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WO2010140145A1 - Procede pour la detection controlee par ordinateur de noyaux de cellules fœtales dans des echantillons cervicaux - Google Patents

Procede pour la detection controlee par ordinateur de noyaux de cellules fœtales dans des echantillons cervicaux Download PDF

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Publication number
WO2010140145A1
WO2010140145A1 PCT/IL2010/000430 IL2010000430W WO2010140145A1 WO 2010140145 A1 WO2010140145 A1 WO 2010140145A1 IL 2010000430 W IL2010000430 W IL 2010000430W WO 2010140145 A1 WO2010140145 A1 WO 2010140145A1
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Prior art keywords
fetal
analysis
probe
chromosomal
cells
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PCT/IL2010/000430
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English (en)
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Aliza Amiel
Moshe Feigin
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Monaliza Medical Ltd.
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Publication of WO2010140145A1 publication Critical patent/WO2010140145A1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
    • G06V20/695—Preprocessing, e.g. image segmentation
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10056—Microscopic image
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30024—Cell structures in vitro; Tissue sections in vitro

Definitions

  • the present invention relates to prenatal diagnosis and screening of fetal genetic material obtained from maternal cervical samples.
  • Prenatal diagnosis involves the identification of fetal malformations or genetic diseases present in a human fetus, as well as determination of fetal gender. Specifically, prenatal diagnosis is helpful for: managing the remaining weeks of the pregnancy; determining the outcome of the pregnancy; anticipating possible complications with the birth process; planning for problems that may occur in the newborn infant; deciding whether to continue the pregnancy and finding conditions that may affect future pregnancies.
  • non-invasive and invasive techniques available for prenatal diagnosis. Each of these techniques can be applied only during specific time periods during the pregnancy for greatest utility.
  • the techniques employed for prenatal diagnosis include: ultrasonography, amniocentesis, chorionic villus sampling, the noninvasive detection and analysis of fetal blood cells in maternal blood, and the different screening tests of Pappa A, maternal serum alpha-fetoprotein, maternal serum beta-HCG, maternal serum Inhibin and maternal serum estriol.
  • Non-invasive prenatal screening and diagnosis has a clear advantage over invasive methods. Intact fetal cells exfoliate in the uterine cavity during early pregnancy and are a potential source of fetal cells for NIPD.
  • NIPD non-invasive prenatal diagnosis
  • Fetal cells or free fetal nucleic acid circulating in the maternal blood and fetal cells obtained from the cervix are the two most important source materials for NIPD (Geifman-Holtzman and Ober Berman, 2008)). Recently, cell-free fetal DNA circulating in the maternal plasma was used for determining fetal gender or Rhesus (RhD) status (Brojer et al., 2005) or point mutations for beta-thalassemia (Li et al., 2008).
  • Fetal cells are present in the uterine cavity from 5-15 weeks of pregnancy (Adinolfi & Sherlock, 2001) and safe retrieval of the exfoliated fetal cells from the uterine cervix has been demonstrated (Fejgin et al., 2001).
  • the use of a cytobrush, among other methods has become a widely used technique (Bischoff & Simpson, 2006) to obtain endocervical cells.
  • Fluorescence microscopy of cells and tissues is well known in the art. Methods have been developed to image fluorescently-stained cells in a microscope and extract information about the spatial distribution and temporal changes occurring in these cells. Some of these methods and their applications are described in Taylor et al (1992). These methods have been designed and optimized for the preparation of a few specimens for high spatial and temporal resolution imaging measurements of distribution, amount and biochemical signal of the fluorescent reporter molecules in the cells.
  • FISH fluorescence in situ hybridization
  • Diagnostic FISH dot counting has been conventionally performed manually. In addition to correctly identifying the dot and its color, other size and shape characteristics must be categorized to correctly identify the chromosomal status. The process has proven to be tedious, lengthy and subject to human error.
  • the present inventors have previously disclosed a method for determining the gender and/or chromosomal abnormality, e.g. chromosomal aneuploidy of a fetus by identifying fetal cells in a transcervical cell sample obtained from a pregnant woman (WO 2004/087863).
  • WO 2008/081451 discloses a method of identifying fetal genetic material in a transcervical specimen using a telomere specific marker.
  • WO 2007/106838 discloses the use of an automated microscope system for analyzing FISH stained samples.
  • U. S Patent No. 7,346,200 discloses a computer-controlled method for detecting and diagnosing rare cell types in tissue samples applicable to cancer cells in blood and tissue biopsy samples.
  • the inventors of the present invention have established a high yield and efficient method for rapid analysis of FISH stained cervical samples using an automated microscopic system, thereby significantly enhancing the detection efficacy of fetal genetic material in the cervical samples.
  • the method of the present invention combines a highly efficient sampling and preparation procedure which results in obtaining high-quality fetal genetic material (e.g. fetal cell nuclei) amenable to genetic analysis and screening, e.g. using FISH techniques, with the use of an automated microscope system (e.g. Ikoniscope®).
