Prospective study of epigenetic alterations responsible for isolated hemihyperplasia/hemihypoplasia and their association with leg length discrepancy

Background Hemihyperplasia and hemihypoplasia result in leg length discrepancy (LLD) by causing skeletal asymmetry. Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS) are opposite growth-affecting disorders caused by opposite epigenetic alterations at the same chromosomal locus, 11p15, to induce hemihyperplasia and hemihypoplasia, respectively. Because of their somatic mosaicism, BWS and SRS show a wide spectrum of clinical phenotypes. We evaluated the underlying epigenetic alterations and potential epigenotype-phenotype correlations, focusing on LLD, in a group of individuals with isolated hemihyperplasia/hemihypoplasia. Results We prospectively collected paired blood-tissue samples from 30 patients with isolated hemihyperplasia/hemihypoplasia who underwent surgery for LLD. Methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) and bisulfite pyrosequencing for differentially methylated regions 1 and 2 (DMR1 and DMR2) on chromosome 11p15 were performed using the patient samples. Samples from patients showing no abnormalities in MS-MLPA or bisulfite pyrosequencing were analyzed by single nucleotide polymorphism (SNP) microarray and CDKN1C Sanger sequencing. We introduced a metric named as the methylation difference, defined as the difference in DNA methylation levels between DMR1 and DMR2. The correlation between the methylation difference and the predicted LLD at skeletal maturity, calculated using a multiplier method, was evaluated. Predicted LLD was standardized for stature. Ten patients (33%) showed epigenetic alterations in MS-MLPA and bisulfite pyrosequencing. Of these, six and four patients had epigenetic alterations related to BWS and SRS, respectively. The clinical diagnosis of hemihyperplasia/hemihypoplasia was not compatible with the epigenetic alterations in four of these ten patients. No patients showed abnormalities in SNP array or their CDKN1C sequences. The standardized predicted LLD was moderately correlated with the methylation difference using fat tissue (r = 0.53; p = 0.002) and skin tissue (r = 0.50; p = 0.005) in all patients. Conclusions Isolated hemihyperplasia and hemihypoplasia can occur as a spectrum of BWS and SRS. Although the accurate differentiation between isolated hemihyperplasia and isolated hemihypoplasia is important in tumor surveillance planning, it is often difficult to clinically differentiate these two diseases without epigenetic tests. Epigenetic tests may play a role in the prediction of LLD, which would aid in treatment planning.

DNA evolution depends on differential methylation patterns in rat speciation

The fixation of phenotypes and underlying alleles is a typical evolutionary process in speciation. As the primary molecular basis of phenotypic plasticity, epigenetic mechanisms also play an essential role in maintaining phenotypes. However, whether and how DNA evolution was shaped by epigenetic alteration remains unknown, especially accompanied DNA fixation in speciation. We used sperm methylomes of three rat subspecies as epigenetic markers and screened out genomic regions that experienced distinct differential methylation. To obtain independent results, they were further filtrated according to genomic locations to guarantee that their evolutionary features were not interactively affected by nearby DMRs (differentially methylated regions) of other datasets. By analyzing intraspecies and interspecies phylogenetic relationships, we showed that, in the same genomic regions, the significantly accelerated DNA evolution only occurred in individuals or lineages that experienced differential methylation. Across the same genomes, differential methylation led to a significant increase of F only in lineage-specific DMRs and a significant increase of π in both individual-specific and lineage-specific DMRs. Correlations among methylation, π and F showed that it was methylation consistency rather than the absolute methylation difference that significantly influenced both π and F. The change of both π and DNA fixation depended on the degree of intraspecies methylation consistency. While the breakdown of methylation consistency facilitated the promotion of π, the maintenance of methylation consistency facilitated the acceleration of DNA fixation.

Prospective Study of Epigenetic Alterations Responsible for Isolated Hemihyperplasia/Hemihypoplasia and Their Association With Leg Length Discrepancy

