Nonrandom X-chromosome Inactivation Research Articles

Clinical features and genetic analysis of 17 Chinese pedigrees affected with X-linked intellectual disability

To analyze the clinical features and genetic etiology of 17 Chinese pedigrees affected with X-linked intellectual disability (XLID). Seventeen pedigrees affected with unexplained intellectual disability which had presented at Henan Provincial People's Hospital from May 2021 to May 2023 were selected as the study subjects. Clinical data of the probands and their pedigree members were collected. Trio-whole exome sequencing (Trio-WES), Sanger sequencing and X chromosome inactivation (XCI) analysis were carried out. Pathogenicity of candidate variants was predicted based on the guidelines from the American College of Medical Genetics and Genomics and co-segregation analysis. The 17 probands, including 9 males and 8 females with an age ranging from 0.6 to 8 years old, had all shown mental retardation and developmental delay. Fourteen variants were detected by genetic testing, which included 4 pathogenic variants (MECP2: c.502C>T, MECP2: c.916C>T/c.806delG, IQSEC2: c.1417G>T), 4 likely pathogenic variants (MECP2: c.1157_1197del/c.925C>T, KDM5C: c.2128A>T, SLC6A8: c.1631C>T) and 6 variants of uncertain significance (KLHL15: c.26G>C, PAK3: c.970A>G/c.1520G>A, GRIA3: c.2153C>G, TAF1: c.2233T>G, HUWE1: c.10301T>A). The PAK3: c.970A>G, GRIA3: c.2153C>G and TAF1: c.2233T>G variants were considered as the genetic etiology for pedigrees 12, 14 and 15 by co-segregation analysis, respectively. The proband of pedigree 13 was found to have non-random XCI (81:19). Therefore, the PAK3: c.1520G>A variant may underlie its pathogenesis. Trio-WES has attained genetic diagnosis for the 17 XLID pedigrees. Sanger sequencing and XCI assay can provide auxiliary tests for the diagnosis of XLID.

A female patient carrying a novel DMD mutation with non-random X-chromosome inactivation from a DMD family

ObjectiveTo analyze the clinical phenotype and genetic characteristics of a female proband carrying a novel mutation in the DMD gene with non-random X-chromosome inactivation in a large pedigree with pseudohypertrophic muscular dystrophy.MethodsClinical information of the female proband, her monozygotic twin sister, and other family members were collected. Potential pathogenic variants were detected with Multiplex Ligation-dependent Probe Amplification (MLPA) and whole-exome sequencing (WES). Methylation-sensitive restriction enzyme (HhaI) was employed for X-chromosome inactivation analysis.ResultsThe proband was a female over 5 years old, displayed clinical manifestations such as elevated creatine kinase (CK) levels and mild calf muscle hypertrophy. Her monozygotic twin sister exhibited normal CK levels and motor ability. Her uncle and cousin had a history of DMD. WES revealed that the proband carried a novel variant in the DMD (OMIM: 300,377) gene: NM_004006.3: c.3051_3053dup; NP_003997.2: p.Tyr1018*. In this pedigree, five out of six female members were carriers of this variant, while the cousin and uncle were hemizygous for this variant. X-chromosome inactivation analysis suggested non-random inactivation in the proband.ConclusionThe c.3051_3053dup (p.Tyr1018*) variant in the DMD gene is considered to be the pathogenic variant significantly associated with the clinical phenotype of the proband, her cousin, and her uncle within this family. Integrating genetic testing with clinical phenotype assessment can be a valuable tool for physicians in the diagnosis of progressive muscular dystrophies, such as Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD).

