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1.  Hypomethylation of the IGF2 DMR in Colorectal Tumors, Detected by Bisulfite Pyrosequencing, is Associated with Poor Prognosis 
Gastroenterology  2010;139(6):1855-1864.
Background & Aims
The insulin-like growth factor 2 (IGF2) gene is normally imprinted. Constitutive loss of imprinting (LOI) of IGF2 has been associated with increased risks of colon cancer and adenoma, indicating its role in carcinogenesis. The conventional LOI assay relies on a germline polymorphism to distinguish between 2 allelic expression patterns but results in many uninformative cases. IGF2 LOI correlates with hypomethylation at the differentially methylated region (DMR)-0. An assay for methylation of the DMR0 could overcome the limitations of the conventional IGF2 LOI assay.
We measured methylation at the IGF2 DMR0 using a bisulfite-pyrosequencing assay with 1178 paraffin-embedded colorectal cancer tissue samples from 2 prospective cohort studies. A Cox proportional hazard model was used to calculate mortality hazard ratio (HR); calculations were adjusted for microsatellite instability, the CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF, and PIK3CA mutations.
Methylation at the IGF2 DMR0 was successfully measured in 1105 (94%) of 1178 samples. Colorectal tumors had significantly less methylation at the DMR0 compared to matched, normal colonic mucosa (P<0.0001; N=51). Among 1033 patients eligible for survival analysis, hypomethylation of the IGF2 DMR0 was significantly associated with higher overall mortality (log-rank P=0.0006; univariate HR=1.41, 95% confidence interval [CI]: 1.16–1.71, P=0.0006; multivariate HR=1.33, 95% CI: 1.08–1.63, P=0.0066).
A bisulfite-pyrosequencing assay to measure methylation of the IGF2 DMR0 is robust and applicable to paraffin-embedded tissue. IGF2 DMR0 hypomethylation in colorectal tumor samples is associated with shorter survival time, so it might be developed as a prognostic biomarker.
PMCID: PMC2995815  PMID: 20682317
epigenetics; clinical outcome; therapeutic target; imprinting control region
2.  Loss of imprinting and marked gene elevation are two forms of aberrant IGF2 expression in colorectal cancer 
Loss of imprinting (LOI) of IGF2 is a common event in many cancers and typically activates the maternally silenced allele. The resulting biallelic IGF2 expression correlates strongly with the hypomethylation of a differentially methylated region (DMR) near its promoter. It has also been shown that IGF2 undergoes overexpression in human malignancies; nevertheless, this phenomenon and its link to aberrant DMR methylation has not been reported in colorectal cancer (CRC). The aim of this study was to determine the relationship between IGF2 LOI, overexpression and DMR hypomethylation in CRC. By analyzing IGF2 and H19 methylation in 97 primary CRC and 64 matched normal colorectal tissues, we have shown a significant correlation between IGF2 LOI and DMR hypomethylation of IGF2 and H19. Additionally, when analyzing Affymetrix expression data of 167 primary CRC tumor and 32 normal tissues, 15% of tumors showed marked IGF2 elevation. We further investigated if substantially elevated IGF2 levels were linked to IGF2 or H19 hypomethylation, but found no significant correlation. However, we demonstrated that noticeable IGF2 overexpression, rather than LOI, negatively correlated with CRC microsatellite instability. These observations indicate that IGF2 expression, particularly when transcribed at significantly high levels, is a result of mechanisms unrelated to LOI. Our results suggest that IGF2 participates in CRC tumorigenesis through two different forms of aberrant gene expression.
PMCID: PMC3270092  PMID: 19957330
CpG methylation; LDR; colorectal cancer; IGF2; imprinting
3.  Distinct Methylation Changes at the IGF2-H19 Locus in Congenital Growth Disorders and Cancer 
PLoS ONE  2008;3(3):e1849.
Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.
Methodology/Principal Findings
We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.
These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.
PMCID: PMC2268001  PMID: 18365005
4.  Insulin-like Growth Factor-2 (IGF2) Loss of Imprinting Marks a Field Defect Within Human Prostates Containing Cancer 
The Prostate  2011;71(15):10.1002/pros.21379.
Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for IGF2, a paracrine growth factor, has been implicated in the development of prostate and other cancers. In the current study, we define IGF2 LOI in histologically normal prostate tissues in relationship to tumor foci and gene expression.
Microdissected tumor associated (TA) adjacent (2 mm) and distant (10 mm) tissues surrounding tumor foci were generated. IGF2 imprinting in informative prostate tissue sets was quantitated using a fluorescent primer extension assay and expression analyzed utilizing quantitative PCR. DNA methylation analyses were performed using quantitative pyrosequencing.
A marked IGF2 LOI was found in adjacent TA tissues (39 ± 3.1%) and did not significantly decrease in tissues distant (38 ± 5.3%) from tumor foci (45 ± 2.9%; P = 0.21). IGF2 imprinting correlated with IGF2 expression in TA tissues, but not within the tumor foci. Hypomethylation of the IGF2 DMR0 region correlated with decreased IGF2 expression in tumors (P < 0.01). The expression of IGF2 and its adjacent imprinted gene H19 were increased in adjacent and distant tissues compared to tumors (P < 0.05) indicating the importance of factors other than LOI in driving IGF2 expression.
