Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Thromb Res. 2009 Sep 27;125(2):e55–e60. doi: 10.1016/j.thromres.2009.08.019

COMBINED CIS-REGULATOR ELEMENTS AS IMPORTANT MECHANISM AFFECTING FXII PLASMA LEVELS

Maria Sabater-Lleal 1, Miguel Chillón 2,3, Carolina Mordillo 4, Ángel Martínez 1, Estel Gil 3, José Mateo 4, John Blangero 5, Laura Almasy 5, Jordi Fontcuberta 4, José Manuel Soria 1
PMCID: PMC2906145  NIHMSID: NIHMS145574  PMID: 19786295

Abstract

Introduction

Factor XII (FXII) deficiency is a recessive Mendelian trait due to mutations in the F12 gene. There is no bleeding associated with FXII deficiency, but FXII deficiency has been reported to be associated with risk of thrombosis in some studies.

Material and Methods

We examined the functional effect of two naturally-occurring mutations in two Spanish FXII deficient families: a C/G substitution at position –8, and a C/T substitution at position –13. Both mutations were located on a putative HNF4 binding site of F12 gene promoter. We also analyzed the F12 C46T polymorphism (rs1801020), associated with a decrease in the FXII levels, which also segregated in both families. A fragment containing each one of both –8 and -13 mutations, was cloned 5′ of a reporter gene. We compared the in vitro expression of these constructs to the wild type expression.

Results

Our analyses confirm that the –8C/G and the –13C/T mutations decreased expression levels, demonstrating that both mutations are involved in the observed FXII deficiency. In addition, electrophoretic shift analyses suggest that they alter the union of nuclear proteins to the promoter. Coinheritance of these mutations with the C46T polymorphism, result in a significant genotype-phenotype correlation.

Conclusions

We have identified two naturally-occurring mutations in the F12 promoter that drastically reduce FXII levels. Knowing rare genetic alterations in the F12 gene, together with the C46T common variant, may yield further understanding about the genetic architecture of FXII levels, which may have a role in the risk of thrombosis.

Keywords: F12, genetic analyses, promoter expression, thrombophilia

Coagulation FXII (Hageman Factor) participates in the initiation of the intrinsic pathway of coagulation cascade. In addition, it has an important role in the fibrinolytic and the complement systems [1]. It is implicated also in the activation of the kallikrein-kinin system [2, 3] resulting in the production and release of the bradykinin, which is a potent vasodilating agent.

FXII deficiency is a recessive Mendelian trait due to mutations in the F12 gene. There is no bleeding associated with congenital FXII deficiency, suggesting that FXII is not essential for the initiation of coagulation. However, the physiological and pathological relevance of FXII remains unclear. Although some studies show that low levels of FXII produce a thrombophilic state clinically characterized by venous [4] and arterial [5, 6] thrombosis, no significant increase in thrombotic risk could be observed in individuals with severe FXII deficiency [7]. In addition, recent work suggests that FXII makes important contributions to the pathologic intravascular thrombi, with FXII-knockout mice being protected from formation of platelet rich occlusive trombi. Moreover, overall survival rates in patients with thrombotic disease are shown to gradually decrease with decreasing FXII activity whereas patients with FXII total deficiency show normal survival rates (FXII levels of 100%). A possible explanation for these controversial results could be due to a parallel effect of the FXII role via the fibrinolytic or other unknown systems [8, 9]. As thrombosis is a common cause of morbidity and mortality in industrialized nations, further studies need to be performed to clarify the effect of FXII concentration on thrombotic risk.