  • the microscope system provides fully automated slide handling, complete slide scanning and real-time image capture and analysis of the FISH results.
  • the automated microscope system exhibits exceptional image quality, increased laboratory throughput and sample capacity.
  • a method of analysis of fetal genetic material in maternal cervical samples obtained during pregnancy comprising:
  • said in situ chromosomal analysis probe is a FISH probe.
  • said method further comprises a step of hypotonic treatment after the digestion with a protease.
  • said protease is tryspsin.
  • the present invention provides a method of analysis of fetal genetic material in maternal cervical samples obtained during pregnancy, comprising:
  • said genetic analysis comprises identifying the gender of said fetus.
  • said genetic analysis comprises identifying male fetal genetic material (e.g. the presence of XY chromosomes) in the cervical samples.
  • said genetic analysis and screening comprises identifying chromosomal aberrations (e.g. specific aneuploidies) in the cervical samples.
  • said chromosomal aberration is trisomy 21.
  • said automated microscopy system comprises an Ikoniscope® robotic microscopy platform, said platform providing high speed and accuracy of slide movement in the x, y, and z space.
  • the Ikoniscope® can acquire images through a high resolution, high sensitivity, and monochrome charge-coupled device (CCD) camera and is able to rapidly and accurately analyze a large number of cells hence is ideally suited to detect rare cell events.
  • CCD monochrome charge-coupled device
  • said method further comprises identifying said fetal genetic material using a fetal specific marker. Said step of identifying the fetal genetic material using the fetal specific marker may be performed prior to, concomitantly or after the genetic analysis of the sample.
  • the present invention provides a kit for analysis of fetal genetic material in maternal cervical samples comprising: a protease, a fixative, an in situ chromosomal analysis probe or in situ DNA analysis probe and instructions for use in analysis of fetal genetic material in maternal cervical samples.
  • said probe is a FISH probe.
  • said protease is trypsin.
  • said kit further comprises an agent for hypotonic treatment, e.g. KCl.
  • an agent for hypotonic treatment e.g. KCl.
  • Fig. l is a photograph demonstrating the identification of trisomy 21 cells in cervical brush samples from trisomy 21 pregnancies.
  • Panel A A male trisomy 21c nucleus exhibiting X signal (arrow head), Y signal (broken arrow) and chromosome 21 signals (arrows).
  • Panel C A female trisomy 21 nucleus exhibiting two X signals (arrow heads) and three chromosome 21 signals (arrows).
  • Panel B and D are the computer generated analysis of the cells depicted in panels A and C, respectively.
  • the present invention relates to prenatal diagnosis. Specifically, the present invention employs a unique sample preparation procedure which is aimed at preserving the integrity of the fetal cell genetic material combined with an automated microscopy system. This combined method is used for molecularly analyzing the genetic material of the fetus.
  • Prenatal diagnosis involves the identification of the fetal gender and major or minor malformations or genetic diseases present in a human fetus.
  • Chromosomal aberrations such as presence of extra chromosomes [e.g., Trisomy 21 (Down syndrome); Klinefelter's syndrome (47, XXY); Trisomy 13 (Patau syndrome); Trisomy 18 (Edwards syndrome); 47, XYY; 47, XXX], the absence of chromosomes [e.g., Turner's syndrome (45, XO)], or various translocations and deletions are currently detected using chorionic villus sampling (CVS) or amniocentesis.
  • CVS chorionic villus sampling
  • fetal nucleated erythrocytes nucleated red blood cells (NRBCs) U.S. Pat. No. 5,750,339; Bischoff, F. Z. et al., 2002
  • trophoblast cells in the maternal blood WO 9915892A1; U.S. Pat. Appl. No. 20050049793; US Pat. Appl. Nos. 20020045196Al, 20030013123 and EP Pat. Appl. No. 1154016A2
  • trophoblast cells in transcervical specimens Miller et al., 1999.
  • the present inventors have previously disclosed non-invasive methods for prenatal diagnosis using trophoblast cells present in transcervical specimens (U.S. Pat. Publication. Nos. 20040197832, 20050003351, 20050181429 and PCT Publication No. WO04087863A2).
  • the trophoblast cells are first identified by immunological or RNA-Zn situ hybridization staining methods using antibodies or probes specific to fetal cells and are then subjected to a molecular analysis (using e.g., FISH, DNA-based analysis) which allows, with relatively high accuracy, the identification of chromosomal and/or DNA abnormalities in the fetus.
  • FISH FISH
  • DNA-based analysis DNA-based analysis
  • the present invention provides a method of analysis of fetal genetic material in maternal cervical samples obtained during pregnancy, comprising: (a) digestion of the cervical sample with a protease; (b) fixation; (c) staining with an in situ chromosomal analysis probe or in situ DNA analysis probe; and (d) identifying stained nuclei of fetal cells using an automated microscopy system.
