Communication
© Schattauer 2010
S153
Molecular genetic background of
haemophilia A patients with
discrepancy between one-stage and
two-stage factor VIII assays
A. Pavlova; D. Delev; S. Pahl; J. Driesen; H. Brondke; J. Oldenburg
Human blood coagulation factor VIII
(FVIII) is an essential protein for the blood
coagulation. Its physiological relevance is
evident from the severe bleeding disorder
haemophilia A associated with FVIII deficiency or dysfunction. The accurate
measurement of FVIII coagulant activity
(FVIII : C) in plasma is important for
● diagnosis of haemophilia,
● defining the severity of the disease and
● follow up the effect of FVIII replacement therapy.
FVIII activity often correlates with the
clinical expression of the disease. It can be
measured by the
● one-stage (FVIII:C1st) or
● the classical two-stage (FVIII:C2st) clotting assay or
● the chromogenic method (FVIII:Cchr).
The most commonly used method is the
FVIII:C1st assay, which is based on the activated partial thromboplastin time (aPTT)
(1). The FVIII:C2st assays consist of two
reactions forming initially activated factor
X (FX), which is either measured in the second stage by a clotting test (FVIII:C2st) (2)
or by using a chromogenic substrate
(FVIII:CChr) (3).
Results from all FVIII:C assays are
usually equivalent for patients with severe,
moderate or mild haemophilia. Several recent
Correspondence to:
Dr. Anna Pavlova
Institut für Experimentelle Hämatologie und
Transfusionsmedizin, Universitätsklinikum Bonn
Sigmund-Freud-Str. 25, 53127 Bonn, Germany
Tel. +49/(0)/228/28 71 97 11,
Fax +49/(0)228/28 71 60 87
E-mail: [email protected]
Hämostaseologie 2010; 30 (Suppl 1): S153–S155
reports have observed discrepancies concerning the haemophilia A phenotype: In approximately one-third of patients with mild haemophilia A discrepancy between results from
the two assays has been reported (4–9). A
small subset of the mutations are characterised in patients with discrepant results.
The aim of our study was to characterise
the mutation profile in patients with discrepancy.
Patients, material, methods
Haemophilia patients
284 patients previously diagnosed in Haemophilia Center Bonn as mild and moderate haemophiacs were studied. Haemophilia was diagnosed on the basis of reduced
FVIII:C (< 30 IU/dl) measured by onestage and chromogrnic methods, normal
value of VWF antigen and VWF: ristocitin
co-factor function and a family history
consistent with sex-linked inheritance. The
FVIII:C represents the mean value of all
measurements in patients who had two and
more multiple sets of analyses. The ratio of
between FVIII:CChr values and FVIII:C1st
values was calculated. Results were considered discrepant at a FVIII:CChr
/FVIII:C1st ratio ≤ 0,6 and the discrepant results remained consistent when tested
more than once.
Coagulation assays
Germany) with FVIII deficient plasma
from Helena (UK). FVIII:C Chromogenic
assay. The chromogenic assay was processed on a BCS coagulation analyser
(Dade Behring, Germany) using reagents
from Siemens Healthcare Diagnostics
(Eschborn, Germany) according to the
manufacturer’s .
VWF:Ag and VWF:RCo
Plasma von Willebrand factor antigen level
(VWF:Ag) was measured by ELISA
(BioMerieux, MarcyI'Etoile, France), vWF
ristocetin cofactor activity (VWF:RCo) was
determined as previously described (10).
DNA isolation, F8 sequence
High molecular weight genomic DNA was
isolated from whole blood by a salting out
procedure (11) DNA concentrations were
standardized to 100 ng/μL.
The entire coding region of the F8 gene
was amplified in all patients and relatives
and direct sequenced using primer and experimental conditions described earlier
(12). DNA sequencing was performed on
both strands, using the BigDye Terminator
Cycle Sequencing V1.1 Ready Reaction kit
and an automated ABI-3130 DNA sequencer (Applied Biosystems). Sequence
Analysis software package (Applied Biosystems) was applied for final sequence reading and mutation documentation.
FVIII:C one-stage assay
The one-stage assay is an aPTT based inhouse assay processed on an KC10A coagulation analyzer (Trinity Biotech, Lemgo,
Results, discussion
284 patients with mild or moderate HA
have been analyzed. In 101 patients
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Hämophilie-Genetik
Institute of Experimental Haematology and Transfusion medicine, University Clinic Bonn, Germany
S154
A. Pavlova et al.: One- and two-stage FVIII assays
(35.5%) discrepant results for FVIII:C were
found; 28 different mutations have been
detected. Eleven of them showed higher
FVIII:C1st results then the FVIII:CChr giving
rise to a ratio larger then 0.6. (씰Tab. 1).
