IGF2 Methylation Research Articles

Replacing sperm with genotyped haploid androgenetic blastomeres to generate cattle with predetermined paternal genomes

Abstract Although meiosis plays an essential role for the survival of species in natural selection, the genetic diversity resulting from sexual reproduction impedes human-driven strategies to transmit the most suitable genomes for genetic improvement, forcing breeders to select diploid genomes generated after fertilization, that is, after the encounter of sperm and oocytes carrying unknown genomes. To determine whether genomic assessment could be used before fertilization, some androgenetic haploid morula-stage bovine embryos derived from individual sperm were biopsied for genomic evaluation and others used to reconstruct “semi-cloned” (SC) diploid zygotes by the intracytoplasmic injection into parthenogenetically activated oocytes, and the resulting embryos were transferred to surrogate females to obtain gestations. Compared to controls, in vitro development to the blastocyst stage was lower and fewer surrogates became pregnant from the transfer of SC embryos. However, fetometric measurements of organs and placental membranes of all SC conceptuses were similar to controls, suggesting a normal post-implantation development. Moreover, transcript amounts of imprinted genes IGF2, IGF2R, PHLDA2, SNRPN and KCNQ1OT1 and methylation pattern of the KCNQ1 DMR were unaltered in SC conceptuses. Overall, this study shows that sperm can be replaced by genotyped haploid embryonic-derived cells to produce bovine embryos carrying a predetermined paternal genome and viable first trimester fetuses after transfer to female recipients.

Male infertility is associated with differential DNA methylation signatures of the imprinted gene GNAS and the non-imprinted gene CEP41.

To investigate whether the DNA methylation profiles of GNAS(20q13.32), MEST(7q32.2), MESTIT1(7q32.2), IGF2(11p15.5), H19 (7q32.2), and CEP41(7q32.2) genes are related to the transcriptomic and epigenomic etiology of male infertility. The DNA methylation levels of spermatozoa were obtained from fertile (n = 30), oligozoospermic (n = 30), and men with normal sperm count (n = 30). The methylation status of each CpG site was categorized as hypermethylated or hypomethylated. Expression levels of target gene transcripts were determined using real-time PCR. The oligozoospermia showed a higher frequency of hypermethylation at GNASAS 1st, 3rd, and 5th CpG dinucleotides (66.7%, 73.3%, 73.3%) compared to the fertile group (33.3%, 33.3%, 40%, respectively). The normal sperm count exhibited a higher frequency of hypermethylation at the 3rd CpG of CEP41 (46.7%) than the fertile group (16.7%). Normal sperm count was predicted by CEP41 hypermethylation (OR = 1.750, 95%CI 1.038-2.950) and hypermethylation of both CEP41 and GNASAS (OR = 2.389, 95%CI 1.137-5.021). Oligozoospermia was predicted solely by GNASAS hypermethylation (OR = 2.460, 95%CI 1.315-4.603). In sperms with decreased IGF2 expression in the fertile group, we observed hypomethylation in the 2nd CpG of IGF2 antisense (IFG2AS), and hypermethylation in the 1st, 2nd, and 4th CpGs of H19. No significant relationship was found between IGF2 expression and methylation status of IGF2AS and H19 in infertile groups. The disappearance of the relationship between IGF2 expression and IGF2AS and H19 methylations in the infertile group provides new information regarding the disruption of epigenetic programming during spermatogenesis. A better understanding of sperm GNASAS and CEP41 hypermethylation could advance innovative diagnostic markers for male infertility.

Methylation Status of IGF-Axis Genes in the Placenta of South Indian Neonates with Appropriate and Small for Gestational Age

Objective Altered methylation patterns of insulin-like growth factor (IGF)-axis genes in small for gestational age (SGA) have been reported in different populations. In the present study, we analyzed the methylation status of IGF-axis genes in the placenta of appropriate for gestational age (AGA) and SGA neonates of South Indian women. Methods Placental samples were collected from AGA (n = 40) and SAG (n = 40) neonates. The methylation of IGF-axis genes promoter was analyzed using MS-PCR. Results IGF2, H19, IGF1, and IGFR1 genes promoter methylation was 2.5, 1.5, 5, and 7.5% lower in SGA compared to AGA, respectively. Co-methylation of IGF-axis genes promoter was 40% and 20% in AGA and SGA, respectively. IGF-axis gene promoter methylation significantly (p < 0.05) influenced the levels of IGFBP3 protein, birth weight, mitotic index, gestational weeks, and IGFR1 and IGFR2 gene expression. Conclusion IGF-axis genes methylation was lower in SGA than in AGA, and the methylation significantly influenced the IGF-axis components.

