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.     

Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.     

Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors: study on a series of 82 tumors

Abnormalities of the 11p15 region with overexpression of the normally imprinted insulin-like growth factor II (IGF-II) gene have been implicated in the pathogenesis of adrenocortical tumors. We evaluated the frequency and distribution of 11p15 loss of heterozygosity (LOH) and IGF-II gene overexpression in a series of 82 sporadic adrenocortical tumors, screened for pathological functional imprinting of the 11p15 region in tumors not exhibiting LOH and evaluated the expression of H19 gene in these tumors. Abnormalities of the 11p15 region as LOH (loss of the maternal allele and duplication of the paternal allele) and/or IGF-II gene overexpression are frequent features of the malignant state and were found in 27 of 29 (93.1%) of the malignant tumors and in only 3 of 35 (8.6%) of the benign tumors. Tumors without abnormality of the 11p15 region (mainly benign tumors) did not exhibit pathological functional imprinting. In tumors with mosaicism for 11p15 LOH, biallelic expression of the IGF-II gene was constant in the tumor cell contingent not undergoing LOH. Abrogation of H19 expression correlated with the loss of the maternal allele (LOH or pathological imprinting), but did not always correlate with overexpression of the IGF-II gene. These data indicate the involvement of dysregulation of the 11p15 region in late steps of adrenocortical tumorigenesis and provide us with new molecular markers for a better diagnostic and prognostic evaluation of adrenocortical tumors.     

Location of enhancers is essential for the imprinting of H19 and Igf2 genes

Genomic imprinting is the process in mammals by which gamete-specific epigenetic modifications establish the differential expression of the two alleles of a gene. The tightly linked H19 and Igf2 genes are expressed in tissues of endodermal and mesodermal origin, with H19 expressed from the maternal chromosome and Igf2 expressed from the paternal chromosome. A model has been proposed to explain the reciprocal imprinting of these genes; in this model, expression of the genes is governed by competition between their promoters for a common set of enhancers. An extra set of enhancers might be predicted to relieve the competition, thereby eliminating imprinting. Here we tested this prediction by generating mice with a duplication of the endoderm-specific enhancers. The normally silent Igf2 gene on the maternal chromosome was expressed in liver, consistent with relief from competition. We then generated a maternal chromosome containing a single set of enhancers located equidistant from 1gf2 and H19; the direction of the imprint was reversed. Thus, the location of the enhancers determines the outcome of competition in liver, and the strength of the H19 promoter is not sufficient to silence Igf2.     

Imprinted M6p/Igf2 receptor is mutated in rat liver tumors

We have previously shown that inactivation of mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is a common early event in both human liver and breast carcinogenesis. The M6p/Igf2r is imprinted in mice while expression is biallelic in most humans. In this investigation the M6p/Igf2r gene is shown to also be imprinted in the liver of Fischer 344, Lewis and Brown Norway rats. In addition, we have identified mutations in the expressed allele of the M6p/Igf2r in 40% of diethylnitrosamine-initiated rat liver tumors. These results provide further evidence that the M6P/IGF2R functions as a liver tumor suppressor gene. They also suggest that mice and rats would be more sensitive than humans to those hepatocarcinogens in which the M6p/Igf2r is mechanistically involved in transformation since one rather than two alleles would need to be inactivated.     

Chromatin conformation of the H19 epigenetic mark

Genomic imprinting in mammals is an epigenetic process that results in differential expression of the two parental alleles. The tightly linked murine H19 and Igf2 genes are reciprocally imprinted: H19 is expressed from the maternal chromosome while Igf2 is expressed from the paternal chromosome. A single regulatory region in the 5' flank of the H19 gene has been implicated in silencing both genes. On the paternal chromosome, this region is heavily methylated at CpG residues, leading to repression of the H19 gene. The mechanism by which the same region in an unmethylated state on the maternal chromosome silences Igf2 is less well understood. We have probed the chromatin structure of the region by assessing its sensitivity to nuclease digestion. Two regions of nuclease hypersensitivity that are specific to the maternal chromosome were identified. These coincide with the region that is most heavily methylated on the paternal chromosome. As is the case with paternal methylation, hypersensitivity is present in all tissues surveyed, irrespective of H19 expression. We suggest that the chromatin structure of the maternal 5' flank of the H19 gene may represent an epigenetic mark involved in the silencing of Igf2.     

