Gene analysis of inherited antithrombin deficiency and functional analysis of abnormal antithrombin protein (N87D)
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Inherited antithrombin (AT) deficiency is one of the most clinically significant forms of congenital thrombophilia and follows an autosomal dominant mode of inheritance. We analyzed SERPINC1 in a patient who developed deep-vein thrombosis and low AT activity during pregnancy, and identified a novel missense mutation c.259A>G (p.Asn87Asp; N87D). Surprisingly, analysis of the parents’ DNA showed that they did not possess this mutant, and thus, it may have been due to a de novo mutation. We also expressed this mutant AT protein in COS-1 cells and compared its intracellular localization and intracellular and extracellular antigen levels with that of wild-type AT. The expression experiment did not reveal a significant difference in the antigen levels of the mutant and wild-type AT in the cell lysate, but the mutant AT antigen level was markedly lower than that of its wild-type counterpart in the COS-1 cell supernatant. Immunofluorescence did not indicate any difference between the mutant and wild-type AT in terms of cytoplasmic localization of fluorescence signals. Our findings suggest that the patient’s AT deficiency may have been caused by impaired extracellular secretion of mutant AT protein p.Asn87Asp.
KeywordsAntithrombin deficiency Venous thrombosis Missense mutation Functional analysis De novo mutation
The authors would like to thank Dr. Tsuneo Imanaka for providing the pcDNA3.1/AT expression plasmid. This study was partly supported by a grant from the Ministry of Health, Labor and Welfare to E.M. (Grant number 26070201), and grants of the Ministry Education, Culture, Sports, Science and Technology of Japan to E.M. (Grant number 15K08643), A.S. (Grant number 25462818) and H.A. (Grant number 15K19176).
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Conflict of interest
The authors declare that they have no conflict of interest.
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