Unknown,Transcriptomics,Genomics,Proteomics

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Single-cell aneuploidy detection by array CGH

ABSTRACT: In this study, we extend array CGH technology by making the accurate detection of chromosomal imbalances possible from a single fibroblast and blastomere following Phi29 DNA polymerase amplification. Firstly, array CGH experiments were performed on four different fibroblast cell lines, derived from patients affected by, respectively, trisomy 13, 18, 21, and monosomy X. For each cell line, three single cells were amplified. Following DNA amplification, all cells showed the expected DNA yields (n=12; 1.87 Âµg plus:minus 0.39). Sex-mismatch array CGH experiments were conducted on amplified DNA samples obtained from each cell. Sex chromosome ploidy levels, as well as all expected autosomal abnormalities were clearly identified. Secondly, we applied single-cell aneuploidy screening for the detection of chromosomal imbalances in preimplantation embryos. DNA from blastomeres from three 7-8 cell-stage embryos was amplified by ï¦29 DNA polymerase. Following DNA amplification, all cells yielded the expected amount of DNA (n=16; 2.45 Âµg plus:minus 0.41). Chromosomal aneuploidies were accurately detected using a simple and rapid array CGH protocol.

ORGANISM(S): Homo sapiens

SUBMITTER: Andre Van Steirteghem

PROVIDER: E-GEOD-3642 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Single-cell chromosomal imbalances detection by array CGH.

Le Caignec Cedric C Spits Claudia C Sermon Karen K De Rycke Martine M Thienpont Bernard B Debrock Sophie S Staessen Catherine C Moreau Yves Y Fryns Jean-Pierre JP Van Steirteghem Andre A Liebaers Inge I Vermeesch Joris R JR

Nucleic acids research 20060512 9

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, arr ...[more]

PMID: 16698960

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Project description:In this study, we extend array CGH technology by making the accurate detection of segmental aneusomies possible from a single lymphoblast and fibroblast following Phi29 DNA polymerase amplification Array CGH experiments were performed on two segmental aneusomic cell lines derived from patients. As a proof of principle, an interstitial 4q-deletion (46, XX, del(4)(q13.1q22.3)) fibroblast cell line and an unbalanced reciprocal translocation involving chromosomes 14 and X (46, XX, der (X) t(X;14)(q21.3;q23.1)) EBV cell line were used. Array CGH experiments were performed using gDNA from the two cell lines to define the exact size of each rearrangement. For the 4q-deletion cell line, the size of the deleted region was 34 Mb corresponding to 39 clones spotted on the array (from RP11-340A13 to RP11-44P19). For the EBV cell line, the size of the 14q-duplication was 47 Mb corresponding to 63 clones (from RP11-62H20 to CTC-820M16) and the Xq-deletion was 58 Mb corresponding to 70 clones (from RP3-380C13 to RP11-218L14). For each cell line, three single cells were amplified. Following DNA amplification, all cells showed the expected DNA yields (n=6; ï?³ = 2.36 Âµg ï?± 0.12). Sex-mismatch array CGH experiments were conducted on amplified DNA samples obtained from the 4q-deletion cell line. Using the chromosome specific threshold obtained from our 18 experiments, sex chromosome and autosome ploidy levels were accurately identified with no false negative and no false positive results. Averaging the 39 clones within the chromosome 4q-deleted region enabled the accurate detection of the deletions. For each of the three amplified single-cell DNA of the unbalanced reciprocal translocation involving chromosomes 14 and X, male and female gDNA were used as references. Sex chromosome and autosome ploidy levels were accurately identified. Averaging intensity ratios of the 63 clones within the chromosome 14 duplicated region enabled the accurate detection of the duplications in the three replicate experiments. When, respectively, male or female gDNA were used as a reference, the average log2 mean ratio of the Xq-deleted region was â??0.04 or â??0.93, close to the theoretical expected value of 0 or minus 1. Interestingly, the log2 mean ratios of chromosomes 1 to 22 were highly similar when the same single cell amplified DNA was used for the two experiments using respectively female and male DNA as a reference. Hence, array CGH intensity ratio profiles were very reproducible.

