Skewed X inactivation in healthy individuals and in different diseases

In female mammalian cells, one of the two X chromosomes is inactivated in early embryonic life. Females are mosaics for two cell populations, one with the maternal and one with the paternal X as the active chromosome. Skewed X inactivation is arbitrarily defined, often as a pattern where 80% or more of the cells show a preferential inactivation of one X chromosome. Inactivation is presumed to be permanent for all descendants of a cell; however, after about 55 years of age, the frequency of skewed X inactivation in peripheral blood cells increases, probably through selection. Unfavourable skewing of X inactivation, where the X chromosome carrying a mutant allele is the predominantly active X, has been found in affected female carriers of several X-linked disorders; however, for many X-linked disorders, a consistent relationship between the pattern of X inactivation and clinical phenotype has been difficult to demonstrate. One reason for this may be that peripheral blood cells are not a representative or relevant tissue in many disorders. In some severe X-linked disorders, post-inactivation selection takes place against the X chromosome carrying the mutant allele, leading to a completely skewed X-inactivation pattern. Skewed X inactivation has also been reported in young females with breast cancer, and may indicate an effect of X-linked genes on the development of this condition.Conclusion: The process of X inactivation and the resultant degree of skewing is clearly important for the expression of genetic diseases. It is also important to consider, however, that under normal conditions the frequency of skewed X inactivation increases with age in peripheral blood cells. Analysis of the expression of a large proportion of the genes on the X chromosome has revealed that X-chromosome inactivation is more heterogeneous than previously thought.     

Disease manifestations and X inactivation in heterozygous females with Fabry disease

Aim: Fabry disease is an X-linked lysosomal storage disorder characterized by an accumulation of neutral glycosphingolipids in multiple organ systems caused by α-galactosidase A deficiency due to mutations in the GLA gene. The majority of heterozygous females show the characteristic signs and symptoms of the disease, and some of them are severely affected. The current hypothesis for the occurrence of disease manifestations in females is skewed X inactivation favouring the mutant GLA allele.
Method: We analyzed the patterns of X inactivation in the leukocytes of 28 biochemically and genetically characterized symptomatic Fabry disease heterozygotes and their correlation with clinical and biochemical disease expression.
Results: X inactivation patterns in symptomatic females who are heterozygous for Fabry disease did not differ from those of female controls of the same age ( p = 0.669). Thirteen (46%) of the 28 females with Fabry disease showed random X inactivation, ten (36%) moderate skewing, and five (18%) highly skewed X inactivation. Segregation analysis was performed in the families of six females who had highly or moderately skewed X inactivation. In four of these females, skewing favoured the wild-type GLA allele and in the other two skewing favoured the mutant allele. Patterns of X inactivation or the extent of skewing were not related to the severity of clinical manifestations or to residual enzyme activity.
Conclusion: In this study we provide evidence that heterozygous females with Fabry disease show random X inactivation. Our data do not support the hypothesis that the occurrence and severity of disease manifestations in the majority of Fabry heterozygotes are related to skewed X inactivation.     

Disease manifestations and X inactivation in heterozygous females with Fabry disease

AIM: Fabry disease is an X-linked lysosomal storage disorder characterized by an accumulation of neutral glycosphingolipids in multiple organ systems caused by alpha-galactosidase A deficiency due to mutations in the GLA gene. The majority of heterozygous females show the characteristic signs and symptoms of the disease, and some of them are severely affected. The current hypothesis for the occurrence of disease manifestations in females is skewed X inactivation favouring the mutant GLA allele. METHOD: We analyzed the patterns of X inactivation in the leukocytes of 28 biochemically and genetically characterized symptomatic Fabry disease heterozygotes and their correlation with clinical and biochemical disease expression. RESULTS: X inactivation patterns in symptomatic females who are heterozygous for Fabry disease did not differ from those of female controls of the same age (p = 0.669). Thirteen (46%) of the 28 females with Fabry disease showed random X inactivation, ten (36%) moderate skewing, and five (18%) highly skewed X inactivation. Segregation analysis was performed in the families of six females who had highly or moderately skewed X inactivation. In four of these females, skewing favoured the wild-type GLA allele and in the other two skewing favoured the mutant allele. Patterns of X inactivation or the extent of skewing were not related to the severity of clinical manifestations or to residual enzyme activity. CONCLUSION: In this study we provide evidence that heterozygous females with Fabry disease show random X inactivation. Our data do not support the hypothesis that the occurrence and severity of disease manifestations in the majority of Fabry heterozygotes are related to skewed X inactivation.     

