First characterization of platelet secretion defect in patients with familial hemophagocytic lymphohistiocytosis type 3 (FHL-3)

To the editor:

Familial hemophagocytic lymphohistiocytosis (FHL), a rare autosomal recessive disorder of lymphocyte cytotoxicity, is caused by mutations in genes encoding perforin (FHL-2) or proteins important for intracellular trafficking/exocytosis of perforin-containing lytic granules: Munc13-4 (FHL-3), syntaxin 11 (FHL-4), and Munc18-2 (FHL-5).¹  FHL-1 is due to an unidentified gene defect located on chromosome 9. Munc13-4 (a Rab27a effector) coordinates exocytosis in hematopoietic cells.^2,3  Munc13-4 deficiency (FHL-3) results in defective cytolytic granule exocytosis.⁴  Interestingly, platelets from Munc13-4–deficient mice showed a severe secretion defect of α- and dense granules.⁵  In patients with FHL-3, bleeding symptoms have been rarely reported because patients may have not been challenged (ie, surgery).⁶  An 8-month-old Chinese FHL-3 patient died because of gastrointestinal hemorrhage.⁷  A platelet secretion defect in patients with FHL-5 has been described previously.^8,9 

Therefore, we analyzed platelet function in 2 unrelated male patients with genetically confirmed FHL-3. Both patients presented with clinical symptoms indicative of hemophagocytic lymphohistiocytosis (HLH) and fulfilled the diagnostic criteria.¹⁰  Functional analyses showed absent degranulation of cytotoxic T cells and reduced degranulation of natural killer cells. Patient 1 (diagnosed at the age of 4 months) showed compound heterozygous mutations in the UNC13D gene accounting for FHL-3: c.551G>A (p.W184X) in exon 6 and c.118-308C>T in intron 1. After treatment according to the HLH 2004 protocol, haploidentical hematopoietic stem cell transplantation was performed at the age of 16 months.¹⁰  Patient 2 (diagnosed at the age of 5 months) had severe central nervous system involvement and showed compound heterozygous splice donor site mutations of UNC13D: c.753+1G>T of exon 9 and c.1389+1G>A of exon 15. The patient received treatment according to the HLH 2004 protocol and underwent hematopoietic stem cell transplantation from a matched unrelated donor at the age of 9 months. He died of HLH relapse due to graft failure 4 months later. No major bleeding episodes were observed in either patient. The platelet studies were performed shortly before transplantation when patients were stable and did not receive medication, which induced secretion defects. Platelet count was normal at the time of platelet studies.

Flow cytometric analyses of platelets from both patients revealed severely diminished to absent platelet α- (CD62P) and dense granule (CD63) secretion in response to thrombin, collagen, and thrombin receptor activating peptide (TRAP) in platelet-rich plasma (Figure 1A-D; TRAP not shown). Collagen-induced adenosine triphosphate secretion (lumiaggregometry) in whole blood was absent (Figure 1E), whereas agonist-induced fibrinogen binding (data not shown) and aggregation (Figure 1F) were normal for a child of that age. In addition, flow cytometric analyses of platelets from patient 2 showed absent mepacrine uptake and release (a marker of uptake and release of dense body contents), as well as absent expression of the lysosomal marker CD107a (LAMP-1) in response to collagen and TRAP (data not shown).

These data demonstrate a selective impairment of platelet granule secretion in patients with FHL-3, and thus support an important role for Munc13-4 in human platelet degranulation. Although bleeding symptoms in FHL-3 patients can be mild, our findings clearly demonstrate that Munc13-4 deficiency is more than a genetic disorder of cytotoxicity.