HIGH-THROUGH METHODS FOR DETECTING CHROMOSOMIC ABERRATIONS AND / OR TELOMERIC ABERRATIONS

Description

[0001] This application relates to a high-throughput method for detecting chromosomal aberrations and / or telomere aberrations.

[0002] Over the past decade, numerous research studies have demonstrated the crucial role of telomeres in genome integrity and stability. With each cell division, the telomeres at the ends of each chromosome shorten. When telomeres become too short, and before genes are affected or chromosomes fuse, the cell stops dividing and enters senescence. Telomeres are therefore considered a biological clock that governs cellular aging. It is now well-documented that telomere length is an important biomarker in age-related diseases such as cancers and atherosclerotic cardiovascular diseases. [Hubert J et al, Circ Res. 16;122(4):616-623 2018; Staerk L et al, J Am Heart Assoc. 2017 Nov 14;6(11)].

[0003] Determining telomere length and chromosomal aberrations not only has practical implications for clinicians in the therapeutic management of patients but also opens up new fields of research and applications in clinical research, and in particular during therapeutic trials.

[0004] The study of telomere length is therefore crucial for understanding cellular mechanisms, patient assessment, and management. To date, several analytical techniques exist.

[0005] Terminal restriction fragmentation (TRF) analysis was the first method developed to determine telomere length and is often considered the gold standard. This method allows for the estimation of the average number of terminal repeats carried by all chromosomes. The procedure involves digesting genomic DNA with one or more restriction enzymes. The fragments are then separated by electrophoresis according to their size. The size of the terminal fragments is estimated by comparison with fragments of known length. [Allshire, Dempster & Hastie, Nucleic Acids Res. 1989 Jun 26;17(12):4611-27]. This method has the disadvantage of being lengthy and tedious and requires large quantities of DNA (>1 µg). Furthermore, it yields an average length value rather than detecting individual telomere shortening or absence.

[0006] Quantitative PCR techniques, such as MMqPCR and aTL qPCR, were developed to overcome the limitation of using large quantities of DNA. However, one of the main drawbacks of this method is the significant variation in measurements observed between different aliquots. Furthermore, as with the TRF method, the measured values ​​represent an average.

[0007] The Q-FISH method (fluorescence in situ hybridization, possibly combined with flow cytometry, allows visualization of telomeres by hybridization using a fluorescence probe on metaphases. [Lansdorp et al. Hum Mol Genet. 1996 May;5(5):685-91.]). The advantage of this technique is that it allows estimation of the individual length of each of the 184 human telomeres and is not limited to average or short telomeres. However, the quantification method on metaphases was very laborious to implement, and the lengths are expressed in relative fluorescence units.

[0008] In 2007, Maria Blasco's team [Canela et al. Proc Natl Acad Sci US A. 2007 Mar 27;104(13):5300-5] described a new technique based on Q-FISH but using interphase nuclei with a 63X magnification objective for overall telomere quantification and a 96-well plate for cell cultures and hybridizations. However, this technique does not allow for telomere quantification of each chromosome or the detection of telomeric aberrations. Furthermore, the technique developed by Maria Blasco's team remains lengthy and laborious, as it only allows for the analysis of approximately 1000 cells in 2 hours.

[0009] Ioanna Karachriston et al (Mutation Research 793(2015) 107-114) describe the use of the "premature chromosome condensation (PCC)" technique by the fusion of human lymphocytes in G0 with mitotic CHO (Chinese Hamster Ovary) cells using a mitogen associated with telomere and centromere labeling for the enumeration of chromosomal aberrations without going through the cell culture phase.

[0010] This technique is very difficult to introduce into routine clinical practice and does not allow the quantification of human lymphocyte telomeres because the sample also contains CHO chromosomes with a telomere size very different from that of human lymphocytes.

[0011] Furthermore, this article does not teach or suggest the overall quantification of telomere length by observation of the total fluorescence intensity of interphase nuclei.

[0012] Zafiroponlous et al. (Int. J. Mod. Phys. Conf. Ser. 2016.44) also describe the use of the "premature chromosome condensation (PCC)" technique associated with telomere and centromere labeling for counting chromosomal aberrations in the context of biological triage dosimetry after accidental irradiation and the need to have the dose absorbed as quickly as possible.

[0013] This document does not describe or suggest the global quantification of telomeres by observation of the total fluorescence intensity of interphase nuclei.

[0014] WO 97 / 014026 describes the Q-FISH technique which consists of quantifying the intensity of telomere fluorescence in metaphase cells after labeling of telomeres and centromeres with PNA type probes.

[0015] CN 108 004 299 describes a Q-FISH method that uses genomic DNA instead of cytogenetically fixed cells (see paragraphs

[0001] ,

[0012] ,

[0013] of this document). This document also describes a telomere-specific PNA probe with a particular sequence. This document does not describe a centromere probe.

[0016] Regarding the detection of chromosomal aberrations, homogeneous chromosome labeling remains the most widely used conventional technique in clinical cytogenetics (chromosome classification), as well as in genetic toxicology and biological dosimetry. However, this technique is time-consuming and requires highly qualified personnel. Indeed, it relies on drawing blood from a heparin-lithium tube of at least 5 ml. This procedure necessitates the patient's visit to the blood collection room or a medical analysis laboratory.

[0017] Cytogenetic approaches, combined with in situ hybridization using DNA probes directed against an entire chromosome or a specific gene, remain the most sensitive and reliable approaches for diagnosing patients and studying the clastogenic effects of chemicals. However, this technique is expensive due to the cost of DNA probes and the hybridization time.

[0018] Furthermore, genomic approaches (transcriptome, CGH-array, SNP, NGS) have been proposed as an alternative for detecting genome alterations. However, these techniques, like other genomic approaches, lose their sensitivity when genome alterations affect repetitive sequences (telomeric and centromeric sequences).

[0019] Therefore, there is still a need to develop a high-throughput method to detect genome aberrations affecting both unique and repetitive sequences using a very small number of cells.

