Search PubMed⌕ Search

Biomedical subjects

W Reik

Publications and source records attributed to W Reik.

At least 55 records · Page 3Linked to original sources
PubMed1999

Overexpression of insulin-like growth factor-II in transgenic mice is associated with pancreatic islet cell hyperplasia.

We have used an insulin-like growth factor (IGF)-II transgenic mouse model in which mouse IGF-II is widely overexpressed, resulting in increased fetal size and selective organ overgrowth, to investigate the effects on the development of the endocrine pancreas. Fetuses examined on day 19.5-20 of gestation had significantly elevated circulating levels of IGF-II, compared with control mice. The pancreatic islets in transgenic animals were of irregular shape and had a mean area five times greater than in controls, whereas the mean number of islets per tissue section was not altered. The size of individual endocrine cells was not altered. Although the islets in animals expressing the IGF-II transgene were considerably larger, immunohistochemistry for insulin and glucagon showed that the relative proportion of beta-cells was significantly less, and that of alpha-cells was higher. Normal islet morphology was disrupted, with alpha-cells appearing in small groups within the islets, as well as on the periphery, whereas beta-cells were often seen at the edge of the islets. Twice as many islet cells (21.9% vs. 11.4%) were involved in cell replication, detected by the presence of immunoreactive proliferating cell nuclear antigen, in pancreata from transgenic mice vs. controls, whereas the number of cells undergoing apoptosis was significantly reduced. Abundant IGF-II messenger RNAwas found within the islets of transgenic animals by in situ hybridization, and the relative area of islets demonstrating immunoreactive IGF-II was significantly greater. Immunoreactive IGF-I was much less abundant and was further reduced in islets of transgenic animals. The area of islets immunopositive for IGF binding protein-2 was unaltered. Despite the presence of islet hyperplasia, circulating insulin levels and serum glucose levels were not significantly different between transgenic and control mice. These results show that an overexpression of IGF-II in fetal life has a profound effect on islet morphology and causes islet hyperplasia while reducing the attrition of islet cells by apoptosis.

Animals↗
PubMed1999

Analysis of germline CDKN1C (p57KIP2) mutations in familial and sporadic Beckwith-Wiedemann syndrome (BWS) provides a novel genotype-phenotype correlation.

Beckwith-Wiedemann syndrome (BWS) is a human imprinting disorder with a variable phenotype. The major features are anterior abdominal wall defects including exomphalos (omphalocele), pre- and postnatal overgrowth, and macroglossia. Additional less frequent complications include specific developmental defects and a predisposition to embryonal tumours. BWS is genetically heterogeneous and epigenetic changes in the IGF2/H19 genes resulting in overexpression of IGF2 have been implicated in many cases. Recently germline mutations in the cyclin dependent kinase inhibitor gene CDKN1C (p57KIP2) have been reported in a variable minority of BWS patients. We have investigated a large series of familial and sporadic BWS patients for evidence of CDKN1C mutations by direct gene sequencing. A total of 70 patients with classical BWS were investigated; 54 were sporadic with no evidence of UPD and 16 were familial from seven kindreds. Novel germline CDKN1C mutations were identified in five probands, 3/7 (43%) familial cases and 2/54 (4%) sporadic cases. There was no association between germline CDKN1C mutations and IGF2 or H19 epigenotype abnormalities. The clinical phenotype of 13 BWS patients with germline CDKN1C mutations was compared to that of BWS patients with other defined types of molecular pathology. This showed a significantly higher frequency of exomphalos in the CDKN1C mutation cases (11/13) than in patients with an imprinting centre defect (associated with biallelic IGF2 expression and H19 silencing) (0/5, p<0.005) or patients with uniparental disomy (0/9, p<0.005). However, there was no association between germline CDKN1C mutations and risk of embryonal tumours. No CDKN1C mutations were identified in six non-BWS patients with overgrowth and Wilms tumour. These findings (1) show that germline CDKN1C mutations are a frequent cause of familial but not sporadic BWS, (2) suggest that CDKN1C mutations probably cause BWS independently of changes in IGF2/H19 imprinting, (3) provide evidence that aspects of the BWS phenotype may be correlated with the involvement of specific imprinted genes, and (4) link genotype-phenotype relationships in BWS and the results of murine experimental models of BWS.

