MEDULLARY THYROID CARCINOMA (MTC) can be

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1 X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(11): Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: /jc Identification of a Novel Point Mutation in the RET Gene (Ala883Thr), Which Is Associated with Medullary Thyroid Carcinoma Phenotype Only in Homozygous Condition ROSSELLA ELISEI, BARBARA COSCI, CRISTINA ROMEI, LAURA AGATE, PAMELA PIAMPIANI, PAOLO MICCOLI, PIERO BERTI, FULVIO BASOLO, CLARA UGOLINI, RAFFAELE CIAMPI, YURI NIKIFOROV, AND ALDO PINCHERA Departments of Endocrinology and Metabolism (R.E., B.C., C.R., L.A., P.P., A.P.), Oncology (F.B., C.U.), and Surgery (P.M., P.B.), University of Pisa, Pisa, Italy; Department of Pathology (R.C., Y.N.), University of Cincinnati, Cincinnati, Ohio 45267; and AMBISEN Center, High Technology Center for the Study of the Environmental Damage of the Endocrine and Nervous Systems (A.P.), University of Pisa, Pisa, Italy The RET protooncogene mutations responsible for multiple endocrine neoplasia type 2 are inherited as autosomic dominant traits. We describe here a novel germline homozygous mutation in exon 15 of the RET gene that determines an amino acid substitution (Ala->Thr) at codon 883. The index case was a 51-yr-old patient with an apparently sporadic form of medullary thyroid cancer (MTC). RET gene mutations screening was performed in exons 10, 11, 13, 14, 15, and 16 by automatic sequence analysis. An unexpected homozygous GCT->ACT point mutation was found at codon 883 in exon 15 and confirmed by restriction analysis (Alu I). The presence of the two chromosomes 10 was confirmed by fluorescence in situ hybridization analysis on lymphocytes. As expected on the basis of the homozygosity of the index case, the parents were consanguineous (second-degree cousins). Eight relatives were further investigated: the mother, two sisters, and the son were positive for heterozygous RET mutation. The mother (82 yr old) showed a nodular goiter but was negative both for basal and pentagastrin stimulated calcitonin. The young son (15 yr old) and the two sisters (63 and 58 yr old) did not show any MEDULLARY THYROID CARCINOMA (MTC) can be sporadic (75%) or familial (25%). The familial form is an autosomal dominant inherited disease characterized by the presence of MTC and/or its precursor lesion, C cell hyperplasia (CCH), plus other endocrine tumors such as pheochromocytoma and/or parathyroid adenomas in multiple endocrine neoplasia 2A, pheochromocytoma, and mucosal neurinomas in MEN 2B. In these syndromes, MTC develops in nearly 100% of the affected individuals, whereas pheochromocytoma and parathyroid adenoma develop in 50 and 20%, respectively (1, 2). MTC alone, lacking the association with other endocrine tumors, may also be inherited (FMTC). Abbreviations: CCH, C cell hyperplasia; Ct, calcitonin; FISH, fluorescence in situ hybridization; FMTC, familial MTC; LOH, loss of heterozygosity; MEN, multiple endocrine neoplasia; MTC, medullary thyroid cancer; Pg, pentagastrin. JCEM is published monthly by The Endocrine Society ( endo-society.org), the foremost professional society serving the endocrine community. clinical and/or biochemical sign of MTC. One brother (59 yr old) was negative both for RET mutation and clinical/biochemical examination. The other brother, 56 yr of age, was positive for both homozygous RET mutation and serum calcitonin. When operated, the histological examination of the thyroid showed the presence of MTC and C cell hyperplasia. In conclusion, we identified a new germline RET gene mutation during a routine RET gene screening of an apparently sporadic MTC case. This mutation showed a very low transforming activity as demonstrated by the absence of MTC phenotype in heterozygous subjects. The possibility that the homozygous gene carriers were indeed carrying a germline loss of heterozygosity was excluded by fluorescence in situ hybridization analysis for RET gene performed on lymphocytes derived from one homozygous patient. The analysis of several RET polymorphisms also confirmed the presence of two mutated alleles in MTC affected patients and both mutated and wild-type allele in heterozygous subjects. (J Clin Endocrinol Metab 89: , 2004) In 1993 two independent groups discovered germline point mutations of the RET protooncogene as a causative event in MEN 2A (3) and FMTC (4). One year later, MEN 2B was also found to be associated with germline RET protooncogene mutations (5). Data regarding RET genetic analysis of a large series of 477 MEN 2-affected families were collected by the International RET Consortium demonstrating that 95% of families with MEN 2B and MEN 2A and 88% of families with FMTC were harboring a heterozygous RET germline mutation (6). After the first pool of RET point mutation was identified, several other mutations have been described and the frequency of RET mutations raised to 96 and 100% according to different series (7, 8). As for sporadic MTC, somatic point mutations of RET gene have been reported in approximately 40% of sporadic MTC (9, 10). These mutations are almost invariably located in exon 16 (codon 918) and can rarely be found in different exons (11, 12). In this paper we report a novel germline homozygous mutation of the RET gene, found by the routine genetic screening for RET mutations in a patient with an apparently 5823

