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An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH.

ABSTRACT: BACKGROUND: Comparative genomic hybridization microarrays for the detection of constitutional chromosomal aberrations is the application of microarray technology coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genomewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient against a normal reference sample. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the "normal" reference instead of a patient aberration. Instead, we apply an experimental loop design that compares three patients in three hybridizations. RESULTS: We develop and compare two statistical methods (linear models of log ratios and mixed models of absolute measurements). In an analysis of 27 patients seen at our genetics center, we observed that the linear models of the log ratios are advantageous over the mixed models of the absolute intensities. CONCLUSION: The loop design and the performance of the statistical analysis contribute to the quick adoption of array CGH as a routine diagnostic tool. They lower the detection limit of mosaicisms and improve the assignment of copy number variation for genetic association studies.

SUBMITTER: Allemeersch J

PROVIDER: S-EPMC2791104 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH.

Allemeersch Joke J Van Vooren Steven S Hannes Femke F De Moor Bart B Vermeesch Joris Robert JR Moreau Yves Y

BMC bioinformatics 20091119

<h4>Background</h4>Comparative genomic hybridization microarrays for the detection of constitutional chromosomal aberrations is the application of microarray technology coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genomewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing ...[more]

PMID: 19925645

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Project description:Comparative genomic hybridization microarrays (array CGH or molecular karyotyping) for the detection of congenital chromosomal aberrations is the application of microarray technology that is coming fastest into routine clinical application. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient sample against a normal reference sample and detecting copy number variations through the deviation of fluorescent signal intensity between patient and normal reference. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the ânormalâ reference instead of a patient aberration. We therefore propose a new experimental loop design that compares three patients in three hybridizations (Patient 1 vs. Patient 3, Patient 3 vs. Patient 2, and Patient 2 vs. Patient 1). We develop and compare two statistical methods (linear models of log ratios and mixed models of absolute measurements). In an analysis of data from 27 patients seen at our genetics center, this new setup together with the linear model analysis significantly overcomes the limitations of the classical setup. Furthermore, we observed that the linear models of the log-ratios had a higher signal-to-noise ratio than the mixed models of the absolute intensities. These improvements are important to guarantee a maximal efficiency of array CGH in a clinical setting and will therefore contribute to its quick adoption as a routine diagnostic tool. The method is implemented as a web application and is available at www.esat.kuleuven.be/loop. Keywords: comparative genomic hybridization The data set consists out of nine loop designs or 27 patients with mental retardation (MR) and multiple congenital anomalies (MCA). The patients were seen at our genetics center (Center for Human Genetics, U.Z.Leuven).

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.

Dataset Information

An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH.

Publications

An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH.

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