Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays

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1 Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays Nathan R. Treff, Ph.D., a,b Jing Su, M.Sc., a Xin Tao, M.Sc., a Brynn Levy, Ph.D., a,c and Richard T. Scott, Jr., M.D. a,b a Reproductive Medicine Associates of New Jersey, Morristown; b Division of Reproductive Endocrinology, Department of Obstetrics Gynecology and Reproductive Science, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New Jersey; and c Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, New York Objective: To develop and validate a whole genome amplification and single nucleotide polymorphism (SNP) microarray protocol for accurate single cell 24 chromosome aneuploidy screening. Design: Prospective, randomized, and blinded study. Setting: Academic reproductive medicine center. Patient(s): Multiple euploid and aneuploid cell lines were obtained from a public repository and blastomeres were obtained after biopsy of cleavage stage embryos from 78 patients undergoing IVF. Main Outcome Measure(s): Accuracy of copy number assignment and consistency of individual SNPs, whole chromosomes, and single cell aneuploidy status were determined. Intervention(s): None. Result(s): Single cells extracted from karyotypically defined cell lines provided 99.2% accuracy for individual SNPs, 99.8% accuracy for whole chromosomes, and 98.6% accuracy when applying a quality control threshold for the overall assignment of aneuploidy status. The concurrence for more than 80 million SNPs in 335 single blastomeres was 96.5%. Conclusion(s): We have established and validated a SNP microarray-based single cell aneuploidy screening technology. Clinical validation studies are underway to determine the predictive value of this methodology. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Aneuploidy screening, microarray, preimplantation genetic diagnosis, single cell, single nucleotide polymorphism Despite the promise that aneuploidy screening could improve outcomes for patients undergoing IVF, all randomized clinical trials evaluating fluorescence in situ hybridization (FISH)-based methodologies of aneuploidy screening have failed to demonstrate the expected benefit (reviewed in Ref. 1). It is possible that the negative impact of embryo biopsy or sampling error due to mosaicism represent the root cause of this failure. Although new technologies may not overcome these important factors, they may improve on other limitations including the lack of comprehensive analysis of all 24 chromosomes. Indeed, current FISH methods of aneuploidy screening evaluate less than half of the human chromosomal complement (2 5) and may result in the transfer of reproductively incompetent embryos with aneuploidy for chromosomes not analyzed. In addition, new technologies may provide more accurate and easily interpretable results than current methods. Some of the most promising progress toward developing a comprehensive 24 chromosome analysis method has been made possible through the combination of whole genome amplification (WGA) and either conventional comparative genome hybridization (CGH) (6 8) or array-based CGH (9 12). Although these methods Received December 7, 2009; revised January 11, 2010; accepted January 20, 2010; published online February 25, N.R.T. has nothing to disclose. J.S. has nothing to disclose. X.T. has nothing to disclose. BL is a consultant and advisory board member for Affymetrix Inc. RTS is supported by grants from EMD Serono, Schering Plough, and Ferring Pharmaceuticals. Reprint requests: Nathan R. Treff, Ph.D., 111 Madison Ave, Suite 100, Morristown, NJ (FAX: ; ntreff@rmanj.com). represent exciting and potentially important advances toward improved preimplantation genetic diagnosis for aneuploidy screening, limited information is available regarding the overall accuracy based on blinded and randomized evaluation of samples with known abnormalities. In addition they have not demonstrated consistency in a large sample size of human blastomeres. To establish these parameters and to begin producing a highly validated methodology, we have combined WGA and high density (>262,000) single nucleotide polymorphism (SNP) microarrays for single cell aneuploidy screening. We have performed a series of prospective, randomized, and blinded studies that should allow full characterization of the precision of a new single cell 24 chromosome aneuploidy screening method. MATERIALS AND METHODS Experimental Design This study was organized into three phases of analysis. Phase I was designed to develop and define a method of analysis that could accurately determine the copy number state of chromosomes in single cells from karyotypically defined cell lines. This phase is effectively a calibration of the methodology. Phase II evaluated the accuracy of the method by testing single cells from karyotypically defined cell lines in a prospective, randomized, and blinded manner. Finally, phase III involved analysis of single human blastomeres to establish similar levels of chromosome-specific SNP copy number assignment concurrence as those observed in cell line single cells. Cell Lines and Patient Samples A total of nine well-characterized cell lines were obtained from the Coriell Cell