Conserved methylation imprints in the human and mouse GRB10 genes with divergent allelic expression suggests differential reading of the same mark

Grb10/GRB10 encodes a cytoplasmic adapter protein which modulates coupling of a number of cell surface receptor tyrosine kinases with specific signalling pathways. Mouse Grb10 is an imprinted gene with maternal-specific expression. In contrast, human GRB10 is expressed biallelically in most tissues, except for maternal-specific expression of one isoform in muscle and paternal expression in fetal brain. Owing to its location in 7p11.2-p12, GRB10 has been considered a candidate gene for the imprinted growth disorder, the Silver-Russell syndrome (SRS), but its predominantly biallelic expression argues against involvement in the syndrome. To investigate the discrepant imprinting between mouse and human, we compared the sequence organization of their upstream regions, and examined their allelic methylation patterns and the splice variant organization of the mouse locus. Contrary to expectation, we detected both maternal and paternal expression of mouse Grb10. Expression of the paternal allele arises from a different promoter region than the maternal and, as in human, is restricted to the brain. The upstream regions are well conserved, especially the presence of two CpG islands. Surprisingly, both genes have a similar imprinted methylation pattern, the second CpG island is a differentially methylated region (DMR) with maternal methylation in both species. Analysis of 24 SRS patients did not reveal methylation anomalies in the DMR. In the mouse this DMR is a gametic methylation mark. Our results suggest that the difference in imprinted expression in mouse and human is not due to acquisition of an imprint mark but in differences in the reading of this mark.     

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.      

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.      

Paternal expression of WT1 in human fibroblasts and lymphocytes

The Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier.      

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.      

<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.      

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).      

Retention of imprinting of the human apoptosis-related gene TSSC3 in human brain tumors

Genomic imprinting is the result of a gamete-specific modification leading to parental origin-specific gene expression in somatic cells of the offspring. Several embryonal tumors show loss of imprinting of genes clustered in human chromosome 11p15.5, an important tumor suppressor gene region, harboring several normally imprinted genes. TSSC3, a gene homologous to mouse TDAG51, implicated in Fas-mediated apoptosis, is also located in this region between hNAP2 and p57 (KIP2). TSSC3 is the first apoptosis-related gene found to be imprinted in placenta, liver and fetal tissues where it is expressed from the maternal allele in normal human development. This study investigated the imprinting status of TSSC3 in human normal, adult brain and in human neuroblastomas, medulloblastomas and glioblastomas. A polymorphism in exon 1 at position 54 was used to analyze the allelic expression of the TSSC3 gene by a primer oligo base extension (PROBE) assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found that the TSSC3 gene is not imprinted in human normal, adult brain and blood. In contrast, strong allelic bias resembling imprinting could be detected in most examined tumor specimens. The results demonstrate for the first time that the tumors under investigation are associated with a retention of imprinting of a potential growth inhibitory gene.     

Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer

IGF2 is the first gene discovered to be imprinted and expressed exclusively from the paternal allele in both human and mouse. IGF2 is also the first imprinted gene displaying loss of imprinting (LOI) or aberrant imprinting in human cancers. Evidently, LOI or reactivation of the maternal allele of IGF2 is associated with an increase of IGF2 expression that may subsequently play an important role in the onset of human cancers. The most important discovery was the association of LOI of IGF2 with the risk of developing human colorectal cancer. LOI occurs not only in colon cancer tissues, but also in matched normal tissues and peripheral blood cells. A pilot study indicated a significant relationship between LOI of IGF2 and family history as well as personal history of colorectal cancer, suggesting that LOI of IGF2 might be a valuable biomolecular marker of predicting an individual's risk for colon cancer. A recent epigenetic progenitor model suggested that human cancers might have a common basis that involves an epigenetic disruption of progenitor cells mediated by "tumor progenitor genes" and proposed that non-neoplastic but epigenetically disrupted progenitor cells might be an important target for cancer risk assessment and prevention.     