LOI of IGF2 occurs not only adjacent to prostate tumor foci, but is widely prevalent even in distant areas within the peripheral zone. These data provide evidence for a widespread epigenetic field defect in histologically normal tissues that might be employed to identify prostate cancer in patients.
PMCID: PMC3825178  PMID: 21432864
field effect; IGF2; H19; prostate cancer
5.  IGF2 differentially methylated region hypomethylation in relation to pathological and molecular features of serrated lesions 
World Journal of Gastroenterology : WJG  2014;20(29):10050-10061.
AIM: To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation in relation to clinicopathological and molecular features in colorectal serrated lesions.
METHODS: To accurately analyze the association between the histological types and molecular features of each type of serrated lesion, we consecutively collected 1386 formalin-fixed paraffin-embedded tissue specimens that comprised all histological types [hyperplastic polyps (HPs, n = 121), sessile serrated adenomas (SSAs, n = 132), traditional serrated adenomas (TSAs, n = 111), non-serrated adenomas (n = 195), and colorectal cancers (CRCs, n = 827)]. We evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1 (LINE-1) in HPs (n = 115), SSAs (n = 120), SSAs with cytological dysplasia (n = 10), TSAs (n = 91), TSAs with high-grade dysplasia (HGD) (n = 15), non-serrated adenomas (n = 80), non-serrated adenomas with HGD (n = 105), and CRCs (n = 794). For the accurate quantification of the relative methylation levels (scale 0%-100%) of IGF2 DMR0 and LINE-1, we used bisulfite pyrosequencing method. Tumor specimens were analyzed for microsatellite instability, KRAS (codons 12 and 13), BRAF (V600E), and PIK3CA (exons 9 and 20) mutations; MLH1 and MGMT methylation; and IGF2 expression by immunohistochemistry.
RESULTS: The distribution of the IGF2 DMR0 methylation level in 351 serrated lesions and 185 non-serrated adenomas (with or without HGD) was as follows: mean 61.7, median 62.5, SD 18.0, range 5.0-99.0, interquartile range 49.5-74.4. The IGF2 DMR0 methylation level was divided into quartiles (Q1 ≥ 74.5, Q2 62.6-74.4, Q3 49.6-62.5, Q4 ≤ 49.5) for further analysis. With regard to the histological type, the IGF2 DMR0 methylation levels of SSAs (mean ± SD, 73.1 ± 12.3) were significantly higher than those of HPs (61.9 ± 20.5), TSAs (61.6 ± 19.6), and non-serrated adenomas (59.0 ± 15.8) (P < 0.0001). The IGF2 DMR0 methylation level was inversely correlated with the IGF2 expression level (r = -0.21, P = 0.0051). IGF2 DMR0 hypomethylation was less frequently detected in SSAs compared with HPs, TSAs, and non-serrated adenomas (P < 0.0001). Multivariate logistic regression analysis also showed that IGF2 DMR0 hypomethylation was inversely associated with SSAs (P < 0.0001). The methylation levels of IGF2 DMR0 and LINE-1 in TSAs with HGD (50.2 ± 18.7 and 55.7 ± 5.4, respectively) were significantly lower than those in TSAs (61.6 ± 19.6 and 58.8 ± 4.7, respectively) (IGF2 DMR0, P = 0.038; LINE-1, P = 0.024).
CONCLUSION: IGF2 DMR0 hypomethylation may be an infrequent epigenetic alteration in the SSA pathway. Hypomethylation of IGF2 DMR0 and LINE-1 may play a role in TSA pathway progression.
PMCID: PMC4123334  PMID: 25110432
BRAF; Colon polyp; Colorectal neoplasia; Colorectum; Genome; Insulin-like growth factor; Long interspersed nucleotide element-1; Microsatellite instability; Serrated pathway
6.  Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2 
BMC Genetics  2011;12:47.
Porcine 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).
Bisulfite 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.
Our 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.
PMCID: PMC3112248  PMID: 21575277
7.  Epigenetic alteration at the DLK1-GTL2 imprinted domain in human neoplasia: analysis of neuroblastoma, phaeochromocytoma and Wilms' tumour 
British Journal of Cancer  2005;92(8):1574-1580.
