Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Identification of biomarkers for tuberculosis disease using a novel dual-color RT–MLPA assay

Genes & Immunity volume 13, pages 71–82 (2012)Cite this article

Abstract

Owing to our lack of understanding of the factors that constitute protective immunity during natural infection with Mycobacterium tuberculosis (Mtb), there is an urgent need to identify host biomarkers that predict long-term outcome of infection in the absence of therapy. Moreover, the identification of host biomarkers that predict (in)adequate response to tuberculosis (TB) treatment would similarly be a major step forward. To identify/monitor multi-component host biomarker signatures at the transcriptomic level in large human cohort studies, we have developed and validated a dual-color reverse-transcriptase multiplex ligation-dependent probe amplification (dcRT–MLPA) method, permitting rapid and accurate expression profiling of as many as 60–80 transcripts in a single reaction. dcRT–MLPA is sensitive, highly reproducible, high-throughput, has an extensive dynamic range and is as quantitative as QPCR. We have used dcRT–MLPA to characterize the human immune response to Mtb in several cohort studies in two genetically and geographically diverse populations. A biomarker signature was identified that is strongly associated with active TB disease, and was profoundly distinct from that associated with treated TB disease, latent infection or uninfected controls, demonstrating the discriminating power of our biomarker signature. Identified biomarkers included apoptosis-related genes and T-cell/B-cell markers, suggesting important contributions of adaptive immunity to TB pathogenesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Atkinson AJ, Colburn WA, DeGruttola VG, DeMets DL, Downing GJ, Hoth DF et al. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther 2001; 69: 89–95.

    Article  Google Scholar 

  2. Bowtell DD . Options available—from start to finish—for obtaining expression data by microarray. Nat Genet 1999; 21: 25–32.

    Article  CAS  Google Scholar 

  3. Lonnroth K, Castro KG, Chakaya JM, Chauhan LS, Floyd K, Glaziou P et al. Tuberculosis control and elimination 2010–50: cure, care, and social development. Lancet 2010; 375: 1814–1829.

    Article  Google Scholar 

  4. World Health Organization. Global tuberculosis control epidemiology, strategy, financing. WHO Report, WHO, Geneva 2009.

  5. Kaufmann SH . Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010; 33: 567–577.

    Article  CAS  Google Scholar 

  6. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G . Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002; 30: e57.

    Article  Google Scholar 

  7. Eldering E, Spek CA, Aberson HL, Grummels A, Derks IA, de Vos AF et al. Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 2003; 31: e153.

    Article  Google Scholar 

  8. White SJ, Vink GR, Kriek M, Wuyts W, Schouten J, Bakker B et al. Two-color multiplex ligation-dependent probe amplification: detecting genomic rearrangements in hereditary multiple exostoses. Hum Mutat 2004; 24: 86–92.

    Article  CAS  Google Scholar 

  9. Bonecini-Almeida MG, Ho JL, Boechat N, Huard RC, Chitale S, Doo H et al. Down-modulation of lung immune responses by interleukin-10 and transforming growth factor beta (TGF-beta) and analysis of TGF-beta receptors I and II in active tuberculosis. Infect Immun 2004; 72: 2628–2634.

    Article  CAS  Google Scholar 

  10. Demissie A, Abebe M, Aseffa A, Rook G, Fletcher H, Zumla A et al. Healthy individuals that control a latent infection with Mycobacterium tuberculosis express high levels of Th1 cytokines and the IL-4 antagonist IL-4delta2. J Immunol 2004; 172: 6938–6943.

    Article  CAS  Google Scholar 

  11. Knudsen TB, Gustafson P, Kronborg G, Kristiansen TB, Moestrup SK, Nielsen JO et al. Predictive value of soluble haemoglobin scavenger receptor CD163 serum levels for survival in verified tuberculosis patients. Clin Microbiol Infect 2005; 11: 730–735.

    Article  CAS  Google Scholar 

  12. Lienhardt C, Azzurri A, Amedei A, Fielding K, Sillah J, Sow OY et al. Active tuberculosis in Africa is associated with reduced Th1 and increased Th2 activity in vivo. Eur J Immunol 2002; 32: 1605–1613.

    Article  CAS  Google Scholar 

  13. Jacobsen M, Repsilber D, Gutschmidt A, Neher A, Feldmann K, Mollenkopf HJ et al. Candidate biomarkers for discrimination between infection and disease caused by Mycobacterium tuberculosis. J Mol Med 2007; 85: 613–621.

    Article  CAS  Google Scholar 

  14. Abebe M, Doherty TM, Wassie L, Aseffa A, Bobosha K, Demissie A et al. Expression of apoptosis-related genes in an Ethiopian cohort study correlates with tuberculosis clinical status. Eur J Immunol 2010; 40: 291–301.

