Is Robotic Surgery the Future for Resectable Esophageal Cancer?: A Systematic Literature Review of Oncological and Clinical Outcomes.

BACKGROUND: Radical esophagectomy for resectable esophageal cancer is a major surgical intervention, associated with considerable postoperative morbidity. The introduction of robotic surgical platforms in esophagectomy may enhance advantages of minimally invasive surgery enabled by laparoscopy and thoracoscopy, including reduced postoperative pain and pulmonary complications. This systematic review aims to assess the clinical and oncological benefits of robot-assisted esophagectomy. METHODS: A systematic literature search of the MEDLINE (PubMed), Embase and Cochrane databases was performed for studies published up to 1 August 2023. This review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocols and was registered in the PROSPERO database (CRD42022370983). Clinical and oncological outcomes data were extracted following full-text review of eligible studies. RESULTS: A total of 113 studies (n = 14,701 patients, n = 2455 female) were included. The majority of the studies were retrospective in nature (n = 89, 79%), and cohort studies were the most common type of study design (n = 88, 79%). The median number of patients per study was 54. Sixty-three studies reported using a robotic surgical platform for both the abdominal and thoracic phases of the procedure. The weighted mean incidence of postoperative pneumonia was 11%, anastomotic leak 10%, total length of hospitalisation 15.2 days, and a resection margin clear of the tumour was achieved in 95% of cases. CONCLUSIONS: There are numerous reported advantages of robot-assisted surgery for resectable esophageal cancer. A correlation between procedural volume and improvements in outcomes with robotic esophagectomy has also been identified. Multicentre comparative clinical studies are essential to identify the true objective benefit on outcomes compared with conventional surgical approaches before robotic surgery is accepted as standard of practice.

Comprehensive analysis of PM20D1 QTL in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complex disorder caused by a combination of genetic and non-genetic risk factors. In addition, an increasing evidence suggests that epigenetic mechanisms also accompany AD. Genetic and epigenetic factors are not independent, but multiple loci show genetic-epigenetic interactions, the so-called quantitative trait loci (QTLs). Recently, we identified the first QTL association with AD, namely Peptidase M20 Domain Containing 1 (PM20D1). We observed that PM20D1 DNA methylation, RNA expression, and genetic background are correlated and, in turn, associated with AD. We provided mechanistic insights for these correlations and had shown that by genetically increasing and decreasing PM20D1 levels, AD-related pathologies were decreased and accelerated, respectively. However, since the PM20D1 QTL region encompasses also other genes, namely Nuclear Casein Kinase and Cyclin Dependent Kinase Substrate 1 (NUCKS1); RAB7, member RAS oncogene family-like 1 (RAB7L1); and Solute Carrier Family 41 Member 1 (SLC41A1), we investigated whether these genes might also contribute to the described AD association. RESULTS: Here, we report a comprehensive analysis of these QTL genes using a repertoire of in silico methods as well as in vivo and in vitro experimental approaches. First, we analyzed publicly available databases to pinpoint the major QTL correlations. Then, we validated these correlations using a well-characterized set of samples and locus-specific approaches-i.e., Sanger sequencing for the genotype, cloning/sequencing and pyrosequencing for the DNA methylation, and allele-specific and real-time PCR for the RNA expression. Finally, we defined the functional relevance of the observed alterations in the context of AD in vitro. Using this approach, we show that only PM20D1 DNA methylation and expression are significantly correlated with the AD-risk associated background. We find that the expression of SLC41A1 and PM20D1-but not NUCKS1 and RAB7L1-is increased in mouse models and human samples of AD, respectively. However, SLC41A1 and PM20D1 are differentially regulated by AD-related stressors, with only PM20D1 being upregulated by amyloid-ß and reactive oxygen species, and with only PM20D1 being neuroprotective when overexpressed in cell and primary cultures. CONCLUSIONS: Our findings reinforce PM20D1 as the most likely gene responsible of the previously reported PM20D1 QTL association with AD.

A KHDC3L mutation resulting in recurrent hydatidiform mole causes genome-wide DNA methylation loss in oocytes and persistent imprinting defects post-fertilisation.

