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Maimiza Zahari
Siti Aishah Sulaiman
Zulhabri Othman
Yasmin Ayob
Faraizah Abd Karim
Rahman Jamal


Factor VIII, Factor IX, genetic mutation, Haemophilia A, Haemophilia B


Background: Haemophilia A (HA) and Haemophilia B (HB) are X-linked blood disorders that are caused by various mutations in the factor VIII (F8) and factor IX (F9) genes respectively. Identification of mutations is essential as some of the mutations are associated with the development of inhibitors. This study is the first comprehensive study of the F8 mutational profile in Malaysia.

Materials and methods: We analysed 100 unrelated HA and 15 unrelated HB patients for genetic alterations in the F8 and F9 genes by using the long-range PCR, DNA sequencing, and the multiplex-ligation-dependent probe amplification assays. The prediction software was used to confirm the effects of these mutations on factor VIII and IX proteins.

Results: 44 (53%) of the severe HA patients were positive for F8 intron 22 inversion, and three (3.6%) were positive for intron 1 inversion. There were 22 novel mutations in F8, including missense (8), frameshift (9), splice site (3), large deletion (1) and nonsense (1) mutations. In HB patients, four novel mutations were identified including the splice site (1), small deletion (1), large deletion (1) and missense (1) mutation.

Discussion: The mutational spectrum of F8 in Malaysian patients is heterogeneous, with a slightly higher frequency of intron 22 inversion in these severe HA patients when compared to other Asian populations. Identification of these mutational profiles in F8 and F9 genes among Malaysian patients will provide a useful reference for the early detection and diagnosis of HA and HB in the Malaysian population.



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1. Bowen DJ. Haemophilia A and haemophilia B: Molecular insights. Mol Pathol 2002;55(2):127-44.

2. de Brasi C, El-Maarri O, Perry DJ, Oldenburg J, Pezeshkpoor B, Goodeve A. Genetic testing in bleeding disorders. Haemophilia 2014;20(0 4):54-8. 10.1111/hae.12409

3. Lakich D, Kazazian HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet 1993;5(3):236-41.

4. Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002;99(1):168-74. 10.1182/blood.V99.1.168

5. Rallapalli PM, Kemball-Cook G, Tuddenham EG, Gomez K, Perkins SJ. 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(7):1329-40. 10.1111/jth.12276

6. EAHAD Coagulation Factor Variant Databases. 2017 [cited 13th March 2017]. Available from:

7. Factor 8 Variant Database. 2014. Available from:

8. Franchini M. The modern treatment of haemophilia: a narrative review. Blood Transfusion 2013;11(2):178-82. 10.2450/2012.0166-11

9. Walsh CE, Soucie JM, Miller CH, and the United States Hemophilia Treatment Center N. Impact of inhibitors on hemophilia a mortality in the United States. Am J Hematol 2015;90(5):400-5. 10.1002/ajh.23957

10. Wight J, Paisley S. The epidemiology of inhibitors in haemophilia A: A systematic review. Haemophilia 2003;9(4):418-35. 10.1046/j.1365-2516.2003.00780.x

11. Lacroix-Desmazes S, Scott DW, Goudemand J, van den Berg M, Makris M, van Velzen AS, Santagostino E, Lillicrap D, Rosendaal FR, Hilger A, Sauna ZE, Oldenburg J, Mantovani L, Mancuso ME, Kessler C, Hay CRM, Knoebl P, Di Minno G, Hoots K, Bok A, Brooker M, Buoso E, Mannucci PM, Peyvandi F. Summary report of the First International Conference on inhibitors in haemophilia A. Blood Transfusion 2017;15(6):568-76. 10.2450/2016.0252-16

12. Kamiya T, Takahashi I, Saito H. Retrospective study of inhibitor formation in Japanese hemophiliacs. Int J Hematol 1995;62(3):175-81.

13. Ljung R. Gene mutations and inhibitor formation in patients with hemophilia B. Acta Haematol 1995;94(Suppl. 1):49-52.

14. Aledort LM, Dimichele DM. Inhibitors occur more frequently in African-American and Latino haemophiliacs. Haemophilia 1998;4(1):68.

15. Carpenter SL, Michael Soucie J, Sterner S, Presley R, Hemophilia Treatment Center Network I. Increased prevalence of inhibitors in Hispanic patients with severe haemophilia A enrolled in the Universal Data Collection database. Haemophilia 2012;18(3):e260-e5. 10.1111/j.1365-2516.2011.02739.x

16. Gouw SC, van den Berg HM, Oldenburg J, Astermark J, de Groot PG, Margaglione M, Thompson AR, van Heerde W, Boekhorst J, Miller CH, le Cessie S, van der Bom JG. F8 gene mutation type and inhibitor development in patients with severe hemophilia A: Systematic review and meta-analysis. Blood 2012;119(12):2922-34. 10.1182/blood-2011-09-379453

17. Malaysian Ministry of Health M. Health technology assessment report: Management of haemophilia. Kuala Lumpur, Malaysia: Ministry of Health, 2012 Contract No.: MOH/P/PAK/258.12(TR).