Repository (CCR, Camden, NJ) and cultured as recommended by the /$36.00 Fertility and Sterility â Vol. 94, No. 6, November doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 FIGURE 1 Graphic representation of copy number changes observed in various aneuploid cell lines using Copy Number Analysis Tool Left. Results from microarray analysis performed on genomic DNA extracted from cultured cell lines. Right. Results from whole genome amplified DNA from single cells derived from the same cell line as shown in the left panel. Each graph indicates the copy number assignments (0, 1, 2, 3, or 4) on the y-axis and the chromosome number on the x-axis. Gains (copy number state >2) and losses (copy number state <2) are seen as bars above and below the copy number state of 2, respectively. The likely karyotype is indicated for each sample and is consistent with the designated karyotype assigned by the Coriell Cell Repository using standard cytogenetic analysis. supplier (CCR). Briefly, lymphocytes were cultured in RPMI 1640 medium (Invitrogen Inc., Carlsbad, CA) with 15% fetal bovine serum (FBS; Invitrogen) and fibroblasts were cultured in Minimum Essential Medium (Invitrogen) with 10% 20% FBS (Invitrogen) at 37 C and 5% carbon dioxide as recommended (CCR). Cell lines included a trisomy 8 female (GM04610), a trisomy 9 male (GM09286), a trisomy 13 male (GM02948), a trisomy 15 male (GM03184), a double trisomy 16 and 21 male (GM04435), a trisomy 18 male (GM01359), a monosomy 21 female (GM01201), a normal female (GM00321), and a trisomy X female (GM04626). Single cells from each cell line were processed by whole genome amplification as described later. Genomic DNA was isolated from approximately cells from each cell line using the QIAgen DNeasy Tissue kit as recommended by the supplier (QIAgen Inc., Valencia, CA), and evaluated (without WGA) by microarray analysis in parallel with single cells as described later. Three hundred and thirty five blastomeres were obtained, as previously described (13), from 235 cleavage stage embryos from 78 IVF patients. Multiple blastomeres were evaluated from the same embryo for 16 that arrested in culture. Arrested embryos were donated for research while the remaining embryos were biopsied for embryo DNA fingerprinting (13). All material was obtained with informed and written consent, and under Institutional Review Board approval. Whole Genome Amplification Fibroblast cell lines were treated with trypsin EDTA solution (Invitrogen) for 3 minutes at 37 C, followed by the addition of media containing FBS to inactivate trypsin as recommended (CCR). Lymphocyte cell lines (which are nonadherent) did not require trypsin treatment to resuspend cells. Cell lines were placed under a dissecting microscope to load single cells in a 1-mL volume into a 0.2-mL nuclease-free polymerase chain reaction (PCR) tube (Ambion Inc., Austin, TX) using a 100-mm stripper tip and pipette (Midatlantic Diagnostics, Mount Laurel, NJ). Blastomeres were also transferred in a 1-mL volume under a dissecting microscope. Samples were brought to an 8-mL volume with nuclease-free water (Sigma-Aldrich, 2018 Treff et al. Single cell 24 chromosome aneuploidy screening Vol. 94, No. 6, November 2010

3 FIGURE 2 Loss of heterozygosity (LOH) probability plots for (A) a single cell derived from a female cell line with monosomy 21, and (B) a single cell derived from a male cell line with trisomy 13. Each upper graph indicates the single nucleotide polymorphism (SNP) copy number assignments (0, 1, 2, 3, or 4) on the y-axis. Each lower graph indicates the SNP LOH probability (0 1) on the y-axis. The chromosome number is indicated for both graphs on the x-axis. Arrows indicate chromosomes with unique LOH probability distributions. In the monosomy 21 cell line, there is only one copy of each SNP present on chromosome 21. This is reflected by a shift in the LOH probability of the SNPs toward 1 on the graph (i.e., a complete LOH). In the trisomy 13 cell line, the number of alleles is increased for all SNPs along chromosome 13 due to the extra copy. This is reflected in a greater frequency and distribution of all probes between 0 and 1 in the graph. Because this cell line is male, the LOH due to the single X chromosome is also evident by a shift in the LOH probability of the SNPs toward 1. St. Louis, MO). Alkaline lysis and neutralization were conducted as previously published (14). The lysate was stored at -20 C and subsequent WGA was performed on the lysate according to the manufacturer s instructions starting with library preparation and using the WGA4 GenomePlex Single Cell Whole Genome Amplification kit (Sigma-Aldrich). The GeneElute PCR Purification kit was used to purify WGA DNA from each reaction (Sigma-Aldrich). SNP Microarrays and Y Chromosome Quantitative Realtime PCR The WGA or genomic DNA was processed for analysis on the 262K NspI SNP genotyping array as recommended by the supplier (Affymetrix Inc., Santa Clara, CA). Hybridization, washing, staining, and scanning was conducted with the GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450, and GeneChip Scanner 7G, respectively, and as recommended by the manufacturer (Affymetrix). Copy number assignments and loss of heterozygosity (LOH) analysis results were obtained using the Copy