Abstract
Imprinted genes play important roles in placental differentiation, growth and function, with profound effects on fetal development. In humans, H19 and IGF2 are imprinted, but imprinting of IGF2R remains controversial. The H19 non-coding RNA is a negative regulator of placental growth and altered placental imprinting of H19-IGF2 has been associated with pregnancy complications such as preeclampsia, which have been attributed to abnormal first trimester placentation. This suggests that changes in imprinting during the first trimester may precede aberrant placental morphogenesis. To better understand imprinting in the human placenta during early gestation, we quantified allele-specific expression for H19, IGF2 and IGF2R in first trimester (6–12 weeks gestation) and term placentae (37–42 weeks gestation) using pyrosequencing. Expression of IGF2R was biallelic, with a mean expression ratio of 49∶51 (SD = 0.07), making transient imprinting unlikely. Expression from the repressed H19 alleles ranged from 1–25% and was higher (P<0.001) in first trimester (13.5±8.2%) compared to term (3.4±2.1%) placentae. Surprisingly, despite the known co-regulation of H19 and IGF2, little variation in expression of the repressed IGF2 alleles was observed (2.7±2.0%). To identify regulatory regions that may be responsible for variation in H19 allelic expression, we quantified DNA methylation in the H19-IGF2 imprinting control region and H19 transcription start site (TSS). Unexpectedly, we found positive correlations (P<0.01) between DNA methylation levels and expression of the repressed H19 allele at 5 CpG’s 2000 bp upstream of the H19 TSS. Additionally, DNA methylation was significantly higher (P<0.05) in first trimester compared with term placentae at 5 CpG’s 39–523 bp upstream of the TSS, but was not correlated with H19 repressed allele expression. Our data suggest that variation in H19 imprinting may contribute to early programming of placental phenotype and illustrate the need for quantitative and robust methodologies to further elucidate the role of imprinted genes in normal and pathological placental development.
Highlights
Genomic imprinting refers to parent-of-origin-dependent allelespecific gene expression
DNA samples from placental tissue was genotyped for single nucleotide polymorphisms (SNPs) H19 rs217727, IGF2 rs680, IGF2 receptor (IGF2R) rs998075 and IGF2R rs1970070 to identify heterozygous individuals
As parental DNA corresponding to term placenta samples was available for 28 cases, we genotyped maternal and paternal DNA for H19 and IGF2 polymorphisms to determine the parental origin of expressed alleles
Summary
Genomic imprinting refers to parent-of-origin-dependent allelespecific gene expression. As imprinted genes appear to facilitate this tugof-war between the maternal and paternal genomes, the conflict hypothesis predicts that imprinted genes are involved in fetal and placental growth and development during pregnancy [2,4,5]. Expressed IGF2 encodes the growth promoting insulin-like growth factor II, a potent mitogen involved in regulating cell proliferation, growth and development. The H19 large intergenic non-coding RNA (lincRNA) is highly expressed in extra-embryonic cell lineages and is a developmental reservoir of miR-675 that suppresses placental growth in the mouse [18]. The IGF2 receptor (IGF2R) mediates endocytosis and clearance or activation of a variety of ligands involved in the regulation of cell growth and motility, including insulin-like growth factor II [19,20,21]
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