Frequent loss of imprinting of the H19 and IGF-II genes in ovarian tumors

Several human imprinted genes have been identified and are implicated in genetic diseases and tumorigenesis. We studied alterations of two imprinted genes, the paternally imprinted H19 and maternally imprinted IGF2, in 15 ovarian tumors with various cell types. To know allele-specific expression of the two genes, we analyzed restriction fragment length polymorphisms (RFLPs) at the 3′-untranslated region (UTR) in their cDNA, compared with those in the respective genomic DNA. As a result, biallelic H19 and IGF2 expression was observed in 8 (62%) of 13 informative (heterozygous) ovarian cancers and in 6 of 11 informative cases, respectively. H19 loss of imprinting (LOI) was most frequently observed in malignant serous cystadenocarcinoma (in four of six cases), whereas IGF2 LOI was not common in malignant epithelial cancers because three of six such LOI events occurred in benign mucinous cystadenomas and noncancerous endometriotic cyst. Our data suggest that the alteration of H19 and IGF2 imprinting plays differential roles in tumorigenesis and progression of ovarian tumors, depending on the tissue type as well as the developmental stage. Our data may argue against tumor suppressor activity of H19 in ovarian cancers. Am. J. Med. Genet. 80:391–395, 1998. © 1998 Wiley-Liss, Inc.     

Deletion of a nuclease-sensitive region between the Igf2 and H19 genes leads to Igf2 misregulation and increased adiposity

The insulin-like growth factor 2 gene (Igf2) is imprinted in most somatic tissues of the mouse with the exception of the choroid plexus and leptomeninges of the brain, where it is expressed from both alleles. The imprinting of Igf2 is dependent upon an imprinting control region (ICR) that lies 90 kb 3' of the gene and acts as a chromatin insulator to block enhancers that lie further 3' on the chromosome. Based on this model we would expect that enhancers of brain-specific expression of Igf2 would lie 5' of the ICR, and thus be insensitive to its action. Here we describe a 12 kb deletion of a region 5' of the ICR that is hypersensitive to nuclease digestion in chromatin. Its deletion results in a biallelic decrease in expression of Igf2, but not H19, in the brain, consistent with the proposal that it encodes a positive regulatory element. In addition, the deletion results in a minor relaxation of Igf2 imprinting in skeletal muscle and tongue. Lastly, the reduction in IGFII expression in the adult is accompanied by increased fat deposition and occasional obesity. Overweight animals are hypophagic, suggesting that IGFII affects fat metabolism rather than feeding behavior in adult mice.     

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.      

Genetic and genetic expression analyses of clear cell sarcoma of the kidney

Clear cell sarcoma of the kidney (CCSK) represents a significant diagnostic and clinical challenge. In search of diagnostically useful or biologically significant genetic abnormalities, we screened 30 CCSKs from the National Wilms Tumor Study Group. Genetic gains and losses were analyzed using comparative genomic hybridization; loss of heterozygosity at 11p15 was studied using microsatellite analysis. Loss of imprinting (LOI) was studied using allele-specific expression or methylation analysis at the ApaI polymorphic site for IGF2, AluI and RsaI sites for H19, and Cfo I site for SNRPN. Comparative genomic hybridization analysis revealed quantitative abnormalities in only 4 of 30 CCSKs. Two showed gain of 1q, one also showed loss of 10q, and the other also showed loss of terminal 4p. The other two cases demonstrated chromosome 19 loss and chromosome 19p gain, respectively. All 22 cases informative for 11p15 showed retention of both alleles. Of 14 CCSKs informative for IGF2, six showed biallelic expression; all three CCSKs informative for H19 exhibited monoallelic expression. The normal imprint pattern was present in all six CCSKs analyzed for SNRPN methylation. These data demonstrate an absence of consistent genetic gains or losses in CCSKs using these methods. The high frequency of LOI for IGF2 in CCSKs (43%) is comparable to that reported in Wilms tumors. The retention of imprinting at the SNRPN and H19 loci confirm that LOI is not a ubiquitous epigenetic change. This suggests that IGF2, a potent growth factor, may play a role in the development or progression of CCSK.     

