Transmission-ratio distortion of X chromosomes among male offspring of females with skewed X-inactivation

We have begun a search for heritable variation in X-chromosome inactivation pattern in normal females to determine whether there is a genetic effect on the imprinting of X-chromosome inactivation in humans. We have performed a quantitative analysis of X-chromosome inactivation in lymphocytes from mothers in normal, three-generation families. Eight mothers and 12 grandmothers exhibited evidence of highly skewed patterns of X-chromosome inactivation. We observed that the male offspring of females with skewed X-inactivation patterns were three times more likely to inherit alleles at loci that were located on the inactive X chromosome (Xi) than the active X chromosome (Xa). The region of the X chromosome for which this phenomenon was observed extends from XP11 to -Xq22. We have also examined X-chromosome inactivation patterns in 21 unaffected mothers of male bilateral sporadic retinoblastoma patients. Six of these mothers had skewed patterns of X-chromosome inactivation. In contrast to the tendency for male offspring of skewed mothers from nondisease families to inherit alleles from the inactive X chromosome, five of the six affected males inherited the androgen receptor alleles from the active X chromosome of their mother. © 1995 Wiley-Liss, Inc.     

X chromosome-inactivation patterns of 1,005 phenotypically unaffected females

X-chromosome inactivation is widely believed to be random in early female development and to result in a mosaic distribution of cells, approximately half with the paternally derived X chromosome inactive and half with the maternally derived X chromosome inactive. Significant departures from such a random pattern are hallmarks of a variety of clinical states, including being carriers for severe X-linked diseases or X-chromosome cytogenetic abnormalities. To evaluate the significance of skewed patterns of X inactivation, we examined patterns of X inactivation in a population of >1,000 phenotypically unaffected females. The data demonstrate that only a very small proportion of unaffected females show significantly skewed inactivation, especially during the neonatal period. By comparison with this data set, the degree of skewed inactivation in a given individual can now be quantified and evaluated for its potential clinical significance.     

Determination of X-chromosome inactivation status using X-linked expressed polymorphisms identified by database searching

The large number of redundant sequences available in nucleotide databases provides a resource for the identification of polymorphisms. Expressed polymorphisms in X-linked genes can be used to determine the inactivation status of the genes, and polymorphisms in genes that are subject to inactivation can then be used as tools to examine X-chromosome inactivation status in heterozygous females. In this study, we have identified six new X-linked single-nucleotide polymorphisms and determined the inactivation status of these genes by examination of expression patterns in female cells previously demonstrated to have skewed inactivation, as well as by analysis of somatic cell hybrids retaining the inactive human X chromosome. Expression was seen from both alleles in females heterozygous for the RPS4X gene, confirming the previously reported expression from the inactive X chromosome. Expression of only a single allele was seen in females heterozygous for polymorphisms in the BGN, TM4SF2, ATP6S1, VBP1, and PDHA1 genes, suggesting that these genes are subject to X-chromosome inactivation.     

Heritability of X chromosome--inactivation phenotype in a large family.

One of the two X chromosomes in each somatic cell of normal human females becomes inactivated very early in embryonic development. Although the inactivation of an X chromosome in any particular somatic cell of the embryonic lineage is thought to be a stochastic and epigenetic event, a strong genetic influence on this process has been described in the mouse. We have attempted to uncover evidence for genetic control of X-chromosome inactivation in the human by examining X chromosome-inactivation patterns in 255 females from 36 three-generation pedigrees, to determine whether this quantitative character exhibits evidence of heritability. We have found one family in which all seven daughters of one male and the mother of this male have highly skewed patterns of X-chromosome inactivation, suggesting strongly that this quantitative character is controlled by one or more X-linked genes in some families.     

A new mutation in exon 7 of NEMO gene: late skewed X-chromosome inactivation in an incontinentia pigmenti female patient with immunodeficiency

Incontinentia pigmenti is an X-linked genodermatosis, lethal in males. Affected females survive because of X-chromosome dizygosity and negative selection of cells carrying the mutant X-chromosome, and for this reason the skewed X inactivation pattern is often used to confirm the diagnosis. The most frequent mutation is a deletion of part of the NEMO gene (NEMOΔ4–10), although other mutations have been reported. Mutations of NEMO which do not abolish NF-κB activity totally permit male survival, causing an allelic variant of IP called hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). We present a non-classical IP female patient who also suffered transient immunodeficiency because of a late and progressive selection against peripheral blood cells carrying an active mutated X-chromosome. This finding suggests that in the absence of known mutation the X-inactivation studies used in genetic counselling can induce mistakes with some female patients. At the age of 3 years and 6 months, all immunodeficiency signs disappeared, and the X-chromosome inactivation pattern was completely skewed. The low T cell proliferation and CD40L expression corroborate the important role of NEMO/ NF-κB pathway in T cell homeostasis. The decreased NEMO protein amount and the impaired IkBα degradation suggest that this new mutation, NM_003639: c.1049dupA, causes RNA or protein instability. To our knowledge, this is the first time that selection against the mutated X-chromosome in X-linked disease has been documented in vivo.     

Extremely skewed X-chromosome inactivation is increased in pre-eclampsia

Pre-eclampsia is a disorder that affects approximately 5% of pregnancies. We tested the hypothesis that skewed X-chromosome inactivation (XCI) could be involved in the pathogenesis of pre-eclampsia. Peripheral blood DNA was obtained from 67 pre-eclampsia patients and 130 control women. Androgen receptor (AR) was analyzed by the HpaII/polymerase chain reaction assay to assess XCI patterns in DNA extracted from peripheral-blood cells. In addition, buccal cells were obtained from seven patients, and the analysis repeated. Extremely skewed XCI was observed in 10 of 46 informative patients (21.74%), and in 2 of 86 informative controls (2.33%, P = 0.0005; χ² test). Our findings support a role for the X-chromosome in the pathogenesis of pre-eclampsia in a subgroup of patients. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A longitudinal twin study of skewed X chromosome-inactivation

X-chromosome inactivation (XCI) is a pivotal epigenetic mechanism involved in the dosage compensation of X-linked genes between males and females. In any given cell, the process of XCI in early female development is thought to be random across alleles and clonally maintained once established. Recent studies, however, suggest that XCI might not always be random and that skewed inactivation may become more prevalent with age. The factors influencing such XCI skewing and its changes over time are largely unknown. To elucidate the influence of stochastic, heritable and environmental factors in longitudinal changes in XCI, we examined X inactivation profiles in a sample of monozygotic (MZ) (n = 23) and dizygotic (DZ) (n = 22) female twin-pairs at ages 5 and 10 years. Compared to MZ twins who were highly concordant for allelic XCI ratios, DZ twins showed much lower levels of concordance. Whilst XCI patterns were moderately stable between ages 5 and 10 years, there was some drift over time with an increased prevalence of more extreme XCI skewing at age 10. To our knowledge, this study represents the earliest longitudinal assessment of skewed XCI patterns, and suggests that skewed XCI may already be established in early childhood. Our data also suggest a link between MZ twinning and the establishment of allelic XCI ratios, and demonstrate that acquired skewing in XCI after establishment is primarily mediated by stochastic mechanisms. These data have implications for our understanding about sex differences in complex disease, and the potential causes of phenotypic discordance between MZ female twins.     

Skewed X-chromosome inactivation is associated with trisomy in women ascertained on the basis of recurrent spontaneous abortion or chromosomally abnormal pregnancies

An increase in extremely skewed X-chromosome inactivation (XCI) (> or = 90%) among women who experienced recurrent spontaneous abortion (RSA) has been previously reported. To further delineate the etiology of this association, we have evaluated XCI status in 207 women who experience RSA. A significant excess of trisomic losses was observed among the women who had RSA with skewed XCI versus those without skewed XCI (P=.02). There was also a significant excess of boys among live births in this group (P=.04), which is contrary to expectations if the cause of skewed XCI was only that these women carried X-linked lethal mutations. To confirm the association between skewed XCI and the risk of trisomy, an independent group of 53 women, ascertained on the basis of a prenatal diagnosis of trisomy mosaicism, were investigated. Only cases for which the trisomy was shown to be of maternal meiotic origin were included. The results show a significantly higher level of extreme skewing (> or = 90%) in women whose pregnancies involved placental trisomy mosaicism (17%) than in either of two separate control populations (n=102 and 99) (P=.02 compared with total control subjects). An additional 11 cases were ascertained on the basis of one or more trisomic-pregnancy losses. When all women in the present study with a trisomic pregnancy (n=103) were considered together, skewed XCI was identified in 18%, as compared with 7% in all controls (n=201) (P=.005). This difference was more pronounced when a cutoff of extreme skewing of 95% was used (10% vs. 1.5% skewed; P=.002). Maternal age was not associated with skewing in either the patient or control populations and therefore cannot account for the association with trisomy. Previous studies have shown that a reduced ovarian reserve is associated with increased risk of trisomic pregnancies. We hypothesize that the association between skewed XCI and trisomic pregnancies is produced by a common mechanism that underlies both and that involves a reduction of the size of the follicular pool.     

