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BACKGROUND: X chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in development and cancer. METHODS: Kdm6a was knocked out (KO) using CRISPR/Cas9 gene editing in hybrid female mouse embryonic stem (ES) cells derived either from a 129 × Mus castaneus (cast) cross or a BL6 x cast cross. In one of the lines a transcriptional stop signal inserted in Tsix results in completely skewed X silencing upon differentiation. The effects of both homozygous and heterozygous Kdm6a KO on Xist expression during the onset of XCI were measured by RT-PCR and RNA-FISH. Changes in gene expression and in H3K27me3 enrichment were investigated using allele-specific RNA-seq and Cut&Run, respectively. KDM6A binding to the Xist gene was characterized by Cut&Run. RESULTS: We observed impaired upregulation of Xist and reduced coating of the Xi during early stages of differentiation in Kdm6a KO cells, both homozygous and heterozygous, suggesting a threshold effect of KDM6A. This was associated with aberrant overexpression of genes from the Xi after differentiation, indicating loss of X inactivation potency. Consistent with KDM6A having a direct role in Xist regulation, we found that the histone demethylase binds to the Xist promoter and KO cells show an increase in H3K27me3 at Xist, consistent with reduced expression. CONCLUSIONS: These results reveal a novel female-specific role for the X-linked histone demethylase, KDM6A in the initiation of XCI through histone demethylase-dependent activation of Xist during early differentiation. X chromosome inactivation is a female-specific mechanism that evolved to balance sex-linked gene dosage between females (XX) and males (XY) by silencing one X chromosome in females. X inactivation begins with the upregulation of the long noncoding RNA Xist on the future inactive X chromosome. While most genes become silenced on the inactive X chromosome some genes escape inactivation and thus have higher expression in females compared to males, suggesting that escape genes may have female-specific functions. One such gene encodes the histone demethylase KDM6A which function is to turn on gene expression by removing repressive histone modifications. In this study, we investigated the role of KDM6A in the regulation of Xist expression during the onset of X inactivation. We found that KDM6A binds to the Xist gene to remove repressive histone marks and facilitate its expression in early development. Indeed, depletion of KDM6A prevents upregulation of Xist due to abnormal persistence of repressive histone modifications. In turn, this results in aberrant overexpression of genes from the inactive X chromosome. Our findings point to a novel mechanism of Xist regulation during the initiation of X inactivation, which may lead to new avenues of treatment to alleviate congenital disorders such as Kabuki syndrome and sex-biased immune disorders where X-linked gene dosage is perturbed.

Altered transcriptional and epigenetic regulation of brain cell types may contribute to cognitive changes with advanced age. Using single-nucleus multi-omic DNA methylation and transcriptome sequencing (snmCT-seq) in frontal cortex from young adult and aged donors, we found widespread age- and sex-related variation in specific neuron types. The proportion of inhibitory SST- and VIP-expressing neurons was reduced in aged donors. Excitatory neurons had more profound age-related changes in their gene expression and DNA methylation than inhibitory cells. Hundreds of genes involved in synaptic activity, including EGR1, were less expressed in aged adults. Genes located in subtelomeric regions increased their expression with age and correlated with reduced telomere length. We further mapped cell-type-specific sex differences in gene expression and X-inactivation escape genes. Multi-omic single-nucleus epigenomes and transcriptomes provide new insight into the effects of age and sex on human neurons.

Background: When the SRY gene is present in a 46,XX fetus, some degree of testicular development is expected. Our laboratory performed prenatal genetic testing for a fetus that had screened positive for Y chromosome material by noninvasive prenatal screening (NIPS) but that had apparently typical female development by ultrasound imaging. The aim of this study was to determine the clinical relevance of the NIPS results. Methods: We analyzed fetal material obtained via amniocentesis procedure by G‐banding, microarray, and fluorescence in situ hybridization (FISH). Optical genome mapping (OGM) was also performed. Results: G‐band analysis revealed a normal 46,XX karyotype. Microarray and FISH analyses together detected an SRY+ gain of 5.7 Mb from terminal Yp that was translocated to terminal Xq, with a loss of 1.6 Mb from terminal Xq. The final karyotype was 46,X,der(X)t(X;Y)(q28;p11.2). Prenatal ultrasound and postnatal physical examination revealed apparently typical female genitalia. The Xq deletion encompassed a gene, IKBKG, that is sensitive to loss of function, suggesting that preferential inactivation of the derivative X chromosome allowed for typical female development. OGM software did not directly identify this translocation. Conclusion: This case demonstrates how the SRY gene may be present in a 46,XX biological female without differences of sexual development. [ABSTRACT FROM AUTHOR]

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types. [ABSTRACT FROM AUTHOR]

Epigenetic control systems are based on chromatin modifications (DNA methylation, histone modifications and nucleosome positioning), which affect the local kinetics of gene expression. They play an important role in maintaining cell fate decisions, X inactivation and genomic imprinting. Aberrant chromatin states that are associated with a deleterious change in gene expression are called epimutations. An epimutation can be a primary epimutation that has occurred in the absence of any genetic change or a secondary epimutation that results from a mutation of a cis-acting regulatory element or trans-acting factor. Epimutations may play a causative role in disease, for example in imprinting disorders, or may be part of the pathogenetic mechanism as in the fragile X syndrome and in syndromes caused by a mutation affecting a chromatin modifier. For several diseases, DNA methylation testing is an important tool in the diagnostic work-up of patients. [ABSTRACT FROM AUTHOR]