Clinical case of Wieacker–Wolff syndrome in a 5-year girl

Wieacker–Wolff syndrome (WWS) (OMIM 314580, 301041) is rare, slowly progressive, X-linked hereditary disorder. It is characterized by fetal akinesia, which results in congenital multiplex arthrogryposis, spasticity, and development delay. WWS is caused by the point mutations or extended deletions in the ZC4H2 gene, located on the long arm of the X chromosome (Xq11.2). Currently, about 100 cases have been described.
 We present the case of WWS 5-year girl. DNA diagnostic was performed using full exome sequencing and confirmed by Sanger sequencing. Determination of non-random X-chromosome inactivation was performed by methyl-sensitive PCR of GAAA-repeat RP2 gene. 
 The main clinical symptoms in our case are stiffness of large and small joints, specific facial phenotype, spasticity and lack of independent walking. We revealed heterozygous mutation с.22_23delAT (p.Met8fs) in ZC4H2 gene. Non-random inactivation of the X chromosome was detected (XCI = 96.1%).
 Conclusions. Clinical symptoms of the disease, the nature of the detected mutation and the literature data indicate to the presence of an X-linked dominant pattern of inheritance of WWS in our patient. We described the case referred to the group of ZC4H2-associated rare disorders.

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.

Novel insights into PORCN mutations, associated phenotypes and pathophysiological aspects

BackgroundGoltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants.ResultsWe report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes.ConclusionsOur combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS.

A female carrier of a novel DMD mutation with slightly skewed X-chromosome inactivation shows a suspected case of Becker muscular dystrophy in a Chinese family

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are both caused by mutations in DMD gene effecting the expression of dystrophin. Generally female carriers are asymptomatic; however, it has been suggested that carriers may exhibit symptoms. We investigated a 6-year-old Chinese girl exhibiting a suspected BMD phenotype, including persistently elevated creatine kinase and creatine kinase isoenzyme levels. The proband harbored a novel heterozygous mutation, c.3458_3459insAA, within exon 26 of the DMD gene inherited from her mother who had a completely normal phenotype and presented with mosaicism in her lymphocytes with 45, X [17%]/46, XX [83%]. In addition, X-chromosome inactivation (XCI) patterns in the peripheral blood of the child were slightly skewed: proband with 62% (mutant allele)/38% (normal allele) when compared with her mother with 32/68%. Amplification of regions of the cDNA revealed different ratios for the expression of these alleles: proband with 50/50% and her mother with 20/80%. Real-time PCR showed that mRNA expression was significantly decreased in both. We proposed that a frameshift or nonsense mutation may contribute to the development of symptoms in carriers. These phenotypes correlate with nonrandom XCI patterns and are compounded by the locus of the mutation. For incompletely skewed XCI patterns, although the mutant allele could suppress the expression of a normal allele, carriers would remain asymptomatic as long as there was adequate compensation from the normal allele. We also proposed a mechanism where mRNA from the mutant allele may be unstable and easily degraded, allowing for phenotypic compensation by the wildtype allele.

Genotype-Phenotype Correlation of G6PD Mutations among Central Thai Children with G6PD Deficiency.

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked inherited erythroenzymopathy in Thailand. The clinical and hematological manifestations of G6PD deficiency are variable. Objective This study aimed to characterize the genotype-phenotype correlation of G6PD mutations in Thai pediatric patients who were followed-up in Phramongkutklao Hospital, a tertiary center in central Thailand. Material and Method. A total of 102 children including 73 males (71.6%) and 29 females (28.4%) were included in our study. Mutation analysis was performed by direct DNA sequencing of all coding exons of the G6PD gene. Ninety-one patients (89.2%) were presented with neonatal hyperbilirubinemia and 11 patients (10.8%) were presented with acute hemolytic anemia beyond the neonatal period. Results Molecular analysis of the G6PD gene in 102 G6PD-deficient Thai children identified 12 different mutations. G6PD Viangchan (871G > A) and G6PD Canton (1376G > T) were the first (46.2%) and the second (15.4%) most common identified mutations among both male and female G6PD-deficient individuals, respectively. All affected males were hemizygous for G6PD mutations and had an average G6PD level of 16.7 ± 11.5 (3–76) IU/ml.RBC. Majority of female patients (27 in 29, 93.1%) were heterozygous for G6PD mutations and had an average G6PD level of 133.6 ± 43.4 (9–195) IU/ml.RBC. Two female patients (6.9%) were either homozygous or compound heterozygous for the mutations and had G6PD level in the affected male range (35 and 10 IU/ml.RBC). Only 1 in 27 heterozygous females (3.7%) had G6PD level in the affected male range (9 IU/ml.RBC) which is possibly explained by nonrandom X-chromosome inactivation. The correlation of genotypes, G6PD levels, and clinical phenotypes was not demonstrated in our study in which all of the included G6PD-deficient patients were presented with neonatal hyperbilirubinemia and acute hemolytic anemia, since the genotype-phenotype correlation is normally demonstrated in chronic nonspherocytic hemolytic anemia (CNSHA) G6PD-deficient individuals. Conclusion This study characterizes the molecular heterogeneity of G6PD variants causing G6PD deficiency in Thai children. Our study demonstrated the efficiency of direct DNA sequencing which can identify 12 missense mutations in Thai children.