[0020] The present invention aims to provide a high-throughput method for detecting both chromosomal and telomere aberrations using a biological sample of 150µl to 200µl, said method comprising: the preparation of a cytogenetic slide from said sample in a microplate, said cell culture being carried out in a microplate well, the ratio of the amount of culture medium to the surface area of ​​the well being 1 ml / cm² to 1.5 ml / cm², the mitotic index in said cytogenetic slide being on average 3 times higher than that of the conventional procedure of culturing cells in flasks with 10 to 20 ml of medium, the simultaneous labeling of telomeres and centromeres by peptide nucleic acid probes with a hybridization time of 30 minutes to 1.5 hours.image flow quantification of telomere fluorescence intensity on interphase nuclei using a 10x magnification objective for global telomere quantification, possibly, quantification of micronuclei and anaphase bridges using a 10x or 40x magnification objective, and automatic capture of metaphase chromosomes to detect chromosomal aberrations and / or telomere aberrations of each chromosome with a 63x magnification objective.

[0021] A chromosomal aberration is defined as an abnormality in the number or structure of a chromosome that leads to an alteration of the karyotype. Examples of structural chromosomal aberrations include dicentric chromosomes, centric rings, acentric chromosomes, chromosomal translocations, isochromosomes, insertions, and deletions. An example of a numerical abnormality is extra chromosomes.

[0022] The method of the invention also makes it possible to detect micronuclei and anaphasic bridges, which are part of chromosomal aberrations.

[0023] The term "micronuclei" refers to fragments of chromosomes or entire chromosomes expelled from the cell nucleus during mitosis and forming small, well-defined entities in the cytoplasm of cells in interphase.

[0024] An anaphasic bridge is defined as DNA in the form of filaments between two daughter cells with or without telomeric and centromeric sequences; an anaphasic bridge indicates the presence of a dicentric chromosome, which is an important marker of chromosomal instability or exposure to a genotoxic agent.

[0025] Telomere aberrations are defined as abnormalities affecting the region containing the telomere repetition sequence located in the terminal regions of chromosomes. Examples of telomere aberrations include the loss of a telomere, the loss of two telomeres on the same arm (deletion), and the formation of telomere duplications.

[0026] The term "mitotic index" refers to the percentage of cells undergoing mitosis in a given cell sample, relative to the total number of cells.

[0027] The mitotic index reflects the rate of cell division.

[0028] The term "mitotic index in said cytogenetic slide being 3 times higher on average than that of the conventional procedure" means the mitotic index measured in a cytogenetic slide of the present invention prepared according to the method described below being 3 times higher on average than that obtained by a conventional method in the same sample from the same subject and implemented by the same practitioner with the same equipment.

[0029] Global telomere quantification refers to the quantification, from images of telomere fluorescence intensity on interphase nuclei obtained with a 10x magnification objective, to obtain data consisting of: the average telomere length and the median telomere length, the frequency of cells with short telomeres, the statistical distributions in order to determine intercellular and interindividual heterogeneities, the biological age of the sample calculated from a standard curve of healthy donors.

[0030] The raw telomere fluorescence intensities on interphase nuclei are normalized to those obtained on control slides and converted to kilobase using a standard curve.

[0031] The overall quantification of telomeres is carried out by a specific software to obtain the aforementioned data (TeloScore).

[0032] Automatic metaphase chromosome capture refers to the automatic quantification, from an image, of the fluorescence intensity of telomeres and centromeres of metaphase chromosomes to obtain data consisting of: the identification of each chromosome based on its size, said size being defined by the distance between the telomeres of the short arm (p) and those of the long arm (q) of the chromosome as well as by the ratio between the size of the p part and the q part of the chromosome (centromeric index), the quantification of the number of centromeres, in order to analyze only the complete metaphases, the quantification of the signal of each telomere of each chromosome in metaphase, i.e. 4 signals per chromosome and 184 signals expected per diploid metaphase).

[0033] This automatic capture allows: Chromosome classification; detection of telomere loss and deletion of each chromosome; detection of structural chromosomal aberrations such as dicentric chromosomes, centric rings, acentric rings and different types of acentric chromosomes; For the detection of simple chromosomal rearrangements, DAPI banding, similar to GTG banding, completes chromosome identification; For the detection of complex rearrangements, M-FISH type labeling, performed on the same metaphases, allows the creation of a multicolor karyotype which makes the analysis more reliable and sensitive.

[0034] Automatic analysis of metaphase chromosomes is performed by specific software (ChromoScore).

[0035] The present invention is implemented through the development of the following techniques: The preparation of a cytogenetic slide in which the proportion of metaphase cells is higher than in a conventional cytogenetic slide; the development of a telomere and centromere labeling method that significantly reduces labeling time compared to a conventional technique requiring 4 to 6 hours of work; the use of a 10x magnification microscope objective that allows for the global quantification of telomeres in 10,000 interphase cells in less than 2 minutes; the automatic capture of metaphases allowing for successive labeling on the same metaphases, as well as automation in the counting of telomeric or chromosomal aberrations; and the automatic detection of micronuclei and anaphase bridges on mononuclear cells on the same cytogenetic slide.

[0036] The present process offers several technical advantages over existing methods.

[0037] First, the method of the invention is implemented with a very small quantity of cells. For example, only 150 µl to 200 µl of whole blood is needed, in other words, a quantity of blood that can be obtained by pricking the index finger using the same principle as measuring blood glucose. This reduction in sample volume facilitates collection and considerably saves analysis time and reagents.

[0038] Secondly, and somewhat unexpectedly, the Inventor observed for the first time that the surface area to volume ratio of a culture vessel can influence the mitotic index, and that the surface area to volume ratio of a microplate well is particularly favorable for increasing this index and producing very rich and easily analyzed cultures. Using a microplate for culturing the sample not only reduces the required sample quantity and culture time, but also allows for the preparation of a cytogenetic slide with a higher mitotic index than a conventional cytogenetic slide. This enables, on the one hand, more reliable cytogenetic analysis and, on the other hand, simultaneous labeling of telomeres and centromeres on interphase and metaphase cells.