Superovulation alters the expression of imprinted genes in the midgestation mouse placenta

Imprinted genes play important roles in embryonic growth and development as well as in placental function. Many imprinted genes acquire their epigenetic marks during oocyte growth, and this period may be susceptible to epigenetic disruption following hormonal stimulation. Superovulation has been shown to affect growth and development of the embryo, but an effect on imprinted genes has not been shown in postimplantation embryos. In the present study, we examined the effect of superovulation/in vivo development or superovulation/3.5dpc (days post-coitum) embryo transfer on the allelic expression of Snrpn, Kcnq1ot1 and H19 in embryos and placentas at 9.5 days of gestation. Superovulation followed by in vivo development resulted in biallelic expression of Snrpn and H19 in 9.5dpc placentas while Kcnq1ot1 was not affected; in the embryos, there was normal monoallelic expression of the three imprinted genes. We did not observe significant DNA methylation perturbations in the differentially methylated regions of Snrpn or H19. Superovulation followed by embryo transfer at 3.5dpc resulted in biallelic expression of H19 in the placenta. The expression of an important growth factor closely linked to H19, Insulin-like growth factor-II, was increased in the placenta following superovulation with or without embryo transfer. These results show that both maternally and paternally methylated imprinted genes were affected, suggesting that superovulation compromises oocyte quality and interferes with the maintenance of imprinting during preimplantation development. Our findings contribute to the evidence that mechanisms for maintaining imprinting are less robust in trophectoderm-derived tissues, and have clinical implications for the screening of patients following assisted reproduction.     

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.     

Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site

The adjacent insulin-like growth factor 2 (IGF2) and H19 genes are imprinted in most normal human tissues, but imprinting is often lost in tumors. The mechanisms involved in maintenance of imprinting (MOI) and loss of imprinting (LOI) are unresolved. We show here that osteosarcoma (OS) tumors with IGF2/H19 MOI exhibit allele-specific differential methylation of a CTCF-binding site upstream of H19. LOI of IGF2 or H19 in OS occurs in a mutually exclusive manner, and occurs with monoallelic expression of the other gene. Bisulfite sequencing reveals IGF2 LOI occurs with biallelic CpG methylation of the CTCF-binding site, while H19 LOI occurs with biallelic hypomethylation of this site. Our data demonstrate that IGF2 LOI and H19 LOI are accompanied by reciprocal methylation changes at a critical CTCF-binding site. We propose a model in which incomplete gain or loss of methylation at this CTCF-binding site during tumorigenesis explains the complex and often conflicting expression patterns of IGF2 and H19 in tumors.     

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.     

不明原因不孕症患者子宫内膜印迹基因H19表达异常

探讨H19基因表达变化在不孕症发病中的作用。应用PCR、RT-PCR以及RsaI酶切位点多态性观察H19特殊等位基因表达，检测不明原因不孕症子宫内膜中的印迹状态。结果显示，25例正常窗口期杂合子子宫内膜中H19皆表达单等位基因，而38例不明原因不孕症患者窗口期子宫内膜中杂合子全部表达双等位基因，提示H19印迹丢失可能与不明原因不孕症的发病有关。这为临床诊断和治疗不明原因不孕症提供了新思路。     

H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas: a pilot case-control analysis

Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern of CTCF binding sites 5 and 6 using methylation-sensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also observed in both tumor and lymphocyte DNA from two patients, and at a high frequency in the control group (29 out of 64 informative controls). Additionally, we found that the C/T transition detected by HhaI RFLP suppressed one dinucleotide CpG in critical CTCF binding site 6, of a mutation showing polymorphic frequencies. Although a heterogeneous methylation pattern was observed after DNA sequencing modified by sodium bisulfite, the biallelic methylation pattern was confirmed in 9 out of 10 HNSCCs. These findings are likely to be relevant in the epigenetic regulation of the DMR, especially in pathological conditions in which the imprinting of IGF2 and H19 genes is disrupted.     

