Random X-chromosome Inactivation Research Articles

Sex-specific single cell-level transcriptomic signatures of Rett syndrome disease progression

Dominant X-linked diseases are uncommon due to female X chromosome inactivation (XCI). While random XCI usually protects females against X-linked mutations, Rett syndrome (RTT) is a female neurodevelopmental disorder caused by heterozygous MECP2 mutation. After 6-18 months of typical neurodevelopment, RTT girls undergo a poorly understood regression. We performed longitudinal snRNA-seq on cerebral cortex in a construct-relevant Mecp2e1 mutant mouse model of RTT, revealing transcriptional effects of cell type, mosaicism, and sex on progressive disease phenotypes. Across cell types, we observed sex differences in the number of differentially expressed genes (DEGs) with 6x more DEGs in mutant females than males. Unlike males, female DEGs emerged prior to symptoms, were enriched for homeostatic gene pathways in distinct cell types over time and correlated with disease phenotypes and human RTT cortical cell transcriptomes. Non-cell-autonomous effects were prominent and dynamic across disease progression of Mecp2e1 mutant females, indicating that wild-type-expressing cells normalize transcriptional homeostasis. These results advance our understanding of RTT progression and treatment.

A range of 30-62% of functioning multiciliated airway cells is sufficient to maintain ciliary airway clearance.

Primary ciliary dyskinesia is a genetic disorder caused by aberrant motile cilia function that results in defective ciliary airway clearance and subsequently leads to recurrent airway infections and bronchiectasis. We aimed to determine: how many functional multiciliated airway cells are sufficient to maintain ciliary airway clearance? To answer this question we exploited the molecular defects of the X-linked recessive primary ciliary dyskinesia variant caused by pathogenic variants in DNAAF6 (PIH1D3), characterised by immotile cilia in affected males. We carefully analysed the clinical phenotype and molecular defect (using immunofluorescence and transmission electron microscopy) and performed in vitro studies (particle tracking in air-liquid interface cultures) and in vivo studies (radiolabelled tracer studies) to assess ciliary clearance of respiratory cells from female individuals with heterozygous and male individuals with hemizygous pathogenic DNAAF6 variants. Primary ciliary dyskinesia male individuals with hemizygous pathogenic DNAAF6 variants displayed exclusively immotile cilia, absence of ciliary clearance and severe primary ciliary dyskinesia symptoms. Owing to random or skewed X-chromosome inactivation in six female carriers with heterozygous pathogenic DNAAF6 variants, 54.3±10% (range 38-70%) of multiciliated cells were defective. Nevertheless, in vitro and in vivo assessment of the ciliary airway clearance was normal or slightly abnormal. Consistently, heterozygous female individuals showed no or only mild respiratory symptoms. Our findings indicate that having 30-62% of multiciliated respiratory cells functioning can generate either normal or slightly reduced ciliary clearance. Because heterozygous female carriers displayed either no or subtle respiratory symptoms, complete correction of 30% of cells by precision medicine could improve ciliary airway clearance in individuals with primary ciliary dyskinesia, as well as clinical symptoms.

A system to analyze the initiation of random X-chromosome inactivation using time-lapse imaging of single cells

In female eutherian mammal development, X-chromosome inactivation (XCI) of one of the two X chromosomes is initiated early. Understanding the relationship between the initiation of XCI and cell fate is critical for understanding early female development and requires a system that can monitor XCI in single living cells. Traditional embryonic stem cells (ESCs) used for XCI studies often lose X chromosomes spontaneously during culture and differentiation, making accurate monitoring difficult. Additionally, most XCI assessment methods necessitate cell disruption, hindering cell fate tracking. We developed the Momiji (version 2) ESC line to address these difficulties, enabling real-time monitoring of X-chromosome activity via fluorescence. We inserted green and red fluorescent reporter genes and neomycin and puromycin resistance genes into the two X chromosomes of PGK12.1 ESCs, creating a female ESC line that retains two X chromosomes more faithfully during differentiation. Momiji (version 2) ESCs exhibit a more stable XX karyotype than other ESC lines, including the parental PGK12.1 line. This new tool offers valuable insights into the relationship between XCI and cell fate, improving our understanding of early female development.