Maternal repression of the human GRB10 gene in the developing central nervous system; evaluation of the role for GRB10 in Silver-Russell syndrome

The GRB10 gene encodes a growth suppressor and maps to human chromosome 7p11.2-p13. Maternal duplication (matdup) of this region has recently been associated with Silver-Russell syndrome (SRS), which is characterised by pre- and postnatal growth restriction, craniofacial dysmorphism and lateral asymmetry. Maternal uniparental disomy for chromosome 7 (mUPD7) occurs in approximately 7% of SRS patients. Exposure of a recessive allele due to isodisomy has been ruled out in five mUPD7 cases, suggesting genomic imprinting as the basis for disease. Assuming SRS patients with matdup of 7p11.2-p13 and mUPD7 share a common aetiology, this would implicate a maternally expressed gene from this interval, which is involved in growth inhibition. Murine Grb10 was identified as a maternally expressed gene by subtractive hybridisation using normal and androgenetic mouse embryos. Grb10 maps to the homologous region of proximal mouse chromosome 11, for which mUPD incurs reduced birthweight. A role for GRB10 in SRS was evaluated by determining its imprinting status in multiple human foetal tissues using expressed polymorphisms, and by screening the coding region for mutations in 18 classic non-mUPD7 SRS patients. Maternal repression of GRB10 was observed specifically in the developing central nervous system including brain and spinal cord, with biallelic expression in peripheral tissues. This is in contrast to mouse Grb10, and represents the first example of opposite imprinting in human and mouse homologues. While a role for GRB10 in mUPD7 SRS cases can not be ruled out on the basis of imprinting status, no mutations were identified in the patients screened.     

Conservation of a maternal-specific methylation signal at the human IGF2R locus

The human 1GF2R gene has been reported to be either blallelicallyor very rarely monoallelically expressed, in contrast to thematernally expressed mouse counterpart. We describe here ananalysis of the 5' portion of the human IGF2R gene and showthat it contains a maternally methylated CpG island in the secondintron. A similar maternally methylated intronic element hasbeen proposed to be the imprinting box for the mouse gene andalthough the relevance of this element has yet to be directlydemonstrated, methylation has been reported to be essentialto maintain allele-specific expression of imprinted genes. Allelicexpression analysis of human IGF2R in 70 lymphoblastoid celllines identified only one iine showing monoallelic expression.Thus, in this tissue monoparental methylatlon of the IGF2R genedoes not correlate with allele-specific expression. We alsoconfirm here that the human IGF2R gene is located in an asynchronouslyreplicating chromosomal region, as are all other imprinted genesso far analyzed. The mouse and human IGF2R intronic CpG islandsboth contain numerous large direct repeats that are methylatedfollowing maternal, but not paternal, transmittance. Thus featuresthat attract maternal-specific methylatlon are conserved betweenthe mouse and human genes. Since these intronic CpG islandsshare organizational rather than sequence homology, this suggeststhat secondary DNA structure may play a role in attracting amaternal methylation imprint.     

Aberrant genomic imprinting in rhesus monkey embryonic stem cells

Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.     

Contribution of GRB10 to the prenatal phenotype in Silver-Russell syndrome? Lessons from 7p12 copy number variations

The growth factor binding protein 10 (GRB10) has been suggested as a candidate gene for Silver-Russell syndrome because of its localization in 7p12, its imprinting status, data from mice models and its putative role in growth. Based on a new patient with normal growth carrying a GRB10 deletion affecting the paternal allele and data from the literature, we conclude that the heterogeneous clinical findings in patients with copy number variations (CNVs) of GRB10 gene depend on the size and the gene content of the CNV. However, evidence from mouse and human cases indicate a growth suppressing role of GRB10 in prenatal development. As a result, an increase of active maternal GRB10 copies, e.g. by maternal uniparental disomy of chromosome 7 or duplications of the region results in intrauterine growth retardation. In contrast, a defective GRB10 copy might result in prenatal overgrowth, whereas the paternal GRB10 allele is not required for proper prenatal growth.