Epigenetics, genomic imprinting and assisted reproductive technology

Epigenetic mechanisms play a key role in regulating gene expression. One hallmark of these modifications is DNA methylation at cytosine residues of CpG dinucleotides in gene promoters, transposons and imprinting control regions. Genomic imprinting refers to an epigenetic marking of genes that results in monoallelic expression depending on their parental origin. There are two critical time periods in epigenetic reprogramming: gametogenesis and early preimplantation development. Major reprogramming takes place in primordial germ cells in which parental imprints are erased and totipotency is restored [1]. Imprint marks are then and re-established during spermatogenesis or oogenesis, depending on sex [1], [2] and [3]. Upon fertilization, genome-wide demethylation occurs followed by a wave of de novo methylation, both of which are resisted by imprinted loci [4]. Epigenetic patterns are usually faithfully maintained during development. However, this maintenance sometimes fails, resulting in the disturbance of epigenetic patterns and human disorders. For example, two fetal growth disorders, the Beckwith-Wiedemann (BWS) and the Silver-Russell (SRS) syndromes with opposite phenotypes, are caused by abnormal DNA methylation at the 11p15 imprinted locus [5], [6] and [7]: respectively loss of methylation at the Imprinting Region Center (ICR2) or gain of methylation at ICR1 in BWS and loss of methylation at ICR1 in SRS. Early embryogenesis is a critical time for epigenetic regulation, and this process is sensitive to environmental factors. The use of assisted reproductive technology (ART) has been shown to induce epigenetic alterations and to affect fetal growth and development [8], [9], [10] and [11]. In humans, several imprinting disorders, including BWS, occur at significantly higher frequencies in children conceived with the use of ART than in children conceived spontaneously [12] and [13]. The cause of these epigenetic imprinting disorders (following ART, unfertility causes, hormonal hyperstimulation, in vitro fertilization-IVF, Intracytoplasmic sperm injection-ICSI, micro-manipulation of gametes, exposure to culture medium, in vitro ovocyte maturation, time of transfer) remains unclear. However, recent data have shown that in patients with BWS or SRS, including those born following the use of ART, the DNA methylation defect involves imprinted loci other than 11p15 [14] and [15] (11p15 region: CTCF binding sites at ICR1, H19 and IGF2 DMRs, KCNQ1OT1 [ICR2], SNRPN [chromosome 15 q11-13], PEG/MEST1 [chromosome 7q31], IGF type2 receptor and ZAC1 [chromosome 6q26 et 6q24 respectively], DLK1/GTL2-IG-DMR [chromosome 14q32] and GNAS locus [chromosome 20q13.3]). This suggests that unfaithful maintenance of DNA methylation marks following fertilization involves the dysregulation of a trans-acting regulatory factor that could be altered by ART.     

Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms' tumor

We have analyzed several cases of Beckwith-Wiedemann syndrome (BWS) with Wilms' tumor in a familial setting, which give insight into the complex controls of imprinting and gene expression in the chromosome 11p15 region. We describe a 2.2-kbp microdeletion in the H19/insulin-like growth factor 2 (IGF2)-imprinting center eliminating three target sites of the chromatin insulator protein CTCF that we believe here is necessary, but not sufficient, to cause BWS and Wilms' tumor. Maternal inheritance of the deletion is associated with IGF2 loss of imprinting and up-regulation of IGF2 mRNA. However, in at least one affected family member a second genetic lesion (a duplication of maternal 11p15) was identified and accompanied by a further increase in IGF2 mRNA levels 35-fold higher than control values. Our results suggest that the combined effects of the H19/IGF2-imprinting center microdeletion and 11p15 chromosome duplication were necessary for manifestation of BWS.     

