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

Summary

In haemophilia B (HB) (factor IX [FIX] deficiency), F9 genotype largely determines clinical phenotype. Aimed to characterise Argentinian families with HB, this study presents F9 genotype frequencies and their specific FIX inhibitor risk and 10 novel F9 mutations. Ninety-one DNA samples from HB patients and relatives were subjected to a new scheme: a primary screen for large deletions, a secondary screen for point mutations using conformation sensitive gel electrophoresis, DNA-sequencing and bioinformatic analysis. Our unbiased HB population (N=52) (77% with severe, 11.5% moderate and 11.5% mild HB) showed 32 missense (61.5%), including three novel mutations predicting specific structural/functional defects in silico, seven nonsense (13.5%) (one novel), five large deletions, four splice including three novel mutations affecting predicted splicing scores, three indels (two novel) and one Leiden mutation. Our comprehensive HB population included five patients with long-lasting FIX inhibitors: three nonsense (p.E35* (novel), p.R75*, p.W240*) and two entire-F9 deletions. Another patient with an indel (p.A26Rfs*14) developed transient inhibitors. A case-control analysis, based on our global prevalence of 3.05% for developing inhibitors in HB revealed that missense mutations were associated with a low risk odds ratio (OR) of 0.05 and a prevalence of 0.39%, whereas nonsense and entire-F9 deletions had significantly higher risks (OR 11.0 and 32.7) and prevalence (14.3% and 44.5%, respectively). Our cost-effective practical approach enabled identification of the causative mutation in all 55 Argentine families with HB, analysis of the molecular pathology of novel F9 defects and determination of mutation-associated FIX inhibitor risks.

