Mol. Hum. Reprod. Advance Access originally published online on January 29, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular Human Reproduction, Vol. 10, No. 4, pp. 283-289, 2004
© European Society of Human Reproduction and Embryology 2004
Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization
Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville Rd, Woodville, South Australia, 5011, Australia
1 To whom correspondence should be addressed. e-mail: nicole.hussey{at}adelaide.edu.au
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
The use of metaphase comparative genomic hybridization (CGH) to screen all human chromosomes for aneuploidy in preimplantation embryos is hindered by the time required to perform the analysis. We report in this paper a novel approach to manufacture a DNA microarray for CGH for the detection of aneuploidy in single cells. We spotted human chromosome-specific libraries on glass slides that were depleted of repetitive sequences and tested our array CGH method in 14 experiments using either single male and/or single female lymphocytes. For the autosomes, the mean normalized ratios were all close to the expected ratio of 1.0 with overall 300/308 (97%) of the normalized ratios falling within the range 0.75 to 1.25. It was possible to deduce the correct copy number of the X chromosome in 13/14 (92.9%) separate array CGH experiments but the Y chromosome in only 4/14 (29%). We tested our microarray CGH method on a single fibroblast from each of three cell lines containing a specific chromosome aneuploidy (trisomy 13, 15 or 18) and in each case our microarray analysis was able to obtain a diagnosis based on the fact that the aneuploid chromosome gave the highest ratio (1.32, 1.27 and 1.27 respectively) with the ratios of all other chromosomes falling within the range 0.75–1.25. Requiring just 30 h, our method may be more suitable for PGD aneuploidy screening than metaphase CGH.
Key words: Key words: aneuploidy screening/array CGH/microarray/preimplantation genetic diagnosis/single cell PCR
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Aneuploidies occur frequently in human preimplantation embryos generated by IVF, and involve almost every human chromosome (Voullaire et al., 2000; Wells and Delhanty, 2000). Aneuploidy screening via preimplantation genetic diagnosis (PGD) is currently carried out using commercially available fluorescence in situ hybridization (FISH) probes for five to nine chromosomes per embryo (Griffin et al., 1993; Gianaroli et al., 1999; Munné et al., 1999, 2002; Verlinsky et al., 1999). The detection of aneuploidy in single cells using metaphase-based comparative genomic hybridization (metaphase CGH) was developed to assess all chromosomes (Voullaire et al., 1999; Wells et al., 1999). Metaphase CGH has been used recently for PGD screening for aneuploidies in single blastomeres (Wilton et al., 2001) and first polar bodies (Wells et al., 2002). However, metaphase CGH takes
3 days, which is too long to allow embryos to be transferred in the cycle of IVF that created them, resulting in the need for the embryos to be cryopreserved until a diagnosis is made. Suitable embryos can then be placed back in a subsequent natural cycle, but not all embryos will survive the freeze–thaw process. By contrast, array CGH is much quicker and has been increasingly used to detect high copy number amplifications of genomic DNA sequence, such as oncogenes in tumour tissues (Albertson et al., 2000; Cai et al., 2002; Pollack et al., 2002). Array CGH has also been used to detect low copy number changes, including heterozygous and homozygous deletions, as well as the presence of partial chromosome and whole chromosome aneuploidy (Pinkel et al., 1998; Pollack et al., 1999; Snijders et al., 2001; Veltman et al., 2002). Array CGH experiments published to date require a DNA sample (0.5–1.0 µg) far in excess of that contained in a single blastomere to obtain a hybridization signal reliable enough for analysis (Pinkel et al., 1998; Pollack et al., 1999; Albertson et al., 2000; Snijders et al., 2001; Cai et al., 2002; Pollack et al., 2002; Veltman et al., 2002). We describe here the development of an array CGH approach using glass slides arrayed with chromosome-specific DNA libraries, applying it for gender determination and diagnoses of trisomies 13, 15 and 18 in single cells. |
Materials and methods |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Chromosome-specific DNA libraries
Chromosome-specific DNA libraries were generated by us using degenerate oligonucleotide-primed PCR (DOP-PCR) amplification of repeat-depleted DNA libraries which were a kind gift from Drs A.Bolzer and J.M.Craig (Bolzer et al., 1999). These chromosome-specific DNA libraries were initially established either by microdissection of the whole chromosome for 10 chromosomes, 1, 3, 6, 7, 9,12, 17, 19, 20 and X, or the q arm of the five acrocentric chromosomes (13–15, 21 and 22) or by flow-sorting for chromosomes 2, 4, 5, 8, 10, 11, 16, 18 and Y. The genomic material was amplified by DOP-PCR and then repetitive sequences were depleted by affinity chromatography in combination with negative subtraction hybridization using human Cot-1 DNA as subtractors (Craig et al., 1997; Bolzer et al., 1999). The depletion of chromosomes 1, 3, 12, 18, 19 and X was not sufficient for their purposes and so each centromere-specific repetitive sequence was used to further deplete these chromosomes. In addition, repetitive sequences were further removed from the chromosome 14 library using itself as a subtractor and for chromosome 22 using both chromosome 14 and 19 libraries as subtractors.
