Analysis of novel RAC2 variants associated with neutrophil dysfunction, lymphopenia, and primary immunodeficiency

Abstract

Abstract RAC2 is a member of Rho-family GTPases, abundant in neutrophils serving roles in NADPH oxidase activation and cytoskeleton dynamics. Patients with dominant, activating mutations in RAC2 (E62K, N92T, Q61R) are associated with neutrophil dysfunction, lymphopenia and immunodeficiency. We now characterize additional disease-related RAC2 variants with in-vitro analysis of RAC2 effector functions including NOX2-based superoxide production, regulation of PAK1-PDB binding, activation of AKT, protein stability, subcellular localization, and F-actin formation. We identified both active and inactive variants associated with lymphopenia and immunodeficiency. Patients with dominant, active RAC2 variants with high protein stability (Q61R, Q61K) presented in the first days of life with severe combined immunodeficiency and absent lymphocytes. Activating variants with lower protein stability (I21S, E62K, N92K, N92S) presented as combined variable immunodeficiency with lymphopenia, detectable by newborn screening but clinically presenting later with recurrent sinopulmonary infections, reduced B-cells and hypogammaglobulinemia. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity (W56*, R68W). Severity of phenotype and initial clinical presentation was correlated with protein stability, with increased stability leading to neonatal presentation and decreased stability of dominant mutations having later clinical onset. Thus, heterologous expression and analysis of RAC2 variants is useful for deciphering the genetic and molecular mechanistic basis for the common underlying clinical phenotype related to lymphopenia and immunodeficiency.