Distribution of F8 Gene Mutations in 81 Inhibitor-Free, Severe Hemophilia a Patients with Full Exposure to FVIII
Authors: Pavlova Anna , Matytsina Irina , Arai Morio , Oldenburg Johannes 
Published: October 2019

Abstract
Introduction: Factor (F) VIII genotype has been shown to be a risk factor for inhibitor development, where different mutation types are associated with different degrees of risk (Oldenburg J, et al.Haemophilia 2006;12 Suppl 6:15–22). However, it is not clear why some patients with a specific mutation develop inhibitors while others with the same mutation do not, including those with high-risk defects such as null mutations. Patients with severe hemophilia A (HA) with low-risk mutations are thought to produce some functional or non-functional FVIII that is sufficient to induce immune tolerance (Oldenburg J, et al.Haemophilia 2002;8 Suppl 2:23–29). In the present study, the distribution of F8 gene mutations in 81 patients fully exposed to FVIII with severe HA and no inhibitors is compared to data on mutation distribution among severe HA patients with/without inhibitors.
Materials & Methods: Turoctocog alfa (NovoEight®) is a new recombinant FVIII product with a truncated B-domain. Previously treated patients (PTPs) with severe HA participated in the pivotal trials guardian™1 (n=150; 12–65 yrs) or guardian™3 (n=63; <12 yrs), in which they received turoctocog alfa as prophylaxis and to treat bleeds. The majority continued in an ongoing extension trial, guardian™2. All patients ≥12 yrs had a history of ≥150 exposure days (EDs) to FVIII products and patients <12 yrs had ≥50 EDs. No patients had inhibitors prior to or after turoctocog alfa treatment. Distribution of mutation types has previously been reported in populations with HA that consisted of or included patients with inhibitors (Salviato R, et al. Haemophilia 2007;13:361–372; Gouw SC, et al.Blood 2012;119:2922–2934). Here we report mutation distribution in a sample of guardian™ patients with severe HA who had long been previously treated with FVIII protein and remained inhibitor free, and compared this distribution with that from 2 published studies (Oldenburg J, et al. 2006; Salviato R, et al. 2007).
Results: Eighty-one patients enrolled in the guardian™ trials had F8 gene mutation analyzed. For 52 patients <12 yrs old, genotype was analyzed during guardian™3. For 29 patients ≥12 yrs old, genotype analysis was done prior to enrollment and obtained from medical records. In 77/81 patients, the F8 gene defect was identifiable. Distribution of gene mutations in guardian™, in a population with severe HA with inhibitors, and in a population with severe HA with/without inhibitors are compared in table 1. It is interesting that the overall distribution of mutations among the populations appears quite similar, pointing toward the multifactorial nature of inhibitor development.
Null mutations (inversions, nonsense mutations, and large deletions) were present in 66% and 75% of patients with severe HA and severe HA with inhibitors, respectively (table 1). In guardian™, 57% (46/81) of patients had null mutations despite never having developed an inhibitor. The difference in distribution of null mutations in the 3 populations is mainly accounted for by large deletions and nonsense mutations, while distribution of inversions was similar. It should be noted that in guardian™, 10/11 patients with missense mutations had these located outside the C1/C2 domain, which reportedly has a lower frequency of inhibitors compared with missense mutations at the C1/C2 domain (Oldenburg J, et al. 2006).
Conclusions: Eighty-one patients in the guardian™ trial with severe hemophilia A had their genotype analyzed and all of them were inhibitor-free over their lifetime. Interestingly, the prevalence of high-risk mutation types does not seem to differ markedly from previously published data in patients with inhibitors, probably indicating the relevance of other factors in inhibitor development.
Table 1. Distribution of F8 gene mutation types guardian™ Additional studies guardian™ patients (N) Distribution (%) in guardian™ patients (severe HA without inhibitors) Distribution (%) in patients with severe HA and inhibitors (Salviato R, et al. 2007)* Distribution (%) in patients with severe HA with/without inhibitors (Oldenburg J, et al. 2006) Total, N 81 81 76 753 Intron 22 inversion 33 40.7 44.7 45.0 Intron 1 inversion 3 3.7 1.3 2.5 Nonsense 8 9.9 18.4 13.5 Large deletion 2 2.5 10.5 5.0 Missense 11 13.6 5.2 14.5 Small del/ins/dupl 16 19.8 10.5 16.0 Splice site 4 4.9 5.2 3.5 Unidentified/ not enough information 4 4.9 3.9 0.0 Total 81 100.0 100.0 100.0
*Only patients with severe HA included.

Disclosures
Matytsina: Novo Nordisk A/S: Employment. Arai:Novo Nordisk Pharma, Ltd: Employment. Oldenburg:Baxter, Bayer, Biogen Idec, Biotest, CSL-Behring, Grifols, Novo Nordisk, Octapharma, Swedish Orphan Biovitrum and Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.