Identification of six novel factor viii gene variants using next generation sequencing and molecular dynamics simulation
Hemophilia A is an X-linked recessive hemorrhagic disorder caused by variants in the F8 gene. To identify known and novel causative variants in hemophilia A, we have carried out genetic analysis among Saudi patients. Twenty-one patients, who were negative for inv-1/inv-22, were selected for analysis by next generation sequencing, thereafter confirmed by Sanger sequencing. In addition, the functionality and structural changes in the variant proteins were assessed using Molecular dynamics (MD) simulation and compared with wild-type and native proteins. In the samples we analyzed, we found 10 variants in 12 individuals; among them, five were novel and five were previously reported. The novel variants were located at positions: c.6130_6131insC, c.5815G>C, c.5493C>G, c.3734_3740delinsATTTCT and c.3744A>T. With the exception of one variant which was silent, the MD simulation revealed that the observed variants were causing severe structural changes when compared to the native protein and resulted in a loss of the protein’s function. The MD analysis is in line with clinical data of patients who had <1% Factor VIII levels (severe hemophilia) with episodic bleeding, and were on more than one treatment. Moreover, some patients presented with chronic joint disability. These results will enrich the spectrum of variants and enlarge the factor VIII protein’s database in the Saudi Arabian population.
Adzhubei, I., D. M. Jordan and S. R. Sunyaev (2013). "Predicting functional effect of human missense mutations using PolyPhen-2." Curr Protoc Hum Genet Chapter 7: Unit7 20.
Al-Allaf, F. A., M. M. Taher, Z. Abduljaleel, A. Bouazzaoui, M. Athar, N. M. Bogari, H. A. Abalkhail and T. M. Owaidah (2017). "Molecular Analysis of Factor VIII and Factor IX Genes in Hemophilia Patients: Identification of Novel Mutations and Molecular Dynamics Studies." Journal of clinical medicine research 9(4): 317.
Antonarakis, S. E., J. P. Rossiter, M. Young, J. Horst, P. de Moerloose, S. S. Sommer, R. P. Ketterling, H. H. Kazazian, Jr., C. Negrier, C. Vinciguerra, J. Gitschier, M. Goossens, E. Girodon, N. Ghanem, F. Plassa, J. M. Lavergne, M. Vidaud, J. M. Costa, Y. Laurian, S. W. Lin, S. R. Lin, M. C. Shen, D. Lillicrap, S. A. Taylor, S. Windsor, S. V. Valleix, K. Nafa, Y. Sultan, M. Delpech, C. L. Vnencak-Jones, J. A. Phillips, 3rd, R. C. Ljung, E. Koumbarelis, A. Gialeraki, T. Mandalaki, P. V. Jenkins, P. W. Collins, K. J. Pasi, A. Goodeve, I. Peake, F. E. Preston, M. Schwartz, E. Scheibel, J. Ingerslev, D. N. Cooper, D. S. Millar, V. V. Kakkar, F. Giannelli, J. A. Naylor, E. F. Tizzano, M. Baiget, M. Domenech, C. Altisent, J. Tusell, M. Beneyto, J. I. Lorenzo, C. Gaucher, C. Mazurier, K. Peerlinck, G. Matthijs, J. J. Cassiman, J. Vermylen, P. G. Mori, M. Acquila, D. Caprino and H. Inaba (1995). "Factor VIII gene inversions in severe hemophilia A: results of an international consortium study." Blood 86(6): 2206-2212.
Boekhorst, J., B. Verbruggen, J. M. Lavergne, J. M. Costa, S. C. Schoormans, P. P. Brons, M. G. van Kraaij, I. R. Novakova and W. L. van Heerde (2005). "Thirteen novel mutations in the factor VIII gene in the Nijmegen haemophilia A patient population." Br J Haematol 131(1): 109-117.
Bolton-Maggs, P. H. and K. J. Pasi (2003). "Haemophilias A and B." Lancet 361(9371): 1801-1809.
Citron, M., L. Godmilow, T. Ganguly and A. Ganguly (2002). "High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype-phenotype correlation." Hum Mutat 20(4): 267-274.
Faisal A Al-Allaf, M. M. T., Zainularifeen Abduljaleel, Mohammad Athar, Faisal A Ba-hammam, Munir Abdulla, Abdellatif and H. A. a. T. M. O. Bouazzaoui (2016). "Mutation Screening of the Factor VIII Gene in Hemophilia A in Saudi Arabia: Two Novel Mutations and Genotype-Phenotype Correlation." J Mol Genet Med 10:2.
