Characterisation of large F9 deletions in seven unrelated patients with severe haemophilia B

Summary

Large deletions in the F9 gene are detected in approximately 5% of patients with severe haemophilia B, but only a few deletion breakpoints have been characterised precisely until now. In this study we identified a total of seven large F9 deletions in the index patients and nine female carriers by the AccuCopy technique. We also successfully characterised the exact breakpoints for each large deletion including four deletions encompassing the entire F9 gene by the genome walking method combined with primer walking strategy. The extents of deletion regions ranged from 11.1 to 884 kb. Microhomologies ranged from 2 to 6 bp were identified in the breakpoint junctions of six deletions. The other deletion occurred between two highly homologous sequences of the same long interspersed nuclear element 1 (LINE/L1). Non-homologous end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) may be the main causative mechanisms for the six large deletions with microhomologies. Non-allelic homologous recombination (NAHR) may mediate the deletion occurred between the two tandem LINEs in the other large deletion. Repetitive elements and non-B DNA forming motifs identified in the junction regions may contribute to DNA breakage leading to large deletions.

Keywords

Copy number variants - deletion breakpoints - DNA end-joining repair - F9 gene - haemophilia B

• References

• 1 Rallapalli PM, Kemball-Cook G, Tuddenham EG. et al. An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B. J Thromb Haemost 2013; 11: 1329-1340.
• 2 Thorland EC, Drost JB, Lusher JM. et al. Anaphylactic response to factor IX replacement therapy in haemophilia B patients: complete gene deletions confer the highest risk. Haemophilia 1999; 05: 101-105.
• 3 Radic CP, Rossetti LC, Abelleyro MM. et al. Assessment of the F9 genotype-specific FIX inhibitor risks and characterisation of 10 novel severe F9 defects in the first molecular series of Argentinian patients with haemophilia B. Thromb Haemost 2013; 109: 24-33.
• 4 Hsu TC, Nakaya SM, Thompson AR. Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: non-homologous recombination associated with a shortened transcript from whole blood. Thromb Haemost 2007; 97: 176-180.
• 5 Chen SH, Scott CR. Recombination between two 14-bp homologous sequences as the mechanism for gene deletion in factor IX Seattle 1. Am J Hum Genet 1990; 47: 1020-1022.
• 6 Ludwig M, Grimm T, Brackmann HH. et al. Parental origin of factor IX gene mutations, and their distribution in the gene. Am J Hum Genet 1992; 50: 164-173.
• 7 Ketterling RP, Vielhaber EL, Lind TJ. et al. The rates and patterns of deletions in the human factor IX gene. Am J Hum Genet 1994; 54: 201-213.
• 8 Li X, Drost JB, Roberts S. et al. Factor IX mutations in South Africans and African Americans are compatible with primarily endogenous influences upon recent germline mutations. Hum Mutat 2000; 16: 371.
• 9 Green PM, Bentley DR, Mibashan RS. et al. Partial deletion by illegitimate recombination of the factor IX gene in a haemophilia B family with two inhibitor patients. Mol Biol Med 1988; 05: 95-106.
• 10 Solera J, Magallon M, Martin-Villar J. et al. Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site. Am J Hum Genet 1992; 50: 434-437.
• 11 Ketterling RP, Ricke DO, Wurster MW. et al. Deletions with inversions: report of a mutation and review of the literature. Hum Mutat 1993; 2: 53-57.
• 12 Peake IR, Matthews RJ, Bloom AL. Haemophilia B Chicago: severe haemophilia B caused by two deletions and an inversion within the factor IX gene. Br J Haematol 1989; 71 (Suppl. 01) 1.
• 13 Hastings PJ, Lupski JR, Rosenberg SM. et al. Mechanisms of change in gene copy number. Nat Rev Genet 2009; 10: 551-564.
• 14 Du R, Lu C, Jiang Z. et al. Efficient typing of copy number variations in a segmental duplication-mediated rearrangement hotspot using multiplex competitive amplification. J Hum Genet 2012; 57: 545-551.
• 15 You GL, Ding QL, Lu YL. et al. Characterisation of large deletions in the F8 gene using multiple competitive amplification and the genome walking technique. J Thromb Haemost 2013; 11: 1103-1110.
• 16 Guo H, Xiong J. A specific and versatile genome walking technique. Gene 2006; 381: 18-23.
• 17 Anson DS, Blake DJ, Winship PR. et al. Nullisomic deletion of the mcf.2 transforming gene in two haemophilia B patients. EMBO J 1988; 07: 2795-2799.
• 18 Siggs OM, Schnabl B, Webb B. et al. X-linked cholestasis in mouse due to mutations of the P4-ATPase ATP11C. Proc Natl Acad Sci USA 2011; 108: 7890-7895.
• 19 Yabas M, Teh CE, Frankenreiter S. et al. ATP11C is critical for the internalisation of phosphatidylserine and differentiation of B lymphocytes. Nat Immunol 2011; 12: 441-449.
• 20 Yamamoto Y, Yoshioka Y, Minoura K. et al. An integrative genomic analysis revealed the relevance of microRNA and gene expression for drug-resistance in human breast cancer cells. Mol Cancer 2011; 10: 135.
• 21 Lieber MR. The mechanism of human non-homologous DNA end joining. J Biol Chem 2008; 283: 1-5.
• 22 Haviv-Chesner A, Kobayashi Y, Gabriel A. et al. Capture of linear fragments at a double-strand break in yeast. Nucleic Acids Res 2007; 35: 5192-5202.
• 23 Chen JM, Cooper DN, Ferec C. et al. Genomic rearrangements in inherited disease and cancer. Semin Cancer Biol 2010; 20: 222-233.
• 24 Hastings PJ, Ira G, Lupski JR. A microhomology-mediated break-induced replication model for the origin of human copy number variation. PLoS Genet 2009; 05: e1000327.
• 25 Lee JA, Carvalho CM, Lupski JR. A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 2007; 131: 1235-1247.
• 26 Gu W, Zhang F, Lupski JR. Mechanisms for human genomic rearrangements. Pathogenetics 2008; 01: 4.
• 27 Argueso JL, Westmoreland J, Mieczkowski PA. et al. Double-strand breaks associated with repetitive DNA can reshape the genome. Proc Natl Acad Sci USA 2008; 105: 11845-11850.
• 28 Bacolla A, Wells RD. Non-B DNA conformations, genomic rearrangements, and human disease. J Biol Chem 2004; 279: 47411-47414.
• 29 Wang G, Vasquez KM. Non-B DNA structure-induced genetic instability. Mutat Res 2006; 598: 103-119.
• 30 Kwon MJ, Yoo KY, Kim HJ. et al. Identification of mutations in the F9 gene including exon deletion by multiplex ligation-dependent probe amplification in 33 unrelated Korean patients with haemophilia B. Haemophilia 2008; 14: 1069-1075.
• 31 Casana P, Haya S, Cid AR. et al. Identification of deletion carriers in hemophilia B: quantitative real-time polymerase chain reaction or multiple ligation probe amplification. Transl Res 2009; 153: 114-117.