    Article  CAS  Google Scholar 

  15. Tibshirani R . Regression shrinkage and selection via the lasso. J Royal Statist Soc B 1996; 58: 267–288.

    Google Scholar 

  16. Berry MP, Graham CM, McNab FW, Xu Z, Bloch SA, Oni T et al. An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature 2010; 466: 973–977.

    Article  CAS  Google Scholar 

  17. Jenner RG, Young RA . Insights into host responses against pathogens from transcriptional profiling. Nat Rev Microbiol 2005; 3: 281–294.

    Article  CAS  Google Scholar 

  18. Mistry R, Cliff JM, Clayton CL, Beyers N, Mohamed YS, Wilson PA et al. Gene-expression patterns in whole blood identify subjects at risk for recurrent tuberculosis. J Infect Dis 2007; 195: 357–365.

    Article  CAS  Google Scholar 

  19. Maertzdorf J, Repsilber D, Parida SK, Stanley K, Roberts T, Black G et al. Human gene expression profiles of susceptibility and resistance in tuberculosis. Genes Immun 2011; 12: 15–22.

    Article  CAS  Google Scholar 

  20. Abebe F, Bjune G . The protective role of antibody responses during Mycobacterium tuberculosis infection. Clin Exp Immunol 2009; 157: 235–243.

    Article  CAS  Google Scholar 

  21. Cooper AM . Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009; 27: 393–422.

    Article  CAS  Google Scholar 

  22. Burl S, Hill PC, Jeffries DJ, Holland MJ, Fox A, Lugos MD et al. FOXP3 gene expression in a tuberculosis case contact study. Clin Exp Immunol 2007; 149: 117–122.

    Article  CAS  Google Scholar 

  23. Shafiani S, Tucker-Heard G, Kariyone A, Takatsu K, Urdahl KB . Pathogen-specific regulatory T cells delay the arrival of effector T cells in the lung during early tuberculosis. J Exp Med 2010; 207: 1409–1420.

    Article  CAS  Google Scholar 

  24. Lund JM, Hsing L, Pham TT, Rudensky AY . Coordination of early protective immunity to viral infection by regulatory T cells. Science 2008; 320: 1220–1224.

    Article  CAS  Google Scholar 

  25. Searle SR, Casella GM, McCulloch CE . Variance components. Wiley-Interscience 1992.

  26. Goeman JJ, Solari A . The sequential rejection principle of familywise error control. Ann Statist 2010; 38: 3782–3810.

    Article  Google Scholar 

  27. Meinshausen N . Hierarchical testing of variable importance. Biometrika 2008; 95: 265–278.

    Article  Google Scholar 

Download references

Acknowledgements

We thank Dr SJ White for his technical support in designing a dual-color RT–MLPA, Dr EMS Leyten and Dr M van Westreenen for their contribution to the Paraguay cohort design and sample collection, and Dr A Geluk and Dr T van Hall for critically reviewing the manuscript. We gratefully acknowledge all the funding that made the work possible. We especially acknowledge the Bill and Melinda Gates Foundation (Grand Challenges in Global Health GC6#74), 6th framework programme TBVAC contract no. LSHP-CT-2003-503367, 7th framework programme NEWTBVAC contract no. HEALTH-F3-2009-241745 (the text represents the authors’ views and does not necessarily represent a position of the Commission that will not be liable for the use made of such information), The Netherlands Organization for Scientific Research (VENI grant 916.86.115) and the Gisela Thier foundation of the Leiden University Medical Center. The funders had no role in study design, data collection and analysis, decision to publish or in preparation of the manuscript.

Author information

Author notes

  1. M Jacobsen

    Present address: 5Current address: Department of Immunology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.,

  2. C Magis-Escurra

    Present address: 6Current address: Department of Pulmonary Diseases, Nijmegen University Medical Center Dekkerswald, Groesbeek, The Netherlands.,

Authors and Affiliations

  1. Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands

    S A Joosten, L Opmeer, K G de Boer, C Magis-Escurra, T H M Ottenhoff & M C Haks

  2. Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands

    J J Goeman & L Finos

  3. Bacterial Diseases Programme, Medical Research Council Laboratories, Banjul, The Gambia

    J S Sutherland & M O C Ota

  4. Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany

    M Jacobsen & S H E Kaufmann

Authors
  1. S A Joosten
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J J Goeman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J S Sutherland
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L Opmeer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K G de Boer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M Jacobsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S H E Kaufmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. L Finos
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. C Magis-Escurra
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M O C Ota
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. T H M Ottenhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. M C Haks
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M C Haks.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Genes and Immunity website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Joosten, S., Goeman, J., Sutherland, J. et al. Identification of biomarkers for tuberculosis disease using a novel dual-color RT–MLPA assay. Genes Immun 13, 71–82 (2012). https://doi.org/10.1038/gene.2011.64

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gene.2011.64

Keywords

This article is cited by

Search

Advanced search

Quick links