In silico and in vitro analysis of rare germline allelic variants of RET oncogene associated with medullary thyroid cancer

Abstract

Germline and somatic RET oncogene mutations are found in 98% hereditary and 40% sporadic medullary thyroid carcinomas. Our aim was to analyse by in silico and in vitro assays the transforming activity of six rare RET mutations (T338I, V648I, M918V, A883T, S904F and M848T). Six known RET mutations were used as controls. The in silico analysis showed the highest score value (i.e. 65) for S904F, M848T, M918T and C634R, whereas L790F, G691S, T338I and V648I had 0 score. Intermediate score values were obtained by A883T (scoreZ55), M918V, V804M and Y791F (scoreZ15). The in vitro focus formation assay showed that cells transfected with S904F, M918T, M848T or C634R generated the largest number of focus formation units (FFU). Intermediate numbers of FFU were observed in cells transfected with M918V, V804M, Y791F or A883T, while cells transfected with L790F, G691S, T338I or V648I showed a number of FFU similar to control cells. A positive correlation between the in silico score and in vitro FFU was found (PZ0.0005). Only cells transfected with M918T or C634R grew faster and generated higher number of colonies in soft agar than control cells. However, the cells that were transfected with V804M produced an intermediate number of colonies. In conclusion, two of the six rare RET mutations, S904F and M848T possessed a relatively high transforming activity but a low aggressiveness; the other four mutations T338I, V648I, M918V and A883T were low or non-transforming, and their ability to induce tumoural transformation might be related to particular genetic conditions.

New insights in the molecular signature of advanced medullary thyroid cancer: evidence of a bad outcome of cases with doubleRETmutations

“…This observation is supported by biological studies showing that M918T point mutations have the highest transforming activity and are thus associated with very aggressive tumours, such as those present in MEN 2B;11 12 mutations at codon 634, although lower in number than M918T, also have a very high transforming ability that is correlated with the rather aggressive behaviour of MTC in MEN 2A 11 13. Nevertheless, no specific recommendations on the need to investigate somatic mutations in sporadic MTC have been provided by the guidelines.…”

2012 European Thyroid Association Guidelines for Genetic Testing and Its Clinical Consequences in Medullary Thyroid Cancer

“…In other cases, rare germline RET mutations have been identified in association with MTC in individual patients with no other clinically affected relatives and characterized by a very low or null transforming activity. For these reasons, their causative involvement in malignant transformation has been questioned [51,52,53]. …”

“…When no germline mutations are found in the 8 exons most frequently affected, but the familial history is strongly suggestive of a hereditary form, an effort should be made to identify a possible association with rare or unknown germline RET mutations [51,52,53]. For this purpose, a search for RET mutations in the remaining 13 exons, rarely but possibly affected by mutations, should be performed before defining the case as an RET -negative hereditary case.…”

“…Many reports indicate that 7–10% of ‘apparently’ sporadic forms are actually misdiagnosed hereditary cases because of the presence of a germline mutation despite a negative family history [23,71,72]. The majority of hereditary MTC discovered by genetic screening represents FMTC, the prevalence of which has been found to be much higher than previously reported, and nowadays FMTC is the most common form of hereditary MTC in many series [23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63]. …”

Análise do gene CDKN1B/p27kip1 em pacientes com neoplasia endócrina múltipla tipo 2

“…Há cinco polimorfismos comuns nesse gene com frequências alélicas menores que 1% e que estão localizados nas regiões dos éxons hotspots, que estão associados com a doença CMT/NEM2: p.G691S, p.L769L, p.S836S, p.S904S, e c.2608-24G>A (Ceccherini et al, 1994;Gimm et al, 1999;Elisei et al, 2004;BaumgartnerParzer et al, 2005 confirmando-se assim que esta variante é um SNP (Cosci et al, 2011).…”