X-chromosome Inactivation Status Research Articles

Role of Skewed X-Chromosome Inactivation in Common Variable Immunodeficiency.

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically <30%. Other proposed mechanisms include digenic, oligogenic, or polygenic inheritance and epigenetic dysregulation. In this study, we aimed to assess the role of skewed X-chromosome inactivation (XCI) in CVID. Within our cohort of 131 genetically analyzed CVID patients, we selected female patients with rare variants in CVID-associated genes located on the X-chromosome. Four patients harboring heterozygous variants in BTK (n = 2), CD40LG (n = 1), and IKBKG (n = 1) were included in the study. We assessed XCI status using the HUMARA assay and an NGS-based method to quantify the expression of the 2 alleles in mRNA. Three of the 4 patients (75%) exhibited skewed XCI, and the mutated allele was predominantly expressed in all cases. Patient 1 harbored a hypomorphic variant in BTK (p.Tyr418His), patient 3 had a pathogenic variant in CD40LG (c.288+1G>A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.

X-Chromosome Inactivation and Related Diseases.

X-chromosome inactivation (XCI) is the form of dosage compensation in mammalian female cells to balance X-linked gene expression levels of the two sexes. Many diseases are related to XCI due to inactivation escape and skewing, and the symptoms and severity of these diseases also largely depend on the status of XCI. They can be divided into 3 types: X-linked diseases, diseases that are affected by XCI escape, and X-chromosome aneuploidy. Here, we review representative diseases in terms of their definition, symptoms, and XCI's role in the pathogenesis of these diseases.

Analysis of X-inactivation status in a Rett syndrome natural history study cohort.

BackgroundRett syndrome (RTT) is a rare neurodevelopmental disorder associated with pathogenic MECP2 variants. Because the MECP2 gene is subject to X‐chromosome inactivation (XCI), factors including MECP2 genotypic variation, tissue differences in XCI, and skewing of XCI all likely contribute to the clinical severity of individuals with RTT.MethodsWe analyzed the XCI patterns from blood samples of 320 individuals and their mothers. It includes individuals with RTT (n = 287) and other syndromes sharing overlapping phenotypes with RTT (such as CDKL5 Deficiency Disorder [CDD, n = 16]). XCI status in each proband/mother duo and the parental origin of the preferentially inactivated X chromosome were analyzed.ResultsThe average XCI ratio in probands was slightly increased compared to their unaffected mothers (73% vs. 69%, p = .0006). Among the duos with informative XCI data, the majority of individuals with classic RTT had their paternal allele preferentially inactivated (n = 180/220, 82%). In sharp contrast, individuals with CDD had their maternal allele preferentially inactivated (n = 10/12, 83%). Our data indicate a weak positive correlation between XCI skewing ratio and clinical severity scale (CSS) scores in classic RTT patients with maternal allele preferentially inactivated XCI (r s = 0.35, n = 40), but not in those with paternal allele preferentially inactivated XCI (r s = −0.06, n = 180). The most frequent MECP2 pathogenic variants were enriched in individuals with highly/moderately skewed XCI patterns, suggesting an association with higher levels of XCI skewing.ConclusionThese results extend our understanding of the pathogenesis of RTT and other syndromes with overlapping clinical features by providing insight into the both XCI and the preferential XCI of parental alleles.

Inferring genes that escape X-Chromosome inactivation reveals important contribution of variable escape genes to sex-biased diseases.

