WO2002088736A2 - Methode de diagnostic prenatal sur cellule foetale isolee du sang maternel - Google Patents
Methode de diagnostic prenatal sur cellule foetale isolee du sang maternel Download PDFInfo
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- WO2002088736A2 WO2002088736A2 PCT/FR2002/001505 FR0201505W WO02088736A2 WO 2002088736 A2 WO2002088736 A2 WO 2002088736A2 FR 0201505 W FR0201505 W FR 0201505W WO 02088736 A2 WO02088736 A2 WO 02088736A2
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- 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
- 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
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
Definitions
- the present invention relates to a new non-invasive prenatal diagnosis method implemented from a maternal blood sample.
- Said method allows prenatal diagnosis on fetal, epithelial, non-apoptotic cells isolated from maternal blood after their enrichment by filtration, their morphological or immunological and genetic analysis.
- the advantages of this method are in particular its harmlessness for the mother or the fetus and the very high sensitivity and specificity of the diagnosis. It allows the early detection of a genetic or chromosomal abnormality of the fetus, of a genetic or infectious pathology (viral, bacterial or parasitic) of the fetus, of a precise genotype, and in particular of the genetic sex of the fetus.
- Prenatal diagnostic methods aim in particular to obtain genetic information about a fetus or an embryo.
- the search for genetic information on a fetus consists either in identifying the presence of a specific allele of a given gene or a combination of alleles in a given sequence of fetal DNA, or in genetically associating a polymorphism of fetal DNA to a particular allele.
- One of the major applications of prenatal genetic diagnosis concerns the detection of congenital anomalies.
- the prenatal genetic diagnostic methods used in the clinic essentially include invasive techniques such as amniocentesis, chorionic villus sampling, fetal blood sampling or tissue biopsies. All of these techniques imply that samples are obtained directly from the fetus or indirectly from ovular structures. Given the highly invasive nature of these methods, they are susceptible to complications for the mother or the fetus. Among these complications, in the case of amniocentesis, mention may be made of a risk of infection, of feto-maternal hemorrhage with possible alloimmunization, loss of amniotic fluid or abdominal pain.
- amniocentesis is 0.2 to 2.1% higher than that of a control group. Therefore, amniocentesis is only offered to women in whom the risk of having a child with a genetic handicap exceeds that of the iatrogenic miscarriage.
- Fetal cells circulating in maternal blood are in particular a source of genetic material potentially usable for prenatal genetic diagnosis (1, 2).
- cell types of fetal origin cross the placenta and circulate in the maternal blood (1).
- cell types include lymphoid and erythroid cells, myeloid precursors, and trophoblastic epithelial cells (cytotrophoblasts and syncyciotrophoblasts).
- the state of the art includes teaching methods of separating circulating fetal cells based on their affinity properties with respect to certain antibodies.
- US Patents 5,714,325 (New England Medical Center Hospitals), US 5,580,724 (Board of Regents, The University of Texas System), and US 5,646,004 (Activated Cell Therapy, Inc.).
- US Patent 5,714,325 teaches a method for enriching fetal granulocytes by separation of fetal granulocytes from maternal cells using antibodies recognizing antigens expressed on the surface of fetal granulocytes, in particular using the combination of an anti-CD71 antibody (transferrin receptor) and an anti-glycophorin A antibody.
- US Patent 5,580,724 describes another method of enriching in fetal cells a population of cells derived from maternal blood by in vitro stimulation of the proliferation of fetal cells using specific growth factors.
- Enrichment in fetal cells can also be carried out by centrifugation of a blood sample in a solution with density gradient (PERCOLL, FICOLL, albumin, sucrose or dextran for example) as described in US Pat. No. 5,646,004.
- PERCOLL PERCOLL, FICOLL, albumin, sucrose or dextran for example
- the invention results from the observation that only a process allowing a sufficient concentration of fetal cells, associated with their morphological examination, and with the genetic analysis targeted at the genome of single cells is likely to overcome the disadvantages of existing methods.
- the inventors have now shown that it is possible to combine the method of filtration of the cells described by Vona et al.,
- the present invention makes it possible to enrich a population of cells originating from maternal blood in fetal cells. more than six million epithelial. It is indeed possible to detect by the method of the invention, from 1 to 7 fetal cells per 2 ml of analyzed maternal blood.
- the method of the invention makes it possible to isolate almost all of the fetal cells present in maternal blood.
- the inventors after filtration and collection of the cells by microdissection, the inventors have shown that it is possible to observe two characteristic cell types, one mononuclear of the cytotrophoblast type with a diameter of between 14 and 20 ⁇ m approximately and the another multinucleate of the syncytiotrophoblast type with a diameter of about 44-47 ⁇ m or more.
- the present invention derives, in part, from the discovery that the fetal cells can be concentrated by the implementation of the filtration process as described in application FR 2 782 730.
