC tend to have multiple genetic changes when regarding mutations, polymorphisms and intronic variants (4/7 vs. 4/29, P = 0.03), which could be a hint for the genetic instability. This could be both either consequence or reason for the malignant behavior. The malignancy is only certain if distant metastases are present.

The prevalence of malignancy in PC ranges from 2.4% to 26%, depending on how malignancy is defined. The highest rate of malignancy is observed in patients with SDHB mutations and reaches 50%. However, the histologic distinction of benign from malignant PC is not reliable. Malignancy is only proven by the presence of distant or lymph node metastases. Efforts have been made to establish molecular markers for malignancy as overexpression of HSP90, human telomerase reverse transcriptase, HIF-2 alpha, tenascin, Ncadherin, COX-2, and others. Comparative genomic hybridization experiments in malignant and benign PC revealed losses of 1p, 3q, 6q and gains of 9q and 17q. Progression to malignant PC was associated to deletion of 6q and 17p [16].

As malignant pheochromocytomas tend to develop multiple genetic alterations, especially intronic variants in the SDHB gene, could be used as an additional marker for malignancy.

In conclusion we confirmed the frequency of germ line mutations in the RET, SDHB, SDHD, and VHL gene in sporadic pheochromocytomas. Polymorphisms in the RET gene are common and occur in about 30% of patients with MEN2A and in 15% of patients with sporadic PC. Multiple genetic alterations including mutations, polymorphisms, and intronic variants are more frequently observed in malignant PC.

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