Keywords

F9 - haemophilia B - mutation - FIX inhibitors - CSGE

• References

• 1 Yoshitake S, Schach BG, Foster DC. et al. Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 1985; 24: 3736-3750. DOI:10.1021/bi00335a049.
• 2 Di Scipio RG, Kurachi K, Davie EW. Activation of human factor IX (Christmas factor). J Clin Invest 1978; 61: 1528-1538. DOI:10.1172/JCI109073.
• 3 Anson DS, Choo KH, Rees DJ. et al. The gene structure of human anti-haemophilic factor IX. EMBO J 1984; 03: 1053-1060.
• 4 Walker I, Pai M, Akabutu J. et al. The Canadian hemophilia registry as the basis for a national system for monitoring the use of the factor concentrates. Transfusion 1995; 35: 548-551. DOI:10.1046/j.1537-2995.1995.35795357875.x.
• 5 Oldenburg J, Schroder J, Brackmann HH. et al. Environmental and genetic factors influencing inhibitor development. Semin Hematol 2004; 41: 82-88. DOI:10.1053/j.seminhematol.2003.11.016.
• 6 Giannelli F, Green PM, Sommer SS. et al. Haemophilia B: database of point mutations and short additions and deletions--eighth edition. Nucleic Acids Res 1998; 26: 265-268. DOI:10.1093/nar/26.1.265.
• 7 Lahiri DK, Nuremberg JI. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991; 19: 5444. DOI:10.1093/nar/19.19.5444.
• 8 Williams IJ, Abuzenadah A, Winship PR. et al. Precise carrier diagnosis in families with haemophilia A: use of conformation sensitive gel electrophoresis for mutation screening and polymorphism analysis. Thromb Haemost 1998; 79: 723-726.
• 9 Rossetti LC, Radic CP, Candela M. et al. Sixteen novel hemophilia A causative mutations in the first Argentinian series of severe molecular defects. Haematologica 2007; 92: 842-845. DOI:10.3324/haematol.11112.
• 10 Goodeve AC, Reitsma PH, McVey JH. Working Group on Nomenclature of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Nomenclature of genetic variants in hemostasis. J Thromb Haemost 2011; 09: 852-855. DOI:10.1111/j.1538-7836.2011.04191.x.
• 11 Guex N, Peitsch MC.. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 1997; 18: 2714-2723. DOI:10.1002/elps.1150181505.
• 12 Pertea M, Lin X, Salzberg S. GeneSplicer: A new computational method for splice site prediction. Nucleic Acids Res 2001; 29: 1185-1190. DOI:10.1093/nar/29.5.1185.
• 13 Vorechovský I. Aberrant 3’ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 2006; 34: 4630-4641. DOI:10.1093/nar/gkl535.
• 14 Buratti E, Chivers M, Krașlovicșovaș J. et al. Aberrant 50 splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 2007; 35: 4250-4263. DOI:10.1093/nar/gkm402.
• 15 World Federation of Hemophilia.. Report on the annual global survey 2009. René Lévesque Boulevard West, Montreal, Quebec H3G1T7 2009; 15-19 pp
• 16 Saad S, Rowley G, Tagliavacca L. et al. First report on UK database of haemophilia B mutations and pedigrees. Thromb Haemost 1994; 71: 563-570.
• 17 Ljung RCR. Gene mutations and inhibitor formation in patients with hemophilia B. Acta Haematologica 1995; 94: 49-52. DOI:10.1159/000204029.
• 18 Parquet A, Laurian Y, Rothschild C. et al. Incidence of factor IX inhibitor development in severe haemophilia B patients treated with only one brand of high purity plasma derived factor IX concentrate. Thromb Haemost 1999; 82: 1247-1249.
• 19 Ljung R, Petrini P, Tengborn L, Sjörin E. Haemophilia B mutations in Sweden: a population-based study of mutational heterogeneity. Br J Haematol 2001; 113: 81-86. DOI:10.1046/j.1365-2141.2001.02759.x.
• 20 Belvini D, Salviato R, Radossi P. et al. Molecular genotyping of the Italian cohort of patients with haemophilia B. Haematologica 2005; 90: 635-642.
• 21 Tagariello G, Belvini D, Salviato R. et al. The Italian haemophilia B mutation database: a tool for genetic counselling, carrier and prenatal diagnosis. Blood Transfus 2007; 05: 158-163.
• 22 Kwon MJ, Yoo KY, Kim HJ, Kim SH. 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. DOI:10.1111/j.1365-2516.2008.01796.x.
• 23 Miller CH, Benson J, Ellingsen D. et al. F8 and F9 mutations in US haemophilia patients: correlation with history of inhibitor and race/ethnicity. Haemophilia 2012; 18: 375-382. DOI:10.1111/j.1365-2516.2011.02700.x.
• 24 Pinotti M, Caruso P, Canella A. et al. Ribosome Readthrough Accounts for Secreted Full-Length Factor IX in Hemophilia B Patients with NonsenseMutations. Hum Mutat. 2012 Epub ahead of print.
• 25 Bhuvanagiri M, Schlitter AM, Hentze MW. et al. NMD: RNA biology meets human genetic medicine. Biochem. J. 2010; 430: 365-377.
• 26 Giannelli F, Choo KH, Rees DJ. et al. Gene deletions in patients with haemophilia B and anti - factor IX antibodies . Nature 1983; 303: 181-182. DOI:10.1038/303181a0.
• 27 Lillicrap D. The molecular basis of haemophilia B. Haemophilia 1998; 04: 350-357. DOI:10.1046/j.1365-2516.1998.440350.x.
• 28 Warrier I, Ewenstein BM, Koerper MA. et al. Factor IX inhibitors and anaphylaxis in hemophilia B. J Pediatr Hematol Oncol 1997; 19: 23-27. DOI:10.1097/00043426-199701000-00003.
• 29 Tagariello G, Belvini D, Salviato R. et al. The Italian haemophilia B mutation database: a tool for genetic counselling, carrier detection and prenatal diagnosis. Blood Transfus 2007; 05: 158-163.
• 30 Vogel F, Motulsky AG. Human Genetics. 3rd ed. Springer-Verlag; Berlin: 1997
• 31 Hartmann L, Theiss S, Niederacher D, Schaal H. Diagnostics of pathogenic splicing mutations: does bioinformatics cover all bases?. Front Biosci 2008; 13: 3252-3272. DOI:10.2741/2924.
• 32 Morgan GE, Figueiredo MS, Winship PR. et al. The high frecuency of the -6G>A factor IX promoter mutation is the result both of a founder effect and recurrent mutation at a CpG dinucleotide. Br J Haematol 1995; 89: 672-674. DOI:10.1111/j.1365-2141.1995.tb08388.x.
• 33 Vidaud D, Tartary M, Costa JM. et al. Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: Consequences for genetic counseling. Hum Genet 1993; 91: 241-244. DOI:10.1007/BF00218264.
• 34 Lenting PJ, Christophe OD, Maat H. et al. Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding. J Biol Chem 1996; 271: 25332-25337. DOI:10.1074/jbc.271.41.25332.
• 35 Ketterling RP, Vielhaber E, Bottema CDK. et al. Germ-Line Origins of Mutation in Families with Hemophilia B: The Sex Ratio Varies with the Type of Mutation. Am J Hum Genet 1993; 52: 152-166.
• 36 Tizzano EF, Domènech M, Baiget M. Inversion of intron 22 in isolated cases of severe hemophilia A. Thromb Haemost 1995; 73: 6-9.