Skewed X-chromosome Inactivation Pattern Research Articles

Role of Circulating X-Chromosome Inactivation and Xist as Biomarkers in Female Carriers of Fabry Disease

Background: Fabry Disease (FD) is an X-linked lysosomal disease, in which, unlike other X-linked disorders, most female carriers manifest signs or symptoms for unknown reasons. Objectives: Herein, we aimed to test the potential role of X-chromosome inactivation (XCI) in leukocytes as a prognostic biomarker of disease in FD female carriers. Moreover, we explored if levels of X-inactive-specific transcript (Xist), a long non-coding RNA driving XCI, were detectable in the leukocytes of FD female carriers. Methods: We tested the XCI pattern in leukocytes on 33 consecutive females carrying pathogenic GLA variants. Disease severity was defined using the Mainz Severity Score Index (MSSI). Xist levels in leukocytes were assessed by real-time PCR and compared to the levels of 22 controls. Results: XCI was obtained for 31 female patients, finding 16 skewed (51.6%) individuals. Global MSSI did not differ in skewed vs. non-skewed FD carriers. In skewed FD females, the renal function and mean cardiologic MSSI subscore were significantly worse, and systemic arterial hypertension was more frequent. Xist levels detected in leukocytes were similar between female patients and controls, and did not differ by phenotype or XCI status. Conclusions: A skewed XCI pattern in leukocytes may represent a prognostic biomarker of worse renal and cardiac outcomes in female FD carriers.

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

Clinical, pathological, and genetic characterization in a large Chinese cohort with female dystrophinopathy

We aimed to investigate the clinical, pathological, and genetic characteristics of Chinese female dystrophinopathy and to identify possible correlations among them. One hundred forty genetically and/or pathologically confirmed female DMD variant carriers were enrolled, including 104 asymptomatic carriers and 36 symptomatic carriers. Twenty of 36 symptomatic and 16 of 104 asymptomatic carriers were sporadic with no family history. Muscle pathological analysis was performed in 53 carriers and X chromosome inactivation (XCI) analysis in 19 carriers. In asymptomatic carriers, the median age was 35.0 (range 2.0–58.0) years, and the serum creatine kinase (CK) level was 131 (range 60–15,745) IU/L. The median age, age of onset, and CK level of symptomatic carriers were 15.5 (range 1.8–62.0) years, 6.3 (range 1.0–54.0) years, and 6,659 (range 337–58,340) IU/L, respectively. Four female carriers with X-autosome translocation presented with a Duchenne muscular dystrophy (DMD) phenotype. Skewed XCI was present in 70.0% of symptomatic carriers. Compared to Becker muscular dystrophy (BMD)-like carriers, DMD-like carriers were more likely to have an early onset age, rapidly progressive muscle weakness, delayed walking, elevated CK levels, severe reduction of dystrophin, and skewed XCI. Our study reports the largest series of symptomatic female DMD carriers and suggests that delayed walking, elevated CK levels, severe reduction of dystrophin, X-autosome translocation, and skewed XCI pattern are associated with a severe phenotype in female dystrophinopathy.

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.

Evaluation of X chromosome inactivation in endemic Tunisian pemphigus foliaceus.

