Allelic DNA Methylation Research Articles

The landscape of allelic expression and DNA methylation at the bovine SGCE/PEG10 locus.

Genomic imprinting is an epigenetic regulation in mammals in which a small subset of genes is monoallelically expressed dependent on their parental origin. A large imprinted domain, SGCE/PEG10 locus, is located on human chromosome 7q21s and mouse proximal chromosome 6. However, genomic imprinting of bovine SGCE/PEG10 cluster has not been systematically studied. In this study, we investigated allele expression of 14 genes of the SGCE/PEG10 locus in bovine somatic tissues and term placenta using a single nucleotide polymorphism (SNP)-based sequencing method. In addition to SGCE and PEG10, two conserved paternally expressed genes in human and mice, five other genes (TFPI2, GNG11, ASB4, PON1, and PON3) were paternally expressed. Three genes, BET1, COL1A2, and CASD1, exhibited tissue-specific monoallelic expression. CALCR showed monoallelic expression in tissues but biallelic expression in the placenta. Three genes, GNGT1, PPP1R9A, and PON2, showed biallelic expression in cattle. Five differentially methylated regions (DMRs) were found to be associated with the allelic expression of TFPI2, COL1A2, SGCE/PEG10, PON3, and ASB4 genes, respectively. The SGCE/PEG10 DMR is a maternally hypermethylated germline DMR, but TFPI2, COL1A2, PON3, and ASB4 DMRs are secondary DMRs. In summary, we identified five novel bovine imprinted genes (GNG11, BET1, COL1A2, CASD1, and PON1) and four secondary DMRs at the SGCE/PEG10 locus.

The conservation of allelic DNA methylation and its relationship with imprinting in maize.

Genomic imprinting refers to allele-specific expression of genes depending on parental origin, and it is regulated by epigenetic modifications. Intraspecific allelic variation for imprinting has been detected; however, the intraspecific genome-wide allelic epigenetic variation in maize and its correlation with imprinting variants remain unclear. Here, three reciprocal hybrids were generated by crossing Zea mays inbred lines CAU5, B73, and Mo17 in order to examine the intraspecific conservation of the imprinted genes in the kernel. The majority of imprinted genes exhibited intraspecific conservation, and these genes also exhibited interspecific conservation (rice, sorghum, and Arabidopsis) and were enriched in some specific pathways. By comparing intraspecific allelic DNA methylation in the endosperm, we found that nearly 15% of DNA methylation existed as allelic variants. The intraspecific whole-genome correlation between DNA methylation and imprinted genes indicated that DNA methylation variants play an important role in imprinting variants. Disruption of two conserved imprinted genes using CRISPR/Cas9 editing resulted in a smaller kernel phenotype. Our results shed light on the intraspecific correlation of DNA methylation variants and variation for imprinting in maize, and show that imprinted genes play an important role in kernel development.

Phase II Study Assessing Safety and Preliminary Efficacy of High Dose Intravenous Ascorbic Acid in Patients with TET2 Mutant Clonal Cytopenias of Undetermined Significance