Number Analysis Tool (Affymetrix). The reference data set consisted of 30 normal female genomic DNA samples. The WGA DNA was also subject to quantitative real time PCR analysis using TaqMan assays for SRY (Hs _s1 and Hs _s1), CYorf15B (Hs _s1), NLGN4Y (Hs _s1), and XKRY (Hs _s1) genes located on the Y chromosome, a 7900HT instrument, and SDS 2.3 software (Applied Biosystems Inc., Foster City, CA). Statistical Analyses In phase I, copy number assignments were compared with the previously established karyotypes of each cell line at three levels of analysis; SNPs, chromosomes, and comprehensive diagnosis of the single cells. The overall copy number assignment for a single chromosome was determined based on the SNP copy number that represented the majority of the assignments within that chromosome. For example, if more than 50% of the SNPs in a given chromosome were assigned a copy number of 3, then that chromosome would be assigned a copy number of 3 and a diagnosis of trisomy. This was performed because the intended type of copy number variation targeted by this assay is whole chromosome aneuploidy. Under this assumption, SNPs within the same chromosome should always be given the same copy number assignment. Those SNP copy number assignments that are not the same as the majority of the copy number assignments on a given chromosome were therefore considered nonconcurrent. The total number of nonconcurrent SNPs from all the chromosomes was divided by the total number of SNPs evaluated for each sample to determine the sample-specific rate of nonconcurrence. Samples in phase I that displayed a rate of nonconcurrence within the third interquartile range of the overall distribution of all the samples were considered outliers and unreliable data. The percentage of nonconcurrence at the interface of the second to third interquartile ranges was used as the cutoff for reliable analysis of samples in phase II. Phase II involved evaluation (as defined in phase I) of randomized, blinded single cells. Upon completion of analysis, the samples were decoded and analyzed for consistency with the known karyotype of the samples. In phase III, blastomeres were evaluated for overall SNP copy number concurrence and, where possible, consistency of results on blastomeres derived from the same embryo. Differences between rates and proportions were evaluated using a c 2 test. Significance was set at P<.05. RESULTS Phase I The combination of WGA4 and 262K SNP array analysis of 72 single cells from 9 cell lines demonstrated 99.2% accuracy of copy number assignment of more than 18 million SNPs. Chromosome copy number assignments (n ¼ 1,656) resulted in 99.8% accuracy. Four of the chromosomes were incorrectly assigned as normal. No false-positive aneuploidy was detected. When the result of each individual chromosome was combined to give the overall aneuploidy status for all chromosomes, 95.8% accuracy was observed. Three of the 72 cells contained 1 or 2 of the 4 incorrectly assigned chromosomes. Because whole chromosome aneuploidy is the only type of copy number variation targeted by this test, we can assess the concurrence within a single cell by looking at how often the chromosomespecific SNP copy number assignments agree within each chromosome. Cells with less than 94% concurrence were determined to be statistical outliers (>3 interquartile range). Ninety-four percent was therefore defined as the cutoff for trustworthy data, independent of knowing whether the data agrees with the known karyotype of the single cell. After applying a 94% concurrence cutoff to the analysis of 72 cells, 2 were identified as statistical outliers, which were also 2 of the 3 misdiagnosed cells, thus resulting in an improved accuracy of 98.6%. Phase II To validate the performance of this technique, we randomized and coded 27 single cells from cell lines with known abnormalities. Twenty-five of the single cells were greater than 94% concurrent, Fertility and Sterility â 2019

4 FIGURE 3 Examples of copy number results from four blastomeres within each of two embryos, embryo A (A) and embryo B (B). Results indicate meiotic aneuploidy of chromosomes 8, 11, and 14 in embryo A, and chromosome 22 in embryo B, as well as mitotic aneuploidy of chromosomes 1, 4, and 14 in embryo B. Shifts in the loss of heterozygosity (LOH) probability of aneuploidy chromosomes (arrows) in a blastomere from embryo A are also shown in C, illustrating the utility of this quality control analysis for verification of copy number assignments. and after decoding, were determined to give 100% accuracy. Not a single misdiagnosis was made for any of the chromosomes evaluated and results were similar to those obtained from genomic DNA isolated from a large number of cells from each cell line (Fig. 1). The overall success rate of obtaining a reliable result from 99 single cells in phases I and II was 96%. Loss of heterozygosity was observed in single cells with monosomic chromosomes including 21 and X (Fig. 2A). The LOH probability patterns in cells with trisomic chromosomes were also clearly distinguishable from disomic chromosomes (Fig. 2B). Phase III Because analysis of blastomeres may differ from analysis of single cells derived from cell lines, we evaluated 335 blastomeres from 235 cleavage stage human