, Volume 62, Issue 8, pp 561–567 | Cite as

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

  • Christine Schwienbacher
  • Alessandro De Grandi
  • Christian Fuchsberger
  • Maurizio F. Facheris
  • Mirija Svaldi
  • Matthias Wjst
  • Peter P. PramstallerEmail author
  • Andrew A. HicksEmail author
Brief Communication


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.


Genomic copy number variants (CNVs) T-cell receptor (TCR) genes Association Hardy–Weinberg equilibrium (HWE) 



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.

Supplementary material

251_2010_459_MOESM1_ESM.pdf (130 kb)
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)
251_2010_459_MOESM2_ESM.pdf (89 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)
251_2010_459_MOESM3_ESM.pdf (77 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)
251_2010_459_MOESM4_ESM.pdf (112 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)
251_2010_459_MOESM5_ESM.pdf (105 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)
251_2010_459_MOESM6_ESM.pdf (128 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)


  1. Coelho FM, Pradella-Hallinan M, Alves GR, Bittencourt LR, Pedrazzoli Neto M, Moreira F, Tufik S (2007) A study of T CD4, CD8 and B lymphocytes in narcoleptic patients. Arq Neuropsiquiatr 65(2B):423–427PubMedGoogle Scholar
  2. Colella S, Yau C, Taylor JM, Mirza G, Butler H, Clouston P, Bassett AS, Seller A, Holmes CC, Ragoussis J (2007) QuantiSNP: an objective bayes hidden-markov model to detect and accurately map copy number variation using SNP genotyping data. Nucleic Acids Res 35(6):2013–2025. doi: 10.1093/nar/gkm076 CrossRefPubMedGoogle Scholar
  3. Conrad DF, Andrews TD, Carter NP, Hurles ME, Pritchard JK (2006) A high-resolution survey of deletion polymorphism in the human genome. Nat Genet 38(1):75–81. doi: 10.1038/ng1697 CrossRefPubMedGoogle Scholar
  4. Cooper GM, Zerr T, Kidd JM, Eichler EE, Nickerson DA (2008) Systematic assessment of copy number variant detection via genome-wide SNP genotyping. Nat Genet 40(10):1199–1203. doi: 10.1038/ng.236 CrossRefPubMedGoogle Scholar
  5. de Villartay JP, Hockett RD, Coran D, Korsmeyer SJ, Cohen DI (1988) Deletion of the human T-cell receptor delta-gene by a site-specific recombination. Nature 335(6186):170–174. doi: 10.1038/335170a0 CrossRefPubMedGoogle Scholar
  6. Diskin SJ, Hou C, Glessner JT, Attiyeh EF, Laudenslager M, Bosse K, Cole K, Mosse YP, Wood A, Lynch JE, Pecor K, Diamond M, Winter C, Wang K, Kim C, Geiger EA, McGrady PW, Blakemore AI, London WB, Shaikh TH, Bradfield J, Grant SF, Li H, Devoto M, Rappaport ER, Hakonarson H, Maris JM (2009) Copy number variation at 1q21.1 associated with neuroblastoma. Nature 459(7249):987–991. doi: 10.1038/nature08035 CrossRefPubMedGoogle Scholar
  7. Fanciulli M, Petretto E, Aitman TJ (2010) Gene copy number variation and common human disease. Clin Genet 77(3):201–213. doi: 10.1111/j.1399-0004.2009.01342.x CrossRefPubMedGoogle Scholar
  8. Fuschiotti P, Pasqual N, Hierle V, Borel E, London J, Marche PN, Jouvin-Marche E (2007) Analysis of the TCR alpha-chain rearrangement profile in human T lymphocytes. Mol Immunol 44(13):3380–3388. doi: 10.1016/j.molimm.2007.02.017 CrossRefPubMedGoogle Scholar