Almost 5% of the world's population develops an autoimmune disease (AID), it is considered the fourth leading cause of disability for women, who represent 78% of cases. The sex ratio when it comes to the most prevalent AID varies from 9:1 in systemic lupus erythematosus (SLE) to 13:1 in endemic Tunisian pemphigus foliaceus (PF). To test the potential involvement of skewed x-inactivation in the pathogenesis of Tunisian PF, we analyzed the methylation status of a highly polymorphic CAG repeat in the androgen receptor gene and evaluated the x chromosome inactivation (XCI) patterns in peripheral blood-leukocyte-derived DNA samples of female patients with PF (n=98) compared to healthy control (HC) subjects (n=150), as well as female patients with SLE (n=98) were enrolled as a reference group. XCI status was informative for 50 of the 98 PF patients (51%) and 70 of the 150 HC women (47%). Extremely skewed XCI patterns were more frequent in PF and SLEwomen than HC, but the difference was statistically significant only in women with SLE. No statistical difference was observed in XCI patterns between PF and SLE patients. PF phenotype-XCI correlation analysis revealed that (i) skewed XCI patterns may be involved in the disease's subtype and (ii) it was more pronounced in the endemic group than the sporadic one. Furthermore, preferential XCI showed an increase in heterozygote genotypes of PF's susceptibility polymorphisms in immunity-related X genes (FOXP3, AR, and TLR7) in PF patients compared to HC. Our results suggest that skewed XCI could lead to hemizygosity of X-linked alleles that might unmask X-linked deleterious alleles.

Two heterozygous mutations in the calcium/calmodulin-dependent serine protein kinase gene (CASK) in cases with developmental disorders.

The calcium/calmodulin-dependent serine protein kinase gene (CASK) is an essential gene in mammals, critical for neurodevelopment. The purpose of this study is to expand the understanding of the diagnosis of CASK-linked disorders. From clinical and genetic mutational analyses, relevant data in 2 Han Chinese patients were collected and analyzed. Real-time quantitative PCR (RT-qPCR) was performed to investigate the CASK expression levels in the patients. The X-chromosome inactivation (XCI) patterns of the patients and their nuclear families were tested by quantitation of methylation of the polymorphic human androgen receptor (HUMARA) locus. Two Han Chinese patients both presented with intellectual disability (ID), microcephaly with pontine and cerebellar hypoplasia (MICPCH). Two de novo mutations of c.82C>T (p.Arg28*) and c.846C>G (p.Tyr282*) in CASK have been investigated and predicted to be deleterious, which have produced truncated proteins. The functional protein association network of STRING (http://string-db.org) generated three-dimensional (3D) atomic models based on protein sequences in CASK and two Arg28 and Tyr282 residues were marked. RT-qPCR showed lower copy numbers of CASK expression in the patients than in their parents, as well as the sex- and age- matched control groups. Patient 1 showed a skewed XCI pattern, while no related changes noted in patient 2. Patients carrying different nonsense variants may have different degrees of different clinical phenotypes. This study expands the spectrum of genotype and phenotype correlations of CASK-linked disorders in the Han Chinese ethnicity and provides new insights into the molecular mechanism.

Preferential X Chromosome Inactivation as a Mechanism to Explain Female Preponderance in Myasthenia Gravis.

Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized by prevalence in young women (3:1). Several mechanisms proposed as explanations for gender bias, including skewed X chromosome inactivation (XCI) and dosage or sex hormones, are often involved in the development of autoimmunity. The skewed XCI pattern can lead to an unbalanced expression of some X-linked genes, as observed in several autoimmune disorders characterized by female predominance. No data are yet available regarding XCI and MG. We hypothesize that the preferential XCI pattern may contribute to the female bias observed in the onset of MG, especially among younger women. XCI analysis was performed on blood samples of 284 women between the ages of 20 and 82. XCI was tested using the Human Androgen Receptor Assay (HUMARA). XCI patterns were classified as random (XCI < 75%) and preferential (XCI ≥ 75%). In 121 informative patients, the frequency of skewed XCI patterns was 47%, significantly higher than in healthy controls (17%; p ≤ 0.00001). Interestingly, the phenomenon was observed mainly in younger patients (<45 years; p ≤ 0.00001). Furthermore, considering the XCI pattern and the other clinical characteristics of patients, no significant differences were found. In conclusion, we observed preferential XCI in MG female patients, suggesting its potential role in the aetiology of MG, as observed in other autoimmune diseases in women.

Analysis of X-inactivation status in a Rett syndrome natural history study cohort.

