Interactions between imprinting effects: summary and review.

Mice with uniparental disomies (uniparental duplications) for defined regions of certain chromosomes, or certain disomies, show a range of developmental abnormalities most of which affect growth. These defects can be attributed to incorrect dosages of maternal or paternal copies of imprinted genes lying within the regions involved. Combinations of certain partial disomies result in interactions between the imprinting effects that seemingly independently affect foetal and/or placental growth in different ways or modify neonatal and postnatal development. The findings are generally in accord with the 'conflict hypothesis' for the evolution of genomic imprinting but do not demonstrate common growth axes within which imprinted genes may interact. Instead, it would seem that any gene that favours embryonic/foetal development, at consequent cost to the mother, will have been subject to evolutionary selection for only paternal allele expression. Reciprocally, any gene that reduces embryonic/foetal growth to limit disadvantage to the mother will have been selected for only maternal allele expression. It is concluded that survival of the placenta is core to the evolution of imprinting.

Imprinting control within the compact Gnas locus.

Mouse distal chromosome 2 was one of the earliest described imprinting regions. Maternal and paternal inheritance of the region is associated with opposite phenotypes affecting growth, development and behaviour. Mis-expression of proteins determined by the imprinted Gnas locus can account for the phenotypes. The imprinting domain in mouse distal chromosome 2 is small, comprising the Gnas locus. This locus is unusually complex, containing biallelic, maternally and paternally expressed transcripts that share exons. It also contains two germline differentially methylated regions that have the characteristics of imprinting control regions. One of these specifically controls the tissue-specific imprinting of the Gnas exon 1 transcript but does not affect the imprinting of other transcripts. Imprinting of other transcripts may be controlled by the other germline differentially methylated region by a mechanism involving antisense RNA.

A reassessment of imprinting regions and phenotypes on mouse chromosome 6: Nap1l5 locates within the currently defined sub-proximal imprinting region.

Previous studies (Beechey, 2000) have shown that mouse proximal chromosome (Chr) 6 has two imprinting regions. An early embryonic lethality is associated with two maternal copies of the more proximal imprinting region, while mice with two maternal copies of the sub-proximal imprinting region are growth retarded at birth, the weight reduction remaining similar to adulthood. No detectable postnatal imprinting phenotype was seen in these earlier studies with two paternal copies of either region. The sub-proximal imprinting region locates distal to the T77H reciprocal translocation breakpoint in G-band 6A3.2 and results reported here show that it does not extend beyond the breakpoint of the more distal T6Ad translocation in 6C2. It has been confirmed that the postnatal growth retardation observed with two maternal copies of the sub-proximal region is established in utero, although placental size was normal. A new finding is that 16.5-18.5-dpc embryos, with two paternal copies of the sub-proximal imprinting region, were larger than their normal sibs, although placental size was normal. As no postnatal growth differences have been observed in these mice, the fetal overgrowth must normalize by birth. The imprinted genes Peg1/Mest, Copg2, Copg2as and Mit1/Lb9 map to the sub-proximal imprinting region and are thus candidates for the observed imprinting phenotypes. Another candidate is the recently reported imprinted gene Nap1l5. Expression studies of Nap1l5 in mice with two maternal or two paternal copies of different regions of Chr 6 have demonstrated that the gene locates within the sub-proximal imprinting region. FISH has mapped Nap1l5 to G-band 6C1, within the sub-proximal imprinting region but several G-bands distal to the Peg1/Mest cluster. This location, and the 30-Mb separation of these loci on the sequence map, makes it probable that Nap1l5 defines a new imprinting domain within the currently defined sub-proximal imprinting region.

The imprinted region on human chromosome 7q32 extends to the carboxypeptidase A gene cluster: an imprinted candidate for Silver-Russell syndrome.

Imprinted gene(s) on human chromosome 7q32-qter have been postulated to be involved in intrauterine growth restriction associated with Silver-Russell syndrome (SRS) as 7-10% of patients have mUPD(7). Three imprinted genes, MEST, MESTIT1, and COPG2IT1 on chromosome 7q32, are unlikely to cause SRS since epigenetic and sequence mutation analyses have not shown any changes. One hundred kilobases proximal to MEST lies a group of four carboxypeptidase A (CPA) genes. Since most imprinted genes are found in clusters, this study focuses on analysing these CPAs for imprinting effects based on their proximity to an established imprinted domain. Firstly, a replication timing study across 7q32 showed that an extensive genomic region including the CPAs, MEST, MESTIT1, and COPG2IT1 replicates asynchronously. Subsequently, SNP analysis by sequencing RT-PCR products of CPA1, CPA2, CPA4, and CPA5 indicated preferential expression of CPA4. Pyrosequencing was used as a quantitative approach, which confirmed predominantly preferential expression of the maternal allele and biallelic expression in brain. CPA5 expression levels were too low to allow reliable evaluation of allelic expression, while CPA1 and CPA2 both showed biallelic expression. CPA4 was the only gene from this family in which an imprinting effect was shown despite the location of this family of genes next to an imprinted cluster. As CPA4 has a potential role in cell proliferation and differentiation, two preferentially expressed copies in mUPD patients with SRS syndrome would result in excess expression and could alter the growth profiles of these subjects and give rise to intrauterine growth restriction.

