大肠癌中胰岛素样生长因子2基因的印迹状态和表达

目的：研究胰岛素样生长因子2（insulin-like growth factor 2,IGF2)基因的印迹状态和表达与大肠癌的关系，为研究大肠癌的发生机理提供线索。方法：用逆转录－聚合酶链反应半定量检测IGF2的表达量，比较其在大肠癌及癌旁组织中有无差异，用限制性片段长度多态检测IGF2的印迹状态，分析印迹状态、表达量与大肠癌的关系。结果：82．4％（28／34）大肠癌有IGF2的表达增加，IGF2的表达量在肿瘤组织与癌旁组织中差异有显著性（P＜0．01，t=3.01)。IGF2在87．5％（14／16）大肠癌组织中发生了印迹丢失，但其相对应的癌旁组织也有71．4％（10／14）存在IGF2的印迹丢失。结论：IGF2的表达增加是大肠癌发生的相关因素，IGF2的印迹丢失可能是大肠癌发生的前期表现。     

Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site

The adjacent insulin-like growth factor 2 (IGF2) and H19 genes are imprinted in most normal human tissues, but imprinting is often lost in tumors. The mechanisms involved in maintenance of imprinting (MOI) and loss of imprinting (LOI) are unresolved. We show here that osteosarcoma (OS) tumors with IGF2/H19 MOI exhibit allele-specific differential methylation of a CTCF-binding site upstream of H19. LOI of IGF2 or H19 in OS occurs in a mutually exclusive manner, and occurs with monoallelic expression of the other gene. Bisulfite sequencing reveals IGF2 LOI occurs with biallelic CpG methylation of the CTCF-binding site, while H19 LOI occurs with biallelic hypomethylation of this site. Our data demonstrate that IGF2 LOI and H19 LOI are accompanied by reciprocal methylation changes at a critical CTCF-binding site. We propose a model in which incomplete gain or loss of methylation at this CTCF-binding site during tumorigenesis explains the complex and often conflicting expression patterns of IGF2 and H19 in tumors.     

LINE-1 hypomethylation in familial and sporadic cancer

Increased and decreased methylation at specific sequences (hypermethylation and hypomethylation, respectively) is characteristic of tumor DNA compared to normal DNA and promotes carcinogenesis in multiple ways including genomic instability. Long interspersed element (LINE), an abundant class of retrotransposons, provides a surrogate marker for global hypomethylation. We developed methylation-specific multiplex ligation-dependent probe amplification assays to study LINE-1 methylation in cases of colorectal, gastric, and endometrial cancer (N?=?276), stratified by patient category [sporadic; Lynch syndrome (LS); familial colorectal cancer type X (FCCX)] and microsatellite instability status. Within each patient group, LINE-1 showed lower methylation in tumor DNA relative to paired normal DNA and hypomethylation was statistically significant in most cases. Interestingly, normal colorectal mucosa samples from different patient groups displayed differences in LINE-1 methylation that mirrored differences between the respective tumor tissues, with a decreasing trend for LINE-1 methylation from patients with sporadic colorectal cancer to LS to FCCX. Despite the fact that the degree of LINE-1 methylation is generally tissue specific, normal colorectal mucosa, gastric mucosa, and endometrium from LS patients showed similar levels of LINE-1 methylation. Our results suggest that the degree of LINE-1 methylation may constitute a "field defect" that may predispose normal tissues for cancer development.     

Global demethylation in loss of imprinting subtype of wilms tumor

Epigenetic abnormalities at the IGF2/H19 locus play a key role in the onset of Wilms tumor. These tumors can be classified into three molecular subtypes depending on the events occurring at this locus: loss of imprinting (LOI), loss of heterozygosity (LOH), or retention of imprinting (ROI). As IGF2 LOI is a consequence of aberrant methylation, we hypothesized that this subtype of Wilms tumors might display global abnormalities of methylation. We therefore analyzed the methylation status of satellite DNA, as a surrogate for global methylation in 50 Wilms tumor patients. Satellite methylation was quantified by a methylation‐sensitive quantitative PCR. We confirmed hypomethylation of both satellite α (Sat α) and satellite 2 (Sat 2) DNA in Wilms tumor samples compared with normal kidney. In addition, we found that LOI tumors, unlike ROI or LOH ones, showed concordant hypomethylation of both Sat α and Sat 2 DNA. This would suggest that the LOI subtype of Wilms tumor, which unlike other subtypes results from an epimutation, has a global deregulation of methylation mechanisms. © 2012 Wiley Periodicals, Inc.     

