Letters to the Editor GNE kinase activity, the transferrin serum glycoforms GNE-related thrombocytopenia: evidence for a analysis revealed a higher level of the asialo, disialo and mutational hotspot in the ADP/substrate domain trisialo forms and a correspondent decrease in the of the GNE bifunctional enzyme tetrasialotransferrin form in both patients (Figure 2A). The hematological and clinical features of P1 and P2 The GNE gene encodes UDP-N-acetylglucosamine are strikingly similar. Except for petechiae and minor (UDP-GlcNAc) 2-epimerase/N-acetylmannosamine post-traumatic bruises (grade 1/5) occurring when the (ManNAc) kinase (GNE), a bifunctional enzyme catalyz- platelet count was below 10-20x109/L, no clinically sig- ing the synthesis of a sialic acid called 5-acetylneu- nificant bleeding was reported regardless of treatment. raminic acid (Neu5Ac).1 Consistent with data in the literature,4 they had Mutations of GNE are responsible for GNE myopathy increased mean platelet volume (MPV) (MPV 11.9 fL in (OMIM #605820), an autosomal recessive late-onset P1 and MPV 10.8 fL in P2) and high immature platelet progressive muscle disorder1 and sialuria (OMIM fraction (IPF) (IPF 50-80% in P1 and IPF 39-89% in P2) #269921), an autosomal dominant disease characterized (Figure 2B). In bone marrow aspirates of both the affect- by a congenital impairment of sialic acid metabolism.1 In ed individuals the number of megakaryocytes was a small set of patients, biallelic mutations of GNE have markedly increased, and several immature small-sized only recently been associated with thrombocytopenia, and hypolobulated megakaryocytes were observed either isolated or combined with muscle weakness.2-6 (Figure 2C to E). Interestingly, this condition mimicked However, it is unclear why only a few of the almost the pathophysiology of another inherited defect of 1,000 individuals carrying biallelic mutations of GNE platelet sialylation, namely SLC35A1 deficiency, which show thrombocytopenia. affects the same biochemical pathway,8 and is partly We studied two families with severe thrombocytope- reminiscent of the peripheral platelet destruction in nia using whole exome sequencing (WES). Family 1 immune thrombocytopenia (ITP). proband (P1) was an 18 months-old boy born to consan- High-dose intravenous immunoglobulin and steroid guineous Egyptian parents with a platelet count of treatment resulted in no improvement of the patients’ 5x109/L at birth. The proband (P2) of family 2 was a 4- platelet count, therefore both probands required regular year-old boy, the third child of consanguineous platelet transfusions in the first year of life (Figure 2F). Moroccan parents, who had scattered petechiae associ- The response to platelet transfusion and the megakary- ated with severe thrombocytopenia (platelet count ocytes features suggest that the patients’ platelets are 4x109/L) in his first hours of life (Figure 1A). In both P1 more rapidly removed from circulation for intrinsic cel- and P2 allo- and auto-antibodies against platelet anti- lular defects, such as sialylation reduction, rather than a gens were not found in the mother's serum. Parents and decreased platelet production.4 siblings were healthy with normal blood counts, and no Both patients were treated with romiplostim to reduce family history of thrombocytopenia was reported in the need for transfusions. P1 responded at low doses (4 either family. Splenomegaly or dysmorphic features µg/kg/week) and his platelet count was higher than were not observed in either of the patients, and the neu- 25x109/L during treatment, which is currently ongoing rological assessment showed normal psychomotor (Figure 2G), while P2 required a high dose of romi- development. Creatine phosphokinase (CPK) level was plostim (up to 10 µg/kg/week) to obtain a substantial, average, and no sign of myopathy was detected. In P1, extremely unstable, response (Figure 2G), thus discour- both karyotyping and comparative genomic hybridiza- aging the continuation. Fluctuating response to romi- tion (CGH) array did not reveal any chromosomal alter- plostim was also reported in patients with ITP further ation. supporting the similarity with GNE-related thrombocy- The