IGF2 is parentally imprinted during human embryogenesis and in the Beckwith-Wiedemann syndrome

The phenomenon of parental imprinting involves the preferential expression of one parental allele of a subset of chromosomal genes and has so far only been documented in the mouse. We show here, by exploiting sequence polymorphisms in exon nine of the human insulin-like growth factor 2 (IGF2) gene, that only the paternally-inherited allele is active in embryonic and extra-embryonic cells from first trimester pregnancies. In addition, only the paternal allele is expressed in tissues from a patient who suffered from Beckwith-Wiedemann syndrome. Thus the parental imprinting of IGF2 appears to be evolutionarily conserved from mouse to man and has implications for the generation of the Beckwith-Wiedemann syndrome.     

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).     

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.     

Microalbuminuria in IDDM is associated with increased expression of monocyte procoagulant activity

Insulin-like growth factor II (IGF-II) plays a key role in mammalian growth, influencing foetal cell division and differentiation and possibly metabolic regulation. The mature 67 amino acid peptide shares sequence homology with both insulin and IGF-I. The liver is the main endocrine source of IGFs, but autocrine/paracrine activity is found in most tissues. The type 1 receptor mediates most of the biological effects of IGF-I and IGF-II; the type 2 receptor is involved with IGF-II degradation. Binding proteins may both localise IGFs to the receptors and regulate their activities. The IGF2 gene is maternally imprinted in mouse and human. Relaxation of IGF2 imprinting occurs in the Beckwith-Wiedemann syndrome of somatic overgrowth, sporadic Wilms' tumour and a number of other cancers. In the general adult population, the IGF2-INS gene cluster may also influence body weight, in which case IGF-II function could become a target for therapeutic intervention in obesity.     

Imprinted chromosomal regions of the human genome display sex-specific meiotic recombination frequencies

BACKGROUND: Meiotic recombination events do not occur randomly along a chromosome, but appear to be restricted to specific regions. In addition, some regions in the genome undergo recombination more frequently in the germ cells of one sex than the other. Genomic imprinting, the process by which the two parental alleles of a gene are differentially marked, is another genetic phenomenon associated with inheritance from only one parent or the other. The mechanisms that control meiotic recombination and genomic imprinting are unknown, but both phenomena necessarily depend on the presence of some DNA signal sequences and/or on the structure of the surrounding chromatin domain. RESULTS: In the present study, we compared the frequencies of sex-specific recombination events in three chromosomal regions of the human genome that contain clustered imprinted genes. Alignment of the genetic and physical maps of the ZNF127-SNRPN-IPW-PAR-5-PAR-1 region on chromosome 15q11-q13 (associated with Prader-Willi and Angelman syndromes) and the IGF2-H19 region on chromosome 11p15.5 (associated with Beckwith-Wiedemann syndrome) shows that both regions recombine with very high frequency during male meiosis, and with very low frequency during female meiosis. A third region around the WT-1 gene on chromosome 11p13 also recombines with higher frequency during male meiosis. CONCLUSIONS: The results show that the two best-known imprinted regions in the human genome are characterized by significant differences in recombination frequency during male and female meioses. A third, less well-characterized, imprinted region shows a similar sex-specific bias. On the basis of these observations, we propose a model suggesting that the region-specific differential accessibility of DNA that leads to differential recombination rates during male and female meioses also leads to the male- and female-specific modification of the signal sequences that control genomic imprinting.     

Igf2r and Igf2 gene expression in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos: absence of regulation by genomic imprinting

Genomic imprinting in mammals is believed to result from modifications to chromosomes during gametogenesis that inactivate the paternal or maternal allele. The genes encoding the insulin-like growth factor type 2 (Igf2) and its receptor (Igf2r) are reciprocally imprinted and expressed from the paternal and maternal genomes, respectively, in the fetal and adult mouse. We find that both genes are expressed in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos. These results indicate that inactivation of imprinted genes occurs postfertilization (most likely postimplantation) and that genomic imprinting and gene inactivation are separate processes. We propose that imprinting marks the chromosome so that regulatory factors expressed in cells at later times can recognize the imprint and selectively inactivate the maternal or paternal allele. For these genes, this finding invalidates models of genomic imprinting that require them to be inactive from the time of fertilization.     

