WO2005078129A2 - Procede pour le diagnostic/pronostic d’une thrombose - Google Patents
Procede pour le diagnostic/pronostic d’une thrombose Download PDFInfo
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- WO2005078129A2 WO2005078129A2 PCT/FR2005/050083 FR2005050083W WO2005078129A2 WO 2005078129 A2 WO2005078129 A2 WO 2005078129A2 FR 2005050083 W FR2005050083 W FR 2005050083W WO 2005078129 A2 WO2005078129 A2 WO 2005078129A2
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Classifications
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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
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to an in vitro method for the diagnosis / prognosis of thrombosis.
- the invention also relates to amplification primers and hybridization probes which can be used in this method, as well as a kit for diagnosis / prognosis of a thrombosis.
- Coagulation is an essential homeostatic system which must be properly regulated in order to avoid hemorrhages as well as thromboses.
- This coagulation involves a cascade of activations of inactive circulating precursors which, by losing one end of their protein chain, give activated factors.
- factors II and factor V there may be mentioned in particular factor II and factor V.
- Factor IL also called prothrombin, is a pro-enzyme synthesized by the liver. It is activated in thrombin, active form, by factors X and V activated in the presence of phospholipids. When activated, it is responsible for the proteolysis of fibrinogen to fibrin.
- This mutation leads to a 25% increase in the activity of thrombin in the plasma and is considered to be a risk factor for venous thrombosis.
- the mutation of nucleotide 1691 of the gene encoding factor V, called the Leiden mutation of factor V is also a risk factor. This mutation increases the risk of venous thrombosis by 4 to 8 times in heterozygous individuals and by 50 to 100 times for homozygotes.
- factor II 20210 mutations and the factor V Leiden mutation are therefore essential in order to prevent the risks of vein thrombosis.
- numerous studies show that the 20210 mutation of factor II and the Leiden mutation of factor V are often consistent and the presence of the two mutations induces an increased risk of venous thrombosis. It is therefore also important to study these two mutations simultaneously.
- US Pat. No. 6,558,913 presents a method for detecting the presence of a genetic risk of thrombosis, based on the detection of the Leiden factor V mutation. Mention may also be made of US Pat. No. 6,043,035 which presents a method for detecting the presence of a genetic risk of thrombosis, based on the detection of the 20210 mutation of factor I
- These methods include in particular the amplification step of the region of the gene in which the mutation is located, by the use of amplification primers , followed by a mutation detection step by the use of a mutation-specific detection probe.
- these methods do not allow the simultaneous detection of the Leiden factor N mutation and the factor H 20210 mutation, which however greatly increases the risk of thrombosis in a patient.
- the early detection of this double mutation which can be done sequentially or separately, makes it possible to warn the patient and offer him appropriate treatment.
- these methods can be optimized by the choice of amplification primers used during the amplification step, in order to improve the detection step, and this all the more so when the amplification step is performed simultaneously with the detection step.
- the present invention proposes to improve the state of the art by proposing a new method for evaluating the risk of thrombosis of a patient.
- This method implements in particular during the amplification step of new amplification primers perfectly suited for the detection of a mutation increasing the risk of thrombosis.
- These new amplification primers can also be used within the same amplification reaction, which in particular makes it possible to determine by a single reaction the presence of the 20210 mutations of factor II and the Leiden mutation of factor V.
- the invention relates to an in vitro method for the diagnosis / prognosis of a thrombosis comprising the following steps: A - extract the nucleic material from a biological sample, B - use at least one pair of amplification primers to obtain amplicons from at least one target sequence of the nucleic material, C - use at least one probe detection method for detecting the presence of said amplicons, characterized in that, during step B, said primer pair comprises at least one amplification primer comprising at least 10 nucleotide units of a nucleotide sequence chosen from SEQ ID N ° l; 3 to 8, 15 and 16.
- diagnosis / prognosis of a thrombosis is understood to mean the establishment of a genetic risk profile for thrombophilia.
- biological sample means any sample capable of containing a nucleic material as defined below.
- This biological sample can be taken from a patient and can be rotatively a sample of tissue, blood, serum, saliva, circulating cells of the patient.
- This biological sample is available by any type of sample known to those skilled in the art.
- the nucleic material comprises a nucleic acid sequence such as a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence.
- the nucleic material comprises a sequence of deoxyribonucleic acids.
- the nucleic material is extracted from a biological sample taken from a patient.
- nucleotide sequence or nucleic acid sequences or nucleotide fragment or oligonucleotide, or polynucleotide
- nucleotide sequence is meant a chain of nucleotide patterns assembled together by phosphoric ester bonds, characterized by the informational sequence of natural nucleic acids, capable of s' hybridize to another nucleic acid sequence, the sequence being able to contain monomers of different structures and to be obtained from a natural nucleic acid molecule and / or by genetic recombination and / or by chemical synthesis.
- nucleotide motif means a derivative of a monomer which can be a natural nucleotide of nucleic acid, the constituent elements of which are a sugar, a phosphate group and a nitrogenous base; in DNA the sugar is deoxy-2-ribose, in TARN the sugar is ribose; depending on whether it is DNA or RNA, the nitrogen base is chosen from adenine, guanine, uracil, cytosine, thymine; or the monomer is a nucleotide modified in at least one of the three constituent elements; by way of example, the modification may take place either at the level of the bases, with modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, dia ⁇ nino-2,6-purine, bromo -5 deoxyuridine or any other modified base capable of hybridization, either at the sugar level, for example the replacement of at least one deoxyrib
- target sequence is meant a sequence the sequence of which in nucleotide units is specific for a target gene, such as preferably the gene coding for factor ⁇ or the gene coding for factor V.
