Journal of Molecular Medicine

, Volume 84, Issue 5, pp 365–377 | Cite as

Quantitative assessment of DNA methylation: potential applications for disease diagnosis, classification, and prognosis in clinical settings

  • Romulo Martin Brena
  • Tim Hui-Ming Huang
  • Christoph PlassEmail author


Deregulation of the epigenome is now recognized as a major mechanism involved in the development and progression of human diseases such as cancer. As opposed to the irreversible nature of genetic events, which introduce changes in the primary DNA sequence, epigenetic modifications are reversible and leave the original DNA sequence intact. There is now evidence that the epigenetic landscape in humans undergoes modifications as the result of normal aging, with older individuals exhibiting higher levels of promoter hypermethylation compared to younger ones. Thus, it has been proposed that the higher incidence of certain disease in older individuals might be, in part, a consequence of an inherent change in the control and regulation of the epigenome. These observations are of remarkable clinical significance since the aberrant epigenetic changes characteristic of disease provide a unique platform for the development of new therapeutic approaches. In this review, we address the significance of DNA methylation changes that result or lead to disease, occur with aging, or may be the result of environmental exposure. We provide a detailed description of quantitative techniques currently available for the detection and analysis of DNA methylation and provide a comprehensive framework that may allow for the incorporation of protocols which include DNA methylation as a tool for disease diagnosis and classification, which could lead to the tailoring of therapeutic approaches designed to individual patient needs.


DNA methylation Epigenetic Biomarker Quantitation Gene expression 



The authors would like to thank Dr. Laura J. Rush and Dr. Joseph Costello for their input and critical reading of this manuscript. The work is supported in part by National Institute of Health grants CA93548 and DE13123, the Leukemia and Lymphoma Society, and the foundation Women Against Lung Cancer.


  1. 1.
    Christman JK (1982) Separation of major and minor deoxyribonucleoside monophosphates by reverse-phase high-performance liquid chromatography: a simple method applicable to quantitation of methylated nucleotides in DNA. Anal Biochem 119:38–48PubMedGoogle Scholar
  2. 2.
    Chiang PK, Gordon RK, Tal J, Zeng GC, Doctor BP, Pardhasaradhi K, McCann PP (1996) S-Adenosylmethionine and methylation. FASEB J 10:471–480PubMedGoogle Scholar
  3. 3.
    Franchina M, Kay PH (2000) Evidence that cytosine residues within 5′-CCTGG-3′ pentanucleotides can be methylated in human DNA independently of the methylating system that modifies 5′-CG-3′ dinucleotides. DNA Cell Biol 19:521–526PubMedGoogle Scholar
  4. 4.
    Malone CS, Miner MD, Doerr JR, Jackson JP, Jacobsen SE, Wall R, Teitell M (2001) CmC(A/T)GG DNA methylation in mature B cell lymphoma gene silencing. Proc Natl Acad Sci U S A 98:10404–10409PubMedGoogle Scholar
  5. 5.
    Clark SJ, Harrison J, Frommer M (1995) CpNpG methylation in mammalian cells. Nat Genet 10:20–27PubMedGoogle Scholar
  6. 6.
    Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R (2000) Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci U S A 97:5237–5242PubMedGoogle Scholar
  7. 7.
    Schmitt F, Oakeley EJ, Jost JP (1997) Antibiotics induce genome-wide hypermethylation in cultured Nicotiana tabacum plants. J Biol Chem 272:1534–1540PubMedGoogle Scholar
  8. 8.
    Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72:141–196PubMedGoogle Scholar
  9. 9.
    Blanchard F, Tracy E, Smith J, Chattopadhyay S, Wang Y, Held WA, Baumann H (2003) DNA methylation controls the responsiveness of hepatoma cells to leukemia inhibitory factor. Hepatology 38:1516–1528PubMedGoogle Scholar
  10. 10.
    Egger G, Liang G, Aparicio A, Jones PA (2004) Epigenetics in human disease and prospects for epigenetic therapy. Nature 429:457–463PubMedGoogle Scholar
  11. 11.