- the invention also derives in part from the demonstration that the morphological analysis of the cells retained on the filter makes it possible to establish a presumption on the fetal or maternal origin of these cells and their genetic analysis allows the demonstration of their fetal or maternal origin.
- a prenatal diagnosis can be carried out on a pure fetal genome, obtained by microdissection of single cells.
- the invention stems from the demonstration that the prenatal diagnostic method can be implemented at an early period of pregnancy and on a limited volume of maternal blood drawn.
- Prenatal diagnosis should be understood to mean both the search for a particular characteristic of the fetus (for example sex) or the search for a genetic abnormality or any type of genetic pathology (DNA damage) , infectious (viral, bacterial or parasitic) or metabolic (alteration of the synthesis of messenger RNAs and / or proteins) which can be detected from the genetic analysis of isolated fetal cells.
- the prenatal diagnosis consists in looking for a genetic or chromosomal anomaly on the DNA of a fetal cell, a genetic or infectious pathology (viral, bacterial or parasitic), or, a precise genotype, and in particular the genetic sex of the fetus.
- minimally invasive or non-invasive method is meant a method which does not involve the removal of fetal tissue or cells by biopsy and / or breach of the placental barrier.
- the invention relates to a method of prenatal diagnosis on fetal cells isolated from maternal blood comprising the following steps: a) filtration of a sample of pure or diluted maternal blood, so as to concentrate on a filter according to their size certain circulating cells and in particular cells of fetal origin; b) analysis of the cells retained on the filter in order to obtain a presumption or an identification of their fetal or maternal origin; and the identification of their epithelial nature, c) the demonstration of the fetal origin of certain cells enriched by genetic analysis of cells isolated individually, d) the search for genetic anomalies targeted specifically at the cellular genomes analyzed individually and whose fetal origin has been demonstrated.
- prenatal diagnosis must be carried out on a pure fetal genome.
- prenatal diagnosis must be carried out on a pure fetal genome.
- the term “presumption” or “presumed” in step (b) therefore indicates the high probability of being in the presence of a cell of fetal origin.
- step (c) the analysis is preferably carried out by genetic analysis using specific markers of the fetal genomes, as is explained in detail below.
- the characteristic (s) of the fetal cells likely to be sought in step (d) can be either a genetic or chromosomal abnormality of the fetus, or a particular genotype of the latter.
- the search for this particular characteristic is preferably carried out by genetic analysis on single cells whose fetal origin has been previously demonstrated.
- a blood sample is taken from the pregnant woman.
- a sample of maternal blood is taken early during pregnancy (for example around the fifth week of pregnancy).
- the taking of the maternal blood sample and the diagnostic method according to the invention can be carried out at any time, from the beginning to the end of the pregnancy.
- the sample and the diagnostic method are carried out between the 7 th and 15 th week of pregnancy.
- the sample and the diagnostic method are carried out between the 10 th and 15 th week of pregnancy.
- 3 and 20 milliliters of maternal blood will be taken, preferably between 5 and 10 milliliters.
- the subsequent analysis of the isolated fetal cells described below can be carried out on a more limited volume of blood sample taken, for example between 1 and 10 milliliters, preferably between 2 and 5 milliliters.
- a more limited volume of blood sample taken for example between 1 and 10 milliliters, preferably between 2 and 5 milliliters.
- This parallel study makes it possible to identify specific genetic markers of the father and the mother, which can be used to demonstrate the fetal origin of the cells isolated by filtration according to the embodiment described below.
- the invention results in particular from the observation that the fetal epithelial cells circulating in the blood have a diameter greater than the maternal leukocyte and erythrocytic cells and can be isolated according to filtration methods adapted from those described for the isolation of circulating pathogenic cells in blood such as those described in patent application FR 2782730.
- the blood sample taken is filtered so as to concentrate on the filter, according to their size, certain circulating cells of fetal or maternal origin and thus to separate them from blood cells, in particular maternal leukocytes.
- any process allowing the enrichment of the cell population resulting from the blood sample, in cells of fetal origin can be implemented.
- an enrichment of the population in fetal cells is achieved by sorting the cells according to the expression of surface markers expressed by the fetal cells to reduce the proportion of cells of maternal origin.
- Cell sorting techniques are, for example, FACS, sorting on immunoaffinity or immunomagnetic column (MACS) or any technique capable of obtaining enrichment of a cell type on the basis of physical (density) or structural characteristics (specific antigens in particular).
- the blood sample is diluted before the filtration step in a filtration solution, said filtration solution consisting of a cell-binding reagent nucleated and / or lysed red blood cells.
- a filtration solution includes a detergent capable of degrading the membrane of red blood cells, such as saponin, and a fixative capable of stabilizing the membrane of nucleated cells, such as formaldehyde.
- the maternal blood sample is diluted approximately 10 to 100 times in the filtration solution.
- the filtration of the sample of pure or diluted maternal blood is carried out using a porous filter making it possible to separate the cells according to their size.
