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Fragouli E.,University of Oxford | Alfarawati S.,University of Oxford | Daphnis D.D.,London Fertility Center | Goodall N.-N.,Reprogenetics LLC | And 4 more authors.
Human Reproduction | Year: 2011

BACKGROUNDRecent studies have suggested that biopsy of several trophectoderm (TE) cells from blastocysts followed by comparative genomic hybridization (CGH) analysis might represent an optimal strategy for aneuploidy detection, but few data on accuracy are available. The main question concerns the rate of mosaicism at the blastocyst stage, and to what extent this might cause misdiagnoses. We assessed blastocyst aneuploidy and mosaicism rates and evaluated the accuracy and efficiency of CGH and microarray-CGH (aCGH) for TE analysis.METHODSA total of 52 blastocysts, from 20 couples, were biopsied and their chromosomes examined by CGH. The remaining cells were spread and tested by fluorescent in situ hybridization (FISH). Of the 52 blastocysts, 20 underwent a second TE biopsy and were tested using aCGH.RESULTSCGH and aCGH produced results for 98 of TE samples. 42.3 of blastocysts were uniformly euploid, 30 were uniformly aneuploid and 32.4 were mosaic. Of the mosaic embryos, 15.4% were found to be composed of a mixture of different aneuploid cell lines, while 17 contained both normal and aneuploid cells. Mosaic diploidaneuploid blastocysts with >30 normal cells accounted for <6 of analysed embryos. CONCLUSIONSComprehensive chromosome screening and follow-up assessment of large numbers of cells provided a unique insight into the cytogenetics of human blastocysts. Meiotic and post-zygotic errors leading to mosaicism were common. However, most mosaic blastocysts contained no normal cells. Hence, CGH or aCGH TE analysis is an accurate aneuploidy detection tool and may assist in identifying viable euploid embryos with higher implantation potential. © 2010 The Author. Source

Howarth K.D.,University of Cambridge | Pole J.C.M.,University of Cambridge | Pole J.C.M.,BlueGnome Ltd | Beavis J.C.,University of Cambridge | And 4 more authors.
Genome Research | Year: 2011

Reciprocal chromosome translocations are often not exactly reciprocal. Most familiar are deletions at the breakpoints, up to megabases in extent. We describe here the opposite phenomenon - duplication of tens or hundreds of kilobases at the breakpoint junction, so that the same sequence is present on both products of a translocation. When the products of the translocation are mapped on the genome, they overlap. We report several of these "overlapping-breakpoint" duplications in breast cancer cell lines HCC1187, HCC1806, and DU4475. These lines also had deletions and essentially balanced translocations. In HCC1187 and HCC1806, we identified five cases of duplication ranging between 46 kb and 200 kb, with the partner chromosome showing deletions between 29 bp and 31 Mb. DU4475 had a duplication of at least 200 kb. Breakpoints were mapped using array painting, i.e., hybridization of chromosomes isolated by flow cytometry to custom oligonucleotide microarrays. Duplications were verified by fluorescent in situ hybridization (FISH), PCR on isolated chromosomes, and cloning of breakpoints. We propose that these duplications are the counterpart of deletions and that they are produced at a replication bubble, comprising two replication forks with the duplicated sequence in between. Both copies of the duplicated sequence would go to one daughter cell, on different products of the translocation, while the other daughter cell would show deletion. These duplications may have been overlooked because they may be missed by FISH and array-CGH and may be interpreted as insertions by paired-end sequencing. Such duplications may therefore be quite frequent. © 2011 by Cold Spring Harbor Laboratory Press. Source

Gray J.C.,University of Cambridge | Hansen M.R.,University of Cambridge | Shaw D.J.,University of Cambridge | Graham K.,University of Cambridge | And 4 more authors.
Plant Journal | Year: 2012

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1- carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd. Source

Newell C.A.,University of Cambridge | Natesan S.K.A.,University of Cambridge | Natesan S.K.A.,BlueGnome Ltd | Sullivan J.A.,University of Cambridge | And 5 more authors.
Plant Journal | Year: 2012

Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively. Fluorescence from GFP-LacI co-localised with plastid DNA in nucleoids in all tissues of transgenic tobacco (Nicotiana tabacum L.) examined and there was no indication of its presence in stromules, not even in hypocotyl epidermal cells, which contain abundant stromules. Fluorescence from Rpl1-GFP and Rps2-GFP was also observed in a punctate pattern in chloroplasts of tobacco and Arabidopsis [Arabidopsis thaliana (L.) Heynh.], and fluorescent stromules were not detected. Rpl1-GFP was shown to assemble into ribosomes and was co-localised with plastid DNA. In contrast, in hypocotyl epidermal cells of dark-grown Arabidopsis seedlings, fluorescence from Rpl1-GFP was more evenly distributed in plastids and was observed in stromules on a total of only four plastids (<0.02% of the plastids observed). These observations indicate that plastid DNA and plastid ribosomes do not routinely move into stromules in tobacco and Arabidopsis, and suggest that transfer of genetic information by this route is likely to be a very rare event, if it occurs at all. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd. Source

Fiorentino F.,GENOMA Molecular Genetics Laboratory | Spizzichino L.,GENOMA Molecular Genetics Laboratory | Bono S.,GENOMA Molecular Genetics Laboratory | Biricik A.,GENOMA Molecular Genetics Laboratory | And 6 more authors.
Human Reproduction | Year: 2011

Background: Fluorescence in situ hybridization (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements in preimplantation embryos but it is known to have several technical limitations. We describe the clinical application of a molecular-based assay, array comparative genomic hybridization (array-CGH), to simultaneously screen for unbalanced translocation derivatives and aneuploidy of all 24 chromosomes. Methods: Cell biopsy was carried out on cleavage-stage embryos (Day 3). Single cells were first lysed and DNA amplified by whole-genome amplification (WGA). WGA products were then processed by array-CGH using 24sure arrays, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on Day 5 of the same cycle. Results: Twenty-eight consecutive cycles of preimplantation genetic diagnosis were carried out for 24 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5) embryos were successfully diagnosed. Embryos suitable for transfer were identified in 17 cycles (60.7), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6 per embryo transfer), with a total of 14 embryos implanted (63.6 per transferred embryo). Three patients delivered three healthy babies, during writing, the other pregnancies (two twins and seven singletons) are ongoing beyond 20 weeks of gestation. Conclusions: The data obtained demonstrate that array-CGH can detect chromosome imbalances in embryos, also providing the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. Array-CGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity and reliability. © 2011 The Author. Source

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