Amplification of DNA libraries
To amplify the DNA of each library, DOP-PCR was carried out in a Minicycler (MJ Research, USA) in a volume of 50 µl, containing 
100 ng of source DNA, 1xPCR buffer (50 mmol/l KCl, 10 mmol/l Tris–HCl, pH 8.3), 2.5 mmol/l MgCl2, 0.25 mmol/l of each dNTP, 2.0 µmol/l of DOP-PCR primer 6MW (5'-CCGACTCGAGNNNNNNATGTGG-3'), and 5 IU of Taq DNA polymerase (Perkin Elmer, USA). Cycling conditions consisted of an initial step of denaturation at 95°C for 4 min followed by 30–35 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 3 min with an addition of 10 s per cycle to the extension time. An extension step of 72°C for 10 min was finally added at the end of cycling. A total of 5 µl of each DOP-PCR product was run on 1% agarose gels in 0.5xTBE to check the quality of amplification. The remaining DOP-PCR product was purified by UltraCleanTM PCR Clean-up DNA purification kit (Mo Bio Laboratories, USA) and finally eluted into 50 µl of UltraPure H2O (Biotech International, Australia). Purified products were either used immediately or stored at –20°C for 1 year without any visible loss of their FISH specificities.
Array fabrication
In preparation for spotting, amplified DNA libraries were dried in wells of a 384-well plate at room temperature and resuspended in 6 µl of 150 mmol/l sodium phosphate to reach a final concentration of 250 ng/µl. Spotting from this plate onto SuperAmine slides (TeleChem, USA) was carried out by a Chipwriter Pro (BioRad) arrayer, which arrayed eight replicate spots for each library per array with two arrays per slide. Post-processing of slides was as follows: slides were allowed to age in a dust-free slide box for several days, then baked at 80°C for 80 min. After cooling to room temperature, slides were washed with constant mixing in 0.2% sodium dodecyl sulphate (SDS) for 4 min followed by three times in MilliQ H2O for 1 min each. Slides were plunged into nearly boiling MilliQ H2O for 2–3 min to denature the DNA, dehydrated in cold ethanol (95%) for 1 min, and finally dried either by spinning at 1000 g for 5 min or by air-drying in the dark. Slides were stored in vacuo at room temperature in the dark for up to 2 months for processed slides and up to 5 months if unprocessed.
Single cells and single-cell sorting
Single lymphocytes were obtained from peripheral blood of a normal male (46,XY) and a normal female (46,XX). Single fibroblasts were obtained from cell lines purchased from Coriell Cell Repositories (USA) including GM01359 (47,XY, +18), GM02948A (47,XY, +13), and GM07189 (47,XY+15). Single cells were isolated exactly as reported (Hussey et al., 1999) except that the autologous plasma was first treated with 20 IU DNase I (Boehringer Mannheim) per 25 µl of plasma for 1 h at 37°C prior to supplementing the Roswell Park Memorial Institute medium. This step was included to remove any DNA present in the autologous plasma before addition to the single cell sorting medium to prevent the cells adhering to the glass slide.
Random whole genome amplification of single cells by DOP-PCR
Lysis of single cells was carried out by the addition of 5 µl of lysis buffer (200 mmol/l KOH, 50 mmol/l dithiothreitol) and incubation at 65°C for 10 min followed by neutralization using 5 µl of neutralization solution (300 mmol/l KCl, 900 mmol/l Tris–HCl, pH 8.3, 200 mmol/l HCl) (Cui et al., 1989). The PCR was carried out in a volume of 50 µl with a final concentration of 50 mmol/l KCl, 100 mmol/l Tris–HCl, pH 8.3, 0.1 mg/ml gelatin, 2.5 mmol/l MgCl2, 200 µmol/l of each dNTP, 2 µmol/l DOP-PCR 6MW primer, along with 5 IU of Taq polymerase (Perkin Elmer, USA), and UltraPure water. The sample was pulse centrifuged, denatured at 95°C for 5 min, and cycled for eight cycles at 94°C for 1 min, 30°C for 1.5 min, 72°C for 3 min with a ramp of 1°C per 4 s between the annealing and the extension steps, followed by 26 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 3 min initially but increased by 14 s for each cycle and a final extension step at 72°C for 10 min.