The X Chromosome plays an important role in human development and disease. However, functional genomic and disease association studies of X genes greatly lag behind autosomal gene studies, in part owing to the unique biology of X-Chromosome inactivation (XCI). Because of XCI, most genes are only expressed from one allele. Yet, ∼30% of X genes “escape” XCI and are transcribed from both alleles, many only in a proportion of the population. Such interindividual differences are likely to be disease relevant, particularly for sex-biased disorders. To understand the functional biology for X-linked genes, we developed X-Chromosome inactivation for RNA-seq (XCIR), a novel approach to identify escape genes using bulk RNA-seq data. Our method, available as an R package, is more powerful than alternative approaches and is computationally efficient to handle large population-scale data sets. Using annotated XCI states, we examined the contribution of X-linked genes to the disease heritability in the United Kingdom Biobank data set. We show that escape and variable escape genes explain the largest proportion of X heritability, which is in large part attributable to X genes with Y homology. Finally, we investigated the role of each XCI state in sex-biased diseases and found that although XY homologous gene pairs have a larger overall effect size, enrichment for variable escape genes is significantly increased in female-biased diseases. Our results, for the first time, quantitate the importance of variable escape genes for the etiology of sex-biased disease, and our pipeline allows analysis of larger data sets for a broad range of phenotypes.

Human induced pluripotent stem cells from two azoospermic patients with Klinefelter syndrome show similar X chromosome inactivation behavior to female pluripotent stem cells.

STUDY QUESTIONDoes the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype?SUMMARY ANSWEROur results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features.WHAT IS KNOWN ALREADYSo far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture.STUDY DESIGN, SIZE, DURATIONThe study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor.PARTICIPANTS/MATERIALS, SETTING, METHODSIn this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives.MAIN RESULTS AND THE ROLE OF CHANCEApplying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS.LIMITATIONS, REASONS FOR CAUTIONDetailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments.WIDER IMPLICATIONS OF THE FINDINGSAs KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI ‘escapee’ genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2–388/2016–40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.

Global Characterization of X Chromosome Inactivation in Human Pluripotent Stem Cells

Dosage compensation of sex-chromosome gene expression between male and female mammals is achieved via X chromosome inactivation (XCI) by employing epigenetic modifications to randomly silence one X chromosome during early embryogenesis. Human pluripotent stem cells (hPSCs) were reported to present various states of XCI that differ according to the expression of the long non-coding RNA XIST and the degree of X chromosome silencing. To obtain a comprehensive perspective on XCI in female hPSCs, we performed a large-scale analysis characterizing different XCI parameters in more than 700 RNA high-throughput sequencing samples. Our findings suggest differences in XCI status between most published samples of embryonic stem cells (ESCs) and induced PSCs (iPSCs). While the majority of iPSC lines maintain an inactive X chromosome, ESC lines tend to silence the expression of XIST and upregulate distal chromosomal regions. Our study highlights significant epigenetic heterogeneity within hPSCs, which may bear implications for their use in research and regenerative therapy.

Xist/Tsix expression dynamics during mouse peri-implantation development revealed by whole-mount 3D RNA-FISH

During peri-implantation development in mice, X chromosome inactivation (XCI) status changes dynamically. Here, we examined the expression of Xist and its antisense partner, Tsix, via whole-mount 3D RNA-FISH using strand-specific probes and evaluated XCI status. The results indicate that Xist expression disappears completely by embryonic day (E) 4.5 without Tsix activation in the ICM and that Xist re-expression occurs at E4.75 in some cells, suggesting that random XCI is already initiated in these cells. Intriguingly, epiblast cells exhibiting biallelic Xist expression were observed frequently (~15%) at E5.25 and E5.5. Immunostaining analysis of epigenetic modifications suggests that global change in epigenomic status occurs concomitantly with the transition from imprinted to random XCI. However, global upregulation of H3K27me3 modifications initiated earlier than other modifications, occurring specifically in ICM during progression of Xist erasure. Although both Xist expression and imprinted XCI are thought to be stable in the primitive endoderm/visceral endoderm and trophectoderm/extraembryonic ectoderm lineages, transient loss of Xist clouds was noted only in a subset of extraembryonic ectodermal cells, suggesting distinct features of Xist regulation among the three different embryonic tissue layers. These results will serve as a basis for future functional studies of XCI regulation in vivo.

Clonal analysis of early-stage bilateral papillary thyroid cancer identifies field cancerization.