BACKGROUND: Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss. Enigmatically, they are also associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). However, the developmental timing, genomic extent and mechanistic basis of these imprinting defects are unknown. The rarity of these disorders and the possibility that methylation defects originate in oocytes have made these questions very challenging to address. METHODS: Single-cell bisulphite sequencing (scBS-seq) was used to assess methylation in oocytes from a patient with BiCHM identified to be homozygous for an inactivating mutation in the human SCMC component KHDC3L. Genome-wide methylation analysis of a preimplantation embryo and molar tissue from the same patient was also performed. RESULTS: High-coverage scBS-seq libraries were obtained from five KHDC3Lc.1A>G oocytes, which revealed a genome-wide deficit of DNA methylation compared with normal human oocytes. Importantly, germline differentially methylated regions (gDMRs) of imprinted genes were affected similarly to other sequence features that normally become methylated in oocytes, indicating no selectivity towards imprinted genes. A range of methylation losses was observed across genomic features, including gDMRs, indicating variable sensitivity to defects in the SCMC. Genome-wide analysis of a pre-implantation embryo and molar tissue from the same patient showed that following fertilisation methylation defects at imprinted genes persist, while most non-imprinted regions of the genome recover near-normal methylation post-implantation. CONCLUSIONS: We show for the first time that the integrity of the SCMC is essential for de novo methylation in the female germline. These findings have important implications for understanding the role of the SCMC in DNA methylation and for the origin of imprinting defects, for counselling affected families, and will help inform future therapeutic approaches.

A case of intraplacental gestational choriocarcinoma; characterised by the methylation pattern of the early placenta and an absence of driver mutations.

BACKGROUND: Gestational choriocarcinoma is a rare malignancy believed to arise from the trophoblast cells of the placenta. Despite the frequently aggressive clinical nature, choriocarcinoma has been routinely curable with cytotoxic chemotherapy for over 50 years. To date little is known regarding the route to oncogenesis in this malignancy. METHODS: In a case of intraplacental choriocarcinoma, we have performed detailed genetic studies including microsatellite analysis, whole genome sequencing (WGS) and methylation analysis of the tumour and surrounding mature placenta. RESULTS: The results of the WGS sequencing indicated a very low level of mutation and the absence of any driver mutations or oncogene activity in the tumour. The methylation analysis identified a distinctly different profile in the tumour from that of the mature placenta. Comparison with a panel of reference methylation profiles from different stages of placental development indicated that the tumour segregated with the first trimester samples. CONCLUSIONS: These findings suggest that gestational choriocarcinoma is likely to arise as a result of aberrations of methylation during development, rather than from DNA mutations. The results support the hypothesis that gestational choriocarcinoma arises from a normally transient early trophoblast cell. At this point in development this cell naturally has a phenotype of rapid division, tissue invasion and sensitivity to DNA damaging chemotherapy that is very similar to that of the mature choriocarcinoma cell.

Characterization of parent-of-origin methylation using the Illumina Infinium MethylationEPIC array platform.

AIM: This study aimed to establish a catalog of probes corresponding to imprinted differentially methylated regions (DMRs) on the Infinium HumanMethylationEPIC BeadChip. MATERIALS & METHODS: Reciprocal uniparental diploidies with low normal biparental mosaic contribution, together with normal diploid controls, were subjected to EPIC BeadChip hybridization. The methylation profiles were assessed for imprinted differential methylation. Top candidates were validated using locus-specific PCR-based assays. RESULTS: Seven hundred and eighty-nine CpG probes coincided with 50 known imprinted DMRs and 467 CpG probes corresponding to 124 novel imprinted DMR candidates were identified. Validation led to identification of several subtle DMRs within known imprinted domains as well as novel maternally methylated regions associated with PTCHD3 and JAKMIP1. CONCLUSION: Our comprehensive list of bona fide-imprinted DMR probes will simplify and facilitate methylation profiling of individuals with imprinting disorders and is applicable to other diseases in which aberrant imprinting has been implicated, such as cancer and fetal growth.

PM20D1 is a quantitative trait locus associated with Alzheimer's disease.

