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.

ALG12-CDG: An unusual patient without intellectual disability and facial dysmorphism, and with a novel variant.

BACKGROUND: Congenital disorder of glycosylation (CDG) type I is a group of rare disorders caused by recessive mutations in up to 25 genes that impair the N-glycan precursor formation and its transfer to proteins resulting in hypoglycosylation of multiple proteins. Congenital disorder of glycosylation causes multisystem defects usually with psychomotor delay that is diagnosed in the infancy. We aim to supply further evidences supporting that CDG may be underestimated. METHODS: Antithrombin and factor XI were studied by chromogenic and coagulometric methods. Hypoglycosylation of plasma proteins was evaluated by western blot, HPLC, Q-TOF, and RP-LC-MRM-MS. Genetic analysis included whole exome, Sanger sequencing, and PCR-allele specific assay. RESULTS: We here present an intriguing patient with an exceptional phenotype: 25-year-old women with a ventricular septal defect and severe idiopathic scoliosis but no facial dysmorphism, who dances as a professional, and has a University degree. Congenital disorder of glycosylation diagnosis started through the identification of antithrombin deficiency without SERPINC1 defect and the detection of hypoglycosylated forms. Increased levels of hypoglycosylated forms of F XI (also with significant deficiency) and transferrin were also detected. Whole exome analysis showed a novel homozygous ALG12 variant c.77T>A, p.(Val26Asp) supporting an ALG12-CDG diagnosis. It also showed three new variants in KMT2D, and a mild, known ALG6 variant. CONCLUSIONS: This novel ALG12-CDG patient (the 13th reported) underlines the heterogeneity of this CDG and broadens its phenotypical spectrum, supports that these disorders are underestimated, and suggests that combination of global hypoglycosylation with specific gene defects might determine the clinical manifestations of CDG patients.

Biochemical and cellular consequences of the antithrombin p.Met1? mutation identified in a severe thrombophilic family.

Nature is always the best inspiration for basic research. A family with severe thrombosis and antithrombin deficiency, the strongest anticoagulant, carried a new mutation affecting the translation-start codon of SERPINC1, the gene encoding antithrombin. Expression of this variant in a eukaryotic cell system produced three different antithrombins. Two downstream methionines were used as alternative initiation codons, generating highly expressed small aglycosylated antithrombins with cytoplasmic localization. Wild-type antithrombin was generated by the use of the mutated AUU as initiation codon. Actually, any codon except for the three stop codons might be used to initiate translation in this strong Kozak context. We show unexpected consequences of natural mutations affecting translation-start codons. Downstream alternative initiation AUG codons may be used when the start codon is mutated, generating smaller molecules with potential different cell localization, biochemical features and unexplored consequences. Additionally, our data further support the use of other codons apart from AUG for initiation of translation in eukaryotes.

Correction: Recurrent mutations in a SERPINC1 hotspot associate with venous thrombosis without apparent antithrombin deficiency.

[This corrects the article DOI: 10.18632/oncotarget.21365.].

Recurrent mutations in a SERPINC1 hotspot associate with venous thrombosis without apparent antithrombin deficiency.

Despite the essential anticoagulant function of antithrombin and the high risk of thrombosis associated with its deficiency, the prevalence of antithrombin deficiency among patients with venous thromboembolism (VTE) is very low. However, increasing evidence suggests that antithrombin deficiency may be underestimated. The analysis of SERPINC1, the gene encoding antithrombin, in 1,304 consecutive Chinese VTE patients and 1,334 healthy controls revealed a hotspot involving residues 294 and 295 that severely increases the risk of VTE. We detected the c.883G>A (p.Val295Met) (rs201381904) mutation in 11 patients and just one control (OR = 13.6; 95% CI: 1.7-107.1); c.881G>T (p.Arg294Leu) (rs587776397) in six patients but no controls; and c.880C>T (p.Arg294Cys) (rs747142328) in two patients but no controls. In addition, c.881G>A (p.Arg294His) (rs587776397) was identified in one control. These mutations were absent in a Caucasian cohort. Carriers of these mutations had normal antithrombin levels and anticoagulant activity, consistent with results obtained in a recombinant model. However, mutation carriers had a significantly increased endogenous thrombin potential. Our results suggest the existence in the Chinese population of a hotspot in SERPINC1 that significantly increases the risk of VTE by impairing the anticoagulant capacity of the hemostatic system. This effect is not revealed by current antigen or in vitro functional antithrombin assays.

Transient desialylation in combination with a novel antithrombin deficiency causing a severe and recurrent thrombosis despite anticoagulation therapy.