2 5824 J Clin Endocrinol Metab, November 2004, 89(11): Elisei et al. Homozygous New Germline RET Mutation sporadic MTC. Eight family members were also studied for the presence of the mutation and its relationship with the MTC phenotype. Fluorescence in situ hybridization (FISH) analysis for RET gene was performed on the lymphocytes of one homozygous affected subject to rule out the possibility of the presence of a germline loss of heterozygosity (LOH). Index case Subjects and Methods A 51-yr-old man was referred to our clinic for the presence of a thyroid nodule with a calcitonin (Ct)-positive immunocytochemistry. At the first evaluation, basal and pentagastrin (Pg)-stimulated Ct levels were 183 and 2853 pg/ml, respectively. The patient was submitted to a further clinical evaluation with a neck ultrasound that showed a thyroid volume of 20 ml and a small hypoechoic nodule of mm in the right lobe. Negative abdomen ultrasound and normal level of plasmatic and urinary epinephrine and norepinephrine excluded the presence of a pheochromocytoma. The normal levels of serum PTH and calcium demonstrated the absence of hyperparathyroidism. No other cases of MTC were present in the family history. The patient underwent total thyroidectomy and central neck dissection. Histological examination showed the presence of one MTC foci of 12 mm in the right lobe and one MTC microfoci of 0.3 mm in the left lobe. No lymph node metastases have been found. As routinely performed in all MTC patients, the index case was submitted to RET mutation genetic screening, which revealed a germline point mutation. The result of the genetic screening determined a reclassification of our apparently sporadic case as hereditary case. Family members As shown in Fig. 1 a total of eight family members were investigated. All of them were examined with neck ultrasound, basal and Pg-stimulated serum Ct, thyroid function (free T 3, free T 4, TSH, thyroglobulin antibodies, thyroperoxidase antibodies), serum PTH and calcium, plasmatic and urinary epinephrine and norepinephrine, and abdomen ultrasound. After signing an informed consent, they were also submitted to the genetic analysis of RET gene mutations. The clinical history of the family was also collected. RET gene mutations analysis Genomic DNA was extracted from blood, amplified by PCR and sequenced following the method previously described (13). To confirm the presence of the Ala883Thr mutation, exon 15 was subjected to digestion using Alu I (Promega, Madison, WI) restriction enzyme. Exon 15 was also amplified with two different pairs of primers to verify the possibility of a preferential annealing to the mutated allele of the firstly used primers pair. Polymorphisms in exons 3, 7, 11, 13, 14, and 15 were also studied. FISH analysis FISH analysis was performed according to a previously reported method (14) and using a 207-kb bacterial artificial chromosome clone RP11-351D16 (accession no. AC010864, human chromosome 10q11) spanning the entire RET gene region. Bovine adrenocortical cell DNA was extracted with a commercial kit (Plasmid Midi, Qiagen, Valencia, CA), and 2 g were directly labeled with SpectrumGreen-deoxyuridine 5-triphosphate using a nick translation kit (Vysis, Abbott Park, IL). Metaphase and interphase nuclei were prepared from heparinized blood sample of the index case according to the previously described method (15). Transfection assay The construct used in this study was cloned in prc/cytomegalovirus. The wild-type RET and RET/C634R were kindly gifted by Dr. Isabella Ceccherini. RET/A883T was generated by site-directed mutagenesis using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). The mutation was confirmed by sequencing. The transfection experiments were performed as previously reported (16). Results First, we screened the index case for germline RET mutation and found a novel homozygous GCT- ACT point FIG. 1. Pedigree of the studied family with new RET gene mutation Ala883Thr (GCT- ACT) showing the consanguinity of the parents of our index case.

