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

Abstract

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.

Hemostatic defects in congenital disorders of glycosylation

“…At the ISTH 2022 congress, Marin-Qilez et al. [ 55 ] presented the clinical and platelet phenotypes of 3 patients for whom new UDP-galactose-4-epimerase variants were discovered by whole-exome sequencing. These variants were likely to reduce the enzyme’s activity and thus lead to a CDG.…”

Bioprinting Soft 3D Models of Hematopoiesis using Natural Silk Fibroin‐Based Bioink Efficiently Supports Platelet Differentiation

“…[ 14 ] Megakaryocyte ploidy, expression of surface markers, and viability were analyzed, as previously described. [ 40 ] All antibodies were from BioLegend. Platelets were analyzed using the same forward and side scatter pattern as human peripheral blood and identified as CD41a + CD42a + (BioLegend) events.…”

Inherited Thrombocytopenia Caused by Variants in Crucial Genes for Glycosylation

“…It has been described that the absence of GPIbα in the MK membrane leads to reduced thrombopoiesis due to aberrant membrane development during MK maturation, impaired formation of the membrane demarcation system (DMS), and disruption of the microtubule cytoskeleton, as described in Bernard Soulier syndrome (BSS) [ 21 ]. In addition, our group has recently described a defect in GPIbα glycosylation that affects thrombopoiesis and actin cytoskeleton remodeling [ 22 ]. These findings highlight the essential role of protein glycosylation during megakaryopoiesis and thrombopoiesis.…”

“…In addition, hypoglycosylated and non-functional platelets prone to apoptosis were observed. Overall, these findings demonstrated the essential role of GALE in glycosylation, platelet formation, function and clearance, providing new clues to understand the biological mechanisms underlying the biology and pathophysiology of the β1 integrin and GPIb-IX-V complex [ 22 ].…”