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. Synopsis: The transcription factor Yin Yang 1 (YY1) is enriched at gene promoters and enhancers on the X chromosome which are silenced more slowly by Xist RNA and contributes towards resistance to pathways of Xist-mediated chromosome silencing. Integrated allelic analysis of gene silencing and chromatin accessibility over time-courses of Xist induction in mouse embryonic stem cell differentiation. Motifs bound by YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. YY1 is evicted relatively slowly from target sites in the course of X chromosome inactivation. YY1 depletion promotes X-linked gene silencing. The transcription factor Yin Yang 1 (YY1) is enriched at gene promoters and enhancers on the X chromosome which are silenced more slowly by Xist RNA and contributes towards resistance to pathways of Xist-mediated chromosome silencing. [ABSTRACT FROM AUTHOR]

There is growing evidence that X-chromosome inactivation is driven by phase-separated supramolecular assemblies. However, among the many proteins recruited to the inactive X chromosome by Xist long non-coding RNA, so far only a minority (CIZ1, CELF1, SPEN, TDP-43, MATR3, PTBP1, PCGF5) have been shown to form Xist-seeded protein assemblies, and of these most have not been analyzed in detail. With focus on CIZ1, here we describe 1) the contribution of intrinsically disordered regions in RNA-dependent protein assembly formation at the inactive X chromosome, and 2) enrichment, distribution, and function of proteins within Xist-seeded assemblies. [ABSTRACT FROM AUTHOR]

Autoimmunity and its associated diseases have been the primary area of focus in research in the past decade; however, several mechanisms are still left unclear. Autoimmune diseases are idiopathic and currently, there are hardly any genetic tools available to predict who is at risk of developing them. With the advancement of artificial intelligence, it could be possible to predict the potential emergence of autoimmune disorders. This review article discusses the disease classification and relevant causes, including environmental, biological, and genetic factors. Additionally, it explores certain mechanisms where the T-regulatory cells fail and lead to disease development. The article delves into the etiology to illustrate how autoimmune diseases could develop in a person as well as therapeutic approaches are also discussed. With the development of multiplex technologies, it is easier to detect autoantibodies in blood while cytokines and chemokines can give a breakthrough in diagnosis and novel therapeutics for these diseases. [ABSTRACT FROM AUTHOR]

There is growing evidence that X-chromosome inactivation is driven by phase-separated supramolecular assemblies. However, among the many proteins recruited to the inactive X chromosome by Xist long non-coding RNA, so far only a minority (CIZ1, CELF1, SPEN, TDP-43, MATR3, PTBP1, PCGF5) have been shown to form Xist-seeded protein assemblies, and of these most have not been analyzed in detail. With focus on CIZ1, here we describe 1) the contribution of intrinsically disordered regions in RNA-dependent protein assembly formation at the inactive X chromosome, and 2) enrichment, distribution, and function of proteins within Xist-seeded assemblies.

In mammals, the second X chromosome in females is silenced to enable dosage compensation between XX females and XY males. This essential process involves the formation of a dense chromatin state on the inactive X (Xi) chromosome. There is a wealth of information about the hallmarks of Xi chromatin and the contribution each makes to silencing, leaving the tantalising possibility of learning from this knowledge to potentially remove silencing to treat X-linked diseases in females. Here, we discuss the role of each chromatin feature in the establishment and maintenance of the silent state, which is of crucial relevance for such a goal. [ABSTRACT FROM AUTHOR]

In female mammals, there are two X chromosomes, and one of them could be inactivated by some mechanisms, and this concept is called X inactivation. The X chromosome, which would be inactivated, is packaged in heterochromatin. Thus, the genes on this X chromosome are prevented from being expressed. The inactivation process maintains dosage compensation between males with one X chromosome and females with two X chromosomes. Although X chromosome inactivation is thought to occur on the entire X chromosome, not all of the genes on the X chromosome are exposed to inactivation, and some genes escape from inactivation. In humans, approximately 12 to 20% of the genes encoded by the inactivated X chromosome can be expressed by escaping the inactivation process. In this review, we will examine the inactivation process and genes that escape inactivation by considering the mechanisms of X inactivation. [ABSTRACT FROM AUTHOR]

In each somatic cell of a female mammal one X chromosome is transcriptionally silenced via X-chromosome inactivation (XCI), initiating early in development. Although XCI events are conserved in mouse and human postimplantation development, regulation of X-chromosome dosage in preimplantation development occurs differently. In preimplantation development, mouse embryos undergo imprinted form of XCI, yet humans lack imprinted XCI and instead regulate gene expression of both X chromosomes by dampening transcription. The long non-coding RNA Xist/XIST is expressed in mouse and human preimplantation and postimplantation development to orchestrate XCI, but its role in dampening is unclear. In this review, we discuss recent advances in our understanding of the role of Xist in X chromosome dosage compensation in mouse and human.