How does the Xist activator Rlim/Rnf12 regulate Xist expression?

The long non-coding RNA (lncRNA) Xist is crucially involved in a process called X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female mammals to achieve X dosage compensation between the sexes. Because Xist RNA silences the X chromosome from which it is transcribed, the activation of Xist transcription marks the initiation of the XCI process and thus, mechanisms and players that activate this gene are of central importance to the XCI process. During female mouse embryogenesis, XCI occurs in two steps. At the 2-4 cell stages imprinted XCI (iXCI) silences exclusively the paternally inherited X chromosome (Xp). While extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo random XCI (rXCI) around implantation. Both iXCI and rXCI are dependent on Xist. Rlim, also known as Rnf12, is an X-linked E3 ubiquitin ligase that is involved in the transcriptional activation of Xist. However, while data on the crucial involvement of Rlim during iXCI appear clear, its role in rXCI has been controversial. This review discusses data leading to this disagreement and recent evidence for a regulatory switch of Xist transcription in epiblasts of implanting embryos, partially reconciling the roles of Rlim during Xist activation.

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy.

Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation. To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs). We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells. Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

363 A CRISPR/dCas9 Epigenetic Therapuetic Approach for CASK-Related MICPCH

OBJECTIVES/GOALS: CASK-related microcephaly with pontine and cerebellar hypoplasia (MICPCH) is a rare X-linked neurodevelopmental disorder caused by mutations in calcium/calmodulin-dependent serine protein kinase (CASK). We aim to rescue CASK expression via an CRISPR/dCas9 epigenetic therapeutic and create iPSC-based CASK relevant in vitro model systems. METHODS/STUDY POPULATION: As females have two X-chromosomes, disease causing mutations present with a 50/50 expression of mutant and wildtype, due to the mosaicism caused by random X-chromosome inactivation (XCI). This project will adapt an established CRISPR/dCas9 epigenetic approach to rescue expression from the silenced, wild-type CASK allele. We aim to accomplish this through testing different dCas9 orthologues and a guide RNA screen targeting the CASK promoter. Constructs will be tested for optimal targeting efficacy in vitro and assessed via RT-qPCR. Additionally, epigenetic modifications from our approach will be analyzed through bisulfite sequencing. We also aim to apply this epigenetic rescue technology in disease relevant cell lines and eventually in engineered patient mutation iPSC-derived neurons. RESULTS/ANTICIPATED RESULTS: Our results show the ability to target CASK and assess gene expression changes with CRISPR/dCas9 paired with an epigenetic modifier and transcriptional activator. Additionally, our fibroblast model with nonpathogenic single nucleotide polymorphisms within CASKallow for allele specific analysis of our targeted reactivation. We anticipate that following an increase of CASK expression, there would be a decrease in region specific promoter methylation. Further, with the identification of clinically described disease-causing point mutations that result in a loss of function of CASK protein, induction of the mutant sequence onto a healthy cell background will result in a similar reduction of CASK protein in our cell model. DISCUSSION/SIGNIFICANCE: This project will demonstrate the first therapeutic avenue for CASK-related MICPCH, and the potential to utilize targeted X-reactivation as a platform approach for X-linked disorders. Further, investigation of smaller dCas9 orthologues prepares our approach for future translational applications such as packaging into AAV for delivery.

A Comparative Analysis of Mouse Imprinted and Random X-Chromosome Inactivation.

