Characterization of DOCK8 as a novel gene associated with hemophagocytic lymphohistiocytosis

Abstract

Abstract Background Hemophagocytic lymphohistiocytosis (HLH) is life threatening, presenting with fever, pancytopenia, coagulopathy, & multi-organ dysfunction (MOD). Familial HLH (fHLH) results from homozygous defects in perforin mediated cytolytic pathway genes used by NK & CD8 T cells. Up to 40% of secondary HLH (sHLH) patients have heterozygous defects in fHLH genes resulting in decreased cytolytic function, prolonged interaction with antigen presenting cells, & subsequent increased pro-inflammatory cytokines resulting in MOD. As NK cell dysfunction is common in HLH, we screened for novel gene associations. Methods By whole genome sequencing, 4 sHLH patients had mutations in DOCK8 (guanine exchange factor) critical to NK cell function. DOCK8 mutations, or wild-type (WT) sequence, were introduced into NK92 cell lines by foamy virus (FV), or by disrupting endogenous NK92 DOCK8 genes (CRISPR). WT & mutant DOCK8 NK92 cells incubated with K562 cells were compared for cytolysis, degranulation (CD107a), & cytokines [interferon-γ (IFNγ), tumor necrosis factor (TNF)]. Results 2 HLH patients had rare heterozygous DOCK8 mutations; 2 had the same DOCK8 polymorphism (D63N). 1 novel mutation was missense (A261V); 1 was a splice acceptor variant (c.54-1G>T, 0.03%). The A261V mutant decreased NK lytic activity by CRISPR or FV (50% vs. WT, p=0.007), & decreased CD107a by >50% (p=0.0129). The A261V DOCK8 NK cells increased expression of IFNγ & TNF by >200% (p=0.0192 & p=0.0027). The D63N NK cells had similar results. The DOCK8 splicing mutant disrupted splicing by exon trapping. Conclusion Heterozygous DOCK8 mutations contribute to HLH by dominant-negative or hypomorphic effects yielding decreased cytolysis & increased inflammatory cytokines.