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.     

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).      

Epigenetic detection of human chromosome 14 uniparental disomy

The recent demonstration of genomic imprinting of DLK1 and MEG3 on human chromosome 14q32 indicates that these genes might contribute to the discordant phenotypes associated with uniparental disomy (UPD) of chromosome 14. Regulation of imprinted expression of DLK1 and MEG3 involves a differentially methylated region (DMR) that encompasses the MEG3 promoter. We exploited the normal differential methylation of the DLK1/MEG3 region to develop a rapid diagnostic PCR assay based upon an individual's epigenetic profile. We used methylation-specific multiplex PCR in a retrospective analysis to amplify divergent lengths of the methylated and unmethylated MEG3 DMR in a single reaction and accurately identified normal, maternal UPD14, and paternal UPD14 in bisulfite converted DNA samples. This approach, which is based solely on differential epigenetic profiles, may be generally applicable for rapidly and economically screening for other imprinting defects associated with uniparental disomy, determining loss of heterozygosity of imprinted tumor suppressor genes, and identifying gene-specific hypermethylation events associated with neoplastic progression.     

Search for imprinted regions on chromosome 14: comparison of maternal and paternal UPD cases with cases of chromosome 14 deletion

Over the past few years, regions of genomic imprinting have been identified on a small number of chromosomes through a search for the etiology of various disorders. Distinct phenotypes have been associated with both maternal and paternal uniparental disomy (UPD) for chromosome 14. This observation indicates that there are imprinted genes present on chromosome 14, although none have been identified to date. In order to focus the search for imprinted genes on chromosome 14, we analyzed cases of maternal and paternal UPD 14 and compared them with cases of chromosome 14 deletions. Cases of paternal UPD were compared with maternal deletions and maternal UPD compared with paternal deletions. The paternal UPD anomalies seen in maternal deletion cases allowed us to associate the following features and chromosomal regions: Hirsute forehead: del(14)(q12q13. 3) and del(14)(q32); blepharophimosis: del(14)(q32); small thorax: del(14)(q11.2q13); and joint contractures: del(14)(q11.2q13) and del(14)(q31). Comparison of maternal UPD and paternal deletion cases revealed fleshy nasal tip to be most often associated with del(14)(q32), scoliosis with del(14) (q23q24.2), and del(14)(q32. 11qter) and small size at birth to be associated with del(14)(q11q13) and del(14)(q32). Our study, in conjunction with a prior study of UPD 14 and partial trisomy 14 cases, and what is known of imprinting in regions of mouse chromosomes homologous to human chromosome 14, leads us to conclude that 14q23-q32 is likely an area where imprinted genes may reside.     

Comparative analysis of isodisomic and heterodisomic segments in cases with maternal uniparental disomy 14 suggests more than one imprinted region

The results of molecular investigations of 21 cases with complete or segmental maternal uniparental disomy (UPD) 14 published in the literature were compared with respect to isodisomic and heterodisomic segments. The aim of the study was to find hints toward imprinted regions other than the recently defined imprinted segment 14q32. Three regions with no isodisomic molecular marker were found. The most distal of these regions located on 14q32.12 and 14q32.13 supports the hypothesis of genomic imprinting as the cause of the maternal UPD 14 phenotype by synteny to the maternally imprinted region on mouse distal chromosome 12 and correlation with the recently defined imprinting cluster on human chromosome 14q32. The other two heterodisomic areas located on 14q11.2-->14q12 and 14q21.1-->14q31.2 are hints toward one or more additional regions of genomic imprinting on human chromosome 14.     

Parental origin effects in human trisomy for chromosome 14q: implications for genomic imprinting.

Parental origin specific congenital anomalies have been noted in patients with uniparental disomy of the long arm of human chromosome 14 (UPD14). This suggests the presence of imprinted genes, consistent with observations of imprinting in the region of syntenic homology in the mouse. It is not known whether the distinct defects reported for paternal and maternal UPD14 are the result of biallelic expression or absence of expression of imprinted genes. Furthermore, identification of the genes responsible would be facilitated by a higher resolution map of the imprinted region(s) involved. Subjects with partial trisomy for chromosome 14 (Ts14) have been reported and hence also have an alteration in the dosage of their parental chromosomes. In this study, we have carried out genotype-phenotype correlations considering the parental origin of the extra chromosome in previously reported cases of maternal and paternal partial Ts14. The analysis has provided evidence of a correlation between distal maternal Ts14 and anomalies including low birth weight, short philtrum, and small hands. The clinical features found in the maternal and paternal trisomies are compared with those associated with maternal and paternal UPD14 and their significance is discussed in relation to genomic imprinting on chromosome 14.     