Coding mutations in p57KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms tumors

The Beckwith-Wiedemann syndrome (BWS) is marked by fetal organ overgrowth and conveys a predisposition to certain childhood tumors, including Wilms tumor (WT). The genetics of BWS have implicated a gene that maps to chromosome 11p15 and is paternally imprinted, and the gene encoding the cyclin-cdk inhibitor p57KIP2 has been a strong candidate. By complete sequencing of the coding exons and intron/exon junctions, we found a maternally transmitted coding mutation in the cdk-inhibitor domain of the KIP2 gene in one of five cases of BWS. The BWS mutation was an in-frame three-amino-acid deletion that significantly reduced but did not fully abrogate growth-suppressive activity in a transfection assay. In contrast, no somatic coding mutations in KIP2 were found in a set of 12 primary WTs enriched for cases that expressed KIP2 mRNA, including cases with and without 11p15.5 loss of heterozygosity. Two other 11p15.5 loci, the linked and oppositely imprinted H19 and IGF2 genes, have been previously implicated in WT pathogenesis, and several of the tumors with persistent KIP2 mRNA expression and absence of KIP2 coding mutations showed full inactivation of H19. These data suggest that KIP2 is a BWS gene but that it is not uniquely equivalent to the 11p15.5 "WT2" tumor-suppressor locus.     

Imprinting of Igf2 and H19 from a 130 kb YAC transgene

A stringent test for imprint control elements is to examine their function at ectopic loci in transgenic experiments. Igf2 and H19 are part of a larger imprinting region and as a first step, we examined these reciprocally imprinted genes in transgenic experiments using a 130 kb YAC clone. After paternal inheritance, H19 was appropriately repressed and Igf2 was expressed, irrespective of copy number or genetic background. After maternal inheritance H19 was consistently expressed, albeit with some variability. The levels of H19 expression per copy of the transgene inversely correlated with Igf2 (-lacZ) expression in cis. The consistent imprinting of H19 from this YAC contrasts with the previously described imprinting of mini-H19 transgenes, which only occurs at multi-copy loci, is inconsistent, and is prone to genetic background effects. We propose a novel model in which silencing of the H19 gene is the default state and its activation after maternal inheritance is the key mechanistic event for imprinting in this region. In addition, in situ analysis of the Igf2-lacZ reporter indicates that additional mesoderm-specific enhancers are present within the YAC clone. No obvious phenotype was detected from the excess gene dosage of H19.     

Mutation of the MENIN gene in sporadic pancreatic endocrine tumors

Pancreatic endocrine tumors occur both sporadically and as part of the multiple endocrine neoplasia type 1 (MEN1) syndrome. MEN1 is an autosomal dominant disease characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. The MEN1 gene called MENIN maps to chromosome 11q13 and is thought to function as a tumor suppressor gene. We previously demonstrated loss of heterozygosity (LOH) at 11q13 in approximately 40% of sporadic pancreatic endocrine tumors and hypothesize that MENIN is involved in the development of these tumors. Thirty-one sporadic pancreatic endocrine tumors were analyzed for mutation of MENIN by nonradioactive single-stranded conformation polymorphism. Twelve mutations were detected in 31 sporadic pancreatic endocrine tumors (34%). Twelve of these 31 tumors previously demonstrated loss of heterozygosity at 11q13. Of the tumors with LOH, seven contained mutations of the MENIN gene (58%). The majority of the MENIN mutations occurred within exon 2. Two independent mutations in MENIN were detected in a gastrinoma that also revealed LOH, leading to the possibility of another tumor suppressor gene locus at 11q13. Mutations were present in both benign and malignant pancreatic endocrine tumors, suggesting that a MENIN gene mutation is a frequent and early event in the tumorigenesis. The high incidence of truncating mutations in tumors with LOH at 11q13 support the hypothesis that MENIN is a tumor suppressor gene.     

