Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Biochem Biophys Res Commun. 2012 Mar 23;419(4):662-9. doi: 10.1016/j.bbrc.2012.02.071. Epub 2012 Feb 20.
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.
骨髓增生异常综合征（MDS）是一种造血干细胞疾病。几种遗传/表观遗传异常与 MDS 的发病机制密切相关。虽然骨髓（BM）抽吸是获得 MDS 细胞以评估其遗传/表观遗传异常的常用策略，但由于疼痛和安全问题，难以重复进行 BM 抽吸以获得连续样本。在这里，我们报告来自 MDS 患者血浆和血清的循环无细胞 DNA 可用于检测遗传/表观遗传异常。发现 BM 中原始细胞计数较高的患者血浆 DNA 浓度相对较高，并证实存在来自单/二核小体的 DNA 片段积累。使用接受低甲基化剂治疗的患者的连续外周血（PB）样本，通过亚硫酸氢盐焦磷酸测序对 LINE-1 启动子的特定 CpG 位点进行了全基因组甲基化分析。结果证实，在用阿扎胞苷治疗后（第 3-9 天），来自血浆、血清和 PB 单核细胞（PBMNC）的 DNA 中甲基化百分比降低。与血清 DNA 和 PBMNC 相比，血浆 DNA 在第 3 天和第 6 天显示出更快的变化趋势。此外，通过焦磷酸测序分析对血浆、血清和 BM 细胞中的 TET2 基因突变进行了定量。突变基因在血浆和血清 DNA 中的存在比例与 BM 中 CD34+/38-干细胞群中的存在比例几乎相当。这些数据表明，使用 PB 循环 DNA 进行遗传/表观遗传分析可以替代使用 BM 细胞，是一种更安全、无痛苦的选择。