Insulin-like growth factor system on adrenocortical tumorigenesis

The insulin-like growth factor (IGF) signaling pathway has many important roles in normal cell growth and development. Remarkably, all of the components of this system (IGFs, receptors, and binding proteins) are expressed in human fetal adrenals. Beckwith-Wiedemann syndrome, a congenital overgrowth disorder characterized by a high risk of development of childhood tumors, is also distinguished by a high incidence of adrenocortical carcinomas. This disease has been associated with structural abnormalities at the 11p15 locus, which harbors the IGF2 gene as well as the genes coding for insulin, H19, and p57kip2. Notably, rearrangements at the 11p15 locus and overexpression of IGF2 were also described in sporadic adrenocortical tumors. In addition, the IGF2 overexpression was exclusively demonstrated in adults with adrenocortical tumors as a frequent feature of the malignant state. More recent studies demonstrated that the interaction of IGF-2 with IGF receptor type 1 (IGF-1R) plays also a pivotal role in adrenocortical tumorigenesis. IGF1R expression levels were significantly higher in pediatric adrenocortical carcinomas, suggesting that IGF1R expression represents a potential prognostic marker in this group of patients. These findings indicate that the IGF system is an important pathway for autonomous growth of adrenocortical cells and potential inhibitors of this system could be a rational therapeutic target for adrenocortical tumors.     

Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15.

Parental origin-specific alterations of chromosome 11p15 in human cancer suggest the involvement of one or more maternally expressed imprinted genes involved in embryonal tumor suppression and the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). The gene encoding cyclin-dependent kinase inhibitor p57KIP2, whose overexpression causes G1 phase arrest, was recently cloned and mapped to this band. We find that the p57KIP2 gene is imprinted, with preferential expression of the maternal allele. However, the imprint is not absolute, as the paternal allele is also expressed at low levels in most tissues, and at levels comparable to the maternal allele in fetal brain and some embryonal tumors. The biochemical function, chromosomal location, and imprinting of the p57KIP2 gene match the properties predicted for a tumor suppressor gene at 11p15.5. However, as the p57KIP2 gene is 500 kb centromeric to the gene encoding insulin-like growth factor 2, it is likely to be part of a large domain containing other imprinted genes. Thus, loss of heterozygosity or loss of imprinting might simultaneously affect several genes at this locus that together contribute to tumor and/or growth- suppressing functions that are disrupted in BWS and embryonal tumors.     

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.     

Limited evolutionary conservation of imprinting in the human placenta

The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.     

IGF-II serum levels are normal in children with Silver-Russell syndrome who frequently carry epimutations at the IGF2 locus

CONTEXT: Epigenetic mutations of 11p15 encompassing IGF2 are present in short children with Silver-Russell syndrome (SRS) with high frequency (31-50%). It has been speculated that these mutations characterized by demethylation of ICR1 cause diminished IGF2 expression. OBJECTIVE: We aimed to determine the prevalence of pathologically low IGF-II serum levels in children with SRS. SUBJECTS: SRS was defined by birth weight or length below the 3rd percentile, lack of postnatal catch-up growth, and the presence of two of the following characteristics: typical face, relative macrocephaly, and skeletal asymmetry. Serum samples of 30 patients were available. Mean age was 5.4 +/- 2.1 yr. METHODS: The serum levels of IGF-I, IGF-II, IGF binding protein (IGFBP)-2, and IGFBP-3 were measured by RIA and compared with age-related reference values and with serum concentrations measured in age- and gender-matched controls born small for gestational age (SGA), but lacking major dysmorphic features. Analysis of genomic DNA was possible in a subgroup of children with SRS: the methylation status of the ICR1 locus on 11p15 and the parental origin of chromosome 7 were analyzed in 9 and 23 children, respectively. RESULTS: Demethylation of ICR1 was found in 44% and uniparental disomy in 17% of the tested children with SRS. The median IGF-II serum level in SRS was 441 microg/liter (range, 238-875). This was significantly higher than in the SGA controls: 387 microg/liter (range, 265-596) (P < 0.03), but below the median value of the age-related reference, which was 532 microg/liter. The four children with SRS and ICR1 demethylation had high-normal and normal IGF-II serum levels that were higher than the levels of their SGA controls. IGF-I, IGFBP-2, and IGFBP-3 serum levels were not different between the SRS children and their SGA controls. CONCLUSIONS: Our data render it unlikely that demethylation of ICR1 on 11p15 does cause diminished IGF-II serum levels in children with SRS. This observation does not exclude deficient IGF-II action before birth.     