WES analysis allowed us to identify two novel topenia.8 Currently, despite a low platelet count (5- homozygous variants (c.1546_1547delinsAG and 10x109/L), patient P2 did not experience any bleeding up c.1724C>G) of the GNE gene, leading to the to the last follow-up. p.Val516Arg and p.Thr575Arg missense substitutions, The absence of severe bleeding despite extremely low respectively (Figure 1A and B). Extensive analysis of the platelet counts was in line with other cases reported in exome data did not yield any other potential pathogenic the literature (Online Supplementary Table S3). Among variant, not even in the inherited thrombocytopenia- these, only two (P6 and P17) experienced severe or life- causing genes (IT-related genes; Online Supplementary threatening hemorrhages. The mild bleeding diathesis Table S1). Moreover, we analyzed the runs of homozy- might be attributed to the abundance of young enlarged gosity (ROH) shared by P1 and P2 searching for poten- platelets, which have a prothrombotic potential.9 These tially deleterious variants. Candidate genes were regard- data suggested that prophylactic treatment might be ed as those whose mutations are associated with throm- needed only in specific conditions (e.g., neonatal period; bocytopenia (n=56 from Online Supplementary Table S1) severe hemorrhage; surgical procedures), and an and those enlisted in the gene ontology (GO) term approach aimed at treating only acute events with “Hemopoiesis” (n=788). Except for the mutations in platelet transfusion might be considered in most GNE, all the other homozygous variants were excluded patients. based on pathogenicity and splicing bioinformatic pre- Several lines of evidence supported the hypothesis dictions (Online Supplementary Table S2). that GNE mutations can cause thrombocytopenia. At Substitutions p.Val516Arg and p.Thr575Arg are rare least to our knowledge, 20 patients from nine unrelated variants affecting well conserved residues during evolu- families of the nearly 1,000 individuals with alterations tion (Figure 1C). Their potential deleterious effect was of this gene have been reported to have thrombocytope- supported by segregation analysis, bioinformatics pre- nia (Online Supplementary Table S3).2-4,10,11 Of the 15 dif- dictions (Online Supplementary Table S2), and significant ferent variants identified in these patients, including P1 reduction of the GNE protein expression, which was and P2, seven have been previously reported in patients likely to maintain some residual activity due to the with GNE myopathy (Figure 3). Except for p.His188Tyr, incompatibility of complete loss of the GNE function which is in cis with a known mutation (p.Asn550Ser) with life (Figure 1D).7 Consistent with alteration of the associated with myopathy,4 the others (p.Asp444Tyr, 750 haematologica | 2022; 107(3) Letters to the Editor p.Gly447Arg, p.Gly506Phe, p.Val516Arg, p.Leu517Pro, We analyzed the structure of N-acetylmannosamine p.Thr575Arg, and p.Gly578Ser) were regarded as specif- kinase in complex with N-acetylmannosamine and ADP ic variants associated with isolated thrombocytopenia. (2yhy)12 to assess the potential effect of the mutations on Of note, these seven variants spread over a region of the structure and function of the enzyme (Figure 3). approximately 130 amino acids of the ManNac kinase Residues Asp444, Gly447 and Thr575 in the ADP pock- domain (Figure 3). et, and Gly506 and Gly578 close to the substrate domain A B C D Figure 1. Identification of novel mutations of the GNE gene. (A) Pedigrees and segregation analysis in the two families F1 and F2. (B) Electropherograms of exon 9 showing the c.1546_1547delinsAG and c.1724C>G substitutions in probands P1 and P2, respectively. Sanger sequencing was performed using the fol- lowing primers: 9F/5’-TTCTAGAAATCTTTAAGGTGCTATGG-3’ and 9R/5’-CCACCTGACCATGTTGAAGA-3’. (C) Protein multiple alignments, showing conservation through different species at residues (in red) affected by the p.Val516Arg and p.Thr575Arg mutations. H. sapiens (NP_001121699.1), P. troglodytes (XP_003312121.1), M. mulatta (XP_001082113.2), C. lupus (XP_003431623.1), B. taurus (NP_001178072.2), M. musculus (NP_056643.3), R. norvegicus (NP_446217.1), G. gallus (NP_001026603.2), D. rerio (NP_957177.1), and X. tropicalis (NP_001072728.1). (D) Western blot and of total lysates from lym- phoblast cells of P1 and P2. Total protein lysates were prepared from these cells using M-PERTM Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). Protein quantification shows only a partial expression (39% and 79%, respectively) of GNE expression compared to wild-type (CTRL) (***P<0,002). Actin was used as a loading control for protein quantification. The antibodies were used as follows: anti-GNE (Santa Cruz Biotechnology, sc-376057, 1:500) and anti-β-actin (Santa Cruz Biotechnology, sc-47778, 1:4,000) as primary antibodies, anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP) (Bethyl, A90-116P, 1:10,000) as a secondary antibody. Statistical analysis was performed using the t-test. Error bars represent the standard deviation of 4 inde- pendent experiments. haematologica | 2022; 107(3) 751 Letters to the Editor A B C D E F G Figure 2. Blood, bone marrow features, and response to transfusion and treatment. (A) Sialotransferrin profile determined by ion-exchange chromatography using a commercial kit (CDT in Serum, Recipe München). The different isoforms were pointed out by UV detection at 460 nm and quantified by the “area percent method” (i.e., the relative abundance of each isoform is expressed as the percentage ratio of the peak area compared to the sum of the areas of all the peaks). (B) Peripheral blood smear of P2 showing enlarged platelets. (C to E) Bone marrow aspirates with an increased number of megakaryocytes at different stages of maturation. May-Grünwald-Giemsa staining; original magnification 100X (B), 10X (C), 20X (D), and 40X (E). (F) Response to platelet transfusion. Median platelet count before platelet transfusion and up to 7 days following transfusion are shown for both patients (P1 and P2’s specific values are indicated in brack- ets). (G) Time course of platelet count in response to treatments for P1 (left) and P2 (right) in response to romiplostim administration at different dosages. The dark grey bar indicates a period of complete transfusion dependency, with transfusion every 5-7 days. Values of platelets measured within 7 days after platelet transfusion are not shown. may directly affect the enzyme active site, impairing the third decade of life, we cannot exclude that patients ADP or substrate binding, respectively (Figure 3). with only thrombocytopenia will develop myopathy Otherwise, p.Val516 and p.Leu517 are localized in the later in their life.2-4 Patients carrying the p.Asp444Tyr hydrophobic core and when mutated into an arginine or (F3), p.Gly447Arg (F5), p.Val516Arg (F1), p.Leu517Pro proline, respectively, may destabilize the fold and the (F7), and p.Thr575Arg (F2) mutations were neonates or conformation of the entire protein. Accordingly, in vitro in their first/second decade of life. Instead, among the mutagenesis of the highly conserved Asp444 residue in six individuals homozygous for p.Gly506Phe (F6) or p. the ADP binding pocket resulted in the complete loss of Gly578Ser (F4) and all between 24-42 years of age, only the kinase function, though retaining the epimerase two have subclinical features of myopathy, suggesting activity.13 Therefore we could hypothesize that that, in addition to a low platelet count, this mutation megakaryocytes and platelets are more sensitive than could correlate with a mild form of muscle wasting of other cells to defective kinetic activity or substrate-bind- late onset. ing affinity, thus explaining the occurrence of thrombo- Trying to explain why only few patients with GNE cytopenia. mutations have a low platelet count, we cannot exclude Nevertheless, whether GNE mutations are responsible that GNE variants cause thrombocytopenia only when co- for thrombocytopenia either isolated or in combination segregating with other genetic factors. Whereas WES with muscle wasting remains to be elucidated. Indeed, analysis did not provide any other plausible candidate in considering that the GNE myopathy typically appears in our families, the recessive transmission of variants in other 752 haematologica | 2022; 107(3) Letters