Inherited Thrombophilia and the Risk of Vascular Events

The definition of thrombophilia includes the impact of hereditary base in the tendency to develop thrombosis and its clinical manifestations. Prothrombotic phenotype results from the interaction of inherited disorders of coagulation and various "clinical" risk factors such as obesity, immobility, major and minor surgery, hormone therapy, malignancy, etc. According to the accepted multicausal model, inherited thrombophilia is a manifestation of mutual influence of gene-gene and gene-environmental factors. Inherited thrombophilia traits include a wide range of disorders. Deficits of some coagulation inhibitors are relatively rare, and their clinical significance is previously known. On the other hand, in genes which control the coagulation cascade, there are many variants that are widespread in the population. The practical significance of these variants, known as genetic polymorphisms, is different, and the subject of numerous epidemiological-genetic, clinical and health economic studies (Novaković et al, 2010; Krcunović et al., 2010; Pavlović et al., 2011). In summary, the most common congenital disorders associated with thrombophilia are: a deficiency of antithrombin, protein C and protein S, variants of factor V Leiden and prothrombin 20210, and mild hyperhomocysteinemia (Table 1). Individually or in combination, these traits are present in about 40% of patients with venous thromboembolism (VTE), and in approximately the same percentage of women with disorders of pregnancy and puerperium, such as fetal loss, fetal growth restriction and preeclampsia (De Stefano et al, 2002).

Leiden varies: is about 1-5% in North America, higher in North and Central Europe, while in the African and Asian population, this polymorphism is almost absent.It is estimated that this polymorphism originated 21-24000 years ago.Under normal conditions, APC protein binds to factor V and cuts it into two inactive fragments.It is determined that the Leiden variant is resistant to APC protein, which prolongs the action of factor V. The result is a continuation of prothrombin activation and continuously maintain coagulation cascade (Robertson et al, 2005, Ho et al, 2006).Table 1.Prevalence of major hypercoagulable states in different patient populations (after De Stefano et al, 2002).

Hypercoagulable
Heterozygous for FVL have a 2-7 times greater risk of VTE event, while the homozygous risk increased even 40-80 times.The first event of VTE often occurs after 45 years of age.The risk is further multiplied in women during pregnancy, as well as due to the intake of oral contraceptives or hormone replacement therapy in menopause (Table 2).Thus, women heterozygous for factor V Leiden who take hormone preparations have 15.6 times higher risk of venous thrombosis, while pregnant women homozygous for this polymorphism show 34 times increased risk of thrombosis.The risk of spontaneous abortion is also increased (Robertson et al, 2005, Ho et al, 2006).Table 2. Results for association of venous thromboembolism with oral contraceptive or hormone replacement use, Factor V Leiden, or both (after Wu et al, 2006).

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Inherited Thrombophilia and the Risk of Vascular Events 61

Prothrombin G20210A
The gene for prothrombin (coagulation factor II) also has a significant polymorphism 20210G> A. This polymorphism is located in the 3' untranslated region of gene, and is supposed to have a regulatory role.Its frequency in European populations is 1-5%, while it is very rare in people of African or Asiatic origin.20210 allele leads to increased activity of prothrombin, and the hypercoagulation state (Robertson et al, 2005, Ho et al, 2006) Meta analyzes show that the risk for the first thrombotic event increased 2-10 times in heterozygotes for factor V Leiden and 2-6 times in heterozygotes for allele 20210A.
Combined heterozygous, possessing both polymorphisms, have as much as 20 times higher risk.These data are particularly important in situations that in themselves predispose thrombosis and thrombus-embolism, such as major orthopedic surgery or malignancy (Robertson et al, 2005, Ho et al, 2006).

