Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization

Abstract

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.

Chromosomal disorders and male infertility

“…More recently, two new testing platforms have come into use, microarray CGH (array-CGH (aCGH)) [96][97][98][99] and single nucleotide polymorphism (SNP) microarrays. [100][101][102][103] These testing platforms allow for comprehensive chromosomal analysis of single cells from day-3 biopsy and yield results in 24 h. The rapid turnaround time for these methods eliminates the need to cryopreserve embryos, while testing is carried out.…”

Whole genome amplification in preimplantation genetic diagnosis

“…The long period required for hybridization (up to 72 h) has limited the widespread use of this technique, as it is necessary to freeze the biopsied embryos, and the survival rate of the thawed embryos has been relatively poor (approximately 50% did not survive the thawing process) (Hu et al, 2004;Escribá et al, 2008). The development of a highly efficient cryopreservation technique is urgently needed to reduce fears concerning the impact of cryopreservation on embryo viability.…”

“…Recently, array-CGH has been developed, and it takes only 30 h to perform the hybridization step (Hu et al, 2004). Array-CGH is much easier to perform and more amenable to automation than CGH.…”

“…It can be used for analyzing a wide variety of genetic mutations. The advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis can be offered by accurate microarray platforms (Hu et al, 2004;Wells et al, 2008). This is a huge improvement over classical karyotyping, and it is considered to have great potential to replace much of the present diagnostic cytogenetics (Vermeesch et al, 2007).…”

Preimplantation genetic diagnosis: present and future

“…Microarray CGH has been successfully applied for the detection of aneuploidies in single cells after whole genome amplification using DOP-PCR or an alternative method known as multiple displacement amplification (MDA) [63][64][65]. Comprehensive chromosome analysis was achieved in less than 48 hours, within the timeframe of a regular embryo transfer after PGD.…”