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Detection limits of DNA copy number alterations in heterogeneous cell populations

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Abstract

Background

Array Comparative Genomic Hybridization (aCGH) is a widely used technique to assess chromosomal copy number alterations. Chromosomal content, however, is often not uniform throughout cell populations. Here we evaluated to what extent aCGH can detect DNA copy number alterations in heterogeneous cell populations. A systematic evaluation is currently lacking, despite its importance in diagnostics and research. The detection limits reported are a compound of analytical software and laboratory techniques and do not account for the number of probes in relation to sample homogeneity.

Methods

Detection limits were explored with DNA isolated from a patient with intellectual disability (ID) and from tumor cell line BT474. Both were diluted with increasing amounts of normal DNA to simulate different levels of cellularity. Samples were hybridized on microarrays containing 180,880 oligonucleotides evenly distributed over the genome (spacing ~17 kb).

Results

Single copy number alterations, represented by down to 249 probes (4 Mb) and present in 10 % of a cell population, could be detected. Alterations encompassing as few as 14 probes (~238 Kb) could also be detected, but for this a 35 % mosaic level was required.

Conclusions

DNA copy number alterations can be detected in cell populations containing 10 % abnormal cells. Detection of sub-megabase alterations requires a higher percentage of abnormal cells or microarrays with a higher probe density.

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Abbreviations

aCGH:

Array comparative genome hybridization

WCP:

Whole chromosome paint

CEP:

Centromere probe

FISH:

Fluorescence in situ hybridization

GEO:

Gene expression omnibus

CNV:

Copy number variation

ID:

Intellectual disability

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Acknowledgements

This study was supported by the VUmc Institute for Cancer and Immunology (VUmc CCA/V-ICI) and performed within the framework of CTMM, the Center for Translational Molecular Medicine, DeCoDe project (grant 03O-101). We would like to thank Els Voorhoeve from the VU university medical center for collecting the samples.

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Authors and Affiliations

  1. Department of Pathology, VU University Medical Center, PO Box 7057, 1007, MB, Amsterdam, The Netherlands

    Oscar Krijgsman, Daniëlle Israeli, Hendrik F. van Essen, Paul P. Eijk, Renske D. M. Steenbergen, Gerrit A. Meijer & Bauke Ylstra

  2. Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

    Aggie W. Nieuwint & Marjan M. Weiss

  3. Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands

    Michel L. M. Berens & Clemens H. M. Mellink

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  1. Oscar Krijgsman
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Electronic supplementary material

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Supplementary_Figure 1

Chromosome 5 profile of BT474. aCGH profiles of a gain at the p-arm of chromosome 5 in BT474 with 100 %, 30 % and 20 % tumor DNA. (JPEG 95 kb)

High resolution image (TIFF 273 kb)

Supplementary_Figure 2

Dilution range of BT474 DNA on the NimbleGen platform. Profiles of all dilutions of BT474 DNA were performed on the NimbleGen platform. (PNG 203 kb)

Supplementary_Figure 3

aCGH profiles for CNVR3771/2 on chromosome 8 in a patient with ID. Panel A shows an overview of chromosome 8 in the undiluted sample. Panels B–D show the detection of CNVR3771/2 (DNA loss) in dilutions to 35 %, 30 % and 25 % patient DNA. CNVR3771/2 (21 probes, ~1 Mb) could be detected with dilutions down to 35 % and 30 %, but not with 25 % patient DNA. The genomic locations of both CNVs are indicated in B. The X-axis represents the genomic locations (in Mb), and the Y-axis represents the log2 ratios for each probe. Grey lines represent the CBS segments [17]. (JPEG 94 kb)

High resolution image (TIFF 253 kb)

Supplementary_Figure 4

HER2/neu amplification in BT474. aCGH profiles of an amplification at 17q12 (30 probes, ~0.3 Mb), which includes HER2/neu. Hybrizations were performed with 100 %, 20 % and 10 % tumor DNA. The X-axis represents the chromosomal locations, and the Y-axis the log2 ratios. Grey lines represent the CBS segments [17]. (JPEG 120 kb)

High resolution image (TIFF 329 kb)

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Krijgsman, O., Israeli, D., van Essen, H.F. et al. Detection limits of DNA copy number alterations in heterogeneous cell populations. Cell Oncol. 36, 27–36 (2013). https://doi.org/10.1007/s13402-012-0108-2

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