Human PEG1/MEST, an imprinted gene on chromosome 7

The mouse Peg1/Mest gene is an imprinted gene that is expressed particularly in mesodermal tissues in early embryonic stages. It was the most abundant imprinted gene among eight paternally expressed genes (Peg 1-8) isolated by a subtraction-hybridization method from a mouse embryonal cDNA library. It has been mapped to proximal mouse chromosome 6, maternal duplication of which causes early embryonic lethality. The human chromosomal region that shares syntenic homology with this is 7q21-qter, and human maternal uniparental disomy 7 (UPD 7) causes apparent growth deficiency and slight morphological abnormalities. Therefore, at least one paternally expressed imprinted gene seems to be present in this region. In this report, we demonstrate that human PEG1/MEST is an imprinted gene expressed from a paternal allele and located on chromosome 7q31-34, near D7S649. It is the first imprinted gene mapped to human chromosome 7 and a candidate for a gene responsible for primordial growth retardation including Silver-Russell syndrome (SRS).      

Genomic structure and parent-of-origin-specific methylation of Peg1

We previously identified Peg1/Mest as a novel paternally expressed gene in the developing mouse embryo. The human PEG1 gene was recently assigned to 7q32 and shown to be imprinted and paternally expressed. Therefore, PEG1 deficiency could participate in the aetiology of pre- and post-natal growth retardation associated with maternal uniparental disomy 7 in humans. We have now initiated the characterization of the Peg1 locus in order to identify and dissect cis-acting elements implicated in its imprinted monoallelic expression. The genomic structure of Peg1 as well as the DNA sequence of the 5'-end of the gene, including 2.4 kb of promoter sequences and covering the first 2 exons, have been determined. Important sequence elements, such as a CpG island spanning exon 1 and direct repeats, are identified and discussed. To address the role of epigenetic modifications in the imprinting of Peg1, a methylation analysis of the Peg1 gene is presented. Partially methylated cytosine residues in 13.5 d.p.c. embryos and undifferentiated ES cells were identified. Using embryos carrying a targetted mutation at the Peg1 locus, we show that this partial promoter methylation pattern reflects a strict parent-of-origin- specific differential methylation: the expressed paternal allele is unmethylated, whereas the silenced maternal allele is fully methylated at the CpG sites studied. That the gametes carry the epigenetic information necessary to lay down this allele-specific methylation pattern is suggested by analysis of DNA isolated from sperm and parthenogenetic embryos.      

Mouse Peg9/Dlk1 and human PEG9/DLK1 are paternally expressed imprinted genes closely located to the maternally expressed imprinted genes: mouse Meg3/Gtl2 and human MEG3

BACKGROUND: Genomic imprinting significantly influences development, growth and behaviour in mammals. Systematic screening of imprinted genes has been extensively carried out to identify the genes responsible for imprinted phenotypes and to elucidate the biological significance of this phenomenon. In this study, we applied DNA chip technology for isolating paternally expressed imprinted genes (Pegs). We compared the resulting expression profiles of parthenogenetic and fertilized control embryos to identify novel imprinted genes. RESULTS: A novel paternally expressed mouse imprinted gene, Peg9/Dlk1, was identified. Consistent with this finding, the paternal expression of its human homologue, PEG9/DLK1, was also confirmed. These two genes form imprinted gene clusters with the reciprocally imprinted mouse Meg3/Gtl2 and human MEG3 genes that we first identified on distal chromosome 12 and chromosome 14q32, respectively. CONCLUSIONS: As DNA chip technology allows us to quickly screen a large number of genes, using this technology to search for imprinted genes could accelerate the identification of genes responsible for human and mouse genetic diseases. Dlk1 and DLK1, which encode transmembrane proteins, have six EGF-like repeats and show homology to the Delta gene in Drosophila melanogaster. Because of its homology to mammalian Delta homologues, PEG9/DLK1 may contribute to the scoliosis phenotype observed in maternal uniparental disomy 14 (mUPD14) patients.     

