A heterozygous RAB27A mutation causes delayed cytolytic granule polarization and hemophagocytic lymphohistiocytosis in 2 unrelated teenagers

Abstract

Abstract Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in natural killer (NK) and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report 2 unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (259 G>C). The contribution of this Rab27A missense (A87P) mutation on NK cell cytotoxicity was studied by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant and wild-type (WT) transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay (PLA), respectively. Over-expression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein was predicted to disrupt binding to WT Munc13-4 by crystal structure modelling, and decreased interaction of Rab27A (A87P) with WT Munc13-4 was shown by PLA in situ and by co-immunoprecipiation in vitro. Finally, Rab27A (A87P) over-expression in NK-92 cells delayed granzyme B polarization toward the immunologic synapse, as noted by confocal microscopy, with a resulting increase in interferon-γ production, a cytokine responsible for HLH. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.