An extended region of biallelic gene expression and rodent-human synteny downstream of the imprinted H19 gene on chromosome 11p15.5

There is increasing evidence for chromosomal domains containing multiple imprinted genes and for domain-wide disruption of imprinting in certain diseases. In a majority of Wilms' tumors (WTs) there is an abnormal bipaternal pattern of expression at three imprinted loci, H19, IGF2 and KIP2, clustered on chromosome 11p15.5. We previously described biallelic expression of L23MRP, 40 kb downstream of H19. Here we map two additional genes, the first encoding a ubiquitously expressed RNA, 2G7, and the second encoding the fast isoform of skeletal muscle troponin-T (TNNT3), in the 55 kb of DNA downstream of L23MRP. 2G7 RNA is spliced and polyadenylated but lacks long open reading frames. 2G7 and TNNT3 are biallelically expressed in mid-fetal and adult human tissues and 2G7 shows persistent expression in WTs. The rat homologue of L23MRP is highly conserved and lies within 85 kb of H19 in a region of rat chromosome 1 which also contains IGF2 and TNNT3. Parallel expression of H19 and TNNT3 in different adult skeletal muscle types suggests that these genes may share an enhancer. These data outline multiple contiguous loci downstream of H19 which escape functional imprinting in humans. The rodent-human synteny of this region may facilitate a search for an imprinting domain boundary.      

IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch

Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith-Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVF-derived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn's advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.     

Development of a monkey model for the study of primate genomic imprinting

An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2, known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres (ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene. Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipulation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or by ICSI. Muscle biopsies followed by RT-PCR and sequence analysis were performed in four heterozygous animals produced by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differentially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells of the monkey.     

Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development

In vitro culture of mouse embryos results in loss of imprinting. The aim of the present study was to examine how two of the techniques commonly used during assisted reproduction, namely embryo culture and embryo transfer, affect genomic imprinting after implantation in the mouse. F1 hybrid mouse embryos were subjected to three experimental conditions: control (unmanipulated), embryo transfer and in-vitro-culture followed by embryo transfer. Concepti were collected on d9.5 of development and allelic expression determination of ten imprinted genes (H19, Snrpn, Igf2, Kcnq1ot1, Cdkn1c, Kcnq1, Mknr3, Ascl2, Zim1, Peg3) was performed. Although control concepti had monoallelic imprinted gene expression in all tissues, both manipulated groups had aberrant expression of one or more imprinted genes in the yolk sac and placenta. Culture further exacerbated the effects of transfer by increasing the number of genes with aberrant allelic expression in extraembryonic, as well as embryonic tissues. Additionally, placentae of both groups of manipulated concepti exhibited reduced levels of Igf2 mRNA and increased levels of Ascl2 mRNA when compared with their unmanipulated counterparts. Furthermore, we show that biallelic expression of Kcnq1ot1 coincided with loss of methylation on the maternal allele of the KvDMR1 locus, a phenotype often associated with the human syndrome Beckwith-Wiedemann. In conclusion, our results show that even the most basic manipulation used during human-assisted reproduction, namely, embryo transfer, can lead to misexpression of several imprinted genes during post-implantation development. Additionally, our results serve as a cautionary tale for gene expression studies in which embryo transfer is used.     

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.     

Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK9 potassium channel gene in mouse and human

Genomic imprinting is the epigenetic marking of gene subsets resulting in monoallelic or predominant expression of one of the two parental alleles according to their parental origin. We describe the systematic experimental verification of a prioritized 16 candidate imprinted gene set predicted by sequence-based bioinformatic analyses. We used Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) and discovered maternal-specific imprinted expression of the Kcnk9 gene as well as strain-dependent preferential expression of the Rarres1 gene in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross mouse embryos. For the remaining 14 candidate imprinted genes, we observed biallelic expression. In adult mouse tissues, we found that Kcnk9 expression was restricted to the brain and also was maternal-specific. QUASEP analysis of informative human fetal brain samples further demonstrated maternal-specific imprinted expression of the human KCNK9 orthologue. The CpG islands associated with the mouse and human Kcnk9/KCNK9 genes were not differentially methylated, but strongly hypomethylated. Thus, we speculate that mouse Kcnk9 imprinting may be regulated by the maternal germline differentially methylated region in Peg13, an imprinted non-coding RNA gene in close proximity to Kcnk9 on distal mouse chromosome 15. Our data have major implications for the proposed role of Kcnk9 in neurodevelopment, apoptosis and tumourigenesis, as well as for the efficiency of sequence-based bioinformatic predictions of novel imprinted genes.