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NASBA® factor V Leiden QL; a new genotype assay to detect the factor V Leiden mutation in anticoagulated whole blood
Fibrinolysis (1996) 10 Suppl 2, 31-32 © Pearson Professional Ltd 1996
N A S B A ® f a c t o r V L e i d e n QL; a n e w g e n o t y p e a s s a y to d e t e c t t h e f a c t o r V L e i d e n m u t a t i o n in a n t i c o a g u l a t e d w h o l e blood F. Roeles, W. Kok, B. Top, H. Adriaanse, N. Tacken, B. van Gemen Organon Teknika BV, Boxtel, The Netherlands.
Summary
NASBA® F a c t o r V Leiden, a new RNA amplification test appeared to be reliable in detecting the Factor V Leiden point mutation in whole blood.
INTRODUCTION
APC resistance is a common and strong hereditary risk factor for venous thrombosis. ] This plasma abnormality is caused in part by a point mutation in the factor V gene, a G---~A transition at nt 1691, the f.V Leiden mutation. 2 The single point mutation lends itself to analysis by a simple and specific genetic assay. This test represents an alternative to plasma-based APC resistance tests, which may be compromised by patient treatment or specimen handling. A protocol utilizing a solid phase extraction methodology, a selective RNA amplification procedure (NASBA), followed by a detection procedure (ELGA enzyme linked gel assay) was investigated. METHODS
NASBA is an isothermal nucleic acid amplification technology based on the simultaneous enzymatic activity of RNaseH, AMV-RT and T7 RNA polymerase, in concerted action with the two specific primers. The NASBA reaction results in the accumulation of single-stranded RNA with amplification factors of approximately 109 in 90 minutes (figure 1). Lymphocytes contain large quantities of factor V RNA 3, which was purified from 100 gl whole blood. An aliquot of the purified nucleic acid eluate, equivalent to 5 tal of the original sample, was subjected to amplification. An RNA
Correspondence to: Frits Floeles, Organen Teknika bv, P.O. Box 84, 5280 AB Boxtel, The Netherlands. Tel. +31-411-654911 ; Fax. +31-411-654201.
system control was added to the isolation mixture to assure blood sample quality as well as isolation and amplification efficiency. A rapid non-radioactive hybridization assay was developed to identify the mutation (polymorphism) at position 1691. In this assay, specific HRP probes for the wild-type and mutant RNA were used in a gel retardation protocol (ELGA). The method was used successfully to identify carriers of the f.V Leiden mutation among samples selected at random from routine coagulation laboratory blood draws. Table 1 NASBA results vs. PCR-MNL1 (n=206) NASBA WT Mut Hetero
WT 137
Mut
Hetero
7 62
Table 2 NASBA results vs. APC ratio (n=60) NASBA < 2.0 wild-type homozygous 4 heterozygous 11 mutant homozygous 1 *tested with Coatest, Chromogenix
APC ratio* > 2.0 38 3 0
2.0 1 2 0
RESULTS
In a direct comparison with PCR-MNLI on 206 samples 100% agreement was achieved (table 1). In another comparison with the APC-R test of Chromogenix (Coatest) on 60 samples 10 samples showed disagreement (table 2). This means that the NASBA® Factor V Leiden assay can clarify overlapping results (ratio 31
32
Roeles et al
ss RNA (sense) Legend: sense
RNA
Primer
1
Primer
Primer 2 m antisense DNA
1
AMV-RT
\ sense
~
DNA H Primer 2 RNase
antisense
RNA
\
B
\
AMV-RT
!
T7 RNA pol
1~ f ss
rVk/V~
m
Primer
2
RNA (antisense)
amplificate
T7 RNA pol
AMV-RT
l
Isothermal amplification
RNase H
Primer I
AMV-RT
-~
Fig. 1
<2.0 for wild-type homozygous samples and >_2.0 for heterozygous samples) in functional APC resistance testing. CONCLUSION
In conclusion, a streamlined protocol was developed for the rapid, reproducible specific detection of heterozygous and homozygous carriers of the f.V Leiden mutation. The method does not require expensive, specialized equipment and therefore holds promise for use in routine coagulation laboratories not specializing in molecular genetics.