Loss of genomic imprinting of insulin-like growth factor 2 is strongly associated with cellular proliferation in normal hematopoietic cells

OBJECTIVE: The human insulin-like growth factor 2 (IGF2) gene was thought to be imprinted and expressed only from the paternal allele in normal tissue. MATERIALS AND METHODS: Initially, we analyzed the imprinting status of IGF2 in bone marrow cells from 49 patients with myelodysplastic syndromes (MDS) utilizing the Apa I polymorphism of IGF2. Thirteen bone marrow and 14 peripheral blood samples from normal individuals served as controls. We utilized normal peripheral blood T lymphocytes to examine the relationship between genomic imprinting and cell proliferation. Expression of IGF2 was quantified by real-time PCR and proliferation of T cells was measured by 3H-thymidine incorporation. Furthermore, methylation status of the imprinting controlling region (ICR) was analyzed by subcloning and sequencing of genomic DNA after sodium bisulfite modification. RESULTS: Among 24 patients who were heterozygous for IGF2, loss of imprinting (LOI) occurred in 22 cases (92%). Surprisingly, LOI of IGF2 occurred in the normal bone marrow cells, but the normal peripheral blood cells showed retention of imprinting (ROI). Unstimulated normal T cells showed ROI. After 24 hours of exposure to PHA, these cells changed their IGF2 imprinting status from ROI to LOI. Concomitantly, their IGF2 RNA levels increased up to sixfold and their proliferation increased 10- to 20-fold. In contrast, normal T cells not stimulated with PHA did not develop LOI of IGF2, had negligible levels of IGF2 RNA, and did not increase their proliferation. In unstimulated T cells, the CpG islands of the ICR were completely methylated on one allele and nearly completely unmethylated on the other allele. After PHA stimulation, the CpG islands at the ICR became completely methylated on both alleles. CONCLUSION: LOI of IGF2 is strongly associated with cell proliferation and is not limited to cancer cells.     

Monoallelic expression and methylation of imprinted genes in human and mouse embryonic germ cell lineages

Imprinting is an epigenetic modification leading to monoallelic expression of some genes, and disrupted imprinting is believed to be a barrier to human stem cell transplantation, based on studies that suggest that epigenetic marks are unstable in mouse embryonic germ (EG) and embryonic stem (ES) cells. However, stem cell imprinting has not previously been examined directly in humans. We found that three imprinted genes, TSSC5, H19, and SNRPN, show monoallelic expression in in vitro differentiated human EG-derived cells, and a fourth gene, IGF2, shows partially relaxed imprinting at a ratio from 4:1 to 5:1, comparable to that found in normal somatic cells. In addition, we found normal methylation of an imprinting control region (ICR) that regulates H19 and IGF2 imprinting, suggesting that imprinting may not be a significant epigenetic barrier to human EG cell transplantation. Finally, we were able to construct an in vitro mouse model of genomic imprinting, by generating EG cells from 8.5-day embryos of an interspecific cross, in which undifferentiated cells show biallelic expression and acquire preferential parental allele expression after differentiation. This model should allow experimental manipulation of epigenetic modifications of cultured EG cells that may not be possible in human stem cell studies.     

A novel deletion of the MEN1 gene in a large family of multiple endocrine neoplasia type 1 (MEN1) with aggressive phenotype

Context The MEN1 syndrome is associated with parathyroid, pancreatic and pituitary tumours and is caused by mutations in the MEN1 gene. In general, there is no genotype–phenotype correlation. Objectives To characterize a large family with MEN1 with aggressive tumour behaviour: malignant pancreatic endocrine tumours were present in five affected subjects and were the presenting features in three subjects. Design The coding region of MEN1 was sequenced. Gene copy number analysis was performed by multiplex ligation‐dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH). Loss of heterozygosity (LOH) in tumour tissue was studied by microsatellite analysis. Insulin‐like growth factor II (IGF‐II) and CDKN1C/p57KIP2 expression were investigated by immunohistochemistry. Results Mutation screening by conventional PCR sequence analysis of patients’ peripheral blood DNA did not reveal any mutation in the MEN1 or CDKN1B gene. Gene copy number analysis by MLPA and aCGH demonstrated a novel monoallelic deletion of 5 kb genomic DNA involving the MEN1 promoter and exons 1 and 2. LOH analysis indicated somatic deletion of maternal chromosome 11, including MEN1 locus (11q13) and 11p15 imprinting control regions (ICR). Methylation analysis of ICR demonstrated ICR1 hypermethylation and ICR2 hypomethylation in the tumour specimens. ICR1 and ICR2 control the expression of IGF‐2 and CDKN1C/p57KIP2, respectively. Immunohistochemistry showed that expression of paternally expressed IGF‐2 was up‐regulated and the maternally expressed CDKN1C/p57KIP2 was lost in the pancreatic endocrine tumours. Conclusions Gene copy number analysis by MLPA should be considered in patients with negative conventional mutation screening. Although large MEN1 deletion causes MEN1, disruption of imprinted CDKN1C/p57KIP2 and IGF‐2 gene expression may contribute to tumour progression and aggressive phenotype.     

