A sensitive functional assay reveals frequent loss of genomic imprinting in human placenta

Abstract

Loss of imprinting (LOI)is the gain of expression from the silent allele of an imprinted gene normally expressed from only one parental copy. LOI has been associated with neurodevelopmental disorders and reproductive abnormalities. The mechanisms of imprinting are varied, with DNA methylation representing only one. We have developed a functional transcriptional assay for LOI that is not limited to a single mechanism of imprinting. The method employs allele-specific PCR analysis of RT-PCR products containing common readout polymorphisms. With this method, we are able to measure LOI at the sensitivity of 1%. The method has been applied to measurement of LOI in human placentas. We found that RNA was stable in placentas stored for more than one hour at 4°C following delivery. We analyzed a test panel of 26 genes known to be imprinted in the human genome. We found that 18 genes were expressed in placenta. Fourteen of the 18 expressed genes contained common readout polymorphisms in the transcripts with a minor allele frequency >20%. We found that 5 of the 14 genes were not imprinted in placenta. Using the remaining nine genes, we examined 93 heterozygosities in 27 samples. The range of LOI was 0%-96%. Among the 93 heterozygosities, we found 23 examples (25%) had LOI >3% and eight examples (9%) had LOI 1-3%. Our results indicate that LOI is common in human placentas. Because LOI in placenta is common, it may be an important new biomarker for influences on prenatal epigenetics.

Differential expression of imprinted genes in normal and IUGR human placentas

“…In the previous report, we concluded that LOI is a common phenomenon in the human placenta based on our LOI measurements of nine genes in 27 placentas. 9 In this report we expanded our investigation. We first examined the imprinting status of 27 out of 52 placentally expressed genes.…”

“…We based this list on information from two sources: (1) 61 experimentally determined imprinted genes listed in two databases, i.e., www.geneimprint.com and http://igc.otago.ac.nz/home.html; and (2) 27 imprinted genes identified by Maynard et al using the ChIP-SNP method. 18 While two genes overlapped between the two sources, we excluded an additional 12 genes that: (1) were shown to be imprinted in other human tissues but not in placenta; 8,9 (2) their sequences could not be found in the NCBI and Ensembl databases (www.ncbi.nlm.nih. gov/sites/entrez; www.ensembl.org); and (3) were not large enough to allow primer design.…”

“…We have developed a highly sensitive and functional assay for measuring LOI that was published previously. 9 Briefly, the assay uses quantitative allele-specific PCR (qASPCR) on reverse transcriptase-PCR (RT-PCR) products containing a readout polymorphism. This mRNA-based assay is independent of the mechanism of imprinting, making it more biologically relevant.…”

“…The exact number of human imprinted genes is however difficult to determine because monoallelic expression of imprinted genes may occur in one specific isoform, only in specific tissue types, or only at a specific stage of development. 8,9 Luedi et al utilized an in silico approach to predict 156 new sequences that are likely to be imprinted in humans; of these 106 correspond to known genes listed by NCBI. 10 Intrauterine growth restriction (IUGR), which accounts for almost 10% of all pregnancies in the US, is associated with major perinatal morbidity and mortality in comparison to their normal size, appropriate for gestational age neonates.…”

“…We also evaluated whether the expression level of these genes in placentas were different between IUGR (less than a third percentile for estimated gestational age) and normal (appropriate-for-gestational-age) pregnancies. Finally by using a functional and quantitative assay recently development in our laboratory, 9 we measured the LOI profiles in normal and IUGR placentas on a subset of imprinted genes. The analysis of expression and LOI for imprinted genes suggests that they may be independently associated with IUGR.…”