[CANCER RESEARCH 60, 6223– 6226, November 15, 2000]
Advances in Brief
Duplication of the Mutant RET Allele in Trisomy 10 or Loss of the
Wild-Type Allele in Multiple Endocrine Neoplasia
Type 2-associated Pheochromocytomas
Steve C. Huang,1 Christian A. Koch,1 Alexander O. Vortmeyer, Svetlana D. Pack, Urs D. Lichtenauer,
Poonam Mannan, Irina A. Lubensky, George P. Chrousos, Robert F. Gagel, Karel Pacak, and
Molecular Pathogenesis Unit, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892 [S. C. H., A. O. V., S. D. P.,
U. D. L., P. M., I. A. L., Z. Z.]; Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Disease, NIH, Bethesda, Maryland 20892
[C. A. K., G. P. C., K. P.]; and University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 [R. F. G.]
Abstract Materials and Methods
Inherited mutations of the RET proto-oncogene are tumorigenic in Patients and Tumors. All five patients are unrelated. Results of biochem-
patients with multiple endocrine neoplasia type 2 (MEN 2). However, it is ical screening for catecholamines combined with abdominal imaging by com-
not understood why only few of the affected cells in the target organs puted tomography were indicative of an intra-adrenal pheochromocytoma.
develop into tumors. Genetic analysis of nine pheochromocytomas from Subsequently, these patients underwent either unilateral or bilateral adrenalec-
five unrelated patients with MEN 2 showed either duplication of the tomy. Blood was drawn for DNA extraction, and tumors from all five patients
mutant RET allele in trisomy 10 or loss of the wild-type RET allele. Our were removed at the time of surgery and frozen at 80°C. DNA was extracted
results suggest a “second hit” causing a dominant effect of the mutant from lymphoblasts and tumor tissue by standard methods.
RET allele, through either duplication of the mutant allele or loss of the Preparation of Tumor Imprints and Interphase Nuclei from Patients’
wild-type allele, as a possible mechanism for pheochromocytoma tumor- Blood for FISH. FISH analysis of tumors was performed using a chromo-
some 10-specific centromeric -satellite probe, as described by Zhuang et al.
igenesis in patients with MEN 2.
(4). Tumor touch preparations were performed by attaching slightly thawed
tumor to a glass slide and air-dried. The air-dried tumor touch preps were fixed
Introduction in an ethanol series (70, 80, 90, and 100% for 10 min each), followed by
collagenase A (0.01%) treatment in Gurr-Ringer buffer for 20 min at room
MEN 23 is an autosomal dominant inherited cancer syndrome,
temperature. Metaphase and interphase slides from patients’ leukocytes were
characterized by the development of pheochromocytoma, medullary made by routine protocol for metaphase harvesting.
thyroid carcinoma, and parathyroid hyperplasia/adenoma. The gene FISH. Chromosome 10-specific -satellite probe, biotin-labeled (Oncor),
responsible for MEN 2, the RET proto-oncogene, has been localized was used for the detection of the chromosome copy number. In situ hybrid-
to chromosome 10q11.2 (1). RET is expressed in human neural ization and detection procedures were carried out as described (5). In brief,
crest-derived and neuronal tissues such as Schwann cells, sympathetic slides were denatured (70% formamide, 2 SSC) at 72°C for 2 min, dehy-
ganglia, adrenal medulla, astrocytes, and cerebral cortical neurons (2). drated in a cold ( 20°C) ethanol series (70, 80, 90, 100%) for 2 min, and
air-dried. -satellite repetitive DNA, specific for chromosome 10, was dena-
In patients with MEN 2, germ-line mutations of RET are commonly
tured for 6 min at 76°C, and overnight hybridization was done in a humidified
identified and are believed to be activating, i.e., causing ligand- chamber at 37°C. Posthybridization washes were at 45°C in 50% forma-
independent dimerization of the receptor. Although the inherited RET mide/2 SSC (3 5 min), 1 SSC (2 5 min), and 0.1 SSC (2 5 min).
mutation is related to tumorigenesis in patients with MEN 2, it is Detection was performed using avidin-FITC (30 min at 37°C), followed by
unknown by which mechanism(s) only few of the affected cells in the washing in 4 SSC/0.1% Tween 20 solution at 45°C and counterstaining with
target organs develop into tumors (3). propidium iodide.
