Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Analysis of imprinted gene expression in normal fertilized and uniparental preimplantation porcine embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.     

Differential differences in methylation status of putative imprinted genes among cloned swine genomes

DNA methylation is a major epigenetic modification in the mammalian genome that regulates crucial aspects of gene function. Mammalian cloning by somatic cell nuclear transfer (SCNT) often results in gestational or neonatal failure with only a small proportion of manipulated embryos producing live births. Many of the embryos that survive to term later succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Aberrant methylation patterns of imprinted genes in cloned cattle and mice have been elucidated, but few reports have analyzed the cloned pig genome. Four surviving cloned sows that were created by ear fibroblast nuclear transfer, each with a different life span and multiple organ defects, such as heart defects and bone growth delay, were used as epigenetic study materials. First, we identified four putative differential methylation regions (DMR) of imprinted genes in the wild-type pig genome, including two maternally imprinted loci (INS and IGF2) and two paternally imprinted loci (H19 and IGF2R). Aberrant DNA methylation, either hypermethylation or hypomethylation, commonly appeared in H19 (45% of imprinted loci hypermethylated vs. 30% hypomethylated), IGF2 (40% vs. 0%), INS (50% vs. 5%), and IGF2R (15% vs. 45%) in multiple tissues from these four cloned sows compared with wild-type pigs. Our data suggest that aberrant epigenetic modifications occur frequently in the genome of cloned swine. Even with successful production of cloned swine that avoid prenatal or postnatal death, the perturbation of methylation in imprinted genes still exists, which may be one of reason for their adult pathologies and short life. Understanding the aberrant pattern of gene imprinting would permit improvements in future cloning techniques.     

Alterations of H19 Imprinting and IGF2 Replication Timing Are Infrequent in Beckwith–Wiedemann Syndrome

Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Oppositely imprinted genes H19 and insulin-like growth factor 2 are coexpressed in human androgenetic trophoblast.

Human uniparental gestations such as gynogenetic ovarian teratomas and androgenetic complete hydatidiform moles provide a model to evaluate the integrity of parent-specific gene expression--i.e., imprinting--in the absence of a complementary parental genetic contribution. We studied expression, in these tissues, of the oppositely imprinted genes H19, which is an embryonic nontranslated RNA, and insulin-like growth factor type 2 (IGF2). Normal gestations only express H19 from the maternal allele and express IGF2 from the paternal allele, whereas neither is expressed from the maternal genome of gynogenetic gestations, and both are expressed from the paternal genome of androgenetic gestations. Coexpression of H19 and IGF2 in the androgenetic tissues was in a single population of cells, mononuclear trophoblast--the same cell type expressing these genes in biparental placentas. These results demonstrate that a biparental genome may be required for expression of the reciprocal IGF2/H19 imprint. Alternatively, biparental expression may be a normal feature of some imprinted genes in specific cell types. Additional experiments with other imprinted genes will clarify whether this reflects global failure of the imprinting process or a change specific to the IGF2/H19 locus.     

Imprinting status of 11p15 genes in Beckwith-Wiedemann syndrome patients with CDKN1C mutations.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by somatic overgrowth, congenital malformations, and predisposition to childhood tumors. Aberrant expression of multiple imprinted genes, including H19, IGF2, KCNQ1OT1, and CDKN1C, has been observed in BWS patients. It has been estimated that mutations in CDKN1C occur in 12-17% of BWS patients. We have screened 10 autosomal dominant pedigrees and 65 sporadic BWS cases by PCR/heteroduplex analysis and DNA sequencing and have identified four mutations, two of which were associated with biallelic IGF2 expression and normal H19 and KCNQ1OT1 imprinting. One patient demonstrated phenotypic expression of paternally transmitted mutation in this maternally expressed gene, a second proband is the child of one of a pair of monozygotic twin females who carry the mutation de novo, and a third patient exhibited unusual skeletal changes more commonly found in other overgrowth syndromes. When considered with other studies published to date, this work reveals the frequency of CDKN1C mutations in BWS to be only 4.9%. This is the first report of an analysis of the imprinting status of genes in the 11p15 region where CDKN1C mutations were associated with loss of IGF2 imprinting and maintenance of H19 and KCNQ1OT1 imprinting.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.     

Imprinting Status of 11p15 Genes in Beckwith–Wiedemann Syndrome Patients with CDKN1C Mutations

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder characterized by somatic overgrowth, congenital malformations, and predisposition to childhood tumors. Aberrant expression of multiple imprinted genes, including H19, IGF2, KCNQ1OT1, and CDKN1C, has been observed in BWS patients. It has been estimated that mutations in CDKN1C occur in 12–17% of BWS patients. We have screened 10 autosomal dominant pedigrees and 65 sporadic BWS cases by PCR/heteroduplex analysis and DNA sequencing and have identified four mutations, two of which were associated with biallelic IGF2 expression and normal H19 and KCNQ1OT1 imprinting. One patient demonstrated phenotypic expression of paternally transmitted mutation in this maternally expressed gene, a second proband is the child of one of a pair of monozygotic twin females who carry the mutation de novo, and a third patient exhibited unusual skeletal changes more commonly found in other overgrowth syndromes. When considered with other studies published to date, this work reveals the frequency of CDKN1C mutations in BWS to be only 4.9%. This is the first report of an analysis of the imprinting status of genes in the 11p15 region where CDKN1C mutations were associated with loss of IGF2 imprinting and maintenance of H19 and KCNQ1OT1 imprinting.     

Imprint status of M6P/IGF2R and IGF2 in chickens

Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.     