Retraction.

Retraction: "Downregulation of long noncoding RNA H19 rescues hippocampal neurons from apoptosis and oxidative stress by inhibiting IGF2 methylation in mice with streptozotocin-induced diabetes mellitus," by Jin-Lu Yu, Chao Li, Li-He Che, Yu-Hao Zhao, and Yun-Bao Guo, J Cell Physiol. 2019; 10655-10670: The above article, published online on 8 December 2018 in Wiley Online Library (https://doi.org/10.1002/jcp.27746), has been retracted by agreement between the authors, the journal's Editor in Chief, Prof. Dr. Gregg Fields, and Wiley Periodicals LLC. The retraction has been agreed because the experimental data in the article could not be reproduced in several attempts during further study of Long noncoding RNA H19 in neurological diseases. The authors state that some of the western blot analysis and RT-qPCR experiments in this study could not be reproduced by their recent work and the conclusions in this article are no longer tenable. The authors apologize for the inconvenience caused to the journal and the readers.

The roles of IGF2 and DNMT methylation and elongase6 related fatty acids in metabolic syndrome.

Background: The prevalence of metabolic syndrome (MetS) has increased along with rapid socio-economic development in China in recent decades, aggravating the burden of the health care system. Both plasma levels of fatty acids (FAs) and aberrant DNA methylation profiles are associated with MetS risk. However, studies exploring the role of DNA methylation and FAs simultaneously in MetS etiology are sparse. Objective: We aimed to explore the association between the gene methylation levels of insulin-like growth factor II (IGF2), H19, DNA methyltransferases 1 (DNMT1), DNA methyltransferases 3a (DNMT3a), and DNA methyltransferases 3b (DNMT3b) and MetS risk, and the etiological role of elongation of very-long-chain fatty acid elongase 6 (ELOVL6) related fatty acids. Method: Plasma levels of FAs were measured using a Gas Chromatography-Flame Ionization Detector (GC-FID) after organic extraction, and gene methylation was quantified using a real-time Quantitative Polymerase Chain Reaction (Q-PCR) detecting system after bisulfite treatment. The C18/C16 ratio was used as the indicator of ELOVL6 activity. Odds Ratio (OR) and 95% Confidence Interval (CI) were estimated with logistic regression. Results: Methylation levels in IGF2 and DNMT3a were not significantly associated with MetS risk. However, when stratified by C18/C16 ratio (high vs. low), positive associations were observed between the risk of MetS and methylation levels (>median) of IGF2a3 (OR = 3.1, 95% CI = 1.3-7.5) and DNMT3a (OR = 2.5, 95% CI = 1.1-5.8) genes, in individuals with lower C18/C16 ratios, while no significant associations were observed in subjects with high C18/C16 ratios. Conclusion: Methylation levels in IGF2 and DNMT3a genes may affect the risk of MetS in an ELOVL6 activity-dependent way among Chinese adults. Further studies in other populations are needed to validate this finding.

The IGF2 methylation score for adrenocortical cancer: an ENSAT validation study.

Adrenocortical carcinoma (ACC) is diagnosed using the histopathological Weiss score (WS), but remains clinically elusive unless it has metastasized or grows locally invasive. Previously, we proposed the objective IGF2 methylation score as diagnostic tool for ACC. This multicenter European cohort study validates these findings. Patient and tumor characteristics were obtained from adrenocortical tumor patients. DNA was isolated from frozen specimens, where after DMR2, CTCF3, and H19 were pyrosequenced. The predictive value of the methylation score for malignancy, defined by the WS or metastasis development, was assessed using receiver operating characteristic curves and logistic and Cox regression analyses. Seventy-six ACC patients and 118 patients with adrenocortical adenomas were included from seven centers. The methylation score and tumor size were independently associated with the pathological ACC diagnosis (OR 3.756 95% CI 2.224-6.343; OR 1.467 95% CI 1.202-1.792, respectively; Hosmer-Lemeshow test P = 0.903), with an area under the curve (AUC) of 0.957 (95% CI 0.930-0.984). The methylation score alone resulted in an AUC of 0.910 (95% CI 0.866-0.952). Cox regression analysis revealed that the methylation score, WS and tumor size predicted development of metastases in univariate analysis. In multivariate analysis, only the WS predicted development of metastasis (OR 1.682 95% CI 1.285-2.202; P < 0.001). In conclusion, we validated the high diagnostic accuracy of the IGF2 methylation score for diagnosing ACC in a multicenter European cohort study. Considering the known limitations of the WS, the objective IGF2 methylation score could potentially provide extra guidance on decisions on postoperative strategies in adrenocortical tumor patients.

