RESEARCH57. 310-313. January 5. 19971 Mutation Analysis of Glial Cell Line-derived Neurotrophic Factor (GDNF), a for the RET/GDNF a Complex, in Sporadic Phaeochromocytomas1

Patricia L. M. Dahia, Sergio P. A. Toledo, Lois M. Mulligan, Eamonn R. Maher, Ashley B. Grossman, and Charis Eng@

Translational Research Laboratory, Division of C'ancer Epidemiology and Control, Dana-Farber C'ancer Institute. Department of Medicine. Harvard Medical School. Boston. Massachusetts 02115 [P. L M. D., C. El: Department of Endocrinology, St. Bartholomew's Hospital, London ECIA 7BE. United Kingdom IP. L M. D., A. B. G.J; Endocrine Unit. Unis'ersirv of Sao Paulo Medical School, Sao Paulo 02146-913, Brazil [S. P. A. TI: Departments of Paediatrics and Patholog-@, Queen ‘sUniversity. Kingston, Ontario. K7L 3N6 Canada IL M. MI; and Human Molecular Genetics Group fE. R. M.J and Cancer Research Campaign Human Cancer Genetics Research Group [C. El, University ()f@Cambridge. Cambridge CB2 2QQ. United Kingdom

ABSTRACT sporadic phaeochromocytomas, unlike the greater proportion (23— 66%) of somatic RET mutations observed in medullary thyroid car Phaeochromocytomas usually occur sporadically but may also be a cinoma, another component tumor of the MEN 2 syndromes (3, 4, feature of three autosomal dominantly inherited cancer syndromes, mul 13—18).Similarly, somatic VHL mutations have been described in tiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and, very rarely, type 1 neurofibromatosis. Germ-line missense mutations in fewer than 2% of sporadic phaeochromocytomas (16, 19, 20). Thus, it the RET proto-, which encodes a , cause can be postulated that other are responsible for the pathogenesis multiple endocrine neoplasia type 2. In VHL, germ-line mutations in one of these tumors. of the three of the VHLtumor suppressor have been found in Recent investigations have shown that GDNF, a member of the the majority offamilies. Whereas somatic mutations in the VHL gene have transforming j3 superfamily, is the natural ligand for the been common in sporadic renal cell carcinoma, a component cancer of RET receptor tyrosine kinase and acts via a multimeric receptor VHL, somatic mutations in the RET and VHL genes together have been complex, which also includes a novel glycosyl-phosphoinositol found in approximately 10% of sporadic phaeochromocytomas. Hence, linked , GDNFR-a (21—24).It has been postulated that GDNF other genes must also contribute to the pathogenesis of sporadic phae is the ligand for a heterotetrameric complex of RET and GDNFR-a in ochromocytomas. Recent data have suggested that glial cell line-derived which GDNFR-cx provides the ligand binding capacity for GDNF, and neurotrophic factor (GDNF) is a ligand for RET and acts via a heterotet RET provides the signaling function. It only takes some extrapolation rameric receptor complex that includes GDNF receptor a, which provides ligand binding capabilities, and RET, which provides the signaling com to postulate that mutations in either GDNF or GDNFR-cx could mimic ponent. Thus, both GDNF and GDNFR-cs are plausible candidate genes the effect of the gain of function RET mutations seen in MEN 2 and for involvement in the pathogenesis of phaeochromocytomas. To investi sporadic component tumors. Therefore, both GDNF and GDNFR-ce gate the role of GDNF in sporadic phaeochromocytomas, we scanned a couldbetargetgenesfor mutationsthatplay a role in thepathogenesis panel of 22 tumors. Among these samples, only a conservative sequence of phaeochromocytomas. To investigate the role of GDNF in the variant was detected in 2 of GDNF. No disease-associated somatic development of sporadic phaeochromocytoma, we scanned a panel of GDNF mutations or gross gene amplification were detected in these 22 such tumors for somatic mutations in GDNF. tumors, suggesting only a minor role for GDNF in the genesis of phae ochromocytomas. MATERIALS AND METHODS