- the target sequence includes a mutation which increases the risk of thrombosis such as in particular the Leiden mutation of factor V or the 20210 mutation of factor II. In the rest of the presentation, we will speak of target sequence whether it is single strand or double strand .
- the nucleic material is extracted from a biological sample by any protocol known to those skilled in the art.
- the extraction of nucleic acids can be carried out by a step of lysis of the cells present in the biological sample, in order to release the nucleic acids contained in the protein and / or lipid envelopes of the cells (such as cellular debris which disrupt subsequent reactions).
- the lysis methods as described in patent applications WO00 / 05338 on mixed magnetic and mechanical lysis, WO99 / 53304 on electrical lysis, and WO99 / 15321 on mechanical lysis can be used.
- This lysis step can also be followed by a purification step, allowing the separation between the nucleic acids and the other cellular constituents released in the lysis step.
- This step generally makes it possible to concentrate the nucleic acids, and can be adapted to the purification of DNA or RNA.
- magnetic particles can be used optionally coated with oligonucleotides, by adsorption or covalence (see on this subject US Patents 4,672,040 and US 5,750,338), and thus purify the nucleic acids which are fixed on these magnetic particles, by a washing step.
- This nucleic acid purification step is particularly advantageous if it is desired to subsequently amplify said nucleic acids.
- a particularly interesting embodiment of these magnetic particles is described in patent applications WO97 / 45202 and WO99 / 35500.
- Another interesting example of a nucleic acid purification method is the use of silica either in the form of a column or in the form of inert particles (Boom R. et al., J. Clin.
- DNA When it is desired to specifically extract DNA from a biological sample, it is possible in particular to carry out an extraction with phenol, chloroform and alcohol to remove proteins and precipitate DNA with alcohol. The DNA can then be pelleted by centrifugation, washed and resuspended.
- step B at least one pair of amplification primers is used to obtain amplicons from at least one target sequence of the nucleic material.
- amplification primer means a nucleic acid sequence comprising from 10 to 100 nucleotide units, preferably from 15 to 25 nucleotide units. This amplification primer comprises at least 10, preferably 15 and even more preferably 20 nucleotide units of a sequence chosen from SEQ ID No. 1; 3 to 8
- an amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of a sequence homologous to SEQ ID No. 1; 3 to SEQ ID No. 8; 15 and 16, ie o the complementary sequence of SEQ ID No. 1; 3 to 8; 15 and 16 o a sequence having sufficient homology to hybridize to SEQ ID No. 1; 3 to SEQ ID No. 8; 15 and 16 or to the sequence complementary to SEQ ID No. 1; 3 to SEQ ID No. 8; 15 and 16, • a sequence comprising a sequence of SEQ ID No. 1; 3 to SEQ ID No. 8; 15 and 16 (or a sequence homologous to SEQ ID No. 1; 3 to SEQ ID No.
- a pair of amplification primers allows the initiation of an enzymatic polymerization, such as in particular an enzymatic amplification reaction.
- enzymatic amplification reaction is meant a process generating multiple copies (or amplicons) of a nucleic sequence by the action of at least one enzyme.
- amplicons is understood to mean the copies of the target sequence obtained during an enzymatic amplification reaction.
- amplification reactions are well known to those skilled in the art and mention may in particular be made of PCR (Polymerase Chain Reaction), as described in patents US-A-4,683,195,
- these enzymatic amphfication reactions generally involve a succession of cycles comprising the following steps: o denaturation of the target sequence if it is in double strand in order to obtain two complementary, target strands, o hybridization of each of the target strands, obtained during the previous denaturation step with at least one amplification primer, the formation from the amplification primers of the strands complementary to the strands on which they are hybridized in the presence of an enzyme polymerase and of nucleoside triphosphate (ribonucleoside triphosphate and / or deoxyribonucleoside triphosphate according to the techniques) this cycle being repeated a number of times determined to obtain the target sequence in a sufficient proportion to allow its detection.
- nucleoside triphosphate ribonucleoside triphosphate and / or deoxyribonucleoside triphosphate according to the techniques
- hybridization is meant the process during which, under appropriate conditions, two nucleic acid sequences such as in particular an amplification primer and a target sequence or a hybridization probe and a target sequence, bind with stable hydrogen bonds and specific to form a double strand.
- These hydrogen bonds are formed between the complementary bases Adenine (A) and thymine (T) (or uracil (U)) (we speak of bond A-T) or between the complementary bases Guanine (G) and cytosine (C) (we speaks of GC liaison).
- the hybridization of two nucleic sequences can be total (we then speak of complementary sequences), that is to say that the double strand obtained during this hybridization comprises only AT bonds and CG bonds.
- This hybridization can be partial (we then speak of sufficiently complementary sequences), that is to say that the double strand obtained comprises AT bonds and CG bonds making it possible to form the double strand, but also bases not linked to a complementary base. .
- the hybridization between two complementary or sufficiently complementary sequences depends on the operating conditions which are used, and in particular on the stringency. Stringency is defined in particular as a function of the base composition of the two nucleic sequences, as well as by the degree of mismatch between these two nucleic sequences. The stringency can also be a function of the parameters of the reaction, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and / or the hybridization temperature.