to the Editor A B Figure 3. Localization of the GNE mutations associated with thrombocytopenia and three-dimensional structure of the GNE enzyme. (A) Among the 15 muta- tions (Online Supplementary Table S3), the 7 associated with myopathy are indicated below the schematic representation of the protein structure; the other 8 are depicted above the protein (in red, the novel mutations reported in this paper). All the mutations identified in patients are in a homozygous state except those represented in matched colored boxes that are heterozygous biallelic GNE mutations found in a single patient. H188Y and N550S (light grey boxes) are homozygous mutations identified in the same patient. UDP-GlcNAc 2-epimerase: UDP-N-acetylglucosamine 2-epimerase; ManNAc kinase: N-acetylman- nosamine; NH2: NH2-terminus; COOH: COOH-terminus; RUF: a region with unknown function; NES: putative nuclear export signal; AS: allosteric site. Nomenclature of mutations was referred to the NM_001128227.3 transcript. (B) The overall structure (left) and the zoom of the enzymatic pocket (right) of GNE. The side chains of the positions affected by the mutations discussed in this article are explicitly indicated. The structure (Protein Data Bank [PDB] entry 2yhy) corresponds to the GNE complex with ManNAc and ADP. The structure was analyzed by PyMOL and MOLMOL graphic support tools. The degree of exposure of the residues affected by mutations was established by DSSP (Define Secondary Structure of Proteins) analysis. genes, such as ANKRD18A, FRMPD1, FLNB, PRKACG, Daniela Simoncini,4 Linda Rossini,2 Fabio Corsolini,7 have been reported in other cases.2,3,5,14 However, their Martina La Bianca,1 Giuseppe Robustelli,4 Maria Gabelli,2 potential impact was not further investigated, except for Massimo Agosti,4 Alessandra Biffi,2 Paolo Grotto,8 Valeria p.Ile74Met in PRKACG, whose functional studies demon- Bozzi,9 Patrizia Noris,9,10 Alberto B. Burlina,11 Adamo Pio strated its effect in thrombocytopenia.14 d'Adamo,1,5 Alberto Tommasini,1,5 Flavio Faletra,1 Annalisa In summary, although the role of GNE mutations is Pastore12,13 and Anna Savoia1,5 well-documented in GNE myopathy and sialuria, we 1 Istitute for Maternal and Child Health – IRCCS Burlo Garofolo, identified two novel GNE variants, which together with Trieste, Italy; 2Pediatric Hematology, Oncology and Stem Cell a few other mutations reported in the literature could Transplant Division, Padua University Hospital, Padua, Italy; explain thrombocytopenia and extend the clinical phe- 3 Fondazione Città della Speranza, Istituto di Ricerca Pediatrica, Padua, notype of the GNE defects. In both patients, as well as Italy; 4Maternal and Child Department, F. Del Ponte Hospital, in those of families F3 and F5 from a literature review, Varese, Italy; 5Department of Medical Sciences, University of Trieste, severe thrombocytopenia was reported since the first Trieste, Italy; 6Department of Genetics and Microbiology, Universitat days of life, when the differential diagnosis of thrombo- Autonoma de Barcelona, Barcelona, Spain; 7LABSIEM - Laboratory cytopenia was broad and included thrombocytopenia for the Study of Inborn Errors of Metabolism, Pediatric Clinic and secondary to sepsis and critical care, neonatal allo- and Endocrinology, Istituto Giannina Gaslini, Genova, Italy; 8Pediatric auto-immune thrombocytopenia, or ITP, such as congen- Department, Hospital of Treviso - Oderzo, Treviso, Italy; ital amegakaryocytic thrombocytopenia.15 Therefore, 9 Biotechnology Research Laboratories, IRCCS Policlinico San Matteo evaluating the sialotransferrin profile in patients with Foundation, Pavia, Italy; 10Department of Internal Medicine and suspected inherited thrombocytopenia, large platelets Medical Therapy, University of Pavia, Pavia, Italy; 11Division of and increased reticulated fraction might provide an Inherited Metabolic Diseases, Regional Center for Expanded Neonatal important diagnostic clue. Screening Department of Women and Children’s Health, University Hospital of Padova, Padova, Italy; 12King’s College London, Roberta Bottega,1* Antonio Marzollo,2,3* Maddalena Department of Clinical Neuroscience, Denmark Hill Campus, London, Marinoni,4 Emmanouil Athanasakis,1 Ilaria Persico,5,6 Anna UK and 13European Synchrotron Radiation Facility 71, Grenoble, Monica Bianco,1 Michela Faleschini,1 Erica Valencic,1 France haematologica | 2022; 107(3) 753 Letters to the Editor *RB and AM contributed equally as co-first authors. identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects. Haematologica. Correspondence: 2016;101(10):1170-1179. ANNA SAVOIA - anna.savoia@burlo.trieste.it 4. Revel-Vilk S, Shai E, Turro E, et al. variants causing autosomal doi:10.3324/haematol.2021.279689 recessive macrothrombocytopenia without associated muscle wasting. Blood. 