WO2008020090A1 - Method and kit for diagnosing the risk of developing a venous thrombotic illness - Google Patents

Method and kit for diagnosing the risk of developing a venous thrombotic illness Download PDF

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WO2008020090A1
WO2008020090A1 PCT/ES2006/000481 ES2006000481W WO2008020090A1 WO 2008020090 A1 WO2008020090 A1 WO 2008020090A1 ES 2006000481 W ES2006000481 W ES 2006000481W WO 2008020090 A1 WO2008020090 A1 WO 2008020090A1
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risk
mutation
factor
developing
thrombosis
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PCT/ES2006/000481
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Spanish (es)
French (fr)
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Antonio Martinez Martinez
Mónica LOPEZ MARTINEZ
Pilar Giraldo Castellano
Miguel Pocovi Mieras
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Laboratorios Indas, S.A.
Progenika Biopharma, S.A.
Instituto Aragonés De Ciencas De La Salud
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Priority to PCT/ES2006/000481 priority Critical patent/WO2008020090A1/en
Publication of WO2008020090A1 publication Critical patent/WO2008020090A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates in general to the diagnosis of thrombotic disorders and in particular it relates to an in vitro method of analysis of biological samples that allows the simultaneous detection of genetic alterations associated with the risk of suffering a venous thrombotic event.
  • Haemostasis is the result of the balance between procoagulant factors and opposing anticoagulant systems, most of these factors are proteins regulated by others that act as activators or inhibitors (Fig. 1).
  • the alteration in the balance of both systems due to genetic defects or due to acquired factors can lead to hemorrhagic or thrombotic phenomena.
  • Individuals with genetic alterations in the factors involved in the anticoagulant system have a biochemical situation that causes a silent activation of the coagulation system even if they remain clinically asymptomatic.
  • Hypercoagulability is a state of activation of procoagulant factors, responsible for the pathogenesis of thromboembolic phenomena.
  • Thrombosis is an abnormal situation whereby there is an interaction between platelets, fibrin and blood vessel wall leading to the formation of an intravascular accumulation that causes a plug that obstructs blood flow.
  • the event that occurs as a consequence of this phenomenon is the activation of the physiological fibrinolysis mechanism that dissolves, rechanges and organizes the thrombus.
  • the DVT affects 1 of every 1,000 individuals every year, being more frequent in the elderly and constituting one of the most important causes of morbidity among the population.
  • the recovery of the health standard after an episode of pulmonary embolism or a central or peripheral post-thrombotic syndrome is generally incomplete, causing sequelae that diminish the quality of life of the population that has suffered episodes of thrombosis.
  • the prevention of the thrombotic phenomenon is a practice that aims to avoid or reduce the incidence of thrombosis in at-risk populations, while reducing healthcare costs.
  • Hereditary hypercoagulable states are most often associated with venous thrombosis; However, they also play a leading role in several forms of arterial thrombosis, the majority associated with an increase in platelet activation and loss of resistance of the vascular endothelium. Arterial thromboembolism can be caused by the release of a thrombus from a DVT. Another association described very recently is spontaneous venous thrombosis and atherosclerotic disease. It is postulated that the atherosclerotic vessel activates the plasma coagulation system as well as platelets causing a prothrombotic state prior to the appearance of a DVT (3).
  • Thrombophilia is the situation in which the conjunction of genetic factors occurs along with acquired factors that precipitate the obstruction of a blood vessel by a clot.
  • the genetic alteration is not responsible for DVT, but it predisposes to it maintaining a constant state of hypercoagulability throughout life.
  • the presence of prothrombotic genetic factors is detected in 50% of the subjects who have suffered an episode of thrombosis.
  • triggers pregnancy, surgery, immobilization
  • the conjunction of two or more genetic factors (prothrombotic mutations) multiplies by 5 the risk of developing a DVT before any trigger.
  • Hypercoagulability can be considered as a chronic and systemic process with a recurrence rate of 17.5% at 2 years and 30.3% at 8 years of the first episode of thrombosis.
  • recurrence occurs in 50% of cases in the contralateral limb. This fact supports that the hypercoagulability situation is systemic and not caused by local factors (6-8).
  • Hypercoagulable states are produced by two fundamental situations:
  • the risk of thrombosis is variable. In a study conducted in a cohort of families with congenital defects of coagulation proteins, it refers to an incidence of DVT in 1.07 x 100 patients / year for Antithrombin III deficiency, 0.54 for protein C deficiency. , 0.50 for protein S deficit and 0.30 for resistance to activated protein C (6).
  • the risk is also different depending on the acquired situations, so the risk is much higher in hip or knee prosthesis surgery than during pregnancy or on long-term flights.
  • Individuals with more than one inherited thrombophilia factor have a much higher risk of thrombosis compared to those who only have one (7,8).
  • the list of related factors increases. Some of them are not true mutations in structural genes, but polymorphisms, characterized by being present in more than 1% of the population studied (9). For example, the polymorphism that affects the substitution of G by A in the 3 'region of the prothrombin gene G20210A, this alteration produces an increase in the prothrombin concentration.
  • Polymorphisms can modulate plasma levels of factors Xl, IX, VIII 1 VII and von Willebrand each of which is associated with a different risk of DVT. Polymorphisms have also been found in the genotype of ABO blood groups that determine an increase in the concentration of von Willebrand factor that is associated with a risk of thrombosis (10). These are individual states in which imbalance occurs in hemostatic systems. It would be desirable to have a system to identify the greatest possible number of risk factors jointly using an easy, reliable and reproducible method to be able to individually assign a genetic profile of hypercoagulability risk. It is known that subjects with hereditary risk factors have a much higher incidence of DVT in acquired risk situations than those who do not.
  • the risk factors for venous thrombosis are pregnancy, heart failure, venous ecstasy due to edema, nephrotic syndrome, postoperative, immobilization, tourist class syndrome, obesity, trauma, pelvic obstruction, estrogen treatment, hormonal treatment, neoplasms, muscle work extreme, varicose veins, vascular anatomical abnormalities and polycythemia / thrombocythemia.
  • genetic or congenital hypercoagulability factors refer to genetic mutations that cause functional alterations in molecules involved in coagulation. .
  • the most important genetic risk factors are the G1691A mutation of Factor V Leiden, the G20210A mutation of Prothrombin II, the MTHFR polymorphism, the C46T polymorphism of Factor 12, the deficiency of the S protein, the deficiency of the C protein, Ia Antithrombin II deficiency, Plasminogen deficiency, and Ia Heparin Cofactor deficiency.
  • the G20210A mutation in the gene encoding prothrombin, a protein involved in the coagulation cascade, is a substitution of a GA base in nucleotide 20210 located in the 3 ' untranslated region of the gene (11). This allelic variable is associated with an increase in plasma concentration and activity of this protein.
  • the C46T polymorphism in the F12 gene (13) is associated with a decrease in plasma concentration and the activity of Factor XII. It is the homozygous form
  • Homocysteinemia (C677T MTHFR) is another inherited factor that leads to an increased risk of thrombosis (17). Patients with hyperhomocysteinemia have an increased risk of thrombosis 2 to 3 times.
  • the causes of hyperhomocisteninemia include nutritional deficiencies of folate, vitamin B6 or vitamin B12 and inherited defects in the enzymes of the metabolic pathway of homocysteine such as cystathionine b tapeza or methylenetetrahydrofolate reductase (MTHFR).
  • MTHFR is a regulatory enzyme involved in the metabolism of homocysteine that catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate.
  • a point mutation changes C for T in nucleotide 677 resulting in a replacement of valine for alanine at position 222. This mutation results in a loss of enzymatic activity (18).
  • the effect of this mutation on venous thrombosis is not well defined and appears to be not an independent risk factor for venous thrombosis.
  • the metabolism of homocysteine is located at the intersection of two metabolic pathways Ia of transulfuration and remethylation Ia: the mutations involved are
  • MTHFR 5-10 methylenetetrahydrofolate reductase (MTHFR: C677T and A1298C genes), methionine synthase and methionine synthase reductase (MTRH: A66G gene).
  • MTHFR C677T and A1298C genes
  • MTORH methionine synthase reductase
  • the increase in homocysteine is accentuated in diets poor in folates and is a risk factor for thrombosis, especially in arterial territory (18).
  • the study of the presence of factors that predispose to thrombosis is important for prevention and possible prophylaxis with anticoagulant treatment.
  • the early identification of patients at risk of developing thrombosis and the application of prophylactic treatment decrease the morbidity and mortality associated with this disease.
  • He Direct study of the subject's DNA could definitely determine the presence of any, one or two copies of one or more genetic defects. It is also the most appropriate method to evaluate asymptomatic members belonging to risk families.
  • prophylaxis give anticoagulant treatment to asymptomatic members with any of these genetic risk factors before specific periods of increased risk of thrombosis, such as surgery, pregnancy, use of oral contraceptives or prolonged immobilization.
  • the method presented allows the detection of G 1691 A genetic alterations in the gene encoding PV, G20210A in gene encoding FII, C677T in gene encoding MTHFR and C46T in gene encoding FXII, alterations that pose a high risk of ETV
  • This method is based on the PCR-ELISA methodology and is a fast, economical, simple, robust method that does not require a high investment in equipment and can easily be included in the hospital routine.
  • the present invention provides a method of diagnosis of individuals with VTE, a very useful diagnostic tool that will be particularly important in the screening of asymptomatic individuals suspected of developing the disease.
  • Asymptomatic individuals from families with a history of VTE can be tested using this method allowing early diagnosis before the onset of symptoms.