- the porosity of the filter is chosen so as to allow the figured elements of the blood to pass, in particular erythrocytes, platelets and maternal leukocytes and to retain certain nucleated cells and in particular large cells (epithelial or hematopoietic precursors) of maternal origin and fetal.
- a filter with a porosity of between 6 and 15 ⁇ m and a density adapted according to the chosen porosity and suitable for retaining the cells while avoiding clogging thereof during filtration Preferably, the filter has substantially cylindrical pores with a diameter of approximately 8 ⁇ m and a density between 5.10 4 and 5.10 5 pores / cm 2 . More preferably, the filter used is calibrated so that all of the pores have a substantially identical diameter.
- a filter that can be used in the process of the invention is a calibrated filter membrane of polycarbonate of the “Track-Etched membrane” type with a pore density of 1x10 5 pores / cm 2 , with a thickness of 12 ⁇ m and of a pore size of 8 ⁇ m, such as that marketed by Whatman®.
- the cells retained on the filter can be observed under a microscope, for example after staining with hematoxylin and eosin so as to analyze their morphology.
- Their epithelial nature can be identified, for example, by immunostaining with an anti-cytokeratin antibody (type KLi). It is at this stage possible to consider recognizing on the basis of morphological characters the cells of fetal origin and in particular, the cytotrophoblastic cells, mononuclear, having a large nucleus, a condensed chromatin and a reduced cytoplasm, of a diameter between 14 and 20 ⁇ m and syncytiotrophoblastic cells with a larger diameter (44-47 ⁇ m or more) and plurinucleated.
- the filtration step will be repeated if no cell suspected of fetal origin is observed on the filter after a first filtration.
- the genetic analysis will be established on all of the cells retained on the filter and comprising certain cells of which the fetal origin is presumed, or on the genome of cells isolated individually.
- the presumption of fetal or maternal origin of the cells retained on the filter is analyzed by looking for the presence of immunological or cytological marker (s) characteristic (s) of fetal cells.
- immunological marker characteristic of fetal cells means any antigen or combination of antigens whose expression is significantly different between fetal cells and maternal cells, and which can be detected using an antibody or a combination antibodies specifically directed against said antigen or combination of antigens.
- immunological markers are in particular the antigens associated with the trophoblastic cells described in patent application WO 90/06509. The search for the presence of immunological markers characteristic of fetal cells consists for example:
- said contacting of the cells with the antibody can be carried out before or after the filtration step.
- the antibodies chosen can be of the polyclonal or monoclonal type.
- An example of an antigen characteristic of fetal cells is the placental alkaline phosphatase antigen.
- the analysis of the presumed fetal origin of the cells can be carried out by determining specific cytological markers of cytotrophoblastic and / or syncyciotrophoblastic cells.
- the cytological markers that can be used include all of the cytological characteristics of the fetal cells making it possible to differentiate them from the other types of circulating cells capable of being retained on the filter, in particular, the size of the cells, the shape of the cells, the presence and the size of particular organelles, size and number of nucleus, structure of chromatin, etc., or any particular combination of these cytological characteristics.
- the cytological characteristics can be observed by staining the cells using dyes conventionally used in cytology, in particular hematoxylin-eosin and by observing the labeled cells under optical microscopy.
- in situ hybridization is carried out of probes specific for the chromosomal or sex abnormality to be detected, on the genome of the cells. retained on the filter, and looking for specific hybridization on the genome of cells whose fetal origin is presumed.
- Probes specific for a chromosomal sequence can in particular be DNA or PNA (peptid nucleic acid) type probes (11).
- An example of an in situ hybridization technique is known as the FISH (Fluorescence In Situ Hybridization) method (10), but any method known to a person skilled in the art which makes it possible to detect an anomaly in the genome of a cell.
- FISH Fluorescence In Situ Hybridization
- chromosomal abnormalities examples include trisomy 13, trisomy 18, trisomy 21, Turner's syndrome, Penta X syndrome, XYY syndrome or Klinefelter syndrome.
- the cells whose fetal origin is presumed are collected individually. By individual collection of cells, it is necessary to understand any method which makes it possible to collect a specific individual cell retained on the filter for its subsequent analysis independently of the other cells retained on the filter. In a preferred embodiment, cells retained on the filter are collected individually by microdissection.
- the individual collection of a cell by microdissection consists in cutting with the laser the part of the filtering membrane on which a cell is retained, or in removing the cell from the filter using the laser, then in recovering the single cell collected in a suitable tube. She is then able to undergo the various analyzes allowing prenatal diagnosis as set out below.
- the individual collection of cells makes it possible to advantageously target genetic analysis on the genome of a single cell. It also makes it possible to carry out the search for a genetic or chromosomal abnormality of the fetus or of a particular genotype thereof on the genome of a single cell whose fetal origin has been previously demonstrated by genetic analysis.
- advantageously pure genetic material that is to say, derived from a single cell, and usable both for the demonstration of the fetal origin of the cell analyzed then for the possible search for a genetic anomaly or a characteristic thereof, or a particular genotype.
