Characterization of an Anti-Factor VIII Inhibitor Related To P.Arg2178Cys Mutation in A Patient with Mild Haemophilia A

  


    
Bleeding context / notable event

    
Date

    
APTT (ratio)

    
Fibrinogen (g/l)

    
FVIII (IU/dL) before treatment

    
Anti-FVIII inhibitor (BU/mL)

    
TGA (ETP nM.min)

    
Eds to rFVIII

    
BPAs

  

  


    
Kite surfing Accident*

    
4/7/2013

    
1.65

    
3.72

    
0.14

    
< 0.6

    


    
5

    


  

  


    
Removal of osteosynthesis material

    
19/08/2013

    
1.48

    
3.52

    
0.13

    


    


    
3

    


  

  


    
One month stay in Brazil

    
15/02/2015

    


    


    


    


    


    


    


  

  


    
Epistaxis resistant Desmopressin and antifibrinolytic drugs / nasal    packing

    
3/12/2015

    
1.46

    
3.43

    
0.26

    
< 0.6

    


    
2

    


  

  


    
Routine follow-up

    
7/1/2016

    
1.51

    
3.3

    
0.26

    
< 0.6

    
Hypocoagulability profile (998)

    


    


  

  


    
Avulsion of 3 impacted wisdom    teeth

    
2/2/2016

    


    


    


    


    


    
2

    


  

  


    
Pressure sore fall

    
10/2/2016

    


    


    


    


    


    
2

    


  

  


    
Immunization againt Flavivirus (Zika virus)

    
13/08/2016

    


    


    


    


    


    


    


  

  


    
ER consultation for isolated headaches (no haemorraging)

    
27/06/2018

    
1.51

    
3.4

    
0.1

    


    


    


    


  

  


    
Epistaxis resistant to Desmopressin and antifibrinolytic drugs

    
27/08/2018

    
1.49

    
3.57

    
0.12

    
Infuffcient blood sample

    


    
1

    


  

  


    
Epistaxis resistant to Desmopressin and antifibrinolytic drugs

    
11/9/2018

    


    


    


    


    


    
1

    


  

  


    
Epistaxis resistant to Desmopressin and antifibrinolytic drugs

    
18/09/2018

    
1.44

    
3.25

    
0.28

    
0.94

    
Hypocoagulability profile

    
2

    


  

  


    
Close follow-up

    
24/09/2018

    
1.47

    
4

    
0.2

    
5

    


    


    


  

  


    
Close follow-up

    
28/09/2018

    


    


    


    


    


    


    


  

  


    
Bilateral nasal cauterization

    
2/10/2018

    
1.54

    
3.45

    
0.16

    
11

    


    


    
1x5 mg and 2x2 mg

  

  


    
Routine follow-up

    
6/2/2020

    
1.45

    
3.46

    
0.25

    
1

    
Hypocoagulability profile (582)

    


    


  

  


    
Routine follow-up

    
9/6/2023

    
1.47

    
3.07

    
0.2

    
0.7

    


    


    


  

  


    
*subcutaneous haematoma extending over the    entire right lower limb, first left metacarpal hand fracture


      TGA = Thrombin Generation Assay


      ETP = Endogenous thrombin potential


      ED = Exposure Day


      rFVIII = Octocog alfa (Advate¤)


      BPA = By Pass    Agents = Eptacog alfa (Novoseven¤) 

  

Table 1: Clinical and Biological chronology.

The inhibitor persisted over time, with a peak at 11 BU/ml with concomitant FVIII at 0.16 IU/dL, at day 15 of its first detection (i.e. at day 10 of the last Octocog alfa injection), before gradually decreasing over a period of more than 4 years of follow-up, without complete disappearance and with no eradication attempt. The inhibitor titre was then at 0.7 BU/ml with concomitant FVIII at 0.20 IU/dL, fibrinogen at 3.07 g/l and APTT at 1.47 (Table 1).

We performed a Thrombin Generation Assay retrospectively on three different samples: one during routine follow-up before immunization and two in the presence of the inhibitor, the results showed a hypocoagulability profile whether the inhibitor is present or absent, but more marked in the presence of the inhibitor. Indeed, without inhibitor and with 0.26 IU/dL level of FVIII, the patient had a normal endogenous thrombin potential (ETP: 998 nM.min). In the presence of 1 IU Bethesda inhibitor and 0.25 IU/dL level of FVIII, a decrease of ETP (582 nM.min) was observed.