  • Individuals who possess any of these mutations may be advised to take the necessary measures that can help them prolong their life.
  • the invention faces the problem of providing an efficient, economical and rapid method to detect the risk of developing a venous thrombotic disease.
  • the solution provided by this invention is based on a methodology capable of detecting the absence / presence of the combination of the following genetic alterations: mutation G1691A of Factor V, mutation G20210A of Ia Prothrombin II, mutation V677T of MTHFR and polymorphism C46T of Factor 12.
  • An aspect of the invention constitutes an in vitro method to diagnose the risk of developing a venous thrombotic disease comprising the detection of the absence / presence of the combination of the following genetic alterations: G1691A mutation of Factor V, mutation G20210A of Prothrombin II, the V677T mutation of MTHFR and the C46T polymorphism of Factor 12.
  • Another aspect of the invention relates to an in vitro method for diagnosing the risk of developing a venous thrombotic disease by detecting the absence / presence of the combination of the following genetic alterations: mutation G1691A of Factor V, mutation G20210A of Ia Prothrombin II, the V677T mutation of MTHFR and the C46T polymorphism of Factor 12, said method comprises the extraction of DNA from a biological sample, the amplification of the DNA fragments and the analysis of the fragments amplified in the previous step by ELISA .
  • Another aspect of the invention constitutes an in vitro method for diagnosing the risk of developing a venous thrombotic disease comprising the simultaneous detection of the absence / presence of the following genetic alterations: G1691A mutation of Factor V, G20210A mutation of Prothrombin II , The V677T mutation of MTHFR and the C46T polymorphism of Factor 12.
  • Another aspect of the invention relates to an in vitro method for diagnosing the risk of developing a venous thrombotic disease by simultaneously detecting the absence / presence of the following genetic alterations: G1691A mutation of Factor V, the G20210A mutation of Prothrombin II, the V677T mutation of MTHFR and C46T polymorphism of Factor 12, said method comprises the extraction of DNA from a biological sample, the amplification of DNA fragments and the analysis of the fragments amplified in the previous step by ELISA.
  • Another aspect of the invention constitutes a kit to diagnose the risk of developing a venous thrombotic disease comprising the reagents necessary to carry out the simultaneous detection of the absence / presence of the following genetic alterations: mutation G1691A of Factor V, the mutation G20210A of the Prothrombin If, the V677T mutation of MTHFR and the C46T polymorphism of Factor 12, by a method comprising the extraction of DNA from a biological sample, the amplification of the DNA fragments and the analysis of the fragments amplified in the previous step by ELISA.
  • Another particular aspect of the invention constitutes a kit to diagnose the risk of developing a venous thrombotic disease comprising the probes with nucleotide sequence according to SEQ ID NO: 1-8
  • FIG. 1 Cascade of coagulation once a lesion in the vascular endothelium is caused.
  • the balance of proteins C and S is critical in the regulation of homeostasis.
  • antithrombin III inhibits factor Il (thrombin) and factors Xa and IXa, so that protein C activated together with its cofactor protein S acts as an anticoagulant due to inactivation of factors Va and VIIa .
  • Figure 2. Analysis by 2% agarose gel electrophoresis of the multiplex PCR that jointly amplifies the four genetic markers object of the methodology developed: factor V (366 bp), prothrombin Il (297 bp), F12 (369 bp) and MTHFR (269 Pb), Figure 3.
  • Figure 4 Part of the electropherogram obtained after the analysis in an automatic AB sequencer that confirms the presence in heterozygous of the G1691A mutation of Factor V, previously detected by the ELISA methodology developed in one of the patients studied.
  • FIG. 5 Analysis by 3% agarose gel electrophoresis corresponding to the cutting technique with the Hinf I restriction enzyme for the validation of the detection of the M67FR C677T polymorphism. This enzyme cuts only the mutated allele generating two fragments of 57 and 176 bp, while the normal allele remains intact with a size of 233 bp.
  • FIG. 6 Melting curves that are obtained as a result of the RT-PCR designed for the genotyping of the C46T polymorphism of Factor XII.
  • the graph above shows a normal homozygous individual, the center one a mutated homozygous individual and the one below a heterozygous individual
  • the present invention has as its main object the development of an in vitro method to simultaneously detect four genetic alterations associated with the risk of venous thrombosis: the G1691A mutation of the V factor, the G20210A mutation of the prothrombin, the C667T polymorphism of MTHFR and the C46T polymorphism of F12.
  • the methodology object of the invention is based in the first place on the development of a multiplex PCR that amplifies the nucleotide sequences corresponding to the regions where the genetic alterations are analyzed and secondly in the design of a series of oligonucleotide probes capable from hybridize with the normal and mutated sequences of each of the four genetic markers included in the diagnosis.
  • PCR polymerase chain reaction
  • the amplification reaction is carried out in a final volume of 50 ⁇ l from 250 ng of DNA in a mixture of 10X PCR buffer (Applied Biosystems), 1.5 rtiM MgCI (Applied Biosystems), 0.1 mM dNTPs, 0.625 nmol dUTP-digoxigenin (Roche), 0.03 ⁇ g / ⁇ l primers Factor V, 0.015 ⁇ g / ⁇ l primers Prothrombin II, 0.01 ⁇ g / ⁇ l primers MTHFR, 0.03 ⁇ g / ⁇ l primers C46T of F12 and 2 U of taq gold polymerase (Applied Biosystems).
  • the amplification cycles used are: 1 cycle of 10 min of denaturation at 96 ° C, 40 cycles: denaturation 30 seconds at 94 ° C, 1 minute hybridization at 58 ° C and elongation at 72 ° C for 2 minutes and a final extension from 72 ° C for 10 minutes.
  • dUTP digoxigenin
  • ELISA - The assay is carried out on a microtriter plate coated with streptavidin
  • oligonucleotide probes (0.2 ng / ⁇ l), labeled with biotin at the 5 ' end that allow the union of the probe to the plate through streptavidin, are added to each corresponding well.
  • the nucleotide sequences of the probes are described in Table 2. Incubate for 15 minutes at room temperature.
  • the wells of the plate are washed 3 times with 1X wash buffer. - The 0.8% SSC hybridization solution and the denatured PCR product are added. - Hybrid 30 minutes at 47 ° C.
  • Diluted conjugate is added and incubated for 30 minutes at room temperature. - Wash 3 times with 1 X wash buffer.
  • the 1X development solution is added and incubated for 30 min at room temperature.
  • the absorbance emitted after the enzymatic reaction at 405 nm is measured in a Multiskan RC plate reader from Labsystems.
  • the first inspection is visual, the green color means a positive signal and if there is no color it means that there is no signal.
  • the genotype is analyzed by optical density by reading the plate at 405 nm.
  • OD (normal) of each sample is the value obtained from the well corresponding to that sample in which reagent 1 (normal probe) has been used.
  • OD (mutated) of each sample is the value obtained from the well corresponding to that sample in which reagent 2 (mutated probe) has been used.
  • OD (normal negative) is the value obtained from the well corresponding to the negative control in which reagent 1 (normal probe) has been used.
  • OD (mutated negative) is the value obtained from the well corresponding to the negative control in which reagent 2 (mutated probe) has been used.
  • the ratio (R) is ⁇ 3 the genotype is normal homozygous If the ratio (R) is ⁇ 2.5 and ⁇ 0.8 the genotype is heterozygous - If the ratio (R) is ⁇ 0.4 the genotype is homozygous mutated
  • This analysis is performed using software that allows filling in the OD data obtained from the plate reader and to which a range of values for each genotype in each factor has previously been supplied.
  • the program automatically generates the genotype of each sample for each of the four risk factors for venous thrombosis analyzed:
  • Example 2 Detection of the heterozygous genotype for the G 2021 OA mutation in the Prothrombin II gene.
  • the heerozygous genotype for the G20210A mutation in the Prothrombin Il has been detected in several individuals using the methodology developed (Fig. 3).
  • the heterozygous genotype of the C677T polymorphism of MTHFR has been observed in a very high percentage of samples studied, while the number of homozygous individuals is smaller (Fig. 3).
  • the heterozygous genotype of the C46T polymorphism of the F12 gene is widely represented in the samples analyzed and the homozygous genotype mutated in several individuals has been observed using the ELISA methodology developed (Fig. 3).
  • Example 5 Simultaneous detection of genetic alteration in more than one thrombotic risk marker.
  • the coexistence of heterozygous genotype in FII and homozygous mutation of MTHFR has been observed.
  • Table 1 Description of the nucleoid position and sequence of the primers used in the multiplex PCR.

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Abstract

The present invention describes an in vitro method for diagnosing the risk of suffering a venous thrombotic event. This method consists of the simultaneous detection of four genetic alterations starting from a sample of DNA of an individual, and subsequent analysis by means of the polymerase chain reaction (PCR) and immuno-enzymatic assay (ELISA). The invention also describes the diagnostic kit for detection of the genetic alterations associated with the risk of suffering a venous thrombotic event.

