X-inactivation Pattern Research Articles

Further clinical delineation of the Börjeson–Forssman–Lehmann syndrome in patients with PHF6 mutations

Börjeson-Forssman-Lehmann syndrome is an X-linked condition caused by PHF6 mutations. The classical description of males with this disorder includes severe intellectual disability with epilepsy, microcephaly, short stature, obesity, hypogonadism, and gynecomastia. We present three males with PHF6 mutations whose features included deep-set eyes, large ears, coarse face, tapering fingers, and truncal obesity. Unlike the original description of the syndrome; however, the males described herein had varying degrees of intellectual disability and hypogonadism, were of normal to tall stature, had normal to large head sizes, and did not have seizures. This departure from the usual clinical description of Börjeson-Forssman-Lehmann syndrome is consistent with recent reports of males with mutations in PHF6. In addition, we describe the phenotype and X-inactivation pattern in two females heterozygous for PHF6 mutations, both of whom have mild features of the syndrome.

Non-random X chromosome inactivation in Aicardi syndrome.

Most females have random X-chromosome inactivation (XCI), defined as an equal likelihood for inactivation of the maternally- or paternally-derived X chromosome in each cell. Several X-linked disorders have been associated with a higher prevalence of non-random XCI patterns, but previous studies on XCI patterns in Aicardi syndrome were limited by small numbers and older methodologies, and have yielded conflicting results. We studied XCI patterns in DNA extracted from peripheral blood leukocytes of 35 girls with typical Aicardi syndrome (AIC) from 0.25 to 16.42 years of age, using the human androgen receptor assay. Data on 33 informative samples showed non-random XCI in 11 (33%), defined as a >80:20% skewed ratio of one versus the other X chromosome being active. In six (18%) of these, there was a >95:5% extremely skewed ratio of one versus the other X chromosome being active. XCI patterns on maternal samples were not excessively skewed. The prevalence of non-random XCI in Aicardi syndrome is significantly different from that in the general population (p < 0.0001) and provides additional support for the hypothesis that Aicardi syndrome is an X-linked disorder. We also investigated the correlation between X-inactivation patterns and clinical severity and found that non-random XCI is associated with a high neurological composite severity score. Conversely, a statistically significant association was found between random XCI and the skeletal composite score. Correlations between X-inactivation patterns and individual features were made and we found a significant association between vertebral anomalies and random XCI.

A possible association between genotypes and early signs of Rett disorder

Introduction: An apparently normal early development was one of the initial criteria for classic Rett syndrome. However, several investigators considered Rett syndrome to be a developmental disorder manifesting very soon after birth. Different types and positions of the MECP2 mutations and a variable pattern of x-inactivation contribute to the range of severity of this disorder. Methods: Videos of 32 Rett cases of various genotypes were carefully assessed for movements, posture and behaviour during the first 6 months of life. All signs, which deviated from the normal standard, were recorded meticulously. Special attention was paid to the face, the hands and to body movements. Results: A detailed analysis clearly demonstrated the following early abnormal signs: long lasting tongue protrusion, asymmetric eye opening and closing, bursts of abnormal facial expressions, bizarre smile, abnormal finger movements, hand stereotypies, an abnormal quality of general movements, postural stiffness, tremor and stereotyped body movements. R168X and R255 mutations were associated with the earliest onset (first days of life) of these abnormal signs. Discussion: Rett disorder is manifest within the first months of life. Those MECP2 mutations associated with the most severe clinical phenotype were also associated with an earlier onset of abnormal signs. (FWF P19581-B02, Lanyar Foundation P325).

A small and active ring x chromosome in a female with features of Kabuki syndrome

A ring X chromosome is found in about 6% of patients with Turner syndrome (TS), often with mosaicism for a 45,X cell line. Patients with this karyotype are reported to have a higher incidence of a more severe phenotype including mental retardation. In fact, some studies have shown a correlation between this severity and the presence or absence of an intact and functional X inactivation center (XIST). However, the phenotype of the individuals with r(X) cannot be entirely defined in terms of their X-inactivation patterns. Nevertheless, a small group of these patients have been described to manifest clinical features reminiscent of the Kabuki syndrome. Here we present a female patient with clinical features resembling Kabuki syndrome and a mos 45,X/46,X,r(X) karyotype. Methylation analyses of polymorphic alleles of the androgen receptor gene showed that both alleles were unmethylated suggesting an active ring chromosome. A specific X chromosome array CGH was performed estimating the size of the ring to be 17 Mb, lacking the XIST gene, and including some genes with possible implications in the phenotype of the patient.