The parent-of-origin effect of 10q22 in pre-eclamptic females coincides with two regions clustered for genes with down-regulated expression in androgenetic placentas

By affected sib-pair linkage analysis of 24 families with pre-eclampsia, we confirm a susceptibility locus on chromosome 10q22.1 in Dutch females: a multipoint non-parametric linkage score of 3.6 near marker D10S1432 was obtained. Haplotype analysis showed a parent-of-origin effect: maximal allele sharing in the affected sibs was found for maternally derived alleles in all families, but not for the paternally derived alleles. As matrilineal inheritance suggests the presence of maternally expressed imprinted genes, while imprinting operates predominantly in (extra)embryonic tissues, all genes (n=132) known on 10q22 between GATA121A08 and D10S580 were screened for seven sequence-related features associated with imprinting and subsequently tested for expression in first trimester placenta. Placental expression of genes selected in this way (n=55) was compared with expression in androgenetic placentas of identical gestational age. Two regions on 10q22 were identified with developmentally co-repressed genes with non-random chromosomal distribution. Interestingly, these two clusters, near CTNNA3 and KCNMA1 and each containing five genes with down-regulated expression in androgenetic placentas, coincided with the regions with maximal maternal allele sharing seen in the pre-eclamptic sisters. Our linkage and expression data are compatible with the concept that pre-eclampsia involves maternally expressed imprinted genes that operate in the first trimester placenta.     

Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes

The Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct syndromes which result from lack of expression of imprinted genes within chromosome 15q11-q13. These two syndromes result from 15q11-q13 deletions, chromosome 15 uniparental disomy (UPD), imprinting centre mutations and, for AS, probable mutations in a single gene. The differential phenotype results from a paternal genetic deficiency in PWS patients and a maternal genetic deficiency in AS patients. Within 15q11-q13, four genes (SNRPN, IPW, ZNF127, FNZ127) and two expressed sequence tags (PAR1 and PAR5) have been found to be expressed only from the paternally inherited chromosome, and therefore all must be considered candidate genes involved in the pathogenesis of PWS. A candidate AS gene (UBE3A) has very recently been identified. The mechanisms of imprinted gene expression are not yet understood, but it is clear that DNA methylation is involved in both somatic cell expression and inheritance of the imprint. The presence of DNA methylation imprints that distinguish the paternally and maternally inherited alleles is a common characteristic of all known imprinted genes which have been studied extensively, including SNRPN and ZNF127. Recently, several PWS and AS patients have been found that have microdeletions in a region upstream of the SNRPN gene referred to as the imprinting centre, or IC. Paternal IC deletions in PWS patients and maternal IC deletions in AS patients result in uniparental DNA methylation and uniparental gene expression at biparentally inherited loci. The IC is a novel genetic element which controls initial resetting of the parental imprint in the germline for all imprinted gene expression over a 1.5-2.5 Mb region within chromosome 15q11-q13.      

Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line

BACKGROUND: Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types. RESULTS: We isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice. CONCLUSIONS: The human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.     

The necdin gene is deleted in Prader-Willi syndrome and is imprinted in human and mouse

Human chromosome 15q11-q13 contains genes that are imprinted and expressed from only one parental allele. Prader-Willi syndrome (PWS) is due to the loss of expression of one or more paternally expressed genes on proximal human chromosome 15q, most often by deletion or maternal uniparental disomy. Several candidate genes and a putative imprinting centre have been identified in the deletion region. We report that the human necdin-encoding gene (NDN) is within the centromeric portion of the PWS deletion region, between the two imprinted genes ZNF127 and SNRPN. Murine necdin is a nuclear protein expressed exclusively in differentiated neurons in the brain. Necdin is postulated to govern the permanent arrest of cell growth of post-mitotic neurons during murine nervous system development. We have localized the mouse locus Ndn encoding necdin to chromosome 7 in a region of conserved synteny with human chromosome 15q11-q13, by genetic mapping in an interspecific backcross panel. Furthermore, we demonstrate that expression of Ndn is limited to the paternal allele in RNA from newborn mouse brain. Expression of NDN is detected in many human tissues, with highest levels of expression in brain and placenta. NDN is expressed exclusively from the paternally inherited allele in human fibroblasts. Loss of necdin gene expression may contribute to the disorder of brain development in individuals with PWS.      

The paternally expressed gene Peg3 regulates sexual experience-dependent preferences for estrous odors

Sexual experience has marked and long-lasting effects on male behavior in mammals, regulating traits such as the anticipation and display of sexual behavior, aggression, and olfaction. The authors conducted urine preference, habituation-dishabituation, and partner choice tests with sexually experienced and naive male mice and found that wild-type males acquire adaptively significant preferences for the odors of receptive, estrous females with sexual experience, and that these preferences are matched by changes in main olfactory system responses involving the piriform cortex, as indicated by c-Fos expression. The authors also report that these experiential effects are disrupted in male mice carrying a knockout of the imprinted gene Peg3. This paternally expressed gene regulates maternal care and offspring development, but the authors here report that Peg3 mutant males suffer a complex olfactory deficit that affects estrous odor preferences and the responses of the main olfactory system to such odors. Peg3 appears to have evolved to regulate the experience-dependent preference for receptive females, an adaptive trait that would enhance male reproductive success and so potentially increase paternal transmission of this paternally expressed gene.     