    Fruhwald MC, O’Dorisio MS, Dai Z, Rush LJ, Krahe R, Smiraglia DJ, Pietsch T, Elsea SH, Plass C (2001) Aberrant hypermethylation of the major breakpoint cluster region in 17p11.2 in medulloblastomas but not supratentorial PNETs. Genes Chromosomes Cancer 30:38–47PubMedGoogle Scholar
  12. 12.
    Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21:163–167PubMedGoogle Scholar
  13. 13.
    Ballabio A, Willard HF (1992) Mammalian X-chromosome inactivation and the XIST gene. Curr Opin Genet Dev 2:439–447PubMedGoogle Scholar
  14. 14.
    Heard E, Clerc P, Avner P (1997) X-chromosome inactivation in mammals. Annu Rev Genet 31:571–610PubMedGoogle Scholar
  15. 15.
    Allaman-Pillet N, Djemai A, Bonny C, Schorderet DF (1998) Methylation status of CpG sites and methyl-CpG binding proteins are involved in the promoter regulation of the mouse Xist gene. Gene Expr 7:61–73PubMedGoogle Scholar
  16. 16.
    Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054PubMedGoogle Scholar
  17. 17.
    Colot V, Rossignol JL (1999) Eukaryotic DNA methylation as an evolutionary device. Bioessays 21:402–411PubMedGoogle Scholar
  18. 18.
    Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol 196:261–282PubMedGoogle Scholar
  19. 19.
    Larsen F, Gundersen G, Lopez R, Prydz H (1992) CpG islands as gene markers in the human genome. Genomics 13:1095–1107PubMedGoogle Scholar
  20. 20.
    Takai D, Jones PA (2002) Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U S A 99:3740–3745PubMedGoogle Scholar
  21. 21.
    Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16:6–21PubMedGoogle Scholar
  22. 22.
    Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33(Suppl):245–254PubMedGoogle Scholar
  23. 23.
    Dai Z, Zhu WG, Morrison CD, Brena RM, Smiraglia DJ, Raval A, Wu YZ, Rush LJ, Ross P, Molina JR, Otterson GA, Plass C (2003) A comprehensive search for DNA amplification in lung cancer identifies inhibitors of apoptosis cIAP1 and cIAP2 as candidate oncogenes. Hum Mol Genet 12:791–801PubMedGoogle Scholar
  24. 24.
    Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926PubMedGoogle Scholar
  25. 25.
    Okano M, Takebayashi S, Okumura K, Li E (1999) Assignment of cytosine-5 DNA methyltransferases Dnmt3a and Dnmt3b to mouse chromosome bands 12A2–A3 and 2H1 by in situ hybridization. Cytogenet Cell Genet 86:333–334PubMedGoogle Scholar
  26. 26.
    Santos F, Hendrich B, Reik W, Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241:172–182PubMedGoogle Scholar
  27. 27.
    Gaudet F, Rideout WM III, Meissner A, Dausman J, Leonhardt H, Jaenisch R (2004) Dnmt1 expression in pre- and postimplantation embryogenesis and the maintenance of IAP silencing. Mol Cell Biol 24:1640–1648PubMedGoogle Scholar
  28. 28.
    Grandjean PW, Crouse SF, Rohack JJ (2000) Influence of cholesterol status on blood lipid and lipoprotein enzyme responses to aerobic exercise. J Appl Physiol 89:472–480PubMedGoogle Scholar
  29. 29.
    Gringras P, Chen W (2001) Mechanisms for differences in monozygous twins. Early Hum Dev 64:105–117PubMedGoogle Scholar
  30. 30.
    Cardno AG, Rijsdijk FV, Sham PC, Murray RM, McGuffin P (2002) A twin study of genetic relationships between psychotic symptoms. Am J Psychiatry 159:539–545PubMedGoogle Scholar
  31. 31.
    Fraga MF, Ballestar E, Paz MF, Ropero S, Setien F, Ballestar ML, Heine-Suner D, Cigudosa JC, Urioste M, Benitez J, Boix-Chornet M, Sanchez-Aguilera A, Ling C, Carlsson E, Poulsen P, Vaag A, Stephan Z, Spector TD, Wu YZ, Plass C, Esteller M (2005) Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci U S A 102:10604–10609PubMedGoogle Scholar
  32. 32.
