SAT-202 DNA Methylation Changes in the GHR, IGF1R and INSR Genes in Foreskin from 46,XY DSD Children Born Adequate (AGA) or Small for Gestational Age (SGA)

Insulin-like growth factor types 1 and 2 (IGF-1; IGF-2) and insulin-like peptides are all members of the insulin superfamily of peptide hormones but bind to several distinct classes of membrane receptor. Like the insulin receptor, the IGF-1 receptor is a heterotetrameric receptor tyrosine kinase, whereas the IGF-2/ mannose 6-phosphate receptor is a single transmembrane domain protein that is thought to function primarily as clearance receptors. We recently reported that IGF-1 and IGF-2 stimulate the ERK1/2 cascade by triggering sphingosine kinase-dependent "transactivation" of G protein-coupled sphingosine-1-phosphate receptors. To determine which IGF receptors mediate this effect, we tested seven insulin family peptides, IGF-1, IGF-2, insulin, and insulin-like peptides 3, 4, 6, and 7, for the ability to activate ERK1/2 in HEK293 cells. Only IGF-1 and IGF-2 potently activated ERK1/2. Although IGF-2 was predictably less potent than IGF-1 in activating the IGF-1 receptor, they were equipotent stimulators of ERK1/2. Knockdown of IGF-1 receptor expression by RNA interference reduced the IGF-1 response to a greater extent than the IGF-2 response, suggesting that IGF-2 did not signal exclusively via the IGF-1 receptor. In contrast, IGF-2 receptor knockdown markedly reduced IGF-2-stimulated ERK1/2 phosphorylation, with no effect on the IGF-1 response. As observed previously, both the IGF-1 and the IGF-2 responses were sensitive to pertussis toxin and the sphingosine kinase inhibitor, dimethylsphingosine. These data indicate that endogenous IGF-1 and IGF-2 receptors can independently initiate ERK1/2 signaling and point to a potential physiologic role for IGF-2 receptors in the cellular response to IGF-2.

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R(flox/flox)) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.

722 IDENTIFICATION OF EPIGENETIC CHANGES IN CHRONIC BLADDER INFLAMMATION

Being born small for gestational age (SGA), a proxy for intrauterine growth restriction (IUGR), and prenatal famine exposure are both associated with a greater risk of metabolic disease. Both associations have been hypothesized to involve epigenetic mechanisms. We investigated whether prenatal growth restriction early in pregnancy was associated with changes in DNA methylation at loci that were previously shown to be sensitive to early gestational famine exposure. We compared 38 individuals born preterm (<32 weeks) and with a birth weight too low for their gestational age (-1SDS) and a normal postnatal growth (>-1SDS at 3 months post term; “AGA”). The SGA individuals were not only lighter at birth, but also had a smaller length (P=3.3x10-13) and head circumference at birth (P=4.1x10-13). The DNA methylation levels of IGF2, GNASAS, INSIGF and LEP were 48.5%, 47.5%, 79.4% and 25.7% respectively. This was not significantly different between SGA and AGA individuals. Risk factors for being born SGA, including preeclampsia and maternal smoking, were also not associated with DNA methylation at these loci. Growth restriction early in development is not associated with DNA methylation at loci shown to be affected by prenatal famine exposure. Our and previous results by others indicate that prenatal growth restriction and famine exposure may be associated with different epigenetic changes or non epigenetic mechanisms that may lead to similar later health outcomes.

