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Departments of Clinical Genetics and Human Genetics (F.H.M.), Pathology (P.J.v.D.), and Internal Medicine (A.W.F.T.T.), Vrije Universiteit Medical Center, 1007 MB Amsterdam, The Netherlands; Department of Internal Medicine (J.M.S.), Medical Center Alkmaar, Alkmaar, The Netherlands; and Departments of Medical Genetics (R.B.v.d.L., I.A.J.d.V.) and Internal Medicine (C.J.M.L.), University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands
Address all correspondence and requests for reprints to: Fred H. Menko, M.D., Ph.D., Department of Clinical Genetics and Human Genetics, Vrije Universiteit Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: fh.menko.humgen{at}med.vu.nl
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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MEN 2B (MIM 162300) is characterized primarily by early onset and aggressive MTC and a typical facial appearance caused by mucosal neurilemmomas (often called ganglioneuromas). Pheochromocytoma develops in about 50% of the patients. Hyperplasia of parathyroid tissue may occur, but clinical hyperparathyroidism is absent. Many MEN 2B patients have severe intestinal and skeletal abnormalities. However, the clinical picture is variable and includes milder phenotypes (1, 2, 3).
In about 95% of the MEN 2B cases, a specific missense mutation of the RET proto-oncogene, M918T, has been detected. Other MEN 2B families had either no detectable RET mutation or the A883F missense alteration. Mutations are de novo in about 50% of the cases (4). Recently, a MEN 2B patient with two RET alterations, V804M and Y806C, was reported. Functional tests suggested that the effect of this double mutation was similar to that of the classical M918T defect (5, 6).
We investigated a kindred in which multiple family members had MTC and mucosal neurilemmomas. Affected individuals carried two germline RET mutations, Val804Met and Ser904Cys, on the same allele. The clinical expression of the syndrome, the expected functional effect of the RET mutations and the implications of our findings for diagnosis and management are discussed.
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Patients and Methods |
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The pedigree of the family is depicted in Fig. 1
. The index patient (pedigree no. II-4; Fig. 2A) had noticed prominent lips from a young age onward. He underwent biopsy examination of tongue lesions at the age of 34 yr. Histologically, several nodular proliferations were diagnosed as multiple neurilemmomas (Schwannomas) (Fig. 2B). Subsequently, the patient was investigated for possible MEN 2B. A pentagastrin test and magnetic resonance imaging of the thyroid gland indicated MTC. Thyroidectomy and central neck dissection were performed. The tumor was a multifocal medullary carcinoma accompanied by C-cell hyperplasia; regional lymph nodes were negative. An elder sister of the index patient (pedigree no. II-3; Fig. 2C) had had right hemithyroidectomy for MTC at the age of 18 yr. At age 39 yr, medullary cancer in her remaining thyroid was diagnosed, and left hemistrumectomy and central neck dissection were performed (Fig. 2D). Regional lymph nodes were negative. The youngest sister (pedigree no. II-5) noticed a tumor in the left neck at the age of 34 yr that proved to be MTC with one positive regional lymph node. In addition, she had hypertension without evidence for pheochromocytoma.
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). Postoperatively, serum calcium values returned to normal. At a later stage, increased urinary cathecholamines prompted 131I-metaiodobenzylguanidine and computed tomography scanning of the abdomen. A left adrenal adenoma was suggested; however, a definite diagnosis of pheochromocytoma was not made. Mutation analysis of the RET gene
For mutation analysis of the RET gene in affected family members and clinically unaffected first-degree relatives, genomic DNA was isolated from 10-ml peripheral blood samples according to standard procedures. Initially, the presence of the M918T and A883F mutations in exons 16 and 15, respectively, was investigated by mutation-specific assays on the basis of digestion of PCR products using the restriction endonucleases FokI (M918T) and DdeI (A883F). Mutation screening of exons 10, 11, 13, 14, 15, and 16 of the RET gene was performed by direct sequencing using an ABI Prism 3700 DNA Analyser (Applied Biosystems, Foster City, CA) with the Sequence Analysis 3.1 software (Applied Biosystems).
Exons 10, 11, 13, 14, 15, and 16 of the RET gene were amplified by PCR using the oligonucleotide primers listed in Table 1. PCRs were performed in a GeneAmp 9600 PCR System (Applied Biosystems) in a 50-µl volume containing 100 ng genomic DNA as template, 50 ng of each oligonucleotide primer, 200 µM of each dNTP, and 1 U AmpliTaq DNA polymerase (Applied Biosystems) in 1x Pol buffer [67 mM Tris (pH 8.8), 6.7 mM MgCl2, 10 mM ß-mercaptoethanol, 6.7 µM EDTA (pH 8.0), and 16.6 mM (NH4)2SO4]. An initial denaturation for 4 min at 94 C was followed by 33 cycles of 1 min at 94 C, 1 min at the appropriate annealing temperature (Table 1), and 2 min at 72 C. Sequencing was performed on an ABI Prism 3700 DNA Sequencer (Applied Biosystems), using either the forward or the reverse oligonucleotide of each primer pair as sequencing primer.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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. Marfanoid habitus was absent in the index patient and his affected sisters (length of index patient, 1.80 m; arm span to height ratio, 1.01; upper segment to lower segment ratio, 0.94). Primary hyperparathyroidism was diagnosed in patient I-2 on the basis of histological features (Fig. 2E) and immunohistochemically assessed cyclin D1 overexpression (7).
