Four Cases of Mucosal Neuroma Syndrome: Multiple Endocrine Neoplasm 2B or Not 2B?1

Catherine M. Gordon, Joseph A. Majzoub, Debbie J. Marsh, John B. Mulliken, Bruce A. J. Ponder2, Bruce G. Robinson and Charis Eng3

Divisions of Endocrinology (C.M.G., J.A.M.), Adolescent/Young Adult Medicine (C.M.G.), and Plastic Surgery (J.B.M.), Children’s 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, Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115.


    Introduction
 Top
 Introduction
 Materials and Methods
 Case Reports and Results
 Discussion
 References
 
The multiple endocrine neoplasia (MEN) type 2 syndromes are disorders involving the neural crest and its derivatives (1, 2) and encompass three clinically related autosomal dominant cancer syndromes (2). MEN 2A is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma in about 50% of cases, and parathyroid hyperplasia or adenoma in 10–35% of patients (3, 4). MEN 2B is similar to MEN 2A, but is distinguished by earlier age of tumor onset and by developmental abnormalities, including mucosal neuromas, intestinal ganglioneuromatosis, unusual facies, and a marfanoid habitus (5). Hypertrophy of corneal nerves, observed with slit-lamp examination, can be helpful in establishing the diagnosis, but is not diagnostic (6). Parathyroid abnormalities in MEN 2B are infrequent and usually subclinical (7).

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-{alpha}, NTNR{alpha}/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.


    Materials and Methods
 Top
 Introduction
 Materials and Methods
 Case Reports and Results
 Discussion
 References
 
Tissue origin

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 2–4).

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 8–16 h with an excess of the restriction endonuclease RsaI under the manufacturer’s 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 (254–312 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 Denhardt’s 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 {alpha}-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 Denhardt’s 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.


    Case Reports and Results
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 Introduction
 Materials and Methods
 Case Reports and Results
 Discussion
 References
 
Patient 1

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 patient’s father, a paternal uncle, and a paternal aunt. A paternal aunt had hypothyroidism; otherwise, there were no endocrinopathies in the family. The patient’s mother underwent a mastectomy for breast cancer at age 48 yr and was in remission at the patient’s 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. 1AGo), with neuromas along the palpebral fissures, nasal alae, buccal mucosa (Fig. 1BGo), 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 25–50th 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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Figure 1. At evaluation at age 15 yr, 11 months, the patient had left-sided facial fullness (A). with neuromas along the palpebral fissures, nasal alae, buccal mucosa (B), left anterior tongue and both upper and lower lips, in the distribution of the three divisions of the left trigeminal nerve.

 
The patient underwent several years of laboratory screening tests for MTC and pheochromocytoma. The workup included annual calcitonin measurements after pentagastrin stimulation since the age of 4 yr; all were within normal limits. Twenty-four-hour urinary total and fractionated catecholamine, metanephrine, and vanillylmandelic acid levels were normal at 16 and 17 yr of age. At age 8 yr, an ophthalmologic slit-lamp examination revealed (1+) medullated corneal nerve fibers, and Lisch nodules were not seen. At age 16 yr, mutational analysis of DNA from peripheral leukocytes, performed in two laboratories [Oncormed (Philadelphia, PA) and Dr. R. F. Gagel’s laboratory, University of Texas (Houston, TX)] was negative for a germline codon 918 mutation. Buccal mucosa cells were also screened simultaneously (Oncormed), but the mutation was not found.

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. 2Go). However, as shown in Fig. 2Go, 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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Figure 2. 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. However, there was no evidence for this mutation in any of the mucosal tumors from the patient.

 
Patient 2

A 12 5/12-yr-old boy was referred for endocrinological consultation after surgical removal of two mucosal neuromas of the tongue. The patient’s 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 patient’s 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 (1–2+ 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 patient’s 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.


    Discussion
 Top
 Introduction
 Materials and Methods
 Case Reports and Results
 Discussion
 References
 
This is the first case series to report results of RETmutation analysis of patients with pure MNS. All four subjects had mucosal neuromas presenting as unilateral lesions and did not have the germline RET codon 918 mutation that is associated with approximately 95% (21) of patients with MEN 2B. As all four patients were negative for a germline mutation, it was hypothesized that the phenotype in these patients was a consequence of either a somatic RET M918T mutation restricted to mucosal nerves or germline mosaicism. PCR-based mutational analysis of the sampled neuromas was also negative in all four patients. Based on this study, we cautiously extrapolate that most, if not all, patients with pure unilateral MNS do not have a germline codon 918 mutation. Furthermore, neuromas in these patients do not appear to be the result of a somatic M918T mutational event.

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-{alpha} (10, 11, 12, 13), NTN, and NTN receptor-{alpha} (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.


    Acknowledgments
 
The authors gratefully acknowledge Dr. Robert F. Gagel for helpful discussions regarding the diagnosis and management of one of these patients, and Dr. S. Jean Emans for her critical review of the final manuscript.


    Footnotes
 
1 This work was supported by the Clinical Investigator Program: Harvard/MIT Health Sciences and Technology-Beth Israel Deaconess Medical Center, in collaboration with Pfizer Inc, and Program Grant MCJ-MA 259195 from the Maternal and Child Health Bureau (to C.M.G.), the Cancer Research Campaign (to C.R.C.), a Dana-Farber fellowship (to C.E.), a program grant from the Clinical Research Center (to B.A.J.P.), the University of Sydney Cancer Research Fund (to D.J.M.), and NIH Training Grant 5T32-DK-07699–15 (to J.A.M.). Back

2 Gibb Fellow of the Clinical Research Center. Back

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. Back

Received July 11, 1997.

Revised September 10, 1997.

Accepted September 25, 1997.


    References
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 Introduction
 Materials and Methods
 Case Reports and Results
 Discussion
 References
 

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