3 Elisei et al. Homozygous New Germline RET Mutation J Clin Endocrinol Metab, November 2004, 89(11): mutation at codon 883 in exon 15, which determines an alanine to threonine substitution. At variance with a normal control, the sequence analysis of the index case showed only the mutated nucleotide but not the wild-type sequence, suggesting either a homozygous mutation or LOH. The analysis of the new Ala883Thr RET mutation in the family members revealed a heterozygous mutation in the 82-yr-old mother, son, and two sisters of the index case. One brother and two first-degree cousins were negative for the mutation. Finally, one brother was found to be homozygous for the Ala883Thr mutation, as the index case. All mutated cases were confirmed by restriction analysis with Alu I digestion. The clinical history of the family showed the absence of other known MTC cases and the consanguinity of the parents of our index case (second-degree cousins). The father died in 1975 for nonthyroid carcinoma, and no archived material was available for genetic analysis. As shown in Table 1, subject III:3 was affected by a nodular goiter (vol 57 ml), but she was negative both for basal and Pg-stimulated Ct. The heterozygous subjects V:1, IV:7, and IV:12, who were 15, 63, and 58 yr old, respectively, did not show any clinical and/or biochemical signs of MTC. Subjects IV:3, IV:4, and IV:6, who were negative for the mutation, were also negative at clinical and biochemical examination. The 56-yr-old brother (IV:2) carrying the homozygous mutation showed a nodular goiter and elevated basal and Pgstimulated Ct levels (18 and 138 pg/ml, respectively). None of the family members was affected by pheochromocytoma and/or parathyroid adenoma. Subject IV:2 underwent thyroidectomy and one MTC microfocus of 0.3 mm associated with CCH was found. The FISH analysis was performed to evaluate the copy number of the RET gene in the index case. It revealed two equally strong RET signals in both metaphases and interphases nuclei, indicating the presence of two RET alleles (Fig. 2). The analysis of RET gene informative polymorphisms showed that subjects IV:1 and IV:2 were homozygous for the exon 7 polymorphism, subjects IV:7 and IV:12 were heterozygous for exon 7 polymorphism, and subject IV:3 was heterozygous for both exon 11 and 15 polymorphisms. Subject III:3 was heterozygous for exon 7, 11, and 15 polymorphisms. According to the distribution of RET polymorphisms and Ala883Thr mutation, we reconstructed the genotype of subject III:2 (Fig. 3). This analysis indirectly confirmed the homozygous status of subjects IV:1 and IV:2 for Ala883Thr mutation. We did not find any association between MTC phenotype and the presence of exon 11 and/or exon 14 polymorphisms. The sequence analysis of the PCR products obtained using two different pairs of primers for exon 15 confirmed the homozygous status in patients IV:I and IV:2 and heterozygous status in subjects IV:7 and IV:12, ruling out the possibility of a preferential annealing of one pair of PCR primers for the mutated allele (data not shown). Finally, the results of transfection experiments indicated that the Ala883Thr mutation did not confer any detectable oncogenic activity to RET. Discussion We report here a germline mutation (Ala883Thr) of the RET gene that was associated with MTC phenotype exclusively in two homozygous subjects, whereas all heterozygous family members had no evidence of MTC. The exon 15 Ala883Thr mutation was never reported in the literature and FIG. 2. FISH analysis of metaphases and interphases of lymphocytes extracted from blood of index case: red arrows indicate two copies of the RET gene in interphases nuclei; yellow arrows indicate two copies of the RET gene in metaphases. TABLE 1. Clinical, biochemical, and genetic features of screened subjects Patient Age (yr) RETmut a Ultrasound Basal and Pgstimulated Ct Tx Histology III:3 82 A/T Nodular goiter Negative No V:1 15 A/T Normal thyroid Negative No IV:7 63 A/T Normal thyroid Negative No IV:12 58 A/T Normal thyroid Negative No IV:4 58 A/A Normal thyroid Negative No IV:6 50 A/A Normal thyroid Negative No IV:3 59 A/A Normal thyroid Negative No IV:2 56 T/T Thyroid nodule Positive Yes MTC IV:1 51 T/T Thyroid nodule Positive Yes MTC Tx, Thyroidectomy. a A, Alanine; T, threonine; A/T, heterozygous; A/A, homozygous normal alleles; T/T, homozygous mutated alleles.

