Fig. 2

Epigenetic dysregulation of CREBBP in JMML and association with clinical parameters indicating poor outcome. a The high-resolution DNA methylation profile of a CpG-rich 437-base pair region upstream of the CREBBP transcription start site revealed high methylation variation between CpG units under physiological conditions and frequent hypermethylation in JMML. The box plots depict the methylation level of 13 CREBBP CpG units in granulocyte DNA from 11 healthy control subjects (open boxes) and 44 patients with JMML (filled boxes). The band inside the box represents the median, and the bottom and top of the box represent the first and third quartiles. The ends of the whiskers correspond to the minimum and maximum values. The significance of differences was calculated using the Mann-Whitney test and is indicated as follows: ns, p > 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. b Methylation of CREBBP CpG sites #8 to #10 was low under physiological conditions but increased dramatically in most JMML cases. The average level of methylation across CpG units #8–#10 is shown for each sample. A JMML sample was categorized as hypermethylated if the methylation level exceeded three standard deviations above the mean observed in healthy controls. The upper limit of normal methylation is illustrated by a dotted line. c In silico prediction of transcription factor binding sites (TFBIND) revealed binding motifs for GATA1/2 and GTF3A at CpG sites #8 to #10. d CREBBP CpG #8–#10 methylation was associated with older age (≥2 years) and elevated fetal hemoglobin (HbF) at diagnosis, two established predictors of poor outcome. ***p ≤ 0.001 (Mann-Whitney test)