Post-weaning diet affects genomic imprinting at the insulin-like growth factor 2 (Igf2) locus

IGF2 loss of imprinting (LOI) is fairly prevalent and implicated in the pathogenesis of human cancer and developmental disease; however, the causes of this phenomenon are largely unknown. We determined whether the post-weaning diet of mice affects allelic expression and CpG methylation of Igf2. C57BL/6JxCast/EiJ F1 hybrid mice were weaned onto (1) a standard natural ingredient control diet, (2) a synthetic control diet or (3) a synthetic methyl-donor-deficient diet lacking folic acid, vitamin B(12), methionine and choline. Maternal Igf2 expression in kidney was negligible at birth, but increased to approximately 10% of total expression after 60 days on the natural control diet. By 60 days post-weaning, both synthetic diets caused significant LOI of Igf2 relative to animals weaned onto the natural control diet. Total Igf2 expression was significantly reduced in these groups, however, indicating that the increase in relative maternal Igf2 expression was caused by specific down-regulation of the paternal allele. The LOI induced by the synthetic-deficient diet persisted during a subsequent 100-day 'recuperation' period on natural ingredient diet. There were no group differences in overall or allele-specific CpG methylation in the H19 differentially methylated region (DMR), Igf2 DMR0 or Igf2 DMR1. At 30 and 60 days post-weaning, however, the paternal allele of Igf2 DMR2 was hypermethylated in the kidneys of mice on the control synthetic diet. These results indicate that post-weaning diet can permanently affect expression of Igf2, suggesting that childhood diet could contribute to IGF2 LOI in humans.     

Specific epigenetic alterations of IGF2-H19 locus in spermatozoa from infertile men

DNA methylation marks, a key modification of imprinting, are erased in primordial germ cells and sex specifically re-established during gametogenesis. Abnormal epigenetic programming has been proposed as a possible mechanism compromising male fertility. We analysed by pyrosequencing the DNA methylation status of 47 CpGs located in differentially methylated regions (DMRs), the DMR0 and DMR2 of the IGF2 gene and in the 3rd and 6th CTCF-binding sites of the H19 DMR in human sperm from men with normal semen and patients with teratozoospermia (T) and/or oligo-astheno-teratozoospermia (OAT). All normal semen samples presented the expected high global methylation level for all CpGs analysed. In the teratozoospermia group, 11 of 19 patients presented a loss of methylation at variable CpG positions either in the IGF2 DMR2 or in both the IGF2 DMR2 and the 6th CTCF of the H19 DMR. In the OAT group, 16 of 22 patients presented a severe loss of methylation of the 6th CTCF, closely correlated with sperm concentration. The methylation state of DMR0 and of the 3rd CTCF was never affected by the pathological status of sperm samples. This study demonstrates that epigenetic perturbations of the 6th CTCF site of the H19 DMR might be a relevant biomarker for quantitative defects of spermatogenesis in humans. Moreover, we defined a methylation threshold sustaining the classification of patients in two groups, unmethylated and methylated. Using this new classification of patients, the observed intrinsic imprinting defects of spermatozoa appear not to impair significantly the outcome of assisted reproductive technologies.     

Familial aggregation of abnormal methylation of parental alleles at the IGF2/H19 and IGF2R differentially methylated regions

Loss of imprinting (LOI) has been observed in many types of human tumors and may be a predisposing event in some colon cancers. LOI is strongly associated with alteration of normal DNA methylation patterns in differentially methylated regions (DMRs) of affected loci but it is not known whether LOI is caused by stochastic, environmental or genetic factors. We have developed a simple, quantitative assay for measurement of allelic methylation ratios based on methylation-sensitive restriction endonuclease digestion of genomic DNA and 'hot-stop' PCR. We examined allelic methylation ratios at DMRs within the IGF2/H19- and IGF2R-loci in a panel of 48 three-generation families. We observed familial clustering of individuals with abnormal methylation ratios at the IGF2/H19 DMR, as well as stability of this trait over a period of nearly two decades, consistent with the possibility that constitutional LOI at this locus is due largely to genetic factors. At the IGF2R DMR, we observed more variability in the allelic methylation ratios over time but also observed familial clustering of abnormal methylation ratios. Overall, our observations at IGF2R suggest that shared genetic factors are responsible for a major fraction of inter-individual variability in parental origin-dependent epigenetic modifications. However, temporal changes also occur in isolated cases, as well as within multiple individuals in the same family, indicating that environmental factors may also play a role.     

Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer

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.     

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.     

Association of genomic instability, and the methylation status of imprinted genes and mismatch-repair genes, with neural tube defects

We studied the genomic instability and methylation status of the mismatch-repair (MMR) genes hMLH1 and hMSH2, and the imprinted genes H19/IGF2, in fetuses with neural tube defects (NTDs) to explore the pathogenesis of the disease. Microsatellite instability (MSI) was observed in 23 of 50 NTD patients. Five NTD patients showed high-degree MSI (MSI-H) and 18 showed low-degree MSI (MSI-L). The frequencies of mutated microsatellite loci were 3/50 (6%) for BatT-25, 10/50 (20%) for Bat-26, 3/50 (6%) for Bat34C4, 6/50 (12%) for D2S123, 4/50 (8%) for D2S119, and 3/50 (6%) for D3S1611. The promoter regions of the hMLH1 and hMSH2 genes were unmethylated in NTD patients, as determined by methylation-specific PCR. The hMLH1 and hMSH2 promoter methylation patterns, the methylation levels of H19 DMR1, and IGF2 DMR0 were detected by bisulfite sequencing PCR, sub-cloning, and sequencing. The hMSH2 promoter sequence was unmethylated, and the hMLH1 promoter showed a specific methylation pattern at two CpG sites. The methylation levels of H19 DMR1 in the NTD and control groups are 73.3% ± 15.9 and 58.3% ± 11.2, respectively. The methylation level of the NTD group was higher than that of the control group (Student's t-test, P<0.05). There is no significant difference in IGF2 DMR0 methylation level between the two groups. All of the results presented here suggest that genomic instability, the MMR system, and hyper-methylation of the H19 DMR1 may be correlated with the occurrence of NTDs.     

Methylation status of T-lymphoma invasion and metastasis 1 promoter and its overexpression in colorectal cancer

T-lymphoma invasion and metastasis 1 has been implicated in tumor invasion and metastasis. However, the regulatory mechanisms underlying aberrant T-lymphoma invasion and metastasis 1 expression in human colorectal cancer have not been well defined. To investigate the relationship between methylation status and expression levels of T-lymphoma invasion and metastasis 1 gene, methylation-specific polymerase chain reaction, and immunohistochemistry staining were performed in 232 matched samples of human colorectal cancer tissue and normal colorectal mucosa. Results showed that T-lymphoma invasion and metastasis 1 protein was overexpressed in colorectal cancer, especially in metastatic cases (P < .001). The degree of T-lymphoma invasion and metastasis 1 promoter methylation was a little lower in cancer tissues than in matched normal mucosa (P < .05), and the expression level of T-lymphoma invasion and metastasis 1 was inversely related to the methylation status in cancer tissues (P < .001). Colon cancer cell lines HT29 and LS174T were treated with demethylating agent 5-aza-2'-deoxycytidine, resulting in promoter hypomethylation accompanied by reexpression of T-lymphoma invasion and metastasis 1 mRNA and protein. In contrast, colon cancer cell lines SW620 and LoVo were treated with hypermethylation agent S-adenosylmethionine, resulting in T-lymphoma invasion and metastasis 1 promoter hypermethylation, accompanied by suppression of T-lymphoma invasion and metastasis 1 expression and inhibition of cell growth, plate colony formation, and migration. The present study demonstrates that overexpression of T-lymphoma invasion and metastasis 1 is associated with hypomethylation status of T-lymphoma invasion and metastasis 1 promoter region in colorectal cancer tissues. It suggests that promotor hypomethylation of T-lymphoma invasion and metastasis 1 may play a role in the progression and metastasis of colorectal cancer. Pharmacologic reversal of T-lymphoma invasion and metastasis 1 promoter hypomethylation may inhibit cell proliferation and migration.     

