Methylation Status of CYP27B1 and IGF2 Correlate to BMI SDS in Children with Obesity.
ABSTRACT: Worldwide increasing childhood obesity is due to interactions between environmental and genetic factors, linked together by epigenetic mechanisms such as DNA methylation.82 obese children (>95th BMI percentile , age: 3-18 years) were included. Anthropometric data, metabolic parameters, 25-OH vitamin D (25OHD), and pubertal status were recorded, 24-hour blood pressure monitoring was performed. BMI standard deviation score (SDS) was calculated. Using candidate gene approach, obesity- (insulin-like growth factor 2 (IGF2), proopiomelanocortin (POMC)) and vitamin D metabolism-related genes (1-alfa-hydroxylase (CYP27B1), VDR) regulated by DNA methylation were selected. After isolating DNA from peripheral blood, bisulfite conversion, bisulfite specific polymerase chain reaction (BS-PCR), and pyrosequencing were carried out.No significant correlation between 25-OHD and metabolic parameters and DNA methylation status, but a tendency of positive correlation between VDR methylation status and 25-OHD (r = 0.2053,p = 0.066) were observed. Significant positive correlations between BMI SDS and CYP27B1 hypermethylation (r = 0.2371,p = 0.0342) and a significant negative correlation between IGF2 hypomethylation and BMI SDS (r = -0.305,p = 0.0059) were found. Conclusions Rate of obesity shows correlation with DNA methylation. Hypomethylation of IGF2 and hypermethylation of CYP27B1 genes might positively influence the rate of BMI observed in obese children.
Project description:BACKGROUND: Insulin like growth factor 2 (IGF2) is an imprinted gene, which has an important role in fetal growth as established in mice models. IGF2 is downregulated through hypomethylation of a differentially methylated region (DMR) in Silver Russell syndrome (SRS), characterised by growth restriction. We have previously reported that severe pre- and post-natal growth restriction associated with insulin resistance and precocious pubarche in a woman without body asymmetry or other SRS features resulted from a balanced translocation affecting the regulation of her IGF2 gene expression. We hypothesised that severe pre- and post-natal growth restriction associated with insulin resistance and precocious pubarche in the absence of SRS are also caused by downregulation of IGF2 through hypomethylation, gene mutation or structural chromosomal abnormalities. METHODS: We performed routine karyotyping, IGF2 gene sequencing and investigated DNA methylation of the IGF2 differentially methylated region (DMR)0 and H19 DMR using pyrosequencing, in four women selected for very low birth weight (<-3 SDS for gestational age), precocious pubarche, short adult stature (<-2 SDS), and insulin resistance (defined as HOMA-IS < 80%); and compared their methylation results to those of 95 control subjects. RESULTS: We identified a 20?year old woman with severe hypomethylation at both DMRs. She was the smallest at birth (birthweight SDS,-3.9), and had the shortest adult height (143?cm). The patient was diagnosed with polycystic ovarian syndrome at the age of 15?years, and had impaired fasting glucose in the presence of a low BMI (19.2 kg/m2). CONCLUSIONS: Our case of growth restriction, premature pubarche and insulin resistance in the absence of body asymmetry or other features of SRS adds to the expanding phenotype of IGF2/H19 methylation abnormalities. Further studies are needed to confirm whether growth restriction in association with premature pubarche and insulin resistance is a specific manifestation of reduced IGF2 expression.
Project description:The prevalence of metabolic disorders, in particular obesity has dramatically increased worldwide. Genetic variants explain only a minor part of the obesity epidemic induced by physical inactivity and over-nutrition. Epidemiological studies in humans and animal models indicate that epigenetic changes associated with adverse parental and/or intrauterine factors may contribute to the missing heritability of metabolic disorders. Possible adverse paternal effects are likely transmitted by sperm to the next-generation. To investigate this hypothesis, we have systematically analyzed the effects of male body mass index (BMI) on sperm epigenome and its association with next-generation fetal cord blood (FCB) DNA methylation. Methylation levels of maternally imprinted (PEG1, PEG4, PEG5, and PEG10), paternally imprinted (H19-IG DMR, IGF2-DMR0, and MEG3-IG DMR) regions, and obesity-related non-imprinted HIF3A gene were quantified by bisulphite pyrosequencing in sperm samples of 294 human donors undergoing in vitro fertilization or intracytoplasmic sperm injection, and in 113 FCBs of the resulting offspring. Multivariable regression analyses revealed that MEG3 intergenic differentially methylated region (IG DMR) showed positive correlation between sperm methylation and donor's BMI. A gender-specific correlation between paternal BMI and FCB methylation was observed for MEG3-IG DMR, HIF3A, and IGF2-DMR0. The former two genes displayed same directional nominal association (as sperm) between paternal BMI and FCB methylation in male offspring. Hypomethylation of IGF2-DMR0 with increased paternal BMI was observed in FCBs from female offsprings. Our results suggest that male obesity is nominally associated with modification of sperm DNA methylome in humans, which may affect the epigenome of the next-generation. Nevertheless, it is important to note that none of the associated p-values survived multiple testing adjustments. Future work should test the effect of associated methylation aberrations in the offspring as DNA methylation was shown to control expression and/or imprint establishment across the studied genes.