Previously, the MEN 2B mutations M918T and A883F had been excluded in the proband. DNA sequence analysis of exons 16, 10, 11, and 13 of the RET proto-oncogene showed no disease-causing mutations. Subsequent examination of exons 14 and 15 by DNA sequence analysis, however, revealed a Val804Met (GTG-to-ATG) mutation in exon 14 and, in addition, a Ser904Cys (TCC-to-TGC) mutation in exon 15 in the index patient and his two affected siblings (Fig. 3). The same abnormalities were detected in a sample taken previously from the mother of these patients. In contrast, the two clinically unaffected siblings of the index patient (pedigree no. II-1 and no. II-2) did not carry either RET variant. One child of patient II-3 had the abnormal facial phenotype and carried the double RET mutation. Six other clinically unaffected children of patients II-3, II-4, and II-5 did not carry either RET variant. Because the combination of germline variants was found to cosegregate with the disease, the V804M and S904C mutations must be located on the same allele of the RET gene.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We identified a kindred with MTC and mucosal neurilemmomas caused by a double mutation in the RET gene not involving codon 918. MTC and neurilemmomas were manifest in all affected family members. However, severe skeletal and intestinal abnormalities were absent. One of the mutation carriers (pedigree no. I-2) had primary hyperparathyroidism, a condition that is usually absent in MEN 2B (2).
Strong correlations have been found between MEN 2 disease phenotypes and specific RET mutations (8). The V804M mutation in the family that we studied was found previously in FMTC kindreds. In these families, marked variability of expression was seen: MTC with metastasis in young family members, and apparent absence of MTC in older mutation carriers (9, 10, 11). Recently, Miyauchi et al. (5) described a MEN 2B patient with a de novo V804M mutation combined with an inherited Y806C mutation. The Y806C variant was also carried by the patient’s father, who was asymptomatic. The transforming activity of RET with both mutations was about 8- to 13-fold higher than that of RET with a single V804M or Y806C mutation. In addition, it appeared that the biological properties of the V804M/Y806C mutation were similar to those of the classic MEN 2B mutation M918T and the A883F mutation associated with the same phenotype (6).
We identified a novel combination of RET missense alterations in affected family members. Whereas the V804M missense mutation has already been described, S904C represents a novel RET variant. The clinical significance of the S904C amino acid substitution is unknown. Both amino acid changes, V804M and S904C, are located within a functionally important region of the RET protein, i.e. the split kinase domain. The V804M mutation is located within the proximal half of the tyrosine kinase domain, within subdomain V of unknown function (12). The mechanism by which the RET protein is affected by the V804M mutation is not known. The S904C mutation is located in the distal half of the split kinase domain, within the highly conserved activation loop of the RET receptor tyrosine kinase. Mutations in the activation loop of other receptor tyrosine kinases, such as c-kit and FGFR3, have been demonstrated to cause ligand-independent activation. Such mutations are hypothesized to affect conformation of the activation loop. Consequently, the dimerization-induced phosphorylation of tyrosine residues in this region (Y905 in RET) is no longer required for activation (13).
Given the location of the mutations, it is unlikely that they will cause constitutive activation of the tyrosine kinase through the formation of disulfide-bonded RET homodimers, such as the MEN 2A type mutations. Probably, the V804M/S904C double mutation produces oncogenic activity through changes in catalytic activity and substrate specificity, analogous to the M918T mutation (1). However, in vitro transformation studies will be required to determine the functional effect of the V804M/S904C mutation.
Our study is the second report of MEN 2B caused by a combination of two missense changes on the same allele of the RET gene. Whereas the report by Miyauchi et al. (5) describes a single MEN 2B patient in whom two RET mutations occurred, in the family reported here several individuals were found to carry a novel combination of RET mutations.
Compared with the M918T and A883F mutations, the presence of multiple RET mutations in the same allele is a rare phenomenon in MEN 2B. In addition to MEN 2B, other diseases associated with germline RET mutations may also be caused by double germline alterations in the same allele; Bartsch et al. (14) identified a double mutation (V804M and R844L) in exon 14 of the RET gene in a FMTC kindred. These findings underscore the importance of investigating additional exons of the RET gene in patients with MEN 2B without M918T or A883F mutations.
In kindreds with the clinical phenotype of MEN 2B, preventive thyroidectomy at a very young age is generally advocated (15). It has been proposed that the age of surgery for MEN 2A and FMTC patients should be based upon the underlying genotype (8).
Apparently, MEN 2B also displays both clinical and genetic heterogeneity. Further insight into this problem may help to determine clinical management for individual MEN 2B families.
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Acknowledgments |
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Footnotes |
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Received January 31, 2001.
Accepted September 25, 2001.
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