title: Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.
abstract: Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.
authors.affiliation: International Agency for Research on Cancer (IARC), Lyon F-69008, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vaissière
authors.forename: Cyrille
authors.initials: C
authors.lastname: Cuenin
authors.forename: Anupam
authors.initials: A
authors.lastname: Paliwal
authors.forename: Paolo
authors.initials: P
authors.lastname: Vineis
authors.forename: G
authors.initials: G
authors.lastname: Hoek
authors.forename: M
authors.initials: M
authors.lastname: Krzyzanowski
authors.forename: L
authors.initials: L
authors.lastname: Airoldi
authors.forename: A
authors.initials: A
authors.lastname: Dunning
authors.forename: S
authors.initials: S
authors.lastname: Garte
authors.forename: P
authors.initials: P
authors.lastname: Hainaut
authors.forename: C
authors.initials: C
authors.lastname: Malaveille
authors.forename: K
authors.initials: K
authors.lastname: Overvad
authors.forename: F
authors.initials: F
authors.lastname: Clavel-Chapelon
authors.forename: J
authors.initials: J
authors.lastname: Linseisen
authors.forename: H
authors.initials: H
authors.lastname: Boeing
authors.forename: A
authors.initials: A
authors.lastname: Trichopoulou
authors.forename: D
authors.initials: D
authors.lastname: Trichopoulos
authors.forename: A
authors.initials: A
authors.lastname: Kaladidi
authors.forename: D
authors.initials: D
authors.lastname: Palli
authors.forename: V
authors.initials: V
authors.lastname: Krogh
authors.forename: R
authors.initials: R
authors.lastname: Tumino
authors.forename: S
authors.initials: S
authors.lastname: Panico
authors.forename: H B
authors.initials: HB
authors.lastname: Bueno-De-Mesquita
authors.forename: P H
authors.initials: PH
authors.lastname: Peeters
authors.forename: M
authors.initials: M
authors.lastname: Kumle
authors.forename: C A
authors.initials: CA
authors.lastname: Gonzalez
authors.forename: C
authors.initials: C
authors.lastname: Martinez
authors.forename: M
authors.initials: M
authors.lastname: Dorronsoro
authors.forename: A
authors.initials: A
authors.lastname: Barricarte
authors.forename: C
authors.initials: C
authors.lastname: Navarro
authors.forename: J R
authors.initials: JR
authors.lastname: Quiros
authors.forename: G
authors.initials: G
authors.lastname: Berglund
authors.forename: L
authors.initials: L
authors.lastname: Janzon
authors.forename: B
authors.initials: B
authors.lastname: Jarvholm
authors.forename: N E
authors.initials: NE
authors.lastname: Day
authors.forename: T J
authors.initials: TJ
authors.lastname: Key
authors.forename: R
authors.initials: R
authors.lastname: Saracci
authors.forename: R
authors.initials: R
authors.lastname: Kaaks
authors.forename: E
authors.initials: E
authors.lastname: Riboli
authors.forename: Pierre
authors.initials: P
authors.lastname: Hainaut
authors.forename: Zdenko
authors.initials: Z
authors.lastname: Herceg
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D001826:Body Fluids / Q000166:cytology; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D019942:Genes, p16; D015894:Genome, Human; D006801:Humans; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D000070957:MutL Protein Homolog 1