Project description:Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.
Project description:Loss of imprinting (LOI) of insulin-like growth factor 2 (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 have 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 tumors 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 2 different forms of aberrant gene expression.
Project description:In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1?-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.
Project description:UNLABELLED: BACKGROUND: Silver-Russell syndrome (SRS) is characterized by severe intrauterine and postnatal growth failure and frequent body asymmetry. Half of the patients with SRS carry a DNA hypomethylation of the imprinting center region 1 (ICR1) of the insulin-like growth factor 2 (IGF2)/H19 locus, and the clinical phenotype is most severe in these patients. We aimed to elucidate the epigenetic basis of asymmetry in SRS and the cellular consequences of the ICR1 hypomethylation. RESULTS: The ICR1 methylation status was analyzed in blood and in addition in buccal smear probes and cultured fibroblasts obtained from punch biopsies taken from the two body halves of 5 SRS patients and 3 controls. We found that the ICR1 hypomethylation in SRS patients was stronger in blood leukocytes and oral mucosa cells than in fibroblasts. ICR1 CpG sites were affected differently. The severity of hypomethylation was not correlated to body asymmetry. IGF2 expression and IGF-II secretion of fibroblasts were not correlated to the degree of ICR1 hypomethylation. SRS fibroblasts responded well to stimulation by recombinant human IGF-I or IGF-II, with proliferation rates comparable with controls. Clonal expansion of primary fibroblasts confirmed the complexity of the cellular mosaicism. CONCLUSIONS: We conclude that the ICR1 hypomethylation SRS is tissue, cell, and CpG site specific. The correlation of the ICR1 hypomethylation to IGF2 and H19 expression is not strict, may depend on the investigated tissue, and may become evident only in case of more severe methylation defects. The body asymmetry in juvenile SRS patients is not related to a corresponding ICR1 hypomethylation gradient, rendering more likely an intrauterine origin of asymmetry. Overall, it may be instrumental to consider not only the ICR1 methylation status as decisive for IGF2/H19 expression regulation.
Project description:Vitamin D is associated with decreased risks of various cancers, including colon cancer. The vitamin D receptor (VDR) is a transcription factor, which plays an important role in cellular differentiation and inhibition of proliferation. A link between VDR and the RAS-mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been suggested. However, the prognostic role of VDR expression or its relationship with PIK3CA or KRAS mutation remains uncertain. Among 619 colorectal cancers in two prospective cohort studies, 233 (38%) tumors showed VDR overexpression by immunohistochemistry. We analyzed for PIK3CA and KRAS mutations and LINE-1 methylation by Pyrosequencing, microsatellite instability (MSI), and DNA methylation (epigenetic changes) in eight CpG island methylator phenotype (CIMP)-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by MethyLight (real-time PCR). VDR overexpression was significantly associated with KRAS mutation (odds ratio, 1.55; 95% confidence interval, 1.11-2.16) and PIK3CA mutation (odds ratio, 2.17; 95% confidence interval, 1.36-3.47), both of which persisted in multivariate logistic regression analysis. VDR was not independently associated with body mass index, family history of colorectal cancer, tumor location (colon versus rectum), stage, tumor grade, signet ring cells, CIMP, MSI, LINE-1 hypomethylation, BRAF, p53, p21, beta-catenin, or cyclooxygenase-2. VDR expression was not significantly related with patient survival, prognosis, or clinical outcome. In conclusion, VDR overexpression in colorectal cancer is independently associated with PIK3CA and KRAS mutations. Our data support potential interactions between the VDR, RAS-MAPK and PI3K-AKT pathways, and possible influence by KRAS or PIK3CA mutation on therapy or chemoprevention targeting VDR.
Project description:The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2-5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.
