Chromosome Inactivation Status Research Articles

Genes that escape from X-chromosome inactivation: Potential contributors to Klinefelter syndrome.

One of the two X chromosomes in females is epigenetically inactivated, thereby compensating for the dosage difference in X‐linked genes between XX females and XY males. Not all X‐linked genes are completely inactivated, however, with 12% of genes escaping X chromosome inactivation and another 15% of genes varying in their X chromosome inactivation status across individuals, tissues or cells. Expression of these genes from the second and otherwise inactive X chromosome may underlie sex differences between males and females, and feature in many of the symptoms of XXY Klinefelter males, who have both an inactive X and a Y chromosome. We review the approaches used to identify genes that escape from X‐chromosome inactivation and discuss the nature of their sex‐biased expression. These genes are enriched on the short arm of the X chromosome, and, in addition to genes in the pseudoautosomal regions, include genes with and without Y‐chromosomal counterparts. We highlight candidate escape genes for some of the features of Klinefelter syndrome and discuss our current understanding of the mechanisms underlying silencing and escape on the X chromosome as well as additional differences between the X in males and females that may contribute to Klinefelter syndrome.

Somatic Cell Nuclear Transfer in Mice: Basic Protocol and Its Modification for Correcting X Chromosome Inactivation Status.

Somatic cell nuclear transfer (SCNT) enables the production of animals from single cell nuclei. Unlike normally fertilized embryos, SCNT-derived embryos ectopically express the Xist gene from the maternal allele, because of the lack of Xist-repressing imprints in the somatic donor genome. This has severely compromised the development of SCNT-derived embryos, at least in mice. Here, we describe the basic protocol of mouse SCNT as well as a Xist knockdown SCNT procedure, which remarkably increases the efficiency of cloning mice.

MLL1: the thin red line divides naïve and primed pluripotency.

From mouse pre-implantation and early post-implantation embryos, stem cells at different pluripotent states can be captured in in vitro culture. For example, naive embryonic stem cells (ESC) are derived from inner cell mass (ICM) of the blastocyst, while primed epiblast stem cells (EpiSC) are captured from the post-implantation embryo epiblast. Though both ESCs and EpiSCs are pluripotent stem cells, they have distinct characters in terms of pluripotent gene expression profile, epigenetic status, metabolic pathways, growth factor requirement, and in female, the X chromosome inactivation status (1). On the other hand, these two pluripotent states are interchangeable, once switching the culture condition of ESCs to that for EpiSCs, naive ESCs convert to EpiSCs. The efficiency of converting EpiSCs to ESCs, however, is much low, which usually requires over-expressing pluripotency-associated genes such as Nanog , Klf4 , Esrrb , Tfcp2l1 and Nr5a2 (2-6). Similar to somatic cell reprogramming to induced pluripotent stem cells, converting EpiSCs to ESCs is an epigenome-resetting process. Characterizing these epigenetic barriers represents an important approach to further improve reprogramming efficiency (7).

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.

Differential X Chromosome Inactivation Patterns during the Propagation of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) represent a potentially useful tool for studying the molecular mechanisms of disease thanks to their ability to generate patient-specific hiPSC clones. However, previous studies have reported that DNA methylation profiles, including those for imprinted genes, may change during passaging of hiPSCs. This is particularly problematic for hiPSC models of X-linked disease, because unstable X chromosome inactivation status may affect the detection of phenotypes. In the present study, we examined the epigenetic status of hiPSCs derived from patients with Rett syndrome, an X-linked disease, during long-term culture. To analyze X chromosome inactivation, we used a methylation-specific polymerase chain reaction (MSP) to assay the human androgen receptor locus (HUMARA). We found that single cell-derived hiPSC clones exhibit various states of X chromosome inactivation immediately after clonal isolation, even when established simultaneously from a single donor. X chromosome inactivation states remain variable in hiPSC clones at early passages, and this variability may affect cellular phenotypes characteristic of X-linked diseases. Careful evaluation of X chromosome inactivation in hiPSC clones, particularly in early passages, by methods such as HUMARA-MSP, is therefore important when using patient-specific hiPSCs to model X-linked disease.