  • the present invention provides a method of analysis of fetal genetic material in maternal cervical samples obtained during pregnancy, comprising: (a) digestion of the cervical sample with a trypsin; (b) hypotonic treatment with KCL; (c) fixation; (d) staining with an in situ chromosomal analysis probe or in situ DNA analysis probe; and (e) identifying stained nuclei of fetal cells using an automated microscopy system.
  • genetic material refers to a chromosome, DNA or RNA.
  • analysis of fetal genetic material refers to the identification of any genetic characteristic of the fetus in chromosomes or DNA derived from the fetus.
  • analysis of the genetic material of the fetus may refer to determining the presence or absence of at least one chromosomal abnormality, determining the presence or the absence of at least one DNA abnormality, determining a paternity of the fetus, determining the gender of the fetus, determining the presence or absence of a specific polymorphic allele, additions or deletions of the DNA and/or analyzing the genetic makeup of the fetus.
  • gender of a fetus refers to the presence or absence of the X and/or Y chromosome(s) in the fetus.
  • detection refers to identification (e.g., visually) of fetal cells or nuclei in a sample.
  • detection of fetal cells or nuclei in a cervical sample is facilitated using an image analysis apparatus (e.g., the Ikoniscope robotic automated microscopy system) and a CCD camera.
  • automated microscopy system relates to a computerized microscope which is adapted to analyze fluorescently labeled tissue samples.
  • present invention employs the Ikoniscope ® automated microscopy system.
  • fetal cell(s) refers to a cell which is derived from the fetus.
  • fetal nucleus refers to a cell nucleus which originates from a fetal cell.
  • Non-limiting examples of fetal cells include trophoblasts, fetal nucleated red blood cells and fetal leukocyte cells.
  • a "cell-free nucleus” refers to a nucleus which has lost its surrounding cytoplasm, such as due to membrane rupture, apoptosis, enhanced osmosis and the like.
  • the cervical sample as used herein is a sample containing fetal biological material (e.g. tissue, cells or cell fragments) obtained from the maternal cervix.
  • fetal biological material e.g. tissue, cells or cell fragments
  • the term "cervical sample” as used in the context of the present invention extends also to samples obtained from the vagina, uterus, or placenta (including placental villi) of a pregnant woman.
  • the sample contains fetal cells or cell nuclei, including for example trophoblast cells, fetal nucleated erythrocytes or amniocytes.
  • the sample is preferably a trophoblast- nuclei containing sample.
  • trophoblast refers to an epithelial cell which is derived from the placenta of a mammalian embryo or fetus; trophoblasts typically contact the uterine wall.
  • trophoblast cells There are three types of trophoblast cells in the placental tissue: the villous cytotrophoblast, the syncytiotrophoblast, and the extravillous trophoblast, and as such, the term “trophoblast” as used herein encompasses any of these cells and cell nuclei.
  • the villous cytotrophoblast cells are specialized placental epithelial cells which differentiate, proliferate and invade the uterine wall to form the villi.
  • Cytotrophoblasts which are present in anchoring villi can fuse to form the syncytiotrophoblast layer or form columns of extravillous trophoblasts (Cohen S. et al., 2003. J. Pathol. 200: 47-52).
  • the sample may be obtained using any of the following known cell collection techniques: cytobrush, aspiration, cotton wool swab, endocervical lavage, biopsies (including needle biopsies) and intrauterine lavage. See Adinolfi, M. and Sherlock, J. (J. Hum. Genet. 2001, 46: 99-104) for comparison of the various sample collection approaches.
  • a cervical sample is obtained by a physician by inserting a hinged metal or plastic speculum into the vagina and collecting a tissue sample.
  • a cervical sample of the present invention may also be obtained by the cytobrush method (e.g. Fejgin, M.D., et al., 2001. Prenat. Diagn. 21: 619-621) in which a Pap smear cytobrush (e.g., MedScand-AB, Malm ⁇ , Sweden) is inserted through the external os to a maximum depth of 2 cm and removed while rotating it a full turn (i.e., 360°).
  • a Pap smear cytobrush e.g., MedScand-AB, Malm ⁇ , Sweden
  • the brush In order to remove the transcervical swab caught on the brush, the brush is shaken into a test tube containing 2-3 ml of a tissue culture medium (e.g., RPMI- 1640 medium, available from Beth Haemek, Israel) in the presence of antibiotics, e.g. 1 % Penicillin, Streptomycin, and Amphotericin B.
  • a tissue culture medium e.g., RPMI- 1640 medium, available from Beth Haemek, Israel
  • antibiotics e.g. 1 % Penicillin, Streptomycin, and Amphotericin B.
  • the sample used by the method according to this aspect of the present invention is obtained from a pregnant woman at any stage of the pregnancy.
  • the cervical sample is obtained from a pregnant woman between the 3 rd and the 15 th week of gestation.