- NASBA is an isothermal amphfication technology for nucleic acid based on the joint action of three enzymes (reverse transcriptase AMV, Rnase-H and polymerase-RNA T7). Associated with amplification primers specific for a target sequence, it amplifies RNA targets more than a billion times in 90 minutes. The amplification reaction occurs at 41 ° C and gives single stranded RNA molecules as the final product. NASBA requires a primer pair, at least one of which includes a promoter for initiating transcription by a bacteriophage T7 polymerase.
- step C at least one detection probe is used to detect the presence of said amplicons.
- this detection probe makes it possible to detect not only the presence of said amplicons, but also makes it possible to detect the presence of a given mutation, likely to be included in the amplicon.
- This detection step can be carried out by all the protocols known to those skilled in the art relating to the detection of nucleic acids.
- hybridization probe is intended to mean a nucleic sequence having a specificity of hybridization under conditions determined to form a hybridization complex with a target nucleic sequence.
- the hybridization probe can comprise a marker allowing its detection.
- detection is meant either a direct detection by a physical method, or an indirect detection by a detection method using a marker.
- marker is meant a tracer capable of generating a signal which can be detected.
- tracers includes the enzymes which produce a detectable signal for example by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta galactosidase, glucose-6-phosphate dehydrogenase; chromophores such as fluorescent, luminescent or coloring compounds; electron density groups detectable by electron microscopy or by their electrical properties such as conductivity, by amperometry or voltammetry methods, or by impedance measurements; the groups detectable by optical methods such as diffraction, surface plasmon resonance, variation of contact angle or by physical methods such as atomic force spectroscopy, tunnel effect, etc. ; radioactive molecules like 32 P, 35 S or 1 5 I.
- the detection probe can in particular be a “molecular beacon” detection probe as described by Tyagi & Kramer (Nature biotech, 1996, 14: 303-308).
- molecular beacons become fluorescent during hybridization. They have a rod-loop structure and contain a fluorophore and an inhibitor or “quencher” group. The binding of the specific loop sequence with its complementary target nucleic acid sequence causes the rod to unwind and the emission of a fluorescent signal upon excitation at the appropriate wavelength.
- the detection probe comprises a fluorophore and a quencher.
- the hybridization probe comprises a FAM (6-carboxy-fluorescein) or ROX (6-carboxy-X-rhodamine) fluorophore at its 5 ′ end and a quencher (Dabsyl ) at its 3 'end.
- a hybridization probe is called “molecular beacon”.
- the presence or absence of the Leiden factor V mutation is detected.
- it is detected during in step C) the presence or absence or of the 20210 mutation of factor H.
- said detection probe comprises at least 10, preferably 15 and even more preferably 20 nucleotide patterns of a nucleotide sequence chosen from SEQ ID No. 9 to 12; 17 and 18.
- SEQ ID No. 9 makes it possible to diagnose the presence of the Leiden mutation of factor V
- SEQ ID N ° 10 makes it possible to diagnose the absence of the Leiden mutation for factor V.
- a detection probe comprising SEQ ID No. 11 makes it possible to detect the presence of the mutation 20210 of factor II
- the use of a detection probe comprising SEQ ID No. 12 makes it possible to detect the absence of the 20210 mutation of factor IL
- the use of a detection probe comprising SEQ ID No. 17 makes it possible to detect the presence of the mutation 20210 of factor ⁇
- the use of a detection probe comprising SEQ ID N ° 18 makes it possible to detect the absence of the 20210 mutation of factor II
- said pair of primers is chosen from the following primer pairs: ⁇ a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 1 and a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 2;
- a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 1
- a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 2
- an amplicon specific for the gene encoding factor V, of a size of 1 is obtained.
- This amplicon may contain the mutated allele responsible for the Leiden mutation or the wild-type allele, the allele being characterized during step C.
- a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 3 and a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 4;
- the first primer has the sequence SEQ ID No. 3
- the second primer has the sequence SEQ ID No.
- an amplicon is obtained, specific for the gene encoding factor V, of a size of 374 base pairs, which corresponds to the sequence 36568-36941 on the sequence of the gene encoding the reference factor V (NT_004668).
- This amplicon may contain the mutated allele responsible for the Leiden mutation or the wild allele, the allele being characterized during step C.
- a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide motifs of the nucleotide sequence SEQ ID N ° 5
- a second primer amplification comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No.
- a first amphfication primer comprising at least 10, preferably 15 and again more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No.
- a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 8;
- an amplicon specific for the gene encoding factor II, of a size of 1 is obtained. 434 base pairs, which corresponds to the sequence 21217-21650 on the sequence of the gene encoding the reference factor II (AF478696).
- This amplicon may contain the mutated allele responsible for the 20210 mutation of factor II or the wild-type allele, the allele being characterized during step C.
- a first amplification primer comprising at least 10, preferably 15 and again more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 15 and a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 16;
- an amplicon specific for the gene encoding factor II, of a size of 1 is obtained.
- 108 base pairs which corresponds to the sequence 21465-21573 on the sequence of the gene encoding the reference factor ⁇ (AF478696).
- This amplicon may contain the allele mutate responsible for the 20210 mutation in factor II or the wild-type allele, the allele being characterized in step C.
- said pair of primer comprises at least one amphfication primer comprising a promoter allowing initiation of transcription by a bacteriophage T7 polymerase.
- this first amplification primer comprises a sequence chosen from the sequences of SEQ ID N ° 13 to 14.
- step B and step C are carried out simultaneously.
- This can be implemented in particular by a NASBA amplification reaction with detection in real time.
