Background: Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of severe hyper-inflammation characterized by immune dysregulation and massive, aberrant activation of cytotoxic T cells and macrophages. While the molecular pathologies underlying secondary HLH are diverse, the primary or monogenic form of HLH is caused by the defects selectively disrupting perforin-mediated cytotoxicity in human lymphocytes. Aims: Aims: In our study we investigated the genetic etiology of and molecular pathomechanisms of the disease in a patient We applied a combination of state-of-the-art genetic and -omics technologies, including whole-exome sequencing (WES) and pharmacological screening combined with a wide range of cellular and molecular techniques to elucidate pathways underlying HLH pathology. Results: Results: We investigated a patient with early-onset HLH who fulfilled all diagnostic criteria who was wildtype for mutations in any of the known HLH genes. We identified biallelic germline mutations in RHOG. Using genetic ablation of the RHOG in a model cell line and primary cytotoxic T lymphocytes (CTLs) from healthy individuals, we confirmed its crucial role in lymphocyte cytotoxicity. To decipher the underlying molecular pathomechanism, we defined the molecular partners of RhoG in human lymphocytes and discovered that RhoG interacts with the regulator of cytotoxic granule (CG) release Munc13-4 inducing docking of Munc13-4-positive CGs to the plasma membrane. This step is required for subsequent fusion of the membranes to release cytolytic cargo toward target cells. Using a pharmacological screening approach, we defined the molecular pathways perturbed in RhoG deficient lymphocytes and assessed in detail their impact on the cytoskeleton. Employing high-resolution microscopy, we showed that RhoG is essential for the reorganization of the synaptic F-actin and control of immune synapse architecture. We also demonstrated that cytoskeletal defects in RhoG deficient lymphocytes could be pharmacologically reconstituted. our dual role of RhoG for CG exocytosis through regulation exocytosis and cytoskeletal machinery.