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Letters to the Editor
GNE-related thrombocytopenia: evidence for a mutational hotspot in the ADP/substrate domain of the GNE bifunctional enzyme
The GNE gene encodes UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE), a bifunctional enzyme catalyz- ing the synthesis of a sialic acid called 5-acetylneu- raminic acid (Neu5Ac).1
Mutations of GNE are responsible for GNE myopathy (OMIM #605820), an autosomal recessive late-onset progressive muscle disorder1 and sialuria (OMIM #269921), an autosomal dominant disease characterized by a congenital impairment of sialic acid metabolism.1 In a small set of patients, biallelic mutations of GNE have only recently been associated with thrombocytopenia, either isolated or combined with muscle weakness.2-6 However, it is unclear why only a few of the almost 1,000 individuals carrying biallelic mutations of GNE show thrombocytopenia.
We studied two families with severe thrombocytope- nia using whole exome sequencing (WES). Family 1 proband (P1) was an 18 months-old boy born to consan- guineous Egyptian parents with a platelet count of 5x109/L at birth. The proband (P2) of family 2 was a 4- year-old boy, the third child of consanguineous Moroccan parents, who had scattered petechiae associ- ated with severe thrombocytopenia (platelet count 4x109/L) in his first hours of life (Figure 1A). In both P1 and P2 allo- and auto-antibodies against platelet anti- gens were not found in the mother's serum. Parents and siblings were healthy with normal blood counts, and no family history of thrombocytopenia was reported in either family. Splenomegaly or dysmorphic features were not observed in either of the patients, and the neu- rological assessment showed normal psychomotor development. Creatine phosphokinase (CPK) level was average, and no sign of myopathy was detected. In P1, both karyotyping and comparative genomic hybridiza- tion (CGH) array did not reveal any chromosomal alter- ation.
The WES analysis allowed us to identify two novel homozygous variants (c.1546_1547delinsAG and c.1724C>G) of the GNE gene, leading to the p.Val516Arg and p.Thr575Arg missense substitutions, respectively (Figure 1A and B). Extensive analysis of the exome data did not yield any other potential pathogenic variant, not even in the inherited thrombocytopenia- causing genes (IT-related genes; Online Supplementary Table S1). Moreover, we analyzed the runs of homozy- gosity (ROH) shared by P1 and P2 searching for poten- tially deleterious variants. Candidate genes were regard- ed as those whose mutations are associated with throm- bocytopenia (n=56 from Online Supplementary Table S1) and those enlisted in the gene ontology (GO) term “Hemopoiesis” (n=788). Except for the mutations in GNE, all the other homozygous variants were excluded based on pathogenicity and splicing bioinformatic pre- dictions (Online Supplementary Table S2).
Substitutions p.Val516Arg and p.Thr575Arg are rare variants affecting well conserved residues during evolu- tion (Figure 1C). Their potential deleterious effect was supported by segregation analysis, bioinformatics pre- dictions (Online Supplementary Table S2), and significant reduction of the GNE protein expression, which was likely to maintain some residual activity due to the incompatibility of complete loss of the GNE function with life (Figure 1D).7 Consistent with alteration of the
GNE kinase activity, the transferrin serum glycoforms analysis revealed a higher level of the asialo, disialo and trisialo forms and a correspondent decrease in the tetrasialotransferrin form in both patients (Figure 2A).
The hematological and clinical features of P1 and P2 are strikingly similar. Except for petechiae and minor post-traumatic bruises (grade 1/5) occurring when the platelet count was below 10-20x109/L, no clinically sig- nificant bleeding was reported regardless of treatment. Consistent with data in the literature,4 they had increased mean platelet volume (MPV) (MPV 11.9 fL in P1 and MPV 10.8 fL in P2) and high immature platelet fraction (IPF) (IPF 50-80% in P1 and IPF 39-89% in P2) (Figure 2B). In bone marrow aspirates of both the affect- ed individuals the number of megakaryocytes was markedly increased, and several immature small-sized and hypolobulated megakaryocytes were observed (Figure 2C to E). Interestingly, this condition mimicked the pathophysiology of another inherited defect of platelet sialylation, namely SLC35A1 deficiency, which affects the same biochemical pathway,8 and is partly reminiscent of the peripheral platelet destruction in immune thrombocytopenia (ITP).
High-dose intravenous immunoglobulin and steroid treatment resulted in no improvement of the patients’ platelet count, therefore both probands required regular platelet transfusions in the first year of life (Figure 2F). The response to platelet transfusion and the megakary- ocytes features suggest that the patients’ platelets are more rapidly removed from circulation for intrinsic cel- lular defects, such as sialylation reduction, rather than a decreased platelet production.4
Both patients were treated with romiplostim to reduce the need for transfusions. P1 responded at low doses (4 μg/kg/week) and his platelet count was higher than 25x109/L during treatment, which is currently ongoing (Figure 2G), while P2 required a high dose of romi- plostim (up to 10 μg/kg/week) to obtain a substantial, extremely unstable, response (Figure 2G), thus discour- aging the continuation. Fluctuating response to romi- plostim was also reported in patients with ITP further supporting the similarity with GNE-related thrombocy- topenia.8 Currently, despite a low platelet count (5- 10x109/L), patient P2 did not experience any bleeding up to the last follow-up.
The absence of severe bleeding despite extremely low platelet counts was in line with other cases reported in the literature (Online Supplementary Table S3). Among these, only two (P6 and P17) experienced severe or life- threatening hemorrhages. The mild bleeding diathesis might be attributed to the abundance of young enlarged platelets, which have a prothrombotic potential.9 These data suggested that prophylactic treatment might be needed only in specific conditions (e.g., neonatal period; severe hemorrhage; surgical procedures), and an approach aimed at treating only acute events with platelet transfusion might be considered in most patients.
Several lines of evidence supported the hypothesis that GNE mutations can cause thrombocytopenia. At least to our knowledge, 20 patients from nine unrelated families of the nearly 1,000 individuals with alterations of this gene have been reported to have thrombocytope- nia (Online Supplementary Table S3).2-4,10,11 Of the 15 dif- ferent variants identified in these patients, including P1 and P2, seven have been previously reported in patients with GNE myopathy (Figure 3). Except for p.His188Tyr, which is in cis with a known mutation (p.Asn550Ser) associated with myopathy,4 the others (p.Asp444Tyr,
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haematologica | 2022; 107(3)