A Novel Point Mutation in the Intracellular Domain of the ret ...

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Mutations in one of five cysteine residues in the extracellular domain have been found in .... Address all correspondence and requests for reprints to: Dr. Robert.
0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 12 Printed in U.S.A.

A Novel Point Mutation in the Intracellular Domain of the ret Protooncogene in a Family with Medullary Thyroid Carcinoma* ROBERT M. W. HOFSTRA, OLIMPIA FATTORUSO, LOREDANA QUADRO, YING WU, ALFONSO LIBROIA, UBERTA VERGA, VITTORIO COLANTUONI, CHARLES H. C. M. BUYS

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Department of Medical Genetics, University of Groningen (R.M.W.H., Y.W., C.H.C.M.B.), 9713 AW Groningen, The Netherlands; and Dipartimento di Biochimica e Biotecnologie Mediche e CEINGE, Universita` di Napoli Federico II (O.F., L.Q., V.C.); Facolta` d’Farmacia, Universita` d Reggio Calabria a Catanzaro (V.C.); and Divisione d’Endocrinologia, Ospedale Niguarda (A.L., U.V.), Milan, Italy ABSTRACT Specific mutations in the ret protooncogene have been found associated with multiple endocrine neoplasia type 2A (MEN 2A) and type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). Mutations in one of five cysteine residues in the extracellular domain have been found in over 95% of families with MEN 2A and 88% of families with FMTC. In MEN 2B patients, a specific mutation at codon 918, substituting a threonine for a methionine, has been found

in 95% of cases. In FMTC, in addition to the mutations of the extracellular cysteines, three intracellular base pair changes have been reported at codons 768 and 804. Here we describe a novel intracellular mutation in exon 15 of the ret gene that leads to the substitution of an alanine for a serine at codon 891 in a family with medullary thyroid carcinoma. This amino acid change may be important in determining substrate specificity or, alternatively, may play a role in ATP binding. (J Clin Endocrinol Metab 82: 4176 – 4178, 1997)

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We report here a new missense mutation of the ret protooncogene in a family affected by FMTC.

ULTIPLE endocrine neoplasia type 2 (MEN 2) includes several autosomal dominant cancer syndromes. MEN 2A is characterized by medullary thyroid carcinoma (MTC), by pheochromocytoma in 50% of cases, and by hyperplasia of the parathyroid glands in 15–30% of cases. In MEN 2B, hyperplasia of the parathyroid is seldomly found, whereas ganglioneuromas of the intestinal tract are common, and some patients show a marfanoid habitus. In familial MTC (FMTC), medullary thyroid carcinoma is the only tumor found (1). To date, mutations in MEN 2A families exclusively occur in the extracellular part of the protein. They affect one of five cysteine residues close to the transmembrane domain in over 95% of all families (2– 4). For MEN 2B, a single mutation, Met918Thr, has been found in the intracellular domain in 95% of cases (4 –7). FMTC patients bear the same mutations as those detected in MEN 2A patients, although in different frequencies (4). In addition, three mutations in the intracellular domain of the protein have been described, one in exon 13, Glu768Asp, and two in exon 14, Val804Leu and Val804Met (8 –10). In 12% of the FMTC families, however, no ret mutations have yet been found (4) (for an overview, see Fig. 1). Received July 9, 1997. Revision received August 6, 1997. Accepted August 15, 1997. Address all correspondence and requests for reprints to: Dr. Robert M. W. Hofstra, Department of Medical Genetics, University of Groningen, A. Deusinglaan 4, 9713 AW Groningen, The Netherlands. E-mail: [email protected]. * This work was supported in part by grants from CNR, Progetto Finalizzato ACRO, Sottoprogetto 4, and Associazione Italiana per la Ricerca sul Cancro.

