Disease-specific biomarkers of pathogenic HRAS variants in human immortalized keratinocytes.

Pathogenic germline variants in HRAS result in the non-mosaic RASopathy Costello syndrome (CS), a rare developmental disorder affecting various tissues including the skin and with > 80% of patients showing the HRAS p.G12S variant. Postzygotic HRAS variants have been identified in disorders of the skin, so-called mosaic RASopathies (e.g. Nevus sebaceous syndrome) and > 85% of patients show the HRAS p.G13R variant. Oncogenic HRAS variants are common in dermatological and other malignancies, with HRAS p.G12V being one of the most common variants. The three disease entities are characterized by different skin manifestations reflecting HRAS function as modulator of epidermal homeostasis and suggesting HRAS variant-specific functional consequences. We analyzed permanent human keratinocytes (HaCaT cells) stably expressing disease-specific HRAS variants (p.G12S, p.G12V, p.G13R) to identify unique biological consequences underlying the phenotypical pleiotropy of HRAS-associated disorders. Immunoblotting experiments revealed significant functional differences between HRAS variants in the expression and/or activation of cell fate markers, signaling proteins, and cell contact molecules. These differences are reflected in different cell biological consequences regarding proliferation, adhesion, and migration. By applying mass spectrometry and collecting data on protein interactions (interactome), protein phosphorylation (phosphoproteome), and protein expression (proteomics), we verified HRAS variant-specific functional consequences and revealed novel and pleiotropic pathophysiological features for each of the three HRAS variants. HRAS p.G13R induced strongest molecular changes suggesting most serious pathobiological consequences, and HRAS p.G12S and p.G12V predominantly interfered with cellular signaling. Taken together, our data underscore the existence of HRAS variant-specific qualities of dysregulation and describe a differential pathophysiological architecture of HRAS-associated phenotypes.