BackgroundRett syndrome (RTT) is a rare neurodevelopmental disorder associated with pathogenic MECP2 variants. Because the MECP2 gene is subject to X‐chromosome inactivation (XCI), factors including MECP2 genotypic variation, tissue differences in XCI, and skewing of XCI all likely contribute to the clinical severity of individuals with RTT.MethodsWe analyzed the XCI patterns from blood samples of 320 individuals and their mothers. It includes individuals with RTT (n = 287) and other syndromes sharing overlapping phenotypes with RTT (such as CDKL5 Deficiency Disorder [CDD, n = 16]). XCI status in each proband/mother duo and the parental origin of the preferentially inactivated X chromosome were analyzed.ResultsThe average XCI ratio in probands was slightly increased compared to their unaffected mothers (73% vs. 69%, p = .0006). Among the duos with informative XCI data, the majority of individuals with classic RTT had their paternal allele preferentially inactivated (n = 180/220, 82%). In sharp contrast, individuals with CDD had their maternal allele preferentially inactivated (n = 10/12, 83%). Our data indicate a weak positive correlation between XCI skewing ratio and clinical severity scale (CSS) scores in classic RTT patients with maternal allele preferentially inactivated XCI (r s = 0.35, n = 40), but not in those with paternal allele preferentially inactivated XCI (r s = −0.06, n = 180). The most frequent MECP2 pathogenic variants were enriched in individuals with highly/moderately skewed XCI patterns, suggesting an association with higher levels of XCI skewing.ConclusionThese results extend our understanding of the pathogenesis of RTT and other syndromes with overlapping clinical features by providing insight into the both XCI and the preferential XCI of parental alleles.

A Novel Missense Mutation of Arginine Vasopressin Receptor 2 in a Chinese Family with Congenital Nephrogenic Diabetes Insipidus: X-Chromosome Inactivation in Female CNDI Patients with Heterozygote 814A>G Mutation.

Background To identify novel clinical phenotypic signatures of congenital nephrogenic diabetes insipidus (CNDI). Methods A Chinese family with CNDI was recruited for participation in this study. The proband and one of his uncles suffered from polydipsia and polyuria since infancy. The results of clinical testing indicated the diagnosis of CNDI. 10 family members had similar symptoms but did not seek medical advice. Genetic testing of mutations in the coding region of the aquaporin 2 (AQP2) gene and the arginine vasopressin receptor 2 (AVPR2) gene were carried out in 11 family members. Somatic DNA from 5 female family members was used to test for methylation of polymorphic CAG repeats in the human androgen receptor (AR) gene, as an index for X-chromosome inactivation pattern (XCIP). Results AQP2 gene mutations were not found in any family members, but a novel missense mutation (814th base A>G) in exon 2 of the AVPR2 gene was identified in 10 individuals. This mutation leads to a Met 272 Val (GAT-GGT) amino acid substitution. Skewed X-chromosome inactivation patterns of the normal X allele were observed in 4 females with the AVPR2 gene mutation and symptoms of diabetes insipidus, but not in an asymptomatic female with the AVPR2 gene mutation. Conclusions Met 272 Val mutation of the AVPR2 gene was identified as a novel genetic risk factor for CDNI. The clinical NDI phenotype of female carriers with heterozygous AVPR2 mutation may be caused by X-chromosome inactivation induced by dominant methylation of the normal allele of AVPR2 gene.

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.

X-chromosome inactivation patterns in females with Fabry disease examined by both ultra-deep RNA sequencing and methylation-dependent assay.

Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A. Males are usually severely affected, while females have a wide range of disease severity. This variability has been assumed to be derived from organ-dependent skewed X-chromosome inactivation (XCI) patterns in each female patient. Previous studies examined this correlation using the classical methylation-dependent method; however, conflicting results were obtained. This study was established to ascertain the existence of skewed XCI in nine females with heterozygous pathogenic variants in the GLA gene and its relationship to the phenotypes. We present five female patients from one family and four individual female patients with Fabry disease. In all cases, heterozygous pathogenic variants in the GLA gene were detected. The X-chromosome inactivation patterns in peripheral blood leukocytes and cells of urine sediment were determined by both classical methylation-dependent HUMARA assay and ultra-deep RNA sequencing. Fabry Stabilization Index was used to determine the clinical severity. Skewed XCI resulting in predominant inactivation of the normal allele was observed only in one individual case with low ⍺-galactosidase A activity. In the remaining cases, no skewing was observed, even in the case with the highest total severity score (99.2%). We conclude that skewed XCI could not explain the severity of female Fabry disease and is not the main factor in the onset of various clinical symptoms in females with Fabry disease.

Analysis and prenatal diagnosis of FMR1 gene mutations among patients with unexplained mental retardation

To analyze the (CGG)n repeats of FMR1 gene among patients with unexplained mental retardation. For 201 patients with unexplained mental retardation, the (CGG)n repeats of the FMR1 gene were analyzed by PCR and FragilEaseTM PCR. Prenatal diagnosis was provided to carriers of pre- and full-mutations. The pattern of X chromosome inactivation (XCI) was determined for women with mental retardation and full mutations. For the 201 patients with unexplained mental retardation, 15 were identified with full mutations of the FMR1 gene. The prevalence of fragile X syndrome (FXS) in patients with unexplained mental retardation was determined as 7.5% (15/201). Prenatal diagnosis was provided for 6 pregnant women with pre- or full mutations. Analysis revealed that women with mental retardation and full FMR1 mutations exhibited a skewed XCI pattern with primary expression of the X chromosome carrying the mutant allele. FXS has a high incidence among patients with unexplained mental retardation. Analysis of FMR1 gene (CGG)n repeats in patients with unexplained mental retardation can facilitate genetic counseling and prenatal diagnosis for their families. FMR1 gene (CGG)n repeats screening should be recommended for patients with unexplained mental retardation.

Spread of X-chromosome inactivation into autosomal regions in patients with unbalanced X-autosome translocations and its phenotypic effects.

Patients with unbalanced X-autosome translocations are rare and usually present a skewed X-chromosome inactivation (XCI) pattern, with the derivative chromosome being preferentially inactivated, and with a possible spread of XCI into the autosomal regions attached to it, which can inactivate autosomal genes and affect the patients' phenotype. We describe three patients carrying different unbalanced X-autosome translocations, confirmed by G-banding karyotype and array techniques. We analyzed their XCI pattern and inactivation spread into autosomal regions, through HUMARA, ZDHHC15 gene assay and the novel 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and identified an extremely skewed XCI pattern toward the derivative chromosomes for all the patients, and a variable pattern of late-replication on the autosomal regions of the derivative chromosomes. All patients showed phenotypical overlap with patients presenting deletions of the autosomal late-replicating regions, suggesting that the inactivation of autosomal segments may be responsible for their phenotype. Our data highlight the importance of the XCI spread into autosomal regions for establishing the clinical picture in patients carrying unbalanced X-autosome translocations, and the incorporation of EdU as a novel and precise tool to evaluate the inactivation status in such patients.

Loss-of-function variants in ARHGEF9 are associated with an X-linked intellectual disability dominant disorder.

ARHGEF9 defects lead to an X-linked intellectual disability disorder related to inhibitory synaptic dysfunction. This condition is more frequent in males, with a few affected females reported. Up to now, sequence variants and gross deletions have been identified in males, while only chromosomal aberrations have been reported in affected females who showed a skewed pattern of X-chromosome inactivation (XCI), suggesting an X-linked recessive (XLR) disorder. We report three novel loss-of-function (LoF) variants in ARHGEF9: A de novo synonymous variant affecting splicing (NM_015185.2: c.1056G>A, p.(Lys352=)) in one female; a nonsense variant in another female (c.865C>T, p.(Arg289*)), that is, also present as a somatically mosaic variant in her father, and a de novo nonsense variant in a boy (c.899G>A; p.(Trp300*)). Both females showed a random XCI. Thus, we suggest that missense variants are responsible for an XLR disorder affecting males and that LoF variants, mainly occurring de novo, may be responsible for an X-linked dominant disorder affecting males and females.

