Molecular Diagnosis of Hemophilia A and Pathogenesis of Novel F8 Variants in Shanxi, China

Abstract

The aim of this study was to perform a molecular diagnosis of hemophilia A (HA) among patients in the Shanxi Province of China. Fifty-two HA patients were tested, including IVS22 (31 samples), IVS1 (3 samples), missense (11 samples), nonsense (3 samples), and 4 cases of frameshift (2 cases of deletion, 1 case of insertion, 1 case of single-base duplication). With the exception of the single-base G duplication variant (p.Ile1213Asnfs*28), this was the hotspot variant reported by research groups at an early stage. The remaining variants were found, for the first time, in the region. The missense variants p.Cys172Ser, p.Tyr404Ser, p.Asp1903Gly, and p.Ser2284Asn, the deletion variant p.Leu2249fs*9, and the insertion variant p.Pro2319fs*97 were novel variants. The application of next-generation sequencing (NGS) molecular diagnosis enriched the variant spectrum of HA, which is greatly significant for individualized genetic counseling, clinical diagnosis, and treatment. NGS and a variety of bioinformatics prediction methods can further analyze the impact of genetic variation on protein structure or function and lay the foundation to reveal the molecular pathogenic mechanism of novel variants.

Keywords

hemophilia A - molecular diagnosis - pathogenic mechanism - novel variants

Publikationsverlauf

Artikel online veröffentlicht:
13. September 2023

• Reference

• 1 Perrin GQ, Herzog RW, Markusic DM. Update on clinical gene therapy for hemophilia. Blood 2019; 133 (05) 407-414
• 2 Al-Allaf FA, Abduljaleel Z, Bogari NM. et al. Identification of six novel factor viii gene variants using next generation sequencing and molecular dynamics simulation. Acta Biochim Pol 2019; 66 (01) 23-31
• 3 Lannoy N, Hermans C. Review of molecular mechanisms at distal Xq28 leading to balanced or unbalanced genomic rearrangements and their phenotypic impacts on hemophilia. Haemophilia 2018; 24 (05) 711-719
• 4 Nair PS, Shetty SD, Chandrakala S, Ghosh K. Mutations in intron 1 and intron 22 inversion negative haemophilia A patients from Western India. PLoS One 2014; 9 (05) e97337
• 5 Lakich D, Kazazian Jr HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet 1993; 5 (03) 236-241
• 6 Radic CP, Rossetti LC, Zuccoli JR, Abelleyro MM, Larripa IB, De Brasi CD. Inverse shifting PCR based prenatal diagnosis of hemophilia-causative inversions involving int22h and int1h hotspots from chorionic villus samples. Prenat Diagn 2009; 29 (12) 1183-1185
• 7 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 (04) 1458-1459
• 8 Bagnall RD, Giannelli F, Green PM. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. J Thromb Haemost 2006; 4 (03) 591-598
• 9 Dutta D, Gunasekera D, Ragni MV, Pratt KP. Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers. Blood Adv 2016; 1 (03) 231-239
• 10 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 (01) 168-174
• 11 McVey JH, Rallapalli PM, Kemball-Cook G. et al. The European association for haemophilia and allied disorders (EAHAD) coagulation factor variant databases: important resources for haemostasis clinicians and researchers. Haemophilia 2020; 26 (02) 306-313
• 12 Swystun LL, James P. Using genetic diagnostics in hemophilia and von Willebrand disease. Hematology (Am Soc Hematol Educ Program) 2015; 2015 (01) 152-159
• 13 Bach JE, Wolf B, Oldenburg J, Müller CR, Rost S. Identification of deep intronic variants in 15 haemophilia A patients by next generation sequencing of the whole factor VIII gene. Thromb Haemost 2015; 114 (04) 757-767
• 14 Bogdanova N, Markoff A, Eisert R. et al. Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A. Hum Mutat 2007; 28 (01) 54-60
• 15 Green PM, Bagnall RD, Waseem NH, Giannelli F. Haemophilia A mutations in the UK: results of screening one-third of the population. Br J Haematol 2008; 143 (01) 115-128
• 16 Johnsen JM, Fletcher SN, Huston H. et al. Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative. Blood Adv 2017; 1 (13) 824-834
• 17 Wang XF, Zhao YQ, Yang RC. et al. The prevalence of factor VIII inhibitors and genetic aspects of inhibitor development in Chinese patients with haemophilia A. Haemophilia 2010; 16 (04) 632-639
• 18 Santacroce R, Acquila M, Belvini D. et al; AICE-Genetics Study Group. Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A. J Hum Genet 2008; 53 (03) 275-284
• 19 Onsori H, Hosseinpour MA, Montaser-Kouhsari S, Asgharzadeh M, Hosseinpour AA. Identification of a novel missense mutation in exon 4 of the human factor VIII gene associated with sever hemophilia A patient. Pak J Biol Sci 2007; 10 (23) 4299-4302
• 20 Fay PJ, Koshibu K, Mastri M. The A1 and A2 subunits of factor VIIIa synergistically stimulate factor IXa catalytic activity. J Biol Chem 1999; 274 (22) 15401-15406
• 21 Pemberton S, Lindley P, Zaitsev V, Card G, Tuddenham EG, Kemball-Cook G. A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin. Blood 1997; 89 (07) 2413-2421
• 22 Lin SW, Lin SR, Shen MC. Characterization of genetic defects of hemophilia A in patients of Chinese origin. Genomics 1993; 18 (03) 496-504
• 23 Lin SY, Su YN, Hung CC. et al. Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM. BMC Med Genet 2008; 9: 53
• 24 Dagil L, Troelsen KS, Bolt G. et al. Interaction between the A3 region of factor VIII and the TIL'E′ domains of the von Willebrand factor. Biophys J 2019; 117 (03) 479-489
• 25 Mazurkiewicz-Pisarek A, Płucienniczak G, Ciach T, Płucienniczak A. The factor VIII protein and its function. Acta Biochim Pol 2016; 63 (01) 11-16
• 26 Ravanbod S, Rassoulzadegan M, Rastegar-Lari G, Jazebi M, Enayat S, Ala F. Identification of 123 previously unreported mutations in the F8 gene of Iranian patients with haemophilia A. Haemophilia 2012; 18 (03) e340-e346
• 27 Wakabayashi H, Fay PJ. Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa. J Biol Chem 2008; 283 (17) 11645-11651
• 28 Newell JL, Fay PJ. Acidic residues C-terminal to the A2 domain facilitate thrombin-catalyzed activation of factor VIII. Biochemistry 2008; 47 (33) 8786-8795
• 29 Jin S, Shang Q, Jin W. et al. A closed-tube nested quantitative PCR assay for rapid detection of intron 22 inversions in the factor VIII gene. Clin Chem 2020; 66 (02) 373-378