Methylation patterns in dysplasia in inflammatory bowel disease patients

Background and aims: Inflammatory Bowel Disease (IBD) with colonic involvement increases colorectal cancer risk. However, the distinction between IBD related and sporadic dysplasia in IBD patients is difficult. Some data favors the importance of abnormal DNA methylation in IBD-related carcinogenesis. We aimed to define methylation patterns in patients with colonic cancer or dysplasia diagnosis following an IBD diagnosis.Methods: Multicentric cross-sectional study-91 samples from colonic mucosa with/without dysplasia from 9 patients with IBD-related dysplasia/cancer and 26 patients with IBD and sporadic dysplasia/cancer were included. Methylation patterns of CpG islands in the promoter regions of 67 genes were studied by Methylation-specific Multiplex Ligation-dependent Probe Amplification.Results: Mean age at IBD diagnosis: 42 ± 16 years;at dysplasia diagnosis: 56 ± 14 years. Twenty-ninepatients had ulcerative colitis. Twenty-five patients had at least 1 lesion endoscopically described as adenoma-like, 4 at least 1 non-adenoma like, 3 had cancer and 3 had dysplasia in flat mucosa. No patient had both adenoma-like and non-adenoma-like lesions. Patients with an IBD-related lesion were significantly younger at IBD diagnosis (p = .003) and at dysplasia/cancer diagnosis (p = .039). Promoter methylation of IGF2, RARB, ESR1, CHFR, CDH13, WT1, GATA5, WIF1genes was significantly associated to dysplasia/cancer; methylation of MSH6, TIMP3 was significantly associated to IBD-related dysplasia/cancer. Promoter methylation of MSH6, MSH3, RUNX3, CRABP1, TP73, RARB, CDH13, PAX5, WT1, THBS1, TP53, SFRP1, WIF1, APAF1, BCL2 genes was significantly associated to active IBD.Conclusions: Methylation analysis, namely of MSH6, may contribute to the classification of dysplastic lesions in IBD– to be further tested in prospective studies.

P829 Methylation patterns in dysplasia in inflammatory bowel disease patients

Abstract Background Inflammatory Bowel Disease (IBD) with colonic involvement increases colorectal cancer risk. However, the distinction between IBD related and sporadic dysplasia in IBD patients is difficult. Some data favours the importance of abnormal DNA methylation in IBD-related carcinogenesis. Our study aimed to define methylation patterns in patients with a colonic cancer or dysplasia diagnosis following an IBD diagnosis. Methods Multicentric cross-sectional study- 91 samples from colonic mucosa with/without dysplasia from 9 patients with IBD-related dysplasia/cancer and 26 patients with IBD and sporadic dysplasia/cancer were included. Methylation patterns of CpG islands in the promoter regions of 67 genes were studied by Methylation-specific Multiplex Ligation-dependent Probe Amplification. Results Mean age at IBD diagnosis: 42±16 years; at dysplasia diagnosis: 56± ± 14 years. Twenty-nine patients had ulcerative colitis. Twenty-five patients had at least 1 lesion endoscopically described as adenoma-like, 4 at least 1 non-adenoma like, 3 had cancer and 3 had dysplasia in flat mucosa. No patient had both adenoma-like and non-adenoma-like lesions. Patients with an IBD-related lesion were significantly younger at IBD diagnosis (p = 0.003) and at dysplasia/cancer diagnosis (p = 0.039). Promoter methylation of IGF2, RARB, ESR1, CHFR, CDH13, WT1, GATA5, WIF1 genes was significantly associated to dysplasia/cancer; methylation of MSH6, TIMP3 was significantly associated to IBD-related dysplasia/cancer. Promoter methylation of MSH6, MSH3, RUNX3, CRABP1, TP73, RARB, CDH13, PAX5, WT1, THBS1, TP53, SFRP1, WIF1, APAF1, BCL2 genes was significantly associated to active IBD. Conclusion Methylation analysis, namely of MSH6, may contribute to the classification of dysplastic lesions in IBD– to be further tested in prospective studies.