Mild hyperhomocysteinemia
In relation to thrombophilia, the importance of some other polymorphisms should be emphasized, such as polymorphism in the gene for methylene-tetrahidrofolat-reductase, MTHFR 677C> T. This polymorphism leads to substitution of alanine to valine at position 222 in the polypeptide, which causes thermolable form of the enzyme with decreased activity.T allele frequency is highest in Asian populations, lower in white European and American, and the lowest in African populations.In Europe, the incidence of TT homozygosity is 5-15%.People with 677TT and 677CT genotype have only 30% and 50% MTHFR enzymatic activity, respectively.MTHFR catalyzes the development of methyltetrahidrofolate, which is the main donor of methyl group in the remethylation of homocysteine to methionine.That is why the MTHFR 677 polymorphism associated with hyperhomocysteinemia, which is considered to be an independent risk factor for thrombophilia, and other disorders, such as atherosclerosis, neural tube defects, etc (Robertson et al, 2005, Wu et al, 2006;Simić-Ogrizović et al. 2006;Todorović et al. 2006;Damnjanović et al. 2010 ).
 Patients with VTE, independently of the age of onset (</> 45 years), the circumstances of thrombosis (provoked /unprovoked), and the severity of the clinical manifestations. As a rule, patients with cancer may be excluded.Prophylaxis: in the case of: surgery, bed immobilization, plastering of the arms or of the legs, long air journeys (more than 4 hours) in the puerperium (all genotypes) throughout the whole pregnancy (severe thrombophilia; the indication for primary antithrombotic prophylaxis with low molecular weight heparin throughout the pregnancy in women with isolated heterozygosity for factor V Leiden or for the prothrombin G20210A is not yet certain).
Box 3. Secondary prophylaxis with oral anticoagulants after VTE in patients with inherited thrombophilia (adapted from de Stefano et al, 2002 andWu et al, 2006) Results of extensive cost-benefit studies do not support universal polymorphism screening prior to the introduction of hormone therapy, during pregnancy or after major orthopedic surgery.Instead, selective testing of patients with a history previous thrombo-embolism is recommended.To prevent spontaneous abortions in women with thrombophilia, low-dose aspirin and heparin is recommended.There is no universal agreement on the need for preventive therapy in elective orthopedic surgery (Robertson et al, 2005, Ho et al, 2006, Wu et al, 2006).

