The proportion of cells with functional X disomy is associated with the severity of mental retardation in mosaic ring X Turner syndrome females

Turner syndrome females (45,X) do not have mental retardation (MR), whereas some mosaic ring X Turner syndrome females, with 45,X/46,X,r(X), have severe MR. The MR is believed to be caused by a failure of X chromosome inactivation (XCI) of the small ring X chromosome, which leads to functional X disomy (FXD), To explore this hypothesis, we examined the proportion of FXD cells in the peripheral blood of four ring X Turner syndrome females with various levels of MR, using two newly developed XCI assays based on DNA methylation of X-linked genes. As a result, the two patients with extremely severe MR showed complete FXD patterns, whereas the remaining two patients with relatively milder MR showed partial FXD patterns. These results indicate that the proportion of FXD cells may be associated with the severity of MR in mosaic ring X Turner syndrome females, although this association should be confirmed by examining brain cells during development. One of the cases with severe MR and a complete FXD pattern neither lacked the XIST gene nor had uniparental X isodisomy, and we discuss the mechanism of the failure of XCI in this case.     

Low-copy-number human transgene is recognized as an X inactivation center in mouse ES cells, but fails to induce cis-inactivation in chimeric mice

X chromosome inactivation is initiated from a segment of the mammalian X chromosome called the X inactivation center. Transgenes from this region of the murine X chromosome are providing the means to identify the DNA needed for cis inactivation in mice. We recently showed that chimeric mice carrying transgenes from the human X inactivation center (XIC) region also provide a functional assay for human XIC activity; approximately 6 copies of a 480-kb human transgene (ES-10) were sufficient to initiate random X inactivation in cells of male chimeric mice (Migeon et al., 1999, Genomics, 59, 113-121). Now, we report studies of another human transgene (ES-5), which contains less than 300 kb of the human XIC region on Xq13.2 including an intact XIST locus and which has inserted in one or two copies into mouse chromosome 6. The ES-5 transgene is recognized as an X inactivation center in mouse embryonic stem cells, but is not sufficient to induce random X inactivation in somatic cells of highly chimeric mice. Human transgenes in chimeric mice provide a means to uncouple the key steps in this complex pathway and facilitate the search for essential components of the human XIC region.     

Studies of X inactivation and isodisomy in twins provide further evidence that the X chromosome is not involved in Rett syndrome.

Rett syndrome (RS), a progressive encephalopathy with onset in infancy, has been attributed to an X-linked mutation, mainly on the basis of its occurrence almost exclusively in females and its concordance in female MZ twins. The underlying mechanisms proposed are an X-linked dominant mutation with male lethality, uniparental disomy of the X chromosome, and/or some disturbance in the process of X inactivation leading to unequal distributions of cells expressing maternal or paternal alleles (referred to as a "nonrandom" or "skewed" pattern of X inactivation). To determine if the X chromosome is in fact involved in RS, we studied a group of affected females including three pairs of MZ twins, two concordant for RS and one uniquely discordant for RS. Analysis of X-inactivation patterns confirms the frequent nonrandom X inactivation previously observed in MZ twins but indicates that this is independent of RS. Analysis of 29 RS females reveals not one instance of uniparental X disomy, extending the observations previously reported. Therefore, our findings contribute no support for the hypothesis that RS is an X-linked disorder. Furthermore, the concordant phenotype in most MZ female twins with RS, which has not been observed in female twins with known X-linked mutations, argues against an X mutation.     

Small marker X chromosomes lack the X inactivation center: implications for karyotype/phenotype correlations.

The abnormal phenotype and/or mental retardation seen in persons with small marker X (mar(X)) chromosomes has been hypothesized to be due to the loss of the X inactivation center (XIC) at Xq13.2, resulting in two active copies of genes in the pericentromeric region. In order to define precisely the DNA content of mar(X) chromosomes and to correlate phenotype with karyotype, we studied small mar(X) chromosomes, using FISH with probes in the juxtacentromeric region. One of the probes was a 40-kb genomic cosmid for the XIST gene, which maps to the smallest interval known to contain the XIC and is thought to be involved in X inactivation. Our findings reveal that small mar(X) chromosomes do not include the XIC and therefore cannot be subject to X inactivation, supporting the premise that abnormal dosage of expressed genes in the pericentromeric region of the X generates the aberrant phenotype seen in patients with small mar(X) chromosomes.     

Seven ring (X) chromosomes lacking the XIST locus, six with an unexpectedly mild phenotype

Small ring (X) chromosomes lacking the XIST gene at Xq13.2 have been associated with a severe phenotype that includes mental retardation, facial dysmorphism and congenital abnormalities. It has been hypothesised that the loss of XIST results in functional disomy for the sequences contained in the ring. We studied 47 females with a 45,X/46,r(X) karyotype and found seven to have an XIST-negative ring. Only one of the seven patients had the severe phenotype. The remaining six patients had physical phenotypes consistent with Turner syndrome. The rings were characterised cytogenetically and molecularly. The severe phenotype in one patient can be explained by the absence of XIST expression, the relatively large amount of Xp material in the ring and, possibly, the concomitant maternal uniparental isodisomy. We propose three explanations for the unexpectedly mild phenotypes in the remaining six patients; (1) the rings contained limited amounts of X-chromosome material, and sequences that, when functionally disomic, result in a severe phenotype were absent; (2) mosaicism resulting in the absence of the ring from tissues, such as the brain, which are important in the severe phenotype and (3) the presence of an inactive X in some tissues at some time, exemplified by the demonstration of XIST expression in one patient.     