X-chromosome inactivation: role in skin disease expression

The occurrence of X inactivation in mammals has the consequence that all women are functional mosaics. In X-linked skin disorders, Lyonization usually gives rise to a mosaic pattern, as manifest by the appearance of the lines of Blaschko. This arrangement of lesions is observed in male-lethal X-linked traits, such as incontinentia pigmenti, focal dermal hypoplasia, Conradi-Hünermann-Happle syndrome, oral-facial-digital syndrome type 1 and MIDAS (microphthalmia, dermal aplasia and sclerocornea) syndrome, as well as in various X-linked non-lethal phenotypes, such as hypohidrotic ectodermal dysplasia of Christ-Siemens-Touraine, IFAP (ichthyosis follicularis-alopecia-photophobia) syndrome and X-linked dyskeratosis congenita. Analogous X-inactivation patterns have been documented in human bones, teeth, eyes and, possibly, the brain. Patterns that are distinct from the lines of Blaschko are also seen, such as the lateralization observed in CHILD (congenital hemidysplasia with ichthyosiform nevus and limb defects) syndrome, and the chequerboard pattern seen in women heterozygous for X-linked congenital hypertrichosis. Exceptional cases of either severe or absent involvement in a woman heterozygous for an X-linked trait can be explained by skewing of X inactivation. Some X-linked skin disorders are caused by genes that escape inactivation, which is why heterozygous female 'carriers' of these disorders do not show mosaicism. A well-known example is X-linked recessive ichthyosis due to steroid sulphatase deficiency, the locus for which is situated at the tip of the short arm of the X chromosome and does not undergo Lyonization. On the other hand, in the case of Fabry disease, the gene encoding alpha-galactosidase A is subject to inactivation. Remarkably, however, the skin lesions of women do not show a mosaic pattern.Conclusion: In the various X-linked skin disorders, affected women show quite dissimilar degrees of involvement and forms of manifestation because X inactivation may give rise to different patterns of functional mosaicism. Paradoxically, no such pattern is observed in women with Fabry disease. Like many X-linked diseases, Fabry disease should neither be called recessive nor dominant, because these dichotomous terms are obscured by the mechanism of X inactivation.     

The impact of concurrent X chromosome anomalies on diagnosis and bleeding phenotype in children with hemophilia: A single-institution case series

Hemophilia is an inherited X-linked bleeding disorder characterized by deficiencies of factors VIII or IX. Concomitant X chromosome disorders can impact bleeding phenotype, complicating timely diagnosis and disease management. Herein, we describe three cases of female and male pediatric patients with hemophilia A or B diagnosed between 6 days and 4 years old in the setting of skewed X chromosome inactivation, Turner syndrome, or Klinefelter syndrome. All of these cases had significant bleeding symptoms, and two patients required initiation of factor replacement therapy. One female patient developed a factor VIII inhibitor similar to that described in males with hemophilia A.     

X-chromosome inactivation and human genetic disease

The inactivation of one X-chromosome in females in early development is the process by which the effective dosage of X-linked genes is equalized between XX females and XY males. The mechanism that brings this about is the subject of intense research. The X-linked gene Xist is a key player, which is necessary but not sufficient for the initiation of X-inactivation. It codes for an untranslated RNA that coats the inactive X-chromosome, which takes on properties characteristic of heterochromatin, but how this change in chromatin is brought about remains unknown. Because of X-inactivation, females heterozygous for X-linked genes are mixtures of two types of cells and show a variable phenotype. The proportion s of the two types of cells can depart from equality due to cell selection either at the tissue or whole organism level. In rare cases, changes in the Xist gene can cause skewing of X-inactivation. A few genes escape from X-inactivation either wholly or partially.
Conclusion : X-chromosome inactivation is a physiological mechanism that equalizes gene-dosage effects on the sex chromosomes. The occurrence of this normal process affects the phenotype seen in females carrying X-linked mutant genes or chromosome anomalies.     

Carrier Detection in Agammaglobulinemia by X Chromosome Inactivation Analysis

Using a recently developed strategy to analyze patterns of X chromosome inactivation in cell populations, we found that two mothers and a sister were carriers in three atypical or sporadic cases of patients with agammaglobulinemia, two of whom were brothers. In this study, a phosphogiycerate kinase 1 (PGK1) gene probe was used to detect patterns of methylation of X-chromosome genes. A random pattern of X inactivation was observed in isolated peripheral blood granulocytes. In contrast, one of the two X chromosomes was preferentially active in the Epstein-Barr virus (EBV)-transformed peripheral B cells of the family members of these patients. The volume of the blood specimen could be significantly reduced using EBV-transfomed B cell lines which contained multiple clones. The analysis described here can be used to distinguish between X-linked agammaglobulinemia (XLA) and other forms of a- or hypogammaglobulinemia as well as to detect the carrier state.     

