Identification of skewed X chromosome inactivation using exome and transcriptome sequencing in patients with suspected rare genetic disease.

BACKGROUND: X-chromosome inactivation (XCI) is an epigenetic process that occurs during early development in mammalian females by randomly silencing one of two copies of the X chromosome in each cell. The preferential inactivation of either the maternal or paternal copy of the X chromosome in a majority of cells results in a skewed or non-random pattern of X inactivation and is observed in over 25% of adult females. Identifying skewed X inactivation is of clinical significance in patients with suspected rare genetic diseases due to the possibility of biased expression of disease-causing genes present on the active X chromosome. The current clinical test for the detection of skewed XCI relies on the methylation status of the methylation-sensitive restriction enzyme (Hpall) binding site present in proximity of short tandem polymorphic repeats on the androgen receptor (AR) gene. This approach using one locus results in uninformative or inconclusive data for 10-20% of tests. Further, recent studies have shown inconsistency between methylation of the AR locus and the state of inactivation of the X chromosome. Herein, we develop a method for estimating X inactivation status, using exome and transcriptome sequencing data derived from blood in 227 female samples. We built a reference model for evaluation of XCI in 135 females from the GTEx consortium. We tested and validated the model on 11 female individuals with different types of undiagnosed rare genetic disorders who were clinically tested for X-skew using the AR gene assay and compared results to our outlier-based analysis technique. RESULTS: In comparison to the AR clinical test for identification of X inactivation, our method was concordant with the AR method in 9 samples, discordant in 1, and provided a measure of X inactivation in 1 sample with uninformative clinical results. We applied this method on an additional 81 females presenting to the clinic with phenotypes consistent with different hereditary disorders without a known genetic diagnosis. CONCLUSIONS: This study presents the use of transcriptome and exome sequencing data to provide an accurate and complete estimation of X-inactivation and skew status in a cohort of female patients with different types of suspected rare genetic disease.

Unraveling the role of Xist in X chromosome inactivation: insights from rabbit model and deletion analysis of exons and repeat A.

X chromosome inactivation (XCI) is a process that equalizes the expression of X-linked genes between males and females. It relies on Xist, continuously expressed in somatic cells during XCI maintenance. However, how Xist impacts XCI maintenance and its functional motifs remain unclear. In this study, we conducted a comprehensive analysis of Xist, using rabbits as an ideal non-primate model. Homozygous knockout of exon 1, exon 6, and repeat A in female rabbits resulted in embryonic lethality. However, X∆ReAX females, with intact X chromosome expressing Xist, showed no abnormalities. Interestingly, there were no significant differences between females with homozygous knockout of exons 2-5 and wild-type rabbits, suggesting that exons 2, 3, 4, and 5 are less important for XCI. These findings provide evolutionary insights into Xist function.

Imprinted X chromosome inactivation at the gamete-to-embryo transition.

In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.

[Analysis of X chromosome inactivation and prenatal diagnosis for a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3].

OBJECTIVE: To explore the correlation between skewed X chromosome inactivation (XCI) and clinical phenotype of a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3. METHODS: A pedigree diagnosed at Taizhou Hospital on November 10, 2021 was selected as the study subject. G-banded chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out to analyze the amniotic fluid and peripheral blood samples from the couple. XCI was detected by PCR amplification of CAG repeats in exon 1 of androgen receptor gene before and after the digestion with methylation-sensitive restriction enzyme Hpa II. Correlation between the genotype and clinical phenotype was analyzed. RESULTS: The karyotypes of the pregnant woman and the fetus were both determined as 46,X,del(X)(q22), and the result of CNV-seq was seq[hg19]del(X)(q22.1q22.3) chrX: g.10046000_105740000del, suggesting that both had harbored a 5.28 Mb deletion on the X chromosome. No obvious abnormality was found in the husband. XCI analysis showed that the activity ratio of the two X chromosomes of the pregnant woman and her fetus was 0 : 100. The X chromosome harboring the q22.1q22.3 deletion was completely inactivated, and the inactivated X chromosome of the fetus was derived from its mother. CONCLUSION: The fetus has harbored a maternally derived inactivated X chromosome del(X)(q22) , and its phenotype is closely associated with the activity of the abnormal X chromosome. Pedigree XCI analysis combined with the clinical phenotype has facilitated recognition of the maternal phenotype and prognosis of female fetus with loss of heterozygosity at Xq22.1q22.3.

