*4.3. RET Analysis*

Genomic DNA was isolated from peripheral blood-EDTA using the QIAmp DNA Blood Mini Kit according the manufacturer's protocol (Qiagen, Valencia, CA, USA).

From 2002 to June 2019, sequencing of exons 5, 8, 10, 11, 13, 14, 15 and 16 of *RET* (RefSeq.NM\_020975.5) was performed through bidirectional Sanger sequencing: briefly, PCR amplifications of target exons were carried out with FastStartTaq DNA polymerase (Roche, Basel, Switzerland), followed by standard dideoxy sequencing, and run on a ABI3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA). PCR and sequencing conditions, as well as the primer sequences, are available upon request. Chromatograms were analyzed for variants using the software Sequencer (Gene Code Corporation, Ann Arbor, MI, USA). According to the ARUP database as of January 2020 (https://arup.utah.edu/database/MEN2/MEN2\_display.php) exons 5, 8, 10, 11, 13, 14, 15 and 16 cover all but one (Exon 7: c.1513\_1518delGAGGGG: p.E505\_G506del) of the *RET* mutations described in the literature.

Since July 2019, the entire RET cds (20 exons) has been included in an NGS panel (IAD177392-ThermoFisher Scientific) comprising 27 genes related to MEN2, pheochromocytoma and renal carcinoma and run on an Ion S5 next-generation sequencing system followed by analysis with the Ion Reporter Software (ThermoFisher Scientific, Waltham, MA, USA). The mean amplicon coverage and the target base coverage at > 100x for multiple experiments were > 800x and > 97%, respectively: these parameters guarantee a sensitivity of > 99% for the test. All the variants of interest were confirmed through Sanger sequencing.

Overall, 157 patients underwent Sanger sequencing analysis and 6 the NGS panel analysis.