Description

MÉTODO Y KlT PARA DIAGNOSTICAR EL RIESGO DE DESARROLLAR UNA ENFERMEDAD TROMBÓTICA VENOSA METHOD AND KlT FOR DIAGNOSING THE RISK OF DEVELOPING A VENOUS THROMBOTHIC DISEASE
DESCRIPCIÓNDESCRIPTION
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
La invención se relaciona en general con el diagnóstico de desórdenes trombóticos y en particular se refiere a un método ¡n vitro de análisis de muestras biológicas que permite Ia detección simultánea de alteraciones genéticas asociadas al riesgo de padecer un evento trombótico venoso.The invention relates in general to the diagnosis of thrombotic disorders and in particular it relates to an in vitro method of analysis of biological samples that allows the simultaneous detection of genetic alterations associated with the risk of suffering a venous thrombotic event.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
La hemostasia es el resultado del equilibrio entre factores procoagulantes y sistemas opuestos anticoagulantes, Ia mayoría de estos factores son proteínas reguladas por otras que actúan como activadores o inhibidores (Fig. 1). (1) La alteración en el equilibrio de ambos sistemas debido a defectos genéticos o por factores adquiridos puede dar lugar a fenómenos hemorrágicos o trombóticos. Los individuos con alteraciones genéticas en los factores que intervienen en el sistema anticoagulante, tienen una situación bioquímica que provoca una activación silente del sistema de coagulación aunque se mantengan clínicamente asintomáticos. La hipercoagulabilidad es un estado de activación de los factores procoagulantes, responsable de Ia patogénesis de los fenómenos tromboembólicos.Haemostasis is the result of the balance between procoagulant factors and opposing anticoagulant systems, most of these factors are proteins regulated by others that act as activators or inhibitors (Fig. 1). (1) The alteration in the balance of both systems due to genetic defects or due to acquired factors can lead to hemorrhagic or thrombotic phenomena. Individuals with genetic alterations in the factors involved in the anticoagulant system have a biochemical situation that causes a silent activation of the coagulation system even if they remain clinically asymptomatic. Hypercoagulability is a state of activation of procoagulant factors, responsible for the pathogenesis of thromboembolic phenomena.
La trombosis es una situación anómala por Ia cual se produce una interacción entre plaquetas, fibrina y pared del vaso sanguíneo dando lugar a Ia formación de un acumulo intravascular que origina un tapón que obstruye el flujo sanguíneo. El acontecimiento que se produce como consecuencia de éste fenómeno es Ia activación de Ia fibrinolisis mecanismo fisiológico que disuelve, recanaliza y organiza el trombo.Thrombosis is an abnormal situation whereby there is an interaction between platelets, fibrin and blood vessel wall leading to the formation of an intravascular accumulation that causes a plug that obstructs blood flow. The event that occurs as a consequence of this phenomenon is the activation of the physiological fibrinolysis mechanism that dissolves, rechanges and organizes the thrombus.
En USA se producen 2 millones de episodios de trombosis anualmente y se estima Ia mortalidad relacionada con embolismo pulmonar en aproximadamente 60.000 casos /año, superior al número de fallecimientos por cáncer de mama. El diagnóstico y tratamiento de Ia trombosis venosa profunda (TVP) en el momento actual supone uno de los objetivos prioritarios de Ia práctica médica ya que constituye un problema de salud máyoritario. (2)In the USA, 2 million episodes of thrombosis occur annually and the mortality related to pulmonary embolism is estimated in approximately 60,000 cases. / year, higher than the number of deaths from breast cancer. The diagnosis and treatment of deep vein thrombosis (DVT) at the present time is one of the priority objectives of medical practice since it constitutes a major health problem. (2)
La TVP afecta cada año a 1 de cada 1.000 individuos, siendo mas frecuente en ancianos y constituyendo una de las causas mas importantes de morbilidad entre Ia población. La recuperación del standard de salud después de un episodio de embolismo pulmonar o de un síndrome postrombótico central o periférico generalmente es incompleta provocando secuelas que merman Ia calidad de vida de ia población que ha sufrido episodios de trombosis. La prevención del fenómeno trombótico es una práctica que pretende evitar o reducir Ia incidencia de trombosis en poblaciones de riesgo, disminuyendo a su vez los costes asistenciales.The DVT affects 1 of every 1,000 individuals every year, being more frequent in the elderly and constituting one of the most important causes of morbidity among the population. The recovery of the health standard after an episode of pulmonary embolism or a central or peripheral post-thrombotic syndrome is generally incomplete, causing sequelae that diminish the quality of life of the population that has suffered episodes of thrombosis. The prevention of the thrombotic phenomenon is a practice that aims to avoid or reduce the incidence of thrombosis in at-risk populations, while reducing healthcare costs.
Los estados de hipercoagulabilidad hereditarios se asocian con mayor frecuencia a trombosis venosas; sin embargo también tienen su protagonismo en varias formas de trombosis arterial, Ia mayoría asociadas a aumento en Ia activación de las plaquetas y a pérdida de resistencia del endotelio vascular. El tromboembolismo arterial puede originarse por liberación de un trombo desde una TVP. Otra asociación descrita muy recientemente es Ia trombosis venosa espontánea y Ia enfermedad ateroesclerótica. Se postula que el vaso ateroescleroso activa el sistema plasmático de Ia coagulación así como a las plaquetas provocando un estado protrombótico previo a Ia aparición de una TVP (3).Hereditary hypercoagulable states are most often associated with venous thrombosis; However, they also play a leading role in several forms of arterial thrombosis, the majority associated with an increase in platelet activation and loss of resistance of the vascular endothelium. Arterial thromboembolism can be caused by the release of a thrombus from a DVT. Another association described very recently is spontaneous venous thrombosis and atherosclerotic disease. It is postulated that the atherosclerotic vessel activates the plasma coagulation system as well as platelets causing a prothrombotic state prior to the appearance of a DVT (3).
La Trombofilia es Ia situación en Ia que se produce Ia conjunción de factores genéticos junto con factores adquiridos que precipitan Ia obstrucción de un vaso sanguíneo por un coágulo. Por sí sola Ia alteración genética no es Ia responsable de Ia TVP, pero predispone a ella manteniendo un estado constante de hipercoagulabilidad a io largo de Ia vida. Así Ia presencia de factores genéticos protrombóticos se detecta en el 50% de los sujetos que han sufrido un episodio de trombosis. En Ia mitad de los episodios de VTP existen factores desencadenantes fácilmente reconocibles (embarazo, cirugía, inmovilización) en los restantes el acontecimiento se considera como idiopático o espontáneo (4,5). La conjunción de dos ó más factores genéticos (mutaciones protrombóticas) multiplica por 5 el riesgo de desarrollar una TVP ante cualquier factor desencadenante. Se puede considerar a Ia hipercoagulabilidad como un proceso crónico y sistémico con una tasa de recurrencia del 17,5% a los 2 años y del 30,3% a los 8 años del primer episodio de trombosis. En el caso de TVP en una extremidad Ia recurrencia se produce en el 50% de los casos en Ia extremidad contralateral. Este hecho apoya que Ia situación de hipercoagulabilidad es sistémica y no provocada por factores locales (6-8).Thrombophilia is the situation in which the conjunction of genetic factors occurs along with acquired factors that precipitate the obstruction of a blood vessel by a clot. On its own, the genetic alteration is not responsible for DVT, but it predisposes to it maintaining a constant state of hypercoagulability throughout life. Thus, the presence of prothrombotic genetic factors is detected in 50% of the subjects who have suffered an episode of thrombosis. In half of the episodes of VTP there are easily recognizable triggers (pregnancy, surgery, immobilization) in the rest the event is considered as idiopathic or spontaneous (4,5). The conjunction of two or more genetic factors (prothrombotic mutations) multiplies by 5 the risk of developing a DVT before any trigger. Hypercoagulability can be considered as a chronic and systemic process with a recurrence rate of 17.5% at 2 years and 30.3% at 8 years of the first episode of thrombosis. In the case of DVT in a limb, recurrence occurs in 50% of cases in the contralateral limb. This fact supports that the hypercoagulability situation is systemic and not caused by local factors (6-8).
Los estados de hipercoagulabilidad se producen por dos situaciones fundamentales:Hypercoagulable states are produced by two fundamental situations:
Defectos cualitativos o cuantitativos de proteínas antitrombóticasQualitative or quantitative defects of antithrombotic proteins
- Déficit de antitrombina III- Antithrombin III deficit
- Déficit de proteína C- Protein C deficit
- Déficit de proteína S - Incremento en Ia concentración de factores protrombóticos- Protein S deficit - Increase in the concentration of prothrombotic factors
Factor V Leiden (cambio G por A en Ia posición 1691 del gen F5) Mutación G20210A en el gen de Ia protrombinaFactor V Leiden (change G by A in position 1691 of the F5 gene) Mutation G20210A in the prothrombin gene
- Mutación C46T en el gen F12- C46T mutation in the F12 gene
Mutaciones en los genes de las enzimas que regulan el metabolismo de Ia homocisteina (variante C677T de Ia metiltetrahidrofolato reductasa)Mutations in the genes of the enzymes that regulate the metabolism of homocysteine (variant C677T of the methyltetrahydrofolate reductase)
Incremento en los niveles plasmáticos de factor VII, Xl, IX, VIII, von WillebrandIncrease in plasma levels of factor VII, Xl, IX, VIII, von Willebrand
El riesgo de trombosis es variable. En un estudio realizado en una cohorte de familias con defectos congénitos de proteínas de Ia coagulación, se refiere a una incidencia de TVP en 1,07 x 100 pacientes / año para Ia deficiencia de Antitrombina III, 0,54 para Ia deficiencia en proteína C, 0,50 para el déficit de proteína S y 0,30 para Ia resistencia a Ia proteína C activada (6).The risk of thrombosis is variable. In a study conducted in a cohort of families with congenital defects of coagulation proteins, it refers to an incidence of DVT in 1.07 x 100 patients / year for Antithrombin III deficiency, 0.54 for protein C deficiency. , 0.50 for protein S deficit and 0.30 for resistance to activated protein C (6).