Disclosure: L. Zoff: None. F.M. Garcia: None. G. Veneruzzo: None. G. Aschettino: None. M. Valdora: None. D. Rodriguez: None. M.C. Mattone: None. N. Perez Garrido: None. R.M. Marino: None. C. Alonso: None. G. Guercio: None. A. Belgorosky: None. M.S. Baquedano: None.About 11% of all children born annually in the world are defined as SGA. Approximately 15% of them will continue to present with low height for their ages, associated with a non-catch-up growth phenotype. However, the pathophysiological mechanism underlying these findings is unknown. Several studies have suggested that the embryonic environmental characteristics of patients SGA could induce changes at the epigenetic level that affect gene transcription and would be stable throughout life. The GH/IGF1 axis and insulin are crucial stimulators of growth, cell proliferation, and metabolism. This study aimed to compare the methylation status of GHR, IGF1R, and INSR genes in peripheral blood in a cohort of boys with idiopathic short stature without GH-treatment who were born SGA (birthweight <10th percentile for the gestation, n=14; 4-13yr) and in age-matched control boys (CTRL, n=19; 4-15yr)using targeted deep-amplicon bisulfite sequencing on a MiSeq system. Sequencing libraries of 4 promoter CpG-rich regions for GHR, 3 for IGF1R and 5 for INSR (104, 103 and 141 CpG sites, respectively) were analyzed for mean methylation values of all CpG sites using amplikyzer2 software. The PCA score plot showed a clear separation between the CTRL and SGA groups. The two principal components explained 92.5% of the variance. The mean CpG methylation levels of the SP1-responsive IGF1R proximal promoter [GRCh38:15:98648387: 98648592:1 (-152 to +53)] were significantly higher in SGA than in CTRL (Mann-Whitney-Wilcoxon test, p<0.05). Compared with CNTRL, the CpG methylation levels of the TATA-less V2 proximal promoter as well as within the V2, V3, and V9 alternative exons in the regulatory 5´-UTR of the GHR gene [GRCh38:5:42423379:42423768:1 (-59 to +330), GRCh38:5:42424357:42424668:1 (+919 a +1230) and GRCh38:5:42424672:42424968:1 (+1234 a +1530)] were lower in SGA (p<0.05). Promoter DNA methylation is a fundamental epigenetic mark associated with transcriptional repression during development. According to our findings, it could be speculated that epigenetic repression of IGF1R might contribute to the lack of catch-up growth in short SGA patients. Finally, the human GHR gene is a complex transcription unit, and the impact or functional relevance of our findings and its relation to lessen activation of IGF1R pathways remains to be elusive.Presentation: Saturday, July 12, 2025

Treatment of non-small-cell lung cancer (NSCLC) patients possessing EGFR-activating mutations with tyrosine kinase inhibitors (TKIs) can confer an initial promising response. However, TKI resistance inevitably arises. Numerous TKI resistance mechanisms are identified including EGFR secondary mutations, bypass receptor tyrosine kinase (RTK) signaling, and cellular transition e.g. epithelial-mesenchymal transition (EMT). To increase the knowledge of TKI resistance we performed an epigenetic screen to identify small non-coding (nc) genes with DNA methylation alterations in HCC827 NSCLC EGFR-mutated cells with acquired TKI resistance. We analyzed Infinium Methylation EPIC 850K Array data for DNA methylation changes present in both TKI-resistant HCC827 cells with EMT and MET-amplification. Hereby, we identified that the polymorphic maternal imprinted gene nc886 (vtRNA2-1) has a decrease in promoter DNA methylation in TKI-resistant cells. This epigenetic change was associated with an increase in the expression of nc886. The induction of EMT did not affect nc886 expression. CRISPR/Cas9-mediated distortion of the nc886 sequence increased the sensitivity of HCC827 cells towards TKI. Finally, nc886 sequence distortion hindered MET RTK activation and instead was EMT the endpoint TKI resistance mechanism. In conclusion, the expression of nc886 contributes to TKI resistance in the HCC827 NSCLC cell line by supporting cell survival and selection of the endpoint TKI resistance mechanism. We propose DNA methylation and expression changes for nc886 to constitute a novel TKI resistance contributing mechanism in NSCLC.

Aim: To investigate DNA methylation changes in placenta tissues associated with small for gestational age (SGA). Materials & methods: A prospective cohort study consisting of 1292 pregnant women from China (including 39 SGA with placenta tissues) was performed, microarray and pyrosequencing were conducted. Results:Total 2012 methylation variable positions stood out from all probes (p<0.05; Δβ>0.2). In SGA cases, a CpG site within ANKRD20B showed lower methylation level (p=0.032) than appropriate for gestational age in validation cohort. Five sites within FAM198A (p=0.047, 0.050, 0.039, 0.026 and 0.043, respectively) had a reduced methylation in male newborns whose mother had preconception folic acid supplementation. Conclusion: DNA methylation changes in placenta tissues may be associated with SGA, maternal preconception folic acid supplementation status and also be fetal sex-specific.

A Phase I Biological Study of Azacitidine (Vidaza) to Determine the Optimal Biological Dose and Route of Administration.