[0039] This approach allows for the rapid acquisition of information regarding both the overall quantification of telomeres on interphase nuclei and information on each chromosome in order to detect chromosomal and telomere aberrations using metaphases. The method of the present invention makes it possible, on the one hand, to establish a relationship between telomere length and the frequency of chromosomal aberrations on the same slide, and on the other hand, to perform chromosome classification (karyotyping) based on chromosomes in metaphase and to search for potential genetic mutations. It also allows for the detection of anaphase bridges indicating the presence of a dicentric chromosome, which is an important marker of chromosomal instability or exposure to a genotoxic agent. Preparation of a cytogenetic slide

[0040] A "cytogenetic slide" is defined as a transparent slide, such as a glass slide, suitable for observation by microscopy, on which the cells to be analyzed are spread and fixed in metaphase and interphase. The mitotic index, which provides an indication of the slide's metaphase richness, has been calculated by the present invention and by the conventional cytogenetic method on the same samples. The ratio of the mitotic index is between 1.5 and 4 between a cytogenetic slide prepared by the present invention and one prepared by the conventional cytogenetic technique.

[0041] In a sample prepared according to a conventional method, a cell suspension culture is carried out in a container with a volume of 10 to 20 ml.

[0042] The increase in the mitotic index is correlated with the increase in the proportion of cells in metaphase.

[0043] Since metaphase chromosomes are more condensed and more visible, the qualitative and quantitative increase in the proportion of metaphase chromosomes not only improves the detection of chromosomal aberrations but also automates the counting of chromosomal aberrations.

[0044] For the purposes of this invention, "biological samples" means preparations of biological fluids or tissues from an animal containing cells to be analyzed. Preferably, these are blood samples, amniotic fluid in the case of prenatal diagnosis, or bone marrow for hematological malignancies. Preferably, the animal is a mammal, and more favorably a human, regardless of sex or age. It may be an embryo, a fetus, a newborn, a child, an adolescent, or an adult.

[0045] In an advantageous embodiment, said biological sample is a sample of whole blood, bone marrow, or a sample of tissue cells.

[0046] The said whole blood sample can be obtained using the same principle as obtaining a few drops of blood for measuring blood glucose.

[0047] The tissue cell sample may be obtained from a tissue fragment, whether tumorous or non-tumorous, which may be taken from a patient by biopsy. The tissue cell sample may be a primary culture or a secondary culture.

[0048] According to the method of the present invention, the step of preparing the cytogenetic slide comprises: the culture of cells in a microplate well, the ratio between the quantity of culture medium and the surface area of ​​the well being 1ml / cm 2< to 1.5ml / cm 2< .

[0049] More specifically, said microplate may be a 24-well microplate, the working volume of each well being 0.5 ml to 1.5 ml.

[0050] The cytogenetic slide prepared from this culture has a mitotic index 3 times higher than a slide prepared by a conventional method performed by the same practitioner using the same initial biological sample and the same experimental equipment.

[0051] Cell culture can be performed with a conventional liquid culture medium for 72 hours. This culture medium can be RPMI1640. After culture, the cells are treated with a mitotic spindle inhibitor, for example colchicine, to block the cell cycle of the chromosomes in metaphase.

[0052] The cells then undergo a hypotonic shock which causes the cells to swell due to the difference in osmotic pressure.

[0053] The cellular components are then fixed using a fixative, for example an acetic acid / methanol mixture. The volume ratio between acetic acid and methanol in such a mixture can be 1V / 3V.

[0054] The aforementioned cell treatments can be performed directly in a microplate well. Marking method

[0055] In an advantageous embodiment of the process of the present invention, the simultaneous labeling of telomeres and centromeres by peptide nucleic acid probes comprises: a single step of treatment of a cytogenetic slide for 1 to 3 minutes, in particular 2 minutes, with a 3-5% formaldehyde solution, in particular a 4% solution.

[0056] Unlike a conventional method which requires multiple applications of formaldehyde fixation, the process of the present invention requires only one treatment step with a formaldehyde solution.

[0057] This treatment allows the three-dimensional structure of the chromosomes to be fixed.

[0058] In a more advantageous embodiment of the process of the present invention, the simultaneous labeling of telomeres and centromeres by peptide nucleic acid probes further comprises, after the formaldehyde treatment step, a single step of treatment of the cytogenetic slide with pepsin for 3-5 minutes, in particular 4 minutes, by immersing a cytogenetic slide previously treated with formaldehyde in a pepsin solution at a concentration of 0.1-0.2ug / ml.

[0059] Pepsin treatment hydrolyzes nuclear proteins and facilitates probe access.

[0060] In a more advantageous embodiment of the process of the present invention, the simultaneous labeling of telomeres and centromeres further comprises, after the pepsin treatment step, the following steps: the dehydration of the cytogenetic slide successively for 1 minute by an aqueous solution of 50% ethanol, an aqueous solution of 70% ethanol and pure ethanol, the denaturation of the chromosomal DNA present on the aforementioned cytogenetic slide obtained at the end of the previous step and the denaturation of the peptide nucleic acid probes for telomeres and centromeres, the hybridization for 10-30 minutes, in particular 20 minutes, at room temperature, between the denatured chromosomal DNA and the denatured peptide nucleic acid probes obtained in the previous step, the successive washings of said cytogenetic slide after hybridization.

[0061] In a particularly advantageous embodiment of the process of the present invention, the simultaneous labeling of telomeres and centromeres by peptide nucleic acid probes comprises or consists of the following steps: the treatment of a cytogenetic slide for 1-3 minutes, in particular 2 minutes, with a 3-5% formaldehyde solution, in particular a 4% formaldehyde solution, the treatment of the cytogenetic slide obtained at the end of the previous step with pepsin for 3-5 minutes, in particular 4 minutes, by immersing said cytogenetic slide in a pepsin solution at a concentration of 0.1-0.2 ug / ml, the dehydration of the cytogenetic slide obtained at the end of the previous step successively for 1 minute by an aqueous solution of 50% ethanol, an aqueous solution of 70% ethanol and pure ethanol, the denaturation of the chromosomal DNA present on the cytogenetic slide obtained at the end of the previous step and the denaturation of the peptide nucleic acid probes for telomeres and centromeres, the hybridization for 10-30 minutes, in particular 20 minutes, at room temperature, between the denatured chromosomal DNA and the denatured peptide nucleic acid probes obtained in the previous step, the successive washings of said cytogenetic slide obtained at the end of the previous step.

