A genome-wide search for new imprinted genes in the human placenta identifies DSCAM as the first imprinted gene on chromosome 21

Abstract

We identified, through a genome-wide search for new imprinted genes in the human placenta, DSCAM (Down Syndrome Cellular Adhesion Molecule) as a paternally expressed imprinted gene. Our work revealed the presence of a Differentially Methylated Region (DMR), located within intron 1 that might regulate the imprinting in the region. This DMR showed a maternal allele methylation, compatible with its paternal expression. We showed that DSCAM is present in endothelial cells and the syncytiotrophoblast layer of the human placenta. In mouse, Dscam expression is biallelic in foetal brain and placenta excluding any possible imprinting in these tissues. This gene encodes a cellular adhesion molecule mainly known for its role in neurone development but its function in the placenta remains unclear. We report here the first imprinted gene located on human chromosome 21 with potential clinical implications.

Fig. 1

Imprinting analysis of the DSCAM gene. a, b Sequences from representative individuals’ genomic DNAs, placental cDNAs and maternal genomic DNAs around polymorphisms (black arrows) rs41395652 in exon 5 (a) and rs34336407 in exon 9 (b). c Maternal to paternal allelic ratios as estimated by pyrosequencing for rs34336407. Control samples consisting in heterozygous and homozygous genomic DNAs were used to check the obtained vs. expected ratios (1 for homozygous and 0.5 for heterozygous samples)

Fig. 2

Epigenetic analysis of the DSCAM gene. a Map of the DSCAM locus on chromosome 21. Upper bands represent exons of the DSCAM (black), DSCAM-AS1 and DSCAM-IT1 (grey) genes. The grey curved arrows represent the transcription starts of DSCAM. CpG islands located within and around the gene are shown as stars. CpG island #22bis (in grey) is detailed with each band representing a CpG dinucleotide (position 40841468–40842223 on chromosome 21 (Genome version hg38)). b Sequencing of the CpG #22bis island region from placental genomic DNA treated by bisulfite. c DNA methylation level of CpG island #22bis by pyrosequencing. The grey squares represent the average of 25 placental samples, ±standard deviation, while black triangles were data obtained from 6 sperm samples, ±standard deviation. d DNA methylation level of CpG island #22bis region by cloning-sequencing from a representative placental sample. White dots represent unmethylated cytosines and black dots, methylated cytosines

Fig. 3

Expression of DSCAM in placental tissues. a–e Immunohistochemistry on placental slides with an anti-DSCAM antibody (60x magnification). The gestational age of placentas is notified in the lower right corner in weeks of amenorrhea. Scale bar 10 μm. ST syncytiotrophoblasts, EC endothelial cells. a, c Staining from a representative sample; b negative control, in the absence of the primary antibody; d negative control in the presence of the blocking DSCAM peptide; e control in the presence of a blocking peptide irrelevant to DSCAM (Sly peptide)

Fig. 4

DSCAM expression levels in placental samples. a Real time RT-PCR in pathological and control placental samples. (IUGR intrauterine growth restriction (n = 13), vIUGR intrauterine growth restriction of vascular origin (n = 15), PE preeclampsia (n = 12), PE + IUGR combination of the maternal and foetal pathologies (n = 5); controls (n = 20)). The expression of DSCAM was normalised using three housekeeping genes. b Real time RT-PCR in human isolated trophoblasts during spontaneous syncytialisation in vitro. (24 h, n = 7; 48 h, n = 6; 72 h, n = 4)