BackgroundCri du chat (also called 5p deletion, or monosomy 5p) syndrome is a genetic disease caused by deletions of various lengths in the short (p) arm of chromosome 5. Genetic analysis and phenotyping have been used to suggest dose-sensitive genes in this region that may cause symptoms when a gene copy is lost, but the heterogeneity of symptoms for patients with similar deletions complicates the picture. The epigenetics of the syndrome has only recently been looked at with DNA methylation measurements of blood from a single patient, suggesting epigenetic changes in these patients. Here, we conduct the deepest epigenetic analysis of the syndrome to date with DNA methylation analysis of eight Cri du chat patients with sibling- and age-matched controls.ResultsThe genome-wide patterns of DNA methylation in the blood of Cri du chat patients reveal distinct changes compared to controls. In the p-arm of chromosome 5 where patients are hemizygous, we find stronger changes in methylation of CpG sites than what is seen in the rest of the genome, but this effect is less pronounced in gene regulatory sequences. Gene set enrichment analysis using patient DNA methylation changes in gene promoters revealed enrichment of genes controlling embryonic development and genes linked to symptoms which are among the most common symptoms of Cri du chat syndrome: developmental delay and microcephaly. Importantly, this relative enrichment is not driven by changes in the methylation of genes on chromosome 5. CpG sites linked to these symptoms where Cri du chat patients have strong DNA methylation changes are enriched for binding of the polycomb EZH2 complex, H3K27me3, and H3K4me2, indicating changes to bivalent promoters, known to be central to embryonic developmental processes.ConclusionsFinding DNA methylation changes in the blood of Cri du chat patients linked to the most common symptoms of the syndrome is suggestive of epigenetic changes early in embryonic development that may be contributing to the development of symptoms. However, with the present data we cannot conclude about the sequence of events between DNA methylation changes and other cellular functions—the observed differences could be directly driving epigenetic changes, a result of other epigenetic changes, or they could be a reflection of other gene regulatory changes such as changed gene expression levels. We do not know which gene(s) on the p-arm of chromosome 5 that causes epigenetic changes when hemizygous, but an important contribution from this work is making the pool of possible causative genes smaller.

It has been 200 years since Parkinson’s disease (PD) was first described, yet many aspects of its etiopathogenesis remain unclear. PD is a progressive and complex neurodegenerative disorder caused by genetic and environmental factors including aging, nutrition, pesticides and exposure to heavy metals. DNA methylation may be altered in response to some of these factors; therefore, it is proposed that epigenetic mechanisms, particularly DNA methylation, can have a fundamental role in gene–environment interactions that are related with PD. Epigenetic changes in PD-associated genes are now widely studied in different populations, to discover the mechanisms that contribute to disease development and identify novel biomarkers for early diagnosis and future pharmacological treatment. While initial studies sought to find associations between promoter DNA methylation and the regulation of associated genes in PD brain tissue, more recent studies have described concordant DNA methylation patterns between blood and brain tissue DNA. These data justify the use of peripheral blood samples instead of brain tissue for epigenetic studies. Here, we summarize the current data about DNA methylation changes in PD and discuss the potential of DNA methylation as a potential biomarker for PD. Additionally, we discuss environmental and nutritional factors that have been implicated in DNA methylation. Although the search for significant DNA methylation changes and gene expression analyses of PD-associated genes have yielded inconsistent and contradictory results, epigenetic modifications remain under investigation for their potential to reveal the link between environmental risk factors and the development of PD.

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

Disclosure: L. Zoff: None. F. Garcia: None. G. Aschettino: None. G. Veneruzzo: None. M.C. Mattone: None. N. Perez Garrido: None. M. Juanes: None. N.I. Saraco: None. C. Alonso: None. G. Guercio: None. A. Belgorosky: None. M.S. Baquedano: None. Growth is influenced by genetic, nutritional, environmental, and hormonal factors, but it proceeds in a predictable pattern characterized by a constant growth deceleration between childhood and adolescence. This growth deceleration is coordinated in tissues and organs in order to maintain body proportions. Growth hormone (GH)/ insulin-like growth factor type 1 (IGF1) axis and insulin are crucial stimulators of growth, cell proliferation and metabolism. However, their levels do not change with age in a pattern that would explain the prepubertal decline in growth rate. DNA methylation represents a fundamental epigenetic mark that is associated with transcriptional repression during development. We hypothesize that the postnatal growth pattern could be orchestrated by epigenetic mechanisms. In order to analyze age related physiological changes, we evaluated promoter methylation in the GHR, IGF1R and INSR genes in peripheral blood from 40 healthy children of both sexes (females (F) n=21 and males (M) n=19), between 3 and 15 years old by using targeted deep-amplicon bisulfite sequencing on a MiSeq system. Sequencing libraries of 4 promoter CpG rich regions for GHR, 3 for IGF1R and 5 for INSR (104, 103 and 141 CpG sites, respectively) were analyzed for mean methylation values of all CpG sites using amplikyzer2 software. The results would suggest a sexual dimorphism in the epigenetic regulation of GH, IGF1 and insulin receptors gene expression. A significant negative correlation between GHR methylation and age was observed in both sexes (Multiple Spearman correlation, p&amp;lt;0.05), but in different GHR promoter regions. Specifically, a decrease in themethylation levels with age were detected in CpG-8, CpG+183 and CpG+243 in F; and in CpG+343, CpG+346, CpG+385, CpG+391, CpG+407 and CpG+487 in M. In contrast, in the IGF1R promoter region, a positive correlation was observed exclusively in F, between mean methylation levels and age in CpG-51, CpG-43, CpG-41, CpG-23, CpG-17, CpG-15, CpG-13, CpG-11, and CpG-9, p&amp;lt;0.05. Male-specific positive correlation between INSR promotermethylation values and age were found in CpG-768, CpG+10, CpG+31, CpG+87, CpG+95, CpG+163, CpG+199, CpG+206, CpG+238, CpG+700, CpG+815 and CpG+900, p&amp;lt;0.05.Taking together, our findings are in agreement with the hypothesis that age-associated DNA promoter methylation changes could be involved in the physiological growth pattern. Finally, the observed sexual dimorphism suggests age- and sex-dependent regulatory mechanisms for normal growth. Presentation: Thursday, June 15, 2023