[0062] Compared to the conventional technique which requires a day's work, the simultaneous labeling of telomeres and centromeres with the method of the present invention can be carried out in 1 hour while maintaining a signal intensity comparable to that obtained by the conventional technique.

[0063] A "peptide nucleic acid probe" is an artificial oligomer similar to a DNA or RNA molecule, whose backbone consists of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds, with purine and pyrimidine bases attached to the backbone by methylcarbonyl bonds. Such a probe has 5 to 30 nucleic acid bases.

[0064] The sequences of peptide nucleic acid probes are complementary to the repetitive sequence in the telomeric region or the repetitive sequence in the centromeric region, respectively. Therefore, peptide nucleic acid probes hybridize to telomeres or centromeres in these regions.

[0065] Peptide nucleic acid probes are labeled with a fluorochrome, such as cyanine-3 derivatives, rhodamine derivatives, or fluorescein derivatives. The fluorescence density emitted by peptide nucleic acid probes hybridizing to telomeres therefore allows for the quantification of telomere length.

[0066] The labeling step of the present invention enables the labeling of telomeres and centromeres with a very strong signal in a hybridization time of 30 minutes to 1.5 hours. The simultaneous labeling of telomeres and centromeres allows for the rapid definition of a chromosome by its centromere and the chromosomal territory by its telomeres, thus making the counting of chromosomal aberrations and the classification of chromosomes more reliable, robust, and independent of the person performing the analysis.

[0067] In a particular embodiment of the process of the present invention, a first peptide nucleic acid probe labels the telomeres and a second peptide nucleic acid probe labels the centromeres, the first probe and the second probe emitting a distinctive fluorescence respectively.

[0068] The sequence of the 1st peptide nucleic acid probe is complementary to the repetitive sequence in the telomeric region.

[0069] The sequence of the 2nd peptide nucleic acid probe is complementary to the repeating sequence in the centromeric region. Quantification of telomere length using image stream

[0070] Unlike the quantification of fluorescent signals in cytogenetics, which has always been performed with a 63X magnification microscopic objective, the present method uses a 10X objective for the overall quantification of telomeres by observing the total fluorescence intensity of interphase nuclei. This allows for the calculation of the mean and median telomere lengths, as well as the heterogeneity within the sample. Observation is performed with a 10X objective, providing a large field of view and a depth of field at least equivalent to the sample thickness. This enables the analysis of a large number of cells while maintaining precision and sensitivity, and avoiding saturation problems in the capture of a fluorescent signal. The speed of the acquisition and analysis system makes this measurement particularly advantageous.With this technique, 10,000 cells can be analyzed in less than 2 minutes. It also provides a histogram of telomere length frequency and data on cell morphology (irregularities, roundness, concavity, etc.) as a function of telomere length or the amount of 4',6-diamidino-2-phenylindole (DAPI) used to label the chromosomes. Internal controls can be introduced with each staining. The sample consists of two lymphoblastoid cell lines with well-established cytogenetic and telomeric characterization. The telomere length of these two cell lines has been measured using several other telomere quantification techniques (TRF, PCR, and Flow FISH). These two controls allow us to convert the fluorescence intensity into kilobase units. In addition to this internal control, there is an external control using fluorescent beads with a precisely defined intensity.This calibration slide is designed to verify the fluorescence intensity of the capture system, including the fluorescence intensity of the lamp. The fluorescence intensity will be adjusted before each capture by modifying several parameters such as the lamp intensity or the camera gain (TeloScore). Detection of micronuclei and anaphasic bridges :

[0071] The detection of micronuclei and anaphasic bridges is performed automatically by fluorescence microscopy using software (BridgeScore) on mononuclear cells. For micronuclei, telomere and centromere labeling allows for the determination of their nature. Micronuclei detected with only telomeric sequences correspond to terminal deletions linked to exposure to a clastogen. Micronuclei detected with both telomeric and centromeric sequences correspond to the elimination of an entire chromosome, which is linked to exposure to an aneugenic agent.

[0072] Anaphasic bridge detection involves the presence of a DNA strand connecting two daughter cells, called an anaphasic bridge. The length of these bridges is measured, as well as the presence or absence of centromeric or telomeric sequences. The presence of telomeric or centromeric sequences in the anaphasic bridge indicates that the dicentric chromosome resulted from the fusion of two chromosomes with telomeric dysfunction. The number of cells that are stuck together is also automatically assessed, as this can indicate a specific configuration of dicentric chromosomes (the two centromeres are very close). In this case, the size of the anaphasic bridge is very small, and the two daughter cells appear stuck together, like binucleated cells. Automatic capture of metaphase chromosomes

[0073] The search and capture of metaphases are performed automatically by fluorescence microscopy using software with a well-defined capture integration time.

[0074] The different parts of the chromosomes are marked by different fluorochromes. Chromosomes can be marked by different fluorochromes, each of which binds strongly to certain nucleic bases: for example, centromeres are marked by FITC carried by a peptide nucleic acid probe targeting centromeres, telomeres are marked by cyanine 3 carried by a peptide nucleic acid probe targeting telomeres, and chromosome backbones are marked by DAPI (4',6-diamidino-2-phenylindole).

[0075] This chromosome marking makes it possible to determine a coding for each chromosome as well as for each aberration and the development of software.

[0076] Using this specialized software, the chromosome skeleton, the distance of each telomere from the end of the skeleton (distance D), and the distance between each telomere and the centromere (distance d) are calculated. The distances d and D are used to identify chromosome pairs based on their size and also on the D / d ratio.