MET represents another proto-oncogene with structural and func- Hybridization signals were scored using a Zeiss Axiophot epifluorescence
microscope, and two-color images were captured on a Photometrics CCD
tional homology to RET. Upon activation, both proto-oncogenes, MET
camera (Sensys) using IPLab Image software (Scanalytics, Inc.).
and RET, act similarly through stimulation of a receptor tyrosine Quantitative PCR Amplification of Microsatellites on Chromosome 10.
kinase. For MET, trisomy 7-harboring nonrandom duplication of the Three polymorphic markers, D10S677, D10S1239, and D10S141 (Research
mutant MET allele has been implicated recently in tumorigenesis of Genetics), were used in quantitative PCR analysis with genomic DNA ex-
patients with hereditary papillary renal carcinoma (4). The functional tracted from peripheral blood and microdissected tumor tissue. PCR amplifi-
homology between MET and RET led us to investigate whether cations in the presence of [ -32P]dCTP (0.1 Ci/ l; DuPont) were performed
activation of the RET proto-oncogene in patients with the familial in a Hybaid Omnigene thermal cycler using Ampli-Taq Gold DNA polymerase
(Perkin-Elmer Roche). PCR conditions were as follows: initial denaturation at
tumor syndrome MEN 2A occurs by a similar mechanism.
95°C for 10 min, then 30 cycles, each with 1 min of denaturation at 95°C, 1
min of annealing at 55°C, and 1 min of extension at 72°C; PCR was completed
Received 7/26/00; accepted 9/26/00. with a final extension at 72°C for 10 min. The amplicons were resolved on a
The costs of publication of this article were defrayed in part by the payment of page 6% polyacrylamide gel. Gels were dried and exposed to Kodak XAR film.
charges. This article must therefore be hereby marked advertisement in accordance with
Quantitative analysis of allelic imbalance was performed using PhosphorImage
18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work. analysis (Molecular Dynamics). All PCR reactions were performed in triplicate
To whom requests for reprints should be addressed, at Surgical Neurology and were repeated twice. Each densitometry measurement was performed four
Branch, National Institute of Neurological Disorders and Stroke, NIH, Building 10, times.
Room 5D37, Bethesda, MD 20892. Phone: (301) 435-8445; Fax: (301) 480-1839;
Sequence Analysis. The primers for sequencing analysis of RET were as
The abbreviations used are: MEN 2, multiple endocrine neoplasia type 2; FISH, follows: exon 10 (IIF, 5 -GGG GGA TTA AAG CTG GCT AT and IR,
fluorescence in situ hybridization; SSCP, single-strand conformational polymorphism. 5 -CTC AGA TGT GCT GTT GAC AC), and exon 11 (IF, 5 -TCA CAC CAC
DUPLICATION OF MUTANT RET ALLELE IN TRISOMY 10
Table 1 Genetic alterations in MEN 2-associated pheochromocytoma SSCP Analysis. To confirm the absence of the wild-type RET allele in
Copy number tumor 5A, we used SSCP analysis. The amplicon (primers: F, 5 -ACA CTG
RET mutation Allelic analysis Phosphorimage of chromosome CCC TGG AAA TAT GG and R, 5 -CTC AGA TGT GCT GTT GAG AC) of
Patient Tumor (WT/mutant) markera ratio ( 0.2) 10 by FISH exon 10 from tumor DNA and a DNA sample extracted from the peripheral
1 1A Codon 634 D10S677 2.1 Trisomy 10 blood of a healthy individual were analyzed on a MDE gel (FMC Bioproducts)
TGC/AGC according to the manufacturer’s protocol.
1B Codon 634 D10S677 1.1 Disomy 10
2 2A Codon 631 D10S1239 1.8 Trisomy 10 Results
2B Codon 631 D10S1239 1.8 Trisomy 10 We studied nine pheochromocytomas from five unrelated patients
3 3A Codon 631 D10S677 1.7 Trisomy 10 with MEN 2A. In all patients, we identified RET germ-line mutations
GAC/TAC in blood DNA (Table 1). Five of the tumors from four patients (1A,
3B Codon 631 D10S677 1.2 Disomy 10
2A, 2B, 3A, and 4; Table 1) were trisomic for chromosome 10 by
4 4 Codon 634 D10S677 2.1 Trisomy 10 FISH analysis with chromosome 10-specific centromeric -satellite
TGC/GGC probes (Fig. 1A). As a control, lymphoblasts from these patients
5 5A Codon 620 D10S141 1.3 Disomy 10
TGC/CGC showed disomy for chromosome 10. To analyze the copy number of
5B Codon 620 D10S141 1.3 Disomy 10 the mutant and the wild-type RET allele in trisomy 10, we performed
quantitative PCR on tumor DNA, using chromosome 10 microsatellite
Representative markers that were informative. markers D10S677 and D10S1239. In two tumors (2A and 2B), the
mutant RET allele on chromosome 10 was identified from the affected
patient’s family pedigree study (Fig. 1B). Upon direct visualization,
CCC CAC CCA CAG and IIR, 5 - TGG TAG CAG TGG ATG CAG AA). the band representing the mutant allele displayed greater intensity
The AmpliCycle sequencing kit (Perkin-Elmer Roche) was used according to than the one representing the wild-type allele. Quantitative analysis
the manufacturer’s protocol. using phosphorimage densitometry revealed an intensity ratio of 2:1
Restriction Enzyme Digestion Analysis. The composition of the wild-
between the mutant and the wild-type alleles in all five trisomic
type and the mutated RET in tumor 1B, bearing a germ-line mutation at codon
tumors (1A, 2A, 2B, 3A, and 4; Table 1). In contrast, blood DNA
634 (TGC/AGC), was resolved by subjecting a 124-bp amplicon (primers: IF,
5 -TCA CAC CAC CCC CAC CCA CAG and IIR, 5 -TGG TAG CAG TGG from the same patients (nos. 1, 2, 3, and 4) revealed equal allelic
ATG CAG AA) of exon 11 to overnight DdeI (C/TNA) restriction enzyme intensities for both chromosome 10 markers (Fig. 1C).