Half Smooth Tongue Sole (Cynoglossus semilaevis) Under Low Salinity Stress Can Change Hepatic igf2 Expression Through DNA Methylation

Salinity is a crucial environmental stress that severely affects fish growth and survival. Under environmental stress, DNA methylation plays an important role in gene expression and genome function. To better understand the epigenetic regulation mechanism of igf2 under low salinity stress, we analyzed the DNA methylation at 5′UTR, exon1, intron1, and exon2, and analyzed the relationship of DNA methylation with mRNA abundance as well as the special single CpG sites methylation patterns of igf2 in the liver of half smooth tongue sole under low salinity (15) for 0, 7, and 60 d. When exposed to low salinity, DNA methylation at 5′UTR and exon2 remained stable, while it was up-regulated firstly and then down-regulated at exon1 and intron1. Some single CpG sites of igf2 associated with low salinity, and most of these sites with significantly changed methylation levels (P 0.05) are located in intron1 area. The discrepant variation of single CpG sites methylation levels and igf2 expression further revealed that females and males showed different response to low salinity. Remarkably, the 38-CpG site of intron1 servers as a sexual marker. Additionally, our integrative analysis demonstrated that regional DNA of igf2 methylation had highly complex interplay on gene expression. The single CpG sites in intron1 were indispensable epigenetic markers under external environmental stress. Above all, to resist the low salinity stress, half smooth tongue sole liver can regulate the expression of igf2 through methylation of CpG sites in intron1.

Associations between in utero exposure to polybrominated diphenyl ethers, pathophysiological state of fetal growth and placental DNA methylation changes

BackgroundPolybrominated diphenyl ethers (PBDEs) are environmental chemicals with harmful effects on pregnancy, but their effects on adverse developmental outcomes are not fully understood. The placental DNA methylation is strongly influenced by prenatal environmental factors and has been linked to fetal growth. ObjectiveTo evaluate the association between in utero PBDEs exposure, placental DNA methylation changes (growth regulatory genes), and pathophysiology of fetal growth (birth outcomes, fetal growth retardation) in a population-based pregnancy cohort study. MethodsThis was a nested case-control study within the prospective Wenzhou Birth Cohort including 130 fetal growth retardation (FGR) cases and 130 healthy controls and their mothers recruited from June 2016 to June 2017. FGR was diagnosed based on the comprehensive evaluation of ultrasound results at 24, 28, and 32 weeks of gestation. Neonatal birth measurements were obtained from medical records. Gestational exposure to 19 PBDEs, including 13 lower BDE congeners (BDE-17–190) and 6 higher brominated BDE congeners (BDE-196–209), were determined by gas chromatography tandem mass spectrometry in the umbilical cord blood. Placental DNA methylation changes of one repetitive element (LINE1) and two candidate genes (HSD11B2, IGF2) were characterized by quantitative polymerase chain reaction-pyrosequencing. Multiple linear regression and logistic regression models were used to examine the associations among PBDEs exposure, fetal growth indicators, and DMR (differential methylation region) methylation fractions. Sobel tests were conducted to assess DNA methylation as a mediator in multivariate models. ResultsAfter excluding women who withdrew from the study or were lost to follow-up or failed to provide placenta or umbilical cord blood, 249 mother-newborn pairs (124 FGR cases, 125 controls) were included in the final analysis. Elevated BDE-206 (OR: 1.569, 95% CIs: 1.053–2.338), BDE-17–190 (OR: 2.860, 95% CIs: 1.233–6.634), BDE-196–209 (OR: 1.688, 95% CIs: 1.024–2.783) and ∑19PBDEs (OR: 2.387, 95% CIs: 1.220–4.668) concentrations were associated with increased risk of FGR in newborns. FGR birth was also associated with increased DNA methylation of HSD11B2 (OR: 1.145, 95% CIs: 1.032–1.270) and decreased DNA methylation of IGF2 (OR: 0.892, 95% CIs: 0.845–0.941). In addition, BDE-17–190 showed significant associations with DNA methylation of HSD11B2 and IGF2 (β: 1.127, 95% CIs: 0.069–2.186; β: −3.452, 95% CIs: −5.512–1.392), indicating placental DNA methylation changes of HSD11B2 and IGF2 were related to both lower BDE congeners exposure and fetal growth. Further mediation analyses showed that IGF2 methylation mediated about 40% of the effects of BDE-17–190 in umbilical cord blood on neonatal FGR. ConclusionWe report an inverse association between in utero exposures to PBDEs and fetal growth and provide evidence supporting epigenetic gene plasticity in these associations. Changes in placental DNA methylation might be part of the underlying biological pathway between prenatal PBDEs exposure and adverse fetal growth.