Molecular characterization of the men1 tumor suppressor gene in sporadic pituitary tumors

Anterior pituitary tumors arise sporadically, and also as part of the inherited multiple endocrine neoplasia type 1 (MEN 1) syndrome. To investigate the role of the recently isolated men1 gene in sporadic pituitary tumorigenesis, the complete coding sequence was screened for mutations in 45 sporadic anterior pituitary tumors, including 14 hormone-secreting tumors and 31 nonsecreting tumors, by dideoxy fingerprinting and sequence analysis. No pathogenic sequence changes were found in the men1 coding region. The men1 gene was expressed in 43 of these tumors with sufficient RNA, including one tumor with loss of heterozygosity (LOH) for several polymorphic markers on chromosomal region 11q13. Furthermore, both alleles were expressed in 19 tumors in which the constitutional DNA was heterozygous for intragenic polymorphisms. Thus, inactivation of the men1 tumor suppressor gene, by mutation or by imprinting, does not appear to play a prominent role in sporadic pituitary adenoma pathogenesis.     

Imprinted control of gene activity in Drosophila

Genetic imprinting is defined as a reversible, differential marking of genes or chromosomes that is determined by the sex of the parent from whom the genetic material is inherited [1]. Imprinting was first observed in insects where, in some species, most notably among the coccoids (scale insects and allies), the differential marking of paternally and maternally transmitted chromosome sets leads to inactivation or elimination of paternal chromosomes [2]. Imprinting is also widespread in plants and mammals [3,4], in which paternally and maternally inherited alleles may be differentially expressed. Despite imprinting having been discovered in insects, clear examples of parental imprinting are scarce in the model insect species Drosophila melanogaster. We describe a case of imprint-mediated control of gene expression in Drosophila. The imprinted gene - the white+ eye-color gene - is expressed at a low level when transmitted by males, and at a high level when transmitted by females. Thus, in common with coccoids, Drosophila is capable of generating an imprint, and can respond to that imprint by silencing the paternal allele.     

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.     

The efficacy of re-TUR after 3 weeks of initial TUR treatment for locally advanced bladder cancer

Epithelioid sarcomas are soft tissue tumors with an indolent, but potentially aggressive, clinical behavior. Distinction from other benign and malignant entities may be a diagnostic dilemma. In this study, we evaluate the presence of loss of heterozygosity (LOH) of chromosome 22q in tumor DNA from 13 epithelioid sarcomas, four epithelioid angiosarcomas, and two epithelioid hemangioendotheliomas, and investigate its possible role in diagnosis. LOH was detected in 6 of 10 (60%) of the informative epithelioid sarcomas. No allele loss was detected in the informative vascular tumors, three angiosarcomas, and two hemangioendotheliomas. Chromosome 22q carries the locus of a tumor suppressor gene, the neurofibromatosis 2 (NF2) gene, which has been shown to be lost or mutated in some NF2-related tumors, sporadic meningiomas, and vestibular schwannomas, as well as a few other tumors. Our data suggest that a region of chromosome 22q may be the locus of a tumor suppressor gene involved in the tumorigenesis of these neoplasms. Genetic alterations of yet-unknown tumor suppressor genes in this region, or even the NF2 tumor suppressor gene, may play a role in epithelioid sarcomas tumorigenesis. The fact that LOH was only detected in epithelioid sarcomas and not in the vascular tumors studied suggests a possible role for this marker in diagnosis.     

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.