Epigenetic alterations in the 11p15.5 imprinted gene cluster are frequent in human cancers and are associated with disordered imprinting of insulin-like growth factor (IGF)2 and H19. Recently, an imprinted gene cluster at 14q32 has been defined and includes two closely linked but reciprocally imprinted genes, DLK1 and GTL2, that have similarities to IGF2 and H19, respectively. Both GTL2 and H19 are maternally expressed RNAs with no protein product and display paternal allele promoter region methylation, and DLK1 and IGF2 are both paternally expressed. To determine whether methylation alterations within the 14q32 imprinted domain occur in human tumorigenesis, we investigated the status of the GTL2 promoter differentially methylated region (DMR) in 20 neuroblastoma tumours, 20 phaeochromocytomas and, 40 Wilms' tumours. Hypermethylation of the GTL2 promoter DMR was detected in 25% of neuroblastomas, 10% of phaeochromocytoma and 2.5% of Wilms' tumours. Tumours with GTL2 promoter DMR hypermethylation also demonstrated hypermethylation at an upstream intergenic DMR thought to represent a germline imprinting control element. Analysis of neuroblastoma cell lines revealed that GTL2 DMR hypermethylation was associated with transcriptional repression of GTL2. These epigenetic findings are similar to those reported in Wilms' tumours in which H19 repression and DMR hypermethylation is associated with loss of imprinting (LOI, biallelic expression) of IGF2. However, a neuroblastoma cell line with hypermethylation of the GTL2 promoter and intergenic DMR did not show LOI of DLK1 and although treatment with a demethylating agent restored GTL2 expression and reduced DLK1 expression. As described for IGF2/H19, epigenetic changes at DLK1/GTL2 occur in human cancers. However, these changes are not associated with DLK1 LOI highlighting differences in the imprinting control mechanisms operating in the IGF2-H19 and DLK1-GTL2 domains. GTL2 promoter and intergenic DMR hypermethylation is associated with the loss of GTL2 expression and this may contribute to tumorigenesis in a subset of human cancers.
PMCID: PMC2362015  PMID: 15798773
imprinting; DLK1; GTL2; methylation
8.  Comprehensive analyses of imprinted differentially methylated regions reveal epigenetic and genetic characteristics in hepatoblastoma 
BMC Cancer  2013;13:608.
Aberrant methylation at imprinted differentially methylated regions (DMRs) in human 11p15.5 has been reported in many tumors including hepatoblastoma. However, the methylation status of imprinted DMRs in imprinted loci scattered through the human genome has not been analyzed yet in any tumors.
The methylation statuses of 33 imprinted DMRs were analyzed in 12 hepatoblastomas and adjacent normal liver tissue by MALDI-TOF MS and pyrosequencing. Uniparental disomy (UPD) and copy number abnormalities were investigated with DNA polymorphisms.
Among 33 DMRs analyzed, 18 showed aberrant methylation in at least 1 tumor. There was large deviation in the incidence of aberrant methylation among the DMRs. KvDMR1 and IGF2-DMR0 were the most frequently hypomethylated DMRs. INPP5Fv2-DMR and RB1-DMR were hypermethylated with high frequencies. Hypomethylation was observed at certain DMRs not only in tumors but also in a small number of adjacent histologically normal liver tissue, whereas hypermethylation was observed only in tumor samples. The methylation levels of long interspersed nuclear element-1 (LINE-1) did not show large differences between tumor tissue and normal liver controls. Chromosomal abnormalities were also found in some tumors. 11p15.5 and 20q13.3 loci showed the frequent occurrence of both genetic and epigenetic alterations.
Our analyses revealed tumor-specific aberrant hypermethylation at some imprinted DMRs in 12 hepatoblastomas with additional suggestion for the possibility of hypomethylation prior to tumor development. Some loci showed both genetic and epigenetic alterations with high frequencies. These findings will aid in understanding the development of hepatoblastoma.
PMCID: PMC3880457  PMID: 24373183
Hepatoblastoma; Genomic imprinting; Differentially methylated region; DNA methylation
9.  Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome 
Journal of Medical Genetics  2000;37(12):921-926.
Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events involving imprinted genes at chromosome 11p15.5. Thus, germline mutations in CDKN1C, uniparental disomy (UPD), and loss of imprinting of IGF2 and other imprinted genes have been implicated. Many familial BWS cases have germline CDKN1C mutations. However, most BWS cases are sporadic and UPD or putative imprinting errors predominate in this group. We have identified previously a subgroup of sporadic cases with loss of imprinting (LOI) of IGF2 and epigenetic silencing of H19 proposed to be caused by a defect in a distal 11p15.5 imprinting control element (designated BWSIC1). However, many sporadic BWS patients show biallelic IGF2 expression in the presence of normal H19 methylation and expression patterns. This and other evidence suggested the existence of a further imprinting control element (BWSIC2) at 11p15.5. Recently, we showed that a subgroup of BWS patients have loss of methylation (LOM) at a differentially methylated region (KvDMR1) within the KCNQ1 gene centromeric to the IGF2 and H19 genes. We have now analysed a large series of sporadic cases to define the frequency and phenotypic correlates of epigenetic abnormalities in BWS. LOM at KvDMR1 was detected by Southern analysis or a novel PCR based method in 35 of 69 (51%) sporadic BWS without UPD. LOM at KvDMR1 was often, but not invariably associated with LOI of IGF2. KvDMR1 LOM was not detected in BWS patients with putative BWSIC1 defects and cases with KvDMR1 LOM (that is, putative BWSIC2 defects) invariably had a normal H19 methylation pattern. The incidence of exomphalos in putative BWSIC2 defect patients was not significantly different from that in patients with germline CDKN1C mutations (20/29 and 13/15 respectively), but was significantly greater than that in patients with putative BWSIC1 defects (0/5, p=0.007) and UPD (0/22, p<0.0001). These findings are consistent with the hypothesis that LOM of KvDMR1 (BWSIC2 defect) results in epigenetic silencing of CDKN1C and variable LOI of IGF2. BWS patients with embryonal tumours have UPD or a BWSIC1 defect but not LOM of KvDMR1. This study has further shown how (1) variations in phenotypic expression of BWS may be linked to specific molecular subgroups and (2) molecular analysis of BWS can provide insights into mechanisms of imprinting regulation.

