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To evaluate loss of imprinting (LOI) and expression of the IGF2 gene in matched esophageal normal and adenocarcinoma tissues, we studied a prospective cohort of 77 patients who underwent esophageal resection between 1998 and 2003. IGF2 imprinting status was determined by reverse transcription-polymerase chain reaction (PCR) following ApaI digestion, and quantitative PCR was used to evaluate IGF2 expression, which was correlated with clinicopathologic findings, disease-free and overall survival. In total, 32% (14/44) of informative tissues showed loss of IGF2 imprinting, with a strong correlation between the tumor and normal esophageal epithelia (Kappa = 0.89, P < 0.01). Normal epithelia with LOI had increased expression of IGF2 [median: 2.91, 95% confidence interval (CI): 0.93-5.06] compared with imprinted normal epithelia (median: 1.13, 95% CI: 0.85-1.39) (P = 0.03). In contrast, tumors with LOI had significantly reduced IGF2 expression (median: 1.87, 95% CI: 0.53-5.21) compared with normally imprinted tumors (median: 6.79, 95% CI: 3.39-15.89) (P = 0.016). Patients below the age of 65 years with normally imprinted tumors had significantly reduced 5 year disease-free survival (DFS) (24%) compared with patients whose tumors had LOI for IGF2 (55%) (P = 0.03). Cox regression analysis showed that IGF2 overexpression was associated with significantly reduced disease-free survival (P = 0.04). We conclude that in a subgroup of younger patients, loss of IGF2 imprinting was associated with improved outcome following esophageal resection. Expression of IGF2 in esophageal adenocarcinoma and normal esophageal epithelia depended on imprinting status and tissue type, suggesting novel molecular regulatory mechanisms in esophageal tumorigenesis.

Loss of genomic imprinting (LOI) of the insulin-like growth factor-2 gene (IGF2) is an epigenetic change involving abnormal activation of the normally silent maternally inherited allele. LOI of IGF2 gene is found in tumor tissue, normal adjoining mucosa and peripheral blood lymphocytes (PBL) of some patients with colorectal cancer (CRC), suggesting that this alteration precedes and is a risk factor for CRC. However, whether LOI of IGF2 is transitory or remains a permanent epigenetic alteration is unknown.Four-hundred patients, mean age 60.7 years (range 15-95), 287 (80%) Caucasian were studied. This included 210 (51.4%) patients with no colorectal neoplasia, and 190 (48.6) with colorectal neoplasia. LOI of IGF2 was present in all age strata examined, and no statistically significant association across age strata (p trend0.05) was noted. Forty-nine patients had repeat analysis of blood imprinting status at a mean follow up time of 38.2 +/- 12.9 months. All but three patients had the same imprinting status at follow up (94% agreement, kappa 0.79, p0.001). Genomic imprinting was stable for patients with and without colorectal neoplasia.Standard RT-PCR assays for imprinting analysis of IGF2 were performed on PBL from ApaI informative individuals recruited at baseline and repeated 1 to 3 years later. Prevalence of LOI of IGF2 was also evaluated according to age strata.LOI of the IGF2 gene in PBL appears to be a stable epigenetic phenomenon in most patients. Furthermore, LOI of IGF2 was not associated with age, suggesting an inherited or congenital epigenetic event. These findings support the concept that LOI of IGF2 may be a useful risk factor for CRC predisposition.

IGF2is the first gene discovered to be imprinted and expressed exclusively from the paternal allele in both human and mouse.IGF2is also the first imprinted gene displaying loss of imprinting (LOI) or aberrant imprinting in human cancers. Evidently, LOI or reactivation of the maternal allele ofIGF2is associated with an increase ofIGF2expression that may subsequently play an important role in the onset of human cancers. The most important discovery was the association of LOI ofIGF2with 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 ofIGF2and family history as well as personal history of colorectal cancer, suggesting that LOI ofIGF2might 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.