Cy3/Cy5 labelling by DOP-PCR
First round DOP-PCR products (5 µl) were labelled in a volume of 50 µl, containing a final concentration of 50 mmol/l KCl, 10 mmol/l Tris–HCl, pH 8.3, 2.5 mmol/l MgCl2, 160 µmol/l of each of dGTP, dCTP and dATP, 120 µmol/l dTTP, 40 µmol/l of either Cy3-dUTP or Cy5-dUTP (PA 53022 or PA 55022; Amersham Phamacia Biotech, USA), 2 µmol/l DOP-PCR 6MW primer, along with 5 IU of Taq polymerase. The sample was pulse centrifuged, denatured at 95°C for 4 min, and cycled for 25 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 3 min initially but increased by 10 s for each cycle and a final extension step at 72°C for 10 min. Labelled products were purified by UltraCleanTM, and eluted in 50 µl of either 10 mmol/l Tris–HCl or UltraPure H2O. A total of 5 µl of the purified product was run on 1% agarose gels in 0.5xTBE prestained with ethidium bromide and photographed. The remaining product was available for use in array CGH and could be stored at –20°C for up to 2 months.
Array CGH
Equal volumes (5 10 µl) of each of Cy3-labelled (test) and Cy5-labelled (reference) DOP-PCR products were mixed with 70 µg of human Cot-1 DNA (Gibco, BRL) and 20 µg of sheared salmon sperm DNA (Gibco, BRL). The resulting mixture was precipitated with cold ethanol (100%) and resuspended in 10 µl of hybridization solution containing 50% deionized formamide, 2xstandard saline citrate (SSC), 0.1% SDS, 10% dextran sulphate and 5xDenhardt’s solution. After denaturation at 80°C for 10 min and preannealing at 37°C for 80 min, the mixture of probes was applied to the array area and covered with a coverslip. Hybridization was carried out at 37°C for 15–20 h in a humid incubator. Post-hybridization washing included twice in 50% formamide/2xSSC at 45°C for 10 min, twice in 2xSSC at 45°C for 5 min, once in 1xSSC at room temperature for 10 min, and three brief rinses in 18.2 M
deionized H2O. Finally, slides were dried in the dark. We found that dried slides could be stored in the dark at room temperature for up to 73 days; we did not test periods longer than this.
Slide scanning and data analysis
Array slides were scanned by a GenePix 4000B microarray reader (Axon Instruments, USA) and the acquired primary data were analysed as 16-bit TIFF files of ratio images for each hybridization using GenePix Pro 4.0.1.12 (Axon Instruments), which produced numerical parameters to interpret the results. We used the median of pixel-by-pixel ratios (Cy3/Cy5) of pixel intensities with the median background subtracted to interpret our results. We filtered the preliminary data using seven parameters, including: (1) Dia.: the diameter in µm of the feature indicator; (2) % >B532 + 2 SD: the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 1 (523 nm, here for Cy3); (3) % >B635 + 2 SD: the percentage of feature pixels with intensities >2 SD above the background pixel intensity, at wavelength 2 (635 nm, here for Cy5); (4) SNR532: the signal-to-noise ratio at wavelength 1 (523 nm, here for Cy3), defined by (Mean Foreground 1 – Mean Background 1)/(SD of Background 1); (5) SNR635: the signal-to-noise ratio at wavelength 2 (635 nm, here for Cy5), defined by (Mean Foreground 1 – Mean Background 1)/(SD of Background 1); (6) F532 % Sat.: the percentage of feature pixels at wavelength 1 (here for Cy3) that are saturated; and (7) F635 % Sat.: the percentage of feature pixels at wavelength 2 (here for Cy5) that are saturated. All these definitions can be found at http://www.axon.com/gn_GenePix_File_Formats.html. Dots were excluded from the analysis if they failed to pass any of the following parameters of (1) Dia. >50, (2) % >B635 + 2 SD >70, (3) % >B532 + 2 SD >70, (4) SNR635 >3.0, (5) SNR532 >3.0, (6) F635 % Sat. = 0, and (7) F532 % Sat. = 0. We first calculated the mean of ratios from all (eight or fewer) replicates of each chromosome. Normalization was then carried out using 22 means of ratios from all autosomes assuming that the mean ratio value of all autosomes in each hybridization was 1.0. This normalization method was recommended by Axon Instruments (http://www.axon.com/mr_Axon_KB_Article.cfm?ArticleID = 50), and can be briefly described as follows: using the afore mentioned criteria, dots are either included or excluded; (i) the median of ratios for all included dots is averaged for each chromosome to give the raw mean, (ii) the log10 value for each raw mean of median of ratios value is determined, (iii) the average of all of the log values is calculated (‘Avglog’), (vi) the true average (TrueAvg) is calculated (TrueAvg = 10 Avglog), (v) the normalization factor (NF) is determined (NF = 1/TrueAvg), and finally (vi) the normalization factor is applied to rescale all raw means of median of ratios (normalized mean of median of ratios = NFxthe raw mean of median of ratios) to give the normalized ratios presented in this paper. Normalized ratios were compared across different array CGH experiments for analysis.