- Any type of genetic analysis method can be used to demonstrate the fetal or maternal origin of a single collected cell or to search for genetic anomalies in the fetus or a particular genotype of it as soon as these methods are sensitive enough to identify on a single cell the character or characters sought.
- a preferred method of analysis for the demonstration of fetal origin is the analysis by DNA allelotyping of the cells collected, in particular by the identification of microsatellite markers specific for paternal and maternal DNA.
- - prenatal diagnosis by looking for genetic or chromosomal abnormalities of the fetus or a particular genotype thereof, by identification on a DNA preparation derived from a single collected cell, from one or more markers (s) / genetic target (s) or polymorphism or of a combination of these markers / targets, or of a particular allelic assay of these markers or of a genetic target.
- the term “genetic target” will be preferred to refer to the genetic characteristic sought, including polymorphism, in the context of prenatal diagnosis and the term “marker” to refer to the elements allowing the demonstration of the origin of the analyzed cells.
- genetic target is intended to mean any genetic characteristic, for example a particular mutation in a gene, associated specifically with a phenotype or with a genetic or infectious pathology of the fetus.
- polymorphism marker it is necessary to understand any identifiable characteristic on DNA whose presence is correlated with a particular genotype. These markers make it possible to distinguish paternal DNA from maternal DNA and therefore to demonstrate the bi-patent composition of DNA. fetal. Examples include restriction fragment length polymorphism (RFLP) markers, SNP (Single Nucleotide Polymorphism) markers, micro-satellite markers, VNTR (Variable Number of Tandem Repeats) or STR (Short Tandem Repeats) markers ).
- RFLP restriction fragment length polymorphism
- SNP Single Nucleotide Polymorphism
- micro-satellite markers micro-satellite markers
- VNTR Very Number of Tandem Repeats
- STR Short Tandem Repeats
- Micro-satellite markers are particularly preferred for the characterization of cells and the implementation of prenatal diagnosis.
- at least one polymorphism marker to be identified is a microsatellite marker, a VNTR (Variable Number of Tandem Repeats) marker or a STR (Short Tandem Repeats) marker.
- VNTR Very Number of Tandem Repeats
- STR Short Tandem Repeats
- the micro-satellite markers, VNTR or STR are made up of tandem repeats, most often of polyCA / GT patterns.
- the allelic variations due to the variation in the number of repetitions, are easily detected by PCR-type amplification, thanks to the use of primers corresponding to the unique sequences flanking the microsatellite.
- microsatellite markers A physical map of these microsatellite markers and the sequence of their associated primers are described by Dib et al. (12). By this methodology, the demonstration of a two-parent contribution to the genotype of the cells analyzed makes it possible to establish with certainty the fetal origin of the cells analyzed. In addition, the presence of particular microsatellite markers can be specifically sought, in particular as a genetic target, for prenatal diagnosis, in particular for the diagnosis of particular chromosomal alterations.
- a marker or a combination of markers or an allelic assay of these markers distinguished from those of the genome of maternal cells, in particular by research on the genome of said collected cell, of a marker or of a combination of markers specific for the DNA of paternal cells. Their presence is necessarily the signature of a fetal origin of the cell in question.
- a specific marker of the DNA of the fetal cells can be a specific genetic marker of the Y chromosome or a combination of such markers.
- said collected cell is lysed and its entire genome is pre-amplified, for example using generic primers covering all possible sequences according to known methods of preamplification by primer extension (PEP) (13) or even the DOP-PCR method. These methods make it possible to amplify the entire genome of a single cell.
- PEP primer extension
- the pre-amplified DNA preparation thus obtained and derived from the DNA of a single cell can then be purified and used as genetic material for the specific detection of genetic markers or of polymorphism and / or the detection of a genetic target.
- the fetal or maternal origin of a collected cell is demonstrated by the amplification of genetic markers or of polymorphism or a combination of these markers, from the preparation of DNA pre- amplified derived from the DNA of a single cell.
- Genetic markers capable of demonstrating the bi-parental contribution to fetal DNA are identified by prior analysis of paternal and maternal DNA.
- PCR Polymerase Chain Reaction
- isothermal amplification methods such as TMA (transcription mediated amplification), NASBA (nucleic acid sequence based amplification), 3SR (self sustained sequence replication) or strand displacement amplification.
- each pre-amplified DNA preparation of a collected cell can be used for the amplification of at least five different genetic markers or targets.
- the amplifications carried out allow the detection of microsatellite markers.
- PCR amplification also makes it possible to detect genetic targets, and in particular point or deletion type mutations, or micro-deletions associated with a genetic character or with a specific pathology.
- the sequences capable of carrying the deletion are amplified and the amplification products are separated according to their size, for example by electrophoresis. The presence of deletions is detected by the presence of an amplification product of size smaller than the amplification products which do not carry a deletion.
- the amplification products can also be sequenced, in particular for precisely characterizing the markers or the genetic targets sought and in particular the mutations or genotypes sought for a prenatal diagnosis.