  • the cervical sample is obtained from a pregnant woman between the 4 th to the 15 th week of gestation, more preferably, between the 5 th and the 15 th week of gestation, more preferably, between the 6 th and the 13 th week of gestation, more preferably, between the 6 th and the 11 th week of gestation, even more preferably between the 6 th and the 10 th week of gestation.
  • proteolysis refers to an enzyme which breaks down proteins, i.e. conducts proteolysis.
  • proteolysis refers to protein catabolism by hydrolysis of the peptide bonds that link the amino acids in the polypeptide chain.
  • the protease used in accordance with the method of the invention is trypsin.
  • trypsin is used at a concentration of between about 0.05% and about 0.5%. In one specific embodiment trypsin is used at a concentration of about 0.25%.
  • the term "digestion” relates to treatment of the samples of the invention with a protease (in particular trypsin) for a limited period of time (e.g. between about 5 to about 45 minutes, preferably between about 10 to about 30 minutes). Such treatment results in breakdown of cell aggregates and facilitates cell separation and processing.
  • a protease in particular trypsin
  • hypotonic treatment refers to incubation of cells in a hypotonic solution which contains a lesser concentration of impermeable solutes than the cellular content. As a consequence of treating the cells with a hypotonic solution water will be acquired by the cells leading to swelling and cell rupture.
  • hypotonic solutions are salt solutions e.g. KCl (specifically 0.075M KCl).
  • fixation relates to a chemical process by which a biological sample (including cells and nuclei) is preserved from decay. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues.
  • fixative relates to a fixation agent, namely a compound capable of fixating a cell or a cell nucleus.
  • a cervical sample is fixed in a manner that preserves the structure of the chromosomes so as to allow genetic analysis.
  • the invention therefore encompasses the use of any fixative which preserves chromosomal structure. Such fixatives are commonly used in cytogenetic procedures.
  • a non limiting example of a suitable fixative in accordance with the present invention is a carnoy fixative, e.g. Methanol: Acetic Acid at a ratio of 3:1.
  • a carnoy fixative e.g. Methanol: Acetic Acid at a ratio of 3:1.
  • the fetal cells and nuclei in the sample exhibit clear FISH signals which can easily identify fetal gender and/or chromosomal abnormalities.
  • the cervical sample obtained in accordance with the present invention is subjected to a molecular analysis of the genetic material.
  • a molecular analysis utilizes an approach such as in situ chromosomal analysis, in situ DNA analysis and/or genetic analysis.
  • in situ chromosomal analysis refers to the analysis of the chromosome(s) within the cell, or the nucleus, using fluorescent in situ hybridization (FISH), and/or multicolor-banding (MCB).
  • FISH fluorescent in situ hybridization
  • MBC multicolor-banding
  • the fluorescent in situ hybridization may be performed using a probe selected from the group consisting of an RNA molecule, a DNA molecule and a PNA oligonucleotide.
  • probes e.g., the CEP X green and Y orange (Abbott cat no. 5Jl 0-51)
  • hybridization buffer e.g., LSIAVCP, Abbott
  • carrier DNA e.g., human Cot 1 DNA, available from Abbott.
  • the probe solution is applied on microscopic slides containing e.g., transcervical specimens and the slides are covered using coverslips.
  • the probe-containing slides are denatured for 4-5 minutes at 71 °C (or 3 minutes at 70 0 C) and are further incubated for 24-60 hours at 37 °C using a hybridization apparatus (e.g., HYBrite, Abbott Cat. No. 2Jl 1-04).
  • a hybridization apparatus e.g., HYBrite, Abbott Cat. No. 2Jl 1-04. It is worth mentioning that although the manufacturer (Abbott) recommends hybridization for 48 hours at 37 0 C, similar results can be obtained if hybridization lasts for 60 hours. According to certain configurations, hybridization takes place between 48-60 hours at 37 °C.
  • the slides are washed for 2 minutes at 70-72 0 C in a solution of 0.3 % NP-40 (Abbott) in 60 mM NaCl and 6 mM NaCitrate (0.4XSSC). Slides are then immersed for 1 minute in a solution of 0.1 % NP-40 in 2XSSC at room temperature, following which the slides are allowed to dry in the dark. Counterstaining is performed using, for example, DAPI II counterstaining (Abbott).
  • MMB multicolor banding
  • in situ DNA analysis refers to DNA-based analysis (e.g., primer extension) which is performed on the fetal cells or fetal nuclei, such as primed in situ labeling (PRINS) or quantitative FISH (Q-FISH).
  • PRINS primed in situ labeling
  • Q-FISH quantitative FISH
  • slides containing interphase chromosomes are denatured for 2 minutes at 71 0 C in a solution of 70 % formamide in 2XSSC (pH 7.2), dehydrated in an ethanol series (70, 80, 90 and 100 %) and are placed on a flat plate block of a programmable temperature cycler (such as the PTC-200 thermal cycler adapted for glass slides which is available from MJ Research, Waltham, Massachusetts, USA).