Statistical analysis
Statistical analyses were performed using Excel 97 (Microsoft Corporation, USA). Differences in ratios of chromosomes were analysed using a single factor analysis of variance. P < 0.05 was considered to be statistically significant.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Preparation of the probes and manufacture of the arrays
To make the DNA array, a complete set of repeat-depleted human chromosome-specific DNA libraries (Bolzer et al., 1999) was obtained and reamplified in our own laboratory by DOP-PCR (Telenius et al., 1992). We confirmed the specificity of each of the chromosomal DNA libraries using FISH to metaphase chromosomes (results not shown). FISH signals of uniform painting were obtained for all target chromosomes and specifically only the q arms of the five acrocentric chromosomes (13–15, 21 and 22) (Guan et al., 1994). Finally, all amplified DNA libraries were spotted onto glass slides with eight replicate spots per chromosome library on each array. Two arrays were printed, separated by
1 cm, in order to perform two experiments per slide.
Array CGH on normal male and female single lymphocytes
To test the possibility of determining the copy number difference between the number of X chromosomes present in male and female single cells, we amplified the single genome within one single male lymphocyte (46,XY) and another single female lymphocyte (46,XX) by one round of DOP-PCR, and then labelled the resulting PCR products by both Cy3-dUTP and Cy5-dUTP using another round of DOP-PCR. We conducted four array CGH experiments of (1) female(Cy3) versus female(Cy5) cell, (2) male(Cy3) versus female(Cy5) cell, (3) male(Cy3) versus male(Cy5) cell, and (4) female(Cy3) versus male(Cy5) cell. Our results (Table I, Section A) show that the normalized ratios (Cy3/Cy5) for all autosomes (except chromosomes 2 and 9) fell within the range 0.75–1.25 in all four comparisons. Therefore, we used this range (0.75–1.25) of ratios obtained from autosomes as the threshold for a theoretical 2/2 ratio. We found that the X chromosome gave a ratio of 0.97 in the female(Cy3)/female(Cy5) comparison, 0.63 in the male(Cy3)/female(Cy5) comparison, 0.94 in the male(Cy3)/male(Cy5) comparison, and 1.58 in the female(Cy3)/male(Cy5) comparison. However, we were unable to obtain a ratio for the Y chromosome outside of the thresholds of 0.75 and 1.25 for both array CGH comparisons of (2) male(Cy3)/female (Cy5) and (4) female(Cy3)/male(Cy5). These results demonstrate the feasibility of our array CGH method on single cells for the diagnosis of all chromosomes except the Y and possibly chromosomes 2 and 9.
|
To test further the reliability of our array CGH method, 10 more experiments of female(Cy3)/male(Cy5) comparisons were carried out using nine single male cells and 10 single female cells (Table I, Section B) and we combined the results with the four previous experiments (Table I, Section A). For the autosomes, the mean normalized ratios were all close to the expected ratio of 1.0 (Table I, Section C). We found that 300 (97%) out of the total 308 (22x14) normalized ratios for all of the autosomes fit within the range 0.75–1.25. Autosomes 1, 3, 5, 6, 7, 8, 10, 12–16, 18, 19, 20 and 21 were always in the range 0.75–1.25. However, for four other autosomes, 2, 4, 11 and 22, 92.9% (13/14), and for the remaining two autosomes, 9 and 17, 85.7% (12/14) were correctly placed in the range 0.75–1.25. For chromosome X, the mean normalized ratio was 1.38 ± 0.14 (mean ± SD) in 11 independent female(Cy3)/male(Cy5) comparisons, and it was possible to deduce the correct copy number of the X chromosome by these normalized ratios in 13/14 (92.9%) separate array CGH experiments (Table I, Section C). The only failure was a normalized ratio of 1.07 for the X chromosome found in a female(Cy3)/male(Cy5) comparison, which could lead to misdiagnosis of a female cell as a male cell (Table I, Section B, Experiment 8). This error would not result in serious clinical consequences but would reduce the number of embryos available for transfer because this embryo would not be transferred in a PGD clinical case where only female embryos are transferred to a woman carrier for an X-linked genetic disorder. By contrast, chromosome Y could be correctly indicated by a normalized ratio (<0.75) in only 2/11 (18%) female(Cy3)/male(Cy5) comparisons (mean = 0.88 ± 0.18) (Table I, Section C).
