Sialylation is known to regulate platelet clearance.1 Sialic acids are terminal sugar components of the oligosaccharide chains of glycoproteins and glycolipids. During platelet ageing, surface modifications such as the loss of sialic acid (i.e., desialylation) expose β-galactose residues. These senescence antigens facilitate platelet clearance, influence the platelet life span, and stimulate thrombopoietin (TPO) production.2 Greater exposure of non-sialylated glycan chains may be associated with accelerated platelet clearance. Sialyltransferases constitute a family of glycosyltransferases that transfer sialic acid from the donor substrate (cytidine-5’-monophosphate (CMP)-sialic acid) to acceptor oligosaccharide substrates.3 Mice in which the St3gal4 sialyltransferase gene has been knocked out are thrombocytopenic; the absence of sialic acid increases β-galactose exposure and thus results in the rapid clearance of platelets from the circulation.4 In sialic acid metabolism, a specific transporter [solute carrier family 35 member A1 (SLC35A1)] transfer CMP-sialic acid to the medial- and trans-Golgi apparatus, where it is used as a substrate for the sialylation of proteins by various sialyltransferases (Figure 1).
Herein, we report on a congenital deficiency in SLC35A1 in two siblings born to consanguineous parents and who displayed moderate macrothrombocytopenia. The sister (II:1, born in 2001) and brother (II:2, born in 2005) shared similar clinical presentations from birth, including delayed psychomotor development, epilepsy, ataxia, microcephaly, choreiform movements, and macrothrombocytopenia. This presentation was highly suggestive of an inherited disease. The hemorrhagic symptoms consisted of easy bruising for both siblings, and menorrhagia (leading to iron deficiency) for II:1. Persistent thrombocytopenia was observed in both siblings, with a mean ± standard error of the mean (SEM) platelet count of 95 ±10 G/L (n=31 analyses between 2002 and 2017) for II:1 and 60 ± 6 G/L (n=21 analysis between 2010 and 2015) for II:2. Comparative with normal values (6.5 to 12 fL), the platelet volume was high (mean ± SEM: 15.7 ± 3.9 fL (n=31) for II:1, and 19.1 and 20.4 fL for II:2). A peripheral blood smear review revealed the presence of giant platelets and macroplatelets (Figure 2A). Fluorescence analysis of the immature platelet fraction (on a Sysmex XE 5000 hematology analyzer) indicated high levels of reticulated platelets: 54% and 66% (n=2) for II:1, and 46% and 60% (n=2) for II:2; the normal range is <5%). Bone marrow aspirate smears of II:1 revealed an elevated megakaryocyte count (>2 megakaryocytes per low-power field), with a predominance of immature forms (characterized by small size, hypolobulation, and a basophilic cytoplasm, as observed in peripheral thrombocytopenia; Figure 2B). The elevated proportion of immature platelets and the bone marrow analysis results were suggestive of a compensatory mechanism for peripheral thrombocytopenia.
By combining whole-exome sequencing with the genetic mapping of disease loci (assuming autosomal recessive inheritance), we found that patient II:1 was homozygous for a missense mutation in the SLC35A1 gene (NM_006416.4: c.439T>C: p.Ser147Pro, Figure 2C). The presence of this variant in both siblings was confirmed by Sanger sequencing (Figure 2C). The variant was absent from the dbSNP150, 1000Genomes or ExAC Browser databases, and was predicted to be pathogenic (Polyphen-2 score: 1).5 Both parents were heterozygous for the mutation (Figure 2C). The SLC35A1 protein comprises 10 transmembrane domains (TMDs), and a topol ogy analysis indicated that the p.Ser147Pro mutation was located in TMD-5. Given that the insertion of a Pro residue into a TMD is predicted to have a profound impact on a protein’s activity, the mutation p.Ser147Pro is expected to be deleterious. The cytoplasmic loop between TMDs 4 and 5 is important for SLC35A1’s transporter activity,6 and so a mutation in TMD-5 might change the protein’s activity via a conformational change in this loop and/or by destabilizing the transmembrane helix. Capillary electrophoresis of patient II:1’s serum transferrin (Figure 2D) showed elevated levels of hyposialylated glycoforms (3-sialo to 1-sialo), which is highly suggestive of a congenital disorder of glycosylation and thus the impaired maturation of protein-linked N-glycans in the Golgi apparatus. SLC35A1 deficiency has been referred to as congenital disorder of N-linked glycosylation IIf (CDG-IIf), with the new nomenclature SLC35A1-CDG.7