    Cantoni GL (1985) The role of S-adenosylhomocysteine in the biological utilization of S-adenosylmethionine. Prog Clin Biol Res 198:47–65PubMedGoogle Scholar
  33. 33.
    Yi P, Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ (2000) Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation. J Biol Chem 275:29318–29323PubMedGoogle Scholar
  34. 34.
    Wainfan E, Poirier LA (1992) Methyl groups in carcinogenesis: effects on DNA methylation and gene expression. Cancer Res 52:2071s–2077sPubMedGoogle Scholar
  35. 35.
    Pogribny IP, Basnakian AG, Miller BJ, Lopatina NG, Poirier LA, James SJ (1995) Breaks in genomic DNA and within the p53 gene are associated with hypomethylation in livers of folate/methyl-deficient rats. Cancer Res 55:1894–1901PubMedGoogle Scholar
  36. 36.
    Pogribny IP, James SJ, Jernigan S, Pogribna M (2004) Genomic hypomethylation is specific for preneoplastic liver in folate/methyl deficient rats and does not occur in non-target tissues. Mutat Res 548:53–59PubMedGoogle Scholar
  37. 37.
    Shivapurkar N, Poirier LA (1983) Tissue levels of S-adenosylmethionine and S-adenosylhomocysteine in rats fed methyl-deficient, amino acid-defined diets for one to five weeks. Carcinogenesis 4:1051–1057PubMedGoogle Scholar
  38. 38.
    Costello JF, Fruhwald MC, Smiraglia DJ, Rush LJ, Robertson GP, Gao X, Wright FA, Feramisco JD, Peltomaki P, Lang JC, Schuller DE, Yu L, Bloomfield CD, Caligiuri MA, Yates A, Nishikawa R, Su Huang H, Petrelli NJ, Zhang X, O’Dorisio MS, Held WA, Cavenee WK, Plass C (2000) Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet 24:132–138PubMedGoogle Scholar
  39. 39.
    Smiraglia DJ, Rush LJ, Fruhwald MC, Dai Z, Held WA, Costello JF, Lang JC, Eng C, Li B, Wright FA, Caligiuri MA, Plass C (2001) Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies. Hum Mol Genet 10:1413–1419PubMedGoogle Scholar
  40. 40.
    Herman JG, Latif F, Weng Y, Lerman MI, Zbar B, Liu S, Samid D, Duan DS, Gnarra JR, Linehan WM et al (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A 91:9700–9704PubMedGoogle Scholar
  41. 41.
    Chen RZ, Pettersson U, Beard C, Jackson-Grusby L, Jaenisch R (1998) DNA hypomethylation leads to elevated mutation rates. Nature 395:89–93PubMedGoogle Scholar
  42. 42.
    Momparler RL, Eliopoulos N, Ayoub J (2000) Evaluation of an inhibitor of DNA methylation, 5-aza-2′-deoxycytidine, for the treatment of lung cancer and the future role of gene therapy. Adv Exp Med Biol 465:433–446PubMedGoogle Scholar
  43. 43.
    Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415–428PubMedGoogle Scholar
  44. 44.
    Esteller M (2003) Cancer epigenetics: DNA methylation and chromatin alterations in human cancer. Adv Exp Med Biol 532:39–49PubMedGoogle Scholar
  45. 45.
    Han SY, Iliopoulos D, Druck T, Guler G, Grubbs CJ, Pereira M, Zhang Z, You M, Lubet RA, Fong LY, Huebner K (2004) CpG methylation in the Fhit regulatory region: relation to Fhit expression in murine tumors. Oncogene 23:3990–3998PubMedGoogle Scholar
  46. 46.
    Kim H, Kwon YM, Kim JS, Lee H, Park JH, Shim YM, Han J, Park J, Kim DH (2004) Tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer. J Clin Oncol 22:2363–2370PubMedGoogle Scholar
  47. 47.
    Kim JS, Lee H, Kim H, Shim YM, Han J, Park J, Kim DH (2004) Promoter methylation of retinoic acid receptor beta 2 and the development of second primary lung cancers in non-small-cell lung cancer. J Clin Oncol 22:3443–3450PubMedGoogle Scholar
  48. 48.