[0077] Parameters such as the number of centromeres, the number of telomeres detected per chromosome, the number of telomeres detected per cell, the sum of fluorescence intensity, the mean of fluorescence intensity, the medium of fluorescence intensity, and the p / q ratio of intensities are calculated.

[0078] This information allows us to classify chromosomes and to indicate chromosomal aberrations or telomere aberrations.

[0079] The method of the present invention can also be combined with other cytogenetic techniques, such as M-FISH (multiplex fluorescence in situ hybridization) and NGS (Next-Generation Sequencing)-exome sequencing, which allows the study of protein-coding regions in the genome. For example, combining the method of the present invention with NGS-exome sequencing can target only the coding regions of the genome involved in rearrangements detected after labeling telomeres and centromeres followed by M-FISH staining. This approach enables targeted NGS.

[0080] Another object of the present invention relates to detection kits.

[0081] The invention proposes a high-throughput detection kit for telomere quantification and the detection of chromosomal aberrations and / or telomere aberrations, comprising: a ready-to-use solution of peptide nucleic acid probes for telomeres and centromeres, wash buffers needed for hybridization, standard slides for telomere quantification, standard slides to control the fluorescence intensity of the microscope.

[0082] The standard slides for telomere quantification are reference slides prepared from lymphoblastoid cell lines derived from healthy subjects. Telomere length was measured using the reference technique TRF to obtain the length in kilobases. These cell lines do not contain any chromosomal aberrations.

[0083] Standard slides for controlling the fluorescence intensity of the microscope are slides with fluorescent beads calibrated with a well-defined intensity.

[0084] The present invention also relates to a method for detecting chromosomal aberrations, said method comprising: the labeling of telomeres and centromeres of metaphase cells with a ready-to-use solution of nucleic acid probes, the automatic capture of fluorescent signals from telomere and centromere labeling, the analysis of the image obtained in the previous step to obtain all the data: counting the number of centromeres in metaphase, identifying each chromosome from its size and the ratio between the short arm (p) and the long arm (q), and quantifying the signal of each telomere of each chromosome in metaphase, the detection of structural chromosomal aberrations such as dicentric chromosomes, centric rings, acentric rings and the different types of acentric chromosomes, possibly DAPI banding, similar to GTG banding, to complete chromosome identification and detect simple translocations,Optionally, M-FISH staining can be performed on the same metaphases to create a multicolor karyotype for detecting complex rearrangements, making the analysis more reliable and simpler.

[0085] In one embodiment, the metaphase cells are those contained in a cytogenetic slide prepared according to the method of the present invention described above.

[0086] The present invention also relates to a method for detecting micronuclei and anaphasic bridges, said method comprising: (i) labeling of telomeres and centromeres of interphase cells with a ready-to-use solution of nucleic acid probes, (ii) automatic capture of fluorescent signals from telomere and centromere labeling, (iii) analysis of the image obtained in step (ii) to obtain the complete data: the total number of micronuclei, the number of micronuclei with only telomeric sequences and the number of micronuclei with telomeric and centromeric sequences (iv) analysis of the image obtained in step (ii) to obtain the complete data: identification of anaphase bridges by detection of the presence of a DNA filament linking two daughter cells, the length of the bridge, detection of the presence or absence of centromeric or telomeric sequences in the bridge, counting the number of stuck cells (very reduced bridge).

[0087] Micronuclei can be identified if there is the presence of only telomeric sequences, which corresponds to terminal deletions linked to exposure to a clastogenic agent, or the simultaneous presence of telomeric and centromeric sequences, which corresponds to the elimination of an entire chromosome which is linked to exposure to an aneugenic agent.

[0088] The presence of anaphasic bridges is correlated with the detection of a DNA strand linking two daughter cells. The length of these bridges is measured, as well as the presence or absence of centromeric or telomeric sequences within the bridge. The presence of these sequences in the anaphasic bridge indicates that the dicentric chromosome originates from the fusion of two chromosomes with telomeric dysfunction. The number of cells that are joined is also investigated and can indicate a specific configuration of dicentric chromosomes (the two centromeres are very close together).

[0089] This method can be performed automatically by fluorescence microscopy using software (BridgeScore).

[0090] The present invention is further explained by the figures and examples below. Figures

[0091] Figure 1 : this figure illustrates a diagram of the high-throughput method for telomere quantification, micronucleus and anaphasic bridge detection and chromosomal and telomeric aberration detection of the present invention. Figure 2 This figure shows a comparison between the quality and quantity of metaphases obtained using the microplate technique and the conventional technique. Images of metaphases from conventional cultures (top frame) and images of metaphases from microplate cultures (bottom frame). This comparison was made under identical conditions with the same cell sample. Figure 3A :This figure shows the comparison of the fluorescence intensity of 6 µm calibration beads measured by a 10x microscopic objective or a 63x microscopic objective with different intensities (100%, 33% and 10%). Figure 3B : this figure shows the distribution of cell fluorescence intensity in quartiles measured by a 10x microscopic objective or a 63x microscopic objective. Figure 4 : This figure shows the comparison of telomere quantification on metaphases (Q-FISH) and on interphases (present invention) using four cell lines. Figure 5 : this figure shows the comparison between the telomere quantification measured by the process of the invention and the result obtained by TRF (A) and the result obtained by PCR (B). Figure 6This figure shows the correlation between the average telomere length measured by the present invention and age in a cohort of 420 healthy donors. The average length is expressed in kb. Figure 7 : reproducibility of the process of the invention with two different measurements of the same sample comprising 420 donors. Figure 8 This figure shows the frequency of micronuclei in healthy donors and cancer patients: (A) all micronuclei; (B) the difference between micronuclei with only telomeric sequences detected by the present invention between patients and donors. Figure 9This figure shows the detection of anaphasic bridges in interphase cells from cancer patients. The method of the invention makes it possible to distinguish between fairly long anaphasic bridges (white arrow) with telomeric and / or centromeric sequences corresponding to the presence of a dicentric chromosome resulting from the fusion of two chromosomes, or very short anaphasic bridges (grey arrow) indicating the presence of a dicentric chromosome with two very close centromeres. Figure 10 This figure shows the detection, using the method of the invention, of an extra marker chromosome corresponding to a centric ring in a prenatal sample. This aberration cannot be detected by the RBG technique or the M-FISH technique alone. Figure 11 :This figure illustrates the detection of a chromosomal translocation and the identification of its nature by conventional techniques (top figure), by chromosome in situ hybridization, by M-banding, and by the method of the invention (bottom figure) within the context of prenatal testing. A decision by genetic counseling will be made if the translocation is not balanced. Because the translocation affects the telomeric portion of chromosome 10q, it was not detected by conventional or molecular cytogenetics. The present invention enables the detection of telomeric sequences and confirmation of the nature of the translocation. Figure 12 : This figure shows the detection of chromosomal aberrations in metaphase cells after labeling of telomeres and centromeres: dicentric chromosomes, centric rings and acentric chromosomes. Figure 13 :This figure shows that sequential analysis after labeling of telomeres and centromeres associated with the M-FISH technique allows for very precise classification of chromosomes and also detection of chromosomal aberrations, such as the dicentric chromosome with a very specific configuration, a chromosomal aberration difficult to detect by a conventional technique. Examples 1. Materials and methods Cell lines

