C620R mutation of the murine ret proto-oncogene: Loss of function effect in homozygotes and possible gain of function effect in heterozygotes Luo Yin 1 , Aldamaria Puliti 2 , Elena Bonora 3 , Cecilia Evangelisti 3 , Valerio Conti 4 , Wei-Min Tong 1 , Jean-Jacques Medard 1 , Marie-France Lavou e 1 , Nathalie Forey 1 , Lily C. Wang 1 , Serge Mani e 5 , G erard Morel 6 , Mireille Raccurt 6 , Zhao-Qi Wang 1 and Giovanni Romeo 1,3 * 1 International Agency for Research on Cancer (IARC), Lyon, France 2 Dipartimento di Pediatria and CEBR, University of Genova and Laboratory of Molecular Genetics, G. Gaslini Institute, Genova, Italy 3 Unita’ di Genetica Medica, Dipartimento di Medicina Interna, Policlinico S. Orsola-Malpighi, Bologna, Italy 4 Laboratory of Molecular Genetics and Renal Child Foundation, G. Gaslini Institute, Genova, Italy 5 Laboratoire de G en etique et Cancer UMR 5641, Facult  e de M edecine, Lyon, France 6 CNRS UMR 5123, Universit  e Claude Bernard-Lyon 1, Villeurbanne, France Germline RET mutations are responsible for different inherited dis- orders: Hirschsprung disease (congenital aganglionic megacolon), caused by loss of function mutations, familial medullary thyroid car- cinoma and multiple endocrine neoplasia type 2, caused by gain of function mutations. Intriguingly, some RET mutations, including C620R, are associated with both types of diseases. To investigate the dual role of such RET mutations, a mouse model with a targeted mutation ret C620R was generated. ret C620R/C620R offspring die during the first postnatal day, and show kidney agenesis and intestinal aganglionosis. Decreased outgrowth of the Ret-positive cells was observed in ret C620R/C620R neuronal cell cultures, which is suggestive of an impaired migration, proliferation or survival of the Ret- expressing cells. Electronmicroscopy revealed the absence of mem- brane-bound Ret in ret C620R/C620R cells as compared to ret 1/1 and ret 1/C620R cells. On the other hand, aged ret 1/C620R mice develop pre- cancerous lesions in the adrenal gland or in the thyroid. Our results suggest that the ret C620R mutation has a loss of function effect in homozygotes and exhibits a dominant gain of function effect with low penetrance causing hyperplasia in heterozygotes. ' 2007 Wiley-Liss, Inc. Key words: targeted mutation; mouse model; neural crests; Hirshsprung disease; renal agenesis; multiple endocrine neoplasia type 2 The RET proto-oncogene encodes a receptor tyrosine kinase (TK) 1,2 composed of extracellular, transmembrane and intracellu- lar TK domains. Four neurotrophic factors, the glial cell line- derived neurotrophic factor (GDNF), neurturin, artemin and perse- phin, have been identified as ligands of Ret. 3,4 The interaction between Ret and the aforementioned ligands requires glycosyl- phosphatidylinositol linked glycoprotein receptors as coreceptors 5. Upon ligand-binding, Ret dimerizes, autophosphorylates and activates downstream molecules, such as PLC-g, PI3, Shc, Enigma, Grb2/7/10, Src and Ras-GAP. 4 Embryonic studies showed that Ret is expressed in the central and peripheral nervous systems, as well as in the excretory systems. 6–8 A mouse model with a targeted mutation abrogating the TK function of Ret (ret.k 2/2 ) showed the absence of superior cervical ganglions (SCG), aganglionosis of the gastrointestinal tract and kid- ney dysgenesis or agenesis. 6 Moreover, ret, gdnf and gfra-1 defi- cient mice manifest strikingly similar phenotypes 6,9–12 further con- firming the critical role of Ret-signaling in the development of the enteric nervous system (ENS) and of the urogenital system. Human heterozygous germline mutations in RET are associated with two distinct types of neurocristopathies 13 :(i) a developmental disorder of the ENS, Hirschsprung disease (HSCR, or congenital intestinal aganglionosis); (ii) neural crest-derived malignancies, namely fami- lial medullary thyroid carcinoma (FMTC), and the dominantly inher- ited cancer syndromes multiple endocrine neoplasia type 2A and type 2B (MEN 2A and MEN 2B). Genetic and biological evidences indicated that inactivation mutations of RET cause HSCR, 14–19 whereas mutations resulting in constitutive activation of Ret are re- sponsible for FMTC, MEN 2A and MEN 2B. 20–25 Interestingly, mutations in the cysteine residues 609, 618 and 620 of Ret are associated with both HSCR and FMTC/MEN 2A. 26,27 HSCR is characterized by the absence of parasympathetic intrinsic ganglion cells in the submucosal and myenteric plexuses of the hind gut, which is attributed to the premature arrest of the cranio-caudal migration of neural crest cells towards the distal gastrointestinal tract. 28 The phenotype of MEN 2A, however, is medullary thyroid carcinoma (MTC, a malignant tumor arising from calcitonin-secret- ing thyroid C-cells), pheochromocytoma (usually benign tumor of adrenal chromaffin cell origin) and parathyroid hyperplasia. 29 In vitro studies showed that certain HSCR-associated mutations in the extracellular domain impair the maturation of Ret and inhibit its transport to the cell membrane. 15,16 As a consequence, GDNF, one of Ret ligands, fails to rescue the apoptosis as it does in wild-type Ret-expressing cells. 18,19 Conversely, the typical FMTC/MEN 2A mutations constitutively activate Ret by forming a covalent dimeri- zation, leading to ligand-independent activation of its TK 20,22 Ret mutants at cysteine residues 609, 618 and 620 were shown to have both effects, 30 providing a plausible model for their dual roles in de- velopment and in tumorigenesis. However, the basis of in vitro sys- tems is the overexpression of the Ret protein transfected into cell lines, which do not reproduce the in vivo conditions. Furthermore, there is no indication on how and when the cell fate is determined and why the same mutations cause FMTC, or HSCR, or both FMTC and HSCR in different individuals. To study these aspects, we have generated a mouse model with a targeted mutation corr- esponding to that of RET C620R in humans (referred hereafter as ret C620R ). Material and methods Gene targeting A 5.6 kb Bam HI fragment containing exons 9, 10 and 11 of ret and an upstream 1.2 kb Taq I/Bam HI fragment were isolated from BAC clones of 129/Sv origin encompassing ret 31 and subcloned into Bluescript vector. A point mutation in exon 10 corresponding to human C620R was introduced into the 5.6 kb Bam HI subclone Grant sponsor: EC; Grant number: QLG1-CT-2001-01646; Grant spon- sor: IARC (Lyon). *Correspondence to: Unita’ di Genetica Medica, Dipartimento di Medicina Interna, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy. Fax: 139-05-16-36-4004. E-mail: romeo@eurogene.org Received 24 February 2006; Accepted after revision 14 August 2006 DOI 10.1002/ijc.22378 Published online 19 March 2007 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: DTA, diphtheria toxin A; ENS, enteric nervous system; ES, embryonic stem; FMTC, familial medullary thyroid carcinoma; GDNF, glial cell line-derived neurotrophic factor; HSCR, Hirschsprung disease; MEN 2A, multiple endocrine neoplasia type 2A; MEN 2B, multi- ple endocrine neoplasia type 2B; MTC, medullary thyroid carcinoma; SCG, superior cervical ganglions; TK, tyrosine kinase. Int. J. Cancer: 121, 292–300 (2007) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer