Chromosome Inactivation Patterns Research Articles

Characteristics and clinical evaluation of X chromosome translocations

BackgroundIndividuals with X chromosomal translocations, variable phenotypes, and a high risk of live birth defects are of interest for scientific study. These characteristics are related to differential breakpoints and various types of chromosomal abnormalities. To investigate the effects of X chromosome translocation on clinical phenotype, a retrospective analysis of clinical data for patients with X chromosome translocation was conducted. Karyotype analysis plus endocrine evaluation was utilized for all the patients. Additional semen analysis and Y chromosome microdeletions were assessed in male patients.ResultsX chromosome translocations were detected in ten cases, including seven females and three males. Infantile uterus and no ovaries were detected in case 1 (FSH: 114 IU/L, LH: 30.90 mIU/mL, E2: < 5.00 pg/ml), and the karyotype was confirmed as 46,X,t(X;22)(q25;q11.2) in case 1. Infantile uterus and small ovaries were both visible in two cases (FSH: 34.80 IU/L, LH: 17.06 mIU/mL, E2: 15.37 pg/ml in case 2; FISH: 6.60 IU/L, LH: 1.69 mIU/mL, E2: 23.70 pg/ml in case 3). The karyotype was detected as 46,X,t(X;8)(q13;q11.2) in case 2 and 46,X,der(X)t(X;5)(q21;q31) in case 3. Normal reproductive hormone levels and fertility abilities were found for cases 4, 6 and 7. The karyotype were detected as 46,X,t(X;5)(p22.3;q22) in case 4 and 46,X,der(X)t(X;Y)(p22.3;q11.2) in cases 6 and 7. These patients exhibited unremarkable clinical manifestations but experienced a history of abnormal chromosomal pregnancy. Normal phenotype and a complex reciprocal translocation as 46,X,t(X;14;4)(q24;q22;q33) were observed in case 5 with a history of spontaneous abortions. In the three male patients, multiple semen analyses confirmed the absence of sperm. Y chromosome microdeletion and hormonal analyses were normal. The karyotypes were detected as 46,Y,t(X;8)(q26;q22), 46,Y,t(X;1)(q26;q23), 46,Y,t(X;3)(q26;p24), respectively.ConclusionsOur study provides insights into individuals with X chromosome translocations. The clinical phenotypes are variable and unpredictable due to differences in breakpoints and X chromosome inactivation (XCI) patterns. Our results suggest that physicians should focus on the characteristics of the X chromosome translocations and provide personalized clinical evaluations in genetic counselling.

Identification and functional analysis of a novel de novo missense mutation located in the initiation codon of LAMP2 associated with early onset female Danon disease.

Danon disease is characterized by the failure of lysosomal biogenesis, maturation, and function due to a deficiency of lysosomal membrane structural protein (LAMP2). The current report describes a female patient with a sudden syncope and hypertrophic cardiomyopathy phenotype. We identified the pathogenic mutations in patients by whole-exon sequencing, followed by a series of molecular biology and genetic approaches to identify and functional analysis of the mutations. Suggestive findings by cardiac magnetic resonance (CMR), electrocardiogram (ECG), and laboratory examination suggested Danon disease which was confirmed by genetic testing. The patient carried a novel de novo mutation, LAMP2 c.2T>C located at the initiation codon. The quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis of peripheral blood leukocytes from the patients revealed evidence of LAMP2 haploinsufficiency. Labeling of the new initiation codon predicted by the software with green fluorescent protein followed by fluorescence microscopy and Western blotting showed that the first ATG downstream from the original initiation codon became the new translational initiation codon. The three-dimensional structure of the mutated protein predicted by alphafold2 revealed that it consisted of only six amino acids and failed to form a functional polypeptide or protein. Overexpression of the mutated LAMP2 c.2T>C showed a loss of function of the protein, as assessed by the dual-fluorescence autophagy indicator system. The mutation was confirmed to be null, AR experiments and sequencing results confirmed that 28% of the mutant X chromosome remained active. We propose possible mechanisms of mutations associated with haploinsufficiency of LAMP2: (1) The inactivation X chromosome carrying the mutation was not significantly skewed. However, it decreased in the mRNA level and the expression ratio of the mutant transcripts; (2) The identified mutation is null, and the active mutant transcript fails to translate into the normal LAMP2 proteins. The presence of haploinsufficiency in LAMP2 and the X chromosome inactivation pattern were crucial factors contributing to the early onset of Danon disease in this female patient.

Nonrandom X Chromosome Inactivation Detection

X chromosome inactivation patterns may be clinically useful in assessing tumor clonality, determining carrier status for certain X-linked disorders, and evaluating the pathogenicity of a genetic variant identified in an X-linked gene. The protocols in this article utilize the highly polymorphic trinucleotide repeat within the first exon of the human androgen receptor gene (AR) and the methylation-sensitive restriction enzyme HpaII to distinguish between the maternal and paternal alleles and simultaneously determine their methylation status. The data obtained from these protocols can be used to calculate the ratio of inactivation between the two alleles that ultimately reflects whether a female has a random or nonrandom pattern of X chromosome inactivation. © 2023 Wiley Periodicals LLC. Basic Protocol 1: X chromosome inactivation assay Basic Protocol 2: PCR amplification and labeling of digested and undigested DNA templates.

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.

XCMAX4: A Robust X Chromosomal Genetic Association Test Accounting for Covariates.

Although the X chromosome accounts for about 5% of the human genes, it is routinely excluded from genome-wide association studies probably due to its unique structure and complex biological patterns. While some statistical methods have been proposed for testing the association between X chromosomal markers and diseases, very a few of them can adjust for covariates. Unfortunately, those methods that can incorporate covariates either need to specify an X chromosome inactivation model or require the permutation procedure to compute the p value. In this article, we proposed a novel analytic approach based on logistic regression that allows for covariates and does not need to specify the underlying X chromosome inactivation pattern. Simulation studies showed that our proposed method controls the size well and has robust performance in power across various practical scenarios. We applied the proposed method to analyze Graves’ disease data to show its usefulness in practice.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

Not Just Loss-of-Function Variations: Identification of a Hypermorphic Variant in a Patient With a CDKL5 Missense Substitution.

Background and ObjectivesCDKL5 deficiency disorder (CDD) is a neurodevelopmental encephalopathy characterized by early-onset epilepsy and impaired psychomotor development. Variations in the X-linked CDKL5 gene coding for a kinase cause CDD. Molecular genetics has proved that almost all pathogenic missense substitutions localize in the N-terminal catalytic domain, therefore underlining the importance for brain development and functioning of the kinase activity. CDKL5 also features a long C-terminal domain that acts as negative regulator of the enzymatic activity and modulates its subcellular distribution. CDD is generally attributed to loss-of-function variations, whereas the clinical consequences of increased CDKL5 activity remain uncertain. We have identified a female patient characterized by mild epilepsy and neurologic symptoms, harboring a novel c.2873C>G nucleotide substitution, leading to the missense variant p.(Thr958Arg). To increase our comprehension of genetic variants in CDKL5-associated neurologic disorders, we have characterized the molecular consequences of the identified substitution.MethodsMRI and video EEG telemetry were used to describe brain activity and capture seizure. The Bayley III test was used to evaluate the patient development. Reverse transcriptase PCR was used to analyze whether the identified nucleotide variant affects messenger RNA stability and/or splicing. The X chromosome inactivation pattern was analyzed determining the DNA methylation status of the androgen receptor (AR) gene and by sequencing of expressed alleles. Western blotting was used to investigate whether the novel Thr958Arg substitution affects the stability and/or enzymatic activity of CDKL5. Immunofluorescence was used to define whether CDKL5 subcellular distribution is affected by the Thr958Arg substitution.ResultsOur data suggested that the proband tends toward a skewed X chromosome inactivation pattern in favor of the novel variant. The molecular investigation revealed that the p.(Thr958Arg) substitution leads to a significant increase in the autophosphorylation of both the TEY motif and residue Tyr171 of CDKL5, as well as in the phosphorylation of the target protein MAP1S, indicating an hyperactivation of CDKL5. This occurs without evidently affecting the kinase subcellular distribution.DiscussionOur data provide a strong indication that the c.2873C>G nucleotide substitution represents an hypermorphic pathogenic variation of CDKL5, therefore highlighting the importance of a tight control of CDKL5 activity in the brain.