Derivation of consensus inactivation status for X-linked genes from genome-wide studies

BackgroundX chromosome inactivation is the epigenetic silencing of the majority of the genes on one of the X chromosomes in XX therian mammals. In humans, approximately 15 % of genes consistently escape from this inactivation and another 15 % of genes vary between individuals or tissues in whether they are subject to, or escape from, inactivation. Multiple studies have provided inactivation status calls for a large subset of the genes on the X chromosome; however, these studies vary in which genes they were able to make calls for and in some cases which call they give a specific gene.MethodsThis analysis aggregated three published studies that have examined X chromosome inactivation status of genes across the X chromosome, generating consensus calls and identifying discordancies. The impact of expression level and chromosomal location on X chromosome inactivation status was also assessed.ResultsOverall, we assigned a consensus XCI status 639 genes, including 78 % of protein-coding genes expressed outside of the testes, with a lower frequency for non-coding RNA and testis-specific genes. Study-specific discordancies suggest that there may be instability of XCI during cell culture and also highlight study-specific variations in call type. We observe an enrichment of discordant genes at boundaries between genes subject to and escaping from inactivation.ConclusionsThis study has compiled a comprehensive list of X-chromosome inactivation statuses for genes and also discovered some biases which will help guide future studies examining X-chromosome inactivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-015-0053-7) contains supplementary material, which is available to authorized users.

Whole exome sequencing in females with autism implicates novel and candidate genes.

Classical autism or autistic disorder belongs to a group of genetically heterogeneous conditions known as Autism Spectrum Disorders (ASD). Heritability is estimated as high as 90% for ASD with a recently reported compilation of 629 clinically relevant candidate and known genes. We chose to undertake a descriptive next generation whole exome sequencing case study of 30 well-characterized Caucasian females with autism (average age, 7.7 ± 2.6 years; age range, 5 to 16 years) from multiplex families. Genomic DNA was used for whole exome sequencing via paired-end next generation sequencing approach and X chromosome inactivation status. The list of putative disease causing genes was developed from primary selection criteria using machine learning-derived classification score and other predictive parameters (GERP2, PolyPhen2, and SIFT). We narrowed the variant list to 10 to 20 genes and screened for biological significance including neural development, function and known neurological disorders. Seventy-eight genes identified met selection criteria ranging from 1 to 9 filtered variants per female. Five females presented with functional variants of X-linked genes (IL1RAPL1, PIR, GABRQ, GPRASP2, SYTL4) with cadherin, protocadherin and ankyrin repeat gene families most commonly altered (e.g., CDH6, FAT2, PCDH8, CTNNA3, ANKRD11). Other genes related to neurogenesis and neuronal migration (e.g., SEMA3F, MIDN), were also identified.

Primed Pluripotent Cell Lines Derived from Various Embryonic Origins and Somatic Cells in Pig

Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1–60 and TRA 1–81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.

X inactivation testing for identifying a non-syndromic X-linked mental retardation gene

The purpose of this study was to identify a gene causing non-syndromic X-linked mental retardation in an extended family, taking advantage of the X chromosome inactivation status of the females in order to determine their carrier state. X inactivation in the females was determined with the androgen receptor methylation assay; thereafter, the X chromosome was screened with evenly spaced polymorphic markers. Once initial linkage was identified, the region of interest was saturated with additional markers and the males were added to the analysis. Candidate genes were sequenced. Ten females showed skewed inactivation, while six revealed a normal inactivation pattern. A maximal lod score of 5.54 at θ = 0.00 was obtained with the marker DXS10151. Recombination events mapped the disease gene to a 17.4-Mb interval between the markers DXS10153 and DXS10157. Three candidate genes in the region were sequenced and a previously described missense mutation (P375L) was identified in the ACSL4/FACL4 gene. On the basis of the female X inactivation status, we have mapped and identified the causative mutation in a gene causing non-syndromic X-linked mental retardation.

