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A Novel Case of Multiple Endocrine Neoplasia Type 2A Associated with Two de Novo Mutations of the RETProtooncogene¹

Dipartimento di Biochimica e Biotecnologie Mediche e Ceinge, Centro di Ingegneria Genetica, Università degli Studi di Napoli Federico II (A.T., M.C., D.V., V.C.), e Istituto di Endocrinologia, Seconda Università di Napoli (A.A.S., D.P., A.B.), 80131 Naples; and Facoltà di Scienze, Università del Sannio (V.C.), 82100 Benevento, Italy

Address all correspondence and requests for reprints to: Vittorio Colantuoni, M.D., Department of Biochemistry and Medical Biotechnologies, Via Sergio Pansini 5, 80131 Naples I, Italy. E-mail: colantuoni{at}dbbm.unina.it

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We report a novel case of multiple endocrine neoplasia type 2A (MEN 2A) associated with two mutations of the protooncogene RET. One affects codon 634 and causes a cysteine to arginine substitution; the second at codon 640 causes an alanine to glycine substitution in the transmembrane region. The two mutations were present on the same RET allele and were detected in germline and tumor DNA. Both mutations were de novo, i.e. they were not found in the DNA of the parents or relatives. Immunohistochemical and RT-PCR analysis showed that the pheochromocytoma expressed calcitonin as well as both RET alleles. A cell line established from the tumor and propagated in culture sustained the expression of RET and calcitonin, as did the original pheochromocytoma. Because the patient presented with medullary thyroid carcinoma and pheochromocytoma without parathyroid gland involvement, we speculate that this clinical picture could be correlated with the two RET mutations and to the unusual calcitonin production. This is the first report of a MEN 2A case due to two mutations of the RET gene and associated with a calcitonin-producing pheochromocytoma.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer syndrome that presents in three clinical subtypes. MEN 2A is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma (50–60% of cases), and hyperplasia of the parathyroid glands (20–30% of cases). MEN 2B patients are affected by medullary thyroid carcinoma, pheochromocytoma, a marfanoid habitus, and neurogangliomatosis of the intestinal tract, but not parathyroid gland involvement. In familial medullary thyroid carcinoma (FMTC), the medullary thyroid carcinoma is the sole disease phenotype (1, 2). In addition, MTC occurs sporadically in 10% of all thyroid tumors (3).

Specific mutations of the protooncogene RET are associated with each of these disease syndromes (4, 5). The RET protooncogene codes for a membrane tyrosine kinase receptor expressed in cells derived from the neural crest (6, 7, 8). It binds the glial-derived neurotropic factor (GDNF) produced by glial cells through an intermediate glycosylphosphatidylinositol-bound molecule, the GDNF receptor (GDNFR). These three components form a complex that transduces mitogenic signals (9, 10, 11, 12).

Most (95%) MEN 2A mutations have been found in exons 10 and 11, which code for the extracellular domain of the receptor. They are missense mutations affecting one of five codons corresponding to cysteine residues positioned in the juxtamembrane cysteine-rich region (4, 5). FMTC patients either have mutations at the same codons but with different amino acid substitutions or mutations at codons in other exons of the gene. MEN 2B is almost uniquely associated with a mutation at codon 918 in exon 16 (for a review, see Refs. 4, 5).

In in vitro systems, MEN 2A mutations confer a dominant transforming potential to the RET allele (13, 14, 15). It has been argued that the cysteine residues of the extracellular domain of the receptor form intramolecular disulfide bridges and that this peculiar structure interacts with similar structural domains present in GDNFR and GDNF. In the mutant receptor, the unpaired cysteine residues form intermolecular disulfide bridges. This causes conformational changes and dimerization of the receptor, followed by activation of its tyrosine-kinase domain and phosphorylation of intracellular substrates. This is believed to be the initial event in stimulating unrestrained growth of C cells. All MEN 2A cases reported to date have this activating mechanism (14).