  • the PRINS reaction is usually performed in the presence of unlabeled primers and a mixture of dNTPs with a labeled dUTP (e.g., fluorescein- 12-dUTP or digoxigenin-11-dUTP for a direct or indirect detection, respectively).
  • a labeled dUTP e.g., fluorescein- 12-dUTP or digoxigenin-11-dUTP for a direct or indirect detection, respectively.
  • sequence-specific primers can be labeled at the 5' end using e.g., 1-3 fluorescein or cyanine 3 (Cy3) molecules.
  • a typical PRINS reaction mixture includes sequence-specific primers (50-200 pmol in a 50 ⁇ l reaction volume), unlabeled dNTPs (0.1 mM of dATP, dCTP, dGTP and 0.002 mM of dTTP), labeled dUTP (0.025 mM) and Taq DNA polymerase (2 units) with the appropriate reaction buffer. Once the slide reaches the desired annealing temperature the reaction mixture is applied on the slide and the slide is covered using a cover slip.
  • Annealing of the sequence-specific primers is allowed to occur for 15 minutes, following which the primed chains are elongated at 72 °C for another 15 minutes. Following elongation, the slides are washed three times at room temperature in a solution of 4XSSC/0.5 % Tween-20 (4 minutes each), followed by a 4-minute wash at PBS. Slides are then subjected to nuclei counterstaining using DAPI or propidium iodide. The fluorescently stained slides can be viewed using a fluorescent microscope and the appropriate combination of filters (e.g., DAPI, FITC, TRITC, FITC-rhodamin).
  • filters e.g., DAPI, FITC, TRITC, FITC-rhodamin
  • the PRINS analysis can be used as a multicolor assay for the determination of the presence, and/or location of several genes or chromosomal loci.
  • FISH also encompasses Quantitative FISH (Q-FISH) which can be used to detect chromosomal abnormalities by measuring variations in fluorescence intensity of specific probes which hybridize to chromosomal DNA.
  • Q-FISH can be performed using Peptide Nucleic Acid (PNA) oligonucleotide probes.
  • PNA Peptide Nucleic Acid
  • the hydrophobic and neutral backbone of PNA probes enables high affinity and specific hybridization to the nucleic acid counterparts (e.g., chromosomal DNA) (Pellestor F and Paulasova P, 2004; Chromosoma 112: 375-380).
  • Q-FISH can be performed by co-hybridizing whole chromosome painting probes (e.g., for chromosomes 21 and 22) on interphase nuclei as described in Truong K et al, 2003, Prenat. Diagn. 23: 146-51.
  • the method according to this aspect of the present invention can diagnose the fetus, i.e., determine fetal gender and/or paternity and identify at least one chromosomal and/or DNA abnormality of the fetus.
  • chromosomal aberration or "chromosomal abnormality” refers to an abnormal number of chromosomes (e.g., trisomy 21, monosomy X) or to chromosomal structure abnormalities (e.g., deletions, translocations, etc).
  • the chromosomal aberration or abnormality can be chromosomal aneuploidy (i.e., complete and/or partial trisomy and/or monosomy), translocation, subtelomeric rearrangement, deletion, microdeletion, inversion and/or duplication (i.e., complete an/or partial chromosome duplication).
  • the trisomy detected by the present invention can be trisomy 21 [using e.g., the LSI 21q22 orange labeled probe (Abbott cat no. 5J13-02)], trisomy 18 [using e.g., the CEP 18 green labeled probe (Abbott Cat No. 5J10-18); the CEP ® 18 (D18Z1, ⁇ satellite) Spectrum OrangeTM probe (Abbott Cat No. 5J08-18)], trisomy 16 [using e.g., the CEP16 probe (Abbott Cat. No. 6J37-17)], trisomy 13 [using e.g., the LSI ® 13 SpectrumGreenTM probe (Abbott Cat. No.
  • chromosome-specific FISH probes PRINS primers, Q-FISH and MCB staining various other trisomies and partial trisomies can be detected in fetal cells according to the teachings of the present invention.
  • trisomy lq32-44 trisomy 9p with trisomy 1Op, trisomy 4 mosaicism, trisomy 17p, partial trisomy 4q26-qter, trisomy 9, partial 2p trisomy, partial trisomy Iq, and/or partial trisomy 6p/monosomy 6q.
  • the method of the present invention can be also used to detect several chromosomal monosomies such as, monosomy 22, 16, 21 and 15, which are known to be involved in pregnancy miscarriage.
  • the monosomy detected by the method of the present invention can be monosomy X, monosomy 21, monosomy 22 [using e.g., the LSI 22 (BCR) probe (Abbott, Cat. No. 5Jl 7-24)], monosomy 16 (using e.g., the CEP 16 (D16Z3) Abbott, Cat. No. 6J36-17) and monosomy 15 [using e.g., the CEP 15 (D15Z4) probe (Abbott, Cat. No. 6J36-15)].