Genetic analysis for carrier diagnosis in hemophilia A and B in the Mexican population: 25 years of experience.

Our 25 years of experience in carrier diagnosis of hemophilia A (HA) and B (HB) in Mexican population comprises linkage analysis of intragenic F8/F9 neutral variants along with, in severe HA (SHA), detection of F8 int22h and int1h inversions. In symptomatic carriers (SCs) we explored Lyonization to explain their symtomatology. From a DNA-Bank of 3,000 samples, intragenic restriction fragment length (RFLPs) and short tandem repeats (STRs) of F8/F9 genes were assessed by PCR-PAGE and GeneScan. In SHA patients, F8 inversions were detected by inverse shifting-PCR/diagnostic and complementary tests. In SCs, we evaluated hemorrhagic symptoms, clotting FVIII/FIX and X-chromosome inactivation (XCI) patterns were assessed by HUMARA assay and the search of XIST promoter pathogenic variants. Informativeness of linkage analysis for HA carrier diagnosis with RFLP's/STR's increased to 74% and reached 80% with five RFLPs for HB. Combined Inv22/Inv1 diagnosed 113 possible carriers, three de novo Inv22-1, and confirmed 45 mothers as obligate or sporadic carriers. Among 21 SCs, four showed extreme skewed XCI pattern (~80:20) but had normal karyotype and no C43G pathogenic variant in XIST promoter. Clotting FVIII/FIX correlated with the active X in leukocytes. Our data integrate the largest comprehensive research worldwide on the molecular diagnosis of HA and HB carriers in terms of the number of studied and diagnosed cases, in addition to the genetic analysis in SCs. Intragenic RFLPs and STRs of F8/F9 genes along with F8 int22h/int1h inversions in SHA emerge as optimal variants for molecular diagnosis in Mexican population. In counseling SCs, inheritance of skewed X-inactivation should be considered.

X-chromosome inactivation pattern of amniocytes predicts the risk of dystrophinopathy in fetal carriers of DMD mutations.

To predict the risk of dystrophinopathy in fetal carriers of dystrophin gene (DMD) mutations. Twenty-three pregnant women, with a total of 25 female fetuses carrying DMD mutations, were recruited. Among them, 13 pregnant women who participated in this study were only used to analyse the incidence of induced abortion after fetuses were diagnosed as dystrophinopathy carriers. Eleven fetal carriers from 10 pregnant women were tested to analyse X-chromosome inactivation (XCI) using amniocytes to assess the risk of dystrophinopathy. Follow-ups were conducted on all cases. Approximately one-third of fetuses were aborted before assessing the risk of dystrophinopathy. XCI analysis of amniocytes showed that 10 fetuses had random XCI patterns, and one fetus exhibited a highly skewed XCI pattern (100:0) with primary expression of the maternal X chromosome that carried the mutant allele. These 11 fetal carriers were born, and follow-up showed that the girl who showed the skewed XCI pattern as a fetus was diagnosed with Duchenne muscular dystrophy (DMD) at the age of four. The others did not present with dystrophinopathy-associated symptoms. XCI was significantly implicated in symptomatic female carriers of dystrophinopathies, and XCI pattern analysis of amniocytes may be useful in predicting the risk of dystrophinopathy in fetal carriers.