Bilaterality is a newly identified indicator for aggressive tumor behavior and poor outcome in papillary thyroid cancer. However, the clonal origin of these bilateral tumors remains unclear. Here we analyzed 28 pairs of early-stage papillary thyroid cancers (stage I-II without extra-thyroidal extension, lymph node metastasis or distant metastasis) that underwent surgery at First Affiliated Hospital of Zhejiang University School of Medicine (Hangzhou, China). Genomic DNA was extracted from paraffin-embedded tissues after microdissection and analyzed for BRAF mutation and X-chromosome inactivation. A total of 16 patients (16/28, 57.1%) harbored different BRAF status in bilateral tumors. Fourteen patients were available for X-chromosome inactivation assay and 10 of them achieved informative results. Bilateral tumors from four cases had distinct patterns of X-chromosome inactivation. Combining the results of X-chromosome inactivation and BRAF analysis, we demonstrated that at least 64.3% (18/28) cases harbored discordant X-chromosome inactivation or BRAF status, indicating their independent clonal origin in bilateral tumors. The present study confirms "field cancerization" in early-stage bilateral thyroid cancers, suggesting that these subtype papillary thyroid cancers should be treated as independent and localized tumors.

Epigenetic reprogramming by naïve conditions establishes an irreversible state of partial X chromosome reactivation in female stem cells.

Female human pluripotent stem cells (PSCs) have variable X-chromosome inactivation (XCI) status. One of the X chromosomes may either be inactive (Xi) or display some active state markers. Long-term cultivation of PSCs may lead to an erosion of XCI and partial X reactivation. Such heterogeneity and instability of XCI status might hamper the application of human female PSCs for therapy or disease modeling. We attempted to address XCI heterogeneity by reprogramming human embryonic stem cells (hESCs) to the naïve state. We propagated five hESC lines under naïve culture conditions. PSCs acquired naïve cells characteristics although these changes were not uniform for all of the hESC lines. Transition to the naïve state was accompanied by a loss of XIST expression, loss of Xi H3K27me3 enrichment and a switch in Xi replication synchronously with active X, except for two regions. This pattern of Xi reactivation was observed in all cells in two hESC lines. However, these cells were unable to undergo classical XCI upon spontaneous differentiation. We conclude that naïve culture conditions do not resolve the variability in XCI status in female human ESC lines and establish an irreversible heterogeneous pattern of partial X reactivation.

RNA-FISH and Immunofluorescence of Mouse Preimplantation and Postimplantation Embryos.

There are two modes of X chromosome inactivation (XCI) in the mouse. One mode is imprinted XCI: it is initiated at around the four-cell stage in favor of the paternal X chromosome, and is maintained in the extraembryonic tissues. The other mode is random XCI, which takes place in the epiblast lineage at the periimplantation stage. X-linked noncoding Xist RNA, which becomes upregulated on the X chromosome to be inactivated at the onset of XCI and plays a critical role in both imprinted and random XCI, and its accumulation in the nucleus have been referred to as one of the hallmarks of the presence of the inactivated X chromosome. RNA-FISH has therefore been an invaluable method for the study of XCI. As XCI status changes dynamically during periimplantation development in the mouse, analysis using samples from these developmental stages is absolutely necessary for elucidation of the molecular basis of XCI mechanisms. However, dissection of the embryos at around the periimplantation stages is not easy, and this impedes in vivo analysis of the kinetics of XCI. Here, we describe our methods for dissecting the periimplantation stage embryo and subsequent procedures for RNA-FISH and immunostaining.