Effects of environmental temperature on IGF1, IGF2, and IGF type I receptor expression in rainbow trout (Oncorhynchus mykiss)

Recently, we have demonstrated in rainbow trout that environmental temperature may, independently of nutritional status, directly stimulate plasma growth hormone (GH) that is recognised as being an insulin-like growth factor (IGF) system regulator. The aim of this study was to determine whether temperature may directly regulate the IGF system or indirectly regulate it through plasma GH or nutritional status. For this purpose, rainbow trout were reared at 8, 12, or 16 degrees C and fed either ad libitum (similar nutritional status) to evidence the global effect of temperature, or with the same ration (1.2% body weight/day), to determine the temperature effect in fish with the same growth rate. Endocrine and autocrine/paracrine regulations of the IGF system were determined by measuring plasma IGF1 and IGF2, liver and muscle IGF1 and IGF2 mRNA as well as IGFRIa, IGFRIb mRNA, and the quantity of IGF type I receptor in muscle. Our results show that neither rearing temperature nor the nutritional status of fish affected the expression of both IGF receptor genes in muscle. Nevertheless, the quantity of IGF type I receptor determined by a binding study, appeared to be inversely proportional (P<0.05) to the rearing temperature without any relationship with nutritional status, suggesting a direct effect of temperature on its turnover. After 2 weeks of treatment, the levels of IGF1 mRNA in muscle at 8 degrees C were 2-fold higher (P<0.05) than at 16 degrees C in both ad libitum and restricted feed fish, whereas after 6 weeks, this difference was no longer observed. In both experiments, the levels of plasma IGF2 were 10-fold higher than the levels of plasma IGF1 (mean 105+/-3.0 versus 13.5+/-0.6 ng/ml), and plasma levels were correlated with their respective mRNA liver concentrations (r2=0.14 and 0.25, respectively; P<0.01). In the ad libitum feeding experiment, plasma and mRNA levels of IGF1 were related to the rearing temperature (P<0.05), while for IGF2 no effect was seen. In contrast, in the restricted feeding experiment, plasma and IGF2 mRNA levels were inversely proportional to the rearing temperature (P<0.0001) while plasma IGF1 was unaltered. Levels of plasma IGF1 were related to the growth rate in both experiments, while levels of plasma IGF2 appeared to be associated with the nutritional status of the fish. Our results suggest that the autocrine/paracrine expression of IGF1 and IGF2 in muscle is not a key regulator of the growth promoting effect of temperature. Conversely, temperature seems to promote growth through IGF1 secretion by the liver following GH stimulation, and impairment of nutritional status would prevent the IGF1 stimulation by temperature. In addition, the growth-promoting effect of temperature did not affect plasma IGF2, which appeared to be more related to the metabolic status of the fish.     

Silver-Russell syndrome

The Silver-Russell syndrome (SRS) is a sporadic clinically and genetically heterogeneous disorder. Diagnosis is based on the variable combination of the following characteristics: intrauterine growth retardation, short stature because of lack of catch-up growth, underweight, relative macrocephaly, typical triangular face, body asymmetry and several minor anomalies including clinodactyly V. Different diagnostic scores have been proposed. The main genetic defects detected are at the epigenetic level: hypomethylation of the imprinting control region 1 (ICR1) on 11p15 in around 44% of cases and maternal uniparental disomy of chromosome 7 (UPD(7)mat) in 5-10% of cases. Severe phenotype is frequently associated with hypomethylation of ICR1 while mild phenotype is more often seen in combination with UPD(7)mat. Origins and biological consequences of these epimutations are still obscure. For genetic testing, we recommend a methylation-specific PCR-approach for both 7p and 7q loci (confirmed by microsatellite typing) for the detection of UPD(7)mat, and the methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) approach for methylation analysis of the 11p15 loci. Short stature in SRS can be treated by use of pharmacological doses of recombinant GH resulting in good short-term catch-up; sufficient information on the therapeutic effect in terms of final height is still missing.