  • monosomy X monosomy 21, monosomy 22 [using e.g., the LSI 22 (BCR) probe (Abbott, Cat. No. 5Jl 7-24)]
  • monosomy 16 using e.g., the CEP 16 (D16Z3) Abbott, Cat. No. 6J36-17
  • monosomy 15 using e.g., the CEP 15 (D15Z4) probe (Abbott, Cat. No. 6J36-15)
  • translocations and microdeletions can be asymptomatic in the carrier parent, yet can cause a major genetic disease in the offspring.
  • a healthy mother who carries the 15ql l-ql3 microdeletion can give birth to a child with Angelman syndrome, a severe neurodegenerative disorder.
  • the present invention can be used to identify such a deletion in the fetus using e.g., FISH probes which are specific for such a deletion.
  • the present invention can be also used to detect any chromosomal abnormality if one of the parents is a known carrier of such abnormality.
  • chromosomal abnormality include, but not limited to, mosaic for a small supernumerary marker chromosome (SMC); t( 11 ; 14)(p 15 ;p 13) translocation; unbalanced translocation t(8;l l)(p23.2;pl5.5); I lq23 microdeletion; Smith-Magenis syndrome 17pl 1.2 deletion; 22ql3.3 deletion; Xp22.3.
  • SMC small supernumerary marker chromosome
  • microdeletion 1Op 14 deletion; 2Op microdeletion; DiGeorge syndrome [del(22)(ql l.2ql 1.23)], Williams syndrome [7ql 1.23 and 7q36 deletions]; Ip36 deletion; 2p microdeletion; neurofibromatosis type 1 (17ql 1.2 microdeletion); Yq deletion; WoIf- Hirschhorn syndrome (WHS, 4pl6.3 microdeletion); Ip36.2 microdeletion; I lql4 deletion; 19ql3.2 microdeletion; Rubinstein-Taybi (16pl3.3 microdeletion); 7p21 microdeletion; Miller-Dieker syndrome (17pl3.3), 17pl 1.2 deletion; 2q37 microdeletion.
  • the present invention can be used to detect inversions [e.g., inverted chromosome X, inverted chromosome 10], cryptic subtelomeric chromosome rearrangements, and/or duplications.
  • inversions e.g., inverted chromosome X, inverted chromosome 10
  • cryptic subtelomeric chromosome rearrangements e.g., cryptic subtelomeric chromosome rearrangements
  • duplications e.g., inverted chromosome X, inverted chromosome 10
  • the fetal cells or nuclei may be subjected to an additional treatment.
  • Such a treatment may include for example, exposing cell nuclei (using e.g., a methanol-acetic acid fixer), or digesting proteins (using e.g., Pepsin).
  • teachings of the present invention can be used to identify chromosomal aberrations in a fetus without subjecting the mother to invasive and risk-carrying procedures.
  • a transcervical sample is obtained from a pregnant woman at 5 th to the 15 th weeks of gestation using a Pap smear cytobrush. The samples are further processed as described in the Example section below.
  • CEP X green and Y orange probes containing the centromere regions Xpl l.l-ql l.l (DXZl) and Ypl l.l-ql l.l (DYZ3) (Abbott Cat no. 5J10-51), can be employed.
  • LSI 21q22 orange labeled probe containing the D21S259, D21S341 and D21S342 loci within the 21q22.13 to 21q22.2 region (Abbott cat no. 5J13-02), may be employed.
  • the teachings of the present invention can be used to significantly reduce the risk of having clinically abnormal babies by providing an efficient method of prenatal diagnosis.
  • the method according to this aspect of the present invention can be also used to diagnose at least one DNA abnormality in the fetus.
  • DNA abnormality refers to a single nucleotide substitution, deletion, insertion, micro-deletion, micro-insertion, short deletion, short insertion, multinucleotide substitution, and abnormal DNA methylation and loss of imprint (LOI).
  • Such a DNA abnormality can be related to an inherited genetic disease such as a single-gene disorder (e.g., cystic fibrosis, Canavan, Tay-Sachs disease, Gaucher disease, Familial Dysautonomia, Niemann-Pick disease, Fanconi anemia, Ataxia telaugiestasia, Bloom syndrome, Familial Mediterranean fever (FMF), X-linked spondyloepiphyseal dysplasia tarda, factor XI), an imprinting disorder [e.g., Angelman Syndrome, Prader-Willi Syndrome, Beckwith- Wiedemann syndrome, Myoclonus-dystonia syndrome (MDS)], predisposition to various cancer diseases (e.g., mutations in the BRCAl and BRCA2 genes), as well as disorders which are caused by minor chromosomal aberrations (e.g., minor trisomy mosaicisms, duplication sub-telomeric regions, interstitial deletions or duplications) which are
  • identification of at least one DNA abnormality is achieved by a genetic analysis.
  • genetic analysis refers to any chromosomal, DNA and/or RNA - based analysis which can detect chromosomal, DNA and/or gene expression abnormalities, respectively in a cell or a cell nucleus of the fetus.