Background & aims: Loss of genomic imprinting (LOI) of insulin-like growth factor II gene ( IGF2 ) involves abnormal activation of the normally silent maternally inherited allele. LOI of IGF2 has been associated with personal and family history of colorectal neoplasia (CRN), supporting a role for LOI in colorectal carcinogenesis. Whether LOI of IGF2 is associated with known environmental risk factors for CRN is unknown. Methods: We performed quantitative hot-stop PCR for imprinting analysis of IGF2 on normal peripheral blood lymphocytes (PBL) of individuals. Environmental exposures including tobacco, alcohol, NSAIDs, and nutrient consumption (calcium, folate, selenium, fiber, and fat) were correlated with LOI expression in PBL. Odds ratios (OR) and 95% CI were calculated. Results: The prevalence of LOI of IGF2 was examined in 172 individuals. Persons with CRN (adenomas/cancer) had 5.1-fold (95% CI: 1.92–13.6) increased risk of having LOI of IGF2 in PBL compared with those without CRN. In contrast, tobacco smoking (OR = 0.96, 95% CI: 0.36–2.55), alcohol consumption (OR = 1.22, 95% CI: 0.45–3.3), and NSAIDs use (OR = 1.21, 95% CI: 0.38–3.94) were not significantly associated with LOI of IGF2 . Nutrient ingestion including calcium ( P = 0.61), folate ( P = 0.23), selenium ( P = 0.19), fiber ( P = 0.63), and fat ( P = 0.14) was not statistically correlated with LOI of IGF2 . Conclusions: Abnormal imprinting of IGF2 gene was strongly associated with CRN but not with any of the environmental exposures examined. LOI of IGF2 does not appear to be an environmentally acquired phenomenon but rather a hereditary risk factor for CRN.

The IGF2 and H19 genes are genomically imprinted and expressed preferentially from the paternal and maternal alleles, respectively, during human prenatal development. The exact role of the parental imprint(s), however, is not known. To explore this issue in some detail, we have examined human androgenetic cells which by definition should be incapable of allelic discrimination given the paternal origin of both genomes. Allele-specific in situ hybridisation analysis of dispermic complete hydatidiform moles shows that IGF2 and H19 can be found to be transcriptionally active in a variegated manner, which results in the generation of random monoallelic expression patterns. This data shows that imprinted genes can be expressed monoallelically in the absence of discriminating parental marks and raises the question whether or not mechanisms underlying monoallelic expression preceded the acquisition of parental imprints during evolution.

The pathophysiologic role of the Philadelphia chromosome translocation in chronic myelogenous leukemia (CML) has been known for nearly 20 years. However, the most significant morbidity and mortality in CML are caused by progression to blast crisis, about which comparatively little is known at the molecular level. Genomic imprinting is a chromosomal modification leading to parental-origin–specific gene expression in somatic cells. Recently, we and others have described loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to biallelic rather than monoallelic expression in a wide variety of solid tumors. We have now examined the imprinting status of IGF2 in samples from CML patients in stable phase, accelerated phase, and blast crisis. Five of six stable-phase patients showed normal imprinting, but LOI was found in all six cases of advanced disease (three accelerated phase, three blast crisis), which was statistically highly significant (P < .01). Thus, LOI represents a novel type of genetic alteration in CML that appears to be specifically associated with disease progression.

IGF2 has been shown to be expressed preferentially from the paternally derived allele, although the maternal allele can be found active during both prenatal and postnatal development as well as in neoplastic tumours in humans. We addressed here whether or not the biallelic expression patterns that can be seen during postnatal human liver development reflected a coordinated change in the activities of the four promoters of human IGF2. We show here that the P2, P3 and P4 promoters, but not the P1 promoter, display monoallelic activity in embryonic, neonatal and younger infant liver specimens. The P2, P3 and P4 promoters can, however, be found active either monoallelically or biallelically or even monoallelically on opposite parental alleles in older infant and adult liver specimens. In contrast, H19, which is closely linked to IGF2, is monoallelically expressed in all postnatal liver samples analysed. We conclude that the functional imprinting status of IGF2 during postnatal liver development appears to be promoter/enhancer-specific and either partly or completely independent of H19.