X Chromosome Inactivation Pattern and Pregnancy Outcome of Female Carriers of Pathogenic Heterozygous X-Linked Deletions.

Prenatal risk assessment of carriers of heterozygous X-linked deletion is a big challenge due to the phenotypic modification induced by X chromosome inactivation (XCI). Herein, we described four Chinese pedigrees with maternal-inherited X-deletions above 1 Mb. The pathogenic evaluation revealed that all X-deletions are harmful to heterozygous carriers; however, the asymptomatic pregnant female carriers in these families tremendously complicate the prognostic assessment of the unborn heterozygous embryos. In this study, we detected the XCI pattern of 11 female carriers of heterozygous X-linked deletions and 4 non-carrier females in these families and performed the first prenatal XCI pattern analysis in a fetal female carrier of heterozygous PCDH19-deletion to make risk prediction. In an adult female who lost one copy of the terminal of X chromosome short arm (Xp), a region enriching a large number of XCI escapees, the expression level of representative XCI escape genes was also detected. Pregnancy outcomes of all families were followed up or retrospected. Our research provides clinical evidence that X-deletions above 1 Mb are indeed associated with extremely skewed XCI. The favorable skewed XCI in combination with potential compensatory upregulation of XCI escapees would protect some but not all female carriers with pathogenic X-deletion from severe clinical consequences, mainly depending on the specific genetic contents involved in the deletion region. For PCDH19-disorder, the XCI pattern is considered as the decisive factor of phenotype expression, of which prenatal XCI assay using uncultured amniocytes could be a practicable way for risk prediction of this disease. These results provide valuable information about the usage of XCI assay in the prenatal risk assessment of heterozygous X-linked deletions.

X Chromosome Inactivation in Carriers of Fabry Disease: Review and Meta-Analysis.

Anderson-Fabry disease is an X-linked inborn error of glycosphingolipid catabolism caused by a deficiency of α-galactosidase A. The incidence ranges between 1: 40,000 and 1:117,000 of live male births. In Italy, an estimate of incidence is available only for the north-western Italy, where it is of approximately 1:4000. Clinical symptoms include angiokeratomas, corneal dystrophy, and neurological, cardiac and kidney involvement. The prevalence of symptomatic female carriers is about 70%, and in some cases, they can exhibit a severe phenotype. Previous studies suggest a correlation between skewed X chromosome inactivation and symptoms in carriers of X-linked disease, including Fabry disease. In this review, we briefly summarize the disease, focusing on the clinical symptoms of carriers and analysis of the studies so far published in regards to X chromosome inactivation pattern, and manifesting Fabry carriers. Out of 151 records identified, only five reported the correlation between the analysis of XCI in leukocytes and the related phenotype in Fabry carriers, in particular evaluating the Mainz Severity Score Index or cardiac involvement. The meta-analysis did not show any correlation between MSSI or cardiac involvement and skewed XCI, likely because the analysis of XCI in leukocytes is not useful for predicting the phenotype in Fabry carriers.

Molecular analysis of low-level mosaicism of the IKBKG mutation using the X Chromosome Inactivation pattern in Incontinentia Pigmenti.

BackgroundIncontinentia pigmenti (IP) is a rare X‐linked disorder affecting the skin and other ectodermal tissues that is caused by mutation of the IKBKG/NEMO gene. Previous studies have reported that the overall mutation detection rate in IP is ~75%. We hypothesized that a low‐level mosaicism existed in the remaining cases.MethodsGenomic variations in the IKBKG gene were examined in 30 IP probands and their family members. Standard mutational analyses were performed to detect common deletions, nucleotide alterations, and copy number variations. To assess skewing of the X chromosome inactivation (XCI) pattern, a HUMARA assay was performed. We compared the results of this analysis with phenotype severity.ResultsPathogenic variants were identified in 20 probands (66.7%), the rate of detection was suboptimal. The remaining 10 probands tended to manifest a mild phenotype with no skewed X chromosome inactivation that is generally observed in IP patients. Quantitative nested PCR and digital droplet PCR were performed for the 10 patients and mosaicism of the common IKBKG deletion were identified in five patients.ConclusionOverall, we detected 25 IKBKG mutations (83.3%). Determination of the XCI value in advance of mutational analyses for IP could improve the mutation detection rate. Our improved detection rate for these mutations, particularly those with a low‐level mosaicism, may present opportunities for appropriate genetic counseling.

Cancer-associated mutations in normal human endometrium: Surprise or expected?

The human endometrium is an essential component in human reproduction that has the unique characteristic of undergoing cyclic regeneration during each menstrual cycle. Vigorous regeneration after shedding may be sustained by stem/progenitor cells, for which molecular markers have not been fully identified. Although clonality analysis using X chromosome inactivation patterns has shown that normal human endometrial glands are composed of a monoclonal cell population, whether clonal expansion is derived from stem/progenitor cells remains unclear. Remarkable advances in next‐generation sequencing technology over the past decade have enabled somatic mutations to be detected in not only cancers, but also normal solid tissues. Unexpectedly frequent cancer‐associated mutations have been detected in a variety of normal tissues, and recent studies have clarified the mutational landscape of normal human endometrium. In epithelial glandular cells, representative cancer‐associated mutations are frequently observed in an age‐dependent manner, presumably leading to growth advantage. However, the extremely high mutation loads attributed to DNA mismatch repair deficiency and POLE mutations, as well as structural and copy number alterations, are specific to endometrial cancer, not to normal epithelial cells. The malignant conversion of normal epithelial cells requires these additional genetic hits, which are presumably accumulated during aging, and may therefore be a rare life event. These discoveries could be expected to shed light on the physiology and pathogenesis of the human endometrium and urge caution against the application of genetic screening for the early detection of endometrial cancer.

PS1498 INCREASED RHOA ACTIVITY DUE TO A DISRUPTED FILAMIN A/ALPHAIIBBETA3 INTERACTION INDUCES MACROTHROMBOCYTOPENIA

Please indicate where the abstract has been published before: BloodBackground:Filamin A (FLNa) is a protein associated to the cytoskeleton, which plays a major role as a mediator between the cell membrane and the cytoskeleton, and it is involved in several processes like cell motility and signal transduction. Heterozygous mutations in the X‐linked FLNA gene are associated with a spectrum of diseases called filaminopathies, which includes macrothrombocytopenia: the affected individuals display two populations of differently sized platelets. One of the possible explanations is the differential expression of the WT or mutated FLNA allele, accordingly to the X chromosome inactivation pattern. As a consequence, any patient sample is a mixture of healthy and mutated cells.Aims:We sought to investigate the molecular basis of this unusual phenotype, using a human‐relevant model able to overcome the limitations described for the murine models. Furthermore, we tried to better understand the role of FLNa in human megakaryopoiesis, which is currently poorly understood.Methods:We developed an isogenic pluripotent stem cell (iPSC) model, via reprogramming of CD34+ cells from the peripheral blood of two female patients, harboring two intragenic deletions. As iPSCs are clonal models, it is possible to segregate cells expressing the wild‐type and the mutated alleles, while preserving the same genomic background.Results:After the in vitro hematopoietic differentiation of the iPS cell lines, we observed the complete absence of the FLNa protein in the mutated megakaryocytes (MKs), which leads to their incomplete maturation, and particularly the severe inability to produce proplatelets. We also observed defective actomyosin contractility, due to the small GTPase RhoA inappropriate activation. This anomaly was surprisingly and specifically associated with fibrinogen, while other extracellular matrices were not triggering this abnormal cytoskeleton remodeling. Further investigation proved a role for the FLNa/a IIb b 3 interaction in the down‐regulation of RhoA, using a collection of gene‐edited iPSC lines, overexpressing a mutant FLNa deleted of the αIIbβ3‐binding domain Ig21, as opposed to deletions of the GPIba‐interacting, the RhoA‐interacting, and the actin‐interacting domains, respectively. Finally, we have been able to show that the chemical inhibition of the RhoA‐associated kinase ROCK1/2 restored a correct proplatelet formation.Summary/Conclusion:Overall, this work suggests a novel physiopathological mechanism underlying the macrothrombocytopenia, associating the increased RhoA activity to the disruption of the integrin αIIbβ3/FLNa axis. This observation could be extended to other inherited macrothrombocytopenias and introduce a new rational basis for pharmacological treatment of these diseases.image

Investigation of the clonal origin of multifocal papillary thyroid carcinoma according to the X-chromosome inactivation pattern.