BackgroundSalivary glands (SG) from Sjögren’s syndrome (SS)-patients show chronic inflammation and altered unfolded protein response (UPR). Pro-inflammatory cytokines induce epigenetic changes including DNA methylation, a dynamic and complex process where...

Replication of influenza virus in the host cells results in production of immune mediators like cytokines. Excessive secretion of cytokines (hypercytokinemia) has been observed during highly pathogenic avian influenza virus (HPAI-H5N1) infections resulting in high fatality rates. The exact mechanism of hypercytokinemia during influenza virus infection is still not known completely. As promoter DNA methylation changes are linked with expression changes in genes, we intend to identify whether changes in promoter DNA methylation have any role in expression of cytokines during influenza A virus infection. A panel of 24 cytokine genes and genes known to be involved in inflammatory response were analyzed for their promoter DNA methylation changes during influenza A virus infections. Four different strains of influenza A viruses, viz. H5N1, H1N1, pandemic (2009) H1N1, and a vaccine strain of H5N1, were used for the study. We found seven of the total 24 inflammatory genes studied, showing significant changes in their promoter methylation levels in response to virus infection. These genes included proinflammatory cytokines CXCL14, CCL25, CXCL6, and interleukines IL13, IL17C, IL4R. The changes in DNA methylation levels varied across different strains of influenza viruses depending upon their virulence. Significant promoter hypomethylation in IL17C and IL13 genes was observed in cells infected with HPAI-H5N1 virus compared with other influenza viruses. This decrease in methylation was found to be positively correlating with the increased expression of these genes. Analysis of IL17C promoter region using bisulfite sequencing resulted in identification of a CpG site within Retinoid X receptor-alpha (RXR-α) transcription factor binding site undergoing demethylation specifically in H5N1-infected cells but not in other influenza-infected cells. Thus, the study could demonstrate that changes in promoter methylation in certain specific cytokine genes actually have a possible role in their expression changes during influenza A virus infection.

Variations in DNA methylation levels in the placenta are thought to influence gene expression and are associated with complications of pregnancy, like fetal growth restriction (FGR). The most important cause for FGR is placental dysfunction. Here, we examined whether changes in DNA methylation, followed by gene expression changes, are mechanistically involved in the etiology of FGR. In this retrospective case-control study, we examined the association between small-for-gestational-age (SGA) children and both DNA methylation and gene expression levels of the genes WNT2, IGF2/H19, SERPINA3, HERVWE1, and PPARG in first-trimester placental tissue. We also examined the repetitive element LINE-1. These candidate genes have been reported in the literature to be associated with SGA. We used first-trimester placental tissue from chorionic villus biopsies. A total of 35 SGA children (with a birth weight below the 10th percentile) were matched to 70 controls based on their gestational age. DNA methylation levels were analyzed by pyrosequencing and mRNA levels were analyzed by real-time PCR. None of the average DNA methylation levels, measured for each gene, showed a significant difference between SGA placental tissue compared to control tissue. However, hypermethylation of WNT2 was detected on two CpG positions in SGA. This was not associated with changes in gene expression. Apart from two CpG positions of the WNT2 gene, in early placenta samples, no evident changes in DNA methylation or expression were found. This indicates that the already reported changes in term placenta are not present in the early placenta, and therefore must arise after the first trimester.