Given the recent discovery of sialylation’s importance in senescent platelet clearance, a congenital deficiency in the CMP-sialic acid transporter might account for the thrombocytopenia identified in the two probands. Since SLC35A1 is expressed in megakaryocytes and platelets (data not shown), we analyzed platelets from patient II:1. We performed a flow cytometry assay for platelet β-galactose exposure by using the lectin Ricinus communis agglutinin (RCA), which is specific for the β-galactose exposed in the absence of sialic acid. The significant elevation (P<0.001) in platelet surface RCA labeling for II:1 was highly suggestive of a sialylation defect (Figure 3A). To take platelet size into account, we also measured the ratio of the mean fluorescence intensity (MFI) of RCA to the MFI of GPIIb. A significant elevation (P<0.01) in II:1 (relative to a control) was still observed (Figure 3B) - indicating the presence of a qualitative defect in platelet sialylation. This defect could not be attributed to anti-GPIb antibodies [which have been reported elsewhere to induce platelet desialylation in immune thrombocytopenia (ITP)]8 because none were present in II:1’s plasma, according to a conventional assay. Moreover, we confirmed the absence of a plasma effect on platelet sialylation; when normal platelets were incubated with either normal plasma or patient II:1’s plasma, no difference in the mean ± SEM RCA binding was observed (21552 ± 1003 and 23080 ± 1930 arbitrary units (AU), respectively; n=3). In contrast, the incubation of normal platelets with control plasma in the presence of botrocetin (a positive control) induced greater RCA binding (38845 ± 880 AU, n=3; P<0.001), as expected.9 Next, we investigated the sialylation defect by Western blotting II:1’s platelet lysate. The RCA intensity was four-fold higher in II:1’s platelets than in a control sample (Figure 3C), and several bands were only present in patient II:1 (at around 100 kDa, 70-72 kDa, and 56-58 kDa). Interestingly, treatment with neuraminidase increased the RCA intensity in control experiments but not in experiments with patient II:1’s platelets – indicating that the latter are already markedly hyposialylated. Taken as a whole, our results suggest that SLC35A1 has a major role in platelet sialylation.
Given that the sialic acid content and β-galactose exposure at the platelet surface are highly relevant for platelet clearance and platelet life span, we next measured the clearance of patient II:1’s hyposialylated platelets in mice. Platelet survival was evaluated in vivo after the injection of washed platelets into the circulation of non-obese diabetic/severe combined immunodeficiency (NOD SCID) gamma mice. As shown in Figure 3D, 46 ± 15% of the control platelets were detected in the circulation 90 minutes after platelet injection. In contrast, II:1’s platelets were virtually undetectable - strongly suggesting that the patient’s platelets had a very short life span. Given that the circulating platelet count results from the balance between platelet clearance and platelet production, we next looked at whether a mutation in SLC35A1 disturbed in vitro proplatelet formation in cultured human megakaryocytes (MKs). Peripheral blood CD34 cells from the controls and patient II:1 were differentiated in vitro in the presence of TPO. We did not observe defective proplatelet formation in the patient’s MKs, which contained normal-sized coiled elements (i.e., future platelets) (Figure 3E). These results suggest that SLC35A1-dependent sialic acid transport in MKs is not involved in platelet formation. Macrothombocytopenia has already been reported in two patients with (respectively) a p.Gln101His mutation10 and a homozygous G>A substitution in the donor splice site of intron 6 in the SLC35A1 gene.11 Furthermore, mutations in the gene coding for glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) (Figure 1) – an enzyme involved in the CMP-SA pathway – has been linked to macrothombocytopenia.1312 However, platelet sialylation and life span were not investigated, and a link between thrombocytopenia and defect in platelet sialylation was not highlighted. Herein, we demonstrated that a severe defect in the sialylation pathway resulted in (i) the hyposialylation of platelet glycoproteins, and (ii) a severely shortened platelet half-life. Furthermore, our results suggest that SLC35A1-regulated sialic acid transfer is not involved in proplatelet formation in MKs or that it is compensated for by another pathway. The presence of giant platelets in patient II:1 was probably not directly due to SLC35A1 dysfunction in MKs. The giant platelets might correspond to a compensatory mechanism in a context of thrombocytopenia, as suggested by elevated reticulated platelet levels and a high MK count in the bone marrow.