    Maruyama R, Sugio K, Yoshino I, Maehara Y, Gazdar AF (2004) Hypermethylation of FHIT as a prognostic marker in nonsmall cell lung carcinoma. Cancer 100:1472–1477PubMedGoogle Scholar
  49. 49.
    Sathyanarayana UG, Padar A, Huang CX, Suzuki M, Shigematsu H, Bekele BN, Gazdar AF (2003) Aberrant promoter methylation and silencing of laminin-5-encoding genes in breast carcinoma. Clin Cancer Res 9:6389–6394PubMedGoogle Scholar
  50. 50.
    Sorm F, Piskala A, Cihak A, Vesely J (1964) 5-Azacytidine, a new, highly effective cancerostatic. Experientia 20:202–203PubMedGoogle Scholar
  51. 51.
    Jones PA, Taylor SM (1980) Cellular differentiation, cytidine analogs and DNA methylation. Cell 20:85–93PubMedGoogle Scholar
  52. 52.
    Goffin J, Eisenhauer E (2002) DNA methyltransferase inhibitors—state of the art. Ann Oncol 13:1699–1716PubMedGoogle Scholar
  53. 53.
    Issa JP, Garcia-Manero G, Giles FJ, Mannari R, Thomas D, Faderl S, Bayar E, Lyons J, Rosenfeld CS, Cortes J, Kantarjian HM (2004) Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies. Blood 103:1635–1640PubMedGoogle Scholar
  54. 54.
    Yan L, Nass SJ, Smith D, Nelson WG, Herman JG, Davidson NE (2003) Specific inhibition of DNMT1 by antisense oligonucleotides induces re-expression of estrogen receptor-alpha (ER) in ER-negative human breast cancer cell lines. Cancer Biol Ther 2:552–556PubMedGoogle Scholar
  55. 55.
    Chuang JC, Yoo CB, Kwan JM, Li TW, Liang G, Yang AS, Jones PA (2005) Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2′-deoxycytidine. Mol Cancer Ther 4:1515–1520PubMedGoogle Scholar
  56. 56.
    Juttermann R, Li E, Jaenisch R (1994) Toxicity of 5-aza-2′-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc Natl Acad Sci U S A 91:11797–11801PubMedGoogle Scholar
  57. 57.
    Ghoshal K, Datta J, Majumder S, Bai S, Kutay H, Motiwala T, Jacob ST (2005) 5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. Mol Cell Biol 25:4727–4741PubMedGoogle Scholar
  58. 58.
    Byrd JC, Stilgenbauer S, Flinn IW (2004) Chronic lymphocytic leukemia. Hematology (Am Soc Hematol Educ Program):163–183Google Scholar
  59. 59.
    Lubbert M (2000) DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action. Curr Top Microbiol Immunol 249:135–164PubMedGoogle Scholar
  60. 60.
    Issa JP, Byrd JC (2005) Decitabine in chronic leukemias. Semin Hematol 42:S43–S49PubMedGoogle Scholar
  61. 61.
    Issa JP (2000) CpG-island methylation in aging and cancer. Curr Top Microbiol Immunol 249:101–118PubMedGoogle Scholar
  62. 62.
    Richardson B (2003) Impact of aging on DNA methylation. Ageing Res Rev 2:245–261PubMedGoogle Scholar
  63. 63.
    Ahuja N, Issa JP (2000) Aging, methylation and cancer. Histol Histopathol 15:835–842PubMedGoogle Scholar
  64. 64.
    Freitas MA, Sklenar AR, Parthun MR (2004) Application of mass spectrometry to the identification and quantification of histone post-translational modifications. J Cell Biochem 92:691–700PubMedGoogle Scholar
  65. 65.
    Cosgrove MS, Wolberger C (2005) How does the histone code work? Biochem Cell Biol 83:468–476PubMedGoogle Scholar
  66. 66.
    Fischle W, Wang Y, Allis CD (2003) Binary switches and modification cassettes in histone biology and beyond. Nature 425:475–479PubMedGoogle Scholar
  67. 67.
