Molecular genetic investigation of placental mesenchymal dysplasia

s / Placenta 36 (2015) A1eA14 A2 JPA2015-01. MOLECULAR GENETIC INVESTIGATION OF PLACENTAL MESENCHYMAL DYSPLASIA Saori Aoki , Ken Higashimoto , Hidenori Hidaka , Hidetaka Watanabe , Yasufumi Ohtsuka , Hiroyuki Mishima , Koh-ichiro Yoshiura , Hitomi Yatsuki , Kenichi Nishioka , Kei-ichiro Joh , Takashi Ohba , Hidetaka Katabuchi , Hidenobu Soejima . Division of Molecular Genetics and Epigenetics, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Japan; Department of Pediatrics, Faculty of Medicine, Taiji School of Biomedical Sciences, Nagasaki, Japan; Department of Obstetrics and Gynecology, Faculty of Life Sciences, Kumamoto University, Japan Placental mesenchymal dysplasia (PMD), a morphological anomaly of the placenta characterized by placentomegaly and multicystic changes, is often associated with fetal growth restriction and death. The incidence of PMD is 0.02% of all pregnancies. Although the etiology of PMD is unknown, about 20% of fetuses with PMD also exhibit clinical features of BeckwitheWiedemann syndrome, a representative imprinting disorder, suggesting the involvement of imprinting disruption. A review of the published literature on PMD in Japan was done to find all articles from 2000 to 2014, and identified all PMD cases that had been confirmed by placental histopathology. We requested additional detailed clinical information and specimens of placental tissue from the authors. To identify the etiology of PMD, we performed genetic and epigenetic analyses on frozen placental specimens. First, we employed DNA microarrays for genetic analysis. We found 11 cases of androgenetic/biparental mosaicism in macroscopic lesions, and four such cases in normal-appearing regions. We detected 5.9±9.0 copy number variations (CNVs) with gain of copy number (average size, 3.5±10.2 Mb) and 3.5±4.3 CNVs with loss of copy number (average size, 7.5±23.7 Mb) by CytoScan HD (Affimetrix). All of these CNVs were found in the Database of Genomic Variants. For epigenetic analysis, we used bisulfite-pyrosequencing to quantitate the DNA methylation status of 57 imprinting-associated differentially methylated regions (DMRs). Analysis of PMDs with biparental genotypes revealed that several DMRs exhibited aberrant methylation relative to normal placentas. These results suggested that abnormal copy number is not associated with PMD pathogenesis, whereas androgenetic/biparental mosaicism or aberrant methylation of imprinted DMRs might be involved. JPA2015-02. A SURVIVED INFANT WITH PLACENTAL MESENCHYMAL DYSPLASIA Rie Oyama, Tanaka Shino, Sasaki Yuri, Tomonobu Kanasugi, Akihiko Kikuchi, Toru Sugiyama. Iwate Medical University Department Obstetrics and Gynecology, Japan Introduction: Mesenchyma-related dysplastic placenta (PMD) has a many type cystic region in placenta, andt then it need the differentiation with a part hydatidiformmole. The frequency is a rare disease estimated to be one case by the 4000-5000 pregnancy, and an onset drop of gene IGF2 overexpression and CDKN1C to imprint by the inpriting abnormality of the 11th chromosomal region is supposed by what the complications of the infant such as Beckwith-Wiedemann syndromes (BWS). In addition, the participation of the VEGF-D gene on the X chromosome relationship with PMD, which is the high-risk pregnancy to be complicated with premature birth and growth retardation and still birth. Case: Patient had cesarean section at 32weeks. An infant was female, 1510g, with clef lips and cyst in liver. Volume of placenta was 2250g, included multiple cyst. pathological diagnosis was PMD Conclusions: It was important that the PMD is similarly a hydatidiform mole, and it was right diagnose by ultrasonography or MRI to prevent from unnecessary termination. JPA2015-03. SKEWED X CHROMOSOME INACTIVATION IN HUMAN PLACENTAL TISSUE Hirotaka Hamada , Hiroaki Okae , Takahiro Arima , Nobuo Yaegashi . Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Japan; Department of Informative Genetics, Tohoku University Graduate School of Medicine, Japan Objective: Female mammals inactivate one of the two X chromosomes for a dosage compensation issue, which is known as X chromosome inactivation (XCI). The status of XCI varies by mammalian species or cell types. For example, in marsupials, the paternal X chromosome is inactive both in embryonic and extra-embryonic tissues, whereas in mice, XCI is random in embryonic tissues and imprinted in extra-embryonic tissues. It remains controversial whether imprinted XCI exists or not in human placental tissue in part because previous studies had analyzed a limited number of X-linked genes using resources containing various types of cells. Material and methods: To assess the XCI status in human placental tissue, we performed a chromosome-wide allele-specific expression analysis using cytotrophoblast cells (CTs) immunomagnetically purified from 1st trimester placentas. We extracted RNA and genomic DNA from CTs and maternal peripheral blood cells, and Exome, Bisulfiteand RNAsequencing were performed. The parental origin was determined using single nucleotides polymorphisms (SNPs), and the allelic expression ratio was calculated for each gene. Results: 18 females’ and 3males’ placentawere analyzed. The purity of CTs averaged 95% (97%-90% in all 21 samples). Among female samples, the average allelic expression ratio from the paternal allele was 48.7±9.5% for autosomes and 39.5±18.7% for the X-chromosome (p<0.0001). The allelic expression ratio of the X-chromosome was highly variable among samples (from 12.8% to 73.2%). We also identified an imprinted gene which is preferentially expressed from the paternal X chromosome. The variation of the XCI status was approximated by a binomial distribution model of 3:2 paternal X inactivation in 5.8±3.2 pooling cells. Conclusions: Our results suggest that XCI in human placental tissue is not randomnor completely imprinted, but is paternally relaxed imprinted. The mechanisms of XCI in placental tissues are preserved, and also diversified among mammalian species. JPA2015-04. THREE POINT MUTATIONS OF PROMOTER I.1 OF MARMOSET AROMATASE GIVES RISE OF HUMAN TYPE EXONI.1 Tatsuya Kobayashi, Hirokazu Usui, Makio Shozu. Reproductive Medicine, Post-graduate School of Medicine, Chiba University, Japan Placental expressions of aromatase in human is regulated by placenta specific promoter, namely promoter I.1. The promoter I.1 and associated exon I.1 sequence is retrotransposone origin, which is introduced into genomic sequence of marmoset CYP19A1 35 million years ago and evolves among hominoidea thereafter. Objective: The objective of this study is to characterize functional significance of evolutional changes in promoter I.1 after New-world monkey common marmoset monkey (callithrix jacchus) diverged. Methods: Common Marmoset term placentae were obtained from Chiba city zoo and Japan SLC. Transcriptional start sites of CYP19A1 were examined using 5’-RACE. A genomic sequence containing marmoset CYP19A1 sequence were cloned into a luciferase vector and transfected into BeWo cells to analyze promoter activity and splicing acceptor site. Result: No of 5’RACE clones were 37, 3, and 1 for Exon I.1, PII (gonadal promoter) and 1f (brain promoter), suggesting that the major promoter used for placental aromatase is promoter I.1 as in human. The splicing