Abstract Background Topologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs has been shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing lead to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains, which are highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains. Results Here we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components and transcription mostly do not affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains. Conclusions In sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

Xp11 translocation renal cell carcinoma (tRCC) is a rare, female-predominant cancer driven by a fusion between the transcription factor binding to IGHM enhancer 3 (TFE3) gene on chromosome Xp11.2 and a partner gene on either chromosome X (chrX) or an autosome. It remains unknown what types of rearrangements underlie TFE3 fusions, whether fusions can arise from both the active (chrXa) and inactive X (chrXi) chromosomes, and whether TFE3 fusions from chrXi translocations account for the female predominance of tRCC. To address these questions, we performed haplotype-specific analyses of chrX rearrangements in tRCC whole genomes. We show that TFE3 fusions universally arise as reciprocal translocations and that oncogenic TFE3 fusions can arise from chrXi:autosomal translocations. Female-specific chrXi:autosomal translocations result in a 2:1 female-to-male ratio of TFE3 fusions involving autosomal partner genes and account for the female predominance of tRCC. Our results highlight how X chromosome genetics constrains somatic chrX alterations and underlies cancer sex differences. [Display omitted] • TFE3 driver fusions usually arise via reciprocal, balanced rearrangements in Xp11 tRCC • Female sex bias of tRCC explained by rearrangements of both the active and inactive X • Active vs. inactive X rearrangements impose sex-specific evolutionary constraints • Inactive X:autosome translocations linked to X-reactivation and/or autosome silencing Whole-genome analysis of a female-predominant kidney cancer reveals the mechanisms by which X chromosome genetics constrains somatic X chromosome alterations and underlies sex bias in tumorigenesis. [ABSTRACT FROM AUTHOR]

Hardikar syndrome (HS) is a MED12‐related ultra‐rare multiple congenital malformation syndrome known to affect the gastrointestinal, cardiac, and genitourinary systems among other features including cleft lip/palate and pigmentary retinopathy. Only 10 patients affected with HS have been previously described in literature, of which seven were molecularly confirmed. We report a 20‐year‐old and a 13‐month‐old patient with HS diagnosed by exome sequencing bringing the total number of clinically diagnosed cases to 12 and MED12 associated to 9. We describe previously unreported molecular and clinical findings associated with HS and review all reported cases to permit prompt diagnosis, appropriate management, and genetic counseling of HS patients. [ABSTRACT FROM AUTHOR]

X chromosome inactivation is a mammalian dosage compensation mechanism, where one of two X chromosomes is randomly inactivated in female cells. Previous studies have suggested that primed human embryonic stem cells (hESCs) maintain an eroded state of the X chromosome and do not express XIST, while in naïve transition, both XIST and the eroded X chromosome are reactivated. However, the pattern of chromosome X reactivation in naïve hESCs remains mainly unknown. In this study, we examine the variations in the status of X chromosome between primed and naïve hESCs by analyzing RNA sequencing samples from different studies. We show that most samples of naïve hESCs indeed reactivate XIST and there is an increase in gene expression levels on chromosome X. However, most of the naïve samples do not fully activate chromosome X in a uniform manner and present a distinct eroded pattern, probably as a result of XIST reactivation and initiation of re-inactivation of chromosome X. This large-scale analysis provides a higher-resolution description of the changes occurring in chromosome X during primed-to-naïve transition and emphasizes the importance of taking these variations into consideration when studying X inactivation in embryonic development. [ABSTRACT FROM AUTHOR]

The long noncoding X-inactivation-specific transcript (Xist gene) is responsible for mammalian X-chromosome dosage compensation between the sexes, the process by which one of the two X chromosomes is inactivated in the female soma. Xist is essential for both the random and imprinted forms of X-chromosome inactivation. In the imprinted form, Xist is paternally marked to be expressed in female embryos. To investigate the mechanism of Xist imprinting, we introduce Xist transgenes (Tg) into the male germ line. Although ectopic high-level Xist expression on autosomes can be compatible with viability, transgenic animals demonstrate reduced fitness, subfertility, defective meiotic pairing, and other germ-cell abnormalities. In the progeny, paternal-specific expression is recapitulated by the 200-kb Xist Tg. However, Xist imprinting occurs efficiently only when it is in an unpaired or unpartnered state during male meiosis. When transmitted from a hemizygous father (+/Tg), the Xist Tg demonstrates paternal-specific expression in the early embryo. When transmitted by a homozygous father (Tg/Tg), the Tg fails to show imprinted expression. Thus, Xist imprinting is directed by sequences within a 200-kb X-linked region, and the hemizygous (unpaired) state of the Xist region promotes its imprinting in the male germ line.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth († Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale. [Display omitted] • 3D genome architecture is preserved in a 52,000-year-old woolly mammoth sample • PaleoHi-C makes it possible to assemble the woolly mammoth's genome • Chromatin compartments also persist, enabling study of mammoth gene expression • We propose that dehydration led to a glass transition arresting molecular movement Three-dimensional genome architecture is shown to be preserved in a 52,000-year-old woolly mammoth sample, enabling genome assembly and analysis of gene expression. Spontaneous freeze-drying of the mammoth carcass in the cold Siberian environment potentially led to a glass transition that preserved sample morphology across eight orders of magnitude in length, from the 3-m carcass to 50-nm chromatin loops. [ABSTRACT FROM AUTHOR]

Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.