4 5826 J Clin Endocrinol Metab, November 2004, 89(11): Elisei et al. Homozygous New Germline RET Mutation FIG. 3. Genotyping of five siblings (IV:1, IV:2, IV:3, IV:7, IV:12) and their mother (III:3) using informative RET gene polymorphisms: the polymorphisms segregation is confirmatory of a homozygous status for the mutated allele in subjects IV:1 and IV:2. The III:2 genotype was reconstructed on the basis of the genotypes of his relatives. not found in a series of 106 MTC and 106 normal controls previously studied in our center (13). The RET mutations described so far in association with MEN 2 and FMTC affected one of the two alleles (heterozygous mutations) and therefore were inherited as autosomic dominant traits (17). The penetrance of MTC is near 100% for the 634 and 918 mutations but lower for the others, which are mainly associated with FMTC. However, it has been shown that positive gene carriers, operated on the basis of the genetic testing and independently from serum Ct levels, showed histological features of CCH and/or MTC micro- or macroscopic disease (18, 19). On the basis of these observations, it has been widely accepted that RET gene acts as a dominant oncogene, and a single allele mutation is sufficient for tumor initiation. To our knowledge, a germline homozygous mutation of RET gene has been reported in only one family affected by FMTC harboring a Val804Met mutation (20). As in our case, this family showed a history of parental consanguinity, heterozygous gene carriers with normal basal and Pg-stimulated serum Ct, and three of four homozygous gene carriers with CCH and/or MTC foci at histology. At variance with the novel RET mutation found in this study, the Val804Met mutation is well known as pathogenic for FMTC and characterized by a high degree of expression with a late onset of the disease whose clinical behavior is usually less aggressive (7, 21, 22). Because the Ala883Thr RET mutation has never been described, there is no evidence that this mutation can lead to the development of the MTC in heterozygous condition. Although it is possible, it is unlikely that the 82-yr-old woman will develop the MTC in her remaining lifetime. Furthermore, all but one heterozygous nonaffected subject were older than the IV:1 and IV:2 affected members. On the basis of these considerations subjects IV:7, IV:12, and IV:2 were expected to have at least a positive Pg stimulation test. In a large series of MTC patients younger than 20 yr, it has been recently demonstrated that whatever the mutation, both extracellular and intracellular, only patients younger than 13 yr had negative histological findings after thyroidectomy (23). In our opinion these results are in agreement with our hypothesis that heterozygous patients should have already developed the disease, at least at the biochemical level. However, the number of heterozygous subjects in our family is quite limited, and assuming that the penetrance of low transforming RET mutations may be incomplete, these subjects might develop the MTC in the future. The follow-up with annual clinical and biochemical evaluation of the heterozygous gene carriers will provide additional information regarding the potential of this novel mutation to promote MTC development. RET gene mutations at codon 883 have been previously described in only four unrelated cases of MEN 2B and one sporadic MTC (24 26). However, the nucleotide mutation in these cases (GCT to TTT) was different from the present case (GCT to ACT) and resulted in a different amino acid change. In the previously reported