    Fischle W, Wang Y, Allis CD (2003) Histone and chromatin cross-talk. Curr Opin Cell Biol 15:172–183PubMedGoogle Scholar
  68. 68.
    Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403:41–45PubMedGoogle Scholar
  69. 69.
    Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA (2001) Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res 61:3573–3577PubMedGoogle Scholar
  70. 70.
    Belinsky SA (2004) Gene-promoter hypermethylation as a biomarker in lung cancer. Nat Rev Cancer 4:707–717PubMedGoogle Scholar
  71. 71.
    Sidransky D (2002) Emerging molecular markers of cancer. Nat Rev Cancer 2:210–219PubMedGoogle Scholar
  72. 72.
    Oakeley EJ, Schmitt F, Jost JP (1999) Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction. Biotechniques 27:744–746, 748–750, 752PubMedGoogle Scholar
  73. 73.
    Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821–9826PubMedGoogle Scholar
  74. 74.
    Gonzalgo ML, Jones PA (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res 25:2529–2531PubMedGoogle Scholar
  75. 75.
    Xiong Z, Laird PW (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25:2532–2534PubMedGoogle Scholar
  76. 76.
    Clark SJ, Harrison J, Paul CL, Frommer M (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22:2990–2997PubMedGoogle Scholar
  77. 77.
    Stanssens P, Zabeau M, Meersseman G, Remes G, Gansemans Y, Storm N, Hartmer R, Honisch C, Rodi CP, Bocker S, van den Boom D (2004) High-throughput MALDI-TOF discovery of genomic sequence polymorphisms. Genome Res 14:126–133PubMedGoogle Scholar
  78. 78.
    Ehrich M, Bocker S, van den Boom D (2005) Multiplexed discovery of sequence polymorphisms using base-specific cleavage and MALDI-TOF MS. Nucleic Acids Res 33:e38PubMedGoogle Scholar
  79. 79.
    Ehrich M, Nelson MR, Stanssens P, Zabeau M, Liloglou T, Xinarianos G, Cantor CR, Field JK, van den Boom D (2005) Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc Natl Acad Sci U S A 102:15785–15790PubMedGoogle Scholar
  80. 80.
    Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28:E32PubMedGoogle Scholar
  81. 81.
    Zeschnigk M, Bohringer S, Price EA, Onadim Z, Masshofer L, Lohmann DR (2004) A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. Nucleic Acids Res 32:e125PubMedGoogle Scholar
  82. 82.
    Afonina I, Zivarts M, Kutyavin I, Lukhtanov E, Gamper H, Meyer RB (1997) Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res 25:2657–2660PubMedGoogle Scholar
  83. 83.
    Galm O, Rountree MR, Bachman KE, Jair KW, Baylin SB, Herman JG (2002) Enzymatic regional methylation assay: a novel method to quantify regional CpG methylation density. Genome Res 12:153–157PubMedGoogle Scholar
  84. 84.
    Cottrell SE, Distler J, Goodman NS, Mooney SH, Kluth A, Olek A, Schwope I, Tetzner R, Ziebarth H, Berlin K (2004) A real-time PCR assay for DNA-methylation using methylation-specific blockers. Nucleic Acids Res 32:e10PubMedGoogle Scholar
  85. 85.
    Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, Knippers R (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61:1659–1665PubMedGoogle Scholar
  86. 86.
    Sozzi G, Conte D, Mariani L, Lo Vullo S, Roz L, Lombardo C, Pierotti MA, Tavecchio L (2001) Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res 61:4675–4678PubMedGoogle Scholar
  87. 87.
    Ronaghi M, Karamohamed S, Pettersson B, Uhlen M, Nyren P (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84–89PubMedGoogle Scholar
  88. 88.
    Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281:363–365PubMedGoogle Scholar
  89. 89.
    Uhlmann K, Brinckmann A, Toliat MR, Ritter H, Nurnberg P (2002) Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis 23:4072–4079PubMedGoogle Scholar
  90. 90.
    Colella S, Shen L, Baggerly KA, Issa JP, Krahe R (2003) Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. Biotechniques 35:146–150PubMedGoogle Scholar
  91. 91.
    Tost J, Dunker J, Gut IG (2003) Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing. Biotechniques 35:152–156PubMedGoogle Scholar
  92. 92.