digestion at 37°C. Only the amplicon from the mutant RET allele has a unique To further characterize the nature of allelic imbalance in the three
DdeI site 40-bp from the 5 end. trisomic tumors (1A, 3A, and 4) without available linkage data, we
Fig. 1. Trisomy 10 with nonrandom duplication of the
mutant RET allele in MEN 2-associated pheochromocy-
toma. A, representative interphase FISH analysis on tumor
touch preparation from patient 2 (tumor 2A). Three copies
of chromosome 10 are shown using a centromeric -satel-
lite probe (FITC, green signal) specific for chromosome 10.
B, combined pedigree and tumor allelic analysis of patient
2 (Pt2). Arrow, patient 2. Filled symbols, individuals with
MEN 2. Genotypes are shown for the chromosome 10
microsatellite marker D10S1239 linked to the RET locus.
Allele 2 of D10S1239 is coinherited with the disease in this
patient’s family. In patient 2, allele 2 shows a greater
intensity in Lanes 2A and 2B (tumors) than allele 1, repre-
senting the wild-type allele, as compared with Lane N2
(blood DNA). Lane N1 (blood DNA) shows equal intensi-
ties of mutant and wild-type allele in the patient’s affected
cousin (C). C, representative results of microsatellite and
phosphorimage analyses. After PCR amplification using
marker D10S1239, quantitative measurement of allelic in-
tensity was performed using phosphorimage analysis. In
tumor tissue (T), allele 2 is more intense than allele 1.
Phosphorimage densitometry shows a 2:1 imbalance be-
tween the two alleles in the tumor (T) as compared with the
normal tissue (N). D, representative results of sequencing
analysis of RET in tumor 3A. Blood DNA from an unaf-
fected/healthy individual (C, left) shows the wild-type RET
sequence (codon 631 GAC). Blood DNA from patient 3 (N,
middle) shows the germ-line mutation (G/T). Tumor DNA
(T, right) shows a higher intensity of the mutant nucleotide
(T ) compared with the wild-type nucleotide (G).
DUPLICATION OF MUTANT RET ALLELE IN TRISOMY 10
Fig. 2. Loss of the wild-type RET sequence in MEN
2-associated pheochromocytoma. A, representative in-
terphase FISH analysis on tumor touch preparation from
tumor 1B. Two copies of chromosome 10 are shown
using a centromeric -satellite probe (FITC, green sig-
nal) specific for chromosome 10. B, representative re-
sults of microsatellite and phosphorimage analyses. Af-
ter PCR amplification of microdissected tumor (1B) and
blood DNA using polymorphic marker D10S677, quan-
titative measurement of allelic intensity was performed
using phosphorimage analysis. In tumor (T) and normal
(N) tissue, allele 2 shows the same densitometry inten-
sity as allele 1. C, representative results of mutation
site-specific restriction enzyme digestion analysis of
tumor 1B, showing loss of wild-type RET. Amplicons
of exon 11 from tumor (1B) and normal DNA were
subjected to overnight restriction enzyme digestion with
DdeI, which exclusively cuts the mutant resulting in two
fragments (84 and 40 bp). Lanes 1 and 2, wild-type
control DNA (124-bp fragment), before (Lane 1) and
after (Lane 2) digestion, respectively; Lane 3, mutant
control DNA after digestion (84 and 40-bp fragments);
Lane 4, blood DNA from patient 1 after digestion,
showing undigested 124-bp fragment (wild-type) and
digested 84- and 40-bp fragments (mutant); Lane 5,
tumor DNA (1B) after digestion, showing only the mu-
tant fragments (84 and 40 bp) and no wild-type frag-
ment (124 bp). D, representative results of sequencing
analysis of RET in patient 1 showing loss of wild-type
RET. Blood DNA from an unaffected/healthy individual
(C, left) shows the wild-type RET sequence (codon 634
TGC). DNA from tumor 1B (T, right) shows exclu-
sively the mutant nucleotide (A) and loss of the wild-
type nucleotide (T ). Blood DNA from patient 1 (N,
middle) shows both nucleotides, the germ-line mutation
(A) and the normal wild-type (T ).
compared sequencing data from the patients’ tumor and blood DNA. either through duplication of the mutant allele in trisomy 10 or loss of
Both the wild-type and the mutant sequence of each affected codon the normal wild-type RET.