Background: Hemihyperplasia and hemihypoplasia result in leg length discrepancy (LLD) by causing skeletal asymmetry. Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are opposite growth-affecting disorders caused by opposite epigenetic alterations at the same chromosomal locus, 11p15, to induce hemihyperplasia and hemihypoplasia, respectively. Because of their somatic mosaicism, BWS and SRS show a wide spectrum of clinical phenotypes. We evaluated the underlying epigenetic alterations and potential epigenotype-phenotype correlations, focusing on LLD, in a group of individuals with pure isolated hemihyperplasia/hemihypoplasia.Results: We prospectively collected paired blood-tissue samples from 30 patients with pure isolated hemihyperplasia/hemihypoplasia who underwent surgery for LLD. Methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) and bisulfite pyrosequencing for differentially methylated regions 1 and 2 (DMR1 and DMR2) on chromosome 11p15 were performed using the patient samples. Samples from patients showing no abnormalities in MS-MLPA or bisulfite pyrosequencing were analyzed by single nucleotide polymorphism (SNP) microarray and CDKN1C Sanger sequencing. We introduced a metric named as the methylation difference, defined as the difference in DNA methylation levels between DMR1 and DMR2. The correlation between the methylation difference and the predicted LLD at skeletal maturity, calculated using a multiplier method, was evaluated. Predicted LLD was standardized for stature. Ten patients (33%) showed epigenetic alterations in MS-MLPA and bisulfite pyrosequencing. Of these, six and four patients had epigenetic alterations related to BWS and SRS, respectively. The clinical diagnosis of hemihyperplasia/hemihypoplasia was not compatible with the epigenetic alterations in four of these ten patients. No patients showed abnormalities in SNP array or their CDKN1C sequences. The methylation difference was strongly correlated with the standardized predicted LLD in patients with epigenetic alterations (r = 0.76; p = 0.01).Conclusions: Pure isolated hemihyperplasia and hemihypoplasia can occur as a spectrum of BWS and SRS. Although the accurate differentiation between isolated hemihyperplasia and isolated hemihypoplasia is important in tumor surveillance planning, it is often difficult to clinically differentiate these two diseases without epigenetic tests. Epigenetic tests may play a role in the prediction of leg length discrepancy, which would aid in treatment planning.

H3K9 methyltransferase EHMT2/G9a controls ERVK-driven non-canonical imprinted genes

Unlike regular imprinted genes, non-canonical imprinted genes are known to not depend on gamete-specific DNA methylation difference. Instead, the paternal allele-specific expression of these genes in the extra-embryonic lineages depends on an H3K27me3-based imprint in the oocyte, but this marking is not maintained beyond pre-implantation development. The maintenance of non-canonical imprinting corresponds to maternal allele-specific DNA methylation and paternal allele-specific H3K4me3 at their somatic DMRs, which occur at ERVK repeats. We hypothesized that EHMT2, the main euchromatic H3K9 methyltransferase, also has a role in this process. Using reciprocal mouse crosses and allele-specific RNA-seq analysis, we found that the maternal allele of each known non-canonical imprinted gene was derepressed from its ERVK promoter in the Ehmt2−/− ectoplacental cone of somite-matched 8.5 dpc embryos. In the Ehmt2−/− embryos, loss of DNA methylation accompanied the derepression of both parental alleles of those ERVK promoters. Our study identifies EHMT2 as an essential player that maintains the repressed chromosomal state in non-canonical imprinting.

Cell-type specific epigenetic links to schizophrenia risk in brain

The importance of cell-type specific epigenetic variation of non-coding regions in neuropsychiatric disorders is increasingly appreciated, yet data from disease brains are conspicuously lacking. We generated cell-type specific whole-genome methylomes (N=95) and transcriptomes (N=89) from neurons and oligodendrocytes from brains of schizophrenia and matched controls. The methylomes of these two cell-types are highly distinct, with the majority of differential DNA methylation occurring in non-coding regions. DNA methylation difference between control and schizophrenia brains is subtle compared to cell-type difference, yet robust against permuted data and validated in targeted deep-sequencing analyses. Differential DNA methylation between control and schizophrenia tends to occur in cell-type differentially methylated sites, highlighting the significance of cell-type specific epigenetic dysregulation in a complex neuropsychiatric disorder. Our resource provides novel and comprehensive methylome and transcriptome data from distinct cell populations from schizophrenia brains, further revealing reduced cell-type epigenetic distinction in schizophrenia.