cases, the MTC had an aggressive clinical behavior, especially if compared with the present case, which was definitively cured by total thyroidectomy. It is well known that different RET gene mutations show various degrees of transforming activity (27, 28), and, in particular, all noncysteine RET mutations are usually associated with a less aggressive phenotype (7). It is possible to postulate that the new mutation (Ala vs. Thr) has the lowest transforming activity among all the noncysteine known mutations. The simultaneous presence of the mutation in both alleles might increase the transforming activity of the RET mutated allele not more counterbalanced by the wild type. Unfortunately, the transfection assay did not demonstrate any in vitro transforming activity of the new mutation. Nevertheless, this experiment cannot distinguish the homozygous from heterozygous status, and the question whether these two conditions may have different biological consequences remains to be answered. To validate this hypothesis, the levels of expression of mrna derived from the mutant and wild-type alleles should be evaluated, but, at least for the moment, thyroid tissues from heterozygous subjects are not available. RET mutations affecting one allele associated with somatic LOH of the other RET allele have been reported in three sporadic MTC cases (29, 30) and one MEN 2A case (31). The sequence analysis of the RET gene exons and of our index case did not reveal any abnormality in the size of PCR fragments, which excluded the possibility of a small deletion. Furthermore, we ruled out the possibility of large losses of DNA in this chromosomal region by FISH analysis that used a probe spanning the entire RET gene. All nuclei showed two distinct and equally strong signals, demonstrating the presence of two copies of RET gene, confirming the homozygous nature of this novel mutation. The analysis of informative RET gene poly-

5 Elisei et al. Homozygous New Germline RET Mutation J Clin Endocrinol Metab, November 2004, 89(11): morphisms also supported the hypothesis to be in front of a homozygous mutation rather than an LOH. In conclusion, we identified a new germline RET gene mutation during a routine RET gene screening of an apparently sporadic case. This mutation showed a very low transforming activity as demonstrated by the absence of MTC phenotype in heterozygous subjects. The possibility to be dealing with a germline LOH has been excluded by FISH analysis for RET gene performed on lymphocytes derived from one homozygous patient. Acknowledgments We thank the family members and in particular our index case for the kind collaboration. We also thank Mrs. Paola Frontini for editorial assistance. Received February 18, Accepted August 5, Address all correspondence and requests for reprints to: R. Elisei, M.D., Department of Endocrinology, University of Pisa, Via Paradisa 2, Pisa, Italy. relisei@endoc.med.unipi.it. This work was supported in part by grants from Ministero dell Istruzione dell Università e della Ricerca (ex 40%) 2002 and Associazione Italiana per la Ricerca sul Cancro. L.A. is a Ph.D. student in Endocrine and Metabolic Sciences. References 1. Williams ED, Brown CL, Doniach I 1966 Pathological and clinical findings in a series of 67 cases of medullary carcinoma of the thyroid. J Clin Pathol 19: Gagel RF, Robinson MF, Donovan DT, Alford BR 1993 Clinical review 44: medullary thyroid carcinoma: recent progress. J Clin Endocrinol Metab 76: Mulligan LM, Kwok JB, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA 1993 Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 363: Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K, Lairmore TC, Howe JR, Moley JF, Goodfellow P, Wells Jr SA 1993 Mutations in the RET protooncogene are associated with MEN2A and FMTC. Hum Mol Genet 2: Eng C, Smith DP, Mulligan LM, Nagai MA, Healey CS, Ponder MA, Gardner E, Scheumann GF, Jackson CE, Tunnacliffe A 1994 Point mutations within the tyrosine kinase domain of the RET protooncogene in multiple endocrine neoplasia type 2B and related sporadic thyroid tumors. Hum Mol Genet 3: Eng C, Clayton D, Schuffnecker I, Lenoir G, Cote G, Gagel RF, van Amstel HK, Lips CJ, Nishisho I, Takai SI, Marsh DJ, Robinson BG, Frank-Raue K, Raue F, Xue F, Noll WW, Romei C, Pacini F, Fink M, Niederle B, Zedenius J, Nordenskjold M, Komminoth P, Hendy GN, Gharib H, Thibodeau SN, Lacroix A, Frilling A, Ponder BAJ, Mulligan LM 1996 The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. JAMA 276: Niccoli-Sire P, Murat A, Rohmer V, Franc S, Chabrier G, Badet L, Maes B, Savagner F, Giraud S, Bezzieau S, Kottler ML, Morange S, Conte-Devolx B 2001 Familial medullary thyroid carcinoma with non cysteine RET mutations: phenotype-genotype relationship in a large series of patients. J Clin Endocrinol Metab 86: Berndt I, Reuter M, Saller B, Frank-Raue K, Groth P, Grussendorf M, Raue F, Ritter MM, Hoppner W 1998 A new hot spot for mutations in the ret protooncogene causing familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2A. J Clin Endocrinol Metab 83: Hofstra RMW, Landsvater RM, Ceccherini I, Rein P, Stelwagen ST, Luo J, Pasini B, Hoppner JWM, van Amstel HKP, Romeo G, Lips CJM, Buys CHCM 1994 A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 367: Eng C, Mulligan M, Smith DP, Healey CS, Frilling A, Raue F, Neumann HP, Pfragner R, Behmel A, Lorenzo MJ, Stonehouse TJ, Ponder MA, Ponder BAJ 1995 Mutation of the RET protooncogene in sporadic medullary thyroid carcinoma. Genes Chromosomes Cancer 12: Romei C, Elisei R, Pinchera A, Ceccherini I, Molinaro E, Mancusi F, Martino E, Romeo G, Pacini F 1996 Somatic mutations of the RET protooncogene in sporadic medullary thyroid carcinoma are not restricted to exon16 and are associated with tumor recurrence. J Clin Endocrinol Metab 81: Eng C, Thomas GA, Neuberg DS, Mulligan LM, Healey CS, Houghton C, Frilling A, Raue F, Williams ED, Ponder BA 1998 Mutation of the RET protooncogene is correlated with RET immunostaining in subpopulations of cells in sporadic medullary thyroid carcinoma. J Clin Endocrinol Metab 83: Elisei R, Cosci B, Romei C, Bottici V, Sculli M, Lari R, Barale R, Pacini F, Pinchera A 2004 RET exon 11 (G691S) polymorphism is significantly more frequent in sporadic medullary thyroid carcinoma than in the general population. J Clin Endocrinol Metab 89: Nikiforova MN, Stringer JR, Blough R, Medvedovic M, Fagin JA, Nikiforov YE 2000 Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. Science 290: Scarpato R, Migliore L, Hirvonen A, Falck G, Norpa H 1996 Cytogenetic monitoring of occupational exposure to pesticides: characterization of a GSTM1, GSTT1 and NAT2 genotypes. Environ Mol Mutagen 27: Orgiana G, Pinna G, Camedda A, De Falco V, Santoro M, Melillo RM, Elisei R, Romei C, Lai S, Carcassi C, Mariotti S 2004 A new germline RET mutation apparently devoid of transforming activity serendipitously discovered in a patient with atrophic autoimmune thyroiditis and primary ovarian failure. J Clin Endocrinol Metab 89: Mulligan LM, Marsh DJ, Robinson BG, Schuffenecker I, Zedenius J, Lips CJM, Gagel RF, Takai SI, Noll WW, Fink M, Raue F, Lacroix