Altered X-chromosome inactivation predisposes to autoimmunity.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

YY1 binding is a gene-intrinsic barrier to Xist-mediated gene silencing.

X chromosome inactivation (XCI) in mammals is mediated by Xist RNA which functions in cis to silence genes on a single X chromosome in XX female cells, thereby equalising levels of X-linked gene expression relative to XY males. XCI progresses over a period of several days, with some X-linked genes silencing faster than others. The chromosomal location of a gene is an important determinant of silencing rate, but uncharacterised gene-intrinsic features also mediate resistance or susceptibility to silencing. In this study, we examine mouse embryonic stem cell lines with an inducible Xist allele (iXist-ChrX mESCs) and integrate allele-specific data of gene silencing and decreasing inactive X (Xi) chromatin accessibility over time courses of Xist induction with cellular differentiation. Our analysis reveals that motifs bound by the transcription factor YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. We further show that YY1 is evicted relatively slowly from target sites on Xi, and that silencing of X-linked genes is increased upon YY1 degradation. Together our results suggest that YY1 acts as a barrier to Xist-mediated silencing until the late stages of the XCI process.

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.

[Clinical features and genetic analysis of 17 Chinese pedigrees affected with X-linked intellectual disability].

OBJECTIVE: To analyze the clinical features and genetic etiology of 17 Chinese pedigrees affected with X-linked intellectual disability (XLID). METHODS: Seventeen pedigrees affected with unexplained intellectual disability which had presented at Henan Provincial People's Hospital from May 2021 to May 2023 were selected as the study subjects. Clinical data of the probands and their pedigree members were collected. Trio-whole exome sequencing (Trio-WES), Sanger sequencing and X chromosome inactivation (XCI) analysis were carried out. Pathogenicity of candidate variants was predicted based on the guidelines from the American College of Medical Genetics and Genomics and co-segregation analysis. RESULTS: The 17 probands, including 9 males and 8 females with an age ranging from 0.6 to 8 years old, had all shown mental retardation and developmental delay. Fourteen variants were detected by genetic testing, which included 4 pathogenic variants (MECP2: c.502C>T, MECP2: c.916C>T/c.806delG, IQSEC2: c.1417G>T), 4 likely pathogenic variants (MECP2: c.1157_1197del/c.925C>T, KDM5C: c.2128A>T, SLC6A8: c.1631C>T) and 6 variants of uncertain significance (KLHL15: c.26G>C, PAK3: c.970A>G/c.1520G>A, GRIA3: c.2153C>G, TAF1: c.2233T>G, HUWE1: c.10301T>A). The PAK3: c.970A>G, GRIA3: c.2153C>G and TAF1: c.2233T>G variants were considered as the genetic etiology for pedigrees 12, 14 and 15 by co-segregation analysis, respectively. The proband of pedigree 13 was found to have non-random XCI (81:19). Therefore, the PAK3: c.1520G>A variant may underlie its pathogenesis. CONCLUSION: Trio-WES has attained genetic diagnosis for the 17 XLID pedigrees. Sanger sequencing and XCI assay can provide auxiliary tests for the diagnosis of XLID.

Distributed X chromosome inactivation in brain circuitry is associated with X-linked disease penetrance of behavior.

The precise anatomical degree of brain X chromosome inactivation (XCI) that is sufficient to alter X-linked disorders in females is unclear. Here, we quantify whole-brain XCI at single-cell resolution to discover a prevalent activation ratio of maternal to paternal X at 60:40 across all divisions of the adult brain. This modest, non-random XCI influences X-linked disease penetrance: maternal transmission of the fragile X mental retardation 1 (Fmr1)-knockout (KO) allele confers 55% of total brain cells with mutant X-active, which is sufficient for behavioral penetrance, while 40% produced from paternal transmission is tolerated. Local XCI mosaicism within affected maternal Fmr1-KO mice further specifies sensorimotor versus social anxiety phenotypes depending on which distinct brain circuitry is most affected, with only a 50%-55% mutant X-active threshold determining penetrance. Thus, our results define a model of X-linked disease penetrance in females whereby distributed XCI among single cells populating brain circuitries can regulate the behavioral penetrance of an X-linked mutation.

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.

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.

[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.

Unbalanced X;Y translocations carrying SRY in prenatal settings: Clinical, molecular, and cytogenetic analysis of three cases.

BACKGROUND: Generally, the translocation of SRY onto one of the X chromosomes leads to 46, XX testicular disorders of sex development, a relatively rare condition characterized by the presence of testicular tissue with a 46, XX karyotype. Three prenatal cases of unbalanced X; Y translocation carrying SRY were identified in this study. METHODS: Structural variants were confirmed using single nucleotide polymorphism array and chromosomal karyotyping. X chromosome inactivation (XCI) was also analyzed. Detailed clinical features of the three cases were collected. RESULTS: We identified two fetuses with maternal inherited unbalanced X; Y translocations carrying SRY and skewed XCI presenting with normal female external genitalia, and one fetus with de novo 46, XX (SRY+) and random XCI manifested male phenotypic external genitalia. CONCLUSION: This study reports that cases with unbalanced X; Y translocations carrying SRY manifested a normal female external genitalia in a prenatal setting. We speculate that the skewed XCI mediates the silence of SRY. In addition, our study emphasizes that combining clinical findings with pedigree analysis is critical for estimating the prognosis of fetuses with sex chromosome abnormalities.

Transcription regulation by long non-coding RNAs: mechanisms and disease relevance.

Long non-coding RNAs (lncRNAs) outnumber protein-coding transcripts, but their functions remain largely unknown. In this Review, we discuss the emerging roles of lncRNAs in the control of gene transcription. Some of the best characterized lncRNAs have essential transcription cis-regulatory functions that cannot be easily accomplished by DNA-interacting transcription factors, such as XIST, which controls X-chromosome inactivation, or imprinted lncRNAs that direct allele-specific repression. A growing number of lncRNA transcription units, including CHASERR, PVT1 and HASTER (also known as HNF1A-AS1) act as transcription-stabilizing elements that fine-tune the activity of dosage-sensitive genes that encode transcription factors. Genetic experiments have shown that defects in such transcription stabilizers often cause severe phenotypes. Other lncRNAs, such as lincRNA-p21 (also known as Trp53cor1) and Maenli (Gm29348) contribute to local activation of gene transcription, whereas distinct lncRNAs influence gene transcription in trans. We discuss findings of lncRNAs that elicit a function through either activation of their transcription, transcript elongation and processing or the lncRNA molecule itself. We also discuss emerging evidence of lncRNA involvement in human diseases, and their potential as therapeutic targets.

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.

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.

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.

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.