Background: Clonal cytopenia(s) of undetermined significance (CCUS) is defined as persistent cytopenias arising in the context of myeloid neoplasm (MN)-associated somatic mutations (MT) in hematopoietic stem cells. Patients (PTs) with TET2 mutant (TET2MT) CCUS have a high probability of progression to MN. To date, no FDA-approved treatment for CCUS exist, with PTs often having similar cytopenias and transfusion needs as those with myelodysplastic syndromes (MDS). High dose intravenous ascorbic acid (HI-AA) demonstrates anti-cancer activity via two mechanisms: (1) hydrogen peroxide-induced oxidative stress and (2) DNA demethylation mediated by TET activation (cofactor for TET1/2/3). TET2 is an AA and oxoglutarate-dependent dioxygenase that catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), providing the rationale to study HI-AA in TET2MT CCUS. Methods: This is an investigator-initiated, single-arm, single-institution, phase II trial assessing the safety and preliminary efficacy of HI-AA for PTs with TET2MT CCUS (NCT03418038). PTs³ 18 years with TET2MT CCUS (≥1 TET2MT with or without additional somatic MTs) and with any of the following laboratory criteria: (1) hemoglobin (Hb) ≤10g/dL, (2) absolute neutrophil count (ANC) ≤1(10^9/L), (3) platelet count (PLT)≤100 (10^9/L) are eligible. HI-AA (1g/kg, maximum 100g) is given 3 times weekly for 12 weeks through a PICC line, with response assessments before each cycle (4 weeks), at 20 weeks, and one year. The primary endpoint is the hematologic response rates determined by the proposed MDS IWG 2018 criteria at week 20. (Platzbecker Blood 2018) and reported as hematologic improvement (HI)- erythropoietic (HI-E), platelets (HI-P), neutrophils (HI-N). Secondary endpoints include safety and adverse events (AEs). AEs were graded using NCI-CTCAE v4.03. Correlative studies include changes in TET2MT variant allele fraction (VAF), TET2 activity, DNA methylation, hydroxymethylation, and 5hmC in bone marrow cells as determined by immunohistochemistry (IHC). Results: As of July 22, 2022, 8 (89%) of 9 enrolled PTs completed 12 weeks of therapy. The mean age was 74 (SD: 5.4) years, 7 (87.5%) males. One PT (PT_X) came off study after cycle one due to a PICC line-associated thrombosis. All PT were TET2MT (44% truncating), with 8 of 9 (89%) having co-mutations (4- 2 MTs, 3- 3 MTs, and 1- 4 MTs; 2 had ≥1 TET2MT). Comutations included SRSF2 (55%), ASXL1 (22%), ZRSR2 and KRAS (11% each). HI-AA was well tolerated with the most common AEs being infusion-related polyuria (56%) and polydipsia (44%, 3 Grade 1, 1 Grade 2). One (11%) PT had constipation and headaches (both Grade 1), and 1 (11%) experienced dyspepsia. No treatment-related Grade 3 or 4 AEs or deaths were observed. Six of 8 (75%) assessable PTs had post-treatment response assessments at week 20, with 3 PTs having completed 1-year assessments. All PTs' baseline Hb was within the normal range, except for 1 (17%) (PT_5- TET2MT/SRSF2MT) PT who continued to remain red blood transfusion dependent. Five (63%) PTs were thrombocytopenic PLT <100 (10^9/L), with 2 (25%) PTs (PT_1 and PT_7) having PLT £50 (10^9/L) and 5 (83%) PTs had ANC<1 (10^9/L) with 1 (17%) (PT_8) with ANC <0.5 (10^9/L). One (17%) PT met criteria for HI-P (PT_6 had an increment to 118 (10^9/L) at week 20- TET2MT/SRSF2MT/KRASMT) and 1 (17%) PT met criteria for HI-N (PT_2, ANC baseline:1.08 vs. week 20: 1.76(10^9/L)-TET2MT/SRSF2MT). PT_1, who had a baseline PLT of 25 (10^9/L) and ANC of 0.72 (10^9/L), had disease progression to MDS-EB-1 (TET2MT/SRSF2MT) at week 20. The 3 PTs with 1 year follow-up have stable disease. Panel 1 demonstrates changes in blood parameters with treatment. Panel 2 shows IHC determined changes in 5hmC changes with two control samples: normal control and TET2MT CMML. In 5 assessable PTs, there were no changes in TET2 VAF at 20 weeks (mean VAF : baseline 37.2% vs. week 20: 36.6%, p=0.3). Additional correlative studies are ongoing. Conclusion: We report safety and preliminary efficacy analysis for the first clinical trial dedicated to TET2MT CCUS. The early results of this pilot demonstrate that HI-AA is safe and has potential for biological and clinical effects. At last follow-up, 2 (33%) PTs met criteria for HI (HI-P-1 and HI-N-1), 3 PTs (50%) had stable disease, and 1 (17%) PT progressed to MDS. Future studies assessing potential synergy of adding HI-AA to standard of care therapies for TET2MT clonal processes are planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DeepLoop robustly maps chromatin interactions from sparse allele-resolved or single-cell Hi-C data at kilobase resolution.

