A simple, cost efficient assay for assessing the functional impact of single and multi-gene variant combinations

Obesity is a major global health burden driven by both genetic and environmental factors. While monogenic forms are rare, their identification is essential given the availability of targeted therapies. However, genetic testing frequenctly uncovers many variants of unknown significance (VUS), highlighting the need for functional assays to enable precision therapeutic decisions. Here, we describe a simple and cost-effective in vitro assay enabling high-throughput functional analysis of genetic variants, including combinations across different genes. As a proof of concept, we applied this platform to systematically assess the most common LEP and LEPR variants based on gnomAD v2.1.1 allele frequencies and benchmarked them against known pathogenic controls. In total, we assessed 35 LEP variants (including 3 controls) and 30 LEPR variants (including 3 controls), measuring in total 2,100 unique variant combinations. This approach identified 19 VUS to be putatively pathogenic. Importantly, 806 combinations of LEP and LEPR variants coexpressed with their respective wild type allele exceeded pathogenicity thresholds, revealing a potential digenic mutational burden. In sum, this assay offers a scalable strategy for the functional characterization of obesity-associated variants and offers a valuable tool for on-demand VUS interpretation in clinical diagnostics.