Skewed X-inactivation Research Articles

WDR45 variants as a major cause for a clinically variable intellectual disability syndrome from early infancy in females.

Pathogenic variants of WD repeat domain 45 (WDR45) cause neurodegeneration with brain iron accumulation 5 (NBIA5), which is characterised by progressive neurological regression and brain iron accumulation in adulthood. Early diagnosis of NBIA5 patients is difficult because they often show only a non-specific developmental delay in childhood, but it is essential for lifelong medical management. We investigated 32 females with developmental delays for coding variants of WDR45 using Sanger sequencing. Whole-genome sequencing (WGS) and X chromosome inactivation (XCI) analysis were also performed. We identified two disease-causing variants, one of which was a novel stop-loss variant, c.1051delG p.(Val351CysfsTer60), in a female with severe developmental delay from early infancy with epileptic spasms. The XCI analysis (which we originally developed) suggested a random pattern in white blood cells. WGS did not reveal any other pathogenic variants, including those in two iron transporter genes. Together with our previous findings in the WGS study, WDR45 variants accounted for 12% (6/51) of the females with developmental delay, suggesting that WDR45 is a major gene in females with developmental delay. Pathogenic variants of WDR45 result in various phenotypes that do not necessarily correlate with variant types or XCI skewing patterns.

High-coverage nanopore sequencing of samples from the 1000 Genomes Project to build a comprehensive catalog of human genetic variation.

Fewer than half of individuals with a suspected Mendelian or monogenic condition receive a precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest in using long-read sequencing (LRS) to streamline clinical genomic testing, but the absence of control datasets for variant filtering and prioritization has made tertiary analysis of LRS data challenging. To address this, the 1000 Genomes Project ONT Sequencing Consortium aims to generate LRS data from at least 800 of the 1000 Genomes Project samples. Our goal is to use LRS to identify a broader spectrum of variation so we may improve our understanding of normal patterns of human variation. Here, we present data from analysis of the first 100 samples, representing all 5 superpopulations and 19 subpopulations. These samples, sequenced to an average depth of coverage of 37x and sequence read N50 of 54 kbp, have high concordance with previous studies for identifying single nucleotide and indel variants outside of homopolymer regions. Using multiple structural variant (SV) callers, we identify an average of 24,543 high-confidence SVs per genome, including shared and private SVs likely to disrupt gene function as well as pathogenic expansions within disease-associated repeats that were not detected using short reads. Evaluation of methylation signatures revealed expected patterns at known imprinted loci, samples with skewed X-inactivation patterns, and novel differentially methylated regions. All raw sequencing data, processed data, and summary statistics are publicly available, providing a valuable resource for the clinical genetics community to discover pathogenic SVs.

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.

Identification of diagnostic candidates in Mendelian disorders using an RNA sequencing-centric approach

BackgroundRNA sequencing (RNA-seq) is increasingly being used as a complementary tool to DNA sequencing in diagnostics where DNA analysis has been uninformative. RNA-seq enables the identification of aberrant splicing and aberrant gene expression, improving the interpretation of variants of unknown significance (VUSs), and provides the opportunity to scan the transcriptome for aberrant splicing and expression in relevant genes that may be the cause of a patient’s phenotype. This work aims to investigate the feasibility of generating new diagnostic candidates in patients without a previously reported VUS using an RNA-seq-centric approach.MethodsWe systematically assessed the transcriptomic profiles of 86 patients with suspected Mendelian disorders, 38 of whom had no candidate sequence variant, using RNA from blood samples. Each VUS was visually inspected to search for splicing abnormalities. Once aberrant splicing was identified in cases with VUS, multiple open-source alternative splicing tools were used to investigate if they would identify what was observed in IGV. Expression outliers were detected using OUTRIDER. Diagnoses in cases without a VUS were explored using two separate strategies.ResultsRNA-seq allowed us to assess 71% of VUSs, detecting aberrant splicing in 14/48 patients with a VUS. We identified four new diagnoses by detecting novel aberrant splicing events in patients with no candidate sequence variants from prior DNA testing (n = 32) or where the candidate VUS did not affect splicing (n = 23). An additional diagnosis was made through the detection of skewed X-inactivation.ConclusionThis work demonstrates the utility of an RNA-centric approach in identifying novel diagnoses in patients without candidate VUSs. It underscores the utility of blood-based RNA analysis in improving diagnostic yields and highlights optimal approaches for such analyses.

