Abstract Number: PB1234
Meeting: ISTH 2022 Congress
Background: Mutations in GATA1 are associated with anemia and thrombocytopenia with defective platelet function and granule content. We discovered a novel uncharacterized hemizygous missense mutation in GATA1 (с.616A>C; p.Asp206His) in a 2-year-old male patient.
Aims: We sought to characterize patient-derived megakaryocytes (MKs) and platelets and their function. We hypothesized that thrombocytopenia and granular defects in our patient may be due to impaired transcriptional control by the novel GATA1 variant.
Methods: MKs were cultured from patient-derived bone marrow. Flow cytometry, confocal and transmission electron microscopy, qPCR, and next-generation sequencing (NGS) were used to characterize patient-derived platelets and MKs. Molecular cloning and mRNA transfection of His-tagged GATA1 protein variants were used to reproduce mutation effects in Meg-01 cells.
Results: NGS analysis revealed the novel hemizygous variant GATA1 с.616A>C; p.Asp206His, confirmed by Sanger sequencing. Mepacrine uptake assays revealed decreased dense granules (38% of normal) in resting platelets with impaired secretion after activation (8% of normal), although the CD62p expression level was not affected. Confocal microscopy of platelets for CD62p, vWF, LAMP2, and CD63 showed diffuse staining with no visible granules. Profound alpha- and dense- granule deficiency was directly confirmed with transmission electron microscopy. Differentiated MKs were largely unable to form proplatelets. Fibrinogen loading assays showed no significant changes in MK granule content after a 24-hour incubation, despite a 60% decrease in NBEAL2 expression revealed by qPCR. Overexpression of mutated GATA1 in Meg-01 also resulted in a similar decrease in NBEAL2 mRNA levels compared to overexpression of the normal variant, with a slight increase in FLI1 and Rab27b expression.
Conclusion(s): Our results reveal that a novel mutation in the GATA1 N-terminal zinc finger impairs late MK development and platelet formation via transcriptional dysregulation of crucial MK genes. Further, we have developed a model that allows for future studies of MK development, thereby reducing the need for limited patient-derived material
To cite this abstract in AMA style:Butov K, Kondrashova Z, Obydennyi S, Ignatova A, Podoplelova N, Kurnikova M, Raykina E, Osipova E, Novichkova G, Donyush E, Machlus K, Panteleev M. Novel Pathological GATA1 Variant Leads to Defective Platelet Biogenesis via Impaired Transcriptional Regulation [abstract]. https://abstracts.isth.org/abstract/novel-pathological-gata1-variant-leads-to-defective-platelet-biogenesis-via-impaired-transcriptional-regulation/. Accessed September 27, 2023.
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