Clonal origin of multifocal hepatocellular carcinoma

Hepatocellular carcinoma is the most common primary tumor of the liver. Patients frequently have multiple histologically similar, but anatomically separate tumors. The clonal origin of multiple hepatocellular carcinomas is uncertain. The authors analyzed 31 tumors from 12 different patients (11 women, 1 man), who had multiple hepatocellular carcinomas involving 1 or both lobes. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue using laser capture microdissection. DNA was analyzed for loss of heterozygosity (LOH), X chromosome inactivation status, and TP53 gene mutations. Ten (83%) of the 12 patients showed LOH in at least 1 of the analyzed microsatellite markers. Concordant LOH patterns between separate hepatocellular carcinomas in individual patients were seen in 8 (80%) of 10 cases, whereas discordant patterns were seen in 2 (20%) of 10 cases. Five (50%) of 10 informative female patients showed identical nonrandom X chromosome inactivation patterns in multiple tumors; 1 case showed discordant nonrandom X chromosome inactivation pattern. TP53 mutations were identified in 8 (67%) of 12 patients. Tumors in 7 (88%) of these 8 patients showed different point mutations. Three patients (Cases 4, 5, and 10) had tumors with additional TP53 point mutations, indicating additional genetic abnormalities in these tumors. The data suggested that the significant proportion of patients with multifocal hepatocellular carcinomas have tumors of common clonal origin.

Escape from X chromosome inactivation is an intrinsic property of the Jarid1c locus

Although most genes on one X chromosome in mammalian females are silenced by X inactivation, some "escape" X inactivation and are expressed from both active and inactive Xs. How these escape genes are transcribed from a largely inactivated chromosome is not fully understood, but underlying genomic sequences are likely involved. We developed a transgene approach to ask whether an escape locus is autonomous or is instead influenced by X chromosome location. Two BACs carrying the mouse Jarid1c gene and adjacent X-inactivated transcripts were randomly integrated into mouse XX embryonic stem cells. Four lines with single-copy, X-linked transgenes were identified, and each was inserted into regions that are normally X-inactivated. As expected for genes that are normally subject to X inactivation, transgene transcripts Tspyl2 and Iqsec2 were X-inactivated. However, allelic expression and RNA/DNA FISH indicate that transgenic Jarid1c escapes X inactivation. Therefore, transgenes at 4 different X locations recapitulate endogenous inactive X expression patterns. We conclude that escape from X inactivation is an intrinsic feature of the Jarid1c locus and functionally delimit this escape domain to the 112-kb maximum overlap of the BACs tested. Additionally, although extensive chromatin differences normally distinguish active and inactive loci, unmodified BACs direct proper inactive X expression patterns, establishing that primary DNA sequence alone, in a chromosome position-independent manner, is sufficient to determine X chromosome inactivation status. This transgene approach will enable further dissection of key elements of escape domains and allow rigorous testing of specific genomic sequences on inactive X expression.

Similar biological characteristics of human embryonic stem cell lines with normal and abnormal karyotypes

BACKGROUNDHuman embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar.METHODSSeven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines.RESULTSAll seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines.CONCLUSIONSThe present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.

Comparison between CD4 and CD8 Lymphocytes as Controls for Methylation-Based Clonal Assay.