X-chromosome inactivation and human genetic disease

The inactivation of one X-chromosome in females in early development is the process by which the effective dosage of X-linked genes is equalized between XX females and XY males. The mechanism that brings this about is the subject of intense research. The X-linked gene Xist is a key player, which is necessary but not sufficient for the initiation of X-inactivation. It codes for an untranslated RNA that coats the inactive X-chromosome, which takes on properties characteristic of heterochromatin, but how this change in chromatin is brought about remains unknown. Because of X-inactivation, females heterozygous for X-linked genes are mixtures of two types of cells and show a variable phenotype. The proportions of the two types of cells can depart from equality due to cell selection either at the tissue or whole organism level. In rare cases, changes in the Xist gene can cause skewing of X-inactivation. A few genes escape from X-inactivation either wholly or partially. CONCLUSION: X-chromosome inactivation is a physiological mechanism that equalizes gene-dosage effects on the sex chromosomes. The occurrence of this normal process affects the phenotype seen in females carrying X-linked mutant genes or chromosome anomalies.     

Severe X-linked chronic granulomatous disease in two unrelated females

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by mutations of one of the subunits of phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leading to decreased or complete absence of neutrophil oxidative burst. We report the clinical and laboratory findings in two young unrelated females 14 and 9 years of age and natives of Tahiti and Reunion Islands, respectively, with severe X-linked granulomatous disease. In both cases, the infectious pattern was unusual, with convergent symptoms suggesting underlying mycobacterial infection. Functional analysis revealed low residual NADPH oxidase activity with about 5–10% of normal neutrophil population. De novo null mutations affecting the CYBB gene that encodes the gp91 protein were found in both cases in the heterozygous state (in patient 1, p.Arg130X in exon 5, and in patient 2, a novel insertion in exon 6, c.632_633insCATC). Methylation analysis confirmed that phenotype expression was linked to skewed X inactivation and showed that the de novo mutation arose on the maternally inherited chromosome in one case and on the paternally inherited chromosome in the other case. In conclusion, X-linked CGD carriers could therefore be at risk for severe infectious diseases depending on the skewed X inactivation pattern and the infectious context.     

Novel DCX mutation‐caused lissencephaly in a boy and very mild heterotopia in his mother

We describe a novel mutation in DCX in a family in which a proband boy had classical lissencephaly and his mother had extremely mild subcortical band heterotopia. No factors that would make the mother's symptoms milder, such as somatic mosaicism or skewed X chromosome inactivation, were observed. From this family, we conclude that a DCX mutation causes a pleiotropic phenotype in the female even if X chromosome inactivation pattern is not skewed, and the novel missense mutation in DCX produced relatively mild dysfunction of the doublecortin protein.     

Fragile X syndrome

Fragile X syndrome is the most common familial form of mental retardation. This X-linked disorder affects one in every 1000 males and one in every 2000 females. The female carrier rate in the general population is estimated to be 1/600. A fragile site at the distal long arm of the X chromosome (Xq 27.3) is the hallmark cytogenetic feature of the syndrome. Clinical features include physical as well as cognitive and neuropsychological deficits. Although fragile X syndrome follows an X-linked pattern of inherltance (which explains the predominance of affected males), females can also beaffected,Many inconsistencies exist between the genetic inheritance pattern of fragile X and traditional Mendelian inheritance tenets of most X-linked diseases. Due to recent molecular advances, our understanding of the perplexing genetic issues surrounding fragile X syndrome has grown and diagnostic techniques have become both reliable and readily available.     

