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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 9 3025-3028
Copyright © 1997 by The Endocrine Society

Original Studies

Mutation Analysis of Glial Cell Line-Derived Neurotrophic Factor, a Ligand for an RET/Coreceptor Complex, in Multiple Endocrine Neoplasia Type 2 and Sporadic Neuroendocrine Tumors

Debbie J. Marsh, Zimu Zheng, Andrew Arnold1, Scott D. Andrew, Diana Learoyd, Andrea Frilling, Paul Komminoth, Hartmut P.H. Neumann, Bruce A.J. Ponder2, Barrett J. Rollins, Geoffrey I. Shapiro, Bruce G. Robinson, Lois M. Mulligan and Charis Eng3

Department of Adult Oncology (D.J.M., Z.Z., B.J.R., G.I.S., C.E.) and Human Cancer Genetics Unit (D.J.M., Z.Z., C.E.), Dana-Farber Cancer Institute, Department of Medicine (D.J.M., Z.Z., A.A., B.J.R., G.I.S., C.E.), Harvard Medical School, Boston, MA; Laboratory of Endocrine Oncology (A.A.), Massachusetts General Hospital, Boston, MA; Molecular Genetics Laboratory, Kolling Institute for Medical Research (S.D.A., D.L., B.G.R.), Royal North Shore Hospital, University of Sydney, St. Leonards, New South Wales, Australia; Department of Surgery (A.F.), Eppendorf University of Hamburg, Hamburg, Germany; Department of Pathology (P.K.), University of Zurich, Zurich, Switzerland; Division of Nephrology (H.P.H.N.), Department of Internal Medicine IV, Albert-Ludwigs University of Freiburg, Freiburg, Germany; CRC Human Cancer Genetics Research Group (B.A.J.P.), University of Cambridge, Cambridge, UK; and Departments of Pathology and Paediatrics (L.M.M.), Queen’s University, Kingston, Ontario, Canada

Address all correspondence and requests for reprints to: Charis Eng, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115-6084.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Causative germline missense mutations in the RET proto-oncogene have been associated with over 92% of families with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). MEN 2A is characterized primarily by medullary thyroid carcinoma (MTC) and pheochromocytoma, both tumors of neural crest origin. Parathyroid hyperplasia or adenoma is also seen in MEN 2A, but rarely in MEN 2B, which has additional stigmata, including a marfanoid habitus, mucosal neuromas, and ganglioneuromatosis of the gastrointestinal tract. In familial MTC, MTC is the only lesion present. Somatic RET mutations have also been identified in a subset of sporadic MTCs, pheochromocytomas, and rarely, small cell lung cancer, but not in sporadic parathyroid hyperplasias/adenomas or other neuroendocrine tumors. Glial cell line-derived neurotrophic factor (GDNF) and its receptor molecule GDNFR-{alpha}, have recently been identified as members of the RET ligand binding complex. Therefore, the genes encoding both GDNF and GDNFR-{alpha} are excellent candidates for a role in the pathogenesis of those MEN 2 families and sporadic neuroendocrine tumors without RET mutations. No mutations were found in the coding region of GDNF in DNA samples from 9 RET mutation negative MEN 2 individuals (comprising 6 distinct families), 12 sporadic MTCs, 17 sporadic cases of parathyroid adenoma, and 10 small cell lung cancer cell lines. Therefore, we find no evidence that mutation within the coding regions of GDNF plays a role in the genesis of MEN 2 and sporadic neuroendocrine tumors.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GERMLINE MISSENSE mutations in the RET proto-oncogene, which encodes a tyrosine kinase receptor, have been identified in over 92% of families who have the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) (1). MEN 2 is characterized by tumors of neuroendocrine origin, specifically medullary thyroid carcinoma (MTC) and pheochromocytoma [reviewed by Eng (2)]. In familial MTC (FMTC), MTC is the only tumor present. In classic MEN 2A, MTC occurs in combination with pheochromocytoma and parathyroid hyperplasia or adenoma. MEN 2B patients develop identical lesions to those found in MEN 2A, with the exception of parathyroid hyperplasia or adenoma, while also manifesting characteristic abnormalities, including a marfanoid habitus, mucosal neuromas, and ganglioneuromatosis of the gastrointestinal tract (3).