There are several well-characterized genetic defects that lead to increased thrombotic risk [10, 11]. Previous studies have reported that mutations in F12 gene that reduce FXII levels significantly should be considered as potential risk factors for thrombosis [12]. Our study examined the functional effect of two naturally-occurring mutations, located in a putative HNF4 binding site of the F12 promoter region in 2 subjects with FXII deficiency. One is a C/G substitution at position –8 identified in a homozygous patient with severe FXII deficiency (<1%) [13], and another is a C/T substitution located at position –13, located in a heterozygous 6-year old girl with mild FXII deficiency (22%) without any personal or familiar history of thrombotic disease. Both mutations have also been described in asymptomatic patients with congenital factor XII deficiency from Northern Italy [14]. Our aim was to analyze the molecular basis of –13C/T and –8C/G mutations in a controlled genetic background. We believe that this study provides a good basis to gain a better understanding of how these mutations, together with the common polymorphism C46T, modulate FXII levels in the Spanish population. Understanding of new natural mutations that reduce FXII levels and their interactions with other genetic effects should provide a better understanding of the molecular basis of the coagulation and fibrinolytic pathways. Such knowledge should shed new light on the mechanisms that affect liability to thrombosis.

Design and Methods

Subjects and samples

One naturally-occurring mutation was found in one patient and another was found in another unrelated patient. Both patients had FXII deficiency. Patient 1 was a 58 year old woman who had suffered an embolism at the age of 57. We recruited also her 2 sisters, who were 60 and 51 years old, a 32 year old daughter, and 2 nieces 25 and 15 years of age respectively. No consanguinity was reported [13]. Patient 2 was a 6 year old girl with FXII deficiency (22% level). Although we obtained phenotypic data from some relatives, only her parents were available for the genetic study (Figure 1). Both patients exhibited normal values for the thrombophilic parameters (including functional antithrombin, amidolytic PC, total free and functional PS, functional assay for FVIII, APCR, the FVL, the G20210A FII, homocysteine levels, lupus anticoagulant, APTT test, anticardiolipin and antiphosphatidylserine antibodies) with the exception of prolonged APTT. Blood samples were collected from the antecubital vein and immediately anticoagulated with 1/10 volume of 0.129-M sodium citrate. Platelet-poor plasma was obtained by centrifugation at 2000 g for 20 min. and was frozen and stored at −40°C until analyzed.

Figure 1.

Figure 1

Open in a new tab

Familiar pedigree of Patient 2, showing the co-segregation of the 46C/T and –13C/T allelic variants. The propositus is indicated by an arrow.

DNA was purified from peripheral blood according to a standard protocol [15].

FXII was assayed using deficient plasma from Diagnostica Stago (Asnières) by automated coagulometry as described elsewhere [16]. All procedures were reviewed and approved by the Institutional Review Board of the Hospital de la Santa Creu i Sant Pau.

We analyzed the whole F12 gene (including the promoter, exons, introns and the 3′-UTR) by PCR and direct sequencing of 4 overlapping fragments as previously described [13]. Numbering refers to +1 position corresponding to the initiation of transcription, i.e, 49bp prior to the ATG codon.

We followed the recommendations of den Dunnen and Antonarakis nomenclature [17] for the gene mutations.

Cloning of the Promoter Fragments

Regions containing either the -13 or -8 mutations, as well as the wild type (wt) sequence were obtained from the patient’s genomic DNA and from one control by PCR amplification with forward primer 5′-TAA AAG TGG GTA TTG TTG TAA GA -3′ and reverse primer 5′-CCG TTG GTC CAG CTG CCT ATC C -3′. The amplification reaction was performed with Biotaq DNA polymerase (Bioline, cat.no. BIO-21039) following recommendations of the provider with a PCR program consisting on 5′ at 95°C, followed by 30 cycles of 30″ at 95°C, 30″ at 55°C and 30″ at 72°C, and with a final 10′ extension time at 72°C. The PCR fragment containing 400bp of the proximal promoter (positions −355 to position 44) was cloned 5′ of an Enhanced Green Fluorescent Protein (GFP) reporter gene from the jellyfish Aquorea victorea in p3T plasmid (MoBiTec) (Figure 2).

Figure 2.

Figure 2

Open in a new tab

Schematic view of the constructs used for the reporter assays with the F12 promoter fragment inserted at 5′ of a GFP coding sequence: negative control without promoter, wild type construct with normal alleles, construct containing the -8C mutation and construct containing the –13T mutation.