X chromosome inactivation analysis to distinguish sporadic cases of X-linked agammaglobulinaemia from common variable immunodeficiency

The X chromosome inactivation analysis of eight female relatives was performed to elucidate the X chromosome gene defect of six male hypogammaglobulinaemic individuals. The patients had diminished numbers of circulating B-cells and no relevant family history. The methylation status of three X-linked genes, phosphoglycerate kinase, hypoxanthine phosphoribosyl transferase and DXS255, was determined on DNA from Epstein-Barr virus-transformed B-cell lines established from the female relatives. The methylation pattern of at least one gene was informative in all eight females examined. While both alleles were equally methylated in four of eight females, the remaining four female relatives of three hypogammaglobulinaemia patients exhibited a non-random methylation pattern in their B-cells, suggesting that these three patients represented sporadic cases of X-linked agammaglobulinaemia (XLA). The clinical or immunological status of these three patients did not differ from the remaining two who had early onset hypogammaglobulinaemia and who were tentatively diagnosed as having common variable immunodeficiency. The sixth patient had recurrent infections after undergoing surgical removal of a brain tumour at 22 years of age, although his immunological features did not distinguish him from the other patients. X chromosome inactivation analysis can be useful in differentiating XLA from hypogammaglobulinaemia in male patients.     

散发Rett综合征患儿新生MECP2突变亲源鉴定和X染色体失活15例分析

Objective Rett syndrome (RTT) is a neurodevelopmental disorder occurring almost exclusively in females as sporadic cases due to de novo mutations in the methyl-CpG-binding protein 2 gene ( MECP2 ). Familial cases of RTT are rare and are due to X-chromosomal inheritance from a cartier mother. Recently, DNA mutations in the MECP2 have been detected in approximately 84.7% of patients with RTT in China. To explain the sex-limited expression of RTT, it has been suggested that de novo X-linked mutations oecttr exclusively in male germ cells resulting therefore only in affected daughters. To test this hypothesis, we have analyzed the parental origin of mutations and the XCI status in 15 sporadic cases with RTT due to MECP2 molecular defects. Methods Allele-specific PCR was performed to amplify a fragment including the position of the mutation. The allele-specific PCR products were sequenced to determine which haplotype contained the mutation. It was then possible to determine the parent of origin by genotyping the single nucleotide polymorphism (SNP) in the parents. The degree of XCI and its direction relative to the X chromosome parent of origin were measured in DNA prepared from peripheral blood leucocytes by analyzing CAG repeat polymorphism in the androgen receptor gene (AR). Results Except for 2 cases who had a frameshifi mutation; all the remaining 13 cases had a C→T transition mutation. Paternal origin has been determined in all cases with the C→T transition mutation. For the two frameshift mutations, paternal origin has been determined in one case and maternal origin in the other. The frequency of male germ-line transmission in mutations is 93.3%. Except for 2 cases who were homozygotic at the AR locus, of the remaining 13 cases, 8 cases had a random XCI pattern; the other five cases had a skewed XCI pattern and they favor expression of the maternal origin allele. Conclusion De novo mutations in sporadic RTr occur almost exclusively on the paternally derived X chromosome and that this is most probably the cause for the high female: male ratio observed in sporadic cases with RTT. Random XCI was the main XCI pattern in sporadic RTT patients. The priority inactive X chromosome was mainly of paternal origin.     

Heterogeneity of clinical severity and molecular lesions in Aicardi syndrome

All patients with Aicardi syndrome are female or have a 47,XXY karyotype. This finding, along with a report of an Aicardi syndrome patient with an Xp22/autosome translocation, led to the hypothesis that Aicardi syndrome might be caused by an X-linked dominant, male-lethal mutation on the short arm of the X chromosome. To study this hypothesis, we investigated X chromosome inactivation patterns in peripheral lymphocytes from seven patients. We used two methods: methylation-sensitive restriction enzyme analysis and segregation of the active X chromosome in somatic cell hybrids. We found that three of seven cytogenetically normal girls with Aicardi syndrome had profoundly skewed X-inactivation in their lymphocytes, supporting the concept that Aicardi syndrome is X linked. Three of the five girls with the greatest degree of psychomotor retardation and the poorest seizure control had skewed X-inactivation. In contrast, the two highest-functioning children had random X-inactivation. We screened DNA using eight polymorphic probes from the Xp22 region but were unable to identify a deletion in any of the seven patients. Nonrandom X-inactivation in lymphocytes and possibly other tissues in some, but not all, patients with Aicardi syndrome may reflect heterogeneity of their molecular lesions.     