Stochasticity in genetics and gene regulation.

Development from fertilized egg to functioning multi-cellular organism requires precision. There is no precision, and often no survival, without plasticity. Plasticity is conferred partly by stochastic variation, present inherently in all biological systems. Gene expression levels fluctuate ubiquitously through transcription, alternative splicing, translation and turnover. Small differences in gene expression are exploited to trigger early differentiation, conferring distinct function on selected individual cells and setting in motion regulatory interactions. Non-selected cells then acquire new functions along the spatio-temporal developmental trajectory. The differentiation process has many stochastic components. Meiotic segregation, mitochondrial partitioning, X-inactivation and the dynamic DNA binding of transcription factor assemblies-all exhibit randomness. Non-random X-inactivation generally signals deleterious X-linked mutations. Correct neural wiring, such as retina to brain, arises through repeated confirmatory activity of connections made randomly. In immune system development, both B-cell antibody generation and the emergence of balanced T-cell categories begin through stochastic trial and error followed by functional selection. Aberrant selection processes lead to immune dysfunction. DNA sequence variants also arise through stochastic events: some involving environmental fluctuation (radiation or presence of pollutants), or genetic repair system malfunction. The phenotypic outcome of mutations is also fluid. Mutations may be advantageous in some circumstances, deleterious in others. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.

Overcoming genetic and cellular complexity to study the pathophysiology of X-linked intellectual disabilities.

X-linked genetic causes of intellectual disability (ID) account for a substantial proportion of cases and remain poorly understood, in part due to the heterogeneous expression of X-linked genes in females. This is because most genes on the X chromosome are subject to random X chromosome inactivation (XCI) during early embryonic development, which results in a mosaic pattern of gene expression for a given X-linked mutant allele. This mosaic expression produces substantial complexity, especially when attempting to study the already complicated neural circuits that underly behavior, thus impeding the understanding of disease-related pathophysiology and the development of therapeutics. Here, we review a few selected X-linked forms of ID that predominantly affect heterozygous females and the current obstacles for developing effective therapies for such disorders. We also propose a genetic strategy to overcome the complexity presented by mosaicism in heterozygous females and highlight specific tools for studying synaptic and circuit mechanisms, many of which could be shared across multiple forms of intellectual disability.

Xist ribonucleoproteins promote female sex-biased autoimmunity.

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.

Dissection of protein and RNA regions required for SPEN binding to XIST A-repeat RNA.

XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction.

Role of sex in immune response and epigenetic mechanisms.

The functioning of the human immune system is highly dependent on the sex of the individual, which comes by virtue of sex chromosomes and hormonal differences. Epigenetic mechanisms such as X chromosome inactivation, mosaicism, skewing, and dimorphism in X chromosome genes and Y chromosome regulatory genes create a sex-based variance in the immune response between males and females. This leads to differential susceptibility in immune-related disorders like infections, autoimmunity, and malignancies. Various naturally available immunomodulators are also available which target immune pathways containing X chromosome genes.

Genes that escape from X-chromosome inactivation and sexual dimorphism of systemic lupus erythematosus.

X chromosome inactivation can balance the effects of the two X chromosomes in females, and emerging evidence indicates that numerous genes on the inactivated X chromosome have the potential to evade inactivation. The mechanisms of escape include modification of DNA, RNA, histone, epitope, and various regulatory proteins, as well as the spatial structure of chromatin. The study of X chromosome inactivation escape has paved the way for investigating sex dimorphism in human diseases, particularly autoimmune diseases. It has been demonstrated that the presence of TLR7, CD40L, IRAK-1, CXCR3, and CXorf21 significantly contributes to the prevalence of SLE (systemic lupus erythematosus) in females. This article mainly reviews the molecular mechanisms underlying these genes that escape from X-chromosome inactivation and sexual dimorphism of systemic lupus erythematosus. Therefore, elucidating the molecular mechanisms underlying sexual dimorphism in SLE is not only crucial for diagnosing and treating the disease, but also holds theoretical significance in comprehensively understanding the development and regulatory mechanisms of the human immune system.

Unfavorable switching of skewed X chromosome inactivation leads to Menkes disease in a female infant.