Mapping chromatin loops from noisy Hi-C heatmaps remains a major challenge. Here we present DeepLoop, which performs rigorous bias correction followed by deep-learning-based signal enhancement for robust chromatin interaction mapping from low-depth Hi-C data. DeepLoop enables loop-resolution, single-cell Hi-C analysis. It also achieves a cross-platform convergence between different Hi-C protocols and micrococcal nuclease (micro-C). DeepLoop allowed us to map the genetic and epigenetic determinants of allele-specific chromatin interactions in the human genome. We nominate new loci with allele-specific interactions governed by imprinting or allelic DNA methylation. We also discovered that, in the inactivated X chromosome (Xi), local loops at the DXZ4 'megadomain' boundary escape X-inactivation but the FIRRE 'superloop' locus does not. Importantly, DeepLoop can pinpoint heterozygous single-nucleotide polymorphisms and large structure variants that cause allelic chromatin loops, many of which rewire enhancers with transcription consequences. Taken together, DeepLoop expands the use of Hi-C to provide loop-resolution insights into the genetics of the three-dimensional genome.

Pyllelic, a Software Suite for Examining Allelic DNA CpG Methylation Patterns in Genomic Datasets

While genome‐wide demethylation of DNA CpG sites is a well‐established early hallmark of cancer, only recently has the presence of allelic methylation patterns associating with pathological gene expression been reported. Such allelic methylation patterns have often been obscured due to statistical averaging procedures applied to bisulfite conversion sequencing (Bis‐Seq) CpG methylation genomic datasets. Here, we present a Python software suite, Pyllelic, that dissects Bis‐Seq genomic datasets at the individual read level, allowing nuanced analyses of methylation patterns of genes, including allelic patterns associated with gene expression. We used this approach to examine the role of allelic methylation in the telomerase reverse transcriptase (TERT) gene, which is pathologically expressed in the majority of human cancers (approximately 80‐90%) and has known allelic expression patterns. Surprisingly, active TERTgene expression in human cancers has been characterized as having a hypermethylated CpG island in the promoter, which is the opposite of general promoter methylation associations with gene expression, including reports of hypomethylation of promoters correlating with overexpression of oncogenes. However, when we used Pyllelic to analyze &gt;600 cancer cell lines from 23 different tissue types, we found that in the proximal region of the TERTpromoter, allele‐specific hypomethylation correlated with TERTexpression. This analysis was performed in cancer cell lines with known activating, monoallelic promoter mutations as well as in wild‐type TERTcancer cell lines. The Pyllelic suite allows both aggregate and individual read‐level access to the underlying methylated reads of genomic datasets, with a data science‐focused implementation to facilitate the path from hypothesis to publishable figures and statistical analyses. Pyllelic thus should serve as an important tool to help identify allelic DNA methylation patterns and associations for a wide variety of genomic regions, with particular relevance for pathological activation in cancers, as well as other tissue or disease methylation analysis.

Analysis of developmental imprinting dynamics in primates using SNP-free methods to identify imprinting defects in cloned placenta.

Our knowledge of genomic imprinting in primates is lagging behind that of mice largely because of the difficulties of allelic analyses in outbred animals. To understand imprinting dynamics in primates, we profiledtranscriptomes, DNA methylomes, and H3K27me3 in uniparental monkey embryos. We further developed single-nucleotide-polymorphism (SNP)-free methods, TARSII and CARSII, to identify germline differentially methylated regions (DMRs) in somatic tissues. Our comprehensive analyses showed that allelicDNA methylation, but not H3K27me3, is a major mark that correlates with paternal-biasedly expressed genes (PEGs) in uniparental monkey embryos. Interestingly, primate germline DMRs are different from PEG-associated DMRs in early embryos and are enriched in placenta. Strikingly, most placenta-specificgermline DMRs are lost in placenta of cloned monkeys. Collectively, our study establishes SNP-free germline DMR identification methods, defines developmental imprinting dynamics in primates, and demonstrates imprinting defects in cloned monkey placenta, which provides important clues for improving primate cloning.

