Functional stratification of YWHAZ variants explains distinct neurodevelopmental disorders

The application of clinical genomics in personalized medical practice increasingly reveals that variants from single loci can lead to phenotypic heterogeneity. This necessitates functional characterization and classification of serial variants to correlate their associated clinical features and molecular properties. Recently, a few putative pathogenic variants in YWHAZ, encoding the 14-3-3ζ protein, have been implicated in clinically variable isolated and syndromic neurodevelopmental phenotypes, based on a few reported affected individuals, but the molecular basis of this heterogeneity has remained unclear. Here, we assembled a large cohort of individuals harboring de novo YWHAZ variants and categorized their clinical stratification into distinct neurodevelopmental conditions, including a typical presentation of cardiofaciocutaneous syndrome and a developmental and epileptic encephalopathy with sleep disorders. By integrating structural analyses, molecular dynamics simulations, in vitro biochemical and cell-based signaling studies, unbiased proteomics analysis, and in vivo phenotypic assays, we define distinct functional classes of pathogenic YWHAZ variants and establish robust genotype-phenotype correlations. We show that disease-associated YWHAZ variants differentially perturb 14-3-3ζ function by variably affecting protein abundance and binding to interacting partners, including the BRAF and RAF1 kinases. Notably, the recurrent p.Ser230Trp (c.689C>G) variant uniquely underlying cardiofaciocutaneous syndrome specifically enhances MAPK signaling, whereas other variants do not significantly impact signal flow through the cascade under the used experimental conditions. Our data establish a mechanistic framework linking allelic variation in YWHAZ to phenotypic heterogeneity.