Improvement of the ratio profile with pooled normal male reference material
To improve the ratio profiles for our array CGH protocol, the normal male reference material was changed from a single cell to a pooled mixture of at least five (but up to 10) single cell DOP-PCR reactions. We hoped in this way that individual PCR variations within the single cell reference material might be avoided. In the first experiment, we compared mixed female cells (Cy3) versus mixed male cells (Cy5) and found that the ratios for all the chromosomes except the Y were as expected, falling in the range 0.75–1.25. Due to the high level of mosaicism within human preimplantation embryos, it is not possible to combine more than one cell for CGH analysis of blastomeres. Therefore we used pooled single cell PCR products for the reference material only (in this case normal male single cells) and a single cell as test sample, and repeated four array CGH experiments that produced deviant results for at least one chromosome (Table I, Section B). We repeated one experiment that did not produce deviant results (Table I, Section B, Experiment 1) and no deviant results were seen with the pooled normal male reference either. For the four that previously produced a deviant result(s), three of these showed an improvement of the deviant results following this change in reference material. The ratio of 1.25 for chromosome 2 decreased to 1.16 and for chromosome 9 the ratio decreased from 1.32 to 1.23 (Table I, Section A, Experiment 2), falling within the threshold of 0.75–1.25. The ratio of 0.71 was corrected to 1.09 for chromosome 17 (Table I, Section B, Experiment 2). Similarly the ratio of 1.25 for chromosome 17 was decreased to 1.13 (Table I, Section B, Experiment 6). For the fourth experiment with a previous deviant result for chromosome 22 of 0.74 (Table I, Section B, Experiment 5), this was corrected by increasing to 1.09; however, four other chromosomes now produced ratios too high from 1.25 to 1.28 (data not shown). Despite this, we obtained better profiles for ratios for three out of the four experiments using pooled normal male or female single cell DOP-PCR products as the reference material. Therefore for all further experiments in this study, we used this method to generate the reference material.
Array CGH on a single cell from each of three trisomic cell lines
We tested our method on a single fibroblast from each of three cell lines, which were shown by conventional cytogenetics to contain a specific chromosome aneuploidy. In each case we compared this cell to pooled normal male reference material and were able to obtain a diagnosis based on the fact that the aneuploid chromosome gave the highest ratio (Table I, Section D). For the trisomy 13 (47,XY,+13) single cell (Figure 1), the chromosome 13 dots gave a ratio of 1.32, well outside the thresholds of 0.75 and 1.25. The range for the other autosomes all fell well within the 0.75–1.25 range, ranging from 0.85 to 1.20. For the trisomy 15 cell (47,XY,+15), a ratio of 1.27 was observed for chromosome 15. This was outside the thresholds 0.75 and 1.25, whereas all the other autosomes fell within the 0.75–1.25 range, varying from 0.94–1.08. The trisomy 18 (47,XY,+18) single cell gave a ratio of 1.27 for chromosome 18, which was outside of the 0.75 and 1.25 thresholds but all the other autosomes fell well within these thresholds, varying between 0.87 and 1.09.
|
Effects of photomultiplier tube gains on ratio profiles
To test the effect of photomultiplier tube (PMT) gains, one slide of a female(Cy3)/male(Cy5) comparison was scanned using six different gain settings (Figure 2). A normalized ratio of
1.25 for the X chromosome was obtained in all cases along with ratios for all other chromosomes falling within the range 0.75–1.25 (the Y chromosome also fell within this range but was an inadmissible result). Ratios of chromosomes obtained from the scanning of six different PMT gain settings were not significantly different (single-factor analysis of variance, F = 0.0032, P = 0.99). This suggests that, for GenePix 4000B, optimization of PMT gains is not required for our array CGH approach as long as PMT gains for Cy3 and Cy5 channels are within the range 500 to 700.