- Real-time detection of amplicons can be carried out using a Nuclisens EasyQ® reader (bioMérieux BV, The Netherlands) and “molecular beacon” detection probes, as defined above.
- the amplification of DNA in NASBA can be carried out in the following way: the DNA is denatured at 95 ° C. to allow the fixing of a "first primer", comprising a polymerase promoter T7 (only one primer is present at this stage). At 41 ° C, the enzyme reverse transcriptase (AMV-RT) is added and allows the extension of the primer.
- AMV-RT enzyme reverse transcriptase
- a "second primer” is added and a new denaturation is carried out.
- An alternative is made by adding the two primers simultaneously, in order to carry out only one denaturation step.
- NASBA enters a cychach phase: the "second primer” hybridizes to a new RNA molecule formed; AMV-RT extends this primer; RNase H degrades RNA from RNA / cDNA hybrids; a "first primer” binds to the cDNA obtained; AMV-RT synthesizes from this a new complementary strand then from this strand, extends the T7 part of the "first primer”; thus T7 RNA polymerase has new matrices with a T7 double tail to generate RNAs.
- the quencher When the “molecular beacon” is in solution, the quencher, near the fluorophore, inhibits its fluorescence. In the presence of the target nucleic acid, the beacon will bind to the sequence complementary to that of the loop. H will therefore change conformation and open, moving the fluorophore away from the quencher and thus allowing the emission of fluorescence. During the amplification, many molecules will be synthesized allowing as many “molecular beacons” to be fixed. The fluorescence will therefore increase during the reaction.
- a suitable device such as EasyQ
- the invention also relates to an amplification primer comprising at least 10, preferably 15, and even more preferably 20 nucleotide units of a nucleotide sequence chosen from SEQ ID No. 1; 3 to 8; 15 and 16.
- the amplification primer comprises a promoter allowing the initiation of transcription by a polymerase of bacteriophage T7.
- amphfication primer comprises a sequence chosen from the sequences of SEQ ID Nos. 13 to 14.
- the invention also relates to a pair of amphfication primer chosen from the following primer pairs: p a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide motifs of the nucleotide sequence SEQ ID N 1 and a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 2; as a guide, when the first primer has the sequence SEQ ID No. 1, and the second primer has the sequence SEQ ID No. 2, an amplicon, specific for the gene encoding factor V, of a size of 1 is obtained.
- a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 3 and a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 units nucleotides of the nucleotide sequence SEQ ID No.
- a amplicon specific for the gene encoding factor V, with a size of 374 base pairs, which corresponds to the sequence 36568-36941 on the sequence of the gene encoding the reference factor V (NT_004668).
- a first amphfication primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 5
- a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 units nucleotides of the nucleotide sequence SEQ ID No.
- the first primer has the sequence SEQ ID N ° 5- and the second primer has the sequence SEQ ID N ° 6, an amplicon is obtained, specific for the gene coding for factor II, of a size of 145 base pairs, which corresponds to the sequence 21455-21599 on the sequence of the gene encoding the reference factor II (AF478696).
- a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 7
- a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 units nucleotides of the nucleotide sequence SEQ ID No.
- a first amplification primer comprising at least 10, preferably 15 and even more preferably 20 nucleotide units of the nucleotide sequence SEQ ID No. 15 and a second amplification primer comprising at least 10, preferably 15 and even more preferably 20 units nucleotides of the nucleotide sequence SEQ ID No.
- said first primer comprises a promoter allowing the initiation of transcription by a polymerase of bacteriophage T7.
- this first amplification primer comprises a sequence chosen from the sequences of SEQ ID N ° 13 to 14.
- the invention also relates to the use of at least one amphfication primer as defined above and / or of a primer pair as defined above during a NASBA amphfication reaction
- the invention also relates to the use of at least one primer as defined above, and / or of at least one pair of primers as defined above for the diagnosis / prognosis of a thrombosis.
- the invention finally relates to a kit for the diagnosis / prognosis of a venous thrombosis comprising at least one primer as defined above and / or at least one pair of primers as defined above.
- Figures 1 and 2 represent the genotyping of different cell lines for the +1691 G / A mutation in Factor V, thanks to the simultaneous presence of the molecular beacons of SEQ ID No.10 and SEQ ID No.9 in the reaction mixture.
- FIG. 1a represents the genotyping of the GM16000C cell line, homozygous for the wild allele (+ 1691-G) of Factor V
- FIG. 1b represents the genotyping of the GM14899 cell line, homozygous for the mutated allele (+ 1691-A) of Factor V
- the white squares represent the detection of the fluorescence of the “molecular beacons” of SEQ ID N ° 9, making it possible to highlight the wild allele of factor V
- the black triangles represent the detection of the fluorecence of the “molecular beacons” of SEQ ID No. 10, allowing the detection of the mutated allele (Leiden mutation).
- FIG. 2 represents the genotyping of the cellular gene GM16028B, heterozygous for the mutation +1691 of Factor V.
- the white squares represent the detection of the fluorescence of the "molecular beacons" of SEQ ID N ° 9, making it possible to highlight the wild factor V allele, while the black triangles represent the detection of the fluorecence of the “molecular beacons” of SEQ ID No. 10, allowing the detection of the mutated allele (Leiden mutation).
- Figures 3 and 4 represent the genotyping of different cell lines for the Factor II mutation +20210 G / A, thanks to the simultaneous presence of the molecular beacons OGH 916 (SEQ ID No. 11, + 20210- A) and OGH 1104 ( SEQ ID No. 12, + 2010-G) in the reaction mixture.