The mammalian sexes are distinguished by the X and Y chromosomes. Whereas males harbor one X and one Y chromosome, females harbor two X chromosomes. To equalize X-linked gene expression between the sexes, therian mammals have evolved X-chromosome inactivation as a dosage compensation mechanism. During X-inactivation, most genes on one of the two X chromosomes in females are transcriptionally silenced, thus equalizing X-linked gene expression between the sexes. Two forms of X-inactivation characterize eutherian mammals, imprinted and random. Imprinted X-inactivation is defined by the exclusive inactivation of the paternal X chromosome in all cells, whereas random X-inactivation results in the silencing of genes on either the paternal or maternal X chromosome in individual cells. Both forms of X-inactivation have been studied intensively in the mouse model system, which undergoes both imprinted and random X-inactivation early in embryonic development. Stable imprinted and random X-inactivation requires the induction of the Xist long non-coding RNA. Following its induction, Xist RNA recruits proteins and complexes that silence genes on the inactive-X. In this review, we present a current understanding of the mechanisms of Xist RNA induction, and, separately, the establishment and maintenance of gene silencing on the inactive-X by Xist RNA during imprinted and random X-inactivation.

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

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. 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. 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. 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.

A Statistical Testing Strategy Accounting for Random and Nonrandom (Skewed) X-Chromosome Inactivation Identifies Lung Cancer Susceptibility Loci among Smokers.

Lung cancer is the most common cancer worldwide in mortality and the second in incidence. Epidemiological studies found a higher lung cancer risk for smoking women in comparison to men, but these sex differences, irrespective of smoking habits, remain controversial. One of the hypotheses concerns the genetic contribution of the sex chromosomes. However, while genome-wide association studies identified many lung cancer susceptibility loci, these analyses have excluded X-linked loci. To account for nongenetic factors, we first presented an association test based on an additive-multiplicative hazard model accounting for random/nonrandom X-inactivation process. A simulation study was performed to investigate the properties of the proposed test as compared with the Wald test from a Cox model with random X-inactivation process and the partial likelihood ratio test proposed by Xu et al. accounting for nonrandom X-inactivation process. Then, we performed an X chromosome-wide association study on 9,261 individuals from the population-based cohort CARTaGENE to identify susceptibility loci for lung cancer among current and past smokers. We adjusted for the PLCOm2012 lung cancer risk score used in screening programs. Simulation results show the good behavior of the proposed test in terms of power and type I error probability as compared to the Xu et al. and the Wald test. Using the proposed test statistic and adjusting for the PLCOm2012 score, the X chromosome-wide statistical analysis identified two SNPs in low-linkage disequilibrium located in the IL1RAPL1 (IL-1 R accessory protein-like) gene: rs12558491 (p = 2.75×10-9) and rs12835699 (p = 1.26×10-6). For both SNPs, the minor allele was associated with lower lung cancer risk. Adjusting for multiple testing, no signal was detected using the Wald or the Xu et al. likelihood ratio tests. By taking into account smoking behavior and the X-inactivation process, the investigation of the X chromosome has shed a new light on the association between X-linked loci and lung cancer. We identified two loci associated with lung cancer located in the IL1RAPL1 gene. This finding would have been overlooked by examining only results from other test statistics.

Random X-Chromosome Inactivation in Adult Hemophilia B Carriers Does Not Attenuate Bleeding Risk