Keywords: Beckwith-Wiedemann syndrome; epigenotype-phenotype correlations; imprinting
PMCID: PMC1734494  PMID: 11106355
10.  Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue 
Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR) which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG) densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes.
Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression). Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ-line and the somatic DMRs.
Our validated pyrosequencing methylation assays can be widely used as a tool to investigate DNA methylation levels of imprinted genes in clinical samples. This first comprehensive analysis of normal methylation levels in adult somatic tissues at human imprinted regions confirm that, despite intra-individual variability and tissue specific expression, imprinted genes faithfully maintain their DNA methylation in healthy adult tissue. DNA methylation levels of a selection of imprinted genes are, therefore, a valuable indicator for epigenetic stability.
PMCID: PMC3038880  PMID: 21281512
11.  Relaxed imprinting of IGF2 in peripheral blood cells of patients with a history of prostate cancer 
Endocrine Connections  2012;1(2):87-94.
Insulin-like growth factor 2 (IGF2) is the predominant IGF in adults and regulates cell growth. In contrast to normal tissues, where IGF2 is imprinted and only expressed from the paternal allele, loss of imprinting (LOI) and biallelic IGF2 expression are observed in many cancers including prostate cancer (PCa). We here studied whether LOI of IGF2 in normal circulating peripheral blood lymphocytes can predict increased PCa risk.
Samples and methods
We analyzed IGF2 protein levels, IGF2 820G/A genotype and imprinting status, as well as methylation status of the IGF2 imprinting control region (ICR) in 113 blood samples of patients with a history of radical prostatectomy (RPE) for PCa by ELISA, restriction-fragment length polymorphism, and bisulfite-DNA sequencing. Results were compared to 249 male blood donors with unknown prostate specific antigen (PSA) status.
The 820G/A genotype was enriched in the RPE group and was associated with younger age at cancer diagnosis. LOI in patients was only slightly more frequent than in controls, but IGF2 levels were significantly higher and uncoupled from the imprinting status. Analysis of the IGF2/H19 ICR revealed marked hypermethylation.
The IGF 820G/A genotype is associated with PCa diagnosis at younger age. Increased IGF2 in patients with PCa appears to be the result of impaired imprinting in non-neoplastic cells rather than a paracrine tumor product. Uncoupling of IGF2 protein levels from imprinting status (not LOI alone) and hypermethylation of the ICR characterized PCa patients and could have the potential to indicate persons at risk in screening programs.
PMCID: PMC3681323  PMID: 23781309
cancer; insulin-like growth factor 2; imprinting; prostate; screening
12.  Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma 
British Journal of Cancer  2008;99(11):1891-1899.
IGF2, a maternally imprinted foetal growth factor gene, is implicated in many childhood tumours including hepatoblastoma (HB); however, the genetic and epigenetic alterations have not comprehensively been studied. We analysed the methylation status of the H19 differentially methylated region (DMR), loss of heterozygosity (LOH) and allelic expression of IGF2 in 54 HB tumours, and found that 12 tumours (22%) with LOH, 9 (17%) with loss of imprinting (LOI) and 33 (61%) with retention of imprinting (ROI). Biallelic and monoallelic IGF2 expressions correlated with hypermethylation and normal methylation of H19 DMR, respectively, in two tumours with LOI and seven tumours with ROI. Quantitative RT–PCR analysis showed minimal expression of H19 mRNA and substantial expression of IGF2 mRNA in tumours with LOH or LOI, and substantial expression of both H19 and IGF2 mRNAs in tumours with ROI. Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers. In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB. Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins.
PMCID: PMC2600691  PMID: 19034281
hepatoblastoma; IGF2; H19; loss of heterozygosity; loss of imprinting
13.  High-throughput detection of aberrant imprint methylation in the ovarian cancer by the bisulphite PCR-Luminex method 
Aberrant DNA methylation leads to loss of heterozygosity (LOH) or loss of imprinting (LOI) as the first hit during human carcinogenesis. Recently we developed a new high-throughput, high-resolution DNA methylation analysis method, bisulphite PCR-Luminex (BPL), using sperm DNA and demonstrated the effectiveness of this novel approach in rapidly identifying methylation errors.
In the current study, we applied the BPL method to the analysis of DNA methylation for identification of prognostic panels of DNA methylation cancer biomarkers of imprinted genes. We found that the BPL method precisely quantified the methylation status of specific DNA regions in somatic cells. We found a higher frequency of LOI than LOH. LOI at IGF2, PEG1 and H19 were frequent alterations, with a tendency to show a more hypermethylated state. We detected changes in DNA methylation as an early event in ovarian cancer. The degree of LOI (LOH) was associated with altered DNA methylation at IGF2/H19 and PEG1.