- the fetal or maternal origin of a collected cell is demonstrated and / or the prenatal diagnosis, in particular the search for a genetic or chromosomal abnormality of the fetus or a particular genotype thereof by sequencing the amplified genetic markers or targets.
- VNTR markers on chromosome 21 such as the markers D21S1414 and D21S1411 and the allelic assay of these markers.
- the fetal or maternal origin of a cell collected individually is demonstrated and then prenatal diagnosis, in particular the search for a genetic or chromosomal abnormality of the fetus or a particular genotype thereof by hybridization of all or part of the preparation of pre-amplified DNA with specific DNA probes.
- the DNA probes are chosen so as to specifically hybridize to the genetic or polymorphic targets for their identification or to the sequences carrying the genetic target (s) to be sought.
- the hybridization of the probes on the genetic targets can be detected according to conventional techniques for detecting hybridization complexes of nucleic acids of the slot blot, Southern blot type or advantageously today by DNA or microarray or macro chips - networks (14).
- Molecular probes can for example be chosen for detection specific for cystic fibrosis, muscular dystrophies, Gaucher disease, hemoglobinopathies, hemophilia, phenylketonuria or cystic fibrosis.
- the DNA probes specific for the genetic targets to be identified are fixed on a support forming a DNA chip or micro-networks or macro-networks.
- the preparation of pre-amplified DNA is for example labeled using a radioactive or fluorescent marker, and brought into contact with the DNA chip or micro-networks or macro-networks comprising the specific probes.
- the intensity of hybridization is measured for each spot containing a specific probe, thus determining with great sensitivity the presence of the markers sought on the DNA of a collected cell.
- genetic targets naturally depends on the genotype to be sought. Examples of genetic targets which can be used according to prenatal diagnoses are described in the state of the art.
- An alternative method for determining chromosomal abnormalities and in particular gains and losses of chromosomes with a view to prenatal diagnosis is the method of comparative genomic hybridization (CGH), consisting of (i) comparing hybridization on a chromosomal, cosmidic preparation or on a DNA chip, a preparation of pre-amplified DNA derived from the genome of a single cell collected after filtration according to the method, and a preparation of pre-amplified DNA derived from cells of maternal origin or non-fetal reference cells, the two preparations having been labeled with different markers and (ii) to identify the differences in hybridization between the DNA of the cell collected after filtration and the maternal DNA (16) .
- CGH comparative genomic hybridization
- the prenatal diagnosis is carried out by a method of comparative genomic hybridization (CGH) of a preparation of pre-amplified DNA derived from DNA. of a single cell collected and of which the fetal origin is demonstrated, and of a preparation of pre-amplified DNA from cells of maternal origin or from non-fetal reference cells.
- CGH comparative genomic hybridization
- Another aspect of the invention relates to a method for obtaining a population of fetal cells derived from a population of cells isolated from maternal blood and concentrated in cells of fetal origin, said method comprising the following steps:
- the method described above results from the observation by the inventors that, unexpectedly, the method of the invention makes it possible to concentrate in a particularly large manner, a population of cells isolated from maternal blood in cells of fetal origin. It also results from the observation that the cells can be filtered even without prior fixation, that is to say with preserved viability.
- the populations of cells obtained by the above method it is possible to obtain pure cultures of fetal cells according to the cloning and expansion techniques known to those skilled in the art.
- the populations pure or enriched in fetal cells obtained by the process find their applications in particular in the preparation of a cell therapy product comprising said fetal cells or cells resulting from their differentiation. All the methods described above are based on the existence and the development of a specific device called ISET (English acronym for Isolation by Size of Epithelial Tumor Cells) and described in EP 0513 139 and comprising on a frame:
- ISET International acronym for Isolation by Size of Epithelial Tumor Cells
- porous filter capable of retaining certain circulating cells according to their size, mounted between two clamping devices, respectively upstream and downstream of the direction of filtration, and intended for sealing the filtration,
- the upstream block comprising means for storing and / or preprocessing the samples to be analyzed
- downstream block comprising perforations opposite the storage means for collecting the waste
- the invention relates to the adaptation and use of a device of this type for the filtration of fetal cells present in maternal blood.
- a porosity filter preferably calibrated, capable of retaining the cells with an average diameter greater than 8 ⁇ m, and better still, greater than 10 ⁇ m, preferably greater than 15 ⁇ m.
- a filter with an average porosity of 8 ⁇ m has been shown to meet these desired characteristics;
- the invention relates more particularly to the use of an ISET type filtration device, for the isolation of fetal cells from maternal blood and comprising a filter with an average porosity of between 6 ⁇ m and 15 ⁇ m, and preferably around 8 ⁇ m.
- the diagnostic method was implemented on cells isolated from the blood of 13 pregnant women.
