After 20 Years, RET genetic screening still identifies new germline and somatic mutations

Objectives: In the last 20 years we performed RET genetic screening in more than 1000 MTC patients either hereditary or sporadic. Methods: RET genetic screening was performed in DNA extracted from blood and/or tissue by direct sequencing. TA cloning was performed to characterize new mutations and deletions. Site-directed mutagenesis, focus formation and soft agar assays were performed to test in vitro the activity of the new mutations. The Align GVGD program was employed for the in silico analysis. Results: in the last year we identified 3 MTC patients with new RET alterations. The first case had a 7bp “somatic” in frame deletion in exon 11 encompassing codon 629-631. The second case showed the simultaneous presence of a “somatic” E616Q mutation in exon 10 and a “somatic” C630G mutation in exon 11 on different alleles. Moreover, in the same patient, we found an alternative splicing causing the in frame skip of exon 10 in the allele carrying the C630G mutation. The third case harboured a new “germline” mutation(E632K in exon 11) although the MTC was apparently sporadic. According to the in vitro and the in silico tests, both E616Q and E632K RET mutations were not transforming while the C630G RET mutation showed a high transforming activity. Conclusions: 1) RET genetic screening should be performed by sequencing analysis in all MTC patients to detect also new RET mutations that would be missed when looking only at the “hot spot” mutations; 2) all new mutations must be evaluated by in silico and/or in vitro analysis to define their transforming ability since in some cases they may be inactive mutations.