New approaches and research strategies in inherited thrombophilia
Until recently, research aimed to elucidate the genetic component of the susceptibility to VTE was based on the traditional approach and employed primarily candidate gene studies; by this approach, the most significant and well-known polymorhisms of coagulation factors V and II (factor V Leiden and prothrombin 20210) were identified.Candidate genes are generally selected with respect to their known role in the traits and processes in interest.In relation to VTE, majority of studies have dealt with genes of coagulation/fibrinolysis cascade.Thus, based on the known biology of clotting, Smith et al. (2007) investigated polymorphisms of 24 candidate genes coding for proteins affecting coagulation (factors II, V, VII, VIII, IX, X, XI, XII, XIIIa1, and XIIIb; fibrinogen , , and ; and tissue factor), anticoagulation (antithrombin, proteins C and S, endothelial protein C receptor, thrombomodulin, and tissue factor pathway inhibitor [TFPI]), fibrinolysis (plasminogen and tissue-type plasminogen activator), and antifibrinolysis (PAI-1 and thrombin activatable fibrinolysis inhibitor [TAFI]).
Although candidate gene studies have yielded some significant results, selection of appropriate candidates may be difficult, and new approaches had to be employed.One of these modern, widely used and successful strategies for identification of new genetic variants associated with susceptibility to multifactorial diseases is GWAS (genome wide association studies).GWAS is based on testing the association of a complex phenotype with large numbers of SNPs in large samples of patients.Recent use of this and other modern strategies resulted in detection of a number of new genetic variants potentially associated with VTE, but further research is needed to elucidate their significance as general risk factors.ABO locus variants were previously found to be associated with susceptibility to VTE: non-O blood group carriers were at higher risk, probably through higher levels of VWF and factor VIII, known risk factors for VTE (O'Donnell et al, 2002).This was confirmed by a recent study - Variation in gene encoding protein C (PROC) responsible for protein C deficiency was initially supposed to be strongly related to thrombosis, but this was not confirmed later (Dahlback, 2008).However, two polymorphisms within the promoter region (C/T at position -2405 and A/G at position -2418) were recently associated with increased risk of VT (Pomp et al. 2009).Carriers of CC/GG genotype, who showed the lowest protein C levels, were at higher risk of VT compared to carriers of the TT/AA genotype.Polymorphism at CADM1, encoding cell adhesion molecule 1, also expressed in endothelial cells, was recently identified as a probable risk factor for VTE in protein C-deficient kindreds (Hasstedt et al, 2009), but it is not clear whether it is a general risk factor also.In gene coding for protein C receptor (PROCR or EPCR) haplotype A3 (Gly219 allele of the Ser219Gly substitution) was associated with increased VTE risk (Qu et al, 2006;Tregouet et al, 2009), especially in highrisk groups of individuals, carriers of other mutations (Morange & Tregouet, 2010).
The association of gene F12 (encoding coagulation factor FXII) variants with VTE has been subject to a long debate.Common F12 variant has attracted much research interest, but the results were not unequivocal.F12 C-46T variant (rs1801020) was reported to be associated with increased VTE risk (homozygous carriers of T allele compared to noncarriers; Tirado et al, 2004).This association was also observed in a number of other studies, e.g. in patients with cerebral VT (Reuner et al, 2008).However, some studies failed to confirm these findings, e.g.Grunbacher et al. (2005) 2010) demonstrated that the F12 gene represents a quantitative trait locus (QTL) that influences factor XII levels, and showed that only the promoter -46C>T variant (rs1801020) accounted for the variance attributed to this QTL.The authors concluded that this variation is evolutionary neutral and that T allele appeared approximately 100,000 years ago, reaching high frequencies by genetic drift.Buil et al. (2010) reported that a new locus, C4BPB/C4BPA (coding for C4-binding protein), is involved in susceptibility to VT through a still unknown, but protein S-independent mechanism.Bezemer et al. (2008) found SNPs significantly associated with VT in CYP4V2/KLKB1/F11 gene cluster, as well as in the GP6 and SERPINC1 genes.Three SNPs were strongly associated with VT: rs13146272 in CYP4V2, rs2227589 in SERPINC1 and rs1613662 in GP6; 4 additional SNPs (in CYP4V2, KLKB1, and F11) were also associated with VT.The effect of CYP4V2 and GP6 loci polymorphisms (SNPs rs1613662 and rs13146272) was further confirmed by Tregouet et al. (2009), and the effect of F11 polymorphisms (SNPs rs2289252 and rs2036914) by Li et al. (2009).Recently, in a multi-stage multi-design study, Antoni et al. (2010) found evidence that BAI3 locus (encoding brain specific angiogenesis inhibitor 3) is associated with early-onset VTE; rs9363864-AA genotype was associated with a lower risk for VTE and low levels of FVIII and VWF.Three variants were recently found to be associated with activated partial thromboplastin time: F12 (rs2731672), KNG1 (rs710446), and HRG (rs9898) (Houlihan et al. 2010); KNG1 Ile581Thr was confirmed as a risk factor for VT (Morange et al., 2011).Morange et al. (2010) in a follow-up study found another new locus involved in susceptibility to VT that was not a part of commonly studied coagulation/fibrinolysis pathway: HIVEP1 (coding for human immunodeficiency virus type 1 enhancer-binding protein).Allele rs169713C was associated with an increased risk for VT.Future research in relation to genetic basis of VTE is aimed to explore other possibilities as well (Morange & Tregouet 2010), such as the effects of CNVs (copy number variations) or changes in epigenetic mechanisms (e.g.DNA methylation patterns).