    Dupont JM, Tost J, Jammes H, Gut IG (2004) De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal Biochem 333:119–127PubMedGoogle Scholar
  93. 93.
    Ronaghi M, Elahi E (2002) Pyrosequencing for microbial typing. J Chromatogr B Analyt Technol Biomed Life Sci 782:67–72PubMedGoogle Scholar
  94. 94.
    Kuppuswamy MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce SL, Bajaj SP (1991) Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes. Proc Natl Acad Sci U S A 88:1143–1147PubMedGoogle Scholar
  95. 95.
    Singer-Sam J, LeBon JM, Dai A, Riggs AD (1992) A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide. PCR Methods Appl 1:160–163PubMedGoogle Scholar
  96. 96.
    Szabo PE, Mann JR (1995) Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms. Genes Dev 9:3097–3108PubMedGoogle Scholar
  97. 97.
    Greenwood AD, Burke DT (1996) Single nucleotide primer extension: quantitative range, variability, and multiplex analysis. Genome Res 6:336–348PubMedGoogle Scholar
  98. 98.
    Thomassin H, Kress C, Grange T (2004) MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome. Nucleic Acids Res 32:e168PubMedGoogle Scholar
  99. 99.
    Agrelo R, Setien F, Espada J, Artiga MJ, Rodriguez M, Perez-Rosado A, Sanchez-Aguilera A, Fraga MF, Piris MA, Esteller M (2005) Inactivation of the lamin A/C Gene by CpG island promoter hypermethylation in hematologic malignancies, and its association with poor survival in nodal diffuse large B-Cell lymphoma. J Clin Oncol 23:3940–3947PubMedGoogle Scholar
  100. 100.
    Raval A, Lucas DM, Matkovic JJ, Bennett KL, Liyanarachchi S, Young DC, Rassenti L, Kipps TJ, Grever MR, Byrd JC, Plass C (2005) TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain mutated and unmutated chronic lymphocytic leukemia. J Clin Oncol 23:3877–3885PubMedGoogle Scholar
  101. 101.
    Song F, Smith JF, Kimura MT, Morrow AD, Matsuyama T, Nagase H, Held WA (2005) Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression. Proc Natl Acad Sci U S A 102:3336–3341PubMedGoogle Scholar
  102. 102.
    Yu L, Liu C, Vandeusen J, Becknell B, Dai Z, Wu YZ, Raval A, Liu TH, Ding W, Mao C, Liu S, Smith LT, Lee S, Rassenti L, Marcucci G, Byrd J, Caligiuri MA, Plass C (2005) Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia. Nat Genet 37:265–274PubMedGoogle Scholar
  103. 103.
    Lewin J, Schmitt AO, Adorjan P, Hildmann T, Piepenbrock C (2004) Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates. Bioinformatics 20:3005–3012PubMedGoogle Scholar
  104. 104.
    Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, Cox AV, Andrews TD, Howe KL, Otto T, Olek A, Fischer J, Gut IG, Berlin K, Beck S (2004) DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol 2:e405PubMedGoogle Scholar
  105. 105.
    Adorjan P, Distler J, Lipscher E, Model F, Muller J, Pelet C, Braun A, Florl AR, Gutig D, Grabs G, Howe A, Kursar M, Lesche R, Leu E, Lewin A, Maier S, Muller V, Otto T, Scholz C, Schulz WA, Seifert HH, Schwope I, Ziebarth H, Berlin K, Piepenbrock C, Olek A (2002) Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Res 30:e21PubMedGoogle Scholar
  106. 106.
    Gitan RS, Shi H, Chen CM, Yan PS, Huang TH (2002) Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res 12:158–164PubMedGoogle Scholar
  107. 107.
    Shi H, Maier S, Nimmrich I, Yan PS, Caldwell CW, Olek A, Huang TH (2003) Oligonucleotide-based microarray for DNA methylation analysis: principles and applications. J Cell Biochem 88:138–143PubMedGoogle Scholar
  108. 108.