18. Moses EJ, Ling SP, Al-Hassan FM, Karim FA, Yusoff NM. Identification of novel mutations in exon 14 of the F8 gene in Malaysian patients with severe Hemophilia A. Indian J Clin Biochem 2012;27(2):207-8. 10.1007/s12291-011-0161-z

19. Balraj P, Ahmad M, Khoo AS, Ayob Y. Factor IX mutations in haemophilia B patients in Malaysia: a preliminary study. Malays J Pathol 2012;34(1):67-9.

20. Ishak R, Zakaria Z. Detection of carrier status of hemophilia B using DNA markers. Southeast Asian J Trop Med Public Health 1997;28(3):629-30.

21. White GC, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J, on behalf of the Factor VIII and Factor IX Subcommittee. Definitions in Hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on thrombosis and haemostasis. Thromb Haemost 2001;85(3):560-.

22. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16(3):1215.

23. Liu Q, Nozari G, Sommer SS. Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in Hemophilia A. Blood 1998;92(4):1458-9.

24. Liu Q, Sommer SS. Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: Application to the inversion hotspot in the factor VIII gene. Biotechniques 1998;25(6):1022-8.

25. Vidal F, Farssac E, Altisent C, Puig L, Gallardo D. Rapid Hemophilia A molecular diagnosis by a simple DNA sequencing procedure: Identification of 14 novel mutations. Thromb Haemost 2001;85(4):580-3.

26. Hinks JL, Winship PR, Makris M, Preston FE, Peake IR, Goodeve AC. A rapid method for haemophilia B mutation detection using conformation sensitive gel electrophoresis. Br J Haematol 1999;104(4):915-8. 10.1046/j.1365-2141.1999.01274.x

27. Vidal F, Farssac E, Altisent C, Puig L, Gallardo D. Factor IX gene sequencing by a simple and sensitive 15-hour procedure for haemophilia B diagnosis: Identification of two novel mutations. Br J Haematol 2000;111(2):549-51. 10.1111/j.1365-2141.2000.02389.x

28. den Dunnen JT, Dalgleish R, Maglott DR, Hart RK, Greenblatt MS, McGowan-Jordan J, Roux A-F, Smith T, Antonarakis SE, Taschner PEM, on behalf of the Human Genome Variation Society, the Human Variome Project, and the Human Genome Organisation. HGVS Recommendations for the description of sequence variants: 2016 Update. Hum Mutat 2016;37(6):564-9. 10.1002/humu.22981

29. Li T, Miller CH, Payne AB, Craig Hooper W. The CDC Hemophilia B mutation project mutation list: A new online resource. Mol Genet Genomic Med 2013;1(4):238-45. 10.1002/mgg3.30

30. Flanagan SE, Patch AM, Ellard S. Using SIFT and PolyPhen to predict loss-of-function and gain-of-function mutations. Genet Test Mol Biomarkers 2010;14(4):533-7. 10.1089/gtmb.2010.0036

31. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protocols 2009;4(8):1073-81.

32. Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One 2012;7(10):e46688. 10.1371/journal.pone.0046688

33. Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: Mutation prediction for the deep-sequencing age. Nat Meth 2014;11(4):361-2. 10.1038/nmeth.2890

34. Shen BW, Spiegel PC, Chang C-H, Huh J-W, Lee J-S, Kim J, Kim Y-H, Stoddard BL. The tertiary structure and domain organization of coagulation factor VIII. Blood 2008;111(3):1240-7. 10.1182/blood-2007-08-109918

35. Zögg T, Brandstetter H. Structural Basis of the Cofactor- and Substrate-Assisted Activation of Human Coagulation Factor IXa. Structure 2009;17(12):1669-78. 10.1016/j.str.2009.10.011

36. Pinto P, Ghosh K, Shetty S. F8 gene mutation profile in Indian hemophilia A patients: Identification of 23 novel mutations and factor VIII inhibitor risk association. Mutat Res 2016;786:27-33.

37. Xue F, Zhang L, Sui T, Ge J, Gu D, Du W, Zhao H, Yang R. Factor VIII gene mutations profile in 148 Chinese hemophilia A subjects. Eur J Haematol 2010;85(3):264-72. 10.1111/j.1600-0609.2010.01481.x

38. Ahmed R, Ivaskevicius V, Kannan M, Seifried E, Oldenburg J, Saxena R. Identification of 32 novel mutations in the factor VIII gene in Indian patients with hemophilia A. Haematologica 2005;90(2):283-4.