Here we describe a novel case of MEN 2A caused by two mutations of the RET gene: a C to T transition at position 634 that causes a cysteine to arginine substitution, and a C to G transversion at codon 640 that causes an alanine to a glycine substitution in exon 11. Interestingly, both mutations lie on the same allele, are de novo mutations, and are not correlated with an earlier onset and a more aggressive clinical course, as occurs in classical MEN 2A. The pheochromocytoma tumor expressed both RET alleles and calcitonin. From the tumor tissue we established a primary cell line that had the characteristics of chromaffin cells and propagated it in culture. The cells retained the capacity to synthesize RET and calcitonin. This is the first report of MEN 2A associated with two RET alterations and calcitonin production by the pheochromocytoma.

	Subjects and Methods

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Case report

A 26-yr-old female presented at the Institute of Endocrinology of the Second University of Naples with headache and paroxysmal hypertension that had started 2 months earlier. Six months previously she had been diagnosed with MTC and subjected to total thyroidectomy and removal of all lymph nodes of the neck. Since surgery she had been receiving L-T₄ suppressive therapy. The family history was negative for relevant thyroid disorders and hypertension. Clinical examination was negative, except for paroxysmal headaches and hypertension (two episodes during the admission period). Biochemical evaluation revealed elevated levels of plasma calcitonin and increased excretion of urinary catecholamines in samples collected over a 24-h period during the spells (Table 1). Ultrasound scan showed no residual thyroid tissue or lymph nodes in the neck, but a mass was detected at the upper pole of the left kidney. Computed tomography confirmed the absence of residual thyroid tissue and nodes in the neck and upper region of the chest and the presence of a large abdominal mass with some necrotic cystic tissue at the level of the left adrenal gland. Metaiodobenzyl-guanidine scanning showed high uptake in the left and modest uptake in the right adrenal region, confirming the presence of a pheochromocytoma on the left side and suggesting concomitant hyperplasia on the right. No uptake was detected in other districts, including the neck. These findings confirmed the diagnosis of MEN 2A with a clinical picture characterized by MTC and pheochromocytoma. An absence of clinical signs, negative computed tomography scan, and normal serum Ca²⁺, phosphate, and PTH values indicated that the parathyroid glands were unaffected. The left adrenal tumor was removed by surgery. Direct palpation and intraoperative ultrasound scan excluded the presence of a mass in the right adrenal gland. Histological examination confirmed the preoperative diagnosis of pheochromocytoma with some areas of colliquative tissue. During a 6-month follow-up, the patient did not experience headache or hypertension. Urinary catecholamines and plasma calcitonin levels were in the normal range 1 and 3 months after surgery (Table 1). Total body computed tomography and metaiodobenzyl-guanidine scintigraphy showed only modest uptake at the right adrenal gland because of persistent hyperplasia.

Genomic DNA was prepared from white blood cells according to standard protocols (16). Oligonucleotide primers to amplify different RET exons were designed on the intronic sequences flanking exons 10 and 11, as previously described (17). PCR reactions were run in a final volume of 50 µL using 100 or 200 ng genomic DNA as reported previously (17). An aliquot of the PCR product was digested with the restriction enzymes HhaI, RsaI, and DdeI at 37 C for 3 h; the product was examined on a 2.5% agarose gel; and the bands visualized by ethidium bromide staining. The presence of the mutation was confirmed by direct sequencing of the PCR product using the Sanger method in an automated sequencer, according to the manufacturer’s instructions (ABI 373A Applied Biosystem Division, Perkin Elmer Corp., Norwalk, CT).

To establish whether the parental alleles were involved in the transmission of the disease, we haplotyped the RET locus using four different intragenic polymorphic markers (18). To prove or exclude paternity, four multiallelic polymorphic loci, D11S905, D12S79, D14S280, and D16S422, were used (19).

