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Antithrombin (AT) deficiency was the first thrombophilia described 50 years ago and so far the strongest one. Up to 78% of cases are explained by point mutations or small deletion/insertions in exons or flanking regions of SERPINC1 that are easily detected by sequencing analysis. A low proportion of cases (2%) is explained by gross gene defects, mainly deletions, which are detected by multiplex ligation-dependent probe amplification (MLPA) analysis. However, the molecular base of AT deficiency is unknown using current methods in 20% of cases.

To identify new SERPINC1 defects causing AT deficiency.

We studied 271 unrelated cases with AT deficiency. Functional and biochemical assays characterized plasma AT. Genetic analyses involved Sanger and Next Generation Sequencing (NGS) (PGM, Ion Torrent), MLPA and specific PCR designs.

Sanger sequencing of PCR amplicons with primers flanking the 7 exons and further analysis with SeqscapeTM detected pathogenic mutations in 173 cases. Whole gene sequencing identified 5 mutations in regulatory regions. MLPA analysis revealed 5 cases with whole or partial deletion of the gene. Moreover, 13 cases had disorders of glycosylation. Interestingly, the analysis of the PCR product and the electropherogram of exon 6 of a 42 year-old male patient (P1) with deep venous thrombosis and 75% of anti-FXa activity with no apparent gene defect by either Sanger sequencing of 7 exons or by NGS analysis of the whole gene using the Ion Torrent platform, revealed a 193 bp insertion, which corresponded to a tandem duplication involving exon 6. Family studies revealed the same duplication in 5 relatives, all with AT deficiency (60-75%). The first MLPA analysis of this case failed to detect the duplication and only after a fine readjustment, it was detected. MLPA analysis under the new conditions of the remaining 59 cases with unknown molecular base for their AT deficiency identified one additional case, P2, with potential duplication of exon 6. P2 was a 17 year-old female with 41% of anti-FXa activity, who developed deep venous thrombosis. Sanger and NGS sequencing also failed to detect any genetic defect in P2. A set of primers specific to detect tandem duplications of exon 6 was designed with forward primer from 3’ end of exon 6, and reverse primer from 5’ of exon 6. This set of primers only rendered amplification in the two cases with exon 6 duplication. The second patient (P2) had a new 863 bp duplication in tandem of exon 6. Sanger sequencing of the specific amplicons in the two cases with tandem duplication of exon 6 revealed Alu sequences surrounding these duplications.

Finally, one out of 5 cases with gene deletions involved breakpoints affecting intron 5 (deletion of exons 2-5).