X-chromosome inactivation pattern and telomere length in recurrent pregnancy loss

Recurrent pregnancy loss is a reproductive disorder affecting about 1 to 5 % of pregnant women worldwide that requires our attention, especially considering that about 50 % of cases are idiopathic. The present study is focused on testing a possible association between extreme skewed X-chromosome inactivation patterns and/or shortened telomeres with idiopathic cases since both are considered non-consensual potential causes underlying recurrent pregnancy loss in the scientific community. For this purpose, two groups of women were analyzed and compared: a group of women with idiopathic recurrent pregnancy loss and a second group of age-matched women with proven fertility, and both X-chromosome inactivation patterns and telomere length were measured and compared from maternal DNA extracted from peripheral blood. Our data showed no statistically significant differences between groups, suggesting no association between extreme skewed X-chromosome inactivation or shortened telomeres with recurrent pregnancy losses. Additionally, the effect of maternal age on both X-chromosome inactivation pattern and telomere length was tested, but no significant correlation was observed between advanced maternal age and extreme skewed X-chromosome inactivation or telomere shortening. This study represents one more valid contribution to the investigation of causes underlying recurrent pregnancy loss suggesting that, new variables may be considered since the pattern of X-chromosome inactivation and telomere length do not seem to be related to this reproductive disorder. Briefly, considering its clinical relevance, it is mandatory a continuous effort in the scientific community to cover new potential recurrent pregnancy loss-related causes.

P-804 Smchd1 is a key regulator of epigenetic cellular memory induced by morphine in mouse embryonic stem cells (mESC)

Abstract Study question Can morphine induce stable epigenetic changes that persist mitotically in mESC? Summary answer SMCHD1 is a key regulator of epigenetic cellular memory induced by morphine, whose deregulation can be memorized by mESC and upon embryo development. What is known already Epigenetic cellular memory induced by environmental factors implies the existence of mitotically heritable changes that influence gene expression without altering DNA sequence, leading to the propagation of heritable changes in phenotype. X chromosome inactivation (XCI) is an important epigenetic process, which takes places during embryo development and is maintained in the adulthood. Since XCI is part of normal development, changes induced by environmental factors might affect embryo development and lead to health problems. As morphine can easily pass through the placental barrier and reach the embryo, our aim is to elucidate whether morphine causes epigenetic regulation on XCI process. Study design, size, duration Epigenetic cellular memory induced by environmental factors implies the existence of mitotically heritable changes that influence gene expression without altering DNA sequence, leading to the propagation of heritable changes in phenotype. To evaluate if morphine can induce cellular epigenetic memory, we analyzed the dynamic epigenetic changes over the time in the absence of morphine. For that purpose, OCT4-reported mESCs were chronically treated with morphine during 24h (10-5 mM) Participants/materials, setting, methods • Transcriptomic analysis by RNA-Sequencing and gene expression validation by qRT-PCR. • Epigenetic analysis by H3K27me3 ChIP-Sequencing and WGBS-Sequencing. • Smchd1 gene silencing by Crisper-Cas9. Main results and the role of chance Morphine induced cellular epigenetic memory upon embryo development. mESCs can memorize morphine exposure and induces transcriptional changes that can persist over the time, even in the absence of morphine. RNA-seq has shown that morphine chronic treatment produces a transcriptional deregulation of 932 genes in P1 just after the treatment and this amount increased after treatment withdrawal (1196 genes, P2 and 2138 in P3). Integrative analyses between P1, P2 and P3 identifies Smchd1 gene as a potential regulator of epigenetic cellular memory induced by morphine. Crisper-Cas9 silencing analyses confirmed that Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1), an architectural factor critical for X-chromosome inactivation (XCI), is a morphine-sensitive target gene. Morphine reduced the gene expression of Smchd1 in mESC at different time points, which is consistent with an increase of H3K27me3 and DNA methylation at promoter. Morphine alters normal X chromosome wide silencing. Our results identified Smchd1 a key regulator of epigenetic cellular memory induced by morphine, whose deregulation can be memorized by the cell, providing an epigenetic mechanism that is maintained into cell-to-cell memory and cell differentiation process, which might cause diseases or health problems during the adulthood. Limitations, reasons for caution To perform the in-vitro analysis in mouse Wider implications of the findings Our results provide insights into the epigenetic mechanisms induced by morphine and establish the bases to understand how environmental factors can cause epigenetic changes that cause health problems or diseases, since a skewed XCI pattern is behind the higher incidence on different neurological diseases such as Alzheimer or Autism Trial registration number not applicable

Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders.