- FIG. 3a represents the genotyping of the GM 14899 cell line, homozygous for the wild-type allele (+ 20210-G) of Factor ⁇ while FIG. 3b represents the genotyping of the GM16000C cellular hgnea, homozygous for Factor II mutated allele (+ 20210- A).
- the crosses represent the detection of the “molecular beacons” of SEQ ID No. 12, allowing the detection of the wild allele, while the circles represent the detection of the “molecular beacons” of SEQ ID No. 11, allowing the detection of the 20210 mutation in factor ⁇ .
- FIG. 4 represents the genotyping of the cell line GM16028B, heterozygous for the +20210 mutation of factor ⁇ .
- the presence of the mutated allele (black circles) and the wild allele (cross curve) is clearly detected.
- the crosses represent the detection of the “molecular beacons” of SEQ ID No. 12, allowing the detection of the wild allele, while the circles represent the detection of the “molecular beacons” of SEQ ID No. 1 1, allowing the detection of the ⁇ factor 20210 mutation.
- FIG. 5 represents the detection of the FAM beacon specific for the allele A of Factor V (G + 1691A) from DNA of the GM14899 lines (A / A homozygous allele mutated; triangles), GM16028B (A / G heterozygous; cross) and GM16000C (G / G homozygous wild allele; round)
- FIG. 6 represents the detection of the ROX beacon specific for the G allele of factor V (G + 1691A) from DNA of the GM14899 lines: A / A (homozygous mutated allele; triangles); GM16028B (A / G heterozygous; cross) and GM 16000C (G / G homozygous wild allele, round)
- FIGS 7 and 8 represent the genotyping of different cellular cells (GM
- FIG. 7 represents the detection of the FAM beacon specific for the G allele of factor II (G + 20210A) from DNA of the GM14899 lines: G / G (homozygous mutated allele; triangles); GM16028B (A / G heterozygous; cross) and GM 16000C (A / A homozygous wild allele, round)
- G / G homozygous mutated allele; triangles
- GM16028B A / G heterozygous; cross
- GM 16000C A / A homozygous wild allele, round
- FIG. 8 represents the detection of the ROX beacons specific for the allele A of factor II (G + 20210A) from DNA of GM14899 lines: G / G (homozygous allele mutated; triangles); GM16028B (A / G heterozygous; cross) and GM 16000C (A / A homozygous wild allele, round)
- Figure 9 is a graphic representation of 72 samples (clinical and cell lines) Factor V. These samples are positioned according to their ratios of fluorescence intensity of FAM (specific for allele A) and ROX (specific for allele G) beacons.
- FIG. 10 is a graphic representation of 130 samples (clinical and cell line) IL factor These samples are positioned according to their fluorescence intensity ratios of the FAM (specific for allele G) and ROX (specific for the beacons) beacons. allele A).
- the method according to the invention has been validated on cellular cells, as well as on chemical samples from patients.
- Cell lines Three lymphoblastoid cell lines of known genotype for the Leiden factor V mutations and the factor II 20210 (Coriell cell repository) mutation were used:
- the GM14899 line expresses only the Leiden mutation for factor V: this gene is homozygous for the Leiden mutation of factor V (A / A) and homozygous for the wild-type allele in position 20120 of factor H (G / G).
- the GM16000C line only expresses the 20210 mutation of factor II: this line is homozygous for the 20210 mutation of factor ⁇ (A / A) and homozygous for the wild-type allele in position 1691 of factor V
- the GM16028B line is heterozygous for the 2 mutations (G / A).
- These cell lines were cultured (37 ° C, CO2 5%) in RPMI 1640 medium, supplemented with fetal beef serum (15%), L glutamine (2mM), Penistreptomycin (penicillin: 200 U / ml ; streptomycin: 0.2 mg / ml).
- DNA extraction was carried out from a GM16000C cell pellet, a GM16028B or GM14899 cell pellet. The pellet cells are subjected to hypotonic lysis and to enzymatic digestion with proteinase K. After deproteinization, the DNA is precipitated with ethanol, dried, and dissolved in water.
- the quantity and quality of DNA were determined by UV spectrophotometry.
- Patient sample - 200 ⁇ l of blood taken from patients whose presence of the Leiden mutation was known was used.
- the genomic DNA was extracted using the NucleoSpin kit (Marcherey-Nagel, Hoerdt, France), and taken up in 20 ⁇ l of DNAse-free water.
- the quantity and quality of the DNA were determined on an agarose gel containing ethidium bromide, and by UV spectrophotometry.
- PCR was carried out using genomic DNA as obtained previously, from cell lines or blood samples, by the use of a pair of amplification primers comprising SEQ ID No. 3, 5 'AGTGCTTAACAAGACCATACTA 3' for the first primer and SEQ ID No. 4, 5 'AACAGACCTGGAATTTGAAACTAA 3' for the second primer.
- the PCR parameters were as follows: 2 min at 95 ° C; 30 cycles of 30 s at 95 ° C, 30 s at 55 ° C, 30 s at 72 ° C; followed by 7 min at 72 ° C).
- Plasmids containing either a nucleotide G or a nucleotide A in position 1691 were amplified and purified by the use of a Plasmid Maxi kit
- a PCR was carried out using genomic DNA as obtained previously, from cellular genes or from blood samples, by the use of a pair of amphfication primers comprising SEQ ID No. 7, 5 '
- PCR were as follows: 2 min at 95 ° C; 30 cycles of 30 s at 95 ° C, 30 s at 55 ° C, 30 s at 72 ° C; followed by 7 min at 72 ° C).