X inactivation Xplained

Random inactivation of one of the two female X chromosomes establishes dosage compensation between XY males and XX females in placental mammals. X inactivation is controlled by the X inactivation center (Xic). Recent advances in genome sequencing show that the Xic has evolved from an ancestral vertebrate gene cluster in placental mammals and has undergone separate rearrangements in marsupials. The Xic ensures that all but one X chromosome per diploid genome are inactivated. Which chromosome remains active is randomly chosen. Pairing of Xic loci on the two X chromosomes and alternate states of the X chromosomes before inactivation have recently been implicated in the mechanism of random choice. Chromosome-wide silencing is then initiated by the noncoding Xist RNA, which evolved with the mammalian Xic and covers the inactive X chromosome.     

Origin and evolution of X chromosome inactivation

Evolution of the mammalian sex chromosomes heavily impacts on the expression of X-encoded genes, both in marsupials and placental mammals. The loss of genes from the Y chromosome forced a two-fold upregulation of dose sensitive X-linked homologues. As a corollary, female cells would experience a lethal dose of X-linked genes, if this upregulation was not counteracted by evolution of X chromosome inactivation (XCI) that allows for only one active X chromosome per diploid genome. Marsupials rely on imprinted XCI, which inactivates always the paternally inherited X chromosome. In placental mammals, random XCI (rXCI) is the predominant form, inactivating either the maternal or paternal X. In this review, we discuss recent new insights in the regulation of XCI. Based on these findings, we propose an X inactivation center (Xic), composed of a cis-Xic and trans-Xic that encompass all elements and factors acting to control rXCI either in cis or in trans. We also highlight that XCI may have evolved from a very small nucleation site on the X chromosome in the vicinity of the Sox3 gene. Finally, we discuss the possible evolutionary road maps that resulted in imprinted XCI and rXCI as observed in present day mammals.     

Fragile X expression and X inactivation

Summary The major concept of fragile X pathogenesis postulates that the fragile site at band Xq27.3 [fra(X)] represents the primary defect. The expression of fra(X) is predicted to be an intrinsic property of the mutated chromosome and, hence, should not be suppressed by X inactivation in females or induced by X-linked trans-acting factors. We made fibroblast clones of a fra(X)-positive female. Monoclonality was demonstrated using the DNA methylation assay at DXS255. The mutated X chromosomes and their states of genetic activity in the different clones were also defined by molecular methods. Five clones were selected to induce expression of fra(X) by 10^-7 M FUdR; two carried an active mutated X chromosome, in the other three the mutated X chromosome was inactivated. Fra(X) was found expressed in both types of clones. The percentages of positive cells were as high as 7–10%, regardless of the genetic activity of the mutated X chromosomes. DNA replicating patterns, obtained by BUdR labelling, demonstrated that expression occurred only on the mutated X chromosomes previously identified by molecular methods. The concept that the fragile site represents the primary mutation is now strongly supported by experimental evidence. The expression of fra (X) in females is independent of X inactivation and other trans-acting factors.     

Sex chromosome elimination,X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae)

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

Abstract. In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara . It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara . Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara. It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara. Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Skewed inactivation of an X chromosome deleted at the dystrophin gene in an asymptomatic mother and her affected daughter

A girl with severe Becker muscular dystrophy and apparently normal chromosomes had a heterozygous deletion for exons 51, 52, and 53 of the dystrophin gene. This deletion was transmitted by her mother, who was unaffected. To differentiate the normal and the deleted X chromosomes, fluorescence in situ hybridization (FISH) was applied to metaphase chromosomes, using probes for both exons 51 and 52, which are only 388 and 113 base pairs long, respectively. FISH signals were observed in one or both chromatids of one chromosome, but never on both chromosomes, suggesting the lack of hybridization on the deleted X chromosome. Using 5-bromodeoxyuridine incorporation to differentiate the late (inactive) and the early replicating (active) X chromosomes, 77% of the signals were observed on the active X chromosomes in the mother. This percentage was only 18% in the daughter, suggesting that skewed inactivation of the X chromosomes was responsible for the phenotypic differences.     

Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas

In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin.     

X inactivation in a mammal species with three sex chromosomes

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.     

Localization of a gene that escapes inactivation to the X chromosome proximal short arm: implications for X inactivation.

The process of mammalian X chromosome inactivation results in the inactivation of most, but not all, genes along one or the other of the two X chromosomes in females. On the human X chromosome, several genes have been described that "escape" inactivation and continue to be expressed from both homologues. All such previously mapped genes are located in the distal third of the short arm of the X chromosome, giving rise to the hypothesis of a region of the chromosome that remains noninactivated during development. The A1S9T gene, an X-linked locus that complements a mouse temperature-sensitive defect in DNA synthesis, escapes inactivation and has now been localized, in human-mouse somatic cell hybrids, to the proximal short arm, in Xp11.1 to Xp11.3. Thus, A1S9T lies in a region of the chromosome that is separate from the other genes known to escape inactivation and is located between other genes known to be subject to X inactivation. This finding both rules out models based on a single chromosomal region that escapes inactivation and suggests that X inactivation proceeds by a mechanism that allows considerable autonomy between different genes or regions on the chromosome.