Tumor tissues from 2 multiple endocrine neo- plasia type 2A patients were analyzed at the DNA and ... M ULTIPLE endocrine neoplasia type 2A (MEN 2A) is an.
0021.972X/94/7902-05SO$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1994 by The Endocrine Society
Frequent Endocrine L. V. R. M.
Vol. 79, No. 2
Printed
RET Protooncogene Mutations Neoplasia Type 2A*
QUADRO, L. PANARIELLO, D. SALVATORE, F. CARLOMAGNO, NUNZIATA, V. COLANTUONI, G. DI GIOVANNI, M. L. BRANDI, GHERI, U. VERGA, A. LIBROIA, N. BERGER, A. FUSCO, GRIECO, AND M. SANTORO
in U.S.A.
in Multiple M. DEL PRETE, M. MANNELLI,
Dipartimento di Biochimica e Biotecnologie Mediche, CEINGE, Centro di Ingegneria Genetica (L.Q., L.P., M.D.P., V.C.); Centro di Endocrinologia e Oncologia Sperimentak de1 Consiglio Nazionale delle Ricer&e, Dipartimento di Biologia e Patologia Cellulare e Molecolnre (D.S., F.C., A.F., M.G., MS.), Istituto di Medicina Interna e Malattie Dismetaboliche (V.N., G.D.G.); and Facoltti di Medicina e Chirurgia, Universitk di Napoli “Federico II, ” 80131 Naples; Dipartimento di Medicina Sperimentale e Clinica, Facoltk di Medicina e Chirurgia di Catanzaro, UniversitZl di Reggio Calabria (A.F., M.G.), 88100 Catanzaro; Unitiz di Endocrinologia, Dipartimento di Fisiopatologia Clinica, Facoltzl di Medicina e Chirurgia, Universitk di Firenze (M.L.B., M.M., R.G.), 50139 Florence; and Divisione di Endocrinologia, Ospedale Niguarda (U.V., A.L.), Milan, Italy; and Laboratoire d’Anatomie Pathologique, Hopital de L’Antiquailk (N.B.), Lyon, France ABSTRACT The occurrence of mutations in the RET protooncogene has been investigated in 12 multiple endocrine neoplasia type 2A families and 18 cases of sporadic thyroid medullary carcinomas and pheochromocytomas. Ten of 12 families showed single base substitutions in the RET protooncogene exons 10 and 11, coding for the extracellular domain of the protein. Tumor tissues from 2 multiple endocrine neoplasia type 2A patients were analyzed at the DNA and ribonucleic acid
M
ULTIPLE endocrine neoplasia type 2A (MEN 2A) is an autosomal dominant cancer syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism (1). By a combination of linkage analysis and genetic mapping, the candidate gene for this diseasewas mapped to lOq11.2, the pericentromeric region of chromosome 10, where the RET protooncogene is located (2-7). This gene has been shown to encode a tyrosine kinase receptor (8), with an amino-terminal signal sequence, a cysteine-rich extracellular region, a transmembrane tract, and a tyrosine kinase domain, interrupted by an insertion of 27 amino acids. The RET protooncogene is expressedin human neuroectodermal tumors, such as neuroblastomas (9), MTC, and pheochromocytomas (10). Moreover, its expression in MTC and neuroblastoma cell lines is induced upon exposure to several differentiating agents, suggesting that RET may play a role in the differentiation and/or proliferation of neural cells (10, 11). In vivo, the RET protooncogene is frequently activated in Received January 26, 1994. Revision received April 21, 1994. Accepted April 28, 1994. Address all correspondence and requests for reprints to: Prof. V. Colantuoni, Dipartimento di Biochimica e Biotecnologie Mediche, Universiti di Napoli, Via S. Pansini 5, 80131 Naples, Italy. * This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), the CNR Progetto Finalizzato Ingegneria Genetica, and P. F. ACRO (Applicazioni Cliniche della Ricerca Oncologica).