A better understanding of the mechanism of thrombocytopenia will have consequences for patient care. Indeed, patients with unexplained macrothrombocytopenia are often misdiagnosed with ITP (an exclusion diagnosis). Even though patient II:1 may have experienced ITP in childhood, hyposialylation was responsible for her basal thrombocytopenia. This knowledge will influence the chosen treatment. Given that thrombocytopenia is associated with a shorter life span for hyposialylated platelets, one can legitimately expect the transfusion of normal platelets to be clinically effective. This was confirmed by Willig et al. report on platelet transfusion.14
Overall, our results demonstrated that sialic acid transport by SLC35A1 is required for platelet life span and sialic acid content in humans. A limitation in our study resides in the fact that due to lack of parental consent for additional blood sampling, megakaryocytes and in vivo platelet clearance experiments could only be performed once. The present case report establishes a link between progress in understanding platelet physiology on one hand and patient care on the other.
- Hoffmeister KM. The role of lectins and glycans in platelet clearance. J Thromb Haemost. 2011; 9(Suppl 1):35-43. PubMedhttps://doi.org/10.1111/j.1538-7836.2011.04276.xGoogle Scholar
- Grozovsky R, Begonja AJ, Liu K. The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling. Nat Med. 2015; 21(1):47-54. PubMedhttps://doi.org/10.1038/nm.3770Google Scholar
- Paulson JC, Colley KJ. Glycosyltransferases. Structure, localization, and control of cell type-specific glycosylation. J Biol Chem. 1989; 264(30):17615-17618. PubMedGoogle Scholar
- Sorensen AL, Rumjantseva V, Nayeb-Hashemi S. Role of sialic acid for platelet life span: exposure of beta-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein receptor-expressing liver macrophages and hepatocytes. Blood. 2009; 114(8):1645-1654. PubMedhttps://doi.org/10.1182/blood-2009-01-199414Google Scholar
- Adzhubei IA, Schmidt S, Peshkin L. A method and server for predicting damaging missense mutations. Nat Methods. 2010; 7(4):248-249. PubMedhttps://doi.org/10.1038/nmeth0410-248Google Scholar
- Chan KF, Zhang P, Song Z. Identification of essential amino acid residues in the hydrophilic loop regions of the CMP-sialic acid transporter and UDP-galactose transporter. Glycobiology. 2010; 20(6):689-701. PubMedhttps://doi.org/10.1093/glycob/cwq016Google Scholar
- Jaeken J, Hennet T, Matthijs G, Freeze HH. CDG nomenclature: time for a change!. Biochim Biophys Acta. 2009; 1792(9):825-826. PubMedhttps://doi.org/10.1016/j.bbadis.2009.08.005Google Scholar
- Li J, van der Wal DE, Zhu G. Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia. Nat Commun. 2015; 6:7737. PubMedhttps://doi.org/10.1038/ncomms8737Google Scholar
- Deng W, Xu Y, Chen W. Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor. Nat Commun. 2016; 7:12863. https://doi.org/10.1038/ncomms12863Google Scholar
- Mohamed M, Ashikov A, Guillard M. Intellectual disability and bleeding diathesis due to deficient CMP--sialic acid transport. Neurology. 2013; 81(7):681-687. https://doi.org/10.1212/WNL.0b013e3182a08f53Google Scholar
- Martinez-Duncker I, Dupre T, Piller V. Genetic complementation reveals a novel human congenital disorder of glycosylation of type II, due to inactivation of the Golgi CMP-sialic acid transporter. Blood. 2005; 105(7):2671-2676. PubMedhttps://doi.org/10.1182/blood-2004-09-3509Google Scholar
- Futterer J, Dalby A, Lowe GC. Mutation in GNE is associated with a severe form of congenital thrombocytopenia. Blood. 2018. Google Scholar
- Manchev VT, Hilpert M, Berrou E. A new form of macrothrombocytopenia induced by a germ-line mutation in the PRKACG gene. Blood. 2014; 124(16):2554-2563. PubMedhttps://doi.org/10.1182/blood-2014-01-551820Google Scholar
- Willig TB, Breton-Gorius J, Elbim C. Macrothrombocytopenia with abnormal demarcation membranes in megakaryocytes and neutropenia with a complete lack of sialyl-Lewis-X antigen in leukocytes--a new syndrome?. Blood. 2001; 97(3):826-828. PubMedhttps://doi.org/10.1182/blood.V97.3.826Google Scholar
- Parente F, Ah Mew N, Jaeken J, Gilfix BM. A new capillary zone electrophoresis method for the screening of congenital disorders of glycosylation (CDG). Clin Chim Acta. 2010; 411(1–2):64-66. PubMedGoogle Scholar