Novel deletions affecting the MEG3-DMR provide further evidence for a hierarchical regulation of imprinting in 14q32

The imprinted region on chromosome 14q32 harbors several maternally or paternally expressed genes as well as two DMRs (differentially methylated regions), the IG-DMR and the MEG3-DMR, which both act as imprinting control centers. Genetic aberrations affecting the imprinted gene cluster in 14q32 result in distinct phenotypes, known as maternal or paternal uniparental disomy 14 phenotypes (upd(14)mat, upd(14)pat). In both syndromes, three types of molecular alterations have been reported: uniparental disomy 14, deletions and epimutations. In contrast to uniparental disomy and epimutations, deletions affecting regulatory elements in 14q32 are associated with a high-recurrence risk. Based on two single deletion cases a functional hierarchy of the IG-DMR as a regulator for the methylation of the MEG3-DMR has been proposed. We have identified two novel deletions of maternal origin spanning the MEG3-DMR, but not the IG-DMR in patients with upd(14)pat syndrome, one de novo deletion of 165 kb and another deletion of 5.8 kb in two siblings. The 5.8 kb deletion was inherited from the phenotypically normal mother, who carries the deletion in a mosaic state on her paternal chromosome 14. The methylation at both DMRs was investigated by quantitative next generation bisulfite sequencing and revealed normal methylation patterns at the IG-DMR in all patients with the exception of certain CpG dinucleotides. Thus, we could confirm that deletions of the MEG3-DMR does not generally influence the methylation pattern of the IG-DMR, which strengthens the hypothesis of a hierarchical structure and distinct functional properties of the two DMRs.     

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.     

Duplication 14(q24.3q31) in a father and daughter: Delineation of a possible imprinted region

A number of clinical reports have described children with a variety of congenital anomalies in association with uniparental disomy (upd) of chromosome 14, suggesting that at least some genes on chromosome 14 are subject to parent of origin, or imprinting, effects. However, little else is known about this putative imprinting of chromosome 14. Both maternal and paternal upd have been observed, but a consistent phenotype has only been suggested for the former. Here we report on a child with developmental delay, microcephaly, distinct facial findings, and who has a duplication of 14q24.3q31. The same cytogenetic abnormality was found in her phenotypically normal father. We hypothesize that this segment of chromosome 14 contains maternally silenced genes, and that this duplicated segment defines an imprinted region on chromosome 14. Alternatively, this cytogenetic duplication may be unrelated to the girl's phenotypic anomalies, and this duplication may contain genes that are not subject to dosage effect. Am. J. Med. Genet. 71:361–365, 1997. © 1997 Wiley-Liss, Inc.     

IMPT1, an imprinted gene similar to polyspecific transporter and multi- drug resistance genes

Human chromosome 11p15.5 and distal mouse chromosome 7 include a megabase-scale chromosomal domain with multiple genes subject to parental imprinting. Here we describe mouse and human versions of a novel imprinted gene, IMPT1 , which lies between IPL and p57 KIP2 and which encodes a predicted multi-membrane-spanning protein similar to bacterial and eukaryotic polyspecific metabolite transporter and multi- drug resistance pumps. Mouse Impt1 and human IMPT1 mRNAs are highly expressed in tissues with metabolite transport functions, including liver, kidney, intestine, extra-embryonic membranes and placenta, and there is strongly preferential expression of the maternal allele in various mouse tissues at fetal stages. In post-natal tissues there is persistent expression, but the allelic bias attenuates. An allelic expression bias is also observed in human fetal and post-natal tissues, but there is significant interindividual variation and rare somatic allele switching. The fact that Impt1 is relatively repressed on the paternal allele, together with data from other imprinted genes, allows a statistical conclusion that the primary effect of human chromosome 11p15.5/mouse distal chromosome 7 imprinting is domain-wide relative repression of genes on the paternal homolog. Dosage regulation of the metabolite transporter gene(s) by imprinting might regulate placental and fetal growth.      

Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation

The evolution of genomic imprinting in mammals occurred more than 100 million years ago, and resulted in the formation of genes that are functionally haploid because of parent-of-origin-dependent expression. Despite ample evidence from studies in a number of species suggesting the presence of imprinted genes on human chromosome 14, their identity has remained elusive. Here we report the identification of two reciprocally imprinted genes, GTL2 and DLK1, which together define a novel imprinting cluster on human chromosome 14q32. The maternally expressed GTL2 (gene trap locus 2) gene encodes for a nontranslated RNA. DLK1 (delta, Drosophila, homolog-like 1) is a paternally expressed gene that encodes for a transmembrane protein containing six epidermal growth factor (EGF) repeat motifs closely related to those present in the delta/notch/serrate family of signaling molecules. The paternal expression, chromosomal localization, and biological function of DLK1 also make it a likely candidate gene for the callipyge phenotype in sheep. Many of the predicted structural and regulatory features of the DLK1/GTL2 domain are highly analogous to those implicated in IGF2/H19 imprint regulation, including two hemimethylated consensus binding sites for the vertebrate enhancer blocking protein, CTCF. These results provide evidence that a common mechanism and domain organization may be used for juxtapositioned, reciprocally imprinted genes.     