[0092] The cell lines tested in the examples are lymphoblastic lines derived from healthy donors; tumor lines from Hodgkin lymphoma, Burkitt lymphoma, and mantle cell lymphomas were used. Q-FISH Test

[0093] The Q-FISH assay, which allows visualization of telomeres by hybridization using a fluorescence probe on metaphases, is implemented according to the method described in [Lansdorp et al. Hum Mol Genet. 1996 May;5(5):685-91.] TRF Test

[0094] TRF analysis is implemented according to the technique described in [Kimura et al., Nat Protoc. 2010 Sep;5(9):1596-607] qPCR test

[0095] The qPCR test is implemented according to the method described in [OCallaghan & Fenech, Clin Nutr. 2012 Feb;31(1):60-4]. Telomere and centromere labeling

[0096] A protocol for marking telomeres and centromeres is illustrated below. 1. Wash the cytogenetic slides in a first phosphate-buffered saline (PBS) solution for 1 minute at room temperature, 2. Fix in a 4% formaldehyde solution for 2 minutes at room temperature, 3. Wash twice in a phosphate-buffered saline (PBS) solution for 1 minute, 4. Treat with a pepsin solution (0.1 µg / ml) at 37°C for 4 minutes, 5. Wash twice in a phosphate-buffered saline solution for 1 minute, 6. Successive dehydrations for 1 minute at 4°C with 50% aqueous ethanol, 70% aqueous ethanol, and pure ethanol, 7. Dry the slides, 8. Denature the probes and chromosomal DNA at 80°C for 3 minutes, 9. Hybridize for 20 minutes at room temperature, 10. Wash the cytogenetic slides, 11. Stain the chromosomes with DAPI and application of a contrast dye. Analysis of chromosomal aberrations

[0097] The analysis is performed on cells in metaphase after labeling of telomeres and centromeres and automatic capture of fluorescent signals from telomeres and centromeres: The identification of each chromosome is based on its size, defined by the distance between the telomeres of the short (p) and long (q) arms of the chromosome, as well as by the ratio between the size of the p and q parts of the chromosome (centromeric index). The number of centromeres is quantified to analyze only complete metaphases. The signal of each telomere is quantified (4 signals per chromosome and 184 signals expected per diploid metaphase). Telomere losses and deletions are detected for each chromosome. Structural chromosomal aberrations, such as dicentric chromosomes, centric rings, acentric rings, and various types of acentric chromosomes, are detected. For the detection of simple chromosomal rearrangements, DAPI banding, similar to GTG banding, complements chromosome identification.For complex rearrangements, M-FISH staining, performed on the same metaphases, allows for the creation of a multicolor karyotype, making the analysis more reliable and sensitive. This software offers a simple interface allowing manual data correction and is capable of "learning" over time. 2. Results 2.1 Technical validation of the invention process Validation of the microplate culture approach

[0098] Microplate cell culture was validated against the conventional technique (25 cm² flask culture) in a cohort of 70 patients with lymphoid disorders, 50 healthy donors, and 150 patients with an inflammatory or proliferative syndrome. Whole blood from 50 patients and bone marrow from 20 patients were cultured using both approaches. No culture failures were observed with the microplate technique, and the metaphase quality allowed for reliable and sensitive cytogenetic analysis. In contrast, several culture failures were observed with the conventional technique (5 bone marrow out of 20 and 7 whole blood out of 50 cancer patients), with less satisfactory metaphase quality that would not allow for automated analysis of chromosomal aberrations. figure 2 ). Interphase nucleus capture by a 10x magnification objective

[0099] The fluorescence capture method using a 10x magnification objective is compared to a method using a 63x magnification objective. The fluorescence intensities emitted by the calibrated beads are measured using a 10x and a 63x microscopic objective, respectively. The results obtained are comparable ( Figures 3A And 3B ). However, for low fluorescent intensities, using the 10x objective allows us to get closer to the exact fluorescence value of the beads.

