HIGH-THROUGHPUT METHOD FOR DETECTING CHROMOSOMAL ABERRATIONS AND/OR TELOMERE ABERRATIONS

Abstract

A high throughput method for detecting chromosomal aberrations and/or telomere aberrations using a biological sample of 150 μL to 200 μL including preparing a cytogenetic slide from the sample in a microplate, the mitotic index in the cytogenetic slide being 3 times higher on average than the conventional procedure of culturing cells in flasks with 10 to 20 mL of medium, simultaneously labeling 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 10× magnification objective for overall telomere quantification, and automatically capturing the metaphase chromosomes to detect chromosomal aberrations and/or telomere aberrations in each chromosome. Also a high-throughput detection kit for quantifying telomeres and detecting chromosomal aberrations and/or telomere aberrations.

Claims

1-12 (canceled)

13. A high throughput method for detecting chromosomal aberrations and/or telomere aberrations using a biological sample of 150 μL to 200 μL, comprising: preparing a cytogenetic slide from said sample in a microplate, the mitotic index in said cytogenetic slide being 3 times higher on average than the conventional procedure of culturing cells in flasks with 10 to 20 mL of medium; simultaneously labeling 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 10× magnification objective for overall telomere quantification; and automatically capturing the metaphase chromosomes to detect chromosomal aberrations and/or telomere aberrations in each chromosome.

14. The method according to claim 13, wherein said chromosomal aberrations are micronuclei, anaphase bridges, dicentric chromosomes, centric rings, acentric chromosomes, chromosomal translocations, isochromosome, chromosome insertions and deletions.

15. The method according to claim 13, said method comprising: preparing a cytogenetic slide from said sample in a microplate, the mitotic index in said cytogenetic slide being 3 times higher on average than the conventional procedure of culturing cells in flasks with 10 to 20 mL of medium; simultaneously labeling the telomeres and centromeres with peptide nucleic acid probes with a hybridisation time from 30 minutes to 1.5 hours; flow image quantifying the telomere fluorescence intensity on interphase nuclei using a 10× magnification objective for overall telomere quantification; quantifying the micronuclei and anaphase bridges using a 10× or even 40× magnification objective; and automatically capturing the metaphase chromosomes to detect chromosomal aberrations and/or telomere aberrations in each chromosome.

16. The method according to claim 13, wherein said biological sample is a whole blood sample, marrow, or tissue cell sample.

17. The method according to claim 13, wherein the step of preparing the cytogenetic slide comprises: culturing the cells in a microplate well, the ratio of the amount of the culture medium to the well surface area of which is 1 mL/cm.sup.2 to 1.5 mL/cm.sup.2.

18. The method according to claim 13, wherein simultaneously labeling the telomeres and centromeres comprises: a single step of treating a cytogenetic slide for 1 to 3 minutes, in particular 2 minutes with a 2-5% formaldehyde solution, in particular a 4% solution.

19. The method according to claim 18, wherein simultaneously labeling 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 0.1-0.2 ug/mL.

20. The method according to claim 19, wherein simultaneously labeling 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; and successively washing said cytogenetic slide after hybridising.

21. The method according to claim 13, wherein 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.

22. The method according to claim 13 for detecting chromosomal aberrations, said method comprising: labeling 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 labeling; 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; and possibly M-FISH labeling on the same metaphases, for multicolour karyotyping to detect complex rearrangements.

23. The method according to claim 13 for detecting micronuclei and anaphase bridges, said method comprising: (i) labeling 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 labeling; (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; and (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.

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

Description

FIGURES

[0144] FIG. 1: This figure illustrates a schematic of the high-throughput method for telomere quantification, micronucleus and anaphase bridge detection and chromosome and telomere aberration detection of the present invention.

[0145] FIG. 2: This figure shows the comparison between the quality and quantity of metaphases obtained by the microplate technique and the conventional technique. Images of metaphases from conventional culture (top frame) and images of metaphases from microplate culture (bottom frame). This comparison was made under the same conditions with the same cell sample.

[0146] FIG. 3A: This figure shows the comparison of the fluorescence intensity of the 6 μm calibration beads measured by a 10× microscopic objective or a 63× microscopic objective with different intensities (100%, 33% and 10%).

[0147] FIG. 3B: This figure shows the distribution of fluorescence intensity of the cells in quartiles measured by a 10× microscope objective or a 63× microscope objective.

[0148] FIG. 4: This figure shows the comparison of telomere quantification on metaphases (Q-FISH) and interphases (present invention) using four cell lines.

[0149] FIG. 5: This figure shows the comparison between the telomere quantification measured by the method of the invention and the result obtained by FISH (A) and the result obtained by PCR (B).

[0150] FIG. 6: This figure shows the correlation between the mean telomere size measured by the present invention and age in a cohort of 420 healthy donors. The mean size is expressed in kb.

[0151] FIG. 7: Reproducibility of the method of the invention with two different measurements of the same sample comprising 420 donors.

[0152] FIG. 8: This figure shows the frequency of micronuclei in healthy donors and cancer patients with (A) the total number of micronuclei. (B) the difference between micronuclei with only telomeric sequences detected by virtue of the present invention between patients and donors.

