Relaxation Of IGF2 Imprinting Research Articles

Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2

BackgroundPorcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD) controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2).ResultsBisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele.ConclusionsOur results indicate that the IGF2 imprinting status is transcript-specific. Biallelic IGF2 expression in adult porcine liver and relaxation of IGF2 imprinting in porcine muscle were a common feature. These results were consistent with the IGF2 promoter P1 usage in adult liver and IGF2 promoter P2, P3 and P4 usages in muscle. The results showed further that bialellic IGF2 expression in liver and relaxation of imprinting in muscle and kidney were not associated with DNA methylation variation at and around at least one CTCF binding site in H19 DMD. The imprinting status in adult liver, muscle and kidney tissues were also not reflected in the methylation patterns of IGF2 DMRs 1 and 2.

Relaxation of Imprinting of Human Insulin-like Growth Factor II Gene,IGF2,in Sporadic Breast Carcinomas

Breast cancer is the most frequent malignancy in women and genetically heterogeneous, and a variety of genetic lesions have been identified that tend to accumulate during the disease progress. In breast cancer, loss of heterozygosity (LOH) has been described in the critical regions of chromosomes 11p15 and 11q22-23. Genomic imprinting is defined as gamete specific modification causing differential expression of the two alleles of a gene, in somatic cells. Human insulin like growth factor II gene (IGF2), located on chromosome 11p15, the same region on which LOH frequently occurred in breast cancer, has been recently identified as a genomic imprinting gene expressing preferentially paternal allele. To determine whether loss ofIGF2imprinting was common in breast cancer we studied 30 patients with sporadic breast carcinoma. A new strategy for detecting intragenicApaI polymorphism in the exon ofIGF2was used to examine allele-specific expression in the breast cancer specimens by reverse-transcription polymerase chain reaction (RT-PCR). Forty percent (12/30) of the breast cancer samples were identified as heterozygous forIGF2and studied further. Nine of the 12 heterozygous patients showed biallelic expression ofIGF2by cDNA-PCR, indicating relaxation of normal imprinting at this chromosomal locus. Conclusively, aberrant imprinting ofIGF2in 30% of the breast cancer patients tested provides strong evidence that pathological loss or relaxation ofIGF2imprinting plays an important role in either tumorigenesis or cytokine dysregulation for breast cancer cells.

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...

Loss of Imprinting of Human Insulin-like Growth Factor II Gene, IGF2, in Acute Myeloid Leukemia

Genomic imprinting is a non-Mendelian form of gene regulation resulting in differential allelic expression of a gene and plays an important role in the development of certain types of cancer and genetic diseases. Imprinting ofIGF2was demonstrated in normal placenta and fetal kidney by showing monoallelic paternal expression. In contrast, several kinds of tumor showed biallelic expression ofIGF2,suggesting that relaxation of the normal imprinting of this growth cytokine may be a feature of some tumors. In this study we wished to determine whether relaxation ofIGF2imprinting was also common in human leukemias. An intragenicApaI polymorphism within the 3′ untranslated region of the gene was used to examine allele-specific transcription in leukemic blasts derived from 24 primary patients with acute myeloid leukemia by RT-PCR. Leukemic samples from 12 of the 24 leukemia patients were identified as heterozygous forIGF2and potentially informative for the RT-PCR assay. RNA-PCR from these informative leukemia samples studied showed biallelic expression ofIGF2,indicating loss of normal imprinting of this gene to be very frequent. In conclusion, constitutional loss of monoallelic expression in 50% of the leukemia samples tested suggests that abnormal imprinting ofIGF2could play an important role in either leukemogenesis or cytokine dysregulation for leukemic cells.

Imprinted and Genotype-specific Expression of Genes at theIDDM2Locus in Pancreas and Leucocytes

One of the loci encoding susceptibility to insulin-dependent diabetes mellitus (IDDM) is IDDM2, mapped to a variable number of tandem repeats (VNTR) polymorphism situated 596 bp upstream of the insulin gene (INS). The shorter alleles (class I) predispose to IDDM, while the longer class III alleles are protective. Besides INS, it is possible that transcription levels of IGF2, the nearby gene encoding the insulin-like growth factor II, may be modulated by allelic forms of the VNTR. In an effort to define the pathophysiologic mechanism of the IDDM2 effect, we examined the effect, in cis, of VNTR genotype on steady-state mRNA levels of INS in samples of human fetal pancreas, and of IGF2 in leucocytes of diabetic children. Relative levels of mRNA transcripts derived from each chromosome carrying a defined VNTR allele were measured by RT-PCR, taking advantage of transcribed polymorphisms at the 3' untranslated region of each gene. In 10 samples of human fetal pancreas, INS transcripts from chromosomes carrying a class III VNTR were slightly but significantly (P = 0.015) lower than those from class I (13% lower, 95% confidence limits 3-21%). In 10 leucocyte samples, mRNA from both IGF2 alleles was seen, indicating relaxation of the parental imprinting of IGF2 in these cells. However, this relaxation was incomplete as maternal allele mRNA was systematically at a lower level than paternal. The paternal/maternal ratio varied widely among individual subjects. Two of the most extreme cases, demonstrating almost complete repression of the maternal allele, were identical twins, suggesting that this variable relaxation of imprinting is genotype-dependent. However, this genotype-dependence cannot be accounted for by the maternal VNTR, as the mean ratios of paternal/maternal IGF2 mRNA levels were not statistically different in individuals with a maternal VNTR of class I vs. class III (3.2 +/- 1.5 vs. 3.89 +/- 0.94). Thus, we present evidence that: (a) class III VNTR alleles are associated with lower INS mRNA in fetal pancreas than class I alleles. The biologic importance of this difference remains to be determined; and (b) the variable relaxation of IGF2 imprinting seen in human leucocytes is not dependent on the presence of a class I vs. a class III VNTR.