Patients with papillary thyroid carcinoma (PTC) usually have multiple tumors, or foci. It remains unclear if these foci originate from independent tumors or a single tumor mass. The present study included 89 female patients with bilateral PTC who had been treated with a total thyroidectomy. An X-chromosome inactivation assay was used to examine the clonal origin of the tumors according to the status of the X-linked human androgen receptor gene. Of the 89 patients, 5 were informative. The X-chromosome inactivation pattern was the same in multiple foci in 3 cases, indicating a monoclonal origin of the tumors. In 1 case, the X chromosome inactivation pattern was different between the tumors. Mixed patterns were observed in 1 case. The results of the present study suggest that in certain cases of multifocal PTC, tumors arise independently, whereas in other cases, separate foci are the outcome of intra-thyroid spread by a single tumor mass.

The power of the Mediator complex-Expanding the genetic architecture and phenotypic spectrum of MED12-related disorders.

MED12 is a member of the large Mediator complex that controls cell growth, development, and differentiation. Mutations in MED12 disrupt neuronal gene expression and lead to at least three distinct X-linked intellectual disability syndromes (FG, Lujan-Fryns, and Ohdo). Here, we describe six families with missense variants in MED12 (p.(Arg815Gln), p.(Val954Gly), p.(Glu1091Lys), p.(Arg1295Cys), p.(Pro1371Ser), and p.(Arg1148His), the latter being first reported in affected females) associated with a continuum of symptoms rather than distinct syndromes. The variants expanded the genetic architecture and phenotypic spectrum of MED12-related disorders. New clinical symptoms included brachycephaly, anteverted nares, bulbous nasal tip, prognathism, deep set eyes, and single palmar crease. We showed that MED12 variants, initially implicated in X-linked recessive disorders in males, may predict a potential risk for phenotypic expression in females, with no correlation of the X chromosome inactivation pattern in blood cells. Molecular modeling (Yasara Structure) performed to model the functional effects of the variants strongly supported the pathogenic character of the variants examined. We showed that molecular modeling is a useful method for in silico testing of the potential functional effects of MED12 variants and thus can be a valuable addition to the interpretation of the clinical and genetic findings.

INCREMENTO EN MUTACIONES DE TIPO TRANSICIONAL PARA ATENUAR EL EFECTO DE SESGO EN LA INACTIVACIÓN DEL CROMOSOMA X CONDUCEN A LA APARICIÓN DE MUTACIONES CON EFECTO PATOLÓGICO EN EL GEN DE LA IDURONATO2 SULFATASA(IDS)(3.1.6.13)

Varios autores han registrado la importancia de la metilación del gen IDS en el proceso de inactivación del cromosoma X. Adicionalmente, algunos pacientes con mucopolisacaridosis muestran un patrón de sesgo en la inactivación del cromosoma X y su relación con una disminución en la actividad de la iduronatosintasa 2. Nuestro estudio registra un patrón consistente de mutaciones de tipo transicional en este gen que podrían estar relacionadas con la génesis de mutaciones de tipo patológico, con el propósito de atenuar los efectos de una inactivación no aleatoria del cromosoma X. Se considera este hallazgo de particular importancia, ya que apoyaría la propuesta de un tamizaje de sitios de metilación en el cromosoma X en mujeres en edad fértil. Este tamizaje podría ayudar a disminuir las frecuencias alélicas de variantes relacionadas con un número variado de enfermedades asociadas al cromosoma X, como son: MPS tipo II, síndrome de Lesch-Nyhan y la distrofia muscular tipo Duchenne.

Two patients with MIRAGE syndrome lacking haematological features: role of somatic second-site reversion SAMD9 mutations

BackgroundMyelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (MIRAGE) syndrome is a recently described congenital disorder caused by heterozygous SAMD9 mutations. The phenotypic spectrum of the syndrome...