A statistical measure for the skewness of X chromosome inactivation based on case-control design

BackgroundSkewed X chromosome inactivation (XCI), which is a non-random process, is frequently observed in both healthy and affected females. Furthermore, skewed XCI has been reported to be related to many X-linked diseases. However, no statistical method is available in the literature to measure the degree of the skewness of XCI for case-control design. Therefore, it is necessary to develop methods for such a task.ResultsIn this article, we first proposed a statistical measure for the degree of XCI skewing by using a case-control design, which is a ratio of two logistic regression coefficients after a simple reparameterization. Based on the point estimate of the ratio, we further developed three types of confidence intervals (the likelihood ratio, Fieller’s and delta methods) to evaluate its variation. Simulation results demonstrated that the likelihood ratio method and the Fieller’s method have more accurate coverage probability and more balanced tail errors than the delta method. We also applied these proposed methods to analyze the Graves’ disease data for their practical use and found that rs3827440 probably undergoes a skewed XCI pattern with 68.7% of cells in heterozygous females having the risk allele T active, while the other 31.3% of cells keeping the normal allele C active.ConclusionsFor practical application, we suggest using the Fieller’s method in large samples due to the non-iterative computation procedure and using the LR method otherwise for its robustness despite its slightly heavy computational burden.

Whole exome sequencing of benign pulmonary metastasizing leiomyoma reveals mutation in the BMP8B gene

BackgroundBenign metastasizing leiomyoma (BML) is an orphan neoplasm commonly characterized by pulmonary metastases consisting of smooth muscle cells. Patients with BML have usually a current or previous uterine leiomyoma, which is therefore suggested to be the most probable source of this tumour. The purpose of this case report was to determine the possible genetic grounds for pulmonary BML.Case presentationWe present a case report in an asymptomatic 44-year-old female patient, who has developed uterine leiomyoma with subsequent pulmonary BML. Whole exome sequencing (WES) was used to detect somatic mutations in BML lesion. Somatic single nucleotide mutations were identified by comparing the WES data between the pulmonary metastasis and blood sample of the same BML patient. One heterozygous somatic mutation was selected for validation by Sanger sequencing. Clonality of the pulmonary metastasis and uterine leiomyoma was assessed by X-chromosome inactivation assay.ConclusionsWe describe a potentially deleterious somatic heterozygous mutation in bone morphogenetic protein 8B (BMP8B) gene (c.1139A > G, Tyr380Cys) that was identified in the pulmonary metastasis and was absent from blood and uterine leiomyoma, and may play a facilitating role in the metastasizing of BML. The clonality assay confirmed a skewed pattern of X-chromosome inactivation, suggesting monoclonal origin of the pulmonary metastases.

Characteristics of induced pluripotent stem cells from clinically divergent female monozygotic twins with Danon disease

RationaleInduced pluripotent stem cells (iPSCs) have been generated from patients with various forms of disease, including Danon disease (DD); however, few reports exist regarding disease-specific iPSCs derived from clinically divergent monozygotic twins. ObjectiveWe examined the characteristics of iPSCs and iPSC-derived cardiomyocytes (iPSC-CMs) generated from clinically divergent monozygotic female twins with DD. Methods and resultsWe generated iPSCs derived from T-cells isolated from clinically divergent, 18-year-old female twins with DD harboring a mutation in LAMP2 at the intron 6 splice site (IVS6+1_4delGTGA). Two divergent populations of iPSCs could prepare from each twin despite of their clinical divergence: one with wild-type LAMP2 expression (WT-iPSCs) and a second with mutant LAMP2 expression (MT-iPSCs). The iPSCs were differentiated into iPSC-CMs and then autophagy failure was observed only in MT-iPSC-CMs by electron microscopy, tandem fluorescent-tagged LC3 analysis, and LC3-II western blotting. Under these conditions, X-chromosome inactivation (XCI) was determined by PCR for the (CAG)n repeat in the androgen receptor gene, revealing an extremely skewed XCI pattern with the inactivated paternal wild-type and maternal mutant X-chromosomes in MT-iPSCs and WT-iPSCs, respectively, from each twin. ConclusionRegardless of their clinical differences, we successfully established two sets of iPSC lines that expressed either wild-type or mutant LAMP2 allele from each monozygotic twin with DD, of which only the populations expressing mutant LAMP2 showed autophagic failure.