The chances to develop Alzheimer's disease (AD) result from a combination of genetic and non-genetic risk factors 1 , the latter likely being mediated by epigenetic mechanisms 2 . In the past, genome-wide association studies (GWAS) have identified an important number of risk loci associated with AD pathology 3 , but a causal relationship remains difficult to establish. In contrast, locus-specific or epigenome-wide association studies (EWAS) have revealed site-specific epigenetic alterations, which provide mechanistic insights for a particular risk gene but often lack the statistical power of GWAS 4 . Here, combining both approaches, we report a previously unidentified association of the peptidase M20-domain-containing protein 1 (PM20D1) with AD. We find that PM20D1 is a methylation and expression quantitative trait locus coupled to an AD-risk associated haplotype, which displays enhancer-like characteristics and contacts the PM20D1 promoter via a haplotype-dependent, CCCTC-binding-factor-mediated chromatin loop. Furthermore, PM20D1 is increased following AD-related neurotoxic insults at symptomatic stages in the APP/PS1 mouse model of AD and in human patients with AD who are carriers of the non-risk haplotype. In line, genetically increasing or decreasing the expression of PM20D1 reduces and aggravates AD-related pathologies, respectively. These findings suggest that in a particular genetic background, PM20D1 contributes to neuroprotection against AD.

Profiling of oxBS-450K 5-hydroxymethylcytosine in human placenta and brain reveals enrichment at imprinted loci.

DNA methylation (5-methylcytosine, 5 mC) is involved in many cellular processes and is an epigenetic mechanism primarily associated with transcriptional repression. The recent discovery that 5 mC can be oxidized to 5-hydromethylcytosine (5hmC) by TET proteins has revealed the "sixth base" of DNA and provides additional complexity to what was originally thought to be a stable repressive mark. However, our knowledge of the genome-wide distribution of 5hmC in different tissues is currently limited. Here, we sought to define loci enriched for 5hmC in the placenta genome by combining oxidative bisulphite (oxBS) treatment with high-density Illumina Infinium HumanMethylation450 methylation arrays and to compare our results with those obtained in brain. Despite identifying over 17,000 high-confidence CpG sites with consistent 5hmC enrichment, the distribution of this modification in placenta is relatively sparse when compared to cerebellum and frontal cortex. Supported by validation using allelic T4 ß-glucosyltransferase assays we identify 5hmC at numerous imprinted loci, often overlapping regions associated with parent-of-origin allelic 5 mC in both placenta and brain samples. Furthermore, we observe tissue-specific monoallelic enrichment of 5hmC overlapping large clusters of imprinted snoRNAs-miRNAs processed from long noncoding RNAs (lncRNAs) within the DLK1-DIO3 cluster on chromosome 14 and SNRPN-UBE3A domain on chromosome 15. Enrichment is observed solely on the transcribed alleles suggesting 5hmC is positively associated with transcription at these loci. Our study provides an extensive description of the 5hmC/5 mC landscape in placenta with our data available at www.humanimprints.net , which represents the most comprehensive resource for exploring the epigenetic profiles associated with human imprinted genes.

NLRPs, the subcortical maternal complex and genomic imprinting.

Before activation of the embryonic genome, the oocyte provides many of the RNAs and proteins required for the epigenetic reprogramming and the transition to a totipotent state. Targeted disruption of a subset of oocyte-derived transcripts in mice results in early embryonic lethality and cleavage-stage embryonic arrest as highlighted by the members of the subcortical maternal complex (SCMC). Maternal-effect recessive mutations of NLRP7, KHDC3L and NLRP5 in humans are associated with variable reproductive outcomes, biparental hydatidiform moles (BiHM) and widespread multi-locus imprinting disturbances. The precise mechanism of action of these genes is unknown, but the maternal-effect phenomenon suggests a function during early pre-implantation development, while biochemical and genetic studies implement them as SCMC members or interacting partners. In this review article, we discuss the role of the NLRP family members and the SCMC proteins in the establishment of genomic imprints and post-zygotic methylation maintenance, the recent advances made in the understanding of the biology involved in BiHM formation and the wider roles of the SCMC in mammalian reproduction.

Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors.

