*4.4. Interpretation of Unclassified Variants*

Rare/novel variants whose associated risk was unknown were evaluated through a review of the information available in the following public databases: gnomAD, ClinVar, varsome. In particular, for each variant, the classification in the mutational databases, the frequency in the population databases, the conservation of the substituted amino acids, the results of in silico predictions about the effect of the variant on the protein and the results of any functional assay were evaluated. All databases were last consulted on 15 September 2020. Moreover, reports about these variants possibly present in

the literature were researched and evaluated. Finally, when possible, segregation of the variant in the family was assessed, in particular for variants classified as probably pathogenic and if/when other cases of MTC were present in the family.
