Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis

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 galactose metabolism and protein glycosylation. Here, we studied three patients from two unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed four variants affecting GALE, three of them previously unreported (Pedigree A: p.Lys78ValfsX32 and p.Thr150Met; Pedigree B: p.Val128Met and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease of N-acetyl-lactosamine (LacNAc), demonstrating a hypoglycosylation pattern, reduced surface expression of GPIbα-IX-V complex, and mature β1 integrin, and increased apoptosis. In vitro studies performed with patients' derived megakaryocytes demonstrated normal ploidy and maturation but decreased proplatelet formation due to the impaired glycosylation of the GPIbα and β1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand Factor were also demonstrated. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasized the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.