  • major and minor chromosomal abnormalities can be detected in interphase chromosomes using conventional methods such as FISH, Q- FISH, MCB and PRINS.
  • the identification of single gene disorders, imprinting disorders, and/or predisposition to cancer can be achieved using any method suitable for identification of at least one nucleic acid substitution such as a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • Direct sequencing of a PCR product is based on the amplification of a genomic sequence using specific PCR primers in a PCR reaction following by a sequencing reaction utilizing the sequence of one of the PCR primers as a sequencing primer.
  • Sequencing reaction can be performed using, for example, the Applied Biosystems (Foster City, CA) ABI PRISM® BigDyeTM Primer or BigDyeTM Terminator Cycle Sequencing Kits.
  • diagnosing according to the present invention also encompasses determining the paternity of a fetus.
  • Current methods of testing a prenatal paternity involve obtaining DNA samples from CVS and/or amniocentesis cell samples and subjecting them to PCR-based or RFLP analyses.
  • the term "paternity" refers to the likelihood that a potential father of a specific fetus is the biological father of that fetus.
  • Paternity testing i.e., identification of the paternity of a fetus
  • Paternity testing is achieved by subjecting the fetal cell or nucleus to a genetic analysis capable of detecting polymorphic markers of the fetus, and comparing the fetal polymorphic markers to a set of polymorphic markers obtained from a potential father.
  • the method further comprises isolating the cell or fetal nucleus prior to employing an in situ chromosomal and/or DNA analysis and /or genetic analysis.
  • the term "isolating” refers to a physical isolation of a fetal cell or nucleus from a heterogeneous population of maternal and fetal cells or nuclei. Fetal cells or nuclei can be isolated in situ (i.e., from a microscopic slide containing such cells or nuclei) using, for example, laser-capture microdissection.
  • Laser-capture microdissection of fetal cells or nuclei is used to selectively isolate a specific cell or nucleus from a heterogeneous population of cells or nuclei contained on a slide. Methods of using laser-capture microdissection are known in the art (see for example, Micke P, et al., 2004. J. Pathol., 202: 130-8).
  • the teachings of the present invention can be used to detect chromosomal and/or DNA abnormalities in a fetus, fetal paternity and/or fetal gender by subjecting fetal cells or nuclei obtained from transcervical specimens to an in situ chromosomal (e.g., FISH, MCB) and/or DNA (e.g., PRINS, Q-FISH) analysis or to nucleus isolation followed by a genetic analysis method such as CGH or any PCR-based detection method, wherein the detection of the stained cell or nucleus is performed by an automated microscopy system, e.g. the Ikonoscope®.
  • an in situ chromosomal e.g., FISH, MCB
  • DNA e.g., PRINS, Q-FISH
  • the slides containing the cervical sample are subjected to FISH analysis (which detects chromosomal abnormalities). Positively stained cells or nuclei are detected by the Ikoniscope system and subsequently isolated by laser micro-dissection. Fetal DNA is isolated and can be further subjected to CGH on either metaphase chromosome derived from a normal individual (i.e., 46, XX or 46, XY) or on a CGH-array.
  • the DNA of the isolated fetal cell or nucleus is subjected to any of the PCR-based genetic analysis methods (e.g., ASO, PCR-RFLP, MS-PCR, MLPA and the like).
  • agents of the present invention which are described hereinabove for preparing the cervical sample and for detecting chromosomal and/or DNA abnormality may be included in a diagnostic kit/article of manufacture preferably along with appropriate instructions for use and labels indicating FDA approval for use in analyzing the genetic material of the fetus.
  • Such a kit can include, for example, at least one container including a protease, a fixative, and a reagent for molecular analysis of the genetic material of the fetus.
  • a reagent can be a FISH probe, a PCR primer for genetic analysis, a PNA probe for Q-FISH and the like.
  • the kit may also include appropriate buffers and preservatives for improving the shelf-life of the kit.
  • the kit may further include DAPI counter stain agents.
  • the present invention provides noninvasive prenatal diagnosis of chromosomal aberrations by analysis of fetal cells that can be accessed in cervical smear samples.
  • the inventors have shown that a FISH-based scanning approach can identify trisomy 21 pregnancies by analysis of routine cervical brushings.
  • Example 1 Identification of XY fetal cells in cervical swabs
  • Cervical samples are obtained during the second half of the first trimester of pregnancy.
  • the endpoint analysis is FISH based scanning using either XY based detection of fetal cells from a male fetus or autosome probe based detection of fetal cells in cases of specific aneuploidies e.g. trisomy 21. Clusters of fetal cells derived from the cervical mucous plug are systematically broken up in the procedure.
  • Cervical samples are collected by the obstetrician.
  • the collecting brush is placed in a vial containing 5 ml RPMI medium containing antibiotics and antifungals (e.g. Penicilin/Sterptavidin/amphotericin B) and transported to a laboratory with minimal delay, preferably within less than 4 hours or up to 24 hours if sample is refrigerated.