Microarray-based survey of CpG islands identifies concurrent hyper- and hypomethylation patterns in tissues derived from patients with breast cancer

Maintenance of CpG island methylation in the genome is crucial for cellular homeostasis and this balance is disrupted in cancer. Our rationale was to compare the methylation of CpG islands in tissues (tumor, healthy breast and blood) from patients with breast cancer. We studied 72 genes in 103 samples using microarray hybridization and bisulfite sequencing. We observed tumor specific hyper- or hypomethylation of five genes; COL9A1, MT1A, MT1J, HOXA5 and FLJ45983. A general drop of methylation in COL9A1 was apparent in tumors, when compared with blood and healthy breast tissue. Furthermore, one tumor displayed a complete loss of methylation of all five genes, suggesting overall impairment of methylation. The downstream, evolutionary conserved island of HOXA5 showed hypomethylation in 18 tumors and complete methylation in others. This CpG island also displayed a semimethylated state in the majority of normal breast samples, when compared to complete methylation in blood. Distinct methylation patterns were further seen in MT1J and MT1A, belonging to the metallothionein gene family. The CpG islands of these genes are spaced by 2 kb, which shows selective methylation of two structurally and functionally related genes. The promoters of FLJ45983 and MT1A were methylated above 25% in 18 primary and metastatic tumors. Concurrently, there was also >10% methylation of healthy breast tissue in 11 and 5 samples, respectively. This suggests that the methylation process for the latter two genes takes place already in normal breast cells. Our results also point to a considerable heterogeneity of epigenetic disturbance in breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Simultaneous Hyper‐ and Hypomethylation at Imprinted Loci in a Subset of Patients with GNAS Epimutations Underlies a Complex and Different Mechanism of Multilocus Methylation Defect in Pseudohypoparathyroidism Type 1b

Most patients with pseudohypoparathyroidism type 1b (PHP‐1b) display a loss of imprinting (LOI) encompassing the GNAS locus resulting in PTH resistance. In other imprinting disorders, such as Russell–Silver or Beckwith–Wiedemann syndrome, we and others have shown that the LOI is not restricted to one imprinted locus but may affect other imprinted loci for some patients. Therefore, we hypothesized that patients with PHP‐1b might present multilocus imprinting defects. We investigated, in 63 patients with PHP‐1b, the methylation pattern of eight imprinted loci: GNAS, ZAC1, PEG1/MEST, ICR1, and ICR2 on chromosome 11p15, SNRPN, DLK1/GTL2 IG‐DMR, and L3MBTL1. We found multilocus imprinting defects in four PHP‐1b patients carrying broad LOI at the GNAS locus (1) simultaneous hypermethylation at L3MBTL1 differentially methylated region 3 (DMR3), and hypomethylation at PEG1/MEST DMR (n = 1), (2) hypermethylation at the L3MBTL1 (DMR3) (n = 1) and at the DLK1/GTL2 IG‐DMR (n = 1), and (3) hypomethylation at the L3MBTL1 DMR3 (n = 1). We suggest that mechanisms underlying multilocus imprinting defects in PHP‐1b differ from those of other imprinting disorders having only multilocus loss of methylation. Furthermore, our results favor the hypothesis of “epidominance”, that is, the phenotype is controlled by the most severely affected imprinted locus.     

Hypermethylation of CpG islands is more prevalent than hypomethylation across the entire genome in breast carcinogenesis

This study aimed to establish a high-throughput, genome-wide and non-gene-specific approach to assess the methylation status of multiple CpG islands in parallel and employ it to detect the CpG island methylation profiling alterations in breast carcinogenesis. We used methylation-sensitive restriction fingerprint (MSRF) to screen the permutations of primers that could detect varied and specific methylation profiling in genomic DNA isolated from four different cell lines. Five permutations of nine arbitrary primers were determined for the following experiments based on the above test. We then examined the methylation profiling alterations of CpG islands in 31 breast cancer tissue samples relative to their adjacent non-neoplastic tissues with modified MSRF that replaced silver staining with denatured high-performance liquid chromatography for size fraction. We found that two pairs of primers could reveal specific alterations of CpG methylation in the examined tissues, and 83.9% (26/31) of breast cancer tissues exhibited specific CpG island methylation profiling relative to their adjacent non-neoplastic tissues. Size fraction analysis revealed that hypermethylation of CpG islands was responsible for the aberrant methylation profiling in breast cancer tissues. Our work not only established a relative high-throughput, genome-wide and economic method to detect methylation alterations of CpG island profiling, but also revealed that hypermethylation of CpG islands was more prevalent than hypomethylation across the entire genome in our examined cancer tissues. The methylation profiling alterations revealed by two primer pairs used in the present study might be a novel marker for breast cancer.     