Project description:Lobular intraepithelial neoplasia grade 3 (LIN) is a recently recognized variant of intraepithelial lobular neoplasia of the breast composed of either uniform, generally small, cells (classic) with massive lobular distension, pleomorphic cells, signet-ring cells, or any cell type with necrosis. In contrast to classic forms of LIN, there is no consensus on therapeutic strategies for LIN3. In part this is due to the paucity of molecular data that could assist in defining the relationship of LIN3 to classic LIN and carcinomas. In this study we have employed array CGH to determine the patterns of chromosomal aberrations in 9 LIN3 lesions. By comparison to array CGH data of 13 classic LIN lesions, we demonstrate that LIN and LIN3 share several recurrent changes, in particular gains of 1q and losses of 16q. Both aberrations are known to appear early in tumorigenesis and to be associated with good prognosis. However, apart from this overlap, there were a number of karyotypic features that were observed exclusively in LIN3. Clearly, this lesion was characterized by a significantly higher number of DNA copy number changes (9 vs. 31 on average), a considerable complexity of chromosomal rearrangements with up to 16 breakpoints in one chromosome and overlapping high copy amplifications. The amplicons encompassed a number of known oncogenes such as ERBB2, CyclinD1 and FGF3 that have already been reported to be overexpressed in breast carcinomas. Our data suggest that, at the genetic level, LIN3 represents a highly advanced lesion with considerable resemblance to carcinomas and, therefore, reflects a lesion on the verge of invasion, and might represent the transition state from an intraepithelial neoplasm to breast carcinoma. In this study, nine cases of pleomorphic/necrotic LIN lesions as well as one classic LIN and the concurrent invasive lobular carcinoma (ILC) were analyzed by array CGH. Sections of formalin-fixed paraffin-embedded (FFPE) specimens cut at 7-8Âµm were mounted on special foil-coated slides (Molecular Devices, San Diego, USA). The sections were then deparaffinized with Xylene, processed in decreasing concentrations of ethanol and stained with haematoxylin. Lasercapture microdissection for both lesions was performed at multiple sites using Veritas Arcturus. For reference-DNA, female mammary tissue without histomorphological changes obtained from reduction mammoplasty specimens was procecessed and laser-microdissected as explained above. Cells were digested in 10Âµl TE, pH 9, and 0.5Âµl proteinase K (20mg/ml) for 48h at 55Â°C. After inactivation of proteinase K at 99Â°C for 10min, the digest was stored at -20Â°C. Without any further purification, the complete digest was used for whole genome amplification by means of the WGA (Whole Genome Amplification) kit from Sigma following the manufacturerâs recommendations. Array CGH was performed as described previously. In brief, two Âµg of amplified tumor and reference DNA were labelled by random priming (BioPrimeÂ® Total Genomic Labeling System, Invitrogen, Carlsbad, CA) with Alexa FluorÂ® 3 and Alexa FluorÂ® 5, respectively, and hybridized onto a tiling path BAC array, consisting of the human 32k BAC Re-Array Set (BACPAC Resources Center; http://bacpac.chori.org/pHumanMinSet.htm; DNA kindly provided by Pieter de Jong) and a 1Mb Resolution BAC set (clones kindly provided by Nigel Carter, Wellcome Trust Sanger Centre). All protocols are provided in detail on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/) and more information concerning this platform have been submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/; GLP: 5114). For the analysis and visualization of array CGH data, our software-package CGH-PRO was employed. No background subtraction was applied. Raw data were normalized by âSubgrid LOWESSâ. For the assessment of copy number gains and losses, we used circular binary segmentation in combination with log2 ratio thresholds of 0.15 and -0.15, respectively. High copy amplifications were defined by focal appearance and a log2 ratio exceeding 0.45. In order to statistically investigate the similarity of aberration profiles we applied Pearson's product-moment correlation to cbs ratios of each of the corresponding arrays.

Dataset Information

Single-cell aneuploidy detection by array CGH

Publications

Single-cell chromosomal imbalances detection by array CGH.

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