*2.1. Clinical Characteristics and Genetic Test Results*

Among subjects submitted to *RET* analysis in our laboratory, 117/163 (71.8%) had personal and/or family history of MTC (114) or CCH (3), while the remaining had personal and/or family history of other pathologies possibly related to *RET* alterations (PCC, PHPT, intestinal ganglioneuromatosis). The list of cases included in the study is reported in Table S1.

A germline *RET* variant was identified in 49/163 (30.1%) subjects: 15/102 (14.7%) index cases, who underwent a complete gene analysis, and 34/61 (55.7%) relatives of a *RET* carrier, who underwent a targeted search for a known variant. Considering only the 72 patients affected by MTC/CCH, a germline *RET* variant was identified in 23 (31.9%), with 15/16 (93.8%) of these having positive family history and 8/56 (14.3%) of sporadic cases. Finally, considering only patients with CCH, 1/3 (33.3%) carried a variant of *RET*.

Mean age at MTC diagnosis was 44.45 years in patients with germline *RET* variants (pathogenetic or considered likely pathogenetic) and 56.42 years in patients with no variants detected (*p* = 0.010). Among patients with known cancer staging, 42.9% of those with *RET* variants and 36.4% of those with negative analysis had locally advanced disease (T > 1). All four MTC patients who also displayed other manifestations of MEN2 were found to carry a pathogenic germline *RET* variant.