Epitope mapping revealed a FVIII specific antibody characterized as IgG isotype (IgG subclasses 1 and 4) specifically binding to the following domains on the Heavy (HC) and Light Chain (LC): respectively A1, A2 and C1.

Discussion

In non-severe haemophilia A, immunization against FVIII is present in 5 to 13% of patients who have been treated with factor VIII concentrates [1,2]. It usually occurs before 150 EDs, but can also be observed at an early stage [3]. The antibody can be directed against both the exogenous non-mutated wild FVIII administered to the patient, and the endogenous mutated self FVIII, thus resulting in worsening the severity of haemophilia phenotype [4,5,6].

As in severe haemophilia A, F8 gene mutations remain the deciding risk factor in immunization. F8 missense mutations are more involved in the development of inhibitors in non-severe haemophilia A patients. The main epitopic regions targeted by the inhibitor are located in the A2, A3 and C2 domains [7]. More specifically, the exon 23 missense mutation p.Arg2169His of the F8 gene (legacy numbering: Arg2150His or R2150H) of the C1 domain has been described in the literature as associated with a 5% incidence rate of immunization in moderate to mild haemophilia A [3]. Due to its location on the C1-C2 junction [5], the developed inhibitor has the specificity of being directed against the C1 domain of the exogenous wild non-mutated FVIII, whilst sparing the endogenous mutated FVIII [6,8,9]. This selective inhibition of exogenous FVIII is also descripted, for example, in the presence of the Glu272Lys mutation of the A1 domain in mild haemophilia A [10].

In our patient, the exon 23 missense mutation p.Arg2178Cys in the F8 gene (legacy numbering: Arg2159Cys or R2159C) of the C1 domain was immunogenic, described in the literature as possibly corresponding to 3 different haplotypes, with an incidence rate of 1.9% [3]. It was not associated with haplotype 1 (despite being white European). Its characterization was very close to the well-known substitution associated with a high risk of inhibitor development, the exon 23 missense mutation p.Arg2169His of the F8 gene (legacy numbering: Arg2150His or R2150H) of the C1 domain, with a difference of only 9 amino acids. Both mutations are located in the C1 domain, on the light chain.

The inhibitor developed after 16 cumulative EDs to Octocog alfa (associated with Haplotype 2). It selectively inhibited the exogenous, non-mutated wild FVIII. In 60% of cases, the antibody disappears after approximately 9 months, with a non-excluded risk of an anamnestic response but rather a scarcer one [5], possibly explained by an immune tolerance induced by the endogenous FVIII despite an impaired function. In our case, the inhibitor is still present after 4 years and 9 months, at a low titre. No FVIII treatment was re-introduced.

Despite its specificity against domains A1, A2 (HC) and C1 (LC), this inhibitor’s mechanism of action has not yet been reported: what we uncovered in this patient was a selective inhibition of wild type FVIII. These findings suggest a possible association of the exon 23 missense mutation p.Arg2178Cys with only the exogenous FVIII specific inhibitor.

This case emphasizes the need for close monitoring and regular testing for inhibitors, even in patients with mild haemophilia, particularly after acute treatment periods [11].

Conclusion

In mild haemophilia A patients, the development of inhibitors A also makes treatment management difficult, especially when desmopressin is ineffective, and increases the risk of bleeding. Genetic testing can be useful to evaluate the risk of developing an inhibitor, and should therefore be considered, regardless of the severity of the haemophilia. In our case, the exon 23 missense mutation p.Arg2178Cys, a close relative of the exon 23 missense mutation p.Arg2169His, played a rather important part in the immunization process, generating a high titre inhibitor, a strong anamnestic response, and the persistent of the inhibitor over years despite any exogenous factor VIII reintroduction. Finally, this case highlights the importance of genetic mutation identification and regular inbitor's monitoring in all patients with mild haemophilia.

Consent was obtained from the patient for the publication of this observation.

Author Statements

Addendum

P. Chamouni and M. Souissi wrote the manuscript, P. Billoir performed biological analyses, M. Fretigny, V. Barbay and V. Le Cam Duchez revised the manuscript.

Acknowledgements

The authors thank Doctor Christoph Königs (Department of Paediatrics, Clinical and Molecular Haemostasis, Frankfurt University Hospital, Frankfurt am Main, Germany) who performed the epitope mapping and is guarantor of the scientific integrity. We are grateful to Nikki Sabourin-Gibbs, Rouen University Hospital, France for her help in editing the manuscript.

Conflict of Interest Disclosure

The authors have no competing interests.

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