    Leu YW, Yan PS, Fan M, Jin VX, Liu JC, Curran EM, Welshons WV, Wei SH, Davuluri RV, Plass C, Nephew KP, Huang TH (2004) Loss of estrogen receptor signaling triggers epigenetic silencing of downstream targets in breast cancer. Cancer Res 64:8184–8192PubMedGoogle Scholar
  109. 109.
    Rein T, DePamphilis ML, Zorbas H (1998) Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res 26:2255–2264PubMedGoogle Scholar
  110. 110.
    Kuo KC, McCune RA, Gehrke CW, Midgett R, Ehrlich M (1980) Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA. Nucleic Acids Res 8:4763–4776PubMedGoogle Scholar
  111. 111.
    Gomes JD, Chang CJ (1983) Reverse-phase high-performance liquid chromatography of chemically modified DNA. Anal Biochem 129:387–391PubMedGoogle Scholar
  112. 112.
    Morris RG (1989) Improved liquid chromatographic fluorescence method for estimation of plasma sotalol concentrations. Ther Drug Monit 11:63–66PubMedGoogle Scholar
  113. 113.
    Ramsahoye BH (2002) Measurement of genome wide DNA methylation by reversed-phase high-performance liquid chromatography. Methods 27:156–161PubMedGoogle Scholar
  114. 114.
    del Gaudio R, Di Giaimo R, Geraci G (1997) Genome methylation of the marine annelid worm Chaetopterus variopedatus: methylation of a CpG in an expressed H1 histone gene. FEBS Lett 417:48–52PubMedGoogle Scholar
  115. 115.
    Huang TH, Perry MR, Laux DE (1999) Methylation profiling of CpG islands in human breast cancer cells. Hum Mol Genet 8:459–470PubMedGoogle Scholar
  116. 116.
    Yan PS, Chen CM, Shi H, Rahmatpanah F, Wei SH, Caldwell CW, Huang TH (2001) Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res 61:8375–8380PubMedGoogle Scholar
  117. 117.
    Lippman Z, Gendrel AV, Colot V, Martienssen R (2005) Profiling DNA methylation patterns using genomic tiling microarrays. Nat Methods 2:219–224PubMedGoogle Scholar
  118. 118.
    Nouzova M, Holtan N, Oshiro MM, Isett RB, Munoz-Rodriguez JL, List AF, Narro ML, Miller SJ, Merchant NC, Futscher BW (2004) Epigenomic changes during leukemia cell differentiation: analysis of histone acetylation and cytosine methylation using CpG island microarrays. J Pharmacol Exp Ther 311:968–981PubMedGoogle Scholar
  119. 119.
    Hatada I, Hayashizaki Y, Hirotsune S, Komatsubara H, Mukai T (1991) A genomic scanning method for higher organisms using restriction sites as landmarks. Proc Natl Acad Sci U S A 88:9523–9527PubMedGoogle Scholar
  120. 120.
    Okazaki Y, Okuizumi H, Sasaki N, Ohsumi T, Kuromitsu J, Hirota N, Muramatsu M, Hayashizaki Y (1995) An expanded system of restriction landmark genomic scanning (RLGS Ver. 1.8). Electrophoresis 16:197–202PubMedGoogle Scholar
  121. 121.
    Kuromitsu J, Kataoka H, Yamashita H, Muramatsu M, Furuichi Y, Sekine T, Hayashizaki Y (1995) Reproducible alterations of DNA methylation at a specific population of CpG islands during blast formation of peripheral blood lymphocytes. DNA Res 2:263–267PubMedGoogle Scholar
  122. 122.
    Motiwala T, Ghoshal K, Das A, Majumder S, Weichenhan D, Wu YZ, Holman K, James SJ, Jacob ST, Plass C (2003) Suppression of the protein tyrosine phosphatase receptor type O gene (PTPRO) by methylation in hepatocellular carcinomas. Oncogene 22:6319–6331PubMedGoogle Scholar
  123. 123.
    Smiraglia DJ, Smith LT, Lang JC, Rush LJ, Dai Z, Schuller DE, Plass C (2003) Differential targets of CpG island hypermethylation in primary and metastatic head and neck squamous cell carcinoma (HNSCC). J Med Genet 40:25–33PubMedGoogle Scholar
  124. 124.