levels and revealed the same heterozygous mutations found in the peripheral blood lymphocytes. This demonstrates that both the normal and mutant alleles are expressed. No mutations in these exons were detected in the 18 cases of sporadic tumors investigated. These data provide further evidence that the mutated RET protooncogene acts in a dominant fashion and is responsible for the pathogenesis of this syndrome. (J Clin Endocrinol Metab 79: 590-594,1994)
human thyroid papillary carcinomas by chromosome rearrangements that involve the fusion of its tyrosine kinase domain to the 5’-terminal region of other genes (H4, or DlOS170, PKA Rla, and the RFG gene in the case of the chimeric oncogenesRET/PTCl, RET/PTC2, and RET/PTC3, respectively) (12-l 6). Two groups have recently identified the RET protooncogene as the gene responsible for the MEN 2A syndrome and for the closely related familial MTC syndrome (17, 18). The point mutations were changes in the codons specifying for some cysteine residues (namely codons 357, 364, 366, and 380) (19) of the RET extracellular domain to different amino acid residues. We have analyzed 12 MEN 2A families and 18 casesof sporadic MTC and pheochromocytomas for the presenceof RET mutations in exons 10 and 11, where cysteine residues 357-380 are located. Here we report the presence of heterozygous mutations in 10 of the MEN 2A families. Nine mutations involved cysteine 380, and only 1 involved cysteine 366. No mutations were found in the sporadic tumors. Materials and Methods Tumor
samples
Three MEN 2A cases, 12 sporadic MTC and 4 sporadic pheochromocytomas were collected at the Laboratoire d’Anatornie Pathologique, Hopital de L’Antiquaille (Lyon, France), and the Laboratoire de Cytolopie, Hopital Jules Courmont (Pierre Benite, France). The rest were
RET MUTATIONS collected in Italy (Florence, Milan, and Naples). The samples were stored frozen until DNA or ribonucleic acid (RNA) extractions were performed. Pedigree resources available for this study and diagnostic procedures can be obtained from the authors. In some families, linkage analysis with several markers of the centromeric region of chromosome 10 was performed and proved to be informative for the diagnosis of MEN 2A.’
Polymerase chain reaction (PCR), reverse transcription-PCR, cloning, and sequence analysis Nucleotide positions are in accordance with the published sequence (8). Numbering of the exons is according to Kwok et al. (19). Oligonucleotide mimer seouences used to amplify exon 10 were: lOF, AGCATT’GTTGGGGGACAC, designed from the complementary DNA seouence (nucleotides 1696-1713): or lOR, AGGCTCAGAGAGGCT&&A, designed from the intronic sequence (see Footnote 1). Oligonucleotide primer sequences used to amplify exon 11 were: 1 lF, ATGAGGCAGAGCATACGCA, designed from the intronic sequence (see Footnote 1); and 1 lR, GGAGTAGCTGACCGGGAA, designed from the complementary DNA sequence (nucleotides 1978-1995).-DNA extraction was performed by standard procedures (20), and total RNA was prepared by a modification of the guanidine-thiocyanate method (21). PCR reactions were performed in a 50-PL final volume, containing 100 or 200 ne eenomic DNA, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1 .S”~mol/L MgCI,; 5 mmol/L NHIC1, 0.5 U ‘Ta9 Polymemse (Polymed, Florence, Italy), 200 rmol/L of each deoxy-NTP, and 2 rmol/ L of each primer. Five cycles of denaturation, annealing, and extension were performed at 95 C for 1 min, 62 C for 1.5 min, and 72 C for 2 min, respectively, followed by 25 cycles of denaturation, annealing, and extension at 95 C for 1 min, 62 C for 1.5 min, and 72 C for 1.5 min, respectively, using an automatic thermocycler (Perkin-Elmer/Cetus, Monza, Italy). The PCR products were purified by agarose gel electrophoresis, followed by purification (JETsorb gel extraction kit, Genomed, Bad Oeynhausen, Germany), and an aliquot was sequenced on both strands by