Heritable rather than age-related environmental and stochastic factors dominate variation in DNA methylation of the human IGF2/H19 locus

Epigenetic variation may significantly contribute to the risk of common disease. Currently, little is known about the extent and causes of epigenetic variation. Here, we investigated the contribution of heritable influences and the combined effect of environmental and stochastic factors to variation in DNA methylation of the IGF2/H19 locus. Moreover, we tested whether this locus was subject to age-related degeneration of epigenetic patterns as was previously suggested for global methylation. We measured methylation of the H19 and IGF2 differentially methylated regions (DMRs) in 196 adolescent and 176 middle-aged twins using a recently developed mass spectrometry-based method. We observed substantial variation in DNA methylation across individuals, underscoring that DNA methylation is a quantitative trait. Analysis of data in monozygotic and dizygotic twins revealed that a significant part of this variation could be attributed to heritable factors. The heritability of methylation of individual CpG sites varied between 20 and 74% for the H19 DMR and was even higher, between 57 and 97%, for the IGF2 DMR. Remarkably, the combined influence of environmental and stochastic factors on DNA methylation was not greater in middle-age than in adolescence, suggesting a limited role for age-related degeneration of methylation patterns at this locus. Single nucleotide polymorphisms in the IGF2/H19 locus were significantly associated with DNA methylation of the IGF2 DMR (P = 0.004). A preliminary analysis suggested an association between H19 DMR methylation and body size (P < 0.05). Our study shows that variation in DNA methylation of the IGF2/H19 locus is mainly determined by heritable factors and single nucleotide polymorphisms (SNPs) in cis, rather than the cumulative effect of environmental and stochastic factors occurring with age.     

Methylation status of <Emphasis Type="Italic">IGF2</Emphasis> DMR and <Emphasis Type="Italic">LINE1</Emphasis> in leukocyte DNA provides distinct clinicopathological features of gastric cancer patients

DNA methylation of leukocyte DNA has been proposed to be a biomarker for cancer that can be used to target patients for appropriate clinical implementation. We investigated IGF2 DMR and LINE1 methylation in the leukocyte DNA and their association with clinicopathological features and prognosis of gastric cancer (GC) patients. Methylation status of IGF2 DMR and LINE1 in the leukocyte DNA was quantified using bisulfite pyrosequencing in 207 GC patients. Methylation of both IGF2 DMR and the LINE1 was significantly higher in the undifferentiated histologic type compared to the differentiated histologic type (both P = 0.0002). Hypermethylation of both the IGF2 DMR and the LINE1 was associated with more aggressive features of GC such as advanced stage (IGF2 DMR, P = 0.0002; LINE1, P < 0.0001), lymphatic invasion positive (IGF2 DMR, P = 0.004; LINE1, P = 0.002), venous invasion positive (IGF2 DMR, LINE1, both P = 0.03), lymph node metastasis positive (IGF2 DMR, P = 0.01; LINE1, P = 0.001), peritoneal dissemination positive (IGF2 DMR, P = 0.04; LINE1, P = 0.002), liver metastasis positive (IGF2 DMR, P = 0.008; LINE1, P = 0.001), and other distant metastasis positive (IGF2 DMR, P = 0.04). Our data suggest that high LINE1 and IGF2 DMR methylation status would be a phenomenon that is observed with the progression of GC, supporting their potential utility as a biomarker in GC patients.     

Exon-specific DNA hypomethylation of the p53 gene of rat colon induced by dimethylhydrazine. Modulation by dietary folate.