Subjects and Methods Family history The pedigree of the family is shown in Fig. 2. Patient I.2 underwent total thyroidectomy for a large nodule in the left lobe of the thyroid (in 1984), which was detected by ultrasound scan. The diagnosis of MTC was based on elevated serum calcitonin (CT) levels and was confirmed histologically. After surgery (in 1987), CT levels were around 6 pg/mL (by immunoradiometric assay, normal values are ,10 pg/mL). The patient died in 1992 from a gastric carcinoma with hepatic metastases, apparently unrelated to the previous MTC. At that time, the CT level was 19/22 pg/mL, and the level of carcinoembryonic antigen was 4.4 ng/mL (normal, ,5 pg/mL). Patient II.2, 48 yr old, had a CT level of 134 pg/mL (by RIA, normal values are ,150 pg/mL) with a pentagastrin stimulation peak of approximately 1000 pg/mL at the time of the initial diagnosis (in 1990). Thyroid ultrasound scan showed nodules in both lobes of the gland. After thyroidectomy, histological examination confirmed the presence of two nodules, one in each lobe, 0.7 cm in diameter; both represented MTC accompanied by diffuse C cell hyperplasia. A recent CT measurement (in 1997) displayed basal values of 12 and 300 pg/mL after pentagastrin stimulation (by the immunoradiometric assay method). All other biochemical and imaging procedures were negative for MTC. Recently, patient II.1, at the age of 45 yr, also consented to undergo a thyroid ultrasound scan. It revealed the presence of two small nodules, both 0.5 cm in diameter. Serum CT determination gave a value of 14 pg/mL. However, the subject refused the pentagastrin stimulation test. All members of the third generation, i.e. patients III.1 (born in 1980), III.2 (born in 1982), and III.3 (born in 1985), were tested routinely for basal and pentagastrin-stimulated CT levels in 1993, 1996, and 1997. The 1997 values were: III.1: basal CT, 1.6 pg/mL; after pentagastrin, 8.7 pg/mL; III.2: basal CT, 1.2 pg/mL; after pentagastrin, 2.2 pg/mL; III.3: basal CT, 1.0 pg/mL; after pentagastrin, 36.0 pg/mL. All family members were also tested routinely for serum PTH and catecholamine levels. Results were in the normal range.

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FIG. 1. Schematic representation of the ret gene, the encoded protein, and the mutations found to date in MEN 2A, FMTC, and MEN 2B (2–10). Indicated are the positions of the mutations in both the protein and the gene (exons).

FIG. 2. Pedigree of the family described in this study. Solid symbols indicate the individuals harboring the Ser891Ala mutation and affected with MTC; hatched symbols indicate gene mutation carriers; open symbols indicate unaffected individuals.

Mutation detection Denaturing gradient gel electrophoresis (DGGE) was carried out for all ret exons using PCR conditions previously described (5). Primers for exon 15 were RET15F GACCGCTGTGCCTGGCCAT and RET15R [40GC]GCTTCCCAAGGACTGCCTGC. For DGGE analysis, a 9% PAA gel (acrylamide-bisacrylamide, 37.5:1) containing a 40 – 80% UF gradient (100% urea formamide gradient gel; 100% UF 5 7 mol/L urea and 40% deionized formamide) was used. Electrophoresis was performed in 0.5 3 TAE [1 3 TAE 5 40 mmol/L Tris, acetic acid (pH 8.0), 20 mmol/L sodium acetate, and 1 mmol/L sodium ethylenediamine tetraacetate] at 11 V/cm and 58 C for 18 h. For documentation, the gel was stained by ethidium bromide. PCR products of exons 10, 11, 13, and 15 were purified using the High Pure PCR Product Purification Kit from Boehringer Mannheim (Indianapolis, IN) and sequenced using the Thermosequenase kit (Amersham Life Science, Arlington Heights, IL) or the Sequenase kit (U.S. Biochemical Corp., Cleveland, OH) as previously described (5). Specific tests were performed to screen for the FMTC mutations in exon 14 (8, 10) and the MEN 2B mutation in exon 16 (5).

Results and Discussion

As no mutations were detected in our routine diagnostic screening of ret exons 10, 11, 13, 14, and 16, DGGE was

FIG. 3. Sequence analysis of ret exon 15 in an affected member of the family. Shown is the relevant part of the sequence containing the Ser891Ala mutation.

performed on all ret exons. Only one aberrant DGGE pattern was detected for exon 15, caused by a T to G transversion at nucleotide 2671 (starting the numbering from the first amino acid), as determined by direct sequencing of the amplicon (see Fig. 3). The transversion results in the replacement of a serine by an alanine at codon 891. This mutation was present in three affected (I.1, II.1, and II.2) and two unaffected members of the family (III.1 and III.3; Fig 2). The unaffected individuals III.1 and III.3 are 17 and 12 yr of age, respectively. The fact that the CT levels, even after PT stimulation, remained in the normal range during the 3-yr screening period may point to an absence of C cell hyperplasia. The subjects were offered an every 4 – 6 months monitoring of basal and pentagastrin-stimulated CT levels and prophylactic thyroid-