the Sanger method, employing the TAQuence Cycle Sequencing kit (U.S. Biochemical Corp., Cleveland, OH). The same oligonucleotides required for the amplification were used as sequencing primers. RNA amplification was performed as previously described (22). The 11R oligo (see above) was used as reverse- primer, and the F6, the sequence of which is CAGCTGCTTGTAACAGTG (nucleotides 142661443), was used as the forward primer. Briefly, 5 fig total RNA were reverse transcribed using the reverse primer and subjected to 35 cycles of I’CR in a thermal cycler. The amplified products were either sequenced directly or cloned in the pT7blue T-vector-kit (Novagen, Milan, Italy) and subsequently sequenced using the Sequenase kit (U.S. Biochemical Corp.). 1
Results For each MEN 2A family, several affected members along with the unaffected relatives were studied. The analysis was conducted by PCR amplification of high mol wt DNA extracted from peripheral blood lymphocytes. The PCR primers spanned exons 10 and 11 and the interposed intron, which corresponded to the extracellular domain of the protein. The amplified bands, about 0.2 kilobasesin length, were isolated and sequencedon both strands by the Sanger method. In 10 of the 12 MEN 2A families investigated, nonconservative mutations were found in codons specifying cysteine residues. Nine of the 10 mutations involved the cysteine residue 380 in exon 11, and only 1 involved the cysteine residue 366 in exon 10 (numbering of the residues is according to the published RET gene sequence) (8). The basepair changes ’ Quadro, L., L. Panariello, D. Salvatore, F. Carlomagno, M. Del Prete, V. Nunziata, V. Colantuoni, G. di Giovanni, M. L. Brandi, M. Mannelli, R. Gheri, U. Verga, A. Libroia, N. Berger, A. Fusco, M. Grieco, and M. Santoro; unpublished data.
IN MEN
2A
591
found are illustrated in Table 1. The most frequent, 4/10, is a Cys to Arg change in a proportion similar to that reported previously (17,lB) (see,for comparison, Table 1). All affected members tested in each family showed the same mutation, whereas the unaffected relatives and 30 unaffected and unrelated individuals displayed a normal pattern. A representative example of the analysis performed is illustrated in Fig. 1. No mutations were found in the RET protooncogene regions examined in two MEN 2A families or in all sporadic MTC and pheochromocytomas analyzed. In two cases(FRl and FR2), the mutation identified in peripheral lymphocytes was also recognized in the DNA extracted from the tumor tissues.Analysis of total RNA was performed by reverse transcription and PCR amplification, using RET protooncogene-specific primers (Fig. 2). The amplified bands were sequencedeither directly or after cloning in the pT7 plasmid. Only the expected mutation was found in the regions examined. In the caseof the FRl family, as in the IT5 family, the G to A transition generated a new site for the restriction enzyme RSAl (GTGC to GTAC), the presence of which was determined by digesting the amplified band with RSAl. In a normal individual, the digestion showed only the full-length band, whereas the MEN 2A patient showed, in addition to this band, two faster migrating bands, which are the digestion products of the mutated allele (see Fig. 1, side panel). In the case of FR3, only one individual with multifocal MTC and pheochromocytoma was examined. As none of the relatives in this family showed symptoms of the disease, this case was considered a de novo MEN 2A mutation. Discussion Twelve separateMEN 2A families and 18 casesof sporadic MTC and pheochromocytomas have been investigated for the presence of mutations in exons 10 and 11 of the RET protooncogene. Ten of the 12 families examined were positive for point mutations, all involving codons for cysteine residues. Nine mutations occurred at the level of codon 380, and 1 at codon 366, a frequency similar to that reported in the families