nce our understanding of the contribution of DNA methylation to the pathogenesis of uterine leiomyoma. leiomyoma and adjacent normal myometrial tissue. Compared with the myometrium, uterine leiomyoma contained 34/55 genes that were hypermethylated and transcriptionally downregulated and 10/55 genes that were hypomethylated and transcriptionally upregulated. Thus, 44/55 genes showed an inverse correlation between promoter region methylation and mRNA expression. We also observed that 15% of the overlapping genes were hypermethylated and transcriptionally upregulated, and a much smaller number were hypomethylated and downregulated. Patterns of differential DNA methylation and mRNA expression in uterine leiomyoma and matched adjacent myometrial tissues We further analyzed the group of 55 genes that overlapped with respect to differential DNA methylation and mRNA expression. The majority of the 18 uterine leiomyoma samples exhibited a homogeneous pattern of DNA hypermethylation, whereas the normal myometrial samples were largely hypomethylated. Intriguingly, while differential mRNA expression in the uterine leiomyoma and adjacent normal myometrial samples exhibited a more heterogeneous pattern, the pattern was a mirror image of the differential DNA methylation pattern. We also performed a functional analysis of the 55 overlapping genes using Ingenuity Pathways Analysis and the Bioconductor GeneAnswers package, and found that based on their p-values level, the top two most significantly enriched gene functions are cancer processes or reproductive system diseases . The genes involved in cancer were DLEC1, KRT19, KLF11, SERPINF1, TEK, APOLD1, LYVE1, CCL2, IL17B, and TNFS10, and genes involved in reproductive system diseases were CRIM1, PCP4, CHRDL2, HOXA5, PLP1, COL9A2, SOX18, BMP, CALCRL, SFRP1. Results Analysis of DNA methylation and mRNA expression in uterine leiomyoma and matched adjacent myometrial tissue Validation of differential DNA methylation using bisulfite genomic sequencing We hypothesized that the 55 overlapping genes with differential DNA methylation and mRNA expression in uterine leiomyoma compared with normal myometrium were likely to be true targets of epigenetic regulation in uterine leiomyoma. Initially, we examined the regulatory CpG islands in the promoter regions of selected genes from the 55 candidates, and characterized the positions of 59 CpG islands and transcriptional start sites using available genome databases. From this set, we then selected three of the hypermethylated genes, Kruppel-like Sodium laureth sulfate transcription factor 11, deleted in lung and esophageal cancer 1, keratin 19 for further analysis based on their known tumor suppressor functions. First, we studied the KLF11 promoter via sequencing of bisulfite-treated genomic DNA from uterine leiomyoma and myometrial tissues from 8 subjects. Four of these were African American that were included in our ” original genome-wide DNA methylation study, and we incorporated four new matched samples from Caucasian subjects. We analyzed the DNA methylation status of a cluster of 16 CpG dinucleotides across a 249-bp region of a CpG island, located approximately 2900 bp to 2500 bp upstream of the KLF11 promoter region. Four to six clones were sequenced from each subject. The detailed CpG methylation level of 
primary leiomyoma and matched myometrial tissues verified the hypermethylated state of the KLF11 promoter 18003836” in uterine leiomyoma compared with adjacent normal myometrium. There was a sig
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