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a XistΔ:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

A finite mixture model for X-chromosome association with an emphasis on microbiome data analysis.

Analysis of the X chromosome has been largely neglected in genetic studies mainly because of complex underlying biological mechanisms. On the other hand, the study of human microbiome data (typically over-dispersed counts with an excess of zeros) has generated great interest recently because of advancements in next-generation sequencing technologies. We propose a novel approach to infer the association between host genetic variants in the X-chromosome and microbiome data. The method accounts for random X-chromosome inactivation (XCI), skewed (or nonrandom) XCI (XCI-S), and escape of XCI (XCI-E). The inference is performed through a finite mixture model (FMM), in which an indicator variable denoting the "true" biological mechanism is treated as missing data. An expectation-maximization algorithm on zero-inflated and two-part models is implemented to estimate genetic effects. We investigate the performance of the FMM along with strategies that assume XCI and XCI-E mechanisms for all subjects compared with alternative approaches. Briefly, an XCI mechanism codes males' genotypes as homozygous females, whereas under XCI-E, males are treated as heterozygous females. By comprehensive simulations, we evaluate tests of the hypothesis under a computationally efficient score statistic. In summary, the FMM renders reduced bias and commensurate power compared to XCI, XCI-E, and alternative strategies while maintaining adequate Type 1 error control. The proposed method has far-reaching applications. In particular, we illustrate its usage on a large-scale human microbiome study, the Genetic, Environmental and Microbial (GEM) project, to test for the genetic association on the X chromosome.

CD40LG duplication-associated autoimmune disease is silenced by nonrandom X-chromosome inactivation

CD40LG duplication-associated autoimmune disease is silenced by nonrandom X-chromosome inactivation

Allele-specific non-CG DNA methylation marks domains of active chromatin in female mouse brain

DNA methylation at gene promoters in a CG context is associated with transcriptional repression, including at genes silenced on the inactive X chromosome in females. Non-CG methylation (mCH) is a distinct feature of the neuronal epigenome that is differentially distributed between males and females on the X chromosome. However, little is known about differences in mCH on the active (Xa) and inactive (Xi) X chromosomes because stochastic X-chromosome inactivation (XCI) confounds allele-specific epigenomic profiling. We used whole-genome bisulfite sequencing in a mouse model with nonrandom XCI to examine allele-specific DNA methylation in frontal cortex. Xi was largely devoid of mCH, whereas Xa contained abundant mCH similar to the male X chromosome and the autosomes. In contrast to the repressive association of DNA methylation at CG dinucleotides (mCG), mCH accumulates on Xi in domains with transcriptional activity, including the bodies of most genes that escape XCI and at the X-inactivation center, validating this epigenetic mark as a signature of transcriptional activity. Escape genes showing CH hypermethylation were the only genes with CG-hypomethylated promoters on Xi, a well-known mark of active transcription. Finally, we found extensive allele-specific mCH and mCG at autosomal imprinted regions, some with a negative correlation between methylation in the two contexts, further supporting their distinct functions. Our findings show that neuronal mCH functions independently of mCG and is a highly dynamic epigenomic correlate of allele-specific gene regulation.

