DNA methylation analysis of multiple imprinted DMRs in Sotos syndrome reveals IGF2-DMR0 as a DNA methylation-dependent, P0 promoter-specific enhancer.

Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.

Clinical manifestations of placental mesenchymal dysplasia in Japan: A multicenter case series.

AIM: This study aimed to evaluate the clinical features and pregnancy outcomes of placental mesenchymal dysplasia (PMD) in Japan. METHODS: We requested detailed clinical information and placental tissue of PMD cases in 2000-2018 from Japanese facilities with departments of obstetrics and gynecology and analyzed the pregnancy course and neonatal outcomes. RESULTS: We collected 49 cases of PMD. Of 18 patients with measured maternal serum alpha-fetoprotein (MSAFP) levels, 15 (83.3%) had elevated levels. Maternal serum human chorionic gonadotropin (MShCG) levels were transiently elevated in five (17.8%) of 28 patients. Forty-seven patients continued their pregnancies. All pregnancies were singleton and 40 (85.1%) were associated with adverse events including fetal growth restriction (FGR), threatened premature delivery, fetal demise, and hypertensive disorder of pregnancy in 34 (72.3%), 14 (29.8%), eight (17.0%), and six (12.8%) patients, respectively. Of 47 infants, there were eight stillbirths. There were 40 (85.1%) female infants, and eight (17.0%) had Beckwith-Wiedemann syndrome. Of 39 live births, 23 (59.0%) were associated with premature induction of labor or cesarean section for obstetric indications related to FGR. Eighteen (46.2%) neonates had complications. PMD-affected placentas were pathologically heterogeneous in both grossly PMD-affected and non-affected areas. CONCLUSIONS: Our study included the largest number of PMD cases with detailed clinical information. PMD is a high-risk condition for both the mother and the child. Elevated MSAFP levels with normal MShCG levels indicate PMD. Conventional perinatal management of FGR in Japan might be effective in reducing the fetal mortality rate.

The extent of DNA methylation anticipation due to a genetic defect in ICR1 in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a representative imprinting disorder. Gain of methylation at imprinting control region 1 (ICR1-GOM), leading to the biallelic expression of IGF2 and silencing of H19, is one of the causative alterations in BWS. Twenty percent of BWS patients with ICR1-GOM have genetic defects in ICR1. Evidence of methylation anticipation in familial BWS patients with ICR1 genetic defects has been reported. However, the precise methylation pattern and extent of anticipation in these patients remain elusive. In addition, although age-related IGF2-DMR0 hypomethylation has been reported in the normal population, the period of its occurrence is unknown. In this study, we analyzed 10 sites (IGF2-DMR0, IGF2-DMR2, CTCF binding sites 1-7, and the H19 promoter) within the IGF2/H19 domain in familial BWS patients harboring a pathogenic variant in ICR1. We found that sites near the variant had relatively higher methylation in the first affected generation and observed methylation anticipation through maternal transmission in the next generation. The extent of anticipation was greater at sites far from the variant than nearby sites. The extended and severe GOM might be due to the insufficient erasure/demethylation of pre-acquired ICR1-GOM in primordial germ cells or during the preimplantation stage. In the normal population, age-related IGF2-DMR0 hypomethylation occurred; it became established by young adulthood and continued to old age. Further studies are needed to clarify (1) the precise mechanism of anticipation in patients with familial BWS and (2) the mechanism and biological significance of constitutive hypomethylation of IGF2-DMR0 and/or other imprinted differentially methylated regions.

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

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.

Comprehensive methylation analysis of imprinting-associated differentially methylated regions in colorectal cancer.

BACKGROUND: Imprinted genes are regulated by DNA methylation at imprinting-associated differentially methylated regions (iDMRs). Abnormal expression of imprinted genes is implicated in imprinting disorders and tumors. In colorectal cancer (CRC), methylation and imprinting status of the IGF2/H19 domain have been studied. However, no comprehensive methylation analysis of iDMRs in CRC has been reported. Furthermore, the relationship between iDMR methylation status and other methylation-related issues, such as CpG island methylator phenotype (CIMP) and long interspersed element-1 (LINE-1) methylation, remains unclear. RESULTS: We analyzed the methylation status of 38 iDMRs in 106 CRC patients. We also investigated CIMP, LINE-1 methylation, KRAS and BRAF gene mutations, and loss of imprinting (LOI) of IGF2. We further examined the relationship between these factors and clinicopathological factors. The overall trend in iDMR methylation was towards hypermethylation, and iDMRs could be grouped into three categories: susceptible, resistant, and intermediate-to-aberrant methylation. The susceptible and resistant iDMRs consisted of all types of iDMR (gametic and somatic, maternally and paternally methylated). Hypermethylation of multiple iDMRs (HyMiD)-positive status was statistically associated with CIMP-positive status, but not associated with mutations in the BRAF and KRAS genes. HyMiD-positive status was inversely associated with LINE-1 methylation. Among four iDMRs within the IGF2/H19 domain, IGF2-DMR0 hypomethylation occurred most frequently, but was not associated with IGF2 LOI. Finally, we statistically calculated predictive prognostic scores based on aberrant methylation status of three iDMRs. CONCLUSION: In CRC tissues, some iDMRs were susceptible to hypermethylation independent of the type of iDMR and genomic sequence. Although HyMiD-positive status was associated with CIMP-positive status, this was independent of the BRAF and KRAS pathways, which are responsible for CIMP. Since IGF2-DMR0 hypomethylation and aberrant methylation of other iDMRs within the IGF2/H19 domain were not associated with IGF2 LOI, dysfunction of any of the molecular components related to imprinting regulation may be involved in IGF2 LOI. The prognostic score calculated based on aberrant methylation of three iDMRs has potential clinical applications as a prognostic predictor in patients. Further study is required to understand the biological significance of, and mechanisms behind, aberrant methylation of iDMRs and IGF2 LOI in CRCs.