An Autosomal Dominant Form of Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2) Is Associated with Haematopoiesis Failure

Abstract

Severe combined immunodeficiencies (SCIDs) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. Reticular dysgenesis (RD) is an autosomal recessive form of human Severe Combined Immunodeficiency (SCIDs) characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, encoding for a mitochondrial protein which regulates the homeostasis of adenine nucleotides.In three newborns presenting a RD-like clinical phenotype as frequent infections and a profound leukopenia, we identified an heterozygous dominant missense mutation in the gene coding for Rac Family Small GTPase 2 (RAC2) protein (p.G12R). Hematopoietic stem cell transplantation (HSCT) performed soon after birth was successful in two out of the three patients attesting that the inherited defect was intrinsic and not micro-environmental. RAC2 protein belongs to the Rac subfamily of RHO small GTPases. In the inactive GDP-bound state, RAC2 is located in the cytosol and upon stimulation, the active RAC2-GTP-bound form translocates to the plasma membrane. Unlike the other members of the Rac subfamily (RAC1 and RAC3), RAC2 is mostly expressed on hematopoietic cells and during T cell differentiation.To gain insight into the disease, we transduced human hematopoietic stem and progenitor cells (HSPCs) with a lentiviral construct containing the RAC2 mutated form. The mutation inhibits HSPCs proliferation and differentiation toward the myeloid and lymphoid lineages reproducing the patients' clinical phenotype. In this condition, we also observed high apoptosis level and an alteration of mitochondrial activity and Ros production. In a biochemical model, we demonstrated that the substitution of the glycine (G) amino-acid by a bulky flexible arginine (R) may prevent GTP hydrolysis. Lastly, our findings suggest that RAC2 gene sequencing must be considered in newborn screening programs for SCID detection.To decipher the mechanisms regulating RAC2 functions, we studied the impact of the p.G12R mutation on a human AML cell line expressing RAC2 protein. Our preliminary data highlight that the cell cycle and mitochondrial activity are disrupted by G12R mutation. By holotomography, we also observed morphological changes and accumulation of lipid droplets into the cells. All these data suggest that RAC2 defective signalling pathway is linked to cell metabolism imbalance and further investigations are ongoing to better understand how RAC2 controls cell energy demand, especially during differentiation. DisclosuresCavazzana: Smart Immune: Other: co-founder.