Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza

Abstract

Background: Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza.

Methods: Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells.

Results: Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS.

Conclusions: This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection.

Keywords: H1N1 influenza; LYST; cytolytic pathway; hemophagocytosis; macrophage activation syndrome; perforin.

© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Figures

Figure 1.

Representative examples of hemophagocytosis characterized by macrophages with engulfed red blood cells in lymph node (A and B) and spleen (C and D) in fatal cases of H1N1 influenza. Hematoxylin-eosin stain was used (1000× [oil; A–C] or 400× [D] original magnification).

Figure 2.

PRF1 p.A91V mutation decreases natural killer (NK) cell cytolytic function. A, eFluor450-labeled K562 target cells were mixed at increasing effector to target (E:T) cell ratios with NK-92 cells transduced with lentiviruses expressing PRF1 wild type (WT; left), or PRF1 p.A91V (right) and were incubated for 4 hours prior to flow cytometry of K562 cell death. Flow cytometry plots were gated on eFluor450-expressing cells, with eFluor450 depicted on the y-axis and near–infrared (IR) staining (dead cells) along the x-axis. One representative experiment is shown, with percentages of lysed K562 cells enumerated. B, Percentage cytotoxicity at 3 different E:T cell ratios for lentivirus-transduced NK-92 cells (PRF1 WT [circles] and PRF1 p.A91V [squares]). Results are plotted as means ± standard error of the mean for 5 independent experiments. Two-way analysis of variance revealed statistically significant differences (P < .05) between the PRF1 WT– and PRF1 p.A91V mutant–transduced NK-92 cells at all 3 E:T cell ratios.