The TATA box-binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of the basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in human males cause X-linked intellectual disability, a neurodevelopmental disorder, and TAF1 is dysregulated in X-linked dystonia-parkinsonism, a neurodegenerative disorder. However, this field has lacked a genetic mouse model of TAF1 disease to explore its mechanism in mammals and treatments. Here, we generated and validated a conditional cre-lox allele and the first ubiquitous Taf1 knockout mouse. We discovered that Taf1 deletion in male mice was embryonically lethal, which may explain why no null variants have been identified in humans. In the brains of Taf1 heterozygous female mice, no differences were found in gross structure, overall expression and protein localisation, suggesting extreme skewed X inactivation towards the non-mutant chromosome. Nevertheless, these female mice exhibited a significant increase in weight, weight with age, and reduced movement, suggesting that a small subset of neurons was negatively impacted by Taf1 loss. Finally, this new mouse model may be a future platform for the development of TAF1 disease therapeutics.

Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 2 Caused by a Novel PIGA Variant Not Associated with a Skewed X-Inactivation Pattern

Germline variants in the phosphatidylinositol glycan class A (PIGA) gene, which is involved in glycosylphosphatidylinositol (GPI) biosynthesis, cause multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) with X-linked recessive inheritance. The available literature has described a pattern of almost 100% X-chromosome inactivation in mothers carrying PIGA variants. Here, we report a male infant with MCAHS2 caused by a novel PIGA variant inherited from his mother, who has a non-skewed pattern of X inactivation. Phenotypic evidence supporting the pathogenicity of the variant was obtained by flow-cytometry tests. We propose that the assessment in neutrophils of the expression of GPI-anchored proteins (GPI-APs), especially CD16, should be considered in cases with variants of unknown significance with random X-inactivation in carrier mothers in order to clarify the pathogenic role of PIGA or other gene variants linked to the synthesis of GPI-APs.

Comprehensive genomic filtering algorithm to expose the cause of skewed X chromosome inactivation. The proof of concept in female haemophilia expression

BackgroundExploring the expression of X linked disorders like haemophilia A (HA) in females involves understanding the balance achieved through X chromosome inactivation (XCI). Skewed XCI (SXCI) may be involved in...

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

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

Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia

BackgroundPrimary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the...

Role of Skewed X-Chromosome Inactivation in Common Variable Immunodeficiency.

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically <30%. Other proposed mechanisms include digenic, oligogenic, or polygenic inheritance and epigenetic dysregulation. In this study, we aimed to assess the role of skewed X-chromosome inactivation (XCI) in CVID. Within our cohort of 131 genetically analyzed CVID patients, we selected female patients with rare variants in CVID-associated genes located on the X-chromosome. Four patients harboring heterozygous variants in BTK (n = 2), CD40LG (n = 1), and IKBKG (n = 1) were included in the study. We assessed XCI status using the HUMARA assay and an NGS-based method to quantify the expression of the 2 alleles in mRNA. Three of the 4 patients (75%) exhibited skewed XCI, and the mutated allele was predominantly expressed in all cases. Patient 1 harbored a hypomorphic variant in BTK (p.Tyr418His), patient 3 had a pathogenic variant in CD40LG (c.288+1G>A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.

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.

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

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

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.