Genetic, physical, and phenotypic characterization of the Del(13)Svea36H mouse.

The Del(13)Svea36H deletion was recovered from a radiation mutagenesis experiment and represents a valuable resource for investigating gene content and function at this region of mouse Chromosome (Chr) 13 and human Chr 6p21.3-23 and 6p25. In this paper we examine the physical extent of chromosome loss and construct an integrated genetic and radiation hybrid map of the deleted segment. We show that embryos which are homozygous for the deletion die at or before implantation and that heterozygotes are subviable, with a substantial proportion of carriers dying after mid-gestation but before weaning. The majority of viable carriers exhibit a variety of phenotypes including decreased size, eyes open at birth, corneal opacity, tail kinks, and craniofacial abnormalities. Both the heterozygous viability and the penetrance of the visible phenotypes vary with genetic background.

Microarray expression profiling of tissues from mice with uniparental duplications of chromosomes 7 and 11 to identify imprinted genes.

Microarray analysis allows the screening of thousands of identifiable genes in a single experiment. The challenge of this approach is to combine the new technology with established genetic tools to associate genes with specific biological function. In this study we have designed a screen to identify imprinted genes from mice with uniparental duplications of proximal Chromosomes (Chrs) 7 and 11, using microarray analysis. By comparing the expression patterns in embryonic and newborn tissues of maternally versus paternally inherited proximal Chrs 7 and 11, we have correctly identified four out of five known imprinted genes represented on a microarray. We have additionally identified two novel imprinted candidate genes as well as a differentially expressed clone that is a potential downstream target. Interpretation of the microarray data requires careful preparation of age- and strain-matched samples and attention to detail in tissue dissection technique.

Peg1/Mest locates distal to the currently defined imprinting region on mouse proximal chromosome 6 and identifies a new imprinting region affecting growth.

Mice with maternal duplication for proximal chromosome 6 (Chr 6) die in utero before 11.5 dpc, an effect that can be attributed to genomic imprinting. Previous studies have defined the region of Chr 6 responsible as lying proximal to the T6Ad translocation breakpoint in G-band 6B3. Evidence presented here with a new Chr 6 translocation T77H has substantially reduced the size of the imprinting region, locating it between G-band 6A3.2 and the centromere. The paternally expressed imprinted gene Mest had been mapped within the original imprinting region and was therefore a candidate for the early embryonic lethality. FISH has shown that Mest locates distal to T77H and therefore outside the redefined imprinting region. This evidence confirms that Mest is not a candidate for the early embryonic lethality found with two maternal copies of proximal Chr 6. Furthermore mice with maternal duplication for Ch 6 distal to T77H (MatDp.dist6) were found to be growth retarded at birth, the weight reduction remaining similar until adulthood. It can be concluded that the growth retardation is established in utero and is maintained at a similar level from birth to adulthood. Therefore Mest locates in a new imprinting region, distal to G-band 6A3.2 which affects growth. A targeted mutation of Mest has been reported that exhibits growth retardation, reduced postnatal survival and abnormal maternal behaviour. Here the phenotype of MatDp.dist6 mice is compared to that of Mest-deficient mutant mice. Unlike the latter, MatDp.dist6 mice have good survival rates and females have normal maternal behaviour. Possible reasons for these differences are discussed.

An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus.

The Gnas locus in distal mouse chromosome (Chr) 2 is emerging as a complex genomic region. It contains three imprinted genes in the order Nesp-Gnasxl-Gnas. Gnas encodes a G protein alpha-subunit, and Nesp and Gnasxl encode proteins of unknown function expressed in neuroendocrine tissue. Together, these genes form a single transcription unit because transcripts of Nesp and Gnasxl are alternatively spliced onto exon 2 of Gnas. Nesp and Gnasxl are expressed from opposite parental alleles, with Nesp encoding a maternal-specific transcript and Gnasxl encoding a paternal-specific transcript. We now identify a further imprinted transcript in this cluster. Reverse transcription-PCR analysis of Nesp expression in 15. 5-days-postcoitum embryos carrying only maternal or paternal copies of distal Chr 2 revealed an isoform that is exclusively paternally, rather than maternally, expressed. Strand-specific reverse transcription-PCR showed that this form is an antisense transcript. The existence of a paternally expressed antisense transcript was confirmed by Northern blot analysis. The sequence is contiguous with genomic sequence downstream of Nesp and encompasses Nesp exons 1 and 2 and an intervening intron. We propose that Nespas is an additional control element in the imprinting region of mouse distal Chr 2; it adds further complexity to the Gnas-imprinted gene cluster.

