Type II antithrombin deficiency caused by a large in-frame insertion: structural, functional and pathological...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Serpins require a metastable native conformation for protease inhibition.
• Missense mutations can destabilize serpins, leading to protein misfolding and disease.
• Antithrombin deficiency is a genetic risk factor for thrombosis.

Purpose of the Study:

• To characterize a novel antithrombin deficiency caused by a large in-frame insertion in the SERPINC1 gene.
• Investigate the structural and functional consequences of this insertion on antithrombin function.

Main Methods:

• Functional, biochemical, and molecular analyses of the affected patient and family members.
• Expression and purification of recombinant mutant antithrombin.
• Calorimetry for stability assessment and X-ray crystallography for structural analysis.

Main Results:

• Identified a 24 bp in-frame insertion in SERPINC1, leading to type II antithrombin deficiency with impaired heparin binding.
• The mutant antithrombin was non-inhibitory, conformationally sensitive, and showed proteolytic cleavage at W49.
• Crystal structure revealed the insertion annealed into beta-sheet A, maintaining a native reactive centre loop (RCL) and a hyperstable conformation.

Conclusions:

• This is the first report of a large in-frame insertion causing a serpin deficiency.
• The mutation allows proper protein folding, glycosylation, and secretion, resulting in a stable, non-inhibitory variant.
• This finding expands the understanding of serpinopathies and their associated thrombotic risks.