The relative ease of BPL method provides a practical method for use within a clinical setting. We suggest that DNA methylation of H19 and PEG1 differentially methylated regions (DMRs) may provide novel biomarkers useful for screening, diagnosis and, potentially, for improving the clinical management of women with human ovarian cancer.
PMCID: PMC3342152  PMID: 22443985
Genomic imprinting; Ovarian cancer; DNA methylation; Bisulphite PCR-Luminex(BPL)method; LOI (loss of imprinting)
14.  Different Epigenetic Alterations Are Associated with Abnormal IGF2/Igf2 Upregulation in Neural Tube Defects 
PLoS ONE  2014;9(11):e113308.
The methylation status of DNA methylation regions (DMRs) of the imprinted gene IGF2/Igf2 is associated with neural tube defects (NTDs), which are caused by a failure of the neural tube to fold and close and are the second-most common birth defect; however, the characterization of the expression level of IGF2/Igf2 in neural tissue from human fetuses affected with NTDs remains elusive. More importantly, whether abnormal chromatin structure also influences IGF2/Igf2 expression in NTDs is unclear. Here, we investigated the transcriptional activity of IGF2/Igf2 in normal and NTD spinal cord tissues, the methylation status of different DMRs, and the chromatin structure of the promoter. Our data indicated that in NTD samples from both human fetuses and retinoic acid (RA)-treated mouse fetuses, the expression level of IGF2/Igf2 was upregulated 6.41-fold and 1.84-fold, respectively, compared to controls. H19 DMR1, but not IGF2 DMR0, was hypermethylated in human NTD samples. In NTD mice, h19 DMR1 was stable, whereas the chromatin structure around the promoter of Igf2 might be loosened, which was displayed by higher H3K4 acetylation and lower H3K27 trimethylation. Therefore, the data revealed that IGF2/Igf2 expression can be ectopically up-regulated by dual epigenetic factors in NTDs. In detail, the upregulation of IGF2/Igf2 is likely controlled by hypermethylation of H19 DMR1 in human NTDs, however, in acute external RA-induced NTD mice it is potentially determined by more open chromatin structure.
PMCID: PMC4244157  PMID: 25423083
15.  Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort 
BMC Medicine  2013;11:29.
Data from epidemiological and animal model studies suggest that nutrition during pregnancy may affect the health status of subsequent generations. These transgenerational effects are now being explained by disruptions at the level of the epigenetic machinery. Besides in vitro environmental exposures, the possible impact on the reprogramming of methylation profiles at imprinted genes at a much earlier time point, such as during spermatogenesis or oogenesis, has not previously been considered. In this study, our aim was to determine associations between preconceptional obesity and DNA methylation profiles in the offspring, particularly at the differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene.
We examined DNA from umbilical cord blood leukocytes from 79 newborns, born between July 2005 and November 2006 at Duke University Hospital, Durham, NC. Their mothers participated in the Newborn Epigenetics Study (NEST) during pregnancy. Parental characteristics were obtained via standardized questionnaires and medical records. DNA methylation patterns at two DMRs were analyzed by bisulfite pyrosequencing; one DMR upstream of IGF2 (IGF2 DMR), and one DMR upstream of the neighboring H19 gene (H19 DMR). Multiple regression models were used to determine potential associations between the offspring's DNA methylation patterns and parental obesity before conception. Obesity was defined as body mass index (BMI) ≥30 kg/m2.
Hypomethylation at the IGF2 DMR was associated with paternal obesity. Even after adjusting for several maternal and newborn characteristics, we observed a persistent inverse association between DNA methylation in the offspring and paternal obesity (β-coefficient was -5.28, P = 0.003). At the H19 DMR, no significant associations were detected between methylation patterns and paternal obesity. Our data suggest an increase in DNA methylation at the IGF2 and H19 DMRs among newborns from obese mothers, but a larger study is warranted to further explore the potential effects of maternal obesity or lifestyle on the offspring's epigenome.
While our small sample size is limited, our data indicate a preconceptional impact of paternal obesity on the reprogramming of imprint marks during spermatogenesis. Given the biological importance of imprinting fidelity, our study provides evidence for transgenerational effects of paternal obesity that may influence the offspring's future health status.
PMCID: PMC3584733  PMID: 23388414
Epigenetics; DNA methylation; IGF2; obesity; offspring; Newborn Epigenetics Study; Epidemiology
16.  Constitutional and somatic methylation status of DMRH19 and KvDMR in Wilms tumor patients 
Genetics and Molecular Biology  2012;35(4):714-724.