Sex chromosome homology and incomplete, tissue-specific X-inactivation suggest that monotremes represent an intermediate stage of mammalian sex chromosome evolution

Female mammals have two X chromosomes and males have a single X and a smaller, male-determining Y chromosome. The dosage of X-linked gene products is equalized between the sexes by the genetic inactivation of one X chromosome in females. The characteristics of the mechanism of X-chromosome inactivation differ in eutherian and metatherian mammals, and it has been suggested that the metatherian system represents a more primitive stage. The present study of monotreme sex chromosomes and X-chromosome inactivation suggests that the prototherian mammals may represent an even more primitive stage. There is extensive G-band homology between the monotreme X and Y chromosomes, and differences in the patterns of replication of the two X chromosomes in females suggest that X inactivation is tissue specific and confined to the unpaired segment of the X. On the basis of these results, we propose a model for the differentiation of mammalian sex chromosomes and the evolution of the mechanism of X-chromosome inactivation. This model involves a gradual reduction of the Y chromosome and an accompanying gradual recruitment of (newly unpaired) X-linked loci under the control of a single inactivation center.     

Skewed X-chromosome inactivation in human embryos with mosaic trisomy 16

The sex ratio and X-chromosome inactivation were analyzed in placental tissues of human spontaneous abortuses with pure and mosaic forms of chromosome 16 trisomy. The sex ratio value was found to decrease with an increase in the share of cells with the trisomic karyotype, which suggests differential survival of embryos belonging to different sexes. The pattern of X-chromosome inactivation in cells of extraembryonic mesoderm in the control group of embryos and in spontaneous abortuses with the level of trisomy 16 below 80% corresponded to random X-inactivation, whereas in most embryos with a frequency of trisomy 16 exceeding 80% skewed inactivation was observed. Our results support the hypothesis about the existence of an autosomal transfactor influencing the initiation of X-chromosome inactivation and suggest its possible localization on chromosome 16.     

Clonality of tuberous sclerosis harmatomas shown by non-random X-chromosome inactivation

Tuberous sclerosis (TSC) is an autosomal dominant condition characterised by tumour-like malformations (hamartomas) in the brain and other organs. A proportion of hamartomas from patients with TSC show loss of heterozygosity (LOH) for DNA markers in the region of either the TSCI gene on chromosome 9834 or the TSC2 gene on 16p13.3. This implies that these lesions are clonal. We have studied X-chromosome inactivation, as a marker of clonality, in 13 hamartomas from females with TSC. The hamartomas comprised five renal angiomyolipomas, three fibromas and seven other lesions. In previous studies, four of the lesions showed LOH. A polymerase chain reaction assay was used to analyse differential methylation of anHpalI restriction site adjacent to the androgen-receptor triplet-repeat polymorphism on Xg11-12. In 12 of the lesions, there was a skewed inactivation pattern with one X chromosome being fully methylated and the other unmethylated. Normal tissue showed a random pattern of inactivation. These data confirm that most TSC hamartomas are clonal in origin. This is an intriguing finding, since these lesions are composed of more than one cell type.     

Analysis of X-chromosome inactivation in X-linked immunodeficiency with hyper-IgM (HIGM1): evidence for involvement of different hematopoietic cell lineages

Summary The pattern of X-chromosome inactivation was analyzed, by means of two different DNA probes (pSPT-PGK and M27), in several cell lineages derived from females belonging to a pedigree with X-linked immunodeficiency with hyper-IgM (HIGM1). Non-random X-chromosome inactivation was demonstrated in T cells, B cells, and neutrophils, but not in fibroblasts, of obligate carriers, suggesting that different hematopoietic cell lineages are primarily involved in HIGM1. Preferential inactivation of the paternally derived X-chromosome was demonstrated by analysis of segregation of the alleles defined by the pSPT-PGK and M27 probes. The possibility that the HIGM1 mutation may confer a proliferative and/or differential advantage to hematopoietic precursors carrying the mutated allele on the active X-chromosome is discussed.     

Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders

Some deleterious X-linked mutations may result in a growth disadvantage for those cells in which the mutation, when on the active X chromosome, affects cell proliferation or viability. To explore the relationship between skewed X-chromosome inactivation and X-linked mental retardation (XLMR) disorders, we used the androgen receptor X-inactivation assay to determine X-inactivation patterns in 155 female subjects from 24 families segregating 20 distinct XLMR disorders. Among XLMR carriers, ~50% demonstrate markedly skewed X inactivation (i.e., patterns 80:20), compared with only ~10% of female control subjects (P<.001). Thus, skewed X inactivation is a relatively common feature of XLMR disorders. Of the 20 distinct XLMR disorders, 4 demonstrate a strong association with skewed X inactivation, since all carriers of these mutations demonstrate X-inactivation patterns 80:20. The XLMR mutations are present on the preferentially inactive X chromosome in all 20 informative female subjects from these families, indicating that skewing is due to selection against those cells in which the XLMR mutation is on the active X chromosome.     

Meiotic sex chromosome inactivation in the marsupial Monodelphis domestica

In eutherian mammals, the X and Y chromosomes undergo meiotic sex chromosome inactivation (MSCI) during spermatogenesis in males. However, following fertilization, both the paternally (Xp) and maternally (Xm) inherited X chromosomes are active in the inner cell mass of the female blastocyst, and then random inactivation of one X chromosome occurs in each cell, leading to a mosaic pattern of X-chromosome activity in adult female tissues. In contrast, marsupial females show a nonrandom pattern of X chromosome activity, with repression of the Xp in all somatic tissues. Here, we show that MSCI also occurs during spermatogenesis in marsupials in a manner similar to, but more stable than that in eutherians. These findings support the suggestion that MSCI may have provided the basis for an early dosage compensation mechanism in mammals based solely on gametogenic events, and that random X-chromosome inactivation during embryogenesis may have evolved subsequently in eutherian mammals.     

X-chromosome inactivation in the Wiskott-Aldrich syndrome: a marker for detection of the carrier state and identification of cell lineages expressing the gene defect

Recently developed techniques for the direct analysis of DNA have made possible the determination of patterns of cellular X-chromosome inactivation. These techniques provide a potential method for carrier detection for several X-linked human disorders in which obligate carriers show nonrandom X inactivation. By using restriction fragment length polymorphic (RFLP) gene-specific probes in conjunction with methylation-sensitive enzymes, we have characterized the patterns of X-chromosome inactivation in cell subsets from females belonging to 10 kindreds segregating for the X-linked immune deficiency disorder Wiskott-Aldrich syndrome (WAS). We show that selective inactivation of the X chromosome distinguishes obligate WAS carriers from noncarrier females and constitutes a valuable marker of the WAS carrier state. Selective inactivation phenomena were observed in the monocytes and T and B lymphocytes of obligate carriers, implying that the WAS gene defect is expressed in each of these cellular lineages. In conjunction with the use of linked DNA markers, RFLP-methylation analysis should render carrier detection feasible for the majority of females from WAS families. The results of such analyses also provide an initial step toward identifying the cellular level and molecular basis for WAS.     

Skewed X-Chromosome Inactivation Is Common in Fetuses or Newborns Associated with Confined Placental Mosaicism

The inactivation of one X chromosome in females is normally random with regard to which X is inactivated. However, exclusive or almost-exclusive inactivation of one X may be observed in association with some X-autosomal rearrangements, mutations of the XIST gene, certain X-linked diseases, and MZ twinning. In the present study, a methylation difference near a polymorphism in the X-linked androgen-receptor gene was used to investigate the possibility that nonrandom X inactivation is increases in fetuses and newborns that are associated with confined placental mosaicism (CPM) involving an autosomal trisomy. Extreme skewing was observed in 7 (58%) of 12 cases with a meiotic origin of the trisomy, but in none of 10 cases examined with a somatic origin of the trisomy, and in only 1 (4%) of 27 control adult females. In addition, an extremely skewed X-inactivation pattern was observed in 3 of 10 informative cases of female uniparental disomy (UPD) of chromosome 15. This may reflect the fact that a proportion of UPD cases arise by "rescue" of a chromosomally abnormal conceptus and are therefore associated with CPM. A skewed pattern of X inactivation in CPM cases is hypothesized to result from a reduction in the size of the early-embryonic cell pool, because of either poor early growth or subsequent selection against the trisomic cells. Since approximately 2% of pregnancies detected by chorionic villus sampling are associated with CPM, this is likely a significant contributor to both skewed X inactivation observed in the newborn population and the expression of recessive X-linked diseases in females.