studied by Mulligan et al. (17, 23). The basepair changes were detected in all affected family members analyzed, but not in the unaffected members, indicating that the mutation is present on the diseased chromosome in each case. The fact that no substitutions were detected at these cysteine residues in 30 normal individuals clearly indicates that the mutations reported are specific for MEN 2A and are not DNA polymorphisms. Our data, therefore, confirm the association of these RET-specific mutations with the MEN 2A syndrome and demonstrate that a single point mutation is an important event for the establishment of this familial neoplasia. The data presented provide further evidence of direct involvement of the RET protooncogene in the pathogenesis of the MEN 2A syndrome. All mutations lie within the extracellular domain, suggesting that they may play an important role in conferring the correct three-dimensional structure to the protein. Their loss may disrupt the RET protein conformation, thereby affecting the ligand-binding proper-
QUADRO
592 TABLE
1. RET
Mutations
mutations
reported
in this
in MEN
Mutations
in other
380 366 380 380 380 380 380 380 380 380 No No
(exll) (exl0) (exll) (exll) (exll) (exll) (exll) (exll) (exll) (exll) mutation mutation
Basepair changes’
TGC TGC TGC TGC TGC TGC TGC TGC TGC TGC
+ + + + + + + + + +
Donis-Keller
Amino acid changes
GGC TAC CGC CGC TAC GGC CGC TAC CGC TAC
Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys
--) + + -+ + + + + -+ +
New
BsrI RsaI SelI, SelI, RsaI BsrI SelI, RsaI SelI, RsaI None None
Gly Tyr Arg Arg Tyr Gly Arg Tyr Arg Tyr
Mutation#/ affected relatives
restriction site
Mutations/ unaffected relatives
212 212 212 111 212 212 212 l/l l/l l/l
H/m1 HhuI
HhnI HhaI
O/l O/l O/l O/l
reports Codon
Mulligan
JCE8zM.1994 Vol79.No2
2A
Codon” (exon)b
indi-
AL.
paper
Family
IT1 IT2 IT3 IT4 IT5 IT6 IT7 FRl FR2 FR3 ITS-IT9 30 unaffected viduals
ET
et al. (17)
et al. (18)
’ Numbered according the published * The exons are numbered according ’ The altered base is shown in bold. d In all the cases, identical mutations
Amino
acid
changes
New
restriction
site
No. of cases
380 380 380 380 380 364
Cys Cys Cys Cys Cys Cys
+ + + + +
Arg Gly Tyr Ser Phe Gly
SelI, HhuI BsrI RsuI Mare1 TaqI None
12 3 2 1 1 1
380 366 366 364 364
Cys Cys Cys Cys Cys
+ + -+ + -+
Arg Arg Tyr Arg Ser
SelI, HhnI Se11 None HhuI None
1 2 2 1 2
RET sequence the published
(8). intron-exon
structure
were present
in different
affected
ties and the transduction of the signal. Interestingly, the RET protooncogene acquires the capacity to transform normal cells in vim in 25% of the casesof human papillary thyroid carcinomas (12-16) by losing its extracellular domain and in vitro during the course of the transfection-transformation assayof cultured cells (24,25). The resulting new RET protein could acquire a different function, which leads to the malignant phenotype. The specificity of the mutations and tissue distribution are reminiscent of the point mutations occurring in other known oncogenes, implying a dominant role of RET in the generation of MEN 2A tumors. Consistent with this is the observation that the normal and mutated allelesare present in the DNA and are expressedat the RNA level. This finding rules out the possibility that mechanismsin which only one allele functions (e.g. imprinting) operate and also indicates that the protein derived from the mutant allele is responsible for the transformed phenotype. In addition, the absence of gross deletions, frame shift, and nonsense mutations indicates a positive action of the activated RET in the generation of MEN 2A tumors. The observation described here and in two previous reports (17, 18) that some casesof MEN 2A are not associated with mutations in the RET protooncogene suggeststhat mutations in other regions of the gene, not investigated in this
of
RET (19).
members
belonging
to the same family.