The fragment was cloned previously into a pGEM plasmid using a commercial Easy Ligation Kit (PGEM-T Easy Vector System, Promega, Cat.#A1360) for ease of sequencing following the recommendations of the provider. All of the fragments were verified by sequencing. The promoter fragment was excised from the pGEM plasmid and introduced into the p3T plasmid (MoBiTec) by restriction digestion with endonucleases SacII and SpeI (both restriction recognition sequences already present in the pGEM polylinker). The GFP gene fragment was introduced by digestion with HindIII and KpnI and it was religated into the p3T containing the fragments of the promoter (Figure 2). A negative control was constructed inserting the GFP gene fragment in a native p3T plasmid without the inserted promoter. A positive control with the GFP gene fragment cloned downstream of the CMV promoter was constructed also to test the efficiency of transfection. Ligation was performed with T4 DNA ligase (New England Biolabs).

Cell Culture, Transfection, and Expression

The constructs (WT, -13, -8) were transiently transfected into the tumoral hepatic cell line HepG2. In addition, a negative control (containing the GFP coding sequence but without the promoter) and a positive control (containing the GFP gene after a CMV promoter) were transfected also. Finally, an irrelevant DNA without GFP was transfected to the posterior calibration of the fluorescence in the cytometer. All of the constructs were transfected in triplicate in each experiment. HepG2 cells were grown in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% Glutamine and 1% Penicillin/Streptomycin (P/S) in a 5% CO2 atmosphere at 37°C. Cells were transferred into six-well plates and grown until they reached 70–80% confluence. Each well was transfected with 5 μg of plasmid. For each transfection experiment, cells were incubated with reduced-serum DMEM medium (1%) with 1% P/S and transfected with 2.25μl of 10μM polyethylenimine (PEI) molecules (22KDa) per μg of DNA [18]. The reagent/DNA mixture was incubated for 30′ at room temperature (22±2 °C) and then added dropwise to the cell medium. After 12 hours, transfection medium was replaced by 10% FBS supplemented medium with 1% P/S was added. Cells were incubated for 72 hours, and then harvested and immediately measured by fluorescence.

Measurement of Cell Fluorescence

Cells were harvested and fixed with 2% paraformaldehyde. Fluorescent quantification was measured in a Fluorescence Activated Cell Sorter (Becton Dickinson FACS Analyzer, SN AL01001) as described previously [19–21]. Fluorescence was analyzed with the MXP software v.2.1. The GFP-related fluorescence was quantified as the mean fluorescence intensity per cell in the cell population displaying fluorescence (cells in region M1) above the autofluorescence limit defined by a cell population transfected with a negative control without GFP.

Electrophoretic Mobility Shift Assays

The double-stranded 30bp DNA oligomers, which corresponded to the putative HNF4 binding site of the F12 promoter surrounded by irrelevant DNA, were constructed by annealing chemically synthesized single-stranded DNA pairs that were biotin-labelled. The duplex DNAs were assayed with the LightShift Chemiluminescent EMSA Kit (PIERCE; 20148) following the recommendations of the provider. We compared the retardation bands of the sequences containing each of the mutations with the WT sequence after addition of 3μg of Human Liver Nuclear Extract (Active Motif; 36042). Forward sequences were FXII-WT-5′-BIOT- TAT TCT CAA GAC CTT TGG CCA GTC CTA TTG-3′, FXII-13G-5′-BIOT- TAT TCT CAA GAG CTT TGG CCA GTC CTA TTG-3′ and FXII-8C-5′-BIOT- TAT TCT CAA GAC CTT TCG CCA GTC CTA TTG-3′. The same sequences were used as unlabelled DNA to the competition experiments. In addition, a random sequence of 30bp was also used as irrelevant DNA to test for unspecific bindings. DNA-protein complexes were incubated for 90 minutes at 37° and then electrophoresed in a 5% non-denaturant polyacrylamide gel in 0.5 Tris-Borate-EDTA running buffer for 1hour at 4°.