In somatic cells from normal female subjects, random inactivation of one of two X chromosomes occurs at an early stage of embryogenesis. Therefore, half of normal females possess an inactivated maternal X chromosome and half possess an inactivated paternal X chromosome. The human androgen receptor gene (HUMARA) contains highly polymorphic CAG trinucleotide repeats and several methyl-sensitive restriction enzyme (me-RE) sites. This polymorphism enables discrimination between paternal and maternal alleles; methylation status can easily be determined using me-RE because unmethylated (activated) sequences are sensitive to me-RE cutting while methylated (inactivated) sequences are resistant. Therefore, in clonal populations, one of the two HUMARA genes should be completely lost.HUMARA-based assays have been applied to evaluate the clonal nature of various hematologic diseases. However, the main drawback of this method is the biased X chromosome inactivation, which is extremely skewed even in normal females. Consequently, HUMARA methylation patterns must be compared with adequate controls and clonal evaluation is impossible when both target and control samples show extremely skewed patterns. In most previous reports, granulocyte lineage clonality is assessed using T-lymphocytes or whole mononuclear cells as controls. To evaluate applicability of CD4 and CD8 cells as controls for clonal analysis, we examined X chromosome inactivation status in 22 healthy heterozygous females. Peripheral CD4+ and CD8+ lymphocytes were separated using the MACS system and DNA was extracted from both samples for PCR-based HUMARA assay. Although the 2 HUMARA peaks were almost identical in the HpaII pre-digested samples, there was skewing in some samples from elderly subjects after HpaII digestion: only 3 females in the CD4 group showed biased X inactivation, while 7 females in the CD8 group showed biased inactivation. These results indicate that CD8+ lymphocytes tend to more frequently show age-dependent skewing when compared with CD4+ lymphocytes.Because it has been reported that the frequency of peripheral naive T lymphocytes is reduced in advanced age, we investigated the correlation between CD28 expression and age in 25 healthy controls (median age of 67), as the majority of CD28-positive cells were naive T cells. The proportion of the naive T cell population that was composed of CD8+ lymphocytes decreased markedly with age when compared with that of CD4+ cells.Subsequently, we investigated age-related T cell receptor repertoire alterations. To evaluate the complexity of the TCR repertoire, the TCR Vβ repertoires of CD4+ and CD8+ lymphocytes were assessed by CDR3 size distribution analysis. The mean complexity score for control CD8+ lymphocytes (72.0) was significantly lower than that for CD4+ ymphocytes (126.5, P=0.011). In conclusion, frequent skewing of X chromosome inactivation was paralleled by severe reductions in naive T lymphocytes and T cell repertoire in CD8+ lymphocytes. Therefore, CD4+ lymphocytes might be better suited as controls for methylation-based clonal assay than CD8+ lymphocytes.

Pregnancy Outcome in Recurrent Miscarriage Patients With Skewed X Chromosome Inactivation

In Brief OBJECTIVE To analyze X inactivation in women with recurrent miscarriage to estimate whether skewed X inactivation is associated with recurrent miscarriage and whether it predicts next pregnancy outcomes. METHODS A multicenter study was performed. A power calculation determined that 101 patients were needed to detect a difference in skewed X inactivation between patients and controls. Patients were entered into a prospective trial of mononuclear-cell immunotherapy and subsequently tested for skewed X inactivation. Age-matched controls had one live birth and no prior miscarriages. Results from our X inactivation assay were compared with those from an independent genetics laboratory. RESULTS Greater than 75% skewing was seen in 22.6% of patients and 26.5% controls (P = .52). Greater than 90% skewing was seen in 6.6% of patients and 3.9% of controls (P = .77). There were 19.8% of primary aborters and 32% of secondary aborters with greater than 75% skewed X inactivation (P = .38). There were 4.9% of primary aborters and 12.0% of secondary aborters with greater than 90% skewed X inactivation (P = .27) Neither greater than 75% nor greater than 90% skewed X inactivation impacted next pregnancy outcomes (odds ratios = 0.87 [95% confidence interval (CI) 0.34, 2.3] and 1.4 [95% CI 0.27, 7.5], respectively). Results of the exchange of samples with an independent laboratory were highly correlated (α = 0.987, P < .001, coefficient of variation = 5.5%). CONCLUSION Skewed X chromosome inactivation is not associated with recurrent miscarriage. A patient's X chromosome inactivation status does not predict next pregnancy outcome. Our assay correlates with another experienced laboratory. This prospective, multicenter study demonstrates that skewed X chromosome inactivation is not associated with recurrent miscarriage, nor is it predictive of a next pregnancy outcome.