- the amplicons thus obtained were cloned into a PCR-Trap vector (GeneHunter, USA) and verified by sequencing.
- the plasmids, containing either a nucleotide G or a nucleotide A at position 1691, were amplified and purified by the use of a Plasmid Maxi kit (Qiagen; Germany). 2) Amplification by NASBA
- an amplification reaction mixture (40m_M Tris HC1 ph 8.5; 12 mM MgC12; 70 mM KC1; 5 mM dithiotheitol; 15% v / v DMSO; 1 mM dNTP) containing the amphfication primers making it possible to amplify either the region capable of containing the Leiden mutation, or the region capable of containing the 20210 mutation of factor II, and detection probes, specific for each mutation has been carried out.
- the reaction medium for detecting the presence of the factor V Leiden mutation included: - 0.2 ⁇ M (final concentration) of a first primer for amplification of SEQ ID No. 2, 5 'AGT GCT TAA CAA GAC CAT ACT A 3 ', - 0.2 ⁇ M (final concentration) of a second amplification primer of SEQ ID No 1.5, AAA TTC TCA GAA TTT CTG AAA GG 3' comprising the promoter of the phage polymerase T7, i.e. an amplification primer whose total sequence corresponds to SEQ ID No.
- the “molecular beacon” of SEQ ID No. 9, marked with a ROX fluorophore (6-carboxy-X-rhodamine) is very specific for the wild-type allele: in fact, the fluorescence was exponential, before the appearance of a plateau, when the reaction was carried out using a cellular signal not expressing the Leiden mutation (curve in white square; FIG. la), while the fluorescence remained basal when the reaction was carried out using a cell line expressing the Leiden mutation (white square curve; FIG. 1 b).
- the “molecular beacons” of SEQ ID No. 10, labeled with a FAM fluorophore (6-carboxy-fluorescein) is specific for the mutated allele.
- the fluorescence was exponential, when the reaction was carried out from a cell line expressing the Leiden mutation (triangle curve; FIG. 1b), while the fluorescence remained basal, when the reaction was carried out from a cell sequence not expressing the Leiden mutation (triangle curve; figure la).
- the “molecular beacon” of SEQ ID No. 9 could be used simultaneously with a “molecular beacon” of SEQ ID No. 10, which made it possible to detect very quickly, and from a single reaction, the presence or absence of the Leiden mutation for factor V.
- the results are presented in FIG. 2, obtained from a heterozygous cell line for the Leiden mutation for factor V.
- Table 1 O / C ratio obtained on cell lines and blood samples expressing or not expressing the Leiden mutation
- the “molecular beacon” of SEQ ID No. 12, marked with a FAM fluorophore (6-carboxy-fluorescein) is very specific for the wild factor H allele. Indeed, the fluorescence was exponential , when the reaction was carried out from a cell line not expressing the 20210 mutation (cross curve; FIG. 3a), while the fluorescence remained basal, when the reaction was carried out from a cell gene expressing this mutation (cross curve; Figure 3b).
- the “molecular beacons” of SEQ ID No. 11, marked with a ROX fluorophore (6-carboxy-X-rhodamine) is specific for the mutated allele.
- the fluorescence was exponential, when the reaction was carried out from a cell line expressing the 20210 mutation (circle curve; FIG. 3b), while the fluorescence remained basal, when the reaction was carried out from a cell line n '' not expressing this mutation (round curve; Figure 3a).
- the “molecular beacon” of SEQ ID N ° 11 could be used simultaneously with a “molecular beacon” of SEQ ID N ° 12, which made it possible to detect very quickly, and from a single reaction, the presence or absence of the 20210 mutation in factor ⁇ .
- This is what is represented in FIG. 4, obtained from a heterorozygous cell line for the mutation 20210 of the actor H. Comparable results were obtained from a clinical sample.
- 203 clinical DNAs were genotypes.
- the GM14899 gene expresses only the Leiden factor V mutation: this line is homozygous for the Leiden mutation of factor V (A / A) and homozygous for the wild alder at position 20120 of factor ⁇ (G / G).
- the GM16000C line only expresses the 20210 mutation for factor II: this line is homozygous for the 20210 mutation in factor II (A / A) and homozygous for the wild animal in position 1691 for factor V (G / G).
- the GM16028B is heterozygous for the 2 mutations (G / A).
- Genomic DNA was extracted using the NucliSens Magnetic Extraction reagent kit and NucliSens Lysis Buffer and the miniMag instrument. (cf Notice miniMag except washing Wash3 (10 sec in 15 hr) and the DNA is eluted in 80 ⁇ L of Elution buffer (10min at 70 ° C + mix at 14000 rpm).
- the quantity and quality of the DNA were determined by UV spectrophotometry.
- Cloning of the region of interest In order to obtain a large amount of DNA from the region capable of expressing the Leiden factor V mutation, a PCR was carried out using genomic DNA as obtained above, from cell lines or blood samples, by the use of a pair of amplification primers comprising SEQ ID No. 3, 5 'AGTGCTTAACAAGACCATACTA 3' for the first primer and SEQ ID No. 4, 5 'AACAGACCTGGAATTTGAAACTAA 3' for the second primer.