A sophisticated mechanistic understanding of physiology and disease requires knowledge of how sex-biasing factors cause sex differences in phenotype. In therian mammals, all sex differences are downstream of the unequal effects of XX vs. XY sex chromosomes. Three major categories of sex-biasing factors are activational and organizational effects of gonadal hormones, and sex chromosome effects operating outside of the gonads. These three types of effects can be discriminated from each other with established experimental designs and animal models. Two important mouse models, which allow conclusions regarding the sex-biasing effects of sex chromosome complement, interacting with gonadal hormone effects, are the Four Core Genotypes model and the XY* model. Chromosome Y consomic strains give information about the role of the Y chromosome. An important recent change in sexual differentiation theory is the increasing realization that sex-biasing factors can counteract the effects of each other, reducing rather than producing sex differences in phenotype. This change in viewpoint rationalizes a change in experimental strategies for dissecting sex chromosome effects. The overall goal is to understand the sexome, defined as the sum of effects of sex-biasing factors on gene systems and networks.

The ability to culture pluripotent stem cells and direct their differentiation into specific cell types in vitro provides a valuable experimental system for modeling pluripotency, development and cellular differentiation. High-throughput profiling of the transcriptomes and epigenomes of pluripotent stem cells and their differentiated derivatives has led to identification of patterns characteristic of each cell type, discovery of new regulatory features in the epigenome and early insights into the complexity of dynamic interactions among regulatory elements. This work has also revealed potential limitations of the use of pluripotent stem cells as in vitro models of developmental events, due to epigenetic variability among different pluripotent stem cell lines and epigenetic instability during derivation and culture, particularly at imprinted and X-inactivated loci. This review focuses on the two most well-studied epigenetic mechanisms, DNA methylation and histone modifications, within the context of pluripotency and differentiation.

To describe a novel unbalanced X;21 translocation resulting in a derivative pseudodicentric chromosome X;21 lacking the critical region for ovarian development and function, in a 16-year-old girl referred for cytogenetic analysis due to primary amenorrhea and Turner-like features. Cytogenetic analysis of the proband and her parents was performed on peripheral blood lymphocytes by GTG banding. Molecular cytogenetic FISH analysis was performed on metaphase preparations, using X chromosome centromeric probe and telomeric and pancentromeric peptide nucleic acid (PNA) analog probes. The HUMARA assay as well as methylation studies for PCSK1N and FMR-1 loci were performed. Cytogenetic analysis revealed a de novo unbalanced X;21 translocation, described as 45,X,der(X)t(X;21)(q22.2;p11.2),-21. FISH analysis showed that the derivative X chromosome carried both the X and 21 centromeres, as well as, the Xp and 21q telomeres. The karyotype was thus reevaluated as 45,X,psu dic(21;X)(21qter→21p13::Xq22.2→Xpter),-21. X inactivation studies revealed that the derivative chromosome was of paternal origin and confirmed the selective inactivation of the derivative X segment of the pseudodicentric chromosome. Primary amenorrhea and other Turner-like characteristics of the proband are apparently due to the loss of the Xq22.2→Xqter critical region which contains critical genes for the ovarian development and function. The chromosome X segment of the derivative pseudodicentric chromosome is selectively inactivated, but inactivation does not seem to spread onto the translocated chromosome 21, accounting probably for the lack of severe clinical consequences which would result from monosomy 21. [ABSTRACT FROM AUTHOR]

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non‐coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2‐dependent H3K27me3 and SETD8‐dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3‐specific intracellular antibody or H3K27me3‐mintbody. By combining live‐cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP‐seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin. SYNOPSIS: H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome, albeit with distinct genomic distributions. H4K20me1 enrichment is dispensable for the induction of X‐linked gene silencing. H4K20me1 and H3K27me3 concurrently accumulate at the inactive X chromosome.H4K20me1 enrichment depends on the B and C repeat regions of Xist RNA.Transcriptional silencing can be initiated in the absence of H4K20me1 accumulation at the inactive X. [ABSTRACT FROM AUTHOR]

Neuroligin 4 X‐linked (NLGN4X) is an X‐linked postsynaptic scaffolding protein, with functional role in excitatory synapsis development and maintenance, that has been associated with neuropsychiatric disorders such as intellectual disability, autism spectrum disorders (ASD), anxiety, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. Chromosomal microarray analysis identified a paternally inherited, 445 Kb deletion on Xp22.3 that includes the entire NLGN4X in a 2.5 year old female (46,XX) with congenital hypotonia, strabismus, ASD, and increased aggressive behavioral issues. Her family history is significant for a mother with learning disabilities, a father with anxiety, major depressive disorder, and substance abuse, as well as two maternal half‐brothers with developmental delays. X‐inactivation studies in the proband's blood showed random X‐inactivation despite the presence of an abnormal X chromosome. Furthermore, trio exome sequencing did not reveal any other deleterious variant that could explain her phenotype. Our report describes the first example of a paternally inherited NLGN4X microdeletion as the genetic etiology of ASD in a female proband, and the psychiatric phenotypes in the father. It also provides further evidence that NLGN4X is sensitive to dosage changes in females, and can contribute to a variety of psychiatric features within the same family. [ABSTRACT FROM AUTHOR]