Introduction: The X-linked bleeding disorder Hemophilia B (HB) results from deficiency in coagulation Factor IX (FIX). In contrast to Hemophilia A, genotype-phenotype correlations in HB males are complex, as some F9 variants in males are associated with low plasma FIX activity (FIX:C) but milder than expected bleeding phenotypes. At least 75% of FIX is extravascular and not adequately measured from plasma. Hemostasis is likely influenced by this extravascular FIX which is bound reversibly to type IV collagen in the subendothelial basement membrane. We predict that genotype-phenotype correlations in female carriers of F9 mutations may have additional complexity as the mutant FIX can competitively interfere with wild type (WT) FIX for extravascular binding sites. F9 expression in female HB carriers is influenced by X-chromosome inactivation (XCI), a process that silences one X in XX females. XCI results in cells that randomly inactivate either the X carrying the mutant or wild type F9, although skewed XCI (>80:20) is found in ~25% of the general population. XCI skewing in HB carriers can lead to preferential expression of the X carrying the F9 mutation, which in turn influences plasma and extravascular levels of FIX and bleeding tendency. How frequently XCI is skewed in HB carriers, and the role of XCI in modulating FIX levels and bleeding phenotypes, remains poorly understood. We sought to determine whether XCI skewing may further differentiate bleeding risk in HB carriers. Methods: Subjects were enrolled in a prospective, observational cross-sectional study with informed consent. Both the Condensed MCMDM-1 bleeding assessment tool (BAT) and Pictorial Blood-loss Assessment Chart (PBAC) were completed at the time of the visit. F9 mutation analysis confirmed carrier status. FIX:C and FIX antigen ELISA assays were performed on all subjects. XCI skewing was assessed using established assays at the Androgen Receptor (AR) and Fragile X Mental Retardation (FMR1) loci. Results: A total of 15 subjects (11 adults) were confirmed F9 mutation carriers; six missense mutations and one frameshift mutation were identified. Abnormal bleeding is frequent in adult HB carriers, with 5/11 (45%) having BAT scores >4. Heavy menstrual bleeding (HMB) is particularly frequent, with all adult HB carriers reporting PBAC > 100, and 8/11 with BAT HMB subcategory score >1. FIX assessment by activity (mean 56% ± 28) and antigen (mean 0.55 IU/mL ± 0.2) assays are highly correlated (r 2= 0.83, p < 0.0001). Notably, only two adult HB carriers with significant bleeding have FIX levels < 50%, while two HB carriers with FIX levels < 50% have BAT scores < 4, indicating that neither FIX:C nor FIX antigen levels adequately predict bleeding risk in all HB carriers. Indeed, for all 11 adult HB carriers, no significant differences in either FIX:C or FIX antigen were observed between those with BAT scores ≥ 4 and < 4. We asked if XCI explained FIX levels or bleeding phenotypes. Nearly half (5/11) of the adult carriers have moderately skewed XCI (> 70:30); only one is extremely XCI skewed (> 80:20), and of the individuals with skewed XCI, 3/5 have BAT scores ≥ 4. Although random XCI is proposed to attenuate HB carrier bleeding phenotype, 2/6 HB carriers with random XCI also have BAT scores ≥ 4. XCI weakly correlated with FIX antigen (n = 15; r 2= 0.27, p < 0.05) but not FIX:C. When assessing this relationship for only the individuals with F9 c.1025C>T (n = 6), there was significant correlation with FIX:C (R 2= 0.73, p < 0.05) but not with FIX antigen or bleeding phenotype. The lack of correlation between XCI and FIX antigen in individuals with an identical F9 mutation argues for additional influences on intravascular, and likely extravascular, FIX levels and will require further investigation. Conclusions: Bleeding tendency in HB carriers is not rare and does not correlate with plasma FIX:C or FIX antigen. Random XCI is not fully protective against abnormal bleeding. Future studies are needed to better understand bleeding phenotypes in HB carriers that consider XCI, F9 mutation type and influences on circulating levels of WT and mutant FIX.

Modelling PCDH19 clustering epilepsy by Neurogenin 2 induction of patient-derived induced pluripotent stem cells.

Loss of function mutations in PCDH19 gene cause an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have normal phenotype. No cure is presently available for this disease. Fibroblasts from a female patient carrying frameshift mutation were reprogrammed into human induced pluripotent stem cells (hiPSC). To create a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, we created mosaic neurons by mixing wild-type (WT) and mutated (mut) human iPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2. We generated functional neurons from patient-derived iPSC using a rapid and efficient method of differentiation through overexpression of Neurogenin 2. Was revealed an accelerated maturation and higher arborisation in the mutated neurons, while the mosaic neurons showed the highest frequency of action potential firing and hyperexcitability features, compared to mutated and WT neurons. Our findings provide evidence that PCDH19 c.2133delG mutation affects proper metaphases with increased numbers of centrosomes in stem cells and accelerates neuronal maturation in premature cells. PCDH19 mosaic neurons showed an elevated excitability, representing the situation in PCDH19-CE brain. We suggest an Ngn-2 hiPSC-derived PCDH19 neurons as an informative experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies.