A Model Incorporating Potential Skewed X‐Inactivation in MZ Girls Suggests that X‐Linked QTLs Exist for Several Social Behaviours Including Autism Spectrum Disorder

Sex differences in the frequency and patterns of behaviours are frequently observed and largely unexplained. We have investigated the possible role of X-linked genes in the aetiology of social behaviour problems, including those involved in autistic spectrum disorders. A novel approach has been implemented. This is based on predictions following from stochastic patterns of X-inactivation of lower concordance of monozygous female (MZF) twins than MZM twins for behaviours underpinned by X-linked QTLs and the converse that DZF twins are expected to correlate more strongly for X-linked traits than DZM twins because unlike males, females always have at least one X chromosome in common. These expectations were tested in an ongoing longitudinal cohort study in which all twins born in England and Wales between 1994 and 1996 were invited to take part. 1000 each of MZF, MZM, DZF and DZM pairs from TEDS were tested at 7 and 8 years of age. The results suggest the persistent influence of X-linked genes on cognition and social behaviour problems, including those involved in autistic spectrum disorders, from early to middle childhood. This emphasises the potential importance of X-linked genes in the developmental trajectories of behaviour and mental health and the need to stratify genetic analysis of behaviours by gender.

Somatic mosaicism for a PDHA1 mutation in a female with pyruvate dehydrogenase deficiency

Somatic mosaicism for a mutation in the X-linked PDHA1 gene was found in a girl who presented with manifestations of pyruvate dehydrogenase deficiency. Mutation in the PDHA1 gene was suggested by a mosaic pattern of E1alpha subunit immunostaining; however, initial screening of cDNA and the exons and intron-exon boundaries yielded only normal sequence, apart from a heterozygous 4 bp insertion in intron 10. This was considered to be a polymorphism as it is also present in her unaffected mother who has normal enzyme activity and uniform E1alpha immunostaining in fibroblasts. Detailed genetic analysis, which included isolation of cloned fibroblasts expressing the mutant X chromosome, resulted in the identification of a base substitution in the acceptor splice site of intron 9 which leads to activation of a cryptic upstream splice site. The proportion of cells expressing the mutation was then determined by direct analysis of the X-inactivation pattern. Genetic diagnosis in this unique case of PDHA1 somatic mosaicism was complicated by the absence of an abnormal transcript in primary fibroblasts, the presence of three different alleles and an X-inactivation pattern favouring expression of the normal, paternal, X chromosome. Although the mutation was only present in a proportion of cells, and only expressed in a subset of these due to random X-inactivation, the resulting enzyme defect was sufficient to be clinically apparent.

A pathogenic dyskerin mutation impairs proliferation and activates a DNA damage response independent of telomere length in mice

Telomeres are nucleoprotein structures that cap the ends of chromosomes, protecting them from exonucleases and distinguishing them from double-stranded breaks. Their integrity is maintained by telomerase, an enzyme consisting of a reverse transcriptase, TERT and an RNA template, TERC, and other components, including the pseudouridine synthase, dyskerin, the product of the DKC1 gene. When telomeres become critically short, a p53-dependent pathway causing cell cycle arrest is induced that can lead to senescence, apoptosis, or, rarely to genomic instability and transformation. The same pathway is induced in response to DNA damage. DKC1 mutations in the disease dyskeratosis congenita are thought to act via this mechanism, causing growth defects in proliferative tissues through telomere shortening. Here, we show that pathogenic mutations in mouse Dkc1 cause a growth disadvantage and an enhanced DNA damage response in the context of telomeres of normal length. We show by genetic experiments that the growth disadvantage, detected by disparities in X-inactivation patterns in female heterozygotes, depends on telomerase. Hemizygous male mutant cells showed a strikingly enhanced DNA damage response via the ATM/p53 pathway after treatment with etoposide with a significant number of DNA damage foci colocalizing with telomeres in cytological preparations. We conclude that dyskerin mutations cause slow growth independently of telomere shortening and that this slow growth is the result of the induction of DNA damage. Thus, dyskerin interacts with telomerase and affects telomere maintenance independently of telomere length.