Abstract Background The aim of Preimplantation Genetic Diagnosis (PGD) on embryos produced in vitro is to identify the embryos without genetic or chromosomal defect from those embryos that will develop the genetic disease or are chromosomally abnormal. In case of PGD for structural chromosome indication (PGR-SR), the normal/balanced embryos are transferred in the maternal uterus. This protocol is valid and widely applied for autosomal chromosome translocation. But which embryo should be transferred after preimplantation genetic diagnosis (PGD-SR) for X-3 reciprocal translocation in male patient? Case presentation The female patient was 26 years old with normal 46,XX karyotype. The male patient had a karyotype with balanced translocation 46,Y,t(X;3)(p11.2;p14)mat, inherited from the mother. The female patient underwent two cycles of ovarian stimulation. In the first cycle, the metaphase II oocytes were vitrified, while in the second cycle they were used as fresh. ICSI was performed on vitrified/warmed and fresh oocytes. Embryos were biopsied at blastocyst stage. Chromosomal analysis was performed by Next Generation Sequencing. Eleven blastocysts were biopsied from 23 vitrified/warmed and fresh metaphase II oocytes. Two embryos were diagnosed 46,XY; two embryos were diagnosed 46,XX; four embryos were diagnosed with unbalanced translocations and three embryos were diagnosed aneuploid. We knew that the two embryos diagnosed as 46,XX inherited the balanced translocation from the father and the two embryos diagnosed as 46,XY had a normal karyotype. It was explain to the couple that the phenotype of balanced translocated female embryos cannot be predicted because of the random inactivation of X chromosome and that could also occur on the der(X). The couple asked to have a 46,XY embryo transferred. Clinical pregnancy was obtained and non invasive prenatal test confirmed PGD-SR result. Conclusions Proposing PGD-SR for gonosome-autosome reciprocal translocation implies the risk to exclude balanced translocated female embryos with a normal phenotype for transfer because the early and late normal development at post-natal stage cannot be predicted based on the only chromosomal analysis.

We previously identified the cyclin dependent kinase Cdk8 as a putative silencing factor for Xist. To investigate its role in X inactivation, we engineered a Cdk8 mutation in mouse embryonic stem cells (ESCs) carrying an inducible system for studying Xist function. We found that Xist repressed X-linked genes at half of the expression level in Cdk8 mutant cells, whereas they were almost completely silenced in the controls. Lack of Cdk8 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 trimethylation but not PRC1 recruitment by Xist. Transgenic expression of wild-type but not catalytically inactive Cdk8 restored efficient gene repression and PRC2 recruitment. Mutation of the paralogous kinase Cdk19 did not affect Xist function, and combined mutations of Cdk8 and Cdk19 resembled the Cdk8 mutation. In mice, a Cdk8 mutation caused post-implantation lethality. We observed that homozygous Cdk8 mutant female embryos showed a greater developmental delay than males on day 10.5. Together with the inefficient repression of X-linked genes in differentiating Cdk8 mutant female ESCs, these data show a requirement for Cdk8 in the initiation of X inactivation. [ABSTRACT FROM AUTHOR]

How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing. [Display omitted] • RNA G-quadruplex (rG4) folding is tightly regulated and developmentally programmed • Folded rG4s avidly bind PRC2 and inhibit its histone methyltransferase activity • rG4 loss in Xist causes PRC2 hyperactivity on the inactive X chromosome • Xist-PRC2 complexes become spatially entrapped, disrupting X inactivation Lee et al. identify RNA G-quadruplexes (rG4s) in Xist RNA and demonstrate that they are developmentally specific. rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of X-linked gene silencing. When the rG4 sequences are mutated in Xist, PRC2 is hyperactivated and becomes spatially entrapped, preventing the progression of X inactivation. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) play an important role in genome regulation. Specifically, many lncRNAs interact with chromatin, recruit epigenetic complexes and in this way affect large-scale gene expression programs. However, the experimental data about lncRNA-chromatin interactions is still limited. The majority of experimental protocols do not provide any insight into the mechanics of lncRNA-based genome-wide epigenetic regulation. Here we present the HiMoRNA (Histone-Modifying RNA) database, a resource containing correlated lncRNA–epigenetic changes in specific genomic locations genome-wide. HiMoRNA integrates a large amount of multi-omics data to characterize the effects of lncRNA on epigenetic modifications and gene expression. The current release of HiMoRNA includes more than five million associations in humans for ten histone modifications in multiple genomic loci and 4145 lncRNAs. HiMoRNA provides a user-friendly interface to facilitate browsing, searching and retrieving of lncRNAs associated with epigenetic profiles of various chromatin loci. Analysis of the HiMoRNA data suggests that several lncRNA including JPX might be involved not only in regulation of XIST locus but also in direct establishment or maintenance of X-chromosome inactivation. We believe that HiMoRNA is a convenient and valuable resource that can provide valuable biological insights and greatly facilitate functional annotation of lncRNAs.

