Adaptable imMKCL-based approach to study the variety of megakaryocyte and platelet mutations

Introduction: Inherited thrombocytopenias (ITs) is a heterogeneous group of disorders that are characterized by impaired megakaryocyte maturation and platelet production or function. These conditions result from diverse genetic mutations affecting various aspects of megakaryopoiesis and thrombopoiesis. Despite recent advancements in genetic sequencing technologies, a significant number of identified variants remain classified as variants of unknown significance (VUS), impeding accurate diagnosis and management. This study aims to develop and validate an in vitro screening tool for assessing the functional impact of potentially pathogenic mutations on megakaryocyte maturation and platelet production. To demonstrate the utility of this approach, we focused on the GP1ba Bolzano mutation (p.A172V), a well-characterized variant associated with Bernard-Soulier syndrome (BSS).

Method: A GP1ba knockout iMK line was first generated from imMK cells using CRISPR/Cas9 technology. A two-plasmid system was employed for targeting the insertion of either wild-type or Bolzano mutant GP1ba, utilizing a safe harbor site. Following transfection, the imMK cells were cultured and differentiated to induce megakaryocyte maturation. Subsequently, the morphological characteristics, glycoprotein expression profiles, and their capacity for platelet production were analyzed. Additionally, functional tests were conducted on the platelets produced.

Results: We successfully established three immortalized megakaryocyte (imMK) cell lines: GP1ba knockout (KO), GP1ba wild-type (WT), and GP1ba Bolzano mutation (Bol) in a safe harbor site. Both GP1ba-KO and GP1ba-Bol megakaryocytes showed impaired formation of the demarcation membrane system, altered marker expression, abnormal morphology, and reduced platelet yield compared to GP1ba-WT. The platelets derived from these cell lines exhibited characteristics consistent with Bernard-Soulier syndrome. These findings indicate that our approach is able to recapitulate the effects of impaired GP1ba on megakaryopoiesis.

Conclusion: In conclusion, this study presents an in vitro pipeline for investigating genetic mutations linked to inherited thrombocytopenia and other platelet disorders. Our proof-of-concept analysis of the GP1ba Bolzano mutation validates this approach. This tool enables the functional characterization of variants of unknown significance and may enhance diagnosis and treatment strategies in hematological disorders.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025