  • antibiotics and antifungals e.g. Penicilin/Sterptavidin/amphotericin B
  • A) Cells are resuspended in 2 ml 0.075M KCl by carefully pipetting up and down. 8 ml 0.075M KCl is added and mixed. The mixture is incubated for 5 to 60 minutes at room temperature or at 37°C in a water bath.
  • Pellet is resuspended in 2 ml Carnoy fixative, and pipetted up and down to break remaining cells and clusters.
  • the cells are washed three times by repeated centrifugation and resuspension in Carnoy fixative (total volume of 10 ml). The centrifugation is performed for 8-10 min with 60Og at 20 0 C, supernatant is remove by aspiration
  • Sample may now be deposited on slides.
  • Chamber slides (Ikonislide ROCO) containing Carnoy fixed cells are stained according to the following protocol: 1.1 Equilibration in PBS
  • Nuclei are fixed on slides in 2% Formaldehyde, 5OmM MgCl 2 in PBS for 10 min.
  • the slides may now be subjected to hybridization with several types of probes.
  • Non-limiting examples include CEP Y and CEP X probes.
  • the hybridization procedure is performed according to protocols well known in the art.
  • DAPI forms fluorescent complexes with natural double-stranded DNA
  • nuclei containing XY chromosomes male nuclei was detected in 60% of the male samples (10 out of 16).
  • Example 2 Prenatal diagnosis of trisomy 21 through detection of trophoblasts in cervical smears
  • a speculum was introduced into the vagina to visualize the uterine cervix. If necessary, the surface of the cervix was wiped clean using a swab.
  • a commercially available cytobrush (Cooper Surgical, Norwalk, CT) was then inserted through the external os to the depth of the brush (1.5 cm to 3.0 cm). Following two full turns the brush was taken out and immersed into the medium containing antibiotic and antifungal agents, and the sample was shaken off briskly. Any remaining mucus was dislodged from the brush using a pair of forceps. All the fetal samples collected were pre- procedural.
  • FISH FISH was then undertaken, essentially as described previously (Kilpatrick et al., 2004, Seppo et al., 2008), using a probe cocktail containing dual-FISH probes for the chromosome of interest, chromosome 21.
  • the FISH probe cocktail comprised a probe for the alpha-repeat region of the X-chromosome, labeled with cy5, a probe for the Y-chromosome alpha- repeat, labeled with SpectrumAqua, and 2 independent chromosome 21 probes; a probe for the AML region, labeled with SpectrumGreen and a 2IqI l probe, labeled with SpectrumOrange.
  • Slides were inspected on a manual fluorescence microscope to ensure adequate signal. Slides were then analyzed using an automated system developed specifically for the rare cell detection and analysis (Kilpatrick et al., 2004; Evans et al., 2006; Wauters et al., 2007). Slides are initially imaged in the 2 chromosome 21 channels with a 2Ox objective, to identify potential trisomy 21 cells. Trisomic cells are then verified by imaging in all channels, with a 10Ox objective.
  • trisomy 21 pregnancies Analysis of 5 samples obtained from trisomy 21 pregnancies identified trisomic cells in all 5 cases. The number of trisomy 21 cells detected ranged from 1 - 27 (Table II). Two examples of the trisomy 21 cells detected, one from a male trisomy 21 fetus t pregnancy and 1 from a female trisomy 21 fetus pregnancy are shown in Figure 1. A set of 28 first trimester control samples from normal fetus pregnancies was also analyzed. In the 28 samples, a single trisomic cell was found in one of the samples To determine the background level of trisomy 21 cells in cervical samples from nonpregnant women, a set of 10 samples were analyzed. In two samples, 1 and 2 trisomic cells were detected respectively.

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Abstract

La présente invention concerne un procédé pour l'analyse par un système microscopique informatisé d'échantillons cervicaux colorés par hybridation in situ en fluorescence (FISH) obtenus à partir de femmes enceintes. Le procédé est destiné au diagnostic prénatal et au criblage de matériau génétique fœtal, particulièrement des noyaux fœtaux. Le procédé selon la présente invention combine un prélèvement hautement efficace et une procédure de préparation cytogénétique qui permet d'obtenir un matériau génétique fœtal de grande qualité (par exemple, des noyaux de cellules fœtales) aptes à l'analyse génétique et au criblage, par exemple, au moyen de techniques FISH, grâce à l'utilisation d'un système de microscope informatisé (par exemple, Ikoniscope®). Le procédé est utile en particulier dans l'analyse du genre fœtal et d'anomalies chromosomiques fœtales, par exemple, la trisomie 21.
PCT/IL2010/000430 2009-06-01 2010-06-01 Procede pour la detection controlee par ordinateur de noyaux de cellules fœtales dans des echantillons cervicaux WO2010140145A1 (fr)

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