embryos. One hundred twenty-five (37.3%) of the 335 blastomeres were euploid for all 24 chromosomes, and there was a significantly larger percentage of abnormal chromosomes in the blastomeres (13.7%; 1,047 of 7,613) studied than there were in the 99 single cells from cell lines (4.2%; 99 of 2,227). The average concurrence within each blastomere was 96.5% 5.2. In the 16 arrested embryos where multiple blastomeres were evaluated, consistent and reciprocal abnormalities were observed indicating successful detection of aneuploidy from both meiotic and mitotic origins (Fig. 3). When mosaicism was observed, the abnormalities made biological sense. That is, a missing chromosome in one blastomere was typically accompanied by an extra copy of the same chromosome in other cells. DISCUSSION Clinical validation of new technologies, like array-based aneuploidy screening, is particularly challenging when dealing with single cell diagnostics. This study demonstrates the first validated comprehensive SNP microarray-based chromosomal aneuploidy screening method. Emphasis has been placed on the ability to determine the correct chromosomal status from single cells derived from karyotypically well-characterized cell lines in a randomized, blinded manner. Furthermore, a high degree of concurrence within individual blastomeres and consistency within embryos provides evidence that the high accuracy observed in single cells from cell lines will translate to the blastomere, making SNP microarray-based aneuploidy 2020 Treff et al. Single cell 24 chromosome aneuploidy screening Vol. 94, No. 6, November 2010

5 screening a likely new diagnostic tool in the IVF arena. Previous studies using WGA and array CGH have performed limited analysis on single cells from cell lines and blastomeres from embryos. Our study evaluated a sample size of 99 single cells in two phases to establish accuracy of diagnosing whole chromosome aneuploidy and 335 blastomeres for consistency of results. We have shown that accurate detection of aneuploidies for even the smaller chromosomes (e.g., 21 and 22) is possible and indicates that diagnosis of partial aneuploidies is well within the ability of this technology. Provided that the number of probes is adequately distributed across any aneusomic region, it is reasonable to assume that patients with balanced translocations will also benefit from SNP microarray-based aneuploidy screening. Translocation patients will have the added advantage of screening for all aneuploidies in addition to the unbalanced derivatives associated with their specific translocation. Because this diagnostic is DNA-based, patients that undergo preimplantation genetic diagnosis for single gene disorders may also screen the same embryo for chromosomal aneuploidies by simultaneous PCR and SNP microarray analysis on lysed cells after trophectoderm biopsy. Finally, preliminary data has demonstrated that SNP microarray analysis can be performed in a timeframe compatible with fresh embryo transfer, resulting in successful delivery of healthy newborns. There are a numerous advantages to using SNP microarrays instead of FISH. First, fixation of individual blastomeres and polar bodies onto glass slides is technically challenging and is often fraught with issues including excessive debris and cytoplasm covering the nucleus. Because the reliability of FISH analysis is linked to the quality of blastomere fixation, poorly fixed blastomeres will impact the ability of the probes to hybridize to their target sequences. In addition, spreading and fixation of trophectoderm tissue is very difficult and often leads to uninterpretable results due to overlapping cells with overlapping signals. These issues are negated in microarray analysis as blastomeres, polar bodies, and trophectoderm tissue can simply be placed directly into a PCR tube and subsequently processed. This should enable broader use in IVF programs as extensive training or experience with fixation is not necessary. Second, analysis of SNP microarray data is performed by computational analysis of signal intensities and not by subjective signal interpretation as occurs with FISH analysis. The SNP microarrays are also amenable to high throughput processing through the use of multiwell plates for DNA amplification and labeling, automation instrumentation for washing, staining, and scanning, and batch analysis for copy number assignments. Third, quality control methods such as copy number concurrence and LOH probability analyses provide the opportunity to eliminate unreliable data and independently confirm aneuploidy status. Additional advantages include the ability to identify possible contamination through DNA fingerprinting (13), determine the stage of meiotic cell division error, and assess the parental origin of aneuploid chromosomes. Additional studies comparing SNP microarrays with FISH, CGH, and array CGH are currently underway. Given the strong link between chromosomal aneuploidy and nonviability, it is anticipated that the introduction of SNP microarray-based 24 chromosome aneuploidy screening may enable the preferential transfer of embryos most likely to form a viable pregnancy and thus lead to improvements in outcomes for patients undergoing IVF. 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