Identification of large gene deletions int the antithrombin gene by multiplex ligation-dependen probe amplification

Antithrombin (AT) is the major inhibitor of thrombin and other serine proteases involved in coagulation. Hereditary AT deficiency is the most severe form of thrombophilia with first thrombotic events often presenting at young age. Thrombotic risk varies with the type of deficiency.
Types and subtypes are defined by plasmatic assays in combination with molecular analysis of the AT gene. In this report, we describe two cases in which sequencing analysis could not identify
the causative mutation.
Genetic testing included complete sequencing of all exons and intron/exon junctions of the AT gene and multiplex ligation-dependent probe amplification (MLPA), a technique which allows exon quantification.
The first patient was a 44-year old woman with a strong personal and familial history of thrombosis. Sequencing revealed a type II Reactive Site mutation at nucleotide position g.13268G>T (p.Ala384Ser), while plasma levels of AT antigen and activity rather suggested type I deficiency. Our second patient was a 13-year old girl with a first event of deep vein thrombosis and positive familial history.
Sequencing could not identify any mutation. MLPA analysis revealed in our first patient a large deletion comprising exons 3A and 3B and in the second patient a deletion of exon 5. Both deletions
were confirmed by long range PCR.
Large deletions/insertions form only a small proportion of all mutations causing hereditary AT deficiency. Southern blot for the detection of large deletions is very time consuming. MLPA, on the other hand, is a much faster and easier method. We suggest performing MLPA whenever inherited AT deficiency is suspected but sequencing fails to identify the molecular defect.