- the method implemented includes the detection of fetal cells by their morphological analysis and specific markers of the male sex (primers for amplification of the Y chromosome sequences). It also includes the demonstration of the fetal origin of the cells retained on the filter by allelotyping by means of PCR amplification of microsatellite markers. The results show that the diagnostic method is particularly sensitive since, in mothers carrying female fetuses, 100% of the cells are negative on the Y chromosome test.
- this example is not limitative, and the method of the invention is applicable to the prenatal diagnosis of any fetal characteristic since this can be identified from an isolated, individualized and characterized fetal cell, according to the means described above.
- Figure 1 polyacrylamide gel of PCR amplification products carried out on trophoblastic cells obtained by the method of the invention.
- the different points numbered 1 to 42 represent:
- Tracks 1 to 21 test of specificity of the primers Y by amplification of the DNA obtained from 20 women (tracks 1 to 12 and 14 to 21) and from a man (track 13, positive control). The test is negative for DNA samples from women, and positive for DNA from a man.
- Tracks 22 to 42 PCR test of the Y primers on individual cells isolated from the blood of a mother carrying a male fetus (tracks 23, 25, 27, 29, 31, 33, 34, 36 and 38).
- the primer Y test is positive for DNA from male fetal cells from lanes 25, 29, 33, 36 and 38.
- the test of the primers Y is negative for the DNA of the maternal cells tracks 23, 27, 31 and 34. Tracks 22, 24, 26, 28, 30, 32, 35, 37 and 39: Result of negative controls corresponding to the buffer without sample inserted in the cell lysis step and leads to the end of the test.
- Tracks 41 and 42 Result of positive controls: A HuH6 cell (lane 40), 5 ng and 2 ng of DNA derived from leukocytes from the blood of 3 men (lanes 41 and 42).
- Figure 2 microscopic analysis of the morphological characteristics of the circulating fetal cells isolated by ISET.
- A Syncitiotrophoblastic polynuclear cells
- Figure 3 genotyping using STR markers of parental and trophoblastic DNA and DNA from cells isolated from maternal blood and collected individually after filtration.
- the STR marker used in this case does not allow the maternal (M) or paternal (F) origin to be distinguished from DNA (heterozygous state, alleles 250 bp and 262 bp).
- trophoblastic DNA (T) presents a homozygous state for an allele (250 bp) and an identical profile is found for a fetal cell (FC).
- MC fetal cell
- the STR marker makes it possible to distinguish the paternal or maternal origin of the cells.
- trophoblastic DNA has an allele of 256 bp, also found on maternal DNA (M); however, the paternal DNA has an allele different from 258 bp.
- the two alleles, 256 bp and 258 bp, were detected on two microdissque cells, thus demonstrating their fetal origin (FC).
- Example of implementation of the prenatal diagnostic method on fetal cells isolated from maternal blood application to the determination of the sex of the fetus
- the blood samples were diluted 10 times in a filter buffer containing 0.175% saponin, 0.2% paraformaldehyde, 0.0372% EDTA and 0.1% BSA, then filtered using a calibrated polycarbonate filter with calibrated cylindrical pores of 8 ⁇ m in diameter.
- the cells retained on the filter were grouped on a circular spot 0.6 cm in diameter. After coloring with eosin and hematoxylin, the spots were analyzed under a microscope and a photograph of each cell was taken at high and low magnification.
- the cell size was determined using Adobe Photoshop software, taking as reference the 8 ⁇ m pore size. The photos allow the cells to be found under the Pixcell II Arcturus microscope (Mountain View, CA).
- Figure 2 illustrates the microscopic analysis obtained by this method.
- the microdissection of each cell was carried out by laser capture without any prior treatment of the filter. To ensure that only one cell is collected each time, photographs were taken of the filter before and after microdissection and of the microdissected cell deposited on the capsule (CapSure TM HS).
- the cell was then lysed in 15 ⁇ l of lysis buffer (100 mM Tris-HCl pH 8, 400 ⁇ g / ml proteinase K) for 16 hours at 37 ° C. The lysate was collected after centrifugation and proteinase K was inactivated at 90 ° C for 10 minutes.
- lysis buffer 100 mM Tris-HCl pH 8, 400 ⁇ g / ml proteinase K
- the lysate was collected after centrifugation and proteinase K was inactivated at 90 ° C for 10 minutes.
- PEP primers
- the DNA was precipitated with ethanol and resuspended in 10 ⁇ l of water.
- Each sample was then
- D16S3031 marker (sense) 5'-TET-ACTTACCACTGTGCCAGTTG-3 'and, (antisense) 5'-ATACATGGGTCCTTAAACCG-3'; D16S539 marker
- the samples were also tested with the primers specific for the Y chromosome (primers Y: Y1.7 and Y1.9 as described in reference 17).
- the PCRs were carried out on a reaction mixture volume of 20 ⁇ l containing 2 ⁇ l of PEP product, 10 mM Tris-HCl, 50 mM KCI, 1.5 mM MgCI 2 , 0.01% gelatin, 200 mM of each deoxynucleotide, 20 picomoles of each primer Y, HLA or STR and 1 U of Taq polymerase (Perkin-Elmer Cetus, Emeryville, CA).
