Identification of the first Alu-mediated large deletion involving the F5 gene in a compound heterozygous patient with severe factor V deficiency

Ilaria Guella; Elvezia Maria Paraboschi; Willem A. van Schalkwyk; Rosanna Asselta; Stefano Duga

doi:10.1160/TH11-03-0149

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2011; 106(02): 296-303
DOI: 10.1160/TH11-03-0149

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Identification of the first Alu-mediated large deletion involving the F5 gene in a compound heterozygous patient with severe factor V deficiency

Authors

Ilaria Guella

¹Dipartimento di Biologia e Genetica per le Scienze Mediche, Università degli Studi di Milano, Milan, Italy
Elvezia Maria Paraboschi

¹Dipartimento di Biologia e Genetica per le Scienze Mediche, Università degli Studi di Milano, Milan, Italy
Willem A. van Schalkwyk

²Department of Haematology, University of Cape Town and National Health Laboratory Service, Red Cross Children’s Hospital, Cape Town, South Africa
Rosanna Asselta

¹Dipartimento di Biologia e Genetica per le Scienze Mediche, Università degli Studi di Milano, Milan, Italy
Stefano Duga

¹Dipartimento di Biologia e Genetica per le Scienze Mediche, Università degli Studi di Milano, Milan, Italy

Abstract

Summary

Factor V (FV) deficiency is a rare autosomal recessive haemorrhagic disorder associated with moderate to severe bleeding symptoms. Conventional mutational screening leads to a complete molecular genetic diagnosis only in about 80–90% of cases. Large gene rearrangements, which could explain at least part of the “missing alleles” have not been reported so far in FV-deficient patients. In this work, we investigated a family with hereditary FV deficiency, in which the proband is compound heterozygous for a 205-Kb deletion, involving the first seven exons of F5, and the entire selectin P, L, and E genes, and for a novel splicing mutation (IVS12+5G>A). The deletion breakpoints, determined by using a combination of semi-quantitative real-time PCR and long PCR assays, occurred within AluY repeat sequences, suggesting an Alu-mediated unequal homologous recombination as the mechanism responsible for the deletion. The in vitro characterisation of the IVS12+5G>A mutation demonstrated that this mutation causes the skipping of exon 12 and the activation of a cryptic splice site. Low levels of residual wild-type splicing were also detectable, in agreement with the notion that the complete absence of FV may be not compatible with life.

Keywords

Coagulation factor V - factor V deficiency - large deletion - Alu sequence - splicing mutation

Full Text

References

References
1 Mann KG, Kalafatis M. Factor V: a combination of Dr Jekyll and Mr Hyde. Blood 2003; 101: 20-30.
2 Giampaolo A, Vulcano F, Macioce G. et al. Factor-V expression in platelets from human megakaryocytic culture. Br J Haematol 2005; 128: 108-111.
3 Asselta R, Peyvandi F. Factor V deficiency. Semin Thromb Hemost 2009; 35: 382-389.
4 Krawczak M, Cooper DN. The human gene mutation database. Trends Genet 1997; 13: 121-122.
5 Lee ST, Kim HJ, Kim DK. et al. Detection of large deletion mutations in the SER-PINC1 gene causing hereditary antithrombin deficiency by multiplex ligation-dependent probe amplification (MLPA). J Thromb Haemost 2008; 6: 701-703.
6 Picard V, Chen JM, Tardy B. et al. Detection and characterisation of large SER-PINC1 deletions in type I inherited antithrombin deficiency. Hum Genet 2010; 127: 45-53.
7 Pintao MC, Garcia AA, Borgel D. et al. Gross deletions/duplications in PROS1 are relatively common in point mutation-negative hereditary protein S deficiency. Hum Genet 2009; 126: 449-456.
8 Castoldi E, Maurissen LF, Tormene D. et al. Similar hypercoagulable state and thrombosis risk in type I and type III protein S-deficient individuals from families with mixed type I/III protein S deficiency. Haematologica 2010; 95: 1563-1571.
9 Yadegari H, Driesen J, Hass M. et al. Large deletions identified in patients with von Willebrand disease by MLPA. J Thromb Haemost 2011; 9: 1083-1086.
10 Caudill JS, Sood R, Zehnder JL. et al. Severe coagulation factor V deficiency associated with an interstitial deletion of chromosome 1q. J Thromb Haemost 2007; 5: 626-628.
11 Lander ES, Linton LM, Birren B. et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860-921.
12 Deininger PL, Batzer MA. Alu repeats and human disease. Mol Genet Metab 1999; 67: 183-193.
13 Baralle M, Baralle D, De Conti L. et al. Identification of a mutation that perturbs NF1 agene splicing using genomic DNA samples and a minigene assay. J Med Genet 2003; 40: 220-222.
14 Dall’Osso C, Guella I, Duga S. et al. Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern. Haematologica 2008; 93: 1505-1513.
15 Spena S, Duga S, Asselta R. et al. Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bbeta-chain gene causing activation of cryptic splice sites. Blood 2002; 100: 4478-4484.
16 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408.
17 Yamazaki T, Nicolaes GA, Sorensen KW. et al. Molecular basis of quantitative factor V deficiency associated with factor V R2 haplotype. Blood 2002; 100: 2515-2521.
18 Rüdiger NS, Gregersen N, Kielland-Brandt MC. One short well conserved region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi. Nucleic Acids Res 1995; 23: 256-260.
19 Montefusco MC, Duga S, Asselta R. et al. A novel two base pair deletion in the factor V gene associated with severe factor V deficiency. Br J Haematol 2000; 111: 1240-1246.
20 van Wijk R, Montefusco M, Duga S. et al. Coexistence of a novel homozygous nonsense mutation in exon 13 of the factor V gene with the homozygous Leiden mutation in two unrelated patients with severe factor V deficiency. Br J Haematol 2001; 114: 871-874.
21 Daly MJ, Rioux JD, Schaffner SF. et al. High-resolution haplotype structure in the human genome. Nat Genet 2001; 29: 229-232.
22 Gabriel SB, Schaffner SF, Nguyen H. et al. The structure of haplotype blocks in the human genome. Science 2002; 296: 2225-2229.
23 Toffolatti L, Cardazzo B, Nobile C. et al. Investigating the mechanism of chromosomal deletion: characterization of 39 deletion breakpoints in introns 47 and 48 of the human dystrophin gene. Genomics 2002; 80: 523-530.
24 Dvorak J, Yang ZL, You FM. et al. Deletion polymorphism in wheat chromosome regions with contrasting recombination rates. Genetics 2004; 168: 1665-1675.
25 Vos HL. Inherited defects of coagulation Factor V: the thrombotic side. J Thromb Haemost 2006; 4: 35-40.
26 Cui J, O’Shea KS, Purkayastha A. et al. Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V. Nature 1996; 384: 66-68.
27 Asselta R, Tenchini ML, Duga S. Inherited defects of coagulation factor V: the hemorrhagic side. J Thromb Haemost 2006; 4: 26-34.
28 Yang TL, Cui J, Taylor JM. et al. Rescue of fatal neonatal hemorrhage in factor V deficient mice by low level transgene expression. Thromb Haemost 2000; 83: 70-77.
29 Duckers C, Simioni P, Spiezia L. et al. Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms. Blood 2010; 115: 879-886.
30 Castoldi E, Duckers C, Radu C. et al. Homozygous F5 deep-intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet-rich plasma. J Thromb Haemost 2011; 9: 959-968.
31 Consortium IH. A haplotype map of the human genome. Nature 2005; 437: 1299-1320.