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

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

Female Hemophilia in a Set of Triplets

Female Hemophilia In A Set Of Triplets Introduction: Hemophilia A (HA) and B (HB) are the most common inherited bleeding disorders. Gene sequencing identifies specific mutations and their associations with plasma factor levels and clinical phenotypes. Both HA and HB are X-linked recessive disorders, where the female carriers pass the gene onto their descendants. Males who inherit the mutated gene from their mothers develop the disease, whereas females are carriers due to having one normal gene, they typically do not display the characteristic features of hemophilia and have moderately low factor VIII levels of less than 40%. Female carriers may have bleeding symptoms, yet they do not experience as severe morbidities as the male hemophilia patients, due to having higher plasma factor VIII levels. Rarely female carriers can be severely affected and may have plasma factor VIII activities comparable to those with severe hemophilia. Here we are presenting a set of triplets, where the male infant and one of his sisters have severe hemophilia while the other sister is a hemophilia carrier. Methods: A set of triplets a male and two female neonates were born after 33 weeks of gestation to a healthy mother. Female neonates were dichorionic but shared the same placenta. The male infant had a circumcision at six weeks of age, which was complicated with prolonged bleeding. He was brought to the hospital and was diagnosed with severe HA. (Table.1). Infant #2 was admitted to the hospital for skin rash at age 8 weeks. She had staphylococcal scalded skin syndrome and was treated with vancomycin and cephalexin. She was noted to have prolonged bleeding from needle puncture sites. She was diagnosed with severe HA. The bleeding was controlled with tight packing. After a week of treatment her rash cleared, and she was discharged home. (Table 1). Infant #3 She was a healthy baby with no bleeding problems. (Table 1). Results and conclusions: We presented three infant-triplets-with factor VIII deficiency; infant #1 and infant #2 have severe HA. Infant #3 is an asymptomatic carrier. They all have the same mutation as their asymptomatic carrier mother. Infant #1 the male infant developed severe HA due to having the hemophilia allele on his “X” chromosome. Infant #3 a female infant is an asymptomatic carrier. She inherited the hemophilia allele on the X chromosome from her carrier mother and has an X chromosome bearing the normal allele, thus is able to produce factor VIII of 40%. Infant #2 the other female infant had severe HA, with a factor VIII activity of &amp;lt;0.3%. Female hemophilia is rare occurrence. Hemophilia treatment center (HTT) population profile (PP) reported that of the 1,676 females attending to HTC, 47.6% of them had less than 40% factor level, 0.48% of them had severe, 1.4% had moderate and 17.9% had mild hemophilia[Haemophilia. 2021:27(6):1037-1044. Doi:10.1111}. This can occur, in the setting of monosomy X (Turner syndrome-XO), due to having one X chromosome carrying the hemophilic gene or in the setting of compound heterozygosity: the female product of a father with hemophilia and the mother being a carrier. Similarly, hemizygosity of the hemophilic gene on both parents would result in hemophilia in their daughters. Another mechanism that plays a role in the pathogenesis of female hemophilia is the “X chromosome inactivation (XCI). This has been described as the most common cause of female hemophilia. One of the two X chromosomes in females is randomly and permanently inactivated in cells to ensure the balance with one X chromosome of males and limit the overproduction of potentially toxic gene proteins. The degree of inactivation determines the plasma levels of the gene products, in hemophilia the factor levels. The chromosome analysis of infant #2 revealed normal 46XX. Structural chromosomal abnormality is ruled out as she did not have Turner syndrome “XO”, or “45XY” with androgen insensitivity syndrome, nor did she have compound heterozygosity or hemizygosity of the X chromosome. The father had normal coagulation studies and factor VIII activity. Therefore, we believe infant #2 had extremely skewed XCI and the inactivation is skewed in favor of hemophilic X chromosome. This mechanism resulted in severe HA in this infant. The infants are now seven months old and have no bleeding problems, as infant #1 and #2 are on prophylaxis with emicizumab (Hemlibra), they both have 1% factor activity by chromogenic assay and do not have inhibitors. (Table 1)

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 (&amp;gt;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 &amp;gt;4. Heavy menstrual bleeding (HMB) is particularly frequent, with all adult HB carriers reporting PBAC &amp;gt; 100, and 8/11 with BAT HMB subcategory score &amp;gt;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 &amp;lt; 0.0001). Notably, only two adult HB carriers with significant bleeding have FIX levels &amp;lt; 50%, while two HB carriers with FIX levels &amp;lt; 50% have BAT scores &amp;lt; 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 &amp;lt; 4. We asked if XCI explained FIX levels or bleeding phenotypes. Nearly half (5/11) of the adult carriers have moderately skewed XCI (&amp;gt; 70:30); only one is extremely XCI skewed (&amp;gt; 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 &amp;lt; 0.05) but not FIX:C. When assessing this relationship for only the individuals with F9 c.1025C&amp;gt;T (n = 6), there was significant correlation with FIX:C (R 2= 0.73, p &amp;lt; 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.

Generation of Induced Pluripotent Stem Cell Line iTAF15Xsk4 from Fibroblasts of a Patient with Microdeletion at Xq24

Differentiation of induced pluripotent stem (iPS) cells from patients and healthy donors allows in vitro study of genetic disorders. We have previously reported a clinical case of recurrent pregnancy loss in a patient with skewed X-chromosome inactivation in peripheral blood lymphocytes, endometrium, and buccal epithelium. We have found a 239 kb microdeletion at Xq24 that affected eight genes including UBE2A. In this work, we produced iPS cell line iTAF15Xsk4 from the patient’s skin fibroblasts using non-integrating episomal vectors. iPS cell line had a normal karyotype, expressed pluripotency markers, and upon differentiation in embryoid bodies expressed markers of all three germ layers. This cell line could be used for the UBE2A deficiency syndrome study.

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

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