Features and mechanisms of canonical and noncanonical genomic imprinting.

Genomic imprinting is the monoallelic expression of a gene based on parent of origin and is a consequence of differential epigenetic marking between the male and female germlines. Canonically, genomic imprinting is mediated by allelic DNA methylation. However, recently it has been shown that maternal H3K27me3 can result in DNA methylation-independent imprinting, termed "noncanonical imprinting." In this review, we compare and contrast what is currently known about the underlying mechanisms, the role of endogenous retroviral elements, and the conservation of canonical and noncanonical genomic imprinting.

Imprinted Genes Impact Upon Beta Cell Function in the Current (and Potentially Next) Generation.

Beta cell failure lies at the centre of the aetiology and pathogenesis of type 2 diabetes and the epigenetic control of the expression of critical beta cell genes appears to play a major role in this decline. One such group of epigenetically-controlled genes, termed ‘imprinted’ genes, are characterised by transgenerational monoallelic expression due to differential allelic DNA methylation and play key functional roles within beta cells. Here, we review the evidence for this functional importance of imprinted genes in beta cells as well as their nutritional regulation by the diet and their altered methylation and/or expression in rodent models of diabetes and in type 2 diabetic islets. We also discuss imprinted genes in the context of the next generation, where dietary overnutrition in the parents can lead to their deregulation in the offspring, alongside beta cell dysfunction and defective glucose handling. Both the modulation of imprinted gene expression and the likelihood of developing type 2 diabetes in adulthood are susceptible to the impact of nutritional status in early life. Imprinted loci, therefore, represent an excellent opportunity with which to assess epigenomic changes in beta cells due to the diet in both the current and next generation.

Lineage and Parent-of-Origin Effects in DNA Methylation of Honey Bees (Apis mellifera) Revealed by Reciprocal Crosses and Whole-Genome Bisulfite Sequencing.

Parent-of-origin methylation arises when the methylation patterns of a particular allele are dependent on the parent it was inherited from. Previous work in honey bees has shown evidence of parent-of-origin-specific expression, yet the mechanisms regulating such pattern remain unknown in honey bees. In mammals and plants, DNA methylation is known to regulate parent-of-origin effects such as genomic imprinting. Here, we utilize genotyping of reciprocal European and Africanized honey bee crosses to study genome-wide allele-specific methylation patterns in sterile and reproductive individuals. Our data confirm the presence of allele-specific methylation in honey bees in lineage-specific contexts but also importantly, though to a lesser degree, parent-of-origin contexts. We show that the majority of allele-specific methylation occurs due to lineage rather than parent-of-origin factors, regardless of the reproductive state. Interestingly, genes affected by allele-specific DNA methylation often exhibit both lineage and parent-of-origin effects, indicating that they are particularly labile in terms of DNA methylation patterns. Additionally, we re-analyzed our previous study on parent-of-origin-specific expression in honey bees and found little association with parent-of-origin-specific methylation. These results indicate strong genetic background effects on allelic DNA methylation and suggest that although parent-of-origin effects are manifested in both DNA methylation and gene expression, they are not directly associated with each other.

Allelic H3K27me3 to allelic DNA methylation switch maintains noncanonical imprinting in extraembryonic cells.

