Skip to main content
SpringerLink
Log in

A Proof-of-Principle Demonstration of a Novel Microarray-Based Method for Quantifying DNA Methylation Levels

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Demethylation of CD11a (ITGAL; GeneID:3683; HGNC: 6148) and CD70 (TNFSF7; GeneID:970; HGNC:11937) regulatory regions in CD4+ T cells contributes to the development of autoreactivity and autoantibody overstimulation in systemic lupus erythematosus (SLE). In this study, we present a novel approach for measuring the methylation status of CD11a and CD70 promoter sequences. The procedure combines the standard method of bisulfite conversion of methylated CpG pairs with high-throughput oligonucleotide microarray-based technology that allows for rapid quantification of deoxycytosine and deoxymethylcytosine content in bisulfite-treated DNA samples. The microarrays were first used to generate a standard curve from fully methylated and fully unmethylated DNA samples using a one-dimensional linear regression equation that calculated fluorescence emission as a function of methylation levels. The methylation status of the CD70 and CD11a promoters in SLE and control CD4+ T cell samples were measured, and the microarray prediction was found to be highly accurate when compared to bisulfite sequencing. Furthermore, the microarrays were able to detect differences in the methylation status between SLE patient and healthy control samples. These results indicate that our new microarray-based assay could prove to be a highly reliable, rapid, and cost effective diagnostic and prognostic test for SLE.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Balada, E., Ordi-Ros, J., Serrano-Acedo, S., et al. (2008). Transcript levels of DNA methyltransferases DNMT1, DNMT3A and DNMT3B in CD4 + T cells from patients with systemic lupus erythematosus. Immunology, 124, 339–347.

    Article  CAS  Google Scholar 

  2. Wilson, A. S., Power, B. E., & Molloy, P. L. (2007). DNA hypomethylation and human diseases. Biochimica et Biophysica Acta, 1775, 138–162.

    CAS  Google Scholar 

  3. Smirnikhina, S. A., & Lavrov, A. V. (2009). Methods for detection of methylated cytosine residues in DNA. Molekuliarnaia Biologiia (Moskva), 43, 387–391.

    CAS  Google Scholar 

  4. Pan, Y., & Sawalha, A. H. (2009). Epigenetic regulation and the pathogenesis of systemic lupus erythematosus. Translational Research, 153, 4–10.

    Article  CAS  Google Scholar 

  5. Paluszczak, J., & Baer-Dubowska, W. (2006). Epigenetic diagnostics of cancer—the application of DNA methylation markers. Journal of Applied Genetics, 47, 365–375.

    Google Scholar 

  6. Tost, J. (2010). DNA methylation: An introduction to the biology and the disease-associated changes of a promising biomarker. Molecular Biotechnology, 44, 71–81.

    Article  CAS  Google Scholar 

  7. Bird, A. P. (1986). CpG-rich islands and the function of DNA methylation. Nature, 321, 209–213.

    Article  CAS  Google Scholar 

  8. Feinberg, A. P., & Tycko, B. (2004). The history of cancer epigenetics. Nature Reviews Cancer, 4, 143–153.

    Article  CAS  Google Scholar 

  9. Nishigaki, M., Aoyagi, K., Danjoh, I., et al. (2005). Discovery of aberrant expression of R-RAS by cancer-linked DNA hypomethylation in gastric cancer using microarrays. Cancer Research, 65, 2115–2124.

    Article  CAS  Google Scholar 

  10. Richardson, B. (2003). DNA methylation and autoimmune disease. Clinical Immunology, 109, 72–79.

    Article  CAS  Google Scholar 

  11. Lau, C. S., & Mak, A. (2009). The socioeconomic burden of SLE. Nature Reviews Rheumatology, 5, 400–404.

    Article  Google Scholar 

  12. Anolik, J., & Sanz, I. (2004). B cells in human and murine systemic lupus erythematosus. Current Opinion in Rheumatology, 16, 505–512.

    Article  Google Scholar 

  13. Lu, Q., Wu, A., & Richardson, B. C. (2005). Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs. Journal of Immunology, 174, 6212–6219.

    CAS  Google Scholar 

  14. Lu, Q., Kaplan, M., Ray, D., et al. (2002). Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus. Arthritis and Rheumatism, 46, 1282–1291.

    Article  CAS  Google Scholar 

  15. Oelke, K., Lu, Q., Richardson, D., et al. (2004). Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors. Arthritis and Rheumatism, 50, 1850–1860.

