Kállai Judit, Gindele Réka, Pénzes-Daku Krisztina, Balogh Gábor, Bogáti Réka, Bécsi Bálint, Katona Éva, Oláh Zsolt, Ilonczai Péter, Boda Zoltán, Róna-Tas Ágnes, Nemes László, Marton Imelda, Bereczky Zsuzsanna
Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
HUN-REN-UD Cell Biology and Signaling Research Group, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Int J Mol Sci. 2024 Mar 1;25(5):2893. doi: 10.3390/ijms25052893.
Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel mutations through in vitro expression studies.
抗凝血酶（AT）是凝血酶（FIIa）和活化因子X（FXa）的主要血浆抑制剂，抗凝血酶缺乏症（ATD）是最严重的血栓形成倾向疾病之一。在本研究中，我们鉴定了9种新的AT突变，并研究了它们的基因型与表型的相关性。收集了患者的临床和实验室数据，并在HEK293细胞中表达了9种突变型AT蛋白（p.Arg14Lys、p.Cys32Tyr、p.Arg78Gly、p.Met121Arg、p.Leu245Pro、p.Leu270Argfs14、p.Asn450Ile、p.Gly456delins_Ala_Thr和p.Pro461Thr）；然后，使用蛋白质印迹法、N-糖苷酶F消化法和酶联免疫吸附测定法检测野生型和突变型AT。进行逆转录定量聚合酶链反应以确定转染细胞中AT mRNA的表达。开展了功能研究（在有和没有肝素的情况下测定AT活性以及使用表面等离子体共振法进行肝素结合研究）。还通过计算机模拟方法对突变进行了研究。在6种突变体中证实了由蛋白质合成改变（p.Cys32Tyr、p.Leu270Argfs14、p.Asn450Ile）或分泌障碍（p.Met121Arg、p.Leu245Pro、p.Gly456delins_Ala_Thr）引起的I型ATD，而在2种突变体中提示了II型肝素结合位点ATD（p.Arg78Gly）和多效性效应ATD（p.Pro461Thr）。最后，p.Arg14Lys的致病作用不明确。我们通过体外表达研究为理解新突变的致病本质提供了证据。