study, may be responsible for a minority of MEN 2A cases. Mutations elsewhere in the gene may also be responsible for sporadic MTCs. In fact, it has recently been reported that 6 of 18 casesof sporadic MTCs showed a mutation at a very conserved Met residue in the tyrosine kinase domain (26). It is also possible that other genes, encoding for proteins along the same pathway of differentiation of neural crest-derived cells, could be similarly involved. The fact that two distinct clinical entities, characterized by different and more severe clinical manifestations, such as the MEN 2A and familial MTC syndrome syndromes, show mutations in the codons for the sameresidues,suggeststhat other genesor molecular events might be responsible for the difference and may also explain the different age of onset and prognosis (18). It remains to be elucidated whether other closely linked genes are affected, thus producing a contiguous gene syndrome. Alternatively, geneslocalized on separatechromosomesmay participate in the establishment of the disease.Loss of heterozygosity of a region of chromosome 1 or other chromosomeshas, in fact, been associatedwith the MEN 2A syndrome (27). This finding contrasts with the “two-hit model” of inherited tumors exemplified by familial retinoblastoma (28) and suggeststhat MEN 2A follows an alternative model. Finally, mutations in cysteine-specifying codons can create novel restriction sites, which can easily be detected on a
RET MUTATIONS
IN MEN
2A
593 3’
MENPA FR-1
8
Norm81
antisense
antisense -“ri
&i-
mu
ACGTACGT
MEN2A IT1
_/ Y
ACGT
ACGT
*I*
ilc
)** ACGT
a-, *_^*
am
MENPA FR-2
T
AC
GT
FIG. 2. Analysis of the MEN 2A tumor tissues. Total RNA extracted from the MTCs of patients FRl and FR2 was subjected to reverse transcription and PCR amplification using as primers oligos covering RET exon 10 or 11. The amplified bands were isolated and cloned in the vector pT7-blue and subsequently sequenced. The result obtained on the antisense strand is illustrated. The amplified band on DNA from a normal individual was similarly cloned and sequenced. Lanes A, C, G, and T show sequencing ladder reaction products; the codon where the mutation occurs is boxed, and the new amino acid residue is indicated.
References 1. Schimke RN. 1984 Genetic Annu Rev Med. 35:25-31. MENOA IT3
Norm81
2. Mathew
aspects
of multiple
neoplasia
3. Simpson NE, Kidd KK, Goodfellow .
l
-*
“d
x
endocrine 328:528-530.
neoplasia
analysis for RETprotooncogene mutations. The genomic DNA extracted from peripheral blood cells was PCR amplified using primers corresponding to RET exons 10 and 11. The amplified products were purified and directly sequenced on both strands. The DNAs from normal individuals from the same families were similarly processed and used as reference for the wild-type RET sequence. The cases reported are representative of mutations occurring at cysteine residue 380 that in IT5 is mutated to a tyrosine residue (G to A transition; upper panel), in IT1 to a glycine residue (T to G transversion; middle panel), and in IT3 to an a&nine residue (T to C transition; lower panel). Lanes A, C, G, and T indicate sequencing ladder reaction products. The codon where the mutation occurs is boxed, and the dots indicate the nucleotides present in the normal and mutated alleles. In the IT5 patient, the mutation generates a new RSAl restriction site, so that the amplified band can be digested in two lower mol wt bands. The undigested band in the DNA from the same patient corresponds to the product derived from the normal allele (side panel).
PCR-amplified band, starting from genomic DNA. Other possible missensemutations in RET exons 10 and 11 can produce the new restriction sitesillustrated in Table 1. Should this be the case, once the mutation has been identified, this rapid and nonradioactive procedure can be used to screen at-risk populations.
Acknowledgment We wish to thank
Jean Gilder
for editing.
genetic 2A on chromosome
PJ, et al. 1987 Assigment
of
10 by linkage.
4. Gardner E, Papi L, Easton DF, et al. 1993 Genetic
studies interval
endocrine neoplasia type lOq11.2. Hum Mol Genet.
5. Mole SE, Mulligan FIG. 1. DNA
type
type 2A to chromosome
map the multiple on chromosome
ACGTACGT
neoplasia.
GCP, Chin KS, Easton DF, et al. 1987 A liked
marker for multiple endocrine 10. Nature. 328:527-528. multiple Nature
endocrine
LM, Healey CS, Ponder BAJ, Tunnacliffe
1993 Localisation of the gene for multiple 2A to a 480 kb region in chromosome Genet. 2~247-252.
6. Ishizaka oncogene 1521.
Y, Itoh F, Tahira mapped
endocrine neoplasia band lOq11.2 Hum
T, et al. 1989 Human
to chromosome
7. Sozzi G, Pierotti MA, Donghi
lOq11.2.
Oncogene.