Beckwith-Wiedemann Syndrome↗
PubMed1998

Analysis and identification of imprinted genes.

Genomic imprinting in mammals results in the unequal expression of the two parental alleles of specific genes. The existence of imprinting in the mouse emerged from nuclear transplantation studies and from the abnormal phenotypes associated with uniparental inheritance of particular chromosome segments. Over the past 5 years, 20 or so imprinted genes have been identified. This has emphasized the important roles played by some imprinted genes in development, permitted a description of the epigenetic properties associated with imprinting, and provided the first insights into the regulation of imprinting. In this article, we discuss the generation of experimental material in which imprinting effects can be analyzed, review the properties of imprinted genes, and discuss how to examine them using state-of-the-art techniques. Finally, we consider the means by which new imprinted genes can be identified.

Alleles↗
PubMed1998

Imprinting mechanisms in mammals.

Imprinting is a genetic mechanism that determines expression or repression of genes according to their parental origin. Some imprinted genes occur in clusters in the genome. Recent work using transgenic mice shows that multiple cis-acting sequences are needed for correct imprinting. Mutation analysis in a normal chromosomal context reveals the importance of imprinting centres for regional establishment or maintenance of imprinting in a cluster. Elements that contribute to the function of imprinting centres and regional propagation of the imprints are CpG-rich differentially methylated regions (that during development retain germline imposed methylation or demethylation), direct repeat clusters, and unusual RNAs (antisense, non-translated etc.). The interaction of these cis elements with transacting factors such as methylase and chromatin factors establishes a hierarchical control system with local and regional effects.

Alleles↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

Imprinting mechanisms.

A number of recent studies have provided new insights into mechanisms that regulate genomic imprinting in the mammalian genome. Regions of allele-specific differential methylation (DMRs) are present in all imprinted genes examined. Differential methylation is erased in germ cells at an early stage of their development, and germ-line-specific methylation imprints in DMRs are reestablished around the time of birth. After fertilization, differential methylation is retained in core DMRs despite genome-wide demethylation and de novo methylation during preimplantation and early postimplantation stages. Direct repeats near CG-rich DMRs may be involved in the establishment and maintenance of allele-specific methylation patterns. Imprinted genes tend to be clustered; one important component of clustering is enhancer competition, whereby promoters of linked imprinted genes compete for access to enhancers. Regional organization and spreading of the epigenotype during development is also important and depends on DMRs and imprinting centers. The mechanism of cis spreading of DNA methylation is not known, but precedent is provided by the Xist RNA, which results in X chromosome inactivation in cis. Reading of the somatic imprints could be carried out by transcription factors that are sensitive to methylation, or by methyl-cytosine-binding proteins that are involved in transcriptional repression through chromatin remodeling.

Animals↗
PubMed1998

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes.

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

Alleles↗
PubMed1997

Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2.

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions flanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

Alleles↗
PubMed1997

Transactivation of Igf2 in a mouse model of Beckwith-Wiedemann syndrome.

The gene IGF2, which encodes a fetal insulin-like growth factor, is imprinted, so only one of two parental copies of the gene is expressed. The altered expression of IGF2 has been implicated in Beckwith-Wiedemann syndrome, a human fetal overgrowth syndrome, which is characterized by overgrowth of several organs and an increased risk of developing childhood tumours. We have introduced Igf2 transgenes into the mouse genome by using embryonic stem cells, which leads to transactivation of the endogenous Igf2 gene. The consequent overexpression of Igf2 results in most of the symptoms of Beckwith-Wiedemann syndrome, including prenatal overgrowth, polyhydramnios, fetal and neonatal lethality, disproportionate organ overgrowth including tongue enlargement, and skeletal abnormalities. These phenotypes establish Igf2 overexpression as a key determinant of Beckwith-Wiedemann syndrome.

