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Technical Briefs |
T) Genotyping by Fluorescence Resonance Energy Transfer in Patients with Coronary Artery Disease or Thrombophilia
1
University Hospital of Leipzig, D-04103 Leipzig, Germany
2
University Hospital of Magdeburg, D-39120 Magdeburg, Germany
3
University Hospital (UKBF) of Berlin, D-12200 Berlin, Germany
aaddress correspondence to this author at: Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Universitätsklinikum Leipzig (AöR), Liebigstrasse 27, D-04103 Leipzig, Germany; fax 49-341-9722209, e-mail orth{at}medizin.uni-leipzig.de
Blood coagulation factor XII (FXII; Hageman factor) is a serine protease. Its NH2-terminal portion binds to negatively charged surfaces, and its COOH-terminal portion contains the enzymatic active site (1). The human FXII gene is located on the chromosomal band 5q33-qter, and 12 kb of the gene (14 exons and 13 introns) have already been sequenced. FXII is converted by activation to a two-chain serine protease with an NH2-terminal heavy chain (Mr 50 000) and a COOH-terminal light chain (Mr 28 000), and activated FXII has been shown in vitro and in vivo to have a pivotal role in several pathways concerned with tissue defense and repair, including the initiation of the intrinsic pathway of blood coagulation and the conversion of plasminogen to plasmin (2).
Hereditary FXII deficiency [with almost no (<1%) FXII coagulant activity (FXIIc) in the homozygous or compound heterozygous state] does not cause a bleeding tendency. However, this deficiency can be detected in vitro because of a prolonged activated partial thromboplastin time (aPTT). Results from previous studies have indicated that FXII is involved in the pathogenesis of thrombophilic diseases and coronary artery disease (CAD): One study (3), but not another (4), indicated that decreased FXIIc is a risk factor for thrombophilia, whereas other studies have reported increased FXIIc in people with CAD (5)(6), suggesting a role of FXIIc in the pathogenesis of atherosclerosis. This hypothesis is supported by findings that human endothelial cells possess receptors for FXII (7) and that FXII is activated by fatty acids in vitro (8) and increases postprandially (9). These effects might explain the particularly high risk for CAD in subjects with a disturbed triglyceride metabolism.
Recently, a common polymorphism in the 5'-untranslated region of the
FXII gene (46C
T) has been described. This polymorphism is associated
with lower FXII antigen concentrations and coagulant activity in
plasma, occurs more frequently in Orientals than in Caucasians
(10), and has been suggested to influence the activity state
of the coagulation pathway (11). The biochemical mechanism
is thought to be by lower translation efficiency when the T allele is
present (10). Genotyping for this polymorphism has been
performed by restriction fragment length polymorphism (RFLP) analysis,
a method that is time-consuming and difficult to automate because of
the postamplification procedures. Another disadvantage of RFLP is that
other mutations close to the mutation of interest may give
false-positive or -negative results. A false-positive result by RFLP
genotyping, e.g., is obtained in carriers of the (silent) factor V
A1692C transversion, which is misinterpreted as factor
VLeiden mutation after MnlI digestion
(12).
Our first aim in this study was to establish a rapid and cost-effective system for FXII genotyping and to examine the effects of the different FXII genotypes on the intrinsic pathway, in particular on FXIIc and aPTT. Our second aim was to examine by a cross-sectional approach whether FXII genotypes differ between a group of 110 subjects with CAD and a group of 193 subjects with thrombophilia.
We studied 110 outpatients [mean (SD) age, 62.1 ± 9.4 years] from the University Hospital Magdeburg with CAD, as diagnosed by angiography. Angiography was performed within 1 year of blood sampling. For the effects of FXII genotype in thrombophilia, 193 subjects with a history of deep vein thrombosis with or without pulmonary embolism were recruited from the University Hospital Benjamin Franklin in Berlin. The protocol was in accordance with the current revision of the Helsinki Declaration of 1975, and all subjects gave informed consent.
As expected, the thrombophilic subjects were younger than the subjects from the CAD study (mean age, 52.0 ± 15.7 years). In all subjects, genomic DNA was isolated from whole blood collected into dipotassium EDTA with the QIAamp DNA Blood Mini Kit (Qiagen). Citrated blood (0.109 mol/L trisodium citrate) was collected from the CAD patients by clean venipuncture and was separated by centrifugation (3000g for 10 min) within 30 min after collection. Plasma was frozen immediately and stored until analysis at -70 °C. For FXIIc determination, we used a clotting assay with FXII-deficient plasma (Dade Behring) and the STA Instrument (Roche Diagnostics). The aPTT was measured using STA APTT LT reagents.