Wild-type MECP2 expression coincides with age-dependent sensory phenotypes in a female mouse model for Rett syndrome.

Rett syndrome is characterized by an early period of typical development and then, regression of learned motor and speech skills in girls. Loss of MECP2 protein is thought to cause Rett syndrome phenotypes. The specific underlying mechanisms from typical developmental trajectory to regression features throughout life are unclear. Lack of established timelines to study the molecular, cellular, and behavioral features of regression in female mouse models is a major contributing factor. Due to random X-chromosome inactivation, female patients with Rett syndrome and female mouse models for Rett syndrome (Mecp2Heterozygous , Het) express a functional copy of wild-type MECP2 protein in approximately half of all cells. As MECP2 expression is regulated during early postnatal development and experience, we characterized the expression of wild-type MECP2 in the primary somatosensory cortex of female Het mice. Here, we report increased MECP2 levels in non-parvalbumin-positive neurons of 6-week-old adolescent Het relative to age-matched wild-type controls, while also displaying typical levels of perineuronal net expression in the barrel field subregion of the primary somatosensory cortex, mild tactile sensory perception deficits, and efficient pup retrieval behavior. In contrast, 12-week-old adult Het express MECP2 at levels similar to age-matched wild-type mice, show increased perineuronal net expression in the cortex, and display significant tactile sensory perception deficits. Thus, we have identified a set of behavioral metrics and the cellular substrates to study regression during a specific time in the female Het mouse model, which coincides with changes in wild-type MECP2 expression. We speculate that the precocious increase in MECP2 expression within specific cell types of adolescent Het may provide compensatory benefits at the behavioral level, while the inability to further increase MECP2 levels leads to regressive behavioral phenotypes over time.

Identification and functional characterization of a novel truncating splicing variant in COL4A5 gene causes X-linked Alport syndrome with astigmatism.

Identification and functional characterization of a novel truncating splicing variant in COL4A5 gene causes X-linked Alport syndrome with astigmatism.

X-chromosome inactivation patterns depend on age and tissue but not conception method in humans

Female somatic X-chromosome inactivation (XCI) balances the X-linked transcriptional dosages between the sexes, randomly silencing the maternal or paternal X chromosome in each cell of 46,XX females. Skewed XCI toward one parental X has been observed in association with ageing and in some female carriers of X-linked diseases. To address the problem of non-random XCI, we quantified the XCI skew in different biological samples of naturally conceived females of different age groups and girls conceived after in vitro fertilization (IVF). Generally, XCI skew differed between saliva, blood, and buccal swabs, while saliva and blood had the most similar XCI patterns in individual females. XCI skew increased with age in saliva, but not in other tissues. We showed no significant differences in the XCI patterns in tissues of naturally conceived and IVF females. The gene expression profile of the placenta and umbilical cord blood was determined depending on the XCI pattern. The increased XCI skewing in the placental tissue was associated with the differential expression of several genes out of 40 considered herein. Notably, skewed XCI patterns (> 80:20) were identified with significantly increased expression levels of four genes: CD44, KDM6A, PHLDA2, and ZRSR2. The differences in gene expression patterns between samples with random and non-random XCI may shed new light on factors contributing to the XCI pattern outcome and indicate new paths in future research on the phenomenon of XCI skewing.

A rat model of a focal mosaic expression of PCDH19 replicates human brain developmental abnormalities and behaviours.

