A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis

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 (c.259G>C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function 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 transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, 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 decreased binding to Munc13-4 and delayed granzyme B polarization towards the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.

Advances in the pathogenesis of primary and secondary haemophagocytic lymphohistiocytosis: differences and similarities

“…Heterozygous mutations in X-linked genes may also cause HLH in female patients, depending on the percentage of wild-type X-chromosome inactivation (Holle et al, 2015;Yang et al, 2015). Moreover, monoallelic missense mutations in STXBP2 or RAB27A have been described to cause late-onset FHL in a (partial) dominantnegative manner (Spessott et al, 2015;Zhang et al, 2016). These findings stretch the traditional notion of primary HLH as a monogenic, homozygous recessive disorder.…”

Macrophage Activation Syndrome: different mechanisms leading to a one clinical syndrome

“…If HLH is considered one clinical syndrome is conceivable that some of the pathogenic mechanisms of pHLH may be involved in other forms of HLH. See Appendix 2: Case 2 for potential impact of heterozygosity for mutations of pHLH genes on development of clinical overt disease and of a defective cytotoxicity [10]. …”

“…Altogether these observations, far from being conclusive, point to the presence of a genetic contribution to MAS similar to that of pHLH. All together these observations suggest that events related to the pathogenesis of pHLH are relevant in sHLH in general, and in MAS in particular: a) accumulation of partial genetic defect in one, or more than one, of the known pHLH related gene b) the possible dominant negative effects of some heterozygous variants [10, 18]. Defective cytotoxicity may impair clearance of infected cells, and/or prolong lymphocyte-APC interactions, both events leading to increased pro-inflammatory cytokine production (e.g.…”

“…Defective cytotoxicity may impair clearance of infected cells, and/or prolong lymphocyte-APC interactions, both events leading to increased pro-inflammatory cytokine production (e.g. IFNγ) by T lymphocytes [10, 19]. …”

“…Indeed, IL-6TG mice challenged with poly(I:C), a substitute for viral dsRNA, display a significant decrease in NK cell cytotoxicity, associated to lower expression levels of perforin and granzyme B, in the absence of altered granule exocytosis [26]. An example of IL-6 leading to decreased cytolytic activity of lymphocytes on top of an underlying genetic defect has been shown [10]. Similarly, in vitro experiments revealed that exposure of human PBMCs to high levels of IL-6, such as those typically present in sJIA, inhibits NK cell cytotoxicity by down-regulating the expression of perforin and granzyme B.…”