Murine Stim1 maps to distal chromosome 7 and is not imprinted

STIM1 (GOK) maps to a region of human Chromosome (Chr) 11p15.5 that is implicated in several embryonal tumors, and some evidence indicates that STIM1 may have a growth suppressor role in rhabdomyosarcoma. In this study we have mapped the murine homolog, Stim1, to the same position as Hbb on distal mouse Chr 7. This region is separated by 20 cM from the region of distal Chr 7 that contains Igf2, H19, and other imprinted genes. Using strain-specific polymorphisms, we have shown that Stim1 is expressed from both parental alleles in fetal and neonatal mouse tissues. Similar analyses of human Wilms' tumor and normal kidney tissues demonstrated biallelic expression of STIM1 in the majority of samples. These data demonstrate that Stim1 expression is not regulated by genomic imprinting in either mouse or human tissues. Thus, if STIM1 is a tumor suppressor at 11p15.5, loss of expression is not due to imprinting effects.     

Association of a redefined proximal mouse chromosome 11 imprinting region and U2afbp-rs/U2af1-rs1 expression

Mice with maternal and paternal disomy for chromosome 11 (Chr 11) show growth retarded and overgrowth phenotypes, respectively, which can be attributed to genomic imprinting. Previous studies have defined the region of Chr 11 responsible (the Chr 11 imprinting region) as lying proximal to the T30H translocation breakpoint at the borders of G-bands 11B1.2 and 11B1.3. Evidence is presented here with two new translocations, T57H and T41Ad, which sequentially reduce the size of the imprinting region and locate it proximal to the T41Ad breakpoint in G-band 11A3.2. It therefore lies close to the centromere. The imprinted gene, U2af1-rs1, is known to be located within the original region and has been regarded as a candidate for the imprinting effects. Meiotic and mitotic chromosome FISH analysis, together with U2af1-rs1 expression studies are now described which show that the gene lies within the newly defined imprinting region and that its expression levels relate to the presence/absence and number of functional paternal alleles. U2af1-rs1 therefore remains a candidate gene for the Chr 11 imprinting effects. However, another recently reported imprinted gene, Meg1/Grb10, that lies within the region is also a good candidate, as it encodes a growth factor receptor. Meg1/Grb10 maps about 15 cM from U2af1-rs1 and is separated by conserved regions showing homology with two different human chromosomes. For these reasons, and because the two human homologues of U2af1-rs1 and Meg1/Grb10 also lie on different chromosomes, it would seem likely that the two genes identify two distinct imprinting domains within the small proximal region of mouse Chr 11.     

The MAS proto-oncogene is imprinted in human breast tissue

The human MAS proto-oncogene is situated at 6q25.3-q26, a region that is homologous to mouse chromosome 17 where two parentally imprinted genes (Mas and Igf2r) have previously been identified. We investigated the imprinting status of MAS in adult lesions to establish the imprinting status of this gene in humans, as certain imprinted genes are known to have altered imprinting phenotypes in cancer. Of 14 breast samples demonstrating a MAS RT-PCR product, 4 were informative for a polymorphic marker. In all 4 cases, expression of the MAS gene was found to be mono-allelic, indicating the presence of a functional imprint at this locus in human breast tissue.     

Mouse U2af1-rs1 is a neomorphic imprinted gene

The mouse U2af1-rs1 gene is an endogenous imprinted gene on the proximal region of chromosome 11. This gene is transcribed exclusively from the unmethylated paternal allele, while the methylated maternal allele is silent. An analysis of genome structure of this gene revealed that the whole gene is located in an intron of the Murr1 gene. Although none of the three human U2af1-related genes have been mapped to chromosome 2, the human homolog of Murr1 is assigned to chromosome 2. The mouse Murr1 gene is transcribed biallelically, and therefore it is not imprinted in neonatal mice. Allele-specific methylation is limited to a region around U2af1-rs1 in an intron of Murr1. These results suggest that in chromosomal homology and genomic imprinting, the U2af1-rs1 gene is distinct from the genome region surrounding it. We have proposed the neomorphic origin of the U2af1-rs1 gene by retrotransposition and the particular mechanism of genomic imprinting of ectopic genes.     

Using the evidence. A need for quantity, not quality?

