A Cdc42 C-terminal Mutation Associated with Life-Threatening Hyperinflammation in Humans Is Well-Tolerated in Mice

Primary immune disorders offer important pathoetiologic insights into human immune dysfunction. Often, studying pathogenic human mutations in murine systems provides an excellent system for probing those mechanisms. Recently, multiple groups found that de novo arginine-to-cysteine missense mutations at position 186 of the human cell division cycle (CDC42) gene, near its C terminus, were associated with a life-threatening hyperinflammatory disorder called NOCARH (neonatal onset of pancytopenia, autoinflammation, rash, and episodes of hemophagocytic lymphohistiocytosis (HLH). NOCARH patients demonstrate excessive activation and expansion of macrophages and CD8+ T lymphocytes. In vitro, human CDC42˄cterm mutants drove cell-intrinsic hyperactivation of the pyrin inflammasome. To better understand the immunobiology of NOCARH, we used CRISPR-Cas9 editing of C57Bl/6 mouse embryos to introduce the R186C transition in murine Cdc42, which is amino acid identical to human. We were successful in generating founders harboring mosaic R186C and R186F alleles, and these alleles transmitted to the germline of offspring upon breeding to wild-type mice. Both R186C and R186F heterozygous and homozygous mice appeared viable, fertile, and were similar to wild-type littermates in growth and development. Mice harboring the mutated alleles (even in homozygosity) also responded to transient LCMV infection similarly to WT. In vitro, activation of the pyrin inflammasome in bone marrow–derived macrophages (BMDM) was unaffected by the presence of Cdc42˄R186C. However, murine pyrin lacks a B30.2 domain present in humans, and this domain is the site of many classical Familial Mediterranean Fever mutations. However, BMDM pyrin activation was unaffected by Cdc42 genotype even in cells from mice also expressing a humanized mouse pyrin allele (Mefv˄B30.2). Murine express an alternative Cdc42 transcript in CNS tissues that utilizes an alternative exon 6 and therefore would not contain the engineered mutation. However, we found no change in the low expression of this alternative transcript in blood cells even in Cdc42˄R186C homozygous mice. These data suggest critical differences in the regulation mouse and human CDC42 that, if understood, could identify novel ways of regulating CDC42˄cterm-driven inflammation in NOCARH patients.