Female mammals express the long noncoding X inactivation-specific transcript ( Xist) RNA to initiate X chromosome inactivation (XCI) that eventually results in the formation of the Barr body. Xist encompasses half a dozen repeated sequence stretches containing motifs for RNA-binding proteins that recruit effector complexes with functions for silencing genes and establishing a repressive chromatin configuration. Functional characterization of these effector proteins unveils the cooperation of a number of pathways to repress genes on the inactive X chromosome. Mechanistic insights can be extended to other noncoding RNAs with similar structure and open avenues for the design of new therapies to switch off gene expression. Here we review recent advances in the understanding of Xist and on this basis try to synthesize a model for the initiation of XCI.

Abstract Background Sex is an important but understudied factor in the genetics of human diseases. Analyses using a combination of gene expression data, ENCODE data, and evolutionary data of sex-biased gene expression in human tissues can give insight into the regulatory and evolutionary forces acting on sex-biased genes. Methods In this study, we analyzed the differentially expressed genes between males and females. On the X chromosome, we used a novel method and investigated the status of genes that escape X-chromosome inactivation (escape genes), taking into account the clonality of lymphoblastoid cell lines (LCLs). To investigate the regulation of sex-biased differentially expressed genes (sDEG), we conducted pathway and transcription factor enrichment analyses on the sDEGs, as well as analyses on the genomic distribution of sDEGs. Evolutionary analyses were also conducted on both sDEGs and escape genes. Results Genome-wide, we characterized differential gene expression between sexes in 462 RNA-seq samples and identified 587 sex-biased genes, or 3.2% of the genes surveyed. On the X chromosome, sDEGs were distributed in evolutionary strata in a similar pattern as escape genes. We found a trend of negative correlation between the gene expression breadth and nonsynonymous over synonymous mutation (dN/dS) ratios, showing a possible pleiotropic constraint on evolution of genes. Genome-wide, nine transcription factors were found enriched in binding to the regions surrounding the transcription start sites of female-biased genes. Many pathways and protein domains were enriched in sex-biased genes, some of which hint at sex-biased physiological processes. Conclusions These findings lend insight into the regulatory and evolutionary forces shaping sex-biased gene expression and their involvement in the physiological and pathological processes in human health and diseases.

Abstract Background Only a small portion of human long non-coding RNAs (lncRNAs) appear to be conserved outside of mammals, but the events underlying the birth of new lncRNAs in mammals remain largely unknown. One potential source is remnants of protein-coding genes that transitioned into lncRNAs. Results We systematically compare lncRNA and protein-coding loci across vertebrates, and estimate that up to 5% of conserved mammalian lncRNAs are derived from lost protein-coding genes. These lncRNAs have specific characteristics, such as broader expression domains, that set them apart from other lncRNAs. Fourteen lncRNAs have sequence similarity with the loci of the contemporary homologs of the lost protein-coding genes. We propose that selection acting on enhancer sequences is mostly responsible for retention of these regions. As an example of an RNA element from a protein-coding ancestor that was retained in the lncRNA, we describe in detail a short translated ORF in the JPX lncRNA that was derived from an upstream ORF in a protein-coding gene and retains some of its functionality. Conclusions We estimate that ~ 55 annotated conserved human lncRNAs are derived from parts of ancestral protein-coding genes, and loss of coding potential is thus a non-negligible source of new lncRNAs. Some lncRNAs inherited regulatory elements influencing transcription and translation from their protein-coding ancestors and those elements can influence the expression breadth and functionality of these lncRNAs.

X inactivation represents a complex multi-layer epigenetic mechanism that profoundly modifies chromatin composition and structure of one X chromosome in females. The heterochromatic inactive X chromosome adopts a unique 3D bipartite structure and a location close to the nuclear periphery or the nucleolus. X-linked lncRNA loci and their transcripts play important roles in the recruitment of proteins that catalyze chromatin and DNA modifications for silencing, as well as in the control of chromatin condensation and location of the inactive X chromosome. A subset of genes escapes X inactivation, raising questions about mechanisms that preserve their expression despite being embedded within heterochromatin. Escape gene expression differs between males and females, which can lead to physiological sex differences. We review recent studies that emphasize challenges in understanding the role of lncRNAs in the control of epigenetic modifications, structural features and nuclear positioning of the inactive X chromosome. Second, we highlight new findings about the distribution of genes that escape X inactivation based on single cell studies, and discuss the roles of escape genes in eliciting sex differences in health and disease.

