P1644: novel variants in gale caused syndromic macrothrombocytopenia disrupting thrombopoiesis and glycosylation

Background: Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme UDP-galactose-4-epimerase, encoded by GALE, is involved in protein glycosylation. There is currently scarce information about the performance of megakaryopoiesis in patients with thrombocytopenia associated with GALE variants. Aims: Assessing the clinical and platelet phenotype in three patients with syndromic macrothrombocytopenia associated to GALE variants, and to investigate the role of GALE in glycosylation and thrombopoiesis. Methods: Three patients from two unrelated families with lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice were enrolled in the Spanish Project of Inherited Platelet Disorders. Biallelic variants in GALE were identified by whole-exome sequencing (WES) (Figure 1). Platelet phenotyping included full blood count, blood film, light transmission aggregometry, flow cytometry analysis of major surface glycoproteins, and agonist-induced granule secretion. UDP-galactose-4-epimerase enzymatic activity was measured by HPLC/MS/MS. GALE and N-acetyl-galactosamine (LacNac) levels were evaluated by immunoblotting. Human megakaryocyte (Mk) culture from peripheral blood samples were assessed in one proband. Results: WES revealed four variants affecting GALE, three of them previously unreported (Figure 1). Patients showed giant and/or grey platelets, impaired platelet aggregation with several agonists, severely reduced alpha (P-selectin) and dense granule (CD63) secretion, and significant decrease of GPIbα-IX-V complex in the platelet surface. UDP-galactose-4-epimerase enzymatic activity was severely decreased in these patients. Immunoblotting showed reduced GALE in platelets and significant reduction of LacNAc, suggesting a hypoglycosylation pattern. In proband B.II.2, CD34+ derived Mks exhibited normal ploidy, and maturation, but impaired proplatelet formation. These Mks showed, similarly to patient platelets, normal surface levels of β3 integrin, but remarkable reduction in the glycosylation and externalization of GPIbα, and delocalization of vWF in the megakaryocyte membrane. Image:Summary/Conclusion: This study expands our knowledge on the GALE related thrombocytopenic disorder, emphasizing its critical role in platelet glycosylation, and providing further evidence that thrombocytopenia derives from altered proplatelet formation developing from GPIbα impairment. Funding PI20/00926, GRS2135/A/2020, GRS2314/A/2021, FMM AP172142019