Potential Spectrum of Missense and Nonsense Factor (F)VIII Gene (F8) Alleles Based on Single Nucleotide Substitutions - How Many Remain To Be Identified?.
Authors: Viel Kevin R. , Khachidze Manana , Almasy Laura , Thompson Arthur R. , Howard Tom E. 
Published: October 2019

Abstract
Regardless of advances in prenatal diagnosis, carrier detection and gene therapy for hemophilia-A, new patients with bleeding diatheses due to inadequate plasma FVIII activity (FVIII:C) levels will still require specialized management at treatment centers. In the ‘post-genome era’ the possibility exists for personalized medicine, in which an individual’s genetic information will be used to tailor prophylactic and/or treatment regimens that will optimize patient outcomes. As listed in the HAMSTeRS database, ~1,000 distinct loss-of-function F8 variants, representing all mutation types including inversions, insertion/deletions and single nucleotide substitutions (SNSs), have been associated with deficiencies of FVIII. To estimate how soon a complete catalogue of every possible mutation affecting FVIII:C levels may become available, we first determined the theoretical number of potential missense and nonsense F8 alleles, whether loss-of-function or not, based on each possible SNS in the coding region as compared to the reference sequence. While the impact of a missense change on FVIII:C, if any, is not always obvious, in contrast to premature-termination codons (PTCs), which are almost always deleterious, findings from a recent resequencing study raises the possibility that non-hemophilic structural differences between a patient’s endogenous FVIII protein and the infused “wildtype” molecule may increase risk of alloimmunization during replacement therapy. Wildtype FVIII contains 2,351 amino acid (aa) residues: 2,332 in the mature protein and 19 in the signal peptide (SP). Appropriate SNSs within the codons for 793 of these residues would create a PTC (UAA, UAG, UGA). Since three distinct base substitutions are possible at each of the three codon positions, 996 nonsense alleles could theoretically arise naturally. As only 123 distinct nonsense mutations are listed in HAMSTeRS, <15% of the theoretical number, many more likely await discovery. Since suitable SNSs within codons for every residue allow for as many as 15,631 naturally-occurring missense variations, of which 462 are in HAMSTeRS, only 2% of all possible alleles, even more mutations of this type likely remain to be identified. The Table presents the number of possible nonsense and missense F8 alleles and the FVIII protein domain/region affected. Although substantial time and diligent surveillance will be required to document the complete allelic architecture of hemophilia-A, since SNS-mutations can occur at every F8 nucleotide, not just those already identified, doing so could potentially have far reaching implications with respect to personalizing both the current strategy of replacement therapy, based on intravenous infusions and future gene-based methods.
Table. Potential nonsense and missense F8 alleles based on the FVIII domain/region and position in codon Nucleotide Position in Codon Domain/Region Amino Acids 1st Codon 2nd Codon 3rd Codon Nonsense (Missense) Nonsense (Missense) Nonsense (Missense) SP 0001–0019 3 (51) 1 (56) 3 (23) A1 0001–0336 61 (911) 27 (981) 38 (317) a1 0337–0372 6 (101) 0 (108) 2 (50) A2 0373–0719 70 (935) 41 (1000) 55 (353) a2 0720–0740 5 (55) 3 (60) 4 (26) B 0741–1648 221 (2400) 139 (2585) 35 (1032) a3 1649–1689 12 (111) 4 (119) 4 (53) A3 1690–2019 71 (887) 15 (975) 45 (364) C1 2020–2172 30 (417) 17 (442) 20 (154) C2 2173–2332 35 (432) 14 (466) 15 (167)