INTRODUCTION Tumors. A total of 22 sporadic phaeochromocytomas from individuals withoutotherstigmataof VHL wereanalyzed.Agesat diagnosisrangedfrom Phaeochromocytomas are usually sporadic, but a significant minor 9—64years. Of the 22 phaeochromocytomas, 18 were benign, and 4 were ity are components of three inherited cancer syndromes, MEN 2,@ malignant, as determined by the finding of metastases in nonchromaffin VHL, and, rarely, NF I. These familial forms of phaeochromocytoma tissues. A phaeochromocytoma was considered sporadic if there was no history have been associated with germ-line mutations in their respective of a first- or second-degree relative with medullary thyroid carcinoma or susceptibilitygenes.Germ-line mutationsin the RET proto-oncogene, phaeochromocytoma and if there were no other features of MEN 2, VHL, or which encodes a receptor tyrosine kinase, account for the great ma NF 1. These phaeochromocytomas have been described previously (16, 25). jority of MEN 2 (1—8).Germ-line mutations in the VHL and the NFl Isolation of DNA and RNA. GenomicDNA wasobtainedfroma fragment tumor suppressor genes cause VHL and NF I , respectively (9—12). of tissue excised from the core of frozen tumor. Corresponding germ-line DNA However, the molecular pathogenesis of the most frequent form of from leukocytes was also obtained. Both were extracted as described previ ously (25). phaeochromocytoma, namely the sporadic tumor, is largely unknown. Preparation of RNA from tumor tissue, first-strand cDNA synthesis, and Somatic RET mutations have been found in approximately 10% of RT-PCR were performed as described previously (26, 27). PCR Amplification and Mutation Analysis. Two pairsof primerswere Received 8/19/96; accepted I 1/8196. designed based on the published GDNF sequence (GenBank accession num The costs of publication of this article were defrayed in part by the payment of page bers, Ll9062 and L19063), encompassing the two coding exons of the gene. charges. This article must therefore be hereby marked advertisement in accordance with I8 U.S.C. Section 1734 solely to indicate this fact. PCR was performed with 50—100ngof DNA, 1.25 mMMgCI,, 200 ,.tMeach @ Supported by the Fundaclo de Amparo a Pesquisa do Estado de Sao Paulo, Brazil deoxynucleotide triphosphate (Promega, Southampton, United Kingdom), 0.4 (FAPESP 94/2025-8), the Lawrence and Susan Marx Investigatorship in Human Cancer @LMeach primer (exon 1 F TGA AOl TAT 000 ATG TCG TOG and exon Genetics, the Markey Charitable Trust, the Charles A. Dana Foundation, and a Patterson I R AOl CAC TGC TCA GCG CGA AGO; Exon 2 F AAA TAT 0CC AGA Fellowship. 2 To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute GGA TFA ICC TGA and exon 2 R CAG ATA CAT CCA CAC Cli' TFA D920C, 44 Binney Street, Boston, MA 021 15-6084. Phone: (617) 632-5632; Fax: (617) OCO), 0.125 unit Taq polymerase (Promega), with the addition of TaqStart 632-4280; E-mail: [email protected]. Antibody (Clontech, Cambridge, United Kingdom), according to the manu 3 The abbreviations used are: MEN 2. multiple endocrine neoplasia type 2; VHL. von facturer'sinstructions.After a 5-mm denaturationstepat 95°C,thesamples Hippel-Lindau disease; NF 1. type I neurofibromatosis; HSCR, Hirschsprung disease; GDNF, glial cell line-derived neurotrophic factor; GDNFR-cs, GDNF receptor a; RT were cycled 30 times at 94'C for 1 mm, 54°C for I mm, and 72°C for 1 mm PCR, reverse -PCR; SSCP. single-strand conformational polymorphism. (for exon 2) and 94°C for 30 s, 61°C for 30 s, and 72°C for 30 s (for exon I), 310

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and both reactions were followed by a 10-mm final extension at 72°C.SSCP analysis was then carried out by labeling the PCR products with 1 pCi I 2 3 4 5 678 [a-32P]dCTP(SA6000 Ci/mmol;ICN Chemicals,Thames,UnitedKingdom) on a three-cyclePCR, as describedpreviously(27). The exon 2 products were additionally subjected to restriction digestion

