Divisions of Endocrinology (C.M.G., J.A.M.), Adolescent/Young Adult Medicine (C.M.G.), and Plastic Surgery (J.B.M.), Childrens Hospital, and the Department of Pediatrics; Department of Adult Oncology, Human Cancer Genetics Unit, Dana-Farber Cancer Institute (D.J.M., C.E.); and the Department of Medicine (D.J.M., C.E.), Harvard Medical School, Boston, Massachusetts 02115; the Molecular Genetics Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney (D.J.M., B.G.R.), St. Leonards, New South Wales, Australia; and the Cancer Research Campaign Human Cancer Genetics Research Group, University of Cambridge (B.A.J.P., C.E.), Cambridge, United Kingdom
Address all correspondence and requests for reprints to: Catherine M. Gordon, M.D., Division of Endocrinology, Childrens Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115.
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Introduction |
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Germline mutations of the RET protooncogene cause MEN 2
[reviewed by Eng (2)]. This gene encodes a transmembrane glycoprotein
receptor tyrosine kinase (8, 9) whose ligands include glial cell
line-derived neurotrophic factor (GDNF) (10, 11, 12, 13) and neuturin (NTN)
(14, 15, 16). Before binding RET, GDNF or NTN must bind to
coreceptor molecules, GDNF receptor-, NTNR
/TrnR2 (14, 15, 16). The
ligand-coreceptor complex, in turn, interacts with and mediates
activation of the RET receptor tyrosine kinase (11, 13, 14, 15, 16). The
precise role of the RET protein in normal embryogenesis is unknown; it
probably plays a role in the development of neural crest, peripheral
neurons, and kidneys (12, 17). The MEN 2B phenotype appears to result
from a specific germline missense mutation in the tyrosine kinase
domain of the RET protooncogene (18, 19, 20). This mutation,
present in 75 of 79 unrelated patients with MEN 2B, is a uniform single
base pair transition within exon 16 (ATG to ACG at codon 918 of the
RET gene) that replaces methionine with threonine, M918T
(21).
Although the great majority (95%) of classic MEN 2B patients carry the codon 918 mutation, it is not known whether patients with partial MEN 2B phenotypes, such as pure mucosal neuroma syndrome (MNS), have the germline codon 918 mutation (21). We report four cases of MNS tested for the presence of germline M918T. In addition, we examined DNA from neuromatous tissue for the presence of a somatic codon 918 mutation.
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Materials and Methods |
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Neuromas obtained by surgical excision were immediately frozen in liquid nitrogen and stored at -80 C until analyzed (patient 1) or were fixed in neutral buffered formalin and embedded in paraffin (patients 24).
DNA preparation and PCR techniques
DNA was extracted from peripheral blood leukocytes or from flash-frozen tumors, as previously described (19, 22). DNA from archival sources was extracted according to the technique of Wright and Manos (23).
PCR amplifications of exon 16 were performed in a total volume of 50 µL with 100 ng genomic DNA and the primers rRET16 and fRET16, as previously described (18) with minor alterations. Modified conditions for PCR were 30 cycles of denaturation at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 72 C for 2 min. Double stranded cycle sequencing was performed according to the manufacturers recommendations [Boehringer Mannheim Corp. (Indianapolis, IN) and Stratagene Cyclist Kit (Cambridge, UK)]. The presence or absence of M918T was assessed with the technique of differential restriction digestion. After amplification with a modified oligonucleotide primer, second round PCR, using identical conditions, was performed using a primer designed to introduce a RsaI site in the presence of the codon 918 mutation ATG to ACG, as previously described (24). The exon 16 PCR product was digested for 816 h with an excess of the restriction endonuclease RsaI under the manufacturers conditions (New England BioLabs, Beverly, MA). PCR fragments and products were analyzed on 3% agarose gels (NuSieve, FMC BioProducts, Rockland, ME) after staining with ethidium bromide and UV transillumination or Southern hybridization (see below).
PCR amplification and cycle sequencing of exons 10, 11, and 13 have been described previously (25, 26, 27).