[0100] The method of capturing interphase nuclei using a 10x magnification objective and the classical method (Q-FISH) of capturing metaphases using a 63x magnification objective are also tested on various human cell lines and on circulating lymphocytes from cancer patients. A strong correlation is observed between the fluorescence intensity obtained from metaphases by the Q-FISH method and the fluorescence intensity obtained from interphase nuclei by the method of the present invention ( figure 4 ). Telomere length analyses are performed using software that provides the average size, the frequency of cells with short telomeres, and the heterogeneity of telomere length. Telomere quantification

[0101] The telomere quantification result obtained by the method of the present invention is also compared with that obtained by the TRF (terminal restricted fragment) technique and that obtained by the PCR technique ( figure 5 ). This comparison shows a significant correlation between the process of the present invention and conventional techniques with regard to telomere quantification and thus validates this new approach in relation to the reference technique (TRF) and also in relation to the most used technique currently (PCR). 2.2 Application of the process of the invention Impact of age on telomere length

[0102] Telomere lengths in circulating lymphocytes obtained from 420 healthy donors aged 1 to 80 years were analyzed using the method of the invention. The donors were classified into 5 age groups. The total fluorescence intensity of the telomeres was quantified. For each sample, more than 10,000 cells were analyzed. The results clearly show a progressive decrease in telomere length with increasing age, with an average loss of 76 bp per year ( figure 6 ). This average telomere regression as a function of age is in perfect correlation with the literature (Vera et al. (2012), Cell Rep 2(4):732-737. ).

[0103] Furthermore, the method of the invention provides good reproducibility with two measurements of telomere length from the same population two years apart ( figure 7 ). Frequency of micronuclei and anaphasic bridges

[0104] The frequency of micronuclei and anaphasic bridges was evaluated in circulating lymphocytes from healthy donors and cancer patients using the method of the invention. The results obtained show a significant increase in the frequency of micronuclei, essentially micronuclei with only telomeric sequences ( Figure 8 ). The present invention has made it possible to detect the nature of these micronuclei which is linked to genotoxic stress.

[0105] Anaphasic bridge detection was also performed on a large cohort of healthy donors and patients. The detection of different anaphasic bridge configurations was compared with data obtained from chromosomal aberrations. The method of the invention makes it possible to detect the mechanisms involved in the formation of anaphasic bridges ( figure9The method of the invention makes it possible to detect long anaphasic bridges, easily detectable on cytogenetic slides, which are linked to the formation of a dicentric chromosome resulting from the fusion of two chromosomes (telomere dysfunction). This method also makes it possible to detect very short anaphasic bridges, which are linked to the presence of a very specific configuration of dicentric chromosomes (the two centromeres being very close together). These different configurations of anaphasic bridges are easily detectable on cytogenetic slides prepared according to the method of the invention described above, allowing for very rapid detection of chromosomal instability. Accuracy in the detection of chromosomal aberrations Example 1

[0106] An extra marker chromosome corresponding to a centric ring, essentially composed of centromeric sequences, can be detected by the method of the invention. The phenotype is infertility. This chromosomal aberration is undetectable by conventional techniques such as molecular cytogenetics or M-FISH alone ( Figure 10 ). Telomere and centromere labeling allowed us to see centromeric sequences and define the nature of the chromosomal aberration. Example 2

[0107] In prenatal diagnosis, the presence of a t(9;10)(q34;q26.3) translocation in the father and fetus, the conventional technique (RBG) as well as molecular cytogenetic techniques (M-Banding and chromosome painting) detect an unbalanced translocation.

[0108] The process of the invention, which leads to the labeling of telomeres and centromeres, made it possible to observe that chromosome 10 only exchanged the telomeric portion with chromosome 9 ( figure 11 The presence of telomeric sequences on chromosome 9 demonstrated that this was therefore a balanced translocation. Compared to conventional techniques, the method of the invention not only detects the chromosomal aberration, but also, with greater precision, the nature of the aberration. Example 3

[0109] The method of the present invention also makes it possible to detect all chromosomal aberrations induced by a genotoxic agent such as irradiation. The method of the invention allows for the evaluation of the number of centromeres, the presence of dicentric chromosomes, translocations, and the different types of acentric chromosomes resulting from a terminal deletion, an interstitial deletion, or the fusion of two terminal deletions. figure 12This figure shows that the method of the invention makes it possible to detect the different configurations of the dicentric chromosome, such as the dicentric chromosome with two linear and well-spaced centromeres, the dicentric chromosome with centromeres coinciding with telomeres, and the dicentric chromosome with two centromeres very close together or coinciding. This figure also shows that the method of the invention can distinguish between centric and acentric rings and characterize acentric chromosomes, such as the acentric chromosome resulting from a terminal deletion, the acentric chromosome resulting from an interstitial deletion, and the acentric chromosome resulting from a fusion of two terminal acentric chromosomes. Example 4

[0110] The method of the present invention also makes it possible to detect a specific configuration of the dicentric chromosome, a configuration that is very difficult to detect by conventional techniques. The method of the invention allows for more precise chromosome classification and more reliable detection of chromosomal aberrations ( figure 13 ). References :

[0111] Benetos A, Toupance S, Gautier S, Labat C, Kimura M, Rossi PM, Settembre N, Hubert J, Frimat L, Bertrand B, Boufi M, Flecher X, Sadoul N, Eschwege P, Kessler M, Tzanetakou IP, Doulamis IP, Konstantopoulos P, Tzani A, Korou M, Gkogkos A, Perreas K, Menenakos E, Samanidis G, Vasiloglou-Gkanis M, Kark JD, Malikov S, Verhulst S, Aviv A. Short Leukocyte Telomere Length Precedes Clinical Expression of Atherosclerosis: The Blood-and-Muscle Model. Circ Res. 2018 Feb 16;122(4):616-623. doi: 10.1161 / CIRCRESAHA.117.311751. Epub 2017 Dec 14. Staerk L, Wang B, Lunetta KL, Helm RH, Ko D, Sherer JA, Ellinor PT, Lubitz SA, McManus DD, Vasan RS, Benjamin EJ, Trinquart L. Association Between Leukocyte Telomere Length and the Risk of Incident Atrial Fibrillation: The Framingham Heart Study. J Am Heart Assoc. 2017 Nov 14;6(11). Allshire RC(1), Dempster M, Hastie ND Human telomeres contain at least three types of G-rich repeat distributed non -randomly. Nucleic Acids Res. 1989 Jun 26;17(12):4611-27.Canela A, Vera E, Klatt P, Blasco MA. High-throughput telomere length quantification by FISH and its application to human population studies. Proc Natl Acad Sci U S A. 2007 Mar 27;104(13):5300-5. Epub 2007 Mar 16. Harley CB(1), Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature. 1990 May 31;345(6274):458-60. Kimura M, Stone RC, Hunt SC, Skurnick J, Lu X, Cao X, Harley CB, Aviv A. Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths. Nat Protoc. 2010 Sep;5(9):1596-607. Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002 May 15;30(10):e47. Cawthon RM. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res. 2009 Feb;37(3):e21. O'Callaghan NJ(1), Toden S, Bird AR, Topping DL, Fenech M, Conlon MA. Colonocyte telomere shortening is greater with dietary red meat than white meat and is attenuated by resistant starch. Clin Nutr.2012 Feb;31(1):60-4. Vera E, Bernardes de Jesus B, Foronda M et al. The rate of increase of short telomeres predicts longevity in mammals. Cell Rep 2012; 2(4):732-737.