Proinsulin-like growth factor-II overexpression does not alter monoallelic H19 gene expression in transfected human embryonic kidney fibroblasts

Insulin-like growth factor-II (IGF-II) is a potent mitogen for cells in culture. The H19 gene is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in tumorigenesis. The H19 gene is closely linked to the human IGF-II gene (IGF2) on chromosome 11p15.5 and these genes are reciprocally imprinted in most fetal tissues. H19 is expressed only from the maternal and IGF2 from the paternal chromosome. We have asked whether overexpression of proIGF-II alters H19 imprinting status and/or expression. Human embryonal kidney fibroblasts (293 cells) were stably transfected with a PCMV5 vector containing the full length human IGF-II cDNA or a control cDNA. Transfectant clones expressed large quantities of IGF-II mRNA and secrete 1-5 ug/ml and 150-230 ng/ml proIGF-II within 24 hours of serum-free culture (transfectant 293-9 and -11 respectively) (1). Cells were genotyped at the exon 5, RsaI restriction fragment length polymorphism (RFLP) and found to be informative (+/-). H19 expression was monoallelic (+) indicating preservation of H19 imprinting in all cell lines. Using quantitative RT-PCR with internal competitors for H19 and for IGF-II cDNA, overexpression of IGF2 in 293-11 and 293-9 cells was confirmed. In contrast, no significant difference with respect to H19 expression was detected between the overexpressing cells and control lines. In conclusion, (1) human embryonal fibroblasts express the H19 gene. (2) H19 imprinting is preserved in these cells. (3) proIGF-II overexpression does not alter H19 expression.     

The origin and evolution of genomic imprinting and viviparity in mammals

Genomic imprinting is widespread in eutherian mammals. Marsupial mammals also have genomic imprinting, but in fewer loci. It has long been thought that genomic imprinting is somehow related to placentation and/or viviparity in mammals, although neither is restricted to mammals. Most imprinted genes are expressed in the placenta. There is no evidence for genomic imprinting in the egg-laying monotreme mammals, despite their short-lived placenta that transfers nutrients from mother to embryo. Post natal genomic imprinting also occurs, especially in the brain. However, little attention has been paid to the primary source of nutrition in the neonate in all mammals, the mammary gland. Differentially methylated regions (DMRs) play an important role as imprinting control centres in each imprinted region which usually comprises both paternally and maternally expressed genes (PEGs and MEGs). The DMR is established in the male or female germline (the gDMR). Comprehensive comparative genome studies demonstrated that two imprinted regions, PEG10 and IGF2-H19, are conserved in both marsupials and eutherians and that PEG10 and H19 DMRs emerged in the therian ancestor at least 160 Ma, indicating the ancestral origin of genomic imprinting during therian mammal evolution. Importantly, these regions are known to be deeply involved in placental and embryonic growth. It appears that most maternal gDMRs are always associated with imprinting in eutherian mammals, but emerged at differing times during mammalian evolution. Thus, genomic imprinting could evolve from a defence mechanism against transposable elements that depended on DNA methylation established in germ cells.     

成年核移植山羊生长相关基因的表达分析

体细胞核移植过程有可能影响克隆动物生长相关基因尤其是印迹基因的表达水平。本研究运用同源引物 PCR 扩增、RACE 技术并结合同源克隆策略, 克隆了 7 个山羊生长相关基因包括 3 个印迹基因(H19、IGF2 和 IGF2R)和 4 个非印迹基因(IGF1、IGF1R、GHR 和 GHSR)的完全 CDS 或者部分 cDNA 序列, 经生物信息学技术确认后, 用荧光实时定量 PCR 对 8只成年克隆山羊中这些基因的表达水平进行分析, 结果表明 3 个印迹基因中 IGF2R 基因表达水平极显著高于对照组的自然繁殖山羊(P<0.01), 而 H19 和 IGF2 的表达则没有很大区别; 4 个非印迹基因中只有 IGF1R 的表达水平极显著高于对照组(P<0.01), IGF1、GHR 和 GHSR 的表达与对照组相似。表明即使在表型正常的成年克隆动物也存在一定的表观遗传异常。通过对获得完全 CDS 和 3′UTR 的 IGF2 基因经过生物信息学分析表明, 山羊 IGF2 基因包含一个 540 bp 的开放阅读框 (ORF)编码 179 个氨基酸。IGF2 基因 cDNA 序列和氨基酸序列以及其它基因部分序列比较分析表明, 山羊所有这些基因与绵羊的同源性要高于同牛的同源性。     

Biallelic expression of<Emphasis Type="Italic"> HRAS</Emphasis> and<Emphasis Type="Italic"> MUCDHL</Emphasis> in human and mouse

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.     

Allelic expression of IGF2 in marsupials and birds

Genomic imprinting, the parent-of-origin- specific expression of genes, has been observed in a variety of eutherian mammals. One gene that has been shown to be imprinted in all eutherians examined is the IGF2 gene. This gene encodes a potent fetal-specific growth factor that is expressed almost exclusively from the paternal chromosome. Several other imprinted genes in the IGF2 pathway are imprinted as well, suggesting that IGF2 is a focal point for the selective pressure leading to imprinted gene expression. This observation is in keeping with a proposal that imprinting arose as the result of a genetic conflict between parents over the allocation of maternal resources to the embryo. One prediction of this model is that imprinting exists in species in which there is at least some contribution of maternal resources to the embryo, and in which polyandry is observed. To test this prediction the allelic expression of the IGF2 gene was examined in two noneutherian species. The IGF2 gene was shown to be expressed in a paternal-specific manner identical to that in eutherians in Monodelphis domestica, a placental South American opossum. In contrast, the IGF2 gene is biallelic in expression in chickens, which are oviparous, and make no postfertilization contribution of maternal resources to the offspring. Received: 24 June 1999 / Accepted: 28 July 1999