A, Thibodeau SN, Frilling A, Ponder BAJ, Eng C 1995 Genotype-phenotype correlation in multiple endocrine neoplasia type 2: report of the International RET Mutation Consortium. J Intern Med 238: Wells SA, Chi DD, Toshima K, Dehner LP, Coffin CM, Dowton SB, Ivanovich JL, DeBenedetti MK, Dilley WG, Moley JF, Norton JA, Donis- Keller H 1994 Predictive DNA testing and prophylactic thyroidectomy in patients at risk for multiple endocrine neoplasia type 2A. Ann Surg 3: Pacini F, Romei C, Miccoli P, Elisei R, Molinaro E, Mancusi F, Iacconi P, Basolo F, Martino E, Pinchera A 1995 Early treatment of hereditary medullary thyroid carcinoma after attribution of multiple endocrine neoplasia type 2 gene carrier status by screening for RET gene mutations. Surgery 118: Lecube A, Hernandez C, Oriola J, Galard R, Gemar E, Mesa J, Simo R 2002 V804M RET mutation and familial medullary thyroid carcinoma: report of a large family with expression of the disease only in the homozygous gene carriers. Surgery 131: Feldman GL, Edmonds MW, Ainsworth PJ, Schuffenecker I, Lenoir GM, Saxe AW, Talpos GB, Robertson J, Petruccelli N, Jackson CE 2000 Variable expressivity of familial medullary thyroid carcinoma (FMTC) due to a RET V804M (GTG- ATG) mutation. Surgery 128: Lombardo F, Baudin E, Chiefari E, Arturi F, Bardet S, Caillou B, Conte C, Dallapiccola B, Giuffrida D, Bidart JM, Schlumberger M, Filetti S 2002 Familial medullary thyroid carcinoma: clinical variability and low aggressiveness associated with RET mutation at codon 804. J Clin Endocrinol Metab 87: Machens A, Niccoli-Sire P, Hoegel J, Frank-Raue K, van Vroonhoven TJ, Roeher HD, Wahl RA, Lamesch P, Raue F, Conte-Devolx B, Dralle H; European Multiple Endocrine Neoplasia (EUROMEN) Study Group 2003 Early malignant progression of hereditary medullary thyroid cancer. N Engl J Med 349: Smith DP, Houghton C, Ponder JB 1997 Germline mutation of RET codon 883 in two cases of de novo MEN 2B. Oncogene 15: Bugalho MJ, Coelho I, Sobrinho LG 2000 Somatic trinucleotide change encompassing codon 882 and 883 of the RET proto-oncogene in a patient with sporadic medullary thyroid carcinoma. Eur J Endocrinol 142: Gimm O, Marsh DJ, Andrew S, Frilling A, Dahia PLM, Mulligan LM, Zayac JD, Robinson BG, Eng C 1997 Germline dinucleotide mutation in codon 883 of the RET proto-oncogene in multiple endocrine neoplasia type 2B without codon 918 mutation. J Clin Endocrinol Metab 82: Santoro M, Carlomagno F, Melillo RM, Billaud M, Vecchio G, Fusco A 1999 Molecular mechanism of RET activation in human neoplasia. J Endocrinol Invest 22: Iwashita T, Kato M, Murakami H, Asai N, Ishiguro Y, Ito S, Iwata Y, Kawai K, Asai M, Kurokawa K, Kajita H, Takahashi M 1999 Biological and biochemical properties of Ret with kinase domain mutations identified in multiple endocrine neoplasia type 2B and familial medullary thyroid carcinoma. Oncogene 18: Jindrichova S, Kodet R, Krskova L, Vlcek P, Bendlova B 2003 The newly detected mutations in the RET proto-oncogene in exon 16 as a cause of sporadic medullary thyroid carcinoma. J Mol Med 81: Uchino S, Noguchi S, Adachi M, Sato M, Yamashita H, Watanabe S, Murakami T, Toda M, Murakami N, Yamashita H 1998 Novel point mutations and allele loss at the RET locus in sporadic medullary thyroid carcinomas. Jpn J Cancer Res 89: Quadro L, Fattoruso O, Cosma MP, Verga U, Porcellini A, Libroia A, Colantuoni V 2001 Loss of heterozygosity at the RET protooncogene locus in a case of multiple endocrine neoplasia type 2A. J Clin Endocrinol Metab 86: JCEM is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community.