Associations between PBDEs exposure during pregnancy, fetal growth and aberrant DNA methylation of human placenta

TPS 711: The exposome, Exhibition Hall, Ground floor, August 26, 2019, 3:00 PM - 4:30 PM Aim: To evaluated the association between in utero PBDEs exposure and fetal growth and examined the potential role of altered DNA methylation of growth regulatory genes in these associations. Methods: A total of 249 mother-newborn pairs comprised of 124 fetal growth restriction (FGR) cases and 125 controls within the Wenzhou Birth Cohort were enrolled in this nested case–control study. In utero exposure of 19 PBDEs congeners, including thirteen lower PBDE congeners (BDE-17~190) and six higher brominate BDE congeners (BDE-196~209), were determined by gas chromatography tandem mass spectrometry in umbilical cord blood. Placental DNA methylation of LINE1, HSD11B2 and IGF2 was measured by quantitative polymerase chain reaction-pyrosequencing at differentially methylated regions (DMRs). Multiple linear regression and logistic regressions models were used to examine associations among PBDEs exposure, fetal growth indicators and DMR methylation fractions. Results: Concentrations of BDE-49, BDE-206, BDE-209, BDE-17~190 and ∑19PBDEs were significantly higher in FGR cases than those in normal controls. Meanwhile, DNA methylation of IGF2 and HSD11B2 showed significant differences between FGR group and control group, respectively. BDE-206 (OR: 1.569, 95% CIs: 1.053-2.338), BDE-17~190 (OR: 2.860, 95% CIs: 1.233-6.634), BDE-196~209 (OR: 1.688, 95% CIs: 1.024-2.783) and ∑19PBDEs (OR: 2.387, 95% CIs: 1.220-4.668) concentrations were associated with increased risk of FGR. Being FGR birth were also associated with increasing DNA methylation of HSD11B2 (OR: 1.145, 95% CIs: 1.032-1.270) and decreasing DNA methylation of IGF2 (OR: 0.892, 95% CIs: 0.845, 0.941). In addition, BDE-17~190 showed significant associations with placental DNA methylation of HSD11B2 and IGF2 (β: 1.127, 95% CIs: 0.069-2.186; β: -3.452, 95% CIs: -5.512--1.392). These results were consistent with the expected direction relating PBDEs exposure, fetal growth and placental DNA methylation. Conclusion: We report an inverse association between in utero exposures to PBDEs and fetal growth and provide evidence supporting epigenetic gene plasticity in these associations.

Combined effect between WT1 methylation and Helicobacter pylori infection, smoking, and alcohol consumption on the risk of gastric cancer.

Peripheral blood leukocyte DNA methylation status has been proposed to be a surrogate marker for evaluating susceptibility to gastric cancer (GC). Helicobacter pylori (Hpylori) infection, smoking, and alcohol consumption are known to induce gene methylation. A case-control study was performed to investigate the interactions between the methylation of two candidate genes and Hpylori infection, smoking, and alcohol consumption in the risk of GC. A total of 400 GC cases and 402 controls were included in this study. The methylation status of WT1 and IGF2 was semiquantitatively determined by using methylation-sensitive high-resolution melting assays. Hpylori IgG antibodies were detected by ELISA method. Based on the area under the curve (AUC), 0% methylated DNA and 0.5% methylated DNA were used as the cutoff values for WT1 and IGF2, respectively. WT1 methylation was significantly associated with increased GC risk (OR=1.65, 95% CI=1.09-2.51, P=.019), especially in males (OR=1.80, 95% CI: 1.10-2.95, P=.019) and older individuals (≥60years) (OR=2.03, 95% CI: 1.15-3.57, P=.014). A significant combination was observed between WT1 methylation and Hpylori infection, alcohol consumption, and smoking for the risk of GC (ORc =2.28, 95% CI=1.47-3.55, P=.003, ORc =2.19, 95% CI=1.37-3.51, P=.001, ORc =2.21, 95% CI=1.39-3.51, P=.001, respectively). However, no association between IGF2 methylation and the risk of GC was found in this study. WT1 methylation may serve as a new potential biomarker for GC susceptibility and can combine with Hpylori infection, smoking, and alcohol consumption to influence GC risk.

Abstract 3159: Do molecular markers differentiate between sessile serrated adenoma/polyps and hyperplastic polyps