Cytogenetic and molecular studies of an X;21 translocation previously diagnosed as complete monosomy 21

We studied a child with apparent monosomy of chromosome 21. Cytogenetic, FISH and microsatellite analyses revealed a 45,X,−21,+der(X)t(X;21)(q25;q21.1) karyotype resulting from a de novo, unbalanced, X;21 non-reciprocal translocation of paternal origin, with partial monosomy of chromosomes 21 and X. An extreme, skewed X-inactivation pattern of the der(X) chromosome was demonstrated. Skewed inactivation probably accounted for a mild phenotype with respect to Xq25 → qter deletion while propagation of inactivation to the adjacent 21q region may account for mild clinical features associated to distal 21q monosomy.

Lyonization pattern of normal human nails

To examine the X-inactivation patterns of normal human nails, we performed the human androgen receptor gene assay of DNA samples extracted separately from each finger and toe nail plates of nine female volunteers. The X-inactivation pattern of each nail was unique and constant for at least 2 years. The frequency of nails with one of the two X-chromosomes exclusively inactivated was 25.9%. In the nails composed of two types of cells with either one X-chromosome inactivated, the two cell types were distributed in patchy mosaics. These findings suggest that the composition of precursor cells of each nail is maintained at each site at least through several cycles of regeneration time, and that the nail plate has a longitudinal band pattern, each band consisting of cells with only one of the two X-chromosomes inactivated. Using the frequency of nails with one of two X-chromosomes exclusively inactivated, we estimated the number of progenitor cells that gave rise to the nail plate during development to be about 3, under the assumption that the process follows the binominal distribution model. A strong correlation observed among the big, index and little fingers, and among the corresponding toes suggests an interesting interpretation concerning their morphogenetic process.

Circulating Angiogenic Precursors in Idiopathic Pulmonary Arterial Hypertension

Vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) involves hyperproliferative and apoptosis-resistant pulmonary artery endothelial cells. In this study, we evaluated the relative contribution of bone marrow-derived proangiogenic precursors and tissue-resident endothelial progenitors to vascular remodeling in IPAH. Levels of circulating CD34+ CD133+ bone marrow-derived proangiogenic precursors were higher in peripheral blood from IPAH patients than in healthy controls and correlated with pulmonary artery pressure, whereas levels of resident endothelial progenitors in IPAH pulmonary arteries were comparable to those of healthy controls. Colony-forming units of endothelial-like cells (CFU-ECs) derived from CD34+ CD133+ bone marrow precursors of IPAH patients secreted high levels of matrix metalloproteinase-2, had greater affinity for angiogenic tubes, and spontaneously formed disorganized cell clusters that increased in size in the presence of transforming growth factor-beta or bone morphogenetic protein-2. Subcutaneous injection of NOD SCID mice with IPAH CFU-ECs within Matrigel plugs, but not with control CFU-ECs, produced cell clusters in the Matrigel and proliferative lesions in surrounding murine tissues. Thus, mobilization of high levels of proliferative bone marrow-derived proangiogenic precursors is a characteristic of IPAH and may participate in the pulmonary vascular remodeling process.

Skewed X-Chromosome Inactivation in Greek Women with Idiopathic Recurrent Miscarriage

Objectives: To investigate if skewed X-chromosome inactivation (XCI) is associated with unexplained recurrent miscarriage (RM) in Greek women. Methods: This was a prospective case-control study. A methylation-sensitive assay was used to investigate the X-inactivation pattern of women with unexplained RM and controls. Results: Fifty-six of the 74 patients (75.7%) and 55 of 80 controls (68.8%) were informative. Among the informative cases, 6/56 (10.7%) women showed extreme XCI (>90%) and among the informative controls, 2/55 (3.6%) showed extreme XCI. Conclusions: In the present study, women with unexplained RM showed a statistically nonsignificant increase in skewed XCI prevalence (10.7%) compared with control women (3.6%; p = 0.271).