Protocadherin 19 gene-related epilepsy or protocadherin 19 clustering epilepsy is an infantile-onset epilepsy syndrome characterized by psychiatric (including autism-related), sensory, and cognitive impairment of varying degrees. Protocadherin 19 clustering epilepsy is caused by X-linked protocadherin 19 protein loss of function. Due to random X-chromosome inactivation, protocadherin 19 clustering epilepsy-affected females present a mosaic population of healthy and protocadherin 19-mutant cells. Unfortunately, to date, no current mouse model can fully recapitulate both the brain histological and behavioural deficits present in people with protocadherin 19 clustering epilepsy. Thus, the search for a proper understanding of the disease and possible future treatment is hampered. By inducing a focal mosaicism of protocadherin 19 expression using in utero electroporation in rats, we found here that protocadherin 19 signalling in specific brain areas is implicated in neuronal migration, heat-induced epileptic seizures, core/comorbid behaviours related to autism and cognitive function.

Complete loss of the X-linked gene CASK causes severe cerebellar degeneration

BackgroundHeterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes developmental delay in girls, while in boys, loss of the only allele of these genes leads to epileptic...

Regulation and importance of factor VIII levels in hemophilia A carriers.

To summarize the recent literature related to female hemophilia A carriers with respect to prevalence in the population, the impact of baseline factor VIII levels and other influences on bleeding phenotype, and clinical management needs. Many female hemophilia A carriers are at risk for abnormal bleeding, yet they are underrecognized by healthcare providers and their bleeding symptoms are underreported. Low FVIII levels are consistently associated with clinically significant bleeding and correlate well with skewed X chromosome inactivation (XCI). Most interestingly, bleeding tendency is also observed in some hemophilia A carriers with normal factor VIII levels and requires further investigation. Well controlled studies investigating peripartum and periprocedural FVIII levels and adequate hemostatic treatment are necessary to inform management guidelines. Prevalence and bleeding tendency of hemophilia A carriers remain underreported, despite a significant proportion having low FVIII levels. Skewed XCI may explain low FVIII but does not explain the bleeding risk encountered in a larger proportion of hemophilia A carriers with random XCI and borderline/normal FVIII.

Random X-Chromosome Inactivation in Hemophilia A Carriers Does Not Fully Attenuate Bleeding Risk