Faithful maintenance of genomic imprinting is essential for mammalian development. While germline DNA methylation-dependent (canonical) imprinting is relatively stable during development, the recently found oocyte-derived H3K27me3-mediated noncanonical imprinting is mostly transient in early embryos, with some genes important for placental development maintaining imprinted expression in the extraembryonic lineage. How these noncanonical imprinted genes maintain their extraembryonic-specific imprinting is unknown. Here, we report that maintenance of noncanonical imprinting requires maternal allele-specific de novo DNA methylation [i.e., somatic differentially methylated regions (DMRs)] at implantation. The somatic DMRs are located at the gene promoters, with paternal allele-specific H3K4me3 established during preimplantation development. Genetic manipulation revealed that both maternal EED and zygotic DNMT3A/3B are required for establishing somatic DMRs and maintaining noncanonical imprinting. Thus, our study not only reveals the mechanism underlying noncanonical imprinting maintenance but also sheds light on how histone modifications in oocytes may shape somatic DMRs in postimplantation embryos.

Endogenous retroviral insertions drive non-canonical imprinting in extra-embryonic tissues

BackgroundGenomic imprinting is an epigenetic phenomenon that allows a subset of genes to be expressed mono-allelically based on the parent of origin and is typically regulated by differential DNA methylation inherited from gametes. Imprinting is pervasive in murine extra-embryonic lineages, and uniquely, the imprinting of several genes has been found to be conferred non-canonically through maternally inherited repressive histone modification H3K27me3. However, the underlying regulatory mechanisms of non-canonical imprinting in post-implantation development remain unexplored.ResultsWe identify imprinted regions in post-implantation epiblast and extra-embryonic ectoderm (ExE) by assaying allelic histone modifications (H3K4me3, H3K36me3, H3K27me3), gene expression, and DNA methylation in reciprocal C57BL/6 and CAST hybrid embryos. We distinguish loci with DNA methylation-dependent (canonical) and independent (non-canonical) imprinting by assaying hybrid embryos with ablated maternally inherited DNA methylation. We find that non-canonical imprints are localized to endogenous retrovirus-K (ERVK) long terminal repeats (LTRs), which act as imprinted promoters specifically in extra-embryonic lineages. Transcribed ERVK LTRs are CpG-rich and located in close proximity to gene promoters, and imprinting status is determined by their epigenetic patterning in the oocyte. Finally, we show that oocyte-derived H3K27me3 associated with non-canonical imprints is not maintained beyond pre-implantation development at these elements and is replaced by secondary imprinted DNA methylation on the maternal allele in post-implantation ExE, while being completely silenced by bi-allelic DNA methylation in the epiblast.ConclusionsThis study reveals distinct epigenetic mechanisms regulating non-canonical imprinted gene expression between embryonic and extra-embryonic development and identifies an integral role for ERVK LTR repetitive elements.

Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs.

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs invivo.

Tissue-Specific Monoallelic Expression of Bovine AXL is Associated with DNA Methylation of Promoter DMR.

The AXL protein is a receptor tyrosine kinase and is often implicated in proliferation, migration and therapy resistance in various cancers. The AXL gene in humans is maternally expressed and paternally imprinted with differentially methylated regions (DMR) surrounding the promoter region. However, the imprinting status and epigenetic regulation of AXL gene in cattle remain unclear. Therefore, we explored the molecular structure along with the patterns of allelic expression and DNA methylation of the bovine AXL gene. First, the complete cDNA sequence of bovine AXL was gathered by Sanger method, from transcripts obtained from RT-PCR, 5' and 3' -RACE. In silico BLAST alignments showed that the longest mRNA sequence of bovine AXL consists of 19 exons and encodes a protein of 887 amino acids. We further analyzed the allelic expression of bovine AXL by employing single-nucleotide polymorphism (SNP)-based sequencing method. A SNP site (GenBank Accession no: rs210020651) found in exon 7 allowed us to distinguish the two parental alleles. Monoallelic expression of AXL was observed in four adult bovine tissues (heart, liver, spleen and fat), while biallelic expression was found in the other adult tissues such as the lung, kidney, muscle, brain and placenta. To determine whether the DNA methylation played a role in the tissue-specific imprinting of bovine AXL, we performed bisulfite sequencing of two regions: region 1 was a CpG island (CGI) in AXL promoter, mapping to 643bp upstream of the transcription start site of AXL 5'-v1 transcripts, while region two was homologous to the region of human AXL DMR, with 10 CpG sites overlapping the first translation start site (TSS1) of bovine AXL. In region 2, DNA from both monoallelic and biallelic expressed tissues were mostly found to be completely unmethylated. However, tissue-specific differential methylation patterns were found in monoallelic expressed tissues such as the heart and liver while hypomethylation was noted in the promoter CpG island in biallelic expressed tissues such as the lung. These observations demonstrated that the tissue-specific monoallelic expression of bovine AXL is dependent on the DNA methylation of its promoter region.

Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction

BackgroundGenome-wide studies have begun to link subtle variations in both allelic DNA methylation and parent-of-origin genetic effects with early development. Numerous reports have highlighted that the placenta plays a critical role in coordinating fetal growth, with many key functions regulated by genomic imprinting. With the recent description of wide-spread polymorphic placenta-specific imprinting, the molecular mechanisms leading to this curious polymorphic epigenetic phenomenon is unknown, as is their involvement in pregnancies complications.ResultsProfiling of 35 ubiquitous and 112 placenta-specific imprinted differentially methylated regions (DMRs) using high-density methylation arrays and pyrosequencing revealed isolated aberrant methylation at ubiquitous DMRs as well as abundant hypomethylation at placenta-specific DMRs. Analysis of the underlying chromatin state revealed that the polymorphic nature is not only evident at the level of allelic methylation, but DMRs can also adopt an unusual epigenetic signature where the underlying histones are biallelically enrichment of H3K4 methylation, a modification normally mutually exclusive with DNA methylation. Quantitative expression analysis in placenta identified two genes, GPR1-AS1 and ZDBF2, that were differentially expressed between IUGRs and control samples after adjusting for clinical factors, revealing coordinated deregulation at the chromosome 2q33 imprinted locus.ConclusionsDNA methylation is less stable at placenta-specific imprinted DMRs compared to ubiquitous DMRs and contributes to privileged state of the placenta epigenome. IUGR-associated expression differences were identified for several imprinted transcripts independent of allelic methylation. Further work is required to determine if these differences are the cause IUGR or reflect unique adaption by the placenta to developmental stresses.

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.

M33 - GENETIC VARIATION AND EPIGENETIC MODIFICATION OF SKA2: ASSOCIATIONS WITH OBSESSIVE-COMPULSIVE DISORDER DISEASE RISK AND SYMPTOM SEVERITY

Background Obsessive-Compulsive Disorder (OCD) has a strong genetic component, is highly comorbid with Major Depressive Disorder (MDD), and is often triggered by environmental stress. Epigenetic modifications are thought to play a significant role in complex psychiatric disorders and may impact the relationship between genes and the environment via the hypothalamic-pituitary-adrenal (HPA) axis. The SKA2 gene interacts with the glucocorticoid receptor, is implicated in mediating HPA axis function, but has yet to be examined in OCD. We hypothesized that genetic and epigenetic variation in SKA2 may play a role in OCD disease risk and symptom severity. Methods A sample of n=64 OCD patients, divided equally by presence/absence of comorbid MDD, and age and gender matched, were selected. Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) score was used to measure disease severity. Genotyping for the SKA2 rs7208505 marker was performed using the Taqman allele specific assay method and DNA methylation levels were quantified using bisulfite pyrosequencing. Methylation percentage was corrected for genotype using previously established methods. Genotypes for rs7208505 were compared to controls of European ancestry (n=365; 1000 Genomes Project Consortium) using Pearson’s chi-squared test. The relationship among the rs7208505 variant, methylation corrected, symptom severity, and MDD status was modeled using linear regression. Results The SKA2 rs7208505 genotype distributions significantly differed between OCD patients and controls (χ2=8.66, p=0.013). Genotypes were associated with symptom severity (p=0.011), in that patients with the CC genotype had lower Y-BOCS scores than the CT/TT genotypes. While OCD patients diagnosed with MDD had more severe Y-BOCS scores (p=0.023), neither genetic nor epigenetic variation of rs7208505 was associated with comorbid MDD status. Methylation corrected for genotype was not significant in the regression model (p>0.05). Discussion These results provide some evidence that SKA2 genetic variation may be associated with OCD risk and symptom severity. Methylation corrected for genotype was not associated with OCD symptom severity; however, patients with TT and CT genotypes had more severe Y-BOCS scores than the CC genotype carriers. Further work will include replication in larger samples and interrogating additional genetic and epigenetic variations in SKA2.