El riesgo también es diferente según las situaciones adquiridas, así el riesgo es muy superior en Ia cirugía de prótesis de cadera o de rodilla que durante el embarazo o en vuelos de larga duración. Los individuos con más de un factor hereditario de trombofilia tienen un riesgo muy superior de trombosis respecto a los que solamente tienen uno (7,8). A medida que se avanza en el conocimiento de las bases genéticas de Ia trombosis, aumenta Ia lista de factores relacionados. Algunos de ellos no son verdaderas mutaciones en genes estructurales, sino polimorfismos, caracterizados por estar presenten en más del 1% de Ia población estudiada (9). Por ejemplo el polimorfismo que afecta a Ia sustitución de G por A en Ia región 3' del gen de Ia protrombina G20210A, esta alteración produce incremento en la concentración de protrombina. Los polimorfismos pueden modular los niveles plasmáticos de los factores Xl, IX, VIII1 VII y von Willebrand cada uno de los cuales se asocia con un riesgo diferente de TVP. También se han encontrado polimorfismos en el genotipo de los grupos sanguíneos ABO que determinan aumento en la concentración del factor von Willebrand al que se asocia riesgo de trombosis (10). Se trata de estados individuales en los que se produce desequilibrio en los sistemas hemostáticos. Sería deseable disponer de un sistema que permitiera identificar el mayor número de factores de riesgo posibles de forma conjunta mediante un método fácil, fiable y reproductible para poder asignar de forma individualizada un perfil genético de riesgo de hipercoagulabilidad. Se sabe que los sujetos con factores de riesgo hereditarios tienen una incidencia muy superior de TVP en situaciones de riesgo adquirido que aquellos que no los tienen.The risk is also different depending on the acquired situations, so the risk is much higher in hip or knee prosthesis surgery than during pregnancy or on long-term flights. Individuals with more than one inherited thrombophilia factor have a much higher risk of thrombosis compared to those who only have one (7,8). As the knowledge of the genetic basis of thrombosis is advanced, the list of related factors increases. Some of them are not true mutations in structural genes, but polymorphisms, characterized by being present in more than 1% of the population studied (9). For example, the polymorphism that affects the substitution of G by A in the 3 'region of the prothrombin gene G20210A, this alteration produces an increase in the prothrombin concentration. Polymorphisms can modulate plasma levels of factors Xl, IX, VIII 1 VII and von Willebrand each of which is associated with a different risk of DVT. Polymorphisms have also been found in the genotype of ABO blood groups that determine an increase in the concentration of von Willebrand factor that is associated with a risk of thrombosis (10). These are individual states in which imbalance occurs in hemostatic systems. It would be desirable to have a system to identify the greatest possible number of risk factors jointly using an easy, reliable and reproducible method to be able to individually assign a genetic profile of hypercoagulability risk. It is known that subjects with hereditary risk factors have a much higher incidence of DVT in acquired risk situations than those who do not.
Al relacionar factores genéticos y adquiridos en Ia génesis de Ia TVP se puede pensar que, probablemente todos los pacientes que desarrollan TVP tienen ya una cierta predisposición genética que es desencadenada por los factores adquiridos correspondientes. El grado de hipercoagulabilidad es individualizado y depende del perfil genético que se posea, siendo de bajo riesgo si solamente presenta un defecto como por ejemplo el factor V Leiden solamente se provocará un episodio de TVP ante un gran estímulo como puede ser un embarazo. Por el contrario individuos con múltiples mutaciones o polimorfismos y un estímulo mucho menor desarrollaran episodios de TVP probablemente de forma recurrente. El conocer Ia situación de riesgo de trombosis individual ayudaría a evitar un número importante de episodios de TVP y por tanto de Ia comorbilidad asociada a los mismos.When relating genetic and acquired factors in the genesis of DVT, it can be thought that, probably all patients who develop DVT already have a certain genetic predisposition that is triggered by the corresponding acquired factors. The degree of hypercoagulability is individualized and depends on the genetic profile that is possessed, being of low risk if it only presents a defect such as factor V Leiden will only cause an episode of DVT before a great stimulus such as pregnancy. On the contrary, individuals with multiple mutations or polymorphisms and a much smaller stimulus will develop episodes of DVT probably recurrently. Knowing the situation of risk of individual thrombosis would help avoid a significant number of episodes of DVT and therefore of the comorbidity associated with them.
Los factores de riesgo de Ia trombosis venosa son embarazo, insuficiencia cardiaca, éxtasis venoso por edemas, síndrome nefrótico, postoperatorio, inmovilización, síndrome de Ia clase turista, obesidad, traumatismo, obstrucción pélvica, tratamiento con estrógenos, tratamiento hormonal, neoplasias, trabajo muscular extremo, varices, anomalías anatómicas vasculares y policitemia/trombocitemia. Durante los últimos años se han establecido varios marcadores moleculares como factores de riesgo genético en el desarrollo de trombosis venosa, como se ha mencionado anteriormente, Los factores genéticos o congénitos de hipercoagulabilidad hacen referencia a mutaciones genéticas que provocan alteraciones funcionales en moléculas implicadas en Ia coagulación. Estas alteraciones genéticas predisponen a Ia persona que las porta a padecer un evento trombótico en cualquier etapa de su vida y en ausencia de estímulos adquiridos (obesidad, embarazo, inmovilidad, traumatismo, otras enfermedades...). Los factores de riesgo genético más importantes son Ia mutación G1691A del Factor V Leiden, Ia mutación G20210A de Ia Protrombina II, el polimorfismo MTHFR, el polimorfismo C46T del Factor 12, Ia deficiencia de Ia proteína S, Ia deficiencia de Ia proteína C, Ia deficiencia de Antitrombina II, Ia deficiencia del Plasminógeno, y Ia deficiencia del Cofactor Il de Ia Heparína.The risk factors for venous thrombosis are pregnancy, heart failure, venous ecstasy due to edema, nephrotic syndrome, postoperative, immobilization, tourist class syndrome, obesity, trauma, pelvic obstruction, estrogen treatment, hormonal treatment, neoplasms, muscle work extreme, varicose veins, vascular anatomical abnormalities and polycythemia / thrombocythemia. During the last years several molecular markers have been established as genetic risk factors in the development of venous thrombosis, as mentioned previously. Genetic or congenital hypercoagulability factors refer to genetic mutations that cause functional alterations in molecules involved in coagulation. . These genetic alterations predispose the person who carries them to suffer a thrombotic event at any stage of their life and in the absence of acquired stimuli (obesity, pregnancy, immobility, trauma, other diseases ...). The most important genetic risk factors are the G1691A mutation of Factor V Leiden, the G20210A mutation of Prothrombin II, the MTHFR polymorphism, the C46T polymorphism of Factor 12, the deficiency of the S protein, the deficiency of the C protein, Ia Antithrombin II deficiency, Plasminogen deficiency, and Ia Heparin Cofactor deficiency.
La mayoría de los defectos congénitos están relacionados con proteínas que intervienen en el equilibrio natural entre procoagulantes y anticoagulantes. La mutación puntual en el gen del F5 (G1691A), que origina un fenotipo denominado resistencia a proteína C activada. El Factor V es una proteína implicada en Ia cascada de Ia coagulación. La presencia de una mutación G-A presente en el nucleótido 1691 (localizado en el exón 10 del gen) en el gen que codifica este factor es Ia responsable de la resistencia a Ia proteína C activada. Esta mutación produce un cambio en Ia proteína en el aminoácido 506, arginina a glutamina, que está implicado en el reconocimiento de Ia proteína C activada. Esta mutación, también conocida como FV Leiden, previene Ia inactivación de este factor por Ia proteína C activada, Io que conduce a un estado de hipercoagulabilidad.Most congenital defects are related to proteins that intervene in the natural balance between procoagulants and anticoagulants. The point mutation in the F5 gene (G1691A), which causes a phenotype called activated protein C resistance. Factor V is a protein involved in the coagulation cascade. The presence of a G-A mutation present in nucleotide 1691 (located in exon 10 of the gene) in the gene encoding this factor is responsible for the resistance to activated protein C. This mutation produces a change in the protein in amino acid 506, arginine to glutamine, which is involved in the recognition of activated protein C. This mutation, also known as FV Leiden, prevents the inactivation of this factor by activated protein C, which leads to a state of hypercoagulability.
La mutación G20210A en el gen que codifica Ia protrombina, proteína implicada en Ia cascada de coagulación, es una sustitución de una base G-A en el nucleótido 20210 localizado en Ia región 3' no traducida del gen (11). Esta variable alélica está asociada con un aumento de Ia concentración plasmática y de Ia actividad de esta proteína.The G20210A mutation in the gene encoding prothrombin, a protein involved in the coagulation cascade, is a substitution of a GA base in nucleotide 20210 located in the 3 ' untranslated region of the gene (11). This allelic variable is associated with an increase in plasma concentration and activity of this protein.
Los individuos heterocigotos para este polimorfismo tienen de 2 a 5 veces aumentado el riesgo de trombosis venosa en ausencia de otros factores de riesgo y este riesgo aumenta si se une a otros factores externos como Ia utilización de anticonceptivos orales. Además, se ha observado que esta mutación confiere un riesgo de recidiva de ETV (12).Heterozygous individuals for this polymorphism have increased the risk of venous thrombosis 2 to 5 times in the absence of other risk factors and this risk increases if you join other external factors such as the use of contraceptives oral In addition, it has been observed that this mutation confers a risk of relapse of VTE (12).
