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Single-cell chromosomal imbalances detection by array CGH.

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.

SUBMITTER: Le Caignec C

PROVIDER: S-EPMC3303179 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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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:BackgroundDevelopment of breast cancer is a multistage process influenced by hormonal and environmental factors as well as by genetic background. The search for genes underlying this malignancy has recently been highly productive, but the etiology behind this complex disease is still not understood. In studies using animal cancer models, heterogeneity of the genetic background and environmental factors is reduced and thus analysis and identification of genetic aberrations in tumors may become easier. To identify chromosomal regions potentially involved in the initiation and progression of mammary cancer, in the present work we subjected a subset of experimental mammary tumors to cytogenetic and molecular genetic analysis.MethodsMammary tumors were induced with DMBA (7,12-dimethylbenz[a]anthrazene) in female rats from the susceptible SPRD-Cu3 strain and from crosses and backcrosses between this strain and the resistant WKY strain. We first produced a general overview of chromosomal aberrations in the tumors using conventional kartyotyping (G-banding) and Comparative Genome Hybridization (CGH) analyses. Particular chromosomal changes were then analyzed in more details using an in-house developed BAC (bacterial artificial chromosome) CGH-array platform.ResultsTumors appeared to be diploid by conventional karyotyping, however several sub-microscopic chromosome gains or losses in the tumor material were identified by BAC CGH-array analysis. An oncogenetic tree analysis based on the BAC CGH-array data suggested gain of rat chromosome (RNO) band 12q11, loss of RNO5q32 or RNO6q21 as the earliest events in the development of these mammary tumors.ConclusionsSome of the identified changes appear to be more specific for DMBA-induced mammary tumors and some are similar to those previously reported in ACI rat model for estradiol-induced mammary tumors. The later group of changes is more interesting, since they may represent anomalies that involve genes with a critical role in mammary tumor development. Genetic changes identified in this work are at very small scales and thus may provide a more feasible basis for the identification of the target gene(s). Identification of the genes underlying these chromosome changes can provide new insights to the mechanisms of mammary carcinogenesis.

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Single-cell chromosomal imbalances detection by array CGH.

Publications

Single-cell chromosomal imbalances detection by array CGH.

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