Transient neonatal diabetes,a disorder of imprinting

Transient neonatal diabetes (TND) is a rare but distinct type of diabetes. Classically, neonates present with growth retardation and diabetes in the first week of life. Apparent remission occurs by 3 months but there is a tendency for children to develop diabetes in later life. Evidence suggests it is the result of overexpression of an imprinted and paternally expressed gene/s within the TND critical region at 6q24. Two imprinted genes, ZAC (zinc finger protein associated with apoptosis and cell cycle arrest) and HYMAI (imprinted in hydatidiform mole) have been identified as potential candidates. Three genetic mechanisms have been shown to result in TND, paternal uniparental isodisomy of chromosome 6, paternally inherited duplication of 6q24, and a methylation defect at a CpG island overlapping exon 1 of ZAC/HYMAI.     

Isolated imprinting mutation of the DLK1/GTL2 locus associated with a clinical presentation of maternal uniparental disomy of chromosome 14

The clinical phenotypes of maternal and paternal uniparental disomy of chromosome 14 (UPD14) are attributed to dysregulation of imprinted genes. A large candidate locus exists within 14q32, under the regulation of a paternally methylated intergenic differentially methylated region (IG-DMR). We present a patient with clinical features of maternal UPD14, including growth retardation, hypotonia, scoliosis, small hands and feet, and advanced puberty, who had loss of methylation of the IG-DMR with no evidence of maternal UPD14. This case provides support for the hypothesis that the maternal UPD14 phenotype is due to aberrant gene expression within the imprinted domain at 14q32.     

Developmentally regulated functions of the H19 differentially methylated domain

Igf2 and H19 are physically linked imprinted genes. In embryonic liver, their reciprocal expression (paternal for Igf2 and maternal for H19) is controlled by a paternally methylated region (H19 DMD) located 5' of H19. This region contains a methylation-sensitive insulator that prevents the Igf2 promoters being activated by downstream enhancers on the maternal chromosome. In adult liver, Igf2 is normally not expressed but is reactivated upon tumour formation. By analysing three deletions of the H19 locus, we investigated the mechanism regulating the imprinted expression of the Igf2 gene in the course of liver tumourigenesis. We observed that the role of the H19 DMD in the control of Igf2 expression changes during tumourigenesis. The H19 DMD is required on the paternal chromosome for Igf2 activation in the early stages while its maternal allele is necessary for maintaining Igf2 imprinting only in the late stages. A positive regulatory function of the paternal H19 DMD is also evident in normal neonatal liver, but its relevance for Igf2 expression becomes higher in the second post-natal week. Our results support a model in which both methylated and non-methylated parental copies of the H19 DMD have active roles in the regulation of Igf2 expression in the liver and these activities are under developmental control.     

Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is caused by paternal deficiency of human chromosome 15q11-q13. There is conflicting evidence from human translocations regarding the direct involvement of SNRPN in the pathogenesis of PWS and it is not known if the phenotypic features result from the loss of expression of a single imprinted gene or multiple genes. In an attempt to dissect genotype/phenotype correlations for the homologous region of mouse chromosome 7C, we prepared three mutant genotypes: (i) mice with a deletion of Snrpn exon 2, which removes a portion of a small, upstream open reading frame (ORF); (ii) mice with double targeting for Snrpn exon 2 and Ube3a; (iii) mice deleted from Snrpn to Ube3a, removing coding exons for both loci and intervening genes. Mice deleted for Snrpn exon 2 have no obvious phenotypic abnormalities and switching of the genomic imprint for the region is conserved. Mice carrying the Snrpn - Ube3a deletion on the paternal chromosome showed severe growth retardation, hypotonia and approximately 80% lethality before weaning. The surviving mice were fertile and were not obese up to 14 months of age. The deletion was transmitted for multiple generations and continued to cause partial lethality when inherited paternally, but not when inherited maternally. The normal imprinted expression and methylation patterns of necdin, a gene outside the deletion region, indicate that the deletion is not an imprinting mutation. The data suggest the presence of a paternally expressed structural gene between Snrpn and Ipw whose deficiency causes lethality, although other possibilities exist, including position effects on expression of imprinted genes or that simultaneous deficiency of both ORFs of Snrpn causes lethality.