Identification of imprinted loci by methylation-sensitive representational difference analysis: application to mouse distal chromosome 2.

Imprinted genes are distinguished by different patterns of methylation on their parental alleles, a property by which imprinted loci could be identified systematically. Here, representational difference analysis (RDA) is used to clone HpaII fragments with methylation differences on the maternal and paternal copies of distal chromosome (Chr) 2 in the mouse. Uniparental inheritance for this region causes imprinting phenotypes whose molecular basis is only partially understood. RDA led to the recovery of multiple differentially methylated HpaII fragments at two major sites of imprinted methylation: paternal-specific methylation at the Nesp locus and maternal-specific methylation at the Gnasxl locus. Nesp and Gnasxl represent oppositely imprinted promoters of the Gnas gene, which encodes the G-protein subunit, Gsalpha. The organization of the Nesp-Gnasxl-Gnas region was determined: Nesp and Gnasxl were found to be 15 kb apart, and Gnasxl was found to be 30 kb upstream of Gnas. Sites of imprinted methylation were also detected at the loci for neuronatin on Chr 2 and for M-cadherin on Chr 8. RDA was highly effective at identifying imprinted methylation, and its potential applications to imprinting studies are discussed.

A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2.

Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as Gnas, which encodes a G protein alpha subunit, and there is clinical and biochemical evidence that the human homologue GNAS1, mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes, Gnasxl and Nesp. Gnasxl determines a variant G protein alpha subunit associated with the trans-Golgi network and Nesp encodes a secreted protein of neuroendocrine tissues. Gnasxl is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas Nesp is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably, Gnasxl, Nesp, and Gnas are all part of the same transcription unit; transcripts for Gnasxl and Nesp are alternatively spliced onto exon 2 of Gnas. This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons.

Localisation of the imprinted gene neuronatin, Nnat, confirms and refines the location of a second imprinting region on mouse chromosome 2.

Nine regions on six mouse autosomes are subject to imprinting and uniparental inheritance of any one of these regions results in mice with phenotypic anomalies. So far on distal Chromosome (Chr) 2 there is a unique imprinting region between 2H3 and 2H4 associated with two behavioural disorders and neonatal lethality. A maternally imprinted gene, Nnat, has been identified which is expressed in the nervous system and maps to distal Chr 2. Nnat has been excluded as a candidate for either or both the behavioural phenotypes as it lies proximal to the 2H3-2H4 imprinting region. Here we have mapped Nnat to band 2H1 which is at least 18 Mb proximal to the previously described imprinting region. It maps close to agouti, some alleles of which show differential expression according to parental origin. The localisation of Nnat to band H1 confirms and refines the map location of a second imprinting region on mouse Chr 2.

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.

Imprinting of distal mouse chromosome 2 is associated with phenotypic anomalies in utero.

Previous studies have shown that the distal region on mouse chromosome (Chr) 2 is subject to imprinting as mice with maternal duplication/paternal deficiency (MatDp.dist2) and the reciprocal (PatDp.dist2) for this region exhibit phenotypic anomalies at birth and die neonatally. We show here that imprinting effects are detectable in utero. Notably PatDp.dist2 embryos show an increase in wet weight compared with normal, which peaks at 16.5 d post coitum (dpc), and diminishes by birth, whereas the wet weight of placenta is slightly reduced in the latter half of gestation. Newborns have increased length of the long bones. By contrast, the wet weight of MatDp.dist2 embryos decreases during the second half of gestation. Measurements of dry weights of embryos at 16.5 dpc have indicated that there is no difference in either PatDp.dist2 or MatDp.dist2 compared with normal so that the wet weight differences are due to fluid retention in PatDp.dist2 but fluid loss in MatDp.dist2. In PatDp.dist2 embryos excess fluid is particularly prominent in the subcuticular skin layer, whereas by birth fluid is evident around the neck and tongue. At 16.5 dpc the PatDp.dist2 embryos are severely oedematous, as the average fluid content per unit dry weight per embryo was increased by 40%, whereas the MatDp.dist2 embryos are dehydrated as the average water content per unit dry weight per embryo was reduced by 6%. A preliminary conclusion is that there is neither growth enhancement in PatDp.dist2 nor growth retardation in MatDp.dist2 offspring.

Genetic and functional analysis of neuronatin in mice with maternal or paternal duplication of distal Chr 2.