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene, and female carriers are usually asymptomatic. We describe a 7-month-old female patient with severe intellectual disability, epilepsy, and low levels of serum copper and ceruloplasmin. While heterozygous deletion of exons 16 and 17 of the ATP7A gene was detected in the proband, her mother, and her grandmother, only the proband suffered from Menkes disease clinically. Intriguingly, X chromosome inactivation (XCI) analysis demonstrated that the grandmother and the mother showed skewing of XCI toward the allele with the ATP7A deletion and that the proband had extremely skewed XCI toward the normal allele, resulting in exclusive expression of the pathogenic ATP7A mRNA transcripts. Expression bias analysis and recombination mapping of the X chromosome by the combination of whole genome and RNA sequencing demonstrated that meiotic recombination occurred at Xp21-p22 and Xq26-q28. Assuming that a genetic factor on the X chromosome enhanced or suppressed XCI of its allele, the factor must be on either of the two distal regions derived from her grandfather. Although we were unable to fully uncover the molecular mechanism, we concluded that unfavorable switching of skewed XCI caused Menkes disease in the proband.

Clinical findings in individuals with duplication of genes associated with X-linked intellectual disability.

Duplication of all genes associated with X-linked intellectual disability (XLID) have been reported but the majority of the duplications include more than one XLID gene. It is exceptional for whole XLID gene duplications to cause the same phenotype as sequence variants or deletions of the same gene. Duplication of PLP1, the gene associated with Pelizaeus-Merzbacher syndrome, is the most notable duplication of this type. More commonly, duplication of XLID genes results in very different phenotypes than sequence alterations or deletions. Duplication of MECP2 is widely recognized as a duplication of this type, but a number of others exist. The phenotypes associated with gene duplications are often milder than those caused by deletions and sequence variants. Among some duplications that are clinically significant, marked skewing of X-inactivation in female carriers has been observed. This report describes the phenotypic consequences of duplication of 22 individual XLID genes, of which 10 are described for the first time.

Unraveling the role of Xist RNA in cardiovascular pathogenesis.

Understanding the molecular pathways behind cardiovascular illnesses is crucial due to the enormous worldwide health burden they impose. New insights into the role played by Xist (X-inactive specific transcript) RNA in the onset and progression of cardiovascular diseases have emerged from recent studies. Since its discovery, Xist RNA has been known for its role in X chromosome inactivation during embryogenesis; however, new data suggest that its function extends well beyond the control of sex chromosomes. The regulatory roles of Xist RNA are extensive, encompassing epigenetic changes, gene expression, cellular identity, and sex chromosomal inactivation. There is potential for the involvement of this complex regulatory web in a wide range of illnesses, including cardiovascular problems. Atherosclerosis, hypertrophy, and cardiac fibrosis are all conditions linked to dysregulation of Xist RNA expression. Alterations in DNA methylation and histones are two examples of epigenetic changes that Xist RNA orchestrates, leading to modifications in gene expression patterns in different cardiovascular cells. Additionally, Xist RNA has been shown to contribute to the development of cardiovascular illnesses by modulating endothelial dysfunction, inflammation, and oxidative stress responses. New treatment approaches may become feasible with a thorough understanding of the complex function of Xist RNA in cardiovascular diseases. By focusing on Xist RNA and the regulatory network with which it interacts, we may be able to slow the progression of atherosclerosis, cardiac hypertrophy, and fibrosis, thereby opening novel therapeutic options for cardiovascular diseases amenable to precision medicine. This review summarizes the current state of knowledge concerning the impact of Xist RNA in cardiovascular disorders.

X-linked chronic granulomatous disease secondary to skewed X-chromosome inactivation in female patients.

BACKGROUND: Chronic granulomatous disease (CGD) is a heterogeneous primary immunodeficiency. X-linked (XL) CGD caused by gene defects of CYBB is the most prevalent type of CGD. OBJECTIVE: We aim to understand the clinical and molecule features of XL-CGD secondary to skewed X-chromosome inactivation (XCI) in female. METHODS: We retrospectively reviewed the medical records of a female patient diagnosed with XL-CGD. Flow cytometry was used to detect the respiratory burst function. After restriction enzyme digestion of DNA, XCI was calculated by detecting fluorescent PCR products with capillary electrophoresis. The previously published female XL-CGD cases secondary to skewed XCI was summarized. RESULTS: Clinical data were available for 15 female subjects. The median age of diagnosis was 16 years. Consistent with XL-CGD in males, infection was the most frequent manifestation in the female patients. Catalase-positive pathogens including Serratia marcescens and Staphylococcus aureus infections were the most common pathogens. Autoimmune/autoinflammation manifestations were observed in five patients. Dihydrorhodamine (DHR) assay showed that median %DHR+ values were 6.5% and the values varying with age were observed in 2 patients. All patients had a skewing XCI and there was no consistency between the daughter and carrier mother. Anti-infective treatment was effective in majority and there was no mortality reported in XL-CGD female patients to date. CONCLUSION: XL-CGD should not be neglected in female patients manifested as CGD phenotype and it is necessary to make periodic clinical evaluation of CGD female carriers as the neutrophil oxidative function may decline with aging and increase the risk for infection.

