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Monitoring mice carrying the RET C620R mutation

Germline RET mutations are responsible for different inherited disorders: Hirschsprung disease (congenital aganglionic megacolon), caused by loss of function mutations, familial medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasia type 2 (MEN2A), caused by gain of function mutations. Intriguingly, some RET mutations, including C620R, are associated with both types of diseases. In order to investigate the dual role of such RET mutations, a mouse model with a targeted mutation retC620R was generated. retC620R/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 retC620R/C620R neuronal cell cultures, which is suggestive of an impaired migration, proliferation, or survival of the Ret-expressing cells. Electronmicroscopy revealed the absence of membrane-bound Ret in retC620R/C620R cells as compared to ret+/+ and ret+/C620R cells. On the other hand, aged ret+/C620R mice develop pre-cancerous lesions in the adrenal gland or in the thyroid. In humans, RET+/C620R is associated with FMTC and MEN 2A. In mice, however, the same genotype does not produce the above phenotypes.

It is worth noting that mice carrying the heterozygous MEN2B-specific mutation M918T (ret+/M918T) obtained by replacing the endogenous wild-type copy of the gene by ES mediated homologous recombination, display merely C-cell hyperplasia (CCH; 55%) and nodular chromaffin cell hyperplasia (16-17%). In addition, when the M918T mutation was targeted to be expressed under the calcitonin gene promoter specifically in thyroid C-cells, in transgenic mice, nodular CCH was observed only in some mice from 8 months on and MTC was detected in 13% of mice from the age of 11 months on. However, no thyroid dysplasia was observed in transgenic mice carrying the wild-type ret allele or non-transgenic controls of same age. MEN 2B manifests a more complex phenotype than MEN 2A and RETM918T has an in vitro tumorigenic capacity much higher than RETC620R. It is therefore not surprising that the C620R mutation in heterozygous mice is not fully capable of transforming the neural crest derivatives.

An increase in C-cell hyperplasia, was observed in 48.4% of ret+/C620R mice of 129/Sv background versus the 22.8% of ret+/+ mice. Although the differences in frequency of C-cell hyperplasia between the two groups do not reach statistical significance (P= 0.077), probably because of the relatively small number of observations allowed by the material available, they suggest that the C620R mutation is predisposing to tumor formation only at late stages in mice. In a recent work by Carniti and coworkers no C-cell hyperplasia or tumor formation was detected in heterozygous mice carrying the same C620R mutation, which were analysed at earlier stages, whereas results similar to ours were reported for the aganglionosis found in the homozygous mice.

It is quite interesting that ret+/C620R mice, in both the129/Sv and 129/Sv X C57BL/6 backgrounds, develop thyroid dysplasia and adenoma, and adrenal gland cortical nodular regeneration or hyperplasia. The above result indicates that the retC620R mutation also causes susceptibility to some cancers of epithelial origin, which are not associated with the human equivalent genotype. How the retC620R transforms the mouse epithelium, which is normally devoid of Ret expression, remains to be elucidated.

In conclusion, we have observed that (1) the retC620R mutation in homozygotes displays a loss of function effect, manifested by the lack of mature Ret protein on the cell membrane, a growth defect of the retC620R/C620R neuronal primary cultures, and consequent total intestinal aganglionosis and kidney agenesis; (2) the retC620R mutation in heterozygotes exerts a possible dominant gain of function effect with low penetrance, which might lead to pre-cancerous lesions in the thyroid and/or adrenal gland.

The low penetrance of these pre-cancerous lesions in ret+/C620R mice suggests that there is a requirement for additional factors, genetic as well as environmental, in order to develop the complete tumour phenotype. Therefore, further studies of ret+/C620R mice will hopefully provide clues for the identification of these additional components.

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UNIVERSITY OF BOLOGNA
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