Molecular mechanisms of antithrombin deficiency in two Chinese families. One novel and one recurrent point mutation in the antithrombin gene causing venous thrombosis.

We investigated the molecular mechanisms responsible for type I congenital antithrombin (AT) deficiency in two unrelated Chinese pedigrees manifesting multiple site venous thrombosis. Phenotype analysis showed both probands had almost 50% of normal AT levels. Direct sequencing of amplified DNA revealed 2757C > T in proband 1 and 13328G > A in proband 2, predicting a heterozygous Thr98Ile (T981) and Ala404Thr (A404T), respectively. No proband had 20210A allele or factor V Leiden mutation. Transient expression of complementary DNA coding for the mutations in COS-7 cells showed impaired secretion of the mutant molecules. Real-time quantitative PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT-198 mutant were stained diffusely without perinuclear enhancement and cells expressing AT-T404 mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. We conclude that the impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular pathogenesis of AT deficiency caused by T981 and A404T mutation for the two families, respectively.