OC 76.3 - Landscape of natural variants affecting the N-glycosylation of antithrombin: biological and clinical impact

Wednesday, June 26, 2024

10:45 – 11:00 ICT

Room: 210 A-D

Co-Author(s)

RC

Rosa Cifuentes-Riquelme

Predoctoral student
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain

Presenting Author(s)

María Eugenia de la Morena-Barrio, PhD

PhD
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain

Co-Author(s)

KM

Kruijt Mirjam

PhD
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden
Leiden, Zuid-Holland, Netherlands
Pedro Garrido-Rodríguez

PhD student
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
Juan José Rojo-Carrillo

PhD Student
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
JP

José Padilla

Tech
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
Belén de la Morena-Barrio, PhD

Post-doc
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
CB

Carlos Bravo-Pérez

PhD MD
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
AM

Antonia Miñano

Tech
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
EN

Esther Navarro

Post-doc
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
ML

María Llamas-López, PhD-Student

Bioinformatician
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
VV

Vicente Vicente

Senior Researcher
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
ML

María Luisa Lozano

Hematologist
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
PS

Per Morten Sandset

Senior Researcher and Medical Doctor
Institute of Clinical Medicine, Faculty of Medicine, University of Oslo
Oslo, Oslo, Norway
Pierre Morange, MD, PhD

Aix-Marseille University, France
LR

L. Renee Ruhaak

PhD, Associate professor
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden
Leiden, Zuid-Holland, Netherlands
Javier Corral

Professor
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain

Background: Antithrombin is an anticoagulant serpin with 4 N-glycans. Glycosylation likely plays a crucial role, as evidenced by the varying heparin affinity among glycoforms resulting from inefficient glycosylation of N167 and the deficiency observed in patients with congenital disorders of glycosylation (CDG).

Aims: Characterization of the biological and clinical impact of natural aberrant N-glycosylation in antithrombin.

Methods: Natural variants deleting or generating SERPINC1 N-glycosylation sequons were screened in 433 unrelated patients with antithrombin deficiency (ATD), the Human Gene Mutation database, and GnomAD. We evaluated the glycosylation, secretion, and function of these variants in plasma and HEK-transfected cells. Levels and glycosylation of antithrombin in 37 unrelated CDG patients was evaluated by chromogenic assays, Western blot and LC-MRM-MS.

Results: We identified 18 natural mutations affecting N-glycosylation sequons. Mutations affecting N167 and N224 generated variants lacking one glycan that were secreted and had normal anti-FXa/FIIa activity. Only p.N224H caused severe thrombosis by affecting anti-FVIIa activity. The variants generating new sequons showed heterogeneous occupancy. Nine variants (creating N-X-C sequons, with conformational effects or located in the signal peptide), were not glycosylated. However, eight variants, primarily situated in helix/strands, had an extra N-glycan with impaired secretion or diminished heparin affinity, leading to severe type I or mild type II HBS deficiency, respectively (Table-1).
CDG patients had ATD (58%) and increased levels of hypoglycosylated antithrombin mainly affecting N167 and N224 (Figure-1).

Conclusion(s): Glycosylation of N128 and N187 seems crucial for antithrombin as these positions are occupied in plasma antithrombin of CDGs and no variant affecting these residues are described among patients with ATD or in the general population. Mutation of N224, while not impacting secretion, affects anti-FVIIa activity resulting in a challenging diagnosis. An extra N-glycan may cause varied functional effects: impaired secretion (severe type I deficiency) or impaired heparin affinity (mild type II HBS deficiency).

Funding: PI21/00174 (ISCIII&UE); 21886/PI/22 (Fundación Séneca)