Abstract 2420: Integrative analyses of gene expression, DNA methylation, genotype and copy number alterations characterize X-chromosome inactivation in ovarian cancer

Abstract Introduction: In females, X-chromosome inactivation (XCI) epigenetically silences transcription of one copy of the X chromosome. Which chromosome is silenced is randomly selected, and is tissue- and cell-specific. While some genes are known to escape XCI under normal conditions, aberrant XCI patterns are thought to occur in female-specific cancers, although the role of XCI in ovarian tumorigenesis and progression is largely unknown. The process of XCI is complex, and integration of gene expression, DNA methylation, and copy number data can inform the XCI status of individual genes and chromosome-wide XCI patterns for individual patients. Methods: We evaluated gene- and chromosome-level patterns of XCI by integrating RNA sequence, copy number alteration, genotype, and DNA methylation data to study XCI escape patterns in tumor samples from 99 ovarian cancer patients. We measured allele-specific expression (ASE) for 397 X-linked genes to identify the active alleles for each tumor. Combining ASE data with knowledge of copy number status, we used a Bayesian beta-binomial mixture model to estimate which genes escaped XCI for each patient, and validated our findings using DNA methylation data. To assess global XCI patterns, we performed cluster analyses on the ASE and methylation data, after adjusting for loss of heterozygosity. We examined the relationship between the clusters and clinical factors, including overall survival and time to recurrence. Results: DNA promoter methylation demonstrated inverse regional correlations with ASE. Cluster analyses using ASE and methylation data demonstrated evidence of two tumor clusters, representing normal XCI and global XCI dysregulation. The dysregulated XCI cluster (N=52) was associated with lower X-inactive specific transcript expression as expected (p&amp;lt;0.01). Patients with XCI dysregulated tumors were higher grade, stage, serous histology and were sub-optimally debulked (p&amp;lt;0.05). These patients also had shorter overall survival time (HR=1.87, p=0.02) and time to recurrence (HR=2.34, p&amp;lt;0.01), although associations were attenuated after covariate adjustment. In 45 tumor samples with sufficient data, we observed escape patterns largely consistent with previous reports of multiple tissue types. When comparing tumor to normal ovarian tissue, eight genes (CXorf23, CXorf36, BRWD3, ELF4, SLITRK4, GABRE, CLCN4, SH3BGRL) showed putative escape in the tumor and two genes (RBBP7, OFD1) showed discrepant tumor inactivation. Conclusions: We identified discrepant gene-level XCI tumor classifications compared to normal tissue and identified a group of patients with chromosome-wide XCI dysregulation associated with worse clinical prognosis. This provides evidence of the role of XCI in ovarian cancer and highlights the need to integrate multiple genomic data types to study XCI. Citation Format: Stacey J. Winham, Nicholas B. Larson, Sebastian M. Armasu, Zachary C. Fogarty, Melissa C. Larson, Kimberly R. Kalli, Kate Lawrenson, Simon Gayther, Brooke L. Fridley, Ellen L. Goode. Integrative analyses of gene expression, DNA methylation, genotype and copy number alterations characterize X-chromosome inactivation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2420. doi:10.1158/1538-7445.AM2017-2420

X chromosome inactivation in human pluripotent stem cells as a model for human development: back to the drawing board?

Human pluripotent stem cells (hPSC), both embryonic and induced (hESC and hiPSC), are regarded as a valuable in vitro model for early human development. In order to fulfil this promise, it is important that these cells mimic as closely as possible the in vivo molecular events, both at the genetic and epigenetic level. One of the most important epigenetic events during early human development is X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female cells. XCI is important for proper development and aberrant XCI has been linked to several pathologies. Recently, novel data obtained using high throughput single-cell technology during human preimplantation development have suggested that the XCI mechanism is substantially different from XCI in mouse. It has also been suggested that hPSC show higher complexity in XCI than the mouse. Here we compare the available recent data to understand whether XCI during human preimplantation can be properly recapitulated using hPSC. We will summarize what is known on the timing and mechanisms of XCI during human preimplantation development. We will compare this to the XCI patterns that are observed during hPSC derivation, culture and differentiation, and comment on the cause of the aberrant XCI patterns observed in hPSC. Finally, we will discuss the implications of the aberrant XCI patterns on the applicability of hPSC as an in vitro model for human development and as cell source for regenerative medicine. Combinations of the following keywords were applied as search criteria in the PubMed database: X chromosome inactivation, preimplantation development, embryonic stem cells, induced pluripotent stem cells, primordial germ cells, differentiation. Recent single-cell RNASeq data have shed new light on the XCI process during human preimplantation development. These indicate a gradual inactivation on both XX chromosomes, starting from Day 4 of development and followed by a random choice to inactivate one of them, instead of the mechanism in mice where imprinted XCI is followed by random XCI. We have put these new findings in perspective using previous data obtained in human (and mouse) embryos. In addition, there is an ongoing discussion whether or not hPSC lines show X chromosome reactivation upon derivation, mimicking the earliest embryonic cells, and the XCI states observed during culture of hPSC are highly variable. Recent studies have shown that hPSC rapidly progress to highly aberrant XCI patterns and that this process is probably driven by suboptimal culture conditions. Importantly, these aberrant XCI states seem to be inherited by the differentiated hPSC-progeny. The aberrant XCI states (and epigenetic instability) observed in hPSC throw a shadow on their applicability as an in vitro model for development and disease modelling. Moreover, as the aberrant XCI states observed in hPSC seem to shift to a more malignant phenotype, this may also have important consequences for the safety aspect of using hPSC in the clinic.

Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation.

Pluripotency, differentiation, and X Chromosome inactivation (XCI) are key aspects of embryonic development. However, the underlying relationship and mechanisms among these processes remain unclear. Here, we systematically dissected these features along developmental progression using mouse embryonic stem cells (mESCs) and single-cell RNA sequencing with allelic resolution. We found that mESCs grown in a ground state 2i condition displayed transcriptomic profiles diffused from preimplantation mouse embryonic cells, whereas EpiStem cells closely resembled the post-implantation epiblast. Sex-related gene expression varied greatly across distinct developmental states. We also identified novel markers that were highly enriched in each developmental state. Moreover, we revealed that several novel pathways, including PluriNetWork and Focal Adhesion, were responsible for the delayed progression of female EpiStem cells. Importantly, we “digitalized” XCI progression using allelic expression of active and inactive X Chromosomes and surprisingly found that XCI states exhibited profound variability in each developmental state, including the 2i condition. XCI progression was not tightly synchronized with loss of pluripotency and increase of differentiation at the single-cell level, although these processes were globally correlated. In addition, highly expressed genes, including core pluripotency factors, were in general biallelically expressed. Taken together, our study sheds light on the dynamics of XCI progression and the asynchronicity between pluripotency, differentiation, and XCI.

Complexities of X chromosome inactivation status in female human induced pluripotent stem cells-a brief review and scientific update for autism research.

Induced pluripotent stem cells (iPSCs) allow researchers to make customized patient-derived cell lines by reprogramming noninvasively retrieved somatic cells. These cell lines have the potential to faithfully represent an individual’s genetic background; therefore, in the absence of available human brain tissue from a living patient, these models have a significant advantage relative to other models of neurodevelopmental disease. When using human induced pluripotent stem cells (hiPSCs) to model X-linked developmental disorders or inherited conditions that undergo sex-specific modulation of penetrance (e.g., autism spectrum disorders), there are significant complexities in the course and status of X chromosome inactivation (XCI) that are crucial to consider in establishing the validity of cellular models. There are major gaps and inconsistencies in the existing literature regarding XCI status during the derivation and maintenance of hiPSCs and their differentiation into neurons. Here, we briefly describe the importance of the problem, review the findings and inconsistencies of the existing literature, delineate options for specifying XCI status in clonal populations, and develop recommendations for future studies.