Pregnancy outcome in recurrent miscarriage patients with skewed X chromosome inactivation.

To analyze X inactivation in women with recurrent miscarriage to estimate whether skewed X inactivation is associated with recurrent miscarriage and whether it predicts next pregnancy outcomes. A multicenter study was performed. A power calculation determined that 101 patients were needed to detect a difference in skewed X inactivation between patients and controls. Patients were entered into a prospective trial of mononuclear-cell immunotherapy and subsequently tested for skewed X inactivation. Age-matched controls had one live birth and no prior miscarriages. Results from our X inactivation assay were compared with those from an independent genetics laboratory. Greater than 75% skewing was seen in 22.6% of patients and 26.5% controls (P =.52). Greater than 90% skewing was seen in 6.6% of patients and 3.9% of controls (P =.77). There were 19.8% of primary aborters and 32% of secondary aborters with greater than 75% skewed X inactivation (P =.38). There were 4.9% of primary aborters and 12.0% of secondary aborters with greater than 90% skewed X inactivation (P =.27) Neither greater than 75% nor greater than 90% skewed X inactivation impacted next pregnancy outcomes (odds ratios = 0.87 [95% confidence interval (CI) 0.34, 2.3] and 1.4 [95% CI 0.27, 7.5], respectively). Results of the exchange of samples with an independent laboratory were highly correlated (alpha = 0.987, P <.001, coefficient of variation = 5.5%). Skewed X chromosome inactivation is not associated with recurrent miscarriage. A patient's X chromosome inactivation status does not predict next pregnancy outcome. Our assay correlates with another experienced laboratory.

Multiple congenital anomalies, brain hypomyelination, and ocular albinism in a female with dup(X)(pter→q24::q21.32→qter) and random X inactivation

We report on an 18-month-old girl with multiple congenital anomalies (prominence of the metopic suture, fine hair, club foot, absence of the 12th rib, brachydactyly) and severe mental retardation. The funduscopic examination showed diffuse retinal hypopigmentation. Brain magnetic resonance image (MRI) showed signs of diffuse hypomyelination. On cytogenetic and molecular evidence, the karyotype was 46,X,dirdup(X) (pter→q24::q21.32→qter). The duplication of the PLP gene, involved in Pelizaeus-Merzbacher disease, was confirmed by fluorescent in situ hybridization (FISH). Both cytogenetic and molecular studies on the X chromosome inactivation status indicated a random pattern in lymphocytes and fibroblasts. This patient appears to be the first case of a female bearing a large duplication of Xq with a random X inactivation. The phenotype of this patient is compared to that of previously reported cases with Xq duplication. Am. J. Med. Genet. 72:329–334, 1997. © 1997 Wiley-Liss, Inc.

Multiple congenital anomalies, brain hypomyelination, and ocular albinism in a female with dup(X)(pter→q24::q21.32→qter) and random X inactivation

We report on an 18-month-old girl with multiple congenital anomalies (prominence of the metopic suture, fine hair, club foot, absence of the 12th rib, brachydactyly) and severe mental retardation. The funduscopic examination showed diffuse retinal hypopigmentation. Brain magnetic resonance image (MRI) showed signs of diffuse hypomyelination. On cytogenetic and molecular evidence, the karyotype was 46,X,dirdup(X) (pter-->q24::q21.32-->qter). The duplication of the PLP gene, involved in Pelizaeus-Merzbacher disease, was confirmed by fluorescent in situ hybridization (FISH). Both cytogenetic and molecular studies on the X chromosome inactivation status indicated a random pattern in lymphocytes and fibroblasts. This patient appears to be the first case of a female bearing a large duplication of Xq with a random X inactivation. The phenotype of this patient is compared to that of previously reported cases with Xq duplication.