title: Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patients with myelodysplastic syndrome.
abstract: Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder. Several genetic/epigenetic abnormalities are deeply associated with the pathogenesis of MDS. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic abnormalities, BM aspiration is difficult to perform repeatedly to obtain serial samples because of pain and safety concerns. Here, we report that circulating cell-free DNAs from plasma and serum of patients with MDS can be used to detect genetic/epigenetic abnormalities. The plasma DNA concentration was found to be relatively high in patients with higher blast cell counts in BM, and accumulation of DNA fragments from mono-/di-nucleosomes was confirmed. Using serial peripheral blood (PB) samples from patients treated with hypomethylating agents, global methylation analysis using bisulfite pyrosequencing was performed at the specific CpG sites of the LINE-1 promoter. The results confirmed a decrease of the methylation percentage after treatment with azacitidine (days 3-9) using DNAs from plasma, serum, and PB mono-nuclear cells (PBMNC). Plasma DNA tends to show more rapid change at days 3 and 6 compared with serum DNA and PBMNC. Furthermore, the TET2 gene mutation in DNAs from plasma, serum, and BM cells was quantitated by pyrosequencing analysis. The existence ratio of mutated genes in plasma and serum DNA showed almost equivalent level with that in the CD34+/38- stem cell population in BM. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a safer and painless alternative to using BM cells.
authors.affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Chisako
authors.initials: C
authors.lastname: Iriyama
authors.forename: Akihiro
authors.initials: A
authors.lastname: Tomita
authors.forename: Hideaki
authors.initials: H
authors.lastname: Hoshino
authors.forename: Mizuho
authors.initials: M
authors.lastname: Adachi-Shirahata
authors.forename: Yoko
authors.initials: Y
authors.lastname: Furukawa-Hibi
authors.forename: Kiyofumi
authors.initials: K
authors.lastname: Yamada
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kiyoi
authors.forename: Tomoki
authors.initials: T
authors.lastname: Naoe
mesh_terms: D000368:Aged; D018899:CpG Islands; D004247:DNA / Q000097:blood*; D019175:DNA Methylation; D004252:DNA Mutational Analysis / Q000379:methods*; D044127:Epigenesis, Genetic; D005260:Female; D005819:Genetic Markers; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D008297:Male; D009154:Mutation; D009190:Myelodysplastic Syndromes / Q000097:blood* / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide
chemical_list: D005819:Genetic Markers; D004247:DNA