Nine mutations have been described for the
first time. All mutations (except one, already described in the literature) are clustered in A1, A2, and A3 domains , in the regions at the interface between subunits
(A1-A2, A1-A3, A2-A3). Additionally, most
of the mutations are presented at the surface of the FVIII protein (씰Fig. 1).This
Hämophilie-Genetik
Tab. 1
render the FVIII less stable so that the activated FVIII is inactivated more quickly
than normal. Seventeen F8 gene alterations
displayed higher FVIII:CChr assay compared to the FVIII:C1st. In these, mainly
mild hemophilia A cases, the effect of the
underlying mutation was only apparent in
the FVIII:C1st clotting assay. The FVIII:CChr
assay yields normal activities. In contrast to
mutation by which the FVIII:C1st is higher
than FVIII:CChr assay, here the mutations
are scattered all over the whole gene. Some
of them share the common feature of clus-
tering in the vicinity of thrombin cleavage
sites. These mutations produce impaired or
delayed thrombin activation and their effect is masked by the usage of unphysiologically high concentrations of thrombin
in the two stage assay. This suggests that abnormal FVIII molecules are changed in the
regions which are important for function
as assessed by FVIII:CChr assay but that
those regions react differently during assessment with FVIII:C1st method. The most
common identified mutations (in 60% of
patients) were presented by Tyr295Ala
Mutation spectrum in haemophilia A patients with discrepancy
FVIII : C
number of
mutation
families patients
1-stage >
chromogenic
1-stage <
chromogenic
1
1
Tyr114Cys
1
1
Thr118Ile
1
1
Asp203Val
1
2
1
domain FVIII : Ag
(U/dl)
FVIII : C (IU/dl)
1-stage
chromogenic
FVIII : Cchromogenic
FVIII : C1-stage
6.9
4
0.57
11.5
1.8
1
0.57
1.7
1.7
1
0.58
Thr275Ile
34.9
3.8
0.9
0.22
1
Asn280Ile
7.1
5.7
2.9
0.50
3
4
Arg531His*
3
3
Arg531Cys*
61.5
1
1
Val663Ala
53.9
27.8
1
1
Arg1749His* A3
59.7
9.4
5.2
0.55
1
2
Pro1825ser
42.6
7.4–15.5
4.2–9.0
0.56
1
1
Arg1941Gln
23.5
7.4
3.9
0.52
1
1
Lys166Thr
70.21
8.7
18
0.48
10
13
The295Ala
18.3
9.44 (6–22)
17.09 (9.9–41.00)
0.55
3
3
Leu412Phe
18
4.5
10
0.45
8
11
1
A1
27.9 (45.3–14.7)
A2
A1
A2
23–30.5
6.5–9.8
13.0–20.0
0.56
3.0–7.0
0.46
18
0.61
Arg527Trp**
89.8 (61.6–138.4)
12.54 (7.7–23.0)
30.6 (20.0–52.5)
0.41
2
Val678Leu
93.9
39
78
0.50
1
1
Met702Leu
10.2
12
13
Glu720Lys*
87.9 (62.0–133.0)
#
2
27
1
1
Arg1689His* a3 (TCS)
1
1
Leu1756Phe
1
1
Leu1756Val
1
1
Arg1781Gly
2
2
Phe2101Leu
Tyr1680Phe
16.2 (9.7–38.6)
a3
A3
C1
3
4
Ser2119Tyr
C1
1
1
Asp2131Asn
C1
4.6
0.41
24.3 (16.0–35.5)
94.15 (54–120.5)
0.25
4.4 (2.2–7.3)
8.7 (4.1–13.5)
0.50
113.2
4.5
41
0.11
17.1
9.5
18.8
0.51
9.3
3.3
8.7
0.38
3.9
7.2
0.54
7.9
#
1.9
15.1 (9.6–24.0)
5.8
2.9 (1.9–3.5)
9.4
13
6.8 (5.0–8.7)
15
0.45
0.42
0.63
#
*previously reported to be associated with FVIII : C discrepancy; VWF binding
Hämostaseologie 4a/2010
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A. Pavlova et al.: One- and two-stage FVIII assays
a)
b)
Fig. 1 Mutations on factor VIII protein in haemophilia A patients with discrepant FVIII : C measured
by 1-stage and chromogenic assays
a) FVIII : C1-stage > FVIII : Cchromogenic; b) FVIII : C1-stage < FVIII : C2-stage
Conclusion
Certain point mutations may escape detection if the residual FVIII:C is tested only by
either the FVIII:C1st or the FVIII:CChr assay.
This can lead to
● misdiagnosis in some cases of mild haemophilia or
● problems of assigning the correct degree
of severity of haemophilia.
References
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4. Parquet-Gernez A, Mazurier C, Goudemand M.
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133 haemophiliacs – characterization of a subgroup
of patients with mild haemophilia A and discrepancy in 1– and 2-stage assays. Thromb Haemost
1988; 59: 202–206.
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demonstrated by a case of mild haemophilia associated with His1954 Leu in theFactor VIIIA3domain.
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Pipe SW, Saenko EL, Eickhorst AN et al. Haemophilia A mutations associated with 1-stage/ 2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa. Blood 2001; 97: 685–691.
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© Schattauer 2010
Hämostaseologie 4a/2010
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Hämophilie-Genetik
(13), Arg527Trp (11), Glu720Lys (13),
Tyr1680Phe (28).
Considering our results (109/284) we
assume that assay discrepancy is quite frequent finding in patients with mild / moderate haemophilia A. Still it is not known
which assay better reflects the haemophilia
severity with regard to clinical manifestation. Based on the existence of the discrepancy phenomena, results in measurements of FVIII activity can be misleading
and fail to diagnose the haemophilia phenotype by certain patients. The strong association between the phenotype and
group of mutations described in this study
suggests that there is genetic basis to the
discrepancy between FVIII:C1st and
FVIII:CChr assay.
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