    Rush LJ, Plass C (2002) Restriction landmark genomic scanning for DNA methylation in cancer: past, present, and future applications. Anal Biochem 307:191–201PubMedGoogle Scholar
  125. 125.
    Costello JF, Smiraglia DJ, Plass C (2002) Restriction landmark genome scanning. Methods 27:144–149PubMedGoogle Scholar
  126. 126.
    Rush LJ, Dai Z, Smiraglia DJ, Gao X, Wright FA, Fruhwald M, Costello JF, Held WA, Yu L, Krahe R, Kolitz JE, Bloomfield CD, Caligiuri MA, Plass C (2001) Novel methylation targets in de novo acute myeloid leukemia with prevalence of chromosome 11 loci. Blood 97:3226–3233PubMedGoogle Scholar
  127. 127.
    Dai Z, Lakshmanan RR, Zhu WG, Smiraglia DJ, Rush LJ, Fruhwald MC, Brena RM, Li B, Wright FA, Ross P, Otterson GA, Plass C (2001) Global methylation profiling of lung cancer identifies novel methylated genes. Neoplasia 3:314–323PubMedGoogle Scholar
  128. 128.
    Kremenskoy M, Kremenska Y, Ohgane J, Hattori N, Tanaka S, Hashizume K, Shiota K (2003) Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses. Biochem Biophys Res Commun 311:884–890PubMedGoogle Scholar
  129. 129.
    Zardo G, Tiirikainen MI, Hong C, Misra A, Feuerstein BG, Volik S, Collins CC, Lamborn KR, Bollen A, Pinkel D, Albertson DG, Costello JF (2002) Integrated genomic and epigenomic analyses pinpoint biallelic gene inactivation in tumors. Nat Genet 32:453–458PubMedGoogle Scholar
  130. 130.
    Nagai H, Kim YS, Yasuda T, Ohmachi Y, Yokouchi H, Monden M, Emi M, Konishi N, Nogami M, Okumura K, Matsubara K (1999) A novel sperm-specific hypomethylation sequence is a demethylation hotspot in human hepatocellular carcinomas. Gene 237:15–20PubMedGoogle Scholar
  131. 131.
    Konishi N, Tao M, Nakamura M, Kitahaori Y, Hiasa Y, Nagai H (1996) Genomic alterations in human prostate carcinoma cell lines by two-dimensional gel analysis. Cell Mol Biol (Noisy-le-grand) 42:1129–1135Google Scholar
  132. 132.
    Lindsay S, Bird AP (1987) Use of restriction enzymes to detect potential gene sequences in mammalian DNA. Nature 327:336–338PubMedGoogle Scholar
  133. 133.
    Ishkanian AS, Malloff CA, Watson SK, DeLeeuw RJ, Chi B, Coe BP, Snijders A, Albertson DG, Pinkel D, Marra MA, Ling V, MacAulay C, Lam WL (2004) A tiling resolution DNA microarray with complete coverage of the human genome. Nat Genet 36:299–303PubMedGoogle Scholar
  134. 134.
    Ching TT, Maunakea AK, Jun P, Hong C, Zardo G, Pinkel D, Albertson DG, Fridlyand J, Mao JH, Shchors K, Weiss WA, Costello JF (2005) Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3. Nat Genet 37:645–651PubMedGoogle Scholar
  135. 135.
    Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37:853–862PubMedGoogle Scholar
  136. 136.
    Misawa A, Inoue J, Sugino Y, Hosoi H, Sugimoto T, Hosoda F, Ohki M, Imoto I, Inazawa J (2005) Methylation-associated silencing of the nuclear receptor 1I2 gene in advanced-type neuroblastomas, identified by bacterial artificial chromosome array-based methylated CpG island amplification. Cancer Res 65:10233–10242PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Romulo Martin Brena
    • 1
    • 2
  • Tim Hui-Ming Huang
    • 2
  • Christoph Plass
    • 2
    • 3
    Email author
  1. 1.Division of Human Cancer Genetics, Department of Molecular GeneticsThe Ohio State UniversityColumbusUSA
  2. 2.Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical GeneticsThe Ohio State UniversityColumbusUSA
  3. 3.Division of Human Cancer GeneticsColumbusUSA

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