Escape from X-chromosome inactivation and sex differences in Alzheimer's disease.

Sex differences exist in the onset and progression of Alzheimer's disease. Globally, women have a higher prevalence, while men with Alzheimer's disease experience earlier mortality and more pronounced cognitive decline than women. The cause of sex differences in Alzheimer's disease remains unclear. Accumulating evidence suggests the potential role of X-linked genetic factors in the sex difference of Alzheimer's disease (AD). During embryogenesis, a remarkable process known as X-chromosome inactivation (XCI) occurs in females, leading to one of the X chromosomes undergoing transcriptional inactivation, which balances the effects of two X chromosomes in females. Nevertheless, certain genes exceptionally escape from XCI, which provides a basis for dual expression dosage of specific genes in females. Based on recent research findings, we explore key escape genes and their potential therapeutic use associated with Alzheimer's disease. Also, we discuss their possible role in driving the sex differences in Alzheimer's disease. This will provide new perspectives for precision medicine and gender-specific treatment of AD.

Roles of the Rlim-Rex1 axis during X chromosome inactivation in mice.

In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2- to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.

Competing endogenous RNA network analysis of Turner syndrome patient-specific iPSC-derived cardiomyocytes reveals dysregulation of autosomal heart development genes by altered dosages of X-inactivation escaping non-coding RNAs.

BACKGROUND: A 45,X monosomy (Turner syndrome, TS) is the only chromosome haploinsufficiency compatible with life. Nevertheless, the surviving TS patients still suffer from increased morbidity and mortality, with around one-third of them subjecting to heart abnormalities. How loss of one X chromosome drive these conditions remains largely unknown. METHODS: Here, we have generated cardiomyocytes (CMs) from wild-type and TS patient-specific induced pluripotent stem cells and profiled the mRNA, lncRNA and circRNA expression in these cells. RESULTS: We observed lower beating frequencies and higher mitochondrial DNA copies per nucleus in TS-CMs. Moreover, we have identified a global transcriptome dysregulation of both coding and non-coding RNAs in TS-CMs. The differentially expressed mRNAs were enriched of heart development genes. Further competing endogenous RNA network analysis revealed putative regulatory circuit of autosomal genes relevant with mitochondrial respiratory chain and heart development, such as COQ10A, RARB and WNT2, mediated by X-inactivation escaping lnc/circRNAs, such as lnc-KDM5C-4:1, hsa_circ_0090421 and hsa_circ_0090392. The aberrant expressions of these genes in TS-CMs were verified by qPCR. Further knockdown of lnc-KDM5C-4:1 in wild-type CMs exhibited significantly reduced beating frequencies. CONCLUSIONS: Our study has revealed a genomewide ripple effect of X chromosome halpoinsufficiency at post-transcriptional level and provided insights into the molecular mechanisms underlying heart abnormalities in TS patients.

EpiTyping: analysis of epigenetic aberrations in parental imprinting and X-chromosome inactivation using RNA-seq.

Human pluripotent stem cells (hPSCs) hold a central role in studying human development, in disease modeling and in regenerative medicine. These cells not only acquire genetic modifications when kept in culture, but they may also harbor epigenetic aberrations, mainly involving parental imprinting and X-chromosome inactivation. Here we present a detailed bioinformatic protocol for detecting such aberrations using RNA sequencing data. We provide a pipeline designed to process and analyze RNA sequencing data for the identification of abnormal biallelic expression of imprinted genes, and thus detect loss of imprinting. Furthermore, we show how to differentiate among X-chromosome inactivation, full activation and aberrant erosion of X chromosome in female hPSCs. In addition to providing bioinformatic tools, we discuss the impact of such epigenetic variations in hPSCs on their utility for various purposes. This pipeline can be used by any user with basic understanding of the Linux command line. It is available on GitHub as a software container ( https://github.com/Gal-Keshet/EpiTyping ) and produces reliable results in 1-4 d.