Two SERPINC1 variants affecting N-glycosylation of Asn224 cause severe thrombophilia not detected by functional assays

Abstract

Antithrombin deficiency, the most severe congenital thrombophilia, might be underestimated, as some pathogenic variants are not detected by routine functional methods. We have identified two new SERPINC1 variants, p.Glu227Lys and p.Asn224His, in four unrelated thrombophilic patients with early and recurrent thrombosis that had normal antithrombin activity. In one case, the mutation was identified by whole genome sequencing, while in the 3 remaining cases, the mutation was identified by sequencing SERPINC1 based on a single functional positive finding supporting deficiency. The two variants shared a common functional defect, an impaired or null N-glycosylation of Asn224 according to a eukaryotic expression model. Carriers had normal anti-FXa or anti-FIIa activities, but impaired anti-FVIIa activity and a detectable loss of inhibitory function when incubating the plasma 1 hour at 41ºC. Moreover, the beta glycoform of the variants, lacking two N-glycans, had reduced secretion, increased heparin affinity, no inhibitory activity, and a potential dominant negative effect. These results explain the increased thrombin generation observed in carriers. Mutation experiments reflected the role that Lysine residues close to the N-glycosylation sequon have in impairing the efficacy of N-glycosylation. Our study shows new elements involved in the regulation of N-glycosylation, a key post-translational modification that, according to our results affects folding, secretion and function, providing new evidence of the pathogenic consequence of an incorrect N-glycosylation of antithrombin. This study supports that antithrombin deficiency is underestimated and encourages the development of new functional and genetic tests to diagnose this severe thrombophilia.

Missense mutation of SERPINC1 (p.Ser426Leu) in a young patient presenting as refractory and recurrent venous thromboembolism: A case report

“…Cases with transient antithrombin deficiency have very recently been described ( 10 , 11 ). Inflammatory processes are always orchestrated by pleiotropic and multifunctional cytokines and chemokines which have pro-thrombotic effects in thrombosis ( 12 ).…”

Thrombosis and hemostasis at the University of Padua: a reappraisal on the occasion of its 800th year of history

“…The large number of newly discovered inherited defects in the last decades both in Padua and all over the world 38 and their clinical impact seems to justify why I have no doubt that the University of Padua will continue to make contribution to the research fields of thrombophilia and hemophilia gene therapy in the years to come.…”

Antithrombin diagnostics by mass spectrometry: Development and analytical validation of a next-generation test

“…However, activity tests may also contribute to an oversimplified view of AT deficiency, as the heterogeneity of molecular forms of AT present in patients is neglected. Specifically, the sensitivity of activity tests is low for AT deficiency caused by heparin-binding-site mutations or when glycosylation is affected [ 3 , 4 ]. Furthermore, current guidelines for diagnosing and treating AT deficiency are based on studies that classify patients with activity tests and do not stratify risks according to specific proteoforms [ [5] , [6] , [7] , [8] ].…”

“…Furthermore, current guidelines for diagnosing and treating AT deficiency are based on studies that classify patients with activity tests and do not stratify risks according to specific proteoforms [ [5] , [6] , [7] , [8] ]. Therefore, diagnosing AT deficiency by molecular characterization of AT may improve patient care by enabling better proteoform-based risk management [ 4 , 9 ].…”

“…Generally, only small quantities of AT carrying other glycoforms occur [ [15] , [16] , [17] ]. Both the macroheterogeneity, ie, the presence of glycans on the protein, and the microheterogeneity have been shown to affect the function of AT, specifically the heparin affinity [ 4 , [18] , [19] , [20] , [21] ]. This effect may lead to difficulties in diagnosing patients harboring affected glycosylation, as the excess heparin used in activity tests may disguise the decreased functionality of glycosylation mutants [ 4 ].…”