The embedded sections were then examined under a dissecting microscope

tional start sites using available genome databases. From this set, we then selected three of the hypermethylated genes, Kruppel-like 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 in uterine leiomyoma compared with adjacent normal myometrium. Six of the eight uterine leiomyoma samples showed increased DNA methylation of the KLF11 promoter. There was a significant statistical difference in DNA methylation levels between the uterine leiomyoma and matched myometrial tissues. Then, we analyzed the promoter region of another tumor suppressor gene, DLEC1, in uterine leiomyoma and myometrial samples from 7 subjects. Three subjects were African Americans included in our original genome-wide DNA methylation study, and we incorporated four new matched samples from Caucasian subjects. We 10455325 sequenced ” a cluster of 18 CpG dinucleotides across a 252-bp region of a CpG island located within a 2100 to 150 bp region of the DLEC1 promoter. Uterine leiomyoma tissues demonstrated a dense methylation pattern at the DLEC1 promoter region in 5 of the 7 subjects. The Neuromedin N site majority of the 18 CpG dinucleotides in the DLEC1 promoter were consistently methylated in uterine leiomyoma, but not in normal myometrial tissues. Overall, there was a significant statistical difference in methylation levels between uterine leiomyoma and matched normal myometrial tissues. Finally, we studied the KRT19 promoter via sequencing of bisulfite-treated genomic DNA from uterine leiomyoma and myometrial tissues from 7 subjects. Four subjects were African Americans included in our original genome-wide DNA methylation study, and we incorporated three new matched samples from Caucasian subjects. We analyzed the DNA methylation status of a cluster of 30 CpG dinucleotides across a 300-bp region of a CpG island, located approximately 2150 bp to 2150 bp around the KRT19 promoter. 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 KRT19 promoter in uterine leiomyoma compared with adjacent normal myometrium. All seven analyzed samples showed increased DNA methylation of the KRT19 promoter. In uterine leiomyoma, the majority of the 30 CpG dinucleotides in the KRT19 promoter were consistently methylated. There was a significant statistical difference in DNA methylation levels between the uterine leiomyoma and matched myometrial tissues. The Illumina platform covers 50 bp regions, whereas bisulfite sequencing evaluates a 250 300 bp region, which overlaps with the 50 bp sequence of interest. These longer fragments enhance fidelity. Impact of DNA methylation on gene expressi