Identification of file novel mutations responsible for type 1 antithrombin deficiency

Inherited antithrombin (AT) deficiency is associated with a predisposition to familial venous thromboembolic disease. Type I AT deficiency is characterized by an approximately 50% reduction of immunological and functional plasma AT levels, with no variant form of protein identifiable. We have investigated the genomic basis underlying this type of inherited deficiency in five unrelated kindreds. DNA studies have been performed using Southern blot analysis, polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) and direct DNA sequencing of the seven exons and flanking intronic sequences. Five novel mutations have been identified.
A nonsense mutation has been observed in exon 2 (np2588), replacing codon 42 by a stop codon.
In exon 6, a frameshift deletion (13286,-A) leads to premature translation termination at codon 395.
Another exon 6 mutation has been found (13258, T to G), predicting an arginine instead of a serine at the PI4 position (codon 380). This residue is located in the proximal hinge of the reactive-centre loop and playing an important role in the mobility of this domain of the antithrombin molecule. In our case it probably results in an unstable form of the protein.
A T to C transition has been observed in exon 2 at np 2462. This missense mutation predicts a Cys to Arg change at codon position -I. The mature AT protein of 432 amino acids is normally cleaved at this point from its N-terminal signal peptide. This 32 amino acid peptide is removed in the hepatocyte, prior to the secretion of AT in the plasma. The described mutation probably disturbs this cleavage with subsequent inhibition of export of the molecule.
In the fifth case we have characterized a partial gene deletion removing a part of intron 3B and a part of exon 4. Using PCR with nested primers spanning this region, we could precisely define the defect of 698 bp.
Studying the molecular defects underlying type I AT deficiency in five unrelated families, we confirm the heterogeneity of the genomic abnormalities. We report 4 novel point mutations and one partial gene deletion, known as a rare basis for AT deficiency.