Fibnnolysis (1996) 10 Suppl 2, 31-32
REFERENCES
1. Dahlbfick B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant respons to activated protein C. Proc.Nat.Acad.Sci. 1993; 90, ! 004-1008. 2. Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood coagulation Factor V associated with resistance to activated protein C. Nature 1994; 369, 64-67. 3. Shen NLL, et al. The serine protease cofactor Factor V is synthesized by lymphocytes. J. Immunol. 1993; 150, 29923001
© Pearson Professional Ltd 1996
N A S B A ® f a c t o r V L e i d e n QL; a n e w g e n o t y p e a s s a y to d e t e c t t h e f a c t o r V L e i d e n m u t a t i o n in a n t i c o a g u l a t e d w h o l e blood F. Roeles, W. Kok, B. Top, H. Adriaanse, N. Tacken, B. van Gemen Organon Teknika BV, Boxtel, The Netherlands.
Summary
NASBA® F a c t o r V Leiden, a new RNA amplification test appeared to be reliable in detecting the Factor V Leiden point mutation in whole blood.
INTRODUCTION
APC resistance is a common and strong hereditary risk factor for venous thrombosis. ] This plasma abnormality is caused in part by a point mutation in the factor V gene, a G---~A transition at nt 1691, the f.V Leiden mutation. 2 The single point mutation lends itself to analysis by a simple and specific genetic assay. This test represents an alternative to plasma-based APC resistance tests, which may be compromised by patient treatment or specimen handling. A protocol utilizing a solid phase extraction methodology, a selective RNA amplification procedure (NASBA), followed by a detection procedure (ELGA enzyme linked gel assay) was investigated. METHODS
NASBA is an isothermal nucleic acid amplification technology based on the simultaneous enzymatic activity of RNaseH, AMV-RT and T7 RNA polymerase, in concerted action with the two specific primers. The NASBA reaction results in the accumulation of single-stranded RNA with amplification factors of approximately 109 in 90 minutes (figure 1). Lymphocytes contain large quantities of factor V RNA 3, which was purified from 100 gl whole blood. An aliquot of the purified nucleic acid eluate, equivalent to 5 tal of the original sample, was subjected to amplification. An RNA
Correspondence to: Frits Floeles, Organen Teknika bv, P.O. Box 84, 5280 AB Boxtel, The Netherlands. Tel. +31-411-654911 ; Fax. +31-411-654201.
system control was added to the isolation mixture to assure blood sample quality as well as isolation and amplification efficiency. A rapid non-radioactive hybridization assay was developed to identify the mutation (polymorphism) at position 1691. In this assay, specific HRP probes for the wild-type and mutant RNA were used in a gel retardation protocol (ELGA). The method was used successfully to identify carriers of the f.V Leiden mutation among samples selected at random from routine coagulation laboratory blood draws. Table 1 NASBA results vs. PCR-MNL1 (n=206) NASBA WT Mut Hetero
WT 137
Mut
Hetero
7 62
Table 2 NASBA results vs. APC ratio (n=60) NASBA < 2.0 wild-type homozygous 4 heterozygous 11 mutant homozygous 1 *tested with Coatest, Chromogenix
APC ratio* > 2.0 38 3 0
2.0 1 2 0
RESULTS
In a direct comparison with PCR-MNLI on 206 samples 100% agreement was achieved (table 1). In another comparison with the APC-R test of Chromogenix (Coatest) on 60 samples 10 samples showed disagreement (table 2). This means that the NASBA® Factor V Leiden assay can clarify overlapping results (ratio 31
32
Roeles et al
ss RNA (sense) Legend: sense
RNA
Primer
1
Primer
Primer 2 m antisense DNA
1
AMV-RT
\ sense
~
DNA H Primer 2 RNase
antisense
RNA
\
B
\
AMV-RT
!
T7 RNA pol
1~ f ss
rVk/V~
m
Primer
2
RNA (antisense)
amplificate
T7 RNA pol
AMV-RT
l
Isothermal amplification
RNase H
Primer I
AMV-RT
-~
Fig. 1
<2.0 for wild-type homozygous samples and >_2.0 for heterozygous samples) in functional APC resistance testing. CONCLUSION
In conclusion, a streamlined protocol was developed for the rapid, reproducible specific detection of heterozygous and homozygous carriers of the f.V Leiden mutation. The method does not require expensive, specialized equipment and therefore holds promise for use in routine coagulation laboratories not specializing in molecular genetics.
Fibnnolysis (1996) 10 Suppl 2, 31-32
REFERENCES
1. Dahlbfick B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant respons to activated protein C. Proc.Nat.Acad.Sci. 1993; 90, ! 004-1008. 2. Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood coagulation Factor V associated with resistance to activated protein C. Nature 1994; 369, 64-67. 3. Shen NLL, et al. The serine protease cofactor Factor V is synthesized by lymphocytes. J. Immunol. 1993; 150, 29923001
© Pearson Professional Ltd 1996