Carrier detection in typical and atypical X-linked agammaglobulinemia

We have recently demonstrated that B cells from obligate carriers of typical X-linked agammaglobulinemia (XLA) exhibit nonrandom X chromosome inactivation. The active X is always the X that does not carry the gene defect. To determine if this were also true in carriers of atypical XLA and to provide carrier detection for all women at risk of being carriers of XLA, we developed a technique that permits analysis of X chromosome inactivation in cells from any woman. This technique combines the production of somatic cell hybrids that selectively retain the active X chromosome with the use of X-linked restriction fragment length polymorphisms that permit the distinction of the two X chromosomes. Three obligate carriers of typical XLA and four women whose sons might be considered to have atypical or sporadic XLA were studied. B cell hybrids from all seven women demonstrated exclusive use a single X as the active X. In addition, B cell hybrids from four of eight women at 25% or 50% risk of being carriers exhibited nonrandom X chromosome inactivation, indicating that these women were also carriers of X-linked forms of hypogammaglobulinemia. These results illustrate a technique that can be used both to help define XLA and to provide carrier detection for all women at risk of being carriers of this disorder.     

Molecular analysis of X-linked agammaglobulinemia with growth hormone deficiency

To address the relationship between the gene (or genes) that causes the syndrome of X-linked hypogammaglobulinemia with isolated growth hormone deficiency and the gene responsible for typical X-linked agammaglobulinemia (XLA), we have used cytogenetics, examination of X chromosome inactivation patterns in potential carriers of the defect, and linkage analysis to study two unrelated families in which the affected males had isolated growth hormone deficiency and immunologic findings indistinguishable from those of typical XLA. A deletion could not be demonstrated in either family by G-banded karyotypes or flow cytometric analysis of metaphase chromosomes. Studies of X inactivation showed that mothers of affected boys from both families exhibited selective use of a single X chromosome as the active X chromosome in B cells but not T cells. This pattern is the same as that seen in obligate carriers of typical XLA. Linkage analysis demonstrated the most likely location for this gene (or genes) to be the midportion of the long arm of the X chromosome between DXS3 and DXS94. This segment of the X chromosome, which constitutes approximately 5% of the total X chromosome, encompasses the gene for XLA. These findings are consistent with the combination of XLA and growth hormone deficiency being caused by a small, contiguous, gene deletion syndrome involving the gene for XLA or an allelic variant of the gene for typical XLA.     

Duchenne muscular dystrophy in a 4-year-old girl due to heterozygous frame shift deletion of the dystrophin gene and skewed X-inactivation

X-linked recessive diseases affect males, whereas female carriers are generally asymptomatic.We report on a 4-year-old girl who presented with a classical phenotype of Duchenne Muscular Dystrophy (DMD), a severe X-linked recessive type of muscular dystrophy affecting boys in early childhood.A thorough diagnostic work-up revealed that this resulted from a heterozygous out-of frame deletion in the DMD-gene in combination with an X-inactivation ratio of <10:90 in blood leukocytes and muscle.The case exemplifies that a skewed X-inactivation pattern has to be taken into account as mechanism causing clinical symptoms in female carriers of X-linked recessive disorders.     

Biology of the X chromosome.

The biology of the X chromosome is unique, as there are two Xs in females and only a single X in males, whereas the autosomes are present in duplicate in both sexes. The presence of only a single autosome, which can occur as a result of an error in meiotic segregation, is invariably an embryonic lethal event. Monosomy for the X chromosome is viable because of dosage compensation, a system found in all organisms with an X:Y form of sex determination, which brings about equality of expression of most X-linked genes in females and males. In mammals, the dosage compensation system involves silencing of most of the genes on one X chromosome; it is called X chromosome inactivation. In this review, we focus first on recent advances in our understanding of the molecular basis of the X inactivation mechanism. Then we consider an unusual feature of X inactivation, the mosaic nature of the female and subsequent exposure to somatic cell selection.     

Advances in X-linked mental retardation

PURPOSE OF REVIEW: Mutations in genes on the X chromosome rival chromosome aberrations as a cause of mental retardation. Progress in the clinical and molecular delineation of X-linked mental retardation has outpaced progress in understanding autosomal mental retardation. This is a result in large part of the identification of large families in which mental retardation has segregated in an X-linked pattern and the greater ease with which molecular technologies can be applied to hemizygosity in males. RECENT FINDINGS: About one-third of the estimated 165 genes associated with syndromal mutations of genes on the X chromosome and one-fourth of the estimated 100 genes associated with nonsyndromal mutations of genes on the X chromosome have been identified. In a number of instances, the same gene is responsible for syndromal and nonsyndromal mutations of genes on the X chromosome. The molecular delineation of mutations of genes on the X chromosome has allowed certain conditions to be lumped together on the basis of allelism and has caused others that appear clinical similar to remain separate. SUMMARY: The clinical and molecular advances have allowed X-linked mental retardation to be more clearly delineated, have provided the means of confirmatory laboratory testing, and have ushered in an era of carrier testing, prenatal diagnosis, and prevention strategies.