|
Effects of rescanning on ratio profiles
We scanned the slide of a trisomic male single cell (47,XY,+18, Cy3) versus a normal male (Cy5) six times over a period of 73 days. A normalized ratio
1.25 for chromosome 18 was obtained in all cases with all normalized ratios for the other chromosomes falling within the range 0.75–1.25 (Figure 3). Ratios of chromosomes obtained from rescanning six times were not significantly different (single-factor analysis of variance, F = 0.0064, P = 0.99). These results suggest that the hybridized slides could be stored at room temperature in the dark for up to 73 days with no change in the resulting diagnosis.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Aneuploidy detection in preimplantation embryos is currently carried out using FISH for a small number of chromosomes (Griffin et al., 1993; Munné et al., 1999; Verlinsky et al., 1999). We and other groups have developed single cell metaphase CGH methods to overcome some of the limitations of FISH, namely that only a small number of chromosomes can be analysed and the difficulty of interpretation of the hybridization signals due to lost DNA, split or overlapping signals (Voullaire et al., 2000; Wells and Delhanty, 2000). The major limitation of metaphase CGH methods is the length of time, up to 72 h, required to perform the hybridization step. Although there are two reports of the clinical application of metaphase CGH for polar body diagnosis and embryo aneuploidy screening (Wilton et al., 2001; Wells et al., 2002) the methods thus far are labour intensive and cannot be scaled up to allow the screening of large numbers of embryos. For blastomere diagnosis, the long length of time to perform the analysis means that embryos must be cryopreserved, a step which may negate the benefits of the screening procedure. The advantage of our method is that it takes only 30 h to perform, making it applicable to PGD for aneuploidy screening, allowing embryo transfer to occur in the fresh IVF cycle. Microarray CGH is easier to perform than single cell metaphase CGH and is more amenable to automation. Compared to single cell FISH, microarray CGH may allow a concomitant diagnosis for single gene disorders, providing the PGD patient at high risk of genetic disorders such as cystic fibrosis with specific information about their genotype as well as information about aneuploidy. Recently it has been reported after DOP-PCR amplification of the first polar body for metaphase CGH aneuploidy screening 10 out of 11 of the samples produced a PCR product for the
F508 locus of the cystic fibrosis gene (Wells et al., 2002). The patient was not a carrier for this mutation and therefore this test could not determine whether the two alleles of the gene had been successfully amplified in all cases. Although the amplification rates seem promising (Wells et al., 1999, 2002), further experiments need to be carried out using heterozygous loci, especially short tandem repeats, to determine the ADO rate. The novelty of our method compared with other CGH microarrays (Pinkel et al., 1998; Pollack et al., 1999; Albertson et al., 2000; Snijders et al., 2001; Cai et al., 2002; Pollack et al., 2002; Veltman et al., 2002) is that we did not spot DNA clones (such as mixtures of PAC or BAC clones) on the microarray. Instead we used DOP-PCR products from chromosome-specific DNA libraries spotted onto glass slides to perform array CGH analysis. Given that some regions of the karyotype in metaphase CGH experiments, such as telomeres, centromeres and heterochromatic regions, showed unexpected variation in the profile of the ratios (Kallioniemi et al., 1994; Voullaire et al., 2000), repeat-depleted DOP-PCR chromosome-specific DNA libraries were used. This strategy aimed to minimize any adverse impact on the profiles of ratios of chromosomes due to the repetitive sequence-rich nature of these regions. In initial experiments we used 30 µg of human Cot-1 DNA per hybridization, but we found that we needed to increase this to 70 µg to suppress the non-specific signals from repetitive sequences produced by DOP-PCR of the samples. Furthermore, we improved the ratio profiles when we changed our washing buffer from SSC to formamide.
Our array CGH was developed specifically to screen aneuploidy across the whole karyotype. As a result, the biggest disadvantage is its inability to detect deletions and duplications involving specific regions of chromosomes, which can be detected by metaphase CGH on genomic DNA (Bentz et al., 1998). Using more spots each from DNA libraries pertaining to smaller regions of the chromosome might solve this problem.
Unexpected deviations are frequently observed for chromosomes 17, 19, 20 and 22 when performing single cell CGH with metaphase spreads (Voullaire et al., 2000). We have also observed unexpected ratio profiles for some chromosomes using our array CGH method. The use of pooled PCR products from normal male single cells as the reference instead of those from just a single cell has the obvious advantage of evening out the signals from the reference material and eliminating extreme variations in the PCR products of the reference material. However, extreme variations in the test sample cannot be avoided in this manner as only a single cell is available for analysis. Although pooled reference material reduced the number of aberrant results, it did not eliminate them. Further refinements in the probes spotted onto the glass slide as well as changes in the amplification technique are likely to yield more accurate results in the future. Not surprisingly we found that DNA libraries generated from microdissected chromosomes gave better results than those from flow-sorted chromosomes. The purity of flow-sorted chromosomes is never likely to be 100% and this may have contributed to the flow-sorted chromosomes producing a wider range of ratios compared to the microdissected ones. This is especially true for the Y chromosome where microdissection of the p arm only would have resulted in a much better probe for this chromosome as it avoids the presence of repetitive sequences in the long arm. Experiments to correct the Y chromosome problem are currently underway in our laboratory.