Although clearly being components of MEN 2, MTC, pheochromocytoma, and parathyroid hyperplasia or adenoma occur as sporadic tumors in the majority of cases (4, 5, 6). Somatic RET mutations have been identified in sporadic MTCs at frequencies varying between 23–86% depending on the sample size and population studied (7, 8, 9, 10, 11, 12, 13, 14). In sporadic pheochromocytoma, somatic RET mutations, and mutations in the gene for von Hippel-Lindau disease (VHL), have been identified in approximately 10% and 1.5% of tumors, respectively (6, 7, 15, 16, 17, 18, 19). Although RET is expressed in the developing parathyroid glands and parathyroid tumors, sequence analysis of the gene has yet to reveal a somatic mutation (20, 21, 22, 23, 24). A similar situation pertains in many other sporadic neuroendocrine tumors such as pituitary tumors and melanoma (23). However, 2 of 60 small cell lung carcinoma (SCLC) cell lines have been shown to have a non-MEN 2-type RET exon 11 mutation (25, 26). Clearly, other etiologies need to be sought.

Recently, glial cell line-derived neurotrophic factor (GDNF), mapping to chromosome 5p13.1-p13.3 (27, 28), and its receptor molecule GDNFR-{alpha} have been shown to participate in RET activation (29, 30, 31). GDNF belongs to the transforming growth factor-ß superfamily and is known to enhance the survival of midbrain dopaminergic neurons (32). Currently, it is believed that GDNF binds GDNFR-{alpha}, which in turn binds RET to form a heterohexameric complex. Because GDNF and GDNFR-{alpha} appear to be involved in the RET signaling pathway, their genes represent excellent candidates for roles in the genesis of sporadic neuroendocrine tumors and RET-negative MEN 2 families.

In this study, we examined RET mutation negative MEN 2 patients, sporadic MTCs, parathyroid adenomas, and SCLC lines to determine whether mutations in the exons of GDNF are involved in their pathogenesis.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients and tumor specimens

The diagnosis of MEN 2 was made according to the classical criteria and those of the International RET Mutation Consortium (1, 33). Constitutive DNA from affected individuals from 6 unrelated MEN 2 families without RET mutation, 2 MEN 2A, 2 MEN 2B and 2 FMTC, was analyzed for mutations in GDNF. Patients with sporadic tumors were those without other family members with neuroendocrine tumors and without known histories of MEN 1, MEN 2, or VHL as outlined previously (6). A total of 39 sporadic neuroendocrine tumors, 12 MTC, 17 parathyroid adenoma, and 10 SCLC lines without RET mutation were analyzed. Sporadic pheochromocytomas have been analyzed for GDNF mutations and reported elsewhere (34, 35). The following SCLC lines, shown previously to lack wild-type RB1, and retain a normal p16Ink4A gene (36, 37, 38), were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 supplemented with 10% FCS, penicillin, and streptomycin: NCI-H82 (HTB-175), NCI-H69 (HTB-119), NCI-H128 (HTB-120), NCI-H446 (HTB-171), NCI-H146 (HTB-173), NCI-H187 (CRL-5804), NCI-H378 (CRL-5808), NCI-N417 (CRL-5809), NCI-H526 (CRL-5811) and NCI-H889 (CRL5817).

All work with human samples has been approved by the IRB under Dana-Farber Cancer Institute Protocol 94–138.

DNA extraction and PCR amplification of GDNF

Constitutional DNA was obtained by extraction from the patients’ peripheral blood leukocytes using standard procedures (39). High molecular weight DNA was obtained from tumor tissue by phenol-chloroform extraction and ethanol precipitation (39).

All PCR products were generated using a Touchdown Thermal Cycler (Hybaid Ltd., Middlesex, UK). A 186-nucleotide PCR product corresponding to the first exon of GDNF was PCR amplified using the oligonucleotide primers GDNF-X1FL (5'-TGTAAAACGACGGCCA-GTTGAAGTTATGGGATGTCGTGGCTGTCT-3') and GDNF-X1R(5'C-AGGAAACAGCTATGACCAGTCACTGCTCAGCGCGAAGG-3') and the following protocol: 95 C x 5 min followed by 35 cycles of 95 C x 1 min, 61 C x 1 min, 72 C x 1 min, and a final extension at 72 C for 10 min. The final concentration of PCR reagents were as follows: 0.8 µM of each primer; 0.2 mM of each deoxynucleotide triphosphate, 1x PCR buffer containing 1.5 mM MgCl2 (Perkin-Elmer Corp., Norwalk, CT) and 2 U Taq polymerase (Perkin-Elmer Corp.) in combination with TaqStart Antibody used as recommended by the manufacturer (Clontech Laboratories, Palo Alto, CA). Exon 2 of GDNF was PCR amplified using the primers GDNF-X2F (5'-TGTAAAACGACGGCCAGTTCCTAGAAGAGAGCGGAATCG-3') or GDNF-2F (5'-TGTAAAACGACGGCCAGTAAATATGCCAGAGGATTATCCTGA-3') and GDNF-3R (5'-CAGGAAACAGCTATGACCCAGATACATCCACACCTTTTAGCG-3') to generate a PCR product of 454 or 485 nucleotides, respectively. The PCR conditions used were similar to those for exon 1 except for annealing at 60 C for 30 sec and a final concentration of 0.4 µM of each primer. All forward primers contained -21 m13F and reverse primers -21 m13R sequences attached to the 5' ends. All PCRs were performed in a final reaction volume of 50 µl.