The IPL gene on chromosome 11p15.5 is imprinted in humans and mice and is similar to TDAG51, implicated in Fas expression and apoptosis

We searched for novel imprinted genes in a region of human chromosome 11p15.5, which contains several known imprinted genes. Here we describe the cloning and characterization of the IPL ( I mprinted in P lacenta and L iver) gene, which shows tissue-specific expression and functional imprinting, with the maternal allele active and the paternal allele relatively inactive, in many human and mouse tissues. Human IPL is highly expressed in placenta and shows low but detectable expression in fetal and adult liver and lung. Mouse Ipl maps to the region of chromosome 7 which is syntenic with human 11p15.5 and this gene is expressed in placenta and at higher levels in extraembryonic membranes (yolk sac), fetal liver and adult kidney. Mouse and human IPL show sequence similarity to TDAG51 , a gene which was shown to be essential for Fas expression and susceptibility to apoptosis in a T lymphocyte cell line. Like several other imprinted genes, mouse and human IPL genes are small and contain small introns. These data expand the repertoire of known imprinted genes and will be helpful in testing the mechanism of genomic imprinting and the role of imprinted genes in growth regulation.      

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith- Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith- Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.      

Identification of a large novel imprinted gene cluster on mouse proximal chromosome 6

Mice with maternal duplication of proximal chromosome 6 die in utero at an early embryonic stage. Recently, two imprinted genes, paternally expressed Sgce and maternally expressed Asb4, were identified in this region. This report analyzes the imprinting status of genes within a 1-Mb region containing these two genes. Peg10, which is next to Sgce, shows complete paternal expression, like Sgce. Conversely, Neurabin, Pon2, and Pon3 show preferential maternal expression at embryonic stages, although they all show biallelic expression in neonatal tissues. These results demonstrate that there is a large novel imprinted gene cluster in this region. 5'-RACE (Rapid Amplification of cDNA Ends) analysis of Peg10 revealed the existence of a novel first exon separate from the second exon, which encoded two putative ORFs similar to the viral Gag and Pol proteins. A differentially methylated region established in sperm and eggs is located just within the region containing the two first exons of Peg10 and Sgce, and may play an important role in regulating the two paternally expressed genes: Peg10 and Sgce.     

<Emphasis Type="Italic">Calcr</Emphasis>, a brain-specific imprinted mouse calcitonin receptor gene in the imprinted cluster of the proximal region of chromosome 6

Expressed sequence tags (ESTs) in the human chromosome 7q21-q31 region were recently used to screen for allelic expression bias in monochromosomal hybrids retaining a paternal or maternal human chromosome 7. Six candidate imprinted genes were identified. In this study, we investigated parent-of-origin-specific expression profiles of their mouse homologues in the proximal region of chromosome 6. An imprinting analysis, using F1 mice from reciprocal crosses between the B6 and JF strains, demonstrated that the mouse calcitonin receptor gene (Calcr) was expressed preferentially from the maternal allele in brain, whereas no allelic bias was detected in other tissues. Our results indicate that Calcr is imprinted in a tissue-specific manner, with a predominant expression from the maternal allele in the brain. Electronic Publication     

Evidence for uniparental, paternal expression of the human GABAA receptor subunit genes, using microcell-mediated chromosome transfer

We have constructed mouse A9 hybrids containing a single normal human chromosome 15, via microcell-mediated chromosome transfer. Cytogenetic and DNA-polymorphic analyses identified mouse A9 hybrids that contained either a paternal or maternal human chromosome 15. Paternal specific expression of the known imprinted genes SNRPN (small nuclear ribonucleoprotein-associated polypeptide N gene) and IPW (imprinted gene in the Prader-Willi syndrome region) was maintained in the A9 hybrids. Using this system, we first demonstrated that human GABAAreceptor subunit genes, GABRB3 , GABRA5 and GABRG3 , were expressed exclusively from the paternal allele and that E6-AP (E6- associated protein or UBE3A ) was biallelically expressed. Moreover, the 5' portion of the GABRB3 gene was found to be hypermethylated on the paternal allele. Our data imply that GABAAreceptor subunit genes are imprinted and are possible candidates for Prader-Willi syndrome, and that this human monochromosomal hybrid system enables the efficient analysis of imprinted loci.