The gene for alpha-stimulating guanine-nucleotide binding polypeptide, Gnas, has been considered as a candidate for the imprinting effects ascribed to distal mouse Chromosome (Chr) 2. Its human homologue (GNAS1) appears, from clinical and biochemical studies of patients with Albright hereditary osteodystrophy, to be paternally imprinted. GNAS1 maps to 20q13, a region that shows linkage conservation with distal mouse Chr 2. We have mapped Gnas within the imprinting region on distal Chr 2 by linkage analysis. To establish if Gnas is imprinted, we have looked for expression differences in tissues taken from mice carrying maternal duplication/paternal deficiency for distal Chr 2 (MatDp2) and its reciprocal (PatDp2). RNA in situ hybridization revealed high levels of Gnas mRNA in glomeruli of PatDp2 embryos at late gestation and lower levels in glomeruli of MatDp2 embryos. These results strongly suggest that Gnas is maternally imprinted and suggest that the mouse gene may be imprinted in a manner opposite that predicted in human.     

A direct repeat sequence at the Rasgrf1 locus and imprinted expression

Genomic imprinting is an epigenetic modification that can lead to parental-specific monoallelic expression of specific autosomal genes. While methylation of CpG dinucleotides is thought to be a strong candidate for this epigenetic modification, little is known about the establishment or maintenance of parental origin-specific methylation patterns. We have recently identified a portion of mouse chromosome 9 containing a paternally methylated region associated with a paternally expressed imprinted gene, Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1). This area of chromosome 9 also contains a short, direct tandem repeat in close proximity to a paternally methylated NotI site 30 kb upstream of Rasgrf1. Short, direct tandem repeats have been found associated with other imprinted genes and may act as important regulatory structures. Here we demonstrate that two rodent species (Mus and Rattus) contain a similar direct repeat structure associated with a region of paternal-specific methylation. In both species, the Rasgrf1 gene shows paternal-specific monoallelic expression in neonatal brain. A more divergent rodent species (Peromyscus) appears to lack a similar repeat structure based on Southern Blot analysis. Peromyscus animals show biallelic expression of Rasgrf1 in neonatal brain. These results suggest that direct repeat elements may play an important role in the imprinting process.     

The role of genomic imprinting in implantation

OBJECTIVES: To outline the possible role of the imprinted genes in early human embryogenesis and implantation. DATA IDENTIFICATION: Literature review. STUDY SELECTION: Studies examining the issues of genomic imprinting, implantation, gestational trophoblastic diseases, placental gene expression, and trophoblast invasion. RESULTS: Certain genes have been shown to be expressed either in the embryo or in the uterine decidua before implantation. Some of these have been shown to be parentally imprinted, that is, expressed either from their paternal or maternal origin. The paternally expressed genes are linked to placental proliferation and invasiveness. CONCLUSIONS: Clinical and basic data from different disciplines indicate that genomic imprinting may be crucial to the process of implantation.     

Chromosome 11p15.5 regional imprinting: comparative analysis of KIP2 and H19 in human tissues and Wilms' tumors

The imprinted H19 gene is frequently inactivated in Wilms' tumors (WTs) either by chromosome 11p15.5 loss of heterozygosity (LOH) or by hypermethylation of the maternal allele and it is possible that there might be coordinate disruption of imprinting of multiple 11p15.5 genes in these tumors. To test this we have characterized total and allele-specific mRNA expression levels and DNA methylation of the 11p15.5 KIP2 gene in normal human tissues, WTs and embryonal rhabdomyosarcoma (RMS). Both KIP2 alleles are expressed but there is a bias with the maternal allele contributing 70-90% of mRNA. Tumors with LOH show moderate to marked reductions in KIP2 mRNA relative to control tissues and residual mRNA expression is from the imprinted paternal allele. Among WTs without LOH most cases with H19 inactivation also have reduced KIP2 expression and most cases with persistent H19 expression have high levels of KIP2 mRNA. In contrast to the extensive hypermethylation of the imprinted H19 allele, both KIP2 alleles are hypomethylated and WTs with biallelic H19 hypermethylation lack comparable hypermethylation of KIP2 DNA. 5-aza-2'-deoxycytidine (aza-C) increases H19 expression in RD RMS cells but does not activate KIP2 expression. These data indicate coordinately reduced expression of two linked paternally imprinted genes in most WTs and also suggest mechanistic differences in the maintenance of imprinting at these two loci.