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Abstract Introduction
Antithrombin (AT) is an N-glycosylated serpin that plays key roles in the coagulation cascade. Thus, AT deficiency sharply increases risk of (recurrent) thrombosis. A cause of hereditary AT deficiency lies in over 350 reported mutations of SERPINC1, the coding gene, which have a varying effect on the concentration and functionality of AT. Likewise, altered glycosylation of the four N-glycosylation sites may also cause AT deficiency. Current diagnostic tests for AT are based on functional assays using chromogenic substrates, a strategy developed over 30 years ago. Underdiagnosis of certain types of AT deficiency and limited information on the impact of specific proteoforms hamper the use of AT activity tests for diagnosing thrombophilia. To address this unmet clinical need, a mass spectrometry test for quantitative and qualitative characterization of AT proteoforms was developed. The test was analytically validated for quantitative precision, and we here present its qualitative potential.
Objectives
This study examined whether molecular characterization of AT using mass spectrometry could identify patients with aberrant AT proteoforms due to hereditary AT deficiency or altered glycosylation caused by congenital disorders of glycosylation (CDGs).
Methods
Two cohorts of anonymized patients with clinically relevant AT proteoforms were analyzed (with local METC approval): a cohort of 91 patients with hereditary AT deficiency and a cohort of 41 patients with AT deficiency caused by CDG type 1. Both cohorts were previously screened for AT activity and genetic mutations in SERPINC1 or CDG-related genes. Citrate plasma samples were measured using our in-house developed MS test for AT, which monitors 23 proteotypic (wildtype) peptides. To confirm alterations in wildtype peptides, mutation-specific transitions were developed.
Results
The study generated an extensive amount of information per patient. To facilitate robust and efficient analysis, an R-script was developed that verifies the overall AT concentration of each sample and identifies peptides that deviate from this concentration and are thus suspected of containing multiple proteoforms. Quantitative defects were observed in 95.6 % of AT deficiency patients and 87.8 % of CDG patients and (median concentration 0.73 and 0.86 µmol/L respectively, ref.int. 1.07 – 1.49 µmol/L). To confirm mutations in patients with AT deficiency, variant specific transitions were established for various mutations, including p.Val30Glu (AT Dublin), p.Arg45Trp, p.Pro73Leu (AT Basel), p.Leu131Phe (AT Budapest3), p.Arg177Cys, p.Arg425Cys. Interestingly, variant peptides were also identified in patients with type-1 AT deficiency, a subtype which is believed to cause a quantitative defect without expression and secretion of the variant proteoform. In CDG-patients the amount of alpha-AT proteoform (containing a glycan at site N167) was significantly decreased compared to healthy controls (71.1 % versus 94.6 %, respectively). Moreover, altered N-glycosylation was observed for a second N-glycosylation site (N224).
Conclusion
The LC-MRM-MS test for AT proves to identify patients with AT deficiency: congenital defects and CDG. By combining quantitative and qualitative proteoform information, a molecular diagnosis can be generated, identifying pathogenic variants in the sample at the amino acid and/or PTM level. We anticipate that quantitation and molecular characterization of AT has potential in precision diagnostics of thrombophilia and will refine the intended uses of AT testing in clinical care pathways. To summarize, by using our next-generation LC-MRM-MS test for AT, we expose the molecular puzzle underlying the clinical heterogeneity of AT deficiency, which cannot be captured by traditional activity tests.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