Allele-Specific Methylome and Transcriptome Analysis Reveals Widespread Imprinting in the Human Placenta

DNA methylation is globally reprogrammed after fertilization, and as a result, the parental genomes have similar DNA-methylation profiles after implantation except at the germline differentially methylated regions (gDMRs). We and others have previously shownthat human blastocysts might contain thousands of transient maternally methylated gDMRs (transient mDMRs), whose maternal methylation is lost in embryonic tissues after implantation. In this study, we performed genome-wide allelic DNA methylation analyses of purified trophoblast cells from human placentas and, surprisingly, found that more than one-quarter of the transient-in-embryo mDMRs maintained their maternally biased DNA methylation. RNA-sequencing-based allelic expression analyses revealed that some of the placenta-specific mDMRs were associated with expression of imprinted genes (e.g., TIGAR, SLC4A7, PROSER2-AS1, and KLHDC10), and three imprinted gene clusters were identified. This approach also identified some X-linked gDMRs. Comparisons of the data with those from other mammals revealed that genomic imprinting in the placenta is highly variable. These findings highlight the incomplete erasure of germline DNA methylation in the human placenta; understanding this erasure is important for understanding normal placental development and the pathogenesis of developmental disorders with imprinting effects.

DNA methylation and mRNA expression of HLA-DQA1 alleles in type 1 diabetes mellitus.

Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analysed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr = 0·041; DRA normalization, Pcorr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA-DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA-DQA1 allele in patients with T1D have been described for the first time.

Analysis of a four generation family reveals the widespread sequence-dependent maintenance of allelic DNA methylation in somatic and germ cells

Differential methylation of the homologous chromosomes, a well-known mechanism leading to genomic imprinting and X-chromosome inactivation, is widely reported at the non-imprinted regions on autosomes. To evaluate the transgenerational DNA methylation patterns in human, we analyzed the DNA methylomes of somatic and germ cells in a four-generation family. We found that allelic asymmetry of DNA methylation was pervasive at the non-imprinted loci and was likely regulated by cis-acting genetic variants. We also observed that the allelic methylation patterns for the vast majority of the cis-regulated loci were shared between the somatic and germ cells from the same individual. These results demonstrated the interaction between genetic and epigenetic variations and suggested the possibility of widespread sequence-dependent transmission of DNA methylation during spermatogenesis.

Gene copy silencing and DNA methylation in natural and artificially produced allopolyploid fish.

Allelic silencing is an important mechanism for coping with gene dosage changes in polyploid organisms that is well known in allopolyploid plants. Only recently, it was shown in the allotriploid fish Squalius alburnoides that this process also occurs in vertebrates. However, it is still unknown whether this silencing mechanism is common to other allopolyploid fish, and which mechanisms might be responsible for allelic silencing. We addressed these questions in a comparative study between Squalius alburnoides and another allopolyploid complex, the Amazon molly (Poecilia formosa). We examined the allelic expression patterns for three target genes in four somatic tissues of natural allo-anorthoploids and laboratory-produced tri-genomic hybrids of S. alburnoides and P. formosa. Also, for both complexes, we evaluated the correlation between total DNA methylation level and the ploidy status and genomic composition of the individuals. We found that allelic silencing also occurs in other allopolyploid organisms besides the single one that was previously known. We found and discuss disparities within and between the two considered complexes concerning the pattern of allele-specific expression and DNA methylation levels. Disparities might be due to intrinsic characteristics of each genome involved in the hybridization process. Our findings also support the idea that long-term evolutionary processes have an effect on the allele expression patterns and possibly also on DNA methylation levels.