An in-depth focused study of specific cases of patients with recurrent thrombosis may help to identify novel circumstances, genetic and acquired factors contributing to the development of this disorder. The aim of this study was to carry out a detailed and sequential analysis of samples from a patient suffering from early and recurrent venous and arterial thrombosis. We performed thrombophilic tests, biochemical, functional, genetic and glycomic analysis of antithrombin and other plasma proteins. The patient carried a new type I antithrombin mutation (p.Ile218del), whose structural relevance was verified in a recombinant model. Experiments with N-glycosidase F and neuraminidase suggested a nearly full desialylation of plasma proteins, which was confirmed by mass spectrometry analysis of transferrin glycoforms. However, partial desialylation and normal patterns were detected in samples collected at other time-points. Desialylation was noticeable after arterial events and was associated with low antithrombin activity, reduced platelet count and glomerular filtration rate. This is the first description of a global and transient desialylation of plasma proteins associated with thrombosis. The decrease in the strong electronegative charge of terminal glycans may modulate hemostatic protein-protein interactions, which in combination with a strong prothrombotic situation, such as antithrombin deficiency, could increase the risk of thrombosis.

High levels of latent antithrombin in plasma from patients with antithrombin deficiency.

Antithrombin is an anticoagulant serpin that efficiently inhibits multiple procoagulant proteases. The cost for the structural flexibility required for this function is the vulnerability to mutations that impact its folding pathway. Most conformational mutations identified in serpins cause polymerisation. Only three mutations in SERPINC1 affecting two residues have been found to favour transformation to the latent conformation of antithrombin, another hyperstable non-anticoagulant form with strong antiangiogenic activity that constitutes 3 % of plasma antithrombin in healthy subjects. The analysis of latent antithrombin in 141 unrelated patients with antithrombin deficiency carrying 89 different SERPINC1 mutations identified four cases with higher levels than that of controls: p.Pro439Thr, p.Pro461Ser, p.Met283Val, and p.His401Tyr, the last also with circulating polymers. Heating of plasma at 42ºC exacerbated the transformation to the latent conformation in p.Pro439Thr and p.Pro461Ser. The conformational effect of p.Met283Val, the mutation associated with the highest levels of latent antithrombin detected in four members of a family, was verified in a recombinant model. Antithrombin deficiency in these cases should be classified as pleiotropic based on the impaired reactivity and low heparin affinity of the variant. Despite high levels of latent antithrombin (up to 80 µg/ml in p.Met283Val carriers), no vascular defects were described in carriers of these mutations. In conclusion, our study identifies new residues involved in the structural stability of antithrombin (and potentially of all serpins). High levels of endogenous latent antithrombin seem to play a minor antiangiogenic effect. Finally, pleiotropic deficiencies may be caused by mutations inducing transformation to the latent conformation.

Correction: Identification of Regulatory Mutations in SERPINC1 Affecting Vitamin D Response Elements Associated with Antithrombin Deficiency.

[This corrects the article DOI: 10.1371/journal.pone.0152159.].

Identification of Regulatory Mutations in SERPINC1 Affecting Vitamin D Response Elements Associated with Antithrombin Deficiency.

Antithrombin is a crucial anticoagulant serpin whose even moderate deficiency significantly increases the risk of thrombosis. Most cases with antithrombin deficiency carried genetic defects affecting exons or flanking regions of SERPINC1.We aimed to identify regulatory mutations inSERPINC1 through sequencing the promoter, intron 1 and 2 of this gene in 23 patients with antithrombin deficiency but without known genetic defects. Three cases with moderate antithrombin deficiency (63-78%) carried potential regulatory mutations. One located 200 bp before the initiation ATG and two in intron 1. These mutations disrupted two out of five potential vitamin D receptor elements (VDRE) identified in SERPINC1 with different software. One genetic defect, c.42-1060_-1057dupTTGA, was a new low prevalent polymorphism (MAF: 0.01) with functional consequences on plasma antithrombin levels. The relevance of the vitamin D pathway on the regulation of SERPINC1 was confirmed in a cell model. Incubation of HepG2 with paricalcitol, a vitamin D analog, increased dose-dependently the levels of SERPINC1transcripts and antithrombin released to the conditioned medium. This study shows further evidence of the transcriptional regulation of SERPINC1 by vitamin D and first describes the functional and pathological relevance of mutations affecting VDRE of this gene. Our study opens new perspectives in the search of new genetic defects involved in antithrombin deficiency and the risk of thrombosis as well as in the design of new antithrombotic treatments.