Ribonucleic acid (RNA) extraction, RT-PCR, and restriction enzyme digestion

Total RNA extracted from tumor tissue with the QIAGEN system (RNeasy mini kit-50, Chatsworth, CA) or from primary cells in culture with Trizol (Life Technologies, Inc., Milan, Italy) was analyzed by electrophoresis to assess its quality and quantity. One microgram of total RNA was reverse transcribed with SuperScript (Life Technologies, Inc.) in a 20-µL reaction volume with random primers. Complementary DNA (cDNA) was amplified using as primers the following oligonucleotides (10F, 5'-GGATTAAAGCTGGCTATGGCA-3'; 11R, 5'-GGAGTAGCTGACCGGAA-3') encompassing exons 10 and 11, corresponding to nucleotides 1952–1972 and 2197–2214 of the RET gene, respectively (6). The band of 262 bp gave rise to segments of 145-, 98-, and 19-bp fragments after digestion with the enzyme HhaI. To check for calcitonin expression, two oligonucleotides corresponding to nucleotides 1861–1881 (CTF, 5'-ATGAAGGCCAGTGAGCTGGA-3') and nucleotides 2140 -2160 (CTR, 5'-AAGGAAAGGGAGGAGTTTAG-3') were synthesized (20). cDNA was synthesized as described above.

Cell culture

Adrenal tissue was minced, and cells were mechanically dispersed. Subsequently, they were seeded directly on plastic culture dishes and incubated with RPMI 1640 supplemented with L-glutamine, 10% (vol/vol) FBS, insulin, and antibiotics (Life Technologies, Inc.) in a humidified incubator (37 C, 5% C0₂). The primary cell cultures were identified as neuroendocrine cells using polyclonal antibodies against chromogranin A and calcitonin. The conditioned medium was used for calcitonin determination by a specific RIA. Total RNA was isolated from the cultures at the first passage, as described above.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The diagnosis of MEN 2A, established from clinical and biochemical parameters, was confirmed by testing the patient’s DNA for RET mutations. High mol wt DNA was extracted from peripheral blood cells, and RET exon 11 was PCR amplified using specific primers. The presence of mutations was detected by digesting the PCR product with HhaI, RsaI, and DdeI restriction enzymes; the only informative digestion was that with HhaI (Fig. 1A). A new site for this enzyme is, in fact, generated by the Cys⁶³⁴Arg mutation; as a consequence, the amplified band of 284 bp is digested in two lower mol wt bands of 164 and 120 bp. Surprisingly, the 164-bp band was absent, and a lower mol wt fragment of about 140 bp appeared, suggesting the presence of an additional restriction site (Fig. 1A, lane 1). The 284-bp band, corresponding to the nonmutated allele, was still detectable, indicating that the mutation was present in a heterozygous state. This unusual digestion product was specific, because the DNA from an unrelated MEN 2A patient, carrying the same Cys⁶³⁴Arg mutation, and DNA from a normal individual, tested in parallel, generated the expected profiles (Fig. 1A, lanes 2 and 3). The MEN 2A pattern revealed the undigested PCR product and two bands of the expected lower mol wt; a single band corresponding to the undigested product appeared in the DNA from the normal individual.

View larger version (60K): [in this window] [in a new window]   Figure 1. The patient’s DNA bears two mutations of the RET protooncogene. A, High mol wt DNA was extracted from the peripheral blood of the propositus (lanes 1, 4, and 6), an unrelated MEN 2A patient (lane 2), and a normal individual (lanes 3, 5, and 7). RET exon 11 was amplified by PCR, and aliquots of the products were digested with the restriction enzymes HhaI, RsaI, and DdeI as indicated. Lane M illustrates the migration of the molecular size marker; the positions of the 396- and 214-bp bands are indicated. The RET sequence encompassing codon 634 is illustrated together with the mutated sequences recognized by the restriction enzymes used. B, RET exon 11 amplified product was directly sequenced in an automated sequencer. Both strands were sequenced, using as primers the oligonucleotides used for PCR. The profile of the sequence relative to the region of interest is illustrated. Only in two positions are two overlapping peaks, corresponding to a C and a T at codon 634 and a C and a G at codon 640, respectively, indicative of the presence of two different nucleotides at that position in the sequence.

The genetic analysis was carried out also on the DNA from the primary MTC and from the pheochromocytoma (Fig. 2A, lanes 2 and 3). Both DNA samples carried the RET mutations identified in the germline DNA (lane 4), supporting the hereditary nature of both mutations.