It has been postulated that imprinting aberrations are common in tumors. To understand the role of imprinting in cancer, we have characterized copy-number and methylation in over 280 cancer cell lines and confirm our observations in primary tumors. Imprinted differentially methylated regions (DMRs) regulate parent-of-origin monoallelic expression of neighboring transcripts in cis. Unlike single-copy CpG islands that may be prone to hypermethylation, imprinted DMRs can either loose or gain methylation during tumorigenesis. Here, we show that methylation profiles at imprinted DMRs often not represent genuine epigenetic changes but simply the accumulation of underlying copy-number aberrations (CNAs), which is independent of the genome methylation state inferred from cancer susceptible loci. Our results reveal that CNAs also influence allelic expression as loci with copy-number neutral loss-of-heterozygosity or amplifications may be expressed from the appropriate parental chromosomes, which is indicative of maintained imprinting, although not observed as a single expression foci by RNA FISH.Altered genomic imprinting is frequently reported in cancer. Here, the authors analyze copy number and methylation in cancer cell lines and primary tumors to show that imprinted methylation profiles represent the accumulation of copy number alteration, rather than epigenetic alterations.

Maternal mutations of FOXF1 cause alveolar capillary dysplasia despite not being imprinted.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare cause of pulmonary hypertension in newborns. Maternally inherited point mutations in Forkhead Box F1 gene (FOXF1), deletions of the gene, or its long-range enhancers on the maternal allele are responsible for this neonatal lethal disorder. Here, we describe monozygotic twins and one full-term newborn with ACD and gastrointestinal malformations caused by de novo mutations of FOXF1 on the maternal-inherited alleles. Since this parental transmission is consistent with genomic imprinting, the parent-of-origin specific monoallelic expression of genes, we have undertaken a detailed analysis of both allelic expression and DNA methylation. FOXF1 and its neighboring gene FENDRR were both biallelically expressed in a wide range of fetal tissues, including lung and intestine. Furthermore, detailed methylation screening within the 16q24.1 regions failed to identify regions of allelic methylation, suggesting that disrupted imprinting is not responsible for ACDMPV.

Human Amniocytes Are Receptive to Chemically Induced Reprogramming to Pluripotency.

Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR (mammalian target of rapamycin) pathway or GSK3ß inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells.

Diagnosis and management of Silver-Russell syndrome: first international consensus statement.

This Consensus Statement summarizes recommendations for clinical diagnosis, investigation and management of patients with Silver-Russell syndrome (SRS), an imprinting disorder that causes prenatal and postnatal growth retardation. Considerable overlap exists between the care of individuals born small for gestational age and those with SRS. However, many specific management issues exist and evidence from controlled trials remains limited. SRS is primarily a clinical diagnosis; however, molecular testing enables confirmation of the clinical diagnosis and defines the subtype. A 'normal' result from a molecular test does not exclude the diagnosis of SRS. The management of children with SRS requires an experienced, multidisciplinary approach. Specific issues include growth failure, severe feeding difficulties, gastrointestinal problems, hypoglycaemia, body asymmetry, scoliosis, motor and speech delay and psychosocial challenges. An early emphasis on adequate nutritional status is important, with awareness that rapid postnatal weight gain might lead to subsequent increased risk of metabolic disorders. The benefits of treating patients with SRS with growth hormone include improved body composition, motor development and appetite, reduced risk of hypoglycaemia and increased height. Clinicians should be aware of possible premature adrenarche, fairly early and rapid central puberty and insulin resistance. Treatment with gonadotropin-releasing hormone analogues can delay progression of central puberty and preserve adult height potential. Long-term follow up is essential to determine the natural history and optimal management in adulthood.

Epigenetic Characterization of CDKN1C in Placenta Samples from Non-syndromic Intrauterine Growth Restriction.

The cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) gene is expressed from the maternal allele and is located within the centromeric imprinted domain at chromosome 11p15. It is a negative regulator of proliferation, with loss-of-function mutations associated with the overgrowth disorder Beckwith-Wiedemann syndrome. Recently, gain-of-function mutations within the PCNA domain have been described in two disorders characterized by growth failure, namely IMAGe (intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome and Silver-Russell syndrome (SRS). Over-expression of CDKN1C by maternally inherited microduplications also results in SRS, suggesting that in addition to activating mutations this gene may regulate growth by changes in dosage. To determine if CDKN1C is involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. We observe higher levels of expression of CDKN1C in IUGR placentas compared to those of controls. All placenta biopsies heterozygous for the PAPA repeat sequence in exon 2 showed appropriate monoallelic expression and no mutations in the PCNA domain were observed. The expression profile was independent of both genetic or methylation variation in the minimal CDKN1C promoter interval and of methylation of the cis-acting maternally methylated region associated with the neighboring KCNQ1OT1 non-coding RNA. Chromatin immunoprecipitation revealed binding sites for CTCF within the unmethylated CDKN1C gene body CpG island and putative enhancer regions, associated with the canonical enhancer histone signature, H3K4me1 and H3K27ac, located ∼58 and 360 kb away. Using 3C-PCR we identify constitutive higher-order chromatin loops that occur between one of these putative enhancer regions and CDKN1C in human placenta tissues, which we propose facilitates expression.

Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting.

Familial recurrent hydatidiform mole (RHM) is a maternal-effect autosomal recessive disorder usually associated with mutations of the NLRP7 gene. It is characterized by HM with excessive trophoblastic proliferation, which mimics the appearance of androgenetic molar conceptuses despite their diploid biparental constitution. It has been proposed that the phenotypes of both types of mole are associated with aberrant genomic imprinting. However no systematic analyses for imprinting defects have been reported. Here, we present the genome-wide methylation profiles of both spontaneous androgenetic and biparental NLRP7 defective molar tissues. We observe total paternalization of all ubiquitous and placenta-specific differentially methylated regions (DMRs) in four androgenetic moles; namely gain of methylation at paternally methylated loci and absence of methylation at maternally methylated regions. The methylation defects observed in five RHM biopsies from NLRP7 defective patients are restricted to lack-of-methylation at maternal DMRs. Surprisingly RHMs from two sisters with the same missense mutations, as well as consecutive RHMs from one affected female show subtle allelic methylation differences, suggesting inter-RHM variation. These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte. In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci. These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

Genomic imprinting in the human placenta.

With the launch of the National Institute of Child Health and Human Development/National Institutes of Health Human Placenta Project, the anticipation is that this often-overlooked organ will be the subject of much intense research. Compared with somatic tissues, the cells of the placenta have a unique epigenetic profile that dictates its transcription patterns, which when disturbed may be associated with adverse pregnancy outcomes. One major class of genes that is dependent on strict epigenetic regulation in the placenta is subject to genomic imprinting, the parent-of-origin-dependent monoallelic gene expression. This review discusses the differences in allelic expression and epigenetic profiles of imprinted genes that are identified between different species, which reflect the continuous evolutionary adaption of this form of epigenetic regulation. These observations divulge that placenta-specific imprinted gene that is reliant on repressive histone signatures in mice are unlikely to be imprinted in humans, whereas intense methylation profiling in humans has uncovered numerous maternally methylated regions that are restricted to the placenta that are not conserved in mice. Imprinting has been proposed to be a mechanism that regulates parental resource allocation and ultimately can influence fetal growth, with the placenta being the key in this process. Furthermore, I discuss the developmental dynamics of both classic and transient placenta-specific imprinting and examine the evidence for an involvement of these genes in intrauterine growth restriction and placenta-associated complications. Finally, I focus on examples of genes that are regulated aberrantly in complicated pregnancies, emphasizing their application as pregnancy-related disease biomarkers to aid the diagnosis of at-risk pregnancies early in gestation.

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Tissue-specific DNA methylation profiles regulate liver-specific expression of the APOA1/C3/A4/A5 cluster and can be manipulated with demethylating agents on intestinal cells.