Statistical Analyses

Autofluorescence of all experiments was corrected by subtracting the average expression of the cell population transfected with the construct containing the GFP gene without promoter. Because the means of fluorescence intensity were compared only for the cell populations displaying fluorescence in region M1 -i.e only the transfected cells- the results are not affected by transfection efficiency. Nevertheless, a positive control was transfected in each experiment to evaluate the transfection efficiency. Variation between experiments was normalized by setting the mean expression of the WT construct in each experiment to 100%. For each experiment, the mean fluorescence intensity was expressed as a percent with respect to the WT of that experiment.

A linear mixed model with the construct mutation as a fixed effect and the experiment as a random effect was used to compare the individually mutated constructs with the WT construct. A significance level of 0.05 was applied in all analyses. The analyses were performed using the statistical environment R (The R Project for Statistical Computing, www.r-project.org).

Results

Familiar Study

Patient 1 was homozigous for the C allele at the -8G/C mutation and presented severe FXII deficiency (<1%). In addition, she was homozygous for the T allele at the 46C/T polymorphism, both in the F12 gene. Familiar segregation of the different allelic variants in the family was described in [13]. Patient 2 was heterozygous for the –13C/T mutation and was also heterozygous for the 46C/T polymorphism. She had mild FXII deficiency (22%). From the analyses in both parents of the Patient, we determined that both allelic variants were located on different alleles; the T allele at the 46C/T polymorphism was inherited from the mother and the T allele at position –13 was inherited from the father (Figure 1).

We found other genetic variants in both patients that were present also in a set of 40 controls from a Spanish population. Thus, we considered these allelic variants as polymorphisms.

Transfection Experiments

A fragment of 400 bp of the F12 proximal promoter containing the observed mutations, obtained from the DNA of each patient, was cloned upstream of a GFP gene in a p3T plasmid. These two constructs and the wt (containing the normal allele for both mutations) were transfected into HepG2 cells (Figure 2). We performed 5 independent transfection experiments in triplicate to analyze differences in expression levels among the different constructs containing each of the naturally-occurring mutations in the F12 promoter. The percentage of the variance between the 3 replicas of the same experiment was 7.65%. The percentage of the variance between experiments, after normalization, was only 0.086%. We constructed a positive control containing the GFP gene regulated by the strong CMV promoter. The percentage of fluorescent cells averaged 95%. This result is a clear indicator of the high efficiency of transfection in our experiments.

The GFP expression mediated by the different promoter constructs was compared by averaging the GFP fluorescence per transfected cell. The effect of each of the different variants was expressed as a percentage decrease in the average GFP intensity per cell compared to the wt construct. The results are shown in Figure 3. The decrease in the promoter expression levels caused by both mutations was highly significant, with a − 75.64 % (p<0.0001) for the –13C/T mutation and − 15.30 % (p<0.0001) for the –8G/C mutation.

Figure 3.

Figure 3

Open in a new tab

Box-Plot (median, 50%, and 95% confidence intervals) showing the differences in the averages of expression levels per cell of the constructs containing each allelic variant. Average expression levels per cell of the different constructs are referred to WT construct (100%). The WT construct includes the common alleles.

EMSA Analyses

We performed an EMSA experiment to test if the -13 and -8 mutations changed the affinity of the proteins from a human hepatocyte nuclear to the HNF4 binding site located in F12 promoter. As it can be seen in Figure 4, a much weaker binding can be observed when the -8 and the -13 mutated sequences were tested. In addition, the competition assays demonstrate that not only there is a binding to the HNF4 binding site, but it is specific for this particular sequence; while it can be deduced in lanes 5–7 that the unlabelled probe effectively competes for the HNF4 site, the random probe (containing the same GC content but altering the order of the bases) was significantly less efficient as competitor (lanes 8–10). Taken together, these results demonstrate that these two mutations disrupt the HNF4 binding site and impair the binding of HNF4, and that this impairment would be responsible for the reduced expression levels in the constructs containing these mutations.

