Methylation of the MLH1 promoter. (A) Depicts the CpG sites with methylation observed in the CD34⁺ cells from 12 donors and the cell line RKO. Black diamonds represent all methylated cytosines observed in bacterial subclones of the 804 bp MLH1 promoter region fragment spanning from −938 bp to −134 bp of the minus strand generated by PCR amplification after bisulfite modification. (B) MLH1 gene promoter methylation observed in the bulk CD34⁺ cell population of individual donors shown as a percentage of CpG sites methylated for each donor sample. (C) Comparison of the percentage of CpG sites methylated in donor CD34⁺ cell samples ≤ 45 years to those of donors > 45 years by 2-tailed unpaired t test (P = .04). (D) Percentage of MSI-H CFCs seen in each individual donor compared with the percentage of CpG sites methylated in the MLH1 promoter region of total CD34⁺ cells from the same individual donor (P = .005).