Identification of a New Mechanism of Antithrombin Deficiency Hardly Detected by Current Methods: Duplication of SERPINC1 Exon 6

Diagnosis of antithrombin (AT) deficiency among patients with thrombophilia is encouraged due to the clinical usefulness of a positive finding.[1] [2] SERPINC1 analysis including sequencing and multiplex ligation-dependent probe amplification (MLPA) identifies gene defects in up to 80% of cases.[3] [4] Up to 315 different SERPINC1 gene defects have been identified in patients with AT deficiency, mostly single nucleotide variants and small deletions or insertions.[5] Gross gene defects are rare (26 different whole or partial deletions, one large duplication, and 4 complex rearrangements).[5] SERPINC1 gene defects not detected by current methods might be responsible for some cases with still unknown molecular base.

The sequence of the 7 exons and flanking regions of SERPINC1 in 223 unrelated cases with AT deficiency (antifactor Xa [anti-FXa] < 80%) identified mutations in 166 cases, with 95 different gene variations. MLPA revealed three whole gene deletions (n = 3) and three partial deletions removing either exon 1, 4, or exons 2 to 5. Sequencing of the promoter and the first two introns revealed three regulatory mutations.

The use of a new set of primers to amplify exon 6 detected one case carrying a heterozygous insertion of 193 bp. P1, a 42-year-old man with deep venous thrombosis, had mild AT deficiency (anti-FXa: 75%). Relatives carrying this insertion also had AT deficiency ([Fig. 1A]). Sequencing of the polymerase chain reaction (PCR) product revealed a tandem duplication of 193 bp comprising exon 6: c.1154–13_1218 + 115dup. Although this duplication of exon 6 was originally not detected by MLPA, fine adjustments of the MLPA assay following the indications of the technical service of MRC-Holland ([Supplementary Material] [online only]) confirmed the duplication ([Fig. 1B]). Next-generation sequencing of the whole SERPINC1 gene performed in a PGM equipment failed to detect this duplication despite two amplicons covered the duplication detected in P1 ([Supplementary Material] [online only]).

Fig. 1 Characterization of new duplications involving SERPINC1 exon 6. (A) Family pedigree of P1 showing the anti-FXa activity and the amplification of exon 6 with the second set of primers with deeper intronic localization (AT6F/AT6B2, see [Supplementary Material] [online only]). The mutated allele with an insertion is indicated by an arrow. (B) MLPA results obtained in P1 using two different adjustments of capillary electrophoresis. Default setting (top): temperature of 60°C, injection time of 18 seconds; modified settings (bottom): temperature of 50°C and injection time of 15 seconds. (C) Detection of different tandem duplication of exon 6 by using a specific set of primers in two patients with antithrombin deficiency, P1 and P2. (D) Alu sequences in SERPINC1. Identification of gross gene defects with breakpoints in intron 5 or 6, rich in Alu repeats. The studies reporting these cases are indicated. Breakpoints of P1 and P2 are indicated by crosses in introns 5 and 6. anti-FXa, antifactor Xa; AT, anti-FXa activity; B, blank; C, control; DVT, deep venous thrombosis; MW, molecular weight marker.

We developed a simple and specific method that only amplified tandem duplications of exon 6. The combination of a forward primer annealing to the 3′-end of exon 6 (5′ − GTCTCAGATGCATTCCATAAGGC-3′) and a reverse primer annealing to the 5′ of exon 6 (5′ − TCTCGGCCTTCTGCAACAAT-3′) do not amplify the wild-type allele, but could amplify tandem duplications involving exon 6 ([Fig. 1C]). This method detected a second case, P2, with a tandem duplication of 893 bp ([Fig. 1C]). Sequencing of this PCR product confirmed the duplication (c.1154–305_1218 + 493dup). P2, a 17-year-old man with deep venous thrombosis, had 41% of plasma anti-FXa activity and reported other relatives with AT deficiency. MPLA under the new adjusted conditions also confirmed this duplication (data not shown).