Figure 4.

Figure 4

Open in a new tab

Electrophoretic Mobility Shift Assays. Lane 1, probe only. Lanes 2 to 4, probes with hepatocyte nuclear extract. Lanes 5 to 7, probes with hepatocyte nuclear extract and unlabeled competitor. Lanes 8 to 10, probes with hepatocyte nuclear extract and unlabeled non-competitor.

Discussion

It has been demonstrated recently that low levels of FXII may produce venous thrombosis [4, 12]probably because of its role in the fibrinolytic system or via other unknown mechanisms. Our study describes two naturally-occurring mutations in the promoter region of F12 gene that were found in two unrelated women with a FXII deficiency. Our in vitro analyses demonstrated that both mutations caused a highly significant reduction in expression levels of the reported gene (−75.64 %, p<0.0001 for the –13T allele and −15.30 %, p<0.0001 for the –8C). In this regard, these results clearly suggest that these mutations have an important effect on FXII levels.

Both mutations are located in the promoter region of F12 gene in a putative binding site for the Hepatocyte Nuclear Factor 4-α transcription factor (HNF4α) (Figure 5). HNF4α is a liver-enriched transcription factor that has a crucial role in the expression of blood coagulation factors [22]. In fact, expression of the mouse FXII and XIIIB is regulated directly by HNF4 via HNF4 binding sites in their promoter regions [23] and HNF4 null mice exhibit a significantly decreased expression of FXII. Our results from the EMSA analyses clearly demonstrate that these mutations impair the binding of the HNF4 binding site, and confirm our hypothesis that the reduced expression levels are due to the modification of the transcription binding site, which results in an alteration in the binding affinity of HNF4α.

Figure 5.

Figure 5

Open in a new tab

Location of the –13C/T and –8G/C mutations in the sequence of the putative HNF4 binding site in humans.

Interestingly, through sequencing the whole F12 gene, our molecular genetic study revealed that the common polymorphic 46C/T was also segregating in both families. The 46C/T is a genetic variant that creates an ATG startcodon located 5bp upstream from the original translation initiation codon ATG. This ATG codon results in a frameshift that produces a 2-aminoacids peptide. This results in reduced levels of FXII in carriers of the T allele [24, 25] that would be consistent with the observed phenotype in the carriers; Patient 1 was heterozygous for the -8G/C mutation and presented severe FXII deficiency (<1%). According to our in vitro studies, this mutation should reduce about 15% the expression levels per mutated allele. However, the patient was also homozygous for the T allele at 46C/T polymorphism in the F12. The combination of these two allelic variants probably leads to the resulting deficient phenotype due to a synergic effect. However, it is also plausible that other polymorphisms found in this patient [13] could contribute also to lowering FXII levels. Patient 2 was heterozygous both for the –13C/T mutation and for the 46C/T polymorphism and she had 22% levels of FXII. Segregation analysis in this family showed that these two alterations came from different alleles, one from the mother and another from the father. Thus, the low expression levels could be explained since one allele was reduced by the –13C/T allele, and the 46C/T genotype was responsible for the reduced expression of the other allele.

In addition, our results from the EMSA analyses clearly demonstrate that these mutations impair the binding of the HNF4 binding site and confirms our hypothesis that the reduced expression levels are due to a clear reduction of the amount of protein that binds the HNF4 binding site.

These results represent a good example of how the combined effect of cis-regulator elements might play a crucial role in the regulation of a complex phenotype that could affect the risk of thrombosis. Indeed, the combination of these mutations probably caused the thrombotic outcome in Patient 1. Therefore, although Patient 2 did not show any thrombotic events at the age of 6, a follow-up would be recommended based on our results, specially considering that older age is a major risk factor for venous thrombosis [26, 27].