View larger version (52K): [in this window] [in a new window]   Figure 2. The RET mutations were present also in the tumor DNA and were de novo mutations. A, The DNA extracted from the primary MTC (lane 2) and from the pheochromocytoma (lane 3) was analyzed for the presence of RET mutations by PCR and HhaI restriction enzyme digestion. Germline DNA from the same individual (lane 4) and DNA from an unrelated MEN 2A patient (lane 1) were used as controls. Lane M illustrates the migration of the molecular size marker; positions of the 396- and 214-bp bands are indicated. B, Germline DNA from the three sisters, one brother, and the two parents (lanes 3–8) were tested for the presence of the two mutations detected in the propositus DNA (lane 2). The DNA from an unrelated MEN 2A patient was used as control. M indicates the migration of the molecular size marker.

To assess whether the mutations were de novo mutations, family screening was performed on all available relatives: three sisters, one brother, and both parents. No clinical symptoms suggestive of MEN 2A were present in any of the relatives tested. Constitutive DNA amplified for RET exon 11 did not bear any mutation (Fig. 2B, lanes 3–6). The patient had no children. The parents were both alive and did not present any sign related to MEN 2A, and the DNA test for RET mutations showed no mutations (Fig. 2B, lanes 7 and 8). To establish which parental allele was involved in the disease, we haplotyped the RET locus, using some of the intragenic polymorphic sites described previously (18). None of them was informative, as both parents were homozygous for the most frequent alleles (data not shown). We, then, used other multiallelic polymorphic microsatellites distributed on different chromosomes and employed for testing cases of uncertain paternity (19). Four of them were informative, indicating that the patient had inherited alleles from the mother and the father, ruling out the possibility of nonpaternity (data not shown). These results confirmed that the mutations were de novo.

The pheochromocytoma tissue was analyzed by immunohistochemistry. A large portion of the tissue was compact in structure and hard in consistency, whereas the rest was mainly formed by colliquative tissue. Both sections were positive for chromogranin A, a specific marker of neuroendocrine cells, and for calcitonin, with a diffuse staining in several serial sections (data not shown). To confirm this result and to verify the expression of the RET double mutant, total RNA was isolated from the tumor tissue and reverse transcribed. The cDNA obtained was subsequently PCR amplified using as primers two oligonucleotides corresponding to exons 3 and 4 of the calcitonin gene (20) and two encompassing exons 10 and 11 of the RET gene (8). Bands of 262 and 252 bp were obtained for the RET and calcitonin genes, respectively, (Fig. 3, lanes 1 and 4), demonstrating that both genes were indeed transcribed. No amplification was obtained in the absence of the reverse transcriptase (lanes 3 and 6), indicating that the bands were specific and not due to DNA contamination. The RET-amplified product was subsequently digested with the HhaI enzyme, and the profile demonstrated that both the allele bearing the two mutations and the wild-type allele were equally transcribed (lane 8). The RT-PCR product obtained from the RNA of a classical MEN 2A patient was HhaI digested as a control (lane 7).

View larger version (63K): [in this window] [in a new window]   Figure 3. The pheochromocytoma and a cell line express both RET alleles and calcitonin. Total RNA was extracted from the pheochromocytoma and from the cells derived from it, reverse transcribed, and PCR amplified with oligonucleotides specific for RET (lanes 1 and 2) and calcitonin, respectively (lanes 4 and 5). The reaction without RT was also carried out to exclude DNA contamination (lanes 3 and 6). The RET RT-PCR products were digested with the enzyme HhaI and analyzed on a 2.5% agarose gel and visualized by ethidium bromide staining (lanes 8 and 9). The RNA from a classical MEN 2A case was investigated under the same experimental conditions and used as control (lane 7). M indicates the migration of the molecular size marker.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We report the identification of a case of MEN 2A associated with two mutations of the RET gene: one at codon 634 and a second at codon 640. Missense mutations at codon 634 coding for one of five cysteine residues of the extracellular part of the protein are frequent in MEN 2A; Cys⁶³⁴Arg, in particular, is the one most frequently associated with this syndrome (4, 5). Duplications of 9 and 12 nucleotides have been described in a few instances (21, 22). In all cases the same pathogenetic mechanism underlies this tumor syndrome: the cysteine residues in the receptor extracellular domain become uneven, allowing dimerization and constitutive, ligand-independent activation of RET (13, 14).

