Analysis of AlphaFold and molecular dynamics structure predictions of mutations in serpins.

Serine protease inhibitors (serpins) include thousands of structurally conserved proteins playing key roles in many organisms. Mutations affecting serpins may disturb their conformation, leading to inactive forms. Unfortunately, conformational consequences of serpin mutations are difficult to predict. In this study, we integrate experimental data of patients with mutations affecting one serpin with the predictions obtained by AlphaFold and molecular dynamics. Five SERPINC1 mutations causing antithrombin deficiency, the strongest congenital thrombophilia were selected from a cohort of 350 unrelated patients based on functional, biochemical, and crystallographic evidence supporting a folding defect. AlphaFold gave an accurate prediction for the wild-type structure. However, it also produced native structures for all variants, regardless of complexity or conformational consequences in vivo. Similarly, molecular dynamics of up to 1000 ns at temperatures causing conformational transitions did not show significant changes in the native structure of wild-type and variants. In conclusion, AlphaFold and molecular dynamics force predictions into the native conformation at conditions with experimental evidence supporting a conformational change to other structures. It is necessary to improve predictive strategies for serpins that consider the conformational sensitivity of these molecules.

Full-length antithrombin frameshift variant with aberrant C-terminus causes endoplasmic reticulum retention with a dominant-negative effect.

Antithrombin, a major endogenous anticoagulant, is a serine protease inhibitor (serpin). We characterized the biological and clinical impact of variants involving C-terminal antithrombin. We performed comprehensive molecular, cellular, and clinical characterization of patients with C-terminal antithrombin variants from a cohort of 444 unrelated individuals with confirmed antithrombin deficiency. We identified 17 patients carrying 12 C-terminal variants, 5 of whom had the p.Arg445Serfs*17 deletion. Five missense variants caused qualitative deficiency, and 7, including 4 insertion-deletion variants, induced severe quantitative deficiency, particularly p.Arg445Serfs*17 (antithrombin <40%). This +1 frameshift variant had a molecular size similar to that of WT antithrombin but possessed a different C-terminus. Morphologic and cotransfection experiments showed that recombinant p.Arg445Serfs*17 was retained at the endoplasmic reticulum and had a dominant-negative effect on WT antithrombin. Characterization of different 1+ frameshift, aberrant C-terminal variants revealed that protein secretion was determined by frameshift site. The introduction of Pro441 in the aberrant C-terminus, shared by 5 efficiently secreted variants, partially rescued p.Arg445Serfs*17 secretion. C-terminal antithrombin mutants have notable heterogeneity, related to variant type and localization. Aberrant C-terminal variants caused by 1+ frameshift, with similar size as WT antithrombin, may be secreted or not, depending on frameshift site. The severe clinical phenotypes of these genetic changes are consistent with their dominant-negative effects.

High penetrance of inferior vena cava system atresia in severe thrombophilia caused by homozygous antithrombin Budapest 3 variant: Description of a new syndrome.

Atresia of inferior vena cava (IVC) is a rare congenital malformation associated with high risk of venous thrombosis that still has unknown etiology, although intrauterine IVC thrombosis has been suggested to be involved. The identification of IVC atresia in a case with early idiopathic venous thrombosis and antithrombin deficiency caused by the homozygous SERPINC1 c.391C > T variant (p.Leu131Phe; antithrombin Budapest 3) encouraged us to evaluate the role of this severe thrombophilia in this vascular abnormality. We have done a cross-sectional study in previously identified cohorts of patients homozygous for the Budapest 3 variant (N = 61) selected from 1118 patients with congenital antithrombin deficiency identified in two different populations: Spain (N = 692) and Hungary (N = 426). Image analysis included computed tomography and phlebography. Atresia of the IVC system was observed in 17/24 cases (70.8%, 95% confidence interval [CI]: 48.9%-87.3%) homozygous for antithrombin Budapest 3 with available computed tomography (5/8 and 12/16 in the Spanish and Hungarian cohorts, respectively), 16 had an absence of infrarenal IVC and one had atresia of the left common iliac vein. All cases with vascular defects had compensatory mechanisms, azygos-hemiazygos continuation or double IVC, and seven also had other congenital anomalies. Short tandem repeat analysis supported the specific association of the IVC system atresia with SERPINC1. We show the first evidence of the association of a severe thrombophilia with IVC system atresia, supporting the possibility that a thrombosis in the developing fetal vessels is the reason for this anomaly. Our hypothesis-generating results encourage further studies to investigate severe thrombophilic states in patients with atresia of IVC.

Pilot Study on the Role of Circulating miRNAs for the Improvement of the Predictive Ability of the 2MACE Score in Patients with Atrial Fibrillation.

Background. Atrial fibrillation (AF) increases the risk for stroke but also for non-stroke major adverse cardiovascular events (MACE). The 2MACE score was recently proposed to predict these events. Since the interest of microRNAs (miRNAs) in cardiovascular diseases is increasing, we aimed to investigate whether miRNA levels may improve the predictive performance of the 2MACE score. Methods. We included consecutive AF patients stable on vitamin K antagonist therapy. Blood samples were drawn at baseline and plasma expression of miRNAs was assessed. During a median of 7.6 (interquartile range (IQR) 5.4-8.0) years, the occurrence of any MACE (nonfatal myocardial infarction/cardiac revascularization and cardiovascular death) was recorded. Results. We conducted a miRNA expression analysis in plasma from 19 patients with and without cardiovascular events. The miRNAs selected (miR-22-3p, miR-107, and miR-146a-5p) were later measured in 166 patients (47% male, median age 77 (IQR 70-81) years) and all were associated with a higher risk of MACE. The addition of miR-107 and miR-146a-5p to the 2MACE score significantly increased the predictive performance (c-indexes: 0.759 vs. 0.694, p = 0.004), and the model with three miRNAs also improved the predictive performance compared to the original score (c-indexes: 0.762 vs. 0.694, p = 0.012). 2MACE models with the addition of miRNAs presented higher net benefit and potential clinical usefulness. Conclusions. Higher miR-22-3p andmiR-107 and lower miR-146a-5p levels were associated with a higher risk of MACE. The addition of these miRNAs to the 2MACE score significantly increased the predictive performance for MACE, which may aid to some extent in the decision-making process about risk stratification in AF.