In addition, this results show that a combination of common and rare variants determine the FXII levels. There is a current debate concerning the genetic architecture of complex diseases such as thromboembolic disease. Whereas the common disease, common variant hypothesis states that a few common allelic variants could account for the genetic variance in disease susceptibility, the rare variant hypothesis states that rare variants actually play an important role in complex diseases. Such rare variants are mostly population specific because of founder effects resulting from genetic drift. What is now accepted is that both of these hypotheses are correct, and that a combination of common and rare variants can explain the genetic basis of most complex traits [28], possibly by common variants acting as significant modifiers of the effects of rare variants. Our study supports this hypothesis, showing that a combination of common and rare variants determines FXII levels.

Mapping the loci that contribute to complex disease -such as thrombophilia- is a major challenge. Our results suggest that rare variants can be important in determining complex phenotypes, which agrees with others who suggest that rare variants can contribute substantially to the multifactorial inheritance of common chronic diseases [29]. This is important for mapping strategies, as the power of Genome Wide Association Studies (GWAS) will be considerably reduced if the rare variants actually play a role in determining the final phenotype. These association studies are designed to detect common variants and are not suitable for detecting rare variants [30]. For this reason, resequencing the entire genomic regions to identify both common and rare variants is now the best approach. With the availability of high throughput DNA sequencing methods and advanced computational methods, the full sequencing of the genome will reveal the true picture of polymorphic structure of human genome.

In conclusion, we have identified two naturally-occurring mutations in the F12 promoter that drastically reduce FXII levels. Our results suggest that knowing rare genetic alterations in the F12 gene, together with the C46T common variant, helps to understand the role of FXII on the risk of thrombosis.

Acknowledgments

We gladly acknowledge the advice and helpful discussion of Professor W.H. Stone. This study was supported partially by grants No. 2 R01 HL070751-05 from the USA NIH, PI-08/0420, PI-08/0756 and Redes Temáticas de Investigación Cooperativa (RETIC) Cardiovascular (RECAVA: Exp-06/0014/0016RD Ministerio de Sanidad y Consumo, Spain) and SAF2008/01859 from Ministerio de Ciencia y Tecnología and FEDER (Spain). J.M. Soria was supported by “Programa d’Estabilització d’Investigadors de la Direcció d’Estrategia i Coordinació del Departament de Salut” (Generalitat de Catalunya). E. Gil is a recipient of a Ministerio de Ciencia y Tecnologia (MCYT) fellowship.