The accuracy of interphase FISH per probe per cell has been estimated to be 91–96% for euploid samples and even lower for trisomic samples (Ruangvutilert et al., 2000). The overall accuracy of our array CGH approach for all 22 autosomes was 97%, which is much higher than that for FISH analyses. In a recent report, DOP-PCR-based array CGH was used to test 500 cells from a cell line containing a trisomy 7 aneuploidy. The mean ratio for the trisomic chromosome was 1.3, ranging from 1.22 to 1.45 (Daigo et al., 2001). In another study, a ratio of 1.28 was used for the diagnosis of duplications of a specific locus (which is equivalent to a trisomic diagnosis) (Veltman et al., 2002). We report here a range of 1.27–1.32 for the aneuploid chromosomes in our three trisomic cells. This falls within the range reported in these studies.
We report in this paper a novel approach to manufacture a DNA microarray for CGH for the detection of aneuploidy. Our microarray overcomes a major limitation of metaphase CGH, which is the length of time required to perform the analysis. Although this study focuses on developing aneuploidy screening for PGD, this technology could also be used for analysing uncultured amniocytes, the limited amount of material in dissected tumour samples, and single fetal cells isolated non-invasively from peripheral blood of pregnant women.
|
Acknowledgements |
|---|
We thank Drs A.Bolzer from the Institute für Anthropologie und Humangenetik, LMU, München, Germany and J.M.Craig, presently at the Murdoch Children’s Research Institute, Melbourne, Australia for their generous gift of repeat-depleted libraries. This work was supported by a grant from the NH&MRC [991345 (GN ID 9938254)] and research grants from The Reproductive Medicine Unit, University of Adelaide, Australia awarded to N.Hussey. Dr Hu was supported by an International Postgraduate Research Scholarship from the University of Adelaide.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Albertson DG, Ylstra B, Segraves R, Collins C, Dairkee SH, Kowbel D, Kuo WL, Gray JW and Pinkel D (2000) Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene. Nat Genet 25,144–146.[CrossRef][Web of Science][Medline]
Bentz M, Plesch A, Stilgenbauer S, Dohner H and Lichter P (1998) Minimal sizes of deletions detected by comparative genomic hybridization. Genes Chromosomes Cancer 21,172–175.[CrossRef][Web of Science][Medline]
Bolzer A, Craig JM, Cremer T and Speicher MR (1999) A complete set of repeat-depleted, PCR-amplifiable, human chromosome-specific painting probes. Cytogenet Cell Genet 84,233–240.[CrossRef][Web of Science][Medline]
Cai WW, Mao JH, Chow CW, Damani S, Balmain A and Bradley A (2002) Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays. Nat Biotechnol 20,393–396.[CrossRef][Web of Science][Medline]
Craig JM, Kraus J and Cremer T (1997) Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography. Hum Genet 100,472–476.[CrossRef][Web of Science][Medline]
Cui XF, Li HH, Goradia TM, Lange K, Kazazian HH Jr, Galas D and Arnheim N (1989) Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers. Proc Natl Acad Sci USA 86,9389–9393.
Daigo Y, Chin SF, Gorringe KL, Bobrow LG, Ponder BA, Pharoah PD and Caldas C (2001) Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers: a new approach for the molecular analysis of paraffin-embedded cancer tissue. Am J Pathol 158,1623–1631.
Gianaroli L, Magli MC, Ferraretti AP and Munné S (1999) Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 72,837–844.[CrossRef][Web of Science][Medline]
Griffin DK, Wilton LJ, Handyside AH, Atkinson GH, Winston RM and Delhanty JD (1993) Diagnosis of sex in preimplantation embryos by fluorescent in situ hybridisation. Br Med J 306,1382.
Guan XY, Meltzer PS and Trent JM (1994) Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection. Genomics 22,101–107.[CrossRef][Web of Science][Medline]
Hussey ND, Donggui H, Froiland DA, Hussey DJ, Haan EA, Matthews CD and Craig JE (1999) Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells. Mol Hum Reprod 5,1089–1094.
Kallioniemi OP, Kallioniemi A, Piper J, Isola J, Waldman FM, Gray JW and Pinkel D (1994) Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosomes Cancer 10,231–243.[Web of Science][Medline]
Munné S et al. (1999) Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 14,2191–2199.
Munné S, Cohen J and Sable D (2002) Preimplantation genetic diagnosis for advanced maternal age and other indications. Fertil Steril 78,234–236.[CrossRef][Web of Science][Medline]
Pinkel D et al. (1998) High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet 20,207–211.[CrossRef][Web of Science][Medline]
Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, Jeffrey SS, Botstein D and Brown PO (1999) Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 23,41–46.[Web of Science][Medline]
Pollack JR, Sorlie T, Perou CM, Rees CA, Jeffrey SS, LonningPE, Tibshirani R, Botstein D, Borresen-Dale AL and Brown PO (2002) Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors. Proc Natl Acad Sci USA 99,12963–12968.