    Article  CAS  Google Scholar 

  16. Lu, Q., Ray, D., Gutsch, D., et al. (2002). Effect of DNA methylation and chromatin structure on ITGAL expression. Blood, 99, 4503–4508.

    Article  CAS  Google Scholar 

  17. Jones, P. A., & Baylin, S. B. (2002). The fundamental role of epigenetic events in cancer. Nature Reviews Genetics, 3, 415–428.

    Article  CAS  Google Scholar 

  18. Feinberg, A. P., Ohlsson, R., & Henikoff, S. (2006). The epigenetic progenitor origin of human cancer. Nature Reviews Genetics, 7, 21–33.

    Article  CAS  Google Scholar 

  19. Hochberg, M. C. (1997). Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis and Rheumatism, 40, 1725.

    Article  CAS  Google Scholar 

  20. Bombardier, C., Gladman, D. D., Urowitz, M. B., et al. (1992). Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis and Rheumatism, 35, 630–640.

    Article  CAS  Google Scholar 

  21. Lu, Q., & Richardson, B. (2004). Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression. Biological Procedures Online, 6, 189–203.

    Article  CAS  Google Scholar 

  22. Anglim, P. P., Alonzo, T. A., & Laird-Offringa, I. A. (2008). DNA methylation-based biomarkers for early detection of non-small cell lung cancer: an update. Molecular Cancer, 7, 81.

    Article  Google Scholar 

  23. Jubb, A. M., Quirke, P., & Oates, A. J. (2003). DNA methylation, a biomarker for colorectal cancer: Implications for screening and pathological utility. Annals of the New York Academy of Sciences, 983, 251–267.

    Article  CAS  Google Scholar 

  24. Kim, M., Long, T. I., Arakawa, K., et al. (2010). DNA methylation as a biomarker for cardiovascular disease risk. Public Library of Science ONE, 5, e9692.21.

    Google Scholar 

  25. Tsou, J. A., Galler, J. S., Siegmund, K. D., et al. (2007). Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma. Molecular Cancer, 6, 70.

    Article  Google Scholar 

  26. Anglim, P. P., Galler, J. S., Koss, M. N., et al. (2008). Identification of a panel of sensitive and specific DNA methylation markers for squamous cell lung cancer. Molecular Cancer, 7, 62.

    Article  Google Scholar 

  27. Hsu, H. S., Chen, T. P., Hung, C. H., et al. (2002). Characterization of a multiple epigenetic marker panel for lung cancer detection and risk assessment in plasma. Cancer, 110, 2019–2026.

    Article  Google Scholar 

  28. Shivapurkar, N., Stastny, V., Suzuki, M., et al. (2007). Application of a methylation gene panel by quantitative PCR for lung cancers. Cancer Letters, 247, 56–71.

    Article  CAS  Google Scholar 

  29. Smiraglia, D. J., & Plass, C. (2002). The study of aberrant methylation in cancer via restriction landmark genomic scanning. Oncogene, 21, 5414–5426.

    Article  CAS  Google Scholar 

  30. Gitan, R. S., Shi, H., Chen, C. M., et al. (2002). Methylation-specific oligonucleotide microarray: A new potential for high-throughput methylation analysis. Genome Research, 12, 158–164.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study received financial support from the National Natural Science Foundation of China (Nos 30730083, 30600152), and National Basic Research Program of China (973 Plan)—(2009CB825605).

Author information

Authors and Affiliations

  1. Department of Dermatology, Epigenetic Research Center, Second Xiangya Hospital, Central South University, No. 139 Renmin Middle Rd., Changsha, Hunan, 410011, People’s Republic of China

    Xiujuan Zhang, Ming Zhao, Yongqi Luo, Peng Zhang & Qianjin Lu

  2. Key Laboratory of Child Development and Learning Science of Ministy of Education of China, Southeast University, Nanjing, 210096, China

    Dongrui Zhou & Zuhong Lu

Authors
  1. Xiujuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dongrui Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ming Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yongqi Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zuhong Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Qianjin Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qianjin Lu.

Additional information

Xiujuan Zhang and Dongrui Zhou are contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, X., Zhou, D., Zhao, M. et al. A Proof-of-Principle Demonstration of a Novel Microarray-Based Method for Quantifying DNA Methylation Levels. Mol Biotechnol 46, 243–249 (2010). https://doi.org/10.1007/s12033-010-9297-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-010-9297-y

Keywords

Search

Navigation