2018;132(17):1851-1854. Received: August 11, 2021. 5. Izumi R, Niihori T, Suzuki N, et al. GNE myopathy associated Accepted: November 11, 2021. with congenital thrombocytopenia: a report of two siblings. Neuromuscul Disord. 2014;24(12):1068-1072. Pre-published: November 18, 2021. 6. Zhen C, Guo F, Fang X, Liu Y, Wang X. A family with distal Disclosures: no conflicts of interest to disclose. myopathy with rimmed vacuoles associated with thrombocy- topenia. Neurol Sci. 2014;35(9):1479-1481. Contributions: RB, AM, MM and AS designed research, inter- 7. Schwarzkopf M, Knobeloch KP, Rohde E, et al. Sialylation is preted data, and wrote the manuscript; EA, AMB, MF, MLB and essential for early development in mice. Proc Natl Acad Sci U S A. APDA performed NGS analysis; IP, EV, FC, ABB and AP per- 2002;99(8):5267-5270. formed experiments; DS, LR, GR, MG, MA, AB, PG, VB, PN, 8. Kauskot A, Pascreau T, Adam F, et al. A mutation in the gene cod- AT and FF collected hematological and other clinical data. All ing for the sialic acid transporter SLC35A1 is required for platelet authors critically revised the paper and approved the final version. life span but not proplatelet formation. Haematologica. 2018;103(12):e613-e617. Acknowledgements: we thank Samuela Francescato and Giulia 9. Bongiovanni D, Santamaria G, Klug M, et al. Transcriptome Bossi for their assistance in the production of the figures, and Dr. analysis of reticulated platelets reveals a prothrombotic profile. Martina Bradaschia for the language revision of the manuscript. Thromb Haemost. 2019;119(11):1795-1806. The authors would like to acknowledge Prof. F. de Cothi and Prof. 10. Li X, Li Y, Lei M, et al. Congenital thrombocytopenia associated A. Dhele for the constructive discussions. with GNE mutations in twin sisters: a case report and literature review. BMC Med Genet. 2020;21(1):224. Funding: this work was supported by IRCCS Burlo Garofolo 11. Mekchay P, Ittiwut C, Ittiwut R, et al. Whole exome sequencing (grant numbers RC 31/17 and RC 02/18) and IRCCS Policlinico for diagnosis of hereditary thrombocytopenia. Medicine San Matteo Foundation (Intramural research grant).We thank (Baltimore). 2020;99(47):e23275. Fondazione Città della Speranza ONLUS (http://cittadellasperan- 12. Martinez J, Nguyen LD, Hinderlich S, et al. Crystal structures of za.org/) and Fondazione Giacomo Ascoli Onlus (https://www.fon- N-acetylmannosamine kinase provide insights into enzyme activ- ity and inhibition. J Biol Chem. 2012;287(17):13656-13665. dazionegiacomoascoli.it/) for their support of our scientific work. 13. Effertz K, Hinderlich S, Reutter W. Selective loss of either the epimerase or kinase activity of UDP-N-acetylglucosamine 2- References epimerase/N-acetylmannosamine kinase due to site-directed mutagenesis based on sequence alignments. J Biol Chem. 1. Hinderlich S, Weidemann W, Yardeni T, Horstkorte R, Huizing M. 1999;274(40):28771-28778. UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE): a master regu- 14. Manchev VT, Hilpert M, Berrou E, et al. A new form of lator of sialic acid synthesis. Top Curr Chem. 2015;366:97-137. macrothrombocytopenia induced by a germ-line mutation in the 2. Futterer J, Dalby A, Lowe GC, et al. Mutation in GNE is associat- PRKACG gene. Blood. 2014;124(16):2554-2563. ed with severe congenital thrombocytopenia. Blood. 15. Sillers L, Van Slambrouck C, Lapping-Carr G. Neonatal thrombo- 2018;132(17):1855-1858. cytopenia: etiology and diagnosis. Pediatr Ann. 2015;44(7):e175- 3. Johnson B, Lowe GC, Futterer J, et al. Whole exome sequencing 180. 754 haematologica | 2022; 107(3)