Functional analysis of evolutionary human methylated regions in schizophrenia patients

BackgroundRecent studies have implicated variations in DNA methylation in the aetiology of schizophrenia. Genome-wide scans in both brain and blood report differential methylated regions (DMRs) and positions (DMPs) between patients with schizophrenia and healthy controls. Previously, we reported that DMRs where human specific methylation (hDMR) has occurred over evolutionary time are enriched for schizophrenia-associated markers (SCZ_hDMR). However, it is unknown whether these human specific DMRs show variable methylation in patients with schizophrenia.MethodsUsing publicly available data, we investigate if human specific DMRs that harbour genetic variants associated with schizophrenia are differentially methylated between cases and controls.ResultsWe find statistically significant (p < 1e-4) methylation difference in schizophrenia associated human specific DMRs (SCZ hDMR) between brain samples of cases and controls. However, we fail to find evidence of similar differences in methylation in blood samples.ConclusionRegions that are evolutionarily important for human species and that are associated with schizophrenia, also show difference in methylation variation in the brain in patients with schizophrenia.

Array-Based Integrative Analysis Of Epigenomic and Transcriptomic Alterations In CD71+ Bone Marrow Erythroprogenitor Cells From Patients With Myelodysplastic Syndromes

Background Myelodysplastic Syndromes (MDS) are heterogeneous groups of hematopoietic malignancies that are highly susceptible to transformation into acute myeloid leukemia and clinically manifest with signs of severe anemia. Numerous studies have revealed recurring molecular alterations in hematopoietic stem/progenitor cells from MDS patients as presumable cause for the persistent dysfunction of their hematopoiesis. However, how these events translate into disturbed regulation of erythropoiesis is poorly understood so far. Characterization of the transcriptional network and associated epigenetic changes in dysplastic erythroprogenitor cells might therefore aid in the elucidation of functional molecular consequences finally leading to impaired erythropoiesis. Methods CD71+ erythroprogenitor cells were isolated from purified bone marrow cells via magnetic cell separation for 15 MDS patients (IPSS low/int-1 risk n=11, int-2/high risk n=4) and 7 age-adjusted healthy donors. Extracted DNA and RNA were processed and hybridized to Affymetrix Exon 1.0 ST Array and Illumina Infinium HumanMethylation450 Beadchips according to manufacturers’ instructions. Data analysis was carried out using Qlucore Omics Explorer 2.3 and in-house scripts for correlation of the two datasets were developed using Python 2.7.5. Results Using a false discovery rate/q-value of ≤ 4% as a cutoff for differential gene expression and CpG DNA methylation, both datasets allowed highly distinct clustering into MDS IPSS low/int-1 risk, int-2/high risk and healthy donor groups. In our MDS low/int-1 risk cohort 63 genes have been identified as significantly up- and 13 as downregulated (fold change ≥1.5, p≤0.001) while 64 genes showed up- and 28 downregulation in the MDS int-2/high risk group (fold change ≥1.5 p≤0.01). Surprisingly, global DNA methylation profiling revealed that 741 CpGs were detected as hypo- but only 19 as hypermethylated in low/int-1 risk, whereas 231 CpGs demonstrated hypo- and 479 hypermethylation in int-2/high risk MDS CD71+ cells relative to healthy controls (mean CpG methylation difference ≥10%, p≤0.0001). In order to discover genes susceptible to DNA methylation associated regulation of transcription, individual CpG methylation values were correlated with corresponding exon probeset expression intensities for every single gene. Using this approach, starting with >23x106 possible CpG/probeset combinations, 4418 displayed significant positive correlation, whereas only 2726 were negatively correlated (R≥0.6 or R≤-0.6, mean methylation difference MDS vs. healthy ≥5%, p≤0.01). Consequently, in MDS low/int-1 risk we identified strong hypomethylation as putative cause for a 5.8-fold upregulation of GDF15, an important regulatory factor involved in iron homeostasis. Moreover, DNA hypermethylation associated 6.6-fold knockdown of the transcription factor GTSF1, which has been associated with increased apoptosis in gametogenesis, was demonstrated for MDS int-2/high-risk. In this cohort, we also observed a 3-fold upregulation of LY6E, which has been shown to result in a strong block of differentiation and maintenance of self-renewal in avian erythroprogenitor cells. Consistently, gene set enrichment analysis identified a stem cell like gene signature as highly enriched in MDS CD71+ BM cells but also gene sets involved in oxidative phosphorylation as well as regulation of cell cycle and apoptosis. Conclusion Our integrative approach reveals novel candidate genes implicated in disturbed erythropoiesis in MDS and allows distinctive separation between healthy donors and MDS risk groups by assessment of epigenomic and transcriptomic landscapes derived from CD71+ bone marrow cells. DNA hypomethylation induced gene upregulation surprisingly appears to be a common event in MDS erythropoiesis which co-occurs with DNA hypermethylation induced gene silencing. In addition, frequent detection of significant positive correlation between DNA methylation and gene expression might add an additional layer of complexity to the current dogma of epigenetic gene regulation. Finally, the distinctively hypermethylated geneset in MDS high-risk as compared to the unexpected global hypomethylation phenotype in MDS low-risk might suggest a mechanistic explanation for the selective efficacy of demethylating substances specifically in the higher risk patient groups. Disclosures: No relevant conflicts of interest to declare.