EI polimorfismo C46T en el gen F12 (13) está asociado con una disminución de Ia concentración plasmática y de Ia actividad de del Factor XII. Es Ia forma homocigotaThe C46T polymorphism in the F12 gene (13) is associated with a decrease in plasma concentration and the activity of Factor XII. It is the homozygous form
(individuos portadores de los dos alelos mutados T/T) Ia que se asocia a un riesgo 5 veces superior de padecer un evento trombótico comparándolo con los individuos no portadores de este genotipo (14). Además, este polimorfismo en su forma homocigota también se asocia a un riesgo 4-5 veces superior de padecer Infarto de miocardio o ictus (infarto cerebral) (15,16).(individuals carrying the two T / T mutated alleles) that is associated with a 5-fold higher risk of suffering a thrombotic event compared to individuals not carrying this genotype (14). In addition, this polymorphism in its homozygous form is also associated with a 4-5 times higher risk of suffering from myocardial infarction or stroke (cerebral infarction) (15,16).
La Homocisteinemia (C677T MTHFR) es otro factor hereditario que conlleva aumento del riesgo de trombosis (17). Los pacientes con hiperhomocisteinemia tienen un riesgo aumentado de trombosis de 2 a 3 veces. Entre las causas de Ia hiperhomocisteninemia se incluyen deficiencias nutricionales de folato, vitamina B6 o vitamina B12 y defectos hereditarios en las enzimas de Ia ruta metabólica de Ia homocisteína como Ia cistationina b cintaza o Ia metilentetrahidrofolato reductasa (MTHFR).Homocysteinemia (C677T MTHFR) is another inherited factor that leads to an increased risk of thrombosis (17). Patients with hyperhomocysteinemia have an increased risk of thrombosis 2 to 3 times. The causes of hyperhomocisteninemia include nutritional deficiencies of folate, vitamin B6 or vitamin B12 and inherited defects in the enzymes of the metabolic pathway of homocysteine such as cystathionine b tapeza or methylenetetrahydrofolate reductase (MTHFR).
La MTHFR es una enzima reguladora implicada en el metabolismo de Ia homocisteína que cataliza Ia reducción de 5,10-metilentetrahidrofolato a 5-metilentetrahidrofolato. Una mutación puntual cambia C por T en el nucleótido 677 dando lugar a una sustitución de valina por alanina en Ia posición 222. Esta mutación se traduce en una pérdida de actividad enzimática (18). El efecto de esta mutación en la trombosis venosa no está bien definido y parece que no es un factor de riesgo independiente de trombosis venosa. El metabolismo de Ia homocisteina se sitúa en Ia intersección de dos vías metabólicas Ia de transulfuración y Ia de remetilación: las mutaciones involucradas sonMTHFR is a regulatory enzyme involved in the metabolism of homocysteine that catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate. A point mutation changes C for T in nucleotide 677 resulting in a replacement of valine for alanine at position 222. This mutation results in a loss of enzymatic activity (18). The effect of this mutation on venous thrombosis is not well defined and appears to be not an independent risk factor for venous thrombosis. The metabolism of homocysteine is located at the intersection of two metabolic pathways Ia of transulfuration and remethylation Ia: the mutations involved are
5-10 methylenetetrahydrofolate reductasa (MTHFR: genes C677T y A1298C), metionin sintasa y Ia metionin sintasa reductasa (MTRH: gen A66G). El aumento de homocisteina se acentúa en dietas pobres en folatos y es un factor de riesgo de trombosis sobre todo en territorio arterial (18).5-10 methylenetetrahydrofolate reductase (MTHFR: C677T and A1298C genes), methionine synthase and methionine synthase reductase (MTRH: A66G gene). The increase in homocysteine is accentuated in diets poor in folates and is a risk factor for thrombosis, especially in arterial territory (18).
El estudio de Ia presencia de factores que predisponen a Ia trombosis es importante para Ia prevención y posible profilaxis con tratamiento anticoagulante. La identificación precoz de pacientes con riesgo de desarrollar trombosis y Ia aplicación del tratamiento profiláctico disminuyen Ia morbilidad y mortalidad asociada a esta enfermedad. El estudio directo del ADN del sujeto podría determinar definitivamente Ia presencia de ninguna, una o dos copias de uno o varios defectos genéticos. Además es el método más adecuado para evaluar a miembros asintomáticos pertenecientes a familias de riesgo.The study of the presence of factors that predispose to thrombosis is important for prevention and possible prophylaxis with anticoagulant treatment. The early identification of patients at risk of developing thrombosis and the application of prophylactic treatment decrease the morbidity and mortality associated with this disease. He Direct study of the subject's DNA could definitely determine the presence of any, one or two copies of one or more genetic defects. It is also the most appropriate method to evaluate asymptomatic members belonging to risk families.
La detección de estos factores de riesgo genéticos en pacientes es de gran importancia en Ia rutina hospitalaria;The detection of these genetic risk factors in patients is of great importance in the hospital routine;
Para prevenir efectos futuros en pacientes con alto riesgo de recurrencia. - Para distinguir los factores de riesgo ambientales de los factores de riesgo adquiridos, que son permanentes e irreversibles.To prevent future effects in patients with high risk of recurrence. - To distinguish environmental risk factors from acquired risk factors, which are permanent and irreversible.
Para establecer tanto Ia duración como Ia intensidad de la terapia anticoagulante que podría depender de sí Ia causa de Ia trombosis es permanente o transitoria. - Para aconsejar profilaxis: dar tratamiento anticoagulante a miembros asintomáticos con alguno de estos factores genéticos de riesgo antes de periodos específicos de riesgo incrementado de trombosis, como cirugía, embarazo, uso de anticonceptivos orales o inmovilización prolongada.To establish both the duration and the intensity of anticoagulant therapy that could depend on whether the cause of the thrombosis is permanent or transient. - To advise prophylaxis: give anticoagulant treatment to asymptomatic members with any of these genetic risk factors before specific periods of increased risk of thrombosis, such as surgery, pregnancy, use of oral contraceptives or prolonged immobilization.
Por Io tanto Ia existencia en los servicios hospitalarios de tests específicos para el estudio de riesgo trombótico (adquirido o heredado) es crucial para Ia evaluación óptima de esta enfermedad. La valoración de los factores de riesgo genético, en particular los procedimientos analíticos para Ia identificación de alteraciones en el DNA genómico, está siendo ya una parte importante en Ia evaluación diagnóstica de pacientes que presentan signos y síntomas de trombosis venosa o en grupos de riesgo.Therefore, the existence of specific tests in the hospital services for the study of thrombotic risk (acquired or inherited) is crucial for the optimal evaluation of this disease. The assessment of genetic risk factors, in particular the analytical procedures for the identification of alterations in genomic DNA, is already being an important part in the diagnostic evaluation of patients presenting with signs and symptoms of venous thrombosis or in risk groups.
Debido a que Ia demanda de estos análisis aumenta, también crece Ia necesidad de métodos rápidos, reproducibles, sencillos, automatizables, robustos y económicos que puedan realizarse con instrumentos frecuentes en cualquier laboratorio de diagnóstico.Because the demand for these analyzes increases, the need for fast, reproducible, simple, automated, robust and economical methods that can be performed with frequent instruments in any diagnostic laboratory also grows.
El hecho de poder detectar las 4 alteraciones genéticas, anteriormente descritas, (G1691A FV, G20210A FII, C677T MTHFR y C46T F12), de forma simultánea, hace que el proceso de genotlpado sea mucho más rápido, eficiente y económico (20). Actualmente no se ha descrito ningún sistema de detección de estos 4 defectos genéticos de forma simultánea. En las patentes US 5830681 , US 5874256, US 6043035, US 6518016, WO /9106861 , WO 95/21938 y WO 96/30546 Se describen métodos de detección de estos defectos genéticos por separado y en las patentes US 5710028 y WO 00/18965 se describen también sistemas capaces de detectar 2 o 3 alteraciones genéticas. Entre las opciones descritas se destacan:The fact of being able to detect the 4 genetic alterations, previously described, (G1691A FV, G20210A FII, C677T MTHFR and C46T F12), simultaneously, makes the genotliding process much faster, more efficient and economical (20). Currently, no detection system for these 4 genetic defects has been described simultaneously. In patents US 5830681, US 5874256, US 6043035, US 6518016, WO / 9106861, WO 95/21938 and WO 96/30546 Methods of detecting these genetic defects are described separately and in systems US 5710028 and WO 00/18965 systems capable of detecting 2 or 3 are also described. genetic alterations Among the options described are:
Métodos basados en las técnicas PCR-RFLP o PCR alelo específica para Ia detección simultánea de FV Leiden y G20210A en protrombina. Además de no detectar más que dos polimorfismos, estos métodos son laboriosos y poco automatizables (analytical evaluation of primer engineered multiplex PCR.) (19). - Métodos basados en las técnica PCR a tiempo real o en microarrays. Estas técnicas son sofisticadas, costosas y requieren una alta inversión en aparatos y en personal cualificado (21 ,22). - Método de análisis de las mutaciones G 1691 A, G20210A, C677T y 844ins68 enMethods based on the PCR-RFLP or PCR allele specific techniques for the simultaneous detection of Leiden and G20210A PV in prothrombin. In addition to not detecting more than two polymorphisms, these methods are laborious and poorly automated (analytical evaluation of first engineered multiplex PCR.) (19). - Methods based on real-time PCR or microarray techniques. These techniques are sophisticated, expensive and require a high investment in equipment and qualified personnel (21, 22). - Method of analysis of mutations G 1691 A, G20210A, C677T and 844ins68 in
CBS, basados en Ia detección fluorescente del producto amplificado, este sistema es costoso y no es un sistema de análisis simultáneo ya que hay que realizar una reacción de amplificación por cada una de las mutaciones que se quiere estudiar (20,23).CBS, based on the fluorescent detection of the amplified product, this system is expensive and is not a simultaneous analysis system since an amplification reaction must be carried out for each of the mutations to be studied (20,23).