Abbreviations

FXII

Factor XII protein

F12

Factor XII gene

PCR

Polymerase Chain Reaction

HNF

Hepatic Nuclear Factor

GFP

Green Fluorescent Protein

DMEM

Dulbecco’s Modified Essential Medium

PEI

Polyethylenimine

WT

Wild Type

Footnotes

Statement of Conflict of Interest: All the authors state that they do not have any financial or personal relationship with other people or organisations that could inappropriately influence (bias) their work.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Furie B, Furie BC. The molecular basis of blood coagulation. Cell. 1988;53(4):505–18. doi: 10.1016/0092-8674(88)90567-3. [DOI] [PubMed] [Google Scholar]
  • 2.Wiggins RC, Bouma BN, Cochrane CG, Griffin JH. Role of high-molecular-weight kininogen in surface-binding and activation of coagulation Factor XI and prekallikrein. Proc Natl Acad Sci U S A. 1977;74(10):4636–40. doi: 10.1073/pnas.74.10.4636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Griffin JH. Role of surface in surface-dependent activation of Hageman factor (blood coagulation factor XII) Proc Natl Acad Sci U S A. 1978;75(4):1998–2002. doi: 10.1073/pnas.75.4.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Halbmayer WM, Mannhalter C, Feichtinger C, Rubi K, Fischer M. The prevalence of factor XII deficiency in 103 orally anticoagulated outpatients suffering from recurrent venous and/or arterial thromboembolism. Thromb Haemost. 1992;68(3):285–90. [PubMed] [Google Scholar]
  • 5.Doggen CJ, Rosendaal FR, Meijers JC. Levels of intrinsic coagulation factors and the risk of myocardial infarction among men: Opposite and synergistic effects of factors XI and XII. Blood. 2006;108(13):4045–51. doi: 10.1182/blood-2005-12-023697. [DOI] [PubMed] [Google Scholar]
  • 6.Santamaria A, Mateo J, Tirado I, Oliver A, Belvis R, Marti-Fabregas J, Felices R, Soria JM, Souto JC, Fontcuberta J. Homozygosity of the T allele of the 46 C->T polymorphism in the F12 gene is a risk factor for ischemic stroke in the Spanish population. Stroke. 2004;35(8):1795–9. doi: 10.1161/01.STR.0000133127.68041.a3. [DOI] [PubMed] [Google Scholar]
  • 7.Zeerleder S, Schloesser M, Redondo M, Wuillemin WA, Engel W, Furlan M, Lammle B. Reevaluation of the incidence of thromboembolic complications in congenital factor XII deficiency--a study on 73 subjects from 14 Swiss families. Thromb Haemost. 1999;82(4):1240–6. [PubMed] [Google Scholar]
  • 8.Gailani D, Renne T. Intrinsic pathway of coagulation and arterial thrombosis. Arterioscler Thromb Vasc Biol. 2007;27(12):2507–13. doi: 10.1161/ATVBAHA.107.155952. [DOI] [PubMed] [Google Scholar]
  • 9.Endler G, Marsik C, Jilma B, Schickbauer T, Quehenberger P, Mannhalter C. Evidence of a U-shaped association between factor XII activity and overall survival. J Thromb Haemost. 2007;5(6):1143–8. doi: 10.1111/j.1538-7836.2007.02530.x. [DOI] [PubMed] [Google Scholar]
  • 10.Lane DA, Mannucci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, Chandy M, Dahlback B, Ginter EK, Miletich JP, Rosendaal FR, Seligsohn U. Inherited thrombophilia: Part 1. Thromb Haemost. 1996;76(5):651–62. [PubMed] [Google Scholar]
  • 11.Lane DA, Mannucci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, Chandy M, Dahlback B, Ginter EK, Miletich JP, Rosendaal FR, Seligsohn U. Inherited thrombophilia: Part 2. Thromb Haemost. 1996;76(6):824–34. [PubMed] [Google Scholar]
  • 12.Soria JM, Almasy L, Souto JC, Bacq D, Buil A, Faure A, Martinez-Marchan E, Mateo J, Borrell M, Stone W, Lathrop M, Fontcuberta J, Blangero J. A quantitative-trait locus in the human factor XII gene influences both plasma factor XII levels and susceptibility to thrombotic disease. Am J Hum Genet. 2002;70(3):567–74. doi: 10.1086/339259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mordillo C, Martinez-Marchan E, Fontcuberta J, Soria JM. Molecular analysis of multiple genetic variants in Spanish FXII-deficient families. Haematologica. 2007;92(11):1569–72. doi: 10.3324/haematol.11388. [DOI] [PubMed] [Google Scholar]