- the PCR parameters were as follows: 2 min at 95 ° C; 30 cycles of 30 s at 95 ° C, 30 s at 55 ° C, 30 s at 72 ° C; followed by 7 min at 72 ° C).
- the amplicons thus obtained were cloned into a PUC19 vector and verified by sequencing.
- the plasmids, containing either a nucleotide G or a nucleotide A at position 1691, were amplified and purified by the use of a Plasmid Maxi kit (Qiagen; Germany).
- a PCR was carried out using genomic DNA as obtained previously, from cell lines or from blood samples, by the use of a pair of amphfication primers comprising SEQ ID No 7, 5 'TCTAGAAACAGTTGCCTGGC 3' for the first primer and SEQ ID No 8, 5 'CTACCAGCGTGCCACCAGGT 3' for the second primer.
- the PCR parameters were as follows: 2 min at 95 ° C; 30 cycles of 30 s at 95 ° C, 30 s at 55 ° C, 30 s at 72 ° C; followed by 7 min at 72 ° C).
- the amplicons thus obtained were cloned into a PCR-Trap vector (GeneHunter, USA) and verified by sequencing.
- the plasmids, containing either a nucleotide G or a nucleotide A at position 1691, were amplified and purified by the use of a Plasmid Maxi kit (Qiagen; Germany). 2) Amplification by NASBA
- “molecular beacons” of SEQ ID N ° 10, 3 'CTG GAC AGG CAA IGA A 3', marked with a fluorophore FAM (6-carboxy-fluorescein) in 5 ', and of a "Quencher” (Dabsyl) in 3 ', (complete sequence: 5' FAM-cgatcg CTGGACAGGCAAIGAAcg ⁇ tC £ -Dabsyl 3 ').
- This “molecular beacon” made it possible to demonstrate the presence of the Leiden mutation.
- a Factor II amplification reaction mixture (40mM tris HC1 ph 8.5; 12mM MgC12; 70mM KC1; 5mM dithiotheitol; 15% v / v DMSO; 1 mM dNTP) containing the amphfication primers making it possible to amplify the region capable of containing the +20210 mutation of the FH:
- Factor II were added to the reaction medium: - 0.2 ⁇ M (final concentration) of a first amplification primer of SEQ ID No.
- This “molecular beacon” made it possible to highlight the absence of the 20210 mutation of factor II
- the “molecular beacon” of SEQ No. 9 marked with a FAM fluorophore is very specific for the mutated allele: in fact, the fluorescence was exponential, before the appearance of a plateau, when the reaction was carried out using cell lines expressing the Leiden mutation (curves in triangle; and cross in FIG. 5), while the fluorescence remained basal, when the reaction was carried out using cell cells expressing the Leiden mutation (curve white circle; figure 5).
- the “molecular beacon” of SEQ ID No. 10, labeled with a ROX fluorophore is specific for the wild allele. Indeed, the fluorescence was exponentieEe, when the reaction was carried out starting from cellular hgussis expressing the wild genotype
- the “molecular beacon” of SEQ ID No. 10 is used simultaneously with a “molecular beacon” of SEQ ID No. 9, which made it possible to detect very quickly, and from a only reaction, the presence and / or absence of the mutation for the two factors of the FactorV gene. Thanks to the analysis of Figures 5 and 6 we can therefore determine if the sample has the genotypes G / G, G / A, or A / A
- results obtained can also be expressed by the ratio between the fluorescence emitted when the plateau is reached and the initial fluorescence (O / C ratios
- Each “square” symbol represents a sample tested and positioned according to the value of F O / C ratio of the FAM beacons and the OC ratio of the ROX beacon for this sample. Thanks to the positivity detection hms of these two beacons, Factor V Leiden typing can be determined graphically for 72 samples. A sample is classified A / A when its O / C ratio beacon FAM specific for the mutated allele (A) is greater than 1.5 and when its O / C ratio beacon ROX specific for the wild allele (G) is less than 2 .
- a sample is classified G / G when its O / C ratio beacon FAM specific for the mutated allele (A) is less than 1.5 and when its O / C ratio beacon ROX specific for wild aele (G) is greater than 2 .
- a sample is classified G / A when its O / C ratio beacon FAM specific for the mutated allele (A) is greater than 1.5 and when its O / C ratio beacon ROX specific for the wild allele (G) is greater than 2 .
- the “molecular beacon” of SEQ No. 18 marked with a FAM fluorophore is very specific for wild honey: in fact, the fluorescence was exponent, before the appearance of a plateau, when the reaction was carried out using cell lines expressing the wild Factor II (curves in triangle; and crosses in FIG. 7), while the fluorescence remained basal, when the reaction was carried out using a cell line expressing the wild factor polymorphism II (round curve; Figure 7).
- the “molecular beacons” of SEQ ID No. 17, marked with a ROX fluorophore is specific for the mutated allele.
- the “molecular beacon” of SEQ ID N ° 17 is used simultaneously with a “molecular beacon” of SEQ ID N ° 18, which made it possible to detect very quickly, and from a only reaction, the presence or absence of the mutation for the Factor IL gene Thanks to the analysis of FIGS. 7 and 8, it can therefore be determined whether the sample has the G / G, G / A or A / A genotype.
- the results of 130 samples including cell lines (squares) and samples extracted from fresh blood (triangle and round) are shown in the figure
- Each symbol represents a sample tested and positioned according to the value of the O / C ratio of the FAM beacons and FOC ratio of the ROX beacon for this sample. Thanks to the positivity detection limits of these two beacons, Factor II typing can be determined graphically for 130 samples.