El método que se presenta permite Ia detección de las alteraciones genéticas G 1691 A en el gen que codifica FV, G20210A en gen que codifica FII, C677T en gen que codifica MTHFR y C46T en gen que codifica FXII, alteraciones que suponen un riesgo elevado de ETV. Este método está basado en Ia metodología PCR-ELISA y es un método rápido, económico, sencillo, robusto, que no requiere una alta inversión en equipo y que puede ser fácilmente incluido en Ia rutina hospitalaria.The method presented allows the detection of G 1691 A genetic alterations in the gene encoding PV, G20210A in gene encoding FII, C677T in gene encoding MTHFR and C46T in gene encoding FXII, alterations that pose a high risk of ETV This method is based on the PCR-ELISA methodology and is a fast, economical, simple, robust method that does not require a high investment in equipment and can easily be included in the hospital routine.
La presente invención proporciona un método de diagnóstico de individuos con VTE, una herramienta diagnóstica muy útil que será particularmente importante en el screening de individuos asintomáticos que se sospecha que puedan desarrollar Ia enfermedad. Los individuos asintomáticos de familias con historia de VTE pueden ser testados usando este método permitiendo el diagnóstico precoz antes de Ia aparición de síntomas. A los individuos que posean alguna de estas mutaciones se les puede aconsejar para que adopten las medidas necesarias que puedan ayudarle a prolongar su vida. COMPENDIO DE LA INVENCIÓNThe present invention provides a method of diagnosis of individuals with VTE, a very useful diagnostic tool that will be particularly important in the screening of asymptomatic individuals suspected of developing the disease. Asymptomatic individuals from families with a history of VTE can be tested using this method allowing early diagnosis before the onset of symptoms. Individuals who possess any of these mutations may be advised to take the necessary measures that can help them prolong their life. SUMMARY OF THE INVENTION
La invención se enfrenta con el problema de proporcionar un método eficaz, económico y rápido para detectar el riesgo de desarrollar una enfermedad trombótica venosa.The invention faces the problem of providing an efficient, economical and rapid method to detect the risk of developing a venous thrombotic disease.
La solución proporcionada por esta invención se basa en una metodología capaz de detectar Ia ausencia/presencia de Ia combinación de las siguientes alteraciones genéticas: mutación G1691A del Factor V, Ia mutación G20210A de Ia Protrombina II, Ia mutación V677T de MTHFR y el polimorfismo C46T del Factor 12.The solution provided by this invention is based on a methodology capable of detecting the absence / presence of the combination of the following genetic alterations: mutation G1691A of Factor V, mutation G20210A of Ia Prothrombin II, mutation V677T of MTHFR and polymorphism C46T of Factor 12.
Un aspecto de Ia invención Io constituye, un método in vitro para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa que comprende Ia detección de Ia ausencia/presencia de Ia combinación de las siguientes alteraciones genéticas: mutación G1691A del Factor V, Ia mutación G20210A de Ia Protrombina II, Ia mutación V677T de MTHFR y el polimorfismo C46T del Factor 12.An aspect of the invention constitutes an in vitro method to diagnose the risk of developing a venous thrombotic disease comprising the detection of the absence / presence of the combination of the following genetic alterations: G1691A mutation of Factor V, mutation G20210A of Prothrombin II, the V677T mutation of MTHFR and the C46T polymorphism of Factor 12.
Otro aspecto de Ia invención se refiere a un método in vitro para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa mediante Ia detección de Ia ausencia/presencia de Ia combinación de las siguientes alteraciones genéticas: mutación G1691A del Factor V, Ia mutación G20210A de Ia Protrombina II, Ia mutación V677T de MTHFR y el polimorfismo C46T del Factor 12, dicho método comprende Ia extracción de ADN a partir de una muestra biológica, Ia amplificación de los fragmentos de ADN y el análisis de los fragmentos amplificados en el paso anterior mediante ELISA.Another aspect of the invention relates to an in vitro method for diagnosing the risk of developing a venous thrombotic disease by detecting the absence / presence of the combination of the following genetic alterations: mutation G1691A of Factor V, mutation G20210A of Ia Prothrombin II, the V677T mutation of MTHFR and the C46T polymorphism of Factor 12, said method comprises the extraction of DNA from a biological sample, the amplification of the DNA fragments and the analysis of the fragments amplified in the previous step by ELISA .
Otro aspecto de Ia invención Io constituye un método in vitro para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa que comprende Ia detección simultanea de Ia ausencia/presencia de las siguientes alteraciones genéticas: mutación G1691A del Factor V, Ia mutación G20210A de Ia Protrombina II, Ia mutación V677T de MTHFR y el polimorfismo C46T del Factor 12.Another aspect of the invention constitutes an in vitro method for diagnosing the risk of developing a venous thrombotic disease comprising the simultaneous detection of the absence / presence of the following genetic alterations: G1691A mutation of Factor V, G20210A mutation of Prothrombin II , The V677T mutation of MTHFR and the C46T polymorphism of Factor 12.
Otro aspecto de Ia invención se refiere a un método ¡n vitro para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa mediante Ia detección simultánea de Ia ausencia/presencia de las siguientes alteraciones genéticas: mutación G1691A del Factor V, Ia mutación G20210A de Ia Protrombina II, Ia mutación V677T de MTHFR y el polimorfismo C46T del Factor 12, dicho método comprende Ia extracción de ADN a partir de una muestra biológica, Ia amplificación de los fragmentos de ADN y el análisis de los fragmentos amplificados en el paso anterior mediante ELISA.Another aspect of the invention relates to an in vitro method for diagnosing the risk of developing a venous thrombotic disease by simultaneously detecting the absence / presence of the following genetic alterations: G1691A mutation of Factor V, the G20210A mutation of Prothrombin II, the V677T mutation of MTHFR and C46T polymorphism of Factor 12, said method comprises the extraction of DNA from a biological sample, the amplification of DNA fragments and the analysis of the fragments amplified in the previous step by ELISA.
Otro aspecto de Ia invención Io constituye un kit para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa que comprende los reactivos necesarios para llevar a cabo Ia detección simultanea de Ia ausencia/presencia de las siguientes alteraciones genéticas: mutación G1691A del Factor V, Ia mutación G20210A de Ia Protrombina If, Ia mutación V677T de MTHFR y el polimorfismo C46T del Factor 12, mediante un método que comprende Ia extracción de ADN a partir de una muestra biológica, Ia amplificación de los fragmentos de ADN y el análisis de los fragmentos amplificados en el paso anterior mediante ELISA.Another aspect of the invention constitutes a kit to diagnose the risk of developing a venous thrombotic disease comprising the reagents necessary to carry out the simultaneous detection of the absence / presence of the following genetic alterations: mutation G1691A of Factor V, the mutation G20210A of the Prothrombin If, the V677T mutation of MTHFR and the C46T polymorphism of Factor 12, by a method comprising the extraction of DNA from a biological sample, the amplification of the DNA fragments and the analysis of the fragments amplified in the previous step by ELISA.
Otro aspecto particular de Ia invención Io constituye un kit para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa que comprende las sondas con secuencia nucleotídica según SEQ ID NO: 1-8Another particular aspect of the invention constitutes a kit to diagnose the risk of developing a venous thrombotic disease comprising the probes with nucleotide sequence according to SEQ ID NO: 1-8
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
Figura 1. Cascada de Ia coagulación una vez provocada una lesión en el endotelio vascular. El equilibrio de las proteínas C y S es crítico en Ia regulación de Ia homeostasis. En condiciones normales, Ia antitrombina III (AT III) inhibe al factor Il (trombina) y a los factores Xa y IXa, de modo que Ia proteína C activada junto con su cofactor Ia proteína S actúa como anticoagulante por inactivación de los factores Va y VIIa.Figure 1. Cascade of coagulation once a lesion in the vascular endothelium is caused. The balance of proteins C and S is critical in the regulation of homeostasis. Under normal conditions, antithrombin III (AT III) inhibits factor Il (thrombin) and factors Xa and IXa, so that protein C activated together with its cofactor protein S acts as an anticoagulant due to inactivation of factors Va and VIIa .
Figura 2. Análisis mediante electroforesis en gel de agarosa al 2% de Ia PCR multiplex que amplifica conjuntamente los cuatro marcadores genéticos objeto de Ia metodología desarrollada: factor V (366 pb), protrombina Il (297 pb), F12 (369 pb) y MTHFR (269 Pb) , Figura 3. Análisis en placa ELISA de los productos obtenidos tras ia amplificación por PCR multiplex. En esta placa se han analizado 11 individuos con antecedentes trombóticos que presentan diversos genotipos correspondientes a cada uno de los factores genéticos analizados. La última columna es un control negativo de cada una de las sondas respectivamente.Figure 2. Analysis by 2% agarose gel electrophoresis of the multiplex PCR that jointly amplifies the four genetic markers object of the methodology developed: factor V (366 bp), prothrombin Il (297 bp), F12 (369 bp) and MTHFR (269 Pb), Figure 3. ELISA plate analysis of the products obtained after multiplex PCR amplification. In this plate 11 individuals with thrombotic antecedents have been analyzed that present diverse genotypes corresponding to each of the genetic factors analyzed. The last column is a negative control of each of the probes respectively.