Background: In contrast to males with the X-linked bleeding disorder Hemophilia A (HA), bleeding in female carriers is attenuated by the presence of a wild type F8 allele. While low FVIII activity (FVIII:C) is more predictive of bleeding tendency, hemophilia carriers with “normal” FVIII:C can have bleeding sequelae. FVIII:C in HA carriers is influenced by X-chromosome inactivation (XCI), a process that silences one X in XX females. XCI results in cells that have randomly inactivated either parental X, although skewed XCI patterns occur commonly in ~25% of the general population. How frequently XCI skewing in HA carriers leads to preferential activation of the X carrying the F8 mutation, increasing bleeding tendency, is poorly understood. We sought to determine whether XCI skewing further differentiates bleeding risk in HA carriers.Methods: Patients were enrolled in this prospective, observational cross-sectional study with informed consent when presenting for F8 genotype analysis through the My Life, Our Future (MLOF) study. Standardized Condensed MCMDM-1 and Pediatric Bleeding Questionnaire (PBQ) bleeding assessment tools (BAT) and Pictorial Blood-loss Assessment Charts (PBAC) were completed at the time of the visit. Hemophilia carrier status was confirmed by documented F8 mutation. One-stage FVIII:C assays and chromogenic FVIII assays were performed on all subjects. XCI skewing was assessed using established assays at the Androgen Receptor (AR) and Fragile X Mental Retardation (FMR1) loci.Results: 98 individuals have been evaluated to date. 65 (46 adults, 19 adolescents and children) are confirmed F8 mutation carriers, while 33 (17 adults) are confirmed non-carriers. 39 (30 adults) have F8 mutations associated with severe HA, and 26 (16 adults) have non-severe F8 mutations. 21/46 (46%) adult HA carriers have BAT scores >4. BAT scores are significantly higher (5 vs. 1.8, p=0.0452) in adult HA carriers compared to age-matched controls (39.9 vs. 35.5, p=0.24). PBAC and BAT correlate well in adult F8 carriers (r =0.62, p<0.0001). Among adolescents, BAT scores are similar between controls and carriers; none of the HA carriers have BAT scores >2.FVIII assessment by one-stage and chromogenic FVIII assays are highly correlated (p<0.0001), with the notable exception of one carrier whose assays are markedly discrepant (19% chromogenic vs 108% one-stage). Her F8 mutation (p.Glu1988Argfs*4), is associated with severe HA and maps within the A3 domain. Although mutations in this domain can result in discrepant one-stage/chromogenic activity, such an association has not been previously reported for this specific mutation. As chromogenic results more accurately reflect bleeding tendency in this carrier, utilization of both activity assays will capture more individuals at potential risk.Notably, only one third (7/21) of adult HA carriers with significant bleeding have FVIII:C below 40%. 4/21 have FVIII:C in the 40-60% range, and 10/21 have “normal” levels >60%, indicating that FVIII:C levels alone cannot predict bleeding risk in all carriers. Indeed, for all adult HA carriers (n=46), chromogenic and one-stage FVIII assays correlate only weakly with BAT (r=-0.29, p=0.053) and PBAC (r=-0.27, p=0.072) scores. However, the correlation for severe F8 mutation carriers (n=30) is stronger between BAT scores and chromogenic assays (r=-0.37, p=0.0464), and PBAC scores and one-stage assays (r=-0.36, p=0.0493).XCI assays are informative in 44 of 46 adult HA carriers. 21/44 adult carriers have skewed XCI (>70:30) and 9 are extremely XCI skewed (>80:20). This level of XCI skewing exceeds that in the general population. Although random XCI is frequently proposed to attenuate carrier bleeding phenotype, 9/23 HA carriers with random XCI have BAT scores >4, including 2 with non-severe F8 mutations predicted to have a mild phenotype. Adolescent HA carriers have similar proportions of XCI skewing and similar FVIII activity to adult HA carriers, yet their reported bleeding is indistinguishable from age matched controls.Conclusion: Contrary to current dogma, bleeding tendency in HA carriers is not rare, and random X-chromosome inactivation is not fully protective. Correlation of bleeding scores and FVIII:C in adult HA carriers is strengthened when stratified by F8 mutation severity. Additional factors must influence hemostasis for HA carriers with random XCI. DisclosuresCygan:Hemostasis and Thrombosis Research Society: Research Funding.

Integrated analysis of Xist upregulation and X-chromosome inactivation with single-cell and single-allele resolution

To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

Immunological Aspects of X-Linked Chronic Granulomatous Disease Female Carriers.

X-linked Granulomatous Disease (XL-CGD) carriers were previously thought to be clinically healthy because random X-chromosome inactivation (XCI) allows approximately half of their phagocytes/monocytes to express functional gp91phox protein. This supports the NADPH oxidase activity necessary for the killing of engulfed pathogens. Some XL-CGD carriers suffer from inflammatory and autoimmune manifestations as well as infections, although the skewed-XCI of a mutated allele is reported to be exclusively determinant for infection susceptibility. Indeed, immune dysregulation could be determined by dysfunctional non-phagocytic leukocytes rather than the percentage of functioning neutrophils. Here we investigated in a cohort of 12 X-CGD female carriers at a particular time of their life the gp91phox protein expression/function and how this affects immune cell function. We showed that 50% of carriers have an age-independent skewed-XCI and 65% of them have a misrepresented expression of the wild-type gene. The majority of carriers manifested immune dysregulation and GI manifestations regardless of age and XCI. Immunological investigations revealed an increase in CD19+ B cells, CD56bright-NK cell percentage, a slightly altered CD107a upregulation on CD4+ T cells, and reduced INFγ-production by CD4+ and CD8+ cells. Notably, we demonstrated that the residual level of ROS robustly correlates with INFγ-expressing T cells, suggesting a role in promoting immune dysregulation in carriers.