The mutation at codon 640 corresponds to an amino acid residue of the transmembrane region (8), and this is the first correlation of this type of mutation with MEN 2 syndromes. The base pair change may affect the activity of the receptor and contribute to the clinical phenotype. The amino acid change, in fact, involves the third alanine of a triad of alanine residues in the context of the transmembrane tract. The replacement with a glycine residue may reduce the hydrophobicity of the region, induce a conformational change, and consequently allow the formation of two additional hydrogen bonds for interactions with other receptors or distinct membrane proteins.

Mutations involving amino acids of the transmembrane domain have been described for other tyrosine kinase receptors. The neu oncogene is frequently activated by point mutations in the transmembrane region, and this alteration correlates with increased tyrosine kinase activity of its gene product (23). Activation occurs only when the original valine is replaced by glutamic acid or glutamine residues, suggesting that a potentially charged amino acid per se cannot activate the Neu protein. The mutated protein may increase or decrease interactions with other receptor molecules, necessary for its activation. Alternatively, the Neu protein might interact with a second distinct protein or other molecules endowed with a transmembrane domain that regulate its kinase activity (24). We cannot say whether similar or different mechanisms operate in the case of the RET mutations described here, and more experiments are required to address this question.

The 640 mutation may also affect the 634 mutation phenotype. The available clinical data do not support a correlation with a more aggressive phenotype; however, this possibility cannot be excluded, because only one patient was examined, and no descendents are available. Experiments in in vitro systems transfecting a RET cDNA carrying only the Ala⁶⁴⁰Gly mutation and analyzing the tyrosine kinase activity in the absence of the ligand will shed some light on the transformation potential associated with this mutation. These experiments are underway.

In classical MEN 2A cases, the specific Cys⁶³⁴Arg mutation is frequently associated with parathyroid gland involvement (4, 5). As our patient’s parathyroid glands were unaffected, this particular RET double mutant might cause early involvement of the adrenal medulla without effects on the parathyroid glands. It appears that the double mutant has a greater effect on the adrenal medullary chromaffin cells than any other known RET mutation.

Calcitonin production by the pheochromocytoma is another intriguing feature of this MEN 2A case. Calcitonin synthesis has been reported in 44% and 21% of pheochromocytomas in two separate series (25, 26). Provided that none of the cases reported in these series is familial, our report represents the first instance of a calcitonin-secreting pheochromocytoma associated with a MEN 2A syndrome. Elevated plasma calcitonin levels in a MEN 2A patient are indicative of the presence of MTC; persistent high values after total thyroidectomy may be due to metastases in regional nodes or distant organs. As we ruled out the presence of residual thyroid tissue, metastasized nodes and tissues, the elevated values resulted exclusively from the pheochromocytoma. Thus, the chromaffin cells of the adrenal medulla can synthesize and release authentic calcitonin. Consequently, physicians should be alert to the fact that elevated plasma calcitonin levels are not necessarily diagnostic of MTC and in patients with pheochromocytoma do not necessarily indicate the presence of underlying MTC or MTC recurrence. In MEN 2A patients, enhanced calcitonin could indicate an unrecognized hyperplasia or a medullary gland tumor.

In conclusion, we described a unique case of MEN 2A with two de novo mutations of the RET gene associated with a peculiar clinical picture that includes unexpected calcitonin production by the pheochromocytoma and the absence of parathyroid gland involvement.

	Acknowledgments

 
We thank Jean Gilder for editing the text.

	Footnotes

 
¹ This work was supported by grants from MURST PRIN ’97 (to V.C.) and the Associazione Italiana per la Ricerca sul Cancro (to A.A.S.).

Received February 1, 1999.

Revised May 17, 1999.

Accepted June 29, 1999.

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