Figura 4. Parte del electroferograma obtenido tras el análisis en un secuenciador automático AB que confirma Ia presencia en heterocigosis de Ia mutación G1691A del Factor V, previamente detectada mediante Ia metodología ELISA desarrollada en uno de los pacientes estudiados.Figure 4. Part of the electropherogram obtained after the analysis in an automatic AB sequencer that confirms the presence in heterozygous of the G1691A mutation of Factor V, previously detected by the ELISA methodology developed in one of the patients studied.
Figura 5. Análisis mediante electroforesis en gel de agarosa al 3% correspondiente a Ia técnica de corte con Ia enzima de restricción Hinf I para Ia validación de Ia detección del polimorfismo C677T de MTHFR. Esta enzima corta sólo el alelo mutado generando dos fragmentos de 57 y 176 pb, mientras que el alelo normal permanece intacto con un tamaño de 233 pb.Figure 5. Analysis by 3% agarose gel electrophoresis corresponding to the cutting technique with the Hinf I restriction enzyme for the validation of the detection of the M67FR C677T polymorphism. This enzyme cuts only the mutated allele generating two fragments of 57 and 176 bp, while the normal allele remains intact with a size of 233 bp.
Figura 6. Curvas de melting que se obtienen como resultado de Ia RT-PCR diseñada para el genotipado del polimorfismo C46T del Factor XII. La gráfica de arriba muestra un individuo homocigoto normal, Ia del centro un individuo homocigoto mutado y Ia de abajo un individuo heterocigotoFigure 6. Melting curves that are obtained as a result of the RT-PCR designed for the genotyping of the C46T polymorphism of Factor XII. The graph above shows a normal homozygous individual, the center one a mutated homozygous individual and the one below a heterozygous individual
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
La presente invención tiene como objeto principal el desarrollo de un método in vitro para detectar de forma simultánea cuatro alteraciones genéticas asociadas al riesgo de trombosis venosa: Ia mutación G1691A del factor V, Ia mutación G20210A de Ia protrombina, el polimorfismo C667T de MTHFR y el polimorfismo C46T de F12.The present invention has as its main object the development of an in vitro method to simultaneously detect four genetic alterations associated with the risk of venous thrombosis: the G1691A mutation of the V factor, the G20210A mutation of the prothrombin, the C667T polymorphism of MTHFR and the C46T polymorphism of F12.
La metodología objeto de Ia invención se basa en primer lugar en el desarrollo de una PCR multiplex que amplifica las secuencias nucleotídicas correspondientes a las regiones donde se encuentran las alteraciones genéticas objeto de análisis y en segundo lugar en el diseño de una serie de sondas oligonucleotídicas capaces de hibridar con las secuencias normales y mutadas de cada uno de los cuatro marcadores genéticos incluidos en el diagnóstico.The methodology object of the invention is based in the first place on the development of a multiplex PCR that amplifies the nucleotide sequences corresponding to the regions where the genetic alterations are analyzed and secondly in the design of a series of oligonucleotide probes capable from hybridize with the normal and mutated sequences of each of the four genetic markers included in the diagnosis.
La metodología a utilizar sería Ia siguiente para el análisis de las cuatro alteraciones genética:The methodology to be used would be the following for the analysis of the four genetic alterations:
PCR multiplex:Multiplex PCR:
- A partir del ADN obtenido de una muestra biológica del individuo (200 μl de sangre), se procede a Ia utilización de Ia técnica de reacción en cadena de Ia polimerasa (PCR) para Ia amplificación de los cuatro fragmentos correspondientes a Ia región génica donde se encuentran cada una de las alteraciones genéticas que van a ser analizadas (Fig. 2), Los cebadores utilizados se describen en Ia tabla 1. - La reacción de amplificación se lleva a cabo en un volumen final de 50 μl a partir de 250 ng de ADN en una mezcla de 10X PCR buffer (Applied Biosystems), 1 ,5 rtiM MgCI (Applied Biosystems), 0,1 mM dNTPs, 0.625 nmol dUTP-digoxigenina (Roche), 0,03 μg/μl cebadores Factor V, 0,015 μg/μl cebadores Protrombina II, 0,01 μg/μl cebadores MTHFR, 0,03 μg/μl cebadores C46T de F12 y 2 U de taq gold polimerasa (Applied Biosystems). Los ciclos de amplificación utilizados son: 1 ciclo de 10 min de desnaturalización a 96° C, 40 ciclos: desnaturalización 30 segundos a 94° C, 1 minuto hibridación a 58° C y elongación a 72° C durante 2 minutos y una extensión final de 72° C durante 10 minutos. - En Ia misma reacción de amplificación se incorpora un nucleótido unido a digoxigenina (dUTP) que constituye un mareaje indirecto para Ia reacción enzimática final con peroxidasa.- From the DNA obtained from a biological sample of the individual (200 μl of blood), the polymerase chain reaction (PCR) technique is used to amplify the four fragments corresponding to the gene region where each of the genetic alterations that are to be analyzed are found (Fig. 2). The primers used are described in Table 1. - The amplification reaction is carried out in a final volume of 50 μl from 250 ng of DNA in a mixture of 10X PCR buffer (Applied Biosystems), 1.5 rtiM MgCI (Applied Biosystems), 0.1 mM dNTPs, 0.625 nmol dUTP-digoxigenin (Roche), 0.03 μg / μl primers Factor V, 0.015 μg / μl primers Prothrombin II, 0.01 μg / μl primers MTHFR, 0.03 μg / μl primers C46T of F12 and 2 U of taq gold polymerase (Applied Biosystems). The amplification cycles used are: 1 cycle of 10 min of denaturation at 96 ° C, 40 cycles: denaturation 30 seconds at 94 ° C, 1 minute hybridization at 58 ° C and elongation at 72 ° C for 2 minutes and a final extension from 72 ° C for 10 minutes. - In the same amplification reaction a nucleotide linked to digoxigenin (dUTP) is incorporated, which constitutes an indirect marking for the final enzymatic reaction with peroxidase.
ELISA - El ensayo se lleva a cabo en una placa microtriter revestida con estreptavidinaELISA - The assay is carried out on a microtriter plate coated with streptavidin
(Labsystems).(Labsystems).
Se añaden las sondas oligonucleótidicas (0,2 ng/μl), marcadas con biotina en el extremo 5' que permiten Ia unión de Ia sonda a Ia placa a través de Ia estreptavidina, a cada pocilio correspondiente. Las secuencias nucleótidicas de las sondas están descritas en Ia tabla 2. Se incuban 15 minutos a Ta ambiente.The oligonucleotide probes (0.2 ng / μl), labeled with biotin at the 5 ' end that allow the union of the probe to the plate through streptavidin, are added to each corresponding well. The nucleotide sequences of the probes are described in Table 2. Incubate for 15 minutes at room temperature.
Se procede a Ia desnaturalización de los productos de Ia PCR multiplex durante 10 minutos a 95? C.Is the denaturation of the multiplex PCR products carried out for 10 minutes at 95? C.
Se lavan los pocilios de Ia placa 3 veces con tampón de lavado 1X. - Se añade Ia solución de hibridación 0,8% SSC y el producto de PCR desnaturalizado. - Se híbrida 30 minutos a 47° C.The wells of the plate are washed 3 times with 1X wash buffer. - The 0.8% SSC hybridization solution and the denatured PCR product are added. - Hybrid 30 minutes at 47 ° C.
Se lava 3 veces con tampón de lavado 1X.Wash 3 times with 1X wash buffer.
Se añade conjugado diluido y se incuba durante 30 minutos a Ta ambiente. - Se lava 3 veces con tampón de lavado 1 X.Diluted conjugate is added and incubated for 30 minutes at room temperature. - Wash 3 times with 1 X wash buffer.
Se añade Ia solución de revelado 1X y se incuba 30 min a Ta ambiente.The 1X development solution is added and incubated for 30 min at room temperature.
Se mide Ia absorbancia emitida tras Ia reacción enzimática a 405 nm en un lector de placas Multiskan RC de Labsystems.The absorbance emitted after the enzymatic reaction at 405 nm is measured in a Multiskan RC plate reader from Labsystems.
TRATAMIENTO DE DATOSDATA TREATMENT
La primera inspección es visual, el color verde significa una señal positiva y si no hay color significa que no hay señal.The first inspection is visual, the green color means a positive signal and if there is no color it means that there is no signal.
Tras Ia inspección visual, el genotipo es analizado por densidad óptica mediante Ia lectura de Ia placa a 405 nm.After the visual inspection, the genotype is analyzed by optical density by reading the plate at 405 nm.
Si estas condiciones preliminares son correctas se puede proceder al análisis de los resultados obtenidos mediante Ia siguiente relación de los valores OD obtenidos tras Ia lectura de Ia placa:If these preliminary conditions are correct, the results obtained can be analyzed by means of the following relation of the OD values obtained after the reading of the plate:
R= OD(normal)-OD(negativo normal)/OD(mutado)- OD(negativo mutado)R = OD (normal) -OD (normal negative) / OD (mutated) - OD (negative mutated)
OD(normal) de cada muestra es el valor obtenido del pocilio correspondiente a esa muestra en el que se ha utilizado el reactivo 1 (sonda normal). OD(mutado) de cada muestra es el valor obtenido del pocilio correspondiente a esa muestra en el que se ha utilizado el reactivo 2 (sonda mutada). OD(negatívo normal) es el valor obtenido del pocilio correspondiente al control negativo en el que se ha utilizado el reactivo 1 (sonda normal).OD (normal) of each sample is the value obtained from the well corresponding to that sample in which reagent 1 (normal probe) has been used. OD (mutated) of each sample is the value obtained from the well corresponding to that sample in which reagent 2 (mutated probe) has been used. OD (normal negative) is the value obtained from the well corresponding to the negative control in which reagent 1 (normal probe) has been used.