@@ with EcoRI (New England Biolabs, Taumus, Gennany) to improve the sensi ‘@•.‘-. w*e@ss@• tivity of the SSCP study by reducing the fragment sizes. The labeled PCR @ products were diluted in denaturing dye and run under two different gel ;‘ ‘ •@t@ @•• @6@1s,s@*= UIIIUI. conditions: 0.5 x mutation detection enhancement (MDE) gel (AT Biochem, Philadelphia, PA) and 8% polyacrylamide gels with 5% glycerol. The mutation detection enhancement gels were run for 12 h at 6 W, and the gels with glycerol were run at 4 W for 16 h, both at room temperature. Gels were Fig. 2. Ethidium bromide-stained agarose gel demonstrating the products of semiquan titative PCR coamplification of an unrelated single-copy gene, the type 3 vasopressin transferred to filter paper, vacuum-dried, and exposed to X-ray film. Nonnal receptor gene (top band), and exon 2 of GDNF (bottom band). Lane 1, PhiXl74/Hinfl controls were run alongside the phaeochromocytoma samples on each gel. marker; Lane 2, control DNA sample; Lanes 3—8,DNA samples from six phaeochromo Samples were sequenced by cloning into pCR2.l vector (TA Cloning ; cytomas. Invitrogen,, theNetherlands)andSequenaseVersion2.0DNA Sequencingkit (USB, Anachem,United Kingdom).At least threeclones from each sample weresequenced. both lysine), most likely representing a neutral polymorphism. This Semiquantitative PCR. Semiquantitative PCR using 28 cycles of ampli variant was not detected in 120 control individuals or in 208 individ ficationwas performedoneach DNA sampleusingthe GDNFexon 2 primers uals with HSCR (this study; Refs. 28 and 29). No abnormal band described previously and a set of primers derived from an unrelated single copy gene coding for the type 3 vasopressin receptor (AlT CTG GGC CTC shifts were observed in any of the remaining samples under either TOT 060 AT andTOC AGC ACG AAC AGO TOC AT). The intensityof SSCP condition. Six random samples were sequenced for each exon, each GDNF PCR product band was compared with that of the coamplified and no mutations were found. vasopressin receptor gene band using densitometry. The absorbance values for Semiquantitative PCR was performed on all 22 samples to deter each were measured using an imaging densitometer (model 05-670; Biorad, mine if there was any evidence of gene amplification. Comparison of Herts, United Kingdom) and the molecular analyst/PC software specific to the the PCR products derived from GDNF exon 2 and those derived from Biorad Image Analysis System. A ratio between the intensities of the two the type 3 vasopressin receptor gene demonstrated equivalent inten bands was obtained for each sample. sities (e.g., Fig. 2). Therefore, it is likely that no genomic amplifica RT-PCR. cDNA (200 ng) derivedfromseven sporadicphaeochmmocyto tion of GDNF has occurred. mas was subjected to RT-PCR using the conditions described previously. The RT-PCR using GDNF exon 2 primers was performed on RNA from GDNF exon 2 primers (0.4 p.M each) and the glyceraldehyde phosphate dehydrogenase gene primers (0.2 @tt@ieach;Ref. 27) were used for coamplifi seven phaeochromocytomas to determine if GDNF is indeed cx cation. The relative intensities of the two bands were measured using densi pressed in these tumors. All seven demonstrated expression (data not tometry as described previously. shown).

RESULTS DISCUSSION

Genomic DNA from 22 sporadic phaeochromocytomas was ana No disease-associated somatic mutations in the coding region of lyzed by SSCP for mutations in the coding sequence of GDNF. One GDNF were found among a series of 22 well-characterized sporadic SsCP variantwas detected under two different conditions (Fig. 1). phaeochromocytomas. In addition, all PCR products were the cx Sequencing of this sample, derived from a benign adrenal phaeochro pected sizes, suggesting no major rearrangements. Gross gene ampli mocytoma, revealed a single-base substitution at nucleotide 519 (nu fication in these tumors is unlikely, although this could not be cx cleotides 1—3areATG), resulting in a silent mutation (AAA to AAG, cluded conclusively because only semiquantitative PCR was used, and

A) B) ACGT

I 2 3 4 5 6 7 8 910 A>GN_ I' @ G I@

Fig. I. A, PCRJSSCP analysis of GDNF exon 2. Samples 1—8,pheochromocytomas; 9, a positive control; 10, a negative control for the assay. Note the aberrant migration pattern of sample2 andthepositivecontrol.B,sequenceofsample2 fromthepreviousgel.A single-basesubstitutioninwhichthe wild-typeAis replacedbya 0 is shown.Seetext. 311