Ruangvutilert P, Delhanty JD, Rodeck CH and Harper JC (2000) Relative efficiency of FISH on metaphase and interphase nuclei from non-mosaic trisomic or triploid fibroblast cultures. Prenat Diagn 20,159–162.[CrossRef][Web of Science][Medline]
Snijders AM et al. (2001) Assembly of microarrays for genome-wide measurement of DNA copy number. Nat Genet 29,263–264.[CrossRef][Web of Science][Medline]
Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BA and Tunnacliffe A (1992) Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13,718–725.[CrossRef][Web of Science][Medline]
Veltman JA, Schoenmakers EF, Eussen BH, Janssen I, Merkx G, van Cleef B, van Ravenswaaij CM, Brunner HG, Smeets D and van Kessel AG (2002) High-throughput analysis of subtelomeric chromosome rearrangements by use of array-based comparative genomic hybridization. Am J Hum Genet 70,1269–1276.[CrossRef][Web of Science][Medline]
Verlinsky Y et al. (1999) Prevention of age-related aneuploidies by polar body testing of oocytes. J Assist Reprod Genet 16,165–169.[CrossRef][Web of Science][Medline]
Voullaire L, Wilton L, Slater H and Williamson R (1999) Detection of aneuploidy in single cells using comparative genomic hybridization. Prenat Diagn 19,846–851.[CrossRef][Web of Science][Medline]
Voullaire L, Slater H, Williamson R and Wilton L (2000) Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet 106,210–217.[CrossRef][Web of Science][Medline]
Wells D and Delhanty JD (2000) Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod 6,1055–1062.
Wells D, Sherlock JK, Handyside AH and Delhanty JD (1999) Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation. Nucleic Acids Res 27,1214–1218.
Wells D, Escudero T, Levy B, Hirschhorn K, Delhanty JD and Munné S (2002) First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril 78,543–549.[CrossRef][Web of Science][Medline]
Wilton L, Williamson R, McBain J, Edgar D and Voullaire L (2001) Birth of a healthy infant after preimplantation confirmation of euploidy by comparative genomic hybridization. N Engl J Med 345,1537–1541.
Submitted on September 30, 2003; resubmitted on November 21, 2003; accepted on November 25, 2003.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. B. Geigl, A. C. Obenauf, J. Waldispuehl-Geigl, E. M. Hoffmann, M. Auer, M. Hormann, M. Fischer, Z. Trajanoski, M. A. Schenk, L. O. Baumbusch, et al. Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays Nucleic Acids Res., August 1, 2009; 37(15): e105 - e105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Wells, S. Alfarawati, and E. Fragouli Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH Mol. Hum. Reprod., December 1, 2008; 14(12): 703 - 710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Obradors, E. Fernandez, M. Oliver-Bonet, M. Rius, A. de la Fuente, D. Wells, J. Benet, and J. Navarro Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: Case Report Hum. Reprod., August 1, 2008; 23(8): 1949 - 1956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Fuhrmann, O. Schmidt-Kittler, N. H. Stoecklein, K. Petat-Dutter, C. Vay, K. Bockler, R. Reinhardt, T. Ragg, and C. A. Klein High-resolution array comparative genomic hybridization of single micrometastatic tumor cells Nucleic Acids Res., April 1, 2008; 36(7): e39 - e39. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Fiegler, J. B. Geigl, S. Langer, D. Rigler, K. Porter, K. Unger, N. P. Carter, and M. R. Speicher High resolution array-CGH analysis of single cells Nucleic Acids Res., February 16, 2007; 35(3): e15 - e15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Donoso, C. Staessen, B.C.J.M. Fauser, and P. Devroey Current value of preimplantation genetic aneuploidy screening in IVF Hum. Reprod. Update, January 1, 2007; 13(1): 15 - 25. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Le Caignec, C. Spits, K. Sermon, M. De Rycke, B. Thienpont, S. Debrock, C. Staessen, Y. Moreau, J.-P. Fryns, A. Van Steirteghem, et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res., January 1, 2006; 34(9): e68 - e68. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Knijnenburg, M. van der Burg, P. Nilsson, H. K. P. van Amstel, H. Tanke, and K. Szuhai Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms Nucleic Acids Res., October 12, 2005; 33(18): e159 - e159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wilton Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization Hum. Reprod. Update, January 1, 2005; 11(1): 33 - 41. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||