Faisal A Al-Allaf, T. M. O., Zainularifeen Abduljaleel, Mohiuddin M Taher, Mohammad Athar, Halah Abalkhail, Wajahatullah Khan and Abdellatif Bouazzaoui ( 2017). "Identification of Four Novel Factor VIII Gene Mutations and Protein Structure Analysis using Molecular Dynamic Simulation." J Genet Syndr Gene Ther 8:317. doi:10.4172/2157-7412.1000317.
Graw, J., H. H. Brackmann, J. Oldenburg, R. Schneppenheim, M. Spannagl and R. Schwaab (2005). "Haemophilia A: from mutation analysis to new therapies." Nat Rev Genet 6(6): 488-501.
Guidelines, B. C. f. S. i. H., www.bcshguidelines.org.
Hamaguchi, M., T. Matsushita, M. Tanimoto, I. Takahashi, K. Yamamoto, I. Sugiura, J. Takamatsu, K. Ogata, T. Kamiya and H. Saito (1991). "Three distinct point mutations in the factor IX gene of three Japanese CRM+ hemophilia B patients (factor IX BMNagoya 2, factor IX Nagoya 3 and 4)." Thromb Haemost 65(5): 514-520.
Higuchi, M., C. Wong, L. Kochhan, K. Olek, S. Aronis, C. K. Kasper, H. H. Kazazian, Jr. and S. E. Antonarakis (1990). "Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA." Genomics 6(1): 65-71.
Hoyer, L. W. (1994). "Hemophilia A." N Engl J Med 330(1): 38-47.
Human Gene Mutation Database ((accessed on 29th Nov 2015)). http://www.hgmd.org/.
Kumar, P., S. Henikoff and P. C. Ng (2009). "Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm." Nature protocols 4(7): 1073-1081.
Lenting, P. J., J. A. van Mourik and K. Mertens (1998). "The life cycle of coagulation factor VIII in view of its structure and function." Blood 92(11): 3983-3996.
Lillicrap, D. (1998). "The molecular basis of haemophilia B." Haemophilia 4(4): 350-357.
Mannucci, P. M. and E. G. Tuddenham (2001). "The hemophilias--from royal genes to gene therapy." N Engl J Med 344(23): 1773-1779.
Nair, P. S., S. D. Shetty, S. Chandrakala and K. Ghosh (2014). "Mutations in intron 1 and intron 22 inversion negative haemophilia A patients from Western India." PLoS One 9(5): e97337.
Peyvandi, F., T. Kunicki and D. Lillicrap (2013). "Genetic sequence analysis of inherited bleeding diseases." Blood 122(20): 3423-3431.
Repesse, Y., M. Slaoui, D. Ferrandiz, P. Gautier, C. Costa, J. M. Costa, J. M. Lavergne and A. Borel-Derlon (2007). "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 5(7): 1469-1476.
Rossetti, L. C., C. P. Radic, I. B. Larripa and C. D. De Brasi (2008). "Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving int22h and int1h hotspots in the factor VIII gene." J Thromb Haemost 6(5): 830-836.
Salviato, R., D. Belvini, P. Radossi and G. Tagariello (2004). "Factor VIII gene intron 1 inversion: lower than expected prevalence in Italian haemophiliac severe patients." Haemophilia 10(2): 194-196.
Schroder, J., O. El-Maarri, R. Schwaab, C. R. Muller and J. Oldenburg (2006). "Factor VIII intron-1 inversion: frequency and inhibitor prevalence." J Thromb Haemost 4(5): 1141-1143.
Vidal, F., E. Farssac, C. Altisent, L. Puig and D. Gallardo (2001). "Rapid hemophilia A molecular diagnosis by a simple DNA sequencing procedure: identification of 14 novel mutations." Thromb Haemost 85(4): 580-583.
White, G., F. Rosendaal, L. Aledort, J. Lusher, C. Rothschild and J. Ingerslev (2001). "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 85: 560.
Acta Biochimica Polonica is an OpenAccess quarterly and publishes four issues a year. All contents are distributed under the Creative Commons Attribution-ShareAlike 4.0 International (CC BY 4.0) license. Everybody may use the content following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use.
Copyright for all published papers © stays with the authors.
Copyright for the journal: © Polish Biochemical Society.