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1997 American Association for Cancer Research. GDNF MUTAT1ONS IN PHAEOCHROMOCYTOMAS insufficient DNA was available for Southern analysis. It is also tyrosine kinase catalytic domain of the RET proto-oncogene is associated with possible that we could have missed a few mutations in the noncoding multiple endocrine neoplasia type 2B. Proc. Natl. Acad. Sd. USA, 91: 1579—1583, I994. regions of this gene. Nevertheless, our data suggest that GDNF plays 6. Eng,C., Smith,D. P., Mulligan,L.M., Healey,C.S., Zvelebil,M.J., Stonehouse, a minor role, at best, in the genesis of phaeochromocytomas, although T. J., Ponder, M. A., Jackson, C. E., Waterfield, M. D., and Ponder, B. A. J. A novel it is expressedinat leastsomeof thesetumors.Interestingly,a similar point mutation in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC. Oncogene, 10: 509—513, conclusion could be drawn from mutation analysis of GDNF in 1995. HSCR. HSCR, a congenital absence of enteric ganglia, is associated 7. Bolino, A., Schuffenecker, I., Luo, Y., Seri, M.. Silengo, M., Tocco, T., Chabrier, 0., Houdent, C., Murat, A., Schlumberger, M., Toumiaire, J., Lenoir, G. M., and Romeo, with inactivating mutations in RET in 10—40%of cases (30, 31). Mice 0. RET mutationsin exons13and 14of FMTC patients.Oncogene,10:2415—2419, null for RET and GDNF lack enteric ganglia (32—35),suggesting that 1995. mutations in RET and GDNF should result equally in human HSCR. 8. Eng. C., Clayton, D., Schuffenecker, I., Lenoir, 0.. Cote, 0., Gagel, R. F.. Ploos van Amstel, H. K., Lips, C. J. M., Nishisho, I., Takai, S-I., Marsh, D. J., Robinson, B. 0., Yet only a single de novo mutation in GDNF was found in a patient Frank-Raue, K., Raue, F., Xue, F., Noll, W. W., Romei, C., Pacini, F., Fink, M., with HSCR in 3 series with a combined total of 314 independent cases Niederle, B., Zedenius, J., Nordenskjöld.M.,Komminoth, P., Hendy, 0. N., Gharib, of familial and sporadic HSCR (28, 29, 36). Four other GDNF H., Thibodeau, S. N., Lacroix, A., Frilling, A., Ponder, B. A. J., and Mulligan, L. M. The relationship between specific RET proto-oncogene mutations and disease phe mutations were detected in two of these series also, but the signifi notype in multiple endocrine neoplasia type 2: Intemational RET Mutation Consor cance remains unclear because these patients seemed to have other tium analysis. J. Am. Med. Assoc., 276: 1575—1579,1996. 9. Latif,F., Tory,K., Gnarra,J., Yao,M., Duh,F-M.,Orcun,M-L.,Stackhouse,T., etiologies to account for their HSCR, e.g., trisomy 21 or the presence Kuzmin, I., Modi, W., Geil, L., Schmidt, L., Thou, F., Weng, Y., Duan, D. R., Dean, of a germ-line RET mutation (28, 36). M.,Glavac,D.,Richards,F.M.,Crossey,P.A.,Ferguson-Smith,M.A.,Le Paslier, Despite the fact that GDNF was a good candidate gene to play a D.,Chumakov,I.,Cohen,D.,Chinault,C.A.,Maher,E.R.,Linehan,W.M.,Zbar, B., and Lerman, M. I. Identification of the von Hippel-Lindau disease tumor sup role in the pathogenesis of phaeochromocytoma, we have found no pressor gene. Science (Washington DC), 260: 1317—1320,1993. disease-associated mutations or gene amplification in 22 sporadic 10. Crossey,P.A.,Richards,F.M.,Foster,K.,Green,J.S.,Prowse,A.,Latif,F.,Lerman, phaeochromocytomas. Given that phaeochromocytoma is an impor M.I.,Thar,B.,Affara,N.A.,Ferguson-Smith,M.A..andMaher,E.R.Identification of intragenic mutations in the Von Hippel-Lindau disease tumour suppressor gene and tant component feature of MEN 2 and VHL, it seemed plausible that correlation with disease phenotype. Hum. Mol. Genet.. 3: 1303—1308.1994. the RET and VHL gene should play a major role in the pathogenesis 11. Wallace, M. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1997 American Association for Cancer Research. Mutation Analysis of Glial Cell Line-derived Neurotrophic Factor (GDNF), a Ligand for the RET/GDNF Receptor α Complex, in Sporadic Phaeochromocytomas

Patricia L. M. Dahia, Sergio P. A. Toledo, Lois M. Mulligan, et al.

Cancer Res 1997;57:310-313.

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