The dynamic changes of X chromosome inactivation during early culture of human embryonic stem cells

X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in human female cells. Several previous reports have described the promiscuous XCI status in long-term cultured female human embryonic stem cells (hESCs), and the majority of them exhibit non-random XCI. However, when and how such female hESCs acquire the aberrant XCI states during culture is unknown. Herein, through comparing the XCI states in 18 paired hES cell lines throughout early culture, we revealed a uniform dynamic change during this culture period under a widely used culture condition. The female initial hESCs (ihESCs, P4-P9) expressed XIST RNA, H3K27me3 punctate enrichment and displayed random XCI pattern. By further culturing, the female early hESCs (ehESCs, P20–P30) lost the expression of XIST RNA, H3K27me3 punctate enrichment and exhibited a completely skewed XCI pattern. Importantly, a subset of X-linked genes was up-regulated in ehESCs, including some cancer-related genes. At last, we found 5% physiological oxygen was beneficial for the expression of XIST and H3K27me3 punctate enrichment, but not for the XCI pattern. We conclude that the XCI dynamic change is a frequent epigenetic instability event during early culture, which is accompanied by the up-regulation of some X-linked genes. Furthermore, we emphasize that physiological oxygen is beneficial for XCI fidelity.

Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore, we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally, differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC, more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. Not applicable. Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen, grant 1502512N), Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel, on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest.

Molecular genetic investigation of placental mesenchymal dysplasia

Molecular genetic investigation of placental mesenchymal dysplasia

Cancer-Testis Antigen Expression in Serous Endometrial Cancer with Loss of X Chromosome Inactivation.

BackgroundCancer-testis antigens (CTAs) are potential targets for cancer immunotherapy. Many CTAs are located on the X chromosome and are epigenetically regulated. Loss of X chromosome inactivation (XCI) is observed in breast and ovarian cancers and is thought to be related to the overexpression of CTAs. We investigated the relation between expression of CTAs and loss of XCI in endometrial cancer.Materials and MethodsWe used data generated by The Cancer Genome Atlas Genome Data Analysis Centers and data for Xist knockout mice available at the Gene Expression Omnibus.ResultsThe status of XCI was estimated by methylation status, and deletion or gain of the X chromosome. The endometrial cancers were classified into the following three groups: preserved inactivated X chromosome (Xi) (n = 281), partial reactivation of Xi (n = 52), and two copies of active X group (n = 38). Loss of XCI was more common in serous adenocarcinoma. Expression of CTAs increased in endometrial cancer with loss of XCI, which was accompanied by global hypomethylation. Expression of CTAs did not increase in Xist knockout mice.ConclusionsLoss of XCI is common in serous adenocarcinoma. Global hypomethylation, and not loss of XCI, is the main mechanism of overexpression of CTAs.

Genetic Evaluation of Copy Number Variations, Loss of Heterozygosity, and Single-Nucleotide Variant Levels in Human Embryonic Stem Cells With or Without Skewed X Chromosome Inactivation.

Human embryonic stem cells (hESCs) exhibiting skewed X chromosome inactivation (XCI) have been reported. The copy number variations (CNVs), loss of heterozygosity (LOH), or single-nucleotide variant (SNV) events in those epigenetically distinct cells remain unknown, and whether such genetic abnormalities will influence the XCI status of hESCs is unclear. In this study, three hESCs with skewed XCI, three with random XCI, and two male hESC lines at different passages were analyzed for CNVs and LOH levels using a high-resolution genotyping microarray. Whole-exome sequencing was used to investigate the potentially damaging SNVs. On average, 17.6 CNVs and 5.3 cases of LOH were identified in the skewed hESCs, which were similar to the rates observed in random hESCs. Five recurrent CNV regions were uniquely identified in the skewed hESCs, but all of them were considered polymorphisms. With the exception of a nongenic CNV, no additional CNVs were detected on the X chromosome in the skewed hESCs. Although the XCI status in two hESC lines was observed to be changed from random to skewed, no significant CNV difference was identified before and after the XCI change. SNV analysis indicated that normal alleles are maintained for most genes within copy-neutral LOH regions. Three types of expression patterns were observed in heterozygous alleles, and the damaging SNVs in skewed hESCs favored the expression of the wild-type alleles. In conclusion, in the present study, we did not find genetic differences in the CNV and LOH levels between hESCs with and without skewed XCI. Wild-type allele expression in the presence of damaging SNVs on the X chromosome in skewed hESCs might alleviate adverse effects in those hESCs.

Escape from X inactivation varies in mouse tissues.

X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3–7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.