Functional analysis of NPR2 variants supports the therapeutic rationale for CNP in short stature.

Human height is a complex trait regulated by genetic and environmental factors. Several components of chondrocyte proliferation and differentiation regulatory pathways are implicated in short-stature disorders; one such component is natriuretic peptide receptor 2 (NPR2), the receptor for C-type natriuretic peptide. We developed a high-throughput GFP reporter assay to evaluate the functional impact of NPR2 missense variants on receptor activity, which was utilized alongside a commercially available CatchPoint assay, and previously published data. Measurements from these sources were integrated and annotated, providing an "activity score" for each variant, based on reduced or increased receptor activity. Unlike variant effect prediction tools that assign scores from benign to pathogenic, our method enabled us to distinguish loss-of-function and gain-of-function variants, categorizing 245 NPR2 missense variants, including 47 loss-of-function, 34 partial loss-of-function, and 14 gain-of-function variants, across the functional domains of NPR2. Activity scores showed a near-linear association with standing height (Pearson's correlation R2 = 0.438, p = 5.8 × 10-10), with the effects additive and apparent across polygenic backgrounds. Moreover, phenome-wide association analysis demonstrated that NPR2 activity was significantly associated only with height and height-associated traits, suggesting that modulating NPR2 activity may modulate height only. To summarize, the relationship between NPR2 activity and height resembles a dimmer switch, whereby subtle variations in activity cause subtle variations in height. We demonstrate the therapeutic potential of modulating NPR2 activity in short-stature conditions, owing to both its strong association with height and its lack of association with traits unrelated to height.