X chromosome inactivation patterns in patients with idiopathic premature ovarian failure

X chromosome aberrations have been reported as the cause of extremely skewed X chromosome inactivation (XCI). The purpose of this study was to investigate whether skewed XCI is associated with idiopathic premature ovarian failure (POF). The XCI status was evaluated in Korean women by the methylation assay of androgen receptor locus in 126 idiopathic POF patients (35.3 +/- 13.9 years old, mean +/- SD) and 126 age-matched controls (35.2 +/- 13.9 years). The incidence of skewed XCI in POF group was compared with that of control. The correlation between age and skewed XCI was also evaluated within both groups. The incidence of extremely skewed XCI (>or=90%) was 3.9 versus 2.7% (P = 0.710) in POF and control group, respectively. No significant differences were found in the incidence of skewed XCI on all three levels (>or=90, >or=80 and >or=70%) compared between these two groups. The calculation of correlation coefficients showed that, in both POF and control group, there were no significant correlations between age and XCI ratio. Neither was there increasing tendency of skewed XCI according to the increase of age in both groups. Furthermore, there were no significant differences when the XCI ratios were analysed according to the age of onset of ovarian failure. The incidence of skewed XCI in Korean POF population was not significantly different from control, implying that skewed XCI may not be associated with idiopathic POF. There were also no significant correlations between age and skewed X-inactivation patterns in both groups.

Correlation between Clinical Phenotypes and X-inactivation Patterns in Six Female Carriers with Heterozygote Vasopressin Type 2 Receptor Gene Mutations

About 90% of patients with congenital nephrogenic diabetes insipidus (NDI) have vasopressin type 2 receptor (V2R) gene mutations that are inherited in an X-linked recessive manner. Although most female carriers are asymptomatic, some female carriers show polydipsia and polyuria. The reason why female carriers show NDI symptoms is explained by skewed X-inactivation. We studied X-inactivation patterns of six female carriers with heterozygote V2R gene mutations. The X-inactivation pattern in peripheral blood leukocytes was examined using methylation analysis of the polymorphic CAG repeat in the androgen receptor gene. Two asymptomatic female carriers showed random X-inactivation (61.9% and 60.7%). Skewed X-inactivation patterns (71.6%, 79.4%, and 91.2%) occurring preferentially to normal X alleles were recognized in three female carriers who showed clinical NDI symptoms. However, in one female carrier who showed clinical NDI symptoms, random X-inactivation (55.4%) was recognized. In conclusion, the clinical NDI phenotypes may correlate with the X-inactivation patterns in female carriers with heterozygote V2R gene mutations. However, in some female carriers, we cannot predict the clinical phenotypes by the evaluation of the X-inactivation patterns in peripheral blood leukocytes, because X-inactivation ratios within an individual are sometimes different between tissues.

In neuroblastic tumours, Schwann cells do not harbour the genetic alterations of neuroblasts but may nevertheless share the same clonal origin

Neuroblastic tumours are composed of variable proportions of neuroblasts and Schwann cells. Whether both components share a common neoplastic origin is highly debated and discrepant results have been reported about the presence of tumour-related genetic alterations in Schwann cells. We have used X-methylation analysis and array-CGH to investigate contiguous Schwannian and neuroblastic areas in tumours with a nodular pattern. A skewed X inactivation was observed in four out of five stromal components. Interestingly, in these four cases, the X-inactivation profiles of the neuroblastic components were identical to the matched stromal areas. However, whereas all neuroblastic areas displayed chromosomal imbalances, no alteration was found in any Schwann cell components. Similarly, no alteration was observed in a series of 19 tumours with a single stroma-rich component, which occasionally exhibited a skewed X-inactivation pattern (3/17 informative tumours). Altogether, this indicates that most stroma-rich tumours display a polyclonal proliferation and that Schwann cells do not derive from neuroblasts. However, in tumours with both stroma-rich and -poor components, our results suggest that cells from both areas share a common progenitor.