OD(negativo mutado) es el valor obtenido del pocilio correspondiente al control negativo en el que se ha utilizado el reactivo 2 (sonda mutada).OD (mutated negative) is the value obtained from the well corresponding to the negative control in which reagent 2 (mutated probe) has been used.
La interpretación de Ia fórmula es Ia siguiente:The interpretation of the formula is the following:
Si el ratio (R) es ≥ 3 el genotipo es homocigoto normal Si el ratio (R) es ≤ 2.5 y ≥ 0.8 el genotipo es heterocigoto - Si el ratio (R) es ≤ 0.4 el genotipo es homocigoto mutadoIf the ratio (R) is ≥ 3 the genotype is normal homozygous If the ratio (R) is ≤ 2.5 and ≥ 0.8 the genotype is heterozygous - If the ratio (R) is ≤ 0.4 the genotype is homozygous mutated
Este análisis es realizado utilizando un software que permite rellenar con los datos de OD que se obtienen del lector de placas y al que previamente se Ie ha suministrado un rango de valores para cada genotipo en cada factor. De este modo tras incluir estos datos, el programa automáticamente genera el genotipo de cada muestra para cada uno de los cuatro factores de riesgo de trombosis venosa analizados:This analysis is performed using software that allows filling in the OD data obtained from the plate reader and to which a range of values for each genotype in each factor has previously been supplied. Thus, after including these data, the program automatically generates the genotype of each sample for each of the four risk factors for venous thrombosis analyzed:
Homocigoto normal Heterocigoto Homocigoto mutadoNormal homozygous Heterozygous mutated Homozygous
Ejemplo 1 :Example 1 :
Detección del genotipo heterocigoto para Ia mutación G 1691 A en el gen del Factor V.Detection of the heterozygous genotype for the G 1691 A mutation in the Factor V gene.
Se ha utilizado Ia metodología anteriormente descrita para el análisis de estos marcadores de riesgo en 100 muestras clínicas correspondientes a individuos con riesgo de padecer una trombosis venosa y a individuos de Ia población. El genotipo heterocigoto para Ia mutación G 1691 A en el Factor V ha sido detectado en varios individuos (Fig. 3).The methodology described above has been used for the analysis of these risk markers in 100 clinical samples corresponding to individuals at risk of having a venous thrombosis and individuals of the population. The heterozygous genotype for the G 1691 A mutation in Factor V has been detected in several individuals (Fig. 3).
Estos resultados han sido validados mediante Ia secuenciación del fragmento de amplificación del Factor V de estos individuos en un secuenciador automático modelo ABI 3100 (Fig. 4).These results have been validated through the sequencing of the Factor V amplification fragment of these individuals in an ABI 3100 model automatic sequencer (Fig. 4).
Ejemplo 2: Detección del genotipo heterocigoto para Ia mutación G 2021 OA en el gen de Ia Protrombina II.Example 2: Detection of the heterozygous genotype for the G 2021 OA mutation in the Prothrombin II gene.
El genotipo heíerocigoto para Ia mutación G20210A en Ia Protrombina Il ha sido detectado en varios individuos mediante Ia metodología desarrollada (Fig.3).The heerozygous genotype for the G20210A mutation in the Prothrombin Il has been detected in several individuals using the methodology developed (Fig. 3).
Estos resultados han sido validados utilizando una técnica alternativa para Ia detección de Ia mutación, Ia digestión selectiva con Ia enzima de restricción Hind III.These results have been validated using an alternative technique for the detection of the mutation, the selective digestion with the restriction enzyme Hind III.
Ejemplo 3:Example 3:
Detección de los genotipos heterocigoto y homocigoto para el polimorfismo C677T en el gen de Ia MTHFR.Detection of heterozygous and homozygous genotypes for C677T polymorphism in the MTHFR gene.
El genotipo heterocigoto del polimorfismo C677T de MTHFR ha sido observado en un porcentaje muy elevado de muestras estudiadas, mientras que el número de individuos homocigotos es menor (Fig. 3).The heterozygous genotype of the C677T polymorphism of MTHFR has been observed in a very high percentage of samples studied, while the number of homozygous individuals is smaller (Fig. 3).
Estos resultados han sido validados utilizando una técnica alternativa para Ia detección del polimorfismo C677T MTHFR, Ia digestión selectiva con Ia enzima de restricción Hinf I (Fig. 5).These results have been validated using an alternative technique for the detection of the C677T MTHFR polymorphism, the selective digestion with the Hinf I restriction enzyme (Fig. 5).
Ejemplo 4:Example 4:
El genotipo heterocigoto del polimorfismo C46T del gen F12 se encuentra ampliamente representado en las muestras analizadas y se ha observado el genotipo homocigoto mutado en varios individuos mediante Ia metodología ELISA desarrollada (Fig. 3).The heterozygous genotype of the C46T polymorphism of the F12 gene is widely represented in the samples analyzed and the homozygous genotype mutated in several individuals has been observed using the ELISA methodology developed (Fig. 3).
Inicialmente Ia validación técnica del Factor XII se realizó mediante una digestión enzimática con SfaNI del producto de PCR donde se encuentra el polimorfismo C46T, pero se observó que sus resultados eran poco reproducibles. Con el fin de utilizar un método más fiable y rápido se procedió a utilizar un protocolo de PCR a tiempo real (RT-PCR) basado en las diferentes Ta de melting de sondas alelo específicas. Las imágenes de los resultados de este protocolo se pueden observar en Ia figura 6.Initially, the technical validation of Factor XII was carried out by means of an enzymatic digestion with SfaNI of the PCR product where the C46T polymorphism is found, but it was observed that its results were poorly reproducible. In order to use a more reliable and faster method proceeded to use a protocol real - time PCR (RT-PCR) based on the different T to the melting of probes specific allele. The images of the results of this protocol can be seen in Figure 6.
Ejemplo 5: Detección simultánea de alteración genética en más de un marcador de riesgo trombótico. En una de las muestras analizadas se ha observado Ia coexistencia de genotipo heterocigoto en el FII y mutación homocigota de MTHFR. Example 5: Simultaneous detection of genetic alteration in more than one thrombotic risk marker. In one of the analyzed samples, the coexistence of heterozygous genotype in FII and homozygous mutation of MTHFR has been observed.
REFERENCIASREFERENCES
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Tabía 1. Descripción de Ia posición nucleoíídica y secuencia de los cebadores utilizados en Ia PCR mulíiplex.Table 1. Description of the nucleoid position and sequence of the primers used in the multiplex PCR.
Figure imgf000021_0001
Figure imgf000021_0001
Tabla 2. Descripción de las sondas utilizadas en ELISA y de Ia secuencia analizada.Table 2. Description of the probes used in ELISA and the sequence analyzed.
Figure imgf000021_0002
Figure imgf000021_0002

Claims

REIVINDICACIONES
1. Método ¡n vitro para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa que comprende Ia detección de Ia ausencia/presencia de Ia combinación de las siguientes alteraciones genéticas: mutación G1691A del Factor V, Ia mutación G20210A de Ia Protrombina II, Ia mutación V677T de MTHFR y el polimorfismo C46T del Factor 12.1. In vitro method to diagnose the risk of developing a venous thrombotic disease comprising the detection of the absence / presence of the combination of the following genetic alterations: mutation G1691A of Factor V, mutation G20210A of Ia Prothrombin II, the mutation V677T of MTHFR and C46T polymorphism of Factor 12.
2. Método in vitro para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa según Ia reivindicación anterior que comprende los siguientes pasos:2. In vitro method for diagnosing the risk of developing a venous thrombotic disease according to the preceding claim comprising the following steps:
Extracción de ADN a partir de una muestra biológica - Amplificación de los fragmentos de ADN - Análisis de los fragmentos amplificados en el paso anterior mediante ELISADNA extraction from a biological sample - Amplification of DNA fragments - Analysis of fragments amplified in the previous step by ELISA
3. Método in vitro para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa según las reivindicaciones anteriores donde Ia detección de Ia ausencia/presencia de dichas alteraciones genéticas se realiza de manera simultanea.3. In vitro method for diagnosing the risk of developing a venous thrombotic disease according to the preceding claims wherein the detection of the absence / presence of said genetic alterations is performed simultaneously.
//
4. Kit para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa que comprende los reactivos necesarios para realizar un método según las reivindicaciones anteriores.4. Kit for diagnosing the risk of developing a venous thrombotic disease comprising the reagents necessary to perform a method according to the preceding claims.
5. Kit para diagnosticar el riesgo de desarrollar una enfermedad trombótica venosa según Ia reivindicación 4, que comprende las sondas con secuencia nucleotídica según SEQ ID NO: 1-8. 5. Kit to diagnose the risk of developing a venous thrombotic disease according to claim 4, comprising the probes with nucleotide sequence according to SEQ ID NO: 1-8.
PCT/ES2006/000481 2006-08-18 2006-08-18 Method and kit for diagnosing the risk of developing a venous thrombotic illness WO2008020090A1 (en)

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WO2012171949A2 (en) * 2011-06-16 2012-12-20 Gendiag.Exe, S.L. Thromboembolic disease
WO2012171949A3 (en) * 2011-06-16 2013-02-21 Gendiag.Exe, S.L. Thromboemoblic disease markers
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US10557170B2 (en) 2011-06-16 2020-02-11 Gendiag.Exe, S.L. Thromboembolic disease markers

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