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de la Morena-Barrio ME, Sevivas TS, Martínez-Martínez I, Miñano A, Vicente V, Jaeken J, Corral J. Congenital disorder of glycosylation (PMM2-CDG) in a patient with antithrombin deficiency and severe thrombophilia. J Thromb Haemost 2012; 10: 2625–7.
Antithrombin deficiency was the first congenital thrombophilic factor identified . Since then, protein C and protein S deficiencies, as well as specific mutations in the factor V and prothrombin genes, have joined antithrombin deficiency as genetic defects predisposing patients to venous thrombosis. However, it is unlikely that new common polymorphisms could play a key role in venous thrombosis . Therefore, rare mutations affecting different elements of the hemostatic system are emerging candidates [3,4]. The analysis of antithrombin in a thrombophilic patient with antithrombin deficiency has allowed us to identify a particular thrombophilic defect that, affecting the N-glycosylation pathway, indirectly disturbs this key anticoagulant serpin.
We report on an 18-year-old male with recurrent left-leg venous thrombosis. The first event happened at 11 years of age. Oral anticoagulation with warfarin was established for 5 months. A second event occurred at the same location at 12 years. Oral anticoagulation with warfarin was re-established. Surprisingly, a new thrombotic event took place 6 months later, under stable oral anticoagulant therapy with an International Normalized Ratio (INR) of 2–3. A fourth thrombotic event was diagnosed at 17 years, again under oral anticoagulant therapy. Currently, the patient is being treated with higher warfarin doses (INR 2.5–3.5).
No familial history of thrombosis was reported. Thrombophilic analysis revealed no prothrombotic polymorphisms (FV Leiden or prothrombin G20210A). No antiphospholipid antibodies were detected. Protein C and protein S levels were within the normal range. The only thrombophilic defect identified was an antithrombin deficiency (anti-FXa activity of 67%). Antigen levels of antithrombin were also low (60%), suggesting a spontaneous heterozygous type I antithrombin deficiency. However, the sequencing of the promoter, as well as the coding and flanking regions of the SERPINC1 gene,  revealed no mutation. Large gene deletions were excluded by multiplex ligation-dependent probe amplification (MLPA) studies. Interestingly, electrophoretic analysis of plasma antithrombin performed under either denaturing or native conditions  revealed the presence of an abnormal antithrombin (Fig. 1A). The increased mobility in SDS suggested abnormal post-translational modifications. We evaluated impaired N-glycosylation, the only post-translational modification described in antithrombin that also affects the function and clearance of this anticoagulant serpin . N-glycan removal with N-glycosidase F  equalized the mobility of both the major and the minor antithrombin species, suggesting an N-glycosylation defect for ∼ 20% of the antithrombin molecules in the plasma of the proband (Fig. 1B). This abnormal antithrombin was not the β-glycoform as, among other findings, this defect was not specific for antithrombin. Thus, smaller forms of α1-antitrypsin were also detected in the plasma of this patient (Fig. 1C). These biochemical and clinical data suggested a congenital disorder of glycosylation (CDG), a category that comprises a broad group of autosomal recessive disorders . Actually, the antithrombin and α1-antitrypsin electrophoretic profiles were very similar to those shown by a patient with the commonest CDG, PMM2-CDG, with mutations in PMM2, the gene encoding phosphomannomutase 2. This is a cytosolic enzyme that catalyzes the conversion of mannose 6-phosphate to mannose 1-phosphate, a substrate for GDP-mannose synthesis, required in N-glycosylation  (Fig. 1C). HPLC analysis of transferrin glycoforms pointed to a CDG-I diagnosis by the presence of asialotransferrin and a significant increase in the amount of disialotransferrin (Fig. 1D). We identified two mutations in PMM2: one of maternal origin in exon 5, and the other in exon 8, responsible for the p.R141H and p.C241S substitutions, respectively. Unfortunately, we do not have samples from the father. The same combination has been reported previously in a PMM2-CDG patient with a mild clinical phenotype . In addition, the proband had mental retardation without ataxia, strabismus, a thyroid nodule, and kyphosis. The digestive, hepatic, renal and cardiovascular systems were normal.
Figure 1. Identification of PMM2-CDG in the proband. (A) Native and SDS-PAGE showing the normal (arrow) and abnormal (dashed arrow) antithrombin present in plasma of the proband, a patient with congenital type I antithrombin deficiency (AT def) (p.R149X) and a control. (B) SDS-PAGE and Western blot of antithrombin from plasma untreated (–) or treated (+) with N-glycosidase F. (C) SDS-PAGE pattern of α1-antitrypsin (α1-AT) and antithrombin (AT) in the proband, his mother, a control, and a PMM2-CDG patient (p.F119L and p.R141H). Arrows mark the abnormal glycoforms of these molecules. (D) HPLC profile of transferrin glycoforms of the proband, his mother, and a PMM2-CDG patient (p.F119L and p.R141H).
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Up to 55% of PMM2-CDG patients show acute events such as ‘stroke-like’ episodes and ischemic cerebral vascular accidents, and at least 10 cases with either venous or arterial thrombosis have been described . Acute vascular events occurred in patients younger than 15 years, especially during fever and prolonged immobilization . These accidents can be recurrent in the same patient . Only a few studies have evaluated hemostatic factors [10,11] and platelets  in PMM2-CDG with the aim of identifying the mechanism(s) involved in the development of these vascular events. PMM2-CDG patients consistently have antithrombin deficiency [10,11], with abnormal hypoglycosylated antithrombin molecules . This might contribute to the increased risk of thrombotic events described in these patients. However, there are no differences in antithrombin levels between patients with and without vascular events .
This is the first report on a PMM2-CDG patient with documented recurrent venous thrombosis. Moreover, the patient suffered from thrombotic events without a clear triggering factor, such as fever, catheterization, or immobilization. Finally, two recurrent thrombotic events occurred under oral anticoagulant therapy. This unusual clinical severity suggests that hemostatic or vascular defects other than those described in PMM2-CDG patients (or additional unknown thrombophilic anomalies) might be present in this patient. This deserves further study.
This case report also highlights another important conclusion concerning the diagnostic procedure in patients with antithrombin deficiency. Direct sequencing and MLPA analysis of SERPINC1 in patients with thrombosis and antithrombin deficiency is highly effective. A recent study including 234 cases with antithrombin deficiency found a high mutation detection rate for SERPINC1 (83.5%) . Our study suggests that a diagnosis of PMM2-CDG might be suspected among cases with antithrombin deficiency and no mutations in SERPINC1, particularly in those cases without a family history of thrombosis, as well as in cases with a lower molecular weight plasma antithrombin.