Carol Houghton

Germline mutation of RET codon 883 in two cases of de novo MEN 2B.

Germline mutations in the RET proto-oncogene are seen in the majority of patients with the dominantly inherited cancer syndromes multiple endocrine neoplasia type 2 (MEN 2). The clinical subtypes of MEN 2 (MEN 2A, MEN 2B and familial MTC) all have medullary thyroid carcinoma, but vary in the involvement of pheochromocytoma, parathyroid adenoma/hyperplasia and developmental abnormalities. A single RET mutation, resulting in the substitution M918T, has been identified in 94% of cases of MEN 2B (which consists of MTC, pheochromocytoma and developmental abnormalities). Here we report the identification of a new germline RET mutation (A883F) in two de novo cases of MEN 2B. Identification of this new mutation will contribute to understanding the molecular basis of MEN 2B, and will assist in the clinical management of families harbouring this mutation.

Mutations in the RET proto-oncogene and the von Hippel-Lindau disease tumour suppressor gene in sporadic and syndromic phaeochromocytomas.

Phaeochromocytomas may occur sporadically, or as part of the inherited cancer syndromes multiple endocrine neoplasia (MEN) type 2, von Hippel-Lindau disease (VHL), and, rarely, in type 1 neurofibromatosis. In MEN 2, germline missense mutations have been found in one of eight codons within exons 10, 11, 13, 14, and 16 of the RET proto-oncogene. In VHL, germline mutations within one of the three exons are responsible for the majority of cases. To determine if somatic mutations similar to those seen in the germline in MEN 2 or VHL disease play a role in the pathogenesis of sporadic or familial phaeochromocytomas, we analysed 48 sporadic tumours and tumours from 17 MEN 2 and five VHL patients for mutations in RET exons 9, 10, 11, 13, 14, 15, and 16, and the entire coding sequence of VHL. Five of 48 sporadic phaeochromocytomas had RET mutations within exons 10, 11, and 16. Of these, one was proven to be germline and two were proven to be somatic mutations. Four of 48 had VHL mutations; these included both the bilateral cases in the series (one was proven to be a germline mutation) and two others, of which one was proven somatic.

RET alternate splicing influences the interaction of activated RET with the SH2 and PTB domains of Shc, and the SH2 domain of Grb2

Activating germline mutations of the RET receptor tyrosine kinase are found in the majority of cases of inherited cancer syndrome MEN 2, and inactivating mutations in some cases of dominantly inherited Hirschsprung disease. Using RET activated by a MEN 2 mutation, we show that both the SH2 and PTB domains of the adaptor protein Shc interact with RET, and we identify the PTB domain interaction site. Interaction with both the SH2 and PTB domains of Shc contributes to the transcriptional activation of a serum response element. RET alternate splicing affects the strength of interaction with both the Shc SH2 and PTB domains. In addition, a splice isoform-specific HSCR missense mutation, which does not inactivate the RET kinase activity, decreases the strength of the PTB domain interaction and the level of RET-dependent Shc phosphorylation.

Cellular inheritance of a Cre‐activated reporter gene to determine paneth cell longevity in the murine small intestine

Here, we exploit an absolute differential between stem and progeny cells in their ability to express Cre from a somatically inducible transgene to determine the longevity of intestinal Paneth cells. In the Ahcre transgenic line induction of Cre recombinase allows constitutive activation of a Cre‐activated reporter in intestinal precursors but not in Paneth cells. The time taken for Paneth cells to inherit the reporter (EYFP) was measured in adult Ahcre/R26R‐EYFP animals. Using confocal microscopy of TOPRO‐3–stained sections, both precursors and Paneth cells were identified and subsequently scored for EYFP expression. It takes up to 57 days for Paneth cells to inherit the reporter, making them three times longer‐lived than previously indicated using nucleotide incorporation and suggesting that such determinations of cell turnover may be significant underestimates. Developmental Dynamics 233:1332–1336, 2005.

C-cell and thyroid epithelial tumours and altered follicular development in transgenic mice expressing the long isoform of MEN 2A RET

Gain-of-function mutations in the gene encoding the receptor tyrosine kinase RET have been identified as the aetiological factor for multiple endocrine neoplasia type 2A (MEN2A). MEN2A is a dominantly-inherited cancer predisposition syndrome characterized by medullary thyroid carcinoma, a tumour of the calcitonin-producing thyroid C-cells. There are three isoforms of RET: RET9, RET43 and RET51, and although in vitro evidence suggests they vary in cellular transformation activities, little is known about their function in tumorigenesis in vivo. To address this, we used RET51 cDNA to construct mice in which the most frequent MEN2A mutation, Cys-634-Arg, was expressed under the control of the human calcitonin promoter (CT-2A mice). These mice developed C-cell tumours resembling human MTC and follicular tumours resembling human papillary thyroid carcinoma (PTC) depending on the founder line examined. One founder line developed compound MTC/PTC at low frequency (8%) and pancreatic cystadenocarcinoma. CT-2A mice also displayed a developmental defect in thyroid follicular structure, in which much of the thyroid was occupied by large irregular cystic follicles thought to be derived from the ultimobranchial body, a developmental precursor of the thyroid gland. The CT-2A mice will provide a suitable model to further study the effects of the MEN 2A RET mutation in vivo.

Mutation analysis of the c-mos proto-oncogene and the endothelin-B receptor gene in medullary thyroid carcinoma and phaeochromocytoma.

The characteristic tumours of MEN 2 are medullary thyroid carcinoma (MTC) and phaeochromocytoma. Somatic RET mutations have been found in only 23-40% of sporadic MTC and 10% of sporadic phaeochromocytomas. Thus, we sought other genes which may play a role in the pathogenesis of these tumours. We carried out direct sequence analysis of human c-mos and human ENRB in a series of sporadic MTC and phaeochromocytomas to determine if somatic mutations in these two genes could account for some of the sporadic MEN 2-related tumours in which no RET mutations are detected. No somatic mutations were found.

Characterization of a heat resistant ß-glucosidase as a new reporter in cells and mice

BackgroundReporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic β-glucosidase.ResultsSYNbglA was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells. SYNbglA expression can be localized in situ or detected quantitatively in colorimetric assays and can be co-localized with E. coli β-galactosidase. Further, we have generated a Cre-reporter mouse in which SYNbglA is expressed following recombination to demonstrate the general utility of SYNbglA for in vivo analyses. SYNbglA can be detected in tissue wholemounts and in frozen and wax embedded sections.ConclusionsSYNbglA will have general applicability to developmental and molecular studies in vitro and in vivo.