Loss of imprinting (LOI), an epigenetic alteration affecting the insulin-like growth factor II gene ( IGF2 ), is found in normal colonic mucosa of about 30% of colorectal cancer (CRC) patients, but it is found in only 10% of healthy individuals. In a pilot study to investigate the utility of LOI as a marker of CRC risk, we evaluated 172 patients at a colonoscopy clinic. The adjusted odds ratio for LOI in lymphocytes was 5.15 for patients with a positive family history [95% confidence interval (95% CI), 1.70 to 16.96; probability P = 0.002], 3.46 for patients with adenomas (95% CI, 1.14 to 11.37; P = 0.026), and 21.7 for patients with CRC (95% CI, 3.48 to 153.6; P = 0.0005). LOI can be assayed with a DNA-based blood test, and it may be a valuable predictive marker of an individual's risk for CRC.

Epigenetic alterations in human cancers include global DNA hypomethylation,gene hypomethylation and promoter hypermethylation, and loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2). A mechanism for LOI described previously is hypermethylation of a differentially methylated region (DMR) upstream of the H19 gene, allowing activation of the normally silent maternal allele of IGF2. Here we show that this mechanism does not apply to colorectal cancers, which show hypomethylation of the H19 DMR as well as a DMR upstream of exon 3 of IGF2. This hypomethylation is found in both colorectal cancers and normal mucosa from the same patients, and in cell lines with somatic cell knockout of DNA methyltransferases DNMT1 and DNMT3B. These data suggest that hypomethylation is a mechanism for LOI, that the popular IGF2-H19 enhancer competition model for IGF2 imprinting does not apply to the human colon, and that an alternative model for LOI would involve a transcriptional repressor acting on the normally silent maternal allele of IGF2.

Since the discovery of epigenetic alterations in cancer 20 years ago by Feinberg and Vogelstein, a variety of such alterations have been found, including global hypomethylation, gene hypomethylation and hypermethylation, and loss of imprinting (LOI). LOI may precede the development of cancer and may thus serve as a common marker for risk, but also as a model for understanding the developmental mechanism for normal imprinting.

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.

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for B rother o f the R egulator of I mprinted S ites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.

An unexpected outcome of the diploid genome is that evolutionary strategies have evolved to express only one of the alleles (1). The rapidly expanding list of genes that are expressed monoallelically fall into three main categories: random inactivation, allelic exclusion, and genomic imprinting. These categories are distinguished by whether the expressed and inactivated alleles are maintained from one cell division to the next (random inactivation occurs with each cell division, compared to stable propagation through subsequent cell divisions as seen for allelic exclusion and genomic imprinting) and whether allele inactivation or expression is determined by parent of origin of inheritance (this differentiates allelic exclusion from genomic imprinting). Genomically imprinted genes are currently more numerous than members of the other categories. This fact is likely to reflect that the persistent (in)activation of one allele in a parent of origin-specific manner has facilitated the detection of monoallelic expression patterns in RNA extracted from homogenized tissue. This crude approach does not take into account, however, different imprinted states within a tissue, or, indeed, random allelic (in)activation. It is not surprising, therefore, that well-known genes, such as Il2 (2), were only recently found to be monoallelically expressed in a random manner (1).

We have developed a simple, quantitative assay for measurement of allele ratios that circumvents the problem of heteroduplex formation skewing the results of restriction endonuclease digestion of PCR products. This assay, 'hot-stop PCR', involves addition of a radiolabelled PCR primer at the final cycle. We applied the assay to analysis of loss of imprinting (LOI) of the insulin-like growth factor II gene (IGF2) in tumours.

Loss of imprinting (LOI) is an epigenetic alteration of some cancers involving loss of parental origin-specific expression of imprinted genes. We observed LOI of the insulin-like growth factor-II gene in twelve of twenty-seven informative colorectal cancer patients (44%), as well as in the matched normal colonic mucosa of the patients with LOI in their cancers, and in peripheral blood samples of four patients. Ten of eleven cancers (91%) with microsatellite instability showed LOI, compared with only two of sixteen tumors (12%) without microsatellite instability (P < 0.001). Control patients without cancer showed LOI in colonic mucosa of only two of sixteen cases (12%, P < 0.001) and two of fifteen blood samples (13%, P < 0.001). These data suggest that LOI in tumor and normal tissue identifies most colorectal cancer patients with microsatellite instability in their tumors, and that LO! may identify an important subset of the population with cancer or at risk of developing cancer.