Southern blot analysis
RET exon 16, after two rounds of PCR amplification and RsaI digestion as described above, was subjected to electrophoresis through a 3% agarose gel and transferred to a nylon membrane by capillary action with 20 x SSC (sodium salt citrate; 3 mol/L sodium chloride and 0.3 mol/L sodium citrate), for 48 h. The gel was denatured by soaking twice for 15 min in 0.5 N NaOH-1.5 mol/L NaCl and neutralized by soaking twice more for 15 min each time in 0.5 mol/L Tris-HCl (pH 7.4)-1.5 mol/L NaCl. The nucleic acids were immobilized onto the membrane by exposure to UV light (254312 nm) for 1 min and 20 s.
The filters were washed with 2 x SSC and prehybridized for 3
h at 65 C in the presence of 0.5% SDS, 5 x Denhardts solution
(0.1% polyvinylpyrrolidine, 0.1% BSA, and 0.1% Ficoll, type 400),
6 x SSC, and 1 mL (400 µg/mL) denatured salmon sperm DNA (Sigma
Chemical Co., St. Louis, MO). A random primed DNA labeling kit
(Boehringer Mannheim Corp.) along with -32P-labeled
deoxy-CTP was used to make a 32P-labeled exon 16 PCR
product. Hybridization was carried out overnight using 1 x
107 cpm probe in 5 x SSC, 0.5% SDS, and 5 x
Denhardts solution at 65 C. The membrane was serially washed in
2 x SSC-0.1% SDS and 0.1 x SSC-0.1% SDS at 60 C. The
filter was rinsed in 2 x SSC-0.1% SDS at 60 C for 30 min,
followed by rinsing in 0.1 x SSC-0.1% SDS at 60 C for 60 min,
and at 65 C for 30 min. Bands were visualized by autoradiography using
Kodak XAR film (Eastman Kodak, Rochester, NY) at -80 C.
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Case Reports and Results |
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A 15 11/12-yr-old adolescent girl was referred to an out-patient pediatric endocrine clinic for evaluation of multiple mucosal neuromas that had been present since infancy. She presented at birth with an irregularity of her upper left lip that became swollen and increasingly disfigured with time. She underwent excision of these facial neuromas at ages 5 and 6 yr. However, mucosal neuromas recurred after each excision, extending along her left eye, nose, lips, and buccal mucosa. A barium enema, performed at age 11 yr during hospitalization for fecal impaction, suggested large bowel neuronal dysplasia, although the diagnosis was not confirmed by biopsy. She had no further bowel problems after increasing her dietary fiber intake. Past medical history was otherwise unremarkable, and growth and development were within normal limits.
Family history was notable for well controlled hypertension in the patients father, a paternal uncle, and a paternal aunt. A paternal aunt had hypothyroidism; otherwise, there were no endocrinopathies in the family. The patients mother underwent a mastectomy for breast cancer at age 48 yr and was in remission at the patients initial evaluation. There were no neuromas in other family members.
At an evaluation at age 15 yr, 11 months, the patient had left-sided
facial fullness (Fig. 1A), with neuromas
along the palpebral fissures, nasal alae, buccal mucosa (Fig. 1B
), left
anterior tongue, and both upper and lower lips in the distribution of
the three divisions of the left trigeminal nerve. Both height and
weight were in the 2550th percentile. Vital signs were within normal
limits. Breasts and pubic hair were both Tanner stage V. The
examination was otherwise within normal limits.
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At age 17 yr, the patient underwent a third resection of neuromas from
the buccal mucosa and lip. DNA was extracted from the tumors and
analyzed for the codon 918 mutation. Somatic mutations in the RETprotooncogene were not identified in any of the four tumors. PCR
amplification of RET exon 16 produced the expected 192-bp
product in all mucosal neuromas. The MEN 2B-type mutation at codon 918,
ATG to ACG, was detected in the positive control after a second round
of PCR amplification and enzymatic digestion with RsaI (Fig. 2). However, as shown in Fig. 2
, there
was no evidence for this mutation in any of the mucosal tumors from the
patient. Southern blot analysis confirmed these findings.
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A 12 5/12-yr-old boy was referred for endocrinological consultation after surgical removal of two mucosal neuromas of the tongue. The patients tumors were removed at the age of 9 yr. There was no involvement of the lips or eyes, and the patient was otherwise healthy. Family history was negative for mucosal neuromas or any components of MEN 2. The patients father had retinitis pigmentosa and is 95% blind; a paternal uncle had a similar problem.