OBJECTIVE: The tissue-specific expression profiles of genes within the APOA1/C3/A4/A5 cluster play an important role in lipid metabolism regulation. We hypothesize that the tissue-specific expression of the APOA1/C3/A4/A5 gene cluster will show an inverse pattern with DNA methylation, and that repression in non- or low-expressing tissue, such as the intestine, can be reversed using epigenetic drugs. METHODS AND RESULTS: We analyzed DNA samples from different human adult tissues (liver, intestine, leukocytes, brain, kidney, pancreas, muscle and sperm) using the Infinium HumanMethyation450 BeadChip array. DNA methylation profiles in APOA1/C3/A4/A5 gene cluster were confirmed by bisulfite PCR and pyrosequencing. To determine whether the observed tissue-specific methylation was associated with the expression profile we exposed intestinal TC7/Caco-2 cells to the demethylating agent 5-Aza-2'-deoxycytidine and monitored intestinal APOA1/C3/A4/A5 transcript re-expression by RT-qPCR. The promoters of APOA1, APOC3 and APOA5 genes were less methylated in liver compared to other tissues, and APOA4 gene was highly methylated in most tissues and partially methylated in liver and intestine. In TC7/Caco-2 cells, 5-Aza-2'-deoxycytidine treatment induced a decrease between 37 and 24% in the methylation levels of APOA1/C3/A4/A5 genes and a concomitant re-expression mainly in APOA1, APOA4 and APOA5 genes ranging from 22 to 600%. CONCLUSIONS: We have determined the methylation patterns of the APOA1/C3/A4/A5 cluster that may be directly involved in the transcriptional regulation of this cluster. DNA demethylation of intestinal cells increases the RNA levels especially of APOA1, APOA4 and APOA5 genes.

Altered expression of the imprinted transcription factor PLAGL1 deregulates a network of genes in the human IUGR placenta.

Genomic imprinting is the epigenetic process that results in monoallelic expression of genes depending on parental origin. These genes are known to be critical for placental development and fetal growth in mammals. Aberrant epigenetic profiles at imprinted loci, such as DNA methylation defects, are surprisingly rare in pregnancies with compromised fetal growth, while variations in transcriptional output from the expressed alleles of imprinted genes are more commonly reported in pregnancies complicated with intrauterine growth restriction (IUGR). To determine if PLAGL1 and HYMAI, two imprinted transcripts deregulated in Transient Neonatal Diabetes Mellitus, are involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. This revealed that despite appropriate maternal methylation at the shared PLAGL1/HYMAI promoter, there was a loss of correlation between PLAGL1 and HYMAI expression in IUGR. This incongruity was due to higher HYMAI expression in IUGR gestations, coupled with PLAGL1 down-regulation in placentas from IUGR girls, but not boys. The PLAGL1 protein is a zinc-finger transcription factor that has been shown to be a master coordinator of a genetic growth network in mice. We observe PLAGL1 binding to the H19/IGF2 shared enhancers in placentae, with significant correlations between PLAGL1 levels with H19 and IGF2 expression levels. In addition, PLAGL1 binding and expression also correlate with expression levels of metabolic regulator genes SLC2A4, TCF4 and PPARγ1. Our results strongly suggest that fetal growth can be influenced by altered expression of the PLAGL1 gene network in human placenta.

Variable maternal methylation overlapping the nc886/vtRNA2-1 locus is locked between hypermethylated repeats and is frequently altered in cancer.

Cancer is as much an epigenetic disease as a genetic one; however, the interplay between these two processes is unclear. Recently, it has been shown that a large proportion of DNA methylation variability can be explained by allele-specific methylation (ASM), either at classical imprinted loci or those regulated by underlying genetic variants. During a recent screen for imprinted differentially methylated regions, we identified the genomic interval overlapping the non-coding nc886 RNA (previously known as vtRNA2-1) as an atypical ASM that shows variable levels of methylation, predominantly on the maternal allele in many tissues. Here we show that the nc886 interval is the first example of a polymorphic imprinted DMR in humans. Further analysis of the region suggests that the interval subjected to ASM is approximately 2 kb in size and somatically acquired. An in depth analysis of this region in primary cancer samples with matching normal adjacent tissue from the Cancer Genome Atlas revealed that aberrant methylation in bladder, breast, colon and lung tumors occurred in approximately 27% of cases. Hypermethylation occurred more frequently than hypomethylation. Using additional normal-tumor paired samples we show that on rare occasions the aberrant methylation profile is due to loss-of-heterozygosity. This work therefore suggests that the nc886 locus is subject to variable allelic methylation that undergoes cancer-associated epigenetic changes in solid tumors.