Long thought to be dispensable after establishing X chromosome inactivation (XCI), Xist RNA is now known to also maintain the inactive X (Xi). To what extent somatic X reactivation causes physiological abnormalities is an active area of inquiry. Here, we use multiple mouse models to investigate in vivo consequences. First, when Xist is deleted systemically in post-XCI embryonic cells using the Meox2-Cre driver, female pups exhibit no morbidity or mortality despite partial X reactivation. Second, when Xist is conditionally deleted in epithelial cells using Keratin14-Cre or in B cells using CD19-Cre, female mice have a normal life span without obvious illness. Third, when Xist is deleted in gut using Villin-Cre, female mice remain healthy despite significant X-autosome dosage imbalance. Finally, when the gut is acutely stressed by azoxymethane/dextran sulfate (AOM/DSS) exposure, both Xist-deleted and wild-type mice develop gastrointestinal tumors. Intriguingly, however, under prolonged stress, mutant mice develop larger tumors and have a higher tumor burden. The effect is female specific. Altogether, these observations reveal a surprising systemic tolerance to Xist loss but importantly reveal that Xist and XCI are protective to females during chronic stress. [ABSTRACT FROM AUTHOR]

Purpose: Chronic granulomatous disease (CGD) is the most common phagocyte defect disease. Here, we describe 114 CGD patients in our center and report a rare female infant with XL-CGD to provide a better understanding of diagnosis, treatment, and prenatal diagnosis of CGD. Method: Patients were diagnosed by DHR-1,2,3 flow cytometry assays and gene analysis. X chromosome inactivation analysis and gp91phox protein test were used for a female infant with XL-CGD. Results: XL-CGD accounts for the majority of cases in China and results in higher susceptibility to some infections than AR-CGD. The DHR assay can help diagnose CGD quickly, and atypical results should be combined with clinical manifestations, genetic analysis, and regular follow-up. For prenatal diagnosis, both gDNA and cDNA genotypes of amniotic fluid cells should be identified, and cord blood DHR assays should be performed to identify female XL-CGD patients. [ABSTRACT FROM AUTHOR]

X inactivation presents two longstanding puzzles: the counting and choice of X chromosomes. Here, we consider counting and choice in the context of pairing, both of the X and of the autosomes. [ABSTRACT FROM AUTHOR]

Our understanding of epigenetics and the mechanisms of gene expression has grown rapidly, requiring the NICU nurse to understand the implications on a neonate's health. Epigenetics is heritable changes of the gene expression without changing the DNA sequence. Though most of the etiologies of birth disorders remain unknown, they could result from interactions of genetics, lifestyle, and environment that make changes in the epigenome landscape. Epigenetics may help explain disorders neonates express including cardiovascular defects, cleft lip and palate, and neural tube defects. As our understanding of epigenetics improves, NICU nurses will need to understand possible epigenetic implications on the neonate's health so as to provide better care and guidance to families. [ABSTRACT FROM AUTHOR]

X chromosome inactivation (XCI) is a female-specific mechanism that serves to balance gene dosage between the sexes whereby one X chromosome in females is inactivated during early development. Despite this silencing, a small portion of genes escape inactivation and remain expressed from the inactive X (Xi). Little is known about the distribution of escape from XCI in different tissues in vivo and about the mechanisms that control tissue-specific differences. Using a new binomial model in conjunction with a mouse model with identifiable alleles and skewed X inactivation we are able to survey genes that escape XCI in vivo. We show that escape from X inactivation can be a common feature of some genes, whereas others escape in a tissue specific manner. Furthermore, we characterize the chromatin environment of escape genes and show that expression from the Xi correlates with factors associated with open chromatin and that CTCF co-localizes with escape genes. Here, we provide a detailed description of the experimental design and data analysis pipeline we used to assay allele-specific expression and epigenetic characteristics of genes escaping X inactivation. The data is publicly available through the GEO database under ascension numbers GSM1014171, GSE44255, and GSE59779. Interpretation and discussion of these data are included in a previously published study (Berletch et al., 2015) [1].

Histone acetylation, one of the first and best studied histone post-translational modifications (PTMs), as well as the factors involved in its deposition (writers), binding (readers) and removal (erasers), have been shown to act at the heart of regulatory circuits controlling essential cellular functions. The identification of a variety of competing histone lysine-modifying acyl groups including propionyl, butyryl, 2-hydroxyisobutyryl, crotonyl, malonyl, succinyl and glutaryl, raises numerous questions on their functional significance, the molecular systems that manage their establishment, removal and interplay with the well-known acetylation-based mechanisms. Detailed and large-scale investigations of two of these new histone PTMs, crotonylation and 2-hydroxyisobutyrylation, along with histone acetylation, in the context of male genome programming, where stage-specific gene expression programs are switched on and off in turn, have shed light on their functional contribution to the epigenome for the first time. These initial investigations fired many additional questions, which remain to be explored. This review surveys the major results taken from these two new histone acylations and discusses the new biology that is emerging based on the diversity of histone lysine acylations.

Biological factors and mechanisms that drive sex differences observed in sleep disturbances are understudied and poorly understood. The extent to which sex chromosome constitution impacts on sex differences in circadian patterns is still a knowledge void in the sleep medicine field. Here we focus on the neurological consequences of X-chromosome functional imbalances between males and females and how this molecular inequality might affect sex divergencies on sleep. In light of the X-chromosome inactivation mechanism in females and its implications in gene regulation, we describe sleep-related neuronal circuits and brain regions impacted by sex-biased modulations of the transcriptome and the epigenome. Benefited from recent large-scale genetic studies on the interplay between X-chromosome and brain function, we list clinically relevant genes that might play a role in sex differences in neuronal pathways. Those molecular signatures are put into the context of sleep and sleep-associated neurological phenotypes, aiming to identify biological mechanisms that link X-chromosome gene regulation to sex-biased human traits. These findings are a significant step forward in understanding how X-linked genes manifest in sleep-associated transcriptional networks and point to future research opportunities to address female-specific clinical manifestations and therapeutic responses., (Copyright © 2023 Elsevier Inc. All rights reserved.)

