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.     

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.     

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.     

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.     

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.     

A new case of autosomal recessive agammaglobulinaemia with impaired pre-B cell differentiation due to a large deletion of the IGH locus

Males with X-linked agammaglobulinaemia (XLA) due to mutations in the Bruton tyrosine kinase gene constitute the major group of congenital hypogammaglobulinaemia with absence of peripheral B cells. In these cases, blockages between the pro-B and pre-B cell stage in the bone marrow are found. The remaining male and female cases clinically similar to XLA represent a genotypically heterogeneous group of diseases. In these patients, various autosomal recessive disorders have been identified such as mutations affecting IGHM, CD79A, IGLL1 genes involved in the composition of the pre-B cell receptor (pre-BCR) or the BLNK gene implicated in pre-BCR signal transduction. In this paper, we report on a young female patient characterised by a severe non-XLA agammaglobulinaemia that represents a new case of Igmu defect. We show that the B cell blockage at the pro-B to pre-B cell transition is due to a large homologous deletion in the IGH locus encompassing the IGHM gene leading to the inability to form a functional pre-BCR. The deletion extends from the beginning of the diversity (D) region to the IGHG2 gene, with all JH segments and IGHM, IGHD, IGHG3 and IGHG1 genes missing. CONCLUSION: alteration in Igmu expression seems to be relatively frequent and could account for most of the reported cases of autosomal recessive agammaglobulinaemia.     

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.     

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.     

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.     

Early diagnosis in X-linked agammaglobulinaemia

The genetic transmission of X-linked agammaglobulinaemia (XLA) can be determined with high probability using closely linked DNA restriction fragment length polymorphisms (RFLP's). In a family known to be at risk for XLA in male offspring, RFLP analysis demonstrated that the mother was an XLA carrier and her newborn son was affected. The infant developed immunological deficiencies a few months later, confirming the diagnosis. RFLP analysis provides a method for carrier detection, prenatal diagnosis and presymptomatic diagnosis of XLA, which plays a significant role in prevention of the disease.Abbreviations XLA X-linked agammaglobulinaemia - RFLP restriction fragment length polymorphism - PBMC peripheral blood mononuclear cells - Ig Immunoglobulin - PWM pokeweed mitogen - SAC Staphylococcus aureus cowan A strain     

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.     

Report on the X-linked lymphoproliferative disease in an Australian family

X-linked lymphoproliferative disease is characterized by immune deficiency, particularly to the Epstein-Barr virus and by a tendency to develop fatal infectious mononucleosis, acquired hypogammaglobulinaemia or malignant lymphoma. This disorder has been diagnosed in three boys, two brothers and a maternally related cousin, residing in Australia. The proband presented at 6 years of age with fulminating infectious mononucleosis. His 9 year old male cousin had developed an ileal Burkitt lymphoma one year earlier. Immunological and molecular genetic evidence is presented to support our view that his younger sibling is also affected with this condition. DNA linkage studies using probes to DXS10 and DXS37 provide confirmatory evidence for the diagnosis in the proband's brother and information on carrier status in female family members.     

Molecular genetic haplotype segregation studies in three families with X-linked lymphoproliferative disease

Three families with X-linked lymphoproliferative disease were studied. Affected males clinically presented with severe or fatal infectious mononucleosis, acquired hypogammaglobulinaemia, hypergammaglobulinaemia M, and malignant lymphoma including Hodgkin disease. Haplotype analysis using various DNA markers from Xq25-q27 allowed the prediction of the carrier status in females and identification of the XLP status in asymptomatic males.     

