Given that the majority of rare missense mutations have little or no functional impact on protein function (Boyko et al., 2008; Yampolsky et al., 2005), a clinical laboratory geneticist on our team who was blinded to participant phenotypes classified each of the 24 observed MC4R variants according to current clinical guidelines (Richards et al., 2015), integrating information from population allele frequency data, computational prediction and conservation scores, functional assay data, and prior reports of the variant segregating with obesity. 4 of these 24 variants met these clinical criteria as pathogenic or likely pathogenic for monogenic obesity, including the p.Tyr35Ter premature stop codon noted above, an inactivating frameshift mutation (p.Phe280AlafsX12), and two missense mutations – p.Arg165Gln and p.Glu61Lys – previously shown to segregate with obesity in family studies and impair receptor activity in functional assays. A summary of the evidence used to classify each of the 24 variants is provided in Table S4.