(codon 634: TGC/AGC in patient 1, TGC/GGC in patient 4; codon In experimental mouse tumors, imbalance between the wild-type
631: GAC/TAC in patient 3) were present in both tumor and blood and the mutant oncogene has been shown to play a mechanistic role
DNA (Table 1). However, the mutant nucleotide (adenine in patient 1, during early tumorigenesis (7). Only recently, such imbalance has also
guanine in patient 4, and thymidine in patient 3) displayed a more been demonstrated in a human hereditary papillary renal cell carci-
intense signal as compared with the wild-type nucleotide (Fig. 1D). noma syndrome (4). Zhuang et al. (4) proposed that inherited muta-
Four tumors (1B, 3B, 5A, and B) from three patients did not reveal tions of MET may render the cells more susceptible to errors in
increased copy numbers of chromosome 10 by FISH analysis (Fig. chromosomal replication during cell division, resulting in nonrandom
2A) and failed to display any imbalance between the two heterozygous chromosomal duplication of the mutant allele in those cells. Our
alleles by microsatellite analysis with markers D10S677 and D10S141 finding of allelic copy changes of RET in MEN 2-associated pheo-
(Fig. 2B). However, loss of the wild-type RET sequence was detected chromocytoma is consistent with previous studies of chromosomal
in two tumors. In tumor 1B (codon 634 TGC/AGC), only the mutant instability in other tumors (8). Cells that contain two mutant RET
sequence was present, as demonstrated by mutation site-specific re- alleles may gain growth advantage and eventually develop into tu-
striction enzyme digestion analysis (Fig. 2C) and sequencing analysis mors. Similarly, loss of the wild-type RET allele may provide a
(Fig. 2D). In tumor 5A (codon 620 TGC/CGC), SSCP and sequencing dominant effect of the mutant RET allele. In support of the loss of the
analyses showed loss of the wild-type RET sequence (data not shown). wild-type RET allele causing tumorigenesis, other investigators have
In the two remaining tumors (3B and 5B), normal wild-type and shown allelic loss on chromosome 10 in MEN 2-associated tumors, in
mutant RET sequences were present and were equally intense by one case with the entire copy of chromosome 10 absent (9). Interest-
phosphorimage densitometry. ingly, various MEN 2-associated tumors from the same patient can
have different genetic alterations; in patient 1, one pheochromocy-
Discussion toma (1A) harbored trisomy 10, and the second tumor (1B) showed
In patients with MEN 2, it is believed that inherited mutations of the loss of the wild-type RET allele (Table 1). This result suggests that the
RET proto-oncogene are responsible for the development of MEN selection between duplication of the mutant RET and loss of the
2-associated tumors, but the mechanism(s) explaining why only few wild-type RET allele is random.
of the affected cells (although all cells have the RET germ-line The reason why the majority of cells carrying a germ-line RET
mutation) undergo tumorigenesis are unknown (3). Furthermore, it mutation in patients with MEN 2A fail to undergo tumorigenesis
remains puzzling why patients with a germ-line mutation in RET do could be that the wild-type RET gene product dimerizes with the
not have MEN 2-associated tumors such as pheochromocytoma from mutant counterpart, exerting a neutralizing effect and thereby partially
birth on, but rather develop these tumors over time, sometimes as late compensating for the activating effects of the mutant RET allele. Once
as the 7th decade (1, 6). In this study, we provide evidence that allelic imbalance occurs with an increased “dosage” of the mutant
activation of RET and subsequent tumor formation may occur by a allele, either by duplication of the mutant allele or by loss of the
“second hit” that causes a dominant effect of the mutant RET allele, wild-type allele, the protective effect of wild-type protein may be
DUPLICATION OF MUTANT RET ALLELE IN TRISOMY 10
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2. Nakamura, T., Ishizaka, Y., Nagao, M., Hara, M., and Ishikawa, T. Expression of the
duplication of the mutant allele in trisomy 10 or loss of the wild-type ret proto-oncogene product in human normal and neoplastic tissues of neural crest
RET allele, may represent an early and fundamental event in the origin. J. Pathol., 172: 255–260, 1994.
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