The utility of quantitative methylation assays at imprinted genes for the diagnosis of fetal and placental disorders.

An imbalance of imprinted gene expression within 11p15.5 is observed in Beckwith-Wiedemann syndrome (BWS), as well as in a variety of placental abnormalities including complete hydatidiform mole (CHM), placental mesenchymal dysplasia (PMD) and triploidy. To facilitate the diagnosis of epigenetic errors and chromosomal imbalance of 11p15.5, we validated a pyrosequencing assay to measure methylation at KvDMR1 using blood samples from 13 BWS cases, 8 of which showed reduced methylation as compared to control blood. An imbalance between maternal and paternal genomes as is found in triploidy, CHM or PMD was also associated with altered KvDMR1 methylation. A reciprocal pattern of methylation was obtained in the triploid cases by assaying the proximal 11p15.5 ICR associated with H19. To distinguish chromosome 11 specific alterations from whole genome imbalance, other imprinted differentially methylated regions (DMRs) can be utilized. Thus, pyrosequencing assays for DMRs associated with SGCE, SNRPN, and MEST were also compared for their utility in diagnosing parental imbalance in placental samples. While each of these assays could successfully distinguish parental origin of triploidy, SGCE showed the clearest separation between groups. The combined use of a chromosome 11p15.5 assay (e.g. KvDMR1 or H19-ICR) and non-chromosome 11 assay (e.g. SGCE) provides a potentially valuable diagnostic tool in the rapid screening of methylation errors in placental disorders. These results also show the maintenance of imprinting status at these loci in the human placenta, even in the presence of abnormal pathology.

Decreased placental methylation at the H19/IGF2 imprinting control region is associated with normotensive intrauterine growth restriction but not preeclampsia.

UNLABELLED: Many genes exhibiting genomic imprinting, parent-of-origin differences in gene expression, are involved in regulating placental and fetal growth. The goal of the present study was to assess whether abnormal regulation of imprinted genes is associated with intrauterine growth restriction (IUGR) and/or preeclampsia (PET). METHODS: Genomic DNA was extracted from at least two whole villi samples from control (N=22), IUGR (N=13), PET (N=17), and PET+IUGR (N=21) placentas. Methylation was assessed using the Illumina GoldenGate Methylation Cancer Panel I array and Pyrosequencing and MS-SNuPE assays. RESULTS: The 11p15.5 ICR1 (associated with H19 and IGF2) methylation showed considerable intra-placental variability. Nonetheless, average methylation at this site was significantly decreased in normotensive IUGR placentas (p<0.001), but not in any other group. Methylation at ICR2 (KvDMR1; associated with CDKN1C and other maternally expressed 11p15.5 genes) was not significantly altered in any group and no significant changes in expression levels were observed in the genes controlled by this region. There were no significant methylation changes observed in any candidate imprinted gene evaluated by the Illumina array. LINE-1 methylation, a marker of whole genome methylation, was also similar in all groups. CONCLUSIONS: Reduced methylation of ICR1 is associated with normotensive IUGR but not IUGR associated with preeclampsia, suggesting a different etiology of IUGR in this group. A reduction in placental IGF2 could be an adaptive response to restrict fetal growth in the presence of abnormal placentation or a response to poor fetal growth itself.