The dynamic changes of X chromosome inactivation during early culture of human embryonic stem cells

X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in human female cells. Several previous reports have described the promiscuous XCI status in long-term cultured female human embryonic stem cells (hESCs), and the majority of them exhibit non-random XCI. However, when and how such female hESCs acquire the aberrant XCI states during culture is unknown. Herein, through comparing the XCI states in 18 paired hES cell lines throughout early culture, we revealed a uniform dynamic change during this culture period under a widely used culture condition. The female initial hESCs (ihESCs, P4-P9) expressed XIST RNA, H3K27me3 punctate enrichment and displayed random XCI pattern. By further culturing, the female early hESCs (ehESCs, P20–P30) lost the expression of XIST RNA, H3K27me3 punctate enrichment and exhibited a completely skewed XCI pattern. Importantly, a subset of X-linked genes was up-regulated in ehESCs, including some cancer-related genes. At last, we found 5% physiological oxygen was beneficial for the expression of XIST and H3K27me3 punctate enrichment, but not for the XCI pattern. We conclude that the XCI dynamic change is a frequent epigenetic instability event during early culture, which is accompanied by the up-regulation of some X-linked genes. Furthermore, we emphasize that physiological oxygen is beneficial for XCI fidelity.

Cervical Carcinomas With Neuroendocrine Differentiation: A Report of 28 Cases With Immunohistochemical Analysis and Molecular Genetic Evidence of Common Clonal Origin With Coexisting Squamous and Adenocarcinomas.

Cervical neuroendocrine carcinomas are rare, aggressive tumors and their immunohistochemical features and clonal relationship to coexisting tumors are incompletely described. Twenty-eight cases were identified (17 small cell, 9 large cell, and 2 mixed), 10 of which had an invasive squamous or adenocarcinoma component. Staining for synaptophysin, chromogranin A, TTF1, c-kit, CD44, and p16 was performed. Analyses for loss of heterozygosity (LOH) at 5 polymorphic microsatellite markers (D3S1300, D9S171, D11S914, D13S319, and TP53) and X-chromosome inactivation were performed. Of 17 cases with available blocks, 13 (76%) were synaptophysin+, 8 (47%) were chromogranin A+, 8 (47%) were TTF1+, 7 (41%) were c-kit+, and 6 (35%) were CD44+. Strong patchy or strong diffuse p16 staining was seen in all cases. LOH and X-chromosome inactivation analysis were performed for 17 cases, 8 of which had a coexisting squamous or adenocarcinoma component. Five of the 8 (63%) cases with 2 components showed allelic loss in both components. All 5 of these cases demonstrated identical LOH between the neuroendocrine and squamous or adenocarcinoma components. Nonrandom X-chromosome inactivation was seen in the neuroendocrine and other components in 4 of the 8 cases. In all 4 cases the pattern of inactivation was identical between the 2 components. Cervical neuroendocrine carcinomas have features similar to other extrapulmonary neuroendocrine carcinomas, including expression of TTF1, c-kit, and CD44. Consistent staining for p16 is also seen. Concordant genetic alterations support common clonal origin for neuroendocrine carcinomas with a coexisting squamous or adenocarcinoma component.