Molecular testing of inherited thrombophilia
Nowadays, direct molecular detection of thrombophilia risk factors including F2 G20210A, factor V Leiden and MTHFR C677T mutations is offered by many clinical diagnostic laboratories.It is possible to discriminate between individuals heterozygous and homozygous for factor V Leiden on the basis of the degree of APCR in functional clottingbased assays.Testing for APC resistance (using an accurate assay) can be helpful in assessing for the presence of FVL, whether used initially as a ''screen'' or if used in conjunction with molecular testing.Therefore the APCR test serves as a phenotypic marker for FVL.A normal modified APCR test excludes the presence of FVL, but if abnormal, the FVL genotype should be confirmed by genetic analysis.Although the presence of PT G20210A is associated with increased levels of prothrombin in plasma, there is no phenotypic screening test for PT G20210A, and detection is always by genotyping.In contrast, there are no analogous functional assays for the detection of the PT G20210A mutation because there is no simple distinction between measurable PT levels in those with and without the mutation.Similarly, although the MTHFR thermolabile variant may be associated with increased levels of homocysteine, there is no other direct functional measure of any effect upon in vitro clotting assays.Detection of thrombophilias by functional testing is associated with a degree of uncertainty because interpretation of results can be influenced by the accuracy of normal ranges and the influence of other factors (both genetic and environmental).Hence repeat testing is often required.Many different DNA-based methods have been described.In every laboratory method is chosen based on required sensitivity, necessary equipment, number of samples and economical capacity.In contrast to functional analyses results obtained by DNA based methods are essentially absolute, provided the requisite controls are in place, such that repeat testing is not required.Most of the methods described rely on DNA amplification by polymerase chain reaction (PCR).Genomic DNA is required for genetic analysis, but quantity and quality of DNA preparation may vary, depending on the requirements of the assay.Most laboratories routinely purify DNA for the analysis, but where only one or two single nucleotide polymorphisms (SNP) are to be evaluated, avoidance of time consuming DNA preparation can be of benefit.PCR-based methods rely on different tools to detect the genotype of the amplified alleles, such as RED, amplification-refractory mutation system (ARMS), enzyme-linked immunosorbent assay (ELISA)-based primer extension assay and fluorescence resonance energy transfer (FRET) assay.Multiplexing allows single procedure to detect more than one mutation.However, when electrophoresis is used, there may be many fragments present and care must be taken to ensure that interpretation of genotypes is clear-cut and not prone to error.Rare mutations at, or very close to the SNP site of interest may suggest different genotypes, depending on methodology.For example, rare silent mutations within the F5 gene (A1692C, G1689A and A1696G) and rare sequence variations at or near the prothrombin (F2) G20210A mutation (C20209T, A20207C, A20218G and C20221T).These will influence genetic analysis in a manner dependent upon the test system used.However, when these sequence variations are rare in the population their influence on the assay may be considered negligible.

Restriction enzyme digestion
Original methods for detection of mutations are based on restriction fragment length polymorphism (RFLP).DNA fragment of interest is amplified by polymerase chain reaction www.intechopen.comThrombophilia 66 (PCR) and than subjected to digestion by specific restriction enzymes whose restriction site is either created or abolished by the presence of mutation.Fragments of different sizes, generated after digestion, are separated by agarose or polyacrylamide gel electrophoresis and visualized under ultraviolet light after staining with ethidium bromide.The number of bands allows the discrimination of possible genotypes.For the FVL mutation restriction enzyme MnlI is used.The fragment generated by PCR contains one restriction enzyme site which serves as a positive internal control.It ensures that the restriction enzyme has been added, and that the restriction digestion has occurred.The second restriction site for MnlI is created by FVL mutation (Bertina, 1994).In the method described detection of the PT G20210A in the presence of the mutant allele an artificial HindIII restriction site had to be created by the mutagenic amplification primer (Poort et al, 1996).Improvement of the original method provides additional restriction site which serves as a control as in the case of FVL detection (Pecheniuk, 2000).The MTHFR C677T mutation is also easily detected by PCR and RFLP analysis since the mutation creates restriction enzyme site for HinfI enzyme (Frosst, 1995).The use of multiplex PCR prior to RFLP and the use of whole blood instead of isolated DNA largely improved the original RFLP analyses (Gomez, 1998).