The massive search of SERPINC1 gene defects among patients with AT deficiency might lead to think on the low probability to identify new gene variations. However, up to 20% of cases with AT deficiency have no SERPINC1 gene defects. Although other genes may be involved in the regulation of the levels of AT in plasma,[6] we speculated that SERPINC1 gene defects not detected by current methods might underlie at least some cases with still unknown molecular defect. The study of our large cohort of patients with AT deficiency reveals that tandem duplication of exon 6 is hardly detected by current molecular methods but represents one-third of gross gene defects associated with AT deficiency. The location of primers used for PCR amplification, usually close to the exons and the length of the duplication may make difficult the detection of duplications involving this exon by classical PCR amplification and sequencing. Moreover, one massive method of sequencing based on small amplicons (Ion Torrent) also failed to detect this genetic defect. In addition, we also revealed technical difficulties to detect the duplication of exon 6 by MLPA. We developed a simple method that specifically detects tandem duplications of exon 6 by a single PCR that might be included in the molecular characterization of AT deficiency.

Finally, our study supports exon 6 as a hot spot for gross gene defects in SERPINC1 associated with AT deficiency through an Alu-related mechanism. The SERPINC1 gene is rich in Alu repeated sequences, with nine complete and one partial repeats that represents approximately 22% of the SERPINC1 intron sequence, which is considerably greater than the estimated 5% of the human genome accounted for by Alu repeats.[7] Interestingly, exon 6 is surrounded by five out of nine complete and one partial repeats Alu sequences[7] ([Fig. 1D]). Picard et al described three gross gene deletions with breakpoints in introns 5 or 6 involving Alu repeats.[8] Additional reports show other SERPINC1 gross deletions with breakpoints in intron 5 or 6[3] [4] [7] [9] [10] [11] [12] [13] ([Fig. 1D]). Interestingly, four cases had deletions involving only exon 6,[7] [8] [9] [12] and extensive studies done in three cases revealed that they had 5′ and/or 3′ breakpoints located within Alu repeat elements.[7] [8] It seems that Alu sequences, particularly those concentrated in introns 5 and 6 may be involved in a proportion of gross gene deletions affecting SERPINC1. In our cohort, one patient also had a gross deletion with breakpoint in intron 6 ([Fig. 1D]). This finding supports the view that Alu repeats in SERPINC1, particularly those located in introns 5 and 6, play an important role in the generation of large SERPINC1 deletions causing AT deficiency. Importantly, our study supports that the same mechanism could underlie duplications of exon 6 ([Fig. 1D]). P2 in our study had breakpoints involving Alu 8 and Alu 9 ([Fig. 1D] and [Supplementary Material] [online only]). In P1, the breakpoints are located 170 bp after Alu 8 and 217 bp of Alu 9 ([Fig. 1D] and [Supplementary Material] [online only]). Mispairing of the homologous Alu sequences has been involved as the main mechanism of gross genomic changes in other genes.[14] Our data strongly suggest that this mechanism may underlie SERPINC1 gene duplications, as it has been previously described for the low-density lipoprotein receptor gene in familial hypercholesterolemia[15] or for the DMD gene involved in Duchenne muscular dystrophy.[16] Indeed, long-term Alu accumulation is associated with DNA duplication initiated by elevated recombinogenic activities in Alu clusters.[17] It is also possible that other gross gene defects, such as tandem duplications with inversion, direct or inverted transpositions, or inversions, not detected by any method used in this study, might also be caused by this mechanism finally leading to AT deficiency. Of note, a cluster of four Alu repeats 5 kb upstream SERPINC1 ([Fig. 1D])[8] might also be involved in some of the not yet fully characterized SERPINC1 gross gene defects.

In summary, this study shows limitations of the current methods used to detect gross gene defects in SERPINC1 (and potentially more genes), which contribute to the small proportion of such mutations among patients with AT deficiency. Our data support that tandem duplication of exon 6 is relatively common (1% of cases with AT deficiency), and it is hardly detected by current methods of diagnosis, although a simple PCR specifically detects it. Other gene defects affecting this region, potentially involving Alu sequences such as inversions or transpositions, may also explain cases with AT deficiency caused by still unknown mechanisms.

Financial Support

This work was supported by PI15/00079 (ISCIII & FEDER), 19873/GERM/15 (Fundación Séneca), Fundación Española de Trombosis y Hemostasia (FETH), and GATRA Grifols Award. MEM-B holds a fellowship from FETH.

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