Wiedman-Beckwith Syndrome, Tumorigenesis and Imprinting

WBS is an overgrowth malformation syndrome characterized by highly variable expressivity, associated with predisposition to different types of pediatric tumors including Wilms' tumor (WT), adrenocortical carcinoma (ADCC), rhabdomyosarcoma (RMS) and hepatoblastoma (HEP). Most cases are sporadic, however 15% of the cases are familial. Cytogenetic, genetic and molecular analysis of the different forms of this syndrome and associated tumors have provided increasing evidence that the gene (or genes) map to 11p15.5 and that genomic imprinting can account for the strange genetics of this syndrome/tumors [1]. Two candidate genes, H19 and Igf2, which are both imprinted, but in opposite direction, map close to but not within one of the two smallest region(s) defined both by constitutional and tumoral rearrangements. These two genes, H19 and Igf2, and their specific parental imprint, may thus account for the pattern of inheritance observed, the variable expressivity, the specific loss of alleles and the loss of imprint. However, that these genes map 400 kb away from one cluster of breakpoints observed in the cytogenetic cases of WBS suggests that other genes could be involved. Indeed, although mapping to a different subregion, a sequence wit the properties of a tumor Suppressor (rhabdomyosarcoma cell line) has recently been isolated [2].Furthermore, neither reduplication of the active Igf2 paternal allele nor relaxation of Igf2 imprinting is sufficient for tumorigenesis, thus indicating that other mutation(s) must occur. The phenotypic consequences of these aberrant expressions will be better understood when the tissues, the stage of development or the state of differentiation are precisely identified [3-5].

Epigenetic changes encompassing the IGF2/H19 locus associated with relaxation of IGF2 imprinting and silencing of H19 in Wilms tumor.

In most tissues IGF2 is expressed from the paternal allele while H19 is expressed from the maternal allele. We have previously shown that in some Wilms tumors the maternal IGF2 imprint is relaxed such that the gene is expressed biallelically. We have now investigated this subset of tumors further and found that biallelic expression of IGF2 was associated with undetectable or very low levels of H19 expression. The relaxation of IGF2 imprinting in Wilms tumors also involved a concomitant reversal in the patterns of DNA methylation of the maternally inherited IGF2 and H19 alleles. Furthermore, the only specific methylation changes that occurred in tumors with relaxation of IGF2 imprinting were solely restricted to the maternal IGF2 and H19 alleles. These data suggest that there has been an acquisition of a paternal epigenotype in these tumors as the result of a pathologic disruption in the normal imprinting of the IGF2 and H19 genes.

13th Immunology of Diabetes Workshop

One of the loci encoding susceptibility to insulin-dependent diabetes mellitus (IDDM) isIDDM2, mapped to a variable number of tandem repeats (VNTR) polymorphism situated 596 bp upstream of the insulin gene (INS). The shorter alleles (class I) predispose to IDDM, while the longer class III alleles are protective. BesidesINS, it is possible that transcription levels ofIGF2, the nearby gene encoding the insulin-like growth factor II, may be modulated by allelic forms of the VNTR. In an effort to define the pathophysiologic mechanism of theIDDM2effect, we examined the effect, incis, of VNTR genotype on steady-state mRNA levels ofINSin samples of human fetal pancreas, and ofIGF2in leucocytes of diabetic children. Relative levels of mRNA transcripts derived from each chromosome carrying a defined VNTR allele were measured by RT-PCR, taking advantage of transcribed polymorphisms at the 3′ untranslated region of each gene. In 10 samples of human fetal pancreas,INStranscripts from chromosomes carrying a class III VNTR were slightly but significantly (P=0.015) lower than those from class I (13% lower, 95% confidence limits 3–21%). In 10 leucocyte samples, mRNA from bothIGF2alleles was seen, indicating relaxation of the parental imprinting ofIGF2in these cells. However, this relaxation was incomplete as maternal allele mRNA was systematically at a lower level than paternal. The paternal/maternal ratio varied widely among individual subjects. Two of the most extreme cases, demonstrating almost complete repression of the maternal allele, were identical twins, suggesting that this variable relaxation of imprinting is genotype-dependent. However, this genotype-dependence cannot be accounted for by the maternal VNTR, as the mean ratios of paternal/maternalIGF2mRNA levels were not statistically different in individuals with a maternal VNTR of class I vs. class III (3.2±1.5 vs. 3.89±0.94). Thus, we present evidence that: (a) class III VNTR alleles are associated with lowerINSmRNA in fetal pancreas than class I alleles. The biologic importance of this difference remains to be determined; and (b) the variable relaxation ofIGF2imprinting seen in human leucocytes is not dependent on the presence of a class I vs. a class III VNTR.