X inactivation as a source of behavioural differences in monozygotic female twins.

Although members of monozygotic twin pairs are identical in genome sequence, they may differ in patterns of gene expression. One early and irreversible process affecting gene expression, which can create differences within pairs of female monozygotic twins, is X inactivation - one twin can express mainly paternally-received genes on the X chromosome while the other twin expresses mainly maternally-received genes. It follows that non-identical X chromosome expression may cause female monozygotic twins to correlate less strongly than male monozygotic twins on complex behavioural traits affected by X-linked loci. We tested this hypothesis using data from around 4000 same-sex twin pairs on 9 social, behavioural and cognitive measures at ages 2, 3 and 4. Consistent with our hypothesis, monozygotic males were generally more similar than monozygotic females. Three of four significant differences were in traits showing higher correlations in males than females, and these traits - prosocial behaviour, peer problems, and verbal ability - have all been proposed previously in the literature as being influenced by genes on the X chromosome. Interestingly, dizygotic twins showed the reverse pattern of correlations for similar variables, which is also consistent with the X inactivation hypothesis; taken together, then, our monozygotic and dizygotic results suggest the presence of quantitative trait loci on the X chromosome.     

ICF syndrome cells as a model system for studying X chromosome inactivation

Mutations in the DNMT3B DNA methyltransferase gene cause the ICF immunodeficiency syndrome. The targets of this DNA methyltransferase are CpG-rich heterochromatic regions, including pericentromeric satellites and the inactive X chromosome. The abnormal hypomethylation in ICF cells provides an important model system for determining the relationships between replication time, CpG island methylation, chromatin structure, and gene silencing in X chromosome inactivation.     

Different patterns of X inactivation in MZ twins discordant for red-green color-vision deficiency.

Two female identical twins who were clinically normal were obligatory heterozygotes for X-linked deuteranomaly associated with a green-red fusion gene derived from their deuteranomalous father. On anomaloscopy, one of the twins was phenotypically deuteranomalous while the other had normal color vision. The color vision-defective twin had two sons with normal color vision and one deuteranomalous son. X-inactivation analysis was done with the highly informative probe M27 beta. This probe detects a locus (DXS255) which contains a VNTR and which is somewhat differentially methylated on the active and inactive X chromosomes. In skin cells of the color vision-defective twin, almost all paternal X chromosomes with the abnormal color-vision genes were active, thereby explaining her color-vision defect. In contrast, a different pattern was observed in skin cells from the woman with normal color vision; her maternal X chromosome was mostly active. However, in blood lymphocytes, both twins showed identical patterns with mixtures of inactivated maternal and paternal X chromosomes. Deuteranomaly in one of the twins is explained by extremely skewed X inactivation, as shown in skin cells. Failure to find this skewed pattern in blood cells is explained by the sharing of fetal circulation and exchange of hematopoietic precursor cells between twins. These data give evidence for X inactivation of the color-vision locus and add another MZ twin pair with markedly different X-inactivation patterns for X-linked traits.     

Expression of an X-linked muscular dystrophy in a female due to translocation involving Xp21 and non-random inactivation of the normal X chromosome

Summary A young female was diagnosed as having X-linked muscular dystrophy of the Duchenne type. Chromosome studies, including trypsin-Giemsa banding, Quinacrine fluorescence, and nucleolus organizer region (NOR) silver staining revealed an X-autosome reciprocal translocation t(X;21) (p21;p12). Utilizing both [³H] thymidine autoradiography and the BrdU-Hoechst 33258-Giemsa technique, lymphocytes and fibroblasts were found to show a preferential inactivation of the normal X suggesting the presence of a single mutant gene on the translocated X. This patient is one of seven reported cases of an X-linked muscular dystrophy associated with an X-autosome translocation. In all seven cases the exchange point in the X chromosome is in band p21 at or near the site of the Duchenne gene.     

X chromosome inactivation in female embryos of a marsupial mouse (Antechinus stuartii)

Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme -galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.     