Clonality assessment of adenomatoid tumor supports its neoplastic nature

Adenomatoid tumor is a relatively rare disease that predominantly involves male and female internal genital tracts. Although its clinical and pathologic features are well characterized, there is still controversy regarding its nature as a true neoplasm or a variant of mesothelial hyperplasia of a reactive nature. We sought to resolve this debate by investigating the clonality of uterine adenomatoid tumor from 13 female cases. The mesothelial cells and surrounding normal myometrium were precisely harvested using laser capture microdissection, and genomic DNA was extracted for clonal analysis by assessing the patterns of X-chromosome inactivation. Fluorescent polymerase chain reaction amplification of a highly polymorphic short tandem repeat of the human androgen receptor (HUMARA) gene with and without methylation-sensitive restriction endonuclease HpaII digestion was performed on DNA extracted from mesothelial cells, using normal myometrium and male blood sample as controls. Of the 13 cases successfully amplified, all 10 informative cases showed concordant nonrandom X-chromosome inactivation pattern consistent with monoclonality. In comparison, surrounding normal myometrium showed a polyclonal pattern of X-chromosome inactivation, and male blood sample failed to be amplified after HpaII treatment. Our results demonstrate that adenomatoid tumor is a monoclonal disease favoring a neoplastic process. This neoplastic rather than reactive nature probably accounts for its frequently observed infiltrative growth pattern and the occurrence of diffuse adenomatoid tumor, especially when host immunity is compromised. Additional studies with larger sample sizes will be needed to conclusively prove our conclusion.

Diagnostic and Treatment Options for Severe IBD in Female X-CGD Carriers with Non-random X-inactivation

X-linked chronic granulomatous disease [X-CGD] due to hemizygous mutations in CYBB is characterised by invasive bacterial and fungal infections and granulomatous inflammation. Inflammatory bowel disease [IBD] is an additional or isolated manifestation. Allogeneic haematopoietic stem cell transplantation [alloHSCT] is the standard curative treatment. X-CGD carriers are usually healthy but those with non-random X-chromosome inactivation [XCI] may develop infectious or autoinflammatory manifestations. We report on two female patients with severe treatment-refractory Crohn-like IBD manifesting at age 23 and 8 years, respectively. NADPH-oxidase activity testing and molecular genetics proved X-CGD carrier status with non-random XCI. As in CGD, histopathology from colonic biopsies disclosed pigment-laden macrophages and reduced CD68(+) macrophages. Following submyelo-ablative conditioning, the younger patient was treated with alloHSCT at age 20 years. She came into remission within 3 months after transplantation and shows complete mucosal healing after 16 months off all medications. We suggest that children and young adults with refractory IBD should mandatorily be tested for CGD. AlloHSCT should be considered as curative therapy in severely diseased female carriers of X-CGD with non-random XCI.

Woman With X-Linked Recessive Dystonia-Parkinsonism

Despite recessive inheritance, X-linked dystonia-parkinsonism (Lubag disease) has also been described in women presenting with a late-onset isolated parkinsonian syndrome. Interestingly, unlike in other populations, there is a slight female predominance in the prevalence of parkinsonism in the Philippines. In a Filipino woman with suspected Parkinson disease, we confirmed the presence of all changes specific for X-linked dystonia-parkinsonism in genomic DNA. Subsequently, we analyzed complementary DNA and evaluated the methylation status of the androgen receptor gene. Owing to extremely skewed (98%:2%) X-chromosome inactivation, the patient expressed almost solely the mutated allele in a disease-specific change, rendering her molecularly comparable with a hemizygously affected man. Skewed X-chromosome inactivation is the likely cause of parkinsonism in this heterozygous mutation carrier. Because women carriers of the genetic changes specific for X-linked dystonia-parkinsonism are common in the Philippines, the epigenetic factor of nonrandom X-chromosome inactivation may contribute to the skewing of the sex prevalence of parkinsonism toward women in this country, warranting further investigation.

X-chromosome genetic association test accounting for X-inactivation, skewed X-inactivation, and escape from X-inactivation.

