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Title: A CPG ISLAND AT THE PROMOTER OF THE PDE8B GENE IS METHYLATED IN PLACENTA AND HYDATIDIFORM MOLES, BUT NOT IN CONTROL DNA

Author
item Van Den Veyver, Ignatia
item SMIRAGLIA, DOMINIC - ROSWELL PARK CANCER INSTI
item PANICHKUL, PRISANA - BAYLOR COLLEGE MED

Submitted to: Journal of the Society for Gynecologic Investigation
Publication Type: Abstract Only
Publication Acceptance Date: 2/1/2004
Publication Date: 2/1/2004
Citation: Van Den Veyver, I.B., Smiraglia, D.J., Panichkul, P.C. 2004. A CpG island at the promoter of the PDE8B gene is methylated in placenta and hydatidiform moles, but not in control DNA [abstract]. Journal of the Society for Gynecologic Investigation. 11(2 Supplement 1):299A.

Interpretive Summary:

Technical Abstract: Objective: We used a genome-wide CpG methylation screen, restriction landmark genome scanning (RLGS) to identify CpG islands that have altered methylation in complete hydatidiform moles (CHM), compared to control genomic DNA. Because CHM are diploid, but of uniparental parental inheritance and unique to humans, we hypothesize that this screen will reveal genes with placental-specific imprinting or genes subject to placenta-specific silencing via DNA methylation of their promoter. Methods: RLGS with the methylation-sensitive enzyme Not1 combined with EcoRV and Hinf1 revealed several spots with altered methylation. Spot 2CO2 was chosen for further analysis. Bioinformatics approaches (including virtual RLGS) were used to identify the sequence associated with spot 2CO2, analyze the genomic region surrounding the alterd Not1 site, and find associated genes. Methylation-sensitive Southern analysis on genomic DNA from lymphoblasts, normal trophoblast, term placenta, and hydatidiform moles was used to confirm differential methylation of the Not1 site. Results: The Not1 site with altered methylation was found by virtual RLGS to be contained in a CpG island on chromosome 5q14.1, upstream of the PDE8B gene which encodes an alternatively spliced cAMP-specific phosphodiesterase. PDE8B is known to be highly expressed in thyroid and weakly in other organs, including brain, spinal cord and placenta. Further analysis of the genomic region harboring PDE8B revealed presence of an antisense transcript WD40.36 encoding a putative beta WD-40 repeat containing G-protein of 51.9 kD that overlaps over 7209 bp with PDE8B. This suggests that this locus may be subject to allelic exclusion, where only mRNA from one strand is stably expressed. Allelic exclusion of overlapping antisense transcripts is a feature of some imprinted loci. There are 4 adjacent Not1 sites in the studied CpG island. Methylation-sensitive genomic Southern analysis with Not1 in combination with Xba1 as flanking enzyme showed approximately 30-50% methylation of at least two of the Not1 sites in all placental tissues examined, but not in control DNA. This supports that the gene may be regulated by promoter methylation in palcenta. Conclusions: RLGS on hydatidiform moles can successfully detect genes with placenta-specific methylation of CpG islands. The promoter region of PDE8B contains CpGs that are methylated in placenta. This finding, combined with the presence of an overlapping antisense transcript, supports that PDE8B deserves further investigation as a novel imprinted gene.