Project description:Human cancer cells often exhibit impaired IGF2 expression and the underlying mechanisms are multifaceted and complex. Besides the well-known imprinting control region IGF2/H19-ICR, the involvement of a differentially methylated region in the promoter P0 of IGF2 gene (IGF2-DMR0) has been suggested. Here, we evaluate several mechanisms potentially leading to up- and/or down-regulation of IGF2 expression in prostate cancer and present a novel role of Kruppel-like factor 4 (KLF4) as a transcriptional regulator of IGF2 binding in IGF2-DMR0.Putative binding sites for transcription factors were identified in IGF2-DMR0 using JASPAR CORE database. Gene expressions were analyzed by RT-qPCR in prostate carcinoma and adjacent benign prostate hyperplasia samples obtained by radical prostatectomy (86 RP-PCa and 47 RP-BPH) and BPH obtained by transurethral prostate resection (13 TUR-BPH). Pyrosequencing and qMSP were used for DNA methylation studies in IGF2-DMR0, IGF2/H19-ICR and Glutathione-S-transferase-P1 (GSTP1) promoter. Loss of imprinting (LOI) was analyzed by RFLP. Copy number variation (CNV) test was performed using qBiomarker CNV PCR Assay. KLF4-binding and histone-modifications were analyzed by ChIP-qPCR in prostate cancer cell lines exhibiting differentially methylated IGF2-DMR0 (LNCaP hypomethylated and DU145 hypermethylated). KLF4 protein was analyzed by western blot. Statistical associations of gene expression to methylation, IGF2 LOI and CNV were calculated by Mann-Whitney-U-test. Correlations between gene expression and methylation levels were evaluated by Spearman's-Rank-Correlation-test.We found a significant reduction of IGF2 expression in the majority of RP-PCa and RP-BPH in comparison to TUR-BPH. Analyzing potential molecular reasons, we found in RP-PCa and RP-BPH in comparison to TUR-BPH a significant hypomethylation of IGF2-DMR0, which coincided with hypermethylation of GSTP1-promoter, a prominent marker of prostate tumors. In contrast, IGF2 LOI and CNV did not associate significantly with up- and/or down-regulation of IGF2 expression in prostate tumors. By analyzing IGF2-DMR0, we detected a consensus sequence for KLF4 with a z-score of 7.6. Interestingly, we found that KLF4 binds to hypomethylated (17%) IGF2-DMR0 enriched with H3K9me3 and H3K27me3 (LNCaP), but does not bind under hypermethylated (85%) and H3K4me3-enriched conditions (DU145). KLF4 expression was detected in TUR-BPH as well as in RP-BPH and RP-PCa and showed a highly significant correlation to IGF2 expression.Our study demonstrated that in human prostate cancer the impairment of IGF2 expression is accompanied by hypomethylation of IGF2-DMR0. We revealed that KLF4 is a putative transcriptional regulator of IGF2, which binds in IGF2-DMR0 in dependence of the prevailing epigenetic state in this region. Herewith we provide complementary new insights into IGF2 dysregulation mechanisms as a critical process in prostate tumorigenesis.
Project description:A central question in genomic imprinting is how parental-specific DNA methylation of imprinting control regions (ICR) is established during gametogenesis and maintained after fertilization. At the imprinted Igf2/H19 locus, CTCF binding maintains the unmethylated state of the maternal ICR after the blastocyst stage. In addition, evidence from Beckwith-Wiedemann patients and cultured mouse cells suggests that two Sox-Oct binding motifs within the Igf2/H19 ICR also participate in maintaining hypomethylation of the maternal allele. We found that the Sox and octamer elements from both Sox-Oct motifs were required to drive hypomethylation of integrated transgenes in mouse embryonic carcinoma cells. Oct4 and Sox2 showed cooperative binding to the Sox-Oct motifs, and both were present at the endogenous ICR. Using a mouse with mutations in the Oct4 binding sites, we found that maternally transmitted mutant ICRs acquired partial methylation in somatic tissues, but there was little effect on imprinted expression of H19 and Igf2. A subset of mature oocytes also showed partial methylation of the mutant ICR, which suggested that the Sox-Oct motifs provide some protection from methylation during oogenesis. The Sox-Oct motifs, however, were not required for erasure of paternal methylation in primordial germ cells, which indicated that the oocyte methylation was acquired post-natally. Maternally inherited mutant ICRs were unmethylated in blastocysts, which suggested that at least a portion of the methylation in somatic tissues occurred after implantation. These findings provide evidence that Sox-Oct motifs contribute to ICR hypomethylation in post-implantation embryos and maturing oocytes and link imprinted DNA methylation with key stem cell/germline transcription factors.