Population-scale datasets of healthy individuals capture genetic and environmental factors influencing gene expression. The expression variance of a gene of interest (GOI) can be exploited to set up a quasi loss- or gain-of-function “in population” experiment. We describe here an approach, huva (human variation), taking advantage of population-scale multi-layered data to infer gene function and relationships between phenotypes and expression. Within a reference dataset, huva derives two experimental groups with LOW or HIGH expression of the GOI, enabling the subsequent comparison of their transcriptional profile and functional parameters. We demonstrate that this approach robustly identifies the phenotypic relevance of a GOI allowing the stratification of genes according to biological functions, and we generalize this concept to almost 16,000 genes in the human transcriptome. Additionally, we describe how huva predicts monocytes to be the major cell type in the pathophysiology of STAT1 mutations, evidence validated in a clinical cohort. Display omitted •Human variation can be exploited to generate gain- or loss-of-function experiments•Huva was used to predict the function of genes central to the immune response•Transcriptome-wide huva analysis uncovers the role of STAT1 in monocytes•Huva is implemented in R as well as accessible via an easy-to-use web interface Clinical genetics; Pathophysiology; Human genetics.