Xq28 duplication including MECP2 in six unreported affected females: what can we learn for diagnosis and genetic counselling?

Duplication of the Xq28 region, involving MECP2 (dupMECP2), has been primarily described in males with severe developmental delay, spasticity, epilepsy, stereotyped movements and recurrent infections. Carrier mothers are usually asymptomatic with an extremely skewed X chromosome inactivation (XCI) pattern. We report a series of six novel symptomatic females carrying a de novo interstitial dupMECP2, and review the 14 symptomatic females reported to date, with the aim to further delineate their phenotype and give clues for genetic counselling. One patient was adopted and among the other 19 patients, seven (37%) had inherited their duplication from their mother, including three mildly (XCI: 70/30, 63/37, 100/0 in blood and random in saliva), one moderately (XCI: random) and three severely (XCI: uninformative and 88/12) affected patients. After combining our data with data from the literature, we could not show a correlation between XCI in the blood or duplication size and the severity of the phenotype, or explain the presence of a phenotype in these females. These findings confirm that an abnormal phenotype, even severe, can be a rare event in females born to asymptomatic carrier mothers, making genetic counselling difficult in couples at risk in terms of prognosis, in particular in prenatal cases.

Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore, we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally, differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC, more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. Not applicable. Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen, grant 1502512N), Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel, on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest.

Differential X Chromosome Inactivation Patterns during the Propagation of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) represent a potentially useful tool for studying the molecular mechanisms of disease thanks to their ability to generate patient-specific hiPSC clones. However, previous studies have reported that DNA methylation profiles, including those for imprinted genes, may change during passaging of hiPSCs. This is particularly problematic for hiPSC models of X-linked disease, because unstable X chromosome inactivation status may affect the detection of phenotypes. In the present study, we examined the epigenetic status of hiPSCs derived from patients with Rett syndrome, an X-linked disease, during long-term culture. To analyze X chromosome inactivation, we used a methylation-specific polymerase chain reaction (MSP) to assay the human androgen receptor locus (HUMARA). We found that single cell-derived hiPSC clones exhibit various states of X chromosome inactivation immediately after clonal isolation, even when established simultaneously from a single donor. X chromosome inactivation states remain variable in hiPSC clones at early passages, and this variability may affect cellular phenotypes characteristic of X-linked diseases. Careful evaluation of X chromosome inactivation in hiPSC clones, particularly in early passages, by methods such as HUMARA-MSP, is therefore important when using patient-specific hiPSCs to model X-linked disease.

Haemophilia A and cardiovascular morbidity in a female SHAM syndrome carrier due to skewed X chromosome inactivation

We have recently described a severe haemophilia A and moyamoya (SHAM) syndrome caused by Xq28 deletions encompassing F8 and the BRCC3 familial moyamoya gene. The phenotype includes haemophilia A, moyamoya angiopathy, dysmorphia and hypertension. The genetic analysis of the family of our SHAM patient demonstrated carrier state in proband's mother and sister.The patient's mother is apparently well, whereas his currently 18-years-old sister presents with mild haemophilia A, coarctation of the aorta, hypertension, and ventricular arrhythmia. We performed X chromosome inactivation assay based on HpaII methylation analysis of a polymorphic short tandem repeat (STR) in the X linked AR (androgen receptor) gene and used quantitative real-time RT PCR to measure the expression of genes from the deleted region in proband's family members. We found an extremely skewed X chromosome inactivation pattern in the female members of the family leading to preferential inactivation of the X chromosome without Xq28 deletion in patient's sister. We demonstrated differential expression of the genes from the deleted region in four members of the family, that tightly correlates with the clinical features.In conclusion, we show that the haematologic and cardiovascular morbidity and the discrepancy between patient's sister and mother despite the same genetic lesion are due to skewed X chromosome inactivation leading to clinically relevant differential expression of SHAM syndrome genes. This report highlights the role for BRCC3 in cardiovascular physiology and disease, and demonstrates that in some complex hereditary syndromes full diagnostics may require the examination of both genetic and epigenetic events.