[0153] FIG. 9: This figure shows the detection of anaphase bridges in interphase cells from cancer patients. The method of the invention allows quite long anaphase bridges (white arrow) to be distinguished with telomeric and/or centromeric sequences corresponding to the presence of a dicentric chromosome resulting from a fusion of two chromosomes or very reduced anaphase bridges (grey arrow) indicating the presence of a dicentric chromosome with two centromeres very close to each other.

[0154] FIG. 10: This figure shows the detection of a supernumerary marker chromosome corresponding to a centric ring in a prenatal sample by the method of the invention. This aberration cannot be detected by the RBG or M-FISH technique alone.

[0155] FIG. 11: This figure shows the detection of a chromosomal translocation and the identification of the nature of this translocation by the conventional technique (top figure), by chromosome in situ hybridisation, by M-banding and by the method of the invention (bottom figure) within the scope of a prenatal examination with a decision of genetic counselling to be taken in case the translocation is not balanced. As the translocation affects the telomeric part of chromosome 10q, it has not been detected by conventional or molecular cytogenetics. The present invention allows detection of telomeric sequences and confirmation of the nature of the translocation.

[0156] FIG. 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.

[0157] FIG. 13: This figure shows that a sequential analysis after telomere and centromere labeling associated with the M-FISH technique allows a very accurate classification of chromosomes and also a 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

[0158] 1. Materials and Methods

[0159] Cell Lines

[0160] The cell lines tested in the examples are lymphoblastic lines from healthy donors, tumour lines from Hodgkin's lymphoma, Burkitt's lymphoma and mantle cell lymphomas were used.

[0161] Q-FISH Assay

[0162] The Q-FISH assay, which allows visualisation of telomeres by hybridisation 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.]

[0163] TRF Assay

[0164] The TRF assay is performed according to the technique described in [Kimura et al, Nat Protoc. 2010 Sep; 5(9):1 596-607]

[0165] qPCR Assay

[0166] The qPCR assay is implemented according to the method described in [OCallaghan & Fenech, Clin Nutr. 2012 Feb; 31(1):60-4].

[0167] Telomere and Centromere Labeling

[0168] A protocol for telomere and centromere labeling is illustrated below.

[0169] 1. washing the cytogenetic slides in a first phosphate-buffered saline (PBS)1 solution for 1 minute at room temperature,

[0170] 2. fixing in 4% formaldehyde solution for 2 minutes at room temperature,

[0171] 3. washing twice in phosphate-buffered saline (PBS) for 1 minute,

[0172] 4. treating with pepsin solution (0.1 ug/mL) at 37° C. for 4 minutes,

[0173] 5. washing twice in phosphate-buffered saline (PBS) for 1 minute,

[0174] 6. successively dehydrating for 1 minute at 4° C. with 50% aqueous ethanol, 70% aqueous ethanol, pure ethanol

[0175] 7. drying the slides,

[0176] 8. denaturing the probes and chromosomal DNA at 80° C. for 3 minutes,

[0177] 9. Hybridising for 20 minutes at room temperature,

[0178] 10. washing the cytogenetic slides

[0179] 11. staining the chromosomes with DAPI and applying a contrast stain.

[0180] Chromosome Aberration Analysis

[0181] The analysis is performed on metaphase cells after telomere and centromere labeling and automatically capturing telomere and centromere fluorescent signals:

[0182] Identification of each chromosome from its size defined by the distance between the telomeres of the short arms (p) and long arms (q) of the chromosome as well as the ratio of the size of the p part to the q part of the chromosome (centromere index).

[0183] Quantification of the number of centromeres to analyse complete metaphases only.

[0184] Quantification of the signal of each telomere (4 signals per chromosome and 184 signals expected per diploid metaphase).

[0185] Detection of telomere losses and deletions on each chromosome.

[0186] Detection of structural chromosome aberrations such as dicentric chromosomes, centric rings, acentric rings and different types of acentric chromosomes.

[0187] For the detection of simple chromosomal rearrangements, DAPI banding, similar to GTG banding close to that of GTG banding, completes the identification of chromosomes.

[0188] For complex chromosomal changes, M-FISH labeling, performed on the same metaphases, allows the production of a multicolour karyotype which makes the analysis more reliable and sensitive.

[0189] This software offers a simple interface allowing manual correction of the data and is capable of “learning” over time.

[0190] 2. Results

[0191] 2.1 Technical Validation of the Method of the Invention

[0192] Validation of the Microplate Culture Approach

[0193] Microplate cell culture was validated against the conventional technique (25 cm2 flask culture) on a cohort of 70 patients with lymphoid diseases, 50 healthy donors and 150 patients with inflammatory or proliferative syndrome. Whole blood from 50 patients and marrow from 20 patients were cultured using both approaches. No culture failures were observed with the microplate technique and with a quality of metaphases allowing reliable and sensitive cytogenetic analysis. In contrast, using the conventional technique, several culture failures were observed (5 out of 20 marrows and 7 whole blood out of 50 cancer patients) with a quality of metaphases that is less interesting and that will not allow an automatic analysis of chromosomal aberrations (FIG. 2).

