Homozygous Mutation of the ETS DNA-Binding Domain of FLI1 Causes a Bleeding Disorder with Giant Alpha Granules Similar to Paris-Trousseau Thrombocytopenia

Deletion of a variable region on chromosome 11q containing FLI1 causes a platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. We report a kindred with the autosomal recessive inheritance of an ETS domain mutation of FLI1, c.970C>T, that causes macrothrombocytopenia with large alpha granule fusion similar to that observed in Paris-Trousseau thrombocytopenia but with no other syndromal features of Paris-Trousseau or Jacobsen syndromes. Affected individuals are moderately thrombocytopenic (mean = 71 x10⁹/L), have absent collagen-induced platelet aggregation and a lifelong mucosal bleeding history. Platelet MYH10 levels were increased in affected members of the kindred consistent with previous reports of elevated MYH10 in Paris-Trousseau thrombocytopenia. Luciferase reporter assays in HEK293 cells demonstrate that the mutant FLI1 transcript is associated with decreased transcription at the FLI target genes GP6, GP9 and ITGA2B compared to the wild-type transcript (23 vs 31, n=9, P<0.01; 3.8 vs 6.5, P<0.01, n=12; 11 vs 14, n=9, P=0.01, respectively). This transcriptional change was consistent with reduced expression of GPVI (P<0.01), GPIbIX (P<0.01) and GPIIbIIIa (P=0.04) observed on western blotting of platelet lysates from affected family members. This mutation replaces the conserved Arg324 with a tryptophan in the DNA-binding loop between the alpha-2 and alpha-3 helices of the FLI1 ETS domain. Protein modelling suggests that Arg324 does not directly bind DNA but may instead make direct contact with an N-terminal autoinhibitory domain of FLI1 that is considered to regulate DNA-binding affinity through a transition between a folded and unfolded state. This mutation may disrupt this important conformational change. These data suggest abnormalities of FLI1 function may be responsible for the complex platelet defect observed in Paris-Trousseau thrombocytopenia and Jacobsen Syndrome and confirm the role of FLI1 as an important transcriptional regulator of normal platelet development.