Animals↗
PubMed1997

Loss of the maternal H19 gene induces changes in Igf2 methylation in both cis and trans.

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.

Alleles↗
PubMed1997

Epigenetic inheritance in the mouse.

Acquired epigenetic modifications, such as DNA methylation or stable chromatin structures, are not normally thought to be inherited through the germline to future generations in mammals [1] [2]. Studies in the mouse have shown that specific manipulations of early embryos, such as nuclear transplantation, can result in altered patterns of gene expression and induce phenotypic alterations at later stages of development [3] [4] [5]. These effects are consistent with acquired epigenetic modifications that are somatically heritable, such as DNA methylation. Repression and DNA methylation of genes encoding major urinary proteins, repression of the gene encoding olfactory marker protein, and reduced body weight can be experimentally induced by nuclear transplantation in early embryos [4]. Strikingly, we now report that these acquired phenotypes are transmitted to most of the offspring of manipulated parent mice. This is the first demonstration of epigenetic inheritance of specific alterations of gene expression through the germline. These observations establish a mammalian model for transgenerational effects that are important for humal health, and also raise the question of the evolutionary importance of epigenetic inheritance.

Amino Acid Sequence↗
PubMed1997

Imprint switch mechanism indicated by mutations in Prader-Willi and Angelman syndromes.

Genomic imprinting is an epigenetic mechanism resulting in the preferential expression of the maternal or paternal alleles of a specific subset of genes in the mammalian genome. A key but relatively unexplored question is how imprints are established in the germline. New observations on two classical imprinting disorders, the Prader-Willi (PWS) and Angelman (AS) syndromes, offer the first genetic insight into this process. Molecular analysis of imprinting mutations that interfere with the appropriate establishment of the maternal and paternal epigenotypes has led to the identification of imprinted transcripts that could be involved in switching imprints in the germlines.

Angelman Syndrome↗
PubMed1997

Imprinting in clusters: lessons from Beckwith-Wiedemann syndrome.

Imprinted genes in mammals can be clustered in the genome. This raises important questions about mechanistic and functional relationships between imprinted genes in a cluster. The insulin-like growth factor II (IGF2) gene is paternally expressed and is surrounded by maternally expressed genes. Loss of imprinting of IGF2 is the most common molecular defect found in the human foetal overgrowth syndrome, Beckwith-Wiedemann syndrome (BWS). Transgenic experiments in the mouse establish that overexpression of IGF2 can result in most of the symptoms of BWS. However, mutations, translocations, or methylation defects in BWS have so far been found in three of the linked maternally expressed genes. We present a model where the paternal growth enhancer IGF2 is surrounded by multiple maternal suppressors, and mutations, or epigenetic alterations, in any of these suppressors could cause BWS. In addition, the precise phenotypic spectrum of BWS might depend on which maternally expressed gene is mutated.

Animals↗
PubMed1997

Imprinting of IGF2 and H19: lack of reciprocity in sporadic Beckwith-Wiedemann syndrome.