[0194] Interphase Nucleus Capturing with a 10× Magnification Objective

[0195] The fluorescence capture method with a 10× magnification objective is compared with a method using a 63x magnification objective. Fluorescence intensities emitted by calibrated beads are measured by a 10× and a 63x microscopic objective respectively. The results obtained are comparable (FIGS. 3A and 3B). However, for low fluorescent intensities, the use of the 10× objective allows one to get closer to the exact fluorescence value of the beads.

[0196] The interphase nucleus capture method using a 10× magnification objective and the classical (Q-FISH) metaphase capture method using a 63× magnification objective are also tested on different 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 (FIG. 4). Telomere size analyses are performed using software for giving the mean size, frequency of cells with short telomeres, and heterogeneity of telomere size.

[0197] Telomere Quantification

[0198] 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 (FIG. 5). This comparison shows a significant correlation between the method of the present invention and conventional techniques with respect to telomere quantification and thus validates this new approach with respect to the reference technique (TRF) and also with respect to the most currently used technique (PCR).

[0199] 2.2 Application of the Method of the Invention

[0200] Impact of Age on Telomere Size

[0201] Telomere sizes in circulating lymphocytes obtained from 420 healthy donors aged between 1 and 80 years were analysed by the method of the invention. The donors are classified into 5 age groups. The total fluorescence intensity of telomeres is quantified. For each sample, more than 10,000 cells were analysed. The result clearly shows a progressive decrease in telomere length with increasing age with a mean loss of 76 bp per year (FIG. 6). This telomere regression mean as a function of age perfectly correlates with the literature (Vera el al. (2012), Cell Rep 2(4):732-737.).

[0202] Furthermore, the method of the invention gives good reproducibility with two measurements of telomere size from the same population two years apart (FIG. 7).

[0203] Frequency of Micronuclei and Anaphase Bridges

[0204] The frequency of micronuclei and anaphase bridges was evaluated in circulating lymphocytes from healthy donors and cancer patients by the method of the invention. The results obtained show a significant increase in the frequency of micronuclei, mainly micronuclei with only telomeric sequences (FIG. 8). The present invention has made it possible to detect the nature of these micronuclei which is related to genotoxic stress.

[0205] The detection of anaphase bridges was also performed on a large cohort of healthy donors and patients. The detection of the different configurations of anaphase bridges was compared with data obtained by chromosomal aberrations. The method of the invention makes it possible to detect mechanisms involved in the formation of anaphase bridges (FIG. 9). The method of the invention makes it possible to detect long anaphase bridges, easily detectable on cytogenetic slides and which are related to the formation of a dicentric resulting from a fusion of two chromosomes (telomere dysfunction). This procedure also allows the detection of very reduced anaphase bridges which are linked to the presence of a very specific configuration of dicentric chromosomes (the two centromeres very close to each other). These different configurations of anaphase bridges are easily detectable on cytogenetic slides prepared according to the method of the invention described above, allowing very rapid detection of chromosomal instability.

[0206] Accuracy in the Detection of Chromosomal Aberrations

Example 1

[0207] A supernumerary marker chromosome corresponding to a centric ring, essentially consisting 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 (FIG. 10). Telomere and centromere labeling allowed centromeric sequences to be seen and the nature of the chromosomal aberration to be defined.

Example 2

[0208] In prenatal diagnosis, presence of a translocation t(9;10)(q34;q26.3) in the father and the fetus, the conventional technique (RBG) as well as molecular cytogenetic techniques (M-Banding and chromosome painting) detect a non-balanced translocation.

[0209] The method of the invention, which leads to labeling of telomeres and centromeres, has made it possible to observe that chromosome 10 has exchanged only the telomeric part with chromosome 9 (FIG. 11). The presence of the telomeric sequences in chromosome 9 showed that this was therefore a balanced translocation. In comparison with conventional techniques, the method of the invention not only allows the detection of the chromosomal aberration, but also more precisely the nature of the aberration.

Example 3

[0210] 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 an evaluation of the number of centromeres, the presence of the dicentric chromosome, translocations, different types of acentric chromosomes resulting from a terminal deletion, interstitial deletion or fusion between two terminal deletions. FIG. 12 shows that the method of the invention makes it possible to detect different configurations of the dicentric chromosome, such as the dicentric with the two linear and well-spaced centromeres, the dicentric with the centromeres identical to the telomeres and the dicentric with the two centromeres very close or identical to each other. This figure shows that the method of the invention can also distinguish between centric and acentric rings and characterise acentric chromosomes, such as acentric ring from a terminal deletion, acentric ring from an interstitial deletion and acentric ring from a fusion between two terminal acentric rings.

Example 4

[0211] The method of the present invention also allows the detection of a specific configuration of the dicentric chromosome, which configuration is very difficult to detect by conventional techniques. The method of the invention allows a more accurate classification of chromosomes and a more reliable detection of chromosomal aberrations (FIG. 13).

REFERENCES

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