  • 14.Lombardi AM, Bortoletto E, Scarparo P, Scapin M, Santarossa L, Girolami A. Genetic study in patients with factor XII deficiency: a report of three new mutations exon 13 (Q501STOP), exon 14 (P547L) and -13C>T promoter region in three compound heterozygotes. Blood Coagul Fibrinolysis. 2008;19(7):639–43. doi: 10.1097/MBC.0b013e32830d8629. [DOI] [PubMed] [Google Scholar]
  • 15.Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Souto JC, Almasy L, Borrell M, Gari M, Martinez E, Mateo J, Stone WH, Blangero J, Fontcuberta J. Genetic determinants of hemostasis phenotypes in Spanish families. Circulation. 2000;101(13):1546–51. doi: 10.1161/01.cir.101.13.1546. [DOI] [PubMed] [Google Scholar]
  • 17.den Dunnen JT, Antonarakis SE. Nomenclature for the description of human sequence variations. Hum Genet. 2001;109(1):121–4. doi: 10.1007/s004390100505. [DOI] [PubMed] [Google Scholar]
  • 18.Goula D, Remy JS, Erbacher P, Wasowicz M, Levi G, Abdallah B, Demeneix BA. Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther. 1998;5(5):712–7. doi: 10.1038/sj.gt.3300635. [DOI] [PubMed] [Google Scholar]
  • 19.Taupin JL, Merville P, McBride T, Potaux L, Moreau JF. Functional quantification of cyclosporine A and FK506 in human whole blood by flow cytometry, using the green fluorescent protein as an interleukin-2 reporter gene. J Immunol Methods. 2001;256(1–2):77–87. doi: 10.1016/s0022-1759(01)00438-0. [DOI] [PubMed] [Google Scholar]
  • 20.Niswender KD, Blackman SM, Rohde L, Magnuson MA, Piston DW. Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. J Microsc. 1995;180 (Pt 2):109–16. doi: 10.1111/j.1365-2818.1995.tb03665.x. [DOI] [PubMed] [Google Scholar]
  • 21.Sabater-Lleal M, Chillon M, Howard TE, Gil E, Almasy L, Blangero J, Fontcuberta J, Soria JM. Functional analysis of the genetic variability in the F7 gene promoter. Atherosclerosis. 2007 doi: 10.1016/j.atherosclerosis.2006.12.031. [DOI] [PubMed] [Google Scholar]
  • 22.Sladek FM, Ruse MD, Jr, Nepomuceno L, Huang SM, Stallcup MR. Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1. Mol Cell Biol. 1999;19(10):6509–22. doi: 10.1128/mcb.19.10.6509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Inoue Y, Peters LL, Yim SH, Inoue J, Gonzalez FJ. Role of hepatocyte nuclear factor 4alpha in control of blood coagulation factor gene expression. J Mol Med. 2006;84(4):334–44. doi: 10.1007/s00109-005-0013-5. [DOI] [PubMed] [Google Scholar]
  • 24.Kanaji T, Okamura T, Osaki K, Kuroiwa M, Shimoda K, Hamasaki N, Niho Y. A common genetic polymorphism (46 C to T substitution) in the 5′-untranslated region of the coagulation factor XII gene is associated with low translation efficiency and decrease in plasma factor XII level. Blood. 1998;91(6):2010–4. [PubMed] [Google Scholar]
  • 25.Endler G, Exner M, Mannhalter C, Meier S, Ruzicka K, Handler S, Panzer S, Wagner O, Quehenberger P. A common C-->T polymorphism at nt 46 in the promoter region of coagulation factor XII is associated with decreased factor XII activity. Thromb Res. 2001;101(4):255–60. doi: 10.1016/s0049-3848(00)00404-7. [DOI] [PubMed] [Google Scholar]
  • 26.White RH. The epidemiology of venous thromboembolism. Circulation. 2003;107(23 Suppl 1):I4–8. doi: 10.1161/01.CIR.0000078468.11849.66. [DOI] [PubMed] [Google Scholar]
  • 27.Anderson FA, Jr, Spencer FA. Risk factors for venous thromboembolism. Circulation. 2003;107(23 Suppl 1):I9–16. doi: 10.1161/01.CIR.0000078469.07362.E6. [DOI] [PubMed] [Google Scholar]
  • 28.Iyengar SK, Elston RC. The genetic basis of complex traits: rare variants or “common gene, common disease”? Methods Mol Biol. 2007;376:71–84. doi: 10.1007/978-1-59745-389-9_6. [DOI] [PubMed] [Google Scholar]
  • 29.Bodmer W, Bonilla C. Common and rare variants in multifactorial susceptibility to common diseases. Nat Genet. 2008;40(6):695–701. doi: 10.1038/ng.f.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hirschhorn JN, Daly MJ. Genome-wide association studies for common diseases and complex traits. Nat Rev Genet. 2005;6(2):95–108. doi: 10.1038/nrg1521. [DOI] [PubMed] [Google Scholar]

RESOURCES