Abstract Background: A significant proportion of colorectal cancers, as much as up to 30%, develop through the alternate serrated pathway. The precursor lesions in this pathway comprise of sessile serrated adenomas or polyps (SSA/SSPs) and hyperplastic polyps (HPs), both with a varying risk-profile associated with them for progression into colorectal cancer. Although both types of polyps can be identified histologically, the clinical challenge for gastroenterologists and pathologists remain for risk-prediction as to which of these polyps have a higher likelihood for subsequent development and progression into colorectal cancer. These data highlight the imperative need for the development of more specific and objective markers that can adequately differentiate between the two types of colonic polyps. Aim: The aim of our study was to determine a variety of molecular biomarkers that may distinguish SSA/Ps and HPs, and to establish biomarker profiles that associate with low vs. high-risk SSA/Ps. Methods: We conducted a retrospective study of all colonoscopies (n=12,085) performed at the Howard University Hospital between January 2010 to December 2015; of which 83% were conducted in patients of African American (AA) descent (n=10,027). Among AAs, pathology reports confirmed 4,070 patients with polyps, including 252 with SSA/Ps. Gene expression and mutation frequency profiles were analyzed in a total of 47 patients which included 62 specimens (29 SSPs, 26 HP, 3 tubular adenomas (TA) and 4 normal colonic tissues). From a panel of 51 candidate transcripts, we validated 4 RNA markers (MUC6, FSCN1, SEMG1, and TRNP1) using qRT-PCR. MSI and BRAF mutations were also analyzed. CIMP analysis was performed for the aberrant methylation of CACNA1G, IGF2, NEUROG1, RUNX, SOCS and MLH1. The frequency of gender, age groups, anatomic location, clinical/pathological symptoms and reason for colonoscopy in SSA/P patients was analyzed. The median age range for SSA/P diagnosis was between 50 to 64 years. Results: MUC6, SEMG1, TRNP1, and FSCN1 expression was significantly higher in SSA/Ps vs. HPs (P &amp;lt; 0.05); with corresponding fold differences of 37.2 10.7, 5.8 and 2.5, respectively. BRAF mutations were found in 55.6% of SSA/Ps as opposed to 12.0% of HPs (P &amp;lt; 0.05). The frequency of CIMP was higher in SSA/Ps and correlated with BRAF mutation, while the degree of MSI was more prevalent in HP (P &amp;gt; 0.05). SSA/P lesions were distal (67%). Conclusion: Our results show that MUC6 and SEMG1 expression and BRAF mutation have the strongest correlation with SSA/Ps in comparison to HPs. In addition, SSA/Ps were predominantly distal in location. These are novel and distinguishing features compared to the published literature in non-AA populations and may help explain why MSI and CIMP, usually linked to proximal lesions, are not optimal molecular biomarkers in AA patients with such serrated lesions. Note: This abstract was not presented at the meeting. Citation Format: Hassan Ashktorab, Saman Azam, Taraneh Tarjoman, Priyanka Kanth, Edward Lee, Mehdi Nouraie, Nazli Atefi, Babak Shokrani, Adeyinka Laiyemo, Ajay Goel, Mark W. Hazel, Ruoxin Yao, Angela Snow, Deborah Neklason, Don Delker, Hassan Brim. Do molecular markers differentiate between sessile serrated adenoma/polyps and hyperplastic polyps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3159.

Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage.

Abnormal imprinted genes methylation in spermatozoa has been shown to be associated with subfertility. However, the relationship between sperm DNA damage and specific imprinted genes methylation remains unclear. In this study, DNA methylation levels were determined at seven imprinted genes loci (H19, INS-IGF2, KCNQ1, MEG3, MEST, PEG3 and SNRPN) in 66 semen samples using the MSRE-qPCR method. The semen samples were divided into two groups according to the threshold value (25%) of DNA fragmentation index (DFI). We found that the mean methylation level at IGF2 (cg17037101) in the group with DFI≥25% was lower than that in the group with DFI<25% (13.7±3% vs. 31.5±5.3%, p=0.0053). However, the methylation levels of other CpGs did not differ from the imprinted genes. Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2 (cg17037101) methylation level was negatively correlated with sperm DFI (r=-0.448, p=0.0038), and the KCNQ1 (cg24932449) methylation level was positively correlated with sperm DFI (r=0.354, p=0.0273). Our results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.

Association of blood leukocyte DNA methylation at LINE-1 and growth-related candidate genes with pubertal onset and progression

ABSTRACTPuberty is a developmentally plastic phase. Variations in pubertal tempo have implications for the risk of later adult diseases. Influences on pubertal tempo have been widely discussed, but the underlying biological mechanisms remain unclear. Epigenetic modifications are known to regulate development processes; they could play an important role in affecting pubertal outcomes. We conducted a population-based analysis to investigate the association of peripubertal blood DNA methylation at LINE-1 and growth-related candidate genes with pubertal onset and progression in healthy adolescents. The analytic sample included 114 males and 129 females aged 10 to 18 years. DNA methylation at growth-related candidate loci IGF2, H19, HSD11B2, as well as LINE-1 repetitive elements were quantified. Cox survival and ordinal regression models were used to examine sex- and locus-specific associations of epigenetic markers with pubertal development using physician-assessed Tanner stages and self-reported menarche, adjusted for covariates. Among boys, DNA methylation at H19 was associated with later pubarche. HSD11B2 methylation was associated with earlier onset of pubic hair and genitalia development and slower pubertal progression. IGF2 was associated with later onset of genital development. Among girls, LINE-1 methylation was associated with later onset of breast development. For each percent increase of methylation at H19, there was 5% increased odds in the earlier onset of breast development. DNA methylation of IGF2 was associated with earlier onset of pubic hair. DNA methylation at genes known to influence early-life growth may also influence pubertal outcomes.