Functional differences between parental genomes are due to differential expression of parental alleles of imprinted genes. Neuronatin (Nnat) is a recently identified paternally expressed imprinted gene that is initially expressed in the rhombomeres and pituitary gland and later more widely in the central and peripheral nervous system mainly in postmitotic and differentiating neuroepithelial cells. Nnat maps to distal chromosome (Chr) 2, which contains an imprinting region that causes morphological abnormalities and early neonatal lethality. More detailed mapping analysis of Nnat showed that it is located between the T26H and T2Wa translocation breakpoints which is, surprisingly, proximal to the reported imprinting region between the T2Wa and T28H translocation breakpoints, suggesting that there may be two distinct imprinting regions on distal chromosome 2. To investigate the potential role of Nnat, we compared normal embryos with those which were PatDp.dist2.T26H (paternal duplication/maternal deficiency of chromosome 2 distal to the translocation breakpoint T26H) and MatDp.dist2.T26H. Expression of Nnat was detected in the PatDp.dist2.T26H embryos, where both copies of Nnat are paternally inherited, and normal embryos but no expression was detected in the MatDp.dist2.T26H embryos with the two maternally inherited copies. The differential expression of Nnat was supported by DNA methylation analysis with the paternally inherited alleles being unmethylated and the maternal alleles fully methylated. Although experimental embryos appeared grossly similar phenotypically in the structures where expression of Nnat was detected, differences in folding of the cerebellum were observed in neonates, and other more subtle developmental or behavioral effects due to gain or loss of Nnat cannot be ruled out.

Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.

Angelman syndrome (AS) is a human genetic disorder characterized by mental retardation, seizures, inappropriate laughter, abnormal galt, tremor and ataxia. There is strong genetic evidence that the disorder is associated with a maternally expressed, imprinted gene mapping to chromosome 15q11-13. Affected patients demonstrate varied molecular abnormalities, including large maternal deletions, uniparental paternal disomy (UPD). Imprinting mutations and loss of function mutations of E6-associated-protein (E6-AP) ubiquitin-protein ligase (UBE3A). All of these abnormalities are associated with loss of maternal expression of UBE3A. Although mutations in UBE3A cause AS, indicating that maternal-specific expression of UBE3A is essential for a normal phenotype, evidence for maternal-specific expression of UBE3A has been lacking. Using mice with partial paternal UPD encompassing Ube3a to differentiate maternal and paternal expression, we found by in situ hybridization that expression of Ube3a in Purkinje cells, hippocampal neurons and mitral cells of the olfactory bulb in UPD mice was markedly reduced compared to non-UPD littermates. In contrast, expression of Ube3a in other regions of the brain was only moderately or not at all reduced in UPD mice. The major phenotypic features of AS correlate with the loss of maternal-specific expression of Ube3a in hippocampus and cerebellum as revealed in the mouse model.

Hindshaker, a novel myelin mutant showing hypomyelination preferentially affecting the spinal cord.

Animals with spontaneous mutations affecting myelin formation have provided useful information about the genetic and cellular mechanisms regulating normal and abnormal myelination. In this paper we describe a novel murine mutation termed hindshaker (hsh), which is inherited in an autosomal recessive manner. Affected mice are characterised by a variable tremor of the hind end which commences at about 2 weeks of age and largely disappears in animals older than 6 weeks. There is hypomyelination affecting predominantly the spinal cord, although the optic nerves and brain are involved to a much lesser degree. The defect of thinly myelinated and naked axons is maximal at 20 days of age and largely resolves with time so that in the adult most axons are myelinated. The myelin structure appears normal and immunostains for the major proteins. Although the distribution of oligodendrocytes in the spinal cord is similar to normal during the period of hypomyelination, there are fewer mature cells. The hsh mutation appears to delay the maturation of oligodendrocytes, particularly in the spinal cord. Additionally, there is a considerable variation in phenotypic expression and in penetrance when the mutation is expressed on different genetic backgrounds, suggesting the hsh locus is subject to the influence of modifying gene(s). Identification of the hsh gene should identify a factor important in the development of oligodendrocytes, particularly those in the spinal cord.

A candidate model for Angelman syndrome in the mouse.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are well-recognized examples of imprinting in humans. They occur most commonly with paternal and maternal 15q11-13 deletions, but also with maternal and paternal disomy. Both syndromes have also occurred more rarely in association with smaller deletions seemingly causing abnormal imprinting. A putative mouse model of PWS, occurring with maternal duplication (partial maternal disomy) for the homologous region, has been described in a previous paper but, although a second imprinting effect that could have provided a mouse model of AS was found, it appeared to be associated with a slightly different region of the chromosome. Here, we provide evidence that the same region is in fact involved and further demonstrate that animals with paternal duplication for the region exhibit characteristics of AS patients. A mouse model of AS is, therefore, strongly indicated.