Timing of Twinning, X-Inactivation and Sex Proportion at Birth

AbstractTwins are not a homogeneous group. According to zygosity and chorionicity essential differences exist. The lower sex proportion at birth is due to the monozygotic twins, especially the monoamnionic variety. X-inactivation patterns in monozygotic twin girls are totally symmetrical in monoamnionic pairs, almost symmetrical in monochorionic diamnionic pairs and can be very asymmetrical in the dichorionic variety.

Dissecting the molecular mechanisms in craniofrontonasal syndrome: differential mRNA expression of mutant EFNB1 and the cellular mosaic

Craniofrontonasal syndrome (CFNS) is an X-linked malformation syndrome with variable phenotype that is caused by mutations in the ephrin-B1 gene (EFNB1). Over 50% of EFNB1 mutations result in premature termination codons that may elicit mRNA degradation by the nonsense-mediated decay pathway. To assess the effects of various mutations at the transcript level, expression of EFNB1 mRNA was studied by RT-PCR in fibroblast cultures established from CFNS female patients. Compared to the wild-type and two missense mutation alleles, severe depletion of transcripts was observed for mutant alleles harbouring either splice site mutation c.407-2A>T at the exon 2/3 boundary or frameshift mutation c.377_384delTCAAGAAG in exon 2. In contrast, escape from mRNA decay was observed for mutation c.614_615delCT, which generates a premature termination codon close to the 3'-end of the penultimate exon 4 disobeying the '50-55 bp' rule. These results suggest differential degradation of mutant EFNB1 transcripts by the nonsense-mediated mRNA decay pathway. Although the clinical phenotypes of the patients were not highly suggestive of a phenotype-genotype correlation, the two female patients were diagnosed with diaphragmatic hernia harbouring putative ephrin-B1 truncating mutations. Previously, disease manifestation in heterozygous females had been attributed mainly to cellular interference of divergent cell populations expressing wild-type or mutant EFNB1, depending on the pattern of X-inactivation. Upon clonal expansion of patient cells with either the wild-type or mutant EFNB1 on the active X-chromosome, we were able to separate mutant and wild-type EFNB1-expressing cells in vitro, further supporting the concept of cellular interference in CFNS.

X chromosome inactivation in females with multiple sclerosis

The aetiology of multiple sclerosis (MS) is unknown. Autoimmune mechanisms are most probably involved. Loss of immunological tolerance to self-antigens is a common feature of autoimmune disorders. Response to X-linked self-antigens could be influenced by X-chromosome inactivation, and contribute to the gender bias observed in autoimmune disorders. Previous studies have indicated an association between skewed X inactivation and autoimmune thyroid disease and scleroderma. To investigate a potential role of X inactivation in MS, we compared the X-inactivation pattern in 568 female MS patients with controls. We found no difference in degree of skewing between patients (median 64%) and controls (median 65%) (P = 0.474). The X-inactivation pattern did thus not explain the female predominance of MS patients in general. As the aetiology of different subgroups of MS may differ, patients were grouped according to disease course: relapsing-remitting (RR-MS), secondary progressive (SP-MS) and primary progressive (PP-MS). A comparison of the X-inactivation pattern between subgroups indicated a possible difference in degree of skewing between patients with a progressive versus a relapsing course (P = 0.05).

Mother and daughter with a terminal Xp deletion: Implication of chromosomal mosaicism and X-inactivation in the high clinical variability of the microphthalmia with linear skin defects (MLS) syndrome

