Identification of a genetic variant causing loss of RHOG expression. (A) Pedigree of the HLH patient family. Solid symbol (▪) indicates affected patient (P1); open symbol (○) corresponds to unaffected family members. The RHOG c.511G>A, p.Glu171Lys variant (RHOG gene reference sequences Ensembl: ENSG00000177105, ENST00000533217, GRCh37.p13) is indicated by E171K, the RHOG ∼33kb deletion is indicated as Del, the WT alleles by WT. ^aIndividual evaluated by whole-exome sequencing, Sanger sequencing, single nucleotide polymorphism (SNP) genotyping, and qPCR analysis. ^bIndividuals evaluated by targeted screening of the RHOG c.511G>A, p.Glu171Lys variant by Sanger sequencing, SNP genotyping, and qPCR analysis. (B) Immunohistochemistry of the patient bone marrow biopsy showing staining for CD68. Arrows indicate histiocytic cells with hemophagocytosis. (C) Abdominal magnetic resonance image revealing hepatosplenomegaly and abnormal signal intensity in both spleen and liver. (D) Amino acid alignment of the selected ρ family GTPase members demonstrating high conservation of the mutated residue. (E) A 3-dimensional model (top) and a scheme (bottom) of RhoG domain organization and localization of the missense mutation (red arrow). (F) Immunoblot analysis of the lysates from the patient and normal donor (ND) expanded T cells and fibroblasts. (G) Immunoblot analysis of the lysates from HEK293 cells untransfected (UT) and transfected with empty vector (EV) or vectors encoding WT and Glu171Lys mutant (MUT) RHOG genes. The difference in WT and mutated RhoG expression levels were quantified by normalization to EGFP expression (graph on right). Data are presented as the mean ± standard error of the mean (SEM). P values were calculated using a multiple Student t test. *****P < .0001.