X inactivation testing for identifying a non-syndromic X-linked mental retardation gene

The purpose of this study was to identify a gene causing non-syndromic X-linked mental retardation in an extended family, taking advantage of the X chromosome inactivation status of the females in order to determine their carrier state. X inactivation in the females was determined with the androgen receptor methylation assay; thereafter, the X chromosome was screened with evenly spaced polymorphic markers. Once initial linkage was identified, the region of interest was saturated with additional markers and the males were added to the analysis. Candidate genes were sequenced. Ten females showed skewed inactivation, while six revealed a normal inactivation pattern. A maximal lod score of 5.54 at θ?=?0.00 was obtained with the marker DXS10151. Recombination events mapped the disease gene to a 17.4-Mb interval between the markers DXS10153 and DXS10157. Three candidate genes in the region were sequenced and a previously described missense mutation (P375L) was identified in the ACSL4/FACL4 gene. On the basis of the female X inactivation status, we have mapped and identified the causative mutation in a gene causing non-syndromic X-linked mental retardation.     

X chromosome inactivation pattern in elderly women over 70 years of age

In the present study we have analyzed X chromosome inactivation patterns in 40 women aged from 74 to 85 years (mean age 78 years). The control group was 36 women (mean age 30 years). The most common AR-assay was used to determine X-inactivation patterns (the study of methylation patterns of HpaII site in human androgen receptor gene (HUMARA) by quantative PCR). The age dependence of X-inactivation was not observed. We have detected skewed X-inactivation in three women among 40 (7.5%) elderly women comparing to two women among 36 (5.5%) women from control group. The difference was not found to be statistically significant. We made a suggestion that higher incidence of skewed X-inactivation in elderly women revealed by previous studies could occur due to some experimental ambiguities as heterogeneity of the group studied; inclusion of women having relatives with genetic abnormalities associated with skewed X-inactivation patterns; the difference of X chromosome inactivation skewing determination. We conclude that present study does not show X chromosome inactivation to be age dependent.     

Genomic and epigenomic approaches to the study of X chromosome inactivation

X chromosome inactivation represents a compelling example of chromosome-wide, long-range epigenetic gene-silencing in mammals. The cis- and trans-acting factors that establish and maintain the patterns and levels of gene expression from the active and inactive X chromosomes remain incompletely understood; however, the availability of the complete genomic sequence of the human X chromosome, together with complementary approaches that explore the computational biology, epigenetic modifications and gene expression-profiling along the chromosome, suggests that the features of the X chromosome that are responsible for its unique forms of gene regulation are increasingly amenable to experimental analysis.     

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.     

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.     

Evidence for the inactivation of an X chromosome early in the development of the human female.

Studies of somatic tissues and cultured cells, including fibroblast clones, from human embryos heterozygous for the electrophoretic variants of glucose-6-phosphate dehydrogenase confirm that one X chromosome is inactivated very early in embryonic development and indicate that X inactivation has occurred in the majority of cells from a variety of tissues at least by 5 weeks from conception.     

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.     

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.     

An X-linked human collagen transgene escapes X inactivation in a subset of cells.

Transgenic mice carrying one complete copy of the human alpha 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle's translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.     

Gene trap as a tool for genome annotation and analysis of X chromosome inactivation in human embryonic stem cells

Human embryonic stem (ES) cells were suggested to be an important tool in transplantation medicine. However, they also play a major role in human genetics. Using the gene trap strategy, we have created a bank of clones with insertion mutations in human ES cells. These insertions occurred within known, predicted and unknown genes, and thus assist us in annotating the genes in the human genome. The insertions into the genome occurred in multiple chromosomes with a preference to larger chromosomes. Utilizing a clone where the integration occurred in the X chromosome, we have studied X-chromosome inactivation in human cells. We thus show that in undifferentiated female human ES cells both X chromosomes remain active and upon differentiation one chromosome undergoes inactivation. In the differentiated embryonic cells the inactivation is random, while in the extra-embryonic cells it is non-random. In addition, using a selection methodology, we demonstrate that in a minority of the cells partial inactivation and XIST expression occur even in the undifferentiated cells. We suggest that X chromosome inactivation during human embryogenesis, which coincides with differentiation, may be separated from the differentiation process. The genetic manipulation of human ES cells now opens new ways of analyzing chromosome status and gene expression in humans.