RUNX1 mutation in a patient with myelodysplastic syndrome and decreased erythrocyte expression of blood group A antigen

Fig. S1. Methylation of CpG dinucleotides of the ABO promoter in individual clones from peripheral blood cells and bone marrow cells obtained from the patient. The top diagram indicates the distribution of CpG dinucleotides from −226 to −7 relative to the translation start site of ABO. Below the CpG dinucleotide plot, ABO promoter methylation is presented by the methylation profile of 19 individual clones in peripheral blood at the top, of 15 individual clones in bone marrow obtained at diagnosis at the middle, and of 14 individual clones in bone marrow prepared eight months later at the bottom, revealing distinct patterns. The presence of a methylated cytosine residue in each CpG dinucleotide is presented as a red vertical line at the individual position of the CpG dinucleotide, whereas the presence of an unmethylated cytosine residue in each CpG dinucleotide is presented as a blue vertical line. Numerals at the right of the individual methylation plots indicate the number of clone(s) with the same methylation plot. Because the percentage of methylated cytosine residues at each CpG site was calculated for each individual site in the −226 to −7 region, the ratios of DNA methylation at each CpG site are presented as the lengths of vertical lines at the relative positions of the CpG dinucleotides below the methylation plot.

Fig. S2. Methylation of CpG dinucleotides of the ABO promoter in individual clones from peripheral blood cells of healthy donors. The top diagram indicates the distribution of CpG dinucleotides from −226 to −7 relative to the translation start site of ABO. Below the CpG dinucleotide plot, the methylation profiles of 12 to 17 individual clones in individual donors with distinct patterns are shown. The presence of a methylated cytosine residue in each CpG dinucleotide is presented as a red vertical line at the individual position of the CpG dinucleotide, whereas the presence of an unmethylated cytosine residue in each CpG dinucleotide is presented as a blue vertical line. Numerals at the right of individual methylation plots indicate the number of clone(s) with the same methylation plot.

Fig. S3. Fractionation of BM cells from the patient. BM cells isolated from the patient were stained in phosphate-buffered saline with 2% fetal calf serum using antibodies against CD13, CD33, CD235a, and CD3. The indicated subfractions were purified with a Bio-Rad S3 Cell Sorter. Panel A shows expression profiles of the surface antigens of the fractionated cells prepared from BM cells harvested at diagnosis, and Panel B displays those obtained eight months later. The subfractions obtained were sorted on the basis of cell surface expression of CD13/CD33, CD235a, and CD3 antigens by flow cytometry to assess the purity of individual fractions. The purities of the CD13⁺CD33⁺, CD235a⁺, and CD3⁺ cell subfractions at diagnosis were 75%, 76%, and 79%, while the corresponding values eight months later were 96%, 99%, and 94%, respectively.

Fig. S4. Methylation of CpG dinucleotides of the ABO promoter in individual clones from BM subfractions obtained from the patient at diagnosis. The top diagram indicates the distribution of CpG dinucleotides from −226 to −7 relative to the translation start site of ABO. Below the CpG dinucleotide plot, the methylation profiles of 14 to 16 individual clones with distinct patterns are shown for CD13⁺33⁺ cells, CD235a⁺ cells and CD3⁺ cells. The presence of a methylated cytosine residue in each CpG dinucleotide is presented as a red vertical line at the individual position of the CpG dinucleotide, whereas the presence of an unmethylated cytosine residue in each CpG dinucleotide is presented as a blue vertical line. Numerals at the right of each individual methylation plot indicates the number of clone(s) with the same methylation plot. The percentage of methylated cytosine residues at each CpG site was calculated for the individual site in the −226 to −7 region using the results from the clones in CD13⁺33⁺ cells, and the ratios of DNA methylation at each CpG site are presented as the lengths of vertical lines at the relative positions of the CpG dinucleotide below the methylation plot.

Fig. S5. Methylation of CpG dinucleotides of the ABO promoter in individual clones from BM subfractions obtained from the patient eight months later. The methylation profiles were obtained from BM subfractions prepared from the patient eight months after diagnosis. The data are presented as shown in Fig. S4, available as supporting information in the online version of this paper.

Table S1. Oligonucleotide primers used for PCR amplification of genomic DNA prior to capillary electrophoresis and pyrosequencing, and oligonucleotide primers used for sequencing in capillary electrophoresis and pyrosequencing

Table S2. Conditions* used for PCR amplification of genomic DNA prior to capillary electrophoresis^† or pyrosequencing^‡