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ectomy as soon as these become elevated. All family members have also been screened for the presence of pheochromocytoma and parathyroid disease. All tests were negative, making this a family with MTC only. Whether this Ser891Ala mutation affects the function of the protein so as to cause tumor formation is not yet known. To date, all mutations detected in MEN 2A, MEN 2B and FMTC are of the activating type (11, 12). A ligand-independent dimerization of the protein has been shown for the RET-MEN 2A proteins. These mutants interact and phosphorylate at high levels the same substrates as the wild type-form (11, 12). In contrast, the MEN 2B mutation, located in the tyrosine kinase domain of the protein, has been shown to produce a constitutively activated protein with changes in both catalytic activity and substrate specificity (11, 13). Several arguments favor the suggestion that the Ser891Ala mutation might have similar effects. The serine 891 residue is conserved among Drosophila (14), mouse (15), and human (16) and lies in subdomain VII (17) of the tyrosine kinase domain of RET in the middle of four amino acid residues highly conserved among tyrosine kinases. Three of these amino acids (Asp892, Phe893, and Gly894) form the most conserved short stretch of the catalytic domain of tyrosine kinases in general and represent a common feature of a number of bacterial phosphotransferases that use ATP as phosphate donor (17). Furthermore, in cAPK-a it was found that Asp184, which corresponds to RET Asp892, may interact with the phosphate groups of ATP through Mg21 salt bridges (18). Finally, the ret mutation in this family with MTC only leads to the replacement of a serine by an alanine, an amino acid frequently found at the corresponding position in nonreceptor tyrosine kinases (17) and absent in 50 control individuals (100 chromosomes). It is, therefore, conceivable that the mutation might cause a change in substrate specificity, although a change in ATP binding cannot be excluded. For a definite answer we have to wait for a functional assay.

References 1. Ponder BAJ, Smith D. 1996 The MEN 2 syndromes and the role of the ret proto-oncogene. Adv Cancer Res. 70:179 –222. 2. Mulligan LM, Kwok JBJ, Healey CS, et al. 1993 Germline mutations of the ret proto-oncogene in multiple endocrine neoplasia type 2A. Nature. 363:458 – 460. 3. Donis-Keller H, Dou S, Chi D, et al. 1993 Mutations in the ret proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet. 7:851– 856. 4. Eng C, Clayton D, Schuffenecker I, et al. 1996 The relationship between specific ret proto-oncogne mutations and disease phenotype in multiple endocrine neoplasia type 2: international ret mutation consortium analysis. JAMA. 276:1575–1579. 5. Hofstra RMW, Landsvater RM, Ceccherini I, et al. 1994 A mutation in the ret proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature. 367:375–376. 6. Carlson KM, Dou S, Chi D, et al. 1994 Single missense mutation in the tyrosine kinase catalytic domain of the ret proto-oncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci USA. 91:1579 –1583. 7. Eng C, Smith DP, Mulligan LM, et al. 1994 Point mutations within the tyrosine kinase domain of the ret proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumors. Hum Mol Genet. 3:237–241. 8. Eng C, Smith DP, Mulligan LM, et al. 1995 A novel point mutation in the tyrosine kinase domain of the ret proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC. Oncogene. 20:509 –513. 9. Bolino A, Schuffenecker I, Luo Y, et al. 1995 ret mutations in exons 13 and 14 of FMTC patients. Oncogene. 10:2415–2419. 10. Fattoruso O, Quadro L, Libroia A, et al. A GTG to ATG novel point mutation at codon 804 in exon 14 of the ret proto-oncogene in two families affected by familial medullary thyroid carcinoma. Hum Mutat. In press. 11. Santoro M, Carlomagno F, Romano A, et al. 1994 Activation of ret as a dominant transforming gene by germline mutations of MEN 2A and MEN 2B. Science. 267:381–383. 12. Borrello MG, Smith DP, Pasini B, et al. 1995 ret activation by germline MEN 2A and MEN 2B mutations. Oncogene. 11:2419 –2427. 13. Songyang Z, Carraway III KL, Eck MJ, et al. 1995 Catalytic specificity of protein tyrosine kinases is critical for selective signalling. Nature. 373:536 –539. 14. Sugaya R, Ishimaru S, Hosoya T, et al. 1994 A drosophila homologue of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells. Mech Dev. 45:139 –145. 15. Iwamoto T, Taniguchi M, Asai N, et al. 1993 DNA cloning of mouse ret proto-oncogene and its sequence similarity to the cadherin superfamily. Oncogene. 8:1087–1091. 16. Takahashi M, Ritz J, Cooper GM. 1985 Activation of novel human transforming gene ret by DNA rearrangement. Cell. 42:581–588. 17. Hanks SK, Quin AM, Hunter T. 1988 The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 241:42–52. 18. Brenner S. 1987 Phosphotransferase sequence homology. Nature. 329:21.