Differential DNA Methylation of TUBB1 Correlates with Tissue-Specific Hβ-1 Tubulin Expression

Abstract 4796 Introduction: Hβ-1 tubulin is essential for normal thrombopoiesis and constitutes the majority of β-tubulin within the platelet marginal band. Hβ-1 tubulin has the highest sequence diversity from other tubulin isotypes, and has expression restricted to hematologic tissues. The best-characterized expression occurs in megakaryocytes, where temporal expression is limited to specific stages of megakaryocyte maturation. While expression of Hβ-1 tubulin has been shown to be dependent on transcription factors such as NF-E2, these factors have a multitude of gene targets and do not provide a full explanation of mechanisms responsible for specific Hβ-1 tubulin expression. Epigenetic regulation of gene transcription is now widely accepted as a mechanism of tissue-specific expression of gene products; however, the role of epigenetic regulation in the expression of tubulin isotypes has not been explored. Given the highly regulated expression of Hβ-1 tubulin and the potential of epigenetic modifications to drive specific gene expression, we hypothesized that epigenetic modulation via DNA methylation may be a mechanism for regulation of Hβ-1 tubulin expression. Methods: We identified 3 CpG-dense areas upstream and within TUBB1 (which encodes Hβ-1 tubulin) based on Human hg19 genome assembly. These areas included the putative promoter region (-2000 to +1bp from the 5'UTR) upstream of the transcriptional start site; a 3kb region spanning most of intron 1; and a region within exon 4. We used Sequenom MassARRAY EpiTYPER on bisulphite-converted DNA to quantitatively determine percent methylation at each CpG within the three CpG-dense regions. Specifically, we extracted genomic DNA from three cell lines expressing Hβ-1 tubulin (K562, MEG-01, and HEL), and six non-expressing cell lines (two hematologic cell lines (REH, KCL-22) and four epithelial cell lines (H1299, PC3, LNCap2, MDA-MB-231)), and performed bisulphite conversion of DNA. A difference of >25% was considered significant when comparing individual CpGs; methylation differences between CpGs across cell lines are reported as mean ± SEM. For each region, median methylation for each CpG was calculated across cell lines within a group, and regional methylation differences compared using the Mann-Whitney test. Results: Between 50–75% of CpGs within the three CpG-dense regions were available for analysis. In exon 4, there was no difference in overall methylation or methylation at any individual CpG between the Hβ-1 tubulin-expressing and non-expressing cell lines. In contrast, within intron 1, Hβ-1 tubulin-expressing cell lines were significantly hypomethylated compared to non-expressing cell lines (p=0.002). This difference was localized to a 1.5kb region within intron 1; mean methylation difference at each CpG within this region was 59±6%. The upstream promoter region similarly showed significant hypomethylation in Hβ-1 tubulin-expressing cell lines (p=0.001); the differentially methylated CpGs were localized to a 350bp region just upstream of the transcription start site, and mean methylation difference was 60±16% at each CpG. Methylation patters were highly similar between CpGs within each group (i.e. Hβ-1 tubulin-expressing or non-expressing cell lines), with only 10% of individual CpGs showing >15% methylation difference between cell lines of the same group. Conclusion: We found significant extra- and intra-genic DNA methylation differences in TUBB1 between Hβ-1 tubulin-expressing and non-expressing cell lines. Methylation changes were localized to two CpG-dense regions, namely the upstream promoter region and intron 1 of TUBB1, while a third region in exon 4 showed no differences in methylation. The overall methylation differences within the regions were attributable to large methylation differences at individual CpGs localized to particular areas within those regions. Taken together, these results suggest that tissue-specific expression of Hβ-1 tubulin may be regulated in part by highly-specific changes in DNA methylation of TUBB1. To our knowledge, this is the first report of epigenetic modulation associated with tissue-specific tubulin isotype expression. Further work is underway to confirm these findings in normal primary hematopoietic tissues and to investigate their associations with temporal expression of Hβ-1 tubulin during megakaryocyte development. Disclosures: No relevant conflicts of interest to declare.