Reactive oxygen species-induced alterations in H19-Igf2 methylation patterns, seminal plasma metabolites, and semen quality.

This study was conducted in order to investigate the effects of reactive oxygen species (ROS) levels on the seminal plasma (SP) metabolite milieu and sperm dysfunction. Semen specimens of 151 normozoospermic men were analyzed for ROS by chemiluminescence and classified according to seminal ROS levels [in relative light units (RLU)/s/106 sperm]: group 1 (n = 39): low (ROS < 20), group 2 (n = 38): mild (20 ≤ ROS < 40), group 3 (n = 31): moderate (40 ≤ ROS < 60), and group 4 (n = 43): high (ROS ≥ 60). A comprehensive analysis of SP and semen parameters, including conventional semen characteristics, measurement of total antioxidant capacity (TAC), sperm DNA fragmentation index (DFI), chromatin maturation index (CMI), H19-Igf2 methylation status, and untargeted seminal metabolic profiling using nuclear magnetic resonance spectroscopy (1H-NMR), was carried out. The methylation status of H19 and Igf2 was significantly different in specimens with high ROS (P < 0.005). Metabolic fingerprinting of these SP samples showed upregulation of trimethylamine N-oxide (P < 0.001) and downregulations of tryptophan (P < 0.05) and tyrosine/tyrosol (P < 0.01). High ROS significantly reduced total sperm motility (P < 0.05), sperm concentration (P < 0.001), and seminal TAC (P < 0.001) but increased CMI and DFI (P < 0.005). ROS levels have a positive correlation with Igf2 methylation (r = 0.19, P < 0.05), DFI (r = 0.40, P < 0.001), CMI (r = 0.39, P < 0.001), and trimethylamine N-oxide (r = 0.45, P < 0.05) and a negative correlation with H19 methylation (r = - 0.20, P < 0.05), tryptophan (r = - 0.45, P < 0.05), sperm motility (r = - 0.20, P < 0.05), sperm viability (r = - 0.23, P < 0.01), and sperm concentration (r = - 0.30, P < 0.001). Results showed significant correlation between ROS levels and H19-Igf2 gene methylation as well as semen parameters. These findings are critical to identify idiopathic male infertility and its management through assisted reproduction technology (ART).

ART manipulation after controlled ovarian stimulation may not increase the risk of abnormal expression and DNA methylation at some CpG sites of H19,IGF2 and SNRPN in foetuses: a pilot study

BackgroundTo examine the effects of IVF, ICSI and FET, as well as in vitro culture, on the safety of offspring, this study was conducted from the perspective of genetic imprinting to investigate whether assisted reproductive technology would influence the parental and maternal imprinting genes.MethodsEighteen foetuses were collected from multifoetal reduction and divided into 6 groups: multifoetal reduction after IVF fresh transferred D3 embryos (n = 3), multifoetal reduction after IVF frozen transferred D3 embryos (n = 3), multifoetal reduction after IVF frozen transferred D5 embryos (n = 3), multifoetal reduction after ICSI fresh transferred D3 embryos (n = 3), multifoetal reduction after ICSI frozen transferred D3 embryos (n = 3), and multifoetal reduction after controlled ovarian hyperstimulation (COH) (n = 3). The imprinted genes H19, IGF2 and SNRPN were selected for analysis. The expression and DNA methylation at some CpG sites of H19, IGF2, and SNRPN were examined using real-time quantitative polymerase chain reaction (PCR) and pyrosequencing.ResultsThere were no significant differences in the mRNA expression levels among the groups. The mean percentage of H19 methylation (eight CpG sites), IGF2 methylation (five CpG sites) and SNRPN methylation (nine CpG sites) did not differ significantly.ConclusionsThe results suggest that ARTs after controlled ovarian stimulation (IVF, ICSI, cryopreservation and duration of in vitro culture) may not increase the risk of abnormal expression and DNA methylation at some CpG sites of H19, IGF2 and SNRPN in foetuses. Further study with strict design, expanded sample size and CpG sites is essential.