X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score--defined as the mean of the allelic expression profiles of the escapees per cell--enables us to discover a heterogeneous and continuous degree of cellular XCIwith extremes represented by "inactive" cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and "escaping" cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI. [ABSTRACT FROM AUTHOR]

Xist, the master regulator of the X chromosome inactivation in mammals, is a 17 kb lncRNA that acts in cis to silence the majority of genes along the chromosome from which it is transcribed. The two key processes required for Xist RNA function, localisation in cis and recruitment of silencing factors, are genetically separable, at least in part. Recent studies have identified Xist RNA sequences and associated RNA-binding proteins (RBPs) that are important for these processes. Notably, several of the key Xist RNA elements correspond to local tandem repeats. In this review, I use examples to illustrate different modes whereby tandem repeat amplification has been exploited to allow orthodox RBPs to confer new functions for Xist-mediated chromosome inactivation. I further discuss the potential generality of tandem repeat expansion in the evolution of functional long non-coding RNAs (lncRNAs). [ABSTRACT FROM AUTHOR]

Key Clinical Message: Unbalanced X‐autosome translocation can result in various phenotypic manifestations. We present the first case of 46,X,der(X)t(X;8)(q28;q13) in a 34‐year‐old female with relatively mild manifestations, including congenital heart disease, epicanthal fold, mild intellectual disability, and menstrual irregularity. Our findings expand the known spectrum of unbalanced X‐autosome translocations, for improved clinical management. [ABSTRACT FROM AUTHOR]

Chronic granulomatous disease (CGD) is a hereditary immunodeficiency syndrome caused by a defect in the NADPH oxidase complex, which is essential for bactericidal function of phagocytes. Approximately 70% of patients with CGD have a mutation in the CYBB gene on the X chromosome, resulting in defective expression of gp91phox, one of the membrane-bound subunits of NADPH oxidase. Although most patients with X-linked CGD are males, owing to transmission of this disease as an X-linked recessive trait, there are female patients with X-linked CGD. Here, we report the case of a teenage girl with X-linked CGD associated with a heterozygous mutation in exon 5 of the CYBB gene (c.389G > C; R130P), which causes skipping of exon 5, resulting in a premature stop codon in exon 6 of CYBB. Accurate pro-mRNA splicing for mature mRNA formation is regulated by several splicing mechanisms that are essential for appropriate recognition of exonic sequences. The c.389G > C mutation disrupts exonic-splicing regulator sequences, thereby resulting in the aberrant skipping of exon 5 in the CYBB transcript of the patient. The patient showed an extremely skewed (≥96%) X inactivation pattern of the HUMARA locus; this inactivation is thought to be responsible for the development of CGD not only in neutrophils but also in monocytic, T-cell, and B-cell lineages and in CD34-positive immature hematopoietic cells. Our case and other reports indicate that the onset of X-linked CGD in female patients tends to occur later in life, and that the symptoms tend to be milder as compared to male patients. [ABSTRACT FROM AUTHOR]

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished. [ABSTRACT FROM AUTHOR]

Background: We report a kindred referred for molecular investigation of severe hemophilia A in a young female in which extremely skewed X-inactivation was observed in both the proband and her clinically normal mother. Methods: Bidirectional Sanger sequencing of all F8 gene coding regions and exon/intron boundaries was undertaken. Methylation-sensitive restriction enzymes were utilized to investigate skewed X-inactivation using both a classical human androgen receptor (HUMARA) assay, and a novel method targeting differential methylation patterns in multiple informative X-chromosome SNPs. Illumina Whole-Genome Infinium microarray analysis was performed in the case-parent trio (proband and both parents), and the proband's maternal grandmother. Results: The proband was a cytogenetically normal female with severe hemophilia A resulting from a heterozygous F8 pathogenic variant inherited from her similarly affected father. No F8 mutation was identified in the proband's mother, however, both the proband and her mother both demonstrated completely skewed X-chromosome inactivation (100%) in association with a previously unreported 2.3 Mb deletion at Xp22.2. At least three disease-associated genes (FANCB, AP1S2, and PIGA) were contained within the deleted region. Conclusions: We hypothesize that true "extreme" skewing of X-inactivation (=95%) is a rare occurrence, but when defined correctly there is a high probability of finding an X-chromosome disease-causing variant or larger deletion resulting in X-inactivation through a survival disadvantage or cell lethal mechanism. We postulate that the 2.3 Mb Xp22.2 deletion identified in our kindred arose de novo in the proband's mother (on the grandfather's homolog), and produced extreme skewing of X-inactivation via a "cell lethal" mechanism. We introduce a novel multitarget approach for X-inactivation analysis using multiple informative differentially methylated SNPs, as an alternative to the classical single locus (HUMARA) method. We propose that for females with unexplained severe phenotypic expression of an X-linked recessive disorder trio-SNP microarray should be undertaken in combination with X-inactivation analysis. [ABSTRACT FROM AUTHOR]