Claims

1. A high throughput method for detecting chromosomal aberrations and / or telomere aberrations, comprising: - preparing a cytogenetic slide from a biological sample of 150µL to 200µL, grown in a microplate, the said growing of cells being carried out in a microplate well, wherein the ratio of the amount of the culture medium to the well's surface area is 1mL / cm2 to 1.5mL / cm2, the mitotic index in said cytogenetic slide being between 1.5 and 4 times higher than the conventional procedure of culturing cells in flasks with 10 to 20 mL of medium, - simultaneously labelling the telomeres and centromeres with peptide nucleic acid probes with a hybridisation time from 30 minutes to 1.5 hours, - flow image quantifying the fluorescence intensity of telomeres on interphase nuclei using a 10x magnification objective for overall telomere quantification, and - automatically capturing the metaphase chromosomes to detect chromosomal aberrations and / or telomere aberrations in each chromosome.

2. The method according to claim 1, wherein said chromosomal aberrations are micronuclei, anaphase bridges, dicentric chromosomes, centric rings, acentric chromosomes, chromosomal translocations, isochromosome, chromosome insertions and deletions, and wherein the telomere aberrations are selected from: the loss of one telomere, the loss of two telomeres from the same arm and the formation of telomere splits.

3. The method according to claim 1 or 2, said method comprising: - quantifying the micronuclei and anaphase bridges using a 10x or even 40x magnification objective, and - automatically capturing the metaphase chromosomes to detect chromosomal aberrations and / or telomere aberrations in each chromosome.

4. The method according to any one of claims 1 to 3, characterized in that said biological sample is a whole blood sample, marrow, or tissue cell sample.

5. The method according to any one of claims 1 to 4, wherein simultaneously labelling the telomeres and centromeres comprises: - a single step of treating a cytogenetic slide for 1 to 3 minutes, in particular 2 minutes with a 3- 5% formaldehyde solution, in particular a 4% solution.

6. The method according to claim 5, wherein simultaneously labelling the telomeres and centromeres further comprises, after the formaldehyde treatment, - a single step of treating the cytogenetic slide with pepsin for 3-5 minutes, in particular 4 minutes, by immersing a cytogenetic slide previously treated with formaldehyde in a pepsin solution at a concentration of 0.1-0.2ug / mL.

7. The method according to claim 6, wherein simultaneously labelling the telomeres and centromeres further comprises, after the pepsin treatment step, the following steps of: - dehydrating the cytogenetic slide successively for 1 minute with a 50% aqueous ethanol solution, a 70% aqueous ethanol solution and pure ethanol, - denaturing the chromosomal DNA present on the above said cytogenetic slide obtained at the end of the previous step and denaturing the peptide nucleic acid probes for telomeres and centromeres, - hybridising for 10-30 minutes, in particular 20 minutes, at room temperature, between the denatured chromosomal DNA and the denatured peptide nucleic acid probes obtained in the previous step, - successively washing said cytogenetic slide after hybridising.

8. The method according to any one of the preceding claims, characterised in that a first peptide nucleic acid probe labels the telomeres and a second peptide nucleic acid probe simultaneously labels the centromeres, the first probe and the second probe emitting a distinctive fluorescence respectively.

9. The method according to any one of the preceding claims for detecting chromosomal aberrations, said method comprising: - labelling the telomeres and centromeres of metaphase cells with a ready-to-use solution of the nucleic acid probes, - automatically capturing the fluorescent signals of the telomere and centromere labelling, - analysing the image obtained in the previous step to obtain the data set: counting the number of metaphase centromeres, identifying each chromosome from its size and the ratio between the short arm (p) and the long arm (q), quantifying the signal of each telomere of each metaphase chromosome, for the detection of structural chromosome aberrations such as dicentric chromosomes, centric rings, acentric rings and different types of acentric chromosomes, - possibly DAPI banding performed on the same metaphases, to complete the identification of chromosomes and detect single translocations, - possibly M-FISH labelling on the same metaphases, for multicolour karyotyping to detect complex rearrangements.

10. The method according to any of the preceding claims for detecting micronuclei and anaphase bridges, said method comprising: (i) labelling the telomeres and centromeres of interphase cells with a ready-to-use solution of the nucleic acid probes, (ii) automatically capturing the fluorescent signals from the telomere and centromere labelling, (iii) analysing the image obtained in step (ii) to obtain the data set : total number of micronuclei, number of micronuclei with only telomeric sequences, and number of micronuclei with both telomeric and centromeric sequences, (iv)analysing the image obtained in step (ii) to obtain the data set: identification of anaphase bridges by detecting the presence of a DNA filament connecting two daughter cells, length of the bridge, detection of the presence or absence of centromeric or telomeric sequences in the bridge, counting the number of cells adhered.

11. A high-throughput detection kit for quantifying telomeres and detecting chromosomal aberrations and / or telomere aberrations according to any of claims 1 to 10, comprising: - a ready-to-use solution of peptide nucleic acid probes for telomeres and centromeres, - wash buffers required for hybridisation, - standard slides for quantifying telomeres, - standard slides for checking the fluorescence intensity of the microscope.