GDNF sequencing

Cycle sequencing was performed using the ABI Prism Dye Primer Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.) and the GeneAmp PCR System 9600 (Perkin-Elmer Corp.). The products of cycle sequencing were electrophoresed on a 6% Long Ranger gel (FMC Bioproducts, Rockland, ME) and analyzed on an Applied Biosystems automated DNA sequencer (Perkin-Elmer Corp.).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Sequence analysis of both exons of GDNF in genomic DNA from 6 unrelated RET mutation-negative MEN 2 individuals did not identify any sequence variants (Table 1Go). Similarly, no GDNF mutations were detected in 12 sporadic MTCs, 17 sporadic parathyroid adenomas, and 10 SCLC cell lines.


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  		Table 1. GDNF mutation analysis in RET mutation-negative MEN 2 families and sporadic neuroendocrine tumors

 

   Discussion
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
No GDNF mutations were detected in 6 unrelated MEN 2 families and 39 sporadic MEN 2-related and other neuroendocrine tumors. In addition, because PCR product sizes were those expected, it is unlikely that gross rearrangements occurred. However, mutations in the promoter region, which is not well characterized, or deep within the intron, would not have been detected in this study.

With recent data demonstrating that GDNF acts as a natural ligand for RET (30, 31, 40), and that the GDNF knockout mouse has a similar phenotype to that for ret, GDNF became an obvious candidate to play a major role in both Hirsch-sprung disease (HSCR) and those MEN 2 families and sporadic related tumors without RET mutations. The major susceptibility gene for HSCR, a disease caused by the congenital absence of enteric neurons (41), is RET (42, 43, 44, 45). True to predictions, the phenotype of the murine knockout model for GDNF is absence of enteric ganglia and renal agenesis (46, 47, 48). Yet, contrary to these data, only 6 of 315 HSCR cases (<2%) had germline GDNF mutations (49, 50, 51).

Mutations of GDNF that cause either gain or loss of function could lead to neoplasia, specifically, MEN 2, or sporadic neuroendocrine tumor. Gain of function mutations could be involved in at least three situations: 1) increased expression, which has been excluded in sporadic pheochromocytomas (34); 2) gene amplification, which, again has been excluded in sporadic pheochromocytomas (34); and 3) missense mutations, which lead to possible better fit of ligand to its receptor and coreceptor. Loss of function mutations might be plausible if we postulate that more than one ligand exists for RET; differential use of competing ligands could lead to a pathological response and hence, neoplasia. A GDNF-like molecule has been shown to exist (52). Interestingly, of 45 sporadic pheochromocytomas without somatic or germline RET or VHL mutations in two series, one was found to have a GDNF R93W mutation that turned out to be germline in origin (34, 35). This is the identical mutation found in two HSCR cases (49, 50). Although somewhat puzzling given the current state of knowledge, this observation could lend some credence to the tissue-specific, differential ligand usage theory.

The current study suggests that if mutation of GDNF were to play a role in the development of MEN 2, its component sporadic tumors, and other endocrine tumors originating from the neural crest, as seems to be the case in HSCR, its role is indeed a subtle one. The very small number of polymorphisms, recognized as conservative amino acid substitutions, provide evidence that sequence alterations in this gene are very infrequent (34, 49, 50). It is perhaps not surprising because of the size of the coding region. Another possibility is that heterozygous activating mutations in GDNF may be highly deleterious to the developing human embryo, resulting in embryonic lethals. This is plausible in view of the widespread expression of GDNF. Whether GDNFR-{alpha}, the third known component of the RET/coreceptor/ligand complex, will harbor mutations resulting in the activation of RET is currently being explored.


   Acknowledgments
 
We gratefully acknowledge Anastasia Satterfield and Anne-Louise Richardson for assistance and Sharice Abdillahi for administrative support.


   Footnotes
 
1 Recipient of an American Cancer Society Faculty Research Award. Back

2 A Gibb Fellow of the Cancer Research Campaign. Back

3 Lawrence and Susan Marx Investigator in Human Cancer Genetics and a Patterson Fellow and is also supported by the Markey Charitable Trust, the Charles A. Dana Foundation, the American Cancer Society (RPG-97–064-01 VM), and the Harvard Nathan Shock Center of Excellence Award in the Basic Biology of Aging (1P30AG13314–01). Back

Received February 24, 1997.

Accepted May 30, 1997.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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