Amplification refractory mutation system (ARMS)
In order to simplify the procedure, increase the efficiency and reduce the costs by eliminating the use of restriction enzymes new methods have been developed.One of them is ARMS based on allele specific amplification of DNA sample.Standard procedure is performed in two separate reactions and with three different primers.In each reaction one of the primers is common (used in both reactions) and the other one is allele specific with 3'end complementary either to wild type allele or mutant allele.Results are obtained by analysis of PCR products on agarose or polyacrylamide gels.The presence or absence of PCR products represents the presence or absence of target alleles.Usually control primers are amplified in the same reaction with allele specific primers to ensure that the PCR reaction is working properly.Multiplex ARMS assay is also widely in use and it allows the analysis of two or more mutations.The Stagen kits for FVL and PT G20210A (Stago, Asnieres, France) amplify ß-2 microglobulin and an unconnected region of F5 (or F2) genes as controls, to ensure efficiency of the PCR reactions.Although this method is simple, efficient and cost effective the specificity of amplification relies highly on careful primer design, careful titration of primer concentrations, and stringent temperature cycling parameters (McGlennen, 2002).

Single strand conformational polymorphism
The other simple and fast method which avoids the need for restriction enzyme digestion is a single-strand conformation polymorphism (SSCP).It relies on the fact that denatured DNA molecules show different migration patterns on electrophoresis gels even when they differ in only one base.After PCR amplification DNA fragment of interest is denatured to single strands and analyzed by gel electrophoresis.Different alleles will form different 3D conformation and different migration patterns will be observed on gels.The advantage of this method is that it may identify rare mutations, such as the silent A1692C transition in the F5 gene, which is falsely identified as FVL when MnlI restriction enzyme is used (Keeney et al., 1999).These authors also described an SSCP multiplex assay to identify FVL and PT G20210A mutations in whole blood samples.Care must be taken on the interpretation of SSCP, as mutations may cause relatively small changes in electrophoretic mobility Cooper & Rezende (2007).

Enzyme-linked immunosorbent assays
ELISA-based methods generally hybridize a biotinylated PCR product to oligonucleotide bound to microtitre plate wells.Streptavidin-horseradish peroxidase conjugate then binds to the bound amplicon and finally, buffered hydrogen peroxide with chromophore detects the bound amplicon.Comparison of colour density generated by reactions with wild-type and mutant oligonucleotide probes determines the genotype.A variation of this test is a multiplex assay, where two separate PCR reactions utilize a common biotinylated primer and reverse primers specific for wild-type or the mutant allele, and PCR products hybridize onto an oligonucleotide probe in microtitre plate wells.The former assay design may be more robust, as a single amplification reaction amplifies DNA irrespective of genotype.An ELISA assay for FVL using a reverse allele-specific oligonucleotide hybridization was described by Kowalski et al. (2000).ELISA-based methods require the use of a thermocycler, but other specialist genotyping apparatus is not required.In comparison to gel technology, chemicals used are less hazardous.The StripA Assay (ViennaLab, Vienna, Austria) is an ELISA, which utilizes the reverse hybridization principle, binding reactants to a membrane strip rather than to microtitre plate wells.The target sequence of DNA is amplified in a single reaction which produces a biotin-labelled amplicon.The amplicon is selectively hybridized to wild-type and mutant oligonucleotide detector bands on an individual membrane strip, and detected by means of streptavidin-alkaline phosphatase with colourforming substrate.Washing and hybridization steps can be automated.Control and test bands are examined by eye or scanned to determine genotype.StripA kits are available for the detection of FVL and PT G20210A mutations and as a multiplex assay.

