Heterozygous nonsense FLI1 p.Met100∗ variant results in thrombocytopenia and increased erythroid gene expression

Megakaryocytes and platelets arise from hematopoietic stem and progenitor cells through a tightly regulated process involving various transcription factors, including erythroblast transformation-specific family member FLI1. Pathogenic variants in FLI1, particularly those affecting the erythroblast transformation-specific DNA-binding domain, have been implicated in inherited platelet disorder and are associated with abnormal giant α granules, as seen in Paris-Trousseau Syndrome. However, the precise pathophysiological mechanisms remain unclear.

In this study, we describe a patient with a de novo heterozygous nonsense mutation, FLI1 p.Met100∗ (c.297del) and investigate its effects on megakaryopoiesis. We hypothesized that this variant generates a truncated protein that exerts a dominant-negative effect on megakaryocyte maturation.

Wild-type (WT) FLI1 and FLI1 p.Met100∗ were expressed in Meg01 cells. Bulk RNA sequencing, proteomics, and flow cytometry were performed to assess their functional consequences.

Western blot analysis confirmed truncated FLI1 p.Met100∗ protein expression in Meg01 cells. Gene set enrichment analysis revealed that WT FLI1 enhanced megakaryocytic phenotypes, while suppressing erythroid phenotypes, whereas FLI1 p.Met100∗ upregulated both. Flow cytometry confirmed that erythroid markers CD235a and KLF1 were downregulated by WT FLI1 but elevated by FLI1 p.Met100∗. Additionally, WT FLI1 promoted megakaryocyte maturation and adhesion, as evidenced by increased expression of the megakaryocyte marker CD61 and adhesion markers CD34 and CD44. Moreover, we demonstrated through immunoprecipitation that both WT FLI1 and FLI1 p.Met100∗ interact with shared cofactors.

Collectively, our findings suggest that FLI1 p.Met100∗ impairs megakaryocyte maturation through a dominant-negative manner, potentially contributing to thrombocytopenia by promoting erythroid features at the expense of proper megakaryocytic development.