中国X连锁无丙种球蛋白血症40例基因型表型相关性分析

目的 通过中国X连锁无丙种球蛋白血症（XLA）患儿临床表现、免疫功能评价、Bruton′s 酪氨酸激酶（BTK）的表达及BTK基因突变分析,分析基因型和表型间可能存在的关系。 方法 选取拟诊为XLA患儿,使用抗BTK单克隆抗体通过流式细胞技术分析单核细胞BTK蛋白表达。采用RT-PCR获得患儿cDNA,使用8对不同引物分2步扩增BTK cDNA,PCR产物测序。突变结果通过对DNA 外显子相应部位扩增、测序证实。并对确诊XLA患儿的母亲及家族中部分亲属进行BTK蛋白表达和BTK基因分析。 结果 ①40/50例原发性低丙种球蛋白血症患儿经BTK基因突变分析确诊为XLA,以错义突变（16例,40.0%）和无义突变（13例,32.5%)为主。②突变类型为错义突变的患儿平均起病年龄为（1.4±1.1）岁,其他突变类型患儿为（1.4±0.7）岁,差异无统计学意义（P=0.45）。错义突变的发生率随年龄的增长呈上升趋势,无义突变的发生率呈下降趋势。③34/40例（85.0%）B细胞<0.1%;4例（10.0%）B细胞在1%~2%,其中错义突变2例,无义突变1例, 剪接突变1例;2例（5.0%）B细胞为2%,均为错义突变。④血清IgG<3 g·L-1患儿BTK基因突变类型以错义突变和无义突变为主。⑤错义突变患儿BTK蛋白表达水平与其他突变类型无显著差异。⑥6/21例（28.6%）2031C/T多态性患儿伴有严重的关节炎,3/19例（15.8％）无多态性患儿有关节炎表现。⑦28/32例（87.5%）XLA患儿母亲为BTK基因杂合型。 结论 错义突变可能与确诊年龄较大有关,且某些位点的错义突变可能与较高的外周血B细胞数量和血清IgG水平及正常的BTK蛋白表达水平有关。BTK基因多态性（2031C/T）可能增加关节炎的风险。     

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.     

Composition of precursor B-cell compartment in bone marrow from patients with X-linked agammaglobulinemia compared with healthy children.

X-linked agammaglobulinemia (XLA) is characterized by a severe B-cell deficiency, resulting from a differentiation arrest in the bone marrow (BM). Because XLA is clinically and immunologically heterogeneous, we investigated whether the B-cell differentiation arrest in BM of XLA patients is heterogeneous as well. First, we analyzed BM samples from 19 healthy children by flow cytometry. This resulted in a normal B-cell differentiation model with eight consecutive stages. Subsequently, we analyzed BM samples from nine XLA patients. Eight patients had amino acid substitutions in the Bruton's tyrosine kinase (BTK) domain or premature stop codons, resulting in the absence of functional BTK proteins. In seven of these eight patients a major differentiation arrest was observed at the transition between cytoplasmic Ig(mu-) pre-B-I cells and cytoplasmic Ig(mu+) pre-B-II cells, consistent with a role for BTK in pre-B-cell receptor signaling. However, one patient exhibited a very early arrest at the transition between pro-B cells and pre-B-I cells, which could not be explained by a different nature of the BTK mutation. We conclude that the absence of functional BTK proteins generally leads to an almost complete arrest of B-cell development at the pre-B-I to pre-B-II transition. The ninth XLA patient had a splice site mutation associated with the presence of low levels of wild-type BTK mRNA. His BM showed an almost normal composition of the precursor B-cell compartment, suggesting that low levels of BTK can rescue the pre-B-cell receptor signaling defect, but do not lead to sufficient numbers of mature B lymphocytes in the peripheral blood.     

Variable expression in focal dermal hypoplasia. An example of differential X-chromosome inactivation

We encountered three women from three generations of the same family with features of focal dermal hypoplasia (FDH). Two of the patients, the proposita and her mother, demonstrated severe manifestations, including skin, dental, skeletal, and visceral abnormalities. The proposita's grandmother, the first family member affected, had very mild expression, with aplasia cutis congenita and dental caries as the only features expressed. This family illustrates both the marked variability of expression and the proposed X-linked dominant mode of inheritance of FDH. We postulate that early embryologic random inactivation of the X chromosome bearing the mutant gene responsible for FDH is the cause of the variable expression.     

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.