Single-cell chromosomal imbalances detection by array CGH - PubMed (original) (raw)

Claudia Spits, Karen Sermon, Martine De Rycke, Bernard Thienpont, Sophie Debrock, Catherine Staessen, Yves Moreau, Jean-Pierre Fryns, Andre Van Steirteghem, Inge Liebaers, Joris R Vermeesch

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Single-cell chromosomal imbalances detection by array CGH

Cedric Le Caignec et al. Nucleic Acids Res. 2006.

Abstract

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.

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Figures

Figure 1

Figure 1

(A–D) Examples of single-cell array CGH profiles performed on aneuploid cell lines. For each panel, the _x_-axis represents the 22 autosomes, followed by the X and Y chromosomes. The _y_-axis marks the log2 mean ratios of all spots of each chromosome. Following ϕ29 DNA polymerase amplification, single cells containing trisomy 13 (A), 18 (B) and 21 (C) were hybridized versus non-amplified gDNA of the opposite sex, and monosomic X single cell (D) versus non-amplified XX gDNA. The checked columns represent the abnormal chromosomes.

Figure 2

Figure 2

Overview of single-cell experiments performed on aneuploid cell lines and analysis using chromosome-specific threshold. The closed circles represent the mean intensity ratios of chromosomes 13, 18 and 21 of each respective trisomy cell line. The open circles represent the mean intensity ratios of X and Y chromosomes of monosomy X cell line. The _x_-axis represents the 22 autosomes, the X and Y chromosomes. The _y_-axis marks the log2 mean ratio of the 18 single-cell experiments for each chromosome. The grey columns represent the mean ratios of the 18 experiments of each chromosome plus or minus three times the standard deviation representing the chromosome-specific threshold.

Figure 3

Figure 3

(A–C) Examples of single-cell array CGH profiles performed on cell lines containing a segmental deletion and/or duplication. (A) Following ϕ29 DNA polymerase amplification, single cells containing an interstitial 4q deletion were hybridized versus non-amplified gDNA of the opposite sex. The _y_-axis marks the log2 mean ratios of all spots of each chromosome. The _x_-axis represents the 22 autosomes, followed by the X and Y chromosomes. Chromosome 4 was divided in three regions: 4a represented the region from the subtelomere 4p to the centromeric breakpoint of the deletion (59.8 Mb), 4b corresponded to the 4q deletion (34 Mb) and 4c the region from the distal breakpoint of the deletion to the subtelomere 4q (97.6 Mb). The deleted region (4b) showed the expected log2 mean ratio. (B and C) Following ϕ29 DNA polymerase amplification, single cells containing an unbalanced reciprocal translocation involving chromosomes X and 14 were hybridized versus non-amplified 46,XX and 46,XY gDNA. The _y_-axis marks the log2 mean ratios of all spots of each chromosome. Chromosome 14 was divided in two regions: 14a represented the region from the centromere to the breakpoint of the translocation (59.4 Mb), 14b corresponded to the 14q duplication (47 Mb). Chromosome X was divided in two regions: Xa represented the region from the subtelomere Xp to the breakpoint of the translocation (59.7 Mb) and Xb corresponded to the Xq deletion. When hybridized versus female gDNA (B), a deletion of Xb was expected, whereas hybridised versus male gDNA (C), a log2 mean ratio corresponding to a duplication of Xa was expected. The checked columns represent the 4q- and the Xq-deleted regions and the 14q-duplicated region.

Figure 4

Figure 4

(A–D) Examples of single-cell array CGH profiles performed on blastomeres. For each, the _x_-axis represents the 22 autosomes, followed by the X and Y chromosomes. The _y_-axis marks the log2 mean ratios of all spots of each chromosome. The checked columns represent the abnormal chromosomes. Following ϕ29 DNA polymerase amplification, (A) one blastomere of embryo 1 was hybridized versus non-amplified female gDNA, (B and D) three blastomeres of embryo 2 versus non-amplified female gDNA. (E) Overview of single-cell experiments performed on embryo 2. The closed circles represent the mean intensity ratios of abnormal chromosomes identified. The _x_-axis represents the 22 autosomes, the X and Y chromosomes. The _y_-axis marks the log2 mean ratios of the 12 single-cell experiments of each chromosome. The grey columns represent the mean ratios of the 12 experiments of each chromosome plus or minus three times the standard deviation used for the chromosome-specific threshold.

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