Folate deficiency enhances colorectal carcinogenesis in dimethylhydrazine-treated rats. Folate is an important mediator of DNA methylation, an epigenetic modification of DNA that is known to be dysregulated in the early stages of colorectal cancer. This study investigated the effect of dimethylhydrazine on DNA methylation of the colonic p53 gene and the modulation of this effect by dietary folate. Sprague-Dawley rats were fed diets containing 0, 2, 8, or 40 mg of folate/kg of diet. Five weeks after diet initiation, dimethylhydrazine was injected weekly for fifteen weeks. Folate-depleted and folate-replete control animals did not receive dimethylhydrazine and were fed the 0- and 8-mg folate diets, respectively. The extent of p53 methylation was determined by a quantitative HpaII-polymerase chain reaction. In exons 6 and 7, significant p53 hypomethylation was observed in all dimethylhydrazine-treated rats relative to controls (P < 0.01), independent of dietary folate. In exon 8, significant p53 hypomethylation was observed only in the dimethylhydrazine-treated folate-depleted rats compared with controls (P = 0.038) and was effectively overcome by increasing levels of dietary folate (P = 0.008). In this model, dimethylhydrazine induces exon-specific p53 hypomethylation. In some exons, this occurs independent of dietary folate, and in others, increasing levels of dietary folate effectively override the induction of hypomethylation in a dose-responsive manner. This may be a mechanism by which increasing levels of dietary folate inhibit colorectal carcinogenesis.     

Increased incidence of aberrant DNA methylation within diverse imprinted gene loci outside of IGF2/H19 in Silver-Russell syndrome

Almost half of the patients with Silver-Russell syndrome (SRS) are affected by DNA hypomethylation of the Imprinting Center Region 1 (ICR1) at the IGF2/H19 locus on 11p15. We searched genome-wide for additional aberrant DNA methylation in such SRS patients that could account for the clinical variability of the disorder. For this purpose, 18 children with SRS (11 with ICR1 hypomethylation) and 9 children small for gestational age (SGA), serving as controls, were recruited. Genomic DNA from whole blood was subjected to microarray analysis with the HumanMethylation27 BeadChip. This array allows investigating 27,500 CpG sites mostly located in the promoter regions of 14,000 genes. Data were validated by the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique for the 11p15 region. SRS patients with ICR1 hypomethylation were significantly more frequently affected by DNA hypo- and hypermethylation of CpG sites from diverse imprinted loci than the SGA controls (p = 0.0048). There was no recurrent specific methylation defect outside of IGF2/H19. These findings suggest as causative in SRS a defective mechanism necessary for establishment or maintenance of imprinting marks, which affects imprinted loci in general with low specificity and the IGF2/H19 locus with high specificity, implying the existence of some structural peculiarities at the IGF2/H19 locus.     

LINE-1 hypomethylation is associated with radiation-induced genomic instability in industrial radiographers

Global DNA hypomethylation is proposed as a potential biomarker for cancer risk associated with genomic instability, which is an important factor in radiation-induced cancer. However, the associations among radiation exposure, changes in DNA methylation, and carcinogenesis are unclear. The aims of this study were (1) to examine whether low-level occupational radiation exposure induces genomic DNA hypomethylation; and (2) to determine the relationships between radiation exposure, genomic DNA hypomethylation and radiation-induced genomic instability (RIGI) in industrial radiographers. Genomic DNA methylation levels were measured in blood DNA from 40 radiographers and 28 controls using the LINE-1 pyrosequencing assay and the luminometric methylation assay. Further, the micronucleus-centromere assay was performed to measure aneuploidy of chromosomes 1 and 4 as a marker of delayed RIGI. Genomic DNA methylation levels were significantly lower in radiographers than those in controls. LINE-1 hypomethylation was not significantly correlated with recent 1-year, recent 3-year, or total cumulative radiation doses in radiographers; however, LINE-1 hypomethylation significantly correlated with the cumulative radiation dose without recent 3-year exposure data (D3dose, r = −0.39, P < 0.05). In addition, LINE-1 hypomethylation was a significant contributor to aneuploidy frequency by D3dose (F (2, 34) = 13.85, P < 0.001), in which a total of 45% of the variance in aneuploidy frequency was explained. Our results provide suggestive evidence regarding the delayed effects of low-dose occupational radiation exposure in radiographers and its association with LINE-1 hypomethylation; however, additional studies using more subjects are needed to fully understand the relationship between genomic DNA hypomethylation and RIGI. Environ. Mol. Mutagen. 60: 174–184, 2019. © 2018 Wiley Periodicals, Inc.