Laboratory investigations included normal 24-h urinary catecholamine levels and ultrasonography of the thyroid gland, which showed no evidence of a mass lesion. At age 9 9/12 yr, pentagastrin-stimulated calcitonin levels were normal. RsaI-based mutational analysis for a codon 918 mutation was negative. In addition, germline mutations in exons 10 and 11 were not found. Finally, the resected neuromas were screened for the presence of a somatic M918T, but it was not found.
Patient 3
An 18 9/12-yr-old woman initially presented at the age of 15 yr with unilateral mucosal neuromas of the tongue and what was felt to be thickened corneal nerves (12+ on an ophthalmalogical exam). No other signs or symptoms suggestive of MTC, pheochromocytoma, or hyperparathyroidism were evident, and no other stigmata of MEN 2B were noted. Family history revealed two healthy siblings and healthy parents and grandparents.
Serial pentagastrin-stimulated calcitonin levels and 24-h urinary catecholamines were within normal limits, as was a serum calcium measurement. Nonetheless, her clinicians felt that this patient had a partially expressed form of MEN 2B at a time when the MEN 2 susceptibility gene was unknown; thus, a prophylactic thyroidectomy was performed. On histopathological examination, no evidence of C cell hyperplasia or tumor nodules was noted.
Direct sequence analysis of this patients DNA subsequently revealed the absence of a germline exon 16 mutation. Specifically, germline RET M918T was absent. In addition, no germline mutations in exons 10, 11, and 13 were noted. Mutational analysis using sequencing and differential allele-specific restriction digestion of DNA extracted from one mucosal neuroma revealed no somatic codon 918 mutation.
Patient 4
This patient presented at age 4 11/12 yr when a biopsy of a small mass at the angles of his mouth revealed neuromas. He was born at a gestational age of 29 weeks with a birth weight of 2 lb, 11 oz. Past medical history was otherwise unremarkable, including no signs or symptoms of MTC or pheochromocytoma, and no other stigmata of classic MEN 2B. Family history was noncontributory.
Serial pentagastrin-stimulated calcitonin levels, 24-h urinary catecholamines and serum PTH levels up to the age of 11 yr were within normal limits. An abdominal computed tomography scan was also normal. Direct sequence analysis revealed the absence of germline mutations in exons 10, 11, 13, and 16. Of note, germline M918T was not found. Mutational analysis using two techniques, sequencing and allele-specific restriction digestion, was negative for a somatic codon 918 mutation in the neuromas.
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Discussion |
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There is less allelic heterogeneity in MEN 2B than in either MEN 2A or familial MTC. A single point mutation, M918T, is present in 95% of patients with MEN 2B (21). A rare minority of phenotypic MEN 2B patients, without clinical distinction, do not carry germline M918T. Furthermore, they have yet to be shown to have any germline RETmutation (21, 29). The International RET Mutation Consortium has begun to address questions regarding the frequency of M918T in classic MEN 2B cases, but has not given information with respect to patients described as having partial MEN 2B-like phenotypes (21, 28). This pilot study, comprising four patients with unilateral mucosal neuromas, begins to address issues related to pure MNS patients that are unanswered by the Consortium. Pure MNS does not appear to be a forme fruste of MEN 2B at the genetic level, nor does it appear to result from mosaicism of the M918T mutation.
Other genes might account for pure MNS. Plausible candidate genes
include those that encode the ligands and coreceptors of RET, GDNF,
GDNF receptor- (10, 11, 12, 13), NTN, and NTN receptor-
(14, 15), as well
as endothelin-3 and its receptor-ß; the latter two are minor
susceptibility loci for Hirschsprung disease (30, 31). Other less
obvious candidate genes could include those that play some role in
neural development or neuroendocrine neoplasia (e.g. the Trk
family, c-MET, and h-ASH). A germline mutation,
germline mosaicism, or a somatic mutation in any of these candidates
might result in MNS or a related entity.
Should a larger series of pure MNS patients confirm these preliminary results, clinicians may be able to distinguish pure MNS from MEN 2B, with all of its implications for patients and their families with regard to surveillance and genetic testing.
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Acknowledgments |
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Footnotes |
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2 Gibb Fellow of the Clinical Research Center.
3 Lawrence and Susan Marx Investigator in Human Cancer Genetics and
supported by a Young Scientist Award from the Markey Charitable Trust
and the Charles A. Dana Foundation.
Received July 11, 1997.
Revised September 10, 1997.
Accepted September 25, 1997.
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References |
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