  9. Hallmayer J, Faraco J, Lin L, Hesselson S, Winkelmann J, Kawashima M, Mayer G, Plazzi G, Nevsimalova S, Bourgin P, Hong SS, Honda Y, Honda M, Hogl B, Longstreth WT Jr, Montplaisir J, Kemlink D, Einen M, Chen J, Musone SL, Akana M, Miyagawa T, Duan J, Desautels A, Erhardt C, Hesla PE, Poli F, Frauscher B, Jeong JH, Lee SP, Ton TG, Kvale M, Kolesar L, Dobrovolna M, Nepom GT, Salomon D, Wichmann HE, Rouleau GA, Gieger C, Levinson DF, Gejman PV, Meitinger T, Young T, Peppard P, Tokunaga K, Kwok PY, Risch N, Mignot E (2009) Narcolepsy is strongly associated with the T-cell receptor alpha locus. Nat Genet 41(6):708–711. doi: 10.1038/ng.372 CrossRefPubMedGoogle Scholar
  10. Hodges E, Krishna MT, Pickard C, Smith JL (2003) Diagnostic role of tests for T cell receptor (TCR) genes. J Clin Pathol 56(1):1–11CrossRefPubMedGoogle Scholar
  11. Koop BF, Rowen L, Wang K, Kuo CL, Seto D, Lenstra JA, Howard S, Shan W, Deshpande P, Hood L (1994) The human T-cell receptor TCRAC/TCRDC (C alpha/C delta) region: organization, sequence, and evolution of 97.6 kb of DNA. Genomics 19(3):478–493. doi: 10.1006/geno.1994.1097 CrossRefPubMedGoogle Scholar
  12. Lefranc MP, Giudicelli V, Ginestoux C, Jabado-Michaloud J, Folch G, Bellahcene F, Wu Y, Gemrot E, Brochet X, Lane J, Regnier L, Ehrenmann F, Lefranc G, Duroux P (2009) IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 37(Database issue):D1006–D1012. doi: 10.1093/nar/gkn838 CrossRefPubMedGoogle Scholar
  13. McCarroll SA, Hadnott TN, Perry GH, Sabeti PC, Zody MC, Barrett JC, Dallaire S, Gabriel SB, Lee C, Daly MJ, Altshuler DM, International HapMap Consortium (2006) Common deletion polymorphisms in the human genome. Nat Genet 38(1):86–92CrossRefPubMedGoogle Scholar
  14. Pattaro C, Marroni F, Riegler A, Mascalzoni D, Pichler I, Volpato CB, Dal Cero U, De Grandi A, Egger C, Eisendle A, Fuchsberger C, Gogele M, Pedrotti S, Pinggera GK, Stefanov SA, Vogl FD, Wiedermann CJ, Meitinger T, Pramstaller PP (2007) The genetic study of three population microisolates in South Tyrol (MICROS): study design and epidemiological perspectives. BMC Med Genet 8:29–43. doi: 10.1186/1471-2350-8-29 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Christine Schwienbacher
    • 1
    • 3
  • Alessandro De Grandi
    • 1
  • Christian Fuchsberger
    • 1
  • Maurizio F. Facheris
    • 1
    • 2
  • Mirija Svaldi
    • 4
  • Matthias Wjst
    • 5
  • Peter P. Pramstaller
    • 1
    • 2
    • 6
    Email author
  • Andrew A. Hicks
    • 1
    Email author
  1. 1.Institute of Genetic MedicineEuropean Academy Bozen/Bolzano (EURAC)BolzanoItaly
  2. 2.Department of NeurologyGeneral Central HospitalBolzanoItaly
  3. 3.Department of Experimental and Diagnostic MedicineUniversity of FerraraFerraraItaly
  4. 4.Department of Haematology and Bone Marrow TransplantationGeneral Central HospitalBolzanoItaly
  5. 5.Institute of Lung Biology and DiseaseGerman Research Center for Environmental Health (Helmholtz Zentrum Muenchen)NeuherbergGermany
  6. 6.Department of NeurologyUniversity of LübeckLübeckGermay

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