Maternal levels of endocrine disrupting chemicals in the first trimester of pregnancy are associated with infant cord blood DNA methylation

ABSTRACTEndocrine disrupting chemicals (EDCs) pose a public health risk through disruption of normal biological processes. Identifying toxicoepigenetic mechanisms of developmental exposure-induced effects for EDCs, such as phthalates or bisphenol A (BPA), is essential. Here, we investigate whether maternal exposure to EDCs is predictive of infant DNA methylation at candidate gene regions. In the Michigan Mother-Infant Pairs (MMIP) cohort, DNA was extracted from cord blood leukocytes for methylation analysis by pyrosequencing (n = 116) and methylation changes related to first trimester levels of 9 phthalate metabolites and BPA. Growth and metabolism-related genes selected for methylation analysis included imprinted (IGF2, H19) and non-imprinted (PPARA, ESR1) genes along with LINE-1 repetitive elements. Findings revealed decreases in methylation of LINE-1, IGF2, and PPARA with increasing phthalate concentrations. For example, a log unit increase in ΣDEHP corresponded to a 1.03 [95% confidence interval (CI): −1.83, −0.22] percentage point decrease in PPARA methylation. Changes in DNA methylation were also inversely correlated with PPARA gene expression determined by RT-qPCR (r = −0.34, P = 0.02), thereby providing evidence in support of functional relevance. A sex-stratified analysis of EDCs and DNA methylation showed that some relationships were female-specific. For example, urinary BPA exposure was associated with a 1.35 (95%CI: −2.69, −0.01) percentage point decrease in IGF2 methylation and a 1.22 (95%CI: −2.27, −0.16) percentage point decrease in PPARA methylation in females only. These findings add to a body of evidence suggesting epigenetically labile regions may provide a conduit linking early exposures with disease risk later in life and that toxicoepigenetic susceptibility may be sex specific.

A novel DNA methylation panel accurately detects colorectal cancer independently of molecular pathway

BackgroundColorectal cancer (CRC) is one of the most incident cancers, associated with significant morbidity and mortality, and usually classified into three main molecular pathways: chromosomal instability, microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). Currently, available screening methods are either costly or of limited specificity, impairing global implementation. More cost-effective strategies, including DNA methylation-based tests, might prove advantageous. Although some are already available, its performance is suboptimal, entailing the need for better candidate biomarkers. Herein, we tested whether combined use of APC, IGF2, MGMT, RASSF1A, and SEPT9 promoter methylation might accurately detect CRC irrespective of molecular subtype.MethodsSelected genes were validated using formalin-fixed paraffin-embedded tissues from 214 CRC and 50 non-malignant colorectal mucosae (CRN). Promoter methylation levels were assessed using real-time quantitative methylation-specific PCR. MSI and CIMP status were determined. Molecular data were correlated with standard clinicopathological features. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.ResultsExcept for IGF2, promoter methylation levels were significantly higher in CRC compared to CRN. A three-gene panel (MGMT, RASSF1A, SEPT9) identified malignancy with 96.6% sensitivity, 74.0% specificity and 91.5 positive predictive value (area under the curve: 0.97), independently of tumor location, stage, and molecular pathway.ConclusionsCombined promoter methylation analysis of MGMT/RASSF1A/SEPT9 displays a better performance than currently available epigenetic-based biomarkers for CRC, providing the basis for the development of a non-invasive assay to detect CRC irrespective of the molecular pathway.

The fetal programming effect of prenatal smoking on Igf1r and Igf1 methylation is organ- and sex-specific

ABSTRACTThe impact of prenatal smoke exposure (PSE) on DNA methylation has been demonstrated in blood samples from children of smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. As the identified differentially methylated genes can be associated with developmental processes, and insulin-like growth factors (IGFs) play a critical role in prenatal tissue growth, we hypothesized that PSE induces fetal programming of Igf1r and Igf1. Using a mouse model of smoking during pregnancy, we show that PSE alters promoter methylation of Igf1r and Igf1 and deregulates their gene expression in lung and liver of fetal (E17.5) and neonatal (D3) mouse offspring. By further comparing female versus male, lung versus liver, or fetal versus neonatal time point, our results demonstrate that CpG site-specific aberrant methylation patterns sex-dependently vary per organ and time point. Moreover, PSE reduces gene expression of Igf1r and Igf1, dependent on organ, sex, and offspring's age. Our results indicate that PSE may be a source of organ-specific rather than general systemic fetal programming. This is exemplified here by gene promoter methylation and mRNA levels of Igf1r and Igf1, together with a sex- and organ-specific naturally established correlation of both parameters that is affected by prenatal smoke exposure. Moreover, the comparison of fetuses with neonates suggests a CpG site-dependent reversibility/persistence of PSE-induced differential methylation patterns.