Objective: Hyperphagia and early-onset, severe obesity are clinical characteristics of rare melanocortin-4 receptor (MC4R) pathway diseases due to loss-of-function (LOF) variants in genes comprising the MC4R pathway. In vitro functional characterization of 12,879 possible exonic missense variants from single-nucleotide variants (SNVs) of LEPR, POMC, and PCSK1 was performed to determine the impact of these variants on protein function.
Methods: SNVs of the three genes were transiently transfected into cell lines, and each variant was subsequently classified according to functional impact. We validated three assays by comparing classifications against functional characterization of 29 previously published variants.
Results: Our results significantly correlated with previously published pathogenic categories (r = 0.623; P = 3.03 × 10-4) of all potential missense variants arising from SNVs. Of all observed variants identified through available databases and a tested cohort of 16,061 patients with obesity, 8.6% of LEPR, 63.2% of PCSK1, and 10.6% of POMC variants exhibited LOF, including variants currently classified as a variant of uncertain significance (VUS).
Conclusions: The functional data provided here can assist in the reclassification of several VUS in LEPR, PCSK1, and POMC and highlight their impact in MC4R pathway diseases.
Keywords: Functional assays; melanocortin signaling; monogenic obesity; variants; variants of uncertain significance.