Genomic imprinting is a novel form of control of gene expression in which the transcription of each allele of an imprinted gene is dependent on the sex of the gamete from which it was derived; to date > 15 genes have been demonstrated to show imprinting. The maintenance of a normal imprinting pattern in many loci has been shown to be essential for normal development and adult life. Many tumours, and some developmental disorders, exhibit loss of imprinting (LOI) in key genes such as insulin-like growth factor 2 (IGF2) which often results in hyperplasia and is associated with cancer. The mechanism by which the genomic imprint is first established, then maintained, is not understood. However, in the case of IGF2, the expression of a neighbouring gene, H19, has been suggested to influence its transcription by competition for a common enhancer, thereby generating a mutually exclusive and allele-specific pattern of gene expression. Associated changes in CpG methylation in discrete areas of both genes have been implicated in maintenance of the imprint. We have examined the allele-specific expression of IGF2 and H19 in fibroblasts derived from patients with sporadic Beckwith-Wiedemann syndrome (BWS), a fetal overgrowth syndrome associated with an imprinted locus on 11p15.5. We report that the majority of karyotypically normal patients show LOI of IGF2 with biallelic expression. In a proportion of these patients, loss of IGF2 imprinting was associated with complete suppression of H19 expression, as predicted by the enhancer competition model. However, in a significant number of cases, IGF2 showed biallelic expression even though H19 expression and methylation status were normal. This indicates that there must be an alternative H19-independent pathway by which allele-specific IGF2 expression is established or maintained.

Beckwith-Wiedemann Syndrome↗
PubMed1997

Epigenetic modification and uniparental inheritance of H19 in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with a characteristic pattern of visceromegaly and predisposition to childhood tumours. BWS is a genetically heterogeneous disorder; most cases are sporadic but approximately 15% are familial and a small number of BWS patients have cytogenetic abnormalities involving chromosome 11p15. Genomic imprinting effects have been implicated in familial and non-familial BWS. We have investigated the molecular pathology of 106 sporadic BWS cases; 17% (14/83) of informative cases had uniparental disomy (UPD) for chromosome 11p15.5. In each case UPD appeared to result from a postzygotic event resulting in mosaicism for segmental paternal isodisomy. The critical region for isodisomy was refined to a 25 cM interval between D11S861 and D11S2071 which contained the IGF2, H19, and p57(KIP2) genes. In three cases isodisomy for 11q markers was detected but this did not extend further than 11q13-q21 suggesting that complete chromosome 11 disomy may not produce a BWS phenotype. The allele specific methylation status of the H19 gene was investigated in 80 sporadic BWS cases. All 13 cases with UPD tested displayed hypermethylation consistent with an excess of paternal H19 alleles. In addition, five of 63 (8%) cases with normal biparental inheritance had H19 hypermethylation consistent with an "imprinting centre" mutation (ICM) or "imprinting error" (IE) lesion. The phenotype of patients with putative ICM/IE mutations was variable and overlapped with that of non-UPD sporadic BWS cases with normal H19 methylation. However, exomphalos was significantly (p < 0.05) more common in the latter group. These findings may indicate differential effects on the expression of imprinted genes in chromosome 11p15 according to the precise molecular pathology. Analysis of H19 methylation is useful for the diagnosis of both UPD or altered imprinting in BWS and shows that a variety of molecular mechanisms may cause relaxation of IGF2 imprinting in BWS.

Aneuploidy↗
PubMed1996

Nucleotide sequence of a 28-kb mouse genomic region comprising the imprinted Igf2 gene.

The mouse insulin-like growth factor II gene (Igf2) is physically linked to the insulin II gene (Ins2) and both are subject to tissue-specific genomic imprinting. The paternal-specific expression of Igf2 has been associated with hypermethylation of some CpG sites in the 5' flanking region and in the body of the gene. As a first step in analyzing the structural features of this imprinted locus, we here report the complete nucleotide sequence of Igf2, including all introns and the intergenic region adjacent to Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80% overall identity with the corresponding rat sequence and has a high GC content of 52%. In addition to the known CpG island within the second Igf2 promoter, another island was identified approximately 2 kb 5' to the first exon. Other features of this locus include a 35-fold tandem repeat of an 11-bp sequence that overlaps Igf2 pseudo-exon 2, and a B2 repeat element in the intergenic region between Ins2 and Igf2. The GC-richness and the presence of CpG islands associated with tandem repeats are common features of imprinted genes and thus may play a role in the imprinting mechanism.

Animals↗
PubMed1996

Dynamic methylation adjustment and counting as part of imprinting mechanisms.

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.

Androgens↗