- the results are shown in Figure 1.
- the PCR conditions for amplification using specific primers of the Y chromosome were established by the amplification of 5 ng of DNA derived from leukocytes from the blood of 3 male individuals then by amplification of the pre-amplified DNA product of filtered and microdissued HuH6 cells (derived from a hepatocellular carcinoma).
- For the PCR using the STR-specific primers after denaturing the DNA at 94 ° C. for 5 minutes, 40 amplification cycles were carried out (94 ° C. 30 ", 54 ° C. 30", 72 ° C 20 ”) followed by an elongation phase at 72 C C for 5 minutes.
- Two microliters of the first PCR product were re-amplified using the same PCR conditions.
- One microliter of the final PCR product was mixed in 20 ⁇ l of deionized formamide and 0.4 ⁇ l of Genescan-500 TAMRA marker then loaded into an ABI Prism 310 automatic sequencer.
- the profiles were analyzed using the Genescan software program (Perkin Elmer, Foster City, CA). Allelotyping was carried out by amplification, using the same primers STR, 1.5 ng of paternal DNA derived from blood leukocyte and / or 1.5 ng of maternal DNA derived from maternal blood or leukocyte before pregnancy and 1.5 ng of trophoblastic DNA obtained from a sample of chorionic villi.
- the specificity of the primers Y was tested by the amplification of 10 ng of DNA derived from leukocytes from the blood of 20 women. The precautions used to avoid contamination of PCR products have been described previously (18).
- a negative control (the buffer without the sample) is inserted for each sample during the lysis step and analyzed like the other samples until the end of the test.
- at least one microdissection of a new filter (without cells) was included, which was carried out in parallel with the samples and the controls. Amplification was repeated with specific primers of the Y chromosome or STR-specific primers on the samples and controls, negative and positive, to verify the specificity of the positive results.
- the specificity of the Y primers for amplification of the DNA obtained from 20 women was determined.
- the test was negative for DNA samples from women, and positive for DNA from a man as a positive control, as shown in Figure 1 when comparing lines 1 to 12, 14 to 21 (women ) on line 13 (male).
- a preliminary test was then carried out on the blood of a mother carrying a male fetus.
- the cells retained on the filter were microdissued and analyzed using the primers specific for chromosome Y and HLA defined in part A.
- the test with the primers HLA was positive for all the cells and the test with the primers Y was positive for half of these cells.
- FIG. 2 illustrates the fact that on the cells found positive for the test, the photographs taken of these cells on the filter reveal two cell types having the following morphological characteristics:
- cytotrophoblast type mononuclear cells, cytotrophoblast type, with a diameter between 14.3 and 19.9 ⁇ m (average value 16.9 +/- 2), a large nucleus with condensed chromatin and little cytoplasm, often with some microvilli on the surface of the membranes,
- the amplification products Y were amplified with the same primers Y. All the controls and the cells negative to the PCR gave a negative result to the test, while the positive cells gave a positive, and a weakly positive cell (track 34 in Figure 1) gave a stronger band.
- Six cells have a fetal profile in accordance with the positive detection of the Y chromosome, and 5 cells have a maternal profile, in accordance with the negative detection of the Y chromosome.
- the STR markers tested do not make it possible to distinguish the paternal alleles (F) from the maternal alleles (M) (heterozygous, 250 and 262 bp), however the trophoblastic DNA (T) has a homozygous state for one of the alleles (250 bp).
- the same profile (homozygous for the 250 bp allele) was detected on a fetal cell (FC) isolated according to the method of the invention, which cell was positive for the Y chromosome detection test.
- FC fetal cell isolated according to the method of the invention, which cell was positive for the Y chromosome detection test.
- another microdissected cell negative on the Y chromosome detection test has a clearly heterozygous profile (maternal cell, MC).
- the trophoblastic DNA (T) has, in addition to the maternal allele (256 bp) also detected from the maternal DNA (M), a paternal allele (258 bp). These two alleles were found on the two microdissque cells (FC), thus determining their fetal origin.
- the inventors have now shown that it is possible to genetically concentrate and analyze the fetal cells collected after their concentration, from only two milliliters of a sample of maternal blood.
- this approach allowed the demonstration of the fetal origin of the cells collected. Taking into account the number of fetal cells circulating in the blood estimated at one cell per milliliter of blood, i.e. one fetal cell for 10 million leukocytes, the enrichment obtained is a factor of about 6.6 million (one fetal cell for 1, 5 large cells retained on the filter).
- this approach provides morphological information about the fetal cells circulating in the blood.
- Mononuclear cells which are probably cytotrophoblast-like cells, and polynuclear cells which are syncytiotrophoblastic cells have been isolated. These two types of cells have never been observed in 22 healthy blood donors and 44 patients with carcinoma analyzed using the same separation and microdissection technique (results not shown).