X-chromosome inactivation (XCI) is the process in which one of the two copies of theX-chromosomeinfemalesis randomly inactivated to achieve the dosage compensation of X-linked genes between males and females. That is, 50% of the cells have one allele inactive and the other 50% of the cells have the other allele inactive. However, studies have shown that skewed or nonrandom XCI is a biological plausibility wherein more than 75% of cells have the same allele inactive. Also, some of the X-chromosome genes escape XCI, i.e., both alleles are active in all cells. Current statistical tests for X-chromosome association studies can either account for random XCI (e.g., Clayton's approach) or escape from XCI (e.g., PLINK software). Because the true XCI process is unknown and differs across different regions on the X-chromosome, we proposed a unified approach of maximizing likelihood ratio over all biological possibilities: random XCI, skewed XCI, and escape from XCI. A permutation-based procedure was developed to assess the significance of the approach. We conducted simulation studies to compare the performance of the proposed approach with Clayton's approach and PLINK regression. The results showed that the proposed approach has higher powers in the scenarios where XCI is skewed while losing some power in scenarios where XCI is random or XCI is escaped, with well-controlled type I errors. We also applied the approach to the X-chromosomal genetic association study of head and neck cancer.

Combined De-Novo Mutation and Non-Random X-Chromosome Inactivation Causing Wiskott-Aldrich Syndrome in a Female with Thrombocytopenia

Disorders linked to mutations in the X chromosomes typically affect males. The aim of the study is to decipher the mechanism of disease expression in a female patient with a heterozygous mutation on the X-chromosome. Clinical data was extracted from the Canadian Inherited Marrow Failure Registry. Genomic ribonucleic acid (DNA) and complementary DNA (cDNA) underwent Sanger sequencing. Protein analysis was performed by flow cytometry. X-inactivation patterns were analyzed by evaluating the DNA methylation status and cDNA clonal expression of several genes on the X-chromosome. SNP array was used for molecular karyotyping of the X-chromosome. A female with thrombocytopenia, eczema and mild T-lymphocyte abnormalities with extensive negative diagnostic testing, was suspected to have Wiskott-Aldrich syndrome (WAS)/X-linked thrombocytopenia. Although the girl had a mutation (c.397G > A, p.E133K) in only one allele, she was found to have an extremely skewed X-inactivation pattern and no expression of the WAS protein. Family studies using DNA methylation analysis and cDNA clonal expression of several genes on the X-chromosome demonstrated that the patient developed de-novo non-random inactivation of the X-chromosome that does not carry the mutation. Genome-wide high-density molecular karyotyping excluded deletions and amplifications as a cause for the non-random inactivation of one X-chromosome. Our study emphasizes the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of a family history.

Relationships between age and epi-genotype of the FMR1 exon 1/intron 1 boundary are consistent with non-random X-chromosome inactivation in FM individuals, with the selection for the unmethylated state being most significant between birth and puberty

Methylation of the fragile X-related epigenetic element 2 (FREE2) located on the exon 1/intron 1 boundary of the FMR1 gene is related to FMRP expression and cognitive impairment in full mutation (FM; CGG>200) individuals. We examined the relationship between age, the size of the FMR1 CGG expansion and the methylation output ratio (MOR) at 12 CpG sites proximal to the exon 1/intron 1 boundary using FREE2 MALDI-TOF MS. The patient cohort included 119 males and 368 females, i.e. 121 healthy controls (CGG<40), 176 premutation (CGG 55-170) and 190 FM (CGG 213-2000). For all CpG units examined, FM males showed a significantly elevated MOR compared with that in hypermethylated FM females. In FM males the MOR for most CpG units significantly positively correlated with both age and CGG size (P< 0.05). In FM females the skewing towards the unmethylated state was significant for half of the units between birth and puberty (P < 0.05). The methylation status of intron 1 CpG10-12 that was most significantly related to cognitive impairment in our earlier study, did not change significantly with age in FM females. These results challenge the concept of fragile X syndrome (FXS)-related methylation being static over time, and suggest that due to the preference for the unmethylated state in FM females, X-inactivation at this locus is not random. The findings also highlight that the prognostic value of FXS methylation testing is not uniform between all CpG sites, and thus may need to be evaluated on a site-by-site basis.

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.