SPRED1 variants reveal differential impacts on signaling dynamics

Germline loss-of-function mutations in SPRED1 impair its negative regulatory function within the RAS-MAPK signaling pathway, leading to pathway hyperactivation. Clinical sequencing of SPRED1 frequently identifies variants of uncertain significance (VUS) in patients suspected of Legius syndrome (LS), yet the functional consequences of most variants, specifically their impact on RAS-MAPK regulation, often remain unresolved. Here, we integrate clinical characterization, 3D structural modeling, stable lentiviral expression systems, real-time live cell imaging and multi-pathway signaling profiling to functionally assess a panel of eleven SPRED1 variants, including two uncharacterized patient-derived alterations, the splice-site mutation c.207+1G>A and the missense substitution c.986A>T (classified as VUS in ClinVar). Unlike c.986A>T, which selectively impairs ERK signaling, the c.207+1G>A variant acts as a broader pathway disruptor, elevating both ERK and p38 phosphorylation, a difference that correlates with its more severe clinical phenotypes. Expanding our analysis across nine additional truncating and missense variants, we uncover reproducible differences in SPRED1 protein abundance and variant-specific signaling dynamics, implicating not only the canonical RAS/ERK axis but also AKT, p38, and p53 pathways. Collectively, these findings reveal genotype-phenotype correlations with differential impacts on signaling dynamics by SPRED1 mutants.