- the fetal lymphoid or myeloid progenitors persist in the bloodstream after pregnancy, but not the trophoblastic cells.
- the advantage of the analysis of trophoblastic cells is therefore also that it makes it possible to associate the result of the genetic test with certainty to the current pregnancy.
- Another advantage of this approach is the possibility of carrying out at least 5 PCR analyzes from the DNA of a single individual cell. This gives the possibility of carrying out a genetic test on cells whose fetal origin has been proven in parallel by molecular analysis, in particularly by analyzing STR polymorphic markers. It is also possible to base the diagnosis on the comparison of independent results obtained on different individual fetal cells isolated from repeated blood samples. Interestingly, it should be noted that the FISH protocol can also be successfully applied to cells isolated according to the filtration method (9).
- the other advantages of the method according to the invention reside, on the one hand, in its sensitivity since nearly 100% of the fetal cells collected on the basis of their morphological observation and analyzed are identified as such by the method of the invention, and secondly, in its specificity since, in particular fetal apoptotic cells and cells of maternal origin can be excluded from the analysis in view of their genetic and morphological characteristics.
- the method of the invention therefore proposes a new approach for carrying out an early prenatal diagnosis, non-invasive and particularly sensitive and specific.
- Bianchi D Fetal cells in the maternai circulation: feasibility for prenatal diagnosis. BrJ Haematol 1999 105: 574-583
Abstract
Description
Claims
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AU2002314224A AU2002314224B2 (en) | 2001-04-30 | 2002-04-30 | Prenatal diagnosis method on isolated foetal cell of maternal blood |
JP2002585985A JP4404553B2 (ja) | 2001-04-30 | 2002-04-30 | 単離された母体血液の胎児細胞に対する出生前診断の方法 |
CA2452099A CA2452099C (fr) | 2001-04-30 | 2002-04-30 | Methode de diagnostic prenatal sur cellule foetale isolee du sang maternel |
EP02740785.7A EP1383926B1 (fr) | 2001-04-30 | 2002-04-30 | Methode de diagnostic prenatal sur cellule foetale isolee du sang maternel |
ES02740785.7T ES2543709T3 (es) | 2001-04-30 | 2002-04-30 | Procedimiento de diagnóstico prenatal en célula fetal aislada de sangre materna |
US10/695,744 US7651838B2 (en) | 2001-04-30 | 2003-10-30 | Prenatal diagnosis method on isolated foetal cell of maternal blood |
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FR0105824A FR2824144B1 (fr) | 2001-04-30 | 2001-04-30 | Methode de diagnostic prenatal sur cellule foetale isolee du sang maternel |
FR01/05824 | 2001-04-30 |
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US10/695,744 Continuation US7651838B2 (en) | 2001-04-30 | 2003-10-30 | Prenatal diagnosis method on isolated foetal cell of maternal blood |
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EP (1) | EP1383926B1 (fr) |
JP (1) | JP4404553B2 (fr) |
AU (1) | AU2002314224B2 (fr) |
CA (1) | CA2452099C (fr) |
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FR2880897A1 (fr) * | 2005-01-18 | 2006-07-21 | Inst Nat Sante Rech Med | Methode de detection, non invasive, prenatale, in vitro de l'etat sain normal, de l'etat de porteur sain ou de l'etat de porteur malade de la mucoviscidose |
WO2006077322A1 (fr) * | 2005-01-18 | 2006-07-27 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Methode de detection, non invasive, prenatale, in vitro de l’etat sain normal, de l’etat de porteur sain ou de l’etat de porteur malade de la mucoviscidose |
EP2345744A2 (fr) | 2005-01-18 | 2011-07-20 | Institut National De La Sante Et De La Recherche Medicale | Methode de detection, non invasive, prenatale, in vitro de l'etat sain normal, de l'etat de porteur sain ou de l'etat de porteur malade de la mucoviscidose |
EP2345744A3 (fr) * | 2005-01-18 | 2011-11-16 | Institut National De La Sante Et De La Recherche Medicale | Methode de detection, non invasive, prenatale, in vitro de l'etat sain normal, de l'etat de porteur sain ou de l'etat de porteur malade de la mucoviscidose |
Also Published As
Publication number | Publication date |
---|---|
AU2002314224B2 (en) | 2007-08-02 |
CA2452099A1 (fr) | 2002-11-07 |
EP1383926A2 (fr) | 2004-01-28 |
FR2824144A1 (fr) | 2002-10-31 |
EP1383926B1 (fr) | 2015-05-06 |
FR2824144B1 (fr) | 2004-09-17 |
WO2002088736A3 (fr) | 2003-09-25 |
US7651838B2 (en) | 2010-01-26 |
JP2004533243A (ja) | 2004-11-04 |
US20050049793A1 (en) | 2005-03-03 |
JP4404553B2 (ja) | 2010-01-27 |
CA2452099C (fr) | 2017-05-09 |
ES2543709T3 (es) | 2015-08-21 |
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