Aberrant hypomethylation at imprinted differentially methylated regions is involved in biparental placental mesenchymal dysplasia

Abstract

Background: Placental mesenchymal dysplasia (PMD) is a morphological abnormality resembling partial hydatidiform moles. It is often associated with androgenetic/biparental mosaicism (ABM) and complicated by Beckwith-Wiedemann syndrome (BWS), an imprinting disorder. These phenomena suggest an association between PMD and aberrant genomic imprinting, particularly of CDKN1C and IGF2. The existence of another type of PMD containing the biparental genome has been reported. However, the frequency and etiology of biparental PMD are not yet fully understood.

Results: We examined 44 placental specimens from 26 patients with PMD: 19 of these were macroscopically normal and 25 exhibited macroscopic PMD. Genotyping by DNA microarray or short tandem repeat analysis revealed that approximately 35% of the macroscopic PMD specimens could be classified as biparental, while the remainder were ABM. We performed a DNA methylation analysis using bisulfite pyrosequencing of 15 placenta-specific imprinted differentially methylated regions (DMRs) and 36 ubiquitous imprinted DMRs. As expected, most DMRs in the macroscopic PMD specimens with ABM exhibited the paternal epigenotype. Importantly, the biparental macroscopic PMD specimens exhibited frequent aberrant hypomethylation at seven of the placenta-specific DMRs. Allelic expression analysis using single-nucleotide polymorphisms revealed that five imprinted genes associated with these aberrantly hypomethylated DMRs were biallelically expressed. Frequent aberrant hypomethylation was observed at five ubiquitous DMRs, including GRB10 but not ICR2 or ICR1, which regulate the expression of CDKN1C and IGF2, respectively. Whole-exome sequencing performed on four biparental macroscopic PMD specimens did not reveal any pathological genetic abnormalities. Clinical and molecular analyses of babies born from pregnancies with PMD revealed four cases with BWS, each exhibiting different molecular characteristics, and those between BWS and PMD specimens were not always the same.

Conclusion: These data clarify the prevalence of biparental PMD and ABM-PMD and strongly implicate hypomethylation of DMRs in the pathogenesis of biparental PMD, particularly placenta-specific DMRs and the ubiquitous GRB10, but not ICR2 or ICR1. Aberrant hypomethylation of DMRs was partial, indicating that it occurs after fertilization. PMD is an imprinting disorder, and it may be a missing link between imprinting disorders and placental disorders incompatible with life, such as complete hydatidiform moles and partial hydatidiform moles.

Keywords: Androgenetic/biparental mosaicism; DNA methylation; Differentially methylated regions; Genomic imprinting; Placental mesenchymal dysplasia.

Fig. 1

Biallelic expression of placenta-specific imprinted genes in specimens of biparental placental mesenchymal dysplasia (PMD). Using single-nucleotide polymorphisms (SNPs), we screened the genotypes of imprinted genes via polymerase chain reaction with genomic DNAs (gPCR) and examined their allelic expression via reverse-transcription PCR (RT-PCR). Two biparental-PMD specimens (PMD-002 and PMD-020) showed biallelic expression. Specimen PMD-024 was a biparental-PMD specimen in which the cystic region had shrunk during pregnancy; this specimen showed biallelic expression of one gene and monoallelic expression of two genes. Specimen PMD-028 was a biparental-normal specimen in which the cystic region had also shrunk during pregnancy; in this specimen, one gene exhibited biallelic expression, two exhibited monoallelic expression, and one was indeterminate (*) because of very low expression of the maternal allele in some of the normal placental samples we used as controls (Additional file 3: Fig. S5). Arrows indicate the positions of the SNPs. bi: biallelic expression; mono: monoallelic expression. Differences in DNA methylation levels between the normal specimens and the PMD specimens are indicated in parentheses. Since the analyses were performed on additional specimens excised from placental tissues, the methylation levels differ from those shown in Table 2, which refer to the original specimens