Genomic imprinting in normal and intrauterine growth restricted human placentas

Genomic imprinting refers to silencing of one parental allele in the zygotes of gametes depending upon the parent of origin. Loss of imprinting (LOI) is the gain of function from the silent allele that theoretically can have a maximum effect of doubling the gene dosage. LOI has been associated with neurodevelopmental disorders and reproductive abnormalities including intrauterine growth restriction (IUGR). Many mechanisms may contribute to regulation of genomic imprinting (DNA methylation, histone modification, RNA interference) with DNA methylation thought to be the primary epigenetic signal (or imprint) controlling the monoallelic expression of imprinted genes.;We developed a functional transcriptional assay for LOI; this RNA-based assay takes into account different mechanisms of imprinting controls such as DNA methylation and histone modification. The method employs allele-specific PCR analysis of RT-PCR products containing common readout polymorphisms. With this method, we are able to measure LOI level as low as 1%. We applied this methodology to measurement of LOI in human placentas. We found that LOI is common in human placentas and it may be an important new biomarker for influences on prenatal epigenetics.;Using placental tissue from 10 normal and 7 IUGR pregnancies, we conducted a systematic survey of the expression of a panel of 74 "putatively" imprinted genes using quantitative RT-PCR. We found that 52 genes were expressed in the placentas. Nine genes were significantly differentially expressed between normal and IUGR placentas. Importantly, there was no correlation between gene expression and LOI profile. Our study suggested that genomic imprinting may play a role in IUGR pathogenesis, but mechanisms other than LOI may contribute to dysregulation of imprinted genes.;We created two DNA pools from the same individuals (10 normal and 7 IUGR pregnancies) from whom we measured expression of imprinted genes and performed methylated DNA immunoprecipitation followed by tiling array (MeDIP-chip). We scanned the promoters, known imprinting control regions (ICR) and intragenic regions of the 74 "experimentally postulated" imprinted genes for the existence of differentially methylated regions (DMR). We found that none of the known ICRs were differentially methylated between the two pools, the promoters of 5 imprinted genes were hypermethylated in IUGR placentas and the promoters of 4 imprinted genes were hypomethylated in IUGR placentas. Among the differentially methylated promoters, only PEG10 was differentially expressed in the placenta. Moreover, we found more hypermethylated or hypomethylated regions in the IUGR placentas in the intragenic regions of the imprinted genes expressed in the placenta than those not expressed genes. Our preliminary results suggested that there was no correlation between DNA methylation and differential gene expression and failed to support a substantial role of DNA methylation in regulation of imprinted genes.;We tested the hypothesis that LOI was an all-or-none phenomenon at the single cell level, wherein partial LOI in tissue would reflect the fraction of cells with complete LOI. Using a quantitative RT-PCR based experimental approach, we measured LOI at the single cell level in human trophoblaste and demonstrated a broad distribution of LOI among cells exhibiting LOI, with the median value of approximately 100% LOI. The width of the distribution reflected the stochastic nature of expression in the cells while the median of the distribution was consistent with our hypothesis that LOI may occur by an all-or-none process. All-or-none LOI could lead to a second distinct cell population that may have a selective advantage, leading to variation of LOI in normal tissues, such as the placenta, or in neoplastic cells.