Rapid identification of promoter hypermethylation in hepatocellular carcinoma by pyrosequencing of etiologically homogeneous sample pools.
Aberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and CTNNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of approximately 1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material.
Dejeux
Emelyne
E
Laboratory for Epigenetics, Centre National de Génotypage, Bâtiment G2, 2 rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France.
Audard
Virginie
V
Cavard
Catherine
C
Gut
Ivo Glynne
IG
Terris
Benoit
B
Tost
Jörg
J
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D018401:Sample Size; D017422:Sequence Analysis, DNA / Q000379:methods*
D004273:DNA, Neoplasm
Nat Genet. 2000 Feb;24(2):132-8
10655057
Cancer Res. 2006 Jul 1;66(13):6546-52
16818626
Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4465-70
11904358
Clin Cancer Res. 2002 Apr;8(4):1087-92
11948118
Biotechniques. 2002 May;32(5):1138-42
12019787
Biotechniques. 2002 May;32(5):1144-6, 1148, 1150 passim
12019788
Nat Rev Genet. 2002 Jun;3(6):415-28
12042769
Hum Genet. 2002 May;110(5):395-401
12073008
Electrophoresis. 2002 Dec;23(24):4072-9
12481262
Br J Cancer. 2003 Feb 24;88(4):521-9
12592365
Nat Rev Cancer. 2003 Apr;3(4):253-66
12671664
Biotechniques. 2003 Jul;35(1):146-50
12866414
Biotechniques. 2003 Jul;35(1):152-6
12866415
J Pathol. 2003 Nov;201(3):473-9
14595760
World J Gastroenterol. 2004 Sep 15;10(18):2740-3
15309731
Anal Biochem. 2004 Oct 1;333(1):119-27
15351288
Hepatology. 1996 Aug;24(2):289-93
8690394
Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8847-51
9671767
Science. 1998 Jul 17;281(5375):363, 365
9705713
Nat Med. 1998 Nov;4(11):1276-80
9809551
Oncogene. 1999 Jan 21;18(3):789-95
9989830
J Mol Diagn. 2005 Feb;7(1):105-10
15681481
Hum Mol Genet. 2005 Apr 15;14 Spec No 1:R65-76
15809275
Mutat Res. 2005 Jun 3;573(1-2):96-102
15829240
J Mol Diagn. 2005 May;7(2):198-205
15858143
Clin Cancer Res. 2005 May 15;11(10):3654-60
15897561
World J Gastroenterol. 2005 Jul 28;11(28):4404-8
16038042
J Mol Diagn. 2005 Aug;7(3):413-21
16049314
Nat Rev Genet. 2005 Aug;6(8):597-610
16136652
Expert Rev Mol Diagn. 2005 Nov;5(6):947-53
16255635
Nat Rev Drug Discov. 2006 Jan;5(1):37-50
16485345
J Mol Diagn. 2006 May;8(2):152-6
16645200
J Mol Diagn. 2006 May;8(2):209-17
16645207
J Mol Med (Berl). 2006 May;84(5):365-77
16416310
Nucleic Acids Res. 2006;34(8):e59
16670426
Cancer Res. 2006 May 15;66(10):5495-503
16707479
Clin Chem. 2006 Jun;52(6):1005-13
16574761
J Mol Diagn. 2006 Sep;8(4):444-8
16931584
J Pathol. 2007 Mar;211(4):410-9
17278115
Biotechniques. 2006 Jun;40(6):721-2, 724, 726
16774114
Hum Mol Genet. 2006 Jul 1;15(13):2038-44
16698880
Mol Carcinog. 2006 Jul;45(7):530-6
16649250
Semin Cancer Biol. 2000 Jun;10(3):185-200
10936068