The microphthalmia with linear skin defects (MLS or MIDAS) syndrome is a rare X-linked dominant inherited disorder with male lethality, associated with segmental aneuploidy of the Xp22.2 region in most of the cases. However, we recently described heterozygous sequence alterations in a single gene, HCCS, in females with MLS. Beside the classical MLS phenotype, occasional features such as sclerocornea, agenesis of the corpus callosum, and congenital heart defects can occur. Although the majority of cases are sporadic, mother-to-daughter transmission has been observed and a high intra- and interfamilial phenotypic variability exists. We describe an asymptomatic mother and her daughter presenting with the typical features of MLS syndrome. By cytogenetic analysis both females were found to have a terminal Xp deletion with the breakpoint in Xp22.2, mapping near to or within the MSL3L1 gene which is located centromeric to HCCS. FISH analysis revealed that the mother is a mosaic with 45,X[11]/46,X,del(X)(p22.2)[89], while in all cells of the MLS-affected daughter a hybridization pattern consistent with a 46,X,del(X)(p22.2) karyotype was detected. By haplotype analysis we identified the paternal X chromosome of the mother to carry the terminal Xp deletion. X-inactivation studies showed a completely skewed pattern in mother and daughter with the deleted X chromosome to be preferentially inactivated in their peripheral blood cells. We suggest that both chromosomal mosaicism as well as functional X chromosome mosaicism could contribute to the lack of any typical MLS feature in individuals with a heterozygous MLS-associated mutation. The 45,X cell population, that most likely is also present in other tissues of the mother, might have protected her from developing MLS. Nonetheless, a non-random X-inactivation pattern in favor of activity of the wild-type X chromosome in the early blastocyte could also account for the apparent lack of any disease sign in this female.

P115 Nilotinib (AMN 107) plus involved field irradiation therapy in extramedullary lymphoid and medullary myeloid blast crisis of chronic myeloid leukemia

Our current understanding of the pathogenesis of the myeloproliferative disorders is based on the assumption that they represent a clonal disorder resulting from transformation of a haematopoietic stem cell. Clonality assays exploit the fact that female cells have only one active X-chromosome. Methods for determining X chromosome inactivation have been devised at the protein (G6PD isoenzymes), DNA (HUMARA; phosphoglycerate kinase (PGK); hypoxanthine-phosphoribosyl transferase (HRPT)) and RNA (G6PD; P55; IDS) levels. In this type of RNA assay the product of the active X chromosome is quantified by studying polymorphisms present in mRNA. The presence of skewed lyonization in normal females is a potential limitation to the method, although the use of T cells as a control makes it possible to distinguish clonal haematopoiesis from skewed lyonization. However, the phenomenon of acquired skewing in normal elderly women means that X-inactivation patterns in elderly patients must be interpreted with caution. Clonality studies have been conducted in polycythaemia vera (PV) and essential thrombocythaemia (ET) patients. They usually demonstrate a clonal X-inactivation pattern in granulocytes and/or platelets but a polyclonal pattern in T cells. However, in both ET and PV a significant minority of patients exhibit polyclonal haematopoiesis with polyclonal patterns in granulocytes/platelets. Female patients with polyclonal haematopoiesis differ from those with clonal haematopoiesis in terms of age and platelet count and possibly in their requirements for treatment. This new technology for the investigation of the MPD seems promising for understanding certain clinical aspects of these diseases and may be introduced for evaluation of new modalities of treatment.

A non‐ancestral RPGR missense mutation in families with either recessive or semi‐dominant X‐linked retinitis pigmentosa

Most X-linked diseases show a recessive pattern of inheritance in which female carriers are unaffected. In X-linked retinitis pigmentosa (XLRP), however, both recessive and semi-dominant inheritance patterns have been reported. We identified an Israeli family with semi-dominant XLRP due to a missense mutation (p.G275S) in the RPGR gene. The mutation was previously reported in two Danish families with recessive XLRP. Obligate carriers from the two Danish families had no visual complaints and normal to slightly reduced retinal function, while those from the Israeli family suffered from high myopia, low visual acuity, constricted visual fields, and severely reduced electroretinogram (ERG) amplitudes. The disease-related RPGR haplotype of the Israeli family was found to be different from the one found in the two Danish families, indicating that the mutation arose twice independently on different X-chromosome backgrounds. A series of genetic analyses excluded skewed X-inactivation pattern, chromosomal abnormalities, distorted RPGR expression level, and mutations in candidate genes as the cause for the differences in disease severity of female carriers. To the best of our knowledge, this is the first detailed analysis of an identical mutation causing either a recessive or a semi-dominant X-linked pattern of disease in different families. Our results indicate that an additional gene (or genes), linked to RPGR, modulate disease expression in severely affected carriers. These may be related to the high myopia concomitantly found in affected carriers from the Israeli family.