Real-time PCR
Real time high resolution melting curve analysis (HRM) employs a new class of fluorescent dyes that intercalate with double-stranded DNA.The intercalating dye is incorporated in PCR reaction and the products are then heated to separate the two strands.Fluorescence levels decrease as the DNA strands dissociate and this melting profile depends on the PCR product size and sequence.HRM appears to be very sensitive and can be used for high throughput mutation screening.Melting temperatures of wild type and mutant allele differ and genotype can be easily interpreted.Whilst Tm values can be extremely useful and reliable in detecting wild-type and common mutations, they cannot be relied upon to characterize unusual mutations.Instrument is relatively expensive and prone to contamination.A multiplex assay for FVL and PT was developed by Van den Bergh et al. (2000).The authors considered their assay to be robust, reliable with high discrimination power.Interpretation of results is based on the presence or absence of peaks with a Tm that is specific for the wild-type or mutant allele.It helps to identify additional sequence variations.This approach is simple allowing the result to be obtained in approximately two hours.Although the instrument is relatively expensive and the method is prone to contamination it is robust and reliable for identification of mutation of interest.(Spector, 2005).

Direct sequencing of DNA
The gold standard method of mutation detection (screening) is bidirectional sequencing.The advantage of this method is that unusual mutations which can cause analytical anomalies can be easily detected.Variation of the standard dideoxy sequencing is Pyrosequencing (Biotage AB, Uppsala, Sweden) which simplifies further the analysis and makes it possible to analyze a large number of PCR products quickly and with minimal effort.

DNA microarray technology
DNA microarray technology provides rapid simultaneous testing for large number of single point mutations.It is a method of choice for laboratories with large number of test requests (Schrijver, 2003).Custom designed oligonucleotide sequences complementary to the normal DNA sequence and known SNP are attached to a chip.The DNA sample screened for mutation is amplified by PCR, fluorescently labeled and hybridized with the oligonucleotides in the microarray.Computer analysis of the color pattern of the microarray generated after hybridization allows rapid automated mutation testing (Turnpeny & Ellard, 2007).Several commercial platforms which include factor V Leiden, PT G20210A and MTHFR C677T mutation are available.However, they are still expensive for most of the laboratories.Low-density arrays with 10 to 20 markers could be a good alternative.In a single test all thrombophilic markers identified through the human genome project could be analysed (McGlennen, 2002).

Invader assay
This assay, unlike most of the other DNA-based genotyping methods does not employ PCR.Analysis is performed with genomic DNA.The Invader technology is based on the generation of a fluorescent signal in the reaction solution following the cleavage of synthetic oligonucleotide probe assembled with a so called invader probe and the DNA template that contains either the normal or mutation nucleotide (McGlennen, 2002).The analysis is performed in microtiter well and the signal is detected by fluorescent plate reader.
The main advantages of the Invader assay are that it does not employ the PCR reaction, therefore, failures resulting from contamination are less likely to occur (Cooper & Rezende, 2007).There is only a requirement for heating block and a fluorometer, but no dedicated apparatus is required for this assay.Genotypes can be obtained reliably directly from genomic DNA.It suffers from two limitations.First it requires relatively large amounts of target DNA.Second, only one SNP can be genotyped per reaction (Olivier, 2005).Modifying of the technology to accommodate multiple SNPs and large numbers of samples simultaneously will significantly improve this assay .

Acknowledgement
This work was supported by Ministry of Science, Republic of Serbia (Grant No. 175091). www.intechopen.com

Relative risk Factor Studies Patients Had VTE Had no VTE (95% CI)
Johnson et al. (2011)genotype was not associated with thrombosis risk, nor with age at first thrombosis.A metaanalysis byJohnson et al. (2011)led to conclusion that evidence for association of this F12 variant with VTE was weak.By genomewide linkage analysis,Calafell et al. ( Some systems use only a single fluorescently labeled oligonucleotide probe (TaqMan hydrolysis probes) that provide very sensitive and specific detection of DNA.For genotyping this technology uses a PCR primer pair and two allele specific hydrolysis probes, one designed to detect the mutated allele and the other wild-type allele.Both allelespecific hydrolysis probes have a quencher dye and different reporter dyes attached.During the extension phase of PCR amplification the probe is hydrolyzed by Taq polymerase, reporter and quencher dyes are separated which causes the increase in fluorescence.After www.intechopen.comInherited Thrombophilia and the Risk of Vascular Events 69 PCR amplification, an endpoint plate reading is performed using real time PCR system.Allelic discrimination is achieved by measuring of the fluorescence values based on the signals from each well