39. Shekari Khaniani M, Ebrahimi A, Daraei S, Derakhshan SM. Genotyping of Intron Inversions and Point Mutations in Exon 14 of the FVIII Gene in Iranian Azeri Turkish Families with Hemophilia A. Indian J Hematol Blood Transfus 2016;32(4):475-80. 10.1007/s12288-016-0699-2
40. Mousavi SH, Mesbah?Namin SA, Rezaie N, Zeinali S. Frequencies of intron 1 and 22 inversions of factor VIII gene: A first report in Afghan patients with severe haemophilia A. Haemophilia 2018;0(0). doi:10.1111/hae.13491

41. Citron M, Godmilow L, Ganguly T, Ganguly A. High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype–phenotype correlation. Hum Mutat 2002;20(4):267-74. 10.1002/humu.10119

42. Guo Z, Yang L, Qin X, Liu X, Zhang Y. Spectrum of Molecular Defects in 216 Chinese Families With Hemophilia A: Identification of Noninversion Mutation Hot Spots and 42 Novel Mutations. Clin Appl Thromb Hemost 2017;24(1):70-8. 10.1177/1076029616687848

43. Lyu C, Xue F, Liu X, Liu W, Fu R, Sun T, Wu R, Zhang L, Li H, Zhang D, Yang R, Zhang L. Identification of mutations in the F8 and F9 gene in families with haemophilia using targeted high-throughput sequencing. Haemophilia 2016;22(5):e427-e34. 10.1111/hae.12924

44. Pezeshkpoor B, Zimmer N, Marquardt N, Nanda I, Haaf T, Budde U, Oldenburg J, El-Maarri O. Deep intronic ‘mutations’ cause hemophilia A: Application of next generation sequencing in patients without detectable mutation in F8 cDNA. J Thromb Haemost 2013;11(9):1679-87. 10.1111/jth.12339

45. RepessÉ Y, Slaoui M, Ferrandiz D, Gautier P, Costa C, Costa JM, Lavergne JM, Borel-Derlon A. Factor VIII (FVIII) gene mutations in 120 patients with hemophilia A: Detection of 26 novel mutations and correlation with FVIII inhibitor development. J Thromb Haemost 2007;5(7):1469-76. 10.1111/j.1538-7836.2007.02591.x

46. Oldenburg J, El-Maarri O, Schwaab R. Inhibitor development in correlation to factor VIII genotypes. Haemophilia 2002;8:23-9. 10.1046/j.1351-8216.2001.00134.x

47. Miller CH, Benson J, Ellingsen D, Driggers J, Payne A, Kelly FM, Soucie JM, Craig Hooper W, The Hemophilia Inhibitor Research Study Investigators. F8 and F9 mutations in US haemophilia patients: Correlation with history of inhibitor and race/ethnicity. Haemophilia 2012;18(3):375-82. 10.1111/j.1365-2516.2011.02700.x

48. Prescott R, Nakai H, Saenko EL, Scharrer I, Nilsson IM, Humphries JE, Hurst D, Bray G, Scandella D. the inhibitor antibody response is more complex in Hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies. Blood 1997;89(10):3663-71.

49. Pipe SW. Functional roles of the factor VIII B domain. Haemophilia 2009;15(6):1187-96. 10.1111/j.1365-2516.2009.02026.x

50. Burke RL, Pachl C, Quiroga M, Rosenberg S, Haigwood N, Nordfang O, Ezban M. The functional domains of coagulation factor VIII:C. J Biol Chem 1986;261(27):12574-8.

51. Pittman D, Alderman E, Tomkinson K, Wang J, Giles A, Kaufman R. Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII. Blood 1993;81(11):2925-35.

52. Shelley N, Miao-Liang L, R. TA. Some factor VIII exon 14 frameshift mutations cause moderately severe haemophilia A. Br J Haematol 2001;115(4):977-82. doi:10.1046/j.1365-2141.2001.03173.x

53. Oldenburg J, Ananyeva NM, Saenko EL. Molecular basis of haemophilia A. Haemophilia 2004;10:133-9. 10.1111/j.1365-2516.2004.01005.x

54. Ogata K, Selvaraj SR, Miao HZ, Pipe SW. Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe Hemophilia A: Implications for genotyping. J Thromb Haemost 2011;9(6):1183-90. 10.1111/j.1538-7836.2011.04268.x

55. Jacquemin M, Lavend'homme R, Benhida A, Vanzieleghem B, d'Oiron R, Lavergne J-M, Brackmann HH, Schwaab R, VandenDriessche T, Chuah MKL, Hoylaerts M, Gilles JGG, Peerlinck K, Vermylen J, Saint-Remy J-MR. A novel cause of mild/moderate hemophilia A: Mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor. Blood 2000;96(3):958-65.

56. Liu Z, Lin L, Yuan C, Nicolaes GAF, Chen L, Meehan EJ, Furie B, Furie B, Huang M. Trp(2313)-His(2315) of factor VIII C2 domain is involved in membrane binding: Structure of a complex between the C2 domain and an inhibitor of membrane binding. J Biol Chem 2010;285(12):8824-9. 10.1074/jbc.M109.080168

57. Foster P, Fulcher C, Houghten R, Zimmerman T. Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine. Blood 1990;75(10):1999-2004.

58. Liu J, Zhang Y, Wang H, Huang W, Cao W, Wang X, Qu B, Wang H, Shao H, Wang Z, Che