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Copy number variation and association over T-cell receptor genes—influence of DNA source

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Abstract

Genomic copy number variants (CNVs) are a common, heritable source of inter-individual differences in genomic sequence. Their influence on phenotypic variability and their involvement in the pathogenesis of several common diseases is well established and the object of many current studies. In the course of examining CNV association to various quantitative traits in a general population, we have detected a strong association of CNVs over the four TCR genes to lymphocyte and neutrophil numbers in blood. In a small replication series, we have further characterized the nature of these CNVs and found them not to be germline, but dependent on the origin of analysed DNA. Germline deletion and rearrangement around the T-cell receptor (TCR) genes naturally occurs in white blood cells. Blood DNA derived from persons with high lymphocyte counts generates variable intensity signals which behave like germline CNVs over these genes. As DNA containing a relative high proportion of these CNV-like events involving the TCR genes has the ability to influence genotype counts of SNPs in the regions of these genes, care should be taken in interpreting and replicating association signals on variants within these genes when blood-derived DNA is the only source of data.

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Acknowledgements

The authors are grateful to the study participants for their participation and collaboration in this research project.The authors acknowledge the help of Stefan Waldner in recruiting the follow-up cohort. We thank Clemens Egger and Yuri D’Elia for technical support, Langes Martin and Roberto Gambato for performing the immunological typing, and Deborah Mascalzoni and Mirko Modenese for their support in ethical and statistical issues. This work was supported by the Ministry of Health of the Autonomous Province of South Tyrol and the South Tyrolean Sparkasse.

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Authors and Affiliations

  1. Institute of Genetic Medicine, European Academy Bozen/Bolzano (EURAC), 39100, Bolzano, Italy

    Christine Schwienbacher, Alessandro De Grandi, Christian Fuchsberger, Maurizio F. Facheris, Peter P. Pramstaller & Andrew A. Hicks

  2. Department of Neurology, General Central Hospital, 39100, Bolzano, Italy

    Maurizio F. Facheris & Peter P. Pramstaller

  3. Department of Experimental and Diagnostic Medicine, University of Ferrara, 44121, Ferrara, Italy

    Christine Schwienbacher

  4. Department of Haematology and Bone Marrow Transplantation, General Central Hospital, 39100, Bolzano, Italy

    Mirija Svaldi

  5. Institute of Lung Biology and Disease, German Research Center for Environmental Health (Helmholtz Zentrum Muenchen), 85764, Neuherberg, Germany

    Matthias Wjst

  6. Department of Neurology, University of Lübeck, 23538, Lübeck, Germay

    Peter P. Pramstaller

Authors
  1. Christine Schwienbacher
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  2. Alessandro De Grandi
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  3. Christian Fuchsberger
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  4. Maurizio F. Facheris
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  6. Matthias Wjst
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  7. Peter P. Pramstaller
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Corresponding authors

Correspondence to Peter P. Pramstaller or Andrew A. Hicks.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online resource 1 electronic supplementary material

QuantiSNP detection of copy number variation over the three T-cell receptor loci. Detailed QuantiSNP (see main text) output for the regions on chromosomes 7 and 14 over the four TCR genes. Boundary SNPs and positions for start and stop of particular CNVs are listed. Copy number scale is relative to normal two copies, such that 0 = homozygous deletion, 1 = deletion on one strand, 3 = sum total of one extra copy and 4 = sum total of two or more extra copies (QuantiSNP does not resolve copy numbers beyond four). (PDF 130 kb)

Online resource 2 electronic supplementary material

Primers for CNV validation. The presence of CNVs was detected by real-time 40-cycle two-step PCR amplifications (50°C for 2 min, 95°C for 10 min; 40 cycles at 95°C for 15 s and 60°C for 1 min; 95°C for 15 min, 60°C for 30 s in a 25 μL volume using 40 ng of genomic DNA, Power SYBR® Green PCR Master Mix (Applied Biosystems, CA, USA) and 20 pmols of each primer. PCR reactions on each sample were performed in triplicate. Gene copy number analysis was performed by the Sequence Detection Software version 1.3.1, the 7300 System SDS Software RQ Study application (Applied Biosystems, CA, USA) and by the Gene Expression Macro™ Version 1.1 (BioRad, Munich, Germany). As reference for the relative quantification an amplicon (ChrXYsgF/chrXYsgR) in a common region of chromosomes X and Y without CNVs was used in our tested samples. (PDF 89.4 kb)

Online resource 3 electronic supplementary material

CNV validation by real-time PCR. 12 samples were analysed with all TCRα/TCRδ probes (TCRA_*), 14 samples for the β probe (TCRB) and 41 and 39 samples, respectively for the TCRγ (TCRG) exonic and intronic probes. (PDF 76 kb)

Online resource 4 electronic supplementary material

Mononuclear blood cell counts in MICROS1 and follow-up collection. α, α–γ, γ: represent presence of CNVs at TCRα/TCRδ, TCRα/TCRδ plus TCRγ, and TCRγ loci predicted by the analysis of the chip-based SNP genotyping platforms. No CNVs: represents samples without chip-predicted CNVs, abnormal lymphocyte and neutrophil counts are highlighted in grey, nd: not determined. (PDF 111 kb)

Online resource 5 electronic supplementary material

Immunological typing in follow-up collection. α, α–γ, γ: represent presence of CNVs at TCRα/TCRδ, TCRα/TCRδ plus TCRγ, and TCRγ loci predicted by the analysis of the chip-based SNP genotyping platforms. No CNVs: represents samples without chip-predicted CNVs. Abnormal cell counts and percent values are highlighted in grey. (PDF 104 kb)

Online resource 6 electronic supplementary material

Multiple independent relative DNA quantification analyses by real-time PCR on randomly selected MICROS1 and follow-up collection DNA samples. To assess the stability of the real-time PCR quantification approach to detect CNVs, analyses for randomly selected samples were performed independently multiple times. Single analyses on each sample were performed in triplicate. Reported values represent means ± SD. 1 is the reference value for two DNA copies. (PDF 127 kb)

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Schwienbacher, C., De Grandi, A., Fuchsberger, C. et al. Copy number variation and association over T-cell receptor genes—influence of DNA source. Immunogenetics 62, 561–567 (2010). https://doi.org/10.1007/s00251-010-0459-7

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  • DOI: https://doi.org/10.1007/s00251-010-0459-7

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