The imprinted H19 gene produces a fully processed transcript that does not exhibit any conserved open reading frame between mouse and man. Although transcriptional control elements associated with the mouse H19 locus have been shown to control the neighboring Igf2 gene incis, the prevailing view is that the cytoplasmicH19 transcript does not display any function. In contrast to earlier reports, we show here that the H19 transcript is associated with polysomes in a variety of cell types, in both mouse and man. A possible trans-function of the H19 gene is suggested by a reciprocal correlation in trans between cytoplasmic H19 and IGF2 mRNA levels, as well as IGF2 mRNA translatability. We discuss these results in terms of their challenge to the prevailing dogma on the function of the enigmatic H19 gene, as well as with respect to the ontogeny of the Beckwith-Wiedemann syndrome, and propose that the human H19 gene is an antagonist of IGF2expressivity in trans.

The IGF2 gene, which encodes a growth factor, is subject to genomic imprinting, The frequently observed loss of IGF2 imprinting in a variety of tumors has been suggested to contribute to neoplasia, Since these reports have not documented the imprinting status of IGF2 at the cellular level, it cannot be excluded that the imprinting status might vary within the tumor, The possibility that loss of IGF2 imprinting in neoplastic cells reflects random imprinting patterns, was therefore addressed, We show here that individual cell populations of the JEG-3 choriocarcinoma cell line display heterogenous imprinting patterns of both IGF2 and H19, In addition, a lack of correlation between IGF2 and H19 imprinting status suggests that any regional parental imprint has been functionally lost, This notion is reinforced by the observation that JEG-3 cell subclones display a range of promoter-specific IGF2 allele usage, Moreover, we observed that the imprinting status of H19 and IGF2 were differentially modulated in JEG-3-derived tumors generated in nude mice, The results suggest that allele-specific expression of IGF2 operates in the absence of a parental imprint, Finally, our observations urge caution with respect to the general interpretation of biallelic expression as `loss of imprinting'.

Genetic imprinting implies the preferential or exclusive expression of one of the parental alleles of a subset of autosomal loci. The insulin-like growth factor II (IGF2) andH19loci are particularly interesting examples of this phenomenon since their products appear to display growth agonistic and antagonistic properties, respectively. In addition,IGF2andH19are only 90 kb apart, are expressed from opposite parental alleles [1,2] and show a striking similarity in their spatial expression patterns during human prenatal development [3]. One exception is the choroid plexus and leptomeninges which express1GF2biallically with no detectableH19expression [3]. Observations like these have fuelled ideas that there is an enhancer competition between theIGF2andH19loci [4]. The imprinting status of theH19locus would then indirectly control the expressivity ofIGF2. This model is likely to be too simple since the P1 promoter ofIGF2is not functionally imprinted during liver development in humans [4]. Moreover, while the liver P2-P4 promoters are expressed primarily from the paternally derived allele during human prenatal development, the P2-P4 promoters can be expressed from both parental alleles in complex patterns during postnatal human development [5]. The enhancer competition model might be put to the test in human and mouse uniparental embryos since the parental origin of their diploid genomes cannot be discerned. Unexpectedly,H19which is expressed preferentially from the maternal allele in mouse [6] and human [7] placenta is expressed in both mouse and human trophoblasts (in complete hydatidiform moles) lacking the maternal genome. In the normal human placenta, the repressed paternalH19allele is more methylated. Interestingly, the CpG methylation pattern ofH19is strikingly similar between normal placenta and complete moles. Hence, both paternalH19alleles are similarly methylated indicating that postzygotic modification events typical of normal development have taken place in complete moles as well in spite of the absence of the maternal genome. In contrast to the normal placenta,H19is expressed biallelically in complete moles as assessed by allele-specific in situ hybridisation analysis of dispermie moles [8]. We discuss these results in relation to current models ofIGF2/H19imprinting mechanism(s).