- a sample is classified A / A when its O / C ratio beacon ROX specific for the mutated allele (A) is greater than 3.5 and when its O / C ratio beacon FAM specific for the wild allele (G) is less than 1.8 .
- a sample is classified G / G when its O / C ratio beacon ROX specific for the mutated allele (A) is less than 3.5 and when its O / C ratio beacon FAM specific for wild alu (G) is greater than 1.8 .
- a sample is classified G / A when its O / C ratio beacon ROX specific for the mutated allele (A) is greater than 3.5 and when its O / C ratio beacon FAM specific for the wild allele (G) is greater than 1.8 .
- the fresh blood samples were genotyped.
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Abstract
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Application Number | Priority Date | Filing Date | Title |
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US10/585,923 US20070160996A1 (en) | 2004-02-12 | 2005-02-10 | Thrombosis diagnosis/prognosis method |
JP2006552671A JP2007521828A (ja) | 2004-02-12 | 2005-02-10 | 血栓症の診断/予測方法 |
EP05726418A EP1713939A2 (fr) | 2004-02-12 | 2005-02-10 | Procede pour le diagnostic/pronostic d'une thrombose |
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FR0450257 | 2004-02-12 | ||
FR0450257A FR2866349B1 (fr) | 2004-02-12 | 2004-02-12 | Procede de diagnostic/pronostic d'une thrombose |
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WO2005078129A2 true WO2005078129A2 (fr) | 2005-08-25 |
WO2005078129A3 WO2005078129A3 (fr) | 2005-11-10 |
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PCT/FR2005/050083 WO2005078129A2 (fr) | 2004-02-12 | 2005-02-10 | Procede pour le diagnostic/pronostic d’une thrombose |
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US (1) | US20070160996A1 (fr) |
EP (1) | EP1713939A2 (fr) |
JP (1) | JP2007521828A (fr) |
FR (1) | FR2866349B1 (fr) |
WO (1) | WO2005078129A2 (fr) |
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RU2499599C2 (ru) | 2007-09-28 | 2013-11-27 | Интрексон Корпорейшн | Конструкции терапевтического переключения генов и биореакторы для экспрессии биотерапевтических молекул и их применение |
Family Cites Families (17)
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US4672040A (en) * | 1983-05-12 | 1987-06-09 | Advanced Magnetics, Inc. | Magnetic particles for use in separations |
US4683202A (en) * | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4683195A (en) * | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4800159A (en) * | 1986-02-07 | 1989-01-24 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences |
US5750338A (en) * | 1986-10-23 | 1998-05-12 | Amoco Corporation | Target and background capture methods with amplification for affinity assays |
US5234809A (en) * | 1989-03-23 | 1993-08-10 | Akzo N.V. | Process for isolating nucleic acid |
CA2020958C (fr) * | 1989-07-11 | 2005-01-11 | Daniel L. Kacian | Methodes d'amplification de sequences d'acide nucleique |
US6335184B1 (en) * | 1993-07-23 | 2002-01-01 | Bio-Rad Laboratories, Inc. | Linked linear amplification of nucleic acids |
ES2166948T3 (es) * | 1994-02-14 | 2002-05-01 | Univ Leiden | Un metodo de seleccion de una anomalia genetica asociada con la trombosis y/o una mala respuesta anticoagulante a la proteina c activada. |
GB9604267D0 (en) * | 1996-02-29 | 1996-05-01 | Royal Infirmary Of Edinburgh N | Mutation assay |
US6043035A (en) * | 1997-11-03 | 2000-03-28 | Rijks University Leiden | Method for determining a risk factor for thrombosis |
US6140054A (en) * | 1998-09-30 | 2000-10-31 | University Of Utah Research Foundation | Multiplex genotyping using fluorescent hybridization probes |
US6936467B2 (en) * | 2000-03-27 | 2005-08-30 | University Of Delaware | Targeted chromosomal genomic alterations with modified single stranded oligonucleotides |
ES2267803T3 (es) * | 2000-08-11 | 2007-03-16 | University Of Utah Research Foundation | Sondas de oligonucleotidos marcados individuales. |
GB0022069D0 (en) * | 2000-09-08 | 2000-10-25 | Pyrosequencing Ab | Method |
CA2426346A1 (fr) * | 2000-10-27 | 2002-06-13 | Abbott Laboratories | Genotypage a l'aide de sondes partiellement complementaires |
DE10237073A1 (de) * | 2002-08-09 | 2004-02-19 | Ogham Gmbh | Methode zur Bestimmung eines erblichen Thromboserisikos mit DNA-arrays |
-
2004
- 2004-02-12 FR FR0450257A patent/FR2866349B1/fr not_active Expired - Fee Related
-
2005
- 2005-02-10 US US10/585,923 patent/US20070160996A1/en not_active Abandoned
- 2005-02-10 JP JP2006552671A patent/JP2007521828A/ja active Pending
- 2005-02-10 WO PCT/FR2005/050083 patent/WO2005078129A2/fr not_active Application Discontinuation
- 2005-02-10 EP EP05726418A patent/EP1713939A2/fr not_active Withdrawn
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US20070160996A1 (en) | 2007-07-12 |
EP1713939A2 (fr) | 2006-10-25 |
WO2005078129A3 (fr) | 2005-11-10 |
FR2866349B1 (fr) | 2008-05-16 |
FR2866349A1 (fr) | 2005-08-19 |
JP2007521828A (ja) | 2007-08-09 |
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