R, et al. 1991 Refined
to contiguous regions of chromosome RET) that form the oncogenic sequence
8. Takahashi
linkage 2 loci to a small 2:241-246.
1Oq of the two PTC. Oncogene.
A. type Mol
RET proto4:1519localization genes (H4, 6:339-342.
M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H, Hiai H.
1988 Cloning and expression of the RET proto-oncogene encoding a tyrosine-kinase with two potential transmembrane domains. Oncogene. 3:571-578. 9. Ikeda I, Ishizaka Y, Tahira T, et al. 1990 Specific expression of the RET proto-oncogene in human neuroblastoma cell lines. Oncogene. 5:1291-1296. 10. Santoro M, Rosati R, Grieco M, et al. 1990 The RET proto-oncogene is consistently expressed in human phaeochromocytomas and thyroid medullary carcinomas. Oncogene. 5:1595-1598. 11. Tahira T, Ishizaka Y, Itoh F, Nakayasu M, Sugimura T, Nagao M. 1991 Expression of the RET proto-oncogene in human neuroblastoma cell lines and its increase during neuronal differentiation induced by retinoic acid. Oncogene. 6:2333-2338. 12. Fusco A, Grieco M, Santoro M, et al. 1987 A new oncogene in human papillary thyroid carcinomas and their lymph-nodal metastases. Nature. 328:170-172. 13. Bongarzone I, Pierotti MA, Monzini N, et al. 1989 High frequency of oncogene activation in human thyroid papillary carcinomas. Oncogene. 4:1457-1462. 14. Grieco M, Santoro M, Berlingieri MT, et al. 1990 PTC is a novel rearranged form of the RET proto-oncogene and is a frequently detected in viva in human thyroid papillary carcinoma. Cell. 60:557-
563.
594
QUADRO
I, Monzini N, Borrello MG, et al. 1993 Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of RET tyrosine-kinase and the regulatory subunit RI alpha of cyclic AMP protein kinase A. Mol Cell Biol. 13:358-366. 16. Santoro M, Dathan NA, Berlingieri MT, et al. 1994 Molecular characterization of RET/PTC3: a novel rearranged version of the RET proto-oncogene in a human thyroid papillary carcinoma. Oncogene. 9:509-516. 17. Mulligan LM, Kwok JBJ, Healey CS, et al. 1993 Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature. 363:458-460. 18. 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. 2851-856. 19. Kwok JBJ, Gardner E, Werner JP, Ponder BAJ, Mulligan LM. 1993 Structural analysis of the human RET proto-oncogene using exon trapping. Oncogene. 8:2575-2582. 20. Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular cloning-a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory. 15.
Bongarzone
ETAL. 21. Chomczynski
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P, Sacchi N. 1987 A single step method of RNA by acid guanidinium thiocyanate-phenol-chloroform exAnal Biochem. 162:156-159. 22. Kawasaki ES. 1990 A guide to methods and applications. In: Innis MA, Gelfand DH, Sninnski JJ, White TJ, eds. PCR protocols. San Diego: Academic Press; 21-38. 23. Mulligan LM. Ene C. Healev CS. et al. 1994 Soecific mutations of the RET proto-onc’ogene are belated to disease bhenotype in MEN 2A and FMTC. Nature Genet. 6:70-74. 24. Takahashi M, Cooper GM. 1985 Activation of a novel transforming gene, RET, by DNA rearrangement. Cell. 42:581-588. 25. Takahashi M, Cooper GM. 1987 RET transforming gene encodes a fusion protein homologous to tyrosine kinases. Mol Cell Biol. 7:1378-1385. 26. Hofstra RMW, Landsvater RM, Ceccherini I, et al. 1994 A mutation in the RET protooncogene associated with multiple endocrine neoplasia type 28 and sporadic medullary thyroid carcinoma. Nature. 3671375-376. 27. Weinberg RA. 1991 Tumor suppressor genes. Science. 254:11381145. 28. Knudson AG. 1971 Mutation and cancer: statistical study of retinoblastoma. Proc Nat1 Acad Sci USA. 68:820-823. isolation traction.