For FXII genotyping, we developed an assay for rapid genotyping that
uses rapid-cycle PCR and fluorescence resonance energy transfer with
the LightCycler System (Roche Molecular Biochemicals)
(13)(14). In this assay, a 326-bp fragment
harboring exon 1 of the FXII gene was amplified from human genomic DNA
with probes matched to published sequence information
(10). The inflection point of the peaks of the negative
derivative of the fluorescence with respect to temperature vs
temperature (-
F/T vs T) was used as a
surrogate marker for the sequence-specific melting point
(Tm). Sequence information for the
probes and cycling conditions are available as a supplement from the
Clinical Chemistry Web Site. The file can be accessed by a
link from the on-line Table
of Contents
(http://www.clinchem.org/content/vol47/issue6/). When
we examined DNA homozygous for the mutated sequence (46T allele), the
empirical Tm as obtained by inflection
point analysis was 62.06 °C (SD, 0.33 °C; n = 40 alleles),
whereas DNA coding for the wild-type allele (46C) produced a
Tm of 70.40 °C (SD, 0.42 °C;
n = 40 alleles). Heterozygous samples contained both types of
targets and thus generated both peaks. Calculated
Tm values of 64.3 and 71.3 °C,
respectively, were obtained for these probes-target hybrids when the
thermodynamic nearest-neighbor model was used (15). The
homogeneous system for genotyping allowed easy and unambiguous
assignment of genotypes to the respective melting curves. We did not
observe melting curve peaks at other temperatures (16). The
fluorescence resonance energy transfer technique, however, can be
suited to detect many of these mutations, if present (16),
in a more selected study group, e.g., in a large group of subjects with
FXII 46C homozygosity and very low FXIIc.
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In a first application of this procedure, we studied the effects of
these polymorphisms in the noncoding region of the FXII gene on FXIIc
and aPTT as well as on the correlation of FXIIc and aPTT. We observed a
profound effect of FXII genotype on FXIIc (Fig. 1
): The 46T allele was associated with significantly lower FXIIc
than the 46C allele (P <0.05, ANOVA). In addition,. FXIIc
and aPTT were negatively correlated [Pearson correlation coefficient
(r) of -0.299; P = 0.02; n = 110].
The effect of FXII genotype on aPTT was not significant
(P = 0.392).
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We next examined whether the distribution of both FXII alleles differs
between subjects with CAD or with thrombophilia. The genotype
distribution was in Hardy-Weinberg equilibrium. The allele frequencies
of the subjects from the CAD study were compared with the allele
frequencies of the subjects from the thrombophilia study. No
significant differences were observed (P = 0.598).
Gender had no effect on genotype distribution (Table 1
). When we compared the combined allele frequencies of our study
(allele frequency of the T allele, 0.222; total of 606 alleles) with
the allele frequencies of an English study (allele frequencies of the T
allele, 0.390; total of 902 alleles) (17), a lower frequency
of the T allele in our middle-European Caucasian population was
noticed. However, a recent very large study (Second Northwick Heart
Study) revealed allele frequencies that were not different from the
frequencies observed in our study (allele frequency of the T allele,
0.25; total of 5248 alleles; P = 0.141,
2 test) (11).
When the results of the studies discussed above are taken together, the role of FXII genotype as a diagnostic or prognostic marker in CAD or thrombophilia is not yet resolved. Although activated FXII has been shown to be an independent risk factor for CAD and although FXII genotype determines FXIIc and activated FXII by mass action (6)(11), an effect of FXII genotype on CAD has not been demonstrated (11). We postulate that confounders, e.g., specific lipoproteins (9), as well as interference from the preanalytical phase of activated FXII analysis obscure strong effects of the genotype on FXIIc or on activated FXII—a situation that resembles the situation of the methylenetetrahydrofolate reductase genotype and homocysteine concentration (18).
In conclusion, we developed an easy-to-perform assay for FXII
genotyping and confirmed in a Caucasian population the pronounced
effect of the 46CT polymorphism on FXIIc. In our study of 296
subjects, we did not detect additional mutations in the vicinity of the
46CT polymorphism of the FXII gene by the hybridization technique
used. Our data do not support the hypothesis that FXII genotyping is
useful in CAD or in thrombophilic diseases.
Acknowledgments
We are grateful to Birgit Gimpel for excellent technical assistance and to Dr. Nicolas von Ahsen (University of Göttingen) for the Meltcalc Program. M.O. was supported in part by the VeRum Foundation.
References
T) polymorphism and in vivo generation of FXII activity—gene frequencies and relationship in patients with coronary artery disease. Thromb Haemost 1999;81:745-747.[ISI][Medline]
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