The most frequent epigenetic alterations in Wilms tumor (WT) occur at WT2, assigned to 11p15. WT2 consists of two domains: telomeric domain 1 (DMRH19) that contains the IGF2 gene and an imprinted maternally expressed transcript (H19) and centromeric domain 2 (KvDMR) that contains the genes KCNQ1, KCNQ1OT1 and CDKN1C. In this work, we used pyrosequencing and MS-MLPA to compare the methylation patterns of DMRH19/KvDMR in blood and tumor samples from 40 WT patients. Normal constitutional KvDMR methylation indicated that most of the epigenetic alterations in WT occur at DMRH19. Constitutional DMRH19 hypermethylation (HM DMRH19) was observed in two patients with Beckwith-Wiedemann syndrome. Pyrosequencing and MS-MLPA showed HM DMRH19 in 28/34 tumor samples: 16/34 with isolated HM DMRH19 and 12/34 with concomitant HM DMRH19 and KvDMR hypomethylation, indicating paternal uniparental disomy. With the exception of one blood sample, the MS-MLPA and pyrosequencing findings were concordant. Diffuse or focal anaplasia was present in five tumor samples and was associated with isolated somatic HM DMRH19 in four of them. Constitutional 11p15 methylation abnormalities were present in 5% of the samples and somatic abnormalities in the majority of tumors. Combined analysis of DMRH19/KvDMR by pyrosequencing and MS-MLPA is beneficial for characterizing epigenetic anomalies in WT, and MS-MLPA is useful and reliable for estimation of DNA methylation in a clinical setting.
PMCID: PMC3526076  PMID: 23271929
epigenetic; histopathology; methylation; MS-MLPA; pyrosequencing
17.  Loss of Imprinting of IGF2 as an Epigenetic Marker for the Risk of Human Cancer 
Disease Markers  2007;23(1-2):105-112.
IGF2 is the first gene discovered to be imprinted and expressed exclusively from the paternal allele in both human and mouse. IGF2 is also the first imprinted gene displaying loss of imprinting (LOI) or aberrant imprinting in human cancers. Evidently, LOI or reactivation of the maternal allele of IGF2 is associated with an increase of IGF2 expression that may subsequently play an important role in the onset of human cancers. The most important discovery was the association of LOI of IGF2 with the risk of developing human colorectal cancer. LOI occurs not only in colon cancer tissues, but also in matched normal tissues and peripheral blood cells. A pilot study indicated a significant relationship between LOI of IGF2 and family history as well as personal history of colorectal cancer, suggesting that LOI of IGF2 might be a valuable biomolecular marker of predicting an individual's risk for colon cancer. A recent epigenetic progenitor model suggested that human cancers might have a common basis that involves an epigenetic disruption of progenitor cells mediated by “tumor progenitor genes” and proposed that non-neoplastic but epigenetically disrupted progenitor cells might be an important target for cancer risk assessment and prevention.
PMCID: PMC3851053  PMID: 17325430
IGF2; loss of imprinting; cancer; biomarker
18.  Leukocyte DNA as Surrogate for the Evaluation of Imprinted Loci Methylation in Mammary Tissue DNA 
PLoS ONE  2013;8(2):e55896.
There is growing interest in identifying surrogate tissues to identify epimutations in cancer patients since primary target tissues are often difficult to obtain. Methylation patterns at imprinted loci are established during gametogenesis and post fertilization and their alterations have been associated with elevated risk of cancer. Methylation at several imprinted differentially methylated regions (GRB10 ICR, H19 ICR, KvDMR, SNRPN/SNURF ICR, IGF2 DMR0, and IGF2 DMR2) were analyzed in DNA from leukocytes and mammary tissue (normal, benign diseases, or malignant tumors) from 87 women with and without breast cancer (average age of cancer patients: 53; range: 31–77). Correlations between genomic variants and DNA methylation at the studied loci could not be assessed, making it impossible to exclude such effects. Methylation levels observed in leukocyte and mammary tissue DNA were close to the 50% expected for monoallellic methylation. While no correlation was observed between leukocyte and mammary tissue DNA methylation for most of the analyzed imprinted genes, Spearman's correlations were statistically significant for IGF2 DMR0 and IGF2 DMR2, although absolute methylation levels differed. Leukocyte DNA methylation levels of selected imprinted genes may therefore serve as surrogate markers of DNA methylation in cancer tissue.
PMCID: PMC3567003  PMID: 23409079
19.  Frequency and characterization of DNA methylation defects in children born SGA 
Various genes located at imprinted loci and regulated by epigenetic mechanisms are involved in the control of growth and differentiation. The broad phenotypic variability of imprinting disorders suggests that individuals with inborn errors of imprinting might remain undetected among patients born small for gestational age (SGA). We evaluated quantitative DNA methylation analysis at differentially methylated regions (DMRs) of 10 imprinted loci (PLAGL1, IGF2R DMR2, GRB10, H19 DMR, IGF2, MEG3, NDN, SNRPN, NESP, NESPAS) by bisulphite pyrosequencing in 98 patients born SGA and 50 controls. For IGF2R DMR2, methylation patterns of additional 47 parent pairs and one mother (95 individuals) of patients included in the SGA cohort were analyzed. In six out of 98 patients born SGA, we detected DNA methylation changes at single loci. In one child, the diagnosis of upd(14)mat syndrome owing to an epimutation of the MEG3 locus in 14q32 could be established. The remaining five patients showed hypomethylation at GRB10 (n=2), hypomethylation at the H19 3CTCF-binding site (n=1), hypermethylation at NDN (n=1) and hypermethylation at IGF2 (n=1). IGF2R DMR2 hypermethylation was detected in five patients, six parents of patients in the SGA cohort and two controls. We conclude that aberrant methylation at imprinted loci in children born SGA exists but seems to be rare if known imprinting syndromes are excluded. Further investigations on the physiological variations and the functional consequences of the detected aberrant methylation are necessary before final conclusions on the clinical impact can be drawn.
PMCID: PMC3722674  PMID: 23232699
IGF2R; imprinting; methylation; SGA; upd(14)mat
20.  The paternally imprinted DLK1-GTL2 locus is differentially methylated in embryonal and alveolar rhabdomyosarcomas 
International Journal of Oncology  2013;44(1):295-300.
Parental imprinting of differentially methylated regions (DMRs) contributes to appropriate expression of several developmentally important genes from paternally or maternally derived chromosomes. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is associated with altered expression of certain parentally imprinted genes. As previously reported, RMS cells display loss of imprinting (LOI) of the DMR at the IGF2-H19 locus, resulting in insulin-like growth factor 2 (IGF2) transcription from both paternally and maternally inherited chromosomes, and overall IGF2 overexpression. As the DLK1-GTL2 locus is structurally similar to the IGF2-H19 locus, the status of parental imprinting of the DLK1-GTL2 locus was studied in RMS. We observed that while both embryonal and alveolar rhabdomyosarcomas (ERMS and ARMS, respectively) show LOI of the DMR at the IGF2-H19 locus, imprinting of the DMR at the DLK1-GTL2 locus varies in association with the histological subtype of RMS. We found that, while ERMS tumors consistently show LOI of the DMR at the DLK1-GTL2 locus, ARMS tumors have erasure of imprinting (EOI) at this locus. These changes in imprinting status of the DLK1-GTL2 locus result in a higher GTL2/DLK1 mRNA ratio in ARMS as compared to ERMS. This difference in imprinting elucidates a novel genetic difference between these two RMS subtypes and may provide a potential diagnostic tool to distinguish between these subtypes.
PMCID: PMC3867365  PMID: 24173021
rhabdomyosarcoma; DLK1-GTL2 locus; genomic imprinting
21.  Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer 
The aim of this study was to determine the clinicopathological features of gastric cancers with loss of imprinting (LOI) of LIT1. Insulin-like growth factor 2 (IGF2) and H19 in Chinese patients.
DNA and RNA from tumours were amplified and then digested with RsaI, ApaI and HinfI, and RsaI respectively to determine the LOI status. The demographic and clinicopathological characteristics in LOI positive and LOI negative patients were compared and tested with Statistical analysis.
Of the 89 patients enrolled for analysis, 22, 40 and 35 were heterozygous and thus informative for LIT1, IGF2 and H19 LOI analyses respectively. The positive rate of LIT1, IGF2 and H19 LOI of gastric cancer tissues were 54.6% (12/22), 45% (18/40) and 8.6% (3/32) in Chinese patients. Gastric corpus cancer (8/10, 80%) were more likely to have LOI of IGF2 in tumours than antrum cancers (10/30, 33.3%){odds ratio (OR) = 8, 95% confidence intervals (CI) = 1.425-44.920, p = 0.018)}. LOI of IGF2 in tumours was also associated with the lymph node metastasis (LNM) (OR = 4.5, 95% CI = 1.084-18.689, p = 0.038).
IGF2 LOI is present in high frequency in Chinese gastric cancer patients, especially those with gastric corpus cancer.
PMCID: PMC2753348  PMID: 19737423
22.  Methylation levels at imprinting control regions are not altered with ovulation induction or in vitro fertilization in a birth cohort 
Human Reproduction (Oxford, England)  2012;27(7):2208-2216.
Do fertility treatments, including ovulation induction (OI), alter epigenetic mechanisms such as DNA methylation at imprinted loci?
We observed small but statistically significant differences in certain imprinting control regions (ICRs) based on the method of conception, however, these small changes in methylation did not correlate to the overall transcriptional levels of the genes adjacent to the ICRs (such as KCNQ1 and SNRPN).
Assisted reproductive technology (ART) has been associated with an increase in the risk of rare childhood disorders caused by loss of imprinting (LOI). This study provides novel epigenetic analyses on infants conceived by OI and examines how methylation levels correlate with gene expression.
Data and biospecimens used in this study were from 147 participants of the Epigenetic Birth Cohort comprising 1941 mother–child dyads recruited between June 2007 and June 2009 at the Department of Obstetrics, Gynecology and Reproductive Biology at Brigham and Women's Hospital (BWH) in Boston, MA, USA. Wilcoxon rank-sum tests were used to examine the differences in median percent methylation at each differentially methylated region (DMR) between the spontaneous conception control group and the fertility treatment groups (OI and IVF).
For each woman who reported IVF we selected a woman who conceived spontaneously matched on age (±2 years). To increase efficiency, we matched the same controls from the spontaneously conceived group to participants who reported OI. If an appropriate control was not identified that had been previously matched to an IVF participant, a new control was selected. The final analytic sample consisted of 61 spontaneous, 59 IVF and 27 OI conceptions.
No functionally relevant differences in methylation levels were observed across five (out of six) imprinted DMRs in either the placenta or cord blood of infants conceived with OI or IVF compared with infants conceived spontaneously. While KCNQ1, SNRPN and H19 DMRs demonstrated small but statistically significant differences in methylation based on the method of conception, expression levels of the genes related to these control regions only correlated with the methylation levels of H19.
Limitations of our study include the limited sample size, lack of information on OI medication used and culture medium for the IVF procedures and underlying reasons for infertility among OI and IVF patients. We did not perform allele-specific expression analyses and therefore cannot make any inferences about LOI.
These results are likely to be generalizable to non-Hispanic white individuals in populations with similar ART and fertility treatments.
This project was supported by the Milton Fund, Harvard University (P.I.: K.B.M) and by Public Health Research Grant 5R21CA128382 from the National Cancer Institute, National Institutes of Health (P.I.: K.B.M.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. No conflict of interests to declare.
Trial registration number
PMCID: PMC3376159  PMID: 22587996
ovulation induction; IVF; DNA methylation; genomic imprinting; fertility treatment
23.  Differentially Methylated Regions of Imprinted Genes in Prenatal, Perinatal and Postnatal Human Tissues 
PLoS ONE  2012;7(7):e40924.
Epigenetic plasticity in relation to in utero exposures may mechanistically explain observed differences in the likelihood of developing common complex diseases including hypertension, diabetes and cardiovascular disease through the cumulative effects of subtle alterations in gene expression. Imprinted genes are essential mediators of growth and development and are characterized by differentially methylated regulatory regions (DMRs) that carry parental allele-specific methylation profiles. This theoretical 50% level of methylation provides a baseline from which endogenously- or exogenously-induced deviations in methylation can be detected. We quantified DNA methylation at imprinted gene DMRs in a large panel of human conceptal tissues, in matched buccal cell specimens collected at birth and at one year of age, and in the major cell fractions of umbilical cord blood to assess the stability of methylation at these regions. DNA methylation was measured using validated pyrosequencing assays at seven DMRs regulating the IGF2/H19, DLK1/MEG3, MEST, NNAT and SGCE/PEG10 imprinted domains. DMR methylation did not significantly differ for the H19, MEST and SGCE/PEG10 DMRs across all conceptal tissues analyzed (ANOVA p>0.10). Methylation differences at several DMRs were observed in tissues from brain (IGF2 and MEG3-IG DMRs), liver (IGF2 and MEG3 DMRs) and placenta (both DLK1/MEG3 DMRs and NNAT DMR). In most infants, methylation profiles in buccal cells at birth and at one year of age were comparable, as was methylation in the major cell fractions of umbilical cord blood. Several infants showed temporal deviations in methylation at multiple DMRs. Similarity of inter-individual and intra-individual methylation at some, but not all of the DMRs analyzed supports the possibility that methylation of these regions can serve as useful biosensors of exposure.
PMCID: PMC3396645  PMID: 22808284
24.  Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues 
Epigenetics  2012;7(12):1355-1367.
Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.
PMCID: PMC3528691  PMID: 23079744
DNA methylation; colorectal cancer; biomarkers; H3K27me3; gene expression; Illumina sequencing
25.  No evidence for copy number and methylation variation in H19 and KCNQ10T1 imprinting control regions in children born small for gestational age 
BMC Medical Genetics  2014;15:67.
There is a substantial genetic component for birthweight variation, and although there are known associations between fetal genotype and birthweight, the role of common epigenetic variation in influencing the risk for small for gestational age (SGA) is unknown. The two imprinting control regions (ICRs) located on chromosome 11p15.5, involved in the overgrowth disorder Beckwith-Wiedemann syndrome (BWS) and the growth restriction disorder Silver-Russell syndrome (SRS), are prime epigenetic candidates for regulating fetal growth. We investigated whether common variation in copy number in the BWS/SRS 11p15 region or altered methylation levels at IGF2/H19 ICR or KCNQ10T1 ICR was associated with SGA.
We used a methylation-specific multiplex-ligation-dependent probe amplification assay to analyse copy number variation in the 11p15 region and methylation of IGF2/H19 and KCNQ10T1 ICRs in blood samples from 153 children (including 80 SGA), as well as bisulfite pyrosequencing to measure methylation at IGF2 differentially methylated region (DMR)0 and H19 DMR.
No copy number variants were detected in the analyzed cohort. Children born SGA had 2.7% lower methylation at the IGF2 DMR0. No methylation differences were detected at the H19 or KCNQ10T1 DMRs.
We confirm that a small hypomethylation of the IGF2 DMR0 is detected in peripheral blood leucocytes of children born SGA at term. Copy number variation within the 11p15